Browsing by Department "Department of Pathology"

Now showing 1 - 20 of 143
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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.
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    Open Access
    A Genome-wide Association Study of Schizophrenia in the South African Xhosa and Generalizability of Polygenic Risk Score across African populations
    (2021) Majara, Lerato Charlotte; Ramesar, Raj; Chimusa, Emile
    African populations are vastly underrepresented in genetic studies despite having the most genetic variation globally and facing wide-ranging environmental exposures. Most of these studies have been conducted in populations of European (EUR) ancestry using GWAS arrays that represent the genetic variation in these populations. Thus, the prediction accuracy of polygenic risk scores (PRS) derived from EUR ancestry populations is less accurate in populations of non-European ancestry, and least accurate in African (AFR) ancestry populations. The extent to which PRS prediction accuracy varies within AFR ancestry populations has not, however, been previously investigated. This study had two aims: the first was to investigate the contribution of common variants to the risk of schizophrenia in the South African Xhosa (SAX) population through genome-wide association study (GWAS) analysis, and to determine if PRS derived from EUR and East Asian (EAS) ancestry populations from the Psychiatric Genomics Consortium (PGC) Schizophrenia Working Group were generalizable to SAX. The second aim was to assess the generalizability of PRS for non-psychiatric phenotypes that were derived from EUR ancestry individuals from the UK Biobank (UKB, n = ~350,000) in the Uganda General Population Cohort (GPC, n = 4,778) and the South African Drakenstein Child Health Study (DHCS, n = 638). To address the first aim, a GWAS was conducted in 2,086 Xhosa individuals from South Africa with and without schizophrenia (ncases = 1,038; ncontrols = 1,048) using a custom-designed Affymetrix GWAS array designed to capture variation in the Xhosa population. The schizophrenia GWAS in SAX yielded one SNP (rs35172303 ; P = 4.74e-08, OR = 0.6004, 95%CI:[0.499,0.721]) in ZFP3 that met genome-wide significance. The association of variants in ZFP3 from the schizophrenia GWAS is consistent with those from an earlier exomesequence study in SAX undertaken by colleagues, but this gene has not previously been associated with schizophrenia in large-scale schizophrenia GWAS of predominantly EUR ancestry. After characterizing the genetic architecture of schizophrenia in SAX, it was found that the heritability was enriched across functional categories involved in the regulation of gene expression. Then, the accuracy of PRS derived from PGC Schizophrenia Working Group from both EUR and EAS ancestries in predicting schizophrenia in SAX was quantified. There was low PRS prediction accuracy using PGC-derived summary statistics in SAX (PGC-EUR: max R2 = 0.0057, P = 0.008; PGC-EAS: max R2 = 0.0059, P = 0.007). These findings are consistent with previous findings that showed that PRS predication accuracy is low when discovery and target cohorts come from different ancestral backgrounds. For the second aim, PRS prediction accuracy was quantified in simulations using data from the African Genome Variation project (AGVP) to represent continental AFR diversity. Samples were categorised by geographical region into West, East and South Africa cohorts. Each cohort was divided into a discovery and target datasets. The West and East African discovery data was used to predict the simulated phenotype in the three target cohorts. Using UKB EUR ancestry individuals, PRS prediction accuracy was assessed for 34 anthropometric and blood panel traits in the Uganda GPC, and then meta-analysed UKB with PAGE (Population Architecture using Genomics and Epidemiology, comprising about 50,000 Latino/Hispanic and African-American individuals) and BBJ (Biobank Japan, n = ~162,000) to assess how the inclusion of diverse sample impacts PRS prediction accuracy. Simulations were limited by sample size but showed that PRS prediction accuracy was highest when the discovery and target cohorts were matched by African region, and for phenotypes with the sparsest genetic architecture. Using empirical data from UKB and the Uganda GPC, a low prediction accuracy was observed across all 34 quantitative traits in GPC when using GWAS data from UKB. There was differential prediction accuracy across AFR ancestry groups within UKB, i.e. the prediction accuracy was highest for the Ethiopian and admixed populations, and lowest for southern African populations. When comparing PRS prediction accuracy of East African individuals from the UKB to that of individuals from GPC, the prediction accuracy was lowest in the Ugandan GPC population, indicating that the difference in environments between the two groups may be contributing to the difference in PRS accuracy. Moreover, the cross-ancestry meta-analyses showed that the inclusion of diverse samples in large scale studies improves PRS prediction accuracy, most especially for phenotypes with population-enriched variants. It was demonstrated for the first time in this thesis that EUR ancestry-derived PRS prediction accuracy varied within continental AFR ancestry groups, and tracks with population history and the evolution of humans. The higher prediction accuracy observed in Ethiopians can be explained by their genetic proximity to Europeans as a result of the back to Africa migration, whereas the southern African populations (including SAX) are more proximal to the ancestral populations that never left the continent. It is therefore imperative to not only include more African samples in future large-scale studies, but to have samples that adequately represent the genetic and environmental diversity on the African continent.
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    Open Access
    A retrospective analysis of post-mortem procedures of sudden unexpected death in the young investigated at Salt River Forensic Pathology Services, Cape Town
    (2022) Hamadziripi, Dirk M; Heathfield, Laura; Mole, Calvin
    Sudden unexpected death in the young (SUDY) is the demise of a seemingly healthy individual aged between one and 40 years. The scope of SUDY investigation varies and there is little research regarding SUDY at Salt River Mortuary (SRM). Accordingly, the objectives of this study were to determine the number of SUDY cases admitted to SRM, document the scope of investigations, and identify candidates for retrospective molecular autopsies. A total of 1088 cases were admitted between 1 January 2010 and 31 December 2015, representing 3.3% (1088/32812) of the entire case load, 30 were excluded as the files were missing. Full autopsies (56.7%; 600/1058) were preferred to partial autopsies (5.6%; 59/1058) and external autopsies (37.7%; 399/1058). The most utilised ancillary tests were LODOX imaging (86.6%, 916/1058) and toxicology (34.8%; 368/1058). Specificity of cause of death was seen to be significantly associated with the extent of autopsy. A total of 35.7% (378/1058) of the cases were established as candidates for molecular autopsy on the criteria of being undetermined, having unspecific causes of death or having specific causes of death that are deemed hereditary. Findings of this study show that SUDY cases do not always undergo all ancillary tests coupled with retention of biological samples. These findings provide insight into the current gaps in the investigation of SUDY cases at SRM and highlight areas for improvement.
