College of Health Sciences

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The awareness and perceptions of sexually transmitted infections among students attending the University of KwaZulu-Natal.
(2021) Mthembu, Funeka Nomvula.; Abbai, Nathlee Samantha.
A high prevalence of sexually transmitted infections (STIs) have been reported among youth globally and this high prevalence calls for global efforts to improve sexual and reproductive health in this population. The prevalence of STIs in young South African women and men is 0.50% and 0.97% for Syphilis, 6.6% and 3.5% for Gonorrhoea and 14.7% and 6.0% for Chlamydia. Increased evidence on behavioural change is dependent on the comprehensive understanding and perception of one’s own risk. Updated evidence of awareness and perception of STIs in university students is needed to inform relevant sex education programmes. The purpose of this study is to assess awareness and perceptions of STIs in students enrolled at the University of KwaZulu-Natal. Methodology The study used a quantitative research approach. This study was conducted at the University of KwaZulu-Natal in Durban, South Africa. The sample consists of 142 undergraduate and postgraduate registered students between the ages of 18 and 35 years. The study used purposive sampling to obtain the sample. A self-administered survey assessing awareness and perceptions of sexual risk behaviour and STIs was administered. Data was analysed using descriptive statistics. Means and standard deviation were used for continuous variables. Analyses were stratified by gender using Chi-square tests as it was expected that there would be differences in awareness and perceptions regarding risky sexual behaviour and STIs.. Analyses were done with STATA version 15.1. Results The study found that 78% of the students were aware of STIs. There was a significant association regarding awareness of Chlamydia infections, p=0.015. Similar to the other infections, a higher proportion of males were aware of Chlamydia when compared to females (96.4% versus 82.8%, p=0.015). Similar to Chlamydia infections, there was a significant association regarding awareness of Trichomonas across the different genders (p=0.011). According to the analysis, females are exposed to awareness of STIs from a younger age when compared to their male counterparts. Most students (34.5%) had reported that they had received information on STIs from social media and from their school teachers. There was a significant difference in the responses related to same sex practices and STI risk (p=0.047). While some students had socially acceptable perceptions, there were some that were not acceptable including sexual debut (34,5%), concern about being at risk of STI (31%), condom-less sex as an STI risk (21.2%), ease of condom negotiation (41.5%), pregnancy being more risky than STIs (28.8%) and alcohol as an STI risk (28.2%). Conclusion This study had revealed the students have high awareness of STIs. Despite the high awareness, the students still have low risk perceptions especially towards condom use, alcohol consumption and age disparate relationships. These distorted attitudes will subsequently impact on the risk behaviours and further research needs to be conducted in order to fill the gap between awareness and perception. This study highlighted the clear discrepancy between the awareness of STIs and the reported perceptions of students. Future research to evaluate STI messaging and assess actual risk versus perceived risk in this population is recommended.
Characterization of Candida isolates from South African pregnant and non-pregnant women.
(2023) Sukali, Gloria.; Abbai, Nathlee Samantha.; Mabaso, Nonkululeko.
Candida infections are a serious health threat to women. Characterization of Candida isolates has become the gold standard method used in determining antimicrobial susceptibility profiles and resistance mechanisms in vaginal Candida infections. However, there is a lack of data on the antimicrobial susceptibility profiles of South African Candida isolates to amphotericin B. This study investigated antimicrobial resistance profiles and genotypes of Candida isolated from South African pregnant and non-pregnant women. This study was a sub-study of a larger study which involved the diagnosis of vaginitis and vaginosis pathogens in women. For the parent study, n=150 women were recruited from the King Edward VIII hospital in Durban, KwaZulu-Natal, South Africa. The women enrolled in the parent study were; 18 years and older, were willing to provide written informed consent and were willing to provide self-collected vaginal swabs. A total of 72 Candida isolates were obtained by culture. Of the 72 isolates, 31 isolates were obtained from pregnant women and 41 isolates were from non-pregnant women. The isolates were typed using the ABC genotyping method. Susceptibility testing was performed using the broth microdilution assay to measure the minimal inhibitory concentrations (MICs) for clinical isolates to amphotericin B. The Candida albicans ATCC 10231 strain was used as a control strain, and untreated cultures of the respective isolates were used as growth controls. Descriptive characteristics of the study participants according to Candida status were presented as frequencies and percentages. Comparisons by Candida status in the descriptive characteristics were performed using Chi square tests with a 5% significance level. P-values ≤0.05 were considered significant. All analyses were conducted using STATA. The prevalence of Candida in the study population was 48.0% (72/150). All the isolates (100%) were confirmed to be C. albicans as per the germ tube test and quantitative polymerase chain reaction (PCR) using primers and probes specific for C. albicans. All 72 isolates (100%) produced positive PCR results for C. albicans. The majority of the isolates (45/72; 62.5%) yielded a 450bp band which was assigned Genotype A. Of the 72 isolates, 19 isolates (26.4%) yielded a band size of 840bp and was assigned Genotype B. A total of 11.1% (8/72) of the isolates yielded band sizes of 450bp and 840bp which was Genotype C. Of the 72 isolates tested, 79.2% (57/72) of the isolates were resistant to amphotericin B (MIC >1ug/ml) and 20.8% (15/72) of the isolates were susceptible to amphotericin B (MIC ≤ 1 ug/ml). When linking MIC patterns to distribution of genotypes, it was observed that the majority (80%) of the isolates which were assigned genotype A were resistant to amphotericin B. When linking clinical symptoms with the distribution of genotypes, it was observed that the majority (58.8%) of women who reported having current symptoms of abnormal vaginal discharge carried genotype A. Genotype A was most prevalent in women who had been treated for vaginal infections in the past and in women who were HIV positive with prevalence of 64.1% and 60.8%, respectively. genotype A was most prevalent in the non-pregnant women with a prevalence of 63.4%. Genotype A was prevalent (61.3%) amongst the pregnant women and the majority (66.7%) of the HIV negative women had Candida infections which belonged to genotype A. The prevalence of Candida was shown to be high in both pregnant and non-pregnant women in this study. This study also found a high level of resistance to the antifungal amphotericin B. Currently in our local setting, resistance patterns to the commonly used antifungals to treat Candida infections are not being monitored. There is a need for antifungal resistance monitoring in order to reduce the risk of future persistent and untreatable infections.
