OpenUCT :: Browsing by Author "Passmore, Jo-Ann"

Browsing by Author "Passmore, Jo-Ann"

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Open Access
A Comparison of Fluorescent Microscopy Methods for the Detection of Chlamydia trachomatis
(2022) Lurie, Micaela; Passmore, Jo-Ann; Bunjun, Rubina
Chlamydia trachomatis (C. trachomatis) is the most common bacterial sexually transmitted pathogen worldwide, especially in low- and middle-income countries, including South Africa. Although frequently asymptomatic, C. trachomatis infections in women cause pronounced genital inflammation. Given that genital inflammation increases women's risk for human immunodeficiency virus (HIV) infection, treating and preventing chlamydia is vital. Thus, there is an urgent need for effective interventions to curb chlamydia infection. Although vaccines are currently in development, none are yet approved for use. New drugs should also be developed and tested given the general rise of antimicrobial resistance. To advance such interventions, expertise in the basic microbiology of C. trachomatis is required. Techniques have indeed advanced over time; however, the standard methods of culture and quantification of C. trachomatis in vitro remain challenging. In South Africa, expertise in C. trachomatis culture and in vitro manipulation is particularly limited. Therefore, I aimed to establish a method to quantify laboratory-adapted stocks of C. trachomatis in in vitro cell culture, to develop research capacity and set the stage for the important future research needed to combat this pathogen. In this study, C. trachomatis was cultured from existing laboratory stocks and used to optimise and compare microscopy-based quantification methods. First, representative C. trachomatis urogenital serovars (E, H) and lymphogranuloma venereum (LGV) serovars (L1 and L2) were propagated in McCoy cells, using an established centrifugation protocol. These stocks were used for all assays comparing three commercially available reagents: (1) Pathfinder's C. trachomatis monoclonal antibody, (2) Invitrogen's C. trachomatis major outer membrane protein (MOMP) antibodies, and (3) Trinity Biotech MicroTrak C. trachomatis culture confirmation kit. In the research setting, fluorescent microscopy techniques are widely used for quantification of C. trachomatis due to their high sensitivity. However, this study showed the Pathfinder C. trachomatis monoclonal antibody kit and Invitrogen's C. trachomatis MOMP Monoclonal Antibody kits had poor sensitivity with high background fluorescent signals. Invitrogen's polyclonal antibody yielded inconsistent results, being either very weakly fluorescent or giving extremely bright signals. Thus, counting bacteria using this polyclonal antibody had limited success and results were not reproducible. MicroTrak's kit, in contrast, allowed for clear visualisation of inclusions and allowed for consistent and successful counting of C. trachomatis bacteria. This study reports inconsistent and/or unreliable results from the kits tested, with two of the three reagents performing poorly. The last, effective kit manufactured by MicroTrak was since discontinued. Thus, molecular methods, particularly qPCRbased methods should be utilised to quantify C. trachomatis in future in vitro cell culture studies.
Open Access
Cellular immune responses to human papillomavirus (HPV) type 16 at the cervix of women with HPV-associated squamous intraepithelial neoplasia
(2005) Milner, Michelle; Passmore, Jo-Ann; Williamson, Anna-Lise
Cervical cancer is the most common cause of cancer-related death in black South African women. Human papillomavirus (HPV) has been found to be a necessary causative agent of cervical cancer and has been reported to be associated with 84% of cervical intraepithelial neoplasia (CIN). HPV type 16 (HPV-16) is the most prevalent HPV type associated CIN and cervical cancer with ±56% of women with cervical disease being infected with HPV 16. Yet studies have shown that 47-85% of CIN regressed, suggesting that perhaps an effective immune response could result in HPV clearance and lesion regression. Since HPV infection does not disseminate and there is no systemic phase of infection, it is hypothesized that local cervical immune responses are important in lesion regression and clearance of HPV infection. There are, however, very few studies of mucosal immune responses to HPV infection. The aim of this study was to determine the type of mucosal immune response elicited by the CD4 and CD8 T cell subsets to HPV infection at the cervix of women diagnosed with varying grades of CIN and to compare these to systemic responses.
