APMIS 116: 1338, 2008 C 2008 The AuthorsPrinted in Denmark . All rights reserved

Journal Compilation C 2008 APMIS

ISSN 0903-4641

Comparison of microscopy, culture andin-house PCR and NASBA assays for diagnosis

ofNeisseria gonorrhoeae in Russia

ELENA SHIPITSYNA,1 ALEXANDER GUSCHIN,2 ANNA MAXIMOVA,3 MARIYA TSESLYUK,2

ALEVTINA SAVICHEVA,1 EVGENIJ SOKOLOVSKY,3 GERMAN SHIPULIN,2

MARIUS DOMEIKA4 and MAGNUS UNEMO5

1D.O. Ott Research Institute of Obstetrics and Gynecology, St. Petersburg, 2Central Research Institute ofEpidemiology, Moscow, 3St. Petersburg State Medical University, St. Petersburg, Russia, 4Department of

Medical Sciences, Uppsala University, Uppsala, and 5National Reference Laboratory for PathogenicNeisseria, Department of Clinical Microbiology, rebro University Hospital, rebro, Sweden

Shipitsyna E, Guschin A, Maximova A, Tseslyuk M, Savicheva A, Sokolovsky E, Shipulin G, Domei-ka M, Unemo M. Comparison of microscopy, culture and in-house PCR and NASBA assays fordiagnosis of Neisseria gonorrhoeae in Russia. APMIS 2008;116:1338.

This study aimed to assess the laboratory diagnosis of Neisseria gonorrhoeae in St. Petersburg, Russia.In total, 334 consecutive symptomatic patients were enrolled. Cervical and urethral specimens fromwomen (n286) and urethral specimens from men (n48) were analyzed by microscopy, culture andtwo in-house NAATs, i.e. polymerase chain reaction (PCR) and nucleic acid sequence-based amplifi-cation (NASBA), developed in Russia. All N. gonorrhoeae-positive samples were confirmed usingporA pseudogene and 16S rRNA gene sequencing. All methods displayed 100% specificity, i.e. positivepredictive values of 100%. Compared to the PCR (most sensitive method in the present study), inwomen the sensitivity of both microscopy and culture was 31.8%, and that of NASBA was 90.9%. Inmen, microscopy, culture and NASBA displayed a sensitivity of 75%, 50% and 100%, respectively. Thenegative predictive values of microscopy, culture, and NASBA were 97.3%, 97.3%, and 99.6% inwomen, and 97.8%, 95.7%, and 100% in men, respectively. According to the PCR, the prevalences ofN. gonorrhoeae were 4.5% (women) and 8.3% (men). In conclusion, both the investigated RussianNAATs displayed a high sensitivity and specificity. However, in general the diagnosis of gonorrhoeain Russia is suboptimal and crucially requires validation, improvements and quality assurance.

Key words: Neisseria gonorrhoeae; microscopy and culture diagnostics; PCR; NASBA; Russia.

Magnus Unemo, Department of Clinical Microbiology, rebro University Hospital, SE-701 85 re-bro, Sweden. e-mail: magnus.unemo/orebroll.se

Neisseria gonorrhoeae is one of the most preva-lent bacterial sexually transmitted pathogensand a public health problem worldwide. Forcontrol of the transmission of gonorrhoea, di-vergent preventive interventions, availability ofeffective diagnostics, antimicrobial treatment,and surveillance of epidemiological character-

Received 21 August 2007.Accepted 15 October 2007.

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istics (antimicrobial susceptibility, phenotypesand genotypes) are crucial.

In most countries, culturing of N. gonorrhoeaeremains the gold standard for diagnosis of go-norrhoea. Under optimized conditions, culture ishighly specific and sensitive and, most import-antly, allows antimicrobial susceptibility testing.Microscopy of Gram-stained smears is still fre-quently used for a presumptive diagnosis of go-norrhoea. However, this method has a subopti-

SHIPITSYNA et al.

mal sensitivity and specificity, especially forsamples from females, and extragenital samplesare not appropriate for microscopic diagnosis(16).

