A trial of the validity of genital smears and cultures with gonococcal vaccine provocation in diagnosing genital gonorrhoea in women

Abstract

In Russia the diagnosis of gonorrhoea in women relied on microscopy, justified by the hypothesis that sensitivity increases using ‘provocation’ techniques. The aim was to test the value of Gonovaccine as provocation in women who would have received it normally. Cervical specimens from 204 women were tested by culture and a ligase chain reaction (LCR) assay before the women were randomized to receive provocation or not. Further cervical specimens were obtained 24, 48 and 72 hours later for microscopy, culture and LCR tests. In both provocation and non-provocation arms, 24 women were positive for gonorrhoea by the LCR assay. Test-by-test, sensitivity of microscopy was 30% in the provocation arm and 13% in the control arm (P = 0.0407, Fisher's exact test). Patient-by-patient, sensitivity of microscopy was 50% in the provocation arm, but only 25% in the control arm (P = 0.0675, Fisher's exact test). The cost per case was greater ($214) using provocation with microscopy than culture and microscopy at the first visit ($150). Thus, although Gonovaccine provocation doubled the sensitivity of microscopy in detecting gonococci, the internationally recommended protocol of microscopy and culture at first visit should be adopted as routine practice in Russia. The findings raise questions about the pathogenesis and natural history of gonorrhoea.

Keywords

gonorrhoea Gonovaccine provocation women diagnosis Russia

INTRODUCTION

Review of studies of the validity of microscopic diagnosis of genital gonococcal infection suggests that, compared with culture, sensitivity is at best 40–70% in women and asymptomatic men.¹ Culture or nucleic acid amplification tests (NAATs) are therefore recommended for routine use of diagnostic and screening in North American² and European³ guidelines. However, in the Russian Federation and much of the Former Soviet Union (FSU), the diagnosis of gonorrhoea relied on microscopy. Such reliance has been based on the widespread belief in FSU countries that the sensitivity of microscopy is increased by the use of ‘provocation’ techniques. Provocation is defined by clinicians as inducing ‘an artificial intensification of the inflammatory process based on tissue irritation, to facilitate gonococcal identification by microscopic or culture techniques’ and is believed to increase the concentration of gonococci at the mucosal surface, making them available for sampling.^4,5 These techniques were used widely in Western Europe before the availability of culture for gonorrhoea.⁶ We estimate that currently over one million patients receive provocation each year.

Current Russian guidelines⁷ and textbooks⁴ recommend the use of ‘provocation’ techniques in conjunction with microscopy for the routine diagnosis of genital gonorrhoea. Provocation may be carried out by chemical, biological (intramuscular injection of a suspension of phenol-killed gonococci in 0.9% NaCl solution – Gonovaccine), thermal and dietary techniques,^4,5,8,9 but such gonococcal vaccine provocation is used in the majority of cases. However, provocation by all techniques is uncomfortable. Administration of Gonovaccine usually causes fever, chills and acute tenderness at the injection site. Allergic reactions can also occur. The unpleasantness of the procedure is widely appreciated by lay Russians and is likely to deter people from attending clinics. Given that gonorrhoea is believed to enhance the sexual transmissibility of HIV,^10–12 compromise of its control could increase the potential for the current large intravenous drug user (IDU)-concentrated HIV epidemics in the region to become generalized.¹³

The three best studies^14–16 concerning the effectiveness of biological provocation suggest that provocation with microscopic examination of genital specimens at three follow-up attendances doubles the number of women diagnosed with gonorrhoea, over that achieved by a single microscopic examination at presentation. In conducting the present trial in women, our primary objective was to define the sensitivity and specificity of microscopy and Russian gonococcal culture systems used with and without Gonovaccine provocation for the diagnosis of gonorrhoea, in comparison with the ligase chain reaction (LCR) assay, and, in so doing, examine the hypothesis that sensitivity was greater in women who had received provocation. Our second objective was to promote a culture of evidence-based clinical practice in Russian STD control and management by the execution, in Russia, of a randomized controlled trial which questioned traditional practice, and which was endorsed by key Russian stakeholders including the Ministry of Health.

