Investigation of carbapenem-resistant Enterobacterales isolates at a tertiary laboratory in Pretoria, South Africa.

This study aimed to investigate phenotypic and genotypic characteristics of carbapenem-resistant Enterobacterales isolates (CRE) at a tertiary laboratory in South Africa. A total of 99 CRE isolates were collected between 2019 and 2021. Carbapenemase production was tested using modified carbapenem inhibitory method. Colistin susceptibility testing was performed using the ComASP™ Colistin broth microdilution method. Conventional PCR assays were conducted for detection of mcr-1 gene and common carbapenemase genes (blaVIM, blaNDM, blaIMP, blaKPC, blaOXA-23, blaOXA-51 and blaOXA-48). Rep-PCR assay was conducted to determine the genetic relatedness of the study isolates. Majority of the isolates were Klebsiella pneumoniae (83%). Carbapenem resistant K. pneumoniae cluster was observed from ICU and surgical ward samples. Colistin resistance was observed in 13% (12/93) of the isolates namely, in 11 K. pneumoniae and one Enterobacter cloacae. The blaOXA-48 (65%) was the most prevalent gene detected followed by blaNDM (25%) and blaVIM (22%). Several K. pneumoniae isolates concomitantly carried multiple carbapenemase genes with one isolate carry up to 5 five genes blaVIM, blaNDM, blaOXA-48, blaOXA-23 and blaOXA-51. The mcr-1 gene was not detected in the isolates. Rep-PCR assay showed that most isolates matched cluster A (50%). The high prevalence of blaOXA-48, blaNDM and emerging colistin resistant isolates is of concern for patient management at this institution and needs close monitoring. Rep-PCR is a valuable tool in establishing infection clusters in resource-limited settings.

Sexually transmitted infections amongst men who have sex with men (MSM) in South Africa.

There is limited data about bacterial STIs in MSM populations in sub-Saharan Africa. Our retrospective analysis used data from the HVTN 702 HIV vaccine clinical trial (October 2016 to July 2021). We evaluated multiple variables. Polymerase chain reaction testing was conducted on urine and rectal samples to detect Neisseria gonorrhoea (NG) and Chlamydia trachomatis (CT) every 6 months. Syphilis serology was conducted at month 0 and thereafter every 12 months. We calculated STI prevalence and the associated 95% confidence intervals until 24 months of follow-up. The trial enrolled 183 participants who identified as male or transgender female, and of homosexual or bisexual orientation. Of these, 173 had STI testing done at month 0, median age was 23 (IQR 20-25) years, with median 20.5 (IQR 17.5-24.8) months follow-up (FU). The clinical trial also enrolled and performed month 0 STI testing on 3389 female participants, median age 23 (IQR 21-27) years, median 24.8 (IQR 18.8-24.8) months FU and 1080 non-MSM males with a median age of 27 (IQR 24-31) years, median 24.8 (IQR 23-24.8) months FU. At month 0, CT prevalence was similar in MSM and females (26.0% vs 23.0%, p = 0.492) but was more prevalent in MSM compared to non-MSM males (26.0% vs 14.3%, p = 0.001). CT was the most prevalent STI among MSM at months 0 and 6 but declined from month 0 to month 6 (26.0% vs 17.1%, p = 0.023). In contrast, NG did not decline in MSM between months 0 and 6 (8.1% vs 7.1%, p = 0.680) nor did syphilis prevalence between months 0 and 12 (5.2% vs 3.8%, p = 0.588). Bacterial STI burden is higher in MSM compared to non-MSM males, and CT is the most prevalent bacterial STI amongst MSM. Preventive STI vaccines, especially against CT, may be helpful to develop.

Sexually transmitted infections and sexual behaviour among men having sex with men from Tshwane, South Africa.

