AABS 3.4 (2016) by PaGe

eISSN: 2349-6991 pISSN: 2455-0396

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

Vol. 3, Issue 4, October-December 2016

DOI : 10.21276/aabs

Pacific Group of eJournals

Editor-in-Chief Dr Shelly Sehgal Dr Dipti Agrawal

Annals of Applied Bio-Sciences Co-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

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Annals of Applied Bio-Sciences 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.

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Table of Contents

Review Articles Microbial cellulases in Industrial applications Original Article

Case Report

Letter to editor

R23-R29 Rajendra Singh, Manoj Kumar, Anshumali Mittal, Praveen Kumar Mehta A Study of comparison of efficacy and safety of intravenous iron sucrose with and A267-A272 without erythropoietin versus blood transfusions in patients with severe iron deficiency anemia. Sakul Sakul, Mega Lahori, Anil Kumar Gupta, Sanjay Bhat, Puneeta Gupta Opportunities of Habitat Connectivity for Tiger (Panthera tigris) between Pench A273-A278 Tiger Reserve and Navegaon-Nagzira Tiger Reserve in Maharashtra, India Rohan Balaram Bhagat, Sanjay Karkare, Saurabh Dande, Gaurav Kadu Modification of antifungal susceptibility testing for Aspergillus species A279-A282 Manga Sirisha Madireddy, Rajasekhar Koppada, K.R.L. Surya Kirani, Rama Krishna Pilli

Results of prompt intravitreal injections in cluster endophthalmitis following A283-A285 cataract surgery Vartika Sobat Anand, Balbir Khan, Meenu Kashyap, Sachin Anand Serum MicroRNA-122 as A Prognostic Biomarker in Patients with Liver A286-A295 Cirrhosis Jehan Hassan Sabry, Osama Saad El-Shaer, Inas Abd El Mneam Ahmed, Ebada Mohamed Said, Amr Mohamed El Hammady, Abdelmoneam Ahmed Abdelmoneam, Asmaa Adel EL- Fallah Spectrum Of Adolescent Tuberculosis in a Tertiary Care Hospital at Shimla, North A296-A300 India. hayam Lal Kaushik, Savita Krishnamurthy, Neelam Grover, Rajni Kaushik Squamous Cell Carcinoma Vulva in a Young Woman C60-C63 Parmjit Kaur, Ruby Bhatia, Aman Dev Singh, Ramiti Gupta Adenomatoid Tumor of Epididymis: A Case report with correlation between histology C64-C67 and cytology Mega Lahori, Sakul Gupta, K C Goswami, Arvind Khajuria A Rare Case of Non Communicating Rudimentary horn with Unicornuate C68-C71 uterus. Puja Jain, Sunita Fotedar, Suman Raje, Rekha Daral Rare site of Metastasis in Cancer Cervix: a Case Report C72-C74 Sweta Khanuja, Vijay Anand Central serous chorioretinopathy leading to sub retinal bleed to postvitrectomy endophthalmitis: Diagnostic Dilemma Balbir Khan, Vartika Sobat Anand, Sachin Anand, Meenu Kashyap Solitary lymph node involvement by Langerhans Cell Hitisocytosis: Cytomorphologic diagnosis and pitfalls on Fine Needle Aspiration Cytology Chhaya Gupta, Neeru Gupta Folic acid supplementation in pregnancy: Hitting many birds with one stone Ajitha Sharma

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III

C75-C77 C78-C81 L5

Review Article Microbial Cellulases in Industrial Applications Rajendra Singh1, Manoj Kumar1, Anshumali Mittal2* and Praveen Kumar Mehta3* Department of Biochemistry, VP Chest Institute, University of Delhi, Delhi-110007, India Division of Structural Biology and Biophysics, Mill Hill laboratory, The Francis Crick Institute, London, UK 3 Centre for Molecular Biology, Central University of Jammu, Jammu -181143, (J&K) India 1

Keywords: Cellulose, Industrial Applications, Enzymes

ABSTRACT Cellulose, most copious constituent of plant cell wall and a renewable resource, is of considerable economic importance due to its potential applications in production of various bioenergy and bio-based products. Cellulose is used as a food source by the wide range of microorganisms and animals. Cellulose degrading enzymes are utilized in numerous applications in several industries, such as biofuel production, food and feed industry, brewing, pulp and paper, textile, laundry, and agriculture. Cellulose degrading enzymes containing microbes are widely spread in nature and isolated from different environments. In this communication we are presenting an overview of microbial cellulases used in different industrial applications.

*Corresponding author: Praveen Kumar Mehta, Centre for Molecular Biology, Central University of Jammu, Jammu -181143, (J&K) India E-mail: mehtapkbiotech@gmail.com Anshumali Mittal, Division of Structural Biology and Biophysics, Mill Hill laboratory, The Francis Crick Institute, London, UK E-mail: anshumalijnu@gmail.com

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

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Introduction

Cellulose is the most abundant organic molecule on the Earth and is the primary structural component of plants. It is fibrous, insoluble and high molecular weight homopolymer of anhydrous glucose units linked by the β-1, 4 glycosidic linkages. It is the major constituent of the lignocellulosic biomass which is an inexpensive and inexhaustible renewable organic material in the nature and has significant potential as alternative source of fuel and bio-based chemicals. In the lignocellulosic biomass, the cellulose fibers are embedded in a matrix of different biopolymers including hemicelluloses and lignin [1]. Structurally, long chains of cellulose are bundled together by numerous cross linkages, such as hydrogen and vander Waal interactions to pack cellulose into microfibrils. The parallel arrangement of cellulose chains result in the development of crystalline region whereas amorphous region of cellulose structure is due to less ordered arrangement of chains [2, 3]. The degree of polymerization of cellulose chain is highly variable ranging from 250 to 10000 and it influences the physiological, mechanical and biological properties of the cellulose. The length of cellulose chain or degree of polymerization depends on the source of material & treatment methods [4-7]. It is the chief constituent in the lignocellulosic biomass and also regarded as the strongest potential candidate for sustainable fuel production due to its environment friendly characteristics, such as renewability, biocompatibility and biodegradability [8, 9]. The cellulases are the third most significant commercial enzyme in the world market. These enzymes are important and essential for hydrolyzing the cellulose into fermentable sugars that can be used for further applications. The cellulose hydrolysis is chiefly carried out by a multi-enzyme system comprising endoglucanase, exoglucanase, and β-glucosidase [10, 11]. Endoglucanase (EC 3.2.1.4; endoβ-1,4-D-glucanase) acts in a random manner on internal but accessible sites in the cellulose chains and hydrolyze β-1,4 linkages to generate oligosaccharides of different lengths with new chain ends. In addition, endoglucanase

also act on cellodextrin and convert them to cellobiose and glucose [12, 13]. Exoglucanase (EC 3.2.1.91; exo-β-1,4-Dglucanase, cellobiohydrolase) acts in a progressive manner on the reducing and non-reducing ends of the cellulose polysaccharide chain to release glucose or cellobiose as major products [13]. β-glucosidase (EC 3.2.1.21) hydrolyse the soluble cellodextrins and cellobiose to glucose units [13, 14]. These three proteins work synergistically and catalyze appropriate hydrolysis for obtaining glucose residues which are used for various applications including production of biofuel, feed stock, single cell protein, and chemicals etc. [3, 13, 15]. Cellulases are inducible enzymes, which are produced by a wide array of microorganisms including fungi and bacteria. Among the microbes, fungi are the major producers of cellulase and accounts for approximately 80% of the cellulose hydrolysis on the Earth [16]. Primarily, ascomycota, basidiomycota and deuteromycota members of the fungi are possessed with efficient cellulolytic activities. Cellulases derived from aerobic fungal microorganism are preferred widely for industrial applications as they are extracellular and secreted in bulk during growth [17]. Trichoderma reesai is the most widely studied fungus and has the ability to convert desired as well as native celluloseto glucose [18]. In addition, fungal species of Aspergillus, Humicola, Penicillum and Sclerotium are considered potential candidates for industrial production of cellulolytic enzymes [11, 19]. Actinomycetes genera such as Cellulomonas, Streptomyces and Thermomonospora have also been involved in the production of cellulolytic enzymes.

Applications

Cellulases, solely or in a mixture with other enzymes, are involved in several industries including biofuel, food, feed, beverages, paper, textile, pharmaceutical, agricultural etc (Table 1.). Enzyme mediated hydrolysis of cellulosic biomass result in the generation of sugars that serve as the starting materials for the production of various value added products of commercial interest, such as bioethanol, organic acids, sugars and animal feeds [11, 20].

Table 1: Applications of cellulases in different industries [6, 10, 11, 21, 22]. Industry Biofuel Food/animal feed processing Textiles Paper and pulp Agriculture Medical others

Applications Production of ethanol, solvents and organic acids; production of energy-rich animal feed and with improved nutritional value Improved yield and extraction of fruit and vegetables juices, clarification of fruit juices, improved maceration, color extraction of fruit and vegetables Biopolishing, biostoning, biofinishing Co-additive in pulp bleaching, improved draining, de-inking Control of plant pathogens and disease, improved soil quality Antibiofilm agent, treatment of phytobezoar Reduction in biomass waste, extraction of olive oil and carotenoids

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Food

Cellulolytic enzyme, solely or in combination with other enzymes, have find significant recognition in food industry. Cellulases are widely used in the preparation of fruit and vegetables juices, animal feed production and in alcohol based industries [20, 23]. The Macerating enzymes, a combination of cellulases, hemicellulases and pectinases, are added to improve process performance and yield to enhance cloud stability & texture and to reduce viscosity of nectars and puree obtained from fruits such as apricot, peach, plum, papaya. The macerization facilitates improved extraction, clarification, stabilization and yield of fruit and vegetables juices [10, 24, 25]. In juice processing, cellulases from Aspergillus and Trichoderma of fungal origin and Bacillus and Paenibacillus of bacterial origin are potentially used for juice clarification [26]. Cellulases are also utilized for extraction of the flavonoids from flowers and seeds. Cellulase mediated extraction is preferred over conventional extraction methods due to increased yield, low process time and less heat damage [11, 27]. They are also involved in the extraction of phenolic compounds from grape pomace [23, 28]. A combination of enzymes including cellulases is used to reduce bitterness of citrus fruits and, consequently, yield products with improved taste and aroma [10, 11]. Î˛-glucosidase and pectinases are utilized in a combination to improve texture, flavour and other sensory properties of fruits and vegetables [23, 28]. Cellulases have also found application in animal feed industry. They are added to monogastric animal feed for improving the digestibility of cereal based food and nutritive value [29-31]. Cellulases from Bacillus subtilis can be used for soya bean hull degradation to enrich its nutritional value for monogasrtic animal feed animals [23, 32]. In wine and beer industry, cellulases are involved in fermentation processes to improve quality and yield of the products. Glucanase is used in mashing process that reduce viscosity of wort and enhance filterability. A mixture of cellulases, hemicellulases, and pectinases is used in wine industry for improving colour extraction, easy clarification & filtration and stabilization. Î˛-glucosidase is utilized to improve the sensory property such as aroma of wine products [10].

AABS; 3(4): 2016 different phase of fabric finishing. Scouring i.e. removal of impurities within raw cotton is carried out more effectively using cellulases in combination with pectinases, proteases and lipases where cellulases hydrolyze the cellulosic surface components and facilitate other enzymes to get in and react on their specific non cellulosic components for fabric finishing [20, 33]. Cellulase mediated wool scouring/pretreatment of wool replaces the conventional use of sulfuric acid or hydrochloric acid and requirement of high temperature (100-110Â°C). Enzyme mediated scouring helps in less emission of CO2 and saving of approximately 20,000 liters of water per ton of fabric [34]. Endocellulases and acid cellulases are used for biopolishing and carbonization of fabrics, respectively. Cellulases utilization for denim finishing results in minimizing use of stone, damage to denim and water pollution. Enzyme mediated carbonization is non-corrosive, non-hazardous and ecosafe. Acid cellulases are employed for shade correction attained by disperses or reactive blends of cotton with polyester [33]. The enzyme based processing in textile industries are economic, eco-friendly, non-hazardous with minimal water consumption. The applications of cellulases have been recognized widely in cellulose based textiles for their advantages over conventional methods as well as for quality improvement and fabric care [10]. Cellulases from microorganisms are efficiently involved for substituting pumice stones for bio-stoning and removal of excess dye to provide softness and faded look to denim. In addition, these cellulolytic enzymes are also used to degrade protruding fiber ends from the fabric for an improved finishing, softening and for colour gradient [11, 35]. Most commercially utilized cellulases in textile industry are derived from fungal microbes, Aspergillus niger and Trichoderma reseei [23]. Cellulases are utilized in detergents and added to laundry detergents to improve fabric softness and brightness. In many formulations, cocktail of different enzymes including protease, amylase, cellulase and lipase are also used for improved washing effect for household purposes [36]. Thermostable cellulases produced by Aspergillus, Trichoderma and Humicola are generally added to enzyme detergent formulations [11].

Paper

Cellulolytic enzymes are utilized in textile processing for effective substitution of conventional chemical methods. Enzyme based processing are more economic, ecofriendly and energy saver. In textile industry, cellulases are employed for scouring, polishing, carbonization and stone wash. Biotechnological development extended the range of application in acid, alkali and neutral medium during

Cellulose degrading enzymes are versatile and can be utilized effectively in paper and pulp industry to replace non eco-friendly conventional methods. Cellulases are used for a wide variety of uses, such as in biomechanical pulping, de-inking, and drainage management, manufacturing biodegradable paper towels, sanitary paper and cardboards. In addition, cellulase mediated processes are more economical, energy saving and low chlorine consuming. They are used as co-additive in pulp bleaching and for biomechanical pulping [10, 22].

Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

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

Textile Industry

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The mechanical pulping methods include grinding and refining of the plant materials resulting in the pulp with high bulk and stiffness whereas cellulase mediated pulping is considerably energy saver and with improved physical properties including inter-fibre bonding and mechanical strength of the product [21, 37, 38]. The recycling of waste papers results in less solid waste and reduces deforestation required to make new paper products. A combination of cellulase and hemicellulase is used for deinking of waste papers that improve the quality of recycled papers [39-41]. The enzymatic deinking leads to minimal or negligible use of alkali, enhanced brightness and strength properties. In addition, enzyme mediated deinking prevent alkaline yellowing and adverse effect on the environment. The cellulase treatment is used to remove fibrils and colloidal substance to improve the drainage problems is paper mills. Besides, cellulases have also find applications in the manufacturing of soft papers and biodegradable cardboards [10, 42-44]. Conversion of Lignocellulosic Biomass into Ethanol Continuous and increased use of fossil fuel will leads eventually to the depletion of fossil fuel reserves as well as increase environmental pollution by releasing toxic gases including greenhouse gases. In the light of fuel crisis and environmental concern, biotechnological development in search of alternative fuels have been accepted and recognized widely. Naturally occurring starch and cellulose have been strongly considered as the potential source of alternative biofuels [11, 36]. Lignocellulosic biomass is available in huge amount in many forms, such as agricultural and forestry residues and wastes generated from different industries including solid municipal wastes. Whereas the principal sources of starch for fuel production are cultivated crops such as maize, tapioca, potato, wheat, oats, barley etc. In addition to huge cultivation cost, utilization of starch containing crops may also lead to the competition with starch based food supply to continuously growing population [3, 9, 45]. Use of lignocellulosic biomass to obtain biofuels is more advantageous than starch due to its easy and low cost availability. The wastes generated every year in huge amount can be utilized for the production of value added products. The production of lignocellulosic biomass is cost effective as well as faster in comparison to other agricultural feedstock, such as corn, sugar cane and soybeans [46-48]. Being abundant and outside the human food chain, makes lignocellulosic materials relatively inexpensive feedstock for ethanol production [49, 50]. The degradation of lignocellulosic biomass is an expensive process and it requires three steps:

physiochemical pretreatment, enzymatic hydrolysis and fermentation [51]. Enzyme mediated processes are specific, low energy consuming and environmental friendly and hence, Cellulases mediated hydrolysis due to its enzymatic nature is preferred over acid/alkali chemical methods. The conversion of biomass to fuel involves hydrolysis of constituents of biomass to fermentable sugars, followed by sugar fermentation to ethanol using appropriate microorganism. The first step involves degradation of the lignocellulosic polymer, deligniďŹ cation to release cellulose and hemicellulose content, followed by hydrolysis of carbohydrate polymers to produce free sugars [46, 52]. According to an evaluation, there is about 40% cost reduction if cellulases are used for bioprocessing and pretreatment [1]. An anaerobic thermophilic bacterium, Clostridium thermocellum, has significant potential to hydrolyze cellulose and, at the same time a potential to ferment sugar to ethanol. This microbe offers many advantages, such as higher growth rate, improved enzyme stability, and the recovery of products is also easier [46, 53]. Saccharomyces cerevisiae has been conventionally utilized for the fermentation of sugars, obtained by cellulosic hydrolysis, to produce ethanol [54, 55].The hexose sugars, such as glucose, galactose, and mannose are readily fermented to alcohol whereas pentoses, such as xylose and arabinose require additional efforts for their hydrolysis to achieve higher production of cellulosic biofuels and other products. In recent years, metabolic engineering of microorganisms has shown significant progress in the efficiently fermentation of hydrolysates containing xylose and arabinose [56-58]. Cellulases produced by various filamentous fungi including Aspergillus, Trichoderma, and Penicillium have been used widely for bioconversion of lignocellulosic biomass into biofuel and others derivatives [11, 59].

Other Applications

Carotenoids, organic pigments, are preferred and used as food colorants owing to natural origin, high versatility, lipo- as well as hydrophilic and negligible toxicity. Solvent extraction method may dissociate pigments from the proteins and may cause water insolubility and their oxidation. Whereas enzyme mediated extraction of pigments, using a mixture of cellulases and pectinolytic enzymes, keep them in the natural state and attached to proteins. Natural state pigments are equipped with desired properties to be used as food colorants [10, 60, 61]. A combination of cellulases, hemicellulases and pectinases has also been used during olive oil extraction to improve oil yield, and maintaining high level of antioxidant and

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AABS; 3(4): 2016

vitamin E content [62]. They are also used to reduce viscosity of oil paste and strengthen the extraction of polyphenolic substances from the olive fruits [10, 63].

2. Saha BC. α-L-Arabinofuranosidases: biochemistry, molecular biology and application in biotechnology. Biotechnol Adv 2000; 18: 403-423.

Cellulolytic enzymes from fungal microbe Aspergillus, Chaetomuim and Trichoderma and actinomycetes have also been used to improve soil quality by decompositing soil cellulose and consequently, reducing the dependency on the mineral fertilizers [10, 64, 65]. Enzyme mixtures containing cellulases, hemicellulases and pectinases have applications in enhancing growth of crops and controlling plant diseases by killing plant pathogens [21, 66].

3. Li X-h, Yang H-j, Roy B, et al. The most stirring technology in future: Cellulase enzyme and biomass utilization. Afr J Biotechnol 2009 8.

In medicinal field, cellulases are used for the treatment of phytobezoars and as antibiofilm agents. Fungal cellulases are utilized to cure phytobezoars, a trapped concretion of indigestible plant materials in the gastrointestinal tract. Bacterial cellulases are potentially degrading cell wall of pathogenic Acanthamoeba, which is responsible for blinding keratitis as well as granulomatous amoebic encephalitis. Cellulase can potentially degrade cellulose component of biofilms and consequently, restrict distribution of pathogenic organisms and drug accessibility to them [23, 67, 68].

5. Klemm D, Heublein B, Fink HP, et al. Cellulose: fascinating biopolymer and sustainable raw material, Angewandte Chemie International Edition. 2005; 44: 3358-3393.

Conclusion

Cellulolytic enzymes are potentially utilized in a wide range of industries due to hydrolytic action on cellulosic biomass. Their activity on biomass lead to the production of fermentable sugars which are further used as a raw material for the production of several value added products, such as biofuel including ethanol, organic acids, sugars etc. Though a wide range of microorganisms have been reported for cellulase production but their application in industries is hindered by the production cost and the low yield. Hence, biotechnological developments to improve production efficiency at a competitive cost are needed to utilize microbial enzyme systems that can have significant industrial impact.

Conﬂict of Interest

The authors declare that they have no conﬂict of interest in the publication.

Funding None

Competing Interests None Declared

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38. Singh A, Kuhad RC, Ward OP. Industrial application of microbial cellulases, Lignocellulose Biotechnology: Future Prospects. 2007; 345-358. 39. Jeffries TW, Klungness JH, Sykes MS et al. Comparison of enzyme-enhanced with conventional deinking of xerographic and laser-printed paper. Tappi journal 1994; 77: 173-179. 40. Lee C, Darah I, Ibrahim C. Enzymatic deinking of laser printed office waste papers: Some governing parameters on deinking efficiency. Bioresour Technol 2007; 98: 1684-1689. 41. Ibarra D, Monte MC, Blanco A, et al. Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system. J Ind Microbiol Biotechnol 2012; 39: 1-9. 42. Kantelinen A, Jokinen O, Sarkki M, et al. Effects of enzymes on the stability of colloidal pitch, in: Proc. 8th Int Symp Wood and Pulping Chemistry, 1995, pp. 605-612. 43. Hsu JC, Lakhani NN. Method of making absorbent tissue from recycled waste paper, in, Google Patents, 2002. 44. Buchert J, Oksanen T, Pere J, et al. Applications of Trichoderma reesei enzymes in the pulp and paper industry, Trichoderma and gliocladium. 1998; 2:343-363.