Open Access
Characterisation of the HIV inhibitory activity of vaginal lactobacilli isolates from young South African women at high risk of HIV acquisition
(2020) Manhanzva, Monalisa Tatenda; Masson, Lindi; Passmore, Jo-Ann; Woodman, Zenda
Bacterial vaginosis (BV) is an important predisposing factor for the acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs) in South African women. However, the microbial causes and the immunomodulatory effects of BV are not yet fully understood, and effective treatment strategies do not exist. BV is associated with upregulated inflammatory cytokine levels in the female genital tract (FGT), which in turn may increase HIV infection risk by recruiting and activating HIV target cells, reducing epithelial barrier function and directly promoting HIV replication. Lactobacillus species on the other hand are thought to protect against HIV by competitive exclusion, producing virucidal hydrogen peroxide (H2O2), maintaining an acidic pH by producing lactic acid and regulating immune responses in the FGT. This dissertation aimed to characterise the relative HIV inhibitory properties of clinical Lactobacillus isolates, to evaluate the immunoregulatory properties of lactobacilli, and determine the mechanisms underlying these relationships. Vaginal Lactobacillus isolates (n=103), including L. crispatus, L. jensenii, L. johnsonii, L. mucosae, L. plantarum, L. ruminis, L. salivarius and L. vaginalis, were isolated from young South African women who participated in the Women's Initiative in Sexual Health (WISH) study. The production of pro-inflammatory cytokines (IL-6, IL-1α, IL-1β), chemokines (IL-8, IP-10, MIP-3α, MIP-1α, MIP-1β) and regulatory IL-1RA by vaginal epithelial cells in response to lactobacilli in the presence or absence of Gardnerella vaginalis ATCC 14018 and Prevotella bivia ATCC 29303, was measured using Luminex. Growth rates, bacterial sizes, adhesion to cervical (Ca Ski) and vaginal epithelial cells (VK2), culture pH changes and D/L-lactate production by the lactobacilli were also measured in vitro. The properties of vaginal Lactobacillus isolates were also compared to those of commercial probiotics and ATCC reference strains. In order to evaluate differences between lactobacilli isolates that induced low (termed “non-inflammatory”) versus high (termed “inflammatory”) levels of inflammatory cytokine production, the proteomic profiles of 22 inflammatory and 22 non-inflammatory Lactobacillus isolates were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the underlying mechanisms leading to the different inflammatory profiles. Lastly, the influence of Lactobacillus culture supernatants (n=16) on HIV infectivity was evaluated using a Luciferase Reporter Gene Assay in TZM-BL cells. Lactobacilli isolated from women with non-optimal microbiota produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1a, IL-1b, MIP-1a and MIP-1b production significantly elevated. Proteomics analysis showed that 164 proteins were differentially abundant between inflammatory lactobacilli and non-inflammatory lactobacilli. Functional analysis revealed that isolates inducing low levels of inflammatory cytokine production had a significantly higher relative abundance of membrane-associated cellular components, metabolic biological processes and enzymatic molecular functions compared to isolates that induced higher levels of inflammation. A subset of sixteen lactobacilli significantly suppressed IL-6 (adjusted p<0.001) and IL-8 (adjusted p=0.0170) responses to G. vaginalis while L. crispatus isolates suppressed inflammatory cytokines responses to P. bivia. Culture supernatants from the same 16 isolates significantly suppressed HIV infectivity in TZM-BL cells (p=0.0078). Lactobacilli adhesion to VK2 cells correlated negatively with IL-6, IL-8, MIP-1a and IL-1RA production. Lactobacillus beneficial characteristics were highly strainspecific and vaginal isolates out-performed commercial probiotics and ATCC strains. Lactobacillus growth rates, bacterial sizes and adhesion to VK2 cells did not differ significantly between isolates from women with non-optimal microbiota versus those from women with optimal microbiota. These findings show that, while cervicovaginal lactobacilli suppressed overall inflammatory responses to G. vaginalis and P. bivia, isolates from women with non-optimal microbiota were more inflammatory, had lower relative protein abundance and produced less antimicrobial lactic acid than isolates from women with optimal microbiota. Additionally, vaginal Lactobacillus isolates performed better than existing commercial probiotics, suggesting room for improvement of current probiotic formulations available on the South African market to improve BV treatment outcomes and reduce inflammation in the FGT.
Open Access
Characterizing the genotypic and phenotypic diversity of Gardnerella vaginalis from vaginal clinical samples
(2018) Masete, Kopano Valerie; Froissart, Rémy; Passmore, Jo-Ann
Bacterial vaginosis (BV) is a common vaginal condition affecting reproductive-age women, especially in sub-Saharan Africa. With poor treatment outcomes, BV has been associated with pregnancy complications, pelvic inflammatory disease as well as acquisition and transmission of sexually transmitted diseases. While the etiology of BV is not well characterized, it is understood that Gardnerella vaginalis plays a critical role in BV by initiating the formation of the polymicrobial biofilm that characterizes BV and by degrading protective vaginal mucus through the release of sialidase. Recent evidence suggests that the G. vaginalis species is more heterogeneous that initially thought and that not all G. vaginalis may be involved BV. The aim of this study was thus to characterize the genotypic and phenotypic diversity of G. vaginalis isolates. This was achieved in vitro, using 109 G. vaginalis isolates that were previously purified from vaginal samples of 109 French women who were BV-positive (n = 75), BV-intermediate (n = 20) or BV-negative (n = 14), as diagnosed by Nugent scoring. To determine the genotypic diversity of G. vaginalis isolates, 90 isolates were successfully genotyped using their chaperonin-60 (cpn60) sequences, revealing the presence of four phylogenetic clades (subgroups A-D) made up of 13 subgroup A, 17 subgroup B, 58 subgroup C and 2 subgroup D isolates. To determine the phenotypic diversity of G. vaginalis isolates, sialidase activity, biofilm formation and susceptibility to antibiotics used to treat BV were measured. Sialidase activity was not detected in subgroup A and D isolates but was detected, at similar levels, in subgroup B and C isolates. Isolates from all subgroups of G. vaginalis could form similar amounts of biofilm. G. vaginalis isolates (n = 45) were largely resistant to metronidazole (71%), but sensitive to clindamycin (100%), moxifloxacin (91%) and augmentin (100%). The presence of prophages in G. vaginalis isolates was also investigated, revealing the presence of bacteriophage (phage)-like particles that could not be classified into any known phage families, whose phage status remains to be confirmed. In conclusion, G. vaginalis subgroup B and C isolates were the only ones that formed biofilm as well as had detectable sialidase activity suggesting that G. vaginalis subgroups B and C are most likely to be involved in BV. These results contribute to our knowledge of BV and could be useful in future studies that aim to design better treatment strategies for BV.