In 2006, in St. Petersburg (about 4,560,000 in-habitants), Russia, the estimated gonorrhoea in-cidence was approximately 38 cases per 100,000inhabitants (7). However, reliable incidence fig-ures in Russia are still lacking, which is mainlydue to suboptimal diagnostics, incomplete epide-miological surveillance, and self-treatment (810). The laboratory diagnosis of N. gonorrhoeaein St. Petersburg, which also reflects the situationin other geographic areas of Russia such as Ark-hangelsk (9, 10), does not adhere to internationalevidence-based recommendations of diagnostics(16) and has previously been reported to be sub-optimal (8). Thus, most of the diagnostics isbased only on microscopy owing to the higher ex-penses associated with culture. The occasionallaboratories that perform any culture diagnost-ics mainly use a non-selective Russian culturemedium (Complegon), mostly do not use speciesconfirmative assays, and rarely perform anti-microbial susceptibility testing (8).

In recent years, however, nucleic acid amplifi-cation tests (NAATs) for diagnosis of N.gonorrhoeae, which in general have a high sensi-tivity and adequate specificity (2, 1113), havebeen used in some municipal, federal, or privatelaboratories in Russia. However, these NAATs,which have been developed and are manufac-tured in Russia, have hitherto not been validatedin relation to any of the internationally well-rec-ognized diagnostic NAATs. In addition, to thebest of our knowledge, no thorough evaluationof either phenotypic or genetic diagnosticmethods used in Russia for diagnosis of N.gonorrhoeae has previously been published.

The aim of the present study wasin a clinicalperspectiveto compare microscopy of Gram-stained genital smears, culture, and two in-houseNAATs, i.e. a polymerase chain reaction (PCR)and a real-time nucleic acid sequence-based am-plification (NASBA), developed in Russia for di-agnosis of N. gonorrhoeae.

MATERIALS AND METHODS

Patients and biological specimensConsecutive symptomatic women (n286) and

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men (n48) attending two dermatovenereologicaldispensaries (VDs) in St. Petersburg, Russia, fromApril to July 2006 were enrolled in the study. Cervicaland urethral specimens from the women and urethralspecimens from the men were sampled. From eachsampling site, one specimen for culture and micro-scopy, and one specimen for NAAT diagnostics (PCRand NASBA) were sampled in random order. ForNAAT analysis, these samples were transported insucrose-phosphate buffer to the Laboratory ofMicrobiology, D.O. Ott Research Institute of Obstet-rics and Gynecology, St. Petersburg within 4 h.

Microscopy and culture diagnosticsBedside culture and microscopy were performed at

the VDs. For microscopy, the genital smears wereGram-stained and subsequently examined for typicalintracellular Gram-negative diplococci in polymor-phonuclear leukocytes. For culture, the non-selectiveRussian Complegon medium (Institute of Vaccinesand Sera, St. Petersburg, Russia) and the selectiveVCA3 medium (bioMerieux, Marcy lEtoile, France)were compared. Complegon is manufactured in threedifferent variants that contain divergent amounts ofagar, rabbit meat extract, Bacto-pepton, NaCl, auto-lysate of yeasts, orotic acid (6-carboxyuracil), bovineserum, and hydrolysate of casein (Institute of Vac-cines and Sera, St. Petersburg, Russia). VCA3 me-dium is a commercially available preprepared me-dium, which contains ordinary chocolate agar, Poly-ViteX (bioMerieux, Marcy lEtoile, France), andVCAT3 (bioMerieux, Marcy lEtoile, France), i.e.vancomycin, colistin, anisomycin, and trimethoprimas selective antimicrobials. In both VDs, only pre-sumptive identification of N. gonorrhoeae, i.e. iso-lation of typical Gram-negative, oxidase-positive dip-lococci, was performed in accordance with routineprocedures. Consequently, suspected gonococcal cul-tures were transported in sucrose-phosphate buffer tothe Laboratory of Microbiology, D.O. Ott ResearchInstitute of Obstetrics and Gynecology within 4 h forspecies confirmation using NAATs (see below).

Isolation of nucleic acid and nucleic acid amplificationtest (NAAT) analysis

Nucleic acid from genital samples and gonococcalcultures was isolated using NucliSens EasyMAG(bioMerieux, Boxtel, The Netherlands) immediatelyprior to NAAT analysis.

All primers and probes for PCR and NASBA weredeveloped and synthesized at the Central ResearchInstitute of Epidemiology, Moscow, Russia.