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    Open Access
    A retrospective descriptive analysis of fatal ground level falls and falls from a height: A 5-year review
    (2022) Chonyera, Rumbidzai Lorraine Stephanie; Mole, Calvin
    Falls have been identified as the second leading cause of accidental deaths in the world and has become a public health issue. Depending on the manner and height at which the fall occurs, different injury patterns are observed, and these are useful for the determination of circumstances surrounding death. The aim of the present study was to determine the demographic characteristics, prevalence and injury patterns associated with ground level falls and falls from a height. A five-year (1 January 2014 - 31 December 2018) retrospective descriptive review of fatal fall cases investigated at Salt River mortuary was conducted. The prevalence and patterns of injuries were assessed with regard to fall height, impacting surface and victim demographics. There were 360 fall related deaths. Fall prevalence in the Western Metropole District of Cape Town is 3.72/ 100 000 population. Ground level falls were prevalent among the elderly while younger individuals fell from greater heights. There is an association between the sex of an individual and height from which they fall. Accidental falls were more common and no association was found between the alleged manner of death and sex. Skeletally, a higher frequency of fractures was observed in ground level falls while the head, chest and pelvis were affected in the high level falls. additionally, an association was observed between injuries sustained and fall heights. There is a significant difference in fracture proportions between the heights in the pelvic and lower extremities and no significant difference in head, spine, chest and upper extremities. As expected, trauma associated with falls varies based on the height of the fall. Lower extremity fractures are common in ground level falls however a challenge remains for falls from a height as there is a need for more studies to focus on the diverse patterns that occur in these.
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    Open Access
    A retrospective study investigating risk factors for sudden unexpected death in the young
    (2022) Oghenechovwen, Ogheneochuko Mary; Heathfield, Laura J; Mole, Calvin
    Sudden unexpected death in the young (SUDY) is the unanticipated demise of individuals aged between 1 and 40 years. In South Africa, these deaths are referred for forensic investigation. The primary aim of this study was to retrospectively investigate the frequency of known risk factors in SUDY cases admitted to Salt River Mortuary in Cape Town and explore differences between males and females. There were 1 088 SUDY cases identified with 0.9% (10/1 088) missing files. Reviewed cases were n=1 078, 62.6% (675/1 078) males, and 37.4% (403/1 078) females; 83.5% (901/1 078) adults and 16.4% (177/1 078) children, accounting for 5.6% of total admissions between 1 January 2010 and 31 December 2015. Despite the predominance of males, significantly more females (61.8%) were obese (p < 0.05). At least one primary medical condition was present in 53.7% of cases, with the leading conditions being tuberculosis (11.9 % of adult males), epilepsy (11.7% of adult males; 10.3% of female children), HIV (10.7% of adult females) and asthma (11.1% of male children). In the subset of the study population where information was available, before death, 74% of individuals were reported to have experienced prodromal symptoms; 37.6% of males and 32.4% of females did not seek medical intervention following symptoms. Information regarding a family history of sudden death was known in 237/1078 cases. In 3.2% of these cases, a family history of sudden death was reported. Significantly more males than females reported the use of tobacco, alcohol, and other illicit drugs (p < 0.05). More females were unemployed (p < 0.05). Interventions based on lifestyle modification, social support, pharmacologic needs, and awareness should be targeted at individuals with the above profiles, especially those with a family history of sudden death, as they may be high-risk groups. Findings from this study contribute new and relevant local reference data for SUDY risk profiles of males and females admitted to Salt River Mortuary.
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    A Study of Genital Human Papillomavirus (HPV) and Evaluation of HPV Testing for Cervical Cancer Screening in Women from the Eastern Cape Province, South Africa
    (2021) Taku, Ongeziwe; Williamson, Anna-Lise; Meiring,Tracy L; Mbulawa, Zizipho Z A
    Introduction: Human papillomavirus (HPV) infection is an important public health problem facing black African women. Persistent infection with high-risk (HR) HPV types is the key factor for the development of cervical cancer. Coinfection of HPV with other sexually transmitted pathogens contributes to the progression of cervical cancer. Preventative measures including screening for and treating pre-cancerous cervical lesions as well as HPV vaccination have been implemented in parts of South Africa. However, in the rural Eastern Cape Province there is limited information on the prevalence of HPV and the HPV types associated with cervical lesions. Two cohorts were chosen to study HPV in the Eastern Cape (South Africa), a community clinic, and a referral hospital for treatment of cervical lesions. This study aimed at determining the prevalence of HPV, risk factors of HPV, coinfection of HPV with sexually transmitted pathogens and evaluate the performance of a number of HPV tests for HPV detection and cervical cancer screening. The objectives of the study were: • To investigate the prevalence of HR-HPV and factors associated with HR-HPV infection among women from rural Eastern Cape, South Africa. • To investigate the distribution of HPV genotypes among women with cervical intraepithelial lesions according to HIV status from Eastern Cape Province, South Africa. • To investigate HR-HPV prevalence and compare agreement between cliniciancollected and self-collected genital specimens as well as two different HPV tests on clinician-collected samples. • To investigate the prevalence of sexually transmitted pathogens and co-infection of with HR-HPV