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LABORATORY DIAGNOSIS OF NEISSERIA GONORRHOEAE IN ST. PETERSBURG, RUSSIA: INVENTORY, PERFORMANCE CHARACTERISTICS AND RECOMMENDED OPTIMISATIONS
M. Unemo 1, A. Savicheva 2, O. Budilovskaya 2,
E. Sokolovskiy 3, M. Larsson 1, M. Domeika 4 ([email protected])
1 National Reference Laboratory for Pathogenic Neisseria, Department of Clinical Microbiology, Orebro University Hospital, Sweden;
2 D.O. Ott Research Institute of Obstetrics and Gynecology, RAMS, St. Petersburg, Russia;
3 Department of Dermatology and Venereal diseases with clinic, Pavlov State Medical University, St. Petersburg, Russia;
4 Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
Objectives: To perform a comprehensive inventory of the number of samples, performance characteristics, and quality assurance of the laboratory diagnosis of Neisseria gonorrhoeae at five laboratories in St Petersburg and Leningradskaya Oblast, Russia, in 2004, and to recommend optimisations for an increased adherence to international evidence based recommendations of diagnostics.
Methods: Surveillance data were obtained with questionnaire and site visits. For evaluation of the culture media utilised at the laboratories, N gonorrhoeae reference strains (n=29) were used.
Results: During 2004 the total numbers of N gonorrhoeae samples analysed at the five laboratories using microscopy of stained smears and culturing were 330 879 (407 positive) and 38 020 (420 positive), respectively. Four laboratories used a Russian non-se-lective culture medium-that is, Complegon, and one laboratory utilised Biocult-GC. Both media seemed suboptimal. Only two of the laboratories used any species confirmative assay. Antibiotic susceptibility test-
ing of N gonorrhoeae was performed at only two of the laboratories and each year only occasional isolates were analysed. None of the laboratories comprised a complete laboratory quality assurance system.
Conclusions: According to international recommendations, the diagnosis of N gonorrhoeae in St Petersburg and Leningradskaya Oblast, Russia, is suboptimal. More samples need to be analysed by culturing on a highly nutritious and selective medium and, furthermore, species confirmation and antibiotic susceptibility testing should be more frequently performed. In addition, the utilised methods for culturing and antibiotic susceptibility testing, including medium and interpretative criteria used, ought to be optimised, standardised, and quality assured using systematic internal and external quality controls.
(Sex Transm Inf, 2006, 82, 41-4; available at: http://www. ncbi.nlm.nih.gov/entrez/query.fcgi?db= pubmed&cmd=Retrieve&dopt=AbstractPlus&list_ uids=16461601&query_hl=1&itool=pubmed_doc-sum)
RESEARCH PROJECTS ON OPTIMIZATION OF LABORATORY DIAGNOSIS OF SEXUALLY TRANSMITTED INFECTIONS
E. V. Shipitsyna 1, K. V. Shalepo 1, A. M. Savicheva 1([email protected]), M. Domeika 2
1 D.O. Ott Research Institute of Obstetrics and Gynecology, RAMS, St. Petersburg, Russia;
2 Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
Introduction
Since 1998, Russian-Swedish project on STI diagnosis and management has been realized in St. Petersburg and Leningrad Oblast. The main goal of the project is to improve the quality of diagnosis and treatment of STIs. To optimize STI diagnostics, it was planned to solve the following tasks:
• development and introduction of standards and
algorithms of STI management;
• optimization and standardization of laboratory diagnosis of STIs, and introduction of a quality control system;
• evaluation of the prevalence of infections affecting the reproductive tract using modern strategies and methods.
Within the project, a number of studies aimed to optimize laboratory diagnosis of STIs in Russia were performed, and the results were presented at international meetings and published in internation-
al and Russian journals. Also, the PhD theses “Validation of laboratory methods used for diagnosis of infections caused by Chlamydia trachomatis” (Sha-lepo K.V.) and “Microbiological aspects of resistance of Chlamydia trachomatis to antimicrobials” (Shipitsyna E.V.).
