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APMIS 116: 1338, 2008

Printed in Denmark . All rights reserved

C 2008 The Authors


Journal Compilation C 2008 APMIS
ISSN 0903-4641

Comparison of microscopy, culture and


in-house PCR and NASBA assays for diagnosis
of Neisseria 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
1

D.O. Ott Research Institute of Obstetrics and Gynecology, St. Petersburg, 2Central Research Institute of
Epidemiology, Moscow, 3St. Petersburg State Medical University, St. Petersburg, Russia, 4Department of
Medical Sciences, Uppsala University, Uppsala, and 5National Reference Laboratory for Pathogenic
Neisseria, Department of Clinical Microbiology, rebro University Hospital, rebro, Sweden

Shipitsyna E, Guschin A, Maximova A, Tseslyuk M, Savicheva A, Sokolovsky E, Shipulin G, Domeika M, Unemo M. Comparison of microscopy, culture and in-house PCR and NASBA assays for
diagnosis 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 from
women (n286) and urethral specimens from men (n48) were analyzed by microscopy, culture and
two in-house NAATs, i.e. polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA), developed in Russia. All N. gonorrhoeae-positive samples were confirmed using
porA pseudogene and 16S rRNA gene sequencing. All methods displayed 100% specificity, i.e. positive
predictive values of 100%. Compared to the PCR (most sensitive method in the present study), in
women the sensitivity of both microscopy and culture was 31.8%, and that of NASBA was 90.9%. In
men, microscopy, culture and NASBA displayed a sensitivity of 75%, 50% and 100%, respectively. The
negative predictive values 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. According to the PCR, the prevalences of
N. gonorrhoeae were 4.5% (women) and 8.3% (men). In conclusion, both the investigated Russian
NAATs displayed a high sensitivity and specificity. However, in general the diagnosis of gonorrhoea
in 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 rebro, Sweden. e-mail: magnus.unemo/orebroll.se

Neisseria gonorrhoeae is one of the most prevalent bacterial sexually transmitted pathogens
and a public health problem worldwide. For
control of the transmission of gonorrhoea, divergent preventive interventions, availability of
effective diagnostics, antimicrobial treatment,
and surveillance of epidemiological characterReceived 21 August 2007.
Accepted 15 October 2007.

istics (antimicrobial susceptibility, phenotypes


and genotypes) are crucial.
In most countries, culturing of N. gonorrhoeae
remains the gold standard for diagnosis of gonorrhoea. Under optimized conditions, culture is
highly specific and sensitive and, most importantly, allows antimicrobial susceptibility testing.
Microscopy of Gram-stained smears is still frequently used for a presumptive diagnosis of gonorrhoea. However, this method has a subopti133

SHIPITSYNA et al.

mal sensitivity and specificity, especially for


samples from females, and extragenital samples
are not appropriate for microscopic diagnosis
(16).
In 2006, in St. Petersburg (about 4,560,000 inhabitants), Russia, the estimated gonorrhoea incidence was approximately 38 cases per 100,000
inhabitants (7). However, reliable incidence figures in Russia are still lacking, which is mainly
due to suboptimal diagnostics, incomplete epidemiological surveillance, and self-treatment (8
10). The laboratory diagnosis of N. gonorrhoeae
in St. Petersburg, which also reflects the situation
in other geographic areas of Russia such as Arkhangelsk (9, 10), does not adhere to international
evidence-based recommendations of diagnostics
(16) and has previously been reported to be suboptimal (8). Thus, most of the diagnostics is
based only on microscopy owing to the higher expenses associated with culture. The occasional
laboratories that perform any culture diagnostics mainly use a non-selective Russian culture
medium (Complegon), mostly do not use species
confirmative assays, and rarely perform antimicrobial susceptibility testing (8).
In recent years, however, nucleic acid amplification tests (NAATs) for diagnosis of N.
gonorrhoeae, which in general have a high sensitivity and adequate specificity (2, 1113), have
been used in some municipal, federal, or private
laboratories in Russia. However, these NAATs,
which have been developed and are manufactured in Russia, have hitherto not been validated
in relation to any of the internationally well-recognized diagnostic NAATs. In addition, to the
best of our knowledge, no thorough evaluation
of either phenotypic or genetic diagnostic
methods used in Russia for diagnosis of N.
gonorrhoeae has previously been published.
The aim of the present study wasin a clinical
perspectiveto compare microscopy of Gramstained genital smears, culture, and two in-house
NAATs, i.e. a polymerase chain reaction (PCR)
and a real-time nucleic acid sequence-based amplification (NASBA), developed in Russia for diagnosis of N. gonorrhoeae.

