Microbiological Labs
Driven by Results Microbiology laboratories are under increasing performancerelated scrutiny. The first of a two-part article addresses the challenges of, as well as techniques for, obtaining optimal results in quantitative urine culture studies One of the important endpoints in any antibiotic clinical trial – though not the most important one – is a set of microbiological data that is both reliable and verifiable. For most trials, the whole dataset comes from a reference or central microbiology laboratory that reidentifies pathogens isolated by local labs, and performs antibiotic susceptibility testing. In a complicated urinary tract infection (cUTI) trial, local micro labs conduct quantitative urine culture research, and the resultant colony count determines both the patient’s eligibility into modified intention-to-treat analysis and the drug efficacy (microbiological cure). A micro lab is able to provide bacterial growth estimations sufficient for a clinical decision, but cannot always generate data for a cUTI trial. Every lab performing primary urine culture should be evaluated for its ability to deliver reliable and verifiable colony count results, such as technical capabilities, including internal quality control for data reliability and data management for data verifiability. Based on this, it is critical to determine the most important aspects for unification of the obtained results in a microbiological clinical trial in advance.
Urine Specimen Stability The recommended stability for unpreserved, non-refrigerated urine is up to two hours (1). Urine kept at room temperature for more than two hours produces significantly higher colony counts (2). The Central Laboratory Manual usually recommends keeping a urine specimen on ice if its plating within two hours is not possible. However, in most cases, the period elapsed from urine collection to plating, as well as the storage temperature, is not verifiable. Even if the site documents the time of urine collection, microbiology labs
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Olga Sazonova, Veronika Khokhlova, Maxim Belotserkovsky and Andrey Karelin at PSI CRO
do not usually schedule the time of plating. Out of 11 local microbiology labs in Poland, Romania, Russia and Ukraine that were evaluated in the last two years, only two labs recorded both urine sampling and plating time; eight labs documented only urine sampling time; one recorded neither sampling nor plating time. The temperature of urine samples was also not documented by local labs, so obligating sites to refrigerate urine does not solve the problem. Preservative tubes are the option of choice here; according to literature, colony counts are stable for up to 48 hours in preserved urine, and the number of negative cultures does not increase over time (2). Therefore, preserved urine is chosen over non-preserved urine. It is also preferable over refrigerated urine without preservative, when transportation to a regional micro lab is needed. This is cost-effective as specimens can be moved under ambient conditions with minimal insulation. Refrigerated transportation is costly in itself, and on top of that, it requires temperature monitoring, which adds further costs.
Contamination Rates Rate of urine contamination with skin flora may reach significant numbers, and depends on the hospital. Based on the College of American Pathologists (CAP) survey carried out in 2008, about 10% of labs demonstrated an extremely high contamination rate of 41.7%, while for the remaining labs, the average contamination rate was 0.8% (3). In a similar manner, in the trials PSI has organised, most hospitals submitted a low number of contaminated urine samples, while a few hospitals demonstrated unusually high contamination rates. It is therefore important to receive timely feedback from micro labs about badquality urine samples. Sites submitting contaminated urine may need to be re-trained, or even blacklisted.
Image: © Zaharia Bogdan Rares – shutterstock.com
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According to the same CAP survey, providing written instructions on a clean-catch procedure to male and female patients decreased contamination rates (3). Literature on the benefit of midstream clean catch in women is controversial, with studies supporting this on the one hand, and studies denying it on the other hand (4,5). Usually, sites are informed on the importance of non-contaminated urine specimen submission, and provided with project coordinators with written reference sheets on the procedure of midstream clean catch.
Quantitative Urine Culture The latest FDA guidance on cUTI trial design sets two important parameters for urine colony counts (6): A single species of bacteria on pure culture identified at 10 5colony-forming units per millilitre (CFU/mL) or greater should be considered a true bacterial pathogen ● No growth of bacteria (or growth at a quantitation of less than 104CFU/mL) should be considered a microbiological success for a midstream clean-catch urine specimen ●
Parameters for urine colony counts – set in the EMA’s guideline addendum on clinical trials for antibacterial drugs (7) – are slightly different: Based on experience and consensus, it would be acceptable that patients deemed to have an infection should have [bacterial growth] > 1 x 105CFU/mL ● Microbiological success should be defined as < 1 x 103CFU/mL ●
Neither FDA nor EMA guidelines specify the technique that should be used for CFU/mL determination, but the assumption is that a micro lab uses a conventional urine culture method available in guidelines issued by American Society for Microbiology (ASM) (1,8). In short, urine is plated onto an agar plate with a calibrated loop of 1mcL or 10mcL by a single streak, followed by perpendicular spreading. This can be done both manually and using an automated device, such as WASP (Copan) or Innova (BD). To obtain CFU/mL, the actual number of colonies needs to be multiplied by the dilution factor; for example, by 1000 or 100. With conventional culture, bacterial growth can be distinguished by <105 versus ≥105CFU/mL, as well as <104 versus ≥104CFU/mL and <103 versus ≥103CFU/ mL. It is advisable to use conventional culture for clinical trial purpose, even if labs normally employ other techniques for urine bacteria growth estimation. To boost method reliability, ASM also recommends a check of colony counts on plates inoculated by calibrated loop, against plates inoculated with a calibrated pipette. Not every lab routinely uses both 1mcL and 10mcL loops for conventional culture. 1mcL loop plating is acceptable if trial results are going to be submitted to FDA only; for EMA submission, parallel plating with a 10mcL loop is needed to demonstrate microbiological success at corresponding visits. Read the next part of this article in ICT May 2016, which compares the various approaches different countries have to optimising quantitative urine culture studies results.
