Methods

The study period was from April 2007 to December 2008. The location was four Community Sexual and Reproductive Health (CSRH) clinics in the borough of Lewisham in South London. Female patients over the age of 16 years with a positive NAATs test for gonorrhoea were recruited and gave informed consent for testing for gonococcal infection.

Specimens were either taken by the subject or the clinician. Subjects were given detailed instructions for collection of self-taken vulvo-vaginal swabs (3 cm into the vagina). High vaginal or cervical swabs were collected if vaginal examination was indicated. The swabs used for NAAT were provided by the manufacturers (BD ProbeTec C trachomatis/N gonorrhoea Amplified DNA Assay Endocervical and Urethral Collection and Dry transport kits, Becton Dickinson).

Tests were performed in the microbiology department at the University Hospital Lewisham. The NAAT used was based on strand displacement amplification assay (SDA, BD ProbeTec, Becton Dickenson). All positive tests were repeated in the laboratory to confirm the results and exclude contamination during testing. A result was reported positive only if both tests were positive.

Subjects with positive NAAT for N gonorrhoeae were recalled to the clinics as soon as possible. Two cervical swabs and a urethral swab from each patient (Transport swab, Amies medium with charcoal, Mediwire, Surrey, UK) were taken by the clinician prior to commencement of treatment for culture and antibiotic sensitivity tests. Participants over the age of 16 years who agreed to take part in the study gave specific informed written consent for the extra cervical swab to be taken.

The swab sent for routine transportation and processing (RTP) was stored at 4°C in the clinic and transported to the laboratory at room temperature on the following day by a routine laboratory courier system, which collected the swabs from the clinics between 11:00–13:00 hours, Monday to Friday. These swabs were delivered and processed in laboratory (for culture and antimicrobial sensitivity) between 14:00–17:00 hours on the same day. Swabs collected from evening clinics on Friday and from Saturday morning clinics were stored at 4°C and processed in the afternoon on Monday. These procedures reflected the routine practice of the community clinics.

The swab sent for urgent transportation at room temperature and processing (UTP) was sent to and processed in the laboratory within 2 hours of collection of the specimen. In the laboratory, the swabs were cultured on selective gonococcus medium (Lysed GC Selective Agar Ref. PB0962A, Oxoid, Basingstoke, UK) for N gonorrhoeae and incubated in 10% CO₂ at 37°C for 48 hours. Oxidase positive colonies were confirmed as N gonorrhoeae by Gram stain, biochemical (Neisseria PET, Bioconnections, Leeds, UK) and immunological tests (Gonogen II, Bioconnections, UK).

The McNemar test was used to compare the probabilities of a misclassification of result between the RTP and UTP methods.

Results

A total of 56 NAAT reactive subjects were recruited into the study. Nine (16.1%) subjects who were culture negative were excluded from final analysis. These included mainly treatment with antibiotics prior to collection of specimens for culture for either having chlamydia infection themselves or being a contact of a subject with chlamydia infection (4/9) or the culture was taken more than 1 month after the NAAT was positive (4/9). In one patient (1/9), both cervical swabs were mistakenly sent by routine transport and processed routinely but the urethral swab, which was sent urgently, was positive by culture. There were no positive tests that failed to be confirmed by repeat testing.

Altogether, 47/56 (83.9%) subjects were included in the final analysis. The clinical notes of two patients could not be traced. Most of the subjects (39/45; 87%) were under 25 years of age. A majority of the subjects were asymptomatic (36/45; 80%). Those who had symptoms (9/45; 20%) included discharge (3), per vaginal bleeding with Implanon (3), dysuria (1), abdominal pain (1) and test of cure for a previous gonococcal infection (1).

Altogether, 41/47 (87.2%) NAAT reactive subjects were positive by culture. Whereas 40/47 (85.1%) were positive by the UTP method, only 27/47 (57.4%) were positive by the RTP method. In one subject, culture by RTP method was positive but negative by UTP method. McNemar’s test with the continuity correction yielded a χ² (1df) = 9.6; p = 0.002.

Of the six subjects (6/47, 12.8%) in whom RTP and UTP cultures were negative, four subjects had no antibiotic treatment prior to culture, one subject was treated with trimethoprim for urinary tract infection (unlikely to affect culture as N gonorrhoeae are resistant to trimethoprim) and in one subject the urethral swab was positive by culture.

Discussion

Use of NAATs such as BD ProbeTec for the detection of gonococcal infection in a low prevalence population may lead to false-positive results.2 To illustrate this, Ison points out that even NAATs with relatively high specificity (99%) may result in relatively low positive predictive value (50%) of the test in populations where the prevalence is only 1%.4 Culture confirmation of the NAAT reactive results in dispersed clinics in the community is challenging because of rapid loss of viability of gonococcus during any delay in transportation and processing of the laboratories.8 In a recent paper describing our experience of screening in the community using BD ProbeTec, we reported an unexpectedly high prevalence (3.8%) of gonococcal infection in the Lewisham community.9 At the time of reporting the results, we were concerned whether some of the NAAT reactive positive results were false-positive as only 67% of these results could be confirmed by culture or whether the cultures were false-negative due to loss of viability of the organisms during transportation and processing. In a recent paper, we showed that over 97% of BD ProbeTec reactive tests could be confirmed by supplementary NAATs.

However, confirmation by supplementary NAATs does not necessarily address the issue of false-negative culture tests in subjects with reactive NAAT tests. We believe that culture not only serves to confirm the results of NAAT but enables determination of antibiotic sensitivity and study of the molecular epidemiology of the infection.

A majority (>80%) of the subjects included in this study were under 25 years and asymptomatic and screened as a part of the Lewisham Chlamydia and Gonococcus Screening Programme. In order to address the issue of false-negative culture tests, we have attempted to evaluate if culture confirmation of NAAT can be improved in community screening programmes by comparing UTP with RTP. Overall, 41/47 (87.2%) NAAT positive subjects were confirmed by culture of cervical swabs.

UTP can significantly increase culture confirmation (40/47; 85.1%) compared with RTP (27/47; 57.4%). We are unable to explain why in one subject the RTP swab was positive for culture and the UTP swab negative. It is conceivable that it may be related to the amount of the specimen material on the swabs. The detection of one subject, where the urethral swab was culture positive and the cervical swabs were negative, confirms that, in at least in some subjects, collection of urethral swabs may further improve the culture confirmation of NAAT where the cervical swab may be negative.10 Including the positive urethral swab, the overall culture confirmation rate was 89.4% (42/47).

Currently, community clinics in Lewisham do not have the resources to directly inoculate swabs on culture media and incubate the inoculated media at 37°C in 10% CO₂ prior to transportation to the microbiology laboratory.

Our study also shows that culture confirmation is feasible in a majority of the subjects even in dispersed clinics if the swabs are transported and processed urgently in the laboratory. We acknowledge that UTP may not be convenient or possible for many dispersed clinics due to the logistics or insufficient resources. Under such circumstances we recommend that periodical “culture” surveys are undertaken in the community when the specimens are transported and processed urgently. This will ensure that there is surveillance of antibiotic resistance in the community strains, which is crucial for determining the empirical treatment of choice for gonococcal infections in the community.

In conclusion, this study shows that culture confirmation of NAAT reactive subjects in community clinics can be significantly improved by urgent transportation and processing of specimens in the laboratory.