AABS 3.2 (2016)

eISSN: 2349-6991 pISSN: 2455-0396

Annals of Applied BioSciences An International, Open access, Indexed, Peer-reviewed Journal

Vol. 3, Issue 2, April-June 2016

Editor-in-Chief Dr Shelly Sehgal Dr Dipti Agrawal

Pacific Group of eJournals

Annals of Applied Bio-Sciences Editor in Chief

Professor & Dean, School of Environment and Sustainable Development, Central University Gujarat, India Dr Igor Iuco Castro-Silva Professor, Clinica Odontologica, Faculdade de Ciencias do Tocantins, Brazil Kapil Agarwal Engineer, Nagoya City, Aichi, Japan Dr Devesh Palharya Consultant Pathologist, Bhopal, Madhya Pradesh, India Dr D A Bhiwgade Dept. of Biotechnology & Bioinformatics, Padmashree Dr. D. Y. Patil University, Navi Mumbai, India Dr Arpana Haritwal Consultant, Obs. & Gynaecology, Saket City Hospital, New Delhi, India Dr Radhika P Kamdar Emory University School of Medicine, Georgia, United States Dr Saba Hasan Asst. Prof. Amity University, Lucknow, Uttar Pradesh, India Dr Manav Kapoor Assistant Professor Neuroscience, Icahn School of Medicine, Mount Sinai, New York, NY, 10029, United States

Dr Shelly Sehgal Specialist Pathologist, Department of Pathology, SDN Hospital, Delhi, India Dr Dipti Agrawal Department of Gynecology and Obstetrics, SDN Hospital, Delhi, India

Co-Editor in Chief

Dr Sompal Singh Specialist Pathologist, Dept. of Pathology, N D M C Medical College & Hindu Rao Hospital, Delhi, India Dr Bhupendra Pushkar Asst. Professor, Dept. of Biotechnology, University of Mumbai, India

Associate Editors

Dr Vijayabhaskar Varadarajalu Chennai, Tamil Nadu, India Dr B K Guha Associate Professor (Anatomy), NSCB Medical College, Jabalpur, Madhya Pradesh, India Dr Hariom Gupta Associate Professor, SSMC and SGMH, Rewa, Madhya Pradesh, India Dr Shashikant Agarwal Associate Prof. JMC, Jhalawar, Rajastjhan, India

Managing Editor

Dr Prashant Goyal Director-Laboratory, Accuprobe Healthcare and Diagnostics, Delhi, India

Editorial Board

Dr Francesco Merolla Advanced Biomedical Sciences, University of Naples, Federico II, Italy Dr Chandrassegar Saravanan Novartis Institutes for Biomedical Research, Technology Square, Cambridge, MA, United States Dr Nandhakumar Balakrishnan College of Veterinary Medicine, North Carolina State University, Raleigh, United States Prof. M H Fulekar

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India) Website: www.pacificejournals.com

www.pacificejournals.com/aabs E-mail: contactus@pacificejournals.com

I

Annals of Applied Bio-Sciences Disclaimer

Annals of Applied Bio-Sciences (AABS) is an international, fast double-blind peer-reviewed, indexed, open access, online and print multidisciplinary journal with high impact factor (2.9254) and IC value (56.90), published by ‘Pacific group of e-Journals’ (PaGe), an ISO 9001:2008 Certified academic publishing house, with a quick post-acceptance online publication.

1.

AABS is a multidisciplinary journal in the all fields of sciences including Medicine(Anatomy, Anesthesia, Cardiology, Community Medicine, Dermatology, Internal Medicine, Neonatology, Oncology, Orthopedics, Pediatrics, Physiology, Plastic Surgery, Psychiatry, Radiology, Surgery, ENT, Nutrition, Anatomy, Forensic , Genetics, Obstetrics And Gynecology, Ophthalmology, Pediatric Surgery, Nephrology, Urology, Neurology etc.) and Dentistry, Nursing and Health Professions, Biological sciences, Biotechnology, Molecular biology, Cell biology, Genetic engineering, Biochemistry, Pharmacology, Pharmaceutical Science, Toxicology, Immunology, Microbiology, Virology, Veterinary Medicine, Veterinary Science, Agricultural, Environmental science, Ecology and Ecotoxicology, Biophysics, Biomedical sciences, Computational biology, Biological anthropology and Biological education etc.

Publication Frequency

Quarterly with continuous publication. Articles are published online in the current volume of the Journal as soon as they are accepted. However, the issue is published in printed form only at the end of the relevant quarter.

2.

Copyright •

•

•

Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work’s authorship and initial publication in this journal.

3.

4.

Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal’s published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.

Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.

II

Thoughts, Language and examples in published research papers are entirely of Authors of Research Papers. It is not necessary that both Editor and Editorial Board are satisfied by the research paper.

The responsibility of the matter of the research paper/ article is entirely of author. Scientific knowledge is ever changing. As new research and experiences broaden our knowledge, old concepts may be required to be looked into afresh. The Authors and editors of the material herein have consulted sources believed to be reliable in their efforts to provide information that is complete and in accord with the standards accepted at the time of publication.

However, in view of the possibility of human error by the authors, editors, or publisher, nor any other who has been involved in the preparation of the work warrants that the information contained herein in every aspect accurate or complete, and they are not responsible for any errors or omissions or the results obtained from the use of such information. Readers are advised to confirm the information contained herein with other sources.

In any condition and due to any reason, if any Educational/Research Institution (College or University) denies accepting the research paper published in AABS, it will not be the responsibility of the Editor, Publisher or the Management.

Only the First author is entitled to receive the print copies of all co-authors.

All the legal disputes related to PaGe are subject to Delhi Jurisdiction only

Table of Contents Review Articles Decontamination of Water Resources through Sustainable Ecological Sanitation of Night Soil With Production of Biofertilizer Pramod Ramkrishna Chaudhari, B K Jha, Sanyogita Verma, Dhiraj Kumar Singh

R11-R16

Original Article Frequency of mucous retention cyst of the maxillary sinus in patients need implants A118-A121 using Cone beam Computed tomography in Tabriz Masoumeh Johari, Farzad Esmaeli, Sahar KhademNejad, Ali Zarandi

Estimation of Sonographic Umbilical Cord Area and Its Correlation with Birth Weight A122-A127 in Gestational Diabetes Mellitus.

Nidhi Jain, Abha Singh Mediastinum: A Pandora’s Box: a study of mediastinal lesions at tertiary hospital of A128-A131 South Canara.

Sheikh Tousif Reza, Kishan Prasad H.L., Jayaprakash Shetty K., Harish S. Permi, Sunil Kumar Y, Swaroop S. Shasidhar Evaluation of the role and utility of neuroimaging in new onset seizures presenting A132-A138 to the Emergency Department Lalit Kumar, Vipin Kumar, Hardeep Singh Gill, G Avasthi, Gagandeep Singh, Rajesh Mahajan Activation Energy and Q10 values at various temperature ranges for alpha amylase A139-A142 activity in Telescopium telescopium Sharvari Nilesh Kukardtar, Prakash V Desai Frequency of Beta thalassemia trait in pediatric age group in a tertiary care hospital A143-A150 in south India B N Krishnamurthy, S R Niveditha, Poornima Shankar Histopathological Spectrum of endometrial changes in women presenting with A151-A157 abnormal uterine bleeding with special reference to endometrial malignancies : A two years hospital based study Riju Rani Deka, Tanma Saikia, Amitabh Handique, Basanta Sonowal Clinical presentation of dengue outbreak 2015, Haryana, India: a prospective A158-A163 observational study Navtej Singh, Jyotsna Singh Effect of Vit. B12 supplementation on auditory neuropathy in type 2 Diabetic A164-A169 patients assesssed by Brainstem Auditory Evoked Response Manish Bansal, Manish Kumar, P K Maheshwari, Prabhat Agrawal, Ritu Agrawal Mycological analysis of 150 cases of dermatophytosis of skin, hair and nail attending A170-A182 the outpatient department of skin and venereology Bindu Mitruka, Amarjit Kaur Gill, Narinder Kaur, Ravinder K Mittal, Anchal Mahajan, Amandeep Kaur Patterns and Demographic Distribution of Hemoglobinopathies in North A183-A188 Maharashtra Manjusha Punjaji Tambse, Maya Suresh Vasaikar, Sunil Santaram Chavan A histopathological study of non-neoplastic gall bladder diseases with special A189-A195 reference to mucin histochemistry Samriddhi Sood, Rajnish Kumar, Anupam Varshney, Veena K Sharma, Alok Mohan, Bharat Wadhwa, Kanchan Kamini

III

Morphological spectrum of changes in gall bladder in correlation to various types of A196-A202 gallstones: a study of 100 cases Meenakumari Gopalakrishnan, Sharmila Thilagavathy, Kamaleshwari ., Jeyanthi ., Shifa ., Raasi .

Comparison of color match to Noritake and Vitapan classical shade guides of three A203-A206 porcelain systems Alireza Pornasrollah, Seyyed Amin Mosavi, Hesam Khandero, ali zarandi Evaluation of Her- 2 neu over expression in morphological variants of cervical A207-2013 carcinomas using immunohistoichemistry: A study of 25 cases.

Nimisha Sharma, Ankit Kaushik, Sumanashree ., Uma Sharma Effects of Vitamin-D Supplementation in Vitamin-D Deficient, near normal HbA1c A214-A117 diabetic patients Prashant Prakash, Manish Kumar Bansal, Ashish Gautam, Abhishek Raj, Shalini Upadhyay, Rosmy Jose

Case Reports

Sudipta Kar’s Modification of Guide Flange Prosthesis for mandibular repositioning Sudipta Kar Peripheral Ossifying Fibroma : A case report Soumya Purkait, Prasanta Bandyopadhyay, Bakul Mallick, Indrasri Das, Dipayan Das Management of Aberrant Frenum: series of cases Suchi Suvra Bagchi, Puja Sarkar, Prasanta Bandyopadhyay Adenomyoepithelioma arising in axillary breast tissue: a diagnostic rarity: case report with literature review Mega Lahori, Sakul Gupta, Bawana Raina, Arvind Khajuria Rare case of Ovarian Ectopic Pregnancy Shikha Joshi, Dipti Agrawal, Sunita Fotedar

Letter to Editor Mucopedermoid Carcinoma of Labial Mucosa

Shahela Tanveeer, Karthik Kv, Nishanth. P, Syed Afroz, Charu Suri, Shravan P

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India)

Website: www.pacificejournals.com

www.pacificejournals.com/aabs

E-mail: contactus@pacificejournals.com

IV

C25-C29 C30-C33 C34-C39 C40-C43 C44-C46 L1-L4

Review Article Decontamination of Water Resources through Sustainable Ecological Sanitation of Night Soil With Production of Biofertilizer Pramod Ramkrishna Chaudhari1*, B.K. Jha1, Sanyogita Verma2, Dhiraj Kumar Singh1 Grass Roots Research and Creation India (P) Ltd., Noida, Uttar Pradesh, India 2 Anand Niketan College, Anandwan, Warora, Maharashtra, India

1

Keywords: Water Pollution, Night Soil, Waterless Sanitation, Biofertilizer

ABSTRACT India and certain other countries have the legacy of using environmental friendly and non-polluting ways of disposing fecal matter and domestic waste by converting them to biofertilizer. Animal and fecal wastes were routinely used to improve the fertility of soil. However, in 20th century due to increased urbanization, these traditional methods were replaced by flush toilets, producing sewage. Sewage is now the number one cause of water pollution. The water pollution decreased the public utility of water resources and resulted in public health problems. The widespread water pollution has left only a few surface and groundwater bodies in good condition. Traditional methods in India and elsewhere and research done in Sweden will be helpful in designing water less management of night soil including composting/digesting fecal matter mixed with domestic and agricultural waste and suitable industrial waste products, to produce fuel gas and biofertilizer. Technological intervention is needed to carry out research on these alternative processes to achieve decontamination of water bodies and to improve the fertility of widely occurring nutrient deficient agricultural soils.

*Corresponding author: Dr. Pramod R. Chaudhari, General Manager, Grass Roots Research and Creation India (P) Ltd., F-375, Sector 63, Noida 201 301 Phone: +91 9766540848 E-mail: pr.chaudhari66@gmail.com, pr.chaudhari@grc-india.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Review Article

R-12

Introduction

Freshwater is a scarce resource and very small part of inland fresh water is available for public use. However, major part of available fresh water is highly polluted and is unsuitable for public use. Around 1.8 billion People in the world don’t have access to safe water; 2.4 billion lack access to adequate sanitation and more than 840,000 people die each year from water-related diseases. Huge amount of infrastructure and enormous cost is now involved in the treatment of sewage to make it suitable for domestic use, however with inadequate success. Experience during River Ganga and River Yamuna cleaning projects showed that even after spending huge amount of public money, it is beyond the capacity of available technology to clean the water bodies to their pristine status. It is unfortunate that the politicians and scientists did not find way to remediate sewage production, the culprit of pollution. Logically the simple solution to prevent sewage production is to avoid use of water to flush and carry night soil. Traditionally, water was never used for management of night soil in India and elsewhere. Preventing sewage production can be achieved by amalgamating the principles of traditional methods and research carried out in Sweden on ecological sanitation. The traditional waterless management of night soil needs to be researched in modified engineered form to suit our modern way of urban and rural life. These modified technologies will be helpful in treating and converting the night soil in useful biofertilizer and to divert pollutants away from water bodies. Organic biofertilizer, the product of night soil treatment, will improve the poor agricultural soils on long term basis, thus helping to improve the regional economy. The issues of water pollution and possible remedy are discussed in this article to find the solution to decontaminate the water bodies. This would save the enormous public money being currently used in river cleaning. The conversion of toilet waste of 1.32 billion population in India would produce huge amount of compost resource for the betterment of the agricultural soils and crop production.

Legal Human Rights for Clean Water The human right to water has been recognized in international law through wide range of international documents, including international human rights treaties, declarations and other standards (http://www2.ohchr. org/english/issues/water/iexpert/standards). In terms of political declarations, the main resolutions were passed by the UN General Assembly and the UN Human Rights Council resolutions both in 2010. The Resolution calls upon States and international organizations to provide financial resources, help capacity-building and technology transfer to help countries, in particular developing countries, to

provide safe, clean, accessible and affordable drinking water and sanitation for all [1].

According to the Indian constitution, every citizen has the right to have the environment protected through reasonable legislative and other measures that promote environmental conservation. In India, there are legal measures for protection and conservation of environmental quality of water resources like Water (Prevention and Control of Pollution) Act, 1974 and implementing agency like Central Pollution Control Board (CPCB) and State Pollution Control Boards (SPCBs) under the Ministry of Environment and Forest and Climate Change (MOEF&CC). The main duty of CPCB is to plan and execute a nation-wide program for the prevention, control or abatement of water and air pollution and all related activities. The other water quality requirements are BIS Water Quality Standards (IS 10500: 2012) related to drinking water, CPCB effluent disposal standards, CPCB Surface Water Quality Classification for various uses, irrigation water standards, etc. all these water quality standards help to maintain the required water quality, if the prevention and control of water pollution is efficient.

Growing Water Demand All the social and economic activities rely heavily on the supply of quality water. As the population and economic activities grow, many countries including India are rapidly reaching conditions of water scarcity and facing limits to economic development. Water scarcity is also due to pollution of water bodies which made them unsuitable for various uses. Water demands are increasing rapidly, with 70-80 per cent required for irrigation, less than 20 per cent for industry and a mere 6 per cent for domestic consumption.

Pollution Potential of Domestic Sewage The largest source of water pollution in India is untreated sewage due to lesser than required number of sewage treatment plants (STPs) (Figure 1) [2]. A 2007 study by CPCB found that discharge of untreated sewage is the single most important source of pollution of surface and groundwater in India. The problem is not only that India lacks sufficient treatment capacity but also that the sewage treatment plants that exist do not operate satisfactorily and are not maintained properly [3].

The scientific analysis of water samples under National Water Quality Monitoring Network of CPCB from 1995 to 2008 indicates that the organic and bacterial contamination is severe in water bodies in India, mostly due to domestic wastewater discharged from urban centers of India. Indian cities produce nearly 40,000 million litres of sewage every

http://www.pacificejournals.com/aabs

R-13

AABS; 3(2): 2016 standards for effluent discharge are made more stringent, as an exercise to control the river pollution. This will increase the cost of new STP development and up gradation of existing STPs. The concern is people have to bear this enormous cost.

Fig. 1: Scenario of Treatment of Wastewater Produced in Towns and Cities in India [2]

day and barely 20 percent of it is treated, and the untreated waste dumped into rivers & seeps into groundwater, thereby creating a ticking health bomb in India according to “Excreta Matters” report [4]. Above observations have shown that pollution control by STPs is not a practicable solution on the basis of its inadequacy on the basis of cost and efficiency, and also the precious fertilizer resource to be generated from fecal matter is lost.

Present Mitigation (Futile) Measures Presently the sewage, which is mix of sullage (Kitchen and bathroom wastewater) and fecal matter from toilet and other waste, is treated and the effluent is discharged in water bodies or used for irrigation. However, many cities don’s have sewage treatment plants of adequate capacities, or do not have any sewage treatment plant, therefore raw sewage, and partially treated or untreated sewage with eutrophication potential are discharged into water bodies. Now the Indian Government is trying to depollute and sanitize the rivers by spending crores of rupees on conventional methods of sewage treatment and sewer construction. The government and the environmental organizations and environmental managers do not realize that how much public money will be spent in the name of restoration of rivers and reservoirs, which is not successful on permanent basis. The money which should be utilized for the socio-economic improvement is flown in drain in the name of cleaning of rivers and reservoirs, which is a futile exercise.

Environmental Friendly Traditional Practices in India and Many Other Countries Without knowing the jargon of environmental theory, the traditional practices used in India and elsewhere for management of domestic waste, fecal matter were environmental friendly and now provide the basis for developing, designing and planning the efficient management system for the waste of different types with the production of fertilizer byproduct, ultimately to save and preserve the rare, life sustaining water resource on the earth. Some of the examples of traditional methods are given below: Traditionally, the waste mater such as fecal matter and urine was never discharged in water bodies but were put through natural degradation process of composting to convert them into useful fertilizer which used to be applied in farms for the crops. This is, in recent terminology, is the conversion of waste into resource and uses it for constructive purpose by the principle of recycle and reuse. In other terms, the materials taken by living component from the earth are returned to earth to make it rich to support biological life further, thus helping the natural cycle of elements through living and non-living components of the earth.

Such treatment at source would be helpful in treating the human waste and animal waste and would not pollute the water bodies; otherwise the demon of water pollution would destroy the ecology and human race. This is as per the natural doctrine that human has no right to deteriorate and convert the rare, life supporting resource into useless commodity. The traditional practices of waste management in India are described below:

Presently Ganga Action Plan is mainly based on development of sewage treatment plants in the cities along the bank of the river. The cost of development of one STP ranges from Rs. 70 lakhs to Rs. 1.2 crores. Now the

● Every village had the system of reserving some area under the jurisdiction of the village for open discharge of fecal matter, separately for men and women. The fecal matter such discharged used to be degraded by the soil bacteria and other microorganisms and convert them into soil humus, making the soil fertile. ● There was also practice to discharge the fecal matter in the agricultural fields and then cover it with soil or ash so that in due course of time the fecal matter is naturally converted into soil humus improving the fertility of the soil. ● Whenever, the large residential areas are concerned, there used to collect the fecal matter by trucks and used to deposit in the trenches made in a barren land away

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article

R-14

from the residential area, which was then covered with soil and allowed to degrade naturally into soil humus and fertilizer, which can then be taken out and used as fertilizer in the agricultural fields. ● The urine is rich in ammonia-nitrogen which is necessary to improve the fertility and can be used as fertilizer when it is discharged in soak pits and the matter in soak pits can be used as nitrogen fertilizer in the agricultural farms. ● Every village farmer used to collect the animal waste of cows, oxen, sheep etc. and other farm waste in a small ditches, and allowed it to get composted and the compost was used in the farms for fertilizer. ● The animals were used to remain in farms during noncrop season, so that the dung was discharged in farms and was naturally converted into fertilizer which used to improve the soil fertility for long time.

Traditional Methods in Other Countries Ancient Attica: The use of sewage as fertilizer was common in ancient Attica. The sewage system of ancient Athens collected the sewage of the city in a large reservoir and then channeled it to the Cephissus river valley for use as fertilizer [5].

Japan: The use of feces as fertilizer was common in Japan.

In Edo City, compost merchant gathered feces to sell to farmers. That was good additional income for apartment owners as their feces were better due to their good diet and more nutrients remained in their excreta. Various historic documents dating from the 9th century detail the disposal procedures for toilet waste [6]. Selling human waste products as fertilizers became much less common after World War II, both for sanitary reasons and because of the proliferation of chemical fertilizers. Modern Japan still has areas with ongoing night soil collection and disposal.

China: Human excreta, or night soil, have been used in China to fertilize crops and feed fish for thousands of years. The night soil has helped China’s land to retain crucial nutrients and fertility for longer duration of time.

Because the night soil was often untreated or partially treated, pathogens could easily be transferred to both humans and food (so eating raw vegetation was seriously frowned upon). However, public health problems due to fecal pathogens were not solved due to this practice. This indicates night soil treatment must be accompanied by the pathogen removal technology.

Before 1979 in China, urban night soil was cleared away jointly by farmers and environmental sanitation bodies.

It was fermented in small-scale storage tanks, with or

without urban domestic waste, in the rural areas. It could be applied to the farmland directly when needed.

After mixing both materials simple piles were made for composting. In 1997, community system was replaced by the family responsibility system. This made it difficult for individuals to collect and transport night soil from cities.

Hence in some cities, night soil had to be disposed of through sewers and this caused environmental pollution.

After a period of disinterest, night soil again gets the attention it deserves, being a valuable resource rather than a contaminant. In recent years, as prices for vegetables and commercial fertilizer rose and the market remained stable, farmers became motivated to use night soil again.

Also, farmers recognize more and more the advantages of using treated night soil in farm lands or fish ponds. But for sanitary reasons, the State now demands that night soil is treated before application. However, to make safe handling possible, treatment of the raw night soil is necessary [7].

Deviation from Traditional System of Composting, Recycle and Reuse Bhangi Mukti Yogana: Bhangi Mukti Yogana was implemented, due to struggle of Harijan Sevak Sangh, with the noble purpose of eliminating the human labour for transporting human excreta from houses to the place of disposal. But this was implemented without planning for environmental friendly method of disposal of human excreta. This issue is explained in detail below.

Traditionally, a section of poor & illiterate community, named as ‘Bhangi’, was assigned the task of transportation of night soil from individual houses to a designated place for onward transportation for composting of night soil.

This task was effectively done for centuries by hated head for elite few whereas, the same task was being performed from time immemorial by the mothers and those who loved you in childhood and also when it was needed most in case of illness, to clean the excreta by disposing it in the soil.

This continued for centuries till the year 1960. This system had its uniqueness of night soil getting treated in no time to valuable bio-fertilizer due to high temperature & humid conditions in most of the area & most of the time. Till this time the water bodies in India were considerably good for domestic use.

Neglected Down-to-Earth Approach Made by Social Workers Central Gandhi Smarak Nidhi, which was established in Delhi in 1956, took to many activities in a move to liberate the scavengers. Finally after 1969, the Nidhi has been working to abolish scavenging by converting the service latrines into pour-flush sanitary latrines of Sulabh Shauchalaya type by constructing new latrines new latrines

http://www.pacificejournals.com/aabs

R-15 which do not required services of scavengers, namely Sopa type and Naigaon type latrines in which arrangement are made to collect the effluent in containers for use as fertilizers in farms. This pioneering experiments in the field were done by the Appa Saheb Patwardhan, the then Chairman of the Central Gandhi Smarak Nidhi, in Gopuri Ashram in Ratnagiri district of Maharashtra. This research was not facilitated by the scientists in the CPCB who were responsible to control water pollution.

Price paid for Neglect of Natural Recycle Process But, in the process of the administrative decision of replacing the transporter of fecal waste (the hated head), water was assigned the task without realizing & understanding the technicality. The blunder which was made in London in 1860 A.D during industrial revolution was repeated in many countries some time or other including India.

Unfortunately, since then the water is being used in larger and larger amount to flush the fecal matter from toilets into sewerages, which ultimately dumped in the final sink i.e. lentic and lotic water bodies and coastal water, thus polluting all the surface and groundwater. This demon has now polluted mostly all the clean water bodies, including sacred ponds and rivers and drinking water resources.

Due to this practice, apart from resulting pollution of the water resources, the night soil did not reach the soil though composting to make it enriched with biofertilizer.

There has been rise in population and industries in 20th century in India, at the same time more water was used to flush out night soil and urine, which ultimately reached to its sink i.e. water bodies. Thus, the asset (night soil and urine) which was to grow with rise in population was lost in increasing the pollution load on the scarcest resource i.e. water.

Probably problem was realized by policy makers and an institution called Central Pollution Control Board (CPCB) was formed framed under Ministry of Environment and Forest & Climate Change (MOEF&CC) with an effective act enacted in March 1974 “Water (Prevention and Control of Pollution) Act, 1974”. But the Institution with its entire technical competency did not act in right direction of searching the technology for transportation & treatment of night soil to a product as usual but allowed water bodies to get polluted with loss of bio-fertilizer, aquatic life etc. They were running after creating better & better technology for effluent & sewage treatment and creating more & more infrastructure for sewage collection system etc with investment of public money. No hesitation in saying that they failed in their task and thus, failed a nation Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 which culturally always worshipped the water body in all its forms. Worshipers of water body culturally, where even a ‘Tyagi’ or “Aghor”, had to carry a ‘Kamandal’ for using water to clean himself by taking water from water body outside its periphery and did not throw night soil in river It is hoped that the institution would realizes and search for better & better technology with old philosophy to achieve the age old task of transporting and treating the night soil with its natural ally i.e. soil, ash, red mud, slag lime & dolomite dust, oil soaked soil from oil industry domestic discards and so many such things.

Habits die hard so we must kill it slowly. Here, we have not to only kill our dirty habit but need to replace with an old habit which may be a bit uncomfortable on various accounts. Now with this in mind, let us discuss some of transportation technology presently being utilized in industry for night soil transportation and using discards of industries and municipal as treatment associate.

Conclusion

It has been recognized that human fecal matter in sewage is the major source of pollution in polluting most of surface and groundwater resources of India. Traditionally, it was used as resource to convert into fertilizer through composting without using water to flush it out in water bodies as is done today. This traditional practice is required to be modified and implemented into water-less technology to collect and convert fecal matter into fertilizer. Specific toilets (based on model developed by Central Gandhi Smarak Nidhi) may be developed for directly converting sewage into fertilizer and fuel gas. Similarly, water less toilets based on experience in Sweden, Japan and China need to be engineered on small and large scale. Many waste byproducts of industries like fly ash, red mud, metal dust, slag & dross, leather discards from tanneries/slaughter houses, oil soaked soil, sludge from oil industry etc. can be mixed with night soil to condition it for composting and then use as fertilizer and soil conditioner in agriculture.

Acknowledgement

The authors wish to thank the Managing Director, Grass Roots Research and Creation India (P) Ltd. for providing facilities for collection of information and preparation this article.

Funding None

Competing Interests None Declared

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Review Article

R-16

References

4. Centre for Science and Environment (CSE), Excreta matters: CSE’s 7th Series of The State of India’s Environment, 2 volumes, 2016 5. Durant, Will, The Life of Greece, pp. 269. Quoted In “Night Soil” by Wikipedia, The Free Encyclopedia (browsed in April 2016).

6. Ebrey P., Walthall A. & Palias J. (2006) Modern East Asia: A Cultural, Social, & Political History. Houghton Mifflin Company. Boston & New York. pp. 337, 2006.

7. Bo Ling, Safe use of treated night soil. ILEIA Newsletter, October1994 (Last modified Feb, 08, 2011). www.agriculturesnetwork.org/magazines/ global/wastes.

1. United Nations Department of Economic and Social Affairs (UNDESA), International Decade for Action ‘WATER FOR LIFE’ 2005-2015, (www.un.org/ waterforlifedecade), 29.05.2014.

2. CPCB, Status of Water Supply, Wastewater Generation and Treatment in Class I Cities and Class II Towns of India. Series: CUPS/70/2009-10. Central Pollution Control Board, India, 2009.

3. CPCB, Evaluation of Operation and Maintenance of Sewage Treatment Plants in India-2007. Central Pollution Control Board, Ministry of Environment & Forests. 2008.

http://www.pacificejournals.com/aabs

Original Article Frequency of Mucous Retention Cyst of The Maxillary Sinus in Patients Need Implants Using Cone Beam Computed Tomography in Tabriz Masoumeh Johari1, Farzad Esmaeli1, Sahar KhademNejad2, Ali Zarandi3* Department of Oral and Maxillofacial Radiology, Faculty of Dentistry, Tabriz University of Medical Sciences, Iran 2 Doctor of Dentist, Faculty of Dentistry, Tabriz University of Medical Sciences, Iran 3 Department of Periodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran

1

Keywords: Retention Cyst, Maxillary Sinus, CBCT.

ABSTRACT Background: Cone Beam Computed Tomography (CBCT) increasingly is a main source of 3D volumetric information in clinical orthodontics. In this study, the maxillary sinus for the presence of the retention cysts in implant patients was evaluated using CBCT. Methods: CBCT images from 320 patients (168 females and 152 males), who were candidate for implant placement in the maxilla, were selected. The single dome shaped image with the soft tissue density on the floor of the maxillary sinus without the cortical boundary was recognized as the retention cyst. The chi –square test was used for the statistical analysis.

Results: maxillary retention cyst was diagnosed in 31 patients (18 females, 13 males). Also, two cases showed the involvement of both sinuses. The incidence was 10.3%, only two patients reported the history of sinusitis. Chi square test did not show any difference between the females and males.

Conclusion: We found an incidental rate of 10.3% of retention cysts in the implant patients.

*Corresponding author: Dr. Ali Zarandi, Department of Periodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran, Phone: +91 98413335596 E-mail: dr.alizarandi@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-119

AABS; 3(2): 2016

Introduction

The most common lesion in maxillary sinus is mucous retention cysts ( MRC) which associated with all types of races, genders and different age groups (1). These cysts are asymptomatic lesions that could determine with panoramic radiography (2, 3). Imaging studies provide chances for dentists to distinguish modifications in the maxillary sinus.

A dentist must be aware of the character of the cysts.

On radiographical images, it appears as rounded edge, radiopaque, well-defined region, dome shaped, different in size, placed in the maxillary sinus, unilateral or bilateral (4, 5).Cone-beam computed tomography (CBCT) could prepare details of scanned images as three-dimensional images. The application of the CBCT method in endodontic treatment could improve treatment design of surgical processes by determining the size and position of the lesion according to another anatomic structure. On the other hand, computed tomography scanning was introduced as a standard method in medicine for imagining the maxillary sinuses due to the ability to prepare bone and soft tissue with thin segmenting (6). About the odontogenic sinusitis, CBCT method could help to improve the treatment process in combination with nonsurgical and surgical dental and medical usages (7). Mucous retention cysts have been evaluated by cone beam computed tomography (CBCT) in different population (5, 8, 9) . In this study, the maxillary sinus for the presence of the retention cysts in implant patients was evaluated using CBCT.

Materials and Methods

In this cross-sectional study, 320 CBCT images were selected who were candidate for dental implants in the maxilla jaw and referred to the private clinic. All the patient CBCT images have been prepared by Planmeca Romexis 3D device (Helsinki, finld) according to the manufactures order. The images obtained in the axial , coronal and cross sectional side with 2mm thickness and evaluated by radiologist expert with three years experience. The single dome shaped image with the soft tissue density on the floor of the maxillary sinus without the cortical boundary was recognized as the retention cyst (Fig 1). The chi â&#x20AC;&#x201C;square test was used for the statistical analysis ( P < 0.05).

Fig 1: a: panomaric images with suggestive of a: bilateral mucous retention cyst ( arrws), b: cross sectional images Table1 : Distribution of mucous retention cysts in relation to gender gender

Bilateral

On one side

MRC

N of patients

female

1

17

18

168

male

1

12

13

152

total

2

29

31

320

Discussion

In this study, MRC was diagnosed in 31 patients (18 females, 13 males). Two cases showed the involvement of both sinuses. The incidence was 10.3%, only two patients reported the history of sinusitis. The frequency of MRC was not significantly difference between the females and males.

A total of 258 patients had thickened sinus mucosa in the maxillary. In addition, 31 radiogrphs images had MRC ( 18 female, 13 male). Of those, 2 cases observed on the bilateral of maxillary sinus and the rest were on one side. Totally, 33 MRC was detected with 10.3 % frequency. The Chi-square test did not show significant differences between the male and female patients. (P = 0.8) ( table 1).

Mucous retention cysts in maxillary sinus are asymptomatic and often diagnosed incidentally in radiographic images during treatment phase of dental process. (10). Sometimes, MRCs are seen as more than one cyst but in the most diagnosis it was determine as single cyst. Cause of etiology and pathology of MRCs is poorly understood (11) Some reports showed the relationship of cyst with periodontal disease, odontogenic infection, allergies, inflammatory, trauma. Also, there is no consensus agreement about an association with age, gender and history of allergies.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Result

Original Article

A-120

(11-15). Celebi and colleagues found that MRCs have an inflammatory cause and are produced by the addition of fluid within the sinus membrane (16). In addition, MRCs are not attending as true cysts since their absence an epithelial coating (17). The frequency of the MRCs differs with the type of radiograph prepared images. In panoramic imaging the frequency is reported about 1-10% on computed tomography scans 12 % and on magnetic resonance imaging (MRI) 21 %.But data on the frequency in cone beam computed tomography (CBCT) scans was not accessible (18). Bosio et al. verified the frequency of MRCs in sequential panoramic radiographs from 173 patients. The result showed that 5.8% of patients presented a MRCs which males had a higher incidence than females (9.1% vs. 3.1%)(P > .05). Total, the frequency of MRCs in orthodontic patients was partly low in comparison with the general population (3).

Cha et al. examined the images from 500 maxillofacial 3D scans which consisted of orthodontic patients, implant patients, endodontic patients, temporomandibular joint (TMJ) disorder patients. The maximum rate of related results was in the airway area, TMJ findings, endodontic findings and others respectively. Only 22% of the airway findings, such as mucosal thickness, polyps, and retention cysts, were associated with clinical symbols and symptoms (19). Wang et al. concluded that MRCs naturally regressed or displayed no significant variation in size during the long period. These results recommend that, strategy of “wait and see” may be the proper management for these cysts (20).

Conclusion

We found an incidental rate of 10.3% of retention cysts in the implant patients.

Acknowledgements

[It should include persons who provided technical help, writing assistance and departmental head that only provided general support. Financial and material support and conflict of interests must be written in this section.]

Funding

3. Bosio JA, Tanaka O, Rovigatti E, de Gruner SK. The incidence of maxillary sinus retention cysts in orthodontic patients. World journal of orthodontics.

2009 Summer;10(2):e7-8. PubMed PMID: 19582248.

Epub 2009/07/08. eng.

4. Ramesh A, Pabla T. Mucous retention cyst of maxillary sinuses. Journal of the Massachusetts Dental Society.

2008 Summer;57(2):14-5. PubMed PMID: 18705208.

Epub 2008/08/19. eng.

5. Donizeth-Rodrigues C, Fonseca-Da Silveira M, Goncalves-De Alencar AH, Garcia-Santos-Silva MA, Francisco-De-Mendonca E, Estrela C. Threedimensional images contribute to the diagnosis of mucous retention cyst in maxillary sinus. Medicina oral, patologia oral y cirugia bucal. 2013 Jan;18(1):e151-7.

PubMed PMID: 23229251. Pubmed Central PMCID: PMC3548636. Epub 2012/12/12. eng.

6. Nishimura T, Iizuka T. Evaluation of odontogenic maxillary sinusitis after conservative therapy using CT and bone SPECT. Clinical imaging. 2002 MayJun;26(3):153-60. PubMed PMID: 11983465. Epub 2002/05/02. eng.

7. Nair UP, Nair MK. Maxillary sinusitis of odontogenic origin: cone-beam volumetric computerized tomography-aided diagnosis. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics. 2010 Dec;110(6):e53-7. PubMed PMID: 20971660. Epub 2010/10/26. eng.

8. Rege IC, Sousa TO, Leles CR, Mendonca EF. Occurrence of maxillary sinus abnormalities detected by cone beam CT in asymptomatic patients. BMC oral health. 2012;12:30. PubMed PMID: 22883529.

Pubmed Central PMCID: PMC3511216. Epub 2012/08/14. eng.

9. Cymerman JJ, Cymerman DH, O’Dwyer RS. Evaluation of odontogenic maxillary sinusitis using cone-beam computed tomography: three case reports.

Journal of endodontics. 2011 Oct;37(10):1465-9.

PubMed PMID: 21924204. Epub 2011/09/20. eng.

None

Competing Interests None declared

Reference

of mucous retention cysts in a Brazilian population.

Dento maxillo facial radiology. 2009 Oct;38(7):4803. PubMed PMID: 19767520. Epub 2009/09/22. eng.

1. Giotakis EI, Weber RK. Cysts of the maxillary sinus: a literature review. International forum of allergy & rhinology. 2013 Sep;3(9):766-71. PubMed PMID: 23677671. Epub 2013/05/17. eng.

2. Rodrigues CD, Freire GF, Silva LB, Fonseca da Silveira MM, Estrela C. Prevalence and risk factors

10. Mardinger O, Manor I, Mijiritsky E, Hirshberg A. Maxillary sinus augmentation in the presence of antral pseudocyst: a clinical approach. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics. 2007 Feb;103(2):180-4. PubMed PMID: 17234532. Epub 2007/01/20. eng.

http://www.pacificejournals.com/aabs

A-121 11. MacDonald A, Newton CW. Pseudocyst of the maxillary sinus. Journal of endodontics. 1993;19(12):618-21.

12. Meer S, Altini M. Cysts and pseudocysts of the maxillary antrum revisited. SADJ: journal of the South African Dental Association= tydskrif van die Suid-Afrikaanse Tandheelkundige Vereniging.

2006;61(1):10-3.

13. Wang JH, Jang YJ, Lee BJ. Natural Course of Retention Cysts of the Maxillary Sinus: Long-Term Follow-Up Results. The Laryngoscope. 2007;117(2):341-4.

14. Donizeth-Rodrigues C, Fonseca-Da Silveira M, Gonçalves-De Alencar AH, Garcia-Santos-Silva MA, Francisco-De-Mendonça E, Estrela C. Threedimensional images contribute to the diagnosis of mucous retention cyst in maxillary sinus. Medicina oral, patologia oral y cirugia bucal. 2013;18(1):e151.

15. Maestre-Ferrín L, Galán-Gil S, Carrillo-García C, Peñarrocha-Diago M. Radiographic findings in the maxillary sinus: comparison of panoramic radiography with computed tomography. International Journal of Oral & Maxillofacial Implants. 2011;26(2).

16. Celebi N, Gonen ZB, Kilic E, Etoz O, Alkan A. Maxillary sinus floor augmentation in patients with

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 maxillary sinus pseudocyst: case report. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2011;112(6):e97-e102.

17. Gardner DG. Pseudocysts and retention cysts of the maxillary sinus. Oral surgery, oral medicine, oral pathology. 1984;58(5):561-7.

18. Tang Z, Wu M, Xu W. Implants placed simultaneously with maxillary sinus floor augmentations in the presence of antral pseudocysts: a case report.

International journal of oral and maxillofacial surgery.

2011;40(9):998-1001.

19. Cha JY, Mah J, Sinclair P. Incidental findings in the maxillofacial area with 3-dimensional cone-beam imaging. American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics.

2007 Jul;132(1):7-14. PubMed PMID: 17628245.

Epub 2007/07/14. eng.

20. Wang JH, Jang YJ, Lee BJ. Natural course of retention cysts of the maxillary sinus: long-term follow-up results. Laryngoscope. 2007 Feb;117(2):341-4.

PubMed PMID: 17277631. Epub 2007/02/06. eng.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Estimation of Sonographic Umbilical Cord Area and Its Correlation with Birth Weight in Gestational Diabetes Mellitus.

Nidhi Jain1*, Abha Singh2 Dept of Gynae endoscopy, Manchandaâ&#x20AC;&#x2122;s Endoscopic Centre, Delhi, India Department of Obstetrics & Gynaecology, Lady Harding Medical College, Delhi, India 1

2

Keywords: Umbilical Cord, Umbilical Cord Area, Gestational Diabetes Mellitus, Macrosomia.

ABSTRACT Background: Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance with its onset or first recognition during pregnancy. It is associated with various maternal and fetal complications of which, macrosomia is the major challenging threat. Prediction of birth weight helps in timely decision and management of these women. A recent way to predict birth weight is by umbilical cord area estimation.

Methods: A prospective cohort study including 50 women with GDM (group I) and 50 without GDM (group II) was conducted in a tertiary hospital over a period of 1 year, after clearance from Institutional Ethical Committee. Women in both groups were subjected to ultrasonographic examination at 30-32 weeks and subsequently at 36-38 weeks. At each examination, umbilical cord area (UCA) was measured in a free loop of umbilical cord. Birth weight of each baby was measured.

Results: At 30-32 weeks, UCA was 239.7 mm2 in group I and 224 mm2 in group II, difference being statistically significant. At 36-38 weeks also, UCA was significantly larger in group I (250.1 mm2) than in group II (228.2 mm2).

Correlation coefficient (r) between umbilical cord area and birth weight was 0.944 in group I and 0.796 in group II at 30-32 weeks and at 36-38 weeks, 0.990 in group I and 0.530 in group II. Conclusions: A strong positive correlation exists between umbilical cord area and birth weight in women with gestational diabetes mellitus, thus it should be estimated during routine antenatal ultrasound for prediction of birth weight in these women.

*Corresponding author: Dr NIDHI JAIN, House no. 655, police line area, near SSP residence, Hisar, Haryana-125001, India Phone: +91 9654822494 E-mail: nidhijain270587@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-123

Introduction

Diabetes, one of the most common medical complications, has become a major challenging threat in a pregnant woman. The prevalence of gestational diabetes mellitus in India varies from 3.8% to 21%1-8.

Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance with its onset or first recognition during pregnancy. It is associated with various maternal and fetal complications which include polyhydramnios, macrosomia, operative interference, shoulder dystocia, birth injuries and perinatal mortality. Of all these, one of the major complication effecting both mother and fetus is macrosomia. Macrosomia is defined as gestational age adjusted birth weight >90th percentile of reference population or as birth weight ≥4.0kg. However, American college of obstetrics & gynaecology (2000) defines it as birth weight ≥4.5kg. Incidence9 of macrosomia is around 1-10%. So, one of the most important perinatal goals in GDM is to predict macrosomia by estimating birth weight, thereby preventing the adverse maternal and fetal outcomes.

Umbilical cord area: A recent way to predict birth weight is by sonographic estimation of umbilical cord area (UCA).

Umbilical cord is made up of three vessels i.e. two arteries and one vein, which are embedded in Wharton’s jelly. It can be classified as: 1. Lean: UCA is below 10th percentile for gestational age. It is seen in conditions such as tobacco smoking10, prematurity, hypertensive disease11 and fetal growth restriction.

2. Large: UCA is above 90th percentile for gestational age. It is found in Gestational diabetes mellitus, fetal structural anomalies like urachal cyst, omphalomesentric cyst and umbilical cord tumor.

Material and Methods

This prospective cohort study was carried out in the department of Obstetrics and Gynaecology in a tertiary hospital over a period of one year after clearance from Institutional ethical committee.

Inclusion criteria: were women with singleton pregnancy, with GDM and who gave consent for participation in the study.

Exclusion criteria: included multiple gestation, obstetrical complications such as preeclampsia, intrauterine growth restriction, oligohydramnios, hydrops, fetal congenital

AABS; 3(2): 2016 malformations, maternal chronic diseases such as overt diabetes, hypertension, renal diseases, cardiac diseases and pulmonary diseases, history of smoking and alcohol, women who were not sure of date of Last menstrual period/ did not have first trimester ultrasound and those who did not give consent foe participation in the study.

Women with singleton pregnancy at 24-28 weeks of gestation, who fulfilled the inclusion criteria, were enrolled from the antenatal clinic. A written informed consent was taken. After detailed history and examination, all women were screened for GDM by 50 grams GCT. In women with GCT ≥140 mg%, 100grams GTT was done. GDM was diagnosed on the basis of Carpenter and Coustan criteria*.

50 women with GDM were included in group I and 50 women with normal GCT were enrolled in group II. All women were subjected to ultrasonographic examination at 30-32 weeks and subsequently at 36-38weeks.

Umbilical cord area was measured in a free loop according to the method used by Binbir12 et al. UCA was measured around outer edges of umbilical cord by using elliptical calibrators (Figure 1). 3 measurements were taken and average value was calculated. Women were followed till the time of delivery to observe maternal and neonatal outcomes. Birth weight of baby was measured. Macrosomia was defined as birth weight ≥4kg.

Data analysis: Data was compiled and analyzed by using SPSS software. p value <0.05 was considered significant.

Results

The mean maternal age in women with GDM, group I (27.8 years) was comparable to that in group II (27.2 years). However maternal weight was higher in group I than in group II, difference being statistically significant.

In both groups, 45 (90%) women were multigravida while 10% were primiparous. Bad obstetric history in term of history of abortions, IUD, big size baby and GDM in previous pregnancies was present in 48%, 20%, 4% and 16% women respectively in group I. Out of 50 women with GDM in group I, 35 (70%) were controlled on diabetic diet only while 15 (30%) women required insulin for glycaemic control.

Among mode of delivery, it was found that in group I, 66% women delivered vaginally, of which 8% had instrumental vaginal delivery and 34% had caesarean section. Caesarean rate was found to be much lower in group II (16%).

*Carpenter & Coustan criteria: Fasting ≥95 mg/dl, 1 hour ≥180 mg/dl, 2 hours ≥155 mg/dl, 3hours ≥140 mg/dl. If ≥2 values are abnormal, woman is diagnosed as GDM.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-124

The mean birth weight was significantly higher in group I (3.15 kg) than in group II (2.90kg). 5 babies (10%) were found to be macrosomic, all belonging to group I. However, no significant difference was found in terms of low Apgar score, birth asphyxia, need of ventilation and neonatal death among two groups.

During ultrasonographic examination at 30-32 weeks, it was found that at 30-32 weeks, UCA was 239.7 mm2 in group I and 224 mm2 in group II, difference being statistically significant. At 36-38 weeks, UCA was significantly larger in group I (250.1 mm2) than in group II (228.2 mm2). Hence, UCA was significantly greater in women with GDM at both the gestations. Estimation value of Umbilical cord area is shown in Table 1.

Correlation coefficient was calculated to see degree of correlation between UCA and birth weight. It was found that in group II, a weak correlation exists between UCA and birth weight at both 30-32 weeks (r = 0.796) and 3638 weeks (r = 0.530). However, in group I (women with GDM), a significant positive relationship was found between umbilical cord area and birth weight at both 30-32 weeks (r = 0.944) and 36-38 weeks (r = 0.990).

leads to development of fetal hyperglycaemia, thus excessive fetal growth, thereby leading to Macrosomia.

Macrosomic babies cause multiple complications during vaginal delivery including shoulder dystocia, brachial plexus injury, meconium aspiration, respiratory distress and low Apgar score. So, to avoid these complications, prediction of birth weight is to be done for timely decision and better management of women with GDM. One of the parameter to predict birth weight is umbilical cord area. Weismann11 et al conducted a study in 368 uncomplicated pregnancies and found that UCA increases with gestational age till it reaches a peak at 36 weeks of gestation and plateau thereafter. Various studies12,13 have observed this peak of UCA at varying gestational age ranging from 32-34 weeks. In the present study also, we found that in women without GDM (group II), UCA was comparable at 30-32 weeks (224.0 mm2) and 36-38 weeks (228.8 mm2). However, in group I, a significant increase was found from 30-32 weeks (239.7 mm2) to 36-38 weeks (250.1 mm2), showing that in women with GDM, UCA increases significantly with advancing gestational age.

Gestational diabetes mellitus is associated with various maternal and fetal complications. Maternal hyperglycaemia

In the present study, fetus of women with GDM was found to have larger umbilical cord area than women without GDM. The proposed mechanism of this larger UCA is that in women with GDM, erosion of endothelial lining of umbilical arteries occurs. This leads to increased permeability with leakage of plasma proteins, causing an expansion of ground substance and thus, increases in area of Whartonâ&#x20AC;&#x2122;s jelly umbilical cord as given in study by Weismann14 et al.

Fig. I: figure showing sonographic measurement of area of umbilical cord, artery, vein and Whartonâ&#x20AC;&#x2122;s jelly.

Fig. II: Scatter diagram showing correlation between UCA and birth weight in group I (r= 0.944) at 30-32 weeks.

Thus, although a positive linear relationship exists between UCA and birth weight in both the groups at both gestations but the correlation is much stronger in women with GDM (Figure II, III, IV, V).

Discussion

http://www.pacificejournals.com/aabs

A-125

AABS; 3(2): 2016

Fig. III: Scatter diagram showing correlation between UCA and birth weight in group II (r = 0.796) at 30-32 weeks.

Fig. IV: Scatter diagram showing correlation between UCA and birth weight in group I (r= 0.990) at 36-38 weeks.

Fig. V: Scatter diagram showing correlation between UCA and birth weight in group II (r= 0.530) at 36-38 weeks.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-126

Table 1: Measured value (Mean) of umbilical cord area in group I and group II at 30-32 weeks and 36-38 weeks.

Period of gestation

UCA in Group I (mm2)

UCA in Group II (mm2)

P value

30-32 wks

239.7

224.0

<0.001

36-38 wks

250.1

228.8

<0.001

Abreviations GDM

Gestational diabetes mellitus

UCA

Umbilical cord area

GCT

Glucose challenge test

GTT

Glucose tolerance test

A significant positive relationship was found between UCA and birth weight at both 30-32 weeks (r = 0.944) and 3638 weeks (r = 0.990) in women with GDM. The results were in accordance with study by Binbir15et al in which a statistically significant correlation was found between umbilical cord area and fetal weight in diabetic group at 3637 weeks. However in a retrospective study by Predanic16 et al, done in 470 women, in which umbilical cord diameter was measured at gestation of 18-23 weeks, no significant correlation was found between umbilical cord diameter and birth weight (p = 0.332).

