Doctoral Degrees (Medical Microbiology)

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The activity of nybomycin against mycobacterium tuberculosis.
(2018) Niehaus, Abraham Johannes.; Moodley, Prashini.; Sturm, Adriaan Willem.
Nybomycin was discovered in 1955, but was never developed for clinical use. The compound was noticed again in recent years when it displayed bactericidal activity against certain fluoroquinolone-resistant bacterial species. The work presented here aims chiefly at describing the effect of nybomycin on Mycobacterium tuberculosis. The study is made up of three parts. In the first part, in vitro nybomycin susceptibility testing was conducted with various fluoroquinolone-susceptible and fluoroquinolone-resistant bacterialspecies. All M. tuberculosis isolates displayed low nybomycin inhibitory concentrations regardless of fluoroquinolone resistance. Similar susceptibility results were obtained for N. gonorrhoeae isolates, but results obtained with other bacterial species were less promising. In the second part, in silico investigations were conducted to elucidate the mechanism of action of nybomycin in M. tuberculosis. Results show that nybomycin binds to M. tuberculosis gyrase enzyme with an affinity at least similar to that of fluoroquinolones. No clear differences in binding affinity were observed when gyrA mutations, commonly associated with fluoroquinolone resistance, were considered. The results suggest that the mechanism of action of nybomycin against M. tuberculosis involves inhibition of gyrase enzyme. In the third part, M. tuberculosis mutants with increased nybomycin minimum inhibitory concentrations were selected and compared with the wild type organism through whole genome sequencing. None of the isolates harbored any mutations commonly linked to known drug resistance mechanisms. This indicates that M. tuberculosis likely employs a novel mechanism of resistance against nybomycin. This may further signify that nybomycin has an additional mechanism of action against M. tuberculosis, besides the action on gyrase enzyme, as suggested by the in silico results from this study. Twenty-two genes were identified through whole genome sequencing that may potentially be linked to the mechanism of resistance and possibly an additional mechanism of action.
Antibiotic resistance in mycobacterium tuberculosis : the role of genetic mutations in resistance conferring genes and efflux transporters.
(2016) Dookie, Navisha.; Moodley, Prashini.
Two decades after the World Health Organisation (WHO) declaration of tuberculosis (TB) as a global emergency, the disease remains a public health crisis of epic proportions. The emergence of drug resistant strains of Mycobacterium tuberculosis, the etiologic agent of TB, and the convergent human immunodeficiency virus (HIV) epidemic places a devastating burden on an already weakened public health care system in South Africa. Rapid and accurate detection of drug resistance to first and second line drugs to guide effective treatment of TB is central to control of the disease and in preventing further dissemination of drug resistant strains. Knowledge of the underlying resistance mechanisms driving drug resistance in M.tuberculosis is pivotal in the design of rapid molecular based assays and will impact of the development of novel drugs and regimens for the disease. The manuscript in chapter 2 of this thesis, entitled Dynamics of antimicrobial resistance in Multi-Drug and Extensively Drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the diversity of the resistance mechanisms amongst the multidrug resistant (MDR) TB strains currently circulating in the KwaZulu-Natal province of South Africa by the analysis of the rpoB, katG, inhA, pncA and embB genes associated with resistance to key drugs used in the treatment of TB. Multiple drug resistance mechanisms in the MDR-TB isolates suggests that the strains emerged separately and acquired resistance mutations independently. The findings of this study also confirms the clonality of the XDR-TB epidemic demonstrated by the predominance of the F15/LAM4/KZN strain family and reveals that MDR-TB strains are evolving and spreading via transmission. The manuscript in chapter 3 of this thesis, entitled Streptomycin resistance in the F15/LAM4/KZN strain of Mycobacterium tuberculosis is mediated by lineage-specific alteration of the gidB gene, demonstrated that streptomycin (STR) resistance