Open Access
Comparing high-throughput methods to measure antibody dependent cellular cytotoxicity during HIV infection
(2014) Mbodo, Iyaloo; Passmore, Jo-Ann; Gamieldien, Hoyam
The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.
Open Access
Comparison of HIV-1 specific T cell immunity in the female genital tract and blood of HIV-infected women : impact of in vitro T cell expansion on HIV-specific T cell specificity, maturational status and functional complexity
(2010) Bere, Alfred; Passmore, Jo-Ann
This study shows that HIV-specific cervical T cells can be isolated by cytobrushing and in vitro polyclonal expansion is a useful approach to increase the number of T cells available from mucosal sites. Dynal beads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the best yields of cervical T cells while anti-CD3 in the presence of IL-2 best conserved the ex vivo T cell profile. Expanded T cell lines, irrespective of expansion method used, generally maintain their cytokine response profile to HIV anti- gens. This study shows that HIV Gag-specific blood and cervical T cells were largely mono-functional with polyfunctional T cells being detected in women with high blood CD4 count and low plasma viral load. This study confirms that HIV-specific Gag T cell responses detected in the polyclonal expanded female genital tract T cells are associated with those measured in blood during HIV infection.
Open Access
Effect of progestin-based hormonal contraceptives on genital inflammation and Th17 cell activation in adolescents at high risk for HIV infection
(2019) Konstantinus, Iyaloo; Passmore, Jo-Ann; Jaspan, Heather; Masson, Lindi
Background: Adolescent girls and young women (AGYW) are at high risk for HIV infection, particularly in Southern Africa. In addition, some hormonal contraceptives (HC), such as progestin only-injectable contraceptives DMPA and NET-EN, have been associated with significantly increased risk for HIV infection. These HC together with sexual immaturity may increase activation of CD4+ T cells in the female reproductive tract (FRT), which are target cells for HIV infection. NuvaRing, also a long-acting progestin-containing contraceptive albeit topical, has recently been introduced to South Africa, and may offer an improved safety profile over NET-EN and DMPA in terms of HIV risk for young women. Recently, Th17 cells have been found to be disproportionally susceptible to HIV infection compared to the other T helper subsets although the impact of HC use on Th17 cell frequency and activation has not been investigated. Here, the impact of NuvaRing, NET-EN and combined oral contraceptive pills (COCPs) on the vaginal microenvironment of the FRT in AGYW was investigated as this relates to HIV risk, with particular focus on cervical Th17 cells and their related cytokines (Th17- related cytokines). Methods: One hundred and thirty HIV-negative adolescent girls between the age of 15 and 19 years were enrolled into a randomized, controlled crossover study comparing NuvaRing (n=45), NET-EN (n=45), and COCPs (n=40) for 16 weeks (~4 menstrual cycles). At crossover (16 weeks), the AGYW changed to another method for the following 16 weeks: 23 of those who used NuvaRing changed to NET-EN while 8 changed to COCPs; 23 of those who used NET-EN and 24 of those who used COCPs changed to NuvaRing. The protocol included three study visits in total (screening, crossover, study completion visits). Of the 130 adolescents enrolled, 107/130 reached the crossover visit and 92/130 reached the study exit visit. All adolescents were screened for STIs (multiplex PCR for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium), BV (by Nugent scoring), and yeast (visible hyphae on gram stain) at all study visits. Data on relative abundance of vaginal microbial community types (CTs) from ectocervical swabs was available for this study, determined by 16S rRNA sequencing of the V4 region. Several genital samples were collected, including Digene cervical cytobrushes (for flow cytometry of cervical T cells) and menstrual cups at each study visit (for measurement of genital cytokine concentrations). Multiparameter flow cytometry was performed on cytobrushes to determine the frequency and activation status (CD38 and HLA-DR) of Th17 cells (defined by expression of CCR6+CCR10-), total CD3+CD4+ T cells and CD3+CD8+ including Tc17 cells (CD8+ CCR6+). A panel of fifteen Th17-related cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-6, IL-1β, IL-23, IL-33, TNF-α, IL-4, IL-10, IL-25, IL-31, IFN-γ and sCD40L) were measured in genital secretions by Luminex. Results have been presented as an intention to treat (ITT) and per protocol (PP; which accounted for early switching of HC products prior to the crossover visit). Unless otherwise stated, all statistical testing was non-parametric, and P≤0.05 were considered significant. The BenjaminiHochberg method was used to adjust for multiple comparisons. Results: In the FRT of adolescents at baseline (before randomization), Th17 cells (CCR6+CCR10-) were found to be the major CD4+ T cell subset in cytobrushes (median 54.4%, IQR 43.7% - 64.3%) compared to CCR6-CCR10- (median 42.2%, IQR 33.5% - 52.6%), CCR6+CCR10+ (median 1.2%, IQR 0.4% - 2.8%) and CCR6-CCR10+ (median 0.8%, IQR 0.2% - 1.9%). Higher frequencies of Th17 cells expressed CCR5 compared to CCR6-CCR10- CD4+ T cells (median 68.0% vs 56.2% respectively, p< 0.0001). However, Th17 cell frequencies did not correlate with genital tract Th17-related cytokines at baseline. The presence of BV or STIs did not appear to influence either the frequencies or activation status of cervical Th17 cells. Although BV (Nugent 7-10) and having a nonLactobacillus-dominated vaginal microbiome (C1) was associated with increased concentrations of all Th17-related cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-6, IL-1β, IL-23, IL-33, TNF-α, IL-4, IL-10, IL-25, IL-31, IFN-γ and sCD40L) compared