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45. Cherubini F. The biorefinery concept: using biomass instead of oil for producing energy and chemicals, Energy Conversion and Management. 2010; 51: 1412-1421. 46. J. Pérez, J. Munoz-Dorado, T. de la Rubia, J. Martinez, Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. ‎Int Microbiol 2002; 5: 53-63. 47. Huber G. Breaking the chemical and engineering barriers to lignocellulosic biofuels: a research roadmap for making lignocellulosic biofuels a practical reality, in: NSF, DOE and American Chemical Society Workshop, 2007. 48. Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review, International journal of molecular sciences. 2008; 9: 1621-1651. 49. Wyman CE, Dale BE, Elander RT, et al. Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover, Bioresour technol 2005; 96: 2026-2032. 50. Chan E, Rudravaram R, Narasu ML, et al. Economics and environmental impact of bioethanol production technologies: an appraisal. Biotechnology and Molecular Biology Reviews 2007; 2: 14-32. 51. Abril D, Abril A. Ethanol from lignocellulosic biomass. Ciencia e investigación agraria 2009; 36 163-176. 52. Lee J. Biological conversion of lignocellulosic biomass to ethanol, ‎J Biotechnol 1997; 56: 1-24. 53. Shoham Y, Lamed R, Bayer EA. The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. Trends in microbiology 1999; 7275-281. 54. Jeffries T, Jin Y-S. Metabolic engineering for improved fermentation of pentoses by yeasts, Appl Microbiol Biotechnol 2004; 63: 495-509. 55. Mosier N, Wyman C, Dale B, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour technol 2005; 96: 673-686. 56. Becker J, Boles E. A modified Saccharomyces cerevisiae strain that consumes L-arabinose and produces ethanol. Appl Environ Microbiol 2003; 69: 4144-4150.

57. Kumar P, Barrett DM, Delwiche MJ, et al. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production, Industrial and engineering chemistry research. 2009; 48: 3713-3729.

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58. Öhgren K, Bengtsson O, Gorwa-Grauslund MF, et al. Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. J biotechnol 2006; 126: 488-498. 59. Adrio JL, Demain AL. Microbial enzymes: tools for biotechnological processes, Biomolecules 2014; 4: 117-139. 60. Çinar I. Effects of cellulase and pectinase concentrations on the colour yield of enzyme extracted plant carotenoids. Process Biochem 2005; 40: 945-949. 61. Bassi R, Pineau B, Dainese P, et al. Carotenoid�binding proteins of photosystem II Eur J Biochem 1993; 212: 297-303. 62. Galante Y, De Conti A, Monteverdi R, Application of Trichoderma enzymes in the food and feed industries, Trichoderma and Gliocladium 1998; 2 327-342. 63. Ranalli A, Pollastri L, Contento S, et al. Enhancing the quality of virgin olive oil by use of a new vegetable enzyme extract during processing, Eur Food Res Technol, 2003; 216: 109-115. 64. Bowen R, Harper S. Decomposition of wheat straw and related compounds by fungi isolated from straw in arable soils, Soil Biology and Biochemistry 1990; 22: 393-399. 65. Fontaine S, Bardoux G, Benest D, et al. Mechanisms of the priming effect in a savannah soil amended with cellulose. Soil Science Society of America Journal 2004; 68: 125-131. 66. Chet I, Benhamou N, Haran S. Mycoparasitism and lytic enzymes, Trichoderma and gliocladium, 1998; 2: 153-172. 67. Martinez AJ, Visvesvara GS. Free�living, amphizoic and opportunistic amebas, Brain Pathology, 1997; 7: 583-598. 68. Loiselle M, Anderson KW. The use of cellulase in inhibiting biofilm formation from organisms commonly found on medical implants, Biofouling 2003; 19: 77-85.

Original Article A Study of Comparison of Efficacy and Safety of Intravenous Iron Sucrose with and Without Erythropoietin Versus Blood Transfusions in Patients with Severe Iron Deficiency Anemia. Sakulm1*, Mega Lahori2, Anil Kumar Gupta1, Sanjay Bhat1 and Puneeta Gupta1 Department of Medicine, Acharya Shri Chander College of Medical Sciences Jammu (J&K), India Department of Pathology, Acharya Shri Chander College of Medical Sciences Jammu (J&K), India

1 2

Keywords: Iron, Anemia, Iron Sucrose, Erythropoietin, Blood Transfusion, Hemoglobin

ABSTRACT Objective: This study was undertaken to compare the efficacy and safety of intravenous iron sucrose with and without erythropoietin versus blood transfusions in raising the hemoglobin level in patients with severe iron deficiency anemia. Study Design: Sixty patients with severe iron-deficiency anemia were randomly assigned to receive intravenously iron sucrose plus recombinant human erythropoietin or iron sucrose alone thrice weekly. Target hemoglobin value was 11.0 g/dL. Efficacy measures were reticulocyte count, increase in hematocrit, and time to target hemoglobin level. Safety profile and cost of therapy was also evaluated. Results: Both regimens were effective, but with adjuvant recombinant human erythropoietin the reticulocyte counts were higher from day 4 (P<.01), increases in hematocrit were greater from day 4 (P <.01), and the median duration of therapy was shorter, with more patients reaching the target hemoglobin earlier. The groups did not differ with respect to adverse effects during therapy. Conclusion: Adjuvant recombinant human erythropoietin safely enhanced the efficacy of iron sucrose in the treatment of severe iron-deficiency anemia but increased the cost of therapy.

*Corresponding author: Dr Sakul, House, No 66, Ward No. 10, Lambi Gali, Udhampur (Jammu & Kashmir, India) Pin code: 182101, Phone: +91 9419216580 Email: sakul68@gmail.com

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

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Introduction

Anemia is defined as a reduction in the number of circulating erythrocytes. It is a common manifestation of primary bone marrow disorders (resulting in impaired production of erythrocytes), primary abnormalities of erythrocytes (resulting in an increased rate of destruction), immunologic disorders, nutritional deficiencies, and a broad spectrum of systemic diseases that secondarily result in anemia1. Iron deficiency occurs as a late manifestation of prolonged negative iron balance and its progression can be divided into 3 stages-Negative iron balance, iron-deficient erythropoiesis and iron-deficiency anemia2. Iron deficiency anemia (IDA) is considered to be among the most important contributing factors to the global burden of disease3. Globally, the most significant contributor to the onset of anemia is iron deficiency so that IDA and anemia are often used synonymously. It is assumed that 50% of the cases of anemia are due to iron deficiency, but the proportion may vary among population groups. Nearly half of the pregnant women in the world are estimated to be anemic: 52% in non-industrialized - as compared with 23% in industrialized countries. In India, for example, up to 88% of pregnant and 74% of non-pregnant women are affected4. Definitive diagnosis requires laboratory tests5. Measurement of hemoglobin or hematocrit is the most cost efficient and commonly used method to screen for anemia. A low serum ferritin (<15 ug/L), in addition to a low hemoglobin or hematocrit, confirms the diagnosis of iron deficiency anemia. However, ferritin levels may be normal or elevated when iron deficiency and infection, chronic inflammation, malignancy or conditions causing organ or tissue damage (e.g., arthritis, hepatitis) occur simultaneously. An elevated serum transferrin receptor concentration (TfR) (>8.5 mg/L) is an early and sensitive indicator of iron deficiency. It is, however, also raised in Thalassemia and hemolytic anemias6. Severe anemia is diagnosed when hemoglobin concentration is less than 7.0g/dl7. Treatment: The traditional approach to anemia has been oral supplementation or blood transfusions. Oral iron therapy, although effective in most cases8, may be limited in many cases due to dose dependent side effects, lack of compliance and insufficient duodenal absorption 9, 10 . Hence, parenteral iron therapy becomes important. Iron-Dextran is the most stable, allowing the total dose to be given at one time11. However, there is a small but significant incidence of anaphylaxis associated with it, which has convinced many practitioners to use either of two alternative preparations â&#x20AC;&#x201C;sodium ferric gluconate12 and iron sucrose13. Rare anaphylactic reactions have been reported with iron sucrose, but they appear to be less

frequent than those with iron dextran. The total dose is calculated from the following formula: Iron to be injected (mg) = weight (kg) x [normal hemoglobin Value â&#x20AC;&#x201C; actual hemoglobin value (g/dl)] x 2.4 + 500mg Blood transfusions are usually that last resort in treatment of iron deficiency anemia but are frequently used in symptomatic patients. There are no definitive guidelines but according to the available literature, usual threshold hemoglobin level for it is 6-7 gm/dl14. Each unit of Red Blood Cells contains approximately 147-278 mg of iron. Hemoglobin equilibrates in 15 minutes after RBC transfusion15. One unit will increase the Hb level in an average-sized individual by approximately 1 g/dl and the Hct by 3%. Erythropoietin (EPO): During the last decade many new and exciting functions have been attributed to EPO and many of these are related to non-erythropoietic effects16. Several functions i.e. inhibition of inflammation and apoptosis, anti-oxidant effects and stimulation of angiogenesis, may be of potential use. Along with potentially beneficial effects attributed to EPO, there are reports indicating that the mortality and morbidity rates are increased in some patient groups17, 18, 19. rhEPO has a direct action on the endothelium increasing the reactivity of the underlying extracellular matrix towards platelets, effect that may be the cause of its thrombogenic potential20. The main mechanism for the increase in hematocrit was long thought to rely exclusively on augmentations in red cell mass, but it was demonstrated that EPO also decreases plasma volume21. The clinical response to IV iron may be attributed to the effect of EPO therapy on iron mobilization from the reticuloendothelial system into RBC precursors22. The success of EPO therapy in correcting the anemia of chronic renal failure has led to substantial clinical experience in iron therapy and erythropoiesis in this setting23. IV iron administration is used commonly in hemodialysis patients undergoing EPO therapy24. Various controlled studies in the past have investigated the efficiency of intravenous iron sucrose with or without erythropoietin in patients of renal failure, pregnant females and cancer associated anemias. In the present study, we will investigate the efficacy of intravenous iron sucrose with or without erythropoietin in patients of severe iron deficiency anemia. The measures of efficacy will be reticulocyte count, increase in hematocrit, time to target hemoglobin and we will evaluate the safety profile and cost of I.V. iron, erythropoietin and blood transfusion.

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Materials And Methods

The study population included 60 patients of both sexes attending the indoor of the Department of Medicine, Acharya Shri Chander College of Medical Sciences and Hospital, Sidhra, Jammu. The subjects were aged between 17 and 62 yrs. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. Criteria For Selection: 1. Hemoglobin concentration<7g/dl 2. PBF showing a microcytic hypochromic picture 3. Serum ferritin<12mcg/dl

AABS; 3(4): 2016 significant. Mean values for individual groups were determined by pooling the results of individual patients and dividing it by 20 (no. of observations). Statistical Analysis: It was done using ANOVA (analysis of variance) and p values were determined. A “p” value < 0.05 was considered statistically significant, <0.01 highly significant, <0.001 very highly significant and <0.0001 extremely significant.

Results

Out of 60 patients, 26 (43%) were males, 34 (57%) were females. In group 1 there were 9 (45%) males and 11 (55%) females. In group 2 also 45% males and 55 % females and in group 3, 8 (40%) were males and 12 (60%) were female patients.

Data are presented as mean ± 1 S D or percentage when appropriate. A “p” Value of < 0.05 was considered

In group A mean age was 37.2 ± 13.5 years and in group B and group C it was 36.45 ± 11.6 and 38.85 ± 11.79 years respectively. The difference in age was nonsignificant (p-value 0.883) between these groups. The difference in baseline parameters was not significant (p-value > 0.05) between these groups. In both the groups hemoglobin levels increased significantly from day 4 (p value < 0.0001). The increment in hemoglobin values continued in both the groups till the end of therapy. However, hemoglobin values were higher in group 2 compared to group 1 on all the days (day 4, 7, 14, 21, 28, 35, and 42). As compared to group 1, group 2 patients had significantly higher increments in hemoglobin throughout the duration of therapy. The difference between two groups was very highly significant (p value < 0.001) at day 7 and highly significant (p value < 0.01) on rest of the days. Reticulocyte count started increasing in both the groups as early as day 4 of therapy. However as compared to group 1, group 2 patients had significantly higher counts (p value < 0.0001) i.e. extremely significant at day 4, day 7 and day 14 of therapy (Table 1). In both the groups hematocrit levels increased significantly from day 4 (p value < 0.0001). The increment in hematocrit values continued in both the groups till the end of therapy. However, hematocrit values were higher in group 2 compared to group 1 on all the days (Table 2). As compared to group 1, group 2 patients had significantly higher increments in hematocrit throughout the duration of therapy. The difference between two groups was extremely significant (p value < 0.0001) at day 4, 7, 14, 21, 28 and 35 and significant (p value < 0.01) at day 42. At the end of the treatment there was a significant rise in serum ferritin values in both the groups (p value < 0.0001) and serum ferritin values came in the normal range in both the groups. There was no statistical difference (p value > 0.05) in the serum ferritin values at the baseline or at the end of treatment among the two groups.

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Exclusion Criteria: 1. History of allergy to iron 2. Patients of anemia due to acute blood loss 3. Patients of chronic renal failure 4. Pregnant anemic females 5. Other causes of microcytic hypochromic anemia 6. Leukemias/Plasma call disorders/Cancers 7. Withdrawal of consent 8. Thalassemia minor After evaluation of all inclusion, exclusion criteria and informed consent, eligible patients entered a randomized parallel group study and were assigned to three treatment groups. The first group received intravenous iron sucrose at a dose of 200 mg thrice weekly for 2 weeks followed by switch to oral iron tablets of 100 mg daily. The second group received iron sucrose as above plus recombinant human erythropoietin in the dose of 6000 IU subcutaneously thrice weekly for two weeks. As above these patients were also switched to oral iron in the same dose after two weeks. The third group received whole blood transfusions on alternate days for 5 times. Patients in the group 1 and 2 were assessed before the start of treatment, then at day 4 and then once weekly, till the target hemoglobin was reached. Reticulocyte counts were measured on day 4, 7 and 14. Hemoglobin and hematocrit were measured on day 4 and then once weekly till the target hemoglobin (11g) was reached. Serum ferritin levels were measured at the baseline and the end of treatment. In group 3 hemoglobin and hematocrit values were checked on alternate days.

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Mean cost of treatment in group 1 was Rs. 1295 ±32 .19, in group 2 it was Rs. 6693 ± 28.63 and in group 3 Rs. 5240 ± 35. There is a significant difference (p value < 0.0001) in the cost of treatment among the three groups (Table 3). In group 1 only 1 patient (5%) reported an adverse reaction to treatment, while in group 2 and group 3, 10 & 20 % of

patients had some adverse effect as a result of treatment (Table 4). It took 43.3 ± 5.5 days in group 1 to reach the target hemoglobin of 11 g / dl while it required 36.6± 3.1 days in group 2 to reach the target hemoglobin level. Time to reach the target hemoglobin was significantly less (p value < 0.001) in group 2 compared to group 1 (Table 5).

Table: 1 Reticulocyte counts (mean ± SD) in % at day 0, 4, 7 and 14

Day 0

Mean 1.63

Day 4 Day 7 Day 14

2.50 3.41 3.54

Group 1

SD 0.44

Mean 1.59

0.28 0.31 0.37

3.09 5.30 4.69

Group 2

SD 0.44

P value 0.775

0.44 0.47 0.45

< 0.0001 < 0.0001 < 0. 0001

Table 2: Mean hematocrit values (%) ± SD at day 0, 4, 7, 14, 21, 28, 35 and 42 Group Time Baseline Day 4 P value Day 7 P value Day 14 P value Day 21 P value Day 28 P value Day 35 P value Day 42 P value

Group 1 Mean 18.67 20.93 < 0.0001 22.66 < 0.0001 26.29 < 0.0001 27.38 < 0.0001 29.42 < 0.0001 31 .20 < 0.0001 32.48 < 0.0001

Group 2 Mean 18.73 22.77 < 0.0001 26.47 < 0.0001 30.08 < 0.0001 31.03 < 0.0001 32.34 < 0.0001 33.47 < 0.0001 34.50 < 0.0001

SD 1.47 1.93 2.13 2.40 2.27 1.92 1.67 1.73

SD 1.33 1.70 2.29 2.58 2.40 2.28 2.02 1.76

Table 3: Total cost of treatment (Rs. ± SD)

Group 1 Group 2 Group 3

Mean 1295 6693 5240

Total cost of treatment

SD 32.19 28.63 35

Table 4: Incidence of side-effects No. of patients reporting side-effects No. % of total (20) 1 5 2 10 4 20

Group 1 Group 2 Group 3 Table 5: Time (Mean ± SD) in days required to reach target Hb

Days to reach target hemoglobin Mean

Group 1

43.3

5.5

Group 2

36.8

3.1

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Discussion

Effect of Adding Erythropoietin with Iron Sucrose on Hematological Parameters: In the present study, patients in second group were given a combination of iron sucrose and rhEPO. Addition of erythropoietin was associated with statistically significant (p value <0.0001) increase in hemoglobin values from 6.19 ± 0.47 to 7.35 ± 0.54, 8.39 ± 0.62, 9.31 ± 0.55 at day 4,7 and 14 respectively, which means there was a rise of 2.19 ±0.59 with in first week of treatment. Increase in Hb values at day 14 of treatment was 3.11 ± 0.55. Similar increments have been reported in previous studies25. Significant rise was also seen in hematocrit values, which increased from a baseline value of 18.73 ± 1.33% to 22.7 ± 1, 70%, 26.47 ± 2.29 % and 30.08 ± 2.585 at day 4, 7 and 14 respectively. This mean increase in hematocrit at day 7 of 7.7 ± 2.1% approximates to two blood transfusion units. Reticulocytosis was observed as early as 4th day of treatment with counts on day 7 of therapy being 5.3 ± 0.4% implying a brisk erythropoietin response. Patients were shifted to oral therapy with iron after 14 days of treatment. Target hemoglobin was reached in this group after 36.8 ± 3.18 days. Effects Of Treatment With Iron Sucrose Alone On Hematological Parameters: Hemoglobin increased from a baseline value of 6.18 ± 0.47 g / dl to 6.91 ± 0.59, 7.52 ± 0.66 and 8.69 ± 0.78 g/dl at day 4, 7 and 14 of treatment respectively. These increments were also consistent with previous studies. Similarly, hematocrit value was also raised to 26.29 ± 2.40% from the initial value of 18.67 ± 1.47% at day 14. Reticulocyte counts also increased during the first 7 days of therapy. It was 3.41 ± 0.31 at day 7 compared to 1.63 ± 0.44% at the start of treatment. Similar to group 2, these patients were also shifted to oral iron after 14 days. It took 43.55 ± 5.53 days to reach target hemoglobin value of 11 g / dl in this group. Effect on Serum Ferritin Levels: In both group 1 and group2, ferritin levels increased significantly from baseline value (p value < 0.0001). Both the treatments were effective in replenishing the iron stores as evident from normalization of the serum ferritin values by the end of treatment. Incidence of Adverse Effects: 2 patients experienced urticarial reaction with i.v. iron sucrose which was managed with anti-histamines and 1 patient developed irritation at the site of administration, discontinuation of therapy was not required. rhEPO has thrombogenic potential. Though due to financial constraints, we could not get any specific tests done; but clinically no patient in the rhEPO group developed any thrombotic complication. In the blood transfusion group, 2 patients developed febrile Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

AABS; 3(4): 2016 reactions requiring discontinuation of that unit of blood, 2 developed mild urticarial reactions which were managed with antihistamines. Efficacy Comparison Among Group 1 and Group 2: Rise of Hb and hematocrit in the rhEPO group were more at all days of follow up compared to group of patients receiving iron sucrose alone, difference of rise among the two groups being statistically highly significant (p <0.001 to <0.0001). Reticulocyte responses were also better in rhEPO group with the differences being statistically extremely significant at day 4 and 7 of therapy (p < 0.0001). Efficacy, Safety and Cost Comparison Among Erythropoietin and Blood Transfusion Group: There was a rise of 2.19 ± 0.59 g/dl within first 7 days of treatment, which is equivalent to rise seen with 2 units of blood transfusion. No adverse effects were attributed to rhEPO in the study while 4 patients in the blood transfusion group developed some adverse reaction. Cost of Treatment: Cost of treatment incurred on the patient receiving iron sucrose alone was approximately Rs.1295 ± 32.19. In the group receiving rhEPO, total cost of therapy was Rs.6693 ± 28.63. Five units of blood were transfused to each patient in group 3, which resulted in expenditure of approximately Rs.5240 ± 35 per patient.

Conclusion

We conclude that addition of rhEPO to I.V Iron was more effective in correcting anemia as estimated by the increase in reticulocyte count, hematocrit and hemoglobin level. Its use might be limited due to cost but if it can be used as a transfusion alternative, it has the potential to reduce the transmission of HIV, Hep B and Hep C and the risk of allogenic immunization which are responsible for heavy economic burden. It also appears to be an attractive option in patients who are Jehovah’s witnesses. Future studies are required to clearly specify the patients who are most likely to benefit from recombinant human erythropoietin therapy.