Conclusion

3.

4.

In conclusion, a significant positive correlation exists between sonographically estimated umbilical cord area and birth weight in women with gestational diabetes mellitus.

Hence, Umbilical cord area should be estimated during routine antenatal ultrasound for prediction of birth weight in women with gestational diabetes mellitus.

5.

Acknowledgements

7.

I am extremely indebted to my guide, Dr. Abha Singh, Director Professor & HOD, Obstetrics & Gynaecology, for her guidance, support and unending encouragement. I am thankful to all my patients and my family and colleagues for being my companion during this arduous path.

6.

8.

Funding: None

Competing Interests

9.

None declared.

References

1. Seshiah V, Balaji V, Balaji M, Panneerselvam A, Kapur A. Pregnancy and Diabetes Scenario around the World: India. Int J Gynaecol Obstet 2009; 104 (1): S35-8.

2. Seshiah V, Balaji V, Madhuri S Balaji, Panneerselvam A, Arthi T,Thamizharasi M et al. Prevalence of GDM

10.

in South India (Tamil Nadu) â&#x20AC;&#x201C; A Community based study. JAPI 2008; 56: 329-33.

Zargar AH, Sheikh MI, Bashir MI, Masoodi SR, Laway BA, Wani AI et al. Prevalence of gestational diabetes mellitus in Kashmiri women from the Indian subcontinent. Diabetes Res Clin Pract. 2004; 66(2): 139- 45.

Grewal E, Kansra S, Khadgawat R, Kachhawa G, Ammini AC, Kriplani A et al. Prevalence of GDM among women attending a Tertiary Care Hospital AIIMS Presented at DIPSI 2009 and 5th DIP Symposium, Sorrento, Italy, 2009.

Singh Dorendra I, Devi Bidhumukhi Th, Devi Ibeyaima Kh, Singh Premchand Th. Scientific Presentation Volume of the First National Conference of the DIPSI, Chennai February 2006.

Yuvaraj M G. Data presented at the First National Conference of DIPSI: Chennai February 2006.

Swami SR, Mehetre R, Shivane V, Bandgar TR, Menon PS, Shah NS. Prevalence of Carbohydrate Intolerance of Varying Degrees in Pregnant Females in Western India (Maharashtra) - A Hospital-based Study. J Indian Med Assoc 2008; 106 (11): 712-4.

Divakar H, Tyagi S, Hosmani P, Manyonda IT. Diagnostic criteria influence prevalence rates for gestational diabetes: implications for interventions in an Indian pregnant population. Perinatology 2008: 10 (6); 155 â&#x20AC;&#x201C; 61.

Martin J, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Kirmeyer S. Births: final data for 2004.

Natl vital stat rep 2006; 55 (1): 1-101.

Milnerowicz-Nabzdyk E, Zimmer M, Tlolka J, Michniewics J, Pomorski M, Wiatrowski A. Umbilical cord morphology in pregnancies complicated by IUGR in cases of tobacco smoking and pregnancyinduced hypertension. Neuro Endocrinol Lett 2010; 31(6): 842-847.

http://www.pacificejournals.com/aabs

A-127

AABS; 3(2): 2016

11. Bankowski E, Sobolewski K, Romanowicz L, Chyczewski L, Jaworski S. Collagen and glycosaminoglycans of Whartonâ&#x20AC;&#x2122;s jelly and their alterations in EPH-gestosis. Eur J Obstet Gynecol Reprod Biol 1996; 66 (2): 109-117.

12. Weissman A, Jakobi P, Bronshtein M, Goldstein I. Sonographic measurements of the umbilical cord and vessels during normal pregnancies. J Ultrasound Med 1994; 13 (1): 11-14.

13. Barbieri C, Cecatti JG, Souza CE, Marussi EF, Costa JV. Sonographic measurement of the umbilical cord and the diameters of its vessels during pregnancy. Journal of Obsetetrics and Gynecology 2012; 32 (3): 230-236.

14. Weissman A, Jakobi P. Sonographic measurements of the umbilical cord in pregnancies complicated by gestational diabetes. J Ultrasound Med 1997; 16 (10): 691-694.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

15. Binbir B, Ozgur Yeniel A, Ergenoglu A, Knzandi M, Akercan F, Sagol S. The role of umbilical cord thickness and HbA1c levels for the prediction of fetal macrosomia in patients with gestational diabetes mellitus. Arch Gynecol Obstet 2012; 285 (3): 635-639.

16. Predanic M, Perni S. Absence of a Relationship between Umbilical Cord Thickness and Coiling Patterns. J Ultrasound Med 2005; 24:1491â&#x20AC;&#x201C;1496.

Original Article Mediastinum: A Pandoraâ&#x20AC;&#x2122;s Box: A Study of Mediastinal Lesions at Tertiary Hospital of South Canara.

Sheikh Tousif Reza*, Kishan Prasad HL, Jayaprakash Shetty K, Harish S Permi Sunil kumar Y, Swaroop Shasidhar Pathology, K. S. Hegde Medical Academy, Deralakatte, Mangalore, Karnataka, India Keywords: Mediastinum, Thymoma, Myasthenia Gravis, Filariasis

ABSTRACT Although the mediastinum is a relatively small anatomic compartment, the diversity of pathologic processes that may reside in it is impressive. Such lesions are both nonneoplastic and neoplastic, and they include proliferations of somatic epithelial, lymphoid and mesenchymal cell types. A retrospective review was undertaken of all cases of mediastinal lesions that presented to the Department of Pathology of K . S. Hegde Medical Academy, Deralakatte, Mangalore, over a 5-year period i.e from 2010 to 2015. 22 mediastinal mass lesions were managed over the period of the review. Thymoma was the most common pathology, being present in 6 (27.2%) cases. A variety of other pathologies were encountered, including neurofibroma , teratoma, thymic cyst , retrosternal goitre , Kimuraâ&#x20AC;&#x2122;s disease and thymic hyperplasia . Patients usually were symptomatic . Chest pain and discomfort was most common symptom, others were dyspnea and cough. MRI has become a useful tool for providing supplemental data in combination with CT. Prompt thoracic surgical referral with view to aggressive, early resection optimizes clinical outcome in the short and mediumterm for patients presenting with mass lesions of the mediastinum.

*Corresponding author: Dr Sheikh Tousif Reza , Flat no. 540, Rosewood Apartment, Pocket A/2, sector 13, Dwarka, New Delhi- 110078, India E-mail: sheikhtousifreza@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-129

Introduction

According to Greek mythology, Pandoraâ&#x20AC;&#x2122;s box refers to a large jar which contained all the evils in the world.

[1] Likewise mediastinum can be considered a small jar if opened reveals wide spectrum of lesions. Mediastinum is a portion of thoracic cavity which is located in between the pleural cavities, extending anterioposteriorly from the sternum to the spine and sagitally from the thoracic inlet to the diaphragm. The mediastinum has been divided into four compartments i.e superior, anterior, middle and posterior compartments.

About half of the patients with mediastinal cysts and tumors are asymptomatic, the lesions being discovered incidentally on chest x-ray films or CT scans done for other reasons. When symptoms develop, they usually result from compression and/or invasion of adjacent structures.[2] Limited approach and the non specific symptoms presented by the patients make the diagnosis more challenging.[3]

Materials and Methods

The study was retrospective and descriptive, done in a 5 year period i.e. from April 2010 to March 2015 in the department of Pathology, K.S. Hegde Medical Academy, Deralakatte, Karnatka. This study comprised of 22 cases including 14 resected specimens and 8 trucut biopsies.

Age and sex distribution, location, histologic types of tumors, symptoms and signs, associated diseases and complications were recorded from patientsâ&#x20AC;&#x2122; files. Patients ranged in age from 2 to 66 years and consisted of 11 males and 11 females. All specimens were routinely fixed in 10% buffered formalin for 12 to 24 hours. Sections of 3mm thickness from the block of each cases were obtained and were routinely stained with hematoxylin-eosin. The study was approved by an internal ethical committee.

AABS; 3(2): 2016 followed by retrosternal goitre (17.4 %), hyperplastic thymic lesions (13.6%), poorly differentiated carcinoma (8.7%) ,thymorelets (4.3%), benign cystic teratoma (4.3%), thymic cyst (4.3%), Kimuraâ&#x20AC;&#x2122;s disease (4.3%), small cell carcinoma (4.3%), neurofibroma (4.3%) and undifferentiated carcinoma (4.3%). A number of associated diseases were observed simultaneously among the patients with mediastinal lesions including plasmacytosis, dermatopathic lymphadenopathy, myasthenia gravis and filariasis.

Myasthenia gravis and thymic lesions: Three patients presented with generalized weakness and easy fatiguability, were diagnosed with myasthenia gravis and confirmed by acetyl choline receptor antibody test. Later they underwent thymectomy which on histopathological revealed 3 cases of thymic hyperplasia, one thymorelet and one thymoma type AB. Hence 1 out of 5 (20%) thymoma and all cases of thymic hyperplasia (100%) showed association with myasthenia gravis.

Thymoma and filariasis: A 53 year old male came with complaints of severe breathlessness and cough since 15 days.

CT scan thorax showed large heterogeneously enhancing anterior mediastinal mass lesion with pericardial effusion.

After that aspiration of pericardial fluid was done which showed plenty of neutrophils, occasional eosinophils, and microfilaria of wutchereria bancrofti against haemorrhagic background with no evidence of malignant cells.

CT guided fine needle aspiration of mediastinal mass showed clusters of spindle cells having bland nucleus with moderate amount of cytoplasm and lymphoid cells in the background. Spindle cell thymoma was considered. Trucut biopsy confirmed the cytology diagnosis. This case showed an unusual association of a thymoma with filariasis.[4]

Results

Discussion

Histopathological evaluation revealed 11 types of different lesions of which the most common is thymoma (27.2%)

Most mediastinal tumors in this series were identified in the sixth decade of life (23.8%) followed by the second decade (19%). The most common location of mediastinal

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

In this duration of 5 year, these mediastinal lesions share 0.02% of the total lesions presented to the Department.

22 patients including 11 males and 11 females with a sex ratio of 1:1 ranging from 2 to 66 years with mean age of 36.5 years entered the study. Most of the medistinal lesions were identified in the sixth decade followed by second decade. In compartment wise, maximum lesions (48.1%) were found in anterior mediastinum followed by superior (33.3%) , middle (11.1%) and inferior (7.4%) mediastinum.

Nonspecific symptoms such as breathlessness and generalised weakness constituted the most commonly presenting complaints followed by cough, pain, dysphagia and fever.

Numerous tumors and cysts occur in the mediastinum and affect people of all ages. They are uncommon and represents 0.02% of total specimens. In this study we encountered important differences in histologic distribution, age range and associated conditions of mediastinal lesions. The precise nature of a lesion in the mediastinum cannot be determined without histologic examination of the tissue.

In this study, male to female ratio was obtained as 1:1, whereas other studies show male predominance like 1.5 :1

by Arman et al[5]., 1.6:1 by Lewis et al.[6] and 1.4:1 by Roy et al[7]. Mohd Vaziri et al[8]. got 0.9:1 ratio showing female predominance in their study.

Original Article

A-130

Table no.1 LOCATION SUPERIOR ANTERIOR MIDDLE POSTERIOR

FREQUENCIES 9 13 3 2

PERCENTAGE 33.3% 48.1% 11.1% 7.4%

Table no.2 Histopathological diagnosis Thymoma Retrosternal goitre Hyperplastic Thymic lesions Poorly differentiated carcinoma Thymorelets Benign cystic teratoma Thymic cyst Kimuraâ&#x20AC;&#x2122;s disease Small cell carcinoma Neurofibroma Undifferentiated carcinoma

Frequencies 6 4 3 2 1 1 1 1 1

Percentage 27.2 % 17.4 % 13.6 % 8.7 % 4.3 % 4.3 % 4.3 % 4.3 % 4.3 %

1 1

4.3 % 4.3 %

Table no. 3. comparatative study of lesions Roy themes et al.

Arman et al.

Present study

Thymic

14.1 %

24.3 %

45.4 %

Cystic Neurogenic Lymphoma Germ cell tumour Vascular Mediastinal goitre Miscellaneous

0% 2.7 % 55.2 % 15.5 % 0% 3.8 % 12.3 %

19.5 % 16.9 % 15.6 % 9.56 % 1.7 % 0% 12.1 %

4.5 % 4.5 % 0% 4.5 % 0% 18.1 % 27.2 %

Fig. 1. a)Thymoma AB (40X) , b).Thymoma B1 (40X) , c). Thymoma B2 (40X) d).Gross picture of thymic cyst ,

http://www.pacificejournals.com/aabs

A-131

AABS; 3(2): 2016

Fig. 2 .a) CT scan showing heterogeneously enhancing anterior mediastinal mass and pericardial effusion. b). Pericardial fluid showing filaria in a haemorrhagic backgroung c).FNAC smear of spindle cell thymoma showing clusters of benign spindle cells and scanty lymphoid cells. d). Trucut biopsy showing spindle cell

lesions in this series was the anterior mediastinum(48.1%).

Even other studies have shown the anterior mediastinum being the most common location i.e 57% by Arman et al.[5] and 65% by Mohd Vaziri et al.[3] In this study, thymic lesions (45.4%) were seen as the majority with thymoma being the maximum similar to Arman et al. study (24.3%)[5]. Roy et al. found lymphoma (55.2%) to be the commonest in their study.[7] We encountered a number of interesting associated diseases with some mediastinal lesions like filariasis with thymoma, myasthenia gravis with thymic hyperplasia, plasmacytosis and dermatopathic lymphadenopathy.

Conclusion

Categorizing the mediastinal lesions according to histopathological features into various types, helps us to know the clinical presentation, treatment, clinical outcome and prognosis of the disease. Prompt thoracic surgical referral with view to aggressive, early resection optimizes clinical outcome in the short and medium-term for patients presenting with mass lesions of the mediastinum.

Funding None

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

Competing Interest None

References

1. https://en.m.wikipedia.org/wiki/Pandora%27_Box. 2. Rosai J. Rosai and Ackermanâ&#x20AC;&#x2122;s Surgical Pathology.

10th edition. Mosby. 2011.472.

3. Vaziri M, Pazooki A, Shoolami LZ. Mediastinal Masses: Review of 105 Cases. Acta Medica Iranica 2009;47(4):297-300.

4. Permi HS, Samaga BN, Subramanyam K, Shetty JS, Teerthnath S, Baikunje V et al. Microfilaria in pericardial effusion coexisting with spindle cell thymoma - a rare case report. NUJHS December 2011;1:40-2.

5. Cohen AJ, Thompson LN, Edwards FH, Bellamy RF. Primarv Cvsts and Tumors of the Mediastinum. Ann Thorac Surg.1991;51:378-86.

6. Lewis JE, Wick MR, Scheithauer BW, Bernatz PE, Taylor WF. Thymoma. A clinicopathologic review.

Cancer December 1987;60(11):2727-43.

7. Temes R. Primary Mediastinal Malignancies:Findings in 219 Patients. WJM March 1999;170(3):161-6.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Evaluation of The Role and Utility of Neuroimaging In New Onset Seizures Presenting To The Emergency Department Lalit Kumar1, Vipin Kumar2, Hardeep Singh Gill3*, G Avasthi4, Gagandeep Singh5, Rajesh Mahajan2 Department of Medicine, Gian Sagar Medical College and Hospital, Rajpura, India Department of Medicine, Dayanand Medical College and Hospital, Ludhiana, India 3 Department of General Surgery, Gian sagar medical college and hospital, Rajpura, India 4 Department of Medicine, SPS Apollo Hospital, Ludhiana, India 5 Department of neurology, Dayanand Medical College and Hospital, Ludhiana, India 1

2

Keywords: CT Scan, Electroencephalography, Epilepsy, MRI, Neuroimaging, Seizure.

ABSTRACT BACKGROUND: Epilepsy is defined as recurrent unprovoked seizures. Initial clinical assessment seldom gives a specific diagnosis and this leads to uncertainty about prognosis and further management. Aim of this study was to evaluate the role and utility of Computerized Tomography Scan and Electroencephalography in the setting of new onset seizures presenting to the emergency department.

METHODS: In this study, all the emergency patients, above 12 years of age, presenting with onset of new seizures within 72 hours prior to presentation, were included for a period of 1½ year. Patients were investigated about any medications taken and EEG was carried out when possible. CSF analysis was done in those seizures patients who had persistently altered mental status, infectious symptoms, elevated WBC count or fever. Patients were subjected to CT scan head and other investigations were carried out based on clinical history and examination.

RESULT: Out of total 110 patients studied, Computerized tomography was done in 88 patients and abnormal findings were found in 44(50%) patients, which is a significant finding.MRI brain was done in only 13 patients and was found to have abnormalities in 12(92.3%) patients. EEG was done in only 20 patients and significant changes were seen in only 3(15%) patients.

CONCLUSION: It was concluded that seizures are one of the common presenting complaints in the emergency department. Imaging studies especially CT scan and MRI are an important part of evaluation of patients with new onset seizures and should be done routinely in these patients.

*Corresponding author: DR. Hardeep Singh Gill , Associate Professor, Department of General Surgery Gian Sagar Medical College And Hospital, Rajpura 140601 Punjab, India Phone: +91 9888507626 E-mail: hardeepgill77@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-133

Introduction

A seizure is defined as discrete spontaneous alteration in behavior or subjective experience occurring due to an abnormal hypersynchronous excessive discharge of a collection of neurons within the brain. Individuals who suffer a seizure are referred to emergency department because of the concern that seizures are potentially serious and dangerous [1].

A community-based study in UK reported that 21% of newly occurring seizures fall into category of acute symptomatic seizures [2].

The incidence of new onset seizures and etiological profile may depend upon referral pattern of patients in a particular location and the availability of tertiary care hospital facilities including availability of routine biochemical investigations and neuroimaging studies.

Till now, very little is known about the etiological profile of new onset seizures presenting to emergency department.

Also the problems presented by seizures in emergency department are neither well defined nor studied. Seizures and epilepsy are not emphasized in studies of ED care and the few specific studies of emergency seizures focus mainly on seizure diagnosis and acute seizure control [1, 3].

The management protocol of these patients and role of neuroimaging studies and EEG in these setting of seizures presenting to the emergency department remain unclear.

Very few studies have been done so far till now to address the role of neuroimaging in these patients.

Earlier electroencephalography was the single most commonly used diagnostic study in patients with seizure and it used to be done in each patient who presented with a seizure activity. EEG may be helpful in diagnosing seizures, classifying the seizure type and monitoring response to treatment. However in cases of new onset seizure presenting to emergency department, its usefulness may be limited to confirm the diagnosis of nonconvulsive status epileptics or to establish the presence of status epilepticus in a patient who has been given long acting paralytic agents to facilitate intubation [4].

CT scan is now considered the imaging modality of choice in patients presenting to the emergency department due to its ease, accuracy and availability. Incidence of abnormal CT scans increases with age. Previous literature had shown abnormal CT scan results in 3 to 41% of patients with first time seizures [5].

AABS; 3(2): 2016 Predictors of abnormal CT scans in new onset seizure patients include recent head trauma, abnormal neurological findings, multiple seizures, previous CNS disorders, focal seizures and history of malignancy.

Presently 1.5 T MRI is the imaging study of choice in patients presenting with epilepsy. It is superior to CT scan in identifying abnormalities of cortical architecture. MRI can detect subtle changes, especially in temporal lobes, which is an important advantage, since abnormalities in this region cause a significant percentage of seizures.

When a patient presents to the emergency department with history of seizures, it is important to get an accurate description of the event. The first step is to determine whether the event was truly a seizure and which diagnostic studies are needed [7]. A proper history should be taken and seizures must be differentiated from confusional states, syncope, arrhythmias or pseudoseizures.

It is a standard practice to do all routine biochemical investigations and get an EEG and CT scan in every patient presenting to emergency department, however there are no standard guidelines.

The role of neurologist in the evaluation and management of new onset seizures varies among different institutions.

Often the internist first sees the patient and later on neurologist is called. Ictal seizure semiology is identified and then seizures are classified in various categories according to classification proposed by international league against epilepsy (ILAE, 1989) [8].

Seizures are usually a manifestation of an underlying pathology which requires thorough evaluation including a careful history, physical examination, laboratory workup and electroencephalographic and neuroimaging studies as dictated by clinical suspicion. The evaluation and treatment of seizures in emergency department must be comprehensive [9].

Eisner and colleagues [3] in 1986 conducted a study on 163 patients to determine the efficacy of standard seizure workup in patients presenting to emergency department with history of seizures. This included detailed clinical examination, routine blood chemistries and cranial CT scan. This study showed that routine serum chemistries in patients presenting to emergency department is of extremely low yield.

William and colleagues (1993) [6] supported CT imaging use only if an abnormal neurologic examination existed or a history of malignancy was present.

Role of Electroencephalography: Electroencephalography is done since long in patients with seizures. However role of EEG in patients presenting to emergency with new onset seizures is less well defined.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-134

EEG helps to define the seizure type and its classification and is also useful in cases when patients are suspected to have nonconvulsive status epilepticus. An urgent EEG in the emergency department is recommended for those patients with persistent altered mental status in whom subtle convulsive or nonconvulsive status epilepticus is suspected. An EEG is also required when a patient motor activity has been suppressed by either paralysis or barbiturate coma and assessment of ongoing seizure activity is needed An EEG performed, soon after the seizures, is more likely to show any abnormalities than one performed later [10].

EEG after sleep deprivation increases the yield in detecting epileptiform abnormalities.

Hopkins A et al [11] in 1998 did a study on 408 patients and it was found that an EEG in adults is of low yield or negligible value in predicting seizure recurrence after an initial seizure. Also a normal EEG does not disapprove the diagnosis.

Mark et al (1998) [12] studied 300 consecutive adults who presented with unexplained seizures. Epileptiform abnormalities were shown in 43% of first EEG records.

Nonepileptiform abnormalities alone were shown in 25% of these EEG records and 3.2% of records were normal.

Also epileptiform abnormalities were observed in 80 of 156(51%) patients who had an EEG within first 24 hours, compared with only 49% of 144 patients who had EEG later on. It was found that higher diagnostic yield was due to performing earlier EEG within 48 hours than later EEG done after 48 hours.

Role of CT scan: CT scan is often recommended in the evaluation of new onset seizures in the emergency department and may be particularly valuable when cause of seizures is inapparent. It is currently unclear whether CT imaging is needed in all emergency department patients who donâ&#x20AC;&#x2122;t have a readily identifiable explanation for their disorder.

Only few studies have been done so far to study the utility of radiologic investigations on patients presenting to emergency department with new onset seizures. The reported incidence of CT abnormalities ranges from 3% to 41% [13, 5]. It has been found to be high in patients with partial seizures and in those with history of seizures after age of 30 years. Predictors of abnormal CT scans in new onset seizures include recent head trauma, abnormal neurologic findings, multiple seizures, previous CNS disorders, focal seizures and history of malignancy.

An evidence based clinical policy regarding neuroimaging of patients with first time seizures was published in 1996

[14]. According to those recommendations, a head CT scan was advised to be done in patients with history of head trauma, in those patients in which some acute intracranial process is suspected, with history of malignancy, immunocompromise, fever, persistent headache or a new focal neurological deficit and age older than 40 years.

In view of the above literature, we can see that very few studies have been done so far to study the etiology of seizures in patients presenting to emergency department.

Some of the studies have suggested routine biochemical investigations in these subsets of patients, while others have advised detailed laboratory workup in these patients.

Also the usefulness of doing imaging studies is not clearly defined in patients with first time seizures. So we undertook this study to do detailed clinical workup in patients presenting to emergency department with new onset seizures along with routine investigations and CT scan in particular in these patients.

Aims and objectives: To evaluate the role and utility of Neuroimaging and Electroencephalography in the setting of new onset seizures presenting to the Emergency Department.

Materials And Methods

All the emergency patients above 12 years of age presenting to emergency department with onset of new seizures within 72 hours prior to presentation were included in the study for a period of 1½ year.

All the patients were questioned about duration of onset of seizures, numbers of seizures and seizure semiology was noted as per Luders classification of Luders et al [15]. An attempt was made to assign an electro-clinical syndrome to the seizures according to the 1989 ILAE classification when possible. Seizure clusters were noted within 24 hours, 48 hours and 72 hours and history regarding postictal neurological deficit, psychosis and postictal headache was obtained.

Pertinent neurological examination was carried out noting the time of examination and the contributory history to find out the cause of seizures was taken. Patients were subjected to all routine investigations i.e. Haemogram, Urine R/E, Stool R/E, blood sugar, LFT, RFT, Serum calcium, serum phosphorus, serum magnesium and ABG. Patients were investigated about any medications taken and EEG was carried out when possible. CSF analysis was done in those seizures patients who had persistently altered mental status, infectious symptoms, elevated WBC count or fever.

Patients were subjected to CT scan head and other investigations, if indicated, were carried out based on clinical history and examination.

http://www.pacificejournals.com/aabs

A-135

AABS; 3(2): 2016

Final diagnosis was made based on ictal, interictal seizure semiology, neurological examination, clinical history and electroclinical classification.

patients. Other abnormalities seen were chronic infarct (11.3%), ICH (11.3%), SAH (4.5%), diffuse cerebral oedema, multiple ischaemic foci and hydrocephalus.

Any treatment given in the form of antiepileptics and other treatment given was noted.

List of patients in which CT scan not done: Table 3 shows list of patients, in which CT scan was not done and diagnosis made in these patients. Total number of such patients was 22. Out of these, cause remained unestablished in five patients. Uraemic seizures were seen in three patients. In another three, diagnosis of acute infarct was made by MRI findings.

Inclusion Criteria 1. Age >12 years.

2. New onset seizures in 72 hours prior presentation to Emergency department.

Exclusion criteria 1. Patients having history of seizures of more than 72 hours prior to presentation in the Emergency department.

2. Diagnosis of non-epileptic seizures.

The patient cohort was analyzed for demographic trends, seizure semiology patterns, Electroclinical classification, Aetiological diagnosis and EEG or imaging abnormalities.

For purpose of analysis appropriate statistical tests were applied.

Result

In this study, total of 110 consecutive patients with new onset seizures who presented to emergency department were included. Patients with age <12 years and with history of head trauma were excluded from the study.

The study was conducted for period of one and a halfyear. The patients were subjected to routine biochemical investigations including renal function tests, liver function tests, computerized tomography scan head, magnetic resonance imaging brain (where ever needed) and electroencephalography (if possible).

Role of imaging and EEG: Table 1 shows that CT scan was done in total of 88 patients and 44(50%) patients had abnormal findings on CT scan.

MRI was done in 13 patients and was found to be abnormal in 12(92.3%) patients. EEG was done in 20 patients and 9 of these patients showed some abnormality. MRI alone was done in 8 patients, out of which 7 (87.5%) patients had abnormal scans. Both MRI and CT scan was done in 5 patients and in this group of patients, CT was normal in two while MRI showed multiple ischaemic foci in one and in another, findings were consistent with diagnosis of acute disseminated encephalomyelitis.

List of EEG findings: Table 4 shows that EEG was done in 20(18.1%) of patients. It was found to be normal in 11(55%) patients.

Five patients had mild diffuse encephalopathy. PLEDS were detected in 3 (15%) patients. Among these three patients, diagnosis of presumed viral encephalitis was in 2 patients. In one patient, EEG was suggestive of some structural lesion in left hemisphere and CT scan in this patient showed chronic infarct.

Discussion

In the present study, we included 110 consecutive patients with history of new-onset seizures who presented to emergency department for a period of one and a half years. We excluded patients who were less than 12 years of age and also had seizures in more than 72 hours prior to admission and with diagnosis of non-epileptic seizures.

Patients with history of head trauma were also excluded from the study.

Epileptic seizures are usually easily distinguishable into provoked and unprovoked seizures. Epilepsy is defined as recurrent unprovoked seizures. Initial clinical assessment seldom gives a specific diagnosis and this leads to uncertainty about prognosis and further management.

However a good history, thorough clinical examination, routine biochemical investigations and routine neuroimaging are usually sufficient to distinguish between provoked and unprovoked seizures.

Role of imaging studies: The indications for performing a CT scan in the emergency department in patients with new onset seizures are controversial and different studies have conflicting results. In our hospital, CT scan is available on site on 24 hours basis without any waiting period while MRI facility is available off site.

List of patients with abnormal CT scan Table 2 is showing abnormalities detected on CT scan.

Forty four (50%) patients showed some abnormality on CT scan. Granulomatous lesion was detected in 13 (29.5%) patients on CT scan. Acute infarct was seen in 9(20.4%)

Sempere et al (1992) [16] reported structural lesion in 34% of patients on CT scan with first seizure in adult.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Henneman et al (1994) [5] in a retrospective analysis of 333 patients with new onset seizures reported CT abnormalities in 41%.

Original Article

A-136

Table 1: Role of imaging and EEG Investigation

Total patients

Normal

Abnormal

CT Scan

88

44(50%)

44(50%)

MRI Brain

13

1(7.7%)

12(92.3%)

EEG

20

11(55%)

9(45%)

MRI alone

8

1(12.5%)

7(87.5%)

MRI +CT Scan

5

CT normal-2

MRI abnormal-5

Table 2: List of patients with abnormal CT scan Abnormality

Number of patients

Granulomatous lesion

13(29.5%)

Acute infarct

9(20.4%)

Chronic infarct

5(11.3%)

Intracranial haemorrhage

5(11.3%)

Hypodensities

3(6.8%)

Venous thrombosis

3(6.8%)

Subarachnoid haemorrhage

2(4.5%)

Hydrocephalus

1(2.2%)

Diffuse cerebral oedema

1(2.2%)

Multiple ischaemic foci

1(2.2%)

Tumour

1(2.2%)

Total

44

Table 3: List of patients in which CT scan not done: Etiological diagnosis

Number of patients

Unestablished

5

Uraemic seizures

3

Presumed viral encephalitis

3

Acute infarct

3

Organo-chlorine poisoning

2

Organophosphorus poisoning

1

Neurocysticercosis

1

Hypertensive encephalopathy

1

Hyperosmolar coma

1

Venous thrombosis

1

Hepatic encephalopathy

1

Total patients

22

Table 4: List of EEG findings: Finding on EEG Normal Mild diffuse encephalopathy PLEDS S/O Structural lesion left hemisphere

Number of patients 11(55%) 5((25%) 3(15%) 1(5%)

http://www.pacificejournals.com/aabs

A-137 In another study by Earnest et al (1988) [17] on 259 patients with suspected alcohol withdrawal seizures, 58% had abnormal CT scan results of which 16 (6%) had a clinically significant lesion.

Shoenenberger and Heim (1994) [18] reported abnormal CT result showing structural lesion in 40 (34%) of patients and concluded that diagnostic yield of CT brain in adults after a first generalized seizure is high.

Ramirez et al (1984) [19] demonstrated structural abnormality by CT scan in 55 (37%) of patients. Our study reported abnormal CT scan results in 44 (50%) patients.

Our study results did not match with view of Hopkins et al (1988) [11] who reported normal CT scan in 87.7% of and concluded that CT scan done in patients with first seizures is of no value.

However our study demonstrated abnormalities in 50% of patients with new onset seizure on performing CT scan, which is a significant result and cannot be ignored.

AABS; 3(2): 2016 Although we did EEG in only 20 patients only, our study results are matching with those of Sempere et al (1992) [16].

An EEG performed, soon after the seizures, is more likely to show any abnormalities than one performed later. EEG after sleep deprivation increases the yield in detecting epileptiform abnormalities.

Conclusion

So, it was concluded in the end that Seizures are one of the common presenting complaints in the emergency department. Imaging studies especially CT scan and MRI are an important part of evaluation of patients with new onset seizures and should be done routinely in these patients.

Acknowledgements None

Funding None

The results of our study are also supported by William et al (2002) [20] who demonstrated some form of CT abnormality in 35% of patients included in his study.

Competing Interests

To conclude, we recommend that routine imaging studies of brain, preferably MRI should be performed in patients with new onset seizures.

1. Day SC, Cook EF, Funkenstein H, Goldman L. Evaluation and outcome of emergency room patients with transient loss of consciousness. Am J Med. 1982; 73:15-23

CT scan is now considered the imaging modality of choice in patients presenting to the emergency department due to its ease, accuracy and availability. Incidence of abnormal CT scans increases with age.

Predictors of abnormal CT scans in new onset seizures include recent head trauma, abnormal neurologic findings, multiple seizures, previous CNS disorders, focal seizures and history of malignancy.

Role of EEG: The role of EEG in the evaluation of patients with new onset seizures in ED is not well studied.

Hopkins et al (1988) [11] on a study of 408 patients concluded that routine EEG should not be performed in patients after their first seizure.

Sempere et al (1992) [16] did EEG in 73 patients out of 98 patients studied after their first seizure. 32 cases (43.8%) had normal results, 24(32.9%) showed focal spikes or focal slowing.

None Declared

References

2. Sander JW, Hart YM, Johnson AL, Shorvon SD. National general practice study of epilepsy; newly diagnosed epileptic seizures in general population.

Lancet. 1990; 336: 1267-1271.

3. Eisner RF, Turnbull TL, Howes DS, Gold IW. Efficacy of a “standard” seizure workup in the emergency department. Ann Emerg Med. 1986; 15(1): 33-9.

4. Kaplan PW. Nonconvulsive status epilepticus in the emergency room. Epilepsia. 1996; 37: 643-650.

5. Henneman PL, DeRoos F, Lewis RJ. Determining the need of admission in patients with new-onset seizures.

Ann of Emerg Med. 1994 Dec; 24(6): 1108-14.

6. William RR, Franz JW, Kavita KE. Seizure patient selection for emergency computed tomography. Ann Emerg Med. 1993; 22-B: 1298-1303.

7. Prego-Lopez M, Devinsky O. Evaluation of a first seizure. Postgrad Med. 2002 Jan; 111(1); 34-6, 43-8.

We performed EEG in 20 patients and 11(55%) patients had normal results, five patients had mild diffuse encephalopathy. PLEDS were detected in 3 cases.

8. Commission on Classification and Terminology of the International League against Epilepsy. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia.1989; 30: 389–399.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-138

9. Jagoda A, Richardson L. The evaluation and treatment of seizures in the emergency department. Mt Sinai J Medicine. 1997 Sep-Oct; 64(4-5): 249-57.

10. Gotman J, Herciani MG. Electroencephalographic spiking activity, drug levels and seizure occurrence in epileptic patients. Ann Neurol. 1985 Jun; 17(6): 597-603.

11. Hopkins A, Garmar A, Clarke C. First seizure in adult life. Lancet. 1988 April; i: 721-726.

12. Mark AK, Newton M, Jackson GD et al. Epileptology of the first-seizure presentation: a clinical, electroencephalographic and magnetic resonance imaging study of 300 consecutive patients. Lancet.

1998; 352: 1007-11.

13. Tardy B, Lafond P, Convers P et al. Adult first generalized seizures: etiology, biological tests, EEG, CT scan, in an ED. Am J Emerg Med.1995; 86: 13-15.

14. American College of Emergency Physicians, American academy of neurology, American Association of Neurological Surgeons, American Society of Neuroradiology. Practice parameter: Neuroimaging in the emergency patient presenting with seizure. Ann Emerg Med. 1996; 27: 114-118.

15. Luders et al. Semiological seizure classification.

Epilepsia. 39(9): 1006-13; 1998.

16. Sempere et al. First seizures in adults: A prospective study for the Emergency department. Acta Neurol Scand.1992 Aug; 86(2): 134-8.

17. Earnest M, Feldmon H, Marx J et al. Intracranial lesions shown by CT in 259 cases of first alcohol related seizures. Neurology. 1988; 38: 1561-1565.

18. Shoenberger RA, Heim SM. Indications for computerized tomography of the brain in patients with first uncomplicated generalized seizures. BMJ. 1994; 309: 986-989.

19. Ramirez Mâ&#x20AC;&#x201C; Lassepas, Cipolle RJ, Morilla LR, Gumnit RJ. Value of Computed Tomographic Scan in the evaluation of adult patient after their first seizure.

Ann Neurol. 1984; 15: 536-543.

20. William RM, Michelle H, Biros, David AT et al.

Selective tomographic imaging of patients with newonset seizure disorder. Academic Emergency Med. 2002; 9: 143-147.

http://www.pacificejournals.com/aabs

Original Article Activation Energy and Q10 Values at Various Temperature Ranges for Alpha- Amylase Activity in Telescopium Telescopium.

Sharvari Nilesh Kudtarkar*, Prakash. V. Desai.

Department of Zoology, Goa University, Taleigao Plateau,Goa. India.

Keywords: Activation energy, Telescopium telescopium, Q10 value, Alpha Amylase Activity, Hepatopancreas

ABSTRACT Enzyme alpha amylase was purified to the electrophoretic homogeneity from hepatopancreas of Telescopium telescopium by ammonium sulphate fractionation procedures, dialysis and chromatographic procedure. Purified enzyme had the activity equivalent to 42.12 µ mol/mg/min. Samples were collected from Choraon Islands of Goa State during 20042006. Energy of Activation was maximum between temperatures 10 0C - 20 0C ,187.38 ±10.43 J/mol; at 60 0C to 70 0C it was equivalent to about 154 J/mol. Q10 value was highest at a temperature range of 10 0C to 20 0C and the enzyme velocity elevated by a factor of 0.065 ± 0.009. As the temperature range was elevated beyond 20 0C up to 70 0C, the lowest Q10 value was obtained at a temperature range of 60 0C to 70 0C and it was equivalent to 0.001 ±0.00.

*Corresponding author: Sharvari Nilesh Kudtarkar. M.Sc.[Oceanography], M.Phill. [Marine Zoology], Department of Zoology, Goa University, Taleigao Plateau,Goa-403 206. India.

E-mail: samikshank@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-140

Introduction

Telescopium telescopium (Linnaeus 1758) is a habitant of mangrove beds, this species is least influenced organism by wide fluctuations in the physicochemical environment of the estuary due to its wide range of tolerance[1]. It belongs to the family Potamiidae popularly known as dominant sediment dwelling prosobranch and are richly represented in the mangrove fauna.[2] In the present study the alpha amylase of hepatopancreas of Telescopium telescopium has been purified and characterized with regard to its temperature stability and energy of activation, Q10 values were calculated.

Enzymes lower the energy required to start these reactions.

Activation energy represents the minimum amount of energy required in order for a process to occur. Q10 term is the factor by which reaction velocity is increased by a rise of 10 0C [3].

As temperature is a measure of molecular agitation [4], it controls the rate of chemical reactions and is one factor limiting energy liberation and organismic growth. Kinetic activity, frequency of molecular collisions is proportional to absolute temperature and increases 3% with 10oC rise in temperature where as enzyme activity increases many times more.

Materials And Methods

Specimens of Telescopium telescopium were collected from Mandovi estuary, Choraon Islands, Goa during 2004 to 2006. Each animal was opened with the help of bone cutter aseptically[5]. Hepatopancreas from intestinal region were isolated; the epithelial sheath was removed carefully using forceps. Wet weight of each animal was measured and recorded. 0.1gm tissue was weighed from each sample and homogenized in chilled mortar and pestle. Homogenate was mixed in 10 ml chilled phosphate buffer and mixed well[6]. Centrifugation was performed in a cooling centrifuge machine at 2000 rpm for 20 mins. , supernant were collected from each samples and stored at 40C and further used for enzyme extraction and purification.

Ammonium sulphate precipitations were performed as described by Deutschen(1990)[7]. Dialysis was performed after getting fractions from precipitation procedure, for salt removal from protein solution. Each fraction was dialyzed twice for 24 hrs at 40C. Volume was measured and recorded. Crude extract dialysate was purified using DEAE cellulose in column chromatography [8]procedure described

by Fagan et. al (1986). The specific activity was determined following the method of Lowry et. al[9] and was expressed as units per mg soluble protein (µ mg -1) subsequently mass of Maltose in mg/gm protein was calculated for temperature regimen of 10,20,30,40,50,60,70 0C.

The temperature dependence of a reaction that is critical incremental energy of enzyme activity is given by Arrhenius equation and is calculated using formula,

Q10 values at various temperatures for amylase activity of hepatopancreas of Telescopium telescopium were calculated using the formula,

Where, K1, K2are velocity constants corresponding the temperature t1 and t2. The activation energy values and Q10 values are graphically expressed in graph number -1 and 2 respectively.

Result

Graph number -1 illustrates the activation energy values of the amylase activity of hepatopancreas of Telescopium telescopium. Energy of activation was maximum between temperatures, 10 oC-20 oC, 187.38±10.43 J/mol. Later from 20 oC to 60 oC the values did not change significantly.

The energy of activation was minimum at 60oC – 70oC temperature and was equivalent to about 154J/mol. The values of energy of activation fluctuated for temperature ranges like 20-30, 30- 40, 40-50, 50-60, and 60-70oC. The energy of activation nearly remained the same for a temperature range of 40-60oC but for a temperature range of 30-40oC it was second highest to that obtained at10-20 o C.On an average, the energy of activation for α-amylase of Telescopium telescopium is equivalent to 163.83±3.33 Jules/mol.

Q10 values are graphically expressed in graph number-2.

Q10 value was highest at a temperature range of 10oC20oC and the enzyme velocity elevated by a factor of 0.065±0.009. As the temperature range was elevated beyond 20oC up to 70oC, the enzyme velocity factor lowered sharply. The lowest Q10 value was obtained at a temperature range of 60-70oC and it was equivalent to 0.001±0.00. For a temperature range of 20-30oC the Q10 value was equivalent to 0.015 while for a temperature range of 30-40oC it was about 0.008, and at temperature range of 40-50oC it was equivalent to 0.004.

Discussion

Heat is a result of molecular agitation and temperature is a measure of heat content. Environmental temperature limits the distribution of living organisms and is an important determinant of their activity. Kinetic activity, frequency of molecular collisions, is proportional to absolute temperature, where as enzyme activity increases

http://www.pacificejournals.com/aabs

A-141

AABS; 3(2): 2016

Graph No-1: Activation energy values of the Alpha amylase activity of hepatopancreas of Telescopium telescopium. The energy of activation is expressed as J/mol.

Graph No- 2 Q10 values for amylase enzyme activity of hepatopancreas of Telescopium telescopium

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-142

many times more. The determination of activation energy helps to understand the energy requirement of the enzyme molecules to get energized and initiate agitation which improves the chances of molecular collisions. The present investigation shows that the energy of activation nearly remained the same at 40-60oC but for a temperature range of 30-40oC it was second highest to that obtained at 1020oC. These finding indicates that at temperature beyond 10oC but less than 20 oC the amylase of Telescopium telescopium requires maximum energy for getting activated viz-a-viz for promoting molecular collision with substrate.

The successive rise in temperature lower the Q10 values of α-amylase of Telescopium telescopium precipitously.

The energy of activation for amylases on an average is 163.83±3.33 Jules/mol and this is probably the first report on energy of activation requirement of amylases from any mollusc studied so far.

Conclusion

Activation Energy is a property of the chemical reaction it varies according to the temperature changes. Present study indicates that beyond 10oC the enzyme requires the maximum energy for getting activated. The energy of activation for α-amylase of Telescopium telescopium is equivalent to 163.83±3.33 J/mol.

Q10 is the ratio of the rate of reaction at one temperature by the rate of the same reaction at a temperature 10oC less. The results of the experiment indicate that the successive rise in temperature the Q10 values of α-amylases of Telescopium telescopium lower subsequently.

Acknowledgements

I take this opportunity to express my profound gratitude to my guide Prof. Dr. P. V. Desai for his guidance and unfailing patience and without whose support and encouragement, this work would not have been completed. I equally thankful to Dr. Shanti Desai for providing valuable assistance in my work. I am also thankful to teaching and non-teaching staff of the Zoology Dept. Goa University.

Funding None

Competing Interests None declared.

References:

1. Das S. Ghosh U.R., Sur R., Ghose K.C. (1982) ‘Comparative studies on food and digestion in Viviparus bengalensis, Acrostoma variable and Telescopium telescopium’ Comparative Physiology and Ecology. Vol7, (3) p. 312-335.

2. Joshi G. V. and B.B. Jamale(1975) ‘Ecological studies in mangroves of Terekhol and vashisti rivers’, p. 751760.Bull Dept. Mar. Science University of Cochin-7, Volume-4.

3. A. E. Stearn. (1949) Kinetics of Biological Reactions, p. 25-41. In Advances in Enzymology and Related areas of Molecular Biology, Vol. 9. Edited by F. F.Nord.,Fordham Uni. N.Y. Interscience Publishers.

4. Benzinger, T. H. (1971), Thermodynamics, Chemical reactions and molecular biology. Nature (London).

229; 100-102.

5. Das S. Manna B. (1993) ‘Alimentary system of Telescopium telescopium’. Environment and Ecology’ Vol 11, p.283-291.

6. David T Plummer (1979). ‘An Introduction to Practical Biochemistry”;2ndedition, p.273-275. Mc Graw-Hill Publishing Co.Ltd., U.K. 7. Deutscher M.P.(1990), ‘Methods in Enzymology’ Vol. 182.(Guide to protein purification) Academic Press N.Y. p380-392.

8. Fagan J. M. (1986)’Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes’.Jr. Biological Chemistry Vol. 262.No 6 pp 2451-2457.

9. Lowry OH, et al. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951:193:265-75.

http://www.pacificejournals.com/aabs

Original Article Frequency of Beta Thalassemia Trait in Pediatric Age Group in a Tertiary Care Hospital in South India B.N. Krishnamurthy1, S.R.Niveditha2*, Poornima Shankar3 Dept of Pathology, Sri Jayadeva Institute of Cardiovascular Sciences and Research, Bengaluru, India 2 Dept of Pathology, Kempegowda Institute of Medical Sciences, Bengaluru, India 3 Dept of Pediatrics, Kempegowda Instiute of Medical Sciences, Bengaluru, India

1

Keywords: Discriminant Functions, Beta Thalassemia Trait, Hb Electrophoresis, Paediatric Age Group

ABSTRACT Background: Approximately 3% of world’s population carry β-thalassemia genes. The prevention of birth of Beta Thalassemia major children lies in effectively screening the carriers. Prevalence of β-thalassemia trait (BTT) varies from 3.5% to 14.9% in India. The objective of the present study was to study the frequency of BTT in pediatric age group at a medical college hospital in Bengaluru and compare the effectiveness of various discriminant functions( DFs) in predicting BTT in microcytic cases.

Methods: From January-2010 to June-2011, 574 pediatric cases with MCV<80fl were screened for possible BTT with five DFs ie., Mentzer index ( DF1) , Shine and Lal (DF2) , Srivastava Index (DF3), RDW index (DF4 ) and Green and King index (DF5). Cases with ≥ 2 positive DFs were subjected to haemoglobin electrophoresis on agarose gel at pH 8.6.

Cases with HbA2 levels ≥4.0%, were diagnosed as BTT. Result: About 195 cases showed positivity for at least two of the DFs suggesting BTT, among whom HbA2 level ≥4.0 was seen in 119 cases ( frequency of 19.76%). Cases with BTT, majority (55.63%) showed normocytic normochromic blood picture with a mean HbA2 of 5.22± 0.79%, mean RBC count of 4.94±0.48 x 1012/l and mean Haemoglobin of 11.38±1.86 gms/dl. DF2, DF4 and DF5 showed greater sensitivity ( 85.84%, 85.85% & 82.3% respectively).

Conclusion: Frequency of BTT was 19.76% among pediatric age group with microcytosis. DF2, DF4 and DF5 along with routine hemogram data in microcytic cases could effectively discriminate between BTT and non-BTT. Being a community based hospital catering predominantly to vokkaliga community, the high frequency of BTT could reflect the increased prevalence, thereby creating a need for awarenesss and conducting regular screening programmes to detect BTT.

*Corresponding author: Dr S.R.Niveditha, Professor, Dept of Pathology, Kempegowda Institute of Medical sciences, BSK 2nd Stage, Bengaluru-560070, India Phone: +91-9845485544 E-mail: srniveditha@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-144

Introduction

β-Thalassemia is the commonest inherited haemoglobinopathy all over the world1 and is the most common single gene disorder in our country.In India every year over 9000 Thalassemic children are born and, an estimated annual average consumption of 27 units of blood and 4, 00,000 Rupees worth of drugs are needed to manage each β-Thalassemia patient according to the recommended standards. Hence the financial burden induced is tremendous especially in a resource restricted country like India. The most effective approach to reduce the disease incidence is, implementation of carrier screening programs, offering genetic counselling, prenatal diagnosis and selective termination of affected fetuses.2 Prevalence of BTT varies from 3.5-14.9% in various regions of India, 1 however most of the Indian studies are based in northern India1,2,. To the best of our knowledge we did not find any studies regarding prevalence of BTT in Karnataka in pediatric age group and hence this study was formulated to study the frequency of BTT at department of pathology, Kempegowda institute of Medical sciences, Bengaluru.

Aims and Objectives:

● To screen all children in the age group of 6 months to 18 years with microcytosis (MCV<80fl) for possible BTT with a set of discriminant indices (DFs). ● To diagnose BTT in children with two positive DFs by haemoglobin electrophoresis. ● To examine the diagnostic accuracy of the discriminations indices (Mentzer’s, Shine and Lal, Srivastava, RDW (RDWI) and Green and King) in the diagnosis of BTT.

Materials and Methods

All pediatric ( 6 months to 18 yrs) blood samples received in the department of pathology for routine hemogram were run on coulter (ABX Pentra-60-HORIBA ABX Diagnostics). Samples with microcytosis ie <80fl were separated and included in study population (n=574) after obtaining informed consent. Mentzer’s , Shine and Lal ‘s, Srivastava’s , RDW and Green and King’s Indices were calculated. The discrimination limits (cut-off point), which suggested BTT and NON-BTT were as suggested in table 1.