METHODS

Overview and participants

A single blind randomized trial design (Figure 1) was used. We recruited women over the age of 18 years attending three dermatovenereology clinics in the Moscow region who tested negative for gonorrhoea on an initial Gram-stained genital smear and who would have received provocation according to Russian clinical guidelines. These guidelines^4,7,17 recommend that women presenting with a history or signs suggesting a possible gonococcal infection should be tested by microscopy performed on a Gram- or methylene blue-stained smear made from a curetted sample of cervical epithelium. Provocation is performed where the initial microscopy result is negative, but either (a) the physician considers that a ‘gonococcal type’ discharge is present or (b) the women is a sexual partner of a patient with gonorrhoea or (c) the woman has evidence of leucocytosis, unusual Gram-negative intracellular organisms or extracellular diplococci in the smear. Following provocation, the patient is re-examined at 24, 48 and 72 hours and microscopy repeated on each occasion. We randomized women to receive, or not to receive provocation and the follow-up procedure was exactly as described above except that culture and LCR tests were also performed on specimens obtained at baseline and all follow-up visits.

Figure 1

Trial procedure

Exclusion criteria included known allergy to Gonovaccine, current or previous tuberculosis, hypertension, cardiovascular, renal or hepatic disease, allergic conditions, current menstruation and pregnancy. The fact that provocation procedures are common practice underpinned the ethical nature of the study. It conformed to good practice clinical guidelines and the Declaration of Helsinki. The protocol and all subsequent amendments were reviewed and ratified by a District Health Authority ethics review board in London, UK and by the ethics review Board of the Central Institute of Skin and Venereal Diseases, Moscow, Russian Federation.

Procedure (Figure 1)

Women were recruited between March 2000 and March 2002. A detailed manual of operations for the study was prepared and provided to each participating clinic. One collaborating doctor (to carry out recruitment and follow-up) and one microbiologist was identified in each clinic, and given detailed training in the recruitment, sampling, specimen transport, clinical management and data management procedures by the study coordinator (EF). Patients meeting inclusion criteria were invited to participate by the study doctor on receipt of the initial negative Gram-stained smear result. Patients were provided with a written information sheet about the study, and asked to sign a consent form indicating their willingness to participate. Consenting patients were then allocated a study number. All women were interviewed by the study doctor using a standard study proforma to obtain subject characteristics and medical and sexual history, and a full clinical examination was carried out and recorded on the proforma. A random allocation sequence was prepared in London and sent to the Moscow-based study coordinator. Allocations were linked to each study number and placed in preprepared study packs in a sealed envelope. Women were randomized immediately to provocation or control arms by opening a sealed envelope containing the allocation group. Those allocated to the provocation arm received an intramuscular injection of killed gonococcal vaccine in the buttock and were monitored by a pretrained clinical nurse for one hour after injection for immediate adverse reactions. Cervical swabs for gonococcal culture and LCR tests were obtained from each woman. All of them were advised to abstain from sexual activity (Figure 1).

At each follow-up visit, women were reviewed clinically and separate cervical swabs were taken for Gram-staining, culture and LCR testing. Gram-stained smears were read immediately by the clinical microbiologist who was blind to the patients' allocation to provocation or control arms. Women who had a positive smear for gonorrhoea at any follow-up visit were treated immediately with ciprofloxacin 500 mg given orally and doxycycline 200 mg twice a day (b.i.d.) for one week. Specimens for culture and LCR tests from these women were retained for analysis, but no further study follow-up visits were undertaken, and further clinical management was carried out according to normal clinic policy. In half of the cases culture results were available at the final trial follow-up visit. Women who were culture and smear-negative at all follow-up visits were all treated with doxycycline 200 mg b.i.d. for one week. If the culture result was positive, women were given ciprofloxacin and doxycycline as above. When culture results were not available at the final study follow-up visit, women were invited to attend for a further visit for treatment if indicated.

Specimen collection and processing and microbiological methods

Cervical specimens were obtained from each woman at each visit and in the following order. A disposable loop specimen was applied to a slide for Gram-staining. A specimen for culture was obtained using a transwab and placed immediately in transport medium. A specimen for the LCR assay was obtained using a thin LCR swab, placed in LCR transport medium and refrigerated immediately at +4°C.

Smears on glass slides were transported immediately to the clinic laboratory, Gram-stained, examined under oil immersion and the results returned to the clinic doctor. Transwab specimens in transport medium were taken on the same day to the microbiology laboratory of the Central Institute of Skin and Venereal Diseases and inoculated onto GC** culture medium. After incubation at 37° C in 5–7% carbon dioxide in moist conditions for at least 48 hours, media were examined and suspicious colonies tested by oxidase reaction and Gram-staining. Oxidase-positive, Gram-negative cocci were sub-cultured onto a fresh culture plate and incubated as before for 24 hours and identified. The culture medium used was a Russian-manufactured base agar, with Russian-manufactured supplements and an antibiotic cocktail (supplied from London). The medium was quality-controlled using the WHO strains A, B, C, D, E and 81–10 with a standard protocol.

