Study location, population, and design.
This pilot study was nested on an HPV case‒control study conducted at the EFSTH. Reproductive aged women (20 – 49 years old) seeking primary health care who met the inclusion criteria were recruited consecutively by trained health care providers. Women with symptoms of STIs were recruited into the case group. Asymptomatic women attending the contraceptive (family planning) clinic were recruited as a control group. The minimum sample size was calculated using a simple Daniel formula [12]:
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Inclusion criteria
The inclusion criteria for cases included the presence of one or more of the following: vaginitis/cervicitis, dysuria (painful and burning urination), lower abdominal pain, bleeding between periods, dyspareunia (pain during sexual intercourse), and vaginal itching/discharge with a fishy or strong odour. The inclusion criteria for controls were asymptomatic.
Consent to participate in the study.
Informed consent was obtained from all participants who met the inclusion criteria, and a participant information sheet was provided for them.
Exclusion criteria
Participants were excluded from the study if they lived outside the study area, were pregnant or were on their menstrual cycle at the time of sampling, had reached menopause or were above 49 years old.
Ethical statement
The study was reviewed and approved by The Gambian Government and Medical Research Council Joint Ethics Committee, The Gambia and the University of Westminster Research Ethics Committee, London.
Sample collection.
A total of two hundred and thirty-two (232) women were recruited consecutively (115 symptomatic and 117 asymptomatic). Endocervical and high vaginal swabs were collected from each participant prior to treatment using the syndromic management treatment guidelines.
The swabs for PCR amplification for Ureaplasma, Mycoplasma genitalium, Chlamydia trachomatis and Neisseria gonorrhoeae were placed immediately into specimen transport media (M4RT, Microtest, Oxoid, Basingstoke, UK). A questionnaire was administered to capture behavioural risk factors that might be associated with STIs in reproductive-aged Gambian women.
Methods
Bacterial vaginosis and Trichomonas vaginalis
Detection of BV and T. vaginalis was carried out at the clinic’s site laboratory. High vaginal swabs were used for direct wet mount microscopy, detection of fishy amine odour (‘Whiff’ test) when mixed with 10% (w/v) potassium hydroxide (KOH) preparation and vaginal pH determination (range 4.0 – 7.0). The wet preparation was viewed microscopically for the presence of ‘clue cells’, yeast cells, and motile T. vaginalis. Bacterial vaginosis was diagnosed using Amsel’s clinical criteria [13].
Neisseria gonorrhoeae isolation and Antibiotic susceptibility Testing
Routine microbiological detection of Neisseria gonorrhoeae was performed in the Department of Medical Microbiology, EFSTH, using standard operating procedures. Endocervical swabs were cultured on chocolate agar (Oxoid, Basingstoke, UK) and incubated overnight at 37°C in 5% carbon dioxide atmospheric conditions. Colonies of interest were subcultured and incubated overnight to generate pure colonies. These were selected for Gram staining and biochemical identification with the oxidase test for presumptive diagnosis of Neisseria gonorrhoeae. Suspected oxidase-positive Neisseria gonorrhoeae isolates were further confirmed using the analytical profile index test API NH (Biomerieux, Basingstoke, UK). Neisseria gonorrhoeae isolates were tested against the following selection of antibiotics: ciprofloxacin (CIP, 5 µg), cefotaxime (CTX, 30 µg), tetracycline (TE, 30 µg), penicillin (P, 10 units), and ceftriaxone (30 µg) using the disc diffusion method. Antibiotic susceptibility testing was interpreted using the European Committee on Antimicrobial Susceptibility Testing [14] and British Society for Antimicrobial Chemotherapy [15] guidelines.
DNA extraction
DNA was extracted from the endocervical and high vaginal swabs using the QIAamp DNA mini extraction kit (Qiagen, Crawley, UK) following the manufacturer’s instructions. Both endocervical and high vaginal swabs were used for Ureaplasma parvum/urealyticum and Mycoplasma genitalium PCR, while only endocervical swabs were used for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae.
Real-time PCR and PCR
All real-time PCRs were performed with Rotorgene-Q (Qiagen, Crawley, UK) in a 20 µl reaction volume containing 0.25 μM Rotorgene probe mastermix kit (Qiagen, Crawley, UK), 1 µM of each primer (unless otherwise stated) and 50 ng -100 ng of DNA template. Two channel wavelengths of carboxyfluorescein (FAM) and 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE) were used to identify the specific species in a duplex real-time PCR. Black hole quencher-1 (BHQ-1) was used for the probes. All samples were tested in duplicate within one run and were considered positive if the cycle threshold (Ct) value was ≤ 35. All samples were tested in duplicate, and those that were not in accordance were repeated.
PCR amplifications were performed in a 25 µl volume containing 5 µM of each primer, 1x Taq PCR master mix containing 2.5 units of Taq DNA polymerase, 0.2 mM deoxynucleotide trisphosphates and 1.5 mM MgCl2 (Qiagen, Crawley, UK) and 50 – 200 ng of DNA template. Extraction control, negative control (molecular grade water) and positive controls (Genekam Biotechnology, Duisburg, Germany) were included in each run.
Detection of Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma genitalium
Real-time PCR was carried out to distinguish between the two Ureaplasma species, U. parvum (parvo) and U. urealyticum (T960). A common primer for the two species and two species-specific probes (Table S1) were used to amplify 90 base pairs (bp) of the urease gene. The 78 (bp) conserved region of the M. genitalium adhesion MgPa gene was targeted using a specific set of primers (Table S1). Thermal cycling and detection of fluorescent signals upon amplification of the specific target sequence were performed as previously described [16, 17].
PCR for the M. genitalium 16S rRNA gene was also carried out using the MG16-45 F and MG16-447 R primers (Table S1), which are in the V1 and V3 hypervariable regions, respectively. The amplification reaction consisted of 40 cycles and was carried out as follows: denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, elongation at 72°C for 30 seconds and a final extension step at 72°C for 5 minutes. Amplified products were resolved by gel electrophoresis using 2% (w/v) agarose gel (Sigma Aldrich, Haverhill, UK).
Detection of Chlamydia trachomatis and Neisseria gonorrhoeae
A duplex real-time PCR was carried out for Neisseria gonorrhoeae and Chlamydia trachomatis using dual-labelled probes (Table S1). In addition to the mastermix, 10 µmol/L of each primer and 10 µmol/L of each probe were added. Both thermal cycling and detection of fluorescent signals were performed as previously described [18].
Primers and probes used for real-time PCR and PCR for the detection of sexually transmitted pathogens (Supplementary Table 1)
Key: UU-Ureaplasma, MG-Mycoplasma genitalium, CT-Chlamydia trachomatis, NG-Neisseria gonorrhoea, F-Forward primer, R-Reverse primer
DNA Sequencing:
PCR amplicons were purified using a PCR purification kit (Sigma Aldrich, Haverhill, UK) and then sequenced using the Sanger chain termination method. An NCBI BLAST [http://www.ncbi. nlm.nih.gov/BLAST/] search was performed for each sequenced product.
Statistical analysis
Data analysis was performed using Epi Info version 7.0 [https://www.cdc.gov/epiinfo]. Descriptive analyses, including frequencies, percentages, and averages, were used when appropriate to describe the study population. A chi-square test was used to assess the association between exposures and outcomes expressed as categorical variables. A confidence interval of 95% (CI) and p value of ≤ 0.05 were used to determine statistical significance. The strength of risk factors associated with STIs as an outcome was measured using the odds ratio (OR). The ORs of the risk characteristics were interpreted as increased odds (>1.0) or decreased odds (<1.0).