Funding None

Competing Interests None Declared

References

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concentration after transfusion in medical inpatients not actively bleeding. Ann Intern Med. 1994 Aug 15; 121(4):278-30. Jelkmann W. Erythropoietin after a century of research: younger than ever. Eur J Haematol. 2007 Mar; 78(3):183-205. Phrommintikul A, Haas SJ, Elsik M & Krum H. Mortality and target haemoglobin concentrations in anaemic patients with chronic kidney disease treated with erythropoietin: a meta-analysis. Lancet. 2007 Feb 3; 369(9559):381-8. Pfeffer MA, Burdmann EA, Chen CY, Cooper ME, de Zeeuw D, Eckardt KU, et al: TREAT Investigators. A Trial of Darbepoetin Alfa in Type 2 Diabetes and Chronic Kidney Disease. N Engl J Med 361, 2019-2032. Dicato M & Plawny L.Erythropoietin in cancer patients: pros and cons. Curr Opin Oncol. 2010 Jul; 22(4):307-11. Fusté B, Serradell M, Escolar G, Cases A, Mazzara R, Castillo R, et al.Erythropoietin Triggers a Signaling Pathway in Endothelial Cells and Increases the Thrombogenicity of their Extracellular Matrices In Vitro. Thromb Haemost. 2002 Oct; 88(4):678-85. Lundby C, Thomsen JJ, Boushel R, Koskolou M, Warberg J, Calbet JAL & Robach P. Erythropoietin treatment elevates haemoglobin concentration by increasing red cell volume and depressing plasma volume. J Physiol. 2007 Jan 1; 578(Pt 1):309-14. Weiss G, Gasche C. Pathogenesis and treatment of anemia in inflammatory bowel disease. Haematologica. 2010; 95(2):175-178. doi:10.3324/haematol.2009.017046. Eschbach JW, Egrie JC. Downing MR, Browne JK, Adamson JW. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med. 1987 Jan 8; 316(2):73-8. Adamson JW, Eschbach JW. Erythropoieitin for endstage renal disease. N Engl J Med. 1998 Aug 27; 339(9):625-7. Breymann C, Visca E, Huch R, Huch A. Efficacy and safety of intravenously administered iron sucrose with and without adjuvant recombinant human erythropoietin for the treatment of resistant irondeficiency anemia during pregnancy. Am J Obstet Gynecol. 2001 Mar; 184(4):662-7.

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Original Article Opportunities of Habitat Connectivity for Tiger (Panthera tigris) between Pench Tiger Reserve and Navegaon-Nagzira Tiger Reserve in Maharashtra, India Rohan Bhagat*, Sanjay Karkare, Saurabh Dande and Gaurav Kadu Conservation Department, Bombay Natural History Society, Mumbai, India Keywords: Connectivity, Panthera Tigris, Central India, Movement Pattern, Fragmentation, Corridor

ABSTRACT Habitat connectivity is essential in sustaining regional populations of Tiger (Panthera tigris), as they require contiguous forest connectivity for dispersal and genetic exchange between populations. An important conservation tool for these carnivores has been to understand connectivity of these fragmented habitats that have helped to identify critical threats to the existing populations. Wildlife corridors have long been a subject of discussion amongst wildlife biologists and conservationists with contrasting schools of thought arguing their merits and demerits. However, it is largely believed that wildlife corridors can help minimize genetic isolation, offset fragmentation problems, improves animal dispersal, restore ecological processes and reduce man animal conflict. This study attempted to evaluate the possibilities of identifying a suitable wildlife corridor between two very important wildlife areas of central India – Pench Tiger Reserve and Navegaon-Nagzira Tiger Reserve – with tiger as the focal species. Geographic information system (GIS), information collected from Forest Department and Local communities was used to identify likely routes for movement of tigers. Results indicate the movement pattern of tiger in the fragmented landscape on the basis of indirect signs and secondary information. It was also found that the potential corridor in fragmented area which is different from corridor marked by the forest department. Resultant maps, displaying bottle necks and weak points in the corridors, are marked to direct ﬁeld-based research and conservation efforts. Field assessment and reﬁnement of the corridors is ongoing. The establishment and proper management of linkages between the habitats is of great importance for future survival of tigers.

*Corresponding author: Rohan Bhagat, Conservation Department, Bombay Natural History Society, Hornbill House, Opp. Lion Gate,Shaheed Bhagat Singh Road, Mumbai-400 001, Maharashtra, India Phone: +91 9594190999 Email: panthera.rohan@gmail.com

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Introduction

The 21st century has brought many conservation challenges to the forest. One very important and significant challenge that has evoked considerable scientific interest is the fragmentation of wildlife habitat. Habitat fragmentation is an umbrella term describing the complete process by which habitat loss results in the division of large, continuous habitats into a greater number of smaller patches of total area, isolated from each other by a matrix of dissimilar habitats, and is not just the pattern of spatial arrangement of remaining habitat. Over a larger time span, species inhabiting isolated habitats also face the risk of extinction through mechanisms such as excessive inbreeding. [1][2][3] [4] It is the most important environmental issues of public concern and the worst threat to biological diversity. In the recent times, considerable scientific and media attention has been focused in India on large mammals – particularly large cats –and their conflict with man largely attributed to shrinking habitat.[5] Today, most wild tigers live in small, isolated Protected Areas within human dominated landscapes in the Indian subcontinent.[6] Future survival of tigers depends on increasing local population size, as well as maintaining connectivity between populations. While significant conservation effort has been invested in increasing tiger population size, few initiatives have focused on landscapelevel connectivity and on understanding the effect different landscape elements have on maintaining connectivity.[6] The idea of wildlife corridors was first time proposed by Wilson and Willis [7] for conserving biodiversity based on theory of island biogeography. A wildlife corridor has been defined as a ‘‘linear landscape element which serves as a linkage between historically connected habitat and natural areas, and is meant to facilitate movement between these natural areas.’’[8]Creation of wildlife corridors has received much global attention during the last two decades. While the utility of wildlife corridors has been debated,[9] it is largely believed that wildlife corridors facilitate animal dispersal from isolated habitats and help counter biological processes that lead to species extinction.[10][11] While the idea of connecting fragmented patches may appear simplistic at first sight, the identification, design and development of wildlife corridors in large landscapes presents unique challenges.Beier & Loe observed that the critical features of a wildlife corridor are not its physical traits but its functionality.[12]The importance of wildlife corridors for tiger conservation in India has also been significantly reiterated by Jhala, Qureshi, Gopal and Sinha[5]

The present study was undertaken to explore possibilities of establishing connectivity between wildlife areas – Pench Tiger Reserve, Maharashtra Navegaon Nagzira Tiger Reserve, Maharashtra in Central Indian Landscape

the two and the

Materials and Methods

Study Area: Pench Tiger Reserve is located in the SatpudaMaikal hills of Nagpur district. It covers an area of 257 sq. km. Nagzira-Navegaon forest is located in Bhandara and Gondia district. Because of dense forests it is rich in wildlife and has good number of tiger population. [5] Status of Connectivity in Study Area: Pench Tiger Reserve & Mansighdeo Wildlife Sanctuary is connected to the forests of Bhandara dist. which includes forests of Deolapar and Paoni range. It comprises of FDCM area which provides great opportunity to the animals for migration due to its connectivity with the Pench Tiger Reserve. The National Highway no. 7 passes through these forest patches which has very heavy vehicular traffic. Movement of many animals likes Tiger, Sloth bear, Chital and Jackel is affected due to this vehicular traffic. There are few small villages with agricultural fields & lakes with fragmented forest which has network of roads. The major forest type of this area is teak dominant. FDCM has cleared some area near Mogarkasa Lake for plantation. The forest of Lendezari Range is well flourished and has connectivity with Paoni range as well as Nakadongri range of Bhandara forest division. Due to Bawanthadi Irrigation Project huge water body is created in this area. Behind this water body, the forest is connected to Balaghat (MP) forest area. Nakadongri range has good forest patch with Chandpur Lake and further connectivity of Chandpur forest is broken by Wainganga River. Further there is a non-forested or degraded forest patch having 15kms aerial distance between Chandpur Forest and Nagzira Wildlife Sanctuary. Moreover according to the study conducted by Wildlife Conservation Trust in 2014 this area inhibits about 9 tigers. Study Aims and objectives: • To study presence and movement of carnivores and other mammals in the fragmented landscape • To demarcate the possible corridor between the protected areas. Methodology • Reconnaissance survey • Gathering secondary data from forest department on tiger presence, man animal conflict and cattle kills. • Questionnaire survey of villages in fragmented landscape.

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Fig. 1: Study Area Map.

(Source: Pench Foundation)

Fig. 2: Threats to the Corridor.

(Source: Pench Foundation)

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The basic data about the village which includes socioeconomic aspects and presence of wild animals was collected from any one of the person viz.; Gram Panchayat, Sarpanch, Gram Sevak, Police Patil, Peon.

Result & Discussion

The area is fragmented and at certain points the forest patch becomes too narrow creating a bottle neck to the connectivity of the path. There are 2 major bottle necks and 2 major threats as shown in Fig. 03 and Fig. 04. The major threats include the fragmented area between Bhandara Forest Division and Buffer area of Navegaon Nagzira tiger reserve and the other threat is National Highway 07 which passes adjacent to the buffer of Pench Tiger Reserve (Maharashtra) all the way to Seoini which also dissects the Kanha-Pench Corridor. The wildlife presence survey carried out in 171 villages present in the deforested rather fragmented region between Tumsar, Tiroda and some area of Gondia in the PenchNagzira corridor; out of which, 46 villages showed big carnivore presence. The major forest type of this area consists of scrubland and grassland inhibited by woodlands surrounding the agricultural patches. Tiger, Leopard, Wild Dog, Jungle Cat, Fox, Hyena, Wolf, Bear, Chital, Sambar, Blue Bull, Wild boar, Langur and Rhesus macaques were the animals seen by the villagers in their surroundings. Amongst these wild boar was found to

be present in most of the villages and Blue Bull was found to be present in very few villages. Out of all the villages surveyed Tiger and Leopard presence was found in 9% of the villages (Fig. 04). On the basis of survey completed in the fragmented landscape Fig. 05 shows the presence of all mammals occurring in the fragmented area along with Tiger and Leopard. The secondary data collected from the forest department about Man-Animal interaction like cattle kill, human kills combined with the data generated from the wild animal survey as well as some direct observation in the fragmented landscape of study area were plotted on Google Earth. The observations show that the corridor shown by the forest department might not be used due to various hindrances. Moreover there are 2 major tehsils Tumsar and Tirora fall on the opposite side of the so called corridor suggested by the forest department. Also many villages occur in this shown corridor which might create another problem. However there arenâ&#x20AC;&#x2122;t any instances of man-animal negative interaction in this patch. Hence this path might not be used by the animals for their movement. As shown in the Fig. 06 the presence of tiger was observed more in the villages towards the Wainganga river side. This presence was found continuous as compared to the presence of tiger in the corridor shown by Forest Department. Additionally there have been instances of cattle kill as well as human kill in the possible corridor suggested by

Fig. 3: Bottle Necks and Major Threats.

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(Source: Google Earth)

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BNHS. During the survey it was also observed that most farmlands of the villagers living near to Wainganga River were towards the river side and far from the main village. Moreover according to the conversation with the villagers,

they visit their farmlands only during daylight and return back with the dusk. Thus this might give the wild animalâ&#x20AC;&#x2122;s easy access to these farmlands and migrate or move using them during the night time.

Fig. 4: Animal Presence in Fragmented Area.

Fig. 5: Animal Presence Map in Fragmented Area.

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(Source: Google Earth)

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Fig. 6: Map Showing Difference in the possible corridor (BNHS) and corridor shown by Forest Department.

Acknowledgements

We would like to thank Maharashtra Forest Department for granting us permission to study the corridor and also for providing us with secondary data. We would also like to thank Tata Steel for funding this project which helped us to understand the landscape in a better way. Finally we would like to thank Dr. Deepak Apte, Director BNHS; Mr. Satish Pradhan for their immense support throughout the study.

Funding

(Source: Google Earth)

5. YV Jhala, Q. Q. (2010). Status of the Tigers, Co-predators, and Prey in India. National Tiger Conservation Authority, Govt. of India, New Delhi and Wildlife Institute of India, Dehradun. 6. Joshi, A., Vaidyanathan, S., Mondol, S., Edgaonkar, A., & Ramakrishnan, U (2013, November 6). Today, most wild tigers live in small, isolated Protected Areas within human dominated landscapes in the Indian subcontinent. Plos One, 8(11). e77980. 7. EO Wilson, EO Willis. (1975). Applied Biogeography. In E. W. EO Wilson, & J. D. ML Cody (Ed.), Ecology and Evolution of Communities (pp. 522-534). Harvard University Press.

Tata Steel

Competing interests None Declared

Reference

1. R Joshi, R Singh. (2008). Asian Elephant (Elephus maximus) and Riparian Wildlife Corridor: A case Study from Lesser Himalayan Zone of Uttarakhnad. The Journal of American Science, 4(1): 63-75. 2. Wiess, M. (2006). The Theory of Island Biogeography. Retrieved from http://web2.uwindsor.ca/courses/ biology/macisaac/55-437/ lecture9.htm 3. Noss, R. (1983). A regional landscape approach to mantain diversity. Bioscience, 33:700-706. 4. Noss, R. (1987). Protecting natural areas in fragmented landscapes. Natural Areas Journal, 7: 2-13.

8. McEuen, A. (1993). The Wildlife Corridor Controversy:A review. Endangered Species update, 10(11 & 12). 9. DS Simberloff, LG Abele. (1976). Island Biogeography Theory and Conservation Practice. Science, 191: 285-286. 10. S Simberloff, J. F. (1992). Consequences and costs of conservation corridors. Conservation Biology, 1: 63-71. 11. RF Noss, LD Harris. (1986). Nodes, networks, and MUMs: preserving diversity at all scales. Environmental Management: 299–309. 12. P Beier, S. L. (1992). A checklist for evaluating impacts to wildlife movement corridors. Wildlife Society Bulletin, 20: 434–440.

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Original Article Modification of Antifungal Susceptibility Testing for Aspergillus Species Manga Sirisha Madireddy*, Rajasekhar Koppada, K.R.L. Surya Kirani and Rama Krishna Pilli Department of Microbiology, Rangaraya Medical College, Kakinada, AP, India Keywords: Aspergillus, AFST Modified, MIC with Injections, Resource Constrained, CLSI Moulds, EUCAST Moulds.

ABSTRACT Background: In resource constrained laboratories, determination of antifungal susceptibility as described by CLSI and EUCAST guidelines is not always feasible. So, we have modified the method of Antifungal susceptibility testing (broth microdilution method) done for Aspergillus sp. to see whether the MIC values obtained are comparable with that of CLSI and EUCAST methods. Methods: MICs for 30 isolates of Aspergillus sp. were determined against locally available drugs like amphotericin B injection (diluent-distilled water) and itraconazole granules (diluent-Dimethyl sulfoxide). SDA broth (pH 7) and RPMI 1640 are used as test medium. Broth microdilution method was followed and MICs were read at 48hrs, compared with references in CLSI and EUCAST standards. Minimum inhibitory concentration is taken as 100% inhibition of growth visually. Result: MIC ranges observed for amphotericin B in Âľg/ml for A.flavus(8) is 0.5-1, A.fumigatus(6) 0.25-0.5, A.niger(10) 0.5-1, A.terreus(6) 0.5-1. Similarly MIC ranges for itraconazole are A.flavus(8) 0.5-1, A.fumigatus(6) 0.5-1, A.niger(10) 0.25-2, A.terreus(6) 0.5-1.Control strains were kept to check the quality control. These ranges are comparable to CLSI and EUCAST standards. Conclusion: MIC ranges obtained in the study are within the ranges as published by CLSI and EUCAST guidelines. The observations in this pilot study will help for extending the method on larger number of isolates of filamentous fungi for standardisation.

*Corresponding author: Dr. Manga Sirisha Madireeddy, Dept. of Microbiology, Rangaraya Medical College, GGH Campus, Kakinada-1, East godavari district, Andhra Pradesh-533006. INDIA Phone: +91 9848133747 Email: drsirishanaidu@gmail.com

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Introduction

Invasive fungal infections have not only increased in frequency but also new fungal species have been reported to cause infection, especially in immunocompromized patients. Concurrent with the increase in fungal infections, a large variety of antifungal drugs are available with different spectrum of activity. Therefore, there is a need to determine the antifungal susceptibility of isolates to available drugs1. Among the fungal infections, invasive aspergillosis has emerged worldwide as an important cause of infection among patients undergoing cancer chemotherapy, hematopoetic stem-cell transplantation, or solid organ transplantation. So, there is a requisite for determination of antifungal susceptibility patterns to know the MIC(Minimum Inhibitory Concentration)s at our community level. CLSI(Clinical Laboratory Standards Institute) has standardised disc diffusion method for yeasts and broth microdilution method for filamentous fungi. To follow the broth microdilution method, procuring pure powder form of drugs is quite expensive, and also to preserve stock solutions of diluted drugs at -70˚C is not feasible in resource constrained laboratories. To overcome these, we made an attempt to simplify the broth microdilution method for Aspergillus sp which is user friendly, done with locally available drugs but yet comparable with that of CLSI and EUCAST2(European committee for Antimicrobial Susceptibility Testing) references. Aims and Objectives: Determination of MICs for Aspergillus species by broth microdilution method using –

locally available drugs.

–

simple basic media.

Comparision of MICs of our study with those of CLSI and EUCAST.

Materials and Methods

MICs for 30 isolates of Aspergillus sp were determined. This includes A.niger 10, A.flavus 8, A.fumigatus 6 and A.terreus 6. Two control strains Aspergillus flavus ATCC 204305, Aspergillus fumigatus ATCC 204304 were used for quality control. The procedure followed was according to CLSI M38A reference method for antifungal susceptibility testing for filamentous fungi with some modifications. According to CLSI M38A, medium used is RPMI 1640 broth, pure powder form of drug is used and the inoculum is set to 0.4x104 - 0.5x105 CFU/ml standardised spectrophotometrically3.

In our study, initially RPMI 1640 broth was used and the procedure was later compared using SDA (Sabourauds Dextrose Agar) broth with pH 7. Antifungal Agents: We used two locally available antifungals amphoterecin B, itraconazole. According to CLSI guidelines, powdered form of pure drug is used with Dimethylsulfoxol (DMSO) as diluent. In our work, we used Injection amphoterecin B(AMP B) with its diluent distilled water. For the second antifungal itraconazole (ITR), we used granular form of drug in capsules with DMSO as diluent. Stock solutions were prepared 1600µg/ ml for both Amp B, ITZ. 20µl of diluted drug solution(each concentration 800, 400, 200...) is added to 1ml of RPMI broth. Inoculum Preparation: Inoculum suspensions are prepared from fresh, mature (2- to 5-day-old) cultures grown on Saborauds dextrose agar slants. Colonies are covered with approximately 1 ml of sterile water supplemented with 0.1% Tween 20. The inoculum is standardized spectrophotometrically to 0.5 Mc Farland. The conidia are collected carefully with a sterile cotton swab and transferred to a sterile tube. Microtitre Plate Inoculation: 100µl of broth diluted drug is added in serial dilutions to the wells. Later 100µl of inoculum is added to each well. Two controls are kept. One is Quality control with 200µl of RPMI broth without inoculum, drug and the other is growth control with 100µl of RPMI broth with 100µl of inoculum without drug. The microtitration plate is incubated in a humid atmosphere in a sealed container or bag at 35˚C for 48 hrs. Minimum Inhibitory concentrations(MIC) were read. Reading and Interpretation of Results: After checking the growth in control tubes, the endpoint is read visually in a good light. The concentration of drug in the first well in which there is no growth is the MIC value. These MIC values are compared with that of CLSI and EUCAST guidelines.

Results

Table 1: Comparison of MIC of standard drugs against control strains. Table 2: Comparison of MICs of RPMI 1640 and SDA broths. Table 3: MIC ranges obtained in the study with comparison to CLSI and EUCAST standards.

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Discussion

and can be procured in all basic laboratories. Initially, AFST was done with modified drug using two control strains, Aspergillus flavus ATCC 204305, Aspergillus fumigatus ATCC 204304. MICs obtained are within the ranges of CLSI and EUCAST (table 1).

Antifungal susceptibility testing has evolved rapidly during the last decade and has now become a relevant tool. To determine the MICs, there are several methods. However, each has its own disadvantages like broth micro dilution method is cumbersome3, E test is relatively expensive, disk diffusion is attractive method but no standard references are available for moulds, AFST with Semi-semisolid agar1 even requires pure form of drug which is expensive.

Later, the medium RPMI 1640 (to be procured) is modified with SDA broth (pH 7) which can be prepared in all laboratories. The MICs obtained are within the ranges of CLSI and EUCAST (table 2).

Coming to our study, we replaced pure powdered form of drug (costly, to be procured, difficult to stock up) with easily available injectables and capsules which are economical

The modified study with local drugs as antifungals and SDA broth (pH 7) as medium was applied to the 30 isolates

Table 1: Comparision of MIC of standard drugs against control strains MIC(µg/dl) Quality control strains

AMP B

ITR

Study

CLSI1

EUCAST1

Study

CLSI1

EUCAST1

Aspergillus flavus ATCC 204305

0.5

0.5-4

0.5-2

0.25

0.25-0.5

0.12-0.5

Aspergillus fumigatus ATCC 204305

0.5

0.5-2

0.25-1

0.5

0.125-1

0.12-0.5

AMP B-amphotericin B, ITR-itraconazole, MIC - minimum inhibitory concentration, CLSI-Clinical Laboratory Standards Institute, EUCAST-European committee for Antimicrobial Susceptibility Testing.

Table 2: Comparison of MICs of RPMI 1640 and SDA broths. MICs for AMP B (µg/ml)

MICs for ITR (µg/ml)

Isolates

RPMI 1640 Broth

SDA broth (pH 7)

CLSI2 (RPMI)

RPMI 1640 Broth

SDA broth (pH 7)

CLSI2 (RPMI)

A. flavus(8)

0.5-1

0.5-2

0.5-1

A. fumigatus(6)

0.25-0.5

0.12-0.5

0.5-2

0.5-1

0.5->8

A. niger(10)

0.5-1

0.5-4

0.25-2

0.25-8

A. terreus(6)

0.5-1

0.5-4

0.5-2

0.5-1

0.5->8

References quoted according to CLSI document M38A3, EUCAST (v 8.0)2. AMP B-amphotericin B, ITR-itraconazole, MIC- minimum inhibitory concentration.