Those cases positive for at least 2 of the above discriminant functions were further subjected to haemoglobin electrophoresis (Horizontal electrophoresis tank and power supply by Helena Laboratories, Beaumount, Texas) on agarose gel at alkaline pH (8.6)using the SAS-MX alkaline Hb-10 kit. 8 Quantitation of HbA2 was done by an

automated densitometer scanner using HELENA software.

Cases with HbA2 ≥ 4.0% were considered diagnostic for BTT. The relative percentages of each haemoglobin type on the gel could be determined by densitometry of the completed gel at 595nm. (fig 1)

Fig. 1: Agarose gel plate showing prominent HbA2 bands (≥4.0%) in all samples except sample no. 5

Statistical methods: Chi-square test was used to test the significance of proportions of lab parameters between BTT and NON-BTT diagnosed based on electrophoresis. Student ‘t’ test (independent) was used to find the significance of mean values of lab parameters and discriminant functions to differentiate between BTT and NON-BTT. The Odd’s ratio was used to find the strength of relationship of BTT and lab parameters. The multivariate Logistic Regression was used to find the significant predictors among lab parameters and discriminant functions of BTT Statistical software: Microsoft Excel 2010 has been used to generate descriptive statistics. The statistical software namely STATA 10.0 IC FOR WINDOWS (COLLEGE STATION, TEXAS USA) was used for the univariate and multivariate analyses.

Result

Of the 574 cases of microcytosis , around 39.9% of study population were in 1-5 years age group followed by 29.62% in the age group >10 years. The study population consisted of 59% males and 41% females. Majority were Hindus (78.22%) by religion followed by Muslims (19.52%) and Christians (1.92%).

Possible BTT was suggested in 195 (34%) out of 574 cases of microcytosis, in whom at least two of the five DFs were beyond the cut off limits. All these 195 cases were subjected to Hb electrophoresis. Of these , 113cases (n=195) showed

http://www.pacificejournals.com/aabs

A-145

AABS; 3(2): 2016 Multivariate Logistic Regression: (table 4) The diagnostic values are obtained independently and will not account for the interaction between the parameters.

Hence multivariate logistic regression analysis was done to evaluate the best combination of DFs to screen cases of BTT. All DFs were significantly associated with BTT. Subjects having DF1<13 were 5.09 times more at risk of having BTT compared to those with DF1≥13. Subjects having DF4≤220 were 7.74times more at risk having BTT compared to those with DF4>220 while subjects having DF5<65 were 4.50 times more at risk having BTT compared to those with DF1≥65.

increased HbA2 in the range of 4.01 to 7.39% and mean 5.22 ± 0.79%. The HbA2levels in non-BTT group ranged between 0.3-3.97 with a mean of 3.01 ± 0.77.

The prevalence of Beta thalassemia trait was 19.69% in the present study .The highest prevalence was observed in Muslims (31/112). As the study population contained majority of Hindus (78.22%) it is but obvious that prevalence of BTT was seen in good number in the same group (79/449) The RBC count was >5.0 million/cu mm in 43.36% of BTT cases while only 9.33% of Non-BTT cases showed increased red cell count, which was statistically significant (p value <0.0001). Mean RBC count was significantly more in BTT (4.94x 10 12cells/L) when compared to NON-BTT (4.44x 10 12 cells/L). However the haemoglobin was above 10 gm% in 85% of BTT cases(mean of 11.65g/dl ). MCH was lower i.e. <26.5 pg in 84.96% cases of BTT with a significant p value of <0.01. RDW was significantly lower in 64.60% of i.e., in 73/113 BTT cases. Hence high RBC count, low MCH and low RDW levels were significantly associated with BTT. (table2)

Discussion

The classic heterozygote carrier of BTT is usually asymptomatic.1 The diagnosis is made through evaluation of positive family history or during population screening.9 Though family history of thalassemia is important, a significant number of patients do not have previously affected family members.10 Given the seriousness of homozygous β-thalassemia, population/risk group screening for BTT is important to enable family screening and genetic counseling.1In this direction, an algorithm was used in the present study to segregate cases of non-BTT and possible BTT by simple, cost effective screening tests like the discriminant indices using the red cell indices.11

On peripheral smear examination majority of BTT cases (55.63%) presented with normocytic normochromic blood picture followed by uniform microcytosis .Target cells and basophilic stippling were also positively associated with BTT. Although this association is significant, the small numbers of subjects hint towards random occurrence of this association.

England and Fraser suggested that DFs should only be used when BTT is suspected in individuals with microcytosis of <80fl.12 It has been proposed that electronic measurement of MCV<80fl should be used as a screening test for BTT as per the BCSH General Hematology Task Force guide lines for investigation of the α and β thalassemia traits.13 Nishi Madan, Meera Sikka et al in 1999 studied 463 BTT cases and 40 controls and found sensitivity of MCV<80fl to be 98.21% and MCV<70fl to be 89.3%.1 Hence in the present study all the pediatric cases with MCV<80fl were screened for possibility of BTT.

Association of Discriminant Functions: (table 3) All the discriminant functions were positively associated with BTT with significant p values. The most sensitive DF was found to be DF4 and DF2 with sensitivity of 85.85% and 85.84%, respectively,however the specificity of DF2 was lower (31.02%). Srivastava index (DF3) showed highest specificity (94.36%), but lowest sensitivity (15.93%). Lowest false positivity (3.47%) was seen with DF3 whereas DF2 showed highest false positivity (68.98%). The DF4 showed highest PPV (53.30%) whereas DF2 showed lowest PPV (22.99%). The DF4 showed highest NPV (95.92%) followed closely by DF5(94.92%).

About 1.5% to 3% of world population carries the β-Thalassemia gene. The overall prevalence of BTT in India is about 3.3%14 to 4.05%,15 however the distribution

Table 1 Discrimination Limits (cut off points) for BTT and NON-BTT Discrimination Functions Mentzer index(MI)

BTT

NON-BTT

MI =MCV/RBC

<13

>13

Shine and Lal (S&L) 4

S & L = (MCV)2xMCHX0.01

<1530

>1530

Srivastava Index

S = MCH/RBC

<3.8

>3.8

RDWI = MCV x RDW/RBC

≤ 220

> 220

G&K = (MCV) x RDW/100xHb

<65

>65

3

5

RDW Index(RDWI) 6 Green and King index (G&K)

7

2

MCV – Mean Corpuscular Volume. RBC – Red Blood Cell count, MCH – Mean Corpuscular Haemoglobin, RDW – Red Cell Distribution Width , Hb – Haemoglobin

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-146

Table 2: Mean pattern of various lab parameters in BTT in different studies1, 19- 21, 25, 29, 30-33.

Study Das Gupta et at (1994) Mohamed M et al (1999) KhinEi Han et al (1992) Nishi Madan et al (1999)

No.of cases (n) n=56 n=382 n=133 n=337

Demir A et al (2002) Ahmed Suliman (2006) Rathod DA et al (2007) Rao et al (2009)

n=37 n=307 n=170 n=145

Hb (g/dl) 11.2±1.4 11.3±1.45 11.5±1.6 11.6±1.6 (p<0.0001) 10.88±0.82 12±1.6 10.34±0.015 10.3±2.1

Patel Jet al (2009) Nishi Madan et al (2010) Present study

n=70 n=449 n-113

10.14 10.9±1.1 11.65±1.71

RBC (x1012/l) 5.6±0.7 5.45±0.71 5.9±1.0 5.56±0.76 (p<0.0001) 5.4±0.41 6.53±0.75 5.52±0.07 5.06±0.9

MCV fl 64.5±3.7 64.81±4.72 62.7±12.1 64.7±4.8

MCH Pg 20±1.2 20.75±1.64 19.9±3.5 20.6±3.6

MCHC %

RDW%

31.2±0.94 29.3±2.2 -

15.1±1.2 16.06±0.97 -

61.66±3.98 61±6.8 62.99±0.52 68.6±7.4

20.54±2.03 20.2±2.4 18.83±0.20 20.5±2.6

32.38±1.09 28.3±1.8

14.91±1.13 15.58±0.28 -

5.37 5.5±0.6 4.94±0.48

63.15 62±7 69.01±7.37

18.75 19.8±2.1 23.62±3.27

29.92 15.04 31.7±2.5 34.17±1.89 14.91±2.63

Table 3: Diagnostic values of Discriminant Functions with respect to Electrophoresis Discriminant Functions

Diagnostic values in relation to Electrophoresis Sensitivity(%)

Specificity (%)

PPV

NPV

DF1(<13) MCV/RBC

30.97

92.62

50.72

84.55

DF2(<1530)(MCV)2xMCHX0.01

85.84

29.50

22.99

89.47

DF3(<3.8) MCH/RBC

15.93

94.36

40.91

82.08

DF4(≤220) MCVxRDW/RBC

85.85

81.56

53.30

95.92

DF5(<65) (MCV)2xRDW/100xHb

82.3

81.13

51.67

94.92

Table 4: Multivariate Logistic Regression for BTT for Discriminant Functions Model Parameters

Estimates of Multivariate Logistic Regression Model Adjusted Odds Ratio

Lower

Higher

DF1(Mentzer)

5.09

1.66

15.64

0.00

DF2 (shine & Lal)

1.97

0.98

3.95

0.06

DF3 (srivastava)

0.22

0.06

0.80

0.02

DF4 (RDW index)

7.74

3.35

17.87

<0.0001

DF5(<65)Green and King index

4.50

2.04

9.93

<0.0001

of β thalassemia gene is not uniform in the Indian subcontinent and therefore has varying frequency in different regions. Different authors have recorded the prevalence of BTT in various geographic areas of India.(table 5) The prevalence of BTT in our study was 19.69% and is in the range of prevalence as studied by various authors in India, in the recent times, as shown above. Prevalence of BTT in general population in Karnataka has not been studied by many authors. In a hospital based study at our institute in the year 2006 24the frequency of BTT was 6.8% in general population. A multicentre trial by D Mohanty et al in 2013 included Bengaluru as one of the centers to address this issue. In their study including Kolkata, Dibrugarh, Ludhiana, Mumbai and Vadodara,

95% Confidence Interval

P value

Bengaluru was also one of the centers.23 They recorded a prevalence of 2.16% in Bengaluru with highest prevalence among Jains(9.6%), followed by Rajputs(6.3%), sunni muslims(5.8%), Shiya muslims(6.3%) and vokkaligas (2.1%). It is interesting to note that in a general population, Vokkaligas recorded a rate of 2.1%, however our hospital caters predominantly to vokkaliga community thereby increasing the frequency rate.

Prevalence of BTT in pediatric age group has been studied by few authors( Demir et al,25- 58.7%, Ambekar et al26 -0.5%, A Earley et al 27 -21% ,Nishi Madan21 - 2.68% in Mumbai and 5.47% in Delhi children).To the best of our knowledge we could not find any literature regarding the prevalence of BTT in pediatric age group, in Karnataka

http://www.pacificejournals.com/aabs

A-147 so as to compare. However Madan N et al recorded a frequency of 6.5% BTT in school going children in Delhi who originally hailed from Karnataka.21 In a diverse country like India, the frequency of BTT has been found to vary in various religions and sub castes.12, 16, 17,21 In the present study 78.22% of patients were Hindus.

We could not correlate the frequency with various sub castes due to non-availability of records.

BTT cases generally have mild anemia with mild decrease in Hb value (table 2). In the present study mean RBC count was higher in BTT as compared to non-BTT, the mean MCV count and mean MCH were lower and comparable to the findings in other studies. However mean MCHC showed no difference as compared to non-BTT. In the present study mean RDW count was 14.91 ± 2.63% in BTT as compared to 15.36 ± 2.75% in non-BTT confirming that the red cells in cases of BTT are indeed microcytic homogeneous.

BTT results from mutations affecting single beta-globin locus.The mutations generally reduce the output of beta globin RNA and therefore the synthesis of beta globin but does not produce structurally abnormal haemoglobin.

Therefore given the same amount of haemoglobin, red cells show microcytosiswith mild anisocytosis in BTT compared to IDA in peripheral smear. Target cells and basophilic stippling are encountered more frequently in BTT.34 peripheral blood film examination usually reveals microcytosiswith mild anisocytosis),target cells and fine basophilic stippling than with IDA given the same level of anemia.34,35 In the present study majority of the cases presented with normocytic, normochromic blood picture followed by uniform microcytic hypochromic blood picture without anisocytosis. BTT cases with microcytic hypochromic red cells showing mild anisocytosis were a minority. BTT cases showed good number of target cells while basophilic stippling was uncommon. The findings of peripheral smears were in concordance with other studies 13,33 in the literature.

The various mathematical formulae using RBC indices have been proposed to differentiate BTT from IDA and to screen for BTT in patients with microcytic anemia1. In the present study 574 pediatric cases with microcytosis (MCV<80fl), were screened with the various DFs. At least two of the five DF’s were positive in 195 cases (33.97%) which suggested BTT. Shine and Lal &RDW indices were the most sensitive DFs(85.84%& 85.85%). Srivastav(DF3) and Mentzer’s (DF1) were the DFs with highest specificity (94.36%& 92.62% respectively). With a high sensitivity, DF 2&4 are good screening tests while DF 3&1 are good Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 diagnostic tests due to their high specificity compared to other DFs.(table3) Mulltivariate logistic regression analysis showed , DFs 1, 2, 3 and 4 were associated with higher risk of BTT with reference to their respective categories. DF3 was however associated with lower risk of BTT and this correlates very well with a very low sensitivity observed in our study.

To our knowledge there is no published literature on multivariate logistic regression analysis of DFs to validate our results.

In conclusion, differentiation of BTT from NON-BTT has important clinical implications in hematology and medicine. The present study demonstrates that a set of linear discriminant functions (DF2 and DF4) using routine hemogram data can effectively discriminate between BTT from non-BTT. The diagnosis of BTT relies on an accurate estimation of HbA2 levels. A raised HbA2 level (≥ 3.9%) 39preferably by Hb electrophoresis on cellulose acetate or by HPLC is the gold standard for the diagnosis of BTT. HbA2 estimation by scanning densitometry is not recommended due to overestimation.28 However as a pilot study , we performed Electrophoresis on agarose gel at alkaline pH on 195 (2 or more DF positive) cases, after screening 574 microcytic cases. Chopra et al 18studied 1032 patients of anemia and conducted electrophoresis on all the cases with alkaline buffered agarose gel and later quantified the various bands using densitometer as in our study. A total of 176 cases were found to be BTT with a cut off >3.5% of HbA2.

The cut off levels of HbA2 to label one as carrier of BTT varies between various studies ranging from >3.0%, >3.5%, >3.9% to >4.0%. Mohamed M et al30 in 1999 conducted study on 382 patients of which 34 patients were diagnosed as BTT based on Hb electrophoresis on cellulose acetate (CAE). HbA2 values more than 3.5% on elution were diagnosed as BTT. Desai S.N. et al43 in 1998 included 95 cases for comparison of CAE electrophoresis with Fast Protein Liquid Chromatography (FPLC). They found both techniques were equally comparable and reproducible.43 In the present study 113cases (n=195) showed increased HbA2- ( ≥4.0%) with a mean HbA2 of 5.22± 0.79 which was higher than non BTT cases (3.01± 0.77).

HbA2 levels are altered by various factors. For eg., decreased HbA2 levels are seen in cases of iron deficiency, sideroblastic anemia,”silent” Beta thalassemia alleles, βδ thalassemia, HbH disease, Hb Lepore and erythroleukemia while increased levels are observed in cases of megaloblastic anemia, in patients on anti-retroviral therapy and hypothyroidism.28 e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-148

Table 5: Frequency of BTT in India by various authors Authors

BTT

year

Balgir RS et al (Orissa)

18.2%

2004

Mulchandani D et al 17 (sindhi community, Nagpur)

16.81%

2008

Chopra GS etal (AFMC, New Delhi )

17.0%

2008

16

18

Patel J et al (Gujarat, India)

16.35%

2009

Rao S et al20 ( New Delhi)

18.1%

2010

Madan N et al ( Mumbai and Delhi)

4.05%

2010

19

21

Vani chandrashekar et al (Chennai)

37.09%

2011

2.16%(Bengaluru)

2013

19.69%

2011

22

D.Mohanty et al(multicentre trial) 23 Bengaluru Present study n=113, p=574

n = number of cases of a particular disease studied, p= number of population of a particular disease studied.

Table 6: Comparison of sensitivity (Number of patients correctly classified as BTT) of BTT with other studies Studies George Klee et al (1976)

Sensitivity (%)

Number of cases

DF1

DF2

DF3

DF4

DF5

30

84.6

-

84.6

-

-

KhinEi Han et al (1992)31

133

71.4

88.7

66.9

-

-

Mamatha et al (1997)

142

66.2

-

-

55.6

-

36

37

Das Gupta et al (1997)

56

60.0

-

-

-

-

Mohamed M et al(1999)30

382

70.6

-

-

-

90.3

Nishi Madan et al (1999)1

337

88.7

97.9

89.0

-

-

Demir A et al (2002)

29

63

86.0

100

60.0

100

97.0

Ntaios G et al (2007)38

373

59.78

-

-

63.8

70.06

Rathod DA et al (2007)14

170

92.0

99.01

89.3

-

91.1

Niazi M et al (2010)39

223

89.0

72.0

61.0

91.0

86.0

Present study (2011)

113

30.21

85.84

16.9

86.62

80.28

25

Table 7: Comparison of Mean HbA2 values in BTT cases with other studies Study KhinEi Han et al

Year

No. of cases

Mean HbA2%

1992

133

5.7±1.3

Desai S.N. et al43

1998

27

5.34±0.467

Mohamed M et al30

1999

134

4.94±0.59

Ahmed Al-Suliman32

2005

307

5.3±1.2

Rathod DA et al

2007

170

5.84±0.06

Rao et al

2009

145

5.5±0.6

2011

113

5.22± 0.79

20

Present study

31

The higher rate of BTT in our study may be attributed to the predominantly vokkaliga community that our hospital is catering to or to the fact that we have used scanning densitometry to quantitate HbA2 levels(following agargel electrophoresis at alkaline pH) which is known to overestimate.28 Nutritional deficiencies affecting HbA2 like megaloblastic anemia and iron deficiency anemia,

hormonal disturbances like hypothyroidism and DNA studies for co-existence of delta mutation could not done.

Conclusion

We conclude that the differentiation of BTT from non-BTT has an important clinical implication in hematology and medicine. The present study demonstrates that set of cost

http://www.pacificejournals.com/aabs

A-149 effective screening tests like DF2, DF4 and DF5 along with routine hemogram data in microcytic cases can effectively filter probable cases of BTT so that they can be subjected to confirmatory tests like HbA2 quantitation.

Prevalence of BTT in the pediatric population attending our hospital is significant suggesting a need to create awareness about the disease and it’s clinical implications highlighting the necessity to set up regular screening programmes. Practice of consanguineous marraiges are higher in southern India, hence family work-up should be performed in positive cases.

AABS; 3(2): 2016

12.

13.

14.

Reference

1. Madan N, Meera S, Satendra S, Rusia U, Kusum Kela.

Red cells Indices and Discriminant Functions in the Detection of Beta-Thalassemia Trait in a Population with High Prevalence of Iron Deficiency Anemia.

Indian J Pathol Microbiol 1999;42(1) :55-61.

2. Maheshwari M, Arora S, Kabra M, Menon PSN. Carrier screening and prenatal Diagnosis of BetaThalassemia. Indian Pediatr 1999;36:1119-1125.

3. Mentzer WC. Differentiation of iron deficiency from thalassemia trait. Lancet 1973;1 : 882.

4. Shine I, Lal S. A Strategy to detect Alpha thalassemia major. Lancet 1977; 1:692-694.

5. Srivastava PC. Differentiation of thalassemia minor from iron deficiency. Lancet 1973; 2:154-155.

6. Jayabose S, Giavanelli J, Levendoglu-Tugal O, Sandoval C, Özkaynak F, Visintainer P.Differentiating iron deficiency index. J Pediatr Hematol 1999; 21: 314.

7. Green R, King R. A new red blood cell discriminant incorporating volume dispersion for differentiating iron deficiency anaemia from thalassemia minor.

Blood Cells. 1989;15:481–495 8. Barbara Wild, Barbara J. Bain: Investigation of abnormal hemoglobins and thalassemia. SM Lewis, BJ Bain, I Bates. Dacie and Lewis Practical Hematology.

10thed, Churchill Livingstone, 2006, 271-310 9. Caterina B, Galanellao R. Thalassemia and Related Disorders: Quntitative disorders of Hemoglobin Synthesis. John P. Gree et al. Wintrobes Clinical Hematology, 12th edn, Lippincott Williams and Wilkins, 2009; 1083-1131 10. Louise L, Sylivia TS. Thalassemia: current approach to an old disease. Pediatr Clin N Am 2002;49:1165-1191 11. Lafferty JD, Mark A, Crowther, Mahmoud A Ali, Levine M. The evaluation of various mathematical RBC Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

15.

16.

17.

18.

19.

20.

21.

22.

indices and their efficacy in discriminating between thalassemic and non-thalassemic microcytosis. Am J Clin Pathol 1996; 106:201-205.

Guidelines for investigation of the Alpha and Beta Thalassemia traits. The thalassemia Working Party of the BCSH General Hematology Task Force. J Clin Pathol 1994;47 : 289-295.

England JM, Fraser PM. Differentiation of iron deficiency from thalassemia trait by routine blood count. Lancet1973; 1:449-452.

Rathod DA, Kaur A, PatelV, Patel K, Kabrawala R, PatelV, Patel M and Shah P. Usefulness of Cell Counter–Based Parameters and Formulas in Detection of β-Thalassemia Trait in Areas of High Prevalence.

Am J Clin Pathol 2007; 128:585-589 Madan N, Sharma S, Sood SK, Colah R, Bhatia HM. Frequency of β-Thalassemia trait and other hemoglobinopathies in northern and western India Ind J Hum Gen. 2010;16(1).16-25 Balqir RS. Spectrum of haemoglobinopahies in the state of Orissa, India. A ten years cohort study. J Asso Phys India.2005:Dec;53:1021-1026.

Mulchandani DV, Fulare MB, Zodpey SP, Vasudeo ND. Prevalence and Some Epidemiological Factors of Beta Thalassemia Trait in Sindhi Community of Nagpur City, India.Indian Journal of Public Health.

2008;.52(1): 11-15.

Chopra GS, Nair V, VSM, Gupta PK, Mishra DK, Sharma A, Mathew OP. Spectrum of Haemoglobinopathies in a Tertiary CareHospital of Armed Forces. MJAFI, 2008; 64(4) ; 311-314.

Patel J, Patel A, Patel J, Kaur A, Patel V: Prevalence of Haemoglobinopathies in Gujarat, India: A CrossSectional Study. The Internet Journal of Hematology.

2009 Volume 5 Number 1.(ISSN:1540-2649 Rao S, Kar R, Gupta Sk, Chopra A &Saxena R. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India. Ind J Med Res. 2010;132;513-519.

Madan N, Sharma S, Sood SK, Colah R, Bhatia HM. Frequency of β-Thalassemia trait and other hemoglobinopathies in northern and western India Ind J Hum Gen. 2010;16(1).16-25.

Chandrashekar V, Soni M. Hemoglobin Disorders in South India.International Scholarly Research Network (ISRN) Hematology Volume 2011, Article ID 748939, 6 pages, doi:10.5402/2011/748939.

23. D. Mohanty, R. B. Colah, A. C. Gorakshakar, R. Z. Patel, D. C. Master,J. Mahanta, S. K. Sharma, U. e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-150

Chaudhari, M. Ghosh, S. Das, R. P. Britt,S. Singh, C. Ross, L. Jagannathan, R. Kaul, D. K. Shukla, and V. Muthuswamy .J Community Genet. 2013 Jan; 4(1): 33–42.

24. Sujatha R , Sreekantha , Niveditha S R, Avinash SS , Remya ,Vinodchandran , Rangaswamy R. The study of recent biochemical and pathological aspects of thalassemia-International Journal of Research in Health Sciences. 2013, 1(3),140-152.

25. Demir A, Yarali N, Fisgin T, et al. Most reliable indices in differentiation between thalassemia trait and iron deficiency anemia. Pediatrics International (2002) 44, 612–616 26. Ambekar SS, Phadke MA, Mokashi GD, Bankar MP, Khedkar VA, Venkat V. Busulkar DG. Pattern of hemoglobinopathesis in Western Maharashtra. Indian Pediatr 2001; 38: 530-534 27. Earley A. Valman HB. Altman DG. Pippard MJ. Microcytosis, iron deficiency in Preschool children.

Archives of Diease in Childhood 1990; 65:610-614.

28. A Mosca, R Paleari, G Ivaldi, R Galanello, P C Giordano.

The role of haemoglobin A2 testing in the diagnosis of thalassaemias and related haemoglobinopathies, J Clin Pathol 2009;62:13–17. doi:10.1136/jcp.2008.056945 29. Gupta AD, Hegde C, Mistri R. Red cell distribution width as a measure of severity of iron deficiency in iron deficiency anemia. Indian J Med Res 1994; 100: 177-183.

30. Mohamed M, Edibany, Kameel F, Ninos J Joseph and Douglas Rhone. Usefulness of certain RBC indices in diagnosing and differentiating thalassemia trait from Iron Deficiency anaemia. Am J ClinPathol 1999; 111:676-682.

31. KhinEi Han, AungMyo Han and TheinMyint.

Thalassemia in the outpatient department of the Yangon children’s hospital in Myanmar: Basic Haematological values of Thalassemia Traits. South East Asian J. Trop Med Public Helath 1992; 23(2):264-268.

32. Al-Suliman A. Prevalence of β-thalassemia trait in premarital screening in Al-Hasan, Saudi Arabia. Ann Saudi Med 2006, 26;(1):14-16

33. Mark C, Walters, Herbert T, Abelson. Interpretation of the Complete blood Count. Pediatr Clin N Am 1996; 43(3) : 559-621.

34. England JM, Fraser PM. Differentiation of iron deficiency from thalassemia trait by routine blood count. Lancet1973; 1:449-452.

35. Klee GG, Fairbabks VF, Pierre RV et al Routine erythrocyte measurements in diagnsosis of iron deficiency anemia and thalassemia minor. Am J Clin Pathol 1976;66:870-877.

36. Manglani M, Lokeshwar MR, Vani VG, Nishi B, Vijay M, ‘NESTROFT’ – An Effective Screening Test for Beta – Thalassemia Trait. Indian Pediatr 1997; 34:702-707.

37. Ntaios G, Chatzinikolaou A, Saouli Z, et al.

Discrimination indices as screening tests for β-thalassemic trait.Ann Hematol (2007) 86:487–491.

38. Niazi M, Tahir M, Raziq F, Hameed A.Usefulness of Red Cell Indices in Differentiating Microcytic Hypochromic Anemias. Gomal J Med Sci 2010;(8):2:125-129.

39. Seema Rao, Rakhee Kar, Sanjeev Kumar Gupta, Anita Chopra & Renu Saxena .

40. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India. Indian J Med Res 132, November 2010, pp 513-519 41. Tzetis M, Trager J, Kanavakis E, Mavromati A, Kattamis C. The molecular basis of normal HbA2 (type 2) beta thalassemia in Greece. Hematol Pathol 1994; 8(1-2): 25-34.

42. Madan N, Sikka M, Sharma S, Rusia U. Hematological parameters and HbA2 levels in beta thalassemia trait with coincident iron deficiency. Indian J Pathol Microbiol 1998; 41(3);309-313.

43. Desai SN, Colah RB, Mohanty D. Comparison of FPLC with cellulose acetate electrophoresis for the diagnosis of beta-thalassemia trait. Indian J Med Res 1998;108: 145-148.

44. Ishwar C. Verma, Renu Saxena & Sudha Kohli,, Past, present & future scenario of thalassaemic care & control in India. Indian J Med Res 2011, 134: 507-521

http://www.pacificejournals.com/aabs

Original Article Histopathological Spectrum of Endometrial Changes in Women Presenting with Abnormal Uterine Bleeding with Special Reference to Endometrial Malignancies : A Two Years Hospital Based Study.

Riju Rani Deka1*, Tanma Saikia2, Amitabh Handique1, Basanta Sonowal1 Dept. of Pathology, Tezpur Medical College, Assam, India Department of O&G, Tezpur Medical College, Assam, India 1

2

Keywords: Abnormal Uterine Bleeding, Clear Cell Carcinoma, Endometrial Hyperplasia, Endometrial Malignancy, Proliferative Endometrium.

ABSTRACT Background: Abnormal uterine bleeding(AUB) is a common symptom of the patients in all age groups attending out patient department of O&G. AUB is caused by a wide variety of causes in different age. Histopathological examination of endometrial biopsy is a major diagnostic tool in evaluation of AUB. Aim: To study the spectrum of endometrial changes in all age groups presenting with AUB and to find the incidence of endometrial malignancies in this area of upper Assam.

Methods: The study was a retrospective cross sectional study of cases presenting with AUB . D&C material and endometrial biopsy specimens were collected in Pathology Department of Tezpur Medical College from 2013 July to 2015 June. Specimens were routinely processed and stained with H&E stain and morphological evaluation was done.

Patients were categorized into reproductive age group(18- 40 yrs), perimenopausal (41yrs -50 yrs) and postmenopausal (> 50years).

Result: A total of 150 specimens of D&C materials and endometrial biopsies were collected of which 18 cases were excluded due to inadequate sampling and finding of 132 cases were analysed . Maximum number of cases were received from reproductive age group (51.6%). Functional causes(64.4%) were the predominating irrespective of age, proliferative phase endometrium(37.2%) was the overall predominant histopathological finding. Most common cause of organic lesion was pregnancy related complications(PRC)(63%).In reproductive age PRC (35.3%) and perimenopausal age groups proliferative endometrium (35.6%) was commonest. In post menopausal age atrophic endometrium(57.8%) was the commonest finding. One case of endometrial carcinoma(0.75%) which was clear cell carcinoma, was recorded in 31-40 years group.

Conclusion: Histopathological evaluation of endometrial samples can be used as first step for diagnosis of abnormal uterine bleeding specially in post menopausal women who are at increased risk of malignancy. However histopathological evaluation is a challenge for the pathologist due to the frequent receipt of inadequate endometrial samples.

*Corresponding author: Dr. Riju Rani Deka, C/O P. C. Deka, Bishnu Nagar, Bishnu Rabha Path, P.O- Tezpur,Dist-Sonitpur,State-Assam, Pin-784001, Phone: +91-09435180795 E-mail: dr.rijurdeka82@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-152

Introduction:

Abnormal uterine bleeding(AUB) is defined as any departure from normal menstruation or from a normal menstrual cycle pattern.1 bleeding is considered abnormal when the pattern is irregular, abnormal duration(>7 days), or menorrhagia or abnormal amount(>80ml/ menses).2 Causes include functional causes like normal cyclical endometrium, abnormal physiological changes of endometrium (atrophic endometrium, weakly proliferative endometrium, disordered proliferative) and organic lesions like chronic endometritis, hyperplasia, polyp, carcinomas and pregnancy related complications. Dysfunctional uterine bleeding is defined as a type of AUB where no underlying cause can be defined. But with passing time more and more causes are being defined.3 therefore diagnosis earlier included in DUB category are now classified4 under 3 headings.

the definition of an adequate endometrial biopsy specimen.9,10,11,12,13,14 Here material was termed inadequate when only a strip of cervical tissue and a very small amount of endometrial tissue composed of endometrial surface lining epithelium only was seen in the sample. A total of 18 cases were excluded from the study considering them as inadequate samples.

The age of the patients were categorized as reproductive (18-40 years), peri-menopausal(41-50years) and postmenopausal(>50 years ) age groups.

Disorders of endometrial origin (disturbances of molecular mechanisms responsible for regulation of the volume of menstrual bleeding )

The histopathological findings were classified as functional and organic causes. Functional causes included physiological cyclical changes i.e proliferative and secretory phases, atrophic and weakly proliferative endometrium, disordered proliferative endometrium,nonspecific degenerative changes. Organic causes included endometrial polyp, chronic endometritis, hyperplasia, carcinomas and pregnancy related complications which again comprises of retained product of conceptus (RPOC) and gestational trophoblastic diseases(GTD).

Disorders of hypothalamic-pituitary-ovarian axis.

Result

Disorders of haemostasis (coagulopathy) These three together are called non-structural causes of abnormal uterine bleeding.5 In reproductive age group heavy menstrual bleeding accounts for about 30% of cases,6 while postmenopausal bleeding accounts for 5% of all gynaecological visits.7 Histopathological examination of endometrial biopsy is a major diagnostic tool in evaluation of abnormal uterine bleeding.8 AIM: Varied causes of AUB and availability of endometrial samples led us to find out the endometrial causes of abnormal uterine bleeding in women of all age groups.

In our study, functional cause (64.4%) was the predominant finding for AUB [figure:1].Majority of cases were observed in reproductive age group(51.6%)[table.1].

Organic lesions(27.3%) were the major cause of AUB in reproductive age group while functional issues were predominating in perimenopausal and post menopausal age groups.[figure:2] Table 1: Cases in different age groups: Age group

Cases

Reproductive

68(51.6%)

Perimenopausal

45(34%)

Postmenopausal

19(14.4%)

Materials and Methods

This study was a retrospective cross sectional study carried out in the Department of Pathology, Tezpur Medical College, Assam for a period of two years from July 2013 to June2015. 150 samples collected mainly were D& C material and endometrial biopsy from specimens from women presenting with AUB, sent to the histopathology section of the Department of Pathology. Samples were fixed in 10% formalin and routinely processed, stained with H& E stain. Histopathological evaluation was done under light microscope.

A recent study showed that there is a considerable controversy among gynaecological pathologists about

Fig. 1: Diagram showing the distribution of causes:

http://www.pacificejournals.com/aabs

A-153

AABS; 3(2): 2016

Fig. 2: Distribution of causes in different age groups:

Fig. 3: Age trend of causes :

In our study it was observed that in early period of life, organic lesions were the main cause of AUB while with increasing age incidence of functional cause predominated. (fig:3)

Functional cause was the main reason of AUB in our study of which proliferative phase endometrial change(32 cases) followed by pregnancy related complications (29cases) which come under the category of organic lesions, were the predominating histopathological finding. This finding of proliferative endometrial change as a leading cause of AUB is comparable to studies by Anuradha Salvi et al (37.2%) and Agrawal et al. Proliferative endometrium was the commonest finding in Khare et al(26.8%) and Saera et al(34.6%) too though cases with pregnancy related complications were not taken into account in their studies.15,16 Again Vaidya et al found secretory phase endometrial change(22.58%) as major cause of AUB in their study but pregnancy related complications were their exclusion criteria.17

The common findings recorded in functional issues were normal cyclical endometrium i.e proliferative phase (37.2%), secretory phase (18.6%) and atrophic endometrium (26.7%) [table.2]. Most commonly seen lesions in the list of organic lesions were pregnancy related complications (63%) and endometrial hyperplasia (32.6%). [table.3]. pregnancy related complications in histopathological sections showed retained product of conceptus (54.3%) and hydatidiform mole (8.7%).

18-20 years of age group had minimum number of cases which were mainly pregnancy related endometrial finding with equal frequency i.e retained product of conceptus(50%) and hydatidiform mole(50%).[table.4] In 21-30 years of age RPOC was the predominant change (13, 46.4%) while in 31-50 years of age proliferative endometrium (30.5%, 35.6%) predominated. In 51-60 years atrophic endometrium(58.3%) was mainly seen.

Atrophic changes were the common finding seen at old age.[table4.]

One case of clear cell carcinoma of endometrium was recorded at 33years of age. Other histopathological finding of endometrium noted in our study were non specific degenerative changes, chronic endometritis. Endometrial hyperplasia which was mainly simple hyperplasia without atypia, seen mainly in perimenopausal age.

Discussion

Our study received maximum number of cases from reproductive age group which is comparable to the study carried out in Meerut by Khare et al who reported 62% and

by Saera et al in Pakistan who reported 64.8% cases from reproductive age group.15,16 But literature have mentioned studies where majority of cases were from perimenopausl age group .17,18,19 Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

In reproductive age group, pregnancy related complications were the commonest finding bellow 30 years of age and in 31-40 years of age cyclical endometrial change was predominant finding which is comparable to the study by Doraiswami et al in 2011.7 In perimenopausal age, cyclical endometrial change, mainly proliferative endometrium (35.6%) was the major cause which is comparable to the findings in Doraiswami et al(41%), Salvi et al(53%), Bhatta et al(29.8%) and also in Damle et al(34%) although the later considered only women above 40 years in their study.7,19,20,21 On the contrary, Vaidya et al found secretory phase as the predominating change in endometrial biopsy in perimenopausal age.17 Again distribution of cases of simple endometrial hyperplasia was highest in this age group. Literature mentioned studies where simple endometrial hyperplasia was one of the leading cause of AUB in perimenopausal age.15,21,22 This supports the anovulatory cycles in this age due to which excessive and prolonged estrogenic stimulus leads to these spectrum of changes in endometrium and hence bleeding.

In this study, atrophic endometrium was the major finding in postmenopausal group. This finding is comparable to the e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-154

Table2: functional causes of abnormal uterine bleeding: Functional causes Proliferative phase

32 (37.2%)

Secretory phase

16 (18.6%)

Atrophic endometrium

23 (26.7%)

Weakly proliferative

04 (4.7%)

Disordered proliferative

10 (11.6%)

Non specific degenerative change

01(1.2%)

Total

86(100%)

Table 3: organic lesions of abnormal uterine bleeding: Organic lesions Retained product of conceptus

25(54.3%)

Chronic endometritis

01(2.2%)

Endometrial hyperplasia

15(32.6%)

Endometrial carcinoma

01(2.2%)

H. mole

04(8.7%)

Total

46(100%)

Table4: distribution of study subjects (n=132) according to histological diagnosis in different age groups.

Histopathological diagnosis

18-20 yrs (%)

21-30 yrs (%)

31-40 yrs (%)

41-50 yrs (%)

51-60 yrs (%)

61-70 yrs (%)

>70yrs (%)

Proliferative endometrium

0

4 (14.3%)

11 (30.5%)

16 (35.6%)

0

1 (33.3%)

0

Secretory endometrium

0

3 (10.8%)

5 (13.9%)

5 (11.1%)

3 (25%)

0

0

Atrophic

0

1 (3.6%)

1 (2.8%)

10 (22.2%)

7 (58.3%)

1 (33.3%)

3 (75%)

Weakly proliferative

0

0

1 (2.8%)

1 (2.2%)

1 (8.3%)

1 (33.4%)

0

Disordered proliferative endometrium

0

01 (3.6%)

4 (11.1%)

5 (11.1%)

0

0

0

Non-specific degenerative change

0

1 (3.6%)

0

0

0

0

0

2 (50%)

13 (46.4%)

9 (25%)

1 (2.2%)

0

0

0

Chronic endometritis

0

0

1 (2.8%)

0

0

0

0

Simple endometrial hyperplasia

0

3 (10.7%)

3 (8.3%)

7 (15.6%)

1 (8.3%)

0

1 (25%)

Endometrial carcinoma

0

0

1 (2.8%)

0

0

0

0

2 (50%)

2 (7.2%)

0

0

0

0

0

04

28

36

45

12

3

4

RPOC

H.mole Total

http://www.pacificejournals.com/aabs

A-155

AABS; 3(2): 2016

Fig. 4: photomicrograph showing proliferative phase endometrium in endometrial biopsy section.(H& E,40X).

Fig. 5: photomicrograph showing section of endometrial biopsy showing papillary structure lined by polygonal cells with clear cytoplasm and enlarged hyperchromatic nuclei, prominent nucleoli.(H&E,40X).

Fig. 6: photomicrograph showing secretory endometrium in histopathological section from endometrial biopsy(H&E,40X)

Fig. 7: photomicrograph of histopathological section from endometrial biopsy showing simple endometrial hyperplasia without atypia. (H&E, 40X)

findings in most of the studies in literature.19,20,21,22 Khare et al found equal number of cases of atrophic endometrium and complex hyperplasia of endometrium without atypia.15 Again Vaidya et al found secretory endometrium and Sweta et al found histopathological finding of proliferative endometrium(45%) to be the major cause of AUB in postmenopausal age.17,18 Mechanism of bleeding due to atrophic endometrium in old age is stated in different studies as sclerotic degeneration of vessel wall or local abnormal haemostatic mechanism.

Incidence of endometrial carcinoma in our study was 0.75% (1) which was seen at 31-40 years of age group.

This finding is comparable to the finding in Mahapatro et al (0.7%), Prajapati et al (0.99%), closely to Sandeepa

et al(1.1%).22,23,24 On the contrary, this finding is lower than the incidence recorded by Doraiswami et al(4.4%), Khare et al(3.7%), Agrawal et al(3.5%) and Bhatta et al(5.7%).7,15,18,20 This dissimilarity may be explained by the majority of the population in our study which was mainly from reproductive age group and only endometrial cause of AUB was considered in our study. The only case of carcinoma was Clear cell carcinoma of endometrium confined to endometrial layer at the time of presentation.

Most of the studies found majority of cases of carcinoma in postmenopusal age.23,25 Karmakar et al found endometriod carcinoma to be the main histopathological variant.25 Incidence of clear cell carcinoma in literature were 20% and 5.5% (26,27) This difference in finding is due to the study

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-156

population in their studies which were mainly the women with endometrial carcinomas and proportion of different histopathological types were mainly evaluated.

Conclusion

Histopathological evaluation of endometrial samples can be used as first step for diagnosis of abnormal uterine bleeding specially in post menopausal women who are at increased risk of malignancy. However histopathological evaluation is a challenge for the pathologist due to the frequent receipt of inadequate endometrial samples.

Acknowledgements NONE

Funding None

Competing Interests None Declared

Abbreviation

AUB: Abnormal uterine bleeding RPOC: Retained product of conceptus GTD: Gestational trophoblastic disease PRC: Pregnancy related complication

Reference

1. Ian S. Fraser, M.D., Hilary O.D. Critchley, M.D., Michael Broder, M.D. and Malcolm G. Munro, M.D. The FIGO recommendations on Terminologies and Definitions for Abnormal uterine bleeding, seminars in reproductive medicine 2011; vol 29(5): pg 383-390.

2. Speroff L, Fritz MA. In : Clinical gynaecologic endocrinology and infertility. 7th edition. Jaypee Brothers Med Publishers (P) Ltd; 2005. Menopause and the peri-menopausal transition; 621–88.

3. Critchley HOD, Maybin JA. Molecular and cellular causes of abnormal uterine bleeding of endometrial origin. Semin Reprod Med 2011;29(5):400–409 4. Munro MG, Critchley HOD, Fraser IS. The flexible FIGO classification concept for underlying causes of abnormal uterine bleeding. Semin Reprod Med 2011;29(5):391–399 5. Dueholm M. Levonorgestrel-IUD should be offered before hysterectomy for abnormal uterine bleeding without uterine structural abnormalities: there are no more excuses! Acta Obstet Gynecol Scand 2009;88(12):1302–1304.

6. Moodley M,Roberts C. Clinical pathway for the evaluation of postmenopausal bleeding with an emphasis on endometrial cancer detection. J Obstet Gynaecol 2004; 24:736

7. Saraswati doraiswami, Thanka Johnson, Shalinee Rao, Aarthi Rajkumar, Jaya Vijayaraghavan and Vinod kumar Panicker. J Obstet Gynaecol India 2011;61(4):426- 430.

8. Market Opinion and Research International (MORI).

Women’s health in 1990. [Research study conducted on behalf of Parke-Davis Laboratories]. London: MORI; 1990 9. Phillips V, McCluggage W G. Results of a questionnaire regarding criteria for adequacy of endometrial biopsies. J Clin Pathol 2005:417–419.

10. Brenner PF. Differential diagnosis of AUB.Am J Obstet Gynecol.1996;175:766-9.

11. Leather AT,Savvas M, Studd W. Endometrial histology and bleeding patterns after 8 years of continuous estrogen and progesterone therapy in postmenopausal women. Obstet Gynecol. 1991;78(6):1008-10.

12. Critchley HO,Warner P, Lee AJ, Breehin S, Guise J, Graham B. Evaluation of abnormal uterine bleeding: Comparision of three out patient procedures within cohorts defined by age and menopausal status. Health technol Assess.2004;8(34):iii-iv,1-139.

13. Chambers JT, Chambers SK.endometrial sampling: when? Where? Why? With what? Clin Obstet Gynecol 1992;35:28-39.

14. Felix YF IU, Stephen PY Yong, Hon –Cheung LLE. Managment of insufficient endometrial biopsy for investigation of premenopausal abnormal uterine bleeding. Hong Kong J Gynaecol Obstet Midwifery 2014;14(1):108-13.

15. Khare A,Bansal R.,Sharma S.,Elhence P.,Makkar N.,Tyagi Y.Morphological spectrum of Endometrium in patients presenting with dysfunctional uterine bleeding. People’s Journal of Scientific Research .2012;5(2):13-16.

16. Saera Afghan,Ara Yasmeen. Abnormal Uterine Bleeding A clinicopathological study of 150 cases.

Ann. Pak.Inst.Med. Sci. 2013;9(4):201-204.

17. Vaidya S,Lakhey M,Vaidya Amatya S.,Sharma P K, Hirachand S,Lama S,KC S.Histopathological pattern of abnormal uterine bleeding in endometrial biopsies.

Nepal Med Coll J .2013;15(1):74-77.

18. Agrawal Sweta,Mathur Asha, Vaishnav Kusum.

Histopathological study of endometrium in abnormal uterine bleeding women of all age groups in Western Rajasthan(400 cases).International Journal of Basic and Applied Sciences.2014;4(3):15-18.

19. Salvi Anuradha, Mital Premlata,Hooja Nupur, Batar Alka, Soni Pradeepkumar, Beniwal Ramesh.Spectrum

http://www.pacificejournals.com/aabs

A-157

20.

21.

22.

23.

of endometrial histopathology in women presenting with abnormal uterine bleeding Sch. J. App Med Sci,2015;3(1A):1-4.

Bhatta S, Sinha AK.Histopathological study of endometrium in abnormal uterine bleeding.Journal of Pathology of Nepal.2012;2:297-300.

Damle Rajshri P,Dravid N.V,Kishor H,Suryawanshi, Gadre Arundhati S, Bagale Priya S, Ahire Neelam.

Clinicopathological spectrum of endometrial changes in perimenopausal amd post-menopausal abnormal uterine bleeding: A 2 year study. J Clin Diagn Res. 2013 ; 7(12): 2774â&#x20AC;&#x201C;2776.

Mahapatro Mitali, Mishra Pratima.Clinicopathological evaluationof abnormal uterine bleeding.Journal of Health Research and Reviews,2015;2(1):45-49.

Prajapati Rujuta, Meena R.,Daveshwar A. Clinicopathological correlation of endometrial pattern

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016

24.

25.

26.

27.

in patients with Abnormal Uterine Bleeding. Int. J. Res Med.2015;4(2):128-132.

Sandeepa Supriya, Jayprakash H.T,Ashwini M.C. Histopathological study of endometrium in Abnormal Uterine Bleeding in different age group. International Journal of Scientific Reasearch.2014;3(11):422-424.

Karmakar Pragati J,Wilkinson Anne, Rathod Mayuri.

Histopathologcal evaluation of postmenopausal bleeding,2014;13(10):53-57.

Wei Jain Ju, Paintal Ajit, Keh Pacita. Histologic and Immunohistochemical analysis of endometrial carcinomas. Arch Pathol Lab Med ,2013;137:1574-83.

Christopherson, William M, Alberhasky,Robert C, Connelly, Patrick J. Carcinoma of the Endometrium: I. A Clinicopathologic Study of Clear-Cell Carcinoma and Secretory Carcinoma. Pathology n laboratory medicine.2013:5(2);122-124.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Clinical Presentation of Dengue Outbreak 2015, Haryana, India- A Prospective Observational Study.”

Navtej Singh1*, Jyotsna Singh2 Dept of General Medicine, BPS Government Medical College for women Khanpur kalan, Sonepat, Haryana, India 2 Dept. of Anatomy, Government Medical College & Hospital, Chandigarh, India

1

Keywords: Dengue, Classical Dengue fever, Dengue Haemorrhagic Fever, Dengue Shock Syndrome.

ABSTRACT Background: Dengue fever is not recognized as a major public health hazard in Delhi and neighbouring areas as Haryana, so there is little evidence and awareness in this regard. A prospective observational study was carried out to determine the group of patients suffering from dengue syndrome; clinical parameters of the subjects for hospitalization and the pattern of presentation of dengue fever in a tertiary care centre of the state from September 2015 to November 2015.

Method: Total 400 serologically positive cases were selected randomly and diagnosed clinically as dengue, and were classified into 3 groups, i.e. 260 cases of classical dengue fever, 120 cases of dengue hemorrhagic fever(DHF) and 20 cases of dengue shock syndrome (DSS).

Results: Classical type of Dengue fever was the commonest (65%) variety. 258(64.5%) were males and 142(35.5%) were females. DHF/DSS were present in adult population (29 yrs). 50.5% of patients had low grade fever of 99˚-100˚F. Diarrhea and abdominal pain were chief complaints in 33.3% cases in group -2. 33.3 % patients of group-2 and 50% of group-3 patients had gum bleeding. 40 patients were diagnosed with acute cholecystitis on ultrasonography. Ascitis with bilateral pleural effusion was a common finding in 95% of the patients of group-2 with platelet counts of <20000.

Mortality rate was less than 1%.

Conclusion: Results of our study indicate that apart from usual manifestations, sometimes unusual but clinically extremely important manifestations can occur which if not detected early can prove fatal requiring vigilant approach.

*Corresponding author: Dr Navtej Singh M.D.(Medicine), Associate Professor, Dept of General Medicine BPS Government medical college for women Khanpur kalan, Sonepat , Haryana ,India Phone: +91-9812031853, 9779741853 E-mail: jyotsnanavtej@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-159

Introduction

Dengue is the most important arthropod-borne viral disease. The virus is transmitted to humans by the bite of infected female mosquitoes of the genus Aedes. The global resurgence of dengue is thought to be due to failure to control the Aedes populations, uncontrolled urbanization, population growth, climate change, and increased airplane travel[1]. Dengue viruses are single stranded RNA viruses of the family Flaviviridae. Dengue is caused by one of the four closely related, but antigenically distinct, virus sero-types 1 to 4(DEN-1, DEN-2, DEN-3, and DEN-4).

