Extragenital Sexually Transmitted Infections Among High-Risk Men Who Have Sex With Men in Johannesburg, South Africa.

BACKGROUND: In South Africa, extragenital etiological sexually transmitted infection (STI) screening among men who have sex with men (MSM) is not routinely available. We aimed to determine the prevalence of STI pathogens at rectal and pharyngeal sites, syphilis seroprevalence, and associated risk factors among a selection of high-risk MSM without symptomatic urethritis attending a men's health clinic in Johannesburg, South Africa. METHODS: A cross-sectional study was conducted in 2022. Enrolled clients self-reported demographic, sexual behavioral risks, and clinical information. Client or clinician-collected rectal and pharyngeal swabs were tested for Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis. C. trachomatis-positive rectal samples were reflex tested for lymphogranuloma venereum. Blood specimens were screened for syphilis. Univariate and multivariate regression models were used to determine factors independently associated with the presence of an extragenital STI or syphilis. RESULTS: Among the 97 participants (median age, 29 years), 24.7% had an extragenital STI and 9.4% had high nontreponemal antibody titers (rapid plasma reagin ≥1:16). Rectal STIs were detected in 26.4% participants: N. gonorrhoeae (14.3%), C. trachomatis (9.9%), and M. genitalium (5.5%). Pharyngeal STIs were less prevalent (4.1%). Overall, the prevalence of any STI was 41%. Sex under the influence of drugs (adjusted odds ratio, 4.94; 95% confidence interval, 1.56-15.69) and engaging in condomless receptive anal intercourse with a casual partner (adjusted odds ratio, 8.36; 95% confidence interval, 1.73-40.28) were independent risk factors for having an extragenital STI. CONCLUSIONS: The high burden of extragenital STIs and active syphilis in asymptomatic MSM underscores the importance of routine etiological screening in this key population, as the syndromic approach would not enable detection or treatment of these infections.

Independent clinic-based evaluation of point-of-care testing for the screening of Chlamydia trachomatis, Neisseria gonorrhoea and Trichomonas vaginalis in women-at-risk in Australia, Guatemala, Morocco, and South Africa.

BACKGROUND: In 2018, the World Health Organization commenced a multi-country validation study of the Cepheid GeneXpert for a range of molecular-based point-of-care (POC) tests in primary care settings. One study arm focused on the evaluation of POC tests for screening 'women at risk' for chlamydia (CT), gonorrhoea (NG) and trichomonas (TV) in four countries - Australia, Guatemala, Morocco and South Africa. METHODS: Study participants completed a pre-test questionnaire which included demographics, clinical information and general questions on POC testing (POCT). Two vaginal swab samples (either self-collected or clinician collected) from each patient were tested on the GeneXpert at the POC and at a reference laboratory using quality-assured nucleic acid amplification tests (NAATs). RESULTS: One thousand three hundred and eighty-three women were enrolled: 58.6% from South Africa, 29.2% from Morocco, 6.2% from Guatemala, and 6.0% from Australia. 1296 samples for CT/NG and 1380 samples for TV were tested by the GeneXpert and the reference NAAT. The rate of unsuccessful tests on the GeneXpert was 1.9% for CT, 1.5% for NG and 0.96% for TV. The prevalence of CT, NG and TV was 31%, 13% and 23%, respectively. 1.5% of samples were positive for all three infections; 7.8% were positive for CT and NG; 2.4% were positive for NG and TV; and 7.3% were positive for CT and TV. Compared to reference NAATs, pooled estimates of sensitivity for the GeneXpert tests were 83.7% (95% confidence intervals 69.2-92.1) for CT, 90.5% (85.1-94.1) for NG and 64.7% (58.1-70.7) for TV (although estimates varied considerably between countries). Estimates for specificity were ≥96% for all three tests both within- and between-countries. Pooled positive and negative likelihood ratios were: 32.7 ([CI] 21.2-50.5) and 0.17 (0.08-0.33) for CT; 95.3 (36.9-245.7) and 0.10 (0.06-0.15) for NG; and 56.5 (31.6-101.1) and 0.35 (0.27-0.47) for TV. CONCLUSION: This multi-country evaluation is the first of its kind world-wide. Positive likelihood ratios, as well as specificity estimates, indicate the GeneXpert POC test results for CT, NG and TV were clinically acceptable for ruling in the presence of disease. However, negative likelihood ratios and variable sensitivity estimates from this study were poorer than expected for ruling out these infections, particularly for TV. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee, as well as local ethics committees from all participating countries.