The present article reviews studies, both completed and current, aimed to evaluate methods used in Russia to diagnose STIs.
Laboratory diagnosis of Neisseria gonor-rhoeae in St. Petersburg, Russia: inventory, performance characteristics and recommended optimizations / Unemo M., Savicheva A., Budilovskaya O., Sokolovskiy E, Larsson M, Domeika M. // Sex Transm Infect. — 2006. — 82(1). — P. 41- 44.
Objectives: The aim of the study was to perform a comprehensive inventory of the number of samples, performance characteristics, and quality assurance of the laboratory diagnosis of Neisseria gonorrhoeae at five laboratories in St. Petersburg and Leningrad Oblast, Russia, in 2004, and to recommend optimizations for an increased adherence to international evidence based recommendations of diagnostics.
Methods: Surveillance data were obtained with questionnaire and site visits. For evaluation of the culture media utilized at the laboratories, N. gonorrhoeae reference strains (n = 29) were used.
Results: During 2004 the total numbers of N. gonorrhoeae samples analyzed at the five laboratories using microscopy of stained smears and culturing were 330 879 (407 positive) and 38 020 (420 positive), respectively. Four laboratories used a Russian non-selective culture medium — that is, Complegon, and one laboratory utilized Biocult-GC. Both media seemed subop-timal. Only two of the laboratories used any species confirmative assay. Antibiotic susceptibility testing of N. gonorrhoeae was performed at only two of the laboratories and each year only occasional isolates were analyzed. None of the laboratories comprised a complete laboratory quality assurance system.
Conclusions: According to international recommendations, the diagnosis of N. gonorrhoeae in St. Petersburg and Leningrad Oblast, Russia, is suboptimal. More samples need to be analyzed by culturing on a highly nutritious and selective medium and, furthermore, species confirmation and antibiotic susceptibility testing should be more frequently performed. In addition, the utilized methods for culturing and antibiotic susceptibility testing, including medium and interpretative crite-
ria used, ought to be optimized, standardized, and quality assured using systematic internal and external quality controls.
Diagnosis of Chlamydia trachomatis in Russia — in-house PCR assays may be effective but overall optimization and quality assurance are urgently needed / Shalepo K., Savitcheva A., Shipitsyna E., Unemo M., Domeika M. // AP-MIS. — 2006. — 114(7 - 8). — P. 500 - 507.
Objectives and methods: In the present study, the performance of the cell culture method, two non-Russian direct immunofluorescence (DIF) assays, and three different in-house polymerase chain reaction (PCR) tests used in St. Petersburg, Russia, for detection of Chlamydia trachomatis in urogenital specimens was evaluated.
Results: A total of 650 patients were examined and it was most disquieting that previous C. trachomatis positivity with Russian DIF assays could — 7 days later — be confirmed only in 26 % of the women and 30 % of the men. Overall, the highest diagnostic sensitivity was obtained using PCR analysis. However, the sensitivity varied significantly: from 79% to 100 % between the different PCR assays, sex of the patients, and type of samples. The highest sensitivity was obtained for female vaginal and male urine samples (100 %). The specificity of the PCR assays varied from 97 % to 100 %. The sensitivity of cell culture and both the examined DIF assays was low, i.e. it varied from 46 % to 56 % and 55 % to 75 %, respectively. Meanwhile, cell culture was 100 % specific and the DIFs showed a specificity varying from 99 % to 100 %.
Conclusions: In a Russian perspective, adequate in-house PCR methods may be used quite effectively for detection of C. trachomatis in invasive as well as non-invasive clinical material. Simultaneous analysis of two different specimens from women resulted in a significantly increased detection rate of C. trachomatis. Nevertheless, in Russia the need for optimization and quality assurance of diagnostic methods for C. trachomatis, especially Russian DIF assays, has to be emphasized.