Specimens in LCR transport medium for LCR tests were taken, on ice, on the same day to the microbiology laboratory of the Central Institute of Skin and Venereal Diseases. On arrival, these transport media were placed immediately at −70°C. Before transportation to London all specimens were thawed, mixed thoroughly and the swab squeezed against the side of the tube and discarded. The sample was then divided into two equal volumes in 2 mL screw-capped vials and the aliquots refrozen at −70°C. One aliquot was left at −70°C and the other transported to London. Aliquots were packed in boxes of 100 vials that were kept cold but thawed during transport. They were, however, refrozen at −70°C within six hours of leaving Russia. All specimens obtained (from all visits) were tested for Neisseria gonorrhoeae using the LCR assay (Abbott Laboratories) according to the manufacturer's instructions. Specimens were categorized as positive, negative or equivocal according to the manufacturer's cut-off. For equivocal results, the tests were repeated at a 1:4 dilution of the specimen. Any remaining equivocal results were considered negative. Box records were kept for all specimens to check for evidence of cross-contamination in transport, but none was found.

Statistical methods

If the proportion of women who were initially negative for gonorrhoea by microscopy was subsequently 20% positive in the provocation arm and 5% in the non-provocation arm, we needed to know the number of women who would be required for this difference to be significant with 80% probability at 95% confidence interval. Assuming a 20% loss to follow-up, we calculated that we would need to recruit 220 women to the study. Data were analysed using SAS version 8.2. CIs on proportions were calculated using SEs derived from normal approximation to the binomial distribution and P values for difference in proportions obtained from LS Fishers exact test.

RESULTS

Patient characteristics and follow-up achieved

Two hundred and four women were recruited to the study, of whom 105 were randomized to the provocation arm. Fourty-three women were excluded at baseline, 31 because they met exclusion criteria and 12 because they did not consent. Provocation and control groups were also closely balanced in clinic location, reason for attendance (check-up, symptoms, contact-tracing, referred or a combination of these), inclusion criteria met (discharge or cervicitis, contact, suspicious smear or a combination of these), clinical features, age and follow-up achieved. Further, groups were closely balanced in terms of sexual history and previous history of sexually transmitted diseases; and there were no statistically significant differences between the groups in any of the characteristics measured. Overall, 152 women (75%) had symptoms and 190 (95%) had clinical signs of genital inflammation. The numbers of women attending different follow-up visits and excluded from further follow-up owing to positive gonorrhoea diagnoses, or loss to follow-up (did not attend) were similar in provocation and control arms. Thus, 94 women (46%) attended all three follow-up examinations; 72 (35%) for two follow-up examinations and 35 (17%) for one follow-up examination.

Results of LCR, culture and smear tests

Among the provocation group of 105 women, 24 (23%), 16 (15%) and 13 (11%) women tested positive for gonorrhoea by LCR, culture and smear, respectively, with specimens obtained at one or more visits. Among 99 controls, 24 (24%), 15 (15%) and six (6%), respectively, were positive in the aforementioned tests. Table 1 shows the distribution of subjects who were positive by the LCR in any specimen according to the number of specimens available and number of those positive by different tests.

Table 1

All positive tests for gonorrhoea by number of visits completed

Numer of specimens available*		LCR number of tests positive				Culture number of tests positive				Smear number of tests positive
Numer of specimens available*		1	2	3	4	1	2	3	4	1	2	3
Four	No. +	4	2	3	3	0	2	1	3
Three	No. +	7	4	6		2	3	6		3	1	0
Two	No. +	4	12			2	11			6	1
One	No. +	3				1				8

* including baseline visit

LCR = ligase chain reaction

Performance of Gram stain and culture compared with the LCR assay

Because there were tests on samples taken on multiple occasions, both test-based and subject-based analyses were undertaken. For the test-based analysis, each test was considered as an independent event and the sensitivity calculated by dividing the number of tests which were concordant with positive LCR status, by the total number of tests carried out among LCR-positive women; and analogously for specificity. For the subject-based analysis, test sensitivity was calculated by dividing the number of subjects who were positive by both the test and the LCR assay, by the total number positive by the LCR; and analagously for specificity.

In the test-based analysis, among 95 smears from LCR-positive women, the sensitivity (95% CI) was 22% (14–30%) and the specificity was 100%. Among 88 cultures from LCR-positive women, the sensitivity (95% CI) was 49% (39–59%) and the specificity was 99%. Findings describing the performance against LCR (any positive) of Gram-stained smear and culture in the two study arms are presented in Table 2. In the test-based analysis, the sensitivity of smear was 30% (16–43%) in the provocation arm and 13% (4–22%) in the control arm. This difference was significant (P = 0.0407, Fisher's exact test). The sensitivity of culture was 55% (40–70%) in the provocation arm and 44% (30–59%) in the control arm (not significant).