to those without BV (Nugent 0-3) or C2/3, while adolescents with any STI had increased concentrations of IL-1β and IL-17A compared to those without an STI. After being randomized on to HC for 16 weeks, cervical cytobrush-derived immune cells were analysed within individuals in each arm (intra-individual) and between individuals in the three contraceptive arms, using both ITT and PP approaches. Although the frequency and activation status of cervical Th17 cells was similar across the three HC arms, adolescents using NuvaRing and NET-EN had significantly increased activation (CD38+HLA-DR+) on Th17 cells compared to their respective baselines (p=0.02 and p=0.03, respectively), which was not evident in those using COCPs. Furthermore, adolescents using NuvaRing had reduced frequencies of Th17 cells compared to baseline (p=0.001), which was not evident in those using NET-EN or COCPs. Although it was hypothesized that NuvaRing would offer some safety advantage over NET-EN in terms of mucosal HIV target cell activation, intra-individual analysis showed a significant increase in the frequency of highly activated cervical Th17 cells in those adolescents who started using the ring. A significant increase in genital tract concentrations of several Th17-related cytokine concentrations (including IL-21, IL-1β, IL-33, TNF-α, IL-4, IFN-γ and sCD40L) was noted in adolescents assigned to NuvaRing after 16 weeks of use, suggesting that the presence of the vaginal ring likely increased genital cytokine responses. Although the frequency and activation of CD8+ T cells was similar across HC arm, intra-individual analysis showed changes in the frequency of activation markers on CD8+ T cells in all HC arms. Moreover, frequencies of Tc17 cells were significantly reduced after 4 months of contraceptive use in each HC arm compared to baseline frequencies. Conclusion: In summary, CCR6+CCR10- Th17 cells were confirmed to be the major CD4+ T cell subset in the FRT of young adolescents. The use of NuvaRing led to decreased frequencies of Th17 cells which were highly activated, and was also associated with an increase in Th17-related cytokines compared to NET-EN and COCPs. All HC altered activation of cervical CD8+ T cells and reduced the frequencies of Tc17 cells. The dramatic alterations observed in cervical immune cells associated with the use of NuvaRing compared to NET-EN and COCPs warrant further investigations.
Open Access
Effects of hormonal contraceptives on the female genital tract microbiota in South African adolescents
(2018) Balle, Christina; Jaspan, Heather; Passmore, Jo-Ann; Lennard, Katie
Background Young women in sub-Saharan Africa are disproportionally affected by HIV and often rely on injectable hormonal contraception (HC) to prevent unintended pregnancies. However, HC might affect HIV-1 risk through changes in the female genital tract (FGT) microbiota. We examined the impact of three different HC methods on the adolescent female genital tract microbiota and related cytokine and HIV target cell levels at the cervical mucosa in a randomized, crossover trial. Study design and methods 131 adolescent females aged 15 to 19 from Cape Town were enrolled into a randomized, crossover study. The participants were randomized into three study arms: 1. progestin-only injectable norethisterone enanthate (Net-En), 2. combined oral contraceptive pills (COCPs) or 3. combined contraceptive vaginal ring (CCVR) for 16 weeks. Participants then switched to one of the other HC options for a final four months. Vaginal samples were collected at baseline, crossover and exit. STI testing and Nugent scoring were performed at all study visits. Vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing, cytokine concentrations were measured by Luminex and CD4+ T cells analysed by flow cytometry. Results Using fuzzy clustering, three major female genital tract bacterial community types were identified. Two of these were dominated by Lactobacillus species (L. crispatus and L. iners, respectively) and the third was comprised of a diverse group of anaerobic bacteria associated with bacterial vaginosis (BV). In an intention-to-treat analysis at crossover, participants randomized to COCP had a significantly less diverse vaginal microbiota compared to participants randomized to either Net-En or CCVR. The same was observed in an according to protocol analysis at crossover. Using differential abundance testing and random forest analyses, we found that species associated with BV and risk of HIV were significantly more abundant in, and predictive of, participants on Net-En (e.g. Prevotella, Sneathia and Dialister) or CCVR (e.g. Prevotella, Mycoplasma and Parvimonas) compared to COCP while L. iners was more common in the COCP group. Cytokine concentrations were positively associated with a diverse vaginal community and with specific bacterial taxa associated with BV and increased risk of HIV including species enriched in participants on Net-En and NuvaRing. In contrast, there were no association of the frequencies of CD4+ T cells expressing CCR5+ with the vaginal community or BV status. There was likewise no significant association with BV or diversity with Th17 cell frequency, yet BVassociated bacteria were more abundant in participants with higher frequencies of Th17 cells. Conclusions Our data generated from a randomized study suggests that COCPs use may exert a positive influence on genital health through an increase in lactobacilli and a decrease in BV-associated bacterial taxa with an accompanying decrease in overall bacterial diversity, vaginal pH and cytokine levels. In contrast, the vaginal microbiota of participants on Net-En and NuvaRing have increased levels of bacteria associated with BV and HIV risk and increased cytokine levels. We did not observe any association of the frequencies of CD4+ T cells expressing CCR5 or Th17-like cells with the vaginal community, BV status or HC use.