Pooling samples: the key to sensitive, specific and cost-effective genetic diagnosis of Chlamydia trachomatis in low-resource countries / Shipits-yna E., Shalepo K., Savicheva A., Unemo M., Domeika M. // Acta Derm Venereol. — 2007. — 87(2).
— P. 140 - 143.
Objectives: The aims of this study were to compare the performance characteristics and cost-effectiveness of pooling endocervical samples for screening and diagnosis of Chlamydia
trachomatis, and to investigate the prevalence of C. trachomatis infection in women in Leningrad Oblast, Russia.
Methods: A total of 1500 endocervical samples were tested individually and when pooled in groups of 5 and 10 samples, respectively. A previously evaluated in-house diagnostic polymerase chain reaction (PCR) assay was utilized.
Results: The sensitivity and specificity of the PCR were not affected by either pooling strategy. The estimated prevalence of genital C. trachomatis infection was 6.6 %, 6.1 % and 6.0 % based on individually tested samples, and pools of 5 and 10, respectively. For diagnosis of individual samples, the pooling strategies resulted in cost savings of 53.3 % (5 samples per pool) and 44.0 % (10 samples per pool).
Conclusion: Pooling samples for PCR detection of C. trachomatis is an accurate and cost-saving approach for diagnosis and large-scale prevalence studies in St Petersburg, Russia.
Antimicrobial resistance of Chlamydia trachomatis in vitro: methodological aspects and clinical relevance / Shipitsyna E. V., Savitcheva A. M., Khusnutdinova Т. А., Shalepo K. V., Misyuri-na O. Yu., Govorun V. M., Domeika M. // Clinical microbiology and antimicrobial chemotherapy — 2004. — Vol. 6, № 1. — P. 54 - 64.
In vitro susceptibility testing of 25 clinical Chlamydia trachomatis strains to antibiotics widely used for chlamydial infection treatment was performed. Resistance to doxycycline was demonstrated by 10 isolates (40 %), and azithromycin — 10 isolates (40 %), 11 isolates (44 %) were defined as resistant to josamycin, 11 (44 %)
— to spiramycin, and 11 (44 %) — to ofloxacin. Multidrug resistance was manifested by 12 strains (48%), 6 of them being resistant to all antibiotics tested. Heterotypic pattern of antimicrobial resistance was shown: a small part of C. trachomatis population (<1 %) survived at the presence of high antibiotic concentrations. The test-of-cure results were juxtapose with susceptibility testing data. There was no association observed between chlamydial infection treatment failure and in vitro resistance of chlamydiae to antibiotics. Of 25 patients 9 were treated with antimicrobials to which chlamydiae isolated before therapy were in vitro resistant. In none of those patients were chlamydiae detected after treatment by cultural method. On the contrary, in the only case of C. trachomatis isolation in cell culture after treatment chlamydiae obtained from that patient before treatment was shown to be susceptible to antibiotic used for therapy. Thus, in vitro susceptibility testing of chlamydiae as a routine test for
choosing antibiotic to treat chlamydial infection is appeared to be inexpedient.
Evaluation of a real-time nucleic acid sequence-based amplification (NASBA) assay for diagnosis of genital chlamydial infection / Shipitsyna E. V., Vorobyova N. E., Savicheva A. M., Sokolovskiy E. V., Guschin A. E., Ryzhikh P. G., Shipulin G. A. // Z. Akus. Zen. Bolezn. — 2005. — Vol. LIV, № 4. — P. 17 - 21.