infection among women from rural Eastern Cape Province, South Africa. Methods: A total of 741 participants were recruited from the Mbekweni Community Clinic (N=417) and the Nelson Mandela Hospital Referral Clinic (N=324) located in the OR Tambo municipality of the Eastern Cape Province. Clinician-collected cervical scrapes from women attending the Community Clinic were screened for HR-HPV prevalence and HR-HPV viral load using Hybrid Capture 2 (HC-2, Qiagen Inc., Gaithersburg, MD; USA); Cervical clinician-collected and vaginal self-collected specimens of women with or without abnormal cytology from both study cohorts were also screened for HR-HPV infection using hpVIR real-time PCR. HPV typing of clinician-collected cervical specimens from women with cervical intraepithelial neoplasia grades 2 and 3 (CIN 2 / 3) was done using Direct Flow Chip HPV kit (Master Diagnostica, Spain). Cervical specimens from the Community Clinic (N=205) were also tested for sexually transmitted infections (STIs) namely Chlamydia trachomatis: CT, Haemophilus ducreyi, Herpes Simplex Virus type 2, Neisseria gonorrhoeae: NG, Treponema pallidum, and Trichomonas vaginalis: TV) and pathobionts (Ureaplasma spp: (UP), Mycoplasma genitalium: MG, and Mycoplasma hominis: MH) using the STD Direct Flow Chip kit (Master Diagnostica, Spain). The univariate and multivariate analysis was used to determine the correlation between HPV infection and potential behavioural risk factors using STATA 14.2 (Stata Corp, College Station, Texas). A chi squared test was used determine the difference in estimated HR-HPV prevalence between self-collected and clinician-collected samples. STIs prevalence and association with behavioural risk factors were analysed using GraphPad Prism v6.01 (GraphPad Software, Inc., San Diego, CA). Results: Of the 417 women from the community clinic, HR-HPV prevalence was significantly higher in HIV-positive women compared to HIV-negative women (40.6%, 63/155 vs 21.4%, 56/262, p< 0.0001). Among women referred to Nelson Mandela Hospital with cervical intraepithelial lesions, HPV prevalence was observed to be significantly higher in HIV-positive than HIV-positive women (98.0% vs 89.1%, p=0.012). Similarly, HIV-positive women (65.3%, 96/147) had higher multiple HPV infections than HIV-negative women (47.8%, 22/46; p=0.034). HPV35 (23.9%), HPV58 (23.9%), HPV45 (19.6%), and HPV16 (17.3%) were the most frequently detected HPV types in CIN2, while HPV35 (22.5%), HPV16 (21.8%), HPV33 (15.6%), HPV58 (14.3%) were commonly detected in women with CIN3 regardless of HIV status. HR-HPV prevalence in clinician-collected samples was equivalent to self-collected samples from both study sites, the community clinic (26.4% vs 27.9%, p=0.601) and the referral clinic (83.6% vs 79.9%, p=0.222). HR-HPV positivity between self-collected and clinician-collected samples showed an agreement of 86.9% for community clinic (k=0.669) and 91.4% for referral clinic (k=0.711). The distribution of HR-HPV genotypes was similar between self-collected and clinician-collected samples from both study sites. The agreement of HR-HPV genotypes between self-collected and clinician ranged from moderate to almost perfect (0.571-0.888). A majority of women reported a high positive response of acceptance for self-collection (community-based clinic: 77.2% and referral clinic: 83.0%). HR-HPV detection agreement between hpVIR real-time PCR and HC-2 was almost perfect (87.7%, k=0.754). The prevalence of the six traditional STIs (CT, TV, NG, HSV-2, TP, and Haemophilus ducreyi) was high (22.9%, 47/205). TV was the most frequently detected STI (15.6%, 32/205). UP (70.2%, 144/205) and MH (36.6%, 47/205) were the most frequently identified pathobionts. Multiple infections/coinfections with more than two STIs/pathobionts was found in 52.7% (108/205) of women with UP/MH (26.9%) and UP/HPV (21.3%) the frequently identified coinfections. HR-HPV infection was significantly associated with HIV infection (p=0.017) and HSV-2 (p=0.026). Conclusion: This study shows that HIV infection and sexual behaviour increased the risk of HPV infection among women from the community clinic. HIV-positive women had significantly higher HPV viral load and multiple HPV type infections compared with HIV-negative women with or without cervical lesions. Since HIV positive women are at higher risk of HPV infection they need to continue to be screened more regularly for cervical lesions and treated when appropriate. In addition, the high prevalence of HPV in the community of HIV negative women indicates that a robust cervical screening programme is needed to implement the cervical screening policy of South Africa. Thus, the women get the allocated three cervical smears in a life time. Distribution of HPV types was similar among women with CIN2 & 3 with HPV35 being the most frequently detected HPV type regardless of HIV status. This highlights the importance for the inclusion of HPV-35 in the next generation of HPV prophylactic vaccines. The findings of this study add to the limited information on genital HPV infection in women from this province. Moreover, our data now acts as a baseline/reference data for future investigations. The data will also contribute to discussions on HPV testing as the primary screening strategy for cervical cancer and HPV vaccination in South Africa. The hpVIR real-time PCR test between self-collected and clinician-collected specimens showed comparable agreement for the detection of HPV infection. The type-specific concordance between self-collected and clinician-collected showed moderate to an excellent agreement, indicating that self-collection can be utilised as the alternative screening tool for cervical cancer. The participants showed a high positive response for the self-collection method, indicating that introducing this method can positively impact the cervical cancer screening program. However, hpVIR real-time PCR is an in-house test which is not practical to introduce on a large scale in South Africa. Therefore, future research should be done to determine what other HPV tests could be done on these types of specimens. Presently, syndromic management is used to treat STI at clinics in South Africa. The high prevalence of sexually transmitted pathogens necessitates the need to enhance the current screening methods for these pathogens.