Quality control and external quality assessment for the independent clinic-based evaluation of point-of-care testing to detect Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in eight countries.

BACKGROUND: Sexually transmitted infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) remain significant global health problems. The World Health Organization (WHO) has recently conducted a multi-faceted, multi-country validation study (ProSPeRo), which included an evaluation of the Xpert CT/NG and Xpert TV assays on the GeneXpert system (Cepheid, Sunnyvale, Ca., USA) in clinic-based settings across eight countries. To support the study, a training and quality management system was implemented and evaluated. METHODS: A comprehensive training program for the study was developed. Quality control (QC) and external quality assessment (EQA) samples were provided by an accredited quality assurance provider. QC testing was conducted at 14 point-of-care testing (POCT) clinics, while EQA samples were tested by the POCT sites and a reference laboratory supporting each clinic. RESULTS: For QC testing, concordance with the expected results for CT and NG was > 99% and rates of unsuccessful tests were < 4%. For TV testing, concordance was similar (97%), but rates of unsuccessful tests were high (18%), particularly in the 'TV negative' sample. For EQA testing initially conducted in 2018, concordance was 100% for CT and NG, and 90% for TV for the reference laboratory group (which used non-GeneXpert systems). Concordance for the POCT group was also high (> 94%) for all analytes, but this cohort (which used GeneXpert systems) exhibited a high rate of unsuccessful TV tests. All but one of these unsuccessful tests was subcategorised as 'invalid'. CONCLUSIONS: The high level of concordance for QC and EQA testing confirm that the trained operators at the POC clinical sites were competent to conduct POC testing and that the training and quality systems implemented for the ProSPeRo study were effective. The quality materials used were satisfactory for CT and NG but exhibited poor performance for TV testing on the GeneXpert system. The WHO should continue to work with industry and EQA providers to provide improved materials that are reliable, stable and cost effective for quality management, as it seeks to rollout molecular-based STI POCT in non-laboratory-based settings. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee.

External quality assessment to support the WHO ProSPeRo study for the evaluation of two dual HIV/syphilis point-of-care tests in seven countries.

BACKGROUND: Sexually transmitted infections (STIs) such as syphilis and HIV remain to be a significant public health issue worldwide. Dual rapid point-of-care tests (POCTs) have shown promise for detecting antibodies to HIV and syphilis but have not been fully evaluated in the field. Our study supported the WHO ProSPeRo study on Sexually Transmitted Infection Point-of-Care Testing (STI POCT) by providing external quality assessment (EQA) for HIV and syphilis testing in reference laboratories and their associated clinical sites in seven countries. METHODS: HIV/syphilis serum liquid and dried tube specimen (DTS) panels were prepared by CDC. Liquid panels were distributed to the reference laboratories for three rounds of testing using commercially and locally available laboratory-based serological tests. DTS panels were sent to the clinical testing sites for 8 rounds of POC testing using the Abbott SD BIOLINE HIV/Syphilis Duo test (hereafter referred to as SD BIOLINE) and the Chembio Dual Path Platform (DPP) HIV-Syphilis assay. EQA panels were tested at CDC using the Rapid Plasma Reagin (RPR) test and the Treponema pallidum Particle Agglutination assay (TP-PA) for syphilis antibodies. Genetic Systems HIV-1/HIV-2 Plus O EIA, Geenius HIV Supplemental Assay and the Oraquick Advance HIV test were used to detect HIV antibodies in the EQA panels. Results from the reference laboratories and POCT sites were compared to those obtained at the CDC and a percentage agreement was calculated. RESULTS: Qualitative RPR and TP-PA performed at the reference laboratories demonstrated 95.4-100% agreement with CDC results while quantitative RPR and TP-PA tests demonstrated 87.7% and 89.2% agreement, respectively. A 93.8% concordance rate was observed for qualitative HIV testing in laboratories. EQA testing at clinical sites using dual tests showed 98.7% and 99.1% agreement for detection of HIV antibodies and eight out of 10 sites had > 95.8% agreement for syphilis testing. However, two clinical sites showed only 65.0-66.7% agreement for SD BIOLINE and 84.0-86.7% for DPP, respectively, for syphilis testing. CONCLUSIONS: Overall, laboratories demonstrated high EQA performance in this study. Both HIV/syphilis POCTs gave expected results in the clinic-based evaluations using DTS. However, testing errors were identified in a few testing sites suggesting the necessity for continuous training and monitoring the quality of POC testing.

