Detection of metronidazole resistance in Trichomonas vaginalis using uncultured vaginal swabs.

Trichomonas vaginalis (T. vaginalis) is the most prevalent sexually transmitted infection (STI) globally. Metronidazole is the drug of choice for treating T. vaginalis infections although metronidazole-resistant T. vaginalis has been reported in clinical isolates. The purpose of this study was to determine the presence of mutations in nitroreductase genes associated with metronidazole resistance in vaginal swabs testing positive for T. vaginalis. This study included 385 human immunodeficiency virus (HIV)-positive pregnant women. Vaginal swabs were collected from consenting pregnant women and used for the detection of T. vaginalis using the TaqMan assay. From the vaginal swabs, nitroreductase genes ntr4 and ntr6 containing mutations associated with metronidazole resistance were amplified using a quantitative polymerase chain reaction (PCR) assay. To validate the PCR assay, T. vaginalis cultured isolates with known metronidazole resistance profiles were used as controls in the mutation detection assays. The prevalence of T. vaginalis in the study population was 12.2% (47/385). Mutations associated with resistance to metronidazole were detected in more than 40% of the samples tested, i.e. 21/47 (45%) and 24/47 (51%) for ntr4 and ntr6, respectively. A total of 19 samples (40%) carried mutations for both ntr4 and ntr6 genes associated with metronidazole resistance. The validation assays showed a positive correlation between phenotypic and genotypic resistance profiles. This study found a high prevalence of mutations associated with metronidazole resistance. This is concerning since metronidazole is currently used in the syndromic management of STIs in South Africa. Molecular-based assays for monitoring metronidazole resistance profiles using nitroreductase genes may serve as a feasible method for antimicrobial surveillance studies for T. vaginalis.

Significant Associations between Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Human Immunodeficiency Virus-Infected Pregnant Women.

There is a lack of data on the burden of Chlamydia trachomatis and Neisseria gonorrhoeae among human immunodeficiency virus- (HIV-) infected pregnant women in South Africa. We conducted a cross-sectional study which included 385 HIV-infected pregnant women attending antenatal clinic at the King Edward VIII Hospital in Durban, South Africa. The women provided vaginal swabs which were tested for C. trachomatis and N. gonorrhoeae. The prevalence of the individual STIs was as follows: C. trachomatis (47/385, 12.2%) and N. gonorrhoeae (16/385, 4.1%). Having a circumcised partner, testing positive for N. gonorrhoeae, and perceiving themselves of being at risk for infection were shown to increase the risk for C. trachomatis infection. Without controlling for the other factors, testing positive for N. gonorrhoeae increased the risk for C. trachomatis infection by 10-fold (OR: 10.17, 95% CI: 3.39-29.66, p < 0.001). Similarly, adjusting for the other factors, the risk for C. trachomatis infection in women who tested positive for N. gonorrhoeae was 9-fold (OR: 9.16, 95% CI: 2.19-40.18, p = 0.003). The following factors were associated with the increased risk of N. gonorrhoeae infection: not knowing their partner's HIV status, partner having other partners, and C. trachomatis infection status. Without controlling for the other factors, testing positive for C. trachomatis increased the risk for N. gonorrhoeae infection by 6-fold (OR: 6.52, 95% CI: 2.22-18.49, p < 0.001). Similarly, adjusting for the other factors, the risk for N. gonorrhoeae infection in women who tested positive for C. trachomatis was 6-fold (OR: 6.09, 95% CI: 1.73-22.03, p = 0.005). We found a significant association between C. trachomatis and N. gonorrhoeae in the pregnant women and the risk factors associated with these pathogens. Future studies are urgently required to investigate the impact of C. trachomatis/N. gonorrhoeae coinfections in HIV pregnant women since this data is lacking in our setting. In addition, etiological screening of C. trachomatis and N. gonorrhoeae during antenatal clinic is urgently required to prevent adverse pregnancy and birth outcomes associated with these infections.