Table 3: MIC ranges obtained in the study with comparison to CLSI and EUCAST standards. MIC(µg/ml) Isolates

AMP B

ITR

Study

CLSI

EUCAST

Study

CLSI

EUCAST

A.flavus(8)

0.5-1

0.5-2

0.5-1

0.25-8

A.fumigatus(6)

0.25-0.5

0.12-0.5

0.5-1

0.5->8

0.25-2

A.niger(10)

0.5-1

0.5-4

0.25-2

0.25-8

0.25-4

A.terreus(6)

0.5-1

0.5-4

0.5-1

0.5->8

0.25->8

References quoted according to CLSI document M38A , EUCAST (v 8.0) . AMP B-amphotericin B, ITR-itraconazole, MIC- minimum inhibitory concentration. 3

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Fig. 1: Microtitre plates showing growth of Aspergillus niger in positive control wells and in wells with lower drug concentrations.

of A.flavus, A.fumigatus, A.niger and A.terreus. The MICs obtained are within the ranges of CLSI and EUCAST (table 3).

Prakash, Professor, PGI Chandigarh for providing me the control strains.

Conclusion

Funding

The MICs obtained with both RPMI 1640 and SDA broth are comparable with CLSI references where they used RPMI 1640 broth. The MICs obtained in the study for both amphotericin B (Injection) and itraconazole (Capsule) are within the reference ranges given by CLSI and EUCAST where pure powder form of drug is used. The procedure adopted in our study is easy to perform and cost-effective. This study is helpful to adopt in all resource constrained laboratories. The observations in this pilot study will help for extending the method on larger number of isolates of filamentous fungi for standardization.

Acknowledgements

I thank my Professor and HOD, Dr.K.R.L.Surya Kirani for allotting me this work and her constant guidance. I thank my Asst.Professor Dr.K.Rajasekhar for his technical support in doing this work. I sincerely thank Dr.M.R.Shiva

None

Competing Interests None declared

References

1. Khan S, Singhal S, Mathur T, Upadhyay DJ, Rattan A. Antifungal susceptibility testing method for resource constrained laboratories. Indian journal of medical microbiology, (2006) 24 (3):171-6. 2. C. Lass-Florl, M. Cuenca-Estrella, D. W. Denning & J. L. Rodriguez-Tudela. Antifungal susceptibility testing in aspergillus spp. according to EUCAST methodology. Medical mycology 2006, 44, s319-s325. 3. Wayne PA. National committee for clinical laboratory standards. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi approved standard. 2002 M38A.

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Original Article Results of Prompt Intravitreal Injections in Cluster Endophthalmitis Following Cataract Surgery Vartika Anand1*, Balbir Khan1, Meenu Kashyap1 and Sachin Anand2 1

Dept of Ophthalmology, Adesh medical college and hospital NH-1, Mohri Ambala, India 2 Shri Gur Har Sahay Diagnostic Centre Sector 22. Chandigarh, India Keywords: Endophthalmitis, Cluster, Phacoemulsification

ABSTRACT The purpose of this study was to determine clinical presentation, microbiological spectrum and visual outcome of cluster endophthalmitis patients after cataract surgery. The records of cluster endophthalmitis patients were retrospectively reviewed. Intravitreal injection was given to all patients, vitreous was sent for culture, smear was sterile in most cases only in 3 cases there was few pus cells. All patients had good visual outcome.

*Corresponding author: Dr. Vartika Anand, House no.169, sector 19-A Phone: +91 9988880755 Email: chandigarh,varti_in@yahoo.com

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Introduction

Postoperative endophthalmitis is a serious and devastating complication after ophthalmic surgery1. The incidence of endophthalmitis has decreased during the past several decades; this decline in incidence is due to improved surgical techniques, improved sterilization methods and better postoperative care and use of broad-spectrum antibiotics2. In spite of optimum precautions taken during ocular surgery, a cluster of cases of endophthalmitis can occur after cataract surgery3. There are numerous reports3, 4 on postoperative endophthalmitis but no report on cluster endophthalmitis patients after cataract surgery from central India where camp cataract surgeries are widely prevalent. This study addresses the clinical profile and visual outcome in these patients.

Materials and Methods

A retrospective review of charts of cluster endophthalmitis patients was done which were referred to our center they were got operated in a camp somewhere outside. Cluster endophthalmitis was defined as five or more cases of endophthalmitis occurring on a particular day in a single operating room in one center. Undiluted vitreous biopsy samples were collected at the beginning before giving intravitreal injections and sent for microbiological evaluation. Good visual outcome was defined as visual acuity of 6/12 or better at final follow-up.

Result

There were total 10 patients in clusters.4 patients were males and 6 patients were females. The age ranged from 55 to 75 years (mean 60.1 years). Pain, blurring of vision and hypopyon with vitritis were the common signs and symptoms of the patients on first presentation. Duration of symptoms ranged immediately on first day after cataract surgery. All 10 patients underwent phacoemulsification with intraocular lens implantation. At presentation only one patient had visual acuity was more than6/18 whereas the remaining patients had visual acuity worse than6/60 or in some cases it was just counting finger close to face. Corneal infiltration was seen in two of 10 patients and hypopyon in 3 of 10patients on presentation. No patient had retinal detachment or choroidal detachment at the time of presentation. All patients had received preoperatively topical ciprofloxacin 0.3% and patients dilated with tropicamide and phenylepinephrine. Intraocular antibiotics and post operative topical steroids were given in all the patients. All 10 developed symptoms and in that intravitreal vancomycin (1 mg/0.1 ml), ceftadizine (2.25 mg/0.1 ml) and dexamethasone (0.4 mg/0.1 ml) was injected. Gradually patients recovered and there was improvement

in symptoms. The duration of follow-up ranged from six to 12 weeks. Good visual outcome was seen in all 10 patients, no surgical intervention was required in any case. The smears negative vitreous samples and only in 3 cases there was presence of few pus cells.

Discussion

The most dreaded complication of any intraocular surgery is the development of endophthalmitis. The incidence of post cataract surgery endophthalmitis in the Indian scenario is 0.05% 5 .In our series all 10 patients had good visual outcome after appropriate intervention. Postoperative outbreaks have been described in association with internal fluid pathways of a phacoemulsifier3 and with contaminated intraocular irrigating solutions6 with a poor visual outcome despite vitreous surgery and intravitreal antibiotics to which isolates were sensitive. In an Indian setup postoperative infections commonly occur in clusters and gram-negative infections7 and fungi are common pathogens isolated in cluster endophthalmitis patients. The microbiology is suggestive of contaminated irrigating solutions as the culprit in causing such outbreaks. Our study was limited by a short follow-up of six weeks as most of the patients were inhabitants of remote tribal interiors of the state. Defects in sterilization, contaminated irrigating solutions, viscoelastics, improper ventilation system, and poor operation room hygiene and hospital construction activity are various factors responsible for cluster postoperative endophthalmitis. Source of infection could not be evaluated in our series. In high-volume cataract surgery, epidemic of endophthalmitis is always possible; we should remain vigilant and follow standardized surgical protocols and sterilization measures even in camp surgeries. Prompt adequate and aggressive treatment by vitreous surgery and intraocular antibiotics may lead to favorable results.

Conclusion

Endophthalmitis is a serious and devastating condition which can lead to vision loss but if diagnosed and managed early it has good visual outcomes even can be cured without surgical intervention.

Acknowledgements None

Funding None

Competing Interests None

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Reference

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4. Das T, Kunimoto DY, Sharma S, Jalali S, Majji AB, Nagaraja Rao T, et al. Relationship between clinical

presentation and visual outcome in postoperative and posttraumatic endophthalmitis in south central India. Indian J Ophthalmol. 2005; 53:5–16. 5. Lalitha P, Rajagopalan J, Prakash K, Ramsasamy K, Prajna NV, Srinivasan M. Post cataract endophthalmitis in South India incidence and outcome. Ophthalmology. 2005; 112:1884–9. 6. Swaddiwudhipong W, Tangkitchot T, Silarug N. An outbreak of Pseudomonas aeruginosa postoperative endophthalmitis caused by contaminated intraocular irrigating solution. Trans R Soc Trop Med Hyg. 1995; 89:288 7. Gupta A, Gupta V, Gupta A, Dogra MR, Pandav SS, Ray P, et al. Spectrum and clinical profile of post cataract surgery endophthalmitis in North India. Indian J Ophthalmol. 2003; 51:139–45.

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1. Aaberg TM, Flynn HW, Schiffman J, Newton J. Nosocomical acute-onset postoperative endophthalmitis survey: A 10-year review of incidence and outcomes. Ophthalmology. 1998; 105:1004–10. 2. Ram J, Kaushik S, Brar GS, Taneja N, Gupta A. Prevention of postoperative infections in ophthalmic surgery. Indian J Ophthalmol. 2001; 49:1–2. 3. Zaluski S, Clayman HM, Karsenti G, Bourzeix S, Tournemire A. Pseudomonas aeruginosa endophthalmitis caused by contamination of the internal fluid pathways of a phacoemulsifier. J Cataract Refract Surg. 1999; 25:540–5

Original Article Serum MicroRNA-122 as a Prognostic Biomarker in Patients with Liver Cirrhosis J.H. Sabry1*, O. S. El-Shaer1, I.A. Ahmed2, E. M. Said3, A. M. El Hammady4, A. AH. Abdelmoneam4 and A.A .El Fallah Clinical and Chemical Pathology Department, Faculty of Medicine, Benha University, Egypt 2 Medical Biochemistry Department, Faculty of Medicine, Benha University, Egypt 3 Hepatology, Gastroenterology and Infectious Diseases Department, Faculty of Medicine, Benha University, Egypt 4 Internal Medicine Department, Faculty of Medicine, Benha University, Egypt 1

Keywords: Micro RNA 122, MELD Score, Liver Cirrhosis, qPCR, Overall Survival

ABSTRACT Background: Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRNAs), a class of endogenous small non-coding RNAs, are becoming increasingly recognized as crucial regulators in gene expression networks. In particular, a low serum miRNA-122 level was associated with hepatic decompensation so it can be used as a prognostic marker for liver decompensation. Circulating miRNA-12 was examined aiming to clarify its prongnostic value in patients with liver cirrhosis and to discover its relation with patientâ&#x20AC;&#x2122;s survival. Methods: Gene expression level of miRNA 122 was extracted and assessed in sera of 100 patients with liver cirrhosis, using quantitative reverse-transcription PCR (qRT-PCR) . MiRNA 122 expression levels were compared to liver function tests, MELD score, overall survival time and to different manifestation of decompensation. Results: Serum samples from patients with hepatic decompensation showed significant down regulation of miRNA122compared to those from patients with compensated liver cirrhosis. Patients with ascites, and hepatorenal syndrome had significantly lower miRNA-122 levels than patients without these complications. A univariate Cox regression analysis revealed a significant association between miRNA-122 levels and overall survival Multivariate Cox regression analysis revealed that only miRNA-122 serum levels and platelet count were independent factor for survival. MiRNA-122 sensitivity and specificity for the predection of decompensation in cirrhotic patients were 100.0% and 90.1% respectively the best cut off point value of 0.892 (AUC= 0.9429) Conclusions: Serum miRNA-122 is a useful new independent prognostic marker in patients with liver cirrhosis.

*Corresponding author: Jehan Hassan Sabry, Assisstant professur of Clinical and Chemical Pathology department , faculty of medicine, Benha University, Egypt Email: jehanrayan@yahoo.com

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

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Introduction

Cirrhosis can be a result of different mechanisms of liver injury that lead to necroinflammation and fibrogenesis. Chronic infection with HCV is the most common cause of chronic liver disease,it is complicated by cirrhosis in about 20 % of western world(1). The morbidity increases rapidly when hepatic decompensation occurs,. After the first manifestation of ascites the one year mortality increases up to 40% (2). With occurrence of spontaneous bacterial peritonitis or hepatorenalsyndrome ,the patients’ prognosis further deteriorates (3). If liver transplantation is indicated , the assessment of the risk of death in these patients will be of great interest and importance to optimize the time point for organ allocation (4). Short term mortality was more precisely evaluated by the development of the Model for End Stage Liver Disease (MELD).MELD best predicts 3 month survival of cirrhotics, irrespective of etiology.Giving priority to patients who are most likely to die without livertransplanationt such as those with hepatorenal failure,it is based on creatinine, bilirubin and INR, but lacks features of portal hypertension, such as ascites..(5) MicroRNAs (miRNAs), a class of endogenous small non-coding RNAs, are becoming increasingly recognized as pivotal regulators in gene expression networks . They can function as translational repressors by binding to the 3’untranslated regions (3’ UTRs) of target messenger RNAs (mRNAs) and inforcing their degradation and/or leading posttranscriptional repression(6). Dysregulated miRNAs play crucial roles in the pathogenesis of various diseases and characteristic miRNA patterns have been found in pathological tissues(7).Playing an important role in cell development, differentiation, and physiological function is not it’s only value, but are also significant in the development of tumors, viral infections, and other closely related diseases.(8,9) MiRNA-122 family is the most abundant type of miRNAs in the liver (10). And is important for the functional condition of the hepatocyte. It organizes many genes in the liver that adjust the cell cycle, differentiation, proliferation and apoptosis(11).Moreover, its Loss in the liver leads to hepatic undifferentiation witha malignant phenotype(12), a finding which is frequent in hepatocellular carcinoma (HCC), and was referred to correlate with migration, invasion and in vivo tumorigenesis(13) Decreased level of miRNA-122 in fibrotic liver biopsies may be explained as the result of compromised normal hepatocytic activity or as the forsaken suppressive function of miRNA-122 that inhibits fibrogenesis.(14) The objectives of this research were studying miRNA-122 as apotential prognostic marker for liver cirrhosis, its correlation with Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

AABS; 3(4): 2016 other standard laboratory parameter,association with complications of liver cirrhosisand discovering its role as a predictor of survival in decompensated patients

Material and Methods

The current study was approved by the Local Ethics Committee of Faculty of Medicine, Benha University and all study participants gave a written informed consent prior to enrollment in the study. The study was designed as a cohort prospective study. It copmrised100 patients with liver cirrhosis, 68 male and 32 female, selected from the departments of Hepatology, Gastroenterology & Infectious Diseases and Internal Medicine, Benha University Hospital between Junuary 2014 and May 2015. The patient were categorized into Group I: with22 patients diagnosed as compensated liver cirrhosis, and Group II: with78 patient with decompensated liver cirrhosis. Exclusion Criteria Included : age below 18 years, former liver transplantation and history of cancer in the last 5 years other than hepatocellular carcinoma. All Individuals in The Study were Subjected to: Full history taking including: age , sex, duration of disease and identification of the causes of liver cirrhosis as Hepatitis C, Hepatitis B, Primary sclerosing cholangitis, Autoimmune hepatitis, Non alcoholic steatohepatitis, Cardiac cirrhosis, Hemochromatosis, Primary biliary cirrhosis, Hepatocellular carcinoma and alcohol abuse . Liver cirrhosis was assessed by: •

1-Histopathological examination or pathognomic result abdominal ultrasound examination. • 2-Coumpter tomography or magnetic resonance imaging. • 3-Biopsies were performed only for patients with inexplicit stage of fibrosis or unclear cause of liver cirrhosis. Assessment of Symptoms of Hepatic Decompensation: (variceal bleeding, sponteannous bacterial perotionitis, ascitis, hepatorenal syndromeandhepatic encephalopathy) was done by clinical, radiological, laparoscopic and laboratory investigation. Diagnostic criteria for hepatorenal syndrome were: 1. A plasma creatinine concentration above 1.5 mg/dl that progresses over days to weeks. 2. The absence of any other apparent cause for the renal disease, including shock, ongoing bacterial infection, e-ISSN: 2349-6991; p-ISSN: 2455-0396

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current or recent treatment with nephrotoxic drugs, and the absence of ultrasonographic evidence of obstruction or parenchymal renal disease. 3. Urine red cell excretion of less than 50 cells per high power field (when no urinary catheter is in place) and protein excretion less than 500 mg/day. 4. Lack of improvement in renal function after volume expansion with intravenous albumin (1 g/kg of body weight per day up to 100 g/day) for at least two days and withdrawal of diuretics.(15) The Patients were followed up until death (=overall survival), Liver Transplantation or Last Contact. Blood Sampling: Peripheral blood samples were withdrawn from all patients under aseptic condition, then each sample was divided into 3 parts: (1.8) ml blood for each 0.2 ml Na citrate for determination of PT and international normalized ratio, 2 ml on EDTA for measurement of platelet count, and 4 ml sample collected in plain vacutainers for separation of serum . Serum was separated by centrifugation at 4ºc at 1500 x g for 10 mins. followed by an additional centrifugation at 4ºc at 2000 x g for 15 mins. The sera were aliquoted into 2 tubes: one was used for chemical analysis of liver function tests using Biosystem A15 autoanalyzer (Biosystem. Spain). The other serum aliquot was used for assessment of miRNA 122 expression level. Data were Used for Calculation of MELD Score. Assessment of miRNA 122 expression level by qPCR: MicroRNA was first extracted from fresh serum samples using the miRNeasy Mini Kit (Qiagen, Germany) Next, the Reverse transcription step was performed using the miScript II RT Kit (Qiagen, Germany) and the cDNA products were kept at -20 till further processing. Finally, qPCR of miRNA122 was performed by StepOne™ Real-Time PCR System (Life Technologies, USA) using miScript SYBR Green PCR Kit supplied by (QIAGEN, Germany)the manufacturer instructions were followed throughout all steps. Briefly, PCR was initially activated at 95°C for 15 mins., then 40 cycles of denaturation at 94°C for 15sec., annealing at 55°C for 30sec.and a final extension step at 70°C for 30sec.The primers for miRNA-122 and housekeeping gene (RNU6B) were supplied by Qiagen, Germany. After PCRmiRNA122quantificationwas analysed using StepOne software (Life Technologies, USA) and expressed as relative mRNA level compared to RNU6B according tothe 2−ΔΔCt method(16)

Statistics: The statistical analysis was conducted using STATA/SE 11.2 for Windows (STATA corporation, College Station, Texas). Tests used were (Chi-square test (χ2), Fisher’s Exact Test (FET) , Student t-test (t) , Mann-Whitney test (z) , Kruskal Wallis test (χ2) and Spearman correlation coefficient (rho; ρ). Survival analysis was carried out using both univariate and multivariate Cox regression models to detect important predictors for patients’ survival based on potential risk factors and liver functions. Receiver Operator Curve (ROC) was used to evaluate the predictability of serum miRNA 122 levels for the presence of liver decompensation in cirrhotic patients. The Area Under the Curve (AUC), the best cut off point and the corresponding sensitivity and specificity were determined. The corresponding P-values were obtained. A P-value < 0.05 was considered statistically significant (S), a P-value < 0.001 was considered statistically highly significant (HS), while a P-value > 0.05 was considered statistically non-significant.

Results

The patients’ characteristics are shown in (Table 1). The follow up period ranged between 200 and 500 days, with median of 343.4±83.44 days and. During the study time 22 out of 100 patients died and 6 patients were dropped out due to loss of contact with them. No patients underwent liver transplantation. Serum miRNA-122expression Levels in Patients with Liver Cirrhosis: Our results showed that serum miRNA-122 was down regulated in patients with hepatic decompensation with a high significance compared to patients with compensated liver disease (P <0.001). (Fig.1) On examining the relation of miRNA 122 with decompensating manifestation, There was significantly lower expression of serum miR-122 levels in patients with hepatorenal syndrome (p=0.03) and, ascitis (P = 0.038) than patients without the respective complications. (Fig.2). Furthermore, serum miRNA 122 expression levels showed no significant differences between patients with and without variceal bleeding (P = 0.81), hepatic encephalopathy (p=0.73), spontaneous bacterial peritonitis (P=0.94) and hepatocelluar carcinoma (p=0.94). (table 2) Correlation of miRNA-122 Levels with Laboratory Parameters: There was a significant negative correlation between serum miRNA 122 expression levels and MELD score (p=0.003) (Fig. 3), serum creatinine (P=0.007), INR (p=0.048), total bilirubin (p=0.002) and GGT (p=0.045),

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and it was highly significant with ALP (p<0.001). Meanwhile, there was significant positive correlation between serum expression of miRNA-122 levels and AST and ALT (p=0.03 and 0.047), respectively.

92.31%and 90.91%respectively with the area under the curve (AUC=0.9161).(Fig 4& 5) The Serum miRNA-122 Level as a Predictor of Survival in Patients with Liver Cirrhosis: The study population were divided into tertiles according to the miRNA-122 levels at cutoff points 0.038 and 0.1805 .The third of patients with the highest miRNA-122 levels was compared to the two thirds of patients with lower miR-122 serum concentration. Aunivariate Cox regression analysis was performed. There was a significant association between miRNA-122 levels and overall survival p<0.001, hazard ratio 0.23, 95% confidence interval (0.10 – 0.52). (Fig. 6) Cox regression analysis using forward stepwise (likelihood ratio) revealed that miRNA-122 serum levels (P =0.02) and platelet count (P =0.02) were independently associated with patients’ survival. (table3).

In contrast, there was no significant correlation between serum miRNA 122expression and neither total protein (p=0.79), albumin (p=0.58) nor platelet count (p=0.64) The Sensitivity and Specificity for the Predection of Decompensation in Cirrhotic Patients: ROC curve showed that at the best cut off value for miRNA 122expression (0.892)the sensitivity and specificity for the prediction of decompensation in cirrhotic patients were 100.0% and 90.1% respectively with the area under the curve (AUC= 0.9429), while at the best cut off value for MELD score (15)the sensitivity and specificity for the prediction of decompensation in cirrhotic patients were Table 1: Patient characteristics. Compensated cirrhosis (No.=22)

Decompensated cirrhosis (No.=78)

No.