Emergence of high-level azithromycin-resistant Neisseria gonorrhoeae causing male urethritis in Johannesburg, South Africa, 2021.

BACKGROUND: In South Africa, Neisseria gonorrhoeae , which is the predominant cause of male urethritis, is treated syndromically using dual ceftriaxone and azithromycin therapy. We determined antimicrobial susceptibilities of N. gonorrhoeae isolates from urethral discharge specimens, and genetically characterised those with elevated minimum inhibitory concentrations (MICs) for first-line antimicrobials. METHODS: Routine antimicrobial susceptibility testing (AST) of N. gonorrhoeae isolates included E-test for ceftriaxone, cefixime and gentamicin and agar dilution for azithromycin and spectinomycin. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) was performed for isolates with elevated MICs to identify antimicrobial resistance (AMR) determinants, and Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) was used to determine strain relatedness. RESULTS: N. gonorrhoeae was cultured from urethral discharge swab specimens obtained from 196 of 238 (82.4%) men presenting to a primary healthcare facility in Johannesburg in 2021. All viable isolates were susceptible to extended-spectrum cephalosporins. Four isolates had high azithromycin MICs ranging from 32mg/L to >256mg/L and grouped into two novel NG-MAST and NG-STAR groups. Two isolates from Group 1 (NG-MAST ST20366, NG-STAR ST4322) contained mutated mtrR (G45D) and 23S rRNA (A2059G) alleles, while the two isolates from Group 2 (NG-MAST ST20367, NG-STAR ST4323) had different mutations in mtrR (A39T) and 23S rRNA (C2611T). CONCLUSIONS: We report the first cases of high-level azithromycin resistance in N. gonorrhoeae from South Africa. Continued AMR surveillance is critical to detect increasing azithromycin resistance prevalence in N. gonorrhoeae , which may justify future modifications to the STI syndromic management guidelines.

Genomic surveillance and antimicrobial resistance determinants in Neisseria gonorrhoeae isolates from Uganda, Malawi and South Africa, 2015-20.

OBJECTIVES: Global antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae is essential. In 2017-18, only five (10.6%) countries in the WHO African Region reported to the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP). Genomics enhances our understanding of gonococcal populations nationally and internationally, including AMR strain transmission; however, genomic studies from Africa are extremely scarce. We describe the gonococcal genomic lineages/sublineages, including AMR determinants, and baseline genomic diversity among strains in Uganda, Malawi and South Africa, 2015-20, and compare with sequences from Kenya and Burkina Faso. METHODS: Gonococcal isolates cultured in Uganda (n = 433), Malawi (n = 154) and South Africa (n = 99) in 2015-20 were genome-sequenced. MICs were determined using ETEST. Sequences of isolates from Kenya (n = 159), Burkina Faso (n = 52) and the 2016 WHO reference strains (n = 14) were included in the analysis. RESULTS: Resistance to ciprofloxacin was high in all countries (57.1%-100%). All isolates were susceptible to ceftriaxone, cefixime and spectinomycin, and 99.9% were susceptible to azithromycin. AMR determinants for ciprofloxacin, benzylpenicillin and tetracycline were common, but rare for cephalosporins and azithromycin. Most isolates belonged to the more antimicrobial-susceptible lineage B (n = 780) compared with the AMR lineage A (n = 141), and limited geographical phylogenomic signal was observed. CONCLUSIONS: We report the first multi-country gonococcal genomic comparison from Africa, which will support the WHO GASP and WHO enhanced GASP (EGASP). The high prevalence of resistance to ciprofloxacin (and empirical use continues), tetracycline and benzylpenicillin, and the emerging resistance determinants for azithromycin show it is imperative to strengthen the gonococcal AMR surveillance, ideally including genomics, in African countries.