in the F15/LAM 4/KZN MDR and XDR-TB strains was mediated by a rare, 130bp deletion within the gidB gene of M.tuberculosis leading to a complete disruption of the gene. Classical mutations in the rpsL gene mediated STR resistance in the remaining strain families. Widespread STR resistance has resulted in the exclusion of the drug from current treatment regimens. The findings of this study support the decision of policymakers and cautions the application of the drug in the absence of drug susceptibility testing. The manuscript in chapter 4 of this thesis, entitled Moxifloxacin resistance in the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis, demonstrated that the F15/LAM4/KZN XDR strain harboured the A90V gyrA mutation associated with high level ciprofloxacin (CPX) and ofloxacin (OFX) resistance and correlated with increased minimum inhibitory concentrations (MIC) for moxifloxacin (MXF). The results of this study cautions the utilization of MXF as part of empiric treatment protocols in the absence of moxifloxacin MIC data of the circulating XDR strains in an area. It also raises concerns regarding the regarding the use of moxifloxacin in KwaZulu-Natal. Furthermore, the current breakpoint defining resistance to MXF is of concern and requires revision. The manuscript in chapter 5 of this thesis, entitled Evaluation of Capreomycin in the treatment of the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis demonstrated that the A1401G rrs mutation was the main mechanism mediating resistance to the aminoglycosides, kanamycin (KAN) and amikacin (AMIK); and to capreomycin (CAP). CAP was reintroduced into TB treatment protocols without prior drug susceptibility testing. This results of this study demonstrates high level resistance to CAP and urges careful consideration in the application of CAP the KwaZulu-Natal province. Furthermore, concerns regarding the high breakpoint value that defines CAP resistance as compared to wild-type MICs for the drug results in misdiagnosis of resistance that results inadequate patient treatment and amplifies resistance. The manuscript in chapter 6 of this thesis, entitled KZN Multidrug and Extensively drug resistant strains of Mycobacterium tuberculosis remain susceptible to Linezolid and para-Amino salicylic Acid, demonstrated that the mechanisms most commonly associated with resistance to the linezolid (LIN) and para-amino salicylic acid (PAS) were absent in the MDR and XDR-TB strains in this study. Mutations detected in the drug targets were lineage specific markers rather than resistance mechanisms. This study also highlights the poor understanding of resistance to these drugs and the need for further study to allow for resistance detection to be incorporated into diagnostic assays, thus prolonging the utility of these drugs. The manuscript in chapter 7 of this thesis, entitled Efflux mediated drug resistance in clinical isolates of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the role of efflux pumps in mediating low level resistance. The results of this study supports the hypothesis that efflux activity leads to decreased intracellular antibiotic concentrations, thereby allowing the survival of a sub-population of bacteria under the sub-inhibitory level of the antibiotic, from which resistant mutants emerge, leading to clinically significant levels of resistance. The results of this study strongly supports the application of efflux pump inhibitors as adjunctive to the current treatment protocols. The results emanating from this thesis has contributed to the body of knowledge of drug resistance in M.tuberculosis, especially in the KwaZulu-Natal province of South Africa. Furthermore, the results can be used to guide treatment protocols and contributes to the future development of molecular based assays aimed at detecting resistance.
Calymmatobacterium granulomatis: culture, electron microscopic studies and molecular analysis.
(1997) Kharsany, Ayesha Bibi Mahomed.; Hoosen, Anwar Ahmed.; Kiepiela, Photini.
Abstract available in PDF.
Development of novel reagents for tuberculosis detection.
(2013) Ngubane, Nqobile Angel Cebile.; Pym, Alexander S.; Khati, Makobetsa.; Rubin, Eric.
Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics.
Diversity of rodent-borne zoonotic pathogens at the human-animal-environment interface in Qatar.
(2021) Islam, Md. Mazharul.; Mkhize-Kwitshana, Zilungile Lynette.; Farag, Elmoubashar Abubaker Abd.