Prevalent Herpes Simplex Virus-2 Increases the Risk of Incident Bacterial Vaginosis in Women from South Africa.

Studies have shown that women diagnosed with herpes simplex virus-2 (HSV-2) have a higher risk for bacterial vaginosis (BV) infection. We investigated the presence of HSV-2 infections as a risk factor for incident BV infections in high risk, Human immunodeficiency virus (HIV) uninfected women enrolled in a HIV prevention trial in Durban, South Africa. The Vaginal and Oral Interventions to Control the Epidemic trial was a multicentre, double blinded, randomized controlled trial which was designed to estimate the effectiveness of daily treatment with vaginal tenofovir gel, oral tenofovir disoproxil fumarate and oral Truvada in preventing HIV-1 infection in women. Women provided samples for the diagnosis of HSV-2 and BV. The presence of HSV-2 antibodies was detected using HerpeSelect™ ELISA IgG. Bacterial vaginosis was diagnosed using the Nugent scoring system. To assess the risk of BV incidence, modelled as a time-dependent variable, we used the Andersen-Gill model with robust variance estimation and Efron methods for ties. Overall, 2750 women were enrolled in the VOICE trial at our study sites. Women who had a HSV-2 infection at enrolment were shown to be at increased risk for incident BV infections (adjusted hazard ratio 1.17, 95% CI 1.08, 1.27, p ≤ 0.001). In addition, being of a young age, being unmarried and having a partner that has other partners were significantly associated with subsequent BV infection. Our findings therefore advocate the need for strengthening STI prevention efforts among women in high burden STI settings.

Low prevalence of macrolide resistance in Mycoplasma genitalium infections in a cohort of pregnant women living with human immunodeficiency virus.

OBJECTIVE: Macrolide resistance in Mycoplasma genitalium (M. genitalium) is increasing as a result of the widespread use of azithromycin in the treatment of sexually transmitted infections (STIs). To date, there are few published studies on macrolide resistance patterns in South African pregnant women. This study now contributes to the growing body of knowledge. METHODS: This study included 385 pregnant women living with HIV. Vaginal swabs were collected from consenting pregnant women and used for the detection of M. genitalium using the TaqMan assay. Macrolide resistance-associated mutations in the 23S rRNA gene were determined for all samples that tested positive for M. genitalium using the AllplexTM MG & AziR assay (Seegene) which allows for the simultaneous detection and identification of M. genitalium and six mutations (A2058C, A2058G, A2058T, A2059C, A2059G and A2059T) responsible for azithromycin resistance. The correlation between the TaqMan assay and AllplexTM MG & AziR assay (Seegene) for the detection of M. genitalium was also performed in a subset of 121 samples. RESULTS: Of the 385 samples tested in this study, 14 samples were positive for M. genitalium estimating a prevalence of 3.6%. The same 14 samples also tested positive on the AllplexTM assay indicating a good correlation between the TaqMan Assay and the AllplexTM. Of the 14 positive samples, one sample carried a mutation at position A2059G denoting macrolide resistance in this pathogen. Mutations in the other regions of the 23S rRNA were not detected. All assay controls used in the mutation scanning produced the desired results showing the validity of the assay. CONCLUSION: In this study, macrolide resistance in M. genitalium was detected. Despite the low prevalence of resistance determinants ongoing antimicrobial resistance surveillance is vital considering that azithromycin is used in the syndromic management for the treatment of vaginal discharge syndrome.

Sexually transmitted infections in pregnant women from sub-Saharan Africa.