Females

27.27

38.46

36.0

Males

72.73

61.54

64.0

Variable No.

Sex Age (years)

Mean ±SD; (range)

52.82±5.29; (40-60)

55.05±6.27; (40-66)

Total (No.=100)

54.56±6.11; (40-66)

Test

χ2= 0.93

0.33

t= 1.52

0.13

Table 2: Relation between miRNA-122 and decompensating manifestations Micro RNA-122

Variable Mean

Spontaneous bacterial peritonitis

Hepatorenal syndrome

Variceal bleeding

Hepatic encephalopathy

Ascites

HCC

± SD

Range

No (No.=72)

0.120

0.183

0.0007-0.892

Yes (No.=6)

0.057

0.025

0.041-0.089

No (No.=46)

0.177

0.13

0.0007-0.589

Yes (No.=32)

0.113

0.23

0.004-0.892

No (No.=46)

0.106

0.185

0.004-0.892

Yes (No.=32)

0.129

0.167

0.0007-0.589

No (No.=64)

0.102

0.134

0.0007-0.589

Yes (No.=14)

0.176

0.307

0.004-0.892

No (No.=22)

0.144

0.163

0.002-0.589

Yes (No.=56)

0.104

0.182

0.0007-0.892

No (No.=72)

0.124

0.181

0.002-0.892

Yes (No.=6)

0.005

0.004

0.0007-0.009

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Test

z= 0.07

0.94

z= 2.11

0.03 (S)

z= 0.24

0.81

z=0.34

0.73

z= 2.07

0.038 (S)

z= 0.07

0.94

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Table 3: Univariate and multivariate analyses of factors associated with overall survival. Univariate analysis

Variable (No.=94)

Multivariate analysis

95% CI

Micro RNA-122 (High vs. low)

0.23

0.10 – 0.52

<0.001

0.36

0.15-0.88

0.02

Meld scores (>18)

5.46

1.89-15.79

0.002

Sex (Male vs. female)

0.91

0.42-1.99

0.82

Age (>65 years)

2.50

0.57-10.84

0.22

Total protein (g/dl)

0.61

0.41 – 0.91

0.01

Platelet count (x103/mm3)

0.98

0.96 – 0.99

0.001

0.98

0.97-1.00

0.02

Albumin (g/dl)

0.49

0.25 – 0.97

0.04

Bilrubin (mg/dl)

1.10

1.04 – 1.17

<0.001

GGT (Iu/l)

1.01

1.00 – 1.01

0.001

ALP (Iu/l)

1.02

1.00 – 1.03

0.008

HR: hazard ratio HR<1 indicates reduced hazard risk and increased survival. HR>1 associated with increased hazard risk and reduced survival.

Fig. 1: Comparison between studied groups as regards microRNA-122.

Fig. 2: Relation between miRNA-122 and ascitis and hepatorenal syndrome.

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Fig. 3: Correlation between miRNA-122 levels and MELD score.

Fig.4: ROC curve analysis of miRNA-122 for the prediction of decompensation in cirrhotic patients.

Fig. 5: ROC curve analysis of MELD scores for the prediction of decompensation in cirrhotic patients.

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Fig. 6: Serum miR-122 levels are associated with survival in patients with liver cirrhosis Distribution of serum miRNA-122 levels throughout the patients. Survival curves for patients with high or low serum miRNA-122 levels. The analysis was performed with the Cox regression model.

Discussion

Liver cirrhosis (LC) is a late stage of progressive hepatic fibrosis characterized by distortion of the architecture and formation of regenerative nodules and different degrees of liver function impairment; these patients are prone to a variety of complications reducing life expectancy markedly(17). In this prospective cross section study we evaluated if the serum levels of miR-122 might be a suitable prognostic parameter in patients with liver cirrhosis. Data obtained from the present study revealed a statistically significant decrease of miRNA 122 in patients with decompensated cirrhosis compared with those with compensated cirrhosis.

indicator of necroinflammatory activity and cell death in the liver. As release from damaged hepatocytes might be the major source of hepatocyte-derived miRNAs(19) . Decompensation of liver reflected the stage of liver fibrosis, in a recent study where reduced level of miRNA-122 in stage F4 fibrosis as compared with stage F0was noted, miRNA-122 showed a negative correlation with fibrosis stage in fibrotic liver samples and, intriguingly, also with liver stiffness (LS values)(20).

Trebicka and colleagues(18)explained the low level of miRNA-122 in decompensate liver cirrhosis by noting that miRNA 122 is present abundantly in hepatocytes with much lower levels in circulation in healthy subjects. With hepatocyte injury miRNA-122 is released in circulation more readily and serum level rise. With eventual loss of hepatocytes and development of fibrosis with proliferation of mylofibroblasts and accumulation of extracellular matrix, the circulating miR-122 levels drop again.

That was most probably due to the fact that miRNA-122 positively regulates the accumulation of cholesterol and triglycerides and the metabolism of fatty acids . Thus, a decreased level of miRNA-122 in fibrotic liver biopsies may be interpreted as the result of compromised normal hepatocytic activity or as the eliminated suppressive function of miRNA-122 that hinders fibrogenesis.(21) Namely, miRNA-122 has been found to suppress the proliferation of hepatic stellate cells(HSCs), resulting in decreased maturation of collagen by down regulating the expression of P4HA1, a key enzyme in collagen maturation (14).

This indicates that in patients with liver cirrhosis, the miRNA-122 serum level might be a marker for hepatic functional capacity, whereas at earlier stages of liver disease, the serum miRNA-122 level is mainly an

The present study further revealed a significant negative correlation between the serum miRNA-122 concentration and MELD score. This was approved by Waidmannet al .(22)and KoÂ¨ berle et al. (23) who found the same results.

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A-293 That correlation could be explained by the suggestion that miRNA-122 serum concentration also reflects residual functional liver tissue in patients with end stage liver disease. In addition, we observed an even stronger positive correlation between miRNA-122 levels and AST and ALT. These findings go on line with another study performed by Kholeif et al.(24)who found a positive significant correlation between serum miRNA-122 and serum levels of ALT and AST in compensated cirrhosis indicating that miRNA-122 represents at least in part ongoing liver damage andcell death. Finding of the inverse correlation between serum concentrations of miRNA-122 and creatinine in this study was agreed by Waidmannet al. (22)who stated that patients with hepatorenal syndrome had highly significant lower miRNA-122 serum levels than patients without this complication. In another publication no correlation between serum miRNA-122 and creatinine levels was found in patients with toxic liver damage(25). Regarding the decompensated cirrhosis group and according to the results of this study correlation tests have revealed a significant negative correlation between miRNA 122 and both bilirubin, GGT and it was highly significant regarding ALP. While no significant correlation was found with albumin, total protein, INR and platelet. Kholeif et al. (24)suggested that serum miRNA-122 level did not reflect overall liver function finding no significant correlation between serum miRNA-122 level and serum albumin, serum bilirubin, platelet count in their study In the present study patients with ascites and hepatorenal syndrome had significantly lower serum levels of miRNA-122 than patients without the respective complication. In contrast, no significant differences were observed between patients with and without variceal bleeding, hepatic encephalopathy, hepatocellular carcinoma or spontaneous bacterial peritonitis, higher volume distribution in patients with ascites could be an explanation(19). This goes on line with a study done by Waidmann et al .(22) who reported that patients with ascites or hepatorenal syndrome had significantly lower serum levels of miRNA-122 in that study.

AABS; 3(4): 2016 (with subsequent upregulation of stem cell-associated proteins such as Pkm2) is a potential step towards hepatocellular carcinoma development.(28) Previous studies suggested that the serum level of miRNA-122 reflects hepatic inflammation and cell death in patients with HBV- or HCV-induced chronic hepatitis, and depending on the context and stage of liver disease(29,30). Supported by a previously reported study, comparison between the diagnostic performance of serum miRNA 122 and MELD score in prediction of decompensation in cirrhotic patients using ROC curve pointed to the high value of serum miRNA 122 level determination as an indicator for impaired liver function and disease severity(31). A univariate Cox regression analysis revealed a significant association between miRNA-122 levels and overall survival and performing Cox regression analysis using forward stepwise (likelihood ratio)to evaluate the predictability of each parameter as independent factor of survival, it revealed that miRNA-122 serum levels and platelet count were independently associated with survival in liver cirrhosis. A previous study on patient with HCC identified serum miRNA-122 as an independent prognostic parameter for overall survival in patients suffering from HCC(23). In that study the miRNA-122 serum level, however, was notan independent factor for overall survival, but correlated with the MELD score and clinical chemistry parameters of hepatic necroinflammation. Few studies shed light on platelet count prognostic value. In a study done by Bureau et al .(32)a platelet count above 75 Ă&#x2014;109/L were predictive of survival inpatients with refractory ascites treated by Transjugular intrahepatic portosystemic shunt (TIPS). Multiple factors can contribute to the development of thrombocytopenia in liver cirrhosis, including splenic platelet sequestration, bone marrow suppression by chronic hepatitis C infection, and antiviral treatment with interferon-based therapy. Reductions in the level or activity of the hematopoietic growth factor thrombopoietin (TPO) may also play a role(33).

Conclusion

In a study done by Abd Elmouttaleb1 et al . (26)the highest serum miRNAâ&#x20AC;&#x201C;122 expression level was in hepatocellular carcinoma (HCC) group, followed by chronic HCV group. The lowest serum level of miRNAâ&#x20AC;&#x201C;122 was in cirrhosis group. The level of miRNA-122 in human serum is also being explored as a potential biomarker to detect hepatocellular carcinoma development and progression(27). MiRNA-122 is hypothesized to regulate terminal hepatocyte differentiation, and loss of its expression

The serum level of the hepatocyte specific miRNA-122 is decreased in patients with hepatic decompensation, and that decrease inmiRNA-122 levels are associated with ascites and hepatorenal syndrome.Suggesting that serummiRNA-122 is an indicator for hepatic functional capacity. Lower miRNA 122 concentration were associated with overall survival of patients with liver cirrhosis . Only low platelet count and low miRNA 122 were independently associated with mortality. Thus, serum miRNA122 level is a useful prognostic parameter in patients with liver cirrhosis.

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Aknowlagments

The authors are very grateful to patients and their family for their participation and cooperation during the study

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25. Starkey PJ, Dear J, Platt V, et al., :Circulating microRNAs as potential markers of human drug induced liver injury. Hepatology.2011, 5: 1767–1776. 26. AbdElmouttaleb AT, Abd-Elatif DM, Soliman GM, Taher MS, Abonar AA. : Serum Micro RNA-122 as a Biomarker for Hepatocellular Carcinoma in Chronic Hepatitis C Virus Patients. Research In Cancer and Tumor. 2015, 4(2): 25-33. 27. Ding X, Ding J, Ning J,Yi F, Chen J, Zhao D, et al., : Circulating microRNA-122 as a potential biomarker for liver injury. Mol Med Rep. 2012, 5(6):1428-1432.

Original Article Spectrum of Adolescent Tuberculosis in a Tertiary Care Hospital at Shimla: North India. Shayam L Kaushik1, Savita Krishnamurthy1, Neelam Grover1 and Rajni Kaushik2 Department of Paediatrics, Indira Gandhi Medical College Shimla, India Department of Pathology, Indira Gandhi Medical College Shimla, India

Keywords: Pulmonary Tuberculosis, Extra-pulmonary Tuberculosis, Adolescent

ABSTRACT Background: Tuberculosis amongst adolescents is distinct from both childhood and adult tuberculosis in terms of incidence, disease manifestations and response. These peculiarities are due to issues related to immunity, hormone imbalance, social interactions and psychology unique to this phase of life. Adolescent tuberculosis remains a less studied area in India. Aims: To study the spectrum of tuberculosis in adolescents. Methods: We analyzed demographic, clinical and paraclinical data from 477 adolescents (10-19 years of age) diagnosed to have Tuberculosis, who reported to the in the Department of paediatrics, Indira Gandhi Medical College, Shimla between the years 2010 and 2014. Results: The incidence of tuberculosis among hospital visiting adolescents was 0.8%.The incidence in females (1.00 %) was significantly more in comparison to males (0.63 %) with females having (OR1.60) 1.60 times more odds of suffering from Tuberculosis than males (p< 0.001). Isolated Pulmonary tuberculosis (PTB) was seen in 52% cases, extrapulmonary tuberculosis (EPTB) in 40 % and combined PTB with EPTB in 8 % cases. The most common forms of extrapulmonary tuberculosis were pleural effusion (25.6%) and central nervous system tuberculosis (24.7%). Infiltration (consolidation) was the most common chest x-ray finding (53.7 %). Conclusion: Pulmonary disease accounted for high proportion of tubercular cases. Among Extrapulmonary forms isolated pleural effusion and central nervous system tuberculosis were the most common closely followed by abdominal tuberculosis, while lymphadenitis was less common. The incidence among females was significantly high in comparison to males (P<.001).

*Corresponding author: Dr Shayam Kaushik, Professor , Department of Paediatrics, Indira Gandhi Medical College Shimla, Himachal Pradesh â&#x20AC;&#x201C; 171001, INDIA Phone: +91 9418001912 Email: shayam.kaushik@live.in

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Introduction

skin tests were recorded and analysed. HIV screening results were recorded wherever available.

Tuberculosis (TB) has plagued the humankind since Neolithic times. Someone somewhere contracts TB every four seconds and one of them dies every 10 seconds.[ 1, 2] India and China together account for almost 40% of the worldâ&#x20AC;&#x2122;s TB cases. . [ 3] In India with 17% of world population we carry 26% of all tuberculosis cases. [ 4]

Mantoux test was done in all the patients with intradermal injection of 0.1 ml PPD stabilized with Tween 80 containing 5 TU, available in government supply and read between 4872 hours. Tuberculosis was classified into pulmonary (PT), extrapulmonary (EPTB) and PT with EPTB features as per RNTCP. [ 10] Cases with either sputum/gastric lavage positive for AFB, or chest x-ray suggestive of TB were classified as pulmonary TB. Cases with both pulmonary and extrapulmonary involvement were grouped separately.

The two main factors determining the risk of progression of TB infection to disease are patient age and immune status. Neonates have the highest risk of progression to disease. Children from 5 to 10 years of age are less likely to develop disease when compared to other age groups, and the risk again increases during adolescence. [ 5] Hormonal changes and altered protein and calcium metabolism associated with adolescent growth contribute to increased risk for tuberculosis. [6, 7]

Result

A total of 61,514 adolescents visited the Hospital during study period. Of these 37,494 (60.95 %) were males and 24,020 (39.05 %) females (M: F: 1.56: 1).Out of these, 477 cases (236 males and 241 females, M: F 1:1.02) were diagnosed to have tuberculosis. The prevalence of tuberculosis in females (1.00 %) was significantly more in comparison to males (0.63 %) with females having of 1.60 times more odds of suffering from Tuberculosis than males (95% CI: 1.33-1.92, p < 0.001) (Table 1).

The endocrinal effect changes the disease nature through immune system and shortens the time interval between initial infection and the development of active disease. [ 8] Adolescents are the major perpetuators of TB in the community due to reluctance in seeking health care and poor adherence to treatment. It leads to appearance of resistant strains and susceptibility of transmission increases due to greater social interaction.

Distribution of TB cases is shown in (Table 2). In all PTB patients chest X-ray was suggestive of TB (Table 3) and sputum positivity was seen in 46% (133/287) cases. Criteria used to diagnose EPTB and distribution of lesions is shown in Table 4. Of the extrapulmonary forms, the most common were tubercular pleural effusions and CNS tuberculosis followed by abdominal TB, Disseminated TB, Tubercular lymphadenitis, Skeletal TB and pericardial tuberculosis (Table 5).

Literature on disease burden of TB in adolescents is scarce in the developing countries including India. This study aims to find out the spectrum of tuberculosis in adolescents.

Materials and Methods

The study cohort comprised of 477 adolescents with tuberculosis, who visited the paediatrics department of Indira Gandhi Medical College, Shimla, between January 2010 and December 2014. [ 9] The data in terms of demographic characters, presenting symptoms, X-rays, bacteriological results, pathological findings and tuberculin

Mantoux test was done in all the patients and was positive in 260 cases (54.6%). Screening for HIV was done in 299 out of 477 patients and was found positive in 8/299 (2.6%) cases.

Table1: Prevalence of tuberculosis among Male and Female adolescents. Tb +ve

Tb -ve

Total

236

37258

37494

Row %

0.63 %

99.37 %

100.00 %

Col %

49.48 %

61.04 %

60.95 %

Female

241

23779

24020

Row %

1.00%

99.00 %

100.00 %

Col %

50.52 %

38.96 %

39.05 %

Total

477

61037

61514

Male

Row %

0.78 %

99.22 %

100.00 %

Col %

100.00 %

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P value

OR (95% CI)

26.601

<0.001

0.63(0.52â&#x20AC;&#x201C; 0.75)

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TABLE 2: Distribution of TB cases. TYPE OF TB

No of cases

Total cases

PTB

247

51.8%

PTB with EPTB

8.4%

287

60.2%

EPTB alone

190

39.8%

190

39.8%

TOTAL

477

100%

Table no. 3: Distribution of radiological lesions in tuberculosis. Radiological findings Infiltration and Consolidation Post primary lesion (cavity, fibrosis, collapse and bronchiectasis) Adenopathy Miliary pattern Pleural effusion

No of lesions (n =363) 154 74

percentage 42.4 % 20.4 %

49 10 59

13.5% 2.8 % 16.3 %

TABLE 4: Distribution of cases based on diagnostic criteria used in cases with Extrapulmonary involvement (n=230). DIAGNOSTIC CRITERIA MET

NO OF CASES

PERCENTAGE

Bacteriological evidence of TB on microscopy or culture of serous fluids

none

Fine needle aspiration cytology (FNAC)/ Histopathological evidence of TB in biopsy

20.9%

Criteria 3 (i)Tuberculin positivity (ii)ADA levels suggestive of TBin serous fluids (iii) CT/ MRI suggestive of tuberculoma or TBmeningitis. (iv)Ultrasound abdomen suggestive of abdominal TB More than one of the above criteria among criteria 3

32 31 3 29

14.0% 13.4% 1.3% 12.6%

37.8%

TOTAL

230

100%

Table 5: Distribution of EPTB cases (n=230). TYPE OF EPTB

No of cases

percentage

Pleural Effusion

25.7%

CNS TB

24.8%

Abdominal TB

22.2%

Disseminated TB

20.4%

TB lymphadenitis

3.1%

Skeletal TB

2.1%

Pericardial TB

1.7%

Discussion

The study reports the incidence and spectrum of tuberculosis in adolescents from Shimla, north India. To our knowledge no such study has been reported from this region till date. There are very few comparable studies which deal with the hospital based incidence of adolescent tuberculosis. Most of the adolescent studies are either grouped with adult or pediatric population hence information specific to this age group is limited.

Incidence of adolescent tuberculosis from various national and international population based studies varies from 0.14 to 0.45%. [11-13] . Higher incidence (0.8%) in the present study may be due to our study cohort being adolescents visiting hospital which is a referral tertiary care hospital. Similar to observation by Jayasree poroor4 incidence of tuberculosis amongst adolescent females was higher in comparison to males (females 1.0 % and males 0.6%, p < 0.001), however, in our study this difference was statistically significant.

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A-299 Frequency of PTB and EPTB in international literature varies from 22 to 78.6% and 15 to 35% respectively. [1419] Our findings of 60% and 40% of PTB and EPTB are in accordance with above however in variance with study by Jaysree pooroor who observed EPTB to be more common amongst adolescents in comparison to PTB 52% vs. 48%. [4] The radiologic findings in adolescents include parenchymal lesions and cavities. In our study, consolidation or infiltrates were the most common x ray finding. Cavitary lesions were seen in 20 % of cases and were low in comparison to reported range of 29 % to 56 % in other studies. [19, 23-25] We observed 46% sputum positivity in the study cohort and it was well in accordance with other study reports of 43 to 53.8%. [4, 19, 26] Extra pulmonary tuberculosis accounts for up to one third of all TB cases and pleural involvement increases from childhood to adolescence. The most common form of EPTB in the present study was pleural effusion, which is similar to the studies conducted in Canada, France, central India and Bucharest while discordant with studies from USA17, South Africa and South India in which peripheral lymphadenopathy was the commonest EPTB form. [12, 16, 18, 20, 17, 13, 4 ] The difference may be due to differences in genetic susceptibility of populations. [ 4] Tuberculosis of the central nervous system (CNS), a highly devastating manifestation of tuberculosis was detected in 25% of our patients. Incidence of CNS TB is quite high in our study in comparison to range of 2.8 % to 13% in other published studies. [18,19,21,22]. In addition we found high occurrence of Abdominal 22.2% and Disseminated TB 20.4% also. Since ours was a hospital based study, high incidence of CNS, abdominal and disseminated TB could be due to the clustering of seriously ill patients who are being referred to medical college. Frequency of tubercular lymphadenitis amongst EPTB cases was 3%, which is higher than observation (0.7%) by Lotfian F while low in comparison to studies by Baghaie et al (7% ) , Shrivastava AK et al (21%), Cruz AT et al (37%) and Jaysree (57%) as a manifestation of EPTB. [19, 27, 18, 17, 4] The low proportion of lymphadenitis in our study could be attributed to the study cohort being from tertiary care hospital and lots of cases of TB lymphadenitis are treated at primary and secondary level.

Conclusion

We have observed higher incidence of TB among females. Commonest forms of EPTB were CNS, abdominal and disseminated forms while Tubercular lymphadenitis was less common. It could be attributed to population differences or clustering of seriously sick patients in Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

AABS; 3(4): 2016 a tertiary care referral hospital. Since the study had limitations of being restricted to hospital wider population based studies are required to identify the definite epidemiological differences.

