Professional Documents
Culture Documents
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.
SHIPITSYNA et al.
134
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
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
SHIPITSYNA et al.
REFERENCES
1. Savicheva A, Sokolovsky E, Frigo N, Priputnevich T, Brilene T, Deak J, et al. Guidelines for laboratory diagnosis of Neisseria gonorrhoeae in
East-European countries. Part 1: Gonorrhoea,
sampling, and microscopy for diagnosis. Acta
Medica Lituanica 2007;14:6574.
2. Savicheva A, Sokolovsky E, Frigo N, Priputnevich T, Brilene T, Deak J, et al. Guidelines for laboratory diagnosis of Neisseria gonorrhoeae in
East-European countries. Part 2. Culture, nonculture methods, determination of antibiotic resistance, and quality assurance. Acta Medica Lituanica 2007;14:12334.
3. Centers for disease control and prevention. Sexually transmitted diseases treatment guidelines.
Available at: http://www.cdc.gov/std/treatment/
4. Tapsall J. Antimicrobial resistance in Neisseria
gonorrhoeae. World Health Organization (WHO)
report 2001. WHO/CDS/CSR/DSR/2001.3.
5. Van Dyck E, Meheus AZ, Piot P, editors. Gonorrhoea. In: Laboratory diagnosis of sexually transmitted diseases. Geneva: World Health Organization 1999:121.
6. Bignell CJ, European Branch of the International
Union against Sexually Transmitted Infection and
the European Office of the World Health Organization. European guideline for the management
of gonorrhoea. Int J STD AIDS 2001;12(Suppl
3):P279.
7. EpiNorthData/Gonorrhoea. EpiNorth, A Cooperation Project for Communicable Disease
Control in Northern Europe. Available at: http://
www.epinorth.org.
8. Unemo M, Savicheva A, Budilovskaya O, Sokolovsky E, Larsson M, Domeika M. Laboratory diagnosis of Neisseria gonorrhoeae in St. Petersburg,
Russia: inventory, performance characteristics
and recommended optimizations. Sex Transm Infect 2006;82:414.
9. Unemo M, Vorobieva V, Firsova N, Ababkova T,
Leniv I, Haldorsen BC, et al. Neisseria gonorrhoeae population in Arkhangelsk, Russia: phenotypic and genotypic heterogeneity. Clin Microbiol Infect 2007;13:8738.
10. Vorobieva V, Firsova N, Ababkova T, Leniv I, Haldorsen BC, Unemo M, et al. Antibiotic suscepti-
138
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.