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    A study of the genital microbiotas of black South African women and men: associations with human papillomavirus and HIV infections
    (2018) Onywera, David Harris; Meiring, Tracy L; Williamson, Anna-Lise
    Persistent genital infection with oncogenic or high-risk human papillomavirus (HPV) is causally associated with cervical cancer in women and some penile cancers in men. The role of the complex genital microbiota in HPV infection has not been extensively addressed. This study characterised the genital microbiotas of heterosexually-active Black South African women and men, predominantly of the Xhosa ethnicity, recruited from a community in Cape Town, South Africa. The association of the genital microbiotas with prevalent HPV, HIV, demographic, behavioural, and clinical characteristics of the participants was examined. In Chapter 2 the bacterial communities in cervicovaginal samples from 62 HIVseronegative South African women were profiled by Ion Torrent PGM sequencing of the V4 hypervariable region of the bacterial 16S rRNA gene (IT-V4 method). The cervicovaginal microbiotas (CVMs) were found to cluster into three distinct community state types (CSTs): Lactobacillus iners-dominated CVMs (CST I (38.7%, 24/62)), unclassified Lactobacillusdominated CVMs (CST II (4.8%, 3/62)), and diverse CVMs (CST III (56.5%, 35/62)) with an array of heterogeneous bacteria, predominantly the bacterial vaginosis (BV)-associated Gardnerella, Prevotella, Sneathia, and Shuttleworthia. The majority of the women had nonLactobacillus-dominated CVMs. Lactobacilli are recognised as protective against sexually transmitted infections. Among the Lactobacillus species detected in the women, L. iners was the most prevalent and abundant. This species is recognised as the least protective amongst the vaginal lactobacilli. Women in CST I were more likely to be on hormonal contraception compared to women in CST III (relative risk (RR): 2.6 [95% CI 1.3-5.3]; p=0.005). Further research is required to confirm this association and to determine the biological mechanism. Microbiome research methodologies are constantly improving and in Chapter 3 the performance of two bacterial 16S rRNA gene amplicon-based methodologies were compared. The CVMs of 19 women were characterised using the IT-V4 method (Chapter 2) and using the Illumina 16S rRNA metagenomics method (IM-V3/V4 method). The latter method involves sequencing the V3 and V4 hypervariable regions of the 16S rRNA gene on the Illumina MiSeq platform. The two methods showed a high degree of correlation (r=0.89, p< 0.0001) in the average relative abundance of shared bacterial taxa. Procrustes analyses of the weighted UniFrac distances further showed a statistically consistent clustering between the two methods (M 2 =0.3, p< 0.0001). The IM-V3/V4 method proved to have a greater throughput, longer read-length, and lower error rates than the IT-V4 method and was therefore used in the subsequent chapters (4 and 5). In Chapter 4, the CVMs of 87 HIV-seronegative women from the same cohort were examined using the IM-V3/V4 method. The CVMs clustered into eight CSTs. Only 23 women (26.4%) had CVMs dominated by a single Lactobacillus species, this included two women (2.3%) with L. crispatus (CST-1), two (2.3%) with L. jensenii (CST-2), and 19 (21.8%) with L. iners (CST-3). The majority of the women (64.4% (56/87)), however, had diverse and heterogeneous CVMs (CST-8) that were associated with BV (p2, p<0.05). In the final experimental chapter, the penile microbiotas of 238 Black South African men were characterised. This is the first large-scale study of the penile microbiota of South African men. Corynebacteriaceae (47.2%) and Prevotellaceae (6.6%) were found to be the most abundant bacterial families. The penile bacterial communities clustered into six CSTs (designated 1-6). A majority of the men (53.4% (127/238)) had Corynebacterium-dominated microbiotas (CST-1). The remaining CSTs (2-6) had lower relative abundances of Corynebacterium than CST-1 and were colonised with several vaginal bacteria. The prevalences of these CSTs (2-6) in men together with their respective most abundant genera (besides Corynebacterium) were as follows: CST-2 (9.2%; unclassified Clostridiales and Porphyromonas), CST-3 (8.8%; Gardnerella), CST-4 (7.6%; Chryseobacterium and Acinetobacter), CST-5 (18.5%; unclassified Clostridiales and Porphyromonas), and CST-6 (2.5%; Lactobacillus). One hundred and thirty (54.6%) and 102 (42.9%) of the men were positive for HPV and high-risk HPV, respectively, as detected by the Roche Linear Array HPV Genotyping assay. Of the 130 HPV-positive men, 37 (28.5%) and 93 (71.5%) had single and multiple HPV types, respectively. Men in CST-1 were less likely to have high-risk HPV and multiple HPV infections relative to men in CSTs 2-6 (RR: high-risk HPV: 0.8 [95% CI 0.6-1.0]; p=0.027 and multiple HPV: 0.8 [95% CI 0.6-1.0]; p=0.042). LefSe revealed that prevalent HPV infection was strongly associated with higher relative abundances of Sneathia, Porphyromonas, Prevotella, Dialister, and Campylobacter (LDA score >3, p3, p< 0.05). The relative abundances of the latter three bacteria together with Peptoniphilus were strongly associated with high-risk HPV infection (LDA score >3, p <0.05). In our cohort, 88 men (37.0%) were positive for HIV. Of these, 71.6% and 60.2% were positive for HPV and highrisk HPV infection, respectively. Among the HIV-negative men (n=150), 44.7% and 32.7% were positive for HPV and high-risk HPV infection, respectively. Although HIV status did not impact the overall composition of the penile microbiotas, HIV-infected men had higher relative abundances of Staphylococcus, Faecalibacterium, Strenotrophominas, Jonquetella, Ruminococcus, Roseburia, and Lamia (LDA score >2, p<0.05) Men with BV-negative female sexual partners (66.5% (157/236)) had higher relative abundances of Lactobacillus in their penile microbiotas than men with BV-positive female partners (p=0.007). Atopobium, Sneathia, and Saccharofermentans were significantly more prevalent in men with BV-positive female partners than men with BV-negative partners (p<0.020). The main limitations of our study include relatively small sample size of women, insufficient participant information such as host genetics, other STIs (e.g., herpes simplex virus) and abnormal vaginal flora (e.g., aerobic vaginitis), using a less sensitive method to diagnose BV in women, and inherent biases evident in any retrospective study. Moreover, we did not adjust for confounding factors in our analysis due to the small sample size. Despite the underscored limitations, our findings provide insight into the baseline genital microbiotas of the Black South African women and men. The associations identified in this cross-sectional study between specific microbiota members and HPV infection, particularly the association between Sneathia and HPV/high-risk HPV infection, identified in both women and men, are hypothesis-generating and warrant further investigation. The study forms a critical starting point for future longitudinal confirmatory association studies and studies examining these bacteria as potential biomarkers or risk factors for HPV infection.
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    Addressing an HIV cure in LMIC
    (2021-08-03) Ismail, Sherazaan D.; Pankrac, Joshua; Ndashimye, Emmanuel; Prodger, Jessica L.; Abrahams, Melissa-Rose; Mann, Jamie F. S.; Redd, Andrew D.; Arts, Eric J.
    HIV-1 persists in infected individuals despite years of antiretroviral therapy (ART), due to the formation of a stable and long-lived latent viral reservoir. Early ART can reduce the latent reservoir and is associated with post-treatment control in people living with HIV (PLWH). However, even in post-treatment controllers, ART cessation after a period of time inevitably results in rebound of plasma viraemia, thus lifelong treatment for viral suppression is indicated. Due to the difficulties of sustained life-long treatment in the millions of PLWH worldwide, a cure is undeniably necessary. This requires an in-depth understanding of reservoir formation and dynamics. Differences exist in treatment guidelines and accessibility to treatment as well as social stigma between low- and-middle income countries (LMICs) and high-income countries. In addition, demographic differences exist in PLWH from different geographical regions such as infecting viral subtype and host genetics, which can contribute to differences in the viral reservoir between different populations. Here, we review topics relevant to HIV-1 cure research in LMICs, with a focus on sub-Saharan Africa, the region of the world bearing the greatest burden of HIV-1. We present a summary of ART in LMICs, highlighting challenges that may be experienced in implementing a HIV-1 cure therapeutic. Furthermore, we discuss current research on the HIV-1 latent reservoir in different populations, highlighting research in LMIC and gaps in the research that may facilitate a global cure. Finally, we discuss current experimental cure strategies in the context of their potential application in LMICs.