Acknowledgements

We express our gratitude to the staff, Medical Records Department, Indira Gandhi Medical College, Shimla for providing the data essential for the study

Funding None

Competing Interests None Declared

Reference

1. Narain, J.P. (ed.): Tuberculosis – epidemiology and control. World Health Organization, Regional Office for South East Asia, New Delhi, India, 2002; SEA/ TB/2002. 248:15,18. 2. Dye C, Scheele S, Dolin P et al: Global burden of disease: estimated incidence, prevalence, and mortality by country. J Am Med Assoc 1999; 282: 677-86. 3. Global tuberculosis report 2012-http://www. who.int/tb/publications/global_report/gtbr12_ executivesummary.pdf. April 2, 2013. 4. Jayasree Poroor, Doye George. ”Tuberculosis in Adolescents and Children: Data on Clinical Presentation and Treatment Outcomes for a Period of 4 Years, from a tertiary care hospital in South India”. Journal of Evidence based Medicine and Healthcare; Volume 2, Issue 18, May 04, 2015; Page: 2752-2757. 5. Swaminathan S, Banu R. Pediatric Tuberculosis: Global overview and challenges.Clin Infect Dis 2010; 50: 184–194. 6. Smith MHD. Tuberculosis ClinPediatr1967; 6:9-15.

adolescents.

7. Wilcox WD, Laufer S. Tuberculosis in Adolescents. A Case Commentary. ClinPediatr1994; 33:258-262. 8. Nelson LJ, Wells CD. Global epidemiology of childhood tuberculosis. Int J Tuberc Lung Dis 2004; 8:636–647. 9. Raina N, Nayar PD, Mehta R, Dawa N. Adolescent Health and Development. In Ghai OP, Paul VK, Bagga A edGhai Essential Pediatrics; 7th edition; New Delhi: CBS Publishers, 2009: p42. 10. Central TB Division. RNTCP at a glance. http://www. tbcindia.nic.in/pdfs/RNTCP%20at%20a%20Glance. pdf. April 2, 2013 e-ISSN: 2349-6991; p-ISSN: 2455-0396

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11. Waako et al. Burden of tuberculosis disease among adolescents in a rural cohort in Eastern Uganda : BMC Infectious Diseases 2013, 13:349 http://www. biomedcentral.com/1471-2334/13/349 12. Uppada et al .Incidence of tuberculosis among school going adolescents in South India : BMC Public Health (2016) 16:641 13. Mahomed H, Ehrlich R, Hawkridge T, Hatherill M, Geiter L, et al. (2013) TB Incidence in an Adolescent Cohort in South Africa. PLoS ONE 8(3): e59652.

20.

21. 22.

14. Franco R, Santana M, Matos E, Sausa V, Lemos AC. Clinical and Radiological Analysis of Children and Adolescents With Tuberculosis in Bahia, Brazil . Braz J Infect Dis2003;7(1): 73-81.

23.

15. Phongsamart, Wanatpreeya MD, Kitai et al. A Population-Based Study of Tuberculosis in Children and Adolescents in Ontario.Pediatr Infect Dis J: 2009; 28 (5): 416-419.

24.

16. De Pontual L, Balu L, Ovetchkine P et al. Tuberculosis in adolescents; A French retrospective study of 52 cases. Pediatr Infect Dis J.2006 ;25(10):930-2. 17. Cruz AT, Hwang KM, Birnbaum GD, Starke JR. Adolescents with tuberculosis: a review of 145 cases. Pediatr Infect Dis J. 2013 Sep;32(9):937-41. http://dx.doi.org/10.1097/INF.0b013e3182933214 PMid:23538527 18. Shrivastava AK, Brahmachari S, Pathak P, Kumar Ratan, SainiaT, Patel U, Mandil A. ClinicoEpidemiological Profile of Extra-pulmonary Tuberculosis in Central India. Int J Med Res Rev 2015;3(2):223-230. 19. Lotfian F., Bolursaz M. R., Khalilzadeh S., Baghaie N., Hassanzad M., Velayati A. Features of Adolescents Tuberculosis at a Referral TB’s Hospital in Tehran,

25.

26.

27.

Iran. Mediterr J Hematol Infect Dis 2016, 8(1): e2016005, Didelescu C, Ibraim E, Tigau M. The epidemiology profile and current evolutionary trends in Tuberculosis in adolescents (15-19 years old). Pneumoftiziologia1997; 46(3): 193-9 Maltezou HC, Spyridis P. Extrapulmonary Tuberculosis in children. Arch Dis Child, 2000;83(4):342-6. R. Bryan Rock, Michael Olin, Cristina A. Baker, Thomas W. Molitor, and Phillip K. Peterson . Central Nervous System Tuberculosis: Pathogenesis and Clinical Aspects. Clin Microbiol Rev. 2008 Apr; 21(2): 243–261 Sant’Anna CC, Schmidt CM, March Mde F, Pereira SM, Barreto ML. Radiologic findings of pulmonary tuberculosis in adolescents. Braz J Infect Dis. 2011 Jan-Feb;15(1):40-4. Sant’Anna C, March MF, Barreto M, Pereira S, Schmidt C. Pulmonary tuberculosis in adolescents: radiographic features.Int J Tuberc Lung Dis. 2009;13(12):1566-8. Weber HC, Beyers N, Gie RP, Schaaf HS, Fish T, Donald PR. The clinical and radiological features of tuberculosis in adolescents. Ann Trop Paediatr. 2000 Mar;20(1):5-10. Nelson LJ, Schneider E, Wells CD, Moore M. Epidemiology of childhood tuberculosis in the United States, 1993-2001: the need for continued vigilance. Pediatrics. 2004 Aug;114(2):333-41. http://dx.doi. org/10.1542/peds.114.2.333 PMid:15286213 Nooshin Baghaie, Soheila Khalilzade, Mohammad Reza Boloursaz, Amir Ali Khodayari, and Ali Akbar Velayati. Extra Pulmonary Tuberculosis in Children: Two Years Study. Acta Medica Iranica 2010; 48(4): 239-243.

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Case Report Squamous Cell Carcinoma Vulva in a Young Woman Parmjit Kaur, Ruby Bhatia, Aman Dev Singh and Ramiti Gupta* Department of Obs & Gynae, GMC Patiala. Punjab, India

Keywords: Vulvar Cancer, Squamous Cell Carcinoma, Pap Smear, Vulvar Biopsy.

ABSTRACT Carcinoma of vulva is an uncommon malignancy accounting for only 2% of all female genital malignancies.[1] It is usually seen in postmenopausal women in age group â&#x2030;Ľ65 yrs. Increased life expectancy has given place to carcinoma vulva among gynaecological malignancies. However in recent years there is an increased incidence (almost doubled) of vulvar cancer in younger women.[2] We report a case of invasive squamous cell carcinoma of vulva stage IIIb in a 36 year old woman, indeed first only in thirty years of gynaecological practical experience. The relative rarity of vulvar cancer in young age and the general lack of awareness of typical signs and symptoms even by medical professionals frequently lead to a delay in diagnosis.

*Corresponding author: Dr. Ramiti Gupta, Junior Resident, Dept of Obs & Gynae, GMC Patiala. Punjab, INDIAx Phone: +91 9876635837 E-mail: gupta.ramiti@gmail.com

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Introduction

Carcinoma of vulva is an uncommon malignancy accounting for only 2% of female genital tract malignancies.[1] It is a disease of postmenopausal women with a median age at diagnosis of about 65 years. In the past three decades the incidence has doubled in younger women in U.K.[2] Squamous cell carcinoma accounts for more than 90% of vulvar cancers, while 10% include melanomas, sarcomas, basal cell carcinomas and adenocarcinoms.[3] Toki and collaborators[4] divided vulvar squamous cell carcinoma into two distinct groups. The first group comprises the vast majority of vulvar malignancies that occur mostly in older women. These neoplasias are keratinizing squamous cell carcinomas (KSCs), and human papillomavirus (HPV) is usually not found in them. The second group comprises a minority of vulvar malignancies occurring in younger women.This second group of tumors usually show a basaloid or warty histology and frequently are positive for HPV DNA.[4] We report a case of keratinizing squamous cell carcinoma vulva in a 36-year-old woman; which is otherwise seen in the postmenopausal age group.

Case Report(S)

A 36 year old obese woman Para2 presented to Out Patient Department of Gynaecology, Government Medical College & Rajindra Hospital Patiala with chief complaint of intense vulval itching & burning sensation with dysuria and burning micturition for last three months. On local examination 5x4 cm angry red, ulcerated, oozing lesion was seen on upper part of right labia majora and minora (fig 1). The growth was involving part of clitoris and right half of urethral meatus. Thick white discharge was oozing out of the lesion. Non tender matted lymph nodes about 3x3cm were palpable in right inguinal region. Vagina, cervix and uterus appeared normal on per speculum and per vaginum examination. There was no history of smoking, diabetes mellitus or hypertension. There was no history of genital malignancy in the family. Human Papilloma Virus Real time PCR was found negative. PAP smear was negative for intraepithelial lesions of malignancy. Patient was negative for VDRL and HIV infection. CT scan abdomen and pelvis depicted enlarged nodes measuring 3.4x3.2 cm in right inguinal region. Subcentimetric sized nodes are seen in left inguinal region. Biopsy from growth confirmed diagnosis of moderately differentiated keratinising squamous cell carcinoma vulva, usually not seen in young women. FNAC from right inguinal nodes show dysplastic cells with metastatic squamous cell carcinoma. A diagnosis of keratinizing squamous cell carcinoma vulva stage IIIb was made as per revised FIGO staging for vulvar cancer 2009.[5] Patient was treated with chemoradiotherapy Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

Fig. 1: Angry red, ulcerated, oozing lesion on upper part of right labia majora and minora

without surgical resection. 57.6 Gy intensity modulated radiotherapy (IMRT) with chemosensitising dose of cisplatin. Patient will be followed every three months for one year, six monthly for next five years and then annually. However patient advised to report earlier in event of any ongoing problem or recurrence of growth.

Discussion

Vulvar cancer is primarily a disease of postmenopausal women yet upto 15 % of vulvar cancers are diagnosed in women less than 40 years .[6] Risk factors for vulvar cancer include cigarette smoking, vulvar dystrophy ( eg lichen sclerosis), vulvar or cervical intraepithelial neoplasia, human papillomavirus infection, immunodeficiency syndromes, a prior history of cervical cancer etc.[7] Our patient was 36 year old with a diagnosis of moderately differentiated squamous cell carcinoma vulva however none of these risk factors were present in our patient. Vulvar carcinoma in situ (VIN) tends to be multifocal with a lower risk of invasive cancer in younger women but higher risk in older women. Thus VIN 3 should be treated with mandatory long term follow up. Two independent pathways of vulvar carcinogenesis are felt to currently exist, the first related e-ISSN: 2349-6991; p-ISSN: 2455-0396

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to mucosal HPV infection and second related to chronic inflammatory (vulvar dystrophy) or autoimmune processes. [8] Women with vulvar carcinoma may present with a vulvar lesion or vulvar pruritis with burning sensation and pain. Cervical cytology and/or cervical examination are recommended, as women with carcinoma of the vulva are at an increased risk of developing other anogenital cancers, particularly cervical cancer.[9,10] In early vulvar tumours the risk of lymph node metastases is reported as low. Clinical assessment of the lymph nodes alone is not recommended. [11] Due to the significant morbidity associated with groin lymphadenectomy, high-resolution imaging may be used preoperatively to stage disease and potentially detect those women who may not require lymphadenectomy. After FNAC when metastasis in inguinal nodes is confirmed disease is staged as stage IIIb (FIGO 2009). Our patient was also stage IIIb vulvar squamous cell carcinoma. Thirty per cent of patients are reported as having lymph node metastasis at presentation.[12] Modified radical vulvectomy with unilateral/bilateral inguinal/femoral lymphadenectomy depending on stage of disease remains standard therapy applied to most patients with vulvar carcinoma. Certain tumour characterstics may preclude an otherwise medically fit patient for undergoing primary surgery. Proximity to important functional structures such as urethra, clitoris and anal sphincter must be considered. If adequate surgical margins can not be obtained without sacrificing such a structure neoadjuvant treatment with radiation and/or chemotherapy should be considered.[1] Our patient was also treated with IMRT with chemosensitising dose of cisplatin. Advanced-stage disease (FIGO IIIâ&#x20AC;&#x201C;IV) may be difficult to manage, especially in cases where primary disease involves the anus, rectum, urethra, bladder or bulky groin nodes. Preoperative radiotherapy may allow for shrinkage of primary tumour (eg when trying to achieve sphincter-preserving surgery). Radical radiotherapy is used in patients for whom surgery is not an option and is usually combined with chemotherapy. [13] Primary tumour factors that appear to have prognostic importance include tumour diameter, depth of invasion or tumour thickness, tumour differentiation, lymphovascular space involvement and surgical margin status. Tumour involvement of distal urethra, vagina or perineum is also an adverse prognostic factor.[1] In our patient growth was involving right half of urethra and clitoris along with involvement of right inguinal lymph nodes. The fiveyear survival rate for women with positive nodal disease (<50 per cent), however, is much lower than for those with node-negative disease (>80 per cent).[6] Groningen international study on sentinel nodes in vulvar cancer (GROINSS-VII)[14], is looking at whether it is safe to have

radiotherapy instead of surgery when vulvar cancer has spread to the sentinel nodes. Invasive squamous cell carcinoma in our patient (young woman) is not completely clear as none of the associated risk factors were present. HPV infection is a known cause of vulvar carcinoma in young patients but our patient was negative for HPV/HIV infection with normal pap smear. she might have an unknown immune deficiency that allowed abnormal cells to proliferate and progress to cancer. Long term follow up after treatment of vulvar cancer is recommended. Every three months for one year, every six months for five years and annually thereafter. Annual cervical or vaginal cuff cytology is warranted as 15-40% patients may have local recurrence.[1]

Conclusion

Vulvar self examination for all women on monthly basis should be promoted for early detection of vulvar cancer as recommended by vulval health awareness campaign (VHAC). Biopsy of any questionable vulvar lesion prior to empirical treatment is warranted. A careful pelvic examination and pap smear screening must be done in patients with vulvar carcinoma as these women have increased incidence of cervical and vaginal neoplasia.

Acknowledgements

We would like to acknowledge the help of pathology department for diagnosis and radiotherapy department for treatment of our patient.

Funding None

Competing Interests None Declared

Reference

1. Mitchel S. Hoffman and Xiaomang B.Stickles: malignancies of vulva. TeLindeâ&#x20AC;&#x2122;s operative gynaecology 11th ed. South Asian Edition: Wolters Kluwer; 2015:1141-79. 2. Cancer Research UK. UK vulva cancer incidence statistics, 2008 (http://info.cancerresearchuk.org/ cancerstats/types/vulva/incidence/?a=5441; accessed 20 August 2009) 15. 3. Julia E, Palmer and Alan M Gillespie Diagnosis and management of squamous cell vulvar carcinoma. Trends in urology, gynaecology and sexual health. March- April 2010;20-5 4. Toki T, Kurman RJ, Park JS, Kessis T, Daniel RW, Shak KV. Probable nonpapillomavirus etiology of squamous cell carcinoma of the vulva in older women:a clinicopathologic study using in situ hybridization

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and polymerase chain reaction.Int J Gynecol Pathol. 1991;10:107–125 Pecorelli S. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J Gynecol Obstet 2009;105:103–4. Royal College of Obstetricians and Gynaecologists. Management of vulval cancer. London: RCOG Press, 2006 Madsen BS, Jensen HL, van den Brule AJ, et al. Risk factors for invasive squamous cell carcinoma of the vulva and vagina--population-based case-control study in Denmark. Int J Cancer 2008; 122:2827. De Koning MN, Quint WG, Pirog EC. Prevalence of mucosal and cutaneous human papillomaviruses in different histologic subtypes of vulvar carcinoma. Mod Pathol 2008; 21:334. Edgren G, Sparen P. Risk of anogenital cancer after diagnosis of cervical intraepithelial neoplasia: a prospective population-based study. Lancet Oncol 2007;8:311–6.

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AABS; 3(4): 2016 10. Kalliala I, Anttila A, Pukkala E, et al.Risk of cervical and other cancers after treatment of cervical intraepithelial neoplasia: retrospective cohort study BMJ2005;331:1183–5. 11. Selman TJ, Luesley DM, Acheson N, et al. A systematic review of the accuracy of diagnostic tests for inguinal lymph node status in vulvar cancer. Gynecol Oncol2005;99:206–14. 12. Royal College of Pathologists. Dataset for histological reporting of vulval neoplasms,version 2, 2nd edn. London: Royal College of Pathologists, 2008. 13. Van Doorn HC, Ansink A, Verhaar-Langereis MMJ, et al.Neoadjuvant chemoradiation for advanced primary vulvar cancer. Cochrane Database Syst Rev 2006, Issue 3. Art. No.: CD003752. DOI:10.1002/14651858. CD003752.pub2 14. www.cancerresearchuk.org/.../a-study-looking-attesting-sentinel-lymph-nodes-radiotherapy-andchemotherapy-for-vulval-cancer.

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Case Report Adenomatoid Tumor of Epididymis: A Case Report with Correlation Between Histology and Cytology Mega Lahori1*, Sakul2, K C Goswami3 and Arvind Khajuria3 Deptt of Pathology, Acharya Shri Chander College of Medical Sciences Jammu (J&K), INDIA Deptt of Medicine, Acharya Shri Chander College of Medical Sciences Jammu (J&K) INDIA 3 Deptt of Pathology, Acharya Shri Chander College of Medical Sciences Jammu (J&K)

Keywords: Adenomatoid Tumor, Epididymal Lesion, Paratesticular Tumors, Mesothelial Neoplasm

ABSTRACT Adenomatoid tumor is a rare and distinctive, benign mesothelial neoplasm of the paratesticular region in males, most commonly occurring at the tail of epididymis and constitutes 30% of paratesticular neoplasms. We present a case of adenomatoid tumor in a 35 year old male, who presented with a mass in right epididymis and was diagnosed by fine needle aspiration cytology and later confirmed on histopathology. The clinical, histopathological and cytological aspects of this rare case are discussed. FNAC plays a pivotal role in the preoperative diagnosis of these tumors as it is a simple, inexpensive, minimally invasive and reliable diagnostic modality. Due to its benign nature, the treatment of choice is local excision. We believe it is important for the physician to be aware of this interesting entity in order to make an accurate diagnosis and prevent unnecessary orchiectomy.

*Corresponding author: Dr Mega Lahori, PG Resident, Deptt of Pathology, Acharya Shri Chander College of Medical Sciences Jammu (J&K) 180017, INDIA Phone: +91 9419177133 E-mail: iammegha00@gmail.com

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AABS; 3(4): 2016

Introduction

Adenomatoid tumor is a rare and benign neoplasm of mesothelial origin seen in male as well as female genital tract along with extragenital sites, but is more common in male adnexa. It is seen usually in the third &fourth decades (mean age is 36 yrs).[1] Adenomatoid tumor was first described in 1945 by Golden and Ash as a small, firm asymptomatic intrascrotal mass, with no pain or tenderness, occurring in third to fifth decades of life. These tumors represent 30% of the paratesticular tumors. [2] Beccia et al studied 314 epididymal tumors, of which 75% were benign and 73% of those were diagnosed as adenomatoid tumors, followed by leiomyomas (11%), and papillary cystadenomas (9%). These tumors are benign in nature (even when they infiltrate into the adjacent sites), with no reported cases of malignant change, metastasis, or relapse after removal, and tumor excision is therefore the treatment of choice. [1]Accurate diagnosis is the key to prevent unnecessary orchiectomy. [3]

Fig. 1: FNAC smear showing monolayered sheet of monomorphic cells with pale, vacuolated cytoplasm (H&E, 100x).

Case Report

A 35 year old male presented at the Urology OPD of our hospital with one year history of a slowly enlarging and painless right sided scrotal mass (felt at the lower pole of epididymis) which was nontender, non fluctuant and non transilluminant on local examination. Personal and family history was unremarkable. There was no history of epididymitis, torsion or trauma. Aspiration from the mass was done using a 10 ml syringe and 24G needle to obtain a scanty, whitish aspirate. FNAC from the mass revealed moderately cellular smears composed of monomorphic, round to oval tumor cells arranged in sheets and multilayered clusters. The cells were round to oval with pale vacuolated cytoplasm, eccentric nucleus having fine chromatin and small, central nucleolus within a background of naked nuclei and stromal bits (Fig 1). A cytological diagnosis of adenomatoid tumor was made; following which the patient underwent a conservative testis sparing surgery with the excision of epididymal nodule which was received in the Histopathology section of our Deptt of Pathology. Grossly, the nodule was globular, measured 1.5x1.3cm in diameter and was grayish-white on cut section (Fig 2a). Histopathological examination revealed a well circumscribed, non encapsulated tumor with characteristic features of adenomatoid tumor composed of tubules and cords of cuboidal cells having large intracytoplasmic vacuoles and gaping spaces (Fig 2b).The cellular vacuoles had a signet ring-like appearance in some fields. There was no mitotic activity or nuclear atypia. Prominent lymphoid collections were seen in a fibroblastic stroma, which is another important clue to the diagnosis. Resection margins were free of tumor cells. This tumor was diagnosed as adenomatoid tumor and the patient underwent no additional treatment. Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

Fig. 2a: Cut section of tumor nodule.

Fig. 2b: Adenomatoid tumor with gaping spaces and cuboidal cells having vacuolated cytoplasm (H&E, 40x).