The evaluation of the Allplex™ BV molecular assay for the diagnosis of bacterial vaginosis in symptomatic South African females.

Bacterial vaginosis (BV) is a dysbiosis of vaginal microbiota characterized by a shift from Lactobacillus species predomination to a heterogeneous mixture of anaerobes. We compared the performance characteristics of the Allplex ™ BV molecular assay with the reference test, Nugent score microscopy, for vaginal swab specimens from symptomatic South African women. A total of 213 patients were enrolled, of whom 99 (46.5%) and 132 (62.0%) were diagnosed with BV by Nugent and Allplex™, respectively. The Allplex™ BV assay displayed a sensitivity of 94.9% (95% CI, 88.7%-97.8%) and a specificity of 66.7% (95% CI, 57.6%-74.6%), with an agreement of 79.8% (95% CI, 73.9%-84.7%) (κ = 0.60). Assay design may be enhanced for improved specificity by accounting for differences in healthy and BV-associated vaginal microbiomes among women of different ethnicities.

Syphilis screening coverage and positivity by HIV treatment status among South African pregnant women enrolled in the 2019 antenatal HIV sentinel survey.

We describe coverage of maternal syphilis screening, syphilis positivity, coverage of treatment and their association with maternal HIV infection and antiretroviral treatment (ART) status among pregnant women attending South African antenatal clinics. The 2019 antenatal care sentinel survey was a cross-sectional survey conducted from 1 October to 15 November 2019 at 1589 sentinel sites in all nine provinces of the country and aimed to enrol 36,000 pregnant women ages 15-49 years regardless of HIV, ART or syphilis status. Data collection procedures included obtaining written informed consent, a brief interview, medical record review and blood specimen collection. Completed data collection forms and specimens were sent to designated regional laboratories for data capture and HIV serology testing. Data analysis determined four outcomes i) syphilis screening coverage ii) syphilis positivity iii) coverage of any treatment and iv) with Benzathine penicillin G (BPG). Multivariable logistic regression models with or without interaction between HIV infection and ART status with province were used to determine factors associated with syphilis positivity. Of the 41 598 women enrolled, 35 900 were included in the analysis for syphilis screening coverage. The weighted syphilis screening coverage was 96.4% [95% Confidence Interval (CI) 95.9-96.7%] nationally and was lowest among HIV positive women not on ART at 93.5% (95% CI 92.2-94.5%). Syphilis positivity was 2.6% (95% CI 2.4-2.9%) nationally. Among those who were syphilis positive, 91.9% (95% CI 89.8-93.7%) had documentation of syphilis treatment status, of whom 92.0% (95% CI 89.8-93.9%) were treated, with the majority treated with one or more doses of BPG [92.2% (95% CI 89.8-94.3%)]. HIV-positive women, not on ART [adjusted odd ratio (aOR) 2.24 (95% 1.71-2.93)] and those on ART [aOR 2.25 (95% CI 1.91-2.64)] were more likely to be syphilis positive compared to those who were HIV negative. The national syphilis screening coverage met the global screening target of 95%. Syphilis positivity was higher among HIV positive women compared to negative women. Introduction of rapid syphilis testing and ensuring a universal supply of appropriate treatment for syphilis will reduce the likelihood of mother-to-child transmission of syphilis.

Mycoplasma genitalium prevalence, antimicrobial resistance-associated mutations, and coinfections with non-viral sexually transmitted infections in high-risk populations in Guatemala, Malta, Morocco, Peru and South Africa, 2019-2021.