BACKGROUND: Sexually transmitted infections (STIs) are a major health problem in most countries of the world, particularly in developing countries where the resources and technology to diagnose and treat them are limited. Currently, there is limited data on STIs and risk factors for these infections in pregnant women living with human immunodeficiency virus (HIV), especially in sub-Saharan Africa (SSA). This review provides data on the prevalence and risk factors for STIs in pregnant women living with HIV from SSA. This review also describes the association between STIs and HIV on pregnancy and birth outcomes as well as highlights the importance of laboratory-based diagnosis of STIs. METHOD: An electronic search of online databases was used to find and collect relevant research articles connected to the prevalence, adverse pregnancy and birth outcomes, health complications and risk factors associated with STIs and HIV in pregnant women from SSA. The search was limited to articles published in English. Relevant studies were identified by searching literature from January 2001 to date. The search yielded 4709 results. RESULTS: In SSA, STIs are highly prevalent in pregnant women and are widely known to be linked with an increased risk of poor maternal and neonatal outcomes. These infections are often asymptomatic and highly prevalent in pregnant women. The screening of STIs in pregnant women living with HIV can reduce the risk of mother-to-child transmission (MTCT) and screening and treatment for STIs can also prevent adverse perinatal outcomes. It is important to recognise regional and national STI epidemics in order to promote STI prevention and control interventions considering the test and treat approach as opposed to syndromic management. CONCLUSION: This review highlights the need to use diagnostic screening methods instead of syndromic STI management in SSA. Moreover, more research into effective prevention and treatment measures for STIs in pregnant women is urgently required.

A review on Trichomonas vaginalis infections in women from Africa.

BACKGROUND: Trichomoniasis is the most common sexually transmitted infection (STI) with an estimated annual incidence of 276.4 million cases globally and about 30 million cases in sub-Saharan Africa. Trichomoniasis has been found to be associated with various health complications including pelvic inflammatory disease (PID), significant pregnancy complications, cervical cancer, prostatitis, infertility and the acquisition of human immunodeficiency virus (HIV). AIM: Despite being a highly prevalent infection in the African continent, there is no review article published that solely focusses on Trichomonas vaginalis (T. vaginalis) infections in women from Africa. This review aims to fill this gap in the literature. METHOD: An electronic search of online databases was used to identify and extract relevant research articles related to the epidemiology, health complications and treatment associated with T. vaginalis in women from Africa. RESULTS: Within the African continent, South Africa has reported the highest prevalence rate for this infection. A combination of sociodemographic, behavioural and biological factors has been shown to be associated with infection. Trichomonas vaginalis infection is associated with the acquisition of HIV, cervical cancer and PIDs in various female populations across the continent. Emerging patterns of resistance to metronidazole have been reported in women from South Africa. Currently, there is no effective vaccine against this pathogen despite efforts at vaccine development. CONCLUSION: Based on the high prevalence and health consequences associated with T. vaginalis, there is a need for improved screening programmes that will lead to early diagnosis, detection of asymptomatic infections and effective treatment regimens.

Lack of resistance to macrolides in Mycoplasma genitalium detected in South African pregnant women.

BACKGROUND: Azithromycin regimens have been considered first-line treatment for Mycoplasma genitalium (M. genitalium), a sexually transmitted infection (STI) associated with adverse pregnancy outcomes. However, recent years have seen rapid emergence of macrolide resistance in M. genitalium as a result of widespread administration of azithromycin. Currently, there are limited data on macrolide resistance in pregnant women from KwaZulu-Natal (KZN), South Africa. This study investigated the prevalence of M. genitalium and emerging patterns of macrolide resistance in pregnant women from KZN. METHODS: This was a sub-study of a larger study which involved laboratory-based detection of STIs in pregnant women. In the main study, pregnant women provided urine samples for detection of STIs. For this study, deoxyribose nucleic acid (DNA) extracted from stored urine was used to determine emerging macrolide resistance by amplification of the 23S ribosomal ribonucleic acid (rRNA) gene of M. genitalium by polymerase chain reaction (PCR) and sequencing of amplicons to identify mutations associated with resistance. The Allplex™ MG & AziR assay was used as a confirmatory assay. RESULTS: The prevalence of M. genitalium in pregnant women was 5.9% (13 out of 221). Sequencing of PCR amplicons did not reveal the presence of the A2059G and A2058G mutations associated with macrolide resistance. These findings were confirmed by the Allplex™ MG & AziR assay. CONCLUSION: Despite the lack of resistance to macrolides in this study population, continued antimicrobial resistance surveillance for M. genitalium in pregnant women is important because azithromycin is now part of the South African national STI syndromic management guidelines for vaginal discharge syndrome.