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Discussion

Most Adenomatoid tumors are asymptomatic masses reported by the patient and, generally remain unchanged in size for years. In males they occur in the epididymis, spermatic cord, prostate and ejaculatory duct. Mostly they arise at the lower pole of the epididymis. In females they occur in uterus, fallopian tubes and ovarian hilus. [1] They are also seen in extragenital sites like adrenal gland, lymph nodes, mediastinum, pleura, pancreas and heart in either sex. They usually present as small (~2cm), solid, firm masses and on cut section, they appear grayish-white & homogeneous (the excised nodule in the present case also had solid, homogeneous cut surface). Occasionally, small cysts may be seen. These tumors are usually well circumscribed but non-encapsulated. They have a plethora of microscopic appearances, represented by four basic patterns: adenomatoid (tubular), angiomatoid (canalicular), plexiform (solid nests) and cystic (mixed) [the tumor in the present case had an adenomatoid (tubular) histological pattern]. Cells are cuboidal to flattened, with weakly eosinophilic & markedly vacuolated cytoplasm. Nucleus is eccentric with fine chromatin and small, central nucleolus and little to no mitotic activity. Cells may form solid cords alternating with channels having dilated luminasimulating vascular structures. Cystically dilated gaping spaces with no evident lining, representing a necrotic tubular component, and smaller gland-like spaces (giving the appearance of vascular spaces) are major clues to the diagnosis. Stroma is prominent and fibrous, with abundant smooth muscle and elastic fibers. Additional stromal features may be-prominent lymphoid follicles, extensive necrosis, abundant desmoplasia and signet-ring like cells (in clusters or individually scattered). The first cytological description of adenomatoid tumors was given by Perez-Guillermo et al in 1989. Cytology reveals sheets, cords, glandular patterns or multilayered clusters of monotonous, round to oval cells with pale, vacuolated cytoplasm. Nucleus is spherical & eccentric with fine chromatin and small central nucleolus.[4] They are considered to be of mesothelial origin (as originally proposed by Masson et al), which is supported by immunohistochemical studies (positivity for HMBE1 &Calretinin) and genetic analysis of WT-1 gene expression. [1]These tumors show positive immunoreactivity forCalretinin, Epithelial markers (AE1AE3, EMA,

Cam5.2, CK5/6, CK7), Vimentin, WT1, HMBE 1, D240 and are negative for Carcinoma markers (CEA, CD15, B72.3, MOC-31, Ber-ep4), Endothelial markers (FVIIIRA, CD31, CD34), Germ cell tumor markers (OCT 3/4, Nanog, Sox-2, AFP, PLAP) and Inhibin.Diagnosis is based mainly on histopathology, aspiration cytology & ultrasonography, which is supported by immunohistochemistry.[5,6] The ultrasound features of adenomatoid tumors may vary but usually include a well-defined, homogeneous, round, hyperechoic nodule. The differential diagnosis includes epithelioid hemangioma, malignant mesothelioma, metastatic adenocarcinoma, papillary cystadenoma of epididymis, large cell calcifying Sertoli cell tumor, Epididymidal carcinoma, testicular rhabdomyosarcoma, and carcinoma of rete testis. Malignant mesotheliomas usually show abundant necrosis and mitoses while papillary cystadenomas can be differentiated on the basis of numerous papillary infoldings which project into cystic spaces. The absence of staining of epithelial markers is of utility in excluding carcinomas (CEA, PSA, MOC 31/BerEP4) and germ cell tumors (0CT 3/4, Sox2, AFP, PLAP) from the differential diagnosis, while negativity of vascular markers (CD34, factor VIII) excludes a diagnosis of epithelioid hemangioma.

Conclusion

Adenomatoid tumor should be suspected in any intrascrotal mass lesion. Its separation from testicular tumors is important as paratesticular tumors have good prognosis compared to testicular tumors. We are highlighting the strong correlation between cytology and histology features. Both diagnostic modalities reveal a monotonous proliferation of tumor cells with similar cellular features. FNAC, as a preoperative diagnostic tool, can help to plan surgery as complete local excision of this benign tumor is both diagnostic as well as therapeutic and has had no reported case of recurrence or metastasis after excision. The main clinical consideration is accurate diagnosis in order to prevent unnecessary orchiectomy and preserve endogenous testicular function.

Acknowledgement

Mr Rajesh Verma for providing technical support

Funding None

Competing Interests None Declared

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References

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1. Amin W, Parvani A V. Adenomatoid tumor of testis. Clin Med Pathol. 2009; 2: 17–22. 2. Moyano CJL, Giraldez PJ, Sanchez de la Vega J, Casanova GD, Lópezet AM. Adenomatoid tumor of the epididymis. ActasUrolEsp 2007; 31: 417. 3. Manjunath GV, Nandini NM. Fine needle aspiration cytology of adenomatoid tumor - a case report with review of literature. Indian J PatholMicrobiol 2005;48:503–4.

4. Gupta S, Garg S, Agarwal R, Sen R. Aspiration cytology of adenomatoid tumor of epididymis: An important diagnostic tool. JCSR. 2012;4:11. 5. Sangoi AR, McKenney JK, Schwartz EJ, Rouse RV, Longacre TA. Adenomatoid tumors of the male & female genital tracts: a clinicopathological and immunohistochemical study of 44 cases. Mod Pathol 2009, 22: 1228-1235. 6. Kalyani R, Das S. Adenomatoid tumor: Cytological diagnosis of two cases. J. Cytol 2009 Jan-Mar; 26: 30-32.

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Case Report A Rare Case of Non Communicating Rudimentary Horn with Unicornuate Uterus Puja Jain*, Sunita Fotedar, Suman Raje and Rekha Daral Department of Obstetrics & Gynecology, Swami Dayanand Hospital Dilshad Garden, Delhi, India Keywords: Unicornuate Uterus, Rudimentary Horn, Endometriosis

ABSTRACT Mullerian duct malformations delineate a miscellaneous group of congenital anomalies that result from the arrested development ,abnormal formation, or incomplete fusion of the mesonephric ducts. Unicornuate uterus with rudimentary horn is a rare mullerian duct anomaly of female genital tract. The frequency of this pathology is 1/100 000. It is responsible for many obstetrics and gynecological complications during reproductive life of women. These uterine anomalies are either diagnosed incidentally or the patient may present with obstetrical or gynecological problems. These patients present with dysmenorrhea,dyspareunia, and rarely chronic pelvic pain because of endometriosis and rarely with acute abdominal symptoms following distention and torsion of the non communicating rudimentary horn. We present a case of pelvic endometriosis due to non communicating but functional rudimentary horn of a uterus in a multiparous woman.

*Corresponding author: Dr Puja Jain, 5204, ATS ADVANTAGE, Ahinsa Khand I, Indirapuram,Ghaziabad, U.P.-201012. INDIA Phone: +91 9899807202 E-mail: dranujpuja@gmail.com

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Introduction

Mullerian duct malformations delineate a miscellaneous group of congenital anomalies that result from arrested development, abnormal formation, or incomplete fusion of mesonephric ducts. [1] The incidence of uterine anomalies in a fertile population is reported to be around 3.2%. [2] Unicornuate uterus with a rudimentary horn is a very rare congenital malformation of female genital tract. The frequency of this pathology is approximately 1/100,000. A rudimentary horn usually develops following incomplete development of one of the mullerian ducts. [3] Besides gynecological complications such as endometriosis, primary infertility and hematometra, anomalies of the urinary system and obstetric problems such as malpresentations, habitual abortions and premature births can occur.[4]

AABS; 3(4): 2016 obvious septations of approximately 10cm by 7.6 cm in size seen in left iliac fossa extending into the left side of pelvis. Left ovary was not seen separately from the lesion .Right ovary and uterus was normal.MRI findings were suggestive of complex ovarian cyst or neoplastic ovarian mass. Her hemoglobin was 11 gms percent and all her serological test were normal. Her CA -125 levels was 243.3u/ml.

This patient 40 year old , para3 with 3 living issues was presented to the gynecology outpatient department with history of progressive pain in left lower abdomen from last 6 months to 1 year duration. Her menstrual cycles were of 30-35 days with slightly decreased flow since last 5-6 months. There was no other menstrual complaints, no history of dyspareunia or bowel and bladder disturbances. She was receiving antispasmodics and NSAIDS for her pain for last one year. Her per abdominal examination were normal. Her clinical pelvic examination revealed a mass of about 6 by 6 cms in the left fornix ,soft to firm in consistency, adherent to uterus. Uterus was normal in size with slightly restricted mobility. There was tenderness over the mass on bimanual examination. Pelvic ultrasound examination was done which revealed 7cm by 6 cm multiloculated space occupying lesion in the left adenexal region. MRI pelvis revealed a large well defined lobulated solid cystic lesion with no

Patient was posted for laprotomy with the possibility of large left ovarian dermoid cyst, tubo ovarian mass or sub serous fibroid. On laprotomy a large cystic lesion was found on the left side of pelvis which was adherent to uterus and also to lateral pelvic wall. Cyst was separated from all sides and from omental and bowel adhesions by blunt and sharp dissections till the base of pedicle was reached which was clamped and removed. Thick endometriotic fluid was present in cyst. On right side another uterus was found with the right tube and ovary. The right round ligament arose from the right uterine cornual region. The left round ligament arose from another mass of about 4 cm in size which was considered a rudimentary horn. The left uterine tube arose from the superior portion of rudimentary horn . So it was diagnosed as a case of Unicornuate uterus with non communicating rudimentary horn with functional endometrium. (Fig 1). Cut section of rudimentary horn showed small cavity with thin endometrial lining. (Fig 2). The decision for hysterectomy was taken in view of perimenopausal age group of patient ,complete family size, risk of recurrence of chronic pelvic endometriosis and refusal of patients consent for fertility conserving surgery. Patient withstood the procedure well and her post op period was uneventfull.On discharge patient was kept on monthly injections of GnRH analogue upto 3 months. Histopathology of cyst wall confirmed the diagnosis of endometrosis.Histopathology of uterine horn confirmed the diagnosis of its functional endometrium.

Fig. 1: rudimentary horn of uterus with Endometriotic cyst.

Fig. 2: Cut Section of Unicornuate uterus with rudimentary horn.

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Discussion

The true incidence of Unicornuate uterus is not known. Current estimates are based only on those few cases which are actually diagnosed and subsequently reported in peer review journals. The reported incidence of uterine anomalies in fertile population is around 3.2%.[2] Unicornuate uterus with rudimentary horn is a rare type of mullerian duct malformation representing only 1-2 % of congenital mullerian anomalies.[5,6] It results from defective fusion of the malformed duct with the contra lateral duct.[7] The uterine anomaly covers a wide range of anatomical variability and is divided into four subgroups according to American Fertility Society classification of mullerian anomalies: (IIa) rudimentary horn with cavity communicating to Unicornuate uterus, (IIb) with cavity non communicating , (IIc) with no cavity,(IId) with no horn. [8] Type IIb is the most common and clinically significant type. A fibrous or fibro muscular band usually connect the horns, but in 80 -90 % of cases there is no communication. This condition is often asymptomatic due to lack of functional endometrium.[9] However when the horn is lined with functional endometrium, the resulting obstructed menstrual flow may cause severe cyclic pain shortly after menarche.[10] Retrograde menstruation is thought to initiate and potentiate endometriosis in women with non communicating uterine anomalies. Endometriosis seen in these cases supports the retrograde menstruation theory. The pain in endometriosis in these cases is usually severe and results in severe dysmenorrhea ,chronic pelvic pain and dyspareunia.[11] The cause of pain in these cases may also be because of hematometra ,causing distention of uterus. Another problem with the non communicating rudimentary horn is rudimentary horn pregnancy. [4] Since myometrial tissue is thin in a rudimentary horn of uterus, uterine rupture is seen frequently in rudimentary horn pregnancies. [4] The presence of gestation in a non communicating rudimentary uterus can be explained by transperitoneal migration of sperm . Although this is uncommon ,these pregnancies may lead to serious complications .Urinary tract abnormalities are commonly associated with mullerian anomalies. This anomaly is associated with ipsilateral renal agenesis (67%) or ipsilateral pelvic kidney. [12] The diagnosis of rudimentary horn is not made until the reproductive age when ruptured rudimentary horn pregnancy or pelvic pain occurs or sometimes it may be diagnosed accidently during laprotomy. The marked lower abdominal pain and pelvic pain usually bring patients for imaging in the form of ultrasound, CT scan or MRI which demonstrate a pelvic mass. In order to facilitate the surgical procedure, it seems important to be prepared

for either presentation. Recent literature has suggested that MRI provides a considerably improved and accurate means of diagnosing and identifying mullerian anomalies. [13] Recently ,three dimensional sonography has been introduced into clinical practice and offers advantages over two dimensional scanning as it provides fine anatomical anatomic details , useful for preoperative planning. [14] The traditional surgical approach to treatment of this problem has been through laprotomy and removal of the dilated non communicating horn. Since the first report by Cannei et el (1990), laparoscopic resection of rudimentary uterine horn has rapidly become the standard treatment of such mullerian dysgenesis ,specially to prevent severe complications as ectopic pregnancy or extensive endometriosis.[15] Though in our case decision for hysterectomy was taken, in considering the perimenopausal age, complete family and refusal for consent for fertility preserving surgery.

Conclusion

Non communicating horn of uterus is a rare cause of pelvic endometriosis. Women in reproductive age groups with dysmenorrhea or lower abdominal and pelvic pain with adenexal mass, the rudimentary horn with functional endometrium should be kept as differential diagnosis. Laparoscopic resection of non communicating rudimentary horn is the preferred treatment. It gives benefit to the patient of shorter hospital stay, significantly reduced postoperative morbidity and speedy recovery.

Acknowledgements None

Funding None

Competing Interests Not Declared

Reference

1. F Raga, C Bauset, J. Remohi, F Bonilla â&#x20AC;&#x201C; Musoles, C. Simon, and A Pellicer, Reproductive impact of congenital mullerian anomalies, Human Reproduction, vol 12, no, pp 2277-2281, 1997. 2. C .Simon, L. Martizeg, F Pardo, M Tortajado, and A Pellicar, â&#x20AC;&#x2DC;Mullerian defects in women and normal reproductive outcome, Fertility and Sterility, volume 12, no, pp. 2277-2281,1997. 3. Atmaca R, German AT, Burak F, Kafkash; A. Acute abdomen in a case with non communicating rudimentary horn and Unicornuate uterus. JSLS 2005; 9(2) 235-237.

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4. Kuskan N K, Lacin S, Kartal O. Rupture of rudimentary horn pregnancy at 15th week of gestation: a case report. Eur J Obstet Gynecol Reprod Biol.2002; 102 :209-210 [Pubmed]. 5. The American Fertility Society classification of adenexal adhesions, distal tubal occlusion, secondary to tubal ligation, tubal pregnancies, mullerian anomalies’ and intrauterine adhesions . Fetil Steril. 1988;49:944-955. 6. Shattman GL, G rifo JA Brinbaun S Laparoscopic resection of non communicating rudimentary uterine horn : Case Report. J Reprod Med 1995; 40 :219-220. 7. Crosby WM, Hill EC. Embryology of the mullerian duct system Review of present day theory. Obstet Gynecol 1962;20:507-15. 8. The American Fertility Society classification of adenexal adhesions, distal tubal occlusion, secondary to tubal ligation, tubal pregnancies, mullerian anomalies and intrauterine adhesions. Fetil Steril. 1988;49:944-955. 9. Speroff L, Glass RH, Kase NG. Clinical Gynecologic Endocrinology and Infertility. 6th ed. Baltimore, Md: Lippincott, Williams & Wilkins;1999:148.

10. March CM. Hysteroscopy and the uterine factor in infertility. In Lobo RA, Mishell DR ,Paulson RJ, Shoupe D, eds. Mishells Textbook of infertility, contraception, Reproductive Endocrinology. 4th ed Malden, Mass: Blackwell Science; 1997:580-603.

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11. Perrotin F, Bertand J Body G Laparoscopic surgery of Unicornuate uterus with rudimentary uterine horn : Case Report. Hum Reprod 1999 ;14(4):931-933. 12. F.F Marshall and D.S. Beisel. ‘The association of uterine and renal anomalies’, ”obstetrics and gynecology , vol 51 , no5, pp559-562, 1978. 13. Amara, D.P. Nezhat, F Gludice, L. etal, 1997. Laproscopic management of a non communicating uterine horn in a patient of an acute abdomen. Surg. Laparoscope. Endorse, 7, 56-59. 14. Heinonen, Pk (1997). Unicornuate uterus and rudimentary horn. Fertil.Steril, 168, 224-230. 15. Canis M, Wattiez, A Pouly. J Letal s (1990). Laparoscopic management of Unicornuate uterus with rudimentary horn and unilateral extensive endometriosis : case report. Hum Repro, 5, 819-820.

Case Report Rare site of Metastasis in Cancer Cervix: A Case Report Sweta Khanuja* and Vijay Anand Department of Radiotherapy, S.N. Medical College, Agra Keywords: Cervical Cancer, Abdominal Wall Metastasis

ABSTRACT Cervical cancer rarely metastasizes to skin. This occurs in <2% of patients. It is more common in patients who do not receive radiotherapy after surgery but can be seen in patients after radiotherapy who have deferred taking radiotherapy by few months. We report a patient who received radiotherapy four months after surgery and remained asymptomatic for 5 months. She then noticed a lump in abdomen for which she underwent excision of the lump and it was reported as squamous cell carcinoma. The patient was concluded to have abdominal wall recurrence of cancer cervix.

*Corresponding author: Dr Sweta Khanuja ,51 Paschim Puri, Agra-282007 Phone: +91 7895617755 E-mail: drswetakhanuja@gmail.com

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Introduction

Cervical cancer is the leading cancer in India and the third most common cancer in the world (1). It spreads through direct local and lymphatic pathways. Hematogenous metastasis is relatively rare and so is the abdominal wall metastasis of cervical cancer. This is associated with poor prognosis, with average survival reported in literature to be around 8-9 months (2).

Case Report

We hereby report a 38-year-old female who presented to our Radiotherapy department in June 2015. Her presenting complaints were pain in abdomen and white discharge per vagina. 18 months ago she had developed similar problems along with bleeding per vagina, for which she underwent hysterectomy elsewhere and later diagnosed to have squamous cell carcinoma of cervix. She was advised Radiotherapy, which she deferred for 4 months. She received external beam radiotherapy along with concurrent chemotherapy followed by intracavitary radiotherapy and remained symptom free for following 5 months.

Fig. 1: Picture showing abdominal lump.

Abdominal examination on this visit revealed 6cm by 5cm globular lump in midline above the pubic symphysis reaching upto the umbilicus (Fig 1). It was hard to palpate but non-tender. Overlying skin was shiny and no colour change was noticed. Margins were clearly defined and surface of the lump was smooth. The lump was found to be irreducible but mobile. Per vaginal examination revealed a smooth cervical vault and no growth was felt. Further work up was advised in the form of blood investigations including complete blood count,renal and liver function tests. Radiological investigations included chest radiograph and contrast enhanced computed tomography. The CT scan revealed hyperdense cystic and solid mass containing approximately 21 cc fluid in anterior abdominal wall. Vaginal vault region appeared mildly bulky with a small 1 cm by 1 cm hypodense area. Fine needle aspiration was done from the abdominal wall mass, which showed features of metastatic squamous carcinoma.

Fig. 2: Histopathology of excised mass confirming squamous carcinoma.

The patient was then referred for surgical opinion. She was taken for exploratory laparotomy. The lump was found involving the rectus sheath. Wide local excision with sheath removal was done. In view of the defect and difficulty in closure of abdominal wall, composite mesh was placed. Histopathology of excised mass confirmed squamouscarcinoma (Fig 2). Resected margins were free of tumour (Fig 3). The pain in abdomen and vaginal discharge persisted and 3 months later she again reported with another palpable mass in paraumblical region. Pap smear and FNAC from Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

Fig. 3: Resected margins free of tumour.

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abdominal lump showed features of malignancy and granulomatous inflammation.

and treatment would improve the chances of survival and quality of life.

Discussion

Acknowledgements

Metastasis to abdominal wall from cancer cervix is fairly an uncommon occurrence. Such metastasis to incisional scar site has been noticed to be higher in patients with either adenocarcinoma or undifferentiated carcinoma than in patients with squamous cell carcinoma. In cases of cervical carcinoma abdominal wall metastasis occurs mostly in cases of tumour recurrences, with cases reported even upto 10 years and averaging less than 1 year(3). There are two possible mechanisms that could explain metastatic recurrence at the incision site. Firstly, it is possible for direct tumor seeding to occur at the time of surgery. Secondly, fibrin platelet deposits in microcirculation of the wound may trap circulating tumor cells. Depending on the different phases of the healing process cancer cells maybe recruited, replicated and selected at the surgical wound. Prognosis in such cases depends upon time interval between the initial diagnosis of primary malignancy and the appearance of abdominal wall metastasis. The earlier is the metastasis, worse is the prognosis. The treatment options remain palliation, either by radiation, chemotherapy or surgery, either alone or in combination (4, 5, 6). The best bet is carefully handling the carcinomatous tissue during surgery and including the surgical scar site during radiotherapy. These days, laparoscopy has also been resorted to during surgery for cervical carcinoma. Port site metastasis has also been reported after laparoscopic lymphadenectomy. Picone et al reported a case of adenocarcinoma cervix in which laparoscopic ovarian transposition was done prior to radiotherapy and patient developed trocar site metastasis after 5 months (7).

Conclusion

Incisional scar site metastasis in cases of cancer cervix is a rare clinical entity and is associated with a poorer prognosis. We propose that cervical carcinoma patients who, develop lumps at scar site, should undergo FNAC/ biopsy at the earliest to rule out metastasis. Early diagnosis

We acknowledge the help, constant support provided by Dr Tabassum, Lecturer, Dr Surabhi Gupta, Associate Professor and Dr A K Arya, Professor & Head, Department of Radiotherapy, SN Medical College, Agra, while managing this case.