The prevalence of Mycoplasma genitalium (MG) and MG antimicrobial resistance (AMR) appear to be high internationally, however, prevalence data remain lacking globally. We evaluated the prevalence of MG and MG AMR-associated mutations in men who have sex with men (MSM) in Malta and Peru and women at-risk for sexually transmitted infections in Guatemala, South Africa, and Morocco; five countries in four WHO regions mostly lacking MG prevalence and AMR data, and estimated MG coinfections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Male urine and anorectal samples, and vaginal samples were tested for MG, CT, NG, and TV (only vaginal samples) using Aptima assays (Hologic). AMR-associated mutations in the MG 23S rRNA gene and parC gene were identified using ResistancePlus MG kit (SpeeDx) or Sanger sequencing. In total, 1,425 MSM and 1,398 women at-risk were recruited. MG was detected in 14.7% of MSM (10.0% in Malta and 20.0% Peru) and in 19.1% of women at-risk (12.4% in Guatemala, 16.0% Morocco, 22.1% South Africa). The prevalence of 23S rRNA and parC mutations among MSM was 68.1 and 29.0% (Malta), and 65.9 and 5.6% (Peru), respectively. Among women at-risk, 23S rRNA and parC mutations were revealed in 4.8 and 0% (Guatemala), 11.6 and 6.7% (Morocco), and 2.4 and 3.7% (South Africa), respectively. CT was the most frequent single coinfection with MG (in 2.6% of MSM and 4.5% of women at-risk), compared to NG + MG found in 1.3 and 1.0%, respectively, and TV + MG detected in 2.8% of women at-risk. In conclusion, MG is prevalent worldwide and enhanced aetiological MG diagnosis, linked to clinical routine detection of 23S rRNA mutations, in symptomatic patients should be implemented, where feasible. Surveillance of MG AMR and treatment outcome would be exceedingly valuable, nationally and internationally. High levels of AMR in MSM support avoiding screening for and treatment of MG in asymptomatic MSM and general population. Ultimately, novel therapeutic antimicrobials and/or strategies, such as resistance-guided sequential therapy, and ideally an effective MG vaccine are essential.

Erratum: Southern African HIV Clinicians Society 2022 guideline for the management of sexually transmitted infections: Moving towards best practice.

[This corrects the article DOI: 10.4102/sajhivmed.v23i1.1450.].

Etiological Surveillance of Male Urethritis Syndrome in South Africa: 2019 to 2020.

BACKGROUND: In South Africa, male urethritis syndrome (MUS) is the most common sexually transmitted infection (STI) syndrome in men. We determined the distribution of STI etiologies and the susceptibility profiles of Neisseria gonorrhoeae isolates from men presenting with MUS to 3 sentinel surveillance health care facilities. Secondary objectives were to determine the seroprevalence of coinfections (HIV, syphilis, herpes simplex virus 2). METHODS: Consecutive, consenting men with symptomatic urethral discharge were enrolled between January 1, 2019, and December 31, 2020. Genital discharge swab and blood specimens were collected and transported to a central STI reference laboratory in Johannesburg, South Africa. RESULTS: Among 769 men enrolled, N. gonorrhoeae was the commonest cause of MUS (674 [87.8%]; 95% confidence interval [CI], 85.2%-89.9%), followed by Chlamydia trachomatis (161 [21.0%]; 95% CI, 18.2%-24.0%). Of 542 cultivable N. gonorrhoeae isolates, all were susceptible to ceftriaxone (modal minimum inhibitory concentration, 0.004 mg/L) and azithromycin (modal minimum inhibitory concentration, 0.128 mg/L). Seroprevalence rates of HIV, syphilis, and HSV-2 were 21.4% (95% CI, 18.5%-24.5%), 2.3%, and 50.1%, respectively. Condom use at last sexual encounter was reported by only 7%, less than 50% had been medically circumcised, and only 66.7% (58 of 87) who self-reported an HIV-positive status were adherent on antiretroviral drugs. CONCLUSIONS: Neisseria gonorrhoeae and C. trachomatis were the predominant causes of MUS. Currently recommended dual ceftriaxone and azithromycin therapy are appropriate for MUS syndromic management; however, surveillance must be maintained to timeously detect emerging and increasing gonococcal resistance. Clinic-based interventions must be intensified in men seeing sexual health care to reduce the community transmission and burden of STI and HIV.