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    Advancements in the Growth and Construction of Recombinant Lumpy Skin Disease Virus (LSDV) for Use as a Vaccine Vector
    (2021-10-04) van Diepen, Michiel; Chapman, Rosamund; Douglass, Nicola; Whittle, Leah; Chineka, Nicole; Galant, Shireen; Cotchobos, Christian; Suzuki, Akiko; Williamson, Anna-Lise
    Attenuated vaccine strains of lumpy skin disease virus (LSDV) have become increasingly popular as recombinant vaccine vectors, to target both LSDV, as well as other pathogens, including human infectious agents. Historically, these vaccine strains and recombinants were generated in primary (lamb) testis (LT) cells, Madin–Darby bovine kidney (MDBK) cells or in eggs. Growth in eggs is a laborious process, the use of primary cells has the potential to introduce pathogens and MDBK cells are known to harbor bovine viral diarrhea virus (BVDV). In this study, data is presented to show the growth of an attenuated LSDV strain in baby hamster kidney (BHK-21) cells. Subsequently, a recombinant LSDV vaccine was generated in BHK-21 cells. Partial growth was also observed in rabbit kidney cells (RK13), but only when the vaccinia virus host range gene K1L was expressed. Despite the limited growth, the expression of K1L was enough to serve as a positive selection marker for the generation of recombinant LSDV vaccines in RK13 cells. The simplification of generating (recombinant) LSDV vaccines shown here should increase the interest for this platform for future livestock vaccine development and, with BHK-21 cells approved for current good manufacturing practice, this can be expanded to human vaccines as well.
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    An Investigation of the SHIV reservoirs in the lymphoid organs of HIV-vaccinated rhesus monkeys after SHIV challenge
    (2022) Benn, Kealan Timothy; Chapman, Rosamund; Hurdayal, Ramona
    The development of an effective vaccine against HIV-1 is thought to be a key component of combating the current HIV epidemic. This study aimed to investigate viral reservoirs in the blood and lymphoid tissues after HIV vaccination and virus challenge using the rhesus macaque model. In addition, the study investigated the envelope sequences of the virus located in the potential reservoir sites in an attempt to understand viral variability. Cryopreserved peripheral blood mononuclear cells (PBMC) and cells isolated from lymphoid tissues obtained at termination of vaccine group (P4, P11, P25, P47, P52) and control group (P67, P70, P71, P72) macaques were processed for determination of viral RNA and proviral DNA loads, and HIV-1 Env amino acid sequences using single genome amplification (SGA) sequencing method. In addition, cryopreserved plasma obtained at key time points pre- and post- vaccination and SHIV challenge were used to measure the levels of HIV Env and SIV Gag antibodies by Western blotting, and HIV-1 Env gp140 ELISA. RNA viral loads were low for all animals with the median of the averages of the viral load for PBMCs being 3.82 copies/107 cells and a range of < 1 copies/107 cells to 61.15 copies/107 cells. For the control group, only P67 had a detectable viral load in the PBMC. The median of the averages for proviral DNA load for PBMCs was 1903.98 copies/107 cells and a range of 696.93 copies/107 cells to 28 663.88 copies/107 cells. Proviral DNA levels were higher than the RNA viral loads indicating a larger amount of integrated provirus and potentially the presence of reservoirs within the macaques. While the low viral load values could mean there are few actively replicating viruses present in the PBMCs. The median of the averages for the RNA viral loads in the inguinal tissue cells was 58.08 copies/107 cells with a range of 8.85 copies/107 cells to 911.61 copies/107 cells. The median of the average for proviral DNA load in the inguinal tissue cells was 3160.36 copies/107 cells with a range of 604.67 copies/107 cells to 73 140.68 copies/107 cells. Proviral DNA levels in the inguinal tissues were higher than the RNA viral load levels which indicates a larger amount of integrated provirus present in the cells of the inguinal tissues and the presence of a potential reservoir. This also indicates that there is less circulating virus. The Western blots showed that the macaques developed binding antibodies to both HIV Env and SIV Gag, however, non-specific binding of antibodies to proteins other than HIV Env and SIV Gag was also seen. The ELISA effectively showed that both the vaccine group and control group macaques were able to produce binding antibodies. Macaques P4, P47, and P52 had the highest endpoint titre for the vaccine group of 5120. Macaque P71 had the highest overall endpoint titre of 20 480. Macaques P11 and P25 of the vaccine group had an endpoint titre of 1280 which was the same endpoint titre for control group macaques P67 and P70, while macaque P72 had an endpoint titre of 5120. Single genome amplification and sequencing showed that there were very few amino acid changes between sequences found in macaques. There was minimal variation within the vaccine group and between the vaccine and control groups with amino acid changes in the variable region 1 (V1), glycan N276 in the conserved region 2 (C2) and variable region 4 (V4) resulting in potential N-glycosylation sites (PNGS) being shifted as well as new glycosylation sites being formed while other lost. This study demonstrates that RNA viral loads and proviral DNA loads were detectable in both macaque groups with no significant difference being present in the viral RNA and proviral DNA loads between the two macaque groups. The lack of changes seen in the sequences of the inguinal and PBMC tissues are due to the early virus seeding the reservoir. Once the reservoir is seeded active replication does not occur in the reservoir resulting in the viruses having similar sequences.