Diagnostic evaluation of the BD Affirm™ VPIII assay as a point-of-care test for the diagnosis of bacterial vaginosis, trichomoniasis and candidiasis in a population of pregnant women from South Africa.
(2020) Dessai, Fazana.; Sebitloane, Hannah Motshedisi.; Abbai, Nathlee Samantha.
OBJECTIVE: Untreated Sexually Transmitted Infections (STIs) and Bacterial vaginosis (BV) pose a serious health risk to mother and child. Limited data exist on the use of the BD Affirm VPIII assay as a point-of-care test. This study compared the BD Affirm VPIII assay to the BD MaxTM Vaginal assay (reference test) for the detection of BV, Trichomonas vaginalis, and Candida spp. The prevalence of single and co-infections are also reported here. METHODS: The study enrolled 273 pregnant women from King Edward VIII hospital in Durban. Socio-demographic, sexual behaviour and clinical data were collected from all consenting women. The women provided two self-collected vaginal swabs for testing. The swabs were tested using the BD Affirm VPIII assay and the BD MaxTM Vaginal assay. The prevalence of BV, trichomoniasis and candidiasis was calculated as the percentage of women who tested positive for BV, T.vaginalis and Candida infection and 95% confidence intervals (CIs) were calculated for these percentages using the formulas for calculating CIs for proportions. The number of co-infections was calculated using chi-square analysis. The diagnostic accuracy of the BD AffirmTM VPIII assay compared to the BD Max assay was assessed through the calculation of sensitivity, specificity, Negative Predictive Value (NPV) and Positive Predictive Value (PPV) and their respective 95% confidence intervals. RESULTS: In this study population, 85% of the participants were unmarried; however, 84% reported having a regular partner, and 96.3% did not use a condom regularly. The prevalence of Bacterial Vaginosis, Candidiasis and Trichomoniasis was 49.4%, 57.2% and 10.3%, respectively. A large proportion of women (78.8%) in this study did not have a discharge despite being positive for one or more pathogens. The BD AffirmTM VPIII assay showed a moderate sensitivity (79.8%) and specificity (80.3%) for diagnosing BV in all participants. The assay had an excellent specificity for Candida and T. vaginalis of 97.4% and 100.0%; respectively, however, it exhibited poor sensitivities of 52.9% and 42.4%, respectively. CONCLUSION: Our findings show a higher prevalence of Bacterial Vaginosis in antenatal attendees than previously reported, while the prevalence of Candidiasis and Trichomoniasis was in keeping with previous reports. The high number of asymptomatic infections detected is of concern and indicates the need for the re-evaluation of the syndromic management approach, especially in the antenatal population. The BD AffirmTM VPIII assay was found to be unsuitable as a screening test for vaginal infections in pregnancy. The assay performed better as a confirmatory test and may serve useful if used in conjunction with other clinical parameters such as vaginal pH.
Evaluation of the Alere Afinion™ AS100 for measuring the levels of C-Reactive Protein in an aged population.
(2020) Mpofana, Innocentia Baxolile.; Abbai, Nathlee Samantha.; Nyirenda, Makandwe.
Introduction The Alere Afinion™ AS100 analyser is a compact bench-top, multi-assay, point-of-care (POC) analyser that provides valuable near patient testing at the point of care. It utilises the latest technology to measure C-Reactive Protein and other analytes to monitor patients’ disease progression. The main objective of the study was to evaluate the performance of the Alere Afinion™ AS100 analyser compared with a reference laboratory test method, ABX Pentra 400 for the measurement of C-Reactive Protein (CRP). Methods This study was a retrospective analysis in which stored serum samples obtained from a cross-sectional study referred to as Sexual Health, HIV infection and comorbidity with non-communicable diseases among Older Persons (SHIOP) were tested for the quantification of the CRP. The primary aim of SHIOP was to describe sexuality, sexual health and the comorbidity of HIV and sexually transmitted infections with chronic non-communicable diseases in adults aged ≥50 years in a setting of high HIV prevalence. Serum stored at -20 ⁰C from participants that consented to long term storage(n=183) was used to perform this evaluation. The serum samples were used to measure CRP on the Alere Afinion™ AS100 and ABX Pentra 400, respectively. Lin’s correlation coefficient was used to assess the agreement between the two analysers for the measurement of CRP. Risk factors associated with elevated CRP levels were assessed through this study. Results A total of 183 serum samples were tested in the study. The mean age of the study participants was 7.62 years (SD 8.15). Male participants were slightly older than female participants (61 vs 58years, p<0.05). Approximately 14.2% of study participants were above 70 years of age. The study population consisted of 77/183 (42%) Black South Africans and 106/183 (57.9%) Indians. The Alere Afinion™ AS100 was able to correctly classify (165/183) >90% of the CRP results when compared to the ABX Pentra 400. Bland-Altman analysis and linear regression analysis showed an excellent agreement (correlation concordance 0.97) between the two analysers. This study showed that being obese (Odds Ratio [OR]: 1.98, 95% Confidence Interval [CI]: 1.3616, 2.889, p<0.001) and having low HDL levels (OR: 1.64, 95% CI: 1.158, 2.307, p= 0.005) were the only significant risk factors that were associated with elevated CRP levels. Conclusion This study showed that the Alere Afinion™ AS100 can be used for the measurement of CRP in instances where CRP is greater than 5mg/L and this may enhance the process of patient care and management in low resource settings. Keywords: C-Reactive Protein (CRP), Affinion AS100, Sexual Health, HIV infection and comorbidity with non-communicable diseases among Older Persons (SHIOP), ABX Pentra 400.
Genetic diversity of Gardnerella vaginalis in pregnant women diagnosed with intermediate and positive bacterial vaginosis.
(2019) Nzimande, Silondiwe Philiswa.; Abbai, Nathlee Samantha.