Etiological Surveillance of Genital Ulcer Syndrome in South Africa: 2019 to 2020.

BACKGROUND: Herpes simplex virus (HSV) has been the leading cause of genital ulcer syndrome (GUS) in South Africa for more than a decade, and acyclovir therapy is incorporated into syndromic management guidelines. We conducted surveillance at 3 sentinel sites to define the common sexually transmitted etiologies of GUS and to determine whether current syndromic management is appropriate. Secondary objectives of surveillance were to determine the seroprevalence of coinfections (HIV, syphilis, HSV-2) in persons presenting with GUS. METHODS: Consecutive, consenting adult men and women presenting with visible genital ulceration were enrolled between January 1, 2019, and December 31, 2020. Genital ulcer swab and blood specimens were collected and transported to a central sexually transmitted infection reference laboratory in Johannesburg. RESULTS: Among 190 participants with GUS, HSV-2 was the most frequently detected ulcer pathogen (49.0%; 95% confidence interval [CI], 41.9%-56.1%). The relative prevalence of the second most common ulcer-derived pathogen, Treponema pallidum, was 26.3% (95% CI, 20.5%-33.1%), with 90% of primary syphilis cases having a positive rapid plasma reagin (RPR) titer. Male sex was independently associated with primary syphilis compared with herpetic ulcers, after adjusting for the effect of casual sex partners and other exposures (adjusted odds ratio, 3.53; 95% CI, 1.35-9.21; P = 0.010). The overall HIV prevalence among participants was 41.3% (78 of 189; 95% CI, 34.2%-48.6%). CONCLUSIONS: Herpes simplex virus 2 remains the predominant cause of GUS, justifying the continued use of acyclovir in syndromic guidelines. Adequate supplies of benzathine penicillin G for syphilis treatment are essential at primary health care level, in addition to the provision of syphilis and HIV risk reduction services.

Etiological Surveillance of Vaginal Discharge Syndrome in South Africa: 2019 to 2020.

BACKGROUND: The syndromic management of vaginal discharge syndrome (VDS) is challenging because of the prevalence of mixed infection with sexually transmitted infection (STI) pathogens and non-STI causes, such as bacterial vaginosis and candidiasis (CA). We aimed to determine the relative prevalence of VDS etiologies in women presenting to sentinel primary health care clinics in South Africa. Secondary objectives were to ascertain the predictive value of speculum findings for the presence of STI pathogens and the proportion of women presenting with clinical features of CA who had identifiable yeast on vaginal smear microscopy. METHODS: Consecutive, consenting women with complaints of abnormal vaginal discharge were enrolled between January 1, 2019, and December 31, 2020. Genital discharge swab and blood specimens were collected and transported to a central STI reference laboratory in Johannesburg. RESULTS: A total of 364 women were enrolled at 3 sentinel sites. Bacterial vaginosis was the most common cause of VDS (163 of 361 [45.2%]; 95% confidence interval [CI], 40.1%-50.3%); however, a significant proportion had STI coinfection (71 of 163 [43.6%]; 95% CI, 35.8%-51.5%). The predominant STI etiology was Chlamydia trachomatis (73 [20.2%]; 95% CI, 16.4%-24.7%). An abnormal speculum finding had poor predictive value for STIs, and Gram stain microscopy showed yeast in only 37.2% of vaginal smears from women with CA symptoms. CONCLUSIONS: Bacterial vaginosis is the predominant cause of VDS in South Africa; however, STI coinfection is common. Clinical findings are poorly predictive of STI etiologies or candidiasis; therefore, a rapid and accurate STI point-of-care test would be useful in optimizing VDS management.