It is a frequent cause of febrile illness in the tropical and subtropical areas of the world[2]. Majority of the infections are asymptomatic and the clinical manifestations occur in two forms- classical dengue fever and dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF/DSS are uncommon in individuals above 15 years and are more common in secondary infection[4] .

Epidemics of dengue fever were first reported from the coast and later from the inland in Southeast Asia in the 19th century[5] . The first description of an outbreak of dengue haemorrhagic fever (DHF) was reported from the Philippines in 1953 . India belongs to category B, where dengue is an emerging disease with cyclical epidemic [6].

Out breaks have been reported in Delhi, India in 1996,1999,2003 and 2009[7-10].In following years, outbreaks did not occur but a high number of cases of suspected dengue infection were reported in rainy season. It has been thought of as a primarily urban disease[4]. Dengue fever was not recognized as a major public health hazard in Delhi and neighbouring areas as Haryana before 1996 outbreak, so there was little evidence and awareness in this regard. A prospective observational study was carried out to determine the group of patients suffering from dengue syndrome; clinical parameters of the subjects for hospitalization and the pattern of presentation of dengue fever in a tertiary care centre of the state from September 2015 to November 2015.

Materials and Methods

The study was conducted at a tertiary care hospital, located in Haryana in India. Serologically confirmed 400 cases of dengue were included for the study out of the 1100 cases who were admitted to our institution during an outbreak of dengue virus infection in Haryana. The diagnosis of dengue infection was based on the clinical presentation & a positive NS1 antigen test on laboratory examination.

Patients were grouped in 03 (three) groups dengue fever (DF), dengue hemorrhagic fever(DHF),and dengue shock syndrome, using the WHO criteria[14]. Subjects were selected according to selection criteria and excluded Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 following the exclusion criteria. The statistical analysis was performed by using frequency and percentage. The patients were treated in the indoor Selection criteria Group- 1(DF): Subjects with acute febrile illness for not more than 2-7 days having the manifestations like sudden onset of fever plus ● 2 or more of the following features i.e. a) severe headache, b) retro orbital pain, c) severe myalgia, arthralgia, backache, and d) leucopoenia and absence of convincing evidence of any other febrile illness.

Group- 2 (DHF): Group 1 manifestations plus hemorrhagic manifestation evidenced by one or more of the followings.

1. Positive tourniquet test.

2. Petechiae, ecchymosis, purpura 3. Bleeding from different sites like epistaxis, gum bleeding, haematemesis, malena, haematuria, menorrhagia and 4. Any evidence of plasma leakage manifested by ● A 20% rise in PCV for the age or sex and / or ● A 20% drop in PCV following treatment with intravenous fluid and/ or ● Pleural effusion, ascites, hypoproteinemia.

Group -3(DSS): Subjects circulatory failure as follows:

with

DHF

manifesting

1. Hypotension for age and/ or 2. Narrow pulse pressure (<20 mmHg) and/ or 3. Profound shock.

Exclusion criteria 1. Pregnancy 2. Patients with other co morbid conditions A detailed history was taken in all patients and a detailed clinical examination was done after an informed consent.

In addition, complete blood count, including platelet count was done. Chest X-ray and abdominal ultrasonography was done when indicated.

Result

The outbreak of dengue started during September, reached its peak in October, and lasted until late November 2015. A total 400 clinically and serologically diagnosed dengue infection were classified into three groups, i.e.

260 (65%) cases of dengue fever (group -1), 120 (30%) cases of dengue hemorrhagic fever/ DHF (group- 2), and 20(5%) cases of dengue shock syndrome/ DSS (group-3).

Classical type of Dengue fever was the commonest (65%) e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-160

variety. The majority of the study subjects were males and male to female ratio was 2.25:1 in group-1, 1.2:1 in group-2 and 1.5:1 in group-3 with a mean of 1.8:1. Mean ages of subjects were 29.16 years. Mean age of subjects were 29.75, 31.7 and 34.23 of group-1, group-2 group -3 respectively. Group- 1(DF) had 31% and group-2 (DHF) had 33% patients of 20-29 year age group. Group -3 (DSS) patients were older patients and 50% were 40 years and above.

All the patients had fever and almost all of them (100%) experienced sudden onset of fever in all groups. Maximum number (50.5%) of patients had low grade fever of 99˚-100˚F, (57.6%, 35.83%, 45%) in group 1,2,and 3 respectively. Fever was accompanied by chills and rigors in 70% of group 1(DF), 35% of group2 (DHF) and 20% of the (DSS) group 3 respectively. Mean duration of fever was 5.5±0.51 days in group 1, 7.12±0.2 days in group2, and 5.00±1.15 in group 3 subjects.

Headache and myalgia were the common clinical presentation. 100% in group-3 patients had complains of myalgia and low back pain whereas 80% complained of headache. Arthralgia was present only in 12.5% group-2 and 30% group-3 subjects. 25% experienced retro-orbital pain & arthritis in group-3, infrequent in group-2 and absent in group-1 respectively. Nausea and vomiting were common in all the groups, 100% in group-3. Diarrhea, abdominal distension and abdominal pain was present in 33.3% cases in group -2, whereas infrequent complains in other groups.

Different type of rash i.e macular, maculopapular and erythmatous rashes over the body were observed. 10% of group-1, 20% of group-2 and 25% of group-3 had macular rash over the trunk and legs. Majority of the patients of group-2(33.3%) and (50%) of group-3 patients had gum bleeding. 25% of the patients of group-2 had more than one bleeding manifestation. Un usual manifestations of dengue hemorrhagic fever (DHF) such as vaginal bleeding & bleeding per rectum were observed in less than 1% of the patients.(Table 3) Organomegaly was observed very rarely in group- 2, but in group - 3, 10% had hepatomegaly and less than 1% presented with splenic & lymph node enlargement. Majority of the group-2 (16.6%) had ascitis with pleural effusion.

Right pleural effusion was reported in 12% of the group-2 patients. Only 3 patients of group-2and 1 patient of group3 patient had jaundice with elevated SGOT/PT levels.

Laborotary investigation revealed thrombocytopenia, haemoconcentration and leucopenia. Thrombocytopenia was found in 100% cases. Platelet count came down below 10000/cumm only in 1% of the patients. Blood transfusion

was required in 20% cases and platelet concentrate was given only in 10% cases.

The present study also aimed to assess severity of disease by ultrasound findings and to correlate USG findings with blood platelet counts. Out of the total 60 patients who complained of abdominal pain 40 patients (10%) were diagnosed with acute acalculous cholecystitis on ultrasonography. The sonographic findings including gall bladder distension, gall bladder wall thickening and stratification (honeycomb appearance), right, left and bilateral pleural effusion with mild to moderate peritoneal fluid and mesenteric inflammation with decreased platelet counts. Ascitis with bilateral pleural effusion was a common finding in (95%) of the patients of group-2 with platelet counts of <20000 . 32% patients had bilateral pleural effusion and organomegaly with platelet count of 21000-40000 and only 2.5% cases on USG with counts of 60000 and above.. As the platelet count decreased, the severity of ascitis also increased. Organomegaly i.e hepatomegaly (60%) and splenomegaly(10%) were found in group-2 patients only. Two patients had inflammation in right perinepheric space and paracolic gutter with internal echoes with mild increase in renal cortical echogenecity.

In one patient moderate free fluid in peritoneal cavity with echogenic threads septations in right upper abdomen and paracolic gutter with internal echoes were seen . In few patients inflammation in perinephric and paranephric space was seen. A patient who presents with sonographically recognizable complications is more likely to have disease that requires immediate and aggressive management.

Three patients of dengue fever developed ARDS(acute respiratory distress syndrome). Inspite of management in ICU on mechanical ventilator, ARDS patients failed to survive (100% mortality). Mortality rate of less than 1% was encountered in the hospital during the outbreak.

In three cases dengue fever coexisted with plasmodium vivax malaria.

Fig:1: Presence of Acute Acalculous Cholecystitis on USG in dengue patients

http://www.pacificejournals.com/aabs

A-161

AABS; 3(2): 2016

Table 1: Distribution of study subjects according to age pain pattern experienced by them

Group-1 (n-260)

Pain pattern

Headache Myalgia Low back pain Retro-orbital pain Arthralgia arthritis

No 80 80 27 -

Group-2 (n=120) % 30.7 30.7 10.3 -

No 54 100 40 1 15 5

Group-3 (no-20) % 45 83.3 33.3 0.8 12.5 4.2

No 16 20 20 5 6 5

% 80 100 100 25 30

25

Table 2: Abdominal complaints of study subjects Abdominal complaint Nausea Vomiting Diarrhea Abdominal distension Abdominal pain

Group-1 (n-260) No 70 65 20 20

Group-2 (n=120) % 26.9 25% 7.6 7.6

No 75 90 39 40 40

Group-3 (no-20) % 62.5 75 32.5 33.3 33.3

No 20 20 04 -

% 100 100 20 -

Table 3: Different types of rash & hemorrhagic manifestations study subjects Type of rashes& hemorrhagic manifestations macular Macula-papular Erythematous Gum bleeding Conjunctival hemorrhage Patechial hemorrhage Epistaxis Haemetemesis malena Haemoptysis Haematuria Vaginal bleeding Bleeding per rectum >1 bleeding manifestation

No 26 06 06 -

Group-1 (n-260)

% 10 2.3 2.3 -

No 24 08 01 40 10 10 15 18 7 10 03 01 01 30

Group-2 (n=120)

% 20 6.7 0.8 33.3 8.3 8.3 12.5 15 5.8 8.3 2.5 0.8 0.8 25

No 5 -

Group-3 (no-20)

Table 4: Organomegaly and collection of fluid in potential spaces in study subjects Organomegaly & ascitis/ pleural effusion Liver Spleen Lymph node ascitis Pleural effusion Ascitis & p. effusion

Group-2 (n=120)

Group-3 (no-20)

No

%

No

%

08 08 00 12 12 30

6.6 6.6 00 16.6 16.6 25

02 1 1 04 00 00

10 0.8 0.8 3.3 00 00

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

% 25 00 00 50 -

Original Article

A-162

Results of our study indicate that apart from usual manifestations, sometimes unusual but clinically extremely important manifestations can occur which if not detected early can prove fatal. So a vigilant and timely approach is warranted. Epidemic has occurred in the post monsoon season requiring extensive preventive measures for vector control

Discussion

In this study, we primarily focused on the clinical manifestations and their associations with the severity of 400 clinically and serologically proved dengue patients.

Mean ages of subjects were 29.16 years, and most patients were in the range of 20-50 years, similar to study by Jhamb R,2010[10] who recorded mean age of subjects was 28 years.

Mean age of subjects were 29.75,31.7,and 34.23 of group-1, group-2 group -3 respectively. Wali JP et al,1999 [8] showed that mean age was 31±5.2 (SD), with most of the patients were in the range of 20-50 years, which is almost similar to this study. DHF/DSS are uncommon in individuals above 15 years and are more common in secondary infections[17,18] but now the pattern seem to change with adult individuals being affected by DHF/DSS. Our findings are consistent [9,10,12] . with published literature Majority (50.5%) of patients had low grade fever of 99˚-100˚F, (57.6% , 35.83%, 45%) in group 1,2,and 3 respectively, but Anuradha S et al ,1998[7]showed that fever was of high grade with chills and rigors. In our study high grade fever accompanied by chills and rigors was present in 70% of group 1(DF), 35% of group2 (DHF) and 20% of the (DSS) group 3 respectively. Mean duration of fever was 5.5±0.51 days in group -1, 7.12±0.2 days in group-2, and 5.00±1.15 in group- 3 subjects in our study was similar to that of Anuradha S et al,1998[7] (4.8±1.32 days) & Jhamb R,2010[10](5.47±2.2) , but differ to that of Wali JP et al,1999[8] (4±1.12 days).

Headache (37.5%) and myalgia (70%) were the common clinical presentation in our study, similar to available literature[7.8] . 100% patients in group-3 had complains of low back pain whereas 80% complained of headache.

Arthralgia was an uncommon presentation, present only in 12.5% group-2 and 30% group-3 subjects in present study, but arthalgia was present in 52.3% of patients of DHF epidemic in 1999[8]. 25% experienced retro-orbital pain & arthritis in group-3, infrequent in group-2 and absent in group-1 respectively. Our findings vary from available literature[7,8] Nausea and vomiting present in 43.75% of cases were common complaints as seen in previous outbreaks[9,10].

Diarrhea and abdominal pain were common complains in group-2 patients, whereas infrequent complains in other

groups, in contrast to Wali JP et al[8] who observed them in group-3 patients in his study. In our study, hemorrhagic manifestations in the form of gum bleeding and epistaxis was observed in 45.8% of cases , higher than previous outbreaks as reported (35.5%, 40%, 28.6%) by Jhamb R;2010[10], Singh N.P et.al;2005[9],and Anuradha S et.al1999[7] respectively. However rashes, patechial hemorrhage and malena was an infrequent occurrence[7,9,10], similar to our study.Unusual manifestations of dengue hemorrhagic fever (DHF) such as vaginal bleeding (menorrhagia) & bleeding per rectum were observed in less than 1% of the patients which has not been documented in previous outbreaks.

25% of the patients of group-2 had more than one bleeding manifestation in present study[7,8] . Organomegaly was observed very rarely in group- 2, but in Group - 3, 10% had hepatomegaly and less than 1% presented with splenic & lymph node enlargement, similar to the study by Singh NP et al ,2005[9]. In our study in group-2, ascitis was reported in 35% of the patients, whereas (16.6%) had ascitis with pleural effusion. Right pleural effusion was reported in 12% of the group-2 patients. Only 3 patients of group-2 and 1 patient of group- 3 patient had jaundice with elevated SGOT/PT levels. Kabra SK et al[13] found ascites in 87%, pleural effusion in 74% in DSS patients, whereas only 27% DHF without DSS had ascites and pleural effusion.

Sai PMV et al [14] on his sonographic study on fifth to seventh day of fever showed that all had gall bladder wall thickening, 21% had hepatomegaly, 7% had splenomegaly, 96% had ascites, 87.5% had right pleural effusion, 66% had left pleural effusion and 28.5% had pericardial fluid .In our study all had gall bladder wall thickening ranging from (5-16 mm) with mean of 6.7 mm. Ascitis was seen in 55% of the patients, 35%patients had right pleural effusion and equal number (35%) had bilateral pleural effusion along with acute acalculous cholecystitis as lumen appeared echo free on USG. Ascitis with bilateral pleural effusion was a common finding in (95%) of the patients of group-2 with platelet counts of <20000.

An unusual presentation was seen in three cases where dengue fever coexisted with plasmodium vivax malaria.

Similar presentation has been encountered in a single case of East Timor where both malaria and dengue lead to the demise of a 7 year old female[15], however in our study all three patients survived due to early diagnosis and proper management. The outbreak occurred in the post monsoon season in the present study similar to earlier outbreaks in North India[7-10], whereas epidemics coincide with the rainy season in South India[4] and other south east Asian countries[12]. Earlier studies in North India[4,8,9] showed that dengue epidemic affected the urban population in contrast it is the rural population that has been afflicted in our study.

http://www.pacificejournals.com/aabs

A-163

Conclusion

During the initial outbreak, the nature of the disease, and unfamiliarity on the part of the general people had made the situation a panic. Patients with dengue syndrome showed varied presentation and the symptoms are non specific. So it necessitates clinical awareness to combat mortality and morbidity. In this study, we tried to find out the clinical parameters with varied presentations to create better awareness and clinically diagnostic skills among the health care providers and people to identify and refer the patients promptly to proper health care facilities to avert the ultimate danger. More stringent measures in the form of vector control; especially in the monsoon and post monsoon season, improved sanitation and health education are needed to decrease health care costs caused by a preventable disease

AABS; 3(2): 2016 6.

7.

8.

9.

10.

11.

Acknowledgements

Dr Ram Chander Siwach M.S.,Director, BPS Government Medical college for Women Khanpur kalan ,Sonepat ,Haryana ,India director bpsgmc@yahoo.com , for the facilities to carry out the study

Funding

12.

13.

NA

Competing Interests

14.

Reference

15.

Conflict of Interest: there is no conflict of interest among the authors 1. Specimens from a Patient Who Had Recently Traveled from a Region Where Dengue Virus 2. Infection Is Endemic. J Clin Microbiol 2007; 45(5): 1523-27.

16.

3. Ahmedul Kabir. et.al The impact of dengue on liver function as evaluated by aminotransferase levels. J Medicine 2008; 9 : 66-68.

17.

4. World Health Organization. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control, 2nd edn. Geneva: WHO, 1997.

18.

5. P. Ggunasekaran .et al. Dengue disease status in Chennai (2006-2008): A retrospective analysis. Indian J.Med Res 2011;133:322-25

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

19.

Smith CEG. The history of dengue in tropical Asia and its probable relationship to the mosquito Aedes aegypti. Trop Med Hyg 1956; 243±51 Ashish Goel et.al Dengue fever- A dangerous Foe. J Indian Academy of Clinical Medicine 2004;5(3):247-58 Anuradha S et.alThe 1996 outbreak of dengue hemorrhagic fever in Delhi, India. South Asian JTrop Med Public Health 1998;29(3):503-6 Wali J.P et al. Dengue hemorrhagic fever in adults: a perspective syudy of 110 cases. Trop. Doct. 1999; 29(1):27-30 Singh N.P et.al The 2003 outbreak of dengue fever in Delhi, India. South East Asian J Trop Med Public Health 2005; 36(5) :1174-8 Jhamb R, Kumat A, Ranga GS, Rathi N. Unusual manifestations in dengue outbreak 2009, Delhi,India.

J Commun Dis. 2010 Dec:42 (4):255-61 World Health Organization. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control, 2nd edn. Geneva: WHO, 1997.

Chareonsook O, Foy HM, Teeraratkul A, Silarug N. Changing epidemiology of dengue haemorrhagic fever in Thailand. Epidemiol infect 1999; 122: 161-6.

Kabra SK, Jain Y, Pandey RM, Singhal T, Tripathi P, Broor S, et al. Dengue haemorrhagic fever in children in the 1996 Delhi epidemic. Trans Royal Soc Trop Med Hyg 1999; 93: 294-8.

Sai PMV, Dev B, Krishnan R. Role of ultrasound in dengue fever. British J Radiol 2005; 78:416-418.

David IW, A case of fatal plasmodium falciparum malaria complicated by acute dengue fever in East Timor. Am J Trop Med Hyg 2006;75(1):182-185.

Shah I, Deshpande GC, Tardeja PN, Outbreak of dengue in Mumbai and predictive markers for dengue shock syndrome. J Trop Pediatr. 2004 Oct;50(5):301-5.

Zuckerman AJ, Banatvala JE, Pattison JR, Griffiths PP, Schoub BD, editors. Principles and practice of clinical virology. 5th ed. West Sussex, England: John Wiley & Sons, Ltd; 2004.

Gregson A, Edelman R. Dengue virus infection. Pediatr Infect Dis J. 2003;22:179–81.[PubMed]

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Effect of Vit. B12 Supplementation on Auditory Neuropathy in Type 2 Diabetic Patients Assesssed by Brainstem Auditory Evoked Response Manish Bansal1, Manish Kumar1*, P K Maheshwari2, Prabhat Agrawal1, Ritu Agrawal3 Dept of Medicine, S. N. Medical College, Agra, India Dept of Neurology, S. N. Medical College, Agra, India 3 Dept of Pathology, G.R. Medical College, Gwalior, India 1

2

Keywords: Diabetes Mellitus, Vitamin B12, BAER, Auditory Neuropathy

ABSTRACT Background: Diabetes mellitus is one of the leading causes of neuropathy across the globe. Peripheral neuropathy is explored in most of the studies so far,In our study we tried to identify abnormalities in auditory nerve making the use of brainstem auditory evoked potential1,2,.

Methods: The patients were categorized into two groups, Group I includes patients who received Vit. B12 and group II includes patients who received placebo. Patients in both the groups were also taking gabapentin and antidiabetic treatment. Details of the patient, symptoms, history of diabetes, Fasting blood sugar,S. Vit. B12 level and Latencies of BAER were monitored at baseline and then periodically till 16 weeks.

Results: We found improvement in latency of BAER of both ears were significant in diabetic patients who received Vit. B12 than those taking placebo.

Conclusion: Abnormalities in brainstem auditory evoked potentials can be detected in diabetic patients even in the absence of clinical symptoms. Improvement in latencies of waves is seen after supplementation of vit. B12 as assessed by brainstem auditory evoked potentials. Although promising, further studies are needed to assess long term treatment of Vit. B12 in large sample of population.

*Corresponding author: Dr Manish Kumar. Tomar Clinic, New Defence Colony, G.T. Road, Muradnagar, Ghaziabad, U.P., INDIA,PIN:201206 Phone: +91-9639682500 E-mail: drmanishtomar@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-165

Introduction

Neuropathy is the one of most common complication, a great source of morbidity with huge expenditure in diabetes patients. The brain stem auditory electric responses identifies impairment of auditory nerve and other central connections at earlier stages before the appearance of any such symptoms1,3,4. Vitamin B12 deficiency is very common in patient with diabetes mellitus,both type 1 and type 25. We did this study using oral Vit. B12 1000mcg per day treatment over 16 weeks to study its effect and found promising results.

AIM: To study the effect of vitamin B12 supplementation on brainstem auditory evoked response in Type 2 diabetic patients with neuropathy.

Objectives

1. To study the Brainstem auditory evoked response in Type 2 diabetic patients.

2. To compare the Brainstem auditory evoked response in Type 2 DM patients after Vit. B12 supplementation to those receiving placebo.

Material and Methods

This study was carried out in PG Department of Medicine Sarojini Naidu Medical College, Agra on diabetics attending indoor and outdoor clinics. The study was conducted over a period of 18 months from March 2014.

Informed consent was taken from every patient included in this study and approval was taken from institutional ethical committee.

Study design : Study design was randomized double blind controlled trial. Baseline BAER was performed. Patients were divided into two groups Group I received Vit. B12 supplementation and Group II received placebo. Vit. B12 supplementation was given in dose of 1000mcg daily oral for 16 weeks. After 16 weeks BAER was repeated. BAER of supplemented group and group receiving placebo was compared. Paired chi square test was applied.

Inclusion criteria A case of diabetes mellitus (known case or recently detected) with sign or symptoms suggestive of diabetic neuropathy.

Exclusion criteria 1. Patient having any other diseases known to cause peripheral neuropathy like chronic renal failure, liver failure, hypothyroidism, leprosy, porphyria etc.

2. Patients with diabetes mellitus, i.e. taking drugs known to cause peripheral neuropathy like isoniazid, streptomycin, ethambutol, chloramphenicol, quinine, phenytoin or who are chronic alcoholics.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 3. Hearing loss from some other cause After dropouts both the study group and control group were having 60 patients each and hence data of total 120 patients was analysed. Out of these 120 cases, 58 were taking oral hypoglycemic drugs, 20 were on insulin and 42 were taking both and these were randomly distributed in both groups. Vit. B12 was given to study group.

Results and Discussion

In Group I – 58.33% of patients belong to the 51-60 age group. 23.33% belongs to 41-50 age group and 18.33% belongs to 61-70 age group of patients. 9 males and 5 females were in the 41-50 age group. 29 males and 6 females were in 51-60 age group.10 males and 1 female was in 61-70 age group. Out of 60, 48 Males (80.00%) and 12 females (20%) were present in group I. In Group II- 51.6% belongs to 51-60 age group. 31.6% belongs to 41-50 age and 16.6% belongs to 61-70 age group. No. 15 males and 4 females were in 41-50 age group. 21 males and 10 females were in 51-60 age group.

9 males and 1 females were in 61-70 age group. Out of 60, 45 males (75%) and 15 females (25%) were present in group II. In TOTAL-out of 120 diabetic patients 93 were males (77.5%), 27 were females (22.5%) Table No. 1 and Figure 1: Depicts the Before treatment values (Baseline) of S. vitamin B12 (pg/ml) and after treatment values for Group I and GROUP II at 4 weeks, 8 weeks, 16 weeks respectively.

In GROUP I; Mean Baseline value for S. vitamin B12 (pg/ ml) is 189.10±1.05 and after treatment value at 16 weeks is 229.38+1.1. This difference was significant(<0.01).

In GROUP II; Mean Baseline value for S. vitamin B12 (pg/ ml) is 188.49±0.96 and after treatment value at 16 weeks is 184.59±0.98. This difference was not significant.

Table No 2 and Figure No. 2 Depicts the baseline values (before treatment) of fasting blood glucose (in mg/dl) and after treatment values for Group I and GROUP II at 4 weeks, 8 weeks, 16 weeks respectively.

In GROUP I; Mean Baseline value for fasting blood glucose (mg/dl) is 144.96±0.95 and after treatment values at 4 weeks is 138.56+0.84, 8 weeks 136.80±0.81, at 16 weeks 130.24±0.79 respectively. This difference was considered significant.

In GROUP II; Mean Baseline value for fasting blood glucose (mg/dl) is 146.14±1.17 and after treatment values at 4 weeks is 140.27±1.02, 8 weeks 138.16±0.98, at 16

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-166

weeks 134.0±0.l93 respectively. This difference was also significant.

In GROUP I Mean Baseline value for Latency (ms) in Auditory Evoked Potential of right ear of waves I, III, V and interwaves I-III, III-V, I-V are 1.78±.14, 4.40±.22, 6.5±.33, 2.5±.3, 2.38±.28, 4.4±.38 respectively and after treatment values at 16 weeks are 1.61±.04, 3.75±.20, 5.78±.34, 2.1±.27, 2.34±.22, 4.2±.25 respectively. The differences were significant (p<0.01) in this group.

Table No. 3 and Figure No.3 depicts the baseline values (before treatment) of Latency (ms) of waves in Brainstem Auditory Evoked Potential of left Ear and after treatment values for Group I and GROUP II at 4 weeks, 8 weeks, 16 weeks respectively.

In GROUP II Mean Baseline value for Latency (ms) in Auditory Evoked Potential of right ear of waves I, III, V and interwaves I-III, III-V, I-V are 1.80±.13, 4.36±.18, 6.5±.3, 2.5±.31, 2.5±.29, 4.6±.22 respectively and after treatment values at 16 weeks are 1.84±.16, 4.26±1.7, 6.61±.29, 2.63±.29, 2.50±.30, 4.58±.22 respectively. The differences were not significant (p>0.05) in this group.

In GROUP I Mean Baseline value for ‘Latency (ms) in Auditory Evoked Potential of left ear of waves I, III, V and interwaves I-III, III-V, I-V are 1.94±.10, 4.48±.20, 6.60±.25, 2.53±.30, 2.36±.25, 4.61±.30 respectively and after treatment values at 16 weeks are 1.60±.12, 3.76±.16, 5.70±.30, 2.11±.21, 2.10±.24, 4.10±.24 respectively. The differences in this group were significant (p<0.01)

Sharma et al (2000)The incidence of delayed wave latencies was 64%, 72%. & 84% at 2KHz,4KHz, & 6KMz respectively suggesting that if brain stem evoked response audiometry is conducted at higher frequency like 6KBz in diabetic patients, the involvement of central neural axis can be detected earlier. This study is the first to demonstrate that brain stem evoked response audiometry is a useful non-invasive test for earlier detection of damage in central neural axis in patients of diabetes mellitus. There is no relationship between the delayed wave latencies and the blood glucose level, however there exists a significant relationship between the delayed wave latencies and the duration of disease & peripheral neuropathy6.

In Group-II Mean Baseline value for Latency (ms) in Auditory Evoked Potential of left ear of waves I, II, III and interwaves I-III, III-V, I-V are 1.84±.14, 4.47±.17, 6.49±.31, 2.56±.32, 2.51±.25, 4.40±.21 respectively and after treatment values at 16 weeks are 1.88±.16, 4.38±1.8, 6.60±.28, 2.61±.28, 2.5±.29, 4.50±.22 respectively. The differences in this group were not significant (p>0.05).

Table No.4 and Figure No.4 depicts the baseline values (before treatment) of Latency(ms) of wave in Auditory Evoked Potential of right ear and after treatment values for Group I and GROUP II at 4 weeks, 8 weeks, 16 weeks respectively.

Table 1: S. Vitamin B12 (pg/ml) BEFORE AND AFTER TREATMENT GROUP I (n=60) Before treatment

4 weeks

8 weeks

189.10

211.29

223.31

S.D

5.53

6.10

S.E.M

1.05

1.32

Mean

% Change Over Baseline p Value

GROUP II (n=60)

After Treatment 16 weeks

Before treatment

After Treatment 4 weeks

8 weeks

16 weeks

229.38

188.49

187.56

182.20

184.59

4.89

4.78

6.35

5.40

5.83

4.83

0.98

1.1

0.96

0.90

1.03

0.98

17.21%

-1.12

<0.01

<0.05

Table 2: FASTING BLOOD GLUCOSE (mg/dl) BEFORE AND AFTER TREATMENT GROUP I (n=60) Before treatment

4 weeks

8 weeks

144.96

138.56

136.80

S.D

7.32

6.81

S.E.M

0.95

0.84

Mean

GROUP II (n=60)

After Treatment 16 weeks

Before treatment

After Treatment 4 weeks

8 weeks

16 weeks

130.24

146.14

140.27

138.16

134.00

6.74

5.92

8.72

8.04

7.78

7.26

0.81

0.79

1.17

1.02

0.98

0.93

% Change Over Baseline

-10.15%

-8.31

p Value

P<0.01

P<0.01

http://www.pacificejournals.com/aabs

A-167

AABS; 3(2): 2016

Table 3: LATENCY (ms) OF WAVES IN B.A.E.P OF LEFT EAR BEFORE AND AFTER TREATMENT Wave

Cases (60) Before treatment

Controls(60)

After treatment

Before treatment

After treatment

4week

8 week

16 week

At 1slt visit

4week

8 week

16 week

I

1.94±.10

1.80±.09

1.70±.08

1.60±.12

1.84±.14

1.88±.16

1.84±.17

‘1.88±.16

III

4.48±.20

4.10±.22

3.90±.22

3.76±.16

4.47±.18

4.42±.20

4.50±..16

4.38±1.8

V

6.60±.25

6.24±.20

5.82±.32

5.70±.30

6.49±.31

6.50±.28

6.52±.29

6.6±.28

I-III IPL

2.53±.30

2.34±.18

2.20±.24

2.11±.21

2.56±.32

2.56±.29

2.54±.32

2.61±.28

III-V IPL

2.36±.25

2.21±.34

‘2.16±.28

2.10±.24

2.51±.25

2.44±.31

2.46±.26

2.50±.29

I-V IPL

4.61±.30

4.40±.24

4.23±.26

4.10±.24

4.40±.21

4.53±.24

4.59±.25

4.50±.22

Table 4: LATENCY (ms) OF WAVES IN B.A.E.P OF RIGHT EAR BEFORE AND AFTER TREATMENT Wave

Cases (60) Before treatment

Controls(60)

After treatment 4weak

8 weak

16 weak

At 1st visit

4weak

8 weak

16 weak

1.78±.14

1.72±.12

1.82±.14

1.61±.04

1.820±.13

1.9±.14

1.86±.15

1.84±.16

4.40±.22

4.1±.22

3.98±.21

3.75±.20

4.36±.18

4.3±.2

4.52±.1.7

4.26±1.7

6.5±.33

6.1±.3

5.9±.31

5.78±.34

6.5±.3

6.61±.30

6.5±.3

6.61±.29

I-III IPL

2.5±.3

2.3±.23

2.24±.27

2.1±.27

2.5±.31

2.56±.28

2.55±.28

2.63±.29

III-V IPL

2.38±.28

2.34±.32

2.20±.25

2.34±.22

2.5±.29

2.42±.32

2.46±.29

2.50±.30

I-V IPL

4.5±.38

4.3±.26

4.26±.25

4.2±.25

4.6±.22

4.5±.34

4.60±.27

4.58±.22

I II III IV V VI

Fig. 1: Serum Vitamin B12 (pg/ml) Before And After Treatment

Fig. 2: Fasting Blood Glucose (Mg/Dl) Before And After Treatment

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-168

Fig. 3: Latency (ms) Wave in Brainstem Auditory Evoked Potential of left ear Before and After Treatment

Fig. 4: Latency (ms) of Waves in Brainstem Auditory Evoked Potential of right Ear Before and After Treatment

Nihat DEMİR et al7 (2015) thirty patients with vitamin B12 deficiency and 30 age-matched healthy controls were included in the study. Hematological parameters, visual evoked potentials, and brainstem auditory evoked potentials tests were performed prior to treatment, 1 week after treatment, and 3 months after treatment. Results: Visual evoked potentials (VEPs) and brainstem auditory evoked potentials (BAEPs) were found to be prolonged in 16 (53.3%) and 15 (50%) patients, respectively. Statistically significant improvements in VEP and BAEP examinations were determined 3 months after treatment. Three months after treatment, VEP and BAEP examinations returned to normal in 81.3% and 53.3% of subjects with prolonged VEPs and BAEPs, respectively. Conclusion: These results demonstrate that vitamin B12 deficiency in infants causes significant impairment in the auditory and visual functioning tests of the brain, such as VEP and BAEP8,9,10.

Jawa AA et al11 in Nutrition-related vitamin B12 deficiency in patients in Pakistan with type 2 diabetes mellitus not taking metformin concluded that vitamin B12 deficiency is common among patients with type 2 diabetes and was related to nutrition in our study group. In addition to intensive glycemic control, vitamin B12 supplementation should be considered for treatment of diabetic neuropathy.

In almost 50% of patients with low vitamin B12 levels, the deficiency was corrected with oral supplementation only. This, indeed, is an important finding, in as much as oral vitamin B12 supplementation is easy, convenient, and readily accepted by patients. Serum vitamin B12 levels were measured in 44 subjects with diabetes and a mean age of 51 years (range, 40 to 70), 21 (48%) of whom had low levels (<200 pg/mL). Of those 21 patients, 10 agreed to enroll in an intervention phase consisting of oral supplementation with mecobalamin, 1,500 microg daily for 3 months. Those patients in whom vitamin B12 levels failed to normalize after oral supplementation alone would be presumed to have vitamin B12 deficiency attributable to malabsorption12.

Conclusion

Abnormalities in brainstem auditory evoked potentials can be detected in diabetic patients even in the absence of clinical symptoms. Improvement in latencies of waves is seen after supplementation of vit. B12 as assessed by brainstem auditory evoked potentials. Although promising, further studies are needed to assess long-term treatment of Vit. B12 given orally in an outpatient setting to assess its potential as a viable treatment option for patients with Diabetes with involvement of auditory nerve and its safety and toxicity profile.

Acknowledgement

I acknowledge the significant contribution of my co-authors and the contents of the manuscript are in agreement with each author mentioned.

Declaration Funding None

Conpeting interest None Declared

References

1. Gupta R, Aslam M, Hasan SA and Siddiqi SS. Type-2 diabetes mellitus and auditory brainstem responses-a hospital based study. Indian J Endocrinol Metab 2010; 14(1): 9-11.

2. Bayazit Y, Yilmaz M, Kepekçi Y, Mumbuç S, Kanlikama M. Use of the auditory brainstem response testing in the clinical evaluation of the patients with diabetes mellitus. J Neurol Sci. 2000;181(l-2):29–32.

3. Durmus C, Yetiser S, Durmus O. Auditory brainstem evoked responses in insulin-dependent (ID) and non-insulin-dependent (NID) diabetic subjects with normal hearing. International J of Audiology.2004;43(1):29–33.

http://www.pacificejournals.com/aabs

A-169 4. Toth, et al. Brainstem auditory evoked potential examinations in diabetic patients. Scandinavian Audiology. 2001;30:156–159.

AABS; 3(2): 2016

7. Demir N, Koc A, Üstyol L, Peker E, Abuhandan M. Clinical and neurological findings of severe vitamin B12 deficiency in infancy and importance of early diagnosis and treatment. J Paediatr Child H 2013; 49: 820–824.

8. Fine EJ, Soria E, Paroski MW, Petryk D, Thomasula L. The neurophysiological profile of vitamin B12 deficiency. Muscle Nerve 1990; 13: 158–164.

9. Markand ON. Brainstem auditory evoked potentials. J Clin Neurophysiol 1994; 11: 319–342.

10. Misra UK, Kalita J, Das A. Vitamin B12 deficiency neurological syndromes: a clinical, MRI and electrodiagnostic study. Electromyo Clin Neur 2003; 43: 57–64.

11. Jawa AA, Akram J, Sultan M, Humayoun MA, Raza R. Nutrition-related vitamin B12 deficiency in patients in Pakistan with type 2 diabetes mellitus not taking metformin. Endocr Pract 2010;16:205-8.

12. Ramachandran A, Snehalatha C, Shetty A, Nanditha A. Trends in prevalence of diabetes in Asian countries. World J Diabetes. 2012;3(6):110.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

5. Pflipsen M, Oh R, Saguil A, Seehusen D, Topolski R. The Prevalence of Vitamin B12 Deficiency in Patients with Type 2 Diabetes: A Cross-Sectional Study. J Am Board Fam Med. 2009;22:528–34.

6. Sharma R, Gupta SC, Tyagi I et al. Brain Stem Evoked Responses in patients with Diabetes Mellitus. Indian J of Otolaryngology and Head and Neck Surgery 2000;52(3); 221- 229.

Original Article Mycological Analysis of 150 Cases of Dermatophytosis of Skin, Hair and Nail Attending The Outpatient Department of Skin and Venereology Bindu Mitruka1*, Amarjit Kaur Gill1, Narinder Kaur1, Ravinder K. Mittal2, Anchal Mahajan1, Amandeep Kaur1 1 Department of Microbiology, Adesh Institute of Medical Sciences and Research, Bathinda, Punjab, India Department of Skin and Venereology, Adesh Institute of Medical Sciences and Research, Bathinda, Punjab, India

2

Keywords: Dermatophytosis, Direct Microscopy, KOH Preparation, LCB Mount, SDA Medium, Tinea Rubrum, Tinea Mentagrophyte.

ABSTRACT Background: Dermatophytes affect millions of people worldwide. Dermatophytosis inflicts a lot of psychosocial trauma. It is not generally appreciated how disabling a skin disease can be since an apparent trivial rash to the observer may be a source of intense discomfort and stigma to the patient.

Methods: The present study involved mycological analysis of 150 cases of dermatophytosis attending the OPD of Skin and Venereology, AIMSR, Bathinda during the period of 1st April 2014 to 30th September 2015. Detailed history was taken. Samples of skin, hair and nail were taken depending upon the part affected. Out of the material collected, part of it was used for direct KOH examination and remaining part was used to inoculate SDA medium with antibiotics for culture. Results of KOH preparation and culture, along with relevant history, were noted in Proforma. The observations and data obtained from the study were compiled and analyzed.

Result: KOH examination was positive in 93 (62%) cases while culture was positive in 77 cases (51.34%) Overall, Trichophyton was the most common genus 76 samples (98.7%) isolated followed by Epidermophyton 1 sample.

Out of the 77 culture positive cases, T. rubrum was the most common isolate in 51 cases (66.23%) followed by T. mentagrophytes in 22 cases (28.57%).

Conclusion: It was concluded that KOH examination gives more positive results as compared to culture. Trichophyton infection is more common than Epidermophyton. T. rubrum is the most common infective dermatophyte out of all varieties.

*Corresponding author: Dr. Bindu Mitruka, Post Graduate Student, Department of Microbiology, Adesh Institute of Medical Sciences and Research, Bathinda, Punjab, India Phone: +91-9417344623 E-mail: hardeepgill77@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-171

Introduction

Although fungi are worldwide, only few of them are considered pathogenic. The pathogenic fungi may give rise to infections in animals and human beings. Most of the agents cause infection of the superficial layers of the integument and only very few give rise to systemic involvement. Recently there has been an increase in the incidence of fungal infections. This increase may be a result of frequent usage of antibiotics, immunosuppressive drugs and various conditions like organ transplantations, lymphomas, leukemias and human immunodeficiency virus (HIV) infections. [1] Dermatophytosis is a major public health problem in the world today affecting millions of people. The estimated lifetime risk of acquiring a dermatophyte infection is between 10- 20 percent. [2] Dermatophytes, with an emerging life risk of 10-20%, affects millions of people worldwide. [3] Dermatophytosis inflicts a lot of psychosocial trauma due to attached social stigma and in case of children, sometimes irritation is so much, that it hampers pupilâ&#x20AC;&#x2122;s concentration in class, as well as representing a potential source of secondary bacterial infection. It is not generally appreciated how disabling a skin disease can be since an apparent trivial rash to the observer may be a source of intense discomfort and stigma for the patient. [4]

AABS; 3(2): 2016 present. Microscopic picture of LCB mount distorted hyphae are present and conidia are rare.

6. M. audouinii: Growth on SDA medium shows velvety, slow growing and brown colored colonies.

Microscopic picture of LCB mount shows thick walled chlamydospores are present but conidia are rare and irregular.

7. M. canis: Growth on SDA medium shows cottony colonies and orange pigment is present on reverse side of the slope. Microscopic picture of LCB mount shows abundant thick walled spindle-shaped macroconidia with up to 15 septa.

8. M. gypseum: Growth on SDA medium shows powdery and buff colored colonies. Microscopic picture of LCB mount shows abundant, thin walled macroconidia with 4-6 septa are present.

9. E. floccosum: Growth on SDA medium shows yellowish-green and powdery colonies. Microscopic picture of LCB mount shows club-shaped macroconidia in clusters.

Lab Diagnosis: Superficial fungal infections are generally strongly suspected on clinical grounds, but at times the diagnosis needs to be confirmed by laboratory support.

These include direct microscopic examination and culture of selected material from the affected area. [6]

Morphological characteristics of commonly encountered dermatophytes [5]1. T. rubrum: Growth on SDA medium shows velvety growth and red pigment is present on reverse side of the slope. Microscopic picture of LCB mount shows tear drop microconidia and few long pencil-shaped macroconidia are present.

2. T. mentagrophytes: Growth on SDA shows white to tan colored, cottony or powdery colonies and variable pigment is present. Microscopic picture of LCB mount shows clusters of microconidia and cigar- shaped macroconidia with terminal rat-tail filaments are present.

3. T. tonsurans: Growth on SDA medium shows cream or yellow colored, powdery colonies with central furrows. Microscopic picture of LCB mount shows abundant microconidia and thick walled irregular macroconidia.

4. T. schoenleinii: Growth on SDA shows smooth, waxy and brown colored colonies. Microscopic picture of LCB mount shows hyphal swellings, favic chandelier and chlamydospores.

5. T. violaceum: Growth on SDA medium very slow growing, waxy colonies and violet to purple pigment is

Collection of Sample: Lal et al (1983) [7] cleaned the skin lesion with 70% alcohol and the scales from the active margin of the lesion were scraped with a sterile scalpel and collected in a sterile petridish. If vesicles were present, their domes were snipped off. In the toe clefts, the material is collected with an epilation forceps. Hairs for examination should be plucked. In black dot variety, material obtained by scraping gives better results. In case of nails, clipping should as proximal as possible. Bindu (2002) [8] described that in tinea capitis, infected and lusterless hair along with scales were collected. In tinea unguim nail scrapings, clippings and subungual debris were collected. The specimen was collected on clean sheets of paper which was folded for transportation [6]. Lari et al (2003) [9] suggested that samples should be taken from patients using scalpels, forceps and glass slides that had been washed in ethanol and sterilized with a Bunsen burner.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Processing of Sample 1. Direct Microscopy: Scrapings and hairs may be mounted for direct examination in 25% KOH mixed with 5% glycerol, heated to emulsify lipids and examined under 400X magnification for fungal structures [10]. Sehgal et al (1985) [11] and Khare et al (1985) [12] have used 10% KOH for wet mounts

Original Article

A-172

for skin, hair and nail. Kumar and Lakshmi (1990) [13] subjected the specimen to routine wet preparation in 10% KOH for the presence of fungal hyphae and arthrospores. Verenkar et al (1991) [14] examined the samples under microscope using 20% KOH. Patwardhan and Dave (1999) [15] suggest that 40% KOH gives a better clearing affect within less time.

2. Culture: According to Weitzman and Summerbell (1995) [10], culture adjunct to direct microscopy and is essential in all nail infections and all those infections which are to be treated by systemic mediation. In all cases, a medium selective against most nondermatophyte moulds and bacteria are used.

Cycloheximide is incorporated as a semi selective agent to reduce the growth of non- dermatophyte fungi, Sabouraud peptone – glucose agar (Emmon’sn Modification) amended with cycloheximide and chloramphenicol is commonly used [10]. Hair and nail were first cut into small pieces and then inoculated. Inoculation was done by using a scalpel and loop (Patwardhan and Dave, 1999). [15] Wide agar slants prepared in large culture tubes or bottles were used. Because growth was slow, four weeks of inoculation was required. However in a majority of dermatophytes, growth and sporulation occurs in five to ten days. When sporulation and pigment formation occurs, lactophenol cotton blue (LCB) mount of the growth was examined.

For identification of dermatophyte, definition of many of its characteristics was essential like colony appearance,color, texture, topography examination of reverse side of colony, topography and arrangement of spores etc. [6] Singh and Beena (2003) incubated the media at 25°C and 37°C for a minimum period of three weeks. [16]

Aims and objectives ● To study dermatophytes in KOH preparation. ● Identification of various species of dermatophytes by culture, LCB mount and other required tests. ● To find out the prevalence of various species of dermatophytes.

Materials and Methods

The present study was conducted on suspected cases of dermatophytosis attending the Dermatology, Venereology & Leprology department of AIMSR, Bathinda from 1st April 2014 to 30th September 2015. Ethical approval from institutional ethical committee of Adesh University was taken before start of the study. Details of the patient and sample collection was taken as per proforma attached.

Method of collection of Skin sample: Skin lesions were sampled from the erythematous, peripheral, actively growing margins of the lesions. Skin was decontaminated with 70% alcohol to remove surface bacterial contamination. An open, sterile 2 ̋ Petri dish was held immediately below the area to be sampled and skin scales were flaked into it by using blunt edge of a sterile surgical blade or microscopic glass slide.16 Whenever there was little scaling as with lesions of the glabrous skin, cellophane tape strips were used to take adequate material. The cellophane strip was pressed against the lesion, peeled off, and was placed adhesive side down on a clean glass microscopic slide on which a drop of 10% KOH solution has been placed. [17, 18] Method of collection of Hair sample In suspected cases of tinea capitis, after cleaning the selected area with spirit, dull lusterless hair and stubs of hairs were chosen and plucked by sterile surgical forceps. Hair stubs were collected by scraping with the blunt edge of the scalpel.

The roots of the hair were included. Skin scrapings were also collected from sites where fungal infection of hairs is suspected.

Method of Collection Of Nail Samples In cases of Tinea unguium the hands and feet were washed with soap and water, with emphasis on the nails. After drying, the nails were decontaminated with 70% alcohol. In majority of cases of tinea unguium, the material for examination was also taken from the distal end of the nail because it was not practicable to take deep samples from the proximal advancing edge. Samples were taken from the nail plate, nail bed and subungual region of the nails with sterile scalpel.

Processing of the sample Skin 1. A small portion of skin scrapings was taken on a clean glass slide. Then 2-3 drops of 10% KOH were added (10% aqueous solution of potassium hydroxide (KOH) was used as a clearing agent).

2. Material was teased with teasing needles.

3. Then clean cover slip was placed on it.

4. Preparation was passed over the flame once or twice to eliminate any bubble which formed during the process.

5. Preparation was kept at room temperature for 30 minutes.

6. Then it was examined under low power of the microscope (10X) for branching and septate hyphae and confirmation was made under 40X of microscope.

http://www.pacificejournals.com/aabs

A-173 Nail 1. In case of nail sample, the material was kept overnight in 40% KOH and then teasing was done.

2. Then cover slip was placed on the material.

3. It was examined under low power and then high power of the microscope.

4. Specimen was examined for branching and septate hyphae.

Hair 1. Hair was cut into 1 cm size and transferred on the glass slide.

2. A few drops of 10% KOH were put on the sample and covered with the cover slip. Hair was not heated as it is delicate and may lose its structure.

3. The hair was examined under low power of the microscope (×10) after 1 minute and then the hair was examined under high power (×40) for branching and septate hyphae.

4. Hair was also examined for ectothrix and endothrix infections.

Culture

Skin, nail and hair samples were inoculated after reducing the size to approximately 1mm. Material was inoculated onto Sabouraud‘s dextrose agar plate containing 0.05 mg/ ml chloramphenicol, gentamicin 0.2 mg/ml and 0.5 mg/ml cycloheximide.

Chloramphenicol and Gentamicin were added to inhibit the bacterial growth and cycloheximide was added to inhibit the growth of saprophytic fungi. Then plates were incubated at 28°C and were examined daily up to 4 weeks for evidence of growth from the edge of the planted material. If no growth appeared, results were declared negative after 4 weeks of incubation.

Medium used for culture: Composition of Sabouraud Dextrose Agar (SDA) Peptone 10gm , Dextrose 40gm , Agar 20gm , Distilled Water 1000ml , Adjust ph at 5.6 Following antibiotics will be added into SDA while boiling but before autoclaving to make it selective for isolation of fungus- Cycloheximide 500 mg , Chloramphenicol 50 mg, Gentamicin 20 mg

AABS; 3(2): 2016 morphology, pigment production on the underside of growth and production of microconidia and macroconidia by making LCB mount.

Method of LCB mount 1. One drop of LCB stain was taken on the glass slide.

2. A small amount of growth was taken from the periphery with the help of bent wire.

3. Growth was gently teased using two teasing needles.

4. Cover slip was applied by avoiding air bubbles.

5. Growth was seen under low power microscope and was confirmed under high power for septate hyphae, arrangement of micro conidia and macro conidia.