The PCRs were run in an iCycler instrument (Bio-Rad, Hercules, USA). The cppB gene, located on thecryptic plasmid of N. gonorrhoeae, and the chromo-somal cytosine DNA methyltransferase gene were iden-tified in separate PCRs and only samples positive forboth genes were considered as N. gonorrhoeae positive.For amplification of the cppB gene target, the primers

DIAGNOSIS OF N. GONORRHOEAE IN RUSSIA

NGA-1 (5-GCTACGCATACCCGCGTTGCTTTG-CT-3) and NGA-2 (5-TTGGCGAAGACCTTCGA-GCAGACATC-3) were used. The cytosine DNAmethyltransferase gene target was amplified using theprimers NGA-3 (5-GTGAACGTGTGCTGATTG-TCGGA-3) and NGA-4 (5-TCTGGCGACGTA-CTTCAGTTGTTG-3). Each PCR mixture (25 ml)contained 1 unit of Taq-polymerase (Central ResearchInstitute of Epidemiology, Moscow, Russia), 4 mMMgCl2, 250 mM of dNTPs (Midigen, Novosibirsk,Russia), a 0.3 mM concentration of each primer (Cen-tral Research Institute of Epidemiology, Moscow, Rus-sia), and 10 ml of purified nucleic acid. The cycling par-ameters of the amplification were as follows: an en-zyme activation step at 95 C for 5 min, followed by 42cycles heating up to 95 C for 10 s, 65 C for 10 s, and72 C for 10 s.

The NASBA was performed using the NucliSensBasic kit (bioMerieux, Boxtel, The Netherlands) ac-cording to the manufacturers instructions. Further-more, N. gonorrhoeae 16S rRNA specific primers(NGNB-T7: 5-AATTCTAATACGACTCACTATA-GGGAGAGAAGGTCCCCTGCTTTCCCTCTCA-A-3 (including a T7 RNA polymerase promoter se-quence) and NGNB-1: 5-GCACAGGGAAGCTT-GCTTCTC-3 (specific for complementary DNA(cDNA) of RNA-transcript)) and probe (NGNB-Z6:Fam-5-CCGGCAGGAACGTACCGGGTAGCGT-GCCGG-3-BHQ1 (molecular beacon)) were used.Briefly, the NASBA reactions were run in a total vol-ume of 20 ml containing 5 ml of purified nucleic acid,5 ml of the enzyme mix, and 10 ml of the amplificationmix, which contained 7.5 pmoles of each primer and2.5 pmoles of probe. The enzyme mix was added tothe reaction mixture during an incubation at 41 Cfor 2 min following heat denaturation of the targetRNA (65 C for 2 min). Subsequent NASBA reactionwith real-time detection of amplified products wasrun at 41 C for 90 min in a NucliSens EasyQ Ana-lyser (bioMerieux, Boxtel, The Netherlands).

In order to ascertain the efficiency of the nucleic acidextraction and identify possible inhibition of the am-plification reactions, internal controls (ICs) compris-ing sequences with annealing sites for the N.gonorrhoeae primers were, before nucleic acid iso-lation, included in both the PCR and NASBA. If nei-ther of the ICs was positive in a N. gonorrhoeae-nega-tive sample, a new portion of the sample was re-tested.

Validation of N. gonorrhoeae-positive resultsFor confirmation of all N. gonorrhoeae-positive

samples, sequencing of the porA pseudogene was per-formed as previously described (14). In addition, 550bp of the 16S rRNA gene was PCR amplified andsubsequently sequenced using the primers NSU(5-GGGTGAGTAACATATCGGAACGTA-3) andNSL (5-CACACTCGAGTCACCCAGTTCAGAA-C-3), the Big-Dye Terminator v1.1 Sequencing Kit(Applied Biosystem, Foster City, USA) and an ABI

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PRISM 3100 Genetic Analyzer (Applied Biosystems,Foster City, USA).

RESULTS

The results of all the diagnostic methods per-formed are summarized in Table 1.

Briefly, in the microscopy of Gram-stainedsmears, four women and three men were posi-tive (Table 1).

Culture of the samples identified five positivepatients (four women and one man) and sixpositive patients (four women and two men)using the Russian non-selective Complegon me-dium and the VCA3 medium, respectively(Table 1). However, one and two of these fourpositive women, respectively, only displayedpositive cervical sample. Accordingly, overallthe number of positive individual samples wasidentical (n8) on the different media (Table 1).Furthermore, Complegon was frequently con-taminated by other microorganisms.

12 women and 4 men, including the micro-scopy and culture-positive ones, were positive inboth the PCR and the NASBA. However, fourof the women were positive in the cervicalsample only. One additional woman was posi-tive in the PCR only (Table 1).