Open Access
Evaluation of probiotic and vaginal Lactobacillus species for the treatment of bacterial vaginosis and promotion of vaginal health in South African women
(2018) Happel, Anna-Ursula; Passmore, Jo-Ann; Froissart, Rémy
Background: Bacterial vaginosis (BV) increases women's risk for adverse reproductive outcomes and acquisition of sexually transmitted infections (STIs), including HIV. The etiology of BV is still unclear, and it has been hypothesized that lytic or temperate Lactobacillus bacteriophages may contribute. The current standard of care for BV are antibiotics like metronidazole or clindamycin, although these are not effective long-term, as rates of BV recurrence are high. There is thus an urgent need for durable treatment of BV to be developed. Probiotics administered adjunctively to antibiotics may improve efficacy and durability of BV treatment, but no randomized trial comparing antibiotic treatment to probiotics as an adjunct to antibiotics has been performed in South Africa, despite BV rates >50%. The South African Health Products Regulatory Authority (SAHPRA) regulates drugs and health supplements, into which probiotics are categorized locally. However, no probiotic product has yet been registered with SAHPRA. Further, there have been no recent surveys of the availability of probiotics for vaginal health in South Africa; neither has their suitability to be used in the treatment for BV been evaluated. Aims: The specific aims of this dissertation were (1) to survey the South African probiotic market and evaluate locally available products marketed explicitly for vaginal health in vitro, (2) to determine the efficacy of the most promising local overthe- counter (OTC) probiotic for vaginal health for BV treatment in South African women in a pilot randomized clinical trial that is approved by the regulatory authorities in South Africa, in order to explore the local regulatory landscape for future trials; (3) to screen and thoroughly characterize vaginal Lactobacillus strains isolated from healthy South African women in vitro for the development of a geographically-specific probiotic for vaginal health; and (4) to evaluate the role of bacteriophages in the etiology of BV and Lactobacillus spp. survival in vitro. Approach and results: To review probiotics available on the South African retail market, a cross-sectional survey using two-stage cluster sampling was conducted in Durban and Cape Town. Of the 104 unique probiotic products identified, only four were explicitly for vaginal health, although they contained bacterial species commonly found in the gastro-intestinal tract (GIT) and not lower female genital tract (FGT). The probiotics marketed for vaginal health were analysed for the bacterial contents, concentration per dose, growth kinetics, influence of bacterial growth on culture pH, adhesion to cervical cells, production of L-and D-lactic acid as well as H2O2, inhibitory activity against G. vaginalis and P. bivia and Group B Streptococcus (GBS), their susceptibility to antibiotics, and mucosal safety (using cytokine biomarkers). Batch testing revealed that they mainly contained the bacterial species and dose as claimed by the manufacturers. The contained bacterial isolates had promising probiotic characteristics, but there was some variability in the biological characteristics of isolates from different lot numbers of some of the products. A single-blind, randomised SAHPRA-approved trial enrolling BV positive, STI negative (including discharge-causing STIs; C. trachomatis, T. vaginalis, M. genitalium and A. vaginae) South African women was initiated to compare standard of care (SOC, MetroGelTM V, n=20) to a combination of metronidazole and a commercially-available probiotic (Vagiforte® PLUS Combo Pack, containing L. rhamnosus, L. acidophilus, B. longum and B. bifidum in oral capsules and vaginal spay, treatment duration 15 days) marketed for vaginal health, including treatment of BV and vaginal thrush, in South Africa (n=30, intervention group). The primary endpoint of the pilot study was BV cure one month after treatment completion. BV was assessed by Nugent scoring, vaginal pH was measured, IL-1α concentrations in cervicovaginal fluid were measured by ELISA as a biomarker of genital inflammation, and quantitative PCR (qPCR) was performed to measure the abundance of several vaginal Lactobacillus spp. (including L. crispatus, L. gasseri, L. jensenii, L. vaginalis, L. mucosae and L. iners), in addition to key BV-associated bacteria (including G. vaginalis, P. bivia, A. vaginae, BVAB2 and Megasphaera 1), the bacterial species contained in Vagiforte® (including L. acidophilus, L. rhamnosus, B. bifidum and B. longum) and Candida spp. (including C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis), as a common side effect of metronidazole treatment is vaginal thrush. An interim analysis of the first 24 participants who have completed the trial is included in this dissertation, as the trial is still ongoing. The probiotic was found to be well accepted and no product related adverse events were reported, although women commonly experienced vaginal Candida infections after topical metronidazole use. In