BACKGROUND: Men having sex with men (MSM) are at increased risk of acquiring sexually transmitted infections (STIs), including extra-urethral infections. This study aimed to provide information on the presence of genital and extra-genital non-viral STIs and associated risk factors among MSM in the Tshwane district of South Africa. METHOD: Samples were collected from 200 MSM in the North-western area of Tshwane. After the completion of a questionnaire including demographics and sexual history and an HIV test, three swabs (pharyngeal, rectal, and urethral) were collected and tested for the presence of Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV). RESULTS: Data were collected from 199 participants and 77/199 (38.7%) participants had at least one infection regardless of specimen site. Of these 34 (17.1%) were infected with NG; 36 (18.1%) with CT, 16 (8.1%) with MG and 14 (7.0%) with TV. NG and CT were most frequently detected in rectal specimens. The HIV prevalence in this study was 66.8% (133/199), with 56 (28.1%) of participants both STI and HIV positive. Being between 18 and 20 years, and difficulty having safe sex (more sex partners and more often condomless anal sex) when high/drunk were significantly associated with having an STI. Factors with increased odds of having an STI were being HIV positive, having two or more sexual partners, depending on partner financially, performing and receiving rimming, or receiving anal sex. CONCLUSIONS: This study has highlighted the high burden of STIs in MSM in the local community, especially the prevalence of these pathogens in extra-genital sites.

Case-fatality and sequelae following acute bacterial meningitis in South Africa, 2016 through 2020.

OBJECTIVES: Providing country-specific estimates of case fatality and sequelae from bacterial meningitis (BM) is important to evaluate and monitor progress toward the World Health Organization's roadmap to "defeating meningitis by 2030". METHODS: From 2016-2020, GERMS-SA conducted enhanced surveillance at 26 hospitals across South Africa. Episodes of laboratory-confirmed BM due to Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis were included. Risk factors for in-hospital death and sequelae at hospital discharge among survivors were analyzed. RESULTS: Of 12,717 invasive bacterial infections reported nationally, 39% (4980) were from enhanced surveillance sites, including 4159 pneumococcal, 640 H. influenzae, and 181 meningococcal infections. BM accounted for 32% (1319/4159) of pneumococcal, 21% (136/640) of H. influenzae, and 83% (151/181) of meningococcal invasive diseases. Clinical data were available for 91% (1455/1606) of BM: 26% (376/1455) were aged <5 years, 50% (726/1455) were female, and 62% (723/1171) with known HIV results, were HIV-infected. In-hospital case fatality was 37% (534/1455), and 24% (222/921) of survivors had adverse sequelae. Risk factors for death included altered mental status, HIV infection, and comorbidities. Risk factors for adverse sequelae included altered mental status and antimicrobial nonsusceptibility. CONCLUSION: BM in South Africa has a high case fatality, and adverse sequelae frequently occur among survivors. Those with comorbidities (including HIV) are at the highest risk.

Analysis of the HIV Vaccine Trials Network 702 Phase 2b-3 HIV-1 Vaccine Trial in South Africa Assessing RV144 Antibody and T-Cell Correlates of HIV-1 Acquisition Risk.

BACKGROUND: The ALVAC/gp120 + MF59 vaccines in the HIV Vaccine Trials Network (HVTN) 702 efficacy trial did not prevent human immunodeficiency virus-1 (HIV-1) acquisition. Vaccine-matched immunological endpoints that were correlates of HIV-1 acquisition risk in RV144 were measured in HVTN 702 and evaluated as correlates of HIV-1 acquisition. METHODS: Among 1893 HVTN 702 female vaccinees, 60 HIV-1-seropositive cases and 60 matched seronegative noncases were sampled. HIV-specific CD4+ T-cell and binding antibody responses were measured 2 weeks after fourth and fifth immunizations. Cox proportional hazards models assessed prespecified responses as predictors of HIV-1 acquisition. RESULTS: The HVTN 702 Env-specific CD4+ T-cell response rate was significantly higher than in RV144 (63% vs 40%, P = .03) with significantly lower IgG binding antibody response rate and magnitude to 1086.C V1V2 (67% vs 100%, P < .001; Pmag < .001). Although no significant univariate associations were observed between any T-cell or binding antibody response and HIV-1 acquisition, significant interactions were observed (multiplicity-adjusted P ≤.03). Among vaccinees with high IgG A244 V1V2 binding antibody responses, vaccine-matched CD4+ T-cell endpoints associated with decreased HIV-1 acquisition (estimated hazard ratios = 0.40-0.49 per 1-SD increase in CD4+ T-cell endpoint). CONCLUSIONS: HVTN 702 and RV144 had distinct immunogenicity profiles. However, both identified significant correlations (univariate or interaction) for IgG V1V2 and polyfunctional CD4+ T cells with HIV-1 acquisition. Clinical Trials Registration . NCT02968849.

Correlation Between CT Features of Active Tuberculosis and Residual Metabolic Activity on End-of-Treatment FDG PET/CT in Patients Treated for Pulmonary Tuberculosis.