Rodents are the most diversified terrestrial mammals in the world. These animals assist with maintaining a healthy ecosystem through the soil structure modification, aeration, and hydration, although 5-10% are regarded as pests and carry zoonotic pathogens. Besides consumption and damage of our food and property, they are responsible for the transmission of several diseases, including plague, typhus, and leishmaniasis. Commensal rodents are the primary source of these pathogens because of their close proximity to humans. Qatar is a small country in the Arabian Peninsula. Four rodent species have been recorded in this country, that includes three commensal (Mus musculus, Rattus norvegicus, and Rattus rattus) and one wild (Jaculus jaculus) species. The zoonotic importance of rodents is yet to be explored. Knowing the pathogens originating from rodents is essential for early preparedness, prevention, and control. Therefore, the current study was undertaken on commensal rodents, rodentborne zoonotic pathogens, and the factors that are associated with pathogen prevalence among rodents, such as rodent sex, age, and trapping location in Qatar. A cross-sectional study was conducted between August 2019 and February 2020, which trapped rodents from different facilities, such as livestock and agricultural farms, bachelor and family accommodations, and industrial and commercial areas of Qatar. After studying the morphological and morphometric characters, blood samples, ectoparasites and visceral samples were collected from the captured rodents. Parasitic, bacterial, and viral pathogens were identified and characterized using gross, necropsy, microscopic, culture, biochemical, immunologic, and molecular methods. Descriptive statistics and univariate analysis were conducted to detect rodents, rodent-borne pathogens abundance, and the related risk factors. The study trapped 148 rodents, most of which were adults (n = 138, 93.2%, 95% CI: 87.92–96.71), and from livestock farms (n = 79, 49%, 95% CI: 41.02–57.65). R. norvregicus was the most prevalent (n = 120, 81%, 95% CI: 73.83–87.05), followed by R. rattus (n=24, 16%, 95% CI:10.68–23.16) and M. musculus (n=4, 3%, 95% CI: 0.74– 6.78) with an average body weight of 18.8 ± 2.2 gm, 264.3 ± 87.5 gm, and 130 ± 71.3 gm, respectively. This is the first morphologic and morphometric study of commensal rodents in Qatar and the Arabian Peninsula that detected the Qatari rodents are relatively smaller than those of Turkey, Tunisia, and Iran. About 63.5% of the rodents were infected with at least one of the 9 species of parasites, viz. Xenopsylla astia, Ornithonyssus bacoti, Hymenolepis diminuta, Taenia taeniaeformis, Capillaria annulosa, Strongyloides spp., Giardia spp., Toxoplasma gondii, Trypanosoma lewisi, and Leishmania spp. Helminths were the most prevalent (46.0%), followed by ectoparasites (31.8%) and protozoa (29.1%). Going by individual species prevalence, X. astia ranked the highest (31.8%), where the lowest prevalent parasite was C. annulosa (0.7%). The prevalence of H. diminuta was positively correlated (OR=4.13; p = 0.00) with the prevalence of X. astia. The study also identified thirteen bacterial species, namely Acinetobacter baumannii, Aeromonas salmonicida, Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, Providencia stuartii, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica, from the intestine samples. The majority of the bacteria were E. coli (54.63%, 95% CI: 44.76-64.24), followed by P. mirabilis (17.59%, 95% CI: 10.94-26.10), and K. pneumoniae (8.33%, 95% CI: 3.88-15.23). The study detected 31.58% (6/19, 95% CI: 12.58-56.55) of the flea pools, and one (1/1) mite pool was positive with Rickettsia spp. S. enterica showed the highest antimicrobial resistance (100% resistant to 8 antimicrobials). The top resistant antimicrobials were from cephalosporin, followed by penicillin and tetracycline groups. E. coli (26.92%, 95% CI:11.57-47.97) and K. pneumonia (50%, 95% CI: 6.76- 93.24) were ESBL (extended-spectrum beta-lactamases) producers. The studied rodents are indicators of the presence and dispersal of zoonotic pathogens in Qatar. Urgent action is needed to prevent future spillover of these pathogens at the human-animal-environment interface. It is essential to understand the biology, epidemiology, and transmission dynamics of these pathogens. Farm biosecurity and integrated pest management approach should be implemented in the farm premises. Implementing the One Health approach to combat rodent-borne zoonoses in order to reduce the risk of the future epidemic in Qatar is strongly recommended.
The effect of HIV and Neisseria gonorrhoeae on the tight junctions of cervical epithelial cells.
(2020) Maharaj, Shevani.; Sturm, Adriaan Willem.; Moodley, Prashini.