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    Antigenic and immunological determinants of acute allergic susceptibility to meat in a uniquely defined cohort in the Eastern Cape
    (2023) Murangi, Tatenda; Levin, Michael; Horsnell William
    Allergic sensitization can occur after allergen exposure through the oral-mucosal or cutaneous route. Allergic remission is associated with a decrease in total and specific IgE levels to allergens. Ascaris lumbricoides is a potent inducer of IgE through the establishment of a strong Th2 environment. IgE induction following A. lumbricoides infection is a risk for allergic sensitization. Tick disruption of host skin during feeding has a systemic effect resulting in the induction of a Th2 phenotype with elevated IgE production. Raised IgE can be driven by exposure to parasite proteins and lipids with complex glycosylation patterns. Our study demonstrates the presence of alpha-gal in both adult and larval developmental stages of A. lumbricoides, Amblyomma hebraeum and Rhipicephalus evertsi. Alpha-gal glycosylation was prominent on 100kDa and 130-250kDa protein bands. A. hebraeum and R. evertsi showed differential expression of alpha-gal glycosylated proteins during feeding with band intensity increasing proportionally to an increase in feeding time in the salivary glands. Immunolocalization of alpha-gal in A. lumbricoides adult worms showed staining in the lining of the gastrointestinal tract while in A. hebraeum and R. evertsi, staining was prominent in the salivary glands. Screening for IgE demonstrated elevated IgE to A. lumbricoides in human research participants with challenge-proven alpha-gal allergy which positively correlated to alpha-gal IgE. Furthermore, non-alpha gal glycosylated A. lumbricoides antigens caused significant activation of a humanized rat basophil RS-ATL8 IgE reporter cell system after incubation with sera from alpha-gal allergic individuals. Interestingly, serum IgG4 from alphagal allergic individuals showed surface labelling of A. lumbricoides larvae invitro. Alpha-gal positive participants also demonstrated raised IgE and IgG4 towards A. hebraeum proteins. Proteomic analysis suggests alpha-gal glycosylation to be present on alpha-2-macroglobulin found in lysates from both A. lumbricoides and A. hebraeum. These findings present A. lumbricoides, A. hebraeum and R. evertsi as potential sources of sensitization to alpha-gal and hypersensitivity reactions including anaphylaxis in humans after the consumption of red meat or use of pharmaceutical products from a mammalian source
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    Assessing the accuracy of the zygoma for estimating ancestry using geometric morphometrics in a South African sample
    (2019) Tawha, Tafadzwa Primrose Rudo; Gibbon, Victoria E; Dinkele, Elizabeth; Mole, Calvin
    The large number of unidentified, decomposed and skeletonised remains found in South Africa (SA) necessitates relevant and reliable methods to assist in victim identification. Ancestry estimation from unknown skeletal remains is essential when reconstructing a demographic profile of a missing person. In the SA population, estimating ancestry is problematic as standards developed internationally rarely apply to the local, biologically heterogenous population. Craniofacial morphology is known to be ancestrally distinct and studies are yet to explore shape and size variation in the zygomatic bone of the SA population. The aim of this study was to assess ancestral variation in zygomatic shape and size in a SA population using three-dimensional geometric morphometric analyses. A sample of 158 individuals were analysed from Bantu-speaking (BA), European (EA) and Mixed Ancestral (MA) South African groups. Males were larger in size than females, but no size differences were observed between ancestral groups. Significant shape differences were observed between ancestral groups, while none were observed between males and females. BA and MA individuals had narrower, shorter and more anteriorly projecting zygomas than EA individuals. The zygoma was shown to accurately distinguish EA (84%) from BA (81%), and MA (80%) from EA (68%) individuals, but unreliably distinguished BA (60%) from MA (66%) individuals. This is likely correlated to the historical peopling of SA and historical forced racial classification. Age-related changes and antemortem tooth loss did not confound the ancestral variation in size, despite minor changes in zygomatic shape being associated with these two factors. These confounders did not impact ancestry estimation accuracies, further suggesting a minor impact on overall zygomatic shape. Furthermore, the patterning of ancestral variation in the zygoma revealed the need for further research to distinguish between the biologically heterogenous ancestral groups in SA.
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    The assessment of molecular markers for skin colour determination in the South African population
    (2017) Slabbert, Nandi; Heathfield, Laura
    Molecular markers associated with skin colour need to be investigated to determine viability and discriminatory power within the SA population. The use of phenotyping as a tool to aid forensic investigation is becoming incorporated internationally and its use within SA needs to be considered. This project proposes to examine the association between skin colour and two markers, which have been identified as potentially problematic within mixed ancestry populations. This will be done through designing and analysing a multiplex SNaPshot genotyping assay and independent pigmentation measurements.
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    Assessment of the suitability of blood samples collected for toxicological analysis for subsequent genetic analysis: A follow-up study one year later
    (2018) Musiyandaka, Fungisai Lorraine; Heathfield, Laura; Davies, Bronwen
    Drug usage, both of a recreational or pharmaceutical nature, is common, however the abuse of such substances is an international problem. In the Western cape, South Africa, the burden of drug-related fatalities is high compared to the rest of the country. The provincial Forensic Pathology Service may encounter cases where drug-related fatalities are unclear whether death was accidental or suicidal, or drug toxicity is inconsistent with the medical/social history. This may be due to genetic alterations with drug metabolism and it has been suggested that genetic analyses may be the next step in these cases. However, toxicology results from the National Forensic Chemistry Laboratory in the Western Cape may be delayed by months to years, meaning that upon interpretation of toxicology results, there is no chance to obtain another blood sample from the deceased individual for genetic analysis. It was therefore important to determine the suitability of blood samples collected and handled in toxicology environments for subsequent genetic tests. Previously, blood samples from 30 post-mortem cases were collected into two red-top (no additives), two grey-top (sodium fluoride/potassium oxalate) and one purple-top (EDTA) tubes. Samples from one red-top and one grey-top tube underwent toxicological analysis, followed by DNA analysis, while the remaining tubes (controls) underwent DNA analysis immediately. All samples were then stored for approximately one year, prior to this study. The DNA analysis was repeated on all blood samples (n = 150) and results were assessed in terms of storage time and tube type. DNA was not significantly degraded in any of the samples; however, DNA from red-top tubes had significantly lower concentrations compared to that from grey-top tubes (p < 0.001), regardless of whether the sample had undergone toxicological analysis. The very low yields of DNA from red-top tubes posed substantial challenges for PCR-based analysis, resulting in poor quality Sanger sequencing results. Some DNA from grey-top tubes, passed the quality assessments and hence further work is required to provide an informed decision on which tube type is better suited for genetic analyses.