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References 1. McCarter YS et al, 'Cumitech 2C: Laboratory Diagnosis of Urinary Tract Infections', ASM Press, 2009 2. Eisinger SW, Schwartz M, Dam L and Riedel S, Evaluation of the BD vacutainer plus urine C&S preservative tubes compared with nonpreservative urine samples stored at 4°C and room temperature, Am J Clin Pathol 140: pp306-313, 2013 3. Bekeris LG, Jones BA, Walsh MK and Wagar EA, Urine culture contamination: A CAP Q-Probes study of 127 laboratories, Arch Pathol Lab Med 132(6): pp913-917, June 2008 4. Leisure MK, Dudley SM and Donowitz LG, Does a clean-catch urine sample reduce bacterial contamination? N Engl J Med 328: pp289290, 1993 5. Jeong I-S, Yang M-G and Oh H-S, Comparison of the bacterial contamination rates according to the urine collection methods in women. Journal of Korean Academy of Fundamentals of Nursing 6(3): p359, 1999 6. Complicated urinary tract infections: Developing drugs for treatment guidance for industry, US Department of Health and Human Services, FDA Centre for Drug Evaluation and Research, February 2015 7. Addendum to the guideline on the evaluation of medicinal products indicated for treatment of bacterial infections, European Medicines Agency, 2013 8. Garcia LS (Ed), Clinical Microbiology Procedures Handbook, ASM Press, 2010, Washington DC
About the authors Olga Sazonova is a Senior Laboratory Specialist at PSI CRO. Before joining the company in 2008, she worked for five years at the Russian Academy of Sciences as a contracted and staff researcher. Email: olga.sazonova@psi-cro.com Veronika Khokhlova has been a Senior Laboratory Specialist at PSI CRO since 2005. Previously, she was employed by the Russian Academy of Science as a Senior Research Associate for over 15 years. Email: veronika.khokhlova@psi-cro.com Maxim Belotserkovskiy holds the position of Head of Medical Affairs at PSI CRO. He has more than 25 years of experience in clinical research as an investigator and clinical research professional. Email: maxim.belotserkovsky@psi-cro.com Andrey Karelin, Director of Laboratory Support Services at PSI CRO, is a graduate of the Department of Biology at the Moscow State University. He worked at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry as Head of Laboratory before joining the company in 2001. Email: andrey.karelin@psi-cro.com
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Microbiological Labs
Driven by Results: Part 2 The first part of this article was dedicated to challenges and techniques in optimising quantitative urine culture studies results; the second part now addresses country-specific differences in achieving this
In the US, most labs generally use conventional urine culture. Twelve local (or hospital) labs within the last year have been evaluated to see if they can readily serve a complicated urinary tract infection (cUTI) clinical trial.
• Seven labs routinely used a 1mcL loop for quantitative plate inoculation
• One used a 10mcL loop • The rest used both 1mcL and 10mcL loops • Six out of 12 labs controlled colony counts – obtained • •
by loop inoculation – against plates inoculated with a calibrated pipette Six different labs had never performed quality control, or had performed it many years ago All labs were ready to undergo quality control for clinical trial purposes
As a result, only one lab was able to start serving clinical trials without any additional training and set-up.
Olga Sazonova, Veronika Khokhlova, Maxim Belotserkovsky and Andrey Karelin at PSI CRO
In contrast, most labs in Central and Eastern Europe employ semi-quantitative culture. In former Soviet countries, hospital labs use a semi-quantitative technique described in the Union of Soviet Socialist Republics’ Ministry of Health Order, number 535. This method has vague colony density interpretation criteria (see Table 1). Implementation of conventional quantitative technique in Eastern European countries frequently requires additional supplies (such as calibrated loops), training and additional supervision. In most cases, hospital labs are not motivated to perform quantitative culture as it is time-consuming, and do not provide additional diagnostic information as compared to routinely used semiquantitative culture. Commercial labs in Georgia, Russia, Ukraine and so forth would usually consent to implementation of conventional technique as per the American Society for Microbiology for clinical trial purposes, although they also need additional training and supervision; most labs further require a fee for implementation of a method that is different from their familiar system.