Introduction: Neisseria gonorrhoeae and HIV are major public health concerns globally. The interaction between these diseases is unclear. To determine the effect that N. gonorrhoeae and HIV have on the tight junctions of cervical epithelial cells, a cervical epithelial cell line was infected with N. gonorrhoeae only, HIV only and with N. gonorrhoeae and HIV simultaneously. Methods: The ME180 cervical cell line was grown to confluence and infected withN. gonorrhoeae only, HIV only and with N. gonorrhoeae and HIV simultaneously. Following infection, N. gonorrhoeae and HIV tansmigration assays and the blue dextran permeability assay were also performedto determine the effect that exposure to the microbes would have on the intact cervical epithelial layer. The tight junction gene expression assays, blue dextran permeability assay and immunofluorescence staining was performed to determine the effect that exposure to the different microbes had on the tight junctions. Results: The results of this study showed that exposure of the cervical epithelial layer to N. gonorrhoeae alone, HIV alone and to N. gonorrhoeae and HIV simultaneously did not affect the paracellular permeability of the epithelial layer. The results showed that a small percentage of N.gonorrhoeaeand HIV was able to migrate across the epithelial layer. With the simultaneous infection of N.gonorrhoeae and HIV, the presence of HIV did not seem to influence the migration of N. gonorrhoeae, as compared to infection with N. gonorrhoeae only, while the presence of N.gonorrhoeae seemed to cause the HIV to pass through the epithelial layer less efficiently than with exposure to HIV only. Discussion: The overall results suggest that since exposure to these microbes does not seem to affect the tight junctions of the intact epithelial layer and does not affect the paracellular permeability, the migration of the microbes across the epithelial layer was possibly through transcytosis.
Effect of HIV status and suppression on SARS-CoV-2 disease severity, vaccine response, and evolution=Umthelela wesifo sesandulela-ngculazi nokucindezelwa kwaso yi-SARS-CoV-2 ubunzima besifo, ukusebenza kwekhambi kanye nokuthuthuka.
(2023) Karim, Farina.; Sigal, Alexander.; Moosa, Mahomed Yunus Suleman.
Abstract SARS-CoV-2 is a global pandemic that has infected 672,115,430 people globally (https://coronavirus.jhu.edu/map.html, accessed 08.02.2023). SARS-CoV-2 is continuously evolving, and new variants pose a continuous threat to curbing this pandemic. Simultaneously, South Africa still struggles to control and manage an enduring HIV pandemic. The synergistic interplay between these two pandemics has necessitated an understanding of how these two viruses interact with each other to tailor an intervention. This thesis investigated the effect of SARS-CoV-2 infection on disease dynamics, differential disease outcomes, vaccine response, as well as SARSCoV- 2 evolution in people living with HIV (PLWH) with different levels of HIV suppression and differing HIV suppression history. The first study investigated the difference in disease severity amongst PLWH in the first and second infection waves in South Africa. COVID-19. Thereafter, we explored persistent SARS-CoV-2 infection in immunocompromised individuals and intra-host evolution in a case study of a participant with advanced HIV infection. Here, we illustrated that advanced HIV disease may lead to prolonged SARS-CoV-2 infection and shedding of infectious virus and results in intra-host evolution of variant mutations, making intra-host evolution in advanced HIV individuals a particular concern within the South African context. Finally, we observed that effectively controlling HIV through ART facilitates SARS-CoV-2 clearance. The last study widened the observations to five participants with advanced HIV disease and showed that vaccination does induce a potent neutralizing antibody response in this group, but only if HIV viremia is first effectively suppressed with antiretroviral therapy. These findings highlight the importance of suppressing HIV infection in eliciting an effective immune response against, and preventing evolution of SARS-CoV-2. Iqoqa I-SARS-CoV-2 yisifo esesigulise umhlaba wonke sase sigulisa inani elingama 672,115,430 abantu emhlabeni jikelele (https://coronavirus.jhu.edu/map.html, accessed 08.02.2023). I-SARS-CoV-2 isaqhubeka nokusabalala kanti izinhlobo zayo ezinye zisaqhubeka nokuba yingozi ukuba isifo singanqandeka. Khona-manjalo, iNingizimu