Molecular Characterization and Detection of Macrolide and Fluoroquinolone Resistance Determinants in Mycoplasma genitalium in South Africa, 2015 to 2018.

BACKGROUND: Antimicrobial resistance in Mycoplasma genitalium is a global concern, as therapeutic options are limited. We aimed to determine the prevalence of macrolide and fluoroquinolone resistance-associated genetic determinants and strain diversity in M. genitalium-positive surveillance specimens from symptomatic primary health care center attendees in South Africa (2015-2018). A secondary objective was to investigate for an association between M. genitalium strain type, HIV serostatus, and antimicrobial resistance. METHODS: A total of 196 M. genitalium-positive specimens from adult males and females presenting with genital discharge to primary health care centers were tested for resistance-associated mutations in 23S rRNA, parC and gyrA. A dual-locus sequence type (DLST) was assigned to M. genitalium strains based on the detection of single nucleotide polymorphisms in the semiconserved 5' region of the mgpB gene (MG191-sequence typing) as well as the enumeration of short tandem repeats within the lipoprotein gene (MG309 short tandem repeat typing). RESULTS: The A2059G mutation in 23S rRNA, associated with macrolide resistance, was detected in 3 of 182 specimens (1.7%; 95% confidence interval, 0.3-4.7). We did not detect gyrA or parC mutations associated with fluoroquinolone resistance in specimens that could be sequenced. Molecular typing with DLST revealed genetic heterogeneity, with DLST 4-11 being the most common M. genitalium strain type detected. There were no associations between DLST and macrolide resistance or HIV infection. CONCLUSIONS: We found a low prevalence of M. genitalium strains with macrolide resistance-associated mutations over a 4-year surveillance period. Ongoing antimicrobial resistance surveillance is essential for informing genital discharge syndromic treatment guidelines.

Phenotypic and genotypic acyclovir resistance surveillance of genital herpes simplex virus 2 in South Africa.

Acyclovir (ACV) is currently included in the syndromic management algorithm for genital ulcer disease in South Africa, and is the recommended first-line treatment for herpes simplex virus 2 (HSV-2). In the majority of cases, HSV-2 resistance to ACV is due to amino acid changes within the viral thymidine kinase (TK). Phenotypic and genotypic ACV resistance surveillance of HSV-2 derived from genital ulcer disease swab specimens was conducted at a primary healthcare facility in Johannesburg between 2018 and 2020. The objectives of this surveillance were to identify ACV resistance-associated mutations and polymorphisms in HSV-2 TK, and to determine the phenotypic ACV resistance profiles of the corresponding clinical HSV-2 isolates. Genotypic analysis of TK from 67 HSV-2 positive genital ulcer swabs revealed 48 specimens with TK mutations, conferring 113 nucleotide changes. No resistance-associated mutations were found, however, we identified nine known natural polymorphisms (R26H, A27T, S29A, G39E, N78D, L140F, T159I, R220K and R284S) and five amino acid changes of unknown significance (R18C, G39K, M70R, P75S and L263P). Phenotypic susceptibility testing of 52 cultivable HSV-2 isolates revealed all to be susceptible to ACV with IC50 values of <2 µg/ml. The five amino acid changes of unknown significance identified by genotypic testing were not correlated to phenotypic ACV resistance, and therefore grouped as natural polymorphisms. We did not detect any unknown or resistance-associated mutations in specimens that could not be phenotypically tested for ACV resistance. Our findings will supplement existing databases of HSV antiviral resistance-associated mutations and polymorphisms that could be used for genotypic ACV resistance screening.

Development and trial of a dried tube specimen (DTS) proficiency testing panel for dual HIV/syphilis rapid diagnostic tests.