Patients who complete a standard course of anti-tuberculous treatment (ATT) for pulmonary tuberculosis and are declared cured according to the current standard of care commonly have residual metabolic activity (RMA) in their lungs on fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG PER/CT) imaging. RMA seen in this setting has been shown to be associated with relapse of tuberculosis. The routine clinical use of FDG PET/CT imaging for treatment response assessment in tuberculosis is hindered by cost and availability. CT is a more readily available imaging modality. We sought to determine the association between CT features suggestive of active tuberculosis and RMA on FDG PET/CT obtained in patients who completed a standard course of ATT for pulmonary tuberculosis. We prospectively recruited patients who completed a standard course of ATT and declared cured based on negative sputum culture. All patients had FDG PET/CT within 2 weeks of completing ATT. We determined the presence of RMA on FDG PET images. Among the various lung changes seen on CT, we considered the presence of lung nodule, consolidation, micronodules in tree-in-bud pattern, FDG-avid chest nodes, and pleural effusion as suggestive of active tuberculosis. We determine the association between the presence of RMA on FDG PET and the CT features of active tuberculosis. We include 75 patients with a mean age of 36.09 ± 10.49 years. Forty-one patients (54.67%) had RMA on their FDG PET/CT while 34 patients (45.33%) achieved complete metabolic response to ATT. There was a significant association between four of the five CT features of active disease, p < 0.05 in all cases. Pleural effusion (seen in two patients) was the only CT feature of active disease without a significant association with the presence of RMA. This suggests that CT may be used in lieu of FDG PET/CT for treatment response assessment of pulmonary tuberculosis.

Oral and oropharyngeal high-risk HPV prevalence, HIV status, and risk behaviours in a cohort of South African men who have sex with men.

Data lag is evident when observing studies focussing on human papillomavirus (HPV) prevalence in the head and neck of men who have sex with men (MSM) in Southern Africa. Sexual behaviours other than anal intercourse, and associated factors are similarly underreported. HPV vaccination has not yet commenced for this population group. One hundred and ninety-nine MSM were enrolled in this study. Participants completed a questionnaire followed by a clinical oral examination, and a rinse-and-gargle specimen in Thinprep® vials containing Preservcyt® solution was collected. Detection and genotyping for high-risk HPV were done by an automated system (Abbott® m2000sp). Six percent of MSM in this cohort had high-risk HPV present in the mouth/oropharynx. This cohort averages 29 years of age, more than half were unemployed (53.3%), and 66.8% were human immunodeficiency virus (HIV) seropositive. The most common sexual practice was anal sex (69.4%) followed by oral sex (28.6%), and by rimming (9.6%). A significant association between oral insertive sex and oral/oropharyngeal HPV status was demonstrated (p = 0.0038; phi coefficient = 0.20). An incidental but significant association between rimming and HIV status was found (p = 0.0046; phi coefficient = 0.19), and HIV seropositive participants had higher oral/oropharyngeal HPV presence. The HPV prevalence of 6% reported in this study is in alignment with global reports. The prevalence of oral/oropharyngeal HPV in this MSM cohort was influenced by sexual practices. MSM participants who practiced rimming appear to be at higher risk of HIV acquisition. Given the transmission routes of HPV in this vulnerable population, vaccination must be urgently studied as an intervention for prevention.

Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa.

Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of bla OXA-51 . Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were bla OXA-51 -positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

HIV Incidence Among Pregnant and Nonpregnant Women in the FACTS-001 Trial: Implications for HIV Prevention, Especially PrEP Use.