Afrikha isathwele kanzima ukulawula isifo sesandulela-ngculazi. Ukuthelelana nokuxhumana phakathi kwalezi zifo kwenze kwanesidingo sokuqonda ukuthi zidlelana kanjani ukwenzela ukuphuma nekhambi. Lolu cwaningo lucwaninge umthelela we SARS-CoV-2 nezinkinga ezikhona ngenxa yazo, imiphumela ehlukene yesifo, ukusebenza kwekhambi, kanjalo nokukhula kwe SARS-CoV-2 ebantwini abaphila naso lesi sifo bebe benesandulela-ngculazi benezinga lesandulela-ngculazi elehlukile kanye nomlando owehlukile. Ucwaningo lokuqala lucubungule umehluko wesankahlu sesifo ezifweni ze-PLWH esiwombeni sokuqala nesesibili salesi sifo eNingizimu Afrikha. Ngakho-ke iye yahlolwa kwalendela umthelela we-SARS-CoV-2 kubantu abanamasosha aphansi omzimba kanye nalokhu esikubiza nge-intra-host evolution ocwaningweni lwesimo kubabambi-qhaza abanesandulela-ngculazi esisezingeni eliphezulu. Lapha kutholakale ukuthi isandulela-ngculazi esisezingeni eliphezulu singaholela ekuguleni isikhathi eside uma unesifo seSARS-CoV-2 kanye nokuthola ezinye izifo ezithelelanayo kanti imiphumela ye-intra-host evolution of variant mutations, okwenza ukukhula kwe-intra-host kulabo abanesandulela-ngculazi ephezulu kusabise isimo saseNingizimu Afrikha. Isiphetho, kutholakale ukuthi ukulawula isandulela-ngculazi nge-ART kwenza iSARS-CoV-2 idambe. Ucwaningo lokugcina luveze ngokubanzi obekubukelwe kubabambiqhaza abanesandulela-ngculazi esiphezulu ngokuthi kuvele ukuthi ikhambi lenza kudambe ukugula kuleli qoqo, kodwa kuphela uma isandulela-ngculazi siqale sacindezelwa nge-antiretroviral therapy. Lemiphumela iveza ukubaluleka kokucindezelwa kwesandulela-ngculazi ukulwa kanye nokucindezela isifo seSARS-CoV-2.
Effect of hybrid immunity on the neutralizing antibody response to emerging SARS-CoV-2 variants including in people living with HIV=Umphumela wamasotsha omzimba ahlobongxube ekutheneni amandla impendulosenzo yezivikelamzimba ekuhlanganiseni izinhlobokuguquka zamagciwane kubandakanya nabantu abaphila ne-HIV.
(2022) Khan, Bibi Khadija.; Sigal, Alexander.
This work focused on the effect of SARS-CoV-2 variants and co-infection with HIV on the neutralizing antibody response elicited by SARS-CoV-2 infection and vaccination. The first study described the effect of HIV status and suppression on antibody neutralization capacity against the Delta variant elicited by previous infection, the Janssen adeno vectored Ad26.CoV2.S vaccine, or hybrid immunity from infection followed by Ad26.CoV2.S vaccination. It was determined that while neutralizing immunity elicited by infection decreased in people living with HIV (PLWH) and particularly in those with unsuppressed HIV viremia, this effect was counteracted with hybrid immunity from infection and Ad26.CoV2.S vaccination, where with hybrid immunity neutralization levels increased and differences between PLWH and HIV negative individuals became non-significant. With the emergence of the Omicron variant, we investigated whether Omicron infection boosts cross-neutralizing antibody levels against other variants. It was determined that this cross-protection does occur but is considerably stronger in people with hybrid immunity consisting of Pfizer BNT162b2 mRNA or the Ad26.CoV2.S vaccination followed by breakthrough infection, implying that hybrid immunity from Omicron variant infection and vaccination may prevent infection with other, more pathogenic variants. Lastly, we demonstrated the escape of the BA.4 and BA.5 subvariants from Omicron BA.1 subvariant elicited immunity. It was found that BA.4 and BA.5 showed substantial escape from BA.1 elicited immunity in the absence of vaccination, but the escape was more moderate in individuals with hybrid immunity from BA.1 infection combined with vaccination. Overall, these studies show the capacity of hybrid immunity, consisting of vaccination and infection, to reduce the negative effects of HIV viremia and emerging variants on the ability of the pre-existing immunity to neutralize SARS-CoV-2. Iqoqo Lo msebenzi wawugxile emiphumeleni yezinhlobokuguquka zamagciwane e-SARS-CoV-2 kanye nesifo eziphila neHIV ekutheneni amandla impendulosenzo yezivikelamzimba ezidalwa isifo i-SARS-CoV-2 kanye nokugomakuhlungisa. Ucwaningo lokuqala lwachaza ngomumo nokucindezela kwesilinganiso sokuthena amandla isivikelimagciwane kuhlobokuguquka iDelta ezidalwa isifo esedlule, iJanssen adeno ithwala isifo somgomo we-Ad26.CoV2.S noma amasotsha omzimba anhlobongxube asuka esifweni esilandelwa ukugomakuhlungisa