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International Clinical Trials

Driven by Results: Part 2

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.

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 (see full PDF). 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:
  • Three labs performed quantitative plate inoculation with 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
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 full PDF)). 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.

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.

Modern Methods


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 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.

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
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

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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.

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.

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.

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.

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Olga Sazonova
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Veronika Khokhlova
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Maxim Belotserkovsky
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Andrey Karelin
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