In European countries, along with a conventional quantitative technique, a number of semi-quantitative culture methods are also used. An overview of the most common is given in Table 1. In Western Europe, the majority of labs use a conventional quantitative technique – although the details may vary – but some labs prefer other methods. For instance, of eight laboratories (five hospitals and two commercial) evaluated in Italy:
All methods are approved for diagnostic use as they provide sufficient information for discerning between significant and non-significant bacteriuria. However, these methods are all unable to distinguish between <103 and >103 colony forming units per millilitre (CFU/mL), so they cannot be used for demonstration of microbiological success in trials with EMA submission.
• Three labs performed quantitative plate inoculation with
Modern Methods
• • • • •
a 10mcL loop One carried out the procedure with a 10mcL and a 1mcL loop One used a 5mcL loop One used a 1mcL loop One utilised a Previ Isola system One used light scattering for determination of bacterial density in urine samples
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Having said this, some semi-quantitative methods (such as DipStreakTM or Previ Isola) might be acceptable for trials with FDA submission – provided they give clear and traceable correlation between colony count and CFU/mL. Interpretation of bacterial growth with DiaSlide is human factor-dependent; therefore, inclusion of a patient into
Table 1: Semi-quantitative urine culture techniques Countries using the technique
Name of technique
Description
Ex-USSR: Belarus Georgia Kazakhstan Moldova Russia Ukraine Uzbekistan
USSR Minzdrav Order #535 (dated 1985)
Quadrant plating with 2mcL loop. Criteria of interpretation are vague; for example, 90 colonies can both be reported as 5x104 and 105CFU/mL; 30 colonies can both be reported as 5x103 and 104CFU/mL
EU (Conformité Européene (CE) approved); Ukraine
DiaSlide® (Novamed)
Urine is streaked onto agar slide with elongated plastic prongs. The manufacturer does not specify the amount of urine that is captured on the elongated prongs. Estimation of bacterial growth is performed by following the manufacturer's reference chart based on the colony’s density. No correlation between number of colonies and CFU/mL is provided
EU (CE-approved); Ukraine
DipStreak (Novamed)
Urine is streaked onto agar slide with elongated plastic prongs. The manufacturer does not specify the amount of urine that is captured on the elongated prongs. Estimation of bacterial growth is performed by following the manufacturer's reference chart. Less than 10 colonies is <104CFU/mL; 10-24 colonies is ≥104CFU/mL; 25-50 colonies is ≥105CFU/mL. Lower limit of quantification is between 500 and 1,000CFU/mL
EU (CE-approved)
Previ Isola (BioMerieux)
Using a circular applicator, 10mcL of urine is pressure-control streaked on agar plate. Growth is estimated by dividing the plate in eight sections. Growth in sections 1 to 2 is <104CFU/mL; growth in sections 1 to 3 means 104 to 5x104CFU/mL; growth in sections 1 to 4 means 5x104 to 105CFU/mL; growth in sections 1 to 5 means >105CFU/mL
modified intention-to-treat (mITT) population, as well as into microbiologically cured population, cannot be verified. Previ Isola provides more explicit interpretation rules (for example, growth only in sector 1 means <104CFU/mL), which makes the results less dependent on the human factor. Independent validation has shown the agreement of semi-quantitative growth estimation with the conventional manual plating (1). According to its reference chart, Previ Isola differentiates <104CFU/mL and ≥104CFU/mL; in other words, microbiological cure versus failure as per the FDA. On the other hand, with Previ Isola, some patients eligible for mITT may be lost, as its reference chart differentiates ≤105CFU/ mL versus >105CFU/mL – whereas according to FDA mITT, population is defined as patients having baseline pathogen growth ≥105CFU/mL. DipStreak provides correlation between the number of colonies per slide and CFU/mL. Reporting intervals allow differentiation between mITT and non-mITT, as well as between microbiological cure and failure as defined by current FDA guidance. At the same time, this method is not able to reliably demonstrate pathogen eradication for EMA submission. According to the latest research, correlation between conventional quantitative plating and DipStreak with CNA/McConkey agar is high (2).
In most cases, Microbiology Manual by Central Lab provides a detailed description of quantitative urine plating and colony count procedure. However, experience shows that each microbiology lab should be carefully evaluated for its ability and willingness to use a technique that is different from their routine, and to perform quality control of colony counts on loop-inoculated plates versus pipetteinoculated plates.