PURPOSE: To develop and trial a dried tube specimen (DTS) panel for proficiency testing of dual HIV/syphilis rapid diagnostic tests (RDTs) at clinical sites. RESULTS: DTS panels were prepared using plasma samples with known HIV and syphilis results, to give varying reactivity for syphilis and HIV test lines on RDTs. Laboratory DTS panels were stable for a minimum 4-week period at ambient temperatures with no inter-reader variability of results. Field testing of panels with Standard Diagnostics Bioline HIV/Syphilis duo showed 100% correlation with laboratory results, and excellent mean pair agreement between the two clinical sites (k = 1.0). With Chembio Dual Path Platform HIV-Syphilis, there were two false negative results for HIV and syphilis, respectively, at one site; and good mean pair agreement between the two sites (k = 0.9). CONCLUSION: It is feasible and practicable to incorporate DTS panels into a field proficiency testing scheme for dual HIV/syphilis RDTs.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Treponema pallidum Macrolide Resistance and Molecular Epidemiology in Southern Africa, 2008 to 2018.

Treponema pallidum macrolide resistance and clinical treatment failure have emerged rapidly within communities where macrolides have been used as convenient, oral therapeutic alternatives to benzathine penicillin G for syphilis or for other clinical indications. Macrolides are not included in the South African syndromic management guidelines for genital ulcer disease; however, in 2015, a 1-g dose of azithromycin was incorporated into treatment algorithms for genital discharge. We determined the prevalence of 23S rRNA macrolide resistance-associated point mutations in 135 T. pallidum-positive surveillance specimens from Botswana, Zimbabwe, and South Africa between 2008 and 2018. Additionally, we investigated the association between macrolide resistance, T. pallidum strain type, and HIV coinfection. A significant increase in the prevalence of the A2058G macrolide resistance-associated point mutation was observed in specimens collected after 2015. There was a high level of molecular heterogeneity among T. pallidum strains circulating in the study communities, with strain type 14d/f being the most predominant in South Africa. Fourteen novel strain types, derived from three new tpr gene restriction fragment length polymorphism patterns and seven new tp0548 gene sequence types, were identified. There was an association between A2058G-associated macrolide resistance and T. pallidum strain types 14d/f and 14d/g but no association between T. pallidum macrolide resistance and HIV coinfection. The majority of T. pallidum strains, as well as strains containing the A2058G mutation, belonged to the SS14-like clade. This is the first study to extensively detail the molecular epidemiology and emergence of macrolide resistance in T. pallidum in southern Africa.

Demographic and Behavioral Risk Factors Associated with Reduced Susceptibility of Neisseria gonorrhoeae to First-Line Antimicrobials in South African Men with Gonococcal Urethral Discharge.

Neisseria gonorrhoeae is the predominant cause of male urethral discharge in South Africa, and escalating prevalence of gonococcal antimicrobial resistance (AMR) is a major health concern both in-country and globally. We analyzed the demographic, behavioral, and clinical characteristics of 685 men presenting with gonococcal urethral discharge to sentinel surveillance clinics over a 3-year period (2017 to 2019) to determine the burden of factors that are known to be associated with N. gonorrhoeae AMR to first-line therapy (defined as group 1 isolates exhibiting resistance or reduced susceptibility to extended-spectrum cephalosporins or azithromycin). Among 685 men with gonococcal urethral discharge, median age was 28 years (interquartile range [IQR], 24 to 32). Only two men (2/632; 0.3%) self-identified as homosexual; however, on further enquiry, another 16 (2%) confirmed that they had sex with men only. Almost 30% practiced oral sex and were at risk for pharyngeal gonococcal infection. In univariate analysis, male circumcision (odds ratio [OR], 0.69; 95% confidence interval [CI], 0.49 to 0.99) and recent sex outside the country (OR, 1.83; 95% CI, 1.21 to 2.76) were significantly associated with having a category 1 N. gonorrhoeae isolate. In a multivariable model, only sex outside South Africa increased the odds of being infected with a decreased susceptible/resistant N. gonorrhoeae isolate (adjusted odds ratio [aOR], 1.64; 95% CI, 1.05 to 2.55). These findings warrant the intensification of N. gonorrhoeae AMR surveillance among recently arrived migrant and overseas traveler populations, as well as the inclusion of extragenital specimens for N. gonorrhoeae AMR surveillance purposes.