BACKGROUND: During pregnancy and postpartum period, the sexual behaviors of women and their partners change in ways that may either increase or reduce HIV risks. Pregnant women are a priority population for reducing both horizontal and vertical HIV transmission. SETTING: Nine sites in 4 South African provinces. METHODS: Women aged 18-30 years were randomized to receive pericoital tenofovir 1% gel or placebo gel and required to use reliable modern contraception. We compared HIV incidence in women before, during, and after pregnancy and used multivariate Cox Proportional hazards models to compare HIV incidence by pregnancy status. RESULTS: Rates of pregnancy were 7.1 per 100 woman-years (95% confidence interval [CI]: 6.3 to 8.1) and highest in those who reported oral contraceptive use (25.1 per 100 woman-years; adjusted hazard ratio 22.97 higher than other women; 95% CI: 5.0 to 105.4) or had 2 children. Birth outcomes were similar between trial arms, with 59.8% having full-term live births. No difference was detected in incident HIV during pregnancy compared with nonpregnant women (2.1 versus 4.3%; hazard ratio = 0.56, 95% CI: 0.14 to 2.26). Sexual activity was low in pregnancy and the early postpartum period, as was consistent condom use. CONCLUSIONS: Pregnancy incidence was high despite trial participation being contingent on contraceptive use. We found no evidence that rates of HIV acquisition were elevated in pregnancy when compared with those in nonpregnant women. Risks from reductions in condom use may be offset by reduced sexual activity. Nevertheless, high HIV incidence in both pregnant and nonpregnant women supports consideration of introducing antiretroviral-containing pre-exposure prophylaxis for pregnant and nonpregnant women in high HIV prevalence settings.

Investigating multi-drug resistant Acinetobacter baumannii isolates at a tertiary hospital in Pretoria, South Africa.

PURPOSE: Antimicrobial resistance is now globally recognised amongst the greatest threat to human health. Acinetobacter baumannii's' (A. baumannii) clinical significance has been driven by its ability to obtain and transmit antimicrobial resistance factors. In South Africa, A. baumannii is a leading cause of healthcare associated infections (HAI). In this study, we investigated the genetic determinants of multi-drug resistant A. baumannii (MDRAB) at a teaching hospital in Pretoria, South Africa. METHODS: One hundred non repetitive isolates of A. baumannii were collected for the study. Antimicrobial susceptibility testing was performed using the VITEK2 system. The prevalence of antibiotic resistance associated genes and AdeABC efflux pump system were investigated using conventional PCR. Genetic relatedness of isolates was determined using rep-PCR. RESULTS: Seventy (70) of 100 isolates collected were confirmed multi-drug resistant and were blaOXA51positive. Phenotypically, the isolates where resistant to almost all tested antibiotics. One isolate showed intermediate susceptibility to tigecycline while all were susceptible to colistin. Oxacillinase gene blaOXA-23 was the most detected at 99% and only 1% was positive for blaOXA-40. For Metallo-betalactamases (MBL), blaVIMwas the most frequently detected at 86% and blaSIM-1 at 3% was the least detected. Fifty-six isolates had the required gene combination for an active efflux pump. The most prevalent clone was clone A at 69% of the isolates. Colistin and tigecycline are the most effective against investigated isolates. CONCLUSION: The major genotypic determinant for drug resistances is oxacillinases blaOXA-23. The study reports for the first time, blaOXA-40 and blaSIM-1 detection in A. baumannii in South Africa.

Vaccine Efficacy of ALVAC-HIV and Bivalent Subtype C gp120-MF59 in Adults.

BACKGROUND: A safe, effective vaccine is essential to eradicating human immunodeficiency virus (HIV) infection. A canarypox-protein HIV vaccine regimen (ALVAC-HIV plus AIDSVAX B/E) showed modest efficacy in reducing infection in Thailand. An analogous regimen using HIV-1 subtype C virus showed potent humoral and cellular responses in a phase 1-2a trial in South Africa. Efficacy data and additional safety data were needed for this regimen in a larger population in South Africa. METHODS: In this phase 2b-3 trial, we randomly assigned 5404 adults without HIV-1 infection to receive the vaccine (2704 participants) or placebo (2700 participants). The vaccine regimen consisted of injections of ALVAC-HIV at months 0 and 1, followed by four booster injections of ALVAC-HIV plus bivalent subtype C gp120-MF59 adjuvant at months 3, 6, 12, and 18. The primary efficacy outcome was the occurrence of HIV-1 infection from randomization to 24 months. RESULTS: In January 2020, prespecified criteria for nonefficacy were met at an interim analysis; further vaccinations were subsequently halted. The median age of the trial participants was 24 years; 70% of the participants were women. The incidence of adverse events was similar in the vaccine and placebo groups. During the 24-month follow-up, HIV-1 infection was diagnosed in 138 participants in the vaccine group and in 133 in the placebo group (hazard ratio, 1.02; 95% confidence interval, 0.81 to 1.30; P = 0.84). CONCLUSIONS: The ALVAC-gp120 regimen did not prevent HIV-1 infection among participants in South Africa despite previous evidence of immunogenicity. (HVTN 702 ClinicalTrials.gov number, NCT02968849.).