Funding None

Competing Interests None Declared

Reference

1. Ferlay J, Shin HR, Forman D, Mathers C, Parkin DM. Estimates of Worldwide burden of Cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; 127:2893-917. 2. Imachi M, Tsukamoto N, Kinoshita S, Nakano H. Skin metastasis from carcinoma of the uterine cervix. Gynecol Oncol 1993;48:349-54. 3. Copas PR, Spann CO, Thoms WW, Horowitz IR. Squamous cell carcinoma of the cervix metastatic to a drain site. Gynecol Oncol 1995;56:102-4. 4. Tharakaram S, Rajendran SS, Premalatha S, Yesudian P, Zahara A. Cutaneous metastasis from Carcinoma Cervix. Int J Dermatol 1985;24:598-9. 5. Diehl LF, Hurwitz MA, Johnson SA, Butler WM, Taylor HG. Skin metastases confined to a field of previous irradiation; report of two cases and review of the literature. Cancer 1984;53:1864-8. 6. Behtash N, Mehrdad N, Shamshirsaz A, Hashemi R, Amouzegar F. Umblical metastasis in cervical cancer. Arch Gynecol Obstet 2008;278(5):489-91. Epub 2008 Apr 1. 7. Picone O, Aucouturier J.S, Louboutin A, Coscas Y, Camus E. Abdominal wall metastasis of cervical adenocarcinoma at Laparoscopic tracer insertion site after ovarian transposition: case report and review of literature. Gynecol Oncol 2003;90:446-49.

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Case Report Central Serous Chorioretinopathy Leading to sub Retinal Bleed to Postvitrectomy Endophthalmitis: Diagnostic Dilemma Balbir Khan1, Vartika Anand1*, Sachin Anand2 and Meenu Kashyap1 Adesh Medical College and Hospital Mohri NH 1 Ambala, India Shri Har Rai Shaib Diagnostic Centre, Sector 22 D, Chandigarh, India 1

Keywords: Endophthalmitis, Subretinal Haemorrhage, Central Serous Retinopathy

ABSTRACT Central serous retinopathy (CSR) is a common cause of visual disturbance in the younger age group. Spontaneous visual recovery occurs in the majority of patients. A minority of patients, however, suffer permanent visual loss commonly caused by chronic retinal pigment epithelial changes. We report a devastating complication of CSR, with sub retinal haemorrhage.

*Corresponding author: Dr. Vartika Anand, House NO.169 Sector 19-A Chandigarh 160019, India. Phone: +91 9988880755 E-mail: varti_in@yahoo.com

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Introduction

Central serous chorioretinopathy (CSCR) is a disease in which a serous detachment of the neurosensory retina occurs over an area of leakage from the choriocapillaris through the retinal pigment epithelium (RPE). Other causes for RPE leaks, such as choroidal neovascularization, inflammation, or tumors, should be ruled out to make the diagnosis.

Case Report

A 34 year old young male presented with a 2 week history of blurred central vision and metamorphopsia affecting his right eye. Ocular examination revealed best corrected visual acuity of 6/12 in right eye and 6/6 in left eye. Funduscopy revealed a neurosensory retinal detachment overlying the right fovea, with retinal pigment epithelial changes. Fundus fluorescein angiography (FFA) confirmed the diagnosis of CSR. He was taking steroids for his nasal problem. Advised him to stop the steroids and put the patient on NSAIDS. He recovered well. Then again after six months he came back with decreased vision in right eye to 6/60.Anterior segment was normal fundus revealed neurosensory detachment overlying the right fovea, diagnosis of CSR

was confirmed. This time 0.05ml of IVA(avastin) was given and he responded well.On further follow up his visual acquity recovered to 6/9. After one year he came back with complaints of decreased vision and some floaters coming in front of eye, visual acquity was counting finger. Fundus examination revealed a massive macular sub retinal haemorrhage in the right eye.FFA shows extensive masking (fig. 1). OCT also shows detachment of neurosensory retina with underlying blood (fig.2). Physical examination and investigations revealed no evidence of underlying systemic disease. Full blood count and coagulation screen were normal. We kept the patient for observation for at least 1 month but haemorrhage didnâ&#x20AC;&#x2122;t resolved so he underwent 23G PPV. To our surprise very next day pt developed endophthalmitis, intravitreal vancomycin with ceftazidine and dexamethasone given endophthalmitis didnâ&#x20AC;&#x2122;t resolved, planned for revitrectomy with silicon oil insertion with intravitreal vanco with ceftazidine. During revitrectomy there was touch to the posterior capsule of lens which leads to development of cataract for which patient underwent phaco with IOL implantation in sulcus and silicon oil removal. After 6 weeks patient recovered 6/12 vision and it remained stable till date.

Fig. 1: Fundus examination revealed a massive macular sub retinal haemorrhage in the right eye. FFA shows extensive masking.

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Fig. 2: OCT also shows detachment of neurosensory retina with underlying blood.

Discussion

Massive sub retinal macular haemorrhage can occur secondary to a number of causes such as choroidal neovascularisation (CNV), retinal artery macro aneurysm, idiopathic polypoidal choroidal vasculopathy, blood dyscrasia, or trauma. Histopathological analysis of patients with age related CNV, complicated by massive sub retinal haemorrhage may be associated with rupture of a large choroidal blood vessel.1 CNV is known to occur infrequently in patients with CSR treated with laser photocoagulation.2 In only two previous cases have CNV developed spontaneously in patients with CSR.3 Massive sub retinal haemorrhage, however, was not the feature in these two reported cases. The pathogenesis of CSR has been disputed. Recent studies with ICG suggest focal choroidal hyper permeability as the possible initial event, leading to the formation of serous retinal pigment epithelial detachment. Excessive fluid accumulation then leads to pressure on the retinal pigment epithelium, resulting in either mechanical disruption or retinal pigment epithelial decompensation.4 The chronic secondary retinal pigment epithelial changes, if extensive, could predispose to the development of CNV. In our patient, the sudden onset of haemorrhage in right eyes may in part be explained by the presence of disorganised and dysfunctional choroidal blood vessels. The latter leads to an initial increase in choroidal hyper permeability (hence the CSR) and later, the tendency to rupture suddenly resulting in massive haemorrhage (as illustrated by our case).

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Conclusion

Still it’s a diagnostic dilemma what was the cause for developing subretinal bleed after CSR, may be there was polyps which we missed as ICG was not done.

Acknowledgements None

Funding None

Competing Interests None

Reference

1. El Baba F, Jarrett WH, Harbin TS, et al. Massive hemorrhage complicating age-related macular degeneration: clinic pathologic correlation and role of anticoagulants. Ophthalmology 198693:1581–1592. 2. Schatz H, Yanuzzi L, Gitter KA. Sub retinal neovascularisation following argon laser photocoagulation treatment for central serous chorioretinopathy: complication or misdiagnosis? Trans Am Acad Ophthalmology Otolaryngology 1977;83:893–906. 3. Gromolin J. Choroidal neovascularisation and central serous retinopathy. Can J Ophthalmol 1989;24:20–23. 4. Guyer DR, Yannuzzi LA, Slakter JS, et al. Digital indocyanine green video angiography of central serous chorioretinopathy. Arch Ophthalmol 1994;112:1057–1062.

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Case Report Solitary Lymph Node Involvement by Langerhans Cell Hitisocytosis: Cytomorphologic Diagnosis and Pitfalls on Fine Needle Aspiration Cytology Chhaya Gupta* and Neeru Gupta Sri Balaji Action Medical Institute, Paschim Vihar, New Delhi, India Keywords: Solitary, Lymph Node, Langerhans Cell Histiocytosis, FNAC

ABSTRACT Langerhans cell histiocytosis (LCH) is a rare disease and when confined to lymph node, it is even rarer. Lymph node involvement in LCH can be seen as a component of the systemic form or it may be the initial and sometimes exclusive manifestation of the disease [1].We present a case of LCH confined to the lymph node diagnosed initially by fine needle aspiration cytology (FNAC) in a 7 year old girl. Highly cellular smears showed fair number of large histiocytic cells (langerhans cells) showing round to oval vesicular nuclei with irregular folded and grooved nuclei with abundant, pale blue cytoplasm admixed with numerous neutrophils, lymphocytes, macrophages, multinucleated giant cells and tingible body macrophages suggesting the diagnosis of LCH. This was confirmed on histopathological and immunohistochemical study of the excised lymph node. The highly characteristic common and rare cytological features are highlighted with focus on differential diagnosis and causes of pitfalls.

*Corresponding author: Dr. Chhaya Gupta, C-6/115A, Lawrence Road, Keshavpuram, New Delhi-110035 INDIA Phone: +91 9910992059 E-mail: chhaya.doc_delhi@rediffmail.com

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Introduction

Langerhans cell histiocytosis (LCH) is a rare disease arising from clonal proliferation of Langerhans cells, which are abnormal cells derived from bone marrow that migrate from skin to lymph node. LCH is considered a neoplasm of the mononuclear phagocytic immunoregulatory system of unknown etiology. This disease is characterized by clonal proliferation of a special kind of histiocyte of the antigenpresenting dendritic type called the Langerhans cell (LC). [1,2] It affects predominantly children and young adults, but it can be found in any age group. LCH remains a complex disease with a wide array of presentations and clinical courses. Approximately two-thirds of children with LCH have single-system disease that most commonly affects bone, but that can also involve skin, lymph nodes or the central nervous system.[3]

AABS; 3(4): 2016 was referred to as histiocytosis X and had three variants: eosinophilic granuloma, Hand-Schuller-Christian disease

Case Report

A seven year old girl presented to surgical oncology OPD with multiple bilateral cervical lymphadenopathy for approximately 20 days. The lymph nodes were non tender, mobile and varying in size from 1 to 2 cm. The patient had no history of fever or weight loss. The liver and spleen were not palpable. Her complete blood counts and urine microscopy were unremarkable. The patient was referred for FNAC. FNAC from lymph node yielded white aspirate and smears were stained with Giemsa and Haematoxylin & Eosin. Highly cellular smears showed numerous atypical histiocytes as the predominant cell type scattered singly and in loosely cohesive clusters. A fair number of these cells showed round to oval vesicular nuclei with irregular folded and grooved nuclei (coffee bean appearance) with abundant, pale blue cytoplasm. Admixed polymorphous population of numerous neutrophils, lymphocytes, macrophages, multinucleated giant cells and tingible body macrophages was seen. Few binucleate and multinucleate forms were also encountered. (Fig. 1) A diagnosis of LCH was suggested and histopathological and immunohistochemical confirmation was advised. Multiple x-rays of whole body (including cervical spine, knee joint with leg, humerus with forearm, pelvis with thigh, skull), ultrasound abdomen and CECT chest and abdomen were done to rule out any other systemic involvement. Excision biopsy of the cervical lymph node was also done which on histopathology (Fig. 2) showed partial effacement of lymphnode architecture with sheets of histiocytes (langerhans cells)admixed with eosinophils, neutrophils and giant cells. These langerhans cells have eosinophilic to clear cytoplasm and contain oval to grooved Annals of Applied Bio-Sciences, Vol. 3; Issue 4: 2016

Fig. 1: Cytomicrograph showing cluster of langerhans cells with oval vesicular to irregularly folded and grooved nuclei (coffee bean appearance) with abundant, pale blue cytoplasm in background of neutrophils, lymphocytes and eosinophils (400x,Giemsa stain). Inset (600x, Giemsa stain).

Fig. 2: Histomorphological picture showing sheets of histiocytes (langerhans cells)admixed with eosinophils ,neutrophils and giant cells.These langerhans cells have eosinophilic to clear cytoplasm and contain oval to grooved nuclei.(600x,H&E stain).

nuclei. These cells were CD68, S-100 and CD1A positive, confirming the diagnosis of LCH in cervical lymph node.

Discussion

LCH is a rare disease with an estimated annual incidence of 0.5-5.4 cases per million.[1] In the past, the disorder e-ISSN: 2349-6991; p-ISSN: 2455-0396

Case Report

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and Letterer-Siwe syndrome. These three conditions are believed to represent different expressions of the same disorder, now collectively known as LCH.[1,4 LCH shows no age bar and effects patients from the neonatal period to adulthood,although it appears to be more common in children aged 0-15 years. There is no significant gender difference.[2,4] A controversial debate exist on the origin of LCH, whether this is a reactive or neoplastic process.[3] The disease is characterized by a clonal proliferation of the antigen-presenting dendritic cell called the Langerhans cell (LC)[.2,3,4] The proliferation may be induced by a viral infection, a defect in T cell macrophage interaction, and/ or a cytokine-driven process mediated by tumor necrosis factor, interleukin 11, and leukemia inhibitory factor. [4] LCH remains a versatile mimicker and diagnosis is often difficult and delayed. The course of the disease varies from spontaneous resolution to a progressive multisystem disorder with organ dysfunction and potential life threatening complications[2] The clinical spectrum of LCH varies from having a single system disease (solitary unifocal and multifocal unisystem) to multifocal multisystem disease. The single system commonly effects bone but can also involve skin, lymphnode or central nervous system.[1-7] Traditionally, the diagnosis of LCH is based on hematologic and histologic criteria and cytology closely reflects histomorphology. Ancillary studies may not be always necessary for diagnosis in appropriate setting. The cytological features of LCH include high cellularity composed of sheets and singly scattered LCs admixed with polymorphous population of numerous eosinophils, neutrophils, lymphocytes, plasma cells, multinucleated giant cells, and macrophages. The key to the diagnosis is to identify the LC through its characteristic features, namely, nuclear grooves (with a coffee bean appearance) and nuclear pseudoinclusions as seen in our case. They show variable degree of pleomorphism and mitotic activity. Sometimes, the LCs are few and nuclear grooves may not be very prominent or lack cytoplasmic processes. Ancillary studies such as immunohistochemistry and ultrastructural study aid the diagnosis in such cases. [1, 2, 4] Charcot-Leyden crystals are crystalloids containing eosinophil membrane protein formed from rupture of eosinophilâ&#x20AC;&#x2122;s granules. Charcot-Leyden crystals singly and in bunches within the macrophages, giant cells, and extracellularly have also been described in cases of LCH. They indicate tissue eosinophilia and may help in drawing attention to the LCH diagnosis.[4] Degree of eosinophil

infiltration varies in different areas of LCH lesion and different organs, thus their number can vary from scant to abundant in cytology smears Their presence can help attract attention to the diagnosis.[4] In our case, predominantly LCs and reactive histiocytes were seen in lymph node smears and eosinophils were scant. Touton type of giant cells have also been described in association with LCH in cytological smears of lymph node.[7] However, we did not encountered such feature in our case. The diagnosis may be missed due to lack of familiarity with its cytological features among pathologists or due to the lack of characteristic cytological findings resulting from a sampling error. The common differential diagnoses in a lymph node should include all those conditions with localized aggregates of LCs such as those observed in association with Dermatopathic lymphadenitis (DL), parasitic infection, Kimuraâ&#x20AC;&#x2122;s disease, hypersensitivity reactions, cat-scratch disease, sinus histiocytosis with massive lymphadenopathy (SHML), and hyperplastic lymph node. [1,4] Dermatopathic lymphadenitis (DL) can be excluded by the absence of pigment in the histiocytes. SHML involves primarily the cervical nodes, but its histiocytes are morphologically quite different from those of LCH. In SHML, the histiocytes have abundant cytoplasm, exhibiting emperipolisis and prominent nucleoli. In addition, in SHML, the histiocytes are S-100 protein (+), lysozyme (-), and CD1a (-), whereas, LCs show positivity for S-100, PNA (peanut agglutinin), MHC class II, CD1a and langerin (CD207). Our case showed positivity for S-100 protein and CD1a. In addition, on rare occasions, LCH can be seen associated with a variety of malignant neoplasms in the same node, i.e., malignant lymphoma or metastatic neoplasms.[8] Other neoplastic conditions with eosinophilic infiltration and nuclear grooving such as Hodgkinâ&#x20AC;&#x2122;s disease (HD), malignant melanoma, papillary thyroid carcinoma, malignant histiocytosis (MH), and other tumor cells with nuclear groovings should be considered in differential diagnosis and ruled out. Malignancies are easily excluded when no malignant cells with obvious cytologic atypia are present. [1] On electron microscopy, Birbeck granules are distinctive ultrastructural hallmark. However, it is time consuming and costly and not considered essential for the diagnosis as suggested by other authors.[ 2, 4] FNA can be a useful diagnostic modality for primary diagnosis as well as to evaluate the extent of the disease or recurrence of LCH. Patients with apparently restricted LCH need careful staging of their disease to ensure that the lesions are not part of a more extensive process.

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Conclusion

AABS; 3(4): 2016

1. Lee L-Y, Kang C-J, Hsieh Y-Y, Hsueh S. Diagnosis of nodal Langerhans cell histiocytosis by fine needle aspiration cytology. Chang Gung Medical Journal. 2005;28(10):735–739.

2. Chandekar SA, Shah VB, Kavishwar V. Cytological diagnosis of Langerhans cell histiocytosis with cutaneous involvement. J Cytol 2013; 30: 81-3. 3. Degar BA, Rollins BJ. Langerhans cell histiocytosis: Malignancy or inflammatory disorder doing a great job of imitating one? Dis Model Mech. 2009;2:436–9. 4. Kumar N, Sayed S, Vinayak S. Diagnosis of Langerhans cell histiocytosis on fine needle aspiration cytology: A case report and review of the cytology literature. Patholog Res Int. 2011;2011:439518. 5. Kumar PV, Mousavi A, Karimi M, Bedayat GR. Fine needle aspiration of langerhans histiocytosis of lymphnodes: a report of six cases.Acta Cytol 2002; 46:753-6. 6. Kakkar S, Kapila K, Verma K. Langerhans cell histiocytosis in lymph nodes cytomorphologic diagnosis and pitfalls. Acta Cytologica. 2001;45(3):327–332. 7. Mukhopadhyay S, Mitra PK, Ghosh S. Touton like giant cell in lymph node in a case of langerhans cell histiocytosis. J Cytol. 2007;24:191–2. 8. Egeler RM, Neglia JP, Puccetti DM, Brennan CA, Nesbit ME. Association of Langerhans Cell Histiocytosis with Malignant Neoplasms. Cancer. 1993;71:865–73.

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To conclude, the present case highlights the role of FNA in the diagnosis of LCH with discussion of differential diagnosis and pitfalls. A high index of suspicion, awareness of characteristic cytological features of LCH, and its differential diagnoses is necessary. This can obviate the need of biopsy, immunohistochemistry and electron microscopy. Immunocytochemistry, if available, can be performed on cytology smears and cell block.

Acknowledgements None

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Reference

Letter to Editor Folic Acid Supplementation in Pregnancy: Hitting Many Birds with one Stone Ajitha Sharma Department of Pharmacology, Kanachur Institute of Medical Sciences, Deralakatte, Mangalore – 575018, Karnataka, India

Dear Sir,

The protective role played by folic acid supplementation in pregnancy against the occurrence of neural tube defects in newborn, specifically anencephaly and spina bifida, is well-documented. In fact folic acid supplementation is being advised routinely to all pregnant women or females who are planning to conceive. [1] Low folate levels have been linked to the development of depression. Due to an increased demand for folate during pregnancy, there may be a dearth of this vitamin, which may be one of the causative factors for depression seen in pregnant women. The beneficial effects of folate in preventing depression has been studied in both animals and humans. It may even confer protection to the mother against depression upto 21 months after pregnancy. [2, 3] In addition, it has been found that risk of development of autism spectrum disorders and developmental delay has been reduced in children by periconceptional maternal intake of folate. [4, 5] Furthermore, it can prevent the occurrence of congenital heart diseases, oral clefts and preterm birth in the fetus, while protecting the mother against anemia, and peripheral neuropathy. [6] Thus folate supplementation seems to have many beneficial effects towards health of both mother and child. This situation may be summarised by the idiom, ‘hitting many birds with a single stone’.

Acknowledgements None

Funding None

Competing Interests None Declared

Reference

1. Pitkin RM. Folate and neural tube defects. Am J Clin Nutr 2007;85(suppl):285S-8S. 2. Brocardo PS, Budni J, Lobato KR, Santos ARS, Rodrigues ALS. Evidence for the involvement of the opioid system in the antidepressant-like effect of folic acid in the mouse forced swimming test. Behav Brain Res 2009;200:122-7. 3. Lewis SJ, Araya R, Leary S, Smith GD, Ness A. Folic acid supplementation during pregnancy may protect against depression 21 months after pregnancy, an effect modified by MTHFR C677T genotype. Eur J Clin Nutr 2012;66:97-103. 4. Schmidt RJ, Tancredi DJ, Ozonoff S, Hansen RL, Hartiala J, Allayee H et al. Maternal periconceptional folic acid intake and risk of autism spectrum disorders and developmental delay in the CHARGE (CHildhood Autism Risks from Genetics and Environment) casecontrol study. Am J Clin Nutr 2012;96:80-9. 5. Suren P, Roth C, Bresnahan M, Haugen M, Hornig M, Hirtz D et al. Association between maternal use of folic acid supplements and risk of autism spectrum disorders in children. JAMA 2013;309:570-7. 6. Greenberg JA, Bell SJ, Guan Y, Yu Y-h. Folic Acid Supplementation and Pregnancy: More Than Just Neural Tube Defect Prevention. Rev Obstet Gynecol 2011;4:52-9.

*Corresponding author: Dr. Ajitha Sharma, Department of Pharmacology, Kanachur Institute of Medical Sciences, Deralakatte, Mangalore – 575018, Karnataka, India Phone: +91 9894338105, 7348845298 E-mail: drajithasharma@gmail.com

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