Bacterial vaginosis (BV) is the main cause of abnormal vaginal discharge in women of reproductive age. Gardnerella vaginalis, has been detected in almost all women with BV. However, there is limited information on the genetic diversity of G. vaginalis isolated from BV intermediate and positive cases. In this study we investigated the genetic diversity of G. vaginalis strains from South African pregnant women. Vaginal swabs were characterized by the Nugent method. A total of n= 87 samples were included in the genetic analysis, (n=50 BV positive) and (n=37 BV intermediate). The presence of G. vaginalis was detected by PCR using bacterium specific 16S rRNA primers. All PCR positive amplicons were sequenced by the Sanger method and the edited sequence data was used for the phylogenetic analysis using the PHYLIP software. The sialidase A gene was amplified by PCR using specific primers and the copy numbers of sialidase A gene was quantified by droplet digital PCR. To assess the diversity of the sialidase A gene, Sanger sequencing was performed. The 16S rRNA gene from G. vaginalis was amplified in all BV positive and BV intermediate samples. All PCR amplicons were successfully sequenced and the nucleotide BLAST results revealed 100% identify to G. vaginalis. The phylogenetic analysis revealed that there is no diversity in G. vaginalis present in BV positive and intermediate cases. The phylogenetic tree of sialidase A sequences from intermediate and positive BV cases revealed two major clades which showed differences related to sialidase A copy number. Quantification of sialidase A showed that the average number of copies per cell was much higher in the BV positive group compared to the intermediate group. Some of the intermediate cases showed high copy numbers for the virulence gene and clustered with the BV positive cases. In the present study the 16S rRNA sequences of the G. vaginalis from BV intermediate and positive women showed that there is no genetic diversity in G. vaginalis detected in BV positive and intermediate samples. The phylogenetic tree of sialidase A gene sequences of intermediate and positive BV revealed two major clades which showed differences related to sialidase A copy number. This data was previously lacking in our setting, especially in a pregnant population. We further demonstrate for the first time that the genetic information present within the sialidase A gene has a direct influence on BV status.
Genotypic and phenotypic analysis of Neisseria gonorrhoeae for the identification of resistance to various antibiotics in pregnant women attending antenatal care at the King Edward VIII Hospital in Durban, KwaZulu-Natal, South Africa.
(2021) Oree, Glynis.; Abbai, Nathlee Samantha.; Ramsuran, Veron.
Introduction: Worldwide antimicrobial resistance (AMR) is making the clinical management of sexually transmitted infections (STIs) increasingly challenging with a particular emphasis on the emergence of antibiotic resistant strains of Neisseria gonorrhoeae (N. gonorrhoeae). In the current study, the detection and emerging patterns of drug resistant clinical isolates of N. gonorrhoeae to previous and current antibiotics to treat cervicitis pathogens as per syndromic management guidelines was investigated. Methodology: This cross-sectional study was conducted at King Edward VIII Hospital and included 307 antenatal attendees. Two endocervical swabs were collected from each enrolled woman. Each enrolled women also provided data on socio-demographic, behavioural and clinical factors. The first swab was placed in Amies Charcoal media immediately after collection. This swab was used to confirm the identification of N. gonorrhoeae clinical isolates using culture based assays. Culture confirmed isolates were grown in Mueller Hinton Broth and were each adjusted to have a bacterial suspension turbidity of a 0.5 McFarland Standard. These isolates were then subjected to antibiotic susceptibility testing to ceftriaxone, tetracycline, spectinomycin, azithromycin, ciprofloxacin, penicillin G and cefixime using the Etest™ method. The second swab was processed for molecular based assays. Extracted DNA from the second swab was subjected to the TaqMan quantitative Polymerase Chain Reaction (qPCR) assay, an in-house 16S ribosomal RNA (rRNA) PCR and PCR detection of the opacity (opa) gene. DNA extracted from the endocervical swabs and cultured isolates were used for the detection of specific targets (genes/plasmids/mutations) associated with resistance to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, azithromycin and ceftriaxone. All statistical analysis performed in this study was conducted in RS Studio. Results: The prevalence of N. gonorrhoeae was 7.8% (24/307) when detected by the TaqMan qPCR assay and 1.9% (6/307) by culture. When compared to culture, PCR for the opa gene and PCR for the 16S rRNA, the TaqMan qPCR assay was a more superior assay demonstrating a diagnostic accuracy of 94.5%. Susceptibility testing of the six isolates obtained after culturing showed resistant phenotypes for penicillin G (12 - 64 mg/L), tetracycline (1.9 - 32 mg/L) and ciprofloxacin (1.16 - 3 mg/L). Isolates displayed either dual or triple resistance to these 3 antibiotics. However, all isolates showed susceptibility to spectinomycin (>64mg/L), azithromycin (1mg/L), ceftriaxone (>0.125 mg/L) and cefixime (>0.125 mg/L). This study also detected the resistance determinants associated with penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, ceftriaxone and azithromycin from the molecular level using the primary endocervical swab sample. Gene mutations and plasmids associated with resistance to tetracycline (tetM gene carried on a plasmid), penicillin G (penicillinase producing plasmid) and ciprofloxacin (Ser-91 mutation) were detected confirming the results obtained with the susceptibility assays. Resistance mutations associated with the remaining antibiotics were not detected. There was a 100% correlation of cultured isolates and endocervical swabs for detecting the specific AMR determinants conferring resistance to tetracycline, penicillin G, and ciprofloxacin. Conclusion: The TaqMan qPCR assay has the ability to serve as a future diagnostic assay for the detection of N. gonorrhoeae. Despite the lack of resistance to spectinomycin, cephalosporins and azithromycin in our study population, continuous surveillance for emerging patterns of resistance to these antibiotics is still required since they form part of the current South African treatment guidelines. The detection of resistance determinants from the molecular level without the need for culture may prove to be more feasible for future epidemiological investigations focused on tracking antimicrobial susceptibility or resistance patterns in N. gonorrhoeae.
Genotyping of Chlamydia trachomatis detected in South African pregnant women.
(2022) Ramnarain, Caitlin.; Abbai, Nathlee Samantha.; Mabaso, Nonkululeko.
Background Chlamydia trachomatis (C. trachomatis) is a common cause of bacterial sexually transmitted infections (STIs). The genetic characterisation of C. trachomatis serovars reveals significant genetic diversity in this organism. Untreated C. trachomatis infection in pregnant women has been linked to miscarriage, low birth weight babies, premature rupture of membranes, postpartum endometritis, and transmission to the new-born babies. Currently, there is limited data and analyses on the serovars of C. trachomatis circulating in South African pregnant women. In this study, the prevalence of C. trachomatis infection was determined, and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the outer membrane protein gene (omp1) was performed in order to identify the different serovars circulating in the population of pregnant women. Methods In this study, 385 vaginal swab samples were analysed for the presence of C. trachomatis. The swabs were collected from human immunodeficiency virus (HIV)-positive pregnant women at the King Edward VIII hospital in Durban, South Africa. Chlamydia trachomatis was detected using commercial primers and probes (TaqMan Assay, assay ID Ba04646249_s1) which targets the gene encoding the translocated actin-recruiting phosphoprotein of C. trachomatis. Genotyping of C. trachomatis positive samples was performed by an omp1 semi-nested polymerase chain reaction (PCR) assay followed by restriction fragment length polymorphism (RFLP) analysis. The omp1 from C. trachomatis was amplified with gene-specific primers in the first round PCR to yield a 1033 base pair (bp) fragment. Following the first round PCR, 1 μL of the first-round PCR product was used for the semi-nested PCR, amplifying a 978 bp fragment. The 978 bp omp1 amplicons were digested with AluI, DdeI and HinfI, and the banding patterns were compared across the three digests for assignment of serovars. Associations between categorical variables was assessed using chi square (𝑥2) tests. All statistical analysis was conducted using RStudio, version 3.6.3. All p-values were considered significant at < 0.05. Results The actin-recruiting phosphoprotein of C. trachomatis was detected in 47/385 swab samples using the TaqMan Assay. The prevalence of C. trachomatis in the study population was 12.2%. All negative no-template controls did not produce any amplification. Factors associated with testing positive for C. trachomatis included, having a low level of education, being unemployed, being unmarried, not cohabitating with sex partner, early age of first sex, high number of lifetime sex partners, partner having other partners, lack of condom use, lacking symptoms of STIs, lacking treatment for STIs and women having a perceived risk of getting STIs. Serovar E (20/43) - 46.5% was the most frequent serovar in our study population, followed by serovar F (9/43) - 20.9%, G (6/43) - 14.0%, D (5/43) - 11.6%, and the least frequent serovar I (2/43) - 4.7% which was detected in two samples. From the five women that carried serovar D, 20.0% (1/5) reported past treatment of STIs. From the 20 women that carried serovar E, 20.0% (4/20) reported having abnormal vaginal discharge. Of the women with serovar E, 20.0% (4/20) reported past treatment of STIs. From the nine women that carried serovar F, 11.1% (1/9) reported having abnormal vaginal discharge and 22.2% (2/9) reported past treatment of STIs. From the six women that carried serovar G, 16.7% (1/6) reported past treatment of STIs. From the two women that carried serovar I, 50.0% (1/2) reported having abnormal vaginal discharge. Conclusion This study detected an overall 12.2% prevalence rate for C. trachomatis in the pregnant women. The identification of factors associated with infection provided evidence on the importance of antenatal clinics to screen women during their routine check-ups for vaginal infections and provide continuous risk reduction counselling to this vulnerable population. Five different serovars were observed in the studied population with serovars E and F being the most prevalent. The observed diversity of serovars reported within specific populations can be challenging for future vaccine design and development for chlamydia. However, many of the South African serovars detected correlated with serovars found in studies conducted throughout the world. This suggests the possibility of conserved C. trachomatis strains from various geographical areas, which may offer some hope for future vaccine development and diagnostic research aimed at the entire C. trachomatis population.
Genotyping of gardnerella vaginalis from pregnant women in Durban by amplified ribosomal DNA restriction analysis.
(2020) Pillay, Kayla.; Abbai, Nathlee Samantha.; Naicker, Meleshni.
Gardnerella vaginalis is one of the most frequently isolated microorganisms associated with bacterial vaginosis (BV). However, limited information concerning the genetic diversity of G. vaginalis isolated from BV positive and intermediate cases, has been documented. This study investigated the diversity of G. vaginalis in pregnant women, a currently under-researched area in South Africa. The study population included pregnant women recruited from a public hospital in Durban, South Africa. The women provided 2 self-collected vaginal swabs for microscopy and the genotyping assays. The BV status of the women was determined using Nugent scoring. A total sample of n=137 specimens was selected for analysis. The 16S ribosomal ribonucleic acid (rRNA) gene of G. vaginalis was used for the genotyping assays. The 16S rRNA gene polymerase chain reaction products were digested with TaqI to generate genotyping profiles and genotypic subtypes were determined by correlating BamHI and HindIII digestion profiles. Phylogenetic analysis was performed on the 16S rRNA gene sequences. The data analysis was performed in R Statistical Computing software, version 3.6.2. Restriction digestion with TaqI revealed the presence of two different genotypes i.e. GT1 and GT2. Within both BV positive and intermediate sample groups, GT1 was the most prevalent genotype (54%). Overall, 4 subtypes (1, 2B, 2AB and C) were shown to be present in the sample population. The most prevalent subtype was 2B (15/37, 40.5%), followed by subtypes 1 (11/37, 29.7%), 2C (4/37, 10.8%) and 2AB (4/37, 10.8%). The phylogenetic analysis of the 16S rRNA genes showed the presence of 5 clusters. The tree displayed clusters which contained groups of specimens from the same BV group with different genotypes and subtypes present. There were also clusters which contained specimens from across the BV groups carrying the same genotype and subtype. Finally, the study did not find a significant association (p>0.05) between reported symptoms of discharge and genotype harboured. This study provides the first report on the diversity of G. vaginalis in South African pregnant women. Diversity assessments of G. vaginalis with respect to genotypes and subtypes may aid in a greater understanding on the pathogenesis of this microorganism.
Genotyping of trichomonas vaginalis in antenatal women from eThekwini.
(2020) Chetty, Rennisha.; Abbai, Nathlee Samantha.
Trichomonas vaginalis is the causative agent of trichomoniasis. The genetic characterisation of T. vaginalis isolates reveals significant genetic diversity in this organism. Data on the prevalence of different genotypes of T. vaginalis in South African populations is lacking. This study investigated the diversity of T. vaginalis in a pregnant population in South Africa. In this study, 362 pregnant women from the King Edward VIII hospital in Durban, South Africa provided vaginal swabs to be tested for the presence of T. vaginalis. T. vaginalis was detected using the TaqMan assay using commercially available primers and probes specific for this protozoa (Pr04646256_s1). The actin gene from T. vaginalis was amplified with gene specific primers. The actin amplicons were digested with HindII, MseI and RsaI and the banding patterns were compared across the three digests for assignment of genotypes. Phylogenetic analysis was conducted using MEGA. The prevalence of T. vaginalis in the study population was 12.9% (47/362). Genotype G was the most frequent genotype in our study population. Genotypes H and I were detected in one sample each. According to the multiple sequence alignments and phylogenetic analysis, a level of diversity was observed across and within genotypes. Four different single nucleotide changes in the actin gene were detected. Sample TV358 (genotype H) contained a single amino acid substitution from Glutamine to Lysine. Sample TV184 (genotype G) contained a single amino acid substitution from Glutamic acid to Arginine. Sample TV357 (genotype G) contained two amino acid substitutions, Arginine to Leucine and Glycine to Aspartic acid. Three different genotypes were observed in the pregnant population. Diversity was observed across and within genotypes. The observed diversity can be challenging for future vaccine design and development of antigen-based rapid diagnostic tests for trichomoniasis.
Identification of mutations in genes associated with metronidazole resistance and susceptibility in Trichomonas vaginalis.
(2021) Mzenda, Tumelo.; Abbai, Nathlee Samantha.
Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen worldwide. Although, metronidazole cure rates are high for T. vaginalis infection, resistance has been reported. Mutations in the nitroreductase genes of T. vaginalis have also been implicated in metronidazole resistance. Therefore, the aim of this study was to detect mutations in the nitroreductase genes and link the mutations to metronidazole resistance patterns in T. vaginalis isolated from South African pregnant women, a currently under-researched area. Vaginal swabs were collected from 362 pregnant women recruited from the King Edward VIII hospital antenatal clinic in Durban from October 2018 to March 2019. The swabs were cultured in Diamonds TYM medium to obtain pure isolates of T. vaginalis. Pure isolates were sub-cultured and subjected to metronidazole susceptibility assays. The susceptibility assays were conducted under aerobic and anaerobic conditions. DNA was extracted from the pure isolates to perform the polymerase chain reaction (PCR) assays for the detection of the nitroreductase genes and the PFOR gene. The PCR amplicons were sequenced using the Sanger approach in order to identify mutations associated with resistance. A total of 21/362 (5.8%) pregnant women tested positive for T. vaginalis infection. Of the 21 T. vaginalis isolates tested for anaerobic metronidazole susceptibility, 9.5% (2/21) had an MIC of 4 μg/ml (resistant), 38.1% (8/21) had an MIC of 2 μg/ml (intermediate) and 52.4% (11/21) had an MIC ≤ 1 μg/ml (susceptible). For the ntr2 gene, susceptible and resistant isolates carried mutations which were absent in the intermediate isolates. The susceptible isolates carried mostly insertion mutations and the resistant isolate carried substitution mutations. Some deletion mutations were also observed in the ntr2 gene. For the ntr3 gene, two substitution mutations were observed in the intermediate isolate only. In the ntr4 gene, substitution mutations were only observed in the susceptible and resistant isolate. For the ntr5 gene, substitution and insertion mutations were observed in the resistant isolate and not in the intermediate or susceptible isolates. For the ntr6 gene, insertion and deletion mutations were observein the intermediate isolate and a single deletion mutation in the resistant isolate. For the PFOR gene, a single substitution mutation was observed in the intermediate and resistant isolate. In this study, mutations in the ntr2, ntr3, ntr4, ntr5 and ntr6 genes were observed across metronidazole susceptibility profiles. Previous studies have not identified mutations in the ntr2, ntr3 and ntr5 genes, so there is not enough data to support the functions of those genes and their association with metronidazole resistance. Future studies aimed at identifying the function of these mutations are needed.
Investigating the prevalence of vaginitis pathogens in HIV infected and uninfected pregnant women In Durban, South Africa.
(2020) Mzimela, Ntokozo.; Swe Swe-Han, Khine.; Abbai, Nathlee Samantha.
BACKGROUND: Vaginitis in pregnancy significantly contributes to obstetric complications such as preterm labor. Human Immunodeficiency Virus (HIV) infection is thought to increase risk of acquiring vaginitis in pregnancy and vice versa. Currently, there are limited data on the prevalence of vaginitis pathogens in vaginal infections and uninfected pregnant women from Durban. This study compared the prevalence of bacterial vaginosis (BV), Candida spp. and Trichomonas vaginalis (T. vaginalis) across HIV infected and uninfected pregnant women, thereby contributing to the body of knowledge in this area. In addition, this study identified risk factors associated with HIV infection, BV, Candida species (spp). and T. vaginalis in the studied population. METHODS: This study was a sub-study of a larger study which enrolled 273 pregnant women from King Edward VIII hospital in Durban. For the larger study, data obtained from eligible women included; sociodemographic, sexual behavior and clinical data. All participants were tested for HIV as part of standard of care at the clinic and two self-collected swabs were obtained from each woman. Permission to obtain data on their HIV status was requested from each women. One vaginal swab was used to test for the presence of vaginal pathogens and the second swab was stored for future use. The BD Max vaginal assay was used to detect BV, T. vaginalis and Candida spp. from a single swab. The prevalence estimates for HIV, BV, T. vaginalis and Candida spp. were calculated using percentage frequencies. Univariate and multiple logistic regressions were used to assess risk factors for HIV status and vaginitis pathogens. All analysis were conducted using RStudio. RESULTS: Of the n=273 enrolled in the larger study, n=128 women provided permission to access data on their HIV statuses. Therefore, for the current study a sample size of n=128 was available for analysis. Of the n=128 women in this study, 52/128 tested positive for HIV resulting in a HIV prevalence of 40.6%. The most prevalent vaginal infection diagnosed in the study population was Candida spp. (73/ 128, 57%), followed by BV (61/ 128, 47.7%) and T. vaginalis (14/128, 10.9%). BV positivity increased the likelihood for HIV by >2-fold (OR: 2.91, 95% CI: 1.08 – 8.63, p= 0.042), being Candida positive increased risk for HIV by 4-fold (OR: 4.04, 95% CI: 1.52 – 11.68, p= 0.007) and testing positive for T. vaginalis increased risk for HIV by 10-fold (OR: 10.15, 95% CI: 2.38 – 55.81, p= 0.018). Having 2-4 lifetime sex partners increased the likelihood of being HIV infected by 9-fold (OR: 9.78, 95% CI: 2.69 – 47.07, p= 0.001) and having >4 lifetime sex partners increased the likelihood of being HIV infected by 33-fold (OR: 33.88, 95% CI: 5.62 – 274.00, p< 0.001). In the adjusted analysis, not knowing if their partner had other partners, increased the risk of being BV positive by 4-fold (OR: 4.05, 95% CI: 1.58 -11.16, p=0.005) and alcohol consumption increased the risk of being BV positive by 4-fold (OR: 4.44, 95% CI: 1.54 -14.96, p=0.009). Having a current abnormal vaginal discharge and HIV infection were significantly associated with Candida infection (OR: 3.63, 95% CI: 1.45- 9.90, p=0.008), and (OR: 5.19, 95% CI: 1.98 – 15.21, p=0.001), respectively. Similarly, to Candida infection, having a current abnormal vaginal discharge increased the risk of T. vaginalis infection by 5-fold (OR: 5.37, 95% CI: 1.39 – 24.59, p=0.019) and HIV infection increased the risk of T. vaginalis infection by 23-fold (OR: 23.25, 95% CI: 4.52 – 174.17, p=0.001). CONCLUSION: In this study, behavioral factors played a significant role in the risk for contracting infections. It is imperative that women must first perceive themselves to be at risk for contracting infections before they can be motivated to reduce that risk. This can be accomplished by conducting future studies which administer a risk assessment tool to the women so that they can be made aware of actual risk versus perceived risk. Older age group (25-34 years) of pregnant women has also been identified to be at higher risk of HIV transmission, therefore this group should be mainly targeted for risk assessment and prompt HIV testing during pregnancy. Lastly, routine screening of BV, Candida & T. vaginalis is recommended during pregnancy, as many women in this particular study population were found to be co-infected with all three pathogens. The screening is highly recommended largely, due to the fact that vaginitis in pregnancy has been identified as a risk factor for HIV infection.
Neuro-immunological investigation of auto-immune thyroid factors in bipolar disease.
(2017) Naicker, Meleshni.; Naidoo, Strinivasen.; Moodley, Kogie.; Abbai, Nathlee Samantha.
Introduction: The co-incidence of thyroid auto-immunity and neuro-psychiatric disorders is well-documented. However, the prevailing school of thought implicating auto-immune thyroid disease (AITD) in bipolar disorder is neither well-understood nor universally accepted. Thus, the lack of recent plausible data linking these two disorders provides the rationale for the present study and lends novelty to our results. We investigated the association between Hashimoto’s disease (the most common cause of auto-immune hypo-thyroidism) and bipolar disorder through the extra-thyroidal localisation of thyroid-specific proteins, thyroid-stimulating hormone receptor (TSH-R) and thyroglobulin (TG) in major limbic regions of bipolar human brain. The limbic system represents the cerebral control centres for human emotional behaviour responses. Interestingly, the extra-thyroidal localisation of TSH-R and TG has been well-documented. Thyroid-stimulating hormone receptors have been identified in mammalian heart, kidneys, bone, lymphocytes, thymus, pituitary, adipose, skin, hair follicles, fish testes and astrocyte culture. Thyroglobulin has been identified in mammalian skin, hair follicles, thymus and kidney. With particular reference to central nervous system (CNS) localisation, the most relevant to the present study are reports of immuno-reactive TSH-R in human anterior pituitary tissue and cultured astrocytes, as well as in rat brain tissue and cultured astrocytes. However, no previous studies have specifically detected TSH-R and TG in other human cerebral cortical regions. Recently, in a pilot study to the present project, we immuno-localised TSH-R and TG to cortical neurons and cerebral vasculature, respectively, within various human non-limbic brain regions including occipital lobe, parieto-occipitotemporal, primary motor cortex and primary sensory cortex. The present study extends this investigation into the distribution of thyroid-specific proteins, specifically in limbic regions of normal and bipolar human brain at both protein and molecular levels. We postulated that changes in thyroid protein expression in bipolar limbic regions may contribute to mood dysregulation associated with bipolar disorder, thereby implying a significant role for the thyroid system in the pathophysiology of bipolar disorder. Thus, we chose to compare the distribution of thyroid-specific mRNA and proteins in major limbic regions between normal and bipolar human adult brain and then use this data to infer whether any regulation of limbic thyroid protein expression could be related to the pathophysiology of bipolar disorder. Methods: Ethical approval for the present study was granted by the University of KwaZulu-Natal (UKZN), Biomedical Research Ethics Committee (Reference EXPO42/06). Forensic human brain limbic tissue samples (frontal cortices, amygdalae, cingulate gyrii, thalamii, hippocampi and hypothalamii) were obtained from five individuals whom had succumbed to causes unrelated to head injury and who had demonstrated no evidence of brain disease or psychological abnormality. Bipolar brain limbic tissue samples of frontal cortices, amygdalae and cingulate gyri were obtained from five confirmed cases of bipolar disorder from a commercial brain bank. In addition, normal thyroid gland tissue was collected for use as a control tissue. Three experimental techniques were used to fulfil the objectives of this project: Commercial polyclonal antibodies raised in rabbit against human thyroid-specific proteins, TSH-R and TG, were used to immuno-localise TSH-R and TG proteins in the major limbic regions of normal and bipolar human brain by standard immuno-histochemical techniques. Quantification of TSH-R and TG immuno-staining was determined by image analysis digitalisation followed by inter- and intra-group statistical comparisons. (ii) Using specific oligonucleotide primers, TSH-R mRNA in normal and bipolar limbic regions was detected by in-situ reverse-transcriptase polymerase chain reaction (in-situ RT-PCR). This cellular distribution of TSH-R mRNA was then graded according to visual intensity of labelling as well as the extent of cellular distribution. (iii) Solution-type reverse-transcriptase quantitative polymerase chain reaction (RTqPCR) was then used to determine fold-change in TSH-R gene expression in the bipolar limbic group, relative to normal controls, and normalised to the endogenous reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The comparative threshold cycle (CT) method was used to calculate gene expression fold-differences between normal and bipolar groups. Results: There were a number of novel results that were demonstrated in this study: Immuno-reactive TSH-R and TG was demonstrated within the neurons and vasculature, respectively, of normal and bipolar limbic regions. Specifically, we immuno-localised TSH-R to large motor neurons whilst TG was evident in smooth muscle cells of the tunica media and tunica adventitia of limbic vasculature. Other results included the presence of TG in limbic neurons of cingulate gyrus and frontal cortex in both normal and bipolar limbic regions. Thyroglobulin was also evident in bipolar amygdala neurons but not in the normal neurons. Further, TSH-R and TG proteins were not detected in any limbic neuronal support cells examined, such as astrocytes, neuroglia, oligodendrocytes and satellite cells. Image analysis intra-group statistical comparisons indicated a significant reduction of TSH-R and TG proteins in the bipolar limbic groups when compared to matched normal groups. In addition, we obtained inter-group statistical comparisons for TSH-R and TG proteins in all limbic regions examined. Inter-regional comparisons for TSH-R in normal limbic regions showed significant differences in the hypothalamus and thalamus groups when compared to the other four limbic regions but not when compared to each other. No appreciable difference was noted amongst the remaining four normal limbic groups. Inter-regional comparisons in all bipolar limbic regions examined, indicated no appreciable difference between any of these categories. Inter-regional comparisons for TG in normal limbic vasculature demonstrated that when compared to the amygdala, there was a significant difference in the cingulate gyrus, frontal cortex and thalamus. None of the other inter-regional comparisons showed statistical differences in these normal groups. Inter-regional comparisons for TG in bipolar limbic vasculature demonstrated that when compared to the frontal cortex, significant differences were noted in the cingulate gyrus and amygdala. The cellular localisation of TSH-R mRNA by in-situ RT-PCR demonstrated evident labelling for TSH-R mRNA in neurons within all matched normal and bipolar limbic regions. However, semi-quantitative comparisons indicated a down-regulated expression of TSH-R mRNA in all bipolar limbic regions when compared to normal controls. In other normal limbic regions examined, TSH-R mRNA was found exclusive to hypothalamic neurons, whilst the thalamus and hippocampus weredevoid of labelling. Further, TSH-R mRNA was not detected in cerebral vasculature or any neuronal support cells. RTqPCR determinations of TSH-R gene expression between normal and bipolar groups confirmed the down-regulated expression of TSH-R mRNA in the bipolar limbic regions. Bipolar inter-group statistical comparisons demonstrated TSH-R expression to be significantly reduced in the cingulate gyrus when compared to the amygdala. None of the other comparisons showed appreciable statistical differences. Discussion and Conclusion: The main study findings that demonstrate the extra-thyroidal expression of TSH-R and TG in limbic regions of normal and bipolar human brain are unique, and are suggestive of the translocation of thyroid-like proteins via the seemingly-impervious blood-brain barrier (BBB). Interestingly, multiple modes of egress beyond the BBB have been previously described, whereby the integrity of the BBB may be altered during neuro-pathological diseases including HIV-associated dementia, multiple sclerosis, Alzheimer’s disease and bipolar disorder. Alteration of the protective BBB increases its permeability to leukocytes and other circulating compounds and triggers signal transduction mechanisms that facilitate the loss of tight junction proteins that constitute the highly specialised cerebral endothelium. Thus, those reports indicating an altered BBB during neuro-pathological conditions, lends support to our findings of TSH-R and TG proteins in the bipolar brain. Further to these novel discoveries is the demonstration of reduced thyroid protein expression in major limbic regions of the bipolar brain. In particular, our findings of reduced TSH-R expression in limbic regions correlate with previous neuro-imaging reports that describe reduced physiological cortico-limbic tissue volumes and neuro-physiological activity during bipolar disorder. In addition, the presence of TG-like proteins, exclusive to bipolar amygdala neurons, may be associated with other neuro-imaging findings of amygdala neuro-physiological hyperactivity and enhanced emotional sensitivity in bipolar disorder. The importance of the thyroid hormones, T3 and T4, in mammalian CNS during various life stages has been reported. It is therefore reasonable to speculate that our findings of thyroid-related proteins, TSH-R and TG, in normal limbic neurons may also exhibit an alternate neuro-physiological role, specifically related to mood control. Further, the reduced expression of TSH-R/TG will likely result in reduced localised thyroid function in limbic neurons with consequent altered neuronal functioning and this may predispose or exhibit some involvement in the pathophysiology of mood dysregulation often observed in bipolar disorder. Interestingly, in the present study, we report the reduced expression of TSH-R and TG in limbic neurons of bipolar brain. Alternatively, we may attribute symptoms of mood dysregulation due to limbic-derived TSH-R providing potential targets for thyroid auto-immunity during Hashimoto’s disease. We speculate that inhibitory-type auto-antibodies associated with Hashimoto’s disease, may agonise TSH-R expressed in limbic neurons. This abnormal interaction may lead to the inactivation or cell-mediated atrophy of limbic neurons with consequential reduction in the levels of expressed TSH-R. Thus, the loss of limbic neurons or diminished neuronal activity may contribute towards mood dysregulation. The neuro-pathology of diminished neuronal functioning or neuronal atrophy, is suggestive of a neuro-degenerative aetiology in bipolar disorder. This is particularly controversial, since current evidence implicating neuronal structural and functional changes in the pathophysiology of bipolar disorder is limited and does not provide enough support to classify bipolar disorder as a neuro-degenerative type disorder. However, our study suggests that neuronal alterations may be due to changes in thyroidal status in bipolar disorder. Moreover, our correlation of reduced thyroid protein expression levels in bipolar limbic regions with previous neuro-imaging findings of reduced cortico-limbic volumes and activity during bipolar disorder, provides further supporting evidence indicating neuro-degeneration of mood-controlling limbic structures in bipolar disorder.
A novel study to detect and quantify microorganisms associated with bacterial vaginosis from urine samples.
(2019) Naicker, Deshanta.; Abbai, Nathlee Samantha.; Naicker, Meleshni.