Benign ethnic neutropenia in a South African population, and its association with HIV acquisition and adverse event reporting in an HIV vaccine clinical trial.

Benign ethnic neutropenia (BEN) is defined as a neutrophil count of <1.5×109 cells/L in healthy individuals and is more common in populations of certain ethnicities, e.g. African or Middle Eastern ethnicity. Neutrophil values are commonly included in eligibility criteria for research participation, but little is known about the relationship between BEN, HIV acquisition, and the occurrence of adverse events during clinical trials. We investigated these relationships using data from an HIV vaccine efficacy trial of healthy adults from 5 South African sites. We analysed data from the double-blind, placebo-controlled, randomized trial HVTN 503, and its follow-on study HVTN 503-S to assess the prevalence of BEN, its association with HIV infection, and adverse event reporting. These data were then compared with a time- and age-matched, non-pregnant cohort from the National Health and Nutrition Examination Survey (NHANES) conducted between 2007-2008 in the United States (US). The 739 South African participants had a median age of 22.0 years (interquartile range = 20-26) and 56% (n = 412) were male. Amongst the US cohort of 845 participants, the median age was 26 (IQR: 21-30) and the majority (54%, 457/745) were also male. BEN was present at enrolment in 7.0% (n = 52) of South African participants (6% in the placebo group versus 8% in the vaccine group); 81% (n = 42) of those with BEN were male. Pretoria North had the highest prevalence of BEN (11.6%, 5/43), while Cape Town had the lowest (0.7%, 1/152). Participants with BEN had a lower median neutrophil count (1.3 vs. 3.2x109 cells/L; p<0.001) and BMI (20.8 vs. 22.3 kg/m2; p<0.001) when compared to those without BEN. A greater proportion of Black South Africans had neutrophil counts <1.5×109 cells/L compared to US non-Hispanic Whites from the NHANES cohort (7% [52/739] vs. 0.6% [3/540]; p<0.001). BEN did not increase the odds for HIV infection (adjusted odds ratio [aOR]: 1.364, 95% confidence interval [95% CI]: 0.625-2.976; p = 0.4351). However, female gender (aOR: 1.947, 95% CI: 1.265-2.996; p = 0.0025) and cannabis use (aOR: 2.192, 95% CI: 1.126-4.266; p = 0.0209) increased the odds of HIV acquisition. The incidence rates of adverse events were similar between participants in the placebo group with BEN, and those without: 12.1 (95% CI: 7.3-20.1) vs. 16.5 (95% CI: 14.6-18.7; p = 0.06) events per 100 person-years (py) were noted in the infections and infestations system organ class, respectively. The vaccine group had an event incidence rate of 19.7 (95% CI: 13.3-29.2) vs. 14.8 (95% CI: 13.0-16.8; p = 0.07) events per 100py in the group with, and without BEN, respectively. BEN is more prevalent in Black South Africans compared to US Non-Hispanic Whites. Our data do not support excluding populations from HIV vaccine trials because of BEN. BEN was not associated with increased risk for HIV infection or Adverse events on a vaccine trial. Predictors of HIV infection risk were females and cannabis use, underlying the continued importance of prevention programmes in focusing on these populations.

18F-FDG PET/CT as a Noninvasive Biomarker for Assessing Adequacy of Treatment and Predicting Relapse in Patients Treated for Pulmonary Tuberculosis.

Microbial culture is the gold standard for determining the effectiveness of tuberculosis treatment. End-of-treatment (EOT) 18F-FDG PET/CT findings are variable among patients with negative microbial culture results after completing a standard regimen of antituberculous treatment (ATT), with some patients having a complete metabolic response to treatment whereas others have residual metabolic activity (RMA). We herein determine the impact of findings on EOT 18F-FDG PET/CT on tuberculosis relapse in patients treated with a standard regimen of ATT for drug-sensitive pulmonary tuberculosis (DS-PTB). Methods: Patients who completed a standard regimen of ATT for DS-PTB and were declared cured based on a negative clinical and bacteriologic examination were prospectively recruited to undergo EOT 18F-FDG PET/CT. Images were assessed for the presence of RMA. Patients were subsequently followed up for 6 mo looking for symptoms of tuberculosis relapse. When new symptoms developed, relapse was confirmed with bacteriologic testing. Repeat 18F-FDG PET/CT was done in patients who relapsed. Results: Fifty-three patients were included (mean age, 37.81 ± 11.29 y), with 62% being male and 75% HIV-infected. RMA was demonstrated in 33 patients (RMA group), whereas 20 patients had a complete metabolic response to ATT (non-RMA group). There was a higher prevalence of lung cavitation in the RMA group (P = 0.035). The groups did not significantly differ in age, sex, presence of HIV infection, body mass index, or hemoglobin level (P > 0.05). On follow-up, no patients in the non-RMA group developed tuberculosis relapse. Three patients in the RMA group developed relapse. All patients who developed tuberculosis relapse had bilateral disease with lung cavitation. Conclusion: A negative EOT 18F-FDG PET/CT result is protective against tuberculosis relapse. Nine percent of patients with RMA after ATT may experience tuberculosis relapse within 6 mo of completing ATT. Bilateral disease with lung cavitation is prevalent among patients with tuberculosis relapse.

Outbreak of multidrug-resistant tuberculosis in South Africa undetected by WHO-endorsed commercial tests: an observational study.

BACKGROUND: Global roll-out of rapid molecular assays is revolutionising the diagnosis of rifampicin resistance, predictive of multidrug-resistance, in tuberculosis. However, 30% of the multidrug-resistant (MDR) strains in an eSwatini study harboured the Ile491Phe mutation in the rpoB gene, which is associated with poor rifampicin-based treatment outcomes but is missed by commercial molecular assays or scored as susceptible by phenotypic drug-susceptibility testing deployed in South Africa. We evaluated the presence of Ile491Phe among South African tuberculosis isolates reported as isoniazid-monoresistant according to current national testing algorithms. METHODS: We screened records of 37 644 Mycobacterium tuberculosis positive cultures from four South African provinces, diagnosed at the National Health Laboratory Service-Dr George Mukhari Tertiary Laboratory, to identify isolates with rifampicin sensitivity and isoniazid resistance according to Xpert MTB/RIF, GenoType MTBDRplus, and BACTEC MGIT 960. Of 1823 isolates that met these criteria, 277 were randomly selected and screened for Ile491Phe with multiplex allele-specific PCR and Sanger sequencing of rpoB. Ile491Phe-positive strains (as well as 17 Ile491Phe-bearing isolates from the eSwatini study) were then tested by Deeplex-MycTB deep sequencing and whole-genome sequencing to evaluate their patterns of extensive resistance, transmission, and evolution. FINDINGS: Ile491Phe was identified in 37 (15%) of 249 samples with valid multiplex allele-specific PCR and sequencing results, thus reclassifying them as MDR. All 37 isolates were additionally identified as genotypically resistant to all first-line drugs by Deeplex-MycTB. Six of the South African isolates harboured four distinct mutations potentially associated with decreased bedaquiline sensitivity. Consistent with Deeplex-MycTB genotypic profiles, whole-genome sequencing revealed concurrent silent spread in South Africa of a MDR tuberculosis strain lineage extending from the eSwatini outbreak and at least another independently emerged Ile491Phe-bearing lineage. Whole-genome sequencing further suggested acquisition of mechanisms compensating for the Ile491Phe fitness cost, and of additional bedaquiline resistance following the introduction of this drug in South Africa. INTERPRETATION: A substantial number of MDR tuberculosis cases harbouring the Ile491Phe mutation in the rpoB gene in South Africa are missed by current diagnostic strategies, resulting in ineffective first-line treatment, continued amplification of drug resistance, and concurrent silent spread in the community. FUNDING: VLIR-UOS, National Research Foundation (South Africa), and INNOVIRIS.

Detection of neonatal unit clusters of Candida parapsilosis fungaemia by microsatellite genotyping: Results from laboratory-based sentinel surveillance, South Africa, 2009-2010.

Neonatal candidaemia is a common, deadly and costly hospital-associated disease. To determine the genetic diversity of Candida parapsilosis causing fungaemia in South African neonatal intensive care units (NICUs). From February 2009 through to August 2010, cases of candidaemia were reported through laboratory-based surveillance. C. parapsilosis isolates from neonatal cases were submitted for identification by internal transcribed spacer (ITS) region sequencing, antifungal susceptibility testing and microsatellite genotyping. Cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Of 1671 cases with a viable Candida isolate, 393 (24%) occurred among neonates. Isolates from 143 neonatal cases were confirmed as C. parapsilosis sensu stricto. Many isolates were resistant to fluconazole (77/143; 54%) and voriconazole (20/143; 14%). Of 79 closely-related genotypes, 18 were represented by ≥2 isolates; 61 genotypes had a single isolate each. Seven clusters, comprised of 82 isolates, were identified at five hospitals in three provinces. Isolates belonging to certain clusters were significantly more likely to be fluconazole resistant: all cluster 7 isolates and the majority of cluster 4 (78%), 5 (89%) and 6 (67%) isolates (P<.001). Candida parapsilosis-associated candidaemia in public-sector NICUs was caused by closely related genotypes and there was molecular evidence of undetected outbreaks as well as intra-hospital transmission.

HIV-Tat immunization induces cross-clade neutralizing antibodies and CD4(+) T cell increases in antiretroviral-treated South African volunteers: a randomized phase II clinical trial.

BACKGROUND: Although combined antiretroviral therapy (cART) has saved millions of lives, it is incapable of full immune reconstitution and virus eradication. The transactivator of transcription (Tat) protein is a key human immunodeficiency virus (HIV) virulence factor required for virus replication and transmission. Tat is expressed and released extracellularly by infected cells also under cART and in this form induces immune dysregulation, and promotes virus reactivation, entry and spreading. Of note, anti-Tat antibodies are rare in natural infection and, when present, correlate with asymptomatic state and reduced disease progression. This suggested that induction of anti-Tat antibodies represents a pathogenesis-driven intervention to block progression and to intensify cART. Indeed Tat-based vaccination was safe, immunogenic and capable of immune restoration in an open-label, randomized phase II clinical trial conducted in 168 cART-treated volunteers in Italy. To assess whether B-clade Tat immunization would be effective also in patients with different genetic background and infecting virus, a phase II trial was conducted in South Africa. METHODS: The ISS T-003 was a 48-week randomised, double-blinded, placebo-controlled trial to evaluate immunogenicity (primary endpoint) and safety (secondary endpoint) of B-clade Tat (30 µg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART (randomised 1:1) with CD4(+) T-cell counts ≥200 cells/µL. Study outcomes also included cross-clade anti-Tat antibodies, neutralization, CD4(+) T-cell counts and therapy compliance. RESULTS: Immunization was safe and well-tolerated and induced durable, high titers anti-Tat B-clade antibodies in 97 % vaccinees. Anti-Tat antibodies were cross-clade (all vaccinees tested) and neutralized Tat-mediated entry of oligomeric B-clade and C-clade envelope in dendritic cells (24 participants tested). Anti-Tat antibody titers correlated positively with neutralization. Tat vaccination increased CD4(+) T-cell numbers (all participants tested), particularly when baseline levels were still low after years of therapy, and this had a positive correlation with HIV neutralization. Finally, in cART non-compliant patients (24 participants), vaccination contained viral load rebound and maintained CD4(+) T-cell numbers over study entry levels as compared to placebo. CONCLUSIONS: The data indicate that Tat vaccination can restore the immune system and induces cross-clade neutralizing anti-Tat antibodies in patients with different genetic backgrounds and infecting viruses, supporting the conduct of phase III studies in South Africa. Trial registration ClinicalTrials.gov NCT01513135, 01/23/2012.

Antigenic distribution of Streptococcus agalactiae isolates from pregnant women at Garankuwa hospital - South Africa.

INTRODUCTION: Streptococcus agalactiae (group B streptococcus; GBS) is globally recognised as one of the leading causes of neonatal sepsis and meningitis. It also causes adverse pregnancy outcomes such as stillbirth and miscarriages. Incidence of invasive disease is increasing in non-pregnant adults with underlying medical conditions (e.g., diabetes mellitus). Epidemiological studies of GBS infections are based on capsular serotyping. Genotyping of the surface anchored protein genes is also becoming an important tool for GBS studies. Currently ten different GBS serotypes have been identified. This study was performed to determine the prevalence of GBS capsular types (CTs) and surface anchored protein genes in isolates from colonized pregnant women attending antenatal clinic, at Dr George Mukhari Academic Hospital, Garankuwa, Pretoria, South Africa. METHODS: The samples were collected over 11 months and cultured on selective media. GBS was identified using different morphological and biochemical tests. Capsular typing was done using latex agglutination test and conventional PCR. Multiplex PCR with specific primers was used to detect the surface anchored protein genes. RESULTS: Of the 413 pregnant women recruited, 128 (30.9%) were colonized with GBS. The capsular polysaccharide (CPS) typing test showed that CPS type III (29.7%) was the most prevalent capsular type followed by CPS type Ia (25.8%), II (15.6%), IV (8.6%), V (10.9%) and Ib (8.6%); 0.7% of the isolates were nontypeable. Multiplex PCR revealed that the surface proteins genes were possessed by all the capsular types: rib (44.5%), bca (24.7%), alp2/3 (17.9%), epsilon (8.6%) and alp4 (4.7%). CONCLUSION: The common capsular types found in this study are Ia, III, and II. The most common protein genes identified were rib and bca, and the distribution of the surface protein genes among the isolates of different capsular types showed similar trends to the distribution reported from previous studies.

Continued Follow-Up of Phambili Phase 2b Randomized HIV-1 Vaccine Trial Participants Supports Increased HIV-1 Acquisition among Vaccinated Men.

BACKGROUND: The Phase 2b double-blinded, randomized Phambili/HVTN 503 trial evaluated safety and efficacy of the MRK Ad5 gag/pol/nef subtype B HIV-1 preventive vaccine vs placebo in sexually active HIV-1 seronegative participants in South Africa. Enrollment and vaccinations stopped and participants were unblinded but continued follow-up when the Step study evaluating the same vaccine in the Americas, Caribbean, and Australia was unblinded for non-efficacy. Final Phambili analyses found more HIV-1 infections amongst vaccine than placebo recipients, impelling the HVTN 503-S recall study. METHODS: HVTN 503-S sought to enroll all 695 HIV-1 uninfected Phambili participants, provide HIV testing, risk reduction counseling, physical examination, risk behavior assessment and treatment assignment recall. After adding HVTN 503-S data, HIV-1 infection hazard ratios (HR vaccine vs. placebo) were estimated by Cox models. RESULTS: Of the 695 eligible, 465 (67%) enrolled with 230 from the vaccine group and 235 from the placebo group. 38% of the 184 Phambili dropouts were enrolled. Enrollment did not differ by treatment group, gender, or baseline HSV-2. With the additional 1286 person years of 503-S follow-up, the estimated HR over Phambili and HVTN 503-S follow-up was 1.52 (95% CI 1.08-2.15, p = 0.02, 82 vaccine/54 placebo infections). The HR was significant for men (HR = 2.75, 95% CI 1.49, 5.06, p = 0.001) but not for women (HR = 1.12, 95% CI 0.73, 1.72, p = 0.62). CONCLUSION: The additional follow-up from HVTN 503-S supported the Phambili finding of increased HIV-1 acquisition among vaccinated men and strengthened the evidence of lack of vaccine effect among women. TRIAL REGISTRATION: clinicaltrials.gov NCT00413725 SA National Health Research Database DOH-27-0207-1539.

Antibiotic resistance of Streptococcus agalactiae isolated from pregnant women in Garankuwa, South Africa.

BACKGROUND: This study was undertaken to determine the susceptibility profile and the mechanism of antibiotic resistance in Group B streptococcus (GBS) isolates detected in vaginal and rectal swabs from pregnant women attending Dr George Mukhari Academic Hospital, a University Teaching Hospital in Pretoria, South Africa. METHODS: The samples were collected over an 11-month period, cultured on selective media (colistin and nalidixic acid agar and Todd-Hewitt broth), and GBS positively identified by using different morphological and biochemical tests. The susceptibility testing was done using the Kirby-Bauer and E test methods according to CLSI guidelines 2012. The D test method was used for the detection of inducible clindamycin resistance. Multiplex PCR with specific primers was used to detect different genes coding for resistance. RESULTS: Out of 413 samples collected, 128 (30.9%) were positive with GBS. The susceptibility testing revealed that 100% of isolates were sensitive to penicillin, ampicillin, vancomycin and high level gentamicin. Erythromycin and clindamycin resistance was 21.1 and 17.2%, respectively, in which 69% had harboured constitutive macrolide, lincosamide and streptogramin B (MLS(B)), 17.4% had inducible MLS(B). The M and L phenotypes were present in 6.8% each. The methylation of target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was also distributed among the isolates. CONCLUSIONS: The study reaffirmed the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it identified the challenges of resistance to macrolides and lincosamides used as alternative drugs for individuals allergic to penicillin. More GBS treatment options for penicillin allergic patients need to be researched on.