kwe-Ad26.CoV2.S. Kwanqunywa ukuthi ngenkathi amasotsha omzimba ethenwa amandla okudalwa ukwehla kwesifo ebantwini abaphila ne-HIV (PLWH) ikakhulukazi kulabo elingacindezelwe egazini igciwane le-HIV, lo mphumela wawuphikisana namasotsha anhlobongxube asuka esifweni kanye nakumgomokuhlungisa we-Ad26.CoV2.S, lapho amazinga okuthena amandla amasotsha anhlobongxube ekhula kanye nomehluko phakathi kwe-PLWH kanye nalabo bantu abangenayo i-HIV abavele babe ngabangekho mqoka. Ukuqhamuka kohlobokuguquka kwegciwane i-Omicron, saphenya ukuba ngabe isifo i-Omicron ikhuthaza amazinga esivikelamzimba esithena amandla kwezinye izinhlobokuguquka zamagciwane. Kwanqunywa ukuthi ukuvikelana akwenzeki kodwa kuthathwa ngokunamandla kubantu abanamasotsha anhlobongxube equkethe i-Pfizer BNT162b2 mRNA noma umgomokuhlunga i-Ad26.CoV2.S okulandelwa isifo esehlula umgomo, okusho ukuthi amasotsha anhlobongxube asuka esifweni sohlobokuguquka kwegciwane i-Omicron kanye nokugomakuhlunga kungasivimba isifo nezinye eziningi zinhlobokuguquka zemindeni yamagciwane. Okokugcina, sabonisa wukuphunyuka kwezinhlokuguquka zamagciwane ezincane i-BA.4 ne-BA.5 kusuka hlobokuguquka kwegciwane okuncane i-Omicron BA.1 eveza amasotsha omzimba. Kwatholakala ukuthi i-BA.4 ne-BA.5 ikhombisa ukuphunyuka okubonakalayo ukusuka ku-BA.1 eveza amasotsha omzimba uma kungenziwanga ukugomakuhlungisa kodwa ukuphunyuka kwakungakukhulu kulabo abanamasotsha omzimba anhlobongxube kusuka esifweni i-BA.1 ihlanganiswe nokugomakuhlungisa. Phezu kwakho konke, lolu cwaningo lwakhombisa isilinganiso samasotsha omzimba anhlobongxube, aqukethe abunjwe umgomokuhlungisa nesifo, ukuze kuncishiswe imiphumela engemihle yamagciwane asegazini e-HIV kanye nezinhlobokuguquka zamagciwane ekukwazini ukuthi amasotsha omzimba abevele ekhona ngaphambilini athene amandla i-SARS-CoV-2.
Effect of the capsular material of cryptococcus neoformans on the interplay between Mmicroglial cells and Neutrophils.
(2019) Bhola, Prathna.; Sturm, Adriaan Willem.
Cryptococcal meningitis is an important opportunistic infection in immunocompromised patients. It has been well established that a distinguishing feature of this form of meningitis is a relatively low neutrophil count in the cerebrospinal fluid (CSF) compared to bacterial meningitis. There has been speculation and research undertaken previously to understand this phenomenon, however, little information is available in human studies. Furthermore, there is insufficient information on expression and function of Toll-like receptors (TLR) in the human central nervous system (CNS). The work presented here investigated the effect of the capsular material of a series of clinical isolates of Cryptococcus neoformans on neutrophil recruitment at the site of infection and determined whether downregulation occurs at the level of TLR expression. This was done in a multiple component study. Clinical information was collected from patients with cryptococcal meningitis and baseline blood and CSF investigations were performed, which included the quantification of neutrophils in CSF and blood specimens. The size of the Cryptococcus capsule was measured in each isolate and shed capsular material was quantified in individual CSF specimens. The extent of neutrophil chemotaxis inhibition by individual strains of C. neoformans was determined by using a Transwell migration assay. Toll-like receptor (TLR)2 and TLR4 gene expression induced by individual C. neoformans isolates in human microglial cells was quantified. The possible associations among these experiments were subsequently evaluated. As anticipated, a paucity of neutrophils in the CSF was observed. The cryptococcal capsule was larger in isolates of patients with lower CSF neutrophil counts. In addition, patients with lower CSF neutrophil counts shed more capsular material in the CSF. Chemotaxis inhibition occurred in close to 70% of tested isolates. The concentration of shed capsular material in this group was higher compared to the group with no chemotaxis inhibition. Patients presenting with fever had higher CSF neutrophil counts as well as elevated intracranial pressures. The majority of isolates expressed downregulation for TLR2 and TLR4 in microglial cells exposed to C. neoformans. CSF neutrophil counts were lower in this group. These findings imply that the capsular components of C. neoformans downregulated recruitment of neutrophils into the CSF. Downregulation of neutrophil recruitment was observed at the level of TLR expression.