This study was aimed to evaluate a new real-time nucleic acid sequence-based amplification (NASBA) assay for diagnosis of urogenital Chlamydia trachomatis infection. A total of 193 patients aged 16 to 42 (mean age, 22.8 years) were examined, with most of them having symptoms of a urogenital infection. Cervical and urethral swabs from women and men, respectively, were investigated with the use of cell culture method, polymerase chain reaction (PCR) and NASBA. C. trachomatis infection was diagnosed in 29 patients (15 %): in 21 patients — by all the three methods, whereas 8 samples were culture negative. Sensitivity of PCR and NASBA methods were 100 %, cell culture method — 78.4 %. Negative predictive value of PCR and NASBA was found to be 100 %, and that of cell culture — 95.3 %. Specificity as well as positive predictive value of all the three methods equaled 100%. Thus, the new realtime NASBA assay was shown to be a very sensitive and specific test, which can be recommended as a confirmatory method for diagnosis of genital chlamydial infection.
Evaluation of nucleic acid amplification tests (NAATs) used in Russia to diagnose STIs (in progress)
Background and objectives: Nucleic acid amplification tests (NAATs) are widely used in Russia for diagnosis of STIs, and the discordance of results of testing for STIs performed in various Russian laboratories with the use of different NAATs, with polymerase chain reaction (PCR) being most widely used, is significant. Consequently, a comprehensive evaluation of different laboratory methods in different clinical specimens is highly needed.
This study is aimed to evaluate the performance of PCR tests used in Russia for diagnosis of infections caused by Chlamydia trachomatis and Neisseria gonorrhoeae.
Preliminary results: Up to date, 446 attendees of youth centers of St. Petersburg — 319 women and 127 men — were examined. In women, cervical and vaginal samples were tested for C. trachomatis DNA, and cervical and urine samples — for N. gonorrhoeae DNA. In men, urethral and urine samples were analyzed for both C. trachomatis and N. gonorrhoeae.
Table 1
Testing urogenital samples for C. trachomatis DNA with the use of five PCR tests*
1 2 3 4 З Number of samples
Women (n=319) Cervical swabs (n=319) + + + + + 34
+ + — — — 2
+ + — — + 1
— — — — — 282
Vaginal smears (n=298) + + + + + 31
+ + — — — 2
+ + — — + 2
— — — — — 263
Men (n=127) Urethral swabs (n=127) + + + + + 13
— — + + + 1
— — — — — 113
Urine (n=127) + + + + + 12
+ + — — — 1
— — — — — 111
* 1 — Conventional PCR (DNA-technology); 2 — Real-time PCR (DNA-technology); 3 — Conventional PCR (Research Institute of Epidemiology); 4 — Real-time PCR (Research Institute of Epidemiology); 5 — Conventional PCR (Lytech)
Table 2
Testing urogenital samples for N. gonorrhoeae DNA with the use of five PCR tests *
1 2 3 4 З Number of samples
Women Cervical swabs + + + + + 5
(n=319) (n=319) + + + + — 1
— — — — — 313
Vaginal smears + + + + + 4
(n=317) + + + + — 1
— — + + — 1
— — — — — 311
Men Urethral swabs + + + + + 4
(n=127) (n=127) — — — + — 1
— — — — — 122
Urine + + + + + 5
(n=127) — + — + — 1
— — — — — 121
*1 - Conventional PCR (DNA-technology); 2 — Real-time PCR (DNA-technology); 3 — Conventional PCR (Research Institute of Epidemiology); 4 — Real-time PCR (Research Institute of Epidemiology); 5 — Conventional PCR (Lytech)
The results are presented in Tables 1 and 2. In general, all the tests displayed a high level of agreement. For example, for 316 of 319 cervical swabs tested for C. trachomatis, the same results were obtained by all the tests, and only in three cases there was a disagreement between different tests.
For discrepancy analysis and evaluation of diagnostic characteristics of the tests, reference methods are planned to be used.
Conclusion
For improvement of the situation with STI diagnosis and management in Russia, careful validation of all diagnostic methods and standardization of all diagnostic procedures should be performed. Hopefully, the results of our studies can form a basis of recommendations for accurate and quality assured diagnosis of STIs in Russia