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    Assessment of the suitability of blood samples collected for toxicology for subsequent genetic analysis: A follow-up study after five years
    (2023) Grobbelaar, Jana; Davies, Bronwen; Pearce Brendon
    Therapeutic and recreational drug use is a common occurrence across the world. However, substance use may sometimes result in adverse drug reactions and death even when typically non-fatal drug doses are administered. This phenomenon may be caused by variants in the genes encoding drug-metabolising enzymes, which leads to altered drug metabolism and at times, toxicity. Cause or manner of death may not be apparent in these cases, even after conducting a standard autopsy and ancillary toxicological and histological investigations. A molecular autopsy may then be performed to identify an underlying genetic cause. Genetic testing is however not routinely conducted in forensic mortuaries, and historic backlogs within the National Forensic Chemistry Laboratories may delay the processing of toxicology samples by months or even years. As such, specimens that have undergone toxicological testing and were stored long-term are sometimes the only samples available to conduct subsequent molecular autopsies, should it be necessary for the cause of death determination. This study therefore aimed to assess whether blood specimens that were used for toxicological analyses could provide suitable DNA for downstream genetic analyses after an extended storage period of five years. In 2017, DNA was analysed from blood samples collected into vials containing sodium fluoride/potassium oxalate preservatives or vials without preservatives (grey and red top tubes, respectively). A subset of these vials underwent preparation for toxicological analyses at the time, prior to DNA extraction, while the remaining tubes underwent DNA extraction immediately and were stored in a molecular laboratory as controls. DNA analysis was then repeated one year later in a separate study, as well as five years later as part of the current study. DNA quantity and quality scores were significantly lower in red top tubes compared to grey top tubes, and toxicological processing did not significantly influence results. DNA concentration and quality also significantly decreased over time for all sample types. PCR amplification and Sanger sequencing results were mostly poor for red top tubes, but grey top tubes showed overall improvements in sequence quality. However, all DNA analysis results generally improved when DNA was extracted using a modified salting out method. Based on these results, it is suggested that forensic laboratories that often experience delays in sample processing should perform molecular autopsies using blood stored in sodium fluoride/potassium oxalate preservative coupled with a salting out DNA extraction method.
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    Association of Faecalibacterium, Lachnospira, Veillonella, and Rothia with childhood wheezing
    (2019) Kanyemba, Saara; Kaba, Mamadou; Tow, Lemese Ah
    Wheezing symptoms among children, present major health and economic problems globally. A recent study conducted in Canada observed a reduction in stool bacterial genera Faecalibacterium, Lachnospira, Veillonella and Rothia (FLVR) in three-month old infants with atopy-wheeze symptoms. It is not known whether this is true in different human populations worldwide. The overall aim of this dissertation was to investigate the contribution of any of the FLVR bacteria or their combination in the occurrence of infant wheezing within the Drakenstein Child Health Study (DCHS), South Africa. To address this aim, I began my thesis's project by conducting a systematic literature review which investigated the association of FLVR bacteria with the occurrence of different respiratory diseases in humans. My review provided evidence for the possible involvement of FLVR bacteria in human respiratory diseases, including asthma, pulmonary tuberculosis and pneumonia. Furthermore, this review highlighted the need for a well-designed and large study to investigate the contribution of the FLVR bacteria in respiratory diseases, in an African setting. Secondly, I optimized SYBR Green based real-time quantitative polymerase chain reaction (qPCR) as well as conventional PCR assays for the detection of FLVR bacteria. Using the optimized assays, I screened 533 stool samples collected from 140 wheezing and 140 nonwheezing infants. The optimized assays demonstrated good performance in the detection of FLVR bacteria from human stool samples. Using qPCR, Rothia, Veillonella, Faecalibacterium and Lachnospira were detected in 90% (479/533), 73% (388/533), 51% (274/533) and 14% (77/533) of the samples, respectively. Conventional PCR permitted the detection of Rothia, Veillonella, Faecalibacterium and Lachnospira in 55% (263/479), 74% (289/388), 53% (145/274) and 0% (0/77) of the qPCR positive samples, respectively. I also determined the factors associated with faecal colonization by FLVR bacteria in the first year of life. I showed that reduced colonisation by Faecalibacterium was associated with male gender (adjusted OR = 0.65, 95%CI: 0.42 - 0.98) and TC-Newman residence (adjusted OR = 0.52, 95%CI: 0.29 - 0.91). Breastfeeding was associated with less colonisation by both Lachnospira, (adjusted OR = 0.17, 95%CI: 0.05 - 0.49) and Veilonella (adjusted OR = 0.32, 95%CI: 0.10 - 0.91). Mother's tertiary education was significantly associated with high Rothia colonisation (adjusted OR = 11.73, 95%CI: 1.36 - 2.58). In the last section of my thesis, I assessed the association of FLVR bacteria with infant wheezing using logistic regression models. I found a significant association of Rothia with reduced risk of infant wheezing (adjusted odds ratio (aOR)=0.54, 95%CI: 0.28-0.93) and recurrent wheezing (aOR=0.29, 95%CI: 0.05-0.88). Using receiver operating characteristic curves (ROC), I showed that among all FLVR bacteria, Lachnospira (AUROC = 0.833, 95%CI: 0.64-1.00) and Rothia (AUC=0.707, 95%CI: 0.62-0.79) could serve as biomarkers for early prediction of infant wheezing. Overall, this is the first study on FLVR bacteria and infant wheezing to be conducted in Africa. Its findings encourage more research to be conducted in order to elucidate the potential protective role of Rothia against childhood wheeze and asthma, as well as the contribution of Lachnospira in asthma development.