Bacterial vaginosis (BV) is the most common vaginal condition found in women of reproductive age. The lack of published data on the detection of BV-associated pathogens from urine, a non-invasive sample, lends novelty to the present study. This study aimed to detect and quantify Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae and Lactobacilllus crispatus from urine, as an alternative non-invasive method to vaginal swabs from pregnant women using droplet digital PCR (ddPCR). A total of n=100 DNA samples (50 paired urine and swabs) were tested. The samples were stratified as BV negative and positive using the BD MAX Vaginal panel assay (Becton Dickinson). Total DNA was extracted from urine (10 ml) and swabs (1 ml) using the PureLink Microbiome Kit (ThermoFisher Scientific). The concentration of extracted DNA for urine and swab samples was determined using the Nanodrop Spectrophotometer (ThermoScientific). Droplet digital PCR was used to determine the absolute quantification of the pathogens using commercially available primer and probe sets. G. vaginalis was observed as the most abundant microorganism, followed by A. vaginae and P. bivia in the BV positive samples. When comparing abundance of microorganisms across urine and swab, it was shown that there was no significant difference across both sample types in the BV negative group. A significant difference in the BV positive group (p=0.004) was only observed for A. vaginae. Good correlation between the urine and swab was observed for G. vaginalis (R=0.63, p<0.0001), L. crispatus (R=0.71, p<0.0001) and P. bivia (R=0.50, p<0.0001). However, a weak correlation across both sample types was observed for A. vaginae (R= 0.21, p=0.001). We observed that urine has the potential to serve as an alternative sample collection method to detect BV-associated bacteria. In addition, the data generated in this study provides a basis for the development of ddPCR as a diagnostic tool for BV.
The prevalence and risk factors for genital mycmoplasmas in Human immunodeficiency virus infected pregnant women from King Edward Vlll hospital.
(2021) Nundlall, Nikita.; Abbai, Nathlee Samantha.; Singh, Ravesh.
Background: Genital mycoplasmas can be found amongst the normal human flora mostly in the respiratory, reproductive and urinary tracts as commensal or pathogenic organisms. These bacteria are sexually transmitted and can be linked to sexually transmitted diseases and other conditions. There are a limited number of studies conducted in South African pregnant women especially from KwaZulu-Natal which have assessed the prevalence, co-infection rates and risk factors for genital mycoplasmas. In this study, the prevalence, co-infection rates and risk factors for Mycoplasma genitalium, M. hominis, Ureaplasma urealyticum and U. parvum were investigated in a cohort of Human immunodeficiency virus (HIV) infected pregnant women. The data generated in this study, therefore adds to the growing body of knowledge on these pathogens. Methods: This study included 264 HIV infected pregnant women attending the King Edward VIII antenatal clinic in eThekwini, South Africa. The women were recruited between October 2020 and April 2021. Each enrolled women provided self-collected vaginal swabs (dry swabs) for detection of the vaginal infections. The consenting women had also completed a questionnaire on socio-demographic, behavioural and clinical factors. DNA was extracted from the vaginal samples using the PureLink Microbiome kit. The individual pathogens were detected using the TaqMan Real-time PCR assays using commercially available primers and probes on a QuantStudio 5 Real-time polymerase chain reaction (PCR) platform. The statistical data analysis was conducted in a freely available Statistical Computing Environment, R software, version 3.6.3 using the RStudio platform. Results: The most prevalent infection in the study population was U. urealyticum, 236/264 (89.4%), followed by M. hominis, 215/264 (81.4%), U. parvum, 203/264 (76.9%) and lastly M. genitalium, 7/264 (2.70%). A total of five women (1.90%) were coinfected with all four microorganisms. Within the group of women who tested positive for Mycoplasma genitalium (M. genitalium), partner having other partners was the only significant behavioral factor in relation with being positive, p=0.031. However, a smaller proportion of positive women reported that their partner had other partners (28.6%) when compared to 57.1% who reported that their partner did not have other partners and 14.3% who did not know if their partner had other partners. Within the group of women who tested positive for Mycoplasma hominis (M. hominis), partner having STI symptoms was a significant clinical factor in relation with being positive, p=0.027. Women that experienced current symptoms of STIs was significantly associated with being positive, p<0.001. In addition, of the M. hominis positive women, a higher proportion, 80.5% tested positive for U. parvum infection compared to 19.5% who tested negative and this was significant, p=0.004. Partner being circumcised was a significant clinical factor in relation with being positive for Ureaplasma urealyticum (U. urealyticum), p=0.028. In addition, partner having symptoms of STIs was a significant clinical factor in relation with being positive, p=0.027. The majority of the positive women were in the third trimester of pregnancy and trimester of pregnancy was significantly associated with being positive for infection, p=0.040. Of the women who tested positive for U. urealyticum, a higher proportion of women also tested positive for M. hominis and this association was significant, p=0.051. Within the group of women who tested positive for Ureaplasma parvum (U. parvum), partners HIV status was significant in relation with being positive, p=0.049. Lifetime number of sex partners was significantly associated with being positive, p=0.012. Partner having other partners was also a significant factor in relation with being positive, p=0.023. Of the U. parvum positive women, a higher proportion of women (85.2%) tested positive for M. hominis. This association was significant, p=0.004. In the adjusted analysis, being employed increased the risk of getting infected with M. hominis p=0.012. In the adjusted analysis, current STI symptoms increased the risk for M. hominis by 95.27 fold, p<0.001. Being U. parvum positive increased the risk for M. hominis by 8.19 fold, p=0.001. Being U. urealyticum positive also increased the risk for M. hominis, p=0.039. In the adjusted analysis, having >4 lifetime sex partners increased the risk of infection with U. parvum by 88.02 fold. This factor was significant, p<0.001. Partner having other partners increased the risk of infection with U. parvum, p=0.008. In the adjusted analysis, being M. hominis positive increased the risk for U. parvum by 4.33 fold, p=0.008. Conclusion: The present study provides information on the risk factors associated with genital mycoplasma infections. The identification of risk factors provides the foundation for the development of prevention interventions. In this study, clinical and behavioral factors were shown to be significantly associated with the risk for infection. Based on this finding, it is evident that a single prevention strategy will not be sufficient, what will be needed is a combination prevention strategy for this vulnerable population.

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