Composition and preparation of LCB Stain Phenol crystals 20g , Lactic acid 20ml , Glycerol 40ml , Distilled water 20 ml , Cotton blue 0.075g . The phenol crystals were dissolved in the liquids by gentle warming and then dye was added.

Identification points of Dermatophytes: The dermatophytes species of common occurrence were identified from the following characteristics.

Trichophyton: Species of Trichophyton attacked the hair, skin and nails.

T. rubrum: In T. rubrum, the cultures were cottony to velvety but in some cases powdery appearance was also seen. Reddish to purple pigmentation developed on the reverse of the colony and diffused to the marginal hyphae.

Primary cultures developed numerous micro conidia in clusters and singly along the hyphae, few macro conidia, racquet hyphae were also present.

T. Mentagrophytes: The cultures in T. Mentagrophytes were powdery to granular, light buff to rose tan in color.

The reverse of the colony was wine to brownish in colour.

These cultures were urease positive within 7 days. These cultures developed macro conidia and numerous micro conidia in clusters and singly on the hyphae. To confirm the diagnosis of T. Mentagrophytes growth was inoculated on Christensen‘s medium which gave positive urease test within 7 days. The tubes, in which no growth appeared up to 4 weeks, were discarded.

Identification of the Dermatophytes: Growth, if and when appeared, was identified from culture characters, colonial

T. Tonsurans: T. Tonsurans culture showed various degrees of heaped or sunken central growth with folding of surface.

The colonies were velvety to powdery and varied in color from white, cream and sulphur yellow. Microscopically, all varieties showed numerous deep stained clavate micro conidia along the sides of hyphae, sessile or on short sterigmata. The hyphae supporting the micro conidia usually remained unstained in LCB preparation. Macro conidia were seen rarely.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Dissolve cycloheximide in 10 ml acetone then add it to the boiling medium and mix properly. Similarly, dissolve chloramphenicol & gentamicin in 10 ml of 95% alcohol and add to the boiling medium. Then remove from heating and mix it well. Dispense the medium into plates and autoclave it at 121 ̊c for 15 minutes. 20

Original Article

A-174

Epidermophyton: This genus attacked skin and nails.

E. Floccosum: The culture of E. floccosum was characteristically velvety to powdery with central radiating furrows and greenish yellow in color. Microscopically, the only conidia produced were the large clavate, multiseptate, smooth and thin walled. Micro conidia were seen in some strains.

Result

The present study involved mycological analysis of 150 clinically suspected cases of dermatophytosis of skin, hair and nail attending the outpatient department of Dermatology and Venereology, AIMSR, Bathinda.

Detailed history was taken. Samples of skin, hair and nail were taken depending upon the part affected.

Out of the material collected, part of it was used for direct KOH examination and remaining part was used to inoculate SDA medium for culture to isolate and identify the pathogenic Dermatophytes.

Results of KOH preparation and culture along with relevant history were noted in proformas.

The observations and data made in the present study were compiled and analyzed and are being presented in the following pages in the form of tables and graphs.

Distribution of Samples Collected According to Site

In the present study of dermatophytosis, out of total 150 samples collected, 123 were skin samples, 15 were nail samples and 12 were hair samples.

Distribution of 150 Clinically Suspected Cases of Dermatophytosis According to Results of KOH Examination and Culture

Out of 150 cases of dermatophytosis, KOH examination was positive for fungal hyphae in total 93 cases (62%) and negative in 57 cases (38%). Culture was positive for dermatophytes in total 77 cases (51.34%) and negative in 73 cases (48.66%). Both KOH examination and culture were positive in 67 cases (44.67%) while both KOH examination and culture were negative in 47 cases (31.33%). 26 cases (17.33%) were KOH positive but culture negative while 10 cases (6.66%) were KOH negative but culture positive.

Total Number of KOH Positive Samples (Only KOH Positive and Both Koh Positive Culture Positive) from 150 Clinically Suspected Cases of Dermatophytosis

In the present study, out of 123 skin samples, 15 nail samples and 12 hair samples, the following observations were made.

From skin samples only KOH positive samples were 17 and both KOH and culture positive samples were 60. So, total KOH positive samples were 77.

From nail samples only KOH positive sample were 5 and both KOH and culture positive samples were 4. So, total KOH positive samples were 9.

From hair samples only KOH positive sample was 4 and both KOH and culture positive samples were 3. So, total KOH positive samples were 7.

Total number of KOH positive samples from skin, hair and nail were 93.

Distribution of Samples in 150 Clinically Suspected Cases of Dermatophytosis According to Culture Positivity (Only Culture Positive and Both Culture & KOH Positive)

In the present study, out of 123 skin samples, 15 nail samples and 12 hair samples, the following observations were made.

From skin samples only culture positive samples were 5 and both culture and KOH positive samples were 60. So, total culture positive samples were 65.

From nail samples only culture positive sample were 3 and both culture and KOH positive samples were 4. So, total culture positive samples were 7.

From hair samples only culture positive sample were 2 and both culture and KOH positive samples were 3. So, total culture positive samples were 5.

Total number of culture positive samples from skin, hair and nail were 77.

Distribution of various types of tinea infections in 150 clinically suspected cases of dermatophytosis according to KOH examination and culture results TABLE 1 shows that in the present study, out of 60 cases of tinea cruris, 37 cases (61.67%) were KOH positive, 31 cases(51.66%) were culture positive, 31 cases(51.66%) were both culture and KOH positive and 23 cases (38.33%) were both KOH and culture negative.

Out of 35 cases of tinea corporis, 23 cases (65.71%) were KOH positive, 19 cases(54.28%) were culture positive, 18 cases(51.42%) were both culture and KOH positive and 11 cases(31.42%) were both KOH and culture negative.

Out of 16 cases of tinea pedis, 11 cases (68.75%) were KOH positive, 9 cases (56.25%) were culture positive, 7

http://www.pacificejournals.com/aabs

A-175

AABS; 3(2): 2016

cases(43.75%) were both culture and KOH positive and 3 cases(18.75%) were both KOH and culture negative.

In 77 culture positive samples, 50 samples were of male patients and 27 samples were of female patients.

Out of 10 cases of tinea capitis, 6 cases (60%) were KOH positive, 5 cases (50%) were culture positive, 3 cases(30%) were both culture and KOH positive and 2 cases(20%) were both KOH and culture negative.

In 50 samples of male patients, 44 culture positive samples were of skin, 2 samples were of nail and 4 samples were of hair.

Out of 15 cases of tinea unguium, 9 cases (60%) were KOH positive, 7 cases (46.66%) were culture positive, 4 cases (26.66%) were both culture and KOH positive and 3cases(20%) were both KOH and culture negative.

Out of 7 cases of tinea manuum, 3 cases (42.85%) were KOH positive, 3 cases (42.85%) were culture positive, 2 cases (28.57%) were both culture and KOH positive and 3 cases(42.85%) were both KOH and culture negative.

Out of 5 cases of tinea faciei, 3 cases (60%) were KOH positive, 3 cases (60%) were culture positive, 2 cases(40%) were both culture and KOH positive and 1(20%) case was both KOH and culture negative.

Out of 2 cases of tinea barbae, 1 case (50%) was KOH positive, none was culture positive and 1 case was both KOH and culture negative.

Distribution of KOH And Culture Positive Samples According to The Age of The Cases

TABLE 2 shows that in KOH positivity, age group 21-30 was predominant with 33 samples, out of which 26 were skin samples and 6 were nail samples followed by age group of 31-40 years with 20 samples, in which 18 samples were of skin and 2 samples were of nail, 11-20 years age group with 11 samples in which 10 samples were of skin and 1 sample was of hair. In culture positivity, the most dominant age group was of 21-30 years, with total number of 24 samples in which 21 samples were of skin, 3 samples were of nail followed by 31-40 years age group with 19 samples, in which 16 samples were of skin and 3 samples were of nail.

Distribution of KOH and Culture Positive Samples According to The Sex Distribution of The Cases

TABLE 3 shows that out of total 93 KOH positive samples, 60 samples were of male patients and 33 samples were of female patients.

In 60 samples of male patients, 52 KOH positive samples were of skin, 4 samples of nail and 4 samples were of hair.

In 27 samples of female patients, 21 culture positive samples were of skin, 5 samples were of nail and 1 sample was of hair.

So in our study we see that male patient samples show more KOH and culture positivity than female patient samples.

Distribution of KOH and Culture Positive Samples According to The Population Staying in Rural and Urban Areas

TABLE 4 shows that out of total 93 KOH positive samples, 41 samples were taken from rural population out of which 32 were skin samples, 4 were nail samples and 5 samples were of hair. In the urban population there were 52 KOH positive samples, out of which 45 were skin samples, 5 were nail samples and 2 were hair samples. In 77 culture positive samples, 35 samples were from rural population, out of which 29 samples were of skin, 3 samples were of nail and hair each. In urban population total culture positive samples were 42, out of which 36 samples were of skin, 4 samples were of nail and 2 samples were of hair.

Distribution of KOH and Culture Positive Samples According to Occupation

TABLE 5 shows that among the 93 KOH positive cases, the most affected profession was of farmers with 34 samples followed by labourers with 20 samples, students with 11 samples, housewives with 10 samples, sportsman with 8 samples, business class with 6 samples, armyman and service class with 2 samples each.

In case of culture positivity farmers formed the most predominant group with 27 samples followed by labourers with 16 samples, housewives with 10 samples. Students with 9 samples, sportsman with 6 samples each, business class with 4 samples, army man with 3 samples and service class with 2 samples.

Species of Fungus Isolated from 77 Culture Positive Cases of Dermatophytosis

Out of 77 culture positive cases, T. rubrum was the most common isolate 51 samples (66.23%) followed by T. mentagrophytes 22 samples (28.57%), T. tonsurans 3 samples (3.89%), E. floccosum 1 sample (1.29%).

In 33 samples of female patients, 25 KOH positive samples were of skin, 5 samples were of nail and 3 were of hair.

Overall, Trichophyton was the most common genus 76 samples (98.7%) isolated followed by Epidermophyton 1 sample (1.29%).

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-176

Table 1: Distribution of Various Types of Tinea Infections in 150 Clinically Suspected Cases of Dermatophytosis According to KOH Examination And Culture Results Type of Tinea

KOH positive Culture negative 6 5 4 3 5 1 1 1 26

Tinea cruris Tinea corporis Tinea pedis Tinea capitis Tinea unguium Tinea manuum Tinea faciei Tinea barbae TOTAL

KOH positive Culture positive 31 18 7 3 4 2 2 0 67

KOH negative Culture positive 0 1 2 2 3 1 1 0 10

KOH negative Culture negative 23 11 3 2 3 3 1 1 47

Total 60 35 16 10 15 7 5 2 150

Table 2: Distribution of KOH and Culture Positive Samples According to The Age of The Cases Age group (in years)

SKIN 0 10 26 18 9 9 3 2 77

0-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 TOTAL

KOH POSITIVE NAIL HAIR 5 1 6 1 2 1 9 7

TOTAL 5 11 33 20 10 9 3 2 93

SKIN 11 21 16 6 8 2 1 65

CULTURE POSITIVE NAIL HAIR 4 1 3 3 1 7 5

TOTAL 4 12 24 19 6 8 2 2 77

TABLE 3: Distribution of KOH and Culture Positive Samples According to The Sex Distribution of The Cases Sex MALE FEMALE TOTAL

SKIN 52 25 77

KOH POSITIVE NAIL HAIR 4 4 5 3 9 7

TOTAL 60 33 93

SKIN 44 21 65

CULTURE POSITIVE NAIL HAIR 2 4 5 1 7 5

TOTAL 50 27 77

TABLE 4: Distribution of KOH and Culture Positive Samples According to The Population Staying in Rural and Urban Areas Sex MALE FEMALE TOTAL

SKIN 52 25 77

KOH POSITIVE NAIL HAIR 4 4 5 3 9 7

TOTAL 60 33 93

SKIN 44 21 65

CULTURE POSITIVE NAIL HAIR 2 4 5 1 7 5

TOTAL 50 27 77

Culture positive NAIL HAIR 1 1 3 4 2 1 -

TOTAL 27 16 10 9 6 4 3 2

Table 5: Distribution of KOH and Culture Positive Samples According to Occupation Occupation Farmers Labourers Housewives Students Sportsmen Business class Army Service class

SKIN 32 18 7 6 7 5 1 1

KOH positive NAIL HAIR 1 1 1 1 3 5 1 1 1 1 -

TOTAL 34 20 10 11 8 6 2 2

SKIN 27 14 7 5 6 4 1 1

http://www.pacificejournals.com/aabs

A-177

AABS; 3(2): 2016

Table 6: Species of Fungus Isolated from Culture Positive Cases of Various Types of Dermatophytosis CLINICAL SITE T.cruris

T.corporis

T.capitis

T.pedis

T.unguium

T. manuum T. faciei

DERMATOPHYTE SP

Culture positive Only

KOH & Culture both positive

Total

Percentage

T. rubrum

-

25

25

80.64%

T. mentagrophytes

-

6

6

19.35%

T. rubrum

-

11

11

57.89%

T. mentagrophytes

-

7

7

36.84%

T. tonsurans

1

-

1

5.26%

T. rubrum

1

1

2

40%

T. mentagrophytes

1

1

2

40%

T. tonsurans

-

1

1

20%

T. rubrum

1

4

5

55.56%

T. mentagrophytes

1

2

3

33.33%

T. tonsurans

-

1

1

11.11%

T. rubrum

2

1

3

42.85%

T. mentagrophytes

1

2

3

42.85%

E. floccosum

0

1

1

14.28%

T. rubrum

1

1

2

66.66%

T. mentagrophytes

0

1

1

33.33%

T. rubrum

1

2

3

100%

T. rubrum predominated as the causative agent of dermatophytosis in the present study.

Species of Fungus Isolated from Culture Positive Cases of Various Types of Dermatophytosis

TABLE 6 shows that out of 60 cases of tinea cruris, 31 samples were culture positive and T. rubrum was the isolated in 25 cases (80.64%) and T. mentagrophytes was isolated in 6 cases (19.35%). T. rubrum was predominant species in tinea cruris cases.

Out of 35 cases of tinea corporis, 19 cases (54.28%) were culture positive and T. rubrum was isolated in 11 cases (57.89%), T. mentagrophytes was isolated in 7 cases (36.84%) and T. tonsurans was isolated in 1 case (5.26%) .

T. rubrum was predominant species in tinea corporis cases.

Out of 10 cases of tinea capitis, 5 cases (50%) were culture positive and T. rubrum, T. mentagrophytes were isolated in 2 cases (40%) each and T. tonsurans was isolated in 1 case (20).

Out of 16 cases of tinea pedis, 9 cases (56.25%) were culture positive and T. rubrum was causative agent in 5 cases (55.56%), T.mentagrophytes was positive in 3 cases (33.33%) and T. tonsurans was positive in 1 case (11.11%).

T. rubrum and T. mentagrophytes were predominant species in tinea pedis cases.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

Out of 15 cases of tinea unguium, 7 cases (46.67%) were culture positive and T. mentagrophytes and T. rubrum were isolated 3 cases (42.85%) each and E. floccosum was isolated in 1 case (14.28%).

Out of 7 cases of tinea manuum, 3 cases (42.86%) were culture positive and T. rubrum was isolated in 2 cases (66.66%) and T. mentagrophytes was isolated in 1 case (33.33%).

Out of 5 cases of tinea faciei, 3 cases (60%) were culture positive. T. rubrum was the only species isolated.

Various Species of Fungus Isolated from Skin, Hair and Nail Samples in 150 Clinically Suspected Cases of Dermatophytosis

In present study, out of 77 culture positive samples T. rubrum was isolated from 51 samples followed by T. mentagrophyte isolated from 22 samples, T. tonsurans from 3 samples and E. floccosum isolated from 1 sample.

Thus, in this study T. rubrum was the prominent dermatophyte species from all the sites

Various Species of Fungus Isolated from Positive Skin/Hair/Nail Samples According to Age Group

In the present study, out of 65 culture positive skin samples, 21 samples were in 21-30 years age group out of which e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-178

15 samples were of T. rubrum followed by 5 samples of T. mentagrophytes and 1 sample of T.tonsurans. In 3140 years age group out of 16 culture positive samples 10 samples were of T. rubrum followed by 6 samples of T. mentagrophytes.

Similarly in other age groups also, T. rubrum was the predominant causative agent of dermatophytosis.

In the present study, out of 5 culture positive hair samples, 0-10 years age group shows 4 culture positive samples in which T. mentagrophyte was isolated in 2 samples and T. rubrum was isolated in 1 sample. In 11-20 years age group 1 sample was culture positive from which T. rubrum was isolated.

Thus T. rubrum and T.mentagrophyte were the most common isolates in hair samples.

In the present study, out of 7 culture positive nail samples, 21-30 years age group shows 3 culture positive samples and T. mentagrophytes was isolated from 2 samples and E.floccosum was isolated from 1 sample. In 31-40 years age group from 3 culture positive sample T.rubrum was isolated from 2 samples and T.mentagrophyte was isolated from 1 sample.

Thus T.rubrum and T.mentagrophyte were the most common isolates in nail samples.

Various Species of Fungus Isolated from Culture Positive Skin/Hair/Nail Samples in Males and Females

In the present study, out of 44 culture positive skin samples in male patients, T. rubrum was isolated in 33 samples followed by 11 samples of T. mentagrophytes. Out of 21 culture positive skin samples in female patients, T. rubrum was isolated in 13 samples followed by 6 samples of T. mentagrophytes and 2 samples of T.tonsurans. In both male and female patients, T. rubrum was predominant dermatophyte species in culture positive skin samples.

In the present study, out of 4 culture positive hair samples in male patients, 2 samples were of T.rubrum and 1 sample each from T. mentagrophytes and T.tonsurans.

Out of 1 culture positive hair sample in female patient,T. mentagrophytes was isolated.

In the present study, out of 2 culture positive nail samples in male patients, T. rubrum and T.mentagrophyte were isolated from 1 sample each. Out of 5 culture positive nail samples in female patients, T. rubrum was isolated in 2 samples, T. mentagrophyte in 2 samples followed by E. floccosum in 1 sample. Thus, T.rubrum was predominant dermatophyte species in culture positive nail samples.

Various Species of Fungus Isolated from Culture Positive Skin/Hair/Nail Samples in Rural & Urban Population

In the present study, out of 29 culture positive skin samples in cases of rural population, T. rubrum was the most commonly isolated dermatophyte in 17 samples followed

by T. mentagrophytes in 12 samples. In urban population out of 36 samples, T. rubrum was isolated in 29 samples followed by 5 samples of T. mentagrophytes & 2 sample of T. tonsurans. In both rural and urban population, T. rubrum was predominant dermatophyte species in culture positive skin samples.

In the present study, out of 5 culture positive hair samples, 3 samples were from rural population and 2 samples were from urban population. In cases of rural population, T.rubrum was the most common isolate in 2 cases followed by T.tonsurans in 1 case. In urban population T. mentagrophyte was isolated in 2 cases. Thus, T.rubrum and T.mentagrophyte were predominant dermatophyte species in culture positive hair samples.

In the present study, out of 3 culture positive nail samples in rural population 1 sample was of T.rubrum and 2 samples were of T.mentagrophyte. Out of 4 culture positive nail samples in urban population, T. rubrum was isolated in 2 samples followed by T. mentagrophytes and T.tonsurans in 1 sample each.

Various Species of Fungus Isolated from Culture Positive Skin/Hair/Nail Samples According to Occupation

Our study of culture positive samples of skin showed that in all the occupations T. rubrum was the most common isolated species and farmers were the most affected group.

Our study of culture positive samples of hair shows that students were the most affected and T. rubrum and T.mentagrophyte were the most common isolated dermatophyte species Our study of culture positive samples of nail shows that housewives were the most affected and T.rubrum was the most common isolated dermatophyte species.

Discussion

The present study involved mycological analysis of 150 cases of dermatophytosis of skin, hair and nail attending the outpatient department of Skin and Venereology, AIMSR, Bathinda. Detailed history was taken. Samples of skin, hair and nail were taken depending upon the part affected. Out of the material collected, part of it was used for direct KOH examination and remaining part was used

http://www.pacificejournals.com/aabs

A-179 to inoculate SDA medium for culture. Results of KOH preparation and culture, along with relevant history, were noted in Proforma. The observations and data obtained from the study were compiled and analyzed.

Mycological Observations

Results of 150 clinically suspected cases which were under mycological study KOH positivity in 150 clinically suspected cases In the present study, out of 123 skin samples, 15 nail samples and 12 hair samples, the following observations were made.

From skin samples only KOH positive samples were 17 and KOH and culture positive samples were 60. So the total KOH positive samples were 77.

From nail samples only KOH positive sample were 5 and KOH and culture positive samples were 4. So the total KOH positive samples were 9.

From hair samples only KOH positive samples were 4 and KOH and culture positive samples were and KOH and culture positive samples were 3. So the total KOH positive samples were 7.

Total number of KOH positive samples from skin, hair and nail were 93.

In the present study, out of the 150 cases examined, 93(62%) were positive for fungal hyphae on direct microscopy of KOH preparation while 57(38%) samples were KOH negative. The KOH positivity was higher in skin samples at 77 cases (62.6%) overall while it was 7 cases (58.33%) in hair samples and 9 cases (60%) in nail samples.

Culture positivity in 150 clinically diagnosed cases The culture was positive in 77(51.33%) samples and negative in 73(48.67%) samples. The culture positivity in skin samples was 65 samples (52.84%), in nail samples it was 7 samples (46.66%) and in hair samples it was 5 samples (41.66%).

Out of the 150 cases, both KOH and culture were positive in 67 cases while both KOH and culture were negative in 47 cases. 26 cases were KOH positive culture negative while 10 cases were culture positive and KOH negative.

From skin samples only culture positive samples were 5 and culture and KOH positive samples were 60. So the total culture positive samples were 65.

From nail samples only culture positive sample were 3 and culture and KOH positive samples were 4. So the total culture positive samples were 7.

AABS; 3(2): 2016 total culture positive samples were 5. KOH and culture positivity rates have a wide range in different studies.

Poria et al (1981) [19] observed a KOH positivity rate of 44.3% and culture was positive in 37.6% samples.

Sharma et al (1983) [20] reported that out of total 114 cases, 101 cases (88.6%) were KOH positive and out of these, 48 gave positive cultures. Of the 13 KOH negative cases, 4 were positive in culture, in all, 52 cultures were positive giving positivity rate of 45.6%.

Jain et al (2008) [21] reported that out of 120 cases of dermatophytosis, 87 cases (72.5%) were KOH positive and 70 cases (58.3%) were culture positive.

Sarma and Borthakur (2007) [22] found that out of 100 cases 90 were KOH positive and 61 were culture positive.

Clearly all the studies are showing higher KOH positivity as compared to culture positivity. These findings are in accordance with the present study.

Various species of dermatophytes isolated In the present study, 77 samples were positive on culture. In all four species of dermatophytes were isolated. T. rubrum was the most common isolate at 66.23 % and the second commonest species, T. mentagrophytes was far behind at 28.57%. Next came T. tonsurans at 3.9% and E.floccosum at 1.29%. Overall, Trichophyton was the most common genus at 98.7% followed by Epidermophyton (1.29%). No Micosporum was isolated.

Present study clearly showed that T. rubrum was predominant in skin samples as the causative agent in 46 samples followed by T. mentagrophytes in 17 samples, T. tonsurans in 2 samples. T.rubrum & T.mentagrophyte were predominant in nail samples as the causative agent in 3 samples each followed by E. floccosum in 1 sample.

T.rubrum & T.mentagrophyte were predominant in hair samples as the causative agent in 2 samples each followed

by T. tonsurans in 1 sample.

Clearly T. rubrum with 51 samples was predominated in the study followed by T. mentagrophytes in 22 samples, T. tonsurans in 3 samples and E. floccosum in 1 sample.

T. rubrum was isolated in all clinical types of tinea and was the most common isolate in all forms Various authors have found T. rubrum to be the most common isolate.

From hair samples only culture positive sample were 2 and culture and KOH positive samples were 3. So the

Poria et al (1981) [19] reported T. rubrum in 44% cases followed by T. mentagrophytes (18.6%), T. violaceum (7.6%), E. floccosum (4.2%) and T. tonsurans (0.8%).

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-180

Lal et al (1983) [7] also found T. rubrum to be the commonest pathogen isolated at 57 % followed by T. mentagrophytes (31.2%), E. floccosum (6.5%) and T. violaceum (3.9%).

Khare et al (1985) [12] also observed T. rubrum as the commonest dermatophyte (78.4%) in their study followed

by T. mentagrophytes (12.4%), E.floccosum (6%) and T. violaceum (3.2%). These findings are well comparable with our study.

Trichophyton genus was the commonest (98.7%) in our study.

This was also observed by Weitzman et al (1998) [10], who reported that Trichophyton remains the most frequently isolated genus, exceeding by far combined genera of Epidermophyton and Microsporum.

Tinea cruris: In our study from 60 cases of tinea cruris, 6 were KOH positive culture negative and 31 were both KOH and culture positive. Out of 31 culture positive cases, T. rubrum constituted 25 cases (80.64%) which were maximum, T. mentagrophytes 6 cases (19.35%). Weitzman and Summerbell (1995) [10] reported T. rubrum, E. floccosum and T. mentagrophytes are commonly associated with Tinea cruris.

Tinea corporis: In our study from 35 cases of tinea corporis, 5 were KOH positive culture negative, 1 was culture positive KOH negative and 18 were both KOH and culture positive. Out of 19 culture positive cases, T. rubrum constituted 11 cases (57.89%) which was maximum, T. mentagrophytes 7 cases (36.84%), T. tonsurans 1 case (5.26.%). According to Weitzman and Summerbell (1995) [10], T. rubrum, E. floccosum and T. mentagrophytes are commonly associated with tinea corporis. Prasad et al (2005) [23] reported that in tinea corporis, T. rubrum is most commonly implicated in 17.3 % cases.

Tinea pedis: In our study from 16 cases of tinea pedis, 4 samples were KOH positive culture negative and 2 samples were culture positive KOH negative. 7 samples were both KOH and culture positive. Out of 9 culture positive cases, T. rubrum constituted 5(55.56%) cases which were maximum, T. mentagrophytes 3 cases (33.33%) and T. tonsurans 1 case (11.11%). According to Weitzman and Summer bell (1995) [10], T. rubrum and T. mentagrophytes are commonly associated with tinea pedis.

Tinea capitis: In our study from 10 cases of tine capitis, 3 samples were KOH positive culture negative, 2 samples were culture positive KOH negative and 3 were both KOH and culture positive. Out of 6 KOH positive hair samples, 4(66.66%) were endothrix type while 2(33.33%) were ectothrix type. Out of 5 culture positive cases, T. rubrum

and T. mentagrophytes constituted 2 cases (40%) each while T.tonsorans constituted 1 case (20%). In a study on tinea capitis, 88% had endothrix while 12% had ectothrix spores (Sehgal et al, 1985). [11] Kalla et al (1995) [24] observed endothrix spores in 78% cases while ectohrix was seen in 22% which is closer to our observation of endothrix (66.66%) and ectothrix (33.33%).

Tinea unguium: In our study from 15 cases of tine unguium, 5 were KOH positive culture negative, 3 were culture positive KOH negative and 4 were both KOH and culture positive. Out of 7 culture positive cases, T.rubrum and T. mentagrophytes constituted 3 cases (42.85%) each and E. floccosum 1 case (14.28%). According to Weitzman and Summer bell (1995) [10], T. rubrum, E. floccosum and T. mentagrophytes are commonly associated with tinea cruris, tinea corporis and tinea pedis while in tinea unguium and tinea manuum, T. rubrum and T. mentagrophytes are often isolated.

Brillowska et al (2007) [25] found in his study on 118 cases of onychomycosis that T. rubrum was the major pathogen.

Tinea manuum: In our study from 7 cases of tine manuum, 1 case was KOH positive culture negative, 1 case was culture positive KOH negative and 2 were both KOH and culture positive. Out of 3 culture positive cases, T. rubrum constituted 2 cases (66.66%) and T. mentagrophytes constituted 1 case (33.33%) only. According to Weitzman and Summerbell (1995) [10] in tinea manuum, T. rubrum and T. mentagrophytes are often isolated.

Tinea faciei: In our study from 5 cases of tine faciei, 1 was KOH positive culture negative, 1 was culture positive KOH negative and 2 were both KOH and culture positive.

T. rubrum was isolated in all three culture positive samples (100%).

Tinea barbae: In our study from 2 cases of tine barbae, 1 was KOH positive culture negative and 1 was both KOH and culture negative.

Positive KOH and culture samples according to age distribution Positive KOH and culture samples according to age distribution shows that in KOH positivity, age group 2130 year was predominant with 33 samples (35.48%), out of which 26 were skin samples and 6 were nail samples followed by age group of 31-40 years with 20 samples (21.50%), 18 samples of skin and 2 of nail. 11-20 years age group with 11 samples (11.82%), 10 samples of skin and 1 of hair followed by other age groups.

In culture positivity, the most dominant age group was of 2130 years, with total number of 24 samples(31.16%) followed

http://www.pacificejournals.com/aabs

A-181 by 31-40 years age group with 19 samples(24.67%). The least common age group were 61-70 & 71-80 years with 2 sample (2.59%) each. In culture positive cases T. rubrum dominated.

Singh and Beena (2003) [16] also found in their study that 45.5% of their patients with positive samples were in the 16-30 years age group.

Mahmoudabadi (2005) [26] observed that 35.7% of the cases with positive samples were in the 21-30 years age group.

Positive KOH and culture samples according to sex distribution In the present study male: female ratio was 1.78:1. Out of total 93 KOH positive samples, 60(64.51%) were of males and 33(35.48%) were of females.

In 77 culture positive samples, 50 (64.93%) were of males and 27(35.06%) were of females.

Clearly our study shows that males dominated KOH and culture positivity in skin and hair, while females dominated KOH and culture positivity in nail samples. Males associated with agriculture and farming class are more exposed to moist conditions and trauma which predisposes to infection, especially during paddy season.

AABS; 3(2): 2016

Conclusion

To conclude, the present study of 150 cases of dermatophytosis at AIMSR, Bathinda shows that: The KOH positivity rate was 62% and culture positivity rate was 51.33%. So, KOH examination gives more positive results as compared to culture.

Trichophyton infection is more common than Epidermophyton.

T. rubrum was the most common isolate followed by T. mentagrophyte.

Acknowledgements None

Funding None

Competing Interests None Declared

References

Positive KOH and culture samples according to occupation In our study we had noticed that farmers and labourers were predominantly affected followed by housewives.

In our country though major part of population stays in villages but in our study only 66 cases (44%) were from rural areas. Persons associated with agriculture showed high positivity for fungal infection that could be attributed to their profession, because they frequently came in contact with soil in the fields, their hands and feet remained moist because they water the fields which predispose to the infection. Secondly housewives were more affected because they wash utensils, clothes and their hands and feet are in contact with water most of the time which predispose to the infection. In case of KOH positive cases, the urban population dominated over the rural population in skin and nail samples, while in hair samples rural population dominated. Same was in the case of culture.

1. Kannan P, Janaki C, Selvi GS. Prevalence of dermatophytes and other fungal agents isolated from clinical samples. Indian Journal of Medical Microbiology. 2006; 24: 212-215.

2. Noble SL, Forbes RC, Stamm PL. Diagnosis and management of common tinea infections. Am Fam Physician. 1998; (10): 2424-32.

3. Sahin I, Kaya D, Parlak AH, Oksuz S, Behcet M. Dermatophytosis in forestry workers and farmers.

Mycoses. 2005; 49(4): 260-4.

4. Bibeka N.J., Vijay K.G., Aggarwal S. Tinea capitis in Eastern Nepal. Int J Dermatol. 2006; 45: 100-03.

5. Chander J. Dermatophytoses. Textbook of Medical Mycology. 2nd edition. Mehta Publishers; 2009: 92-105.

6. Kanwar AJ, Mamta, Chander J. Superficial fungal infections. IADVL textbook and atlas of dermatology.

Valia RG, Valia AR, Sidappa K, editors. 2nd ed.

Mumbai: Bhalani Publishing House. 2001; 215-58.

7. Lal S, Rao RS, Dhandapani R. Clinico-mycological study of dermatophytosis in a coastal area. Ind J Dermatol Venereol Leprol. 1983; 49(2): 71-75.

8. Bindu V and Pavithran K. Clinico-mycological study of dermatophytosis in Calicut. Ind J Dermatol Venereol Leprol. 2002; 68: 259-61.

9. Lari AR, Akhlaghi L, Falahati M, Alaghebandan R. Characteristics of dermatophytoses among children in an area south of Tehran, Iran. Mycoses. 2003; 48: 32-37.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

In culture positive cases T. rubrum dominated in both sexes.

Patwardan and Dave (1999) [15] have observed a male to female ratio of 2:1 in cases of dermatophytosis.

Singh and Beena, (2003) [16], noted male: female ratio as 1.57: 1.

Original Article

A-182

10. Weitzman I, Summerbell RC. The dermatophytes.

Clin Microbiol Rev. 1995; 8(2): 240-59.

11. Sehgal VN, Saxena AK, Kumari S. Tinea capitis. A clinicoetiologic correlation. Int J Dermatol. 1985; 24(2): 116-9.

12. Khare AK, Singh G, Pandy SS, Sharma BM , Kaur P. Pattern of dermatophytoses in and around Varanasi.

Ind J Dermatol Venereol Leprol. 1985; 51: 328-31.

13. Kumar GA, Lakshmi N. Tinea capitis in Tirupati. Ind J Pathol Microbiol. 1990; 33 (4): 360-3.

14. Verenkar MP, Pinto MJW, Rodrigues S, Roque WP, Singh I. Clinico-microbiological study of dermatophytes. Ind J Pathol Microbiol. 1991; 34(3):186-92.

15. Patwardhan N, Dave R. Dermatomycosis in and around Aurangabad. Ind J Pathol Microbiol. 1999; 42(4): 455-62.

16. Singh S, Beena PM. Profile of dermatophyte infections in Baroda. Ind J Dermatol Venereol Leprol. 2003; 69(4): 281-83.

17. Tasic S, Stojanovic S, Poljacki M. Etiopathogenesis, clinical picture and diagnosis of onychomycoses. Med Pregl. 2001; 54(1-2): 45-51.

18. Faergemann J, Baran R. Epidemiology, clinical presentation and diagnosis of onychomycosis. Br J Dermatol. 2003; 149(Supl 65): 1-4.

19. Poria VC,Samuel A, Acharya KM, Tilak SS. Dermatomycoses in and around Jamnagar . Ind J Dermatol Venereol Leprol. 1981; 47(2): 84-7.

20. Sharma NL, Gupta ML, Sharma RC, Singh P, Gupta N. Superficial mycoses in Simla. Ind J Dermatol Venereol Leprol. 1983; 49(6): 266-9.

21. Jain Neetu, Sharma Meenakshi, Saxena V.N. Clinicomycological profile of dermatophytosis in Jaipur, Rajasthan. Ind J Dermatol Venereol Leprol. 2005; 74(3): 274-75.

22. Sarma S and Borthakur A.K. A clinic-epidemiological study of dermatophytosis in Northeast India. Ind J Dermatol Venereol Leprol. 2007; 73(6): 427.

23. Prasad PVS, Priya K, Kaviarasan PK, Aananthi C, Sarayu L. A study of chronic dermatophyte infection in a rural hospital. Ind J Dermatol Venereol Leprol.

2005; 71(2): 129-30.

24. Kalla G, Begra B, Solanki A, Goyal A, Batra A. Clinicomycological study of tinea capitis in desert district of Rajasthan. Ind J Dermatol Venereol Leprol.

1995; 61: 342-5.

25. Browska A, Marie Saunte Ditte, Cavling arendrup Maiken. Five hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum. Journal of Clinical Microbiology. 2007; 45(4): 1200-04.

26. Mahmoudabadi AZ. A study of dermatophytosis in South West of Iran (Ahwaz). Mycopathologia. 2005; 160: 21-4.

http://www.pacificejournals.com/aabs

Original Article Patterns and Demographic Distribution of Hemoglobinopathies in North Maharashtra Manjusha Punjaji Tambse*, Maya Suresh Vasaikar, Sunil Santaram Chavan Department of Pathology, Shri Bhausaheb Hire Government Medical College, Dhule.

Keywords: Hemoglobinopathies, Sickle Cell Anaemia, Thalassemia, HPLC.

ABSTRACT Background: The main aim of the study is to determine patterns and demographic distribution of hemoglobinopathies in North Maharashtra using HPLC testing system. Another aim is to find the categories at high risk of hemoglobinopathies in North Maharashtra.

Methods: It is a prospective study carried out in GMC Dhule over a period of 6 years. Here patients come from Dhule, Nandurbar,Nashik & Jalgaon districts having high prevalence rate of sickle cell anaemia. A total 10081 patients included in the study. All the samples were sent for HPLC study.

Result: A female preponderance was noted (54%). The most common age group was below 20yrs (73%).

Hemoglobinopathies were most prevalent in ST category (79%), followed by OBC category (8%). The most common abnormal haemoglobin pattern detected was SA (63.1%), followed by SS (6.6%). Prevalence of Beta thalassemia heterozygous was2.3% .

Conclusion: Pawara and Bhill community formed the major chunk of this study in whom sickle cell disorders are common. Prevalence of Beta heterozygous thalassemia in this study was 2.3%, which in contrast to other studies is much less. This might be due to demographic variation as this area shows prevalence of sickle cell disorder. HPLC has helped in identification of compound heterozygous disorders like SA-BTT, SA-HBQ India, SA- Hereditary persistance of fetal Hb, HBE-BTT and HBD-SA. To the best of our knowledge, it is the first of its kind study reported from North Maharashtra.

*Corresponding author: Dr. Manjusha Punjaji Tambse. Saisandesh Childrens Hospital, Shrirang Colony, Deopur, Dhule. Pin code-424005.

Phone: +91-2562-273100 E-mail: varsha.tambse@gmail.com.

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-184

Introduction:

Haemoglobinopathies are the most prevalent genetic defect worldwide.[1] The World Health Organization has suggested that about 5% of the world population are carriers for different inherited disorders of hemoglobin.

[2] Hemoglobinopathy is a group of inherited disorders characterized by structural variations of the hemoglobin molecule; result from either production of an abnormal haemoglobin chain, such as substitution of one amino acid, or underproduction of a given globin chain. [3] Amongst all hemoglobinopathies in India, sickle cell anaemia and thalassemia contributes the major bulk.

According to some studies it is stated that sickle cell gene is widely spread in the region of eastern and northern Maharashtra and some parts of Marathwada region.[4&5] Sickle cell disease is an inherited haemoglobinopathy resulting from a mutation which occurs in beta-globin gene, on chromosome 11. [6] There is a substitution of glutamate with valine in position 6 of the beta globin resulting in the formation of haemoglobin S. [7] Amongst all abnormal hemoglobins, Sickle Cell Hemoglobin, is more deleterious, since in hypoxic condition desaturation of HbS results in the polymerization of haemoglobin, forming large aggregates called tactoids, which deform the red cells into the typical sickle shape. It leads to early destruction of the cells and sometime clogging of the sickled red cells in the microcapillaries, producing tremendous, unbearable pain.

Homozygous (SS) cells begin to sickle at a much higher oxygen saturation, typically 85% (PaO25.2â&#x20AC;&#x201C;6.5 kPa) than heterozygous (SA) cells, which usually do so well below the saturation of venous blood40% (PaO2 3.2â&#x20AC;&#x201C;4.0 kPa).

Thalassemias characterised by reduced rate of synthesis of one or more globin chains. Thalassemia syndromes are sub classified based on the gene involved i.e. Alpha and beta.

The heterozygous state is known as thalassaemia minor and results in a mild hypochromic, microcytic anaemia with haemoglobin levels 2â&#x20AC;&#x201C;3 g/dl below normal for age. The homozygous disease is known as thalassaemia major or Cooley Anaemia. It results in profound anaemia requiring repeated blood transfusions.

Conditions in which foetal haemoglobin synthesis persists beyond the neonatal period, known as hereditary persistence of foetal haemoglobin.

The need to screen hemoglobinopathies is that though they are not curable but they can be prevented by population screening, genetic counselling and prenatal diagnosis.

Hence it can reduce a huge burden on the families and ultimately on the nation.

Cation exchange HPLC offers a tool for early, accurate detection of hemoglobinopathies there by aiding in its prevention and management.

Materials and Methods

Study design: It is a hospital based prospective study carried out in GMC Dhule, a tertiary care centre over a period of 6 years. Here patients come from Dhule, Nandurbar , Nashik & Jalgaon district. All these districts have high prevalence rate of sickle cell anaemia.

Aim of study: 1.To determine patterns and demographic distribution of hemoglobinopathies in North Maharashtra using HPLC testing system. 2.To find the catagories at high risk of hemoglobinopathies in North Maharashtra so as to target this population for cost effective prevention and management of hemoglobinopathies.

Period of study: Six years from Year 2008 to 2013.

Study subject: A total 10081 patients having positive screening tests or negative screening tests but having clinical suspicion of hemoglobinopathies were included in the study. These patients included, patients coming to OPD for investigations of anaemia, IPD patient in wards, ANC patients and patients from various camps taken in primary and secondary units.

Ethical consideration: Informed consent was taken before enrolling the patients in the study.

Data analysis Screening tests: Solubility test for sickle cell anemia and Nestroft (Naked Eye single tube red cell osmotic fragility) test for thalassemia was done. All patients having positive screening test or having negative screening test but having clinical suspicion of hemoglobinopathies were subjected for HPLC studies. Diagnosis of hemoglobinopathies was made based on laboratory tests and clinical findings as mentioned in literature. [8] Demographic study: Age, sex and category wise distribution of patients and patterns of hemoglobin were observed.

Procedure: 2-3ml of blood samples were collected after obtaining informed consent using Ethlene Diamine Tetra acetic acid as an anticoagulant from each patient ensuring that they were free of blood transfusion for at least one month.First samples were run on cell counter to note Hb% and red blood cell indices.Samples were tested within a week of collection and stored at 2-80c.The samples were run on BIO-RAD Variant Hemoglobin Testing System which utilizes the principle of HPLC.An HbA2F calibrator and two levels of controls (BIO-RAD) were analyzed at the beginning of each run. The total area acceptable was

http://www.pacificejournals.com/aabs

A-185

AABS; 3(2): 2016

Result

between one to three million. [9]The value more than 3.5% of A2 fraction of hemoglobin was taken as cut off point for determining the thalassemia trait and more than 10% was assumed to be haemoglobin E. [7]

From table 4 it is seen that most common abnormal haemoglobin pattern was SA. SS was the common abnormal haemoglobin pattern after SA.

Table 1: Sex distribution of cases: Year 2008 2009 2010 2011 2012 2013 Total

Male 357 992 765 770 1105 686 4675(46%)

Female 698 855 851 860 1242 900 5406(54%)

Total 1055 1847 1616 1630 2347 1586 10081

From table 1 it is seen that 46% patients were male and 54% patients were female.

Table 2: Age wise distribution of cases: Year 2008 2009 2010 2011 2012 2013 Total

Paediatric age group (0-12yrs) 215 872 835 864 939 713 4438(44%)

12-20yrs

21-30yrs

31-40yrs

41-50yrs

>50yrs

Total

388 533 679 408 516 365 2889(29%)

352 235 65 277 610 381 1920(19%)

60 116 16 49 164 79 484(5%)

27 60 15 24 70 32 228(2%)

13 31 6 8 48 16 122(1%)

1055 1847 1616 1630 2347 1586 10081

From table 2 it is seen that predominant age group affected was paediatrics (0-12yrs) followed by 12-20 yrs.

Table 3: Category wise distribution of cases: Year 2008 2009 2010 2011 2012 2013 Total

SC 158 166 97 31 188 95 735(7%)

ST 707 1515 1356 1369 1760 1205 7912(79%)

OBC 106 92 81 181 211 174 845(8%)

VJ,NT 21 19 17 16 70 33 176(2%)

Open 63 55 65 33 118 79 413(4%)

Total 1055 1847 1616 1630 2347 1586 10081

From table 3 it is seen that 79% patients belong to ST category.

Table 4: Patterns of hemoglobinopathies: Haemoglobin variant Total samples Normal SA SS BTT BTM BT intermedia SA-BTT SA-HbQ INDIA SA-Hereditary persistence of fetal Hb HbE-BTT Hereditary persistence of fetal Hb HbD trait HbE trait

2008 1055 419 524 89 18 1

2009 1847 542 1106 164 28 2

1

3 1

1 1 1

Year 2010 1616 447 1050 68 40 3 1 5

2011 1630 365 1103 105 45 3 1 5 1 1

1 1

1

1

2012 2347 485 1649 110 60 12 22 1 2 1 2 1 2

2013 1586 458 932 131 37 4 3 20

1

Total 10081(N) 2716 (26.9%) 6364 (63.1%) 667 (6.6%) 228 (2.3%) 25 (0.2%) 5 (0.05%) 56 (0.6%) 3 (0.03%) 3 (0.03%) 3 (0.03%) 2 (0.02%) 4 (0.04%) 5 (0.05%)

SA-Sickle cell heterozygous, SS- Sickle cell homozygous, BTT- Beta thalassemia trait, BTM- Beta thalassemia major.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-186

In patients detected as Sickle cell heterozygous, HBS ranged between 30-40% and HBS value less than HBA value. Hb ranged between10-12gm%.

A2 region. HbE being>40%. In patients detected to have delta heterozygous thalassemia, HbF is raised but less compared to homozygous thalassemia.

In patients detected as Sickle cell homozygous, HBS was >70%, increased HbF value (5-20%) and normal HbA2 value. In patients detected as Beta heterozygous thalassemia HBA2 was >3.5%, Hb <9gm% and reduced red blood cell indices. In patients detected to have Beta homozygous thalassemia, HbF level was very high>90%, HbA<4gm%, reduced red blood cell indices. These patients required frequent blood transfusion.

Discussion

The general incidence of heterozygous thalassemia and sickle cell hemoglobinopathies in India varies between 3-17% and 1-44% respectively [8], [9],[10]. The highest frequency of beta-thalassemia is reported in Gujarat (10.0%â&#x20AC;&#x201C;15.0%) followed by Calcutta(10.2%), Punjab (6.5%), Delhi (5.5%), Tamil Nadu (4.0%),Bengal (3.5%), Mumbai (2.6%), Maharashtra (1.9%), and Kerala (0.6%).

[11] The highest frequency of sickle cell gene in India is reported in Orissa (9%), followed by Assam (8.3%), Madhya Pradesh (7.4%), Uttar Pradesh (7.1%), Tamil Nadu (7.1%) and Gujarat(6.4%) [12-14]. But in light of population migration it is becoming a worldwide phenomenon. Mean prevalence of carriers of beta thalassemia is 3.3%. [15]

Patients detected to have double heterozygous for SA-BTT, showed HbS>70%, HbA2 ranging from5-7% and HbF620%. Patients detected to have HbD Punjab heterozygous had unknown peak at D window. HbD ranged between 3040%. Patients detected to have HbE showed raised peak in Table 5: Comparison of male: female ratio with other studies Study Present study RS Balgir

10

Mannan etal16 Uddin etal

Compared to other studies

17

[10, 16, 17]

Male %

Female %

46

54

53

47

53

47

57

43

our study shows a slight female preponderance (54%).

Table 6: Age distribution of cases in comparison with other studies Age group

R S Balgir[10]

Uddin etal[16]

Age group

Mannan etal[17]

Our study

0-15 yrs

38

56

0-20 yrs

68

73

16-45 yrs

57

39

20-40 yrs

26

24

>45yrs

5

5

>40yrs

6

3

These observations show that hemoglobinopathies are more common at birth.

Table 7: Category wise distribution of cases in comparison with other study.

Category

R S Balgir[10]

Our study

General casts

79

14

SC

17

7

ST

4

79

In contrast to study by Balgir etal, our study showed hemoglobinopathies were more prevalent in reservation category, especially in ST and SC categories. Similar results were observed by Bhasin etal. [18].

High risk of hemolytic genetic disorders in a particular area and particular community might be due to caste and area endogamy, consanguinity and lack of education.

Our study showed 26.9% patients showing normal pattern of haemoglobin, which is comparable to study

by Balgir etal (34.3%).[10] In our study the most common hemoglobinopathy observed was sickle cell heterozygous (63.1%).When compared to other studies it is very high.

But a study by Ajjack etal showed SA pattern in 52.9% patients. [19] In our area Pawara and Bhill community are the predominant tribal population and it formed the major chunk (study subject) of the study. According to study by Kate etal[20] the prevalence of sickle cell carrier in Pawara community is 25% and prevalence in Bhill community

http://www.pacificejournals.com/aabs

A-187

AABS; 3(2): 2016

Table8: Patterns of hemoglobinopathies in comparison with other studies.

Hemoglobin Variant Normal SA SS BTT BTM BT Intermedia SA-BTT SA- HBQ India SA-Hereditary persistance of fetal Hb HBE-BTT Hereditary persistance of fetal Hb HBD trait HBE trait HBE disease Delta-Beta Thalassemia HBD-SA Total

Our study 26.9% 63.1% 6.6% 2.3% 0.2% 0.05% 0.6% 0.03% 0.03%

Balgir etal[10] 34.3% 29.8% 7.6% 18.2% 5.3% 1.7% -

Uddin etal[17] 42.2% 21.3% 0.5% -

0.03% 0.02% 0.04% 0.05% 100%

0.7% 0.2% 0.9% 0.3% 0.9% 0.2% 100%

13.5% 12.1% 9.2% 0.5% 0.7% 100%

is 20%. This explains the high prevalence of sickle cell heterozygous in this study.

Sickle cell homozygous patients showed HBS values more than 70% along with raised HBF levels. Similar findings were observed by Kar etal. [21] Prevalence of Beta heterozygous thalassemia in this study was 2.3%, which in contrast to other studies is much less. This might be due to demographic variation as this area shows prevalence of sickle cell disorder.