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    At-risk individual's perspectives of Spinocerebellar Ataxia (SCA) Presymptomatic Testing (PT)
    (2021) Lloyd, Deanah; Wessels, Tina-Marie; Greenberg, Jacquie
    Since the introduction of presymptomatic testing for Spinocerebellar Ataxia in South Africa, no research has looked at the impact, perceptions or acceptance of such testing within this diverse population. Despite the relatively high frequencies of late onset autosomal dominant conditions in South Africa, the uptake of presymptomatic testing by those at-risk of inheriting these conditions has been lower than that seen internationally. This research project sought to understand these low levels of utilisation, by exploring the perceptions of those at-risk of inheriting Spinocerebellar Ataxia towards presymptomatic testing. In depth semi-structured interviews were conducted with six individuals at-risk of inheriting Spinocerebellar Ataxia. The interviews were transcribed verbatim and thematically analysed. The four themes that emerged from the data included: 1) Caregiving, 2) Relationships, 3) Being At-Risk and 4) Presymptomatic Testing (PT) Perceptions. These themes explore the significant and long-lasting burdens faced both physically and emotionally by the affected individual as well as their relatives. With no currently available way of preventing or curing the condition, those atrisk described being left with a sense of hopelessness and anxiety about their future. The at-risk individuals' perceptions and fears were often linked to their and their family's experiences of the condition. Additionally, their perceptions of presymptomatic testing, although positive, did not correlate with testing utilisation amongst the participants. As such, the current underutilisation of presymptomatic testing in South Africa was found to be due to the at-risk individuals' fears of the result and its' perceived consequences, rather than a negative perception of presymptomatic testing. This is significant as it indicates that the current lack of uptake of presymptomatic testing is due to external factors unrelated to the test itself. As such, genetic counsellors should focus their efforts on counselling the individual through their fears as opposed to primarily offering presymptomatic testing. Although these findings contribute to our understanding of this previous understudied population, they cannot be extrapolated to apply to the entire South African at-risk population due to the small sample size of the study. This knowledge however, may assist in improving the presymptomatic testing process by providing greater insight into the population's experiences and perspectives. Thus, it is recommended that future studies explore ways that genetic counselling sessions and the presymptomatic testing process could be altered to incorporate this knowledge.
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    Barriers to tuberculosis drug discovery: the mycobacterial cell wall
    (2023) Whittaker, Caitlin; Warner, Digby; Egan Timothy John
    The mycobacterial cell wall is a highly complex macromolecular structure that provides intrinsic resistance to several anti-tuberculosis drugs, making it critical in the success of Mycobacterium tuberculosis infection. Multiple layers encapsulate the cell membrane, including arabinogalactan chains and mycolic acids that are covalently bonded to the peptidoglycan layer, resulting in a highly selectively permeable barrier that is unique to this genus. The current treatment for tuberculosis (TB) utilizes antibiotics that weaken the structural integrity of the cell wall to allow easier access for drugs that have intracellular targets. Although this approach is theoretically effective, patient adherence is often poor owing to the lengthy treatment times and negative side effects associated with the multidrug combination regimen. As such, rational drug design to develop more potent, faster-acting anti-TB compounds requires a comprehensive understanding of the composition and functioning of the mycobacterial cell envelope to ensure effective penetration through this barrier. Bioinformatic approaches to compound validation provide a crucial foundation for drug development, but empirical validation of these molecules can present a serious bottleneck in the drug discovery pipeline. Here, we investigated fluorescent click chemistry as a rapid and inexpensive means of ascertaining molecular properties that impact compound permeation of the mycobacterial cell envelope. The variability in permeation of different click-reactive moieties could be rapidly determined using fluorescent read-outs; this, in combination with the availability of a wide array of click-reactive side chains, presents a potentially powerful platform for establishing the properties required by a compound to effectively cross the mycomembrane. Enzymatic degradation of cell wall components further revealed the resilience of mycobacteria as the resulting organisms, spheroplasts, were capable of surviving in the absence of this seemingly essential protective layer. This presents a potentially novel form of intrinsic resistance whereby stripping of the cell wall could allow for tolerance to cell wall active antibiotics, a previously under-appreciated strategy that has been reported in other pathogenic bacteria. Together, these findings highlight the highly dynamic nature of the mycobacterial cell envelope and the need for further investigation into the properties of this structure that allow for such efficient antibiotic evasion.
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    Bystander influence of nematode exposure on subsequent herpesvirus infections in vivo
    (2019) Chetty, Alisha; Horsnell, William; Dewals, Benjamin
    Parasitic worms have the ability to modulate the hosts immune response to promote host control of the infection and also parasite survival in the host. Helminth infections classically induce a potent Th2-biased and regulatory immune imprint. This immune response also influences unrelated inflammatory processes in the host. Studies have shown helminth infections have bystander influences on unrelated conditions such as allergy and autoimmunity. Additionally, helminth infections can alter susceptibility to other infections. In this thesis, we investigate the systemic influences of murine nematode Nippostrongylus brasiliensis infection on host immunity in colonized and non-colonized tissues, and the implications of these effects on susceptibility to subsequent herpesvirus infections in vivo. We show that prior N. brasiliensis infection enhanced control of acute respiratory murid gammaherpesvirus (MuHV-4) infection, with an increase in viral-specific CD8+ T cells in colonized lung tissue. Enhanced effector cytokine responses by cytotoxic T cells were also observed with prior helminth exposure. Conversely, despite enhanced primary control, prior helminth exposure was associated with earlier and heightened genital reactivation of MuHV4. This demonstrates differences in local bystander and systemic effects of helminth exposure on the host, and on unrelated viral infections. We also show that N. brasiliensis infection, which transits the respiratory and gastrointestinal tracts, also systemically influences immunity in the female genital tract (FGT) in vivo. Here, helminth infection induced Th2-type immunity in the FGT, namely increased tissue IL-4, IL-5 and long-lasting eosinophilia. We further demonstrated that systemic influences of N. brasiliensis infection results in exacerbated genital pathology and inflammation, following subsequent intravaginal herpes simplex virus type II (HSV-2) infection. Increased HSV-2 pathology with prior helminth exposure was associated with diminished innate anti-viral immunity, increased IL-33, ILC2 and IL-5 responses, as well as significant eosinophilia. Interestingly, abolition of canonical Th2 immune signalling by the lack of IL-4Rα expression, enhanced innate anti-viral defences and provided protection from HSV-2 pathology. However, N. brasiliensis-induced exacerbation of HSV-2 illness was IL-4Rαindependent, associated with significant genital eosinophilia. Furthermore, antibody-depletion of eosinophils ameliorated nematode-exacerbated HSV-2 pathology, suggesting that nematode-induced genital eosinophilia mediates increased HSV-2 pathology in coinfected mice. We have therefore shown that helminth infections can induce local and systemic bystander immunity to lymphoid and myeloid immune compartments, which alters susceptibility to subsequent herpesvirus infections.
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    Catching a glimpse: the visualization of Mycobacterium tuberculosis from TB patient bioaerosols
    (2023) Dinkele, Ryan; Warner, Digby; Gessner, Sophia
    Transmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission.