Improvement in Neisseria gonorrhoeae culture rates by bedside inoculation and incubation at a clinic for sexually transmitted infections

Inclusion and exclusion criteria

For a sample to be included, both case definition and technical criteria had to be fulfilled. Patients visiting the clinic with gonorrhoea fell naturally into two case definitions according to clinical presentation. Symptomatic patients were patients who referred themselves with symptoms and/or clinical signs, with microscopy findings suggestive of gonorrhoea and these patients were treated on the same day. The symptomatic site was cultured before the PCR result was known. Asymptomatic patients were patients without symptoms but with a recent N. gonorrhoeae-positive PCR result. The gonococcal PCR-positive site was cultured. Notably, during the study period no N. gonorrhoeae-negative PCR samples (n = 177) had a positive gonococcal culture.

The technical criteria for a sample to be included were two plates (standard and bedside culture) and a N. gonorrhoeae-positive PCR taken on the day of culture. To avoid dependency, only samples from the first visit during the study period were included.

Patients who received any antibiotic treatment in the 4 weeks before culture or developed symptoms between the screening PCR and the day of treatment, were excluded. Patients who had an initial screening PCR result from other laboratory than the Department of Microbiology at Oslo University Hospital, were also excluded. Finally, samples that proved PCR negative on the day of culture were excluded.

Sampling methods

Opti-Swab 1 ml Liquid Amies Transport Medium with polyester HydraFlock swab (3506-H, Puritan) was used to sample all anatomical sites. The swab (not the liquid) was then immediately used to inoculate the agar plate for the culture at the clinic. All culture samples were taken before antibiotic treatment was administered.

Two swabs were taken from each sample site in random order.

Urethra: The external urethral meatus was swabbed. While urine is the material of choice for screening with PCR, no urine samples were cultured.

Pharynx: The posterior nasopharynx and the tonsillar arches were swabbed.

Anorectum: The swab was inserted 2–3 cm, moved back and forth/rotated.

Cervix: A vaginal speculum was used to provide a clear view of the cervix. The cervix was swabbed.

CLAT selective agar plates (Heart infusion agar (Difco), defibrinated horseblood, yeast autolysate supplement (OXOID SR 0105B) and LCAT selective supplement (Oxoid SR 0095B)) were used for both bedside and standard culture of samples. LCAT selective supplement contains colistin 3.0 mg, lincosamide 0.5 mg, amphotericin B 0.5 mg and trimethoprim 3.25 mg per 500 ml of medium, to inhibit growth of non-gonococcal bacterial and fungal species.

Study structure

Patients with symptoms:

1) Swab for standard culture: The swab taken from the symptomatic site was placed directly in the transport medium, and stored immediately at 4 °C. Samples were transported twice daily in insulated boxes to the Department of Microbiology, a 10 min drive from the clinic. At the Department of Microbiology the liquid transport medium was vortexed and separated into two parts; one part was examined with a standard PCR for N. gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium, and the other part was cultured on CLAT plates.

2) Swab for bedside culture: An incubator (set to 36 °C, 5% CO2-enriched atmosphere) was installed at the STI clinic. CLAT plates were prewarmed in the incubator. After sampling the symptomatic site, the swab was immediately placed and rolled on approximately 1/3 of the plate, then streaked out further with an inoculation loop. Plates were immediately incubated. The swab was then placed in its transport medium, stored at 4 °C and sent the same day for PCR.

Patients without symptoms:

1) Swab for standard culture: The swab from the N. gonorrhoeae PCR positive site was placed directly in the transport medium, stored at 4 °C and transported to the Department of Microbiology where the liquid was cultured according to the standard routine. As the PCR status of the sampling site was assumed to be positive from the earlier screening test, no PCR was performed from this swab.

2) Swab for bedside culture: This was performed in the same manner as for patients with symptoms, described above, including confirmatory PCR test.

CLAT plates from standard culture were inspected for growth on day 1 and day 2.

Incubation and transportation of the bedside culture plates

Plates from Monday, Tuesday and Wednesday were incubated for 2 days at the STI clinic, and were first inspected for N. gonorrhoeae colonies at the Department of Microbiology on day 2. Samples plated on Thursday and Friday were all transported to the Department of Microbiology at the last transportation on Friday. Therefore Thursday plates were incubated for 1 day and Friday plates for a maximum of 4 h (0 days) at the clinic. At the Department of Microbiology the plates were inspected for N. gonorrhoeae colonies, the two day incubation period was completed and identification and antibiotic susceptibility testing were performed.

Transportation of plates was optimized by using special plastic pouches (Thermo Scientific Compact W-Zip Seal Pouches) containing a CO2 generator (Thermo Scientific Oxoid™ CO2Gen™ Compact Sachet). The sealed plastic pouches with plates were transported in a heated (36 °C) transport box (Portable Vaccine and Sample Carrier from LABCOLD) to the Department of Microbiology.

N. gonorrhoeae was species-verified using MALDI-TOF MS (Microflex LT MALDI-TOF with MALDI Biotyper software v.2.0, Bruker Daltonics, Bremen, Germany).

PCR

PCR analysis was performed at the Department of Microbiology. A previously described duplex PCR targeting the porA pseudogene and opa genes was used [9]. If only one of the targets was positive, a confirmatory PCR (Fast-Track Diagnostics, Luxembourg) targeting another area of the opa genes and the pilin gene was applied. For a gonorrhoea diagnosis to be made, at least two different gonococcal targets had to be detected.

Demographic and clinical characteristics

Microbiology results, relevant epidemiological and clinical information were retrospectively extracted from the electronic patient journal, and stored in an anonymised database.

Statistical analysis

Sample size was calculated with a significance level α = 0.05, power 80%, one-sided superiority test. Culture results from 2014 were set as the baseline, and expected culture rates set at 50% for urethral, cervical and anorectal samples, and 10% for pharyngeal samples. For adequately powered analysis we required 252 urethral, 50 cervical, 17 anorectal and 79 pharyngeal samples. The null hypothesis that the intervention has no impact on the likelihood of obtaining a successful culture, was examined by McNemar’s test and the effect size of the intervention was examined by Odds Ratio (OR).

A linear logistic regression model with interaction was used to investigate differences in culture method success rate according to sample site. To facilitate the interpretation of significant differences between the two culture methods, estimated marginal means, i.e. the modelled culture rate, and their 95% confidence intervals were calculated and Wald tests were used for pairwise comparisons between methods within each sample site. An adjustment for 4 pairwise comparisons (performed on the log odds ratio scale) was performed using Holm method.

Data handling and analysis were performed in Statistical Package for the Social Sciences (SPSS) ‘IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, N.Y., USA), VassarStats Website for Statistical Computation (R. Lowry, http://vassarstats.net/) and R version 4.0.2. (R Core Team, http://www.R-project.org/).

Ethics

The study was approved by the Privacy and Data Protection Officer at Oslo University Hospital, study reference: 2015–19428. Individual patient informed consent was not required.

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