Table 2

Sensitivity and specificity of Gram-stained smear and culture in women compared with ligase chain reaction (LCR; any positive at any visit) by study arm

Test comparison		Provocation				No Provocation
Test comparison		Value	L 95	U 95	n	Value	L 95	U 95	n
All smears vs. LCR	Sensitivity	30	16	43	43	13	4	22	52
All smears vs. LCR	Specificity	99	40	70	181	100	30	58	167
Any smear vs. LCR	Sensitivity	50	30	70	24	25	8	42	24
Any smear vs. LCR	Specificity	99			80	100			75
Smear 1 only	Sensitivity	25	8	42	24	13	−1	26	24
Smear 1 only	Specificity	99			80	100			75
Smears 1 and 2	Sensitivity	46	26	66	24	21	5	37	24
Smears 1 and 2	Specificity	99			80	100			75
All culture vs. LCR	Sensitivity	55			40	44			48
All culture vs. LCR	Specificity	100			180	100			165
Any culture vs. LCR	Sensitivity	67	48	86	24	58	39	78	24
Any culture vs. LCR	Specificity	100			80	99			75
Culture 0 only	Sensitivity	63	43	82	24	46	26	66	24
Culture 0 only	Specificity	100			80	100			75
Culture 0 and 1	Sensitivity	67	48	86	24	46	26	66	24
Culture 0 and 1	Specificity	100			80	100			75
Culture 0–2	Sensitivity	67	48	86	24	58	39	78	24
Culture 0–2	Specificity	100			80	100			75
Smear 1 vs. LCR	Sensitivity	25	8	42	24	13	−1	26	24
Smear 1 vs. LCR	Specificity	99			80	100			75
Smear 2 vs. any LCR	Sensitivity	21	5	37	24	13	−1	26	24
Smear 2 vs. any LCR	Specificity	100			80	100			75
Smear 3 vs. any LCR	Sensitivity	8	−3	19	24	4	−4	12	24
Smear 3 vs. any LCR	Specificity	100			80	100			75
Culture 0 vs. any LCR	Sensitivity	63	43	82	24	46	26	66	24
Culture 0 vs. any LCR	Specificity	100			80	100			75
Culture 1 vs. any LCR	Sensitivity	58	39	78	24	46	26	66	24
Culture 1 vs. any LCR	Specificity	100			80	100			75
Culture 2 vs. any LCR	Sensitivity	17	2	32	24	25	8	42	24
Culture 2 vs. any LCR	Specificity	100			80	100			75
Culture 3 vs. any LCR	Sensitivity	17	2	32	24	17	2	32	24
Culture 3 vs. any LCR	Specificity	100			80	99			75

U 95 = upper limit of 95% CI; L 95 = lower limit of 95% CI

In the subject-based analysis, among 48 LCR-positive women, sensitivity (95% CI) of the smear was 38% (24–51%) and among 155 LCR-negative women, the specificity was 100%. For culture based on the same numbers of LCR-positives and -negatives, the sensitivity was 63% (49–76%), and the specificity was 99%. Sensitivity (95% CI) of the smear was 50% (30–70%) in the provocation arm, but only 25% (8–42%) in the non-provocation arm (P = 0.0675, Fisher's exact test). Specificity of the smear was 100% in both arms. Sensitivity (95% CI) of the culture was 67% (48–86%) in the provocation arm and 58% (39–78%) in the non-provocation arm. Specificity of the culture was 99% in the provocation arm and 100% in the non-provocation arm. The cumulative effect of repeat smears on sensitivity was also examined. Among women with LCR-positive tests who received provocation, the first smear identified 25% of LCR-positive subjects, the second a further 21% and the third a further 3%. This pattern of increased diagnostic yield with the second smear, but a marginal increase with the third smear was similar among LCR-positive women who did not receive provocation. However, the gonorrhoea diagnoses were consistently about two-fold more in the provocation group than in the control group.