'Mycoplasma hominis does not share common risk factors with other genital pathogens': Findings from a South African pregnant cohort.

BACKGROUND: The role of Mycoplasma hominis (M. hominis) as a genital tract pathogen was still debatable. This study identified the risk factors associated with the prevalence of M. hominis in South African pregnant women. METHODS: This was a cross-sectional analysis of n = 221 prenatal patients attending a Durban hospital during November 2017 to April 2018. M. hominis was detected from urine samples using the quantitative polymerase chain reaction. The population characteristics were described using frequencies stratified by the infection status of M. hominis. In addition, a univariate analysis was used to assess the relationship between each risk factor and infection status. The analysis further considered logistic regression to assess the influence of these risk factors univariately and in the presence of other factors. The coinfection rate between M. hominis and bacterial vaginosis (BV), Trichomonas vaginalis (T. vaginalis), Mycoplasma genitalium (M. genitalium) and Candida species was also determined. All the tests were conducted at 5% level of significance. RESULTS: The prevalence of M. hominis in this study population was 48% (106/221). In the univariate analysis, factors significantly associated with M. hominis positivity included having past abnormal vaginal discharge (p = 0.037), having current abnormal vaginal discharge (p = 0.010) and a borderline significance (p = 0.052), which were noted for previous pre-term delivery. However, none of these factors were sustained in the multivariate analysis. There was a statistically significant association between M. hominis and BV positivity (p < 0.001). Similarly, M. hominis and M. genitalium positivity was significant (p = 0.006). CONCLUSION: This study showed that M. hominis does not share common risk factors with known genital tract pathogens in a population of pregnant women and therefore cannot be considered a genital tract pathogen.

Comparison of methods for the detection of Neisseria gonorrhoeae from South African women attending antenatal care.

The detection of Neisseria gonorrhoeae using culture assays is challenging. This study aims to compare different assays for the detection of N. gonorrhoeae. This cross-sectional study was conducted at King Edward VIII Hospital and included 307 antenatal attendees, each willing to provide two endocervical swabs. The first swab was used for culture identification of N. gonorrhoeae, and the second swab was processed for the detection of the pathogen by the TaqMan quantitative polymerase chain reaction (qPCR) assay, an in-house 16S ribosomal RNA (rRNA) PCR and PCR detection of the opa gene. Culture and the nucleic acid amplification assays were each used as comparator tests in the analysis. Sensitivity and specificity were calculated using RS Studio. The prevalence of N. gonorrhoeae was 7.8%. When compared to the TaqMan assay, the 16S rRNA PCR exhibited the highest sensitivity of 62%, with a substantial level of agreement (kappa level of agreement: 0.60), followed by the opa PCR (38%) with a moderate level of agreement (0.52) and culture exhibiting the lowest sensitivity of 25% with a fair level of agreement (0.38). The diagnostic accuracy of all the assays was >90%. The TaqMan qPCR assay has the ability to serve as a future diagnostic assay for the detection of N. gonorrhoeae.

Prevalent bacterial vaginosis infection - a risk factor for incident sexually transmitted infections in women in Durban, South Africa.