HPLC has helped in identification of compound heterozygous disorders like SA-BTT, SA-HBQ India, SAHereditary persistence of fetal Hb, HBE-BTT, HBD-SA. These were diagnosed based on quantification of levels of HBE, HBS and HBA. In India HbD is seen in Sindhi, Punjabi and Gujrathi communities. There were 4 patients detected as HBD trait.

They showed band in SDG region and decrease in A2 level (1.5-2.5%). Similar findings were noted by Lele etal. [5] Prevalence of HBD is more in Punjab. [22] These cases were seen in two families whose native places were in Punjab.

HbE is more prevalent in Bengali, Assami and Muslim communities. There were 5 patients of HBE Trait. Out of them 4 patients were belonging to Muslim families.

Conclusion

Our study showed female preponderance (54%) over male (46%). The most common age group was paediatric (0-12yrs) i.e. 44%, followed by 12-20 yrs and least common was age more than 50 yrs.The most Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

common hemoglobinopathy in the study was Sickle cell heterozygous (63%), followed by Sickle cell homozygous (6.6%). Prevalence of Beta thalassemia heterozygous was 2.3%, and Beta thalassemia homozygous was0.2%.

We encountered some rare compound heterozygous conditions like SA-HBQ India, SA-Hereditary persistence of foetal hemoglobin.

Hemoglobinopathy was most prevalent in ST category (79%) as Pawara and Bhill community in this region belongs to this category. HPLC provides rapid and accurate tool for diagnosis of hemoglobinopathies as identification of abnormal hemoglobin based on electrophoretic mobility is only presumptive and have to be confirmed by another technique applying different principle.

To the best of our knowledge, it is the first of its kind study reported from North Maharashtra.This study provides patterns and demographic distribution of hemoglobinopathies in North Maharashtra. This can be helpful to target specific age group and categories in which hemoglobinopathies are most prevalent. So that it can be helpful to control the disease in cost effective way by screening programme, genetic counselling and public education.

Acknowledgement

Great regard to Staff-Sickle Cell Project, Shri Bhausaheb Hire Government Medical college, Dhule. Mr. Sunil Pawar- Technician. e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-188

Funding

12. Verma IC, Choudhury VP, Jain PK. Prevention of thalassemia : a necessity in India. Indian J Pediatr 1992; 59: 649-654.

None

Competing Interests

13. Manglani M, Lokeshwar MR, Vani VG, BhatiaN, Mhaskar V. ‘NESTROFT-an effective test for b-thalassemia trait. Indian Pediatr 1997; 34: 703 -708.

None declared.

References

1. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem.

Bulletin of the World Health Organization, 2001, 79:704–712.

2. Angastinosis M, Modell B. Global epidemiologyof hemoglobin disorders. Proc Natl Acad Sci USA 1998; 850: 251.

3. Wilson M, Forsyth P, Whiteside J: Haemoglobinopathy and sickle cell disease. Continuing Education in Anaesthesia, Critical Care & Pain 2009, 10.

4. Kate S L Health problems of tribal population groups from state of Maharashtra Ind J Med Sci 2001; 5(2): 99-108.

5. 3 Lele R d, Solanki B R, Bhagwat R B, Hemoglobinopathies in Aurangabad region. Journal Association Physicians India 10:263 6. Herrick J: Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia. Arch Intern Med 1910, 6: 517-521.

7. Serjeant GR, Serjeant BE: Sickle Cell Disease, 3rd edn: Oxford: Oxford University Press.; 2001.

8. 8.Balgir RS. The burden of hemoglobinopathies in India and the challenges ahead. Curr Sci 2000; 79:1536-1547.

9. Balgir RS. The genetic burden ofhemoglobinopathies with special reference tocommunity health in India and the challengahead. Indian J Hematol Blood Transfus 2002;20: 2-7.

10. Balgir RS. Spectrum of hemoglobinopathies inthe state of Orissa, India : A ten years cohort study. J Assoc Physicians India 2005; 53:1021-1026.

11. Verma IC. Burden of genetic disorders in India. Indian J Paediatr2000;67:893–8.

14. Wild BJ, Bain BJ. Investigation of abnormalhemoglobins and thalassemia. In: Lewis SM, Bain BJ, Bates I (Eds.), Dacie and Lewis Practical Hematology, 9th Edition. London :Churchill Livingstone. 2001; 231-268.

15. Chouhan V. Epidemiology: symposium on thalssaemia.

Indian J Hematol Blood Transf 1992;10:1–20.

16. M. Mesbah Uddin etal. Pattern of B- Thalassemia and other haemoglobinopathies: A cross- Sectional Study in Bangladesh. International Scholarly Research Network ISRN Hematology 2012;659191:1-6 17. Mannan A, J Kawsar etal. A demographic approach for understanding the prevalence of B Thalassemia Patterns and other hemoglobinopathies: Selective Study in Chittagong City Perspective. Asian Journal of Biological Sciences 2013;6(2): 124-130.

18. Bhasin MK, Walter H, Danker-Hopfe H. Glucose-6phosphate dehydrogenase deficiency and abnormal hemoglobins (S andE) in people of India. J Hum Ecol 1994;3:131-59 19. Eman A. Ajjack, Hiba A. Awooda etal.

Hemoglobin patterns in Patients with Sickle Cell Hemoglobinopathies. International Journal of Haematological Disorders, 2014;1(1):8-11.

20. Kate SL, Lingojwar DP. Epidemiology of Suckle Cell Disorder in the State of Maharashtra. Int J Gene 2002; 2(3):161-167.

21. Kar B C, Devis, Clinical profile of sickle cell disease in Orissa. Indian journal of Pediatric, 64:73- 77, (1997).

22. Kate SL, Health problems of tribal population groups from the state of Maharashtra. 2000; Oct 23rd: 1 to 9 from Immunology Bulletin.

http://www.pacificejournals.com/aabs

Original Article A Histopathological Study of Non Neoplastic Gall Bladder Diseases with Special Reference to Mucin Histochemistry Samriddhi Sood*, Rajnish Kumar, Anupam Varshney, Veena K. Sharma, Alok Mohan, Bharat Wadhwa, Kanchan Kamini Deptt. of Pathology , Muzaffarnagar Medical College and Hospital, Muzaffarnagar, UP, India Keywords: Gall bladder, Cholecystitis, Cholesterolosis, Gall stones, Mucin histochemistry, PAS.

ABSTRACT Background: Cholelithiasis and even silent gallstones which are asymptomatic produce a series of epithelial pathological changes in gall bladder mucosa, which could be precursor lesions of gall bladder carcinoma.

Aim: To study the non neoplastic gall bladder diseases with special reference to mucin histochemistry.

Material and Methods: Formalin-fixed paraffin-embedded surgically resected gallbladder tissue samples of last 5 years were sectioned. All sections were stained with Hematoxylin and Eosin, Alcian Blue (AB) at pH 2.5 and pH 1.0 and Periodic acid-Schiffâ&#x20AC;&#x2122;s (PAS).

Result: A total of 1420 cholecysteclomy cases were observed. Maximum cases were of chronic cholecystitis with gall stones 91.55 % (1300/1420) cases. The stones were mainly of mixed type 45.19% (474/1049) cases. Metaplasia was found in 182 out of 1420 cases. Mucin histochemistry in metaplstic group showed PAS positivity in all 182 cases, AB/ PAS positivity at pH 2.5 in 97.25%(177/182) cases and AB/PAS positivity at pH 1 in 78.57%(143/182) cases.

Conclusion: Cholelithiasis is the most important entity in gall bladder lesions and that there is a definite association between metaplasia-dysplasia-carcinoma sequence. Early detection of mucin histochemistry in metaplastic cases can lead to early diagnosis of carcinoma gallbladder.

*Corresponding author: Dr Samriddhi Sood, Deptt. Of Pathology, Muzaffarnagar Medical College and Hospital, Muzaffarnagar, UP, India Phone: +91 7417763644 E-mail: dr.samriddhi.sood@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-190

Introduction

Gall bladder is one of the organs having a wide spectrum of diseases ranging from congenital anomalies, calculi and its complications, non-inflammatory, inflammatory to neoplastic lesions. So the classification of various histomorphological types of gallbladder lesions is important to categorize into non neoplastic and neoplastic lesions of gallbladder.

The Non neoplastic lesions mainly includes cholelithiasis, cholecystitis, adenomyomatosis and cholesterolosis .

Neoplastic category includes adenoma, carcinoma and mesenchymal tumours. The early malignancies have to be diagnosed on histopathology as they present as cholecystitis clinically and for proper management & better prognosis.

The study was performed to find out the prevalence of metaplasia in and around Muzaffarnagar.

Material and Methods

A retrospective and prospective study from July 2011 to June 2015 was conducted. A total of 1420 cholecystectomy specimen received in histopathology section of Pathology Department were analyzed. After gross examination, tissues were subjected to formalin fixation, routine processing and paraffin embedding. For cases without any gross abnormality, standard 3 sections including fundus, body and neck were taken. In cases with any growth, irregular mucosa , thickened wall, etc more sections were taken. Five microns thick sections on three to four slides were prepared from each specimen. Apart from routine hematoxylin and eosin stain, special stains like PAS and Alcian Blue at pH 1.0 and 2.5 were used whenever needed.

Gross and microscopic features of all incidentally detected cases were studied in detail.

Observations

Age of the patient in the present study ranged from 1180 years and commonest age group with maximum gall bladder diseases was in 4th decade i.e. 26.12% (371/1420) cases. Female to Male ratio was 4.5:1.

Maximum cases were of chronic cholecystitis 91.54% (1300/1420) cases followed by acute cholecystitis 3.31% (47/1420cases), follicular cholecystitis and eosinophilic cholecystitis. In cases of eosinophilic cholecystitis cases, there was no history of local or allergic reaction to substances released at foci of inflammation, local diathesis involving gall stones, acalculus cholecystitis, parasites, hypereosinophilic syndrome, eosinophilic gastroenteritis and eosinophilic myalgia syndrome, drugs such as cephalosporins and herbal medicines.

Gall stones were present in 73.87% (1049/1420) cases and 26.13% (371/1420) cases had no gall stones. Most common histomorphological lesion associated with gall stones was chronic cholecystitis 73.07% (950/1300 cases) followed by acute cholecystitis 63.82% (30/41 cases), eosinophilic cholecystitis 70% (7/10) cases (table 1).

Gall stones observed in maximum number of cases were multiple stones accounting for 80% (839/1049 cases) in non neoplastic lesions.

Out of 1049 gall stone cases, gall stones were more common in females 85.32% (895/1049cases) than in males 14.68% (154/1049) cases respectively. Most common gall stone was mixed stone 45.19% (474/1049) cases followed by cholesterol and pigment stone 36.51% (383/1049 cases) and 18.30% (192/1049) cases respectively (Table 2 & photomicrograph 1).

In present study, metaplasia was seen in 12.82% (182/1420) cases. Most common histomorphological lesion associated with metaplasia was Chronic Cholecystitis 12% (156/1300) cases (Table 3 & photomicrograph 2). Out of 156 cases of Chronic Cholecystitis with metaplasia, 143 cases were associated with stones which were mainly of solitary type 61.54%(88/143) cases.

Out of 1420 cholecystectomy specimens, mucin histochemistry was done in 182 cases showing metaplasia. In metaplatic group PAS positivity was seen in all cases. Different grades of positivity for AB/ PAS at pH 2.5 was seen in 47.25% (177/182) showed positivity for acid mucin (AB/PAS at pH2.5) followed by sulphomucin(AB/PAS at pH1) in 78.57% (143/182) cases. However out of 177 cases, 34 cases were negative for AB/PAS at pH1 indicating the type of acid mucin was only in sialoform. Remaining 143 cases were taken as positive for sulpho and sialomucin in different concentration interpreted according to staining intensity.

Thus metaplastic lesions showed predominantly neutral mucin followed by sialomucin and sulphomucin (table 4 and photomicrographs 3&4).

In this study, the control group (normal gall bladder and randomly selected cases of chronic cholecystitis without metaplasia) showed predominance of sulphomucin followed by neutral mucin. However none of the case in control group showed presence of sialomucin.

Discussion

Cholecystitis is defined as inflammation of the gall bladder that occurs mostly commonly because of an obstruction of the cystic duct from cholelithiasis.1 Cholelithiasis produces diverse changes in the gall bladder mucosa namely acute

http://www.pacificejournals.com/aabs

A-191

AABS; 3(2): 2016

Table 1: Histomorphological lesions associated with gallstones S.N.

Histopathological Lesion

No. of cases

With gallstones

Without gallstones

1

Acute Cholecystitis

47(3.31%)

30(63.82%)

17(36.17%)

2

Eosinophilic Cholecystitis

10(0.70%)

07(70%)

03(30%)

3

Chronic cholecystitis

1300(91.55%)

950(73.07%)

350(26.92%)

4

Follicular cholecystitis

13(0.92%)

13(100%)

--

5

Cholesterolosis

24(1.69%)

24(100%)

--

6

Xanthogranulomatous Cholecystitis

05(0.35%)

04(100%)

01(20%)

7

Mucocele

01(0.07%)

01(100%)

--

8

Adenomyomatosis

15(1.06%)

15(100%)

--

9

Dysplasia

05(0.35%)

05(100%)

--

1420

1049(73.87%)

371(26.13%)

Total

Table 2: Gross morphological types of gall stones (n=1049) Gross morphological types of gall stones

Male

Female

Total

Percentage

Cholesterol

33

350

383

36.51%

Mixed

89

385

474

45.19%

Pigment

32

160

192

18.30%

Total

154(14.68%)

895(85.32%)

1049

100

Table 3: Histomorphological spectrum of gall bladder lesions associated with metaplasia (n=1420) S.N.

Histopathological Lesion

No. of cases

Metaplasia (%)

Metaplsia with stones(%)

1

Acute Cholecystitis

47

03(6.38%)

3(100%)

2

Eosinophilic Cholecystitis

10

--

--

3

Chronic cholecystitis

1300

156(12%)

143(91.67%)

4

Follicular cholecystitis

13

--

--

5

Cholesterolosis

24

03(12.50%)

03(100%)

6

Xanthogranulomatous Cholecystitis

05

01(20%)

01(100%)

7

Mucocele

01

--

--

8

Adenomyomatosis

15

15(100%)

15(100%)

9

Dysplasia

05

04(80%)

4(100%)

1420

182(12.82%)

169(92.86%)

Total

Table 4: Alcian blue/Periodic Acid Schiff (AB/PAS) stain results in metaplastic cases Condition Metaplastic (182)

Total

Grading

At pH 2.5

At pH 1

No. of cases

%age

No. of cases

%age

No staining

5

2.75

39

21.43

+

50

27.47

71

39.01

++

85

46.70

57

31.32

+++

42

23.08

15

8.24

182

100

182

100

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-192

hotomicrograph 1: Cholesterolosis showing prominent foamy cells in mucosa x400,H&E

Photomicrograph 2: Pyloric gland metaplasia showing prominent pyloric glands in the mucosa x400 H&E

Photomicrograph 3: Sulphomucin in Chronic Cholecystitis with metaplasia x400, AB-PAS pH=1

Photomicrograph 4: Metaplastic glands showing acid mucin x400, AB-PAS pH=2.5

inflammation, chronic inflammation, granulomatous inflammation, cholesterolosis and glandular hyperplasia.2 The pathological changes related to gallstone formation are still the focus of intensive research. Gallstones mainly injure the mucosal columnar epithelium and thus cause changes like metaplasia, dysplasia and neoplasia.3

In the present study on histopathological examination of gallbladder lesions, maximum cases were of chronic cholecystitis 91.54% (1300/1420) followed by acute cholecystitis 3.31% (47/1420cases). These observations were almost similar to Selvi etal7 who reported it to be 85.8%. However percentages varying from 45 to 64.59 had been reported by various workers09-13

In present study, age of the patients ranged from 11-80 years. Maximum cases(27.16%) of gall bladder disease were seen in 4th decade of life, followed by ( 23.96%) in 5th & ( 20.91% ) in 3rd decade. The mean age in the present study was 43.56 years where as in a study by Banerjee et al (2015), it was observed to be 39.3 years.4 Male to Female ratio was 1:4.5. This was in accordance with various studies5-8 where M:F ratio ranged from 1:3.2 to 1:6.5.

After chronic cholecystitis, acute cholecystitis cases accounted for 3.26% which was in concordance with study

by Kaur et al14 (2012) who showed it to be 2.60% cases while another study by Vahini et al15 (2015) and Terada16 (2013) showed 18.3% and 1.5% cases of acute cholecystitis which was in contrast to present study. This might be due to large sample size in this study.

http://www.pacificejournals.com/aabs

A-193 In this study, cases of cholesterolosis were 1.69% (24/1420 cases) which was similar to study by Vahini et al15 (2015) who reported it to be 1.8% cases. In a study

by Kaur et al14(2012), much higher percentage of cases of cholesterolosis (12.25%) were found. This may be due to different dietary habits and different religions.

In this study, adenomyomatosis was found in 1.06% (15/1420) cases. Similar study by Terada16 (2013) showed 3%(16/540) cases which was higher than this study. The reason is due to the increased sample size.

In present study, eosinophilic cholecystitis was found in 0.70%(10/1420) cases. Similar studies conducted by Kaur et al14 (2012) and Vahini et al15 (2015) and showed 0.78% and 0.90% respectively which was in concordance with this study. The prevalence of Eosinophilic Cholecystitis ranges from 0.25% to 6.4% as reported in other study.17 In a study,17 it was also proposed that local or allergic reaction to substances released at foci of inflammation, local diathesis involving gall stones, acalculus cholecystitis, parasites, hypereosinophilic syndrome, eosinophilic gastroenteritis and eosinophilia myalgia syndrome, drugs such as cephalosporins and herbal medicines had been implicated in Eosinophilic cholecystitis. No such correlation was found in these cases probably it may be due to idiopathic cholecystitis and histopathology remains the mainstay for the diagnosis of eosinophilic cholecystitis as there is no specific clinical presentation. A subset of idiopathic Eosinophilic cholecystitis had also been repoted, after excluding the above etiologies. The reason is mainly uncertain.

In this study, 0.35% (5/1440) cases of xanthogranulomatous cholecystitis were found. Other studies showed xanthogranulomatous cases ranging from 1% to 3% which was higher percentage as compared to this study. This may be due to change in dietary habits and geographical regions.

In present study, 0.35%(5/1420 cases) showed dysplastic changes which was in concordance with the study by Costa et al(2010) which observed 0.2% cases of dysplasia out of 727 cases. This was in discordance with other studies04,14,18 probably due to their small sample size.

In this study, chronic cholecystitis cases were mostly associated with cholelithiasis 73.08% (950/1300) cases.

Similar findings were noted by Baig et al10, Mathur SK et al11, Mohan H et al14 and which was also similar to the present study. Also, maximum cases were associated with multiple stones i.e. 80% (839/1049) cases.

AABS; 3(2): 2016 of non neoplastic lesions with stones respectively which was in concordance with our study.

Mixed stone was the commonest stone 45.19% (474/1049) cases. Cholesterol and pigment stones were 36.51% (383/1049) and 18.30% (192/1049) respectively. Similar results were noted in other studies, however percentage of cholesterol and pigment stones were variable 7,09-12.This variation might be due difference in dietary habits.

In this study, out of 156 cases of chronic cholecystitis with metaplasia, 143 cases (91.67%) were associated with gallstones which were mainly of solitary type 61.54%(88/143) cases. This was also in concordance with other studies11,20 In present study of mucin histochemistry, it was found that gall bladder with cholelithiasis showed sulfomucins, that were predominant in supranuclear region. In deeper parts sialomucins were found and neutral mucins were seen sparsely. So present studies are comparable with similar study done by Halagowder et al.21 Increased mucin secretion by gallbladder mucosa during gallstone formation has been described in various studies.22-26 Earlier studies have shown that mucins, in addition to being a structural component of gallstones,27 also play an acceleratory role in lithogenesis.28 Also, mucin histochemistry in metaplastic group showed neutral mucin (PAS) in all 182 cases. 97.25% (177/182) showed positivity for acid mucin (AB/PAS at 2.5pH) followed by sulphomucin(AB/PAS at pH 1) in 78.57% (143/182) of cases. However out of 177 cases, 34 cases were negative for AB/PAS at pH1 indicating the type of acid mucin was only in sialoform. Remaining 143 cases were taken as positive for sulpho and sialomucin in different concentration interpreted according to staining intensity. Thus metaplastic lesions showed predominantly neutral mucin followed by sialomucin and sulphomucin.

Similar findings were reported by Gupta et al.3

Conclusion

Cholelithiasis is the most important entity in gall bladder lesions and that there is a definite association between metaplasia-dysplasia-carcinoma sequence.

Early detection of mucin histochemistry in metaplastic cases can lead to early diagnosis of carcinoma gallbladder.

Bibliography

In present study, non neoplastic lesions of gall bladder were found to be 80% (839/1049) cases with multiple stones. Similar study was also conducted by Goyal et al19 (2014) which also observed that multiple stones are far more common than single stone accounting for 72% cases

1. Abro AH, Haider IH, Ahmad S. Helicobacter pylori Infection in patients with Calcular Cholecystitis : A Hospital Based Study. Journal Ayub Med Coll Abbottabad 2011;23(1).

2. Zaki M, Al-Refeidi A. Histological changes in the Human Gallbladder Epithelium associated with gallstones. Oman Medical Journal 2009 Oct;24(4).

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-194

3. Gupta SC, Misra V, Singh PA, Anu Roy, Misra SP, Gupta AK. Gallstones and carcinoma gallbladder.

Indian J Pathol Microbiol 2000;43(1):147-54.

4. Banerjee A, Tapadar A. Spectrum of histopathological changes in cholecystitis.Int J Biol Med Res 2015;6(1):4769-74 5. Mohan H, Punia RPS, Dhawan SB, Ahal S, Sekon MS. Morphological spectrum of gall stone in 1100 cholecystectomies in North India. Indian Jounal of surgery 2005;67:140-2.

6. Tyagi SP, Tyagi N, Maheshwari V, Ashraf SM, Sahoo P. Morphological changes in diseased gall bladder: A study of 415 cholecystectomies at Aligarh. Journal Indian Med Assoc 1992 Jul;90(7):178-81.

7. Selvi T.R, Sinha P, Subramaniam P.M, Konapur P.G, Prabha C.V. A clinicopathological study of cholecystitis with special reference to analysis of cholelithiasis. International Journal of Basic Medical Science 2011(4):68-72.

8. Pradhan SB, Joshi MR, Vaidya A. Prevalence of different types of gallstone in the patients with cholelithiasis at Kathmandu Medical College, Nepal. Kathmandu University Medical Journal 2009;27(7):268-71.

9. Tyagi SP, Tyagi N, Maheshwari V, Ashraf SM, Sahoo P. Morphological changes in diseased gall bladder: A study of 415 cholecystectomies at Aligarh. Journal Indian Med Assoc 1992 Jul;90(7):178-181.

10. Baig SJ, Biswas S, Das S, Basu K, Chattopadhyay G. Histopathological changes in gall bladder mucosa in cholelithiasis: correlation with chemical composition of gall stones. Trop Gastroenterol 2002;23(1):25-27.

11. Mathur SK, Duhan A, Sunita S, Aggarwal M, Aggarwal G, Sen R, Sinsh S, Garg S. Correlation of gallstone characteristics with mucosal changes in gall bladder. Tropical gastroenterology 2012;33:39-44.

12. Pradhan SB, Joshi MR, Vaidya A. Prevalence of different types of gallstone in the patients with cholelithiasis at Kathmandu Medical College, Nepal. Kathmandu University Medical Journal 2009;27(7):268-71.

13. Costa ALA, Bresciani CJ, Perez RO, Bresciani BH, Aparcedia S, Siqueira C, Cecconello I. Are histological alterations observed in the gall bladder precancerous lesions. CLINICS 2010;65(2):143-50.

14. Kaur A, Dubey V.K.,Mehta K.S.Gallbladder Mucosal Changes Associated with Chronic Cholecystitis and Their Relationship with Carcinoma Gallbladder. J.k science2012:14(2).

15. Vahini G, Premalatha P, Mathi A, Krishna R, Renuka I.V. A Clinicopathological Study of Gallbladder Lesions. IOSR-JDMS 2015;14(2)III:15-20.

16. Terada T. Histopathologic features and frequency of gall bladder lesions in consecutive 540 cholecystectomies.

Int J Clin Exp Pathol 2013;6(1):91-96 17. Mohan H, punia RP, dhawan SB, Ahal S, Sekhon MS. Morphological Spectrum of gall stone disease in 1100 cholecystectomies in North India. Indian J Surg.2005;67:140-142.

18. Duarte I, Llanos O, Domke H, Harz C, Valdivieso V. Metaplasia and precursor lesions of gallbladder carcinoma. Cancer 1993;72:1878-84.

19. Goyal S. Sharma S. Duhan A. Correlation between gallstones characteristics and gallbladder mucosal changes: A retrospective study of 313 patients. Clinical cancer Investigation Journal 2014:3(2):157-61.

20. Domeyer PJ, Sergentanis TN, Zagouri F, Tzilalts B, Mouzakioti E, Parasi A, et al. Chronic cholecystitis in elderly patients. Correlation of the severity of inflammation with the number and size of the stones. In Vivo 2008;22:269-72.

21. Smith BF, Lamont JT, Moore JRL, et al. Role of gall bladder mucin in pathophysiology of gall stones.

The Journal of the American Medical Association 2008;4:51-56.

22. Shiffman ML, Sugerman HJ, Kellum HJ, Moore EW. Changes in gallbladder bile composition following gallstone formation and weight reduction.

Gastroenterology 1992 Jul;103(1):214–1.

23. Shalin S, Ahlberg J, Einarsson K, Henriksson R, Danielson A. Quantitative ultrastructural studies of gallbladder epithelium in gallstones free subjects and patients with gall stones. Gut 1990 Jan;31(1):100–5.

24. Madrid JF, Hernandez F, Ballesta J. Characterisation of glycoproteins in the epithelial cells of human and other mammalian gallbladder. A Review. Pathol Int 1996 Apr;46:261–6.

25. Swobodnik W, Wenk H, Janowitz P, et al. Total biliary protein, mucus glycoproteins, cyclic-AMP and apolipoprotein in the gall bladder bile of patients with cholesterol stonesand stone-free controls. Scand J Gastroenterol 1991 Jul;26(7):771–8.

26. Hakkinen I, Laitio M. Epithelial glycoproteins of human gallbladder. Immunological characterization.

Arch Pathol 1970 Aug;90(2):137–42.

http://www.pacificejournals.com/aabs

A-195

AABS; 3(2): 2016

27. Maki T, Matsushiro T, Suzuki N, Nakamura N. Role of sulphated glycoprotein in gallstone formation. Surg Gynecol Obstet 1971 May;132(5):846â&#x20AC;&#x201C;54.

28. Lee SP. Hypersecretion of mucus glycoproteins by the gallbladder epithelium in experimental cholelithiasis.

J Pathol 1981 Jul;134(3):199â&#x20AC;&#x201C;207.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Morphological Spectrum of Changes in Gall Bladder in Correlation to Various Types of Gallstones â&#x20AC;&#x201C; A Study of 100 Cases Meenakumari Gopalakrishnan, Sharmila Thilagavathy, Kamaleshwari, Jeyanthi, Shifa, Raasi Department Of Pathology, Madurai Medical College, Madurai, Tamil Nadu, India Keywords: Cholesterol, Hyperplasia, Metaplasia, Carcinoma.

ABSTRACT Background: Gallstones are a common cause of morbidity world wide. Presence of gallstones in gallbladder results in diverse histological changes. Some of them could be precursor lesions for malignancy. This study was aimed to correlate the various histological changes in the gallbladder to the chemical composition of gallstones whether it was cholesterol, mixed or pigment variety.

Method: We analysed gallbladders of 100 patients who underwent cholecystectomy for gall stones.The age ,sex distribution and the incidence of different types of gallstones were studied.The histological changes in the gallbladders were observed and correlation with the type of gallstones was evaluated.

Result: Gallstones were more common in the 40 -49 age group with increased incidence in females. Many histological changes including hyperplasia ,lymphoid follicles ,prominent RokitanskyAschoff sinuses, muscular hypertrophy, metaplasia and carcinoma were observed. Most of these histological changes were seen in the gallbladders with cholesterol stones. Hyperplasia was observed in 31.5%,lymphoid follicles in 31.5% ,RokitanskyAshoff sinus in 36.8%, muscular hypertrophy in 47.3%,pyloric metaplasia in 26% of gallbladders with cholesterol stones. Intestinal metaplasia was commonly associated with pigment stones(11%) and carcinoma was seen in gallbladders with mixed stones (6.7%).

Conclusion: Gallstones are common in the adult population with a female predominance. Mixed stones were the common stones encountered . Correlation of histological changes with the chemical composition of gallstones showed increased incidence of changes in gallbladders with cholesterol stones.This could be due to the larger size of the cholesterol stones leading to more irritation and chemical injury produced by lithogenic bile.

*Corresponding author: Dr. G. Meena Kumari MD., 23, Surya Nagar First Street, K. Pudur, Madurai, Tamilnadu, India Phone: +91 9994901811 E-mail: meenakumariilango1971@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-197

Introduction

Gallstone disease is a common health problem throughout the world affecting 10% to 20% of adult population.[1] Majority of the gallstones are asymptomatic. Gallstones are classified into cholesterol stones containing more than 80 %crystalline cholesterol monohydrate, pigment stones composed predominantly of bilirubin calcium salts and mixed stones with components of both types.[2] Cholesterol stones are more common in the West whereas pigment stone is the predominant type in non-western population.

Hypomotility of gallbladder,accumulation of lipids in bile and mucus hypersecretion contribute to the formation of cholesterol stones.[3] Pigment stones occur in disorders associated with elevated unconjugated bilirubin in bile.[4] Gallstones produce a spectrum of morphological changes in gallbladder that ranges from inflammation, hyperplasia, metaplasia and carcinoma. The aim of this study is to evaluate the various histological changes in gallbladder in correlation with the chemical composition of gallstones.

Materials and Methods

Gallbladder of 100 patients who underwent cholecystectomy for gallstone disease between November 2014 and August 2015 were included in this study. Each gallbladder was serially sectioned from neck to fundus.

The sections obtained were fixed in 10% formalin, processed and cut into 5 microns thickness and stained in H and E. The histological changes in all the layers of the gallbladder such as hyperplasia, lymphoid follicles, RokitanskyAschoff sinuses, muscular hypertrophy, pyloric gland metaplasia and intestinal metaplasia were observed and noted. Biochemical analysis of the gallstones was done. The various histological changes observed in the gallbladder were analysed in correlation with the type of gallstones.

Result

Out of the 100 cholecystectomy specimens studied, 29 were from males and 71 from females with a male to female ratio of 1:2.4.[Table :1 ]. The age ranged from 20 to 75 years. Maximum number of patients belonged to the age group of 40 â&#x20AC;&#x201C; 49 years. .[Table :2 ]. Most of the patients had mixed gallstones (45%) followed by pigment stones (36%) and cholesterol stones (19%). Among the many histological changes observed in the present study, mucosal hyperplasia was seen in 21 cases. .[Table :3,4.Fig 1 ].It occurred in 31.5 % of gallbladder with cholesterol stones followed by those with pigment stones (19.44%) and mixed stones (17.7%). Lymphoid follicles in the lamina propria and muscle layer was seen in 31.57% of gallbladder with cholesterol stones.[Fig :2]. It was less obvious in gallbladders with mixed stones(22.22%) and Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 pigment stones (19.44%). RokitanskyAschoff sinuses were seen in 36.8% of gallbladders with cholesterol stones,31% in mixed stones and 27.7% in pigment stones. Muscle hypertrophy was common in gallbladders with cholesterol stones(47.36%) followed by mixed stones (35.55%) and pigment stones (22.2%) [Fig :3]. Metaplastic changes were seen in 18% of the cases. Pyloric gland metaplasia was observed commonly in gallbladders with cholesterol stones (26%), 13% of gallbladders with pigment stones and 6.6% of those with mixed stones. [Fig : 4]. Intestinal metaplasia was common in gallbladders with pigment stones (11%) followed by those with mixed stones (6.6%) and cholesterol stones (5.2%). [Fig :5,6]. Adenoarcinoma was observed in three cases. All the three were elderly females and had mixed stones.(6.7%) [Fig :7,8,9]. Associated intestinal metaplastic changes were observed in all the gallbladders with carcinomatous change.

Discussion

Gall stones are a common cause of morbidity throughout the world. The prevalence of gallstones disease in India ranges from 2 to 29%.[5,6]. It is seven times more common in North India (stone belt) than in South.[7] This may be attributed to the dietary differences in the two regions.[8] In our study, majority of the patients belonged to the 40 â&#x20AC;&#x201C; 49 years age group. This correlated with other studies.[9,10] In the present study, gallstones were more common in females with a male to female ratio of1:2.4 .Such female preponderance was noted in many other studies.[10].This increased incidence may be attributed to the female sex hormones and sedentary habits.[11] Gallstones are of three types : cholesterol,pigment and mixed stones. Factors that increase hepatic secretion of cholesterol like pregnancy, oral contraceptives and Table 1 : Sex Ratio In This Study

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-198

Table 2 : Age and Sex Wise Distribution AGE (YRS)

PIGMENT STONE

MIXED STONE

CHOLESTEROL STONE

TOTAL

M

F

M

F

M

F

M

F

20–29

1

6

1

–

–

3

2

9

11

30–39

2

8

–

7

–

3

2

17

20

40–49

2

6

7

9

1

5

10

19

30

50–59

5

3

3

6

2

3

10

13

22

60–69

2

–

2

7

–

1

4

9

12

70–79

–

1

1

2

–

1

1

4

5

Total

12

24

14

31

3

16 29

71

100

36

45

19

Table 3 : Frequency of Histological Changes in Each Stones HISTOLOGICAL CHANGES

CHOLESTEROL STONE

PIGMENT STONE

MIXED STONE

MUCOSAL HYPERPLASIA

31.5 %

19.4%

17.7%

LYMPHOID FOLLICLE

31.5%

19.4%

22.2%

ROKITANSKY ASCHOFF SINUSES

36.8%

27.7%

31%

MUSCULAR HYPERTROPHY

47.3%

22.2%

35.5%

PYLORIC METAPLASIA

26%

13%

6.7%

INTESTINAL METAPLASIA

5.2%

11%

6.7%

–

–

6.7%

CARCINOMA

Table 4: Frequency of Histological Changes in Each Stone

http://www.pacificejournals.com/aabs

A-199

Fig. 1 : Photomicrograph of gallbladder mucosa showing hyperplasia. (H & E - 10X )

Fig. 2 : Photomicrograph of gallbladder wall showing lymphoid follicles. (H & E - 10X )

AABS; 3(2): 2016

Fig. 4: Photomicrograph of gallbladder mucosa showing pyloric metaplasia. (H & E-10X

Fig. 5: Photomicrograph of gallbladder mucosa showing intestinal metaplasia.H & E-10X

Fig. 3 : Photomicrograph of gallbladder wall showing muscle hypertrophy. (H & E -10X )

Fig. 6 : Photomicrograph of gallbladder mucosa showing intestinal metaplasia with the goblet cells showing strong reaction to Alcian blue stain . (Alcian blue â&#x20AC;&#x201C; 10X )

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-200

Fig. 7: Photomicrograph of gallbladder showing carcinomatous growth along with multiple small greyish black gallstones.

Fig. 9: Photomicrograph showing adenocarcinoma of gallbladder. (H & E â&#x20AC;&#x201C; 40X )

FIG 8: Photomicrograph showing adenocarcinoma of gallbladder. (H & E- 10X )

http://www.pacificejournals.com/aabs

A-201 rapid weight loss enhance cholesterol stone formation.

Conditions like chronic haemolytic anaemia and bacterial contamination of biliary tree increase the risk of pigment stones. In the present study.mixed stone was the most common variety encountered.This correlated with the study by Mathur et.al.[10,20] Gallstones produce a series of histological changes in gallbladder and some could be precursor lesions for gallbladder carcinoma. In the present study, epithelial hyperplasia with disruption was observed in 21 cases.

Putz and Willens suggests cholelithiasis induces active proliferation of the epithelium in response to chronic irritation.[12] According to Albores â&#x20AC;&#x201C;Saavedra et al, a small number of hyperplasia evolves into atypical hyperplasia progressing into insitu carcinoma and finally into invasive carcinoma.[13] In our study epithelial hyperplasia was more common in gallbladder with cholesterol stones(31.57 %) as in the study by MunaZahir et.al.[14] Prominent RokitanskyAchoff sinuses which are outpouchings of the mucosal epithelium through the wall was observed in 31 cases. As in the study by MunaZahir et.al, it was commonly associated with cholesterol stones(36.84%). This could be due to the fact that cholesterol is a more potent stimulus leading to its formation.[14] Lymphoid follicles in the lamina propria and muscular layer were seen in 23 cases. It was found mainly in the gallbladder with cholesterol stones ( 31.57%) as in the study by MunaZahir et.al.[14].

Muscular hypertrophy was found in 33 cases. It was more common in gallbladders with cholesterol stones (47.36%).

But in the study by MunaZahir et.al, it was found mainly associated with pigment stones.[14] However no specific explanation can be given for this variation.

In the present study, metaplastic changes were observed in 18 % of the cases. Chang HG in his study stated mucous hypersecretion enhance stone formation and then the stone itself would produce metaplastic changes along with inflammation and physical injury to the epithelium.

(15) In our study pyloric gland metaplasia was commonly observed in gallbladders with cholesterol stones(26%).

This is in correlation with the study by Mathur et.al and Chang HG.[10,15].

Intestinal metaplasia was observed in 8 cases which correlated with the study by Mathuret.al.[10]. In the present study, intestinal metaplasia was common in gallbladders with pigment stones (11%) followed by mixed stones (6.7%).

Intestinal metaplasia is considered as a precancerous lesion in contrast to pyloric gland metaplasia which is considered Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016 a benign lesion.[16] . In our study too, intestinal metaplasia was observed in gallbladders harbouring malignancy.

Carcinoma of gallbladder is the most common malignancy of the biliary tract.[17]. The incidence is more in women when compared to men.[18]. Cholelithiasis is one of the most important risk factor for gallbladder cancer. The relationship of gallstones to carcinoma is attributed to the chronic stimulation leading to metaplasia and carcinoma.

[19].

In the present study, carcinomatous changes were seen in three cases. During the period of this study only four cases of carcinoma gallbladder were reported in our centre.

Out of the four cases, three were associated with gallstones indicating its significance. All the three cases were elderly females which is comparable to other studies.[10,20]. The increased risk of gallbladder carcinoma in females might be due to the higher incidence of gallstones in females and to female sex hormones.[20] All the three cases in the present study had mixed stones as in the retrospective study of 313 cases by Sunder Goyal et.al. [20]

Conclusion

Gallstones produce diverse histopatholological changes in the gallbladder including hyperplasia, metaplasia and carcinoma. In the present study most of the changes were associated with cholesterol stones. Cholesterol stones which are usually larger in size may lead to more irritation.

Moreover the toxic effect of lithogenic bile produce chemical injury to the mucosa. But carcinomatous changes were observed in mixed stones, The pathogenesis of this needs further studies. Moreover various preventable risk factors are attributed to gallstones. Proper counselling regarding dietary modification, weight reduction and obesity management goes a long way in preventing gallstone formation and its complications.

Acknowledgements None

Funding None

Competing Interests None declared

Reference

1. Weedon D. Diseases of the gallbladder. In: McSween RMN, Antony PP, Bua AD, et al, eds. Pathology of the Liver, 3rd edition. New York: Churchill Livingstone; 1994:514-534. 2.

2. Meyer G, GuizzardiF,Rodighiero S et.al.Ion transport across the gallbladder epithelium.Curr Drug Targets Immune Endocr Metabolic Disorders 2005;5:143-151. e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-202

3. Moser AJ, Abedin MZ, Roslyn JJ. The pathogenesis of gallstone formation. AdvSurg 1993; 26:357-386. 4.

4. Donovan JM, Carey MC. Physical-chemical basis of gallstone formation. GastroenterolClin North Am 1991;20:47-66.

5. Prakash A.Chronic cholecystitis and cholelithiasis in India.IntSurg 1968;49:79-85.

6. KhurroMS,MahajanR,ZargarSA,JavidG.Prevalence of biliary tract disease in India: a sonographic study in adult population in Kashmir.Gut.1989;30:201—05.

7. Jayanthi V, Palanivelu C, Prasanthi R,Methew S, Srinivasan V. Composition of gall stones in Coimbatore district of TamilnaduState. Ind J Gastroenterol 1998;17:134-35.

8. Malhotra SL.Epidemiological study of cholelithiasia among railroad workers in India with special reference to causation.Gut 1968;9:290-95.

9. Khanna Rahul,ChansuriaRashmi,Kumar Mohan et.al.

Histological changes in gallbladder due to stone disease;Indian Journal of Surgery 2006;68:201-204.

10. SK Mathur,AmritaDuhan,Sunita Singh et al.Correlation of gallstone charecterstics with mucosal changes in gallbladder.Tropical Gastroenterology 2012;33(1):39-44.

11. Mohan H,PuniaRPS,Dhavan SB et.al.Morphological spectrum of gallstone disease in 1100 cholecystectomies North India.Indian J Surg 2005; 67: 140 -2.

12. Putz P and Willems G.Cell proliferation in the human gallbladder epithelium: effect of distension.Gut 2002;20:246-248.

13. Albores –Saavedra J,MolbergK,HensonDE. Unusual malignant epithelial tumors of gallbladder.

SeminDiagnPathol 1996;13:326-38.

14. MunaZahair,RanaMumtaz,KhalidaShaya.Histological changes of gallbladder mucosa:Correlation with various types of cholelithiasis.Iraqi J Comm.Med,July .2011;24(3):234- 240.

15. Albores –Saavedra J,Henson DE .Pyloric gland metaplasia with perineural invasion of the gallbladder.A lesion that can be confused adenocarcinoma .Cancer 1999;1586(12):2625-2631.

16. FernandesJEF,FranceMIF,SuzukiRKet.al.Intestinal metaplasia in gallbladders:Prevalencestudy.Sao Paulo Medical Journal 2008;126(4):131-151.

17. Crawford JM:The liver and the biliary system.

In Robbins Pathologic basis of disease eds.

CotranRS,Kumar SL.5thedition.WB Sanders Philadelphia.1994;831-896.

18. Diehl AK:Epidemiology of gallbladder cancer:A synthesis of recent data.J Natl Cancer Inst.

1980;65:1209-14.

19. Usha,Gupta S: Mucosal metaplasia in cholecystitis and carcinoma of the gallbladder.Ind J Pathol Microbiol.1990;33:92-95.

20. Sunder Goyal,Sanjeevsingle,AmritaDuhan.

Correlation between gallstones charecterestics and gallbladder mucosal changes : A retrospective study of 313 cases.Clin Cancer Investig J 2014 ;3:157 -161.

http://www.pacificejournals.com/aabs

Original Article Comparison of Color Match to Noritake and Vitapan Classical Shade Guides of Three Porcelain Systems Alireza Pornasrollah1, Seyyed Amin Mosavi1, Hesam Khandero2, Ali Zarandi1* Department of Prosthodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz , Iran 2 Department of Dentistry, Tabriz University of Medical Sciences, Tabriz , Iran

1

Keywords: Esthetics, Prosthodontics, implant design.

ABSTRACT Objective: This study intended to evaluate the color match of porcelain samples of Ceramco, Noritake, and Vita by two shade guides of their systems from the viewpoints of the teachers in Department of Prosthodontics.

Methods: In this study, five professors of Prosthodontics determined the color of four samples made out of three different types of porcelain, using Vitapan Classical and Noritake shade guides. Data were analyzed using descriptive statistics, t-test, and ANOVA (P< 0.05).

Results: Samples made out of Noritake porcelain had a better color match with both determination systems; however based on ANOVA test results, these findings were not statistically significant. The average of color match in Vitapan Classical system and Noritake system was 40% and 43%, respectively. Although clinically, Noritake system compared to Vitapan classical system had better color match, t-test showed this difference was not statistically significant.

Conclusion: Porcelain type and shade selection system had no effect on tooth color selection.

*Corresponding author: Ali Zarandi, Assistant Professor, Department of Periodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran E-mail: dr.alizarandi@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-204

Introduction

Shade matching is one of the key aspects in achieving beautiful dental restoration. Understanding the scientific principle of color is more critical to make the restoration natural and attractive (1, 2). Selecting the color of restoration to be matched with shade of the patient’s teeth is also of importance (3, 4) , otherwise, it can cause complications such as dental cost for replacement of treatment, patient’s non-satisfaction, as well as reputational consequences for the dentist (3, 5).

It was reported that color vision is a major component of restorative and esthetic dentistry, however in the researches, color vision of the dentists, as a group, has not been tested at any time during their jobs (6). Carsten et Al. in their study concluded that dentists should learn how to control the internal and external factors that affect the human vision through the selection of correct color(7). The results of this research showed that the object reflects only the light which falls on them and the eyes can sense hue, intensity, and saturation of light. Therefore, accurate and high-intensity lighting is required for correct shade matching (4, 7). In another study, Vitapan Classical shade guide was used to assess the effect of different arrangements of a shade guide on the repeatability and accuracy of visual shade choice by restorative dentists. They found that these two characters were similar when different arrangements (hue/chromaordered and value-ordered) of this shade guide were used (8). In addition, restorative dentists ought to identify the related characteristics of porcelain with accurate matching color in selected restoration. The present study aimed to evaluate the color accordance of porcelain samples of Ceramco, Noritake, and Vita by shade guides of their systems from the viewpoints of the teachers in Department of Prosthodontics.

Materials and Methods

The present study was designed as a descriptive cross sectional study. Three porcelain systems were used and four samples were investigated in each system: 1. Ceramco ( ceramco-Dentsply, Burlington, NJ,USA) 2. Noritake ( Kuraray Noritake Dental Inc, Tokyo, Japan) 3. VITA VM13 ( Vita, Zahnfabrik, Germany) Four colors from the shade guide (A2, B2, C2, D2) were selected for testing. Four samples of each porcelain system were fired in chromium metal ( president dental, Munchen, Germany) according to the manufacturer’s instructions for the size of premolar teeth (12×10). After identification coding of fired porcelain samples ( n=12), color selection and lack of color blindness (based on Ishihara test(9))

were performed by five male observers who were assistant professors of Department of Prosthodontics.

Selection procedure was done in gray-colored 3×4 rooms and between the same hours of the day (10 to 12 a.m.).

They were asked to be placed behind the window, 50 cm far from the samples and to be color matched using Vita system. During the color selection, observers looked blue sheet for 5 sec to avoid errors during the visual selection of color (10).

The color selection test was repeated for Noritake system in the same conditions. The results was recorded in separate questionnaires. Percentage of each selected color, in each porcelain system, was calculated by the following formula: Percentage of color matching = (numebr of correct colors / 5 ) × 100 Statistical analysis The results were analysed by descriptive method. T-test and ANOVA test were also conducted (P < 0.05).

Result

Percentage of color matching in three porcelain systems by two shade guides is shown in Table 1. In a clinical viewpoint, samples made by Noritake porcelain were more matched with practical color determination method.

ANOVA test showed that no significant differences were obtained for the accuracy of shade selection results achieved with two different shade guides,Vitapan Classical and Noritake ( Table 2). Acoording to the results, accurate color match was related to Noritake porcelain by Noritake shade guide which was determined 100% , 40% , 80%, and 60% in A2,B2,C2, and D2,respectively. The maximum mismatch was recorded for ceramco porcelain which was zero in B2 and D2 in both Noritake and Vitapan Classical shade guides. Moreover, Vita porcelain could not be determined in D2 samples by Vitapan Classical shade guide.

Discussion

Tooth shade selection has acted as the main factor in the restorative dentistry in which various factors such as light source, observed object, and observer affect color perception(8). Different shade guides have been introduced from which the first shade guide, Vitapan Classical, consists of four arrangements (A to D). Another popular shade guide which is not so systematically arranged is Ivoclar Vivadent Chromascop which has been categorized based on the hue (1 to 5) and increasing chroma ( 10 to 40).

Having developed dental color sciences, Vita 3D Master was introduced consisting of 11 types of ceramic porcelains and samples ranged from the lightest (value= 1) to the

http://www.pacificejournals.com/aabs

A-205

AABS; 3(2): 2016

Table 1: Percentage of color matching in three porcelain systems by two shade guides Sample A2

B2

C2

D2

Porcelain Color detection by observers VC* N#

Ceramco A2 B1

C2

D2

D3

Noritake A2

40% 20% 20% 20% 20% 20% 40% 20%

Color detection by A2 B2 observers VC 40% 0 N 40% 0

C2

D2

D3

80% 20% 100% B2

A2

A3

20% 20% 20% 20% 40% 20% 40%

Color detection by C2 C4 C3 observers VC 80% 20% N 80% 20%

C2

B1

80% 20% 80%

Color detection by D2 A1 A3 C1 C3 D2 A1 observers VC 40% 20% 20% 20% 20% 20% N 0 20% 40% 20% 20% 60% -

*:Vitapan classical shade guide, # : Noritake guide

Vita A2

A3

B1

C1

D3

40% 20% 40% 40% 20% 20% 20% C2

D2

B2

A1

A2

C2

D2

20% 20% 20% 20% 20% 20% 20% 20% 60% 20% 20% 20% 20% 20% D2

C2

60%

60% 20% 20% 80% 20%

B1

B2

40% 20% 40% -

A2

C4

C2

D3

D2

C1

C3

A2

0 0

20% 60% 20% 20% 40% 20% 20%

Table2: Mean (SD) of color match in porcelain samples Color selection/ Porcelain

Noritake shade guide

Vitapan Classical

P value (t-test)

Vita Noritake Ceramco Total

35(34.15) 70(25.81) 25(37.85) 43(36.01)

30(25.81) 50(34.64) 40(32.65) 40(29.54)

0.82* 0.39* 0.57* 0.80*

* Statistically non-significant

darkest (value =5). This shade guide used three dimensions of color-including value, chroma, and hue to systematically determine the shade with the consistent criteria(11). In order to decrease the color mismatch because of visual determination, two categories were developed based on the electronic instruments: colorimeters (using a filter with simulation of the human eye) and spectrophotometers (measurement of reflection/ transmission of an object in 350-800 nm as visible frequency range)(12, 13). Although the shade guides do not fully express the natural color of teeth, they are still the main evaluation mechanisms and color transferors in dentistry.