MATERIALS AND METHODS


Patients and biological specimens
Consecutive symptomatic women (n286) and

134

men (n48) attending two dermatovenereological


dispensaries (VDs) in St. Petersburg, Russia, from
April to July 2006 were enrolled in the study. Cervical
and urethral specimens from the women and urethral
specimens from the men were sampled. From each
sampling site, one specimen for culture and microscopy, and one specimen for NAAT diagnostics (PCR
and NASBA) were sampled in random order. For
NAAT analysis, these samples were transported in
sucrose-phosphate buffer to the Laboratory of
Microbiology, D.O. Ott Research Institute of Obstetrics and Gynecology, St. Petersburg within 4 h.
Microscopy and culture diagnostics
Bedside culture and microscopy were performed at
the VDs. For microscopy, the genital smears were
Gram-stained and subsequently examined for typical
intracellular Gram-negative diplococci in polymorphonuclear leukocytes. For culture, the non-selective
Russian Complegon medium (Institute of Vaccines
and Sera, St. Petersburg, Russia) and the selective
VCA3 medium (bioMerieux, Marcy lEtoile, France)
were compared. Complegon is manufactured in three
different variants that contain divergent amounts of
agar, rabbit meat extract, Bacto-pepton, NaCl, autolysate of yeasts, orotic acid (6-carboxyuracil), bovine
serum, and hydrolysate of casein (Institute of Vaccines and Sera, St. Petersburg, Russia). VCA3 medium is a commercially available preprepared medium, which contains ordinary chocolate agar, PolyViteX (bioMerieux, Marcy lEtoile, France), and
VCAT3 (bioMerieux, Marcy lEtoile, France), i.e.
vancomycin, colistin, anisomycin, and trimethoprim
as selective antimicrobials. In both VDs, only presumptive identification of N. gonorrhoeae, i.e. isolation of typical Gram-negative, oxidase-positive diplococci, was performed in accordance with routine
procedures. Consequently, suspected gonococcal cultures were transported in sucrose-phosphate buffer to
the Laboratory of Microbiology, D.O. Ott Research
Institute of Obstetrics and Gynecology within 4 h for
species confirmation using NAATs (see below).
Isolation of nucleic acid and nucleic acid amplification
test (NAAT) analysis
Nucleic acid from genital samples and gonococcal
cultures was isolated using NucliSens EasyMAG
(bioMerieux, Boxtel, The Netherlands) immediately
prior to NAAT analysis.
All primers and probes for PCR and NASBA were
developed and synthesized at the Central Research
Institute of Epidemiology, Moscow, Russia.
The PCRs were run in an iCycler instrument (BioRad, Hercules, USA). The cppB gene, located on the
cryptic plasmid of N. gonorrhoeae, and the chromosomal cytosine DNA methyltransferase gene were identified in separate PCRs and only samples positive for
both 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-GCTACGCATACCCGCGTTGCTTTGCT-3) and NGA-2 (5-TTGGCGAAGACCTTCGAGCAGACATC-3) were used. The cytosine DNA
methyltransferase gene target was amplified using the
primers NGA-3 (5-GTGAACGTGTGCTGATTGTCGGA-3) and NGA-4 (5-TCTGGCGACGTACTTCAGTTGTTG-3). Each PCR mixture (25 ml)
contained 1 unit of Taq-polymerase (Central Research
Institute of Epidemiology, Moscow, Russia), 4 mM
MgCl2, 250 mM of dNTPs (Midigen, Novosibirsk,
Russia), a 0.3 mM concentration of each primer (Central Research Institute of Epidemiology, Moscow, Russia), and 10 ml of purified nucleic acid. The cycling parameters of the amplification were as follows: an enzyme activation step at 95 C for 5 min, followed by 42
cycles heating up to 95 C for 10 s, 65 C for 10 s, and
72 C for 10 s.
The NASBA was performed using the NucliSens
Basic kit (bioMerieux, Boxtel, The Netherlands) according to the manufacturers instructions. Furthermore, N. gonorrhoeae 16S rRNA specific primers
(NGNB-T7: 5-AATTCTAATACGACTCACTATAGGGAGAGAAGGTCCCCTGCTTTCCCTCTCAA-3 (including a T7 RNA polymerase promoter sequence) and NGNB-1: 5-GCACAGGGAAGCTTGCTTCTC-3 (specific for complementary DNA
(cDNA) of RNA-transcript)) and probe (NGNB-Z6:
Fam-5-CCGGCAGGAACGTACCGGGTAGCGTGCCGG-3-BHQ1 (molecular beacon)) were used.
Briefly, the NASBA reactions were run in a total volume of 20 ml containing 5 ml of purified nucleic acid,
5 ml of the enzyme mix, and 10 ml of the amplification
mix, which contained 7.5 pmoles of each primer and
2.5 pmoles of probe. The enzyme mix was added to
the reaction mixture during an incubation at 41 C
for 2 min following heat denaturation of the target
RNA (65 C for 2 min). Subsequent NASBA reaction
with real-time detection of amplified products was
run at 41 C for 90 min in a NucliSens EasyQ Analyser (bioMerieux, Boxtel, The Netherlands).
In order to ascertain the efficiency of the nucleic acid
extraction and identify possible inhibition of the amplification reactions, internal controls (ICs) comprising sequences with annealing sites for the N.
gonorrhoeae primers were, before nucleic acid isolation, included in both the PCR and NASBA. If neither of the ICs was positive in a N. gonorrhoeae-negative sample, a new portion of the sample was re-tested.
Validation of N. gonorrhoeae-positive results
For confirmation of all N. gonorrhoeae-positive
samples, sequencing of the porA pseudogene was performed as previously described (14). In addition, 550
bp of the 16S rRNA gene was PCR amplified and
subsequently sequenced using the primers NSU
(5-GGGTGAGTAACATATCGGAACGTA-3) and
NSL (5-CACACTCGAGTCACCCAGTTCAGAAC-3), the Big-Dye Terminator v1.1 Sequencing Kit
(Applied Biosystem, Foster City, USA) and an ABI

PRISM 3100 Genetic Analyzer (Applied Biosystems,


Foster City, USA).

RESULTS
The results of all the diagnostic methods performed are summarized in Table 1.
Briefly, in the microscopy of Gram-stained
smears, four women and three men were positive (Table 1).
Culture of the samples identified five positive
patients (four women and one man) and six
positive patients (four women and two men)
using the Russian non-selective Complegon medium and the VCA3 medium, respectively
(Table 1). However, one and two of these four
positive women, respectively, only displayed
positive cervical sample. Accordingly, overall
the number of positive individual samples was
identical (n8) on the different media (Table 1).
Furthermore, Complegon was frequently contaminated by other microorganisms.
12 women and 4 men, including the microscopy and culture-positive ones, were positive in
both the PCR and the NASBA. However, four
of the women were positive in the cervical
sample only. One additional woman was positive in the PCR only (Table 1).
All positive samples were confirmed to contain N. gonorrhoeae DNA using the porA
pseudogene and 16S rRNA gene sequencing.
Thus, all the methods displayed 100% specificity
and, accordingly, positive predictive values
(PPVs) of 100% (Table 2). However, compared
to the confirmed results of the PCR that was
the most sensitive method in the present study,
the sensitivity of the other methods varied significantly (Table 2). The sensitivity of microscopy was 31.8% and 75% in women and men,
respectively. Culture displayed a sensitivity of
31.8% and 50% in women and men, respectively.
The NASBA displayed 90.9% and 100% sensitivity in women and men, respectively. Consequently, 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 was
4.5% (13/286) and 8.3% (4/48) in the women and
men, respectively.
135

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
Culture
Complegona
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%)
a
Non-selective Russian culture medium manufactured by St. Petersburg Institute of Vaccines and Sera, St.
Petersburg, Russia (8).
b
VCA3 (chocolate agar and PolyViteX VCAT3), selective culture medium manufactured by bioMerieux, Marcy
lEtoile, France.
c
Bedside microscopy of Gram-stained genital smears.
d
PCR, polymerase chain reaction; NASBA, nucleic acid sequence-based amplification.
e
The numbers of culture-positive cervical samples correspond to positive women. However, some of these
positive women were negative in their urethral sample.

TABLE 2. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of microscopy, 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
97.3
Men
75.0
100
100
97.8
Culture

Women
Men

PCRa,b

Women
Men

31.8
50.0
100
100

100
100

100
100

100
100

100
100

97.3
95.7
100
100

NASBAb

Women
90.9
100
100
99.6
Men
100
100
100
100
a
The confirmed results of the PCR, which was the most sensitive method, were considered as the gold standard.
b
PCR, polymerase chain reaction; NASBA, nucleic acid sequence-based amplification.

DISCUSSION
The present study unambiguously emphasizes
that the laboratory diagnosis of gonorrhoea in
St. Petersburg, which may reflect the situation
in many other geographic areas of Russia, is
suboptimal and crucially requires improvements. This is obvious even though the present
study is relatively small and consequently needs
to be interpreted with some caution. Both
microscopy and culture, which are mainly used
for diagnostics, had a disquietingly low sensitivity, especially for diagnosis in women.
In the present study, the sensitivity of microscopy was only 31.8% in women and 75% in
men. Overall, microscopy of Gram-stained urethral smears, which may provide a definitive
136

diagnosis of N. gonorrhoeae in symptomatic


men (16), is inexpensive and valuable for rapid
diagnosis and antimicrobial treatment. However, this method has a significantly lower sensitivity for urethral and especially cervical
specimens from women, and moreover is not at
all appropriate for extragenital, such as pharyngeal and rectal, samples (16, 15). Furthermore, early and asymptomatic infection as well
as some auxotypes and serological variants of
N. gonorrhoeae may be less likely to be detected
(4, 6, 15).
In the present study, the sensitivity of the culture diagnostics was extremely low, i.e. 31.8% in
women and 50% in men. This was most feasibly
attributed to a combination of factors involving
inadequate sampling and suboptimal culti-