Effectiveness of a monovalent human rotavirus vaccine among children of 5 years and under in KwaZulu-Natal.
(2016) Asowata, Osaretin Emmanuel.; Moodley, Prashini.
Human rotavirus infection is the leading cause of gastroenteritis in infants and young children worldwide. In South Africa, gastroenteritis is a major cause of childhood morbidity and mortality in children less than 5 years, and rotavirus infection has been documented as causing one-third of all gastroenteritis related hospital admissions. Vaccination is the major public health intervention to control rotavirus disease. The Rotarix® is the only rotavirus vaccine included in the national immunization program of South Africa. The effectiveness of this vaccine is questionable due to the continual outbreaks of rotavirus infection in South Africa, including KwaZulu-Natal, regardless of the high vaccination coverage. This study focused on evaluating the factors influencing the effectiveness of the Rotarix® vaccine in children 5 years and under in KwaZulu-Natal, South Africa. After obtaining written informed consent from parents or guardians, stool and blood specimens where collected from children 5 years and under presenting to King Edward VIII hospital (KEH VIII) in Durban, South Africa. The study was conducted between June 2014 and June 2015. Demographic and clinical information was collected using a well-structured questionnaire. Enzyme immunoassay (EIA) was performed to detect rotavirus antigen in the stool and rotavirus immunoglobulin G (IgG) in the serum. Selected EIA positive and negative samples were confirmed using G-types and P-types consensus primers in a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The RT-PCR positives were genotyped using genotypes specific primers. The avidity of the rotavirus specific IgG was determined using the urea elution technique. Rotarix® vaccines stored at optimum temperatures were collected from the provincial pharmaceutical store. The effect of sub-optimal temperatures on the potency of the Rotarix® vaccine were determined using the plaque assay. Three hundred and sixty-five (365) stool specimens were collected. Rotavirus antigen was detected in 83 (22.7%) patients from stool specimens. The stratification of rotavirus cases by vaccination status was not significant (p=0.4). The distribution of rotavirus was not significantly associated with HIV status of the children (p=0.7). We observed that seasonality was a significant driving force influencing the prevalence of rotavirus infection in our setting (p<0.001). We recorded the highest rotavirus prevalence in the winter months of the year with 79 (45.9%) positive cases of rotavirus associated diarrhoea. Blood specimens were only collected in 35 patients. From the corresponding stool specimens [21 (60%) EIA positives and 14 (40%) EIA negatives)], 29 (82.9%) were positive for rotavirus using conventional RT-PCR. Genotyping revealed G9P[8] (20.7%) to be the most prevalent genotype followed by G9P[4] (13.8%), G12P[4] (10.3%), G9P[6] (6.9%) and a 3.4% prevalence was recorded for each of G4/G8P[6], G4P[6], G12P[6], G8P[10] and G9P[10]. We were unable to fully genotype some of the rotavirus strains (non-typeable) by the available primers. 2 (6.9%) and 4 (13.8%) were non-typeable for the G and P types respectively. However, all 35 serum samples were positive for rotavirus IgG. We observed that the rotavirus specific IgG had no significant effect on the prevalence of rotavirus detection in stool (p=0.8). There was no significant difference in the mean avidity of IgG in the 3 vaccination strata (p=0.3). Exposure of the Rotarix® vaccine to the seasonal temperatures and to extreme temperatures of 40oC for 3 to 72 hours as well as -20oC and -80oC for 12 hours did not affect the potency of the vaccine beyond its expected standard. Our study highlighted the genetic diversity of rotaviruses and poor immunogenicity of the vaccine as key factors affecting the effectiveness of the rotavirus vaccine. Whether the vaccine is able to induce homotypic and heterotypic protection in immunized children is critical in predicting the long range effectiveness of this vaccine against uncommon regional rotavirus strains. Interventions targeted at improving socio-economic conditions in low income countries might be a starting point towards the control and prevention of rotavirus infection in these settings.