All positive samples were confirmed to con-tain N. gonorrhoeae DNA using the porApseudogene and 16S rRNA gene sequencing.Thus, all the methods displayed 100% specificityand, accordingly, positive predictive values(PPVs) of 100% (Table 2). However, comparedto the confirmed results of the PCR that wasthe most sensitive method in the present study,the sensitivity of the other methods varied sig-nificantly (Table 2). The sensitivity of micro-scopy was 31.8% and 75% in women and men,respectively. Culture displayed a sensitivity of31.8% and 50% in women and men, respectively.The NASBA displayed 90.9% and 100% sensi-tivity in women and men, respectively. Conse-quently, the negative predictive values (NPVs)of microscopy, culture and NASBA were 97.3%,97.3% and 99.6% in women, and 97.8%, 95.7%and 100% in men, respectively (Table 2).

According to the most sensitive method, i.e.the PCR, the prevalence of N. gonorrhoeae was4.5% (13/286) and 8.3% (4/48) in the women andmen, respectively.

SHIPITSYNA et al.

TABLE 1. Number (%) of Neisseria gonorrhoeae-positive samples in consecutive symptomatic women (n286)and men (n48) using different diagnostic methods in St. Petersburg, Russia

CultureComplegona VCA3b Microscopyc PCRd NASBAd

Women (n286)Cervixe 4 (1.4%) 4 (1.4%) 4 (1.4%) 13 (4.5%) 12 (4.2%)Urethrae 3 (1.0%) 2 (0.7%) 3 (1.0%) 9 (3.1%) 8 (2.8%)

Men (n48)Urethra 1 (2.1%) 2 (4.2%) 3 (6.2%) 4 (8.3%) 4 (8.3%)aNon-selective Russian culture medium manufactured by St. Petersburg Institute of Vaccines and Sera, St.Petersburg, Russia (8).bVCA3 (chocolate agar and PolyViteX VCAT3), selective culture medium manufactured by bioMerieux, MarcylEtoile, France.cBedside microscopy of Gram-stained genital smears.dPCR, polymerase chain reaction; NASBA, nucleic acid sequence-based amplification.eThe numbers of culture-positive cervical samples correspond to positive women. However, some of thesepositive women were negative in their urethral sample.

TABLE 2. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of micro-scopy, culture, PCR and NASBA for diagnosis of Neisseria gonorrhoeae in consecutive symptomatic women (n

286) and men (n48) in St. Petersburg, Russia

Sensitivity Specificity Positive predictive Negative predictive(%) (%) value (%) value (%)

Microscopy Women 31.8 100 100 97.3Men 75.0 100 100 97.8

Culture Women 31.8 100 100 97.3Men 50.0 100 100 95.7

PCRa,b Women 100 100 100 100Men 100 100 100 100

NASBAb Women 90.9 100 100 99.6Men 100 100 100 100

aThe confirmed results of the PCR, which was the most sensitive method, were considered as the gold standard.bPCR, polymerase chain reaction; NASBA, nucleic acid sequence-based amplification.

DISCUSSION

The present study unambiguously emphasizesthat the laboratory diagnosis of gonorrhoea inSt. Petersburg, which may reflect the situationin many other geographic areas of Russia, issuboptimal and crucially requires improve-ments. This is obvious even though the presentstudy is relatively small and consequently needsto be interpreted with some caution. Bothmicroscopy and culture, which are mainly usedfor diagnostics, had a disquietingly low sensi-tivity, especially for diagnosis in women.

In the present study, the sensitivity of micro-scopy was only 31.8% in women and 75% inmen. Overall, microscopy of Gram-stained ur-ethral smears, which may provide a definitive

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diagnosis of N. gonorrhoeae in symptomaticmen (16), is inexpensive and valuable for rapiddiagnosis and antimicrobial treatment. How-ever, this method has a significantly lower sensi-tivity for urethral and especially cervicalspecimens from women, and moreover is not atall appropriate for extragenital, such as pha-ryngeal and rectal, samples (16, 15). Further-more, early and asymptomatic infection as wellas some auxotypes and serological variants ofN. gonorrhoeae may be less likely to be detected(4, 6, 15).