In this study, four colors of each porcelain system were evaluated. Porcelain samples made out of Noritake, Ceramco, and Vita had the better match with Vitapan classical guide, respectively. Moreover, it was found that Noritake, Vita, and Ceramco had a high match with Noritake guide. Although clinically, Noritake porcelain showed the highest match with two shade guides, this observation was not statistically significant. Moreover, significant difference was not found in color determination results by observers in comparison of two shade guides.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

Bayindir et al. compared the coverage errors of three different shades and found that Vitapan 3D Master shade guide had the lowest error compared to other guides (14).

In another study, Vita 3D-Master shade guide with the conventional guide was investigated. The results showed that restorations which were determined with the 3D-Master could be placed without any further shade corrections.

However, almost 17% of restorations identified with the classical system required following shade modifications.

Finally, they reported that the match of the selection shades with the 3D-Master was observed significantly better by the clinicians (15). Paul et al. tested the shade match of single porcelain restorations with the conventional visual shade and a new spectrophotometric system and found that Vita Classical did not cover all natural teeth colors (16). Öngül et al. found that the ceramic crowns which were made with the Vita Tooth guide 3D-Master shade guide showed a closer color match with the natural teeth in comparison to Vitapan Classical guide. Additionally, the ΔE values and the observer’s scores were determined within the range of clinical satisfactory for both investigated shade guides (17). e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-206

Chen et al. compared the coverage errors of five shade guides (VITA Classical, VITA 3D-Master, Chromascop, Shofu Vintage Halo NC, and E Noritake) in anterior vital normal teeth. They resulted that Shofu Vintage Halo NC could better show color match of natural teeth in different regions (cervical, body, and incisal)(18). Matching of the shade among the 15 beverage and porcelain modifications by four shade tabs was also studied (Vita Akzent, Vita Interno, Shofu Vintage and Unibond and Noritake Super Porcelain EX-3 ) and found that excluding the hue, the color arrangement of four kinds of shade tabs was the same as that investigated for 15 kinds of beverage. On the other hand, the beverage color could be closely matched via any type of porcelain modification by means of mixing the porcelain powder of proper hue, value, and chroma (19).

Conclusion

To conclude, porcelain system and shade guide systems could not affect the successful color selection. Two applied shade guides (Vitapan Classical and Noritake) could determine a matched color lower than 50 %.

Acknowledgements

[It should include persons who provided technical help, writing assistance and departmental head that only provided general support. Financial and material support and conflict of interests must be written in this section.]

Funding

8. Yilmaz, B.; Yuzugullu, B.; Cinar, D.; Berksun, S. Effects of shade tab arrangement on the repeatability and accuracy of shade selection. The Journal of prosthetic dentistry 2011, 105, 383-386.

9. Ishihara, S. Ishihara tests for colour blindness.

Shepherd and Newman: Sydney, 1943.

10. Shillingburg, H.T. prosthodontics. 2006.

Fundamentals

of

fixed

11. Paravina, R.D. Performance assessment of dental shade guides. Journal of dentistry 2009, 37 Suppl 1, e15-20.

12. Hunter, R.S. The measurement of appearance. John wiley: 1975.

13. Brewer, J.D.; Wee, A.; Seghi, R. Advances in color matching. Dental Clinics of North America 2004, 48, 341-358.

14. Bayindir, F.; Kuo, S.; Johnston, W.M.; Wee, A.G. Coverage error of three conceptually different shade guide systems to vital unrestored dentition. The Journal of prosthetic dentistry 2007, 98, 175-185.

15. Hassel, A.J.; Koke, U.; Schmitter, M.; Beck, J.; Rammelsberg, P. Clinical effect of different shade guide systems on the tooth shades of ceramicveneered restorations. The International journal of prosthodontics 2005, 18, 422-426.

None

Competing interests None declared

Reference

7. Carsten, D.L. Successful shade matching--what does it take? Compendium of continuing education in dentistry (Jamesburg, N.J. : 1995) 2003, 24, 175-178, 180, 182 passim; quiz 188.

1. Hammad, I.A. Intrarater repeatability of shade selections with two shade guides. The Journal of prosthetic dentistry 2003, 89, 50-53.

2. Sikri, V.K. Color: Implications in dentistry. Journal of conservative dentistry: JCD 2010, 13, 249.

3. Fondriest, J. Shade matching in restorative dentistry: The science and strategies. The International journal of periodontics & restorative dentistry 2003, 23, 467-479.

4. Alomari, M.; Chadwick, R.G. Factors influencing the shade matching performance of dentists and dental technicians when using two different shade guides.

British dental journal 2011, 211, E23.

5. Browning, W.D.; Contreras-Bulnes, R.; Brackett, M.G.; Brackett, W.W. Color differences: Polymerized composite and corresponding vitapan classical shade tab. Journal of dentistry 2009, 37 Suppl 1, e34-39.

6. Wasson, W.; Schuman, N. Color vision and dentistry.

Quintessence international (Berlin, Germany : 1985) 1992, 23, 349-353.

16. Paul, S.J.; Peter, A.; Rodoni, L.; Pietrobon, N. Conventional visual vs spectrophotometric shade taking for porcelain-fused-to-metal crowns: A clinical comparison. The International journal of periodontics & restorative dentistry 2004, 24, 222-231.

17. Ongul, D.; Sermet, B.; Balkaya, M.C. Visual and instrumental evaluation of color match ability of 2 shade guides on a ceramic system. The Journal of prosthetic dentistry 2012, 108, 9-14.

18. Chen, L.; Tan, J.G.; Liu, M.Y.; Zhou, J.F.; Liu, X.Q. [coverage errors of five shade guide systems to vital natural teeth]. Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 2013, 48, 449-452.

19. Yao, J.W.; Li, S.G.; Lin, J.Y. [study on the matching of the shade between beverage and modifying porcelain shade guide]. Hua xi kou qiang yi xue za zhi = Huaxi kouqiang yixue zazhi = West China journal of stomatology 2007, 25, 481-484.

http://www.pacificejournals.com/aabs

Original Article Evaluation of Her-2 neu Over Expression in Morphological Variants of Cervical Carcinoma: A Study of 25 Cases.

Nimisha Sharma1*, Ankit Kaushik1, Sumanashree2, Uma Sharma2 1

Dept. of Pathology, VMMC and Safdarjung Hospital, Delhi, India 2 Dept. of Pathology, SGT Medical College, Gurgaon, India

Keywords: Her- 2 neu, cervical carcinomas, immunohistoichemistry

ABSTRACT Introduction: Carcinoma of uterine cervix is the second most prevalent cancer among women worldwide and remains a major gynecological malignancy in developing countries. HER-2/neu, a pro-oncogene is a cell membrane surface bound tyrosine kinase receptor and is involved in signal transduction pathway leading to cell growth and differentiation.

Aim: The present study is conducted to evaluate the over-expression of HER-2/neu in carcinoma cervix, its pattern of membranous staining and correlation with histological type, grade of tumor and stage of disease wherever available.

Methods: A part retrospective and part prospective study was done on a total of 25 cervical biopsies and hysterectomies specimen. Tissue blocks with extensive necrosis or hemorrhage were excluded from the study and immunohistochemistry for HER-2/neu was done. The percentage of cells showing the membranous positivity for HER2/neu, extent of its positivity in the cervical epithelium in different lesions of the cervix and membranous intensity of HER2/neu positive cells was noted. The HER2/neu expression was then correlated with morphological diagnosis, grade of lesion, invasion and lymph node metastasis in hysterectomies.

Results: 18 cases were reported to be SCC (72%) ,while Adenocarcinoma (12%) and Adenosquamous (16%) carcinoma constituted small subgroups of 3 and 4 cases, respectively, 13(52%) cases of carcinoma cervix were found to be positive for HER 2/neu while 12/25 cases(48%) were negative. 11 cases of SCC were positive for HER2/neu, 2/4 adenosquamous carcinoma cases were positive. No adenocarcinoma cases were found to be positive for HER2/neu.

12 carcinoma cervix cases show <10% cells staining for HER2 /neu (48%). 9 cases involved 10-20% of cells (36%), while only four cases had positive cell count > 20% (6%). Of the 19 of carcinoma cervix with known staging, (9) were of stage 1, 1/9 cases of stage I showed HER2/neu positivity (2+), 4/6 of stage II were moderately positive (2+) for the marker 2/ 2 of stage IV cases showed 2+ and 3+ positivity.

The present study showed SCC to be the most common carcinoma with 52% of carcinoma cervix cases expressed positivity for HER-2/neu. Maximum number of carcinoma cases showed 2+ HER-2/neu intensity. HER-2/neu positivity was found to be highest in SCC, followed by adenosquamous and adenocarcinoma. Her 2/neu was found to be over expressed in higher stage with high percentage positivity.

Conclusion: The high incidence of HER-2/neu amplification in cervical cancer suggests the role of this gene in tumorigenesis and this study emphasize HER-2/neu overexpression in cervical carcinoma.

*Corresponding author: Dr. Nimisha, 3067, B4, Ridge View Apartment, Vasant Kunj, New Delhi, 110070 Phone: +91-9650682846 E-mail: nims427@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Original Article

A-208

Introduction

Carcinoma of uterine cervix is the second most prevalent cancer among women worldwide and remains a major gynecological malignancy in developing countries.1 It is common in lower socio-economic group, in women with early initial sexual activity and/or multiple sexual partners and in smokers. 2Invasive carcinoma cervix continues to be one of the most common cancer.3 Most common histological subtype of carcinoma cervix is Squamous cell carcinoma 80% followed by Adenocarcinoma 15%.

Adenosquamous and neuroendocrine tumors account for remaining 5%.4 Development of cancer is a multi factorial process which includes sequential activation of oncogenes and other genetic derangements.5 Despite most optimal combination of cytotoxic drugs, surgery and radiation, testing of molecular targeted therapies against cervical cancer is a logical step to follow.6 HER-2/neu also known as c-erbB-2, CD 340 and p 185, stands for â&#x20AC;&#x153;Human Epidermal Growth Factor Receptor 2â&#x20AC;?. It is a cell membrane surface bound receptor tyrosine kinase and is involved in signal transduction pathway leading to cell growth and differentiation7,8. It is encoded by proto-oncogenes HER-2/neu located on long arm of human chromosome 17(17q21q22). Its over expression has been detected in CIN and cervical cancer and is believed to be associated with poor prognosis, aggressive biological behavior and enhanced metastatic potential 9,10. There is now a general agreement that the membranous IHC staining pattern of HER-2/neu correlates with receptor protein expression and prognosis.10 There are variable reports on expression of HER-2/neu in cervical carcinoma either due to heterogeneity of the lesion or due to antigen retrieval.11,12 More studies are required in India to assess correlation of over expression of HER-2/neu with various histological subtypes and stage of carcinoma cervix, as it is the leading cause of death.

The present study is conducted to evaluate the overexpression of HER-2/neu in carcinoma cervix, its pattern of membranous staining and correlation with histological type, grade of tumor and stage of disease wherever available.

Materials and Methods

The study was carried out in study between the time period of 18 months starting from September 2010 to March 2012, in Histopathology unit, Department of Pathology, VMMC and Safdarjung hospital, New Delhi. A part retrospective and part prospective study was done on a total of 25 cervical biopsies and hysterectomies specimen.

Biopsy specimen was studied for different type of cervical carcinoma. Patients with a coexistent pathology of any other organ like uterus and ovary etc. in addition to cervix were excluded. Tissue blocks with extensive necrosis or

hemorrhage were also excluded from the study. 4-5 micron thick sections were cut for H & E staining, and for HER-2/ neu immunohistochemistry, sections were taken on polyL-lysine coated slides.

For HER2/neu immunohistochemistry, representative sections from paraffin blocks were taken on poly- L-lysine coated glass slides.

1. The sections were first deparaffinized the sections with xylene, with two changes of 5 minutes each .

2. The sections were later immersed in descending levels of alcohol 90%, 80%, 70% 3. Slides were washed in tap water and then rinsed in distilled water.

4. Antigen were retrieved by microwave heat method for 15-20 minutes using citrate acid buffer at pH 6.0 and a temperature of 95 degree Celsius.

5. Sections were then rinsed in phosphate buffer saline and excess PBS was drained off.

6. Endogenous peroxidise activity was blocked by using peroxidase block 9 hydrogen peroxide(3%) and methanol for 20 minutes.

7. Sections were then with PBS for 5 minutes and cubated with protein block for 5 minutes.

8. Slides were washed in Phosphate Buffer Saline (PBS).

9. Optimally diluted primary antibody was applied for 60 minutes 10. Washed in PBS 11. Incubated with post primary block for 30 minutes 12. Washed in PBS 13. Incubated with polymer for 30 minutes.

14. Washed in PBS 15. Incubated in diaminobenzenzidine solution for 10 minutes.

16. Slides were rinsed in PBS and transferred to running water 17. Counterstaining was done with hematoxylin 18. Sections were dehydrated in graded alcohols and xylene.

19. Clearing and mounting was done in disterene 80 dibutyl phthalate xylene (DPX) The strength of staining was calculated as defined in table below: Positive control in the form of known positives of cervical cancer for Her 2 neu was run and negative controls staining was obtained by substituting the primary antibody

http://www.pacificejournals.com/aabs

A-209

AABS; 3(2): 2016

by antibody of irrelevant specificity. The percentage of cells showing the membranous positivity for HER2/ neu, extent of its positivity in the cervical epithelium in different lesions of the cervix and membranous intensity of HER2/neu positive cells was noted (table 1). The HER2/ neu expression was then correlated with morphological diagnosis, grade of lesion, invasion and lymph node metastasis in hysterectomies.

P value <0.05 will be taken as statistically significant.

Result

On H & E staining done on 25 paraffin sections, 18 cases were reported to be SCC (72%) ,while Adenocarcinoma (12%) and Adenosquamous (16%) carcinoma constituted small subgroups of 3 and 4 cases, respectively. As observed in our study, maximum number of carcinoma cases fall in 51-60 age group (9) followed by 31-40 age bracket (6).

Fourth and sixth decade include 5 and 3 cases each. Only 2 cases were studied in seventh decade. 52% of all the carcinoma cases fall in perimenopausal age group.

12 carcinoma cervix cases show <10% cells staining for HER2 /neu (48%). 9 cases involved 10-20% of cells (36%).

While only four cases had positive cell count > 20% (6%) 6 cases of carcinoma cervix had lymph node involvement.

3 cases showed HER2/neu positivity (50%), while 3 cases did not express the marker (50%) .

Of the 25 cases included, stage could be studied in 19 cases of carcinoma cervix. Maximum number of cases (9) were of stage 1, which is a microscopic diagnosis. Out of 9 cases of stage I , 1 showed HER2/neu positivity (2+), 2 out of 2 of stage IV cases showed 2+ and 3+ positivity. 66% of stage II were moderately positive (2+) for the marker (4/6). 1 out of 2 stage III patient had HER2 positivity (2+). Therefore there appears to be an obvious correlation between increasing stage of the disease in patients and HER2/neu expression.

On applying chi square test, the P value was found to be 0.043, and the association is significant.

Most common symptom was vaginal bleeding, followed by discharge per vaginum and other less common complaints were post coital bleeding, leg pain, pelvic pain and abdominal tenderness.

13(52%) cases of carcinoma cervix were found to be positive for HER 2/neu(Figure 1) with no specific age related distribution while 12/25 cases(48%) were negative.

Of the 25 cases no staining was seen in 2 cases (8%). 1+, 2+ and 3+ intensity is seen in 10 (40%), 12 (48%) and 1 (4%) cases. This observation was not significant statistically, as the P value was >0.050.

11 cases of SCC were positive for HER2/neu out of 18 cases (61%) . Out of 4 Adenosquamous carcinoma cases 2 were positive (50%) (Figure 2). None of the adenocarcinoma cases were found to be positive for HER2/neu (Figure 3).

Fig. 1: HER2/neu Immunostaining in poorly differentiated squamous cell carcinoma, cervix (3+) (IHC;Ă&#x2014;400)

Table 1 : Her-2/Neu scoring system13, Staining Pattern No staining or weak membrane staining in < 10% of tumor cells Weak membrane staining in >10% of tumor cells Moderate membrane staining in > 10% of tumor cells Strong membrane staining in > 10% of tumor cells

Score 0 1 2 3

HER-2/neu Negative Negative Positive Positive

Table 2: Correlation of HER2 positivity in various stages of carcinoma cervix.

Stage I II III IV Total

Total Cases 9 6 2 2 19

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

HER2 /neu + 1 (11.11%) 4 (66.7%) 1 (50%) 2 (100%) 8 (42%)

HER2/neu 8 (88%) 2 (33.3%) 1 (50%) 0(0%) 11(58%)

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-210

Fig. 2: HER2/neu immunostaining in adenosquamous carcinoma, cervix (1+) (IHC;Ă&#x2014;400)

Fig. 3: HER2/neu immunostaining in adenocarcinoma, cervix (2+) (IHC;Ă&#x2014;400)

Discussion

Study by Gupta N. et al58 mentions that in various studies HER2 positive staining in invasive cervical carcinoma ranged from 14 -100%.12,13 . Gupta et al included 65 cervical carcinoma and 10 CIN cases in their study and found out 2+ and 3+ membrane staining of HER2/ neu in 33 (44%) of carcinoma cases and 1 (10%) of CIN cases (P <0.05) 13 while Hale R J et al found definite membrane staining in 24 (38.7%) out of 62 cases of stage IB/IIA cervical carcinoma.21 Califano et al in study on 65 cervical carcinoma patients, found 2+ positivity in 10 patients (15%). 15 patients (23%) showed 1+ staining22.

Ndubisi et al evaluated archival tissue from 150 cervical carcinoma patients, HER2/neu expression was seen in 34 (22%) tumors with no statistically significant difference in survival of patients with HER2 positivity (P=0.05)23 Blanco et al stated that only 1 out of 35 primary cervical cancer lines expressed 2+, 3+ HER2 receptor. Whereas 2 out of 4 (50%) recurrent tumor were positive.24 Kersemaeker et al found focal positivity of HER2/neu in 12 (8.8%) patients out of 136 with stage I and II of cervical cancer.25

We conducted a study between the time period of 18 months starting from September 2010 to March 2012, in Histopathology unit, Department of Pathology, VMMC and Safdarjung hospital. In present study squamous cell carcinoma formed the largest group (72%) with 18 cases followed by adenosquamous (16%) and adenocarcinoma (12%) corresponding to 4 and 3 cases each, corresponding to available text which states that most common histological subtype of carcinoma cervix is SCC (80%) followed by adenocarcinoma (15%) and adenosquamous carcinoma (5%).14 The peak age was found in 36% cases in fifth decade indicating maximum incidence in perimenopausal age group(51-60). No particular trend could be observed in other groups, because of limited number of cases. Ray et al15 and Graham et al16 also found similar age group to be affected by cervical carcinoma.

HER2 (also known as c-erbB-2) is a transmembrane receptor protein with tyrosine kinase activity, is over expressed in a number of solid tumors17,18 HER-2/neu has been shown to be over expressed in breast and female genital tract malignancies.19 It plays an important role in coordinating the complex Erb B signaling network that is responsible for regulating cell growth and differentiation.20 The amplification of this gene is associated with resistance to treatment and poor survival, suggesting that cells over expressing HER-2/neu may manifest a more aggressive biological behavior and may have a selective growth advantage over HER-2/neu-negative tumor cells. 19 Immunostaining was done in all 25 cases in the present study, 52% of carcinoma cases expressed positive membrane staining for HER2/neu( 13/25). P value by Chi square test was 0.035 which shows significant association.

A definite association could not be proved between overall histologic subtypes and HER2/neu expression in carcinoma cervix in this study. Considering our study, findings in case of SCC and adenosquamous carcinoma are comparable, but in case of adenocarcinoma no positivity was seen in our study. It may be due to small number of cases of this type.

In the present study SCC formed the largest group with 18 cases. Of this 11(61%) were positive for HER2/neu.

Among the SCC cervix, maximum number of cases belonged to moderately differentiated (MD) group(8) (44.4%), followed closely by poorly differentiated (PD) (7) (38.9%), well differentiated(WD) carcinoma were only 3

http://www.pacificejournals.com/aabs

A-211 cases (16.7%). On performing immunohistochemistry the positivity was 5/8 (62.5%) for MD SCC, 5/7 (71.4%) for PD SCC and 1/3 (33.3%) for WD SCC. On applying Chi square , P value was 0.52. Hence no significant association could be proved between HER2/neu positivity and grades of squamous cell carcinoma. In a study by Wong Y.F. et al 11(16%) of 70 cases of invasive cervical squamous cell carcinoma showed an amplification of HER2/neu by differential PCR but there was no association between amplification and tumor histologic grading.(P=0.408).26 Ray et al found HER2/neu positivity in 13 out of 50 cases of squamous cell carcinoma cervix (26%). No statistical association was seen between HER2/neu positivity and histological grade (P=0.91).15 Brumm et al reported a high rate (75%) of expression of HER2/neu in SCC cervix.27 Gupta N et al states that out of 48 cases of squamous cell carcinoma 7 cases (14.5%) showed 2+ staining and 14 cases (29.2%) showed 3+ staining. Therefore, 21/48 (43.7%) cases were positive for HER2/neu . Gupta N et al13 correlated HER2/neu expression in various grades of 48 cases of SCC uterine cervix. Positivity was found to be 0/5 (0%) in well differentiated, 18/33 (55%) in moderately differentiated and 8/10 (80%) in poorly differentiated type of SCC. Hale et al found significant correlation between positive staining and poor prognosis in Squamous cell carcinoma. (P=0.043)21 3 cases of adenocarcinoma were included in our studies. Of them , none was found to be strongly positive. All the three cases showed 1+ membrane positivity i.e, mild positivity.

Kihana et al studied 44 cases of cervical adenocarcinoma and found 1+ and 2+ positivity in 34 (77%) cases. Gupta N et al found positivity in 8 cases of the total 13 cases (60%) of adenocarcinoma13. 4 cases showed 2+ positivity and the other 4 cases were 3+, 1+ positivity was seen in 3/13 cases (23%), whereas 2 (15.3%) cases were negative for HER2/neu expression. This association was not found to be significant.

In the study conducted by Hale et al 5 out of 6 adenocarcinoma (83%) showed definite membranous positivity.21 Our study does not corresponds to this finding due to small sample size. Out of 4 adenosquamous carcinomas included in the study 2 showed 2+ positivity (50%). As 2/4 (50%) cases were negative (1+), there was no significant association between the adenosquamous subtype and HER2/neu expression. Gupta N et al13 observed 75% (3/4) of 2+ membrane positivity in 4 cases of adenosquamous carcinoma studied.1 case showed 1+ positivity (25%).

AABS; 3(2): 2016 Considering our study, findings in case of SCC and adenosquamous carcinoma are comparable, but in case of adenocarcinoma no positivity was seen in our study. Of the total cases of carcinoma cervix , stage could be studied in 19 cases of carcinoma cervix. Out of 9 cases of Stage I, Her2/neu was found to be positive in 1 case(11.11%) only.

2 cases (33.33%) were positive for HER2/neu in 6 stage II carcinoma cervix. Of the cases in stage III, 1 out of 2 (50%) was positive. Both (2/2) the cases in stage IV were positive for HER2/neu (100%). As stage advances percentage of cases showing HER2/ neu positivity increases. P value was < 0.05, hence a significant association is seen between stage of disease and HER2/neu expression.

A significant correlation was observed by Gupta et al13, between positive HER2/neu expression and higher clinical stage of presentation. Positivity was 48% (14/29) in stage I. In stage II 13/18 (72%) cases showed HER2/neu positivity.

In stage III this positivity was increased upto 72% (13/18).

2 cases were there in stage IV , both of which were positive for the IHC marker.

Kihana et al observed expression of HER-2/neu protein on cell membrane, which was more often seen at clinical stage II or III(9/23) (39%) than at stage I (2/21) (9%). P value was calculated by Fisher’s exact test and was found to be less than 0.05.28 Wong et al in his retrospective study on 70 women with invasive cervical squamous cell carcinoma showed amplification of HER-2/neu in 16%(11/70) of cases but there was no relation between gene amplification and clinical staging (P= 0.180).26 Rosty et al stated in their study that there was no correlation between presence of immunolabeling and age, histologic type or clinical stage.20 The conflicting result may be due to difference in institutional treatment standards and due to varied subjective interpretation of staining intensity and distribution between centers. Because staining intensity is judged on a continuum differences in institutional “cut off” for positivity which is likely to also effect correlation with histologic results.

Conclusion

In the study by Hale et al21 on 62 patients of cervical carcinoma 21 were that of adenosquamous carcinoma.

The present study showed SCC to be the most common carcinoma with 52% of carcinoma cervix cases expressed positivity for HER-2/neu. Maximum number of carcinoma cases (48%) showed 2+ HER-2/neu intensity. HER-2/ neu positivity for SCC, Adenosquamous carcinoma and Adenocarcinoma was 61% ,50% and 0% respectively.

The high incidence of HER-2/neu amplification in cervical cancer suggests the role of this gene in tumorigenesis and this study emphasize HER-2/neu overexpression in cervical carcinoma.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-212

Acknowledgements

12. Nakano T, Oka K, Morita S. Correlation of cervical carcinoma c-erbB2 oncogene with cell proliferation parameters in patients treated with radiation therapy for cervical carcinoma. Cancer 1997;79:513-20.

None

Funding None

13. Gupta N, Singh S, Marwah N, Kumar S, Chabra S, Sen R.HER-2/neu Expression in Lesion of Uterine Cervix; Is It Reliable and Consistent? Indian J Pathol Micro. 2009; 52(4):482-485.

Competing interests None declared

Reference

1. Shanta V, Krishnamurthy S, Gajalakshmi CK, Swaminathan R, Ravichandran K. Epidemiology of cancer of the cervix: global and national perspective. J Indian Med Assoc; 2000;98:49-52.

2. Rotkin, I. D. Relation of adolescent coitus to cervical cancer risk. Journal of the American Medical Association;1962;179,486.

3. Fauci S, Braunwald E , Kasper D, Hauser SL, Lengo D, Jameson JL et al. Gynecologic Malignancy in Harrison’s Principles of Internal Medicine.

Seventeenth Ed.USA: Mc Graw-Hill; 2008;604-609.

4. Kumar V, Abbas AK, Fausto N, Astor JC,. Female Genital Tract in Robbins and Cotran Pathologic Basis of Disease. Eighth Ed. Philadelphia: Elsevier; 2010;1020-1025.

5. Hung MC, Matin A, Zhang Y, Xing X, Sorgi F, Huang L, et al. HER-2/neu-targeting gene therapy-a review.

Gene 1995;159:65-71.

6. Duenas-Gonzalez A, Cetina l, Mariscal I, Garza J. Cancer Treat Rev. 2003;29(5):389-99.

7. Ray A, Naik SL, Sharma BK. Distribution of prognostically unfavourable product of C-erb B-2 oncogene and EGFR in carcinomas of breast and the uterine cervix. Indian J Physiol Pharmacol 2002;46:423-33.

8. Popescu, N. C., King, C. R., and Kraus, M. H. Localization of the human erbB2 gene on normal and rearranged chromosomes 17 to bands q12.2l.32.

Genomics, 4:362- 366,1989.

9. Bargmann CL, Hung MC, Weinberg RA. The new oncogene encodes an epidermal growth factor receptor-related protein. Nature;1986;4:362-6.

10. Nevin J, Liang D, Kaye P, McCulloch T. The Significance of ErbB-2 Immunostaining in Cervical Cancer. Gyenecologic Oncology. 1999;73:354-358.

11. Mitra A B,Murty VVVS, Pratap M, Sodhani P, Chaganti R S K.ErbB-2 (HER2/neu) Oncogene is frequently amplified in Squamous Cell Carcinoma of the Uterine Cervix. Cancer Research. 1994; 54: 637-639.

14. Marwah N, Garg M, Singh S, Sethi D, Sen R. Unusual form of squamous cell carcinoma of the cervix extending in situ into the endometrium: Three case reports and review of literature. Int J App Basic Med Res 2012;2:139-41 15. Ray A, Naik S. L. D. and Sharma B. K. Distribution of prognostically unfavourable product of c-erbB-2 oncogene and EGF-R in carcinomas of the breast and uterine cervix. Indian J Physiol Pharmacol 2002; 46(4):423-33.

16. Graham J.B. Characteristics of women with various gynaecological cancers.Obstetrics and Gynecology, 1964;23,176.

17. Horton J: Trastuzumab use in breast cancer: clinical issues. Cancer Control 2002, 9:499-507.

18. Bethwaite P, Yeong M 1, Holloway 1 et al1992. The prognosis of adenosquamous carcinoma of the uterine cervix. Br J Obstet Gynaecol99: 745-750.

19. Goud KI, Dayakar S, Vijyalaxmi K, Babu SJ, Vijay ARP. Evaluatiion of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay. The Indian Journal of Medical Research.2012;135(3):312-7.

20. Rosty C, Couturier J, Genin P. et al. Overexpression/ amplification of HER-2/neu is uncommon in invasive carcinoma of uterine cervix. Int J Gynecol Pathol.

2004;23(1):13-17.

21. Hale R J,Buckley C H,Fox H,Williams J.Prognostic value of c-erbB-2 in expression of Uterine Cervical Carcinoma. J Clin Pathol. 1992; 45: 594-596.

22. Califano D, Losito S, Pisano C. et al. Significance of erbB-2 immunoreactivity in cervical cancer.Front Biosci, 2006 sep 1;11:2071-6.

23. Nadubisi B, Sanz S et al. The prognostic value of HER-2/neu oncogene in cervical cancer.Ann Clin Lab Sci. 1997;27:396-401.

24. Alma Chavez-Blanco, Victor Perez-Sanchez et al.

HER-2/neu expression in cervical cancer as a potential therapeutic target. BMC cancer 2004;4:59.

http://www.pacificejournals.com/aabs

A-213

AABS; 3(2): 2016

25. Kersemaeckers AF, Fleuren G J, Kenter G G, Broek LV, Uljee SM, Hermans J et al.Oncogene overexpression of Epidermal Growth Factor Receptor is associated with poor prognosis.Clinical Cancer Research. 1999; 5: 577-586.

26. Wong, A. J., Ruppert, J. M., Eggleston, J., Hamilton, S. R., Baylin, S. B., and Vogelstein, B. Gene amplification of C-myc and N-myc in small cell carcinoma of the lung. Science (Washington DC), 233: 461—464, 1986.”8201,991.

27. Brumm C, Riviere A, Wilckens C: Immunohistochemical investigation and northern blot analysis of c-erb B2 expression in normal,premalignant and malignant tissue of cervix uteri.Virchow archeive pathol Anat. 417:477-484,1990.

28. Kihana T, Tsuda H,Teshima S, Nomoto K, Tsugane S, Sonada T et al.Prognostic Significance of over expression of c-erbB-2 Protein in Adenocarcinoma of Uterine Cervix. Cancer. 1994; 73(1): 148-153.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article Effects of Vitamin-D Supplementation in Vitamin-D Deficient, Near Normal HbA1c Diabetic Patients Prashant Prakash, Manish Kumar Bansal, Ashish Gautam, Abhishek Raj, Shalini Upadhyay*, Rosmy Jose Department of Medicine, S.N. Medical College, Agra, India Keywords: Vitamin D, HbA1c, Diabetes Mellitus

ABSTRACT Background: The study was conducted with aim to evaluate the effects of vitamin D supplementation on diabetic patients with subnormal serum vitamin D levels and minimally elevated HbA1c on their current regimen. Objective was to find association between vitamin D deficiency and type 2 diabetes.

Methods: Type 2 diabetic patients who were regular and compliant on their current prescription were investigated for their recent HbA1c level. Patients with HbA1c between 7.5 and 8.0 were further investigated for serum vitamin D level.

Vitamin D deficient patients (serum vitamin D < 25ng/ml) were enrolled for the study and randomised one to one in two groups. Group 1 in whom oral vitamin D supplementation done at dose of 60.000IU weekly for 3 months. Group 2 in whom no vitamin D supplementation done. In both groups current medications and other advices were continued without any alteration for 3 months. HbA1c was estimated again at end of 3 month in both groups.

Results: 24 patients in each group completed the study period with regular compliance. Group 1; 15 female and 9 male, average age 54 years (SD 9.55), average HbA1c 7.7, average serum vitamin D was 16.8ng/ml (SD 4.51). Group 2; 10 female and 14 male, average age 50 years (SD 7.99), average HbA1c 7.8, average serum vitamin D was 17.3 ng/ml (SD 4.20). Group 1 average HbA1c after 3 months was 7.4 whereas in group 2 remain same 7.8. The difference was statistically significant (t-value is 3.51725. The p-value is 0.000497. The result is significant at p < 0.05) Conclusion: A judicious supplementation of vitamin D among vitamin D deficient type 2 diabetic patients with near normal HbA1c can reduce the blood sugar level. HbA1c reduce to target levels if it is minimally elevated.

*Corresponding author: Dr Shalini Upadhyay, Junior Resident, P.G. Department of Medicine, S.N. Medical College, Agra, India E-mail: ushalini19@yahoo.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-215

Introduction

Diabetes mellitus is a complex, polygenic, and multifactorial disease. Both genetic predisposition and environmental factors contribute in the development and pathogenesis of T2DM. Several reports in literature have already shown that Vitamin D deficiency is a risk factor for glucose intolerance, causes decreased insulin secretion, and finally results in T2DM, suggesting the role for vitamin D in the pathogenesis of diabetes mellitus There are several mechanisms whereby vitamin D may influence insulin secretion and sensitivity. The three important mechanism1 are Impaired pancreatic β-cell function (Presence of vitamin D receptors in beta cells and vitamin D response element in human insulin gene support a direct effect of vitamin D on insulin synthesis and secretion), insulin resistance (vitamin D stimulate the expression of the insulin receptor in peripheral tissues and thereby increase glucose transport2) and low-grade systemic inflammation. Vitamin D attenuates the expression of pro-inflammatory cytokines involved in insulin resistance such as interleukins, IL-1, IL-6, TNF-a, also down regulates NF-Kb (Nuclear factor) activity.

Vitamin D appears to affect exclusively the insulin response to glucose stimulation, it does not appear to influence basal insulinemia. Diabetic patients have a higher incidence of hypovitaminosis D. The prevalence of Vitamin D deficiency in Diabetics ranged from 54% to 78%3.After adjustment for age, sex, blood pressure, lifestyle, family history, seasonal change, parathyroid hormone, and highsensitivity C-reactive protein, the participants with 25(OH) D deficiency had an increased risk of T2DM independent of BMI4.

Material and Methods

AABS; 3(2): 2016

compliant patients, patient with hepatic diseases, severely sick patients requiring hospitalisation, patient having other comorbidity like tuberculosis or any other chronic infections, patients taking drugs that can alter vitamin D level were excluded from the study. Enrolled patients were divided into two groups, group 1 and group 2. 31 patients were selected in group 1 and 30 in group 2. Randomisation was done one to one. Oral vitamin D supplementation with dose of 60,000 international units per week as per prescribed instructions was done in patients of group 1 for 12 weeks. Besides this no changes is done in the current prescription and advices for diet and life style modification remains unchanged. Patient strictly advised to follow the instructions and avoid major changes in diet and physical activity. Patients further instructed to consult immediately, if appear any new symptom or complication or there is any major variation on SMBG. Similar instructions were given to patient in group 2 except the vitamin D supplementation. Patients were called every month for routine clinical examination and to check compliance. This is also done to monitor any untoward manifestation of vitamin D supplementation. After 12 weeks of regular follow up 24 patients form each group were selected for HbA1C testing. The selected patients were those who regular followed the instruction and were strict compliant to the treatment.

Observation and Results

24 patients in each group completed the study period with regular compliance. Characteristics of both groups summarized in table 1.

Group 1- 15 female and 9 male, average age 54 years (SD 9.55), average HbA1c 7.7, average serum vitamin D was 16.8ng/ml (SD 4.51).

Type 2 diabetic patients attending medicine OPD at S N Medical College, Agra were investigated for their recent HbA1c levels and serum vitamin D levels. The selection is done irrespective of their current treatment regimen and previous vitamin D supplementation.

The patients who were having HbA1c between 7.5 and 8.0 and lower serum vitamin D level (serum vitamin level<25ng/ml) were enrolled for the study.

Patients having diabetic nephropathy more than stage 2 were excluded. Besides this unwilling, poorly

Group 2- 10 female and 14 male, average age 50 years (SD 7.99), average HbA1c 7.8, average serum vitamin D was 17.3 ng/ml (SD 4.20) figure 1. After 3 months average HbA1c in group 1 was 7.4 whereas in group 2 remain same 7.8. The difference was statistically significant (t-value is 3.51725, p-value is 0.000497.

Result is significant at p < 0.05) figure 2. Serum vitamin D cannot be rechecked due to financial constrains but it is presumed that levels might have improved to normal level after recommended supplementation.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Original Article

A-216

Although the absolute difference in HbA1C due to Vitamin D supplementation may appear to be small (0.38%), such a difference could have a large effect at the population level, especially in individuals who are pre-diabetics . For example, in the Diabetes Prevention Program trial, which targeted a population very similar to our population, the difference in HbA1c between the active lifestyle intervention and placebo throughout the entire duration of the study was ≈0.15%, which was associated with a 58% decrease in incident diabetes 5.

Kositsawatet al6. Conducted cross- sectional analyses of data from 9,773 adults who participated in the 2003– 2006 National Health and Nutrition Survey (NHANES), and found that Serum 25-hydroxyvitamin D concentration was inversely associated with Hba1c level in individuals 35–74 years old, but not among the younger or older adults. Lu L. et al.7found Vitamin D deficiency and insufficiency is common (69.2 and 24.4%, respectively) in the middle-aged and elderly Gagnon C, Lu ZX, (the Australian Diabetes, Obesity and Lifestyle study)8 found that each 25 nm/ ml increment in serum 25OHD was associated with a 24% reduced risk of diabetes. Dietary calcium intake was not associated with reduced diabetes risk. Parini PATEL et al9 randomized Subjects with T2DM and serum 25-hydroxyvitamin D (25(OH) D) concentrations <25 ng/mL to receive 400 IU (Group 1) or 1200 IU (Group 2) cholecalciferol for 4 months. Mean 25(OH) D levels increased in both groups, but not to optimal levels.

Fig. 1: Change in HbA1c in Group 1 and Group 2

Fig. 2: Average vitamin D level in Group 1 and Group 2

Fig. 3: Relation between HbA1c change and vitamin D level in group 1 (Series 1 is change in HbA1c and series 2 is vitamin D levels)

Fig. 4: Relation between HbA1c change and vitamin D level in group 2 (Series 1 is change in HbA1c and series 2 is vitamin D levels)

There is no static relationship between change in HbA1c levels and vitamin D supplementation. As depicted in figure 3 and figure 4 the difference in HbA1c is unpredictable after vitamin D supplementation.

The observation period remain uneventful and no new symptoms or complications developed with supplementation.

Discussion

http://www.pacificejournals.com/aabs

A-217

AABS; 3(2): 2016

In conclusion, our study showed that Vitamin D supplementation had a significant effect in glycemic control in diabetic patients especially among those deficient in vitamin D. The glycemic effect of vitamin D is more prominent in postprandial blood sugar control probably by decreasing insulin resistance.

Patients with minimally elevated HbA1c and vitamin D deficiency, mare supplementation of vitamin D improves glycemic control without modification in present diabetic treatment.

TABLE 1: Characteristics of Group 1 and Group 2 CHARACTERISTICS

Group 1

Group 2

Sex M/F

9/15

14/10

Average age in years (SD)

54 (9.55)

50 (7.99)

Vitamin D level

16.8 ng/ml

17.3 ng/ml

Average HbA1c before treatment

7.7

7.8

Average HbA1c after treatment

7.4

7.8

Conclusion

Judicious vitamin D supplementation among vitamin D deficient type 2 diabetic patients with minimally deranged HbA1c can promote better sugar control as evident with improved HbA1c levels. Placebo control trials with double blinding required to strengthen the present evidences.

3.

4.

5.

6.

7.

8.

References

1. Anastassios G. Pittas, Joseph Lau. Review: The Role of Vitamin D and Calcium in Type 2 Diabetes.

A Systematic Review and Meta-Analysis .J ClinEndocrinolMetab, June 2007, 92(6):2017–2029.

2. Maestro B, CampionJ,(2000) Stimulation by1, 25-dihydroxyvitamin D3 of insulin receptor

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

9.

expression and insulin responsiveness for glucose transport in U-937 human promonocytic cells. Endocr J 47:383–391.

Lim S, Kim MJ, Association of vitamin D deficiency with incidence of type 2 diabetes in high-risk Asian subjects. Am J ClinNutr. 2013 Mar; 97(3):524-30. doi: 10.3945/ajcn.112.048496. Epub 2013 Jan. Daga RA, LawayBA.High prevalence of vitamin D deficiency among newly diagnosed youthonset diabetes mellitus in north India. Arq Bras EndocrinolMetabol 2012 Oct;56(7):423-8.

Delahanty LM, Nathan DM. Implications of the diabetes prevention program and Look AHEAD clinical trials for lifestyle interventions. J Am Diet Assoc. 2008 Apr;108(4 Suppl 1):S66-72. DOI: 10.1016/j.jada.2008.01.026.

Kositsawat, J., Freeman, v . L., Gerber, B. s. &Geraci, s. Association of A1c levels with vitamin D status in US adults: data from the National Health And Nutrition Examination Survey. Diabetes Care 33, 1236–1238 (2010).

Lu, L. et al. Plasma 25-hydroxyvitamin D concentration and metabolic syndrome among middle- aged and elderly Chinese individuals. Diabetes Care 32, 1278– 1283 (2009).

Gagnon C1, Lu ZX, Magliano DJ, Dunstan DW , Shaw JE, Zimmet PZ, Sikaris K, Grantham N, Ebeling PR, Daly RM Serum 25-hydroxyvitamin D, calcium intake, and risk of type 2 diabetes after 5 years : results from a national , population- based p rospective study (the A australian Diabetes, Obesity and Lifestyle study). Diabetes Care. 2011 May;34(5).

PATEL, P., PORETSKY, L. and LIAO, E. (2010), Lack of effect of subtherapeutic vitamin D treatment on glycemic and lipid parameters in Type 2 diabetes: A pilot prospective randomized trial. Journal of Diabetes, 2: 36–40.

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report Sudipta Kar’s Modification of Guide Flange Prosthesis for Mandibular Repositioning Sudipta Kar Department of Pedodontics & Preventive Dentistry, Guru Nanak Institute of Dental Sciences & Research, Kolkata, West Bengal, India.

Keywords: Guide Flange Prosthesis, Condylectomy, Deviation, Paediatric.

ABSTRACT The unilateral surgical removal condyle due to irreparable traumatic injury may result in mandibular deviation towards the surgical side and ultimately causes unstable occlusion. Unilateral condylectomy may also result in impaired speech articulation, and facial disfigurement. A corrective device known as ‘guide flange prosthesis’ is used to limit this manifestation. Restoration of acceptable functional occlusion is one of the goals of our treatment modality. Dentition can be used to confirm proper realignment of the mandibular deviation. This can be achieved by the use of guide flange prosthesis. This case report delineates the rehabilitation of a paediatric patient having deviation of the mandible following condylectomy using maxillary guide flange prostheses. Guide flange prosthesis can be considered as a training device. It guides the patient to reposition the mandible into the proper intercuspal position.

*Corresponding author: Dr. Sudipta Kar, 21F, Charakdanga Road, Uttarpara, Hooghly – 712258, West Bengal, India E-mail: dr.sudiptakar@yahoo.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Case Report

C-26

Introduction

Discontinuity of the mandible after unilateral surgical resection of condyle destroys the balance and symmetry of mandibular function ultimately leading to loss of occlusion at the unresected extremity. This deviation of mandible is mainly due to uncompensated functional effect of contralateral musculature specially the internal pterygoid muscle[1,2] Guide flange prosthesis may be helpful in reducing the unavoidable consequence resulting from surgical resection of mandible like ontraction of muscle, occlusal plane mutilation, scar contracture etc.[3] A well designed vertical extension from the buccal and or lingual aspect of a maxillary prosthesis extends to contact the buccal and or lingual surface of the opposing mandibular teeth. This necessary extension maintains the mandible in the proper position though it makes the appliance little heavier. The GFP may often be discontinued if the patient can successfully maintain the position for acceptable period of time. Some patients may continue with a guide flange for indefinite period of time so for them periodic monitoring is needed. It may be designed and incorporated immediately after operative procedure or 7-10 days after resection for restoring mandibular function.[4,5] Delay in the initiation of treatment may results in tight wound closure causing delayed success rate. This modification of GFP can provide more accurate and predictable result.

inferiorly and labiolingually on the lingual surface of the mandibular molars and the premolars [Figure 7 and 8]. Care was taken to protect mandibular teeth and gingiva from trauma during opening and closing of mandible. Finished and polished prosthesis was then evaluated and inserted intraorally [Figure 9]. and periodic check up was done for any inconvenience. The patient was instructed to use the guide flange prosthesis throughout the day, except at night and during meals.

He was also advised to practice simple physiotherapy exercises which were started 2 weeks after the surgery.

It consists of simple opening and closure of mandible with and without the appliance and patient was advised to grasp the chin and shift the mandible away from the surgical site.[6,7] These exercises tend to reduce scar contracture, and trismus as well as improve inter maxillary relationships [Figure 10]. The patient was recalled every 4 weeks for periodic check up. The aim of treatment through GFP was to re-educate the muscles of mandible to re-establish a comfortable, acceptable and hermonic interocclusal relationship for the residual mandible, so that the treated individual can govern the closing and opening of the mandibular movements repeatedly and correctly lifelong. This dual flange helps to maintain occlusal relationship more firmly.

Case Report

A 12 years old male pediatric patient reported to our Department of Pediatric and preventive dentistry with chief complaint of difficulty in mouth opening, mastication and speech [Figure 1 and 2]. He had a history of road traffic accident. Orthopantomogram revealed fractured left condyle [Figure 3].

Condylectomy was performed by retromandibular approach resulting in mandibular deviation [Figure 4 and 5]. Extraoral examination showed facial asymmetry. Clinical examination revealed deviation of mandible towards the left side of face with lack of proper contact between both maxillary and mandibular teeth. Intra oral examination showed no missing teeth [Figure 6]. The patient was evaluated for fabrication of duel guide flange prosthesis. It was observed that the childâ&#x20AC;&#x2122;s mandible could be manually manipulated into the centric occlusion without applying excessive external force. Palatal based guide flange prosthesis was fabricated as a training appliance on the non-defect (right) side and another guide flange was fabricated in the defected side (left) buccally. The retention in the appliance was provided by adams and c clasps. The size and shape of the flange was dependent upon the degree of mandibular deviation the guide flange extended

Fig. 1: Pre-operative photograph

http://www.pacificejournals.com/aabs

C-27

AABS; 3(2): 2016

Fig. 2 : Pre-operative photograph

Fig. 5: Fractured condyle

Fig. 3: Orthopantomogram of the patient

Fig. 6: Deviation of mandible

Fig. 4: Surgical site through retromandibular approach

Fig. 7: Unpolished guide flange prosthesis

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-28

Fig. 8: Finished & polished guide flange prosthesis

Fig. 9: Intraoral placement of guide flange prosthesis

Fig. 10: Corrected deviation

Discussion

Loss of continuity of the mandible due to surgical intervention leads to deviation of the residual affected segment towards the surgical site and alteration in muscle function. Guide flange prosthesis is indicated to limit clinical manifestation of mandibular deviation and restores mandibular function. The literature describes various types

of guide flange prostheses.[8-14] There are various types of cast metal guide flange prostheses but they are complex, technique sensitive and costly and they require a number of appointments. The acrylic guide flange prosthesis is a simple and cost effective method. The advantage of it is, ease of adjustment and require fewer appointments.

Robinson et al. (1964) [8] suggested that for correcting lacking mandibular motor control a cast mandibular resection restoration is appropriate. They also indicated that fabrication of a provisional guide plane facilitates the fabrication of a definitive restoration. Moore DJ et al. (1976) [15] described a combined technique with crowns and maxillary prosthesis to guide the mandible into a functional occlusion. Hasanreisoglu et al. (1992) [9] suggested that palatal guide flange or mandibular guide flange prostheses are indicated for dentate patients.

Beumer et al. (1996) [10] suggested that if the mandible can be manipulated comfortably into an acceptable occlusion a cast metal guidance ramp would be treatment of choice.

If some resistance is encountered in acceptable positioning the mandible, a guide flange of acrylic resin was suggested because of its adjustability. Nasrin Sahin et al. (2005) [16] described the fabrication of a cast metal guidance prosthesis with supporting flanges and retentive flanges. Joshi P et al (2008) [17] suggested that the fabrication of a removable mandibular guide flange prosthesis was an effective alternative for most of the patients with mandibular defects and considering economic feasibility. Prencipe MA et al (2009) [18] described a technique by which only 1 mandibular prosthesis can be used both for physiotherapy and eating, by simply inserting and removing the guide flange. This clinical report illustrates the prosthetic management of a patient who had undergone condylectomy after traumatic injury. The acrylic guide flange prosthesis presented here was a simple and cost effective method for managing the deviation of mandible in a pediatric patient. This was less time consuming and can be easily adjusted and relined according to the need of the patient. This prosthesis also helped the pediatric patient to maintain chewing efficacy from both sides equally and effectively. So this guide flange prosthesis can be regarded as a training appliance. If the patient can successfully repeat the proper, acceptable, and comfortable intermaxillary occlusion the prosthesis can be discontinued. Some patients may continue guide flange for an indefinite period of time but a periodic monitoring is required in every case. An optimum result is achieved when the patient can comfortably and repeatedly occlude maxillary and mandibular teeth without using guide flange prosthesis.[19] Advantages of guide flange prosthesis is it improves masticatory efficacy, reduce the scar contracture and decrease trismus, effectively realigns the residual

http://www.pacificejournals.com/aabs

C-29 mandible to proper intercuspation, ease of fabrication, good patient compliance, reprogram the remaining musculature and improve the maxillo-mandibular relationship, and economical.