DIAGNOSIS OF N. GONORRHOEAE IN RUSSIA

vation conditions. Overall, the selective VCA3


medium and the non-selective Russian medium
Complegon displayed similar sensitivity. However, Complegon was frequently contaminated
by other microorganisms, which may interfere
with the growth and detection of gonococci.
Neither of the two participating VDs uses any
species confirmative assays, which most likely
reflects the situation in many diagnostic laboratories in Russia. Consequently, none of these
are able to provide any definitive laboratory diagnosis of gonorrhoea, especially not for
women and extragenital samples (16). Accordingly, in St. Petersburg and several other geographic areas of Russia it is crucial to establish and implement effective and quality-assured culture diagnostics, including use of selective medium, appropriate species confirmative assays and antimicrobial susceptibility testing.
In recent years, NAATs for diagnosis of N.
gonorrhoeae have been implemented in some
municipal, federal, or private laboratories in
Russia. NAATs offer several advantages, such
as high sensitivity and usually specificity, rapidity, the possibility of non-invasive sampling, opportunity 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 viable
organisms and consequently can be valuable in
settings where optimal sampling, transportation
and culturing are problematic. However, the disadvantages of N. gonorrhoeae NAATs have
been higher expense per sample, risk of contamination and inhibition, suboptimal specificity, and most importantly inability to provide
antimicrobial resistance data (14, 6, 1113). At
present, in Russia it is not usually recommended
that NAATs be used as the sole method for diagnosis of gonorrhoea (1, 2). Nevertheless, for
screening populations or core groups of highfrequency transmitters, or using in settings
where effective sampling, transportation and
culturing are not available, effective NAATs can
be very useful. Meanwhile, as far as we know,
the present study is the first ever internationally
reported assessment of any Russian N.
gonorrhoeae NAATs. Both the PCR and the
NASBA had a very much higher sensitivity than
microscopy and culture. Regarding the present
PCR, however, great awareness of the existence

of N. gonorrhoeae strains lacking the cryptic


plasmid and consequently the cppB gene target
is crucial. In some geographic areas, this genetic
target has been shown to be absent in up to 10%
of the N. gonorrhoeae strains (1618). In Russia,
according to present knowledge, cryptic
plasmid-free N. gonorrhoeae strains have been
absent or at least their prevalence has been negligible. Regarding the specificity of the examined Russian PCR and NASBA, in the present
study both assays displayed 100% specificity. All
positive samples were also ascertained to be true
N. gonorrhoeae positive by sequencing of the
16S rRNA gene as well as the porA pseudogene,
which previously has been reported to be exceedingly conserved and displaying 100% specificity for N. gonorrhoeae (14, 1821). Nevertheless, the specificity of many international N.
gonorrhoeae NAATs has previously been shown
to be suboptimal (2, 3, 11, 13, 17, 18) and both
the targets of the Russian PCR, i.e. the cppB
gene and especially the cytosine DNA methyltransferase gene, can be detected in some commensal Neisseria species. Furthermore, previously published NASBA assays targeting N.
gonorrhoeae-specific 16S rRNA have displayed
rare false-positive as well as false-negative results (22). Accordingly, larger studies that evaluate, quality assure, and validate the presently investigated NAATs as well as other NAATs used
for diagnosis of N. gonorrhoeae in Russia in relation to internationally well-recognized diagnostic NAATs are crucial.
In conclusion, to the best of our knowledge
the present study is the first ever to thoroughly
compare phenotypic and particularly NAAT diagnostic methods used in Russia for diagnosis
of N. gonorrhoeae. Thus, it is essential to establish and implement effective and quality-assured
culture diagnostics, for example, in order to
examine the antimicrobial susceptibility of N.
gonorrhoeae. The investigated Russian PCR and
NASBA assays seemed to have high sensitivity
and specificity. However, comprehensive evaluation and quality assurance of all the NAATs
used in Russia in relation to internationally
validated diagnostic NAATs are crucial. At
present, such studies that evaluate the performance characteristics of all the commercially
available NAATs for the diagnosis of N.
gonorrhoeae as well as Chlamydia trachomatis in
Russia are in progress.
137

SHIPITSYNA et al.

We express our gratitude to the head and all the staff


of the two included dermatovenereological dispensaries. We especially want to thank Svetlana Sakhartseva
(VD 3) and Irina Afonina (VD 11). This study was
supported by grants from the East Europe Committee of the Swedish Health Care Community, Sweden.

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