School of Laboratory Medicine & Medical Sciences

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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.
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.
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 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.
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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