Costs of management through provocation algorithm and through single culture carried out at baseline

The average health service costs per case diagnosed were calculated for two diagnostic protocols: (a) provocation with smear at three follow-up visits; (b) culture and smear at baseline only. Costs were estimated using a bottom-up approach¹⁷ to calculate the price of basic resource inputs, including staff, materials and building service costs. The cost of a single visit including smear was estimated at $2.48; with additional costs of $2.80 for a culture test and $0.43 for a gonococcal vaccine injection.¹⁸ In this study, women in the provocation arm made, on average, 3.2 visits per person incurring travel costs of $0.40 per visit. Visits took the patient on average four hours door-to-door, and required 15 minutes of staff time. Costs of patient time estimated as an opportunity cost were $5.00 per visit. Average total per-patient cost for management with the provocation protocol was $8.36 for direct health service costs and $17.28 incurred by the patient. For the baseline culture and smear protocol, the health service cost was $5.71 with $10.80 incurred by the patient. Using the provocation protocol, the total cost (health service to-patient) per case was $213 ($70, $144) at the gonorrhoea prevalence of 24% found in this population, leaving 12 infected individuals undiagnosed. Using the non-provocation protocol, the total cost per case (health service to-patient) was $150 ($52, $98), leaving 13 infected individuals undiagnosed.

DISCUSSION

Gonovaccine provocation increased the overall sensitivity of microscopic examination of Gram-stained cervical smears in the diagnosis of genital gonorrhoea infection in women from 25% to 50%, achieving a sensitivity close to that of single baseline culture. This level of sensitivity was, however, achieved only through having the patient make at least two follow-up visits; the sensitivity of a single follow-up smear was low (25%) in the provocation arm, and very low (13%) in the non-provocation arm. This doubling of sensitivity was consistent, whether sensitivity was calculated for any smear-positive, or for each smear considered individually, suggesting that this method of provocation does indeed have a significant positive effect. However, Gonovaccine provocation appears to have little effect on the sensitivity of culture which was estimated at 63% overall and 47% for a single culture performed at the initial visit. This level of sensitivity may seem poor, but women with gonorrhoea diagnosed on initial microscopy were excluded and these were very likely to have been culture-positive. Thus, a group with a presumably lower gonococcal load was selected for the study which may explain the perceived relatively poor sensitivity of culture.

The health service and patient-incurred costs per case diagnosed were higher ($70, $144) for the protocol comprising provocation with three follow-up visits, than for the internationally recommended protocol of culture and smear at the initial visit ($52, $98). Moreover, there are considerable disadvantages for the patient entailed with provocation, including the pain and discomfort of the injection and multiple time-consuming visits. It is likely that patients would be willing to contribute to the cost of a culture test at baseline in order to save the time and expense associated with multiple attendances. We conclude that a protocol comprising quality-controlled culture and smear at first visit should be adopted in routine practice to replace the use of provocation in the diagnosis of genital gonorrhoea in women in the Russian Federation and, indeed, wherever else this practice is used. Of course, if financial restrictions eased then the use of a NAAT would certainly have a place. We used LCR as the ‘gold’ standard because it was an assay with which we were familiar. However, it is no longer commercially available, but others such as the Gen-Probe Aptima Combo 2 assay, that are available have even greater sensitivity.¹⁹

Despite the relatively small number of women examined, we were interested to find that Gonovaccine provocation significantly improved the sensitivity of microscopic diagnosis of genital gonorrhoea infection in women, a phenomenon seen in each clinic. It is difficult to offer a plausible and biologically satisfactory explanation for the mechanism through which this effect might be mediated. However, Russian clinical practice has always identified a category of patients with ‘chronic gonorrhoea’²⁰ and Russian research studies on specimens from such patients²¹ have shown by electron microscopy sub-epithelial bodies, consistent with Neisseria gonorrhoeae bacteria, in a so-called metabolically closed-down state. It is plausible, perhaps, that provocation techniques modify the host-pathogen interaction through influencing the immune response, perhaps by stimulating a brief cell-mediated response in which the increased inflammation causes enhanced exposure of gonococci at the mucosal surface. However, this is mere speculation, and further detailed studies, including those in animal models^22,23 would be required to elucidate any such effect. Also somewhat but perhaps less mystifying is the reason(s) why there is little or no enhancing effect of provocation on culture and LCR results. So far as culture is concerned, there was a trend for more culture-positive results in the provocation arm, which might mean that some metabolically inactive, not easily seen, bacteria, if they truly exist, became active and culturable. So far as the LCR is concerned, it might be expected that the sensitivity of the technique would enable the DNA of inactive bacteria to be detected and so account for the lack of difference between the provocation and non-provocation arms in terms of LCR-positives.

Footnotes

ACKNOWLEDGEMENTS

The authors are grateful to the Wellcome Trust for a grant to undertake this study and also thank Natasha Anwar for technical assistance.

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