Colony Count Reporting In the clinical trial setting, lab reports are usually defined as source documents for case report forms (CRFs). However, if quantitative urine culture results appear in the report as rounded CFU/mL (104CFU/mL or 3x103CFU/mL, for example), they cannot be regarded as a source but rather as an interpretation of colony count. Source data is the exact number of colonies per quantitative plate documented elsewhere (from one colony to 100 colonies; numbers above 100 are registered as >100). Reporting rounded CFU/mL can lead to inconsistencies between results obtained by different labs, which may use a variety of rounding rules. One lab, for instance, might report 60 colonies (1mcL loop) as ≥104CFU/mL, whereas another lab could report it as 105CFU/mL.
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In addition to different rounding rules, labs may establish varying reporting intervals for CFU/mL. For example, some labs place round colony counts with the smaller order of magnitude rather than with the same: ≤103CFU/mL; >103 to ≤104CFU/mL; and so on. The FDA’s definitions of baseline pathogen and microbiological cure place round colony counts with the same order of magnitude (pathogenic bacteria growth is ≥105CFU/mL, for example). The safest option to avoid the above discrepancies is reporting the number of colonies (from one to 100 and >100) multiplied by the dilution factor. This may not be easily achievable, however, as in most labs, the reports are generated by laboratory information systems (LIS). In many labs, LIS allows entry of rounded results only; in some cases, LIS may perform rounding automatically using its own rules that cannot be re-programmed for the trial purpose. Having said this, if the trial needs are clearly explained to a laboratory during its evaluation and set-up, the lab usually agrees to add the exact number of colonies in the comment field of the report. Even if bacterial growth is captured in the CRF in a rounded format, trained study monitors will be able to re-check the data against raw colony counts available in the lab reports.
Local versus Regional Evaluation of microbiology labs is a standard step in any antibiotic trial. A project-specific questionnaire is developed for each study in order to cover all specific logistics, testing and data management requirements. In countries where conventional quantitative culture is routinely used, and overall performance and data management are of a high quality, it is appropriate to use local (hospital) labs, as any gaps discovered during their evaluation can be discussed and addressed in most cases. In a cUTI trial carried out in the US and Germany, for example, local labs were most frequently asked to use both 1mcL and 10mcL loops (as per protocol); to provide the exact number of colonies in the report; and to perform quality control checks of colony counts on loop-inoculated plates versus pipetteinoculated plates. In countries where semi-quantitative culture is routinely used and overall quality of local labs for clinical trials is questionable, the preferred choice is regional (or commercial) labs. With preservative tubes, specimen logistics is not an issue. Regional labs in countries like Bulgaria, Romania, Russia and Ukraine have the following advantages:
• General management is of higher quality than at local labs • • • • •
(more reliable performance) No gaps in data management, which makes the results verifiable Higher flexibility in culture techniques and results reporting format Reliable isolate management Dedicated personnel to supervise the study Real time feedback on submitted samples quality, such as urine contamination
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In countries undergoing transit to EU laboratory standards (such as Poland, Hungary or Slovenia), parallel evaluation of local and regional labs may help to choose the optimal scenario.
From All Angles Based on past experience in cUTI clinical trials with microbiology labs located in the US and Europe, the following conclusion can therefore be drawn: to obtain uniform, reliable and verifiable pathogenic growth results in a global cUTI trial, one should take into consideration the country-specific features of different microbiological labs’ practices. Failure to take these variations into account may lead to lost data due to unnoticed ‘gaps’ and ‘bugs’ in culture techniques, reporting, quality and data management. References 1. Bustamante V, Meza P, Román JC and García P, Evaluation of an automated streaking system of urine samples for urine cultures, Rev Chilena Infectol 31(6): pp670-675, December 2014 2. Colodner R and Keness Y, Evaluation of DipStreak containing CNAMacConkey agar: A new bedside urine culture device, Isr Med Assoc J 2(7): pp563-565, July 2000
About the authors Olga Sazonova is a Senior Laboratory Specialist at PSI CRO. Before joining the company in 2008, she worked for five years at the Russian Academy of Sciences as a contracted and staff researcher.
Email: olga.sazonova@psi-cro.com Veronika Khokhlova has been a Senior Laboratory Specialist at PSI CRO since 2005. Previously, she was employed by the Russian Academy of Science as a Senior Research Associate for over 15 years. Email: veronika.khokhlova@psi-cro.com Maxim Belotserkovsky holds the position of Head of Medical Affairs at PSI CRO. He has more than 25 years of experience in clinical research as an investigator and clinical research professional.
Email: maxim.belotserkovsky@psi-cro.com Andrey Karelin, Director of Laboratory Support Services at PSI CRO, is a graduate of the Department of Biology at the Moscow State University. He worked at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry as Head of Laboratory before joining the company in 2001. Email: andrey.karelin@psi-cro.com