In the present study, the sensitivity of the cul-ture diagnostics was extremely low, i.e. 31.8% inwomen and 50% in men. This was most feasiblyattributed to a combination of factors involvinginadequate sampling and suboptimal culti-

DIAGNOSIS OF N. GONORRHOEAE IN RUSSIA

vation conditions. Overall, the selective VCA3medium and the non-selective Russian mediumComplegon displayed similar sensitivity. How-ever, Complegon was frequently contaminatedby other microorganisms, which may interferewith the growth and detection of gonococci.Neither of the two participating VDs uses anyspecies confirmative assays, which most likelyreflects the situation in many diagnostic labora-tories in Russia. Consequently, none of theseare able to provide any definitive laboratory di-agnosis of gonorrhoea, especially not forwomen and extragenital samples (16). Accord-ingly, in St. Petersburg and several other geo-graphic areas of Russia it is crucial to estab-lish and implement effective and quality-as-sured culture diagnostics, including use of sel-ective medium, appropriate species confirma-tive assays and antimicrobial susceptibility test-ing.

In recent years, NAATs for diagnosis of N.gonorrhoeae have been implemented in somemunicipal, federal, or private laboratories inRussia. NAATs offer several advantages, suchas high sensitivity and usually specificity, rapid-ity, the possibility of non-invasive sampling, op-portunity for cost-efficient pooling of samples,and simultaneous detection of several agents,e.g. N. gonorrhoeae and Chlamydia trachomatis.Most importantly, NAATs do not require viableorganisms and consequently can be valuable insettings where optimal sampling, transportationand culturing are problematic. However, the dis-advantages of N. gonorrhoeae NAATs havebeen higher expense per sample, risk of con-tamination and inhibition, suboptimal speci-ficity, and most importantly inability to provideantimicrobial resistance data (14, 6, 1113). Atpresent, in Russia it is not usually recommendedthat NAATs be used as the sole method for di-agnosis of gonorrhoea (1, 2). Nevertheless, forscreening populations or core groups of high-frequency transmitters, or using in settingswhere effective sampling, transportation andculturing are not available, effective NAATs canbe very useful. Meanwhile, as far as we know,the present study is the first ever internationallyreported assessment of any Russian N.gonorrhoeae NAATs. Both the PCR and theNASBA had a very much higher sensitivity thanmicroscopy and culture. Regarding the presentPCR, however, great awareness of the existence

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of N. gonorrhoeae strains lacking the crypticplasmid and consequently the cppB gene targetis crucial. In some geographic areas, this genetictarget has been shown to be absent in up to 10%of the N. gonorrhoeae strains (1618). In Russia,according to present knowledge, crypticplasmid-free N. gonorrhoeae strains have beenabsent or at least their prevalence has been neg-ligible. Regarding the specificity of the exam-ined Russian PCR and NASBA, in the presentstudy both assays displayed 100% specificity. Allpositive samples were also ascertained to be trueN. gonorrhoeae positive by sequencing of the16S rRNA gene as well as the porA pseudogene,which previously has been reported to be ex-ceedingly conserved and displaying 100% speci-ficity for N. gonorrhoeae (14, 1821). Neverthe-less, the specificity of many international N.gonorrhoeae NAATs has previously been shownto be suboptimal (2, 3, 11, 13, 17, 18) and boththe targets of the Russian PCR, i.e. the cppBgene and especially the cytosine DNA methyl-transferase gene, can be detected in some com-mensal Neisseria species. Furthermore, pre-viously published NASBA assays targeting N.gonorrhoeae-specific 16S rRNA have displayedrare false-positive as well as false-negative re-sults (22). Accordingly, larger studies that evalu-ate, quality assure, and validate the presently in-vestigated NAATs as well as other NAATs usedfor diagnosis of N. gonorrhoeae in Russia in re-lation to internationally well-recognized diag-nostic NAATs are crucial.

In conclusion, to the best of our knowledgethe present study is the first ever to thoroughlycompare phenotypic and particularly NAAT di-agnostic methods used in Russia for diagnosisof N. gonorrhoeae. Thus, it is essential to estab-lish and implement effective and quality-assuredculture diagnostics, for example, in order toexamine the antimicrobial susceptibility of N.gonorrhoeae. The investigated Russian PCR andNASBA assays seemed to have high sensitivityand specificity. However, comprehensive evalu-ation and quality assurance of all the NAATsused in Russia in relation to internationallyvalidated diagnostic NAATs are crucial. Atpresent, such studies that evaluate the perform-ance characteristics of all the commerciallyavailable NAATs for the diagnosis of N.gonorrhoeae as well as Chlamydia trachomatis inRussia are in progress.

SHIPITSYNA et al.

We express our gratitude to the head and all the staffof the two included dermatovenereological dispensar-ies. We especially want to thank Svetlana Sakhartseva(VD 3) and Irina Afonina (VD 11). This study wassupported by grants from the East Europe Commit-tee of the Swedish Health Care Community, Sweden.

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