the interim analysis, BV cure rates were similar between the SOC and intervention group, as was vaginal pH and the abundances of Lactobacillus spp. and most BV-associated bacteria. Women randomized to the intervention group had higher levels of B. bifidum and B. longum after treatment, which tended to go along with increased levels of Candida spp. and some BV-associated bacteria, while L. crispatus levels were lower in these women. This shows the urgent need to develop a vaginal probiotic containing Lactobacillus strains that are commensal to the FGT, to ensure achieving the desired effect of adjunctive probiotics in BV treatment. Thus, the characteristics of the commercially in South Africa available probiotic strains were compared to clinical vaginal Lactobacillus strains isolated from healthy South African women. A weighted scoring system was developed to select candidate strains for the development of a vaginal probiotic. Towards this aim, 57 Lactobacillus strains were isolated from healthy South African women (including 10 L. crispatus, 9 L. gasseri, 18 L. jensenii, 8 L. vaginalis, and 12 L. mucosae strains) which were distinct to the commercially available probiotic strains, and several isolates exhibited better probiotic characteristics in vitro than the commercially available probiotic bacterial strains (such as ability to lower pH and adherence to cervical cells), although this appeared to be highly strain- and not species specific. Based on weighted scores, two L. crispatus, two L. jensenii, and one L. vaginalis and one L. gasseri strain isolated from BV and STI negative South African women were selected for the development of a local probiotic for vaginal health. The presence of bacteriophages that target vaginal Lactobacillus spp. in cervicovaginal secretions of women with and without BV was evaluated using serial bacteriophage transfer and plaque assays. No lytic bacteriophages that targeted vaginal Lactobacillus spp. (including L. crispatus, L. gasseri, L. jensenii, L. vaginalis and L. mucosae) were isolated from FGT secretions, although CRISPR loci were common in publically available full Lactobacillus genome sequences. However, temperate bacteriophages were induced from the majority (71.8%) of the clinical Lactobacillus strains and 61.1% of the probiotic Lactobacillus strains screened using Mitomycin C, which was confirmed by transmission electron microscopy. Based on their morphology, these Lactobacillus bacteriophages belonged to the families of Sipho-, Myo- and Podoviridae. Conclusions: There are very few probiotics for vaginal health on the South African market, and the development of a probiotic containing commensals of the lower FGT should urgently be considered. Lytic bacteriophages targeting Lactobacillus spp. were not found in this study, although temperate bacteriophages were common and could influence Lactobacillus survival in vivo. Screening women with vaginal discharge for the SAHPRA-acknowledged pilot probiotic trial of Vagiforte PLUS® confirmed a high burden of STIs in Cape Town, South Africa, and that the symptom vaginal discharge is a very poor predictor for BV. The pilot trial showed that large doubleblind, randomized, placebo-controlled trials with adequate screening and enrolment algorithms and sample sizes, using a product containing vaginal Lactobacillus spp., are needed to determine the efficacy of adjunctive probiotics on BV cure and recurrence in South African women. Finally, while the products currently being marketed for vaginal health in South Africa and worldwide mostly do not contain Lactobacillus spp. commonly found in the lower FGT, several promising candidates from the FGTs of healthy, young, HIV- and BV- South African women were isolated and characterized that may prove more efficacious in treating BV. These have the potential to make a big impact on reproductive outcomes and HIV risk in young South African women.
Open Access
Evolution of HIV-1 subtype C immune responses during acute and chronic HIV infection
(2011) Gamieldien, Hoyam; Passmore, Jo-Ann
The aim of this study was to compare the magnitude and breadth of HIV-specific T cell responses to HIV Gag and Nef mounted during acute HIV infection with those that emerged during chronic infection and to investigate the association of these responses with subsequent HIV disease progression (CD4 counts and plasma viral loads).
Open Access
HIV pathogenesis in the female genital tract during chronic HIV infection : the impact of inflammation, T cell memory differentiation status and homeostatic cytokines on mucosal T cell immunity
(2010) Gumbi, Pamela; Passmore, Jo-Ann
The female genital tract serves as the major portal of entry for human immunodeficiency virus (HIV). Local immune factors unique to the mucosal micro-environment such as the genital tract cytokine milieu or the activation/differentiation status of T cells may play a significant role in heterosexual transmission of HIV and subsequent pathogenesis. Elucidation of the mechanisms underlying the persistent recruitment, activation and differentiation of mucosal T cells will give crucial insight into potential therapeutic targets to restore effective local immunity.