The association between bacterial vaginosis (BV) and incident sexually transmitted infections (STIs) in a cohort of high-risk women from Durban, South Africa was investigated in this study. We undertook a secondary analysis of the Methods for Improving Reproductive Health in Africa trial that assessed effectiveness of the latex diaphragm and lubricant gel on HIV prevention among women. During study visits, urine specimens were collected for testing for Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis The presence of BV was based on vaginal pH and wet mount test assessments. The association between BV and the risk for incident STIs was determined using the Cox proportional hazards model. Prevalence of BV was 31% in a cohort of 435 women tested at baseline. Among these women, BV was significantly associated with incident Trichomonas vaginalis (14.6 per 100 PY, p = 0.03) and Chlamydia trachomatis infections (15.8 per 100 PY, p = 0.04). BV remained a significant predictor for Trichomonas vaginalis infections even after adjusting for potential confounders such as age and marital status (HR: 1.60, 95% CI: 1.00, 2.57, p = 0.04). Our study showed an association between baseline BV infections and incident Trichomonas vaginalis and Chlamydia trachomatis infections. Women with BV infections should be counselled on the use of condoms and the risk of new STIs.

Analysis of hydrocarbon-contaminated groundwater metagenomes as revealed by high-throughput sequencing.

The tendency for chlorinated aliphatics and aromatic hydrocarbons to accumulate in environments such as groundwater and sediments poses a serious environmental threat. In this study, the metabolic capacity of hydrocarbon (aromatics and chlorinated aliphatics)-contaminated groundwater in the KwaZulu-Natal province of South Africa has been elucidated for the first time by analysis of pyrosequencing data. The taxonomic data revealed that the metagenomes were dominated by the phylum Proteobacteria (mainly Betaproteobacteria). In addition, Flavobacteriales, Sphingobacteria, Burkholderiales, and Rhodocyclales were the predominant orders present in the individual metagenomes. These orders included microorganisms (Flavobacteria, Dechloromonas aromatica RCB, and Azoarcus) involved in the degradation of aromatic compounds and various other hydrocarbons that were present in the groundwater. Although the metabolic reconstruction of the metagenome represented composite cell networks, the information obtained was sufficient to address questions regarding the metabolic potential of the microbial communities and to correlate the data to the contamination profile of the groundwater. Genes involved in the degradation of benzene and benzoate, heavy metal-resistance mechanisms appeared to provide a survival strategy used by the microbial communities. Analysis of the pyrosequencing-derived data revealed that the metagenomes represent complex microbial communities that have adapted to the geochemical conditions of the groundwater as evidenced by the presence of key enzymes/genes conferring resistance to specific contaminants. Thus, pyrosequencing analysis of the metagenomes provided insights into the microbial activities in hydrocarbon-contaminated habitats.

Pyrosequence analysis of unamplified and whole genome amplified DNA from hydrocarbon-contaminated groundwater.

Pyrosequence data was used to analyze the composition and metabolic potential of a metagenome from a hydrocarbon-contaminated site. Unamplified and whole genome amplified (WGA) sequence data was compared from this source. According to MG-RAST, an additional 2,742,252 bp of DNA was obtained with the WGA, indicating that WGA has the ability to generate a large amount of DNA from a small amount of starting sample. However, it was observed that WGA introduced a bias with respect to the distribution of the amplified DNA and the types of microbial populations that were accessed from the metagenome. The dominant order in the WGA metagenome was Flavobacteriales, whereas the unamplified metagenome was dominated by Actinomycetales as determined by RDPII and CARMA databases. According to the SEED database, the subsystems shown to be present for the individual metagenomes were associated with the metabolic potential that was expected to be present in the contaminated groundwater, such as the metabolism of aromatic compounds. A higher percentage (4.4) of genes associated with the metabolism of aromatic compounds was identified in the unamplified metagenome when compared to the WGA metagenome (0.66%). This could be attributed to the increased number of hydrocarbon degrading bacteria that had been accessed from this metagenome (Mycobacteria, Nocardia, Brevibacteria, Clavibacter, Rubrobacter, and Rhodoccocus). Therefore, it was possible to relate the taxonomic groups accessed to the contamination profile of the metagenome. By collating the sequencing data obtained pre- and post-amplification, this study provided insight regarding the survival strategies of microbial communities inhabiting contaminated environments.