Conclusion

AABS; 3(2): 2016 with segmental mandibulectomy: A clinical report. J Prosthet Dent 2005;93(3):217-20.

8. Robinson JE, Rubright W.C. Use of a guide plane for maintaining the residual fragment in partial or hemimandibulectomy. J Prosthet Dent 1964;14:992-9.

A well fabricated dual guide flange prosthesis and an appropriate mandibular exercise regimen is an important adjunct for achieving proper function of the masticatory apparatus. Child’s physiological, psychological and perpetual well being is truly restored in this way. This modification can also restrict aberrant muscle forces and redirect mandible to maintain its proper position in an easier way.

9. Hasanreisoglu U, Uçtasli S, Gurbuz A. Mandibular guidance prosthesis following resection procedures: Three case reports. Eur J Prosthodont Rest Dent 1992;1:69-72.

Acknowledgements

11. Sahin N, Hekimoglu C, Aslan Y. The fabrication of cast metal guidance flange prostheses for a patient with segmental mandibulectomy: a clinical report. J Prosthet Dent. 2005;93:217-20.

Nil

Funding Nil

Competing Interests Nil

Reference

10. Beumer. J III, Curtis T.A, Marunick MT. Maxillofacial Rehabilitation.

Prosthodontic and surgical consideration. St. Louis : Ishiyaku. Euro America.

1996. p 113 –224.

12. Cantor R, Curtis TA. Prosthetic managements of edentulous mandibulectomy patients. Part 1.

Anatomic, physiologic and psychologic consideration.

J Prosthet Dent 1971;25:446-57.

1. Moore DJ, Mitchell DL. Rehabilitating dentulous hemimandibulectomy patients. J Prosthet Dent 1976; 35:202-6.

2. Robinson JE ,RubrightWC. Use of a guide plane for maintaining the residual fragment in partial or hemimandibulectomy. J Prosthet Dent 1964;14:992-9.

3. Principles, concepts, and practices in prosthodontics-1994. Academy of Prosthodontics. J Prosthet Dent 1995;73:73-94.

4. Schneider RL, Taylor TD. Mandibular resection guidance prostheses: a literature review. J Prosthet Dent 1986; 55:84-6.

5. Fattore L, Marchmont-Robinson H, Crinzi RA, Edmonds DC. Use of a two piece gunning splint as a mandibular guide appliance for a patient treated for ameloblastoma. Oral Surg Oral Med Oral Patho 1988; 66:662-5.

6. Beumer J, Curtis TA, Firtell DN. Maxillofacial rehabilitation: Prosthodontics and Surgical Considerations.St Louis,1979,The CV Mosby Co,p 130-156.

7. Sahin N, Hekimoglu C, Aslan Y. The fabrication of cast metal guidance flange prostheses for a patient

13. Prakash V. Prosthetic rehabilitation of edentulous mandibulectomy patient. A clinical report. Indian J Dent Res 2008;19:257-60.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

14. Parel SM. Overdentures in the maxillofacial prosthetic practice. Part I: the cancer patient. J Prosthet Dent 1983;50:522-9.

15. Moore DJ, Mitchell DL. Rehabilitating dentulous hemimandibulectomy patients. J Prosthet Dent 1976:35:202–6.

16. Sahin N, Hekimoglu C, Aslan Y. The fabrication of cast metal guidance flange prostheses for a patient with segmental mandibulectomy: a clinical report. J Prosthet Dent. 2005;93:217-20.

17. Joshi P, Saini G, Shetty P, Bhat S Prosthetic rehabilitation following segmental mandibulectomy.

Journal of Indian Prosthodontic Society 2008;VOL8(2) 108-111.

18. Prencipe MA, Durval E, De Salvador A, Tatini C, Roberto B. Removable partial prosthesis (RPP) with acrylic resin flange for the mandibular guidance therapy. J Maxillofac Oral Surg2009; 8:19-21.

19. Taylor TD, editor. Clinical maxillofacial prosthetics.

Chicago: Quintessence Publishing; 2000. pp.155-170.

Case Report Peripheral Ossifying Fibroma : A case report Soumya Purkait1*, Prasanta Bandyopadhyay1, Bakul Mallick1, Indrasri Das1, Dipayan Das2 Dept. of Periodontics, Dr. R. Ahmed Dental College & Hospital, Kolkata, India 2 Dept. of Pathology, Medical College & Hospital, Kolkata, India

1

Keywords: Peripheral ossifying fibroma, gingival overgrowth

ABSTRACT Peripheral ossifying fibroma (POF) is a reactive lesion of the gingival tissue that predominantly affects women. It originates from the cells of the periodontal ligament. The definitive diagnosis is established by histological examination.

In this article, we have presented a case report with the histopathological evaluation and management.

*Corresponding author: Dr. Soumya Purkait, 6A, Temple Lane, Dhakuria, Kolkata-700031, West Bengal, India Phone : +91 09038608022 E-mail: soumyapurkaitpublication@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-31

AABS; 3(2): 2016

Introduction

The peripheral ossifying fibroma is one of the triad of lesions that is present as gingival mass usually emerging from interdental gingiva and seemingly from PDL. The other two are pyogenic granuloma and peripheral giant cell granuloma. It is a relatively common gingival overgrowth that is considered to be reactive rather than neoplastic in nature.1 They are exophytic, pedunculated or sessile, nodular masses, and mostly affecting the gingiva.2 The pathogenesis of this lesion is uncertain.

Some investigators believe that the lesion is nevertheless odontogenic in origin, being derived from the periodontal ligament, especially since it only occurs in the gingiva and may contain oxytalan fibers within the mineralized matrix of some lesions.3,4 The lesion is reactive in nature and is not the extraosseous counterpart of a central ossifying fibroma (COF) of the maxilla and mandible. It is more common in children and young adults, although may occur at any age with a female predilection (2:1 to 3:2) It may be sessile or pedunculated and surface may be intact or ulcerated It most commonly appears as broad based / pedunculated mass, usually in the anterior quadrant of either arches , emerging from PDL space, mostly seen in young adults. It is mostly fibrotic in nature and is not as red as pyogenic granuloma. In most of the cases there is no radiographic evidence of underlying bone involvement.

pedunculated growth Fig. 01: shows a 9Ă&#x2014;10 mm pedunculated growth

elevated and proper curettage is done. She underwent an uneventful recovery (FIG 03). The patient was on regular follow up for the next 6 months and no recurrence of the lesion is found.

Case Report

A 40 year old female patient reported to the Dept. Of Periodontics, Dr. R. Ahmed Dental College and Hospital, with the chief complaint of pedunculated gingival enlargement in respect to #32 & #33 for last 4 months.

Fig. 02: shows the excised gingival

On intraoral examination, a pedunculated growth of 9mm Ă&#x2014; 10mm dimension with pale pink colour, smooth, nonhaemorrhagic and non-lobulated surface, non-tender and firm in consistency was found in relation to #32 & #33, which were vital (Fig. 1) Radiologically no bone resorption was evident. The patient had non-contributary medical history. There was no h/o deleterious oral habits. Oral hygiene was poor.

Differential diagnosis includes as Pyogenic Granuloma, Peripheral Giant Cell Granuloma (PGCG) and Peripheral Ossifying Fibroma (POF).

Surgical procedure: At first, PHASE -I periodontal therapy was done thoroughly. Intra-oral irrigation is done with Chlorhexidine gluconate 0.2% and the mass is surgically excised under local anaesthesia (fig 02 ) and sent for H/P evaluation. Full thickness muco-periosteal flap is

Fig. 03: shows 1 week post-op photograph with uneventful healing

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

photograph with uneventful healing

Case Report

C-32

H/P EVALUATION:

Discussion

Section stained with Haematoxylin & Eosine (H&E) revealed the presence of stratified squamous epithelium backed by fibro-vascular connective tissue (FIG 04 & 05).

In the stroma, an area of marked proliferation of fibroblasts with formation of a bony trabeculae (FIG 06) is found.

Beside this, a marked chronic inflammatory cell infiltration in the connective tissue was also noted. Hence the overall H/P evaluation of the lesion is suggestive of Peripheral Ossifying Fibroma (POF).

Fig. 04: shows H/P picture showing non-ulcerated stratified squamous epithelium with fibrous proliferation: (4X magnification)

Fig. 05: shows surface epithelium & underlying loose fibrous connective tissue (40X magnification)

Fig. 06: shows an area of mineralisation in the stroma (40X magnification)

In 1982, Gardner coined the term peripheral ossifying fibroma for a lesion that is reactive in nature and is not the extra osseous counterpart of a central ossifying fibroma (COF) of the maxilla and mandible.5 The term fibroma means tumour of fibrous connective tissue (Latin fibra means ‘fiber’ and Greek oma means ‘tumour’). But POF is considered not as a true neoplasm.6 Apparently, the name POF represents a misnomer.

Considerable confusion exists over the nomenclature of this lesion. In the past, the terms peripheral odontogenic fibroma & peripheral ossifying fibroma often were used synonymously, but former is now considered as a separate and distinct entity.1 The use of a variety of terminologies for POF indicates a great amount of confusion regarding the lesion and its pathogenesis. Ossifying fibroid epulis, calcifying fibroma, peripheral fibroma with calcification, peripheral fibroma with cementogenesis, peripheral cemento-ossifying fibroma, peripheral cementifying fibroma, ossifying fibroepithelial polyp, peripheral fibroma with osteogenesis, calcifying or ossifying fibrous epulis and calcifying fibroblastic granuloma are all terms that have been used to refer to peripheral ossifying fibroma.4 There are two types of ossifying fibromas: the central type and the peripheral type. The central type arises from the endosteum or the periodontal ligament adjacent to the root apex and causes the expansion of the medullary cavity. The peripheral type occurs solely on the soft tissues covering the tooth-bearing areas of the jaws.7 Central ossifying fibroma was found to exhibit increased proliferative activity compared to peripheral ossifying fibroma.8 The radiographic findings showed normal underlying bone structure in 26 cases out of 27 and one lesion revealed cupping out of alveolar bone.2 Here, in the H/P picture, there was presence of normal stratified squamous gingival epithelium (FIG 07 & 09), and hence it can be differentiated from central ossifying fibroma (COF), which will lack the presence of epithelium.

The H/P slide examination also revealed the presence of bony trabeculae with a marked osteoblastic rimming (FIG 08). This feature differentiates it from fibrous dysplasia histologically. Dispersed dystrophic calcifications represented by clusters of basophilic granules are seen.

The connective tissue was more collagenized and the inflammatory infiltrate was diminished.

An origin in the periodontal ligament has been suggested for considering the periodontal ligament as the origin of POF due to its exclusive occurrence in the gingiva

http://www.pacificejournals.com/aabs

C-33 (interdental papilla), and the presence of oxytalan fibers within the mineralized matrix of some lesions. A possible hormonal influence has also been considered mainly because POFs are rare in prepubertal patients. However, a few study failed to demonstrate the expression of oestrogen and progesterone receptors in proliferating cellular components.2 The rare manifestation of multicentric occurrence suggests a possible role of genetics in the pathogenesis of the diseasre.4 The treatment of choice is complete surgical excision with the removal of the irritating factors. The mass should be excised down to periosteum as it shows a tendency of recurrence if any part of the lesion is allowed to remain.

In addition, adjacent teeth should be thoroughly scaled to eliminate all possible irritants. Periodontal plastic surgery may be necessary to repair the gingival defect in an aesthetic manner. Although excision is usually curative, a recurrence rate of 8% - 16% has been reported.1

Conclusion

This report highlights the common clinical feature and H/P picture of POF along with the management. Peripheral ossifying fibroma has a high rate of recurrence, making postoperative follow-up mandatory. It is also necessary to use consistent and specific nomenclature in the literature to avoid confusion and the loss of important data.

Acknowledgements

We acknowledge all the staffs of Deptt. Of Periodontiocs of Dr. R. Ahmed dental college & hospital for their sincere supports.

Funding None

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

AABS; 3(2): 2016

Competing Interests None declared

References

1. Nevile BW, Damm DD, Allen CM, Bouquot JE / Oral and Maxillofacial Pathology/ 3rd edition/ page-520 2. Giovanni Mergoni, Marco Meleti, Simone Magnolo, Ilaria Giovannacci, Luigi Corcione, Paolo Vescovi; Peripheral ossifying fibroma: A clinicopathologic study of 27 cases and review of the literature with emphasis on histomorphologic features ; Journal of Indian Society of Periodontology- vol 19, Issue 1, JanFeb 2015 3. Shafer’s Textbook of Oral Pathology/ 7th edition/ page-133 4. Kumar SK, Ram S, Jorgensen MG, Shuler CF, Sedghizadeh PP; Multicentric peripheral ossifying fibroma; J Oral Sci 2006;48:239‑43.

5. Gardner DG; The peripheral odontogenic fibroma: an attempt at classification; Oral Surg Oral Med Oral Pathol 1982; 54:40 6. Subramanyam RV; Misnomers in oral pathology; Oral Dis 2010;16:740‑6.

7. Keluskar V, Byakodi R, Shah N; Peripheral ossifying fibroma; J Indian Acad Oral Med Radiol 2008; 20:54-6 8. Mesquita RA, Suzana Catanhede Orsini, Machado Sousa, Araujo NS; Proliferative Activity in peripheral ossifying fibroma and ossifying fibroma; J Oral Pathol Med 1988; 27:64

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report Management of Aberrant Frenum: Series of Cases Suchi Suvra Bagchi*, Puja Sarkar, Prasanta Bandyopadhyay Department of Periodontics, Dr. R. Ahmed Dental College & Hospital, Kolkata, India

Keywords: Frenum, Frenectomy, Mucogingival techniques

ABSTRACT The frenum is a mucous membrane fold that attaches the lip and the cheek to the alveolar mucosa, the gingiva, and the underlying periosteum. A frenum that encroaches on the margin of the gingiva may interfere with plaque removal and cause tension. Frenectomy is the complete removal of the frenum that can be made by scalpels or with soft tissue lasers.

This article describes 3 case reports of different frenectomy techniques used for management of aberrant frenum.

*Corresponding author: Dr Suchi Suvra Bagchi, W-14, Cluster-3, Purbachal Housing Estate, Salatlake City, Kolkata-97, India Phone : +91 9434513366 E-mail: suchisuvra76@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-35

Introduction

A freum may be described as a triangular fold of mucous membrane that attaches the lip and the cheek to the alveolar mucosa, the gingiva and the underlying periosteum.

Sometimes it creates problem and hamper the plaque control by interfering in tooth brushing1.Midline diastema may be caused by maxillary frenum thereby compromising the orthodontic result leading to aesthetic problems2.

These aberrant frena may be removed either by frenectomy or frenotomy. Various techniques like simple excision with the help of scalpel or electrosurgery, even LASERs may be advocated for frenectomy or frenotomy.

The procedure of complete removal of the frenum, including its attachment to the underlying bone is known as frenectomy while the incision and the relocation of the frenal attachment is known as frenotomy.

The conventional technique involves excision of the frenum using a scalpel. Depending upon the type of frenum various modifications of this conventional procedure3,4,5 have been recommended like Millerâ&#x20AC;&#x2122;s technique, V-Y plasty and Z-plasty.

AABS; 3(2): 2016

Case Report(S)

CASE 1:- A female patient aged about 40 years reported to the department of periodontia of Dr.R.Ahmed Dental College & Hospital with gingival recession in lower anterior tooth region for last 2-3 yrs. She was otherwise healthy with a negative drug history.

Intraoral examination revealed high attachment of lower labial frenum, extended into the marginal gingiva. The classical frenectomy procedure was planned to remove the frenum.

Frenum was engaged with a haemostat which was inserted into the depth of the vestibule [Fig-1(a)] and incisions were placed on the upper and undersurface of the haemostat until the tissue became free. The triangular resected portion of the frenum was removed along with the haemostat. A blunt dissection was done to relieve the fibrous attachments.

Interrupted sutures were applied at the edges of diamond shaped wound [Fig-1(b)] with 4-0 black silk [Fig-1(c)]. The area was covered with a periodontal pack (Coe-pack). The pack and the sutures were removed 1 week postoperatively

This article presents three different surgical techniques to treat aberrant labial frenum.

CASE 2 :- A female patient of 25yrs reported in the department of Periodontics of Dr R. Ahmed Dental College & Hospital with a midline diastema. The patient was systemically healthy with a negative drug history.

Fig-1(a)Engagement with haemostat

Fig-1(b) Excision of Frenum

Fig-1 (c) After suturing

Fig-1 (d) 1month postoperative view

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-36

On intra oral examination it was found that there was aberrant upper labial frenum extending into the interdental papilla between upper central incisors. Case was operated with Z-plasty technique.

Scalpel incision was given along the whole length of the frenum. At each end of the incision another two incisions of equal length was made with angulation between 60° and 90°. The submucosal tissues were dissected beyond the base of each flap into the loose non-attached tissue

planes. Care should be taken not to damage the apices of the flaps. These flaps were then mobilised and moved through 90° to close the previous vertical incisions [Fig2(d)]. Stabilisation of the flaps was done by placing black silk (4-0) sutures beginning from the apices of the flaps to ascertain the adequacy of the flap repositioning. Then sutures were evenly spaced along the edges of the flaps to close the wound along the cut edges [Fig-2(e)]. A periodontal dressing was placed. After 1 week the dressing and sutures were removed.

Schematic Diagram of Z-plasty technique

Fig-2(a) Preoperative view

Fig-2(b) Excision of the frenum

Fig-2(c) Incisions given

Fig-2(d) Apposition of the flaps

http://www.pacificejournals.com/aabs

C-37

AABS; 3(2): 2016

Fig-2(e) After suturing

Fig-2(f) 1month postoperative view

CASE 3 :- A female patient of 25yrs was referred from the department of orthodontics of Dr R. Ahmed Dental College & Hospital for removal of frenum. The patient was systemically healthy with a negative drug history.

in a case of a papilla type frenal attachment. The frenum was held with the haemostat and an incision was made in the form of V on the undersurface of the frenal attachment.

The frenum was relocated at an apical position and V shaped incision was converted into a Y, after suturing.

The sutures were removed at 1 week of follow-up. After 1 month the frenal attachment was found to be relocated at an apical position with uneventful healing.

On intra oral examination it was found that there was aberrant upper labial frenum extending into the interdental papilla between upper central incisors. Frenectomy by V-Y plasty was done in this case. This technique was employed

Schematic Diagram of V-Y plasty technique

Fig-3(a)-Preoperative

Fig-3(a)-Preoperative

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

Fig-3(b)- Engagement with haemostat

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-38

Fig-3(c)- V shaped incision given

Fig-3(d) Incision extended

Fig-3(e) Sutures placed

Fig-3(f) 1 month postoperative view

Discussion

1) An aberrant frenal attachment is present, which causes a midline diastema.

2) A flattened papilla with the frenum closely attached to the gingival margin is present, which causes a gingival recession and a hindrance in maintaining the oral hygiene.

3) An aberrant frenum with an inadequately attached gingiva and a shallow vestibule is seen.

Knox & Young histologically studied the frenulum, and reported presence of both elastic and muscle fibres (Orbicularis oris – horizontal bands and oblique fibres). However, Henry, Levin and Tsakins have found considerably dense collagenous tissue and elastic fibres without any muscle fibres1.

The labial frenal attachments have been classified by Placek et al in 19746 into 1) Mucosal – when the frenal fibres are attached up to the mucogingival junction.

2) Gingival – when the fibres are inserted within the attached gingiva.

3) Papillary – when the fibres are extending into the interdental papilla.

4) Papilla penetrating – when the frenal fibres cross the alveolar process and extend up to the palatine papilla.

The abnormal frena are detected visually by applying tension over the frenum to see the movement of papillary tip or the blanch which is produced due to ischaemia in the region The frenum is characterised as pathogenic and is indicated for removal when

Resection of aberrant frena was initially included under the term mucogingival surgery given by Friedman in 19577. Later it was included under the broad heading of periodontal plastic surgery. As mentioned earlier scalpel method, electrosurgery as well as LASERs may be used to treat these aberrant frena. The classical scalpel technique was introduced by Archer (1961) and Kruger (1964). After introduction of this technique various modifications were proposed, like Z-plasty, V-Y-plasty and Miller’s technique.

Till date the classical technique remains the most widely used method. But the classical technique may leave a longitudinal surgical scar which may lead to periodontal problems and an unaesthetic appearance.

The Z-plasty technique was found to be ideal for broad, thick hypertrophic frenum associated with midline diastema

http://www.pacificejournals.com/aabs

C-39 and a short vestibule. This procedure enables us to remove the fibrous band and also helps in vertical lengthening of the vestibule. Overall the Z-plasty procedure is considered to be safe. Cost effective and results in better functional and aesthetic appearance. This procedure allows for soft tissue healing by primary intentions; increasing recovery and reducing the risk of tissue contractures.

V-Y plasty can be used in case of broad frena in the premolar molar region. It allows the lengthening of that area.

AABS; 3(2): 2016

Acknowledgements None

Funding None

Competing Interests None

Reference

An aberrant frenum can be removed by any of the modification techniques but a functional and aesthetic result can be achieved by proper technique selection based on the type of frenal attachment.

1. Jhaveri H. The Aberrant Frenum. In: Dr HiralJhaveri(ed), Dr PD Miller the father of periodontal plastic surgery, 2006;29-34.

2. Huang WJ, Creath CJ. The midline diastema: a review on its etiology and treatment. Pediatric Dentistry 1995;17:171-9.

3. Coleton SH. The mucogingival surgical procedures which were employed in re-establishing the integrity of the gingival unit. Thefrenectomy and the free mucosal graft. Quintessence Int.1977;8(7):53-61 4. Kahnberg KE. Frenum Surgery.A comparison of three surgical methods.Int J Oral Surg.1977;6:328-33 5. Ito T, Johnson JD. Frenectomy and frenotomy.Color Atlas of Periodontal Surgery. In: Ito T, Johnson JD, editors. London: Mosby Wolfe; 1994.pp.225-39.

6. Dibart S, Karima M. Labial frenectomy alone or in combination with a free gingival autograft. In: Serge Dibart, MamdouthKarima (eds). Practical Periodontal Plastic Surgery. Germany. Blackwell Munksgaard p-53.

7. Friedman N. Mucogingival Surgery. Texas Dent J 75:358,39,1957.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

The Millerâ&#x20AC;&#x2122;s technique was advocated by Miller PD in 1985. This technique was proposed for the post-orthodontic diastema cases. The ideal time for performing this surgery is after the orthodontic movement is complete and about 6 months before the appliances are removed. In this technique after excision of frenum a laterally positioned split thickness pedicle graft is obtained and is sutured across the midline. The advantage of this technique are 1) Post operatively there is a continuous collagenous band of gingival across the midline. This gives bracing effect and chances of relapse is less.

2) The transseptal fibres are not disrupted surgically and so there is no loss of interdental papilla.

In this orthodontic stability is achieved without compromising aesthetics.

Conclusion

Case Report Adenomyoepithelioma Arising in Axillary Breast Tissue- A Diagnostic Rarity: case Report with Literature Review Mega Lahori*, Sakul, Bawana Raina, Arvind Khajuria Deptt of Pathology, Acharya Shri Chander College of Medical Sciences, Jammu, J&K, India Keywords: Adenomyoepithelioma, Myoepithelial Cells, Axillary Breast Tissue

ABSTRACT Adenomyoepithelioma is an uncommon, benign tumor of biphasic nature which arises from myoepithelial and epithelial cells. It has been recognized mainly in breast tissue, along with skin adnexa, lungs and salivary glands. The typical histologic appearance consists of acinar structures composed of an inner layer of epithelial cells with eosinophilic cytoplasm and a prominent peripheral layer of myoepithelial cells with clear cytoplasm. Prognosis of patients with AME is usually good, but it has a potential for local recurrence and rarely, malignant transformation with distant metastases to lung, brain, and liver. We present the rare case of a 42 year old female with left sided axillary lump that was diagnosed as adenomyoepithelioma arising from accessory breast tissue in the axilla. This of interest not only because of the rarity of the lesion, but also due to the peculiarity of its origin from accessory mammary gland tissue of axillary location.

*Corresponding author: Dr Mega Lahori, 17/1-B, K B Nagar, Bantalab Jammu, J&K 180023 Phone : +91 09419177133 E-mail: iammegha00@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-41

Introduction

Adenomyoepithelioma (AME) is an uncommon, diagnostically challenging tumor which demonstrates dual differentiation into luminal cells and myoepithelial cells.

It was first described in breast by Hamperl in 1970. While AMEâ&#x20AC;&#x2122;s have been reported in primary breast tissue, their location in axillary breast tissue is a diagnostic rarity as well as curiosity. Although AMEs are benign in majority of cases, sporadic malignant AMEs with distant metastases have also been reported. Recognition of this unique entity, its accurate diagnosis, and the knowledge of its expected behavior are important in guiding the most appropriate patient management 1.

Case Report

A 42 year old female presented in the rural outreach opd of our hospital with a 3x3 cm sized painless, mobile lump in the left axilla for 1.5 years. There was no tenderness, erythema, warmth or discharge associated the mass.

Bilateral breasts and contralateral axilla revealed no abnormality on clinical examination. Family history was unremarkable. Routine hematological parameters, chest X ray and ultrasound of abdomen revealed no abnormality.

Local excision was done and the lump was submitted to the pathology department. Grossly, a 2x1.5x1cm sized, rounded firm mass with irregular outer surface and greyishbrown cut surface was seen (Fig 1). Histopathological examination revealed a well circumscribed tumor with pushing margins composed of closely packed ducts lined by a bilayer of inner cuboidal to columnar cells and outer polygonal cells with clear cytoplasm in a fibrous stroma

AABS; 3(2): 2016 (Fig 2). Adjacent foci revealed presence of mammary gland tissue showing adenosis. Deep and lateral resection margins were free of tumor. Rest of the tumor sample was sent for IHC to another center, and it was positive for S-100, SMA, pan-Cytokeratin (AE1/AE3), estrogen receptor (ER) and progesterone receptor (PR). Based on the histopathology and IHC, the tumor was diagnosed as Adenomyoepithelioma of left axillary breast tissue.

Postoperatively, her recovery was uneventful. She has been on follow up for the past 7 months with no evidence of tumor recurrence till date.

Discussion

Adenomyoepithelioma is an uncommon, benign tumor which arises from myoepithelial and epithelial cells. It has been recognized mainly in breast tissue, along with skin adnexa, lungs and salivary glands. In skin, it is known as apocrine mixed tumor and in salivary glands as epithelialmyoepithelial carcinoma. AME has a biphasic nature and the typical histologic appearance consists of acinar structures composed of an inner layer of epithelial cells with eosinophilic cytoplasm and a prominent peripheral layer of myoepithelial cells with clear cytoplasm 2. Epithelial cells are cuboidal to columnar and tend to have hyperchromatic nuclei with dense eosinophilic cytoplasm, when compared with myoepithelial cells.

AME diagnosed in axillary lump in the present case can be speculated to be arising from skin adnexa, axillary breast tissue or as a metastatic lesion from breast, salivary glands or lung. But the presence of adjacent foci of uninvolved breast tissue along with positivity for hormone receptors

Fig. 1: Gross image of lump resected from left axilla showing a firm nodule with greyish-brown cut surface and irregular margins

Fig. 2: Photomicrograph of adenomyoepithelioma showing closely packed ducts lined by a bilayer of inner cuboidal cells and outer polygonal cells with clear cytoplasm (H&E Ă&#x2014;100)

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

C-42

and failure in detection of any primary source in bilateral breasts, lungs or salivary glands clinched the diagnosis in favor of AME arising from accessory breast tissue of axillary location.

The histogenesis of this tumor is still obscure. All cases have been sporadic and no familial aggregation has been observed. Kiaer et al3 reported a case of sequential changes from adenomyoepithelial adenosis into benign AME which eventually became low grade malignant AME during the course of 18 years. From this observation, Choi et al4 proposed that adenomyoepithelioma was derived from a long-standing underlying breast disease such as adenosis and fibroadenoma. Tavassoli has described three variants of AMEs- tubular variant (proliferation of rounded tubules, as well as unusually prominent and hyperplastic myoepithelial cells), spindle cell variant (predominantly spindled myoepithelial cell proliferation admixed with a few columnar epithelium-lined tubules) and lobular variant (solid nests of myoepithelial cells proliferating around compressed tubules) 5. Combinations of growth patterns sometimes exist within the same tumor.

The interplay between epithelial and myoepithelial cell elements is highlighted by immunohistochemical staining with antibodies specific for these 2 components.

The cytoplasm of epithelial cells uniformly reacts with antibodies to cytokeratins and the luminal surfaces of the glandular cells are positive for the epithelial membrane antigen. Luminal cells also possess receptors for ovarian steroid hormones(ER and PR). The myoepithelial component is highlighted by p63, smooth muscle myosin heavy chains, CK5, CD10, calponin, actin, and S100 6, 7.

Because of the rarity and varied nature of AMEâ&#x20AC;&#x2122;s, they can be confused with other neoplasms. The differentiation of intraductal papilloma with prominent myoepithelial cells from AMEs can be made based on architecture, pattern, and degree of myoepithelial proliferation.

Myxochondroid matrices produced by the myoepithelial cells may also be noted, as seen in pleomorphic adenomas.

Clear cell carcinoma may also mimic AMEs, but that may be differentiated by the presence of both epithelial and myoepithelial cell types, and confirmed with immunohistochemistry7. Some areas of an AME may resemble adenoid cystic carcinoma, but that has infiltrative borders, a characteristic cribriform architecture with smaller, more hyperchromatic, and basaloid appearing myoepithelial cells than AME 8. In case of small biopsy, the sampled tissue may even be mistaken for invasive carcinoma. The presence of regularly spaced, rounded or ovoid glands; unidirectional streaming of the glands; and

prominent clear cell or spindle cell myoepithelium are some morphologic clues to the diagnosis of AME 9.

Prognosis of patients with AME is usually good, but it has a potential for local recurrence, especially in the tubular and lobulated variants. Total surgical excision with an adequate margin of resection is therefore recommended.

Malignant transformation has also been reported along with distant metastases to lung, brain, and liver 3,5,8.

Carcinomas may arise from ductal epithelial cells, myoepithelial cells, or both. Atypical features include infiltrative margins, increased mitotic activity, cytologic atypia with nuclear pleomorphism, prominent nucleoli, hyperchromasia, and necrosis 5,6.

Conclusion

This unique case of adenomyoepithelioma arising from axillary breast tissue is of interest not only for its rarity, but also due to the peculiarity of its origin from accessory mammary gland tissue of axillary location. Its diagnosis and optimal therapy are problematic issues for the clinicians due to the possibility for local recurrence and rare chance of metastatic spread. Recognition of biphasic cellular elements and the overall tumor architecture in combination with immunohistochemistry are important when diagnosing AMEs. Any case with significant cytologic atypia, rapid growth, infiltrating margins, necrosis, and brisk mitotic rates raises the likelihood of the lesion being malignant which is associated with the potential of metastasis.

Hence, the histopathologist should duly report any atypical features and recommend complete excision with adequate margins so as to decrease any possibility of recurrence and metastasis in future.

Funding None

Competing Interests None Declared

References

1. Yoon JY, Chitale D. Adenomyoepithelioma of the Breast: A Brief Diagnostic Review. Archives of Path and Lab Med. May 2013; 137 (5): 725-729 2. Rosen PP. Adenomyoepithelioma of the breast. Hum Pathol. 1987; 18(12):1232â&#x20AC;&#x201C;1237.

3. Kiaer H, Nielsen B, Paulsen S, Sorensen I, Dyreborg U, Blichert-Toft M: Adenomyoepithelial adenosis and low-grade malignant adenomyoepithelioma of the breast. Virchows Arch A Pathol Anat Histopathol; 1984; 405: 55-67.

4. Choi JS, Bae JY, Jung WH. Adenomyoepithelioma of the breast. Yonsei Med J 1996;37:284-289.

http://www.pacificejournals.com/aabs

C-43

AABS; 3(2): 2016

5. Tavassoli FA. Myoepithelial lesions of the breast: myoepitheliosis, adenomyoepithelioma, and myoepithelial carcinoma. Am J Surg Pathol.

1991;15(6):554–568.

6. Loose JH, Patchefsky AS, Hollander IJ, et al.

Adenomyoepithelioma of the breast: a spectrum of biologic behavior. Am J Surg Pathol. 1992; 16(9):868–876.

7. McLaren BK, Smith J, Schuyler PA, Dupont WD, Page DL. Adenomyoepithelioma: clinical,

histologic, and immunohistologic evaluation of a series of related lesions. Am J Surg Pathol.

2005;29(10):1294–1299.

8. Hayes MM. Adenomyoepithelioma of the breast: a review stressing its propensity for malignant transformation. J Clin Pathol. 2011;64(6):477–484.

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

9. Hoda SA, Rosen PP. Observations on the pathologic diagnosis of selected unusual lesions in needle core biopsies of breast. Breast J. 2004;10(6):522–527.

Case Report Rare Case of Ovarian Ectopic Pregnancy Shikha Joshi, Dipti Agrawal, Sunita Fotedar Dept. of Gynecology and Obstetrics, Swami Dayanand Hospital Dilshad Garden, Delhi, India Keywords: Ovarian Pregnancy, Intra Uterine Devices, Laparotomy

ABSTRACT Ovarian pregnancy is a rare form of ectopic pregnancy. It constitutes to 1% of all ectopic pregnancies which usually ends with rupture before the end of the first trimester.

We report here one such uncommon case of ovarian ectopic pregnancy. 38 years old P2L2 woman with one previous cesarean section and one VBAC with history of IUCD for 5 years presented with hypovolemic shock. During laparotomy, ruptured right ovarian ectopic pregnancywith 1.5 litres of haemoperitoneum was found , and wedge resection of the ovary was done. Histopathological examination confirmed it to be an ovarian ectopic pregnancy.

Intra uterine devices (IUD) is one of contraceptive methods which prevents intra-uterine implantation in 99.5%, if implant occurs with IUD, it is tubal implantation in 95% of cases, and it is very rare in other places such as ovary. The most important risk factor of ovarian ectopic pregnancy is IUD as in this study it was showed.

*Corresponding author: Dr Shikha Joshi, 27/76 Gali No 8 Vishwas Nagar Shahdara Delhi 110032, India Phone : +91 09582647083 E-mail: drshikhajoshi@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-45

Introduction

Ovarian pregnancy is estimated to be 3% of diagnosed ectopic pregnancies [1]. We report a case of ruptured right ovarian pregnancy with previous LSCS followed by VBAC with history of Intra uterine contraceptive devices (IUCD) for 5 years presented to casualty with hypovolemic shock and was treated with laparotomy with wedge resection of ovary and volume replacement. The preoperative diagnosis of ovarian pregnancy is difficult though diagnosis of ovarian pregnancy should be suspected from elevated beta HCG, lack of intrauterine gestation, a complex ovarian mass on USG, IUCD usage, history of PID, in addition to the Spiegelberg criteria [2]. Spiegelberg’s criteria includes : (1) an intact ipsilateral tube, clearly separate from the ovary; (2) a gestation occupying the normal position of the ovary; (3) a gestational sac connected to the uterus by the utero-ovarian ligament; (4) ovarian tissue in the wall of the gestational sac.Ovarian pregnancy during surgery is difficult to differentiate from a hemorrhagic corpus luteum intraoperatively.[3] Environmental conditions favouring tubal ectopic gestation such as pelvic inflammatory disease, previous surgery, and history of infertility are very rare in ovarian pregnancies. However, a few risk factors seem to be present for ovarian pregnancies: endometriosis and intrauterine device usage are reported to contribute in the majority of cases.

Case Presentation

AABS; 3(2): 2016 appeared to be normal. Rent was observed from the right ovary, which was bleeding suggestive of ruptured ectopic pregnancy of around (5 cm × 4 cm) size (figure 2). Wedge resection of ovary was done and rest of the ovarian tissues was preserved. Intraoperatively 2 units blood was given.

Postoperatively patient was stable Hb was 7.5 gm% and she received one more unit of blood.Patient was discharged three days later.

Discussion

Ovarian pregnancy is a rare variant of ectopic pregnancy [1]. One in every nine ectopic pregnancies among Intra uterine devices (IUD) users is an ovarian pregnancy [3,4]. Preoperative diagnosis remains challenging, and it is diagnosed generally during surgery [5]. Rupture in the first trimester is the usual rule in ovarian ectopic pregnancy. Diagnosis of ovarian pregnancy should be suspected from elevated beta HGC, lack of intrauterine gestation, a complex ovarian mass on USG, patient’s history , signs, symptoms, in addition to the Spiegelberg criteria [2].Comstock et al [7]reviewed six cases of ovarian pregnancy to review common ultrasonographic findings in all. The study demonstrated a common feature in five of six patients: A hypoechogenic ring was seen on/within the surface of the ovary. Only one of these contained a yolk sac. The patient in which this finding was not seen was found to have a ruptured ectopic at surgery.

A 38-year-old P2L2 with one cesarean followed by vaginal delivery woman presented to casualty of our department complaining of anxiousness with severe pain in lower abdomen. She had amenorrhea of 4 week 6 days with faintly positive UPT. Physical examination revealed initial blood pressure 90/60 mmHg, pulse 110 beats/min, and body temperature 98.4° F. Per abdominal examination revealed patient had tenderness in lower abdomen.

On speculum examination there was a small amount of blood but the cervical os was closed. On bimanual clinical examination she was found to have a normal size anteverted uterus, no cervical motion tenderness but right adnexal tenderness was present. Urine pregnancy test was faintly positive, Transvaginal examination showed an empty uterus, right side TO mass of about 5 x 4 cm with fluid in the cul de sac(figure 1). Her laboratory results were: White Blood Cells (WBC): 6500, Hemoglobin (Hb): 6 g/dL, Hematocrit (Hct): 30%, Platelets (Plt): 250 × 109/L. The patient was admitted with a diagnosis of suspected ruptured tubal pregnancy and decision for emergency laparotomy was taken with 3 units of blood and high risk consent for ICU admission. On laparotomy around 1.5 litre haemoperitoneum was present .Both fallopian tubes

The difference between ultrasonographic appearance of a corpus luteum and ovarian pregnancy is that while both have a ring-like appearance, in the majority of cases, a corpus luteum appears less echogenic than the ovary itself. This is in contrast to an ovarian pregnancy in which the ring-like structure appears more echogenic than the

Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

e-ISSN: 2349-6991; p-ISSN: 2455-0396

Figure 1: USG Findings

Case Report

C-46

Fig. 2: Intra-operative images showing ruptured right ovarian pregnancy

ovary. This is attributed to the fact it enables a histological diagnosis. Hallat confirms this in his study of 25 cases of ovarian pregnancy where he reports that a correct diagnosis at surgery was only made in 28% cases. The study correctly highlighted the difficulty in distinguishing between an ovarian pregnancy and a haemorrhagic cyst/corpus luteum.

Funding

In our case the ovarian pregnancy was misdiagnosed as a tubal pregnancy, as USG was not useful for distinguishing between ovarian and tubal pregnancy. Medical therapy with methotrexate was not possible due to the occurrence of massive bleeding. The most common surgical treatment consists of ovarian wedge resection or oophorectomy [8], either laparoscopic or via minilaparotomy. Patient was haemodyanamically low so we took decision for laparotomy along with all resuscitative measures. Wedge resection of ovary was done and tissue was sent for histopathology and later confirmed for ovarian ectopic.

Patient was stable postoperatively and discharged on day 4.

1. Fritz MA, Speroff L. Clinical Gynecologic Endocrinology and infertility. 8th Ed. Philadelphia: 2011.

2. O. Spiegelberg, “Zur casuistic der ovarial Schwangerschft,” Archiv für Gynaekologie, 1873;13:73–76, 3. Hallet JG. Primary ovarian pregnancy. A case report of twenty five cases. Am J Obstet Gynecol.1982;143(1):55–60] 4. Grimes H, Nosal RA, Gallagher JC. Ovarian pregnancy.

A series of 24 cases. Obstet Gyncol.1983;61:174–180.

5. S. Panda, L. M. Darlong, S. Singh, and T. Borah, “Case report of a primary ovarian pregnancy in a primigravida,” Journal of Human Reproductive Sciences, vol. 2, no. 2, pp. 90–92, 2009.

6. D. Jurkovic and D. Mavrelos, “Catch me if you scan: ultrasound diagnosis of ectopic pregnancy,”Ultrasound in Obstetrics and Gynecology, 2007;30:1–7,.

7. Comstock C, Huston K, Lee W. The ultrasonographic appearance of ovarian ectopic pregnancies. Am College Obstet Gynaecol 2005;105:42–5.

8. Sidek S Lai SF, Lim-Tan SK.Primary ovarian pregnancy: current diagnosis and management.

Singapore med j 1994 Feb;35(1):71-3

Conclusion

Ovarian ectopic pregnancy is a rare variant of ectopic gestation. The diagnosis is made often at surgery and requires histologic confirmation. Fertility after conservative surgical procedures does not appear to be affected and ovarian wedge resection or ovarian cystectomy is the treatment of choice.

Acknowledgment

We would like to thank our Head Dr Sunita Fotedar for always helping and guiding us and also Dr kalpana for her constant support

None

Competing interests None declared

References

http://www.pacificejournals.com/aabs

Letter to Editor Mucopedermoid Carcinoma of Labial Mucosa Shahela Tanveer, Karthik Kv, Nishanth.P, Syed Afroz, Charu Suri, Shravan D Dept. of Oral Pathology, Sri Sai Dental College, Vikarabad, India Keywords: Minor Salivary Glands, Mucoepidermoid Carcinoma, Adenoid Cystic Carcinoma,Labial Mucosa.

Dear Sir Although mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma is the most common malignant salivary gland neoplasm in minor salivary gland, it is rarely seen on the labial mucosa . The clinical presentation of a salivary gland malignancy in an intraoral minor salivary gland will often be mistaken for a benign or inflammatory process .

Although it is rare before age 20, it is the most common salivary gland malignancy in the pediatric and adolescent populations . We present a unique case of a 29yr male with diffuse swelling on upper labial mucosa. This solitary, movable lesion present a diagnostic dilemma to the pathologist with rarer manifestations.

A 29 year old male patient came to the op depatrment with a painless diffuse swelling on upper inside of lip since 3 months. Paresthesia present over the swelling. On intra oral palpation the swelling was firm with well defined borders on palpation. There was no pain associated with the swelling. No history of trauma. The clinical appearance was that of a fibroma. On examination the swelling was of the same color as that of normal mucosa, Ovoid stretched and shiny ,soft to firm on palpation measuring about 2x2 cm (FIG 1).The differentialdiagnosis included fibroma, peripheral ameloblastoma, lipoma, neurofibroma, and salivary gland neoplasm .

Excisional biopsy was done and we received a tissue grayish white in color with hemmarrhoge measuring about 2x 2 x 5 mm in size. On sectioning, a smooth glistening white material was seen. (FIG 2) The lesion was aspirated and tissue was then excised for biopsy The aspirated lesion cytology stained with H and E

showed areas of inflammatory cell and very sparse mucin pooling at few areas (FIG 3).Pale mucoid background with cells in clusters were seen . Cells were arranged in groups the nuclear and cytological features were not appreciable in the smear, but mild pleomorphism of cells was noticed.

At one foci scattered individual cells were seen which were showing spindle shape and vacoulated .(FIG 4) Tissue was processed and the H&E stained section showed a well defined capsule around sheets and islands of tumour cells with connective tissue (FIG 5). Islands of tumour tissues shows two populations of cells, one clear cell and one cell with inconspicuous nucleolie and granular cytoplasm surrounded by pale stain eosinopilic area. (FIG 6) . At one foci near the tumor capsule a lymphoid component forming a germinal centre was seen .

(FIG 7) The histological differential diagnosis of the tumor included mucoepidermoid carcinoma, adenoid cystic carcinoma, polymorphous low grade adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma Histomorphogenic diversity within the salivary gland lesions are common. A careful clinical, histopathological and cytological study is necessary to rule out, whether the lesion can be cateogerized as inflammatory ,benign or malignant lesion1. Also in contrast to previously existing literature, the incidence of malignancies in minor salivary glands was higher than benign salivary gland neoplasms [2].

In our case with clinical evaluation of the lesion as a firm and movable lesion we further proceeded to aspiration cytology. The aspirated smear showed vacuolated cells, few squamous cells in a dirty background suggestive of mucin with pleomorphism of cells. Scattered inflammatory cells

*Corresponding author: Dr. Shahela Tanveer, H. No 9-4-87/B/43, Minar Colony, Hyderabad 5000008, India Phone: +91 8790144486 E-mail: drshahela17@gmail.com

This work is licensed under the Creative commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

L-2

Letter to editor

Fig. 1: A firm and movable lesion on upper labial mucosa

Fig. 2: Grossed lesion showing white glistening material

Fig. 3: Smear showing mucoid background with few squamous cells in clusters (4X & 10X )

Fig. 4: Squamoid cells with mild pleomorphism and few scattered vacoulated cells (40X)

http://www.pacificejournals.com/aabs

L-3

AABS; 3(2): 2016

Fig. 5: Two population of cells vacoulated cells and few epidermoid like cells (10x,40x)

Fig. 7: Tumour capsule with germinal centre at one foci (10x)

predominantly lymphocytes were seen. The presence of such cluster of cells with mucin background could suggest a variety of salivary gland lesion. Also with inflammation it could also suggest an inflamed or obstructive salivary gland lesion [3,4].

Histological sections in our case showed lesional tissue arranged in sheets and islands with intervening hyalinization in connective tissue stroma.Two populations of cells, one clear cell and one cell with inconspicious nucleolie and granular cytoplasm was seen. With such features Mucoepidermiod carcinoma was given as final diagnosis.

Mucoepidermoid carcinoma (MEC) is a malignant epithelial neoplasm composed of varying proportions of mucous, epidermoid, intermediate, columnar, and clear cells and often demonstrates prominent cystic growth.[1] Annals of Applied Bio-Sciences, Vol. 3; Issue 2: 2016

MEC have been categorized as either classical MEC or variant (non-classical)MEC. Classical MECs are tri- or biphasic neoplasms composed of well recognizable mucous cells with prominent single goblet cells or contiguous goblet cells forming mucinous gland-like or cystic spaces in addition to variable proportions of epidermoid (squamoid) and intermediate cells.

Variant or non classical MECs have predominant squamoid , eosinophilic, or clear cell morphology . Few tumors are subvariant MEC which shows focal acantholytic, pseudoglandular or pseudovascular pattern. Another pattern seen in this group was the presence of differentiated squamoid sheets with numerous microcystic lumina filled with blue mucin and imparting a pseudocribriform pattern to the tumor. [5] Once diagnosed as a malignant lesion ,a grading system becomes important.There is lot of grading systems . e-ISSN: 2349-6991; p-ISSN: 2455-0396

L-4

Letter to editor According to the grading schemes for mucoepidermoid carcinoma, histological features scoreAFIP(Goode et al, 1998) Brandweinet al (2001) with cystic component <25% without neural invasion ,mitoses and anaplasia and nuclear atypia this tumour is regarded as low grade tumour [6]. Based on wide morphological diversity there is no particular prognostic indicator, however size is of concern as large lesions tend to have poor prognosis regardless of grade and high grade lesions might do well when small.[7] The pattern at the tumour edge, a lymphocytic infiltrate with possible germinal centre formation can mimic nodal invasion ,it represents part of the concept of tumorassociated lymphoid proliferation (TALP) [8].

Acknowledgements None

Funding None

3. Review of Fine-NeedleAspiration Cytology of SalivaryGland Neoplasms, With Emphasis on Differential DiagnosisAm J Clin Pathol 2002;118(Suppl 1):S100-S115 4. Dardick.I, Birek C,Mark W. Differentiation and the cytomorphology of salivary gland tumors with specific reference to oncocytic metaplasia . Vol. 88 No. 6 December 1999 .

5. SchwarzS, Stiegler.C, MĂźller.Salivary Gland Mucoepidermoid Carcinoma is a Clinically,Morphologically and Genetically Heterogeneous Entity. A Clinicopathologic Study of 40 Cases with Emphasis on Grading, Histological Variants and Presence of the t(11;19) Translocation.

Histopathology 58, 4 (2011) 557 6. Brandwein MS, Ivanov K, et al. Mucoepidermoid carcinoma. A clinicopathologic study of 80 patients with special reference to histological grading. Am J Surg Pathol. 2001; 25: 835-845

Competing interests None declared

Reference

of Salivary Gland Tumors: A 5 Year Indian Experience International Journal of Oral & Maxillofacial Pathology. 2012;3(1):15-22

1. Reichart P., Sidransky D. World Health Organization Classification of Tumours. Pathology and Genetics of Head and Neck Tumours. Lyon: IARC 2. Vineet Gupta, Pratibha Ramani, Chandrasekar T .A Clinico-Pathological and Immunohistochemical Study

7. PM Speight, AW Barrett. Salivary glands and Saliva.

Oral diseases (2002) 8, 229â&#x20AC;&#x201C;240 8. Auclair PL.Tumor-associated lymphoid proliferation in the parotid gland. A potential diagnostic pitfall. Oral Surg Oral Med Oral Pathol. 1994 Jan;77(1):19-26.

http://www.pacificejournals.com/aabs

Annals of Pathology and Laboratory Medicine Annals of Pathology and Laboratory Medicine (APALM) is an international, Double-blind peer-reviewed, indexed, open access, online and print journal with an Impact Factor (IFJ): 2.8952 and IC Value (ICV 2014): 67.16

Annals of Woman and Child Health

Pacific Group of eJournals ISO 9001:2008 Certified academic publishing house

www.pacificejournals.com

Printed at: Allied Traders, New Delhi

Annals of Woman and Child Health (AWCH) is an International, Fast Double-blind peer-reviewed, Indexed, Open access, online multidisciplinary journal, related to woman and child health