Open Access
Impact of HIV infection on the frequency and phenotype of Th17 cells in the female genital tract
(2010) Salkinder, Amy Leia; Passmore, Jo-Ann
T helper (Th) 17 cells have recently been implicated in regulating gut mucosal immunity during HIV infection by sustaining gut mucosal barrier integrity, although they do not respond to HIV directly. Depletion of Th17 cells from the gut mucosa during HIV infection has been suggested to contribute to elevated microbial translocation and immune activation. The role of Th17 cells in regulating genital mucosal immunity during HIV infection is less well described. The aims of this study were (1) to compare the frequency and phenotype of Th17 cells in the female genital tract and blood in uninfected compared to HIV-infected women; and (2) to investigate the role of inflammatory/regulatory cytokines and bacterial burden in modulating Th17 cell frequencies in genital secretions and plasma.
Open Access
Impact of immune activation and inflammation on the susceptibility to HIV infection and disease progression in HIV serodiscordant and seroconcordant couples
(2014) Jaumdally, Shameem Zaer; Passmore, Jo-Ann; Gumbi, Pamela; Little, Francesca
The biological correlates of protection against HIV infection remain poorly characterized, hindering the development of an effective prevention strategy. Studies of individuals who resist HIV infection or progress more slowly after being infected are important for the conception of appropriate approaches for mimicking the effective responses against HIV infection or progression. The role of immune activation and chronic inflammation in the modulation of HIV acquisition risk and/or rate of HIV disease progression has been proposed as one of the most important mechanisms determining risk and pathogenesis but is not fully understood. A state of immune quiescence has been associated with protection against HIV infection and slower disease progression. To explore potential risk factors associated with HIV transmission and HIV disease progression, this dissertation investigates the relationship between clinical and biological biomarkers and resistance to HIV infection or disease progression (including viral load, CD4 counts, cellular activation, soluble inflammatory and regulatory cytokines, and HIV co-receptor expression) in stable long-term HIV seroconcordant and serodiscordant couples.
Open Access
The impact of sexually transmitted infections and inflammation in the female genital tract and blood on susceptibility to HIV-1 infection and disease progression
(2011) Masson, Lindi; Passmore, Jo-Ann; Williamson, Carolyn; Little, Francesca
Background. In sub-Saharan Africa, which has the highest prevalence of HIV-1 worldwide, most newHIV-1 infections occur by sexual transmission to women. Recent studies in non-human primates have demonstrated that pro-inflammatory cytokine production in the genital tract is necessary for immune cell recruitment and establishment of simian immunodeficiency virus (SIV) infection following vaginal inoculation. The aims of this study were to evaluate the relationships between inflammation in the female genital tract and (i) susceptibility to HIV-1 infection and (ii) subsequent disease progression in women who became infected. Additionally, genital inflammation was investigated as a mechanism for breakthrough HIV-1 infections in women who became infected even though they were using 1% tenofovir (TFV) microbicide. In the systemic compartment, the level of T cell activation and soluble markers of immune activation during HIV-1 infection are associated with disease outcome. Therefore, the relationships between plasma cytokine concentrations during early HIV-1 infection and disease progression were evaluated Methods. The participants of this study included 230 HIV-uninfected women from the CAPRISA 002cohort who were followed longitudinally for HIV-1 infection, 49 women who were enrolled during acuteHIV-1 infection and followed until 12 months post-infection and 166 HIV-uninfected women who were enrolled in the CAPRISA 004 1% TFV microbicide trial (62 of whom later became HIV-1-infected).Cytokine concentrations were measured in cervicovaginal lavage (CVL) and plasma samples from these women using Luminex and ELISA.
Open Access
Isolation and Characterization of Bacteriophages Targeting Uropathogenic E. coli
(2019) Magwai, Thandi; Passmore, Jo-Ann; Froissart, Remy; Gamieldien, Hoyam
Urinary tract infections (UTI)are one of the most common human bacterial infections, caused by uropathogenic E. coli (UPEC). Conventionally, UTI’s are treated with antibiotics, such as sulpha-mathoxazole/trimethoprim and ampicillin. However, a significant proportion of UPEC strains have become resistant to these antibiotics, resulting in a significant burden on the health care system worldwide. Bacteriophages (phages) that target E. coli strains could provide an alternative treatment for UTIs, particularly those resistant to antibiotics. This study aimed to screen a variety of environmental samples for phages that target a UPEC strains and characterize these phenotypically and genotypically, towards the development of a candidate phage preparation for treatment for antibiotic-resistant UPEC. Environmental samples (including faeces cattle, sheep, horse, goat faeces, river water, raw cow milk and mud) were collected from various sources around the City of Cape Town and screened for phages using an E. coli laboratory strain; K-12 MG1655. Lytic phages against E. coli MG1655 were purified and host-range testing was conducted against a panel of 11 UPEC strains form the E. coli reference collection (ECOR). These lytic phages were characterized phenotypically using transmission electron microscopy (TEM) using negative staining and genotypically using restriction enzyme HaeIII. In total, 41 phages that were lytic against MG1655 were isolated from six different environmental samples. Of these, 31/41 (76%) were lytic against 7/11 (64%) UPEC strains tested. Four phages (16-3, 16-4, 16-5 and 16-7; all isolated from raw milk) had the broadest host range of all the phages screened, being lyticagainst4/11(36%) UPEC strains. UPEC strain ECOR-40 was the most susceptible of the E. coli strains tested, being susceptible to 18/31 (58%) of the lytic phages isolated. In contrast, four UPEC strains (including ECOR-14, -60, -62 and -64) were not susceptible to any of the phages isolated. Ninety percent (28/31) of the isolated phages were structurally similar to four known phage families; including Myoviridae, Siphoviridae, Inoviridae and Rudiviridae. The remaining 10% (3/31) had structures that did not resemble any ofthe known phage families. DNA was x isolated from all 31 phage isolates to screen for similarities in restriction patterns of isolates. Of these, RFLP banding was clear for 5/31 samples; which showed that 5/5 phages were unique based on their banding patterns. In conclusion, this study demonstrated the existence of several unique E. coli phages in nature and their ability to target several of the UPEC strains known to cause UTI. Although time did not allow for sequencing of the full genomes of those isolates with unique characteristics in this study, this should be a priority for this research going forward.
Open Access
More men than women make mucosal IgA antibodies to Human papillomavirus type 16 (HPV-16) and HPV-18: a study of oral HPV and oral HPV antibodies in a normal healthy population
(BioMed Central Ltd, 2006) Marais, Dianne; Sampson, Candice; Jeftha, Anthea; Dhaya, Dherendra; Passmore, Jo-Ann; Denny, Lynette; Rybicki, Edward; Van Der Walt, Eric; Stephen, Lawrence; Williamson, Anna-Lise
BACKGROUND:We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV. METHODS: The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study. RESULTS: Oral HPV infection was highest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09-12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97-6.2, P = 0.04). CONCLUSION: The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation.
Open Access
Papanicolaou smears and cervical inflammatory cytokine responses
(BioMed Central Ltd, 2007) Passmore, Jo-Ann; Morroni, Chelsea; Shapiro, Samual; Williamson, Anna-Lise; Hoffman, Margaret
In a case-control study among 2064 South African women to investigate the risk of clinically invasive cancer of the cervix, we found a marked reduction in the risk of cervical cancer among women who gave a history of ever having undergone even a single Pap smear, and a statistically significant decline in the HPV positivity rate correlated with the lifetime number of Pap smears received. HPV infections and their associated low-grade lesions commonly regress, indicating that most often there is an effective host immune response against HPV infection. We hypothesized that act of performing a Pap smear is associated with inflammatory responses at the site of trauma, the cervix, and that this inflammatory signalling may be an immunological factor initiating these productive anti-HPV responses. In the present study, a randomized controlled trial, we enrolled 80 healthy young women to investigate the impact of performing a Pap smear on cervical inflammation. Forty one women, in the intervention group, received a Pap smear at enrollment and cervicovaginal lavages (CVLs) were collected at baseline and 2 weeks later. Thirty nine women received no intervention at enrollment (control group) but CVLs were collected at enrolment and 2 weeks later. We assessed various markers of inflammation including IL-12 p70, TNF-alpha, IL-8, IL-6, IL-10, and IL-1beta in CVL specimens. While CVL levels of IL-8, IL-1beta and IL-6 remained unchanged following a Pap smear, markers of cell mediated immunity (IL-12 p70 and TNF-alpha) and T cell regulation (IL-10) were significantly elevated.
Open Access
Phenotypic and functional characterisation of cervical and peripheral HIV-1 specific T cell responses
(2007) Liebenberg, Lenine Julie; Passmore, Jo-Ann; Burgers, Wendy
Distinct HIV variants occur at the genital mucosa compared to in blood, which may similarly result in differences in HIV T cell responses. There have been no studies of the maturational status of HIV-specific T cells present at the female genital mucosa. This study aimed to characterise HIV-specific cervical immune responses and to determine if compartmentalized immune responses occur in chronic HIV infection by comparing the characteristics of T cells at the cervical mucosa to those in blood.
Open Access
Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents
(2021-11-23) Happel, Anna-Ursula; Balle, Christina; Maust, Brandon S; Konstantinus, Iyaloo N; Gill, Katherine; Bekker, Linda-Gail; Froissart, Rémy; Passmore, Jo-Ann; Karaoz, Ulas; Varsani, Arvind; Jaspan, Heather
The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact.
Open Access
Role of HAART in reconstituting T-cell function and HIV inhibitory activity in the female genital tract during chronic HIV infection
(2014) Dabee, Smritee; Passmore, Jo-Ann
Includes abstract. Includes bibliographical references.