Enterococcus sp. contamination surveillance in different levels of healthcare in eThekwini District, KwaZulu-Natal (KZN) South Africa.
(2021) Shobo, Christiana Omowunmi.; Bester, Linda Antionette.; Essack, Sabiha Yusuf.
Hospital-acquired infections (HAIs) have been identified as long-standing setbacks affecting hospitals' quality of health care. While one of the major challenges related to HAIs is controlling cross-transmission, the role and significance of the inanimate hospital environment chain of transmission are yet to be unequivocally elucidated. Therefore, this study investigated the functional profile and diverseness of bacteria from various inanimate environmental sources, from two different wards in public hospitals at various healthcare levels in eThekwini District, KwaZulu-Natal, South Africa. True to the study focus on investigating the dissemination of bacteria from equipment within the hospital, the study further used Enterococcus as well-known HAI as target bacteria and described the molecular and genomic profiles of this specie isolated from the hospital environments. Samples were collected for a period of three months (September – November 2017) from the four levels of healthcare in eThekwini district, KwaZulu-Natal. The intensive care unit and peadiatic ward were employed in this study. An overall of 620 swabs were collected from areas frequently touched by healthcare workers (HCWs) and patients. These sites include the occupied bed linen, unoccupied bed linen, drip stands, patient files, ward phones, ventilators, nurses' tables, blood pressure apparatus, sinks, linen room door handle and mops. Swabs were placed in Amies transport medium and transported in a cooler box to the laboratory facility to be processed within four hours. The collected swabs (n=620) were pooled and incubated in tryptone soya broth containing 6.5% NaCl at 36.5oC for 24 hrs and subsequently plated on enterococci chromogenic media. The microbial diversity and functional profiles from the sites were identified using 16S rRNA metagenomics. Positive colonies were sub-cultured on bile esculin azide agar, and screened using standard microbiological methods, including haemolytic, oxidase and catalase, and API. Identifications were confirmed with polymerase chain reaction (PCR) with the added genus-specific tuf-gene and species-specific sodA-gene. Antibiotic resistance patterns in the Enterococcus spp. isolates were determined by the Kirby-Bauer disk diffusion method against 14 antibiotics as recommended by the Clinical and Laboratory Standard Institute (CLSI) guidelines. Thirty-seven samples from E. faecalis showed intermediate Resistance to vancomycin and were further analyzed using molecular tools viz. whole-genome sequencing (WGS) and bioinformatics analyses. This enabled determining the resistome, mobile genetic elements (MGEs), and clonal lineages circulating across the sites, wards, and hospitals. Metagenomics identified a total of 288 species, 190 genera, 105 families, 50 orders, 29 classes and 11 phyla from the samples analyzed. The dominant functional metabolic pathways implicated in causing human infection discovered were the signal transduction mechanisms, citrate cycle (TCA), transcription-factor bisphenol degradation, tyrosine metabolism. A total of 295 Enterococcus spp. isolates were recovered from the hospitals` environmental sites, 83% (n=245) were identified as Enterococcus faecium, 13% (n=38) as Enterococcus faecalis, 2% (n=6) Enterococcus gallinarum and another 2% (n=6) Enterococcus casseliflavus. Notably, the pediatric wards had the highest isolation rate compared to ICU, 64% and 36%, respectively. Overall, the sites with the highest isolation rate were occupied beds and mops (to clean ward floors) with 14.9% (n=44) each. The tertiary hospital were the most affected. The most prominent MDR antibiogram for E. faecium was CIP-RIF-NIT-TET-ERY and for WGS analysis of the E. faecalis samples confirmed that the tet(M) and erm(C) genes were the prevalent antibiotic resistance genes found in hospitals. The isolates harboured mobile genetic elements consisting of plasmids (n =11) and prophages (n=14), predominantly clonally specific. The 37 isolates analyzed consisted of 15 clonal lineages with six major sequence types (ST). Phylogenomic analysis showed that major lineages were mostly conserved within specific hospital environments. This study highlighted the inanimate hospital environment as a possible source of opportunistic nosocomial pathogens using Enterococcus as an illustrative example and emphasized the urgent necessity to optimize infection prevention and control measures to intercept/moderate the spread of bacteria in the hospital environments.
Epidemiology and alternative approaches for SARS-CoV-2 testing within limited resources settings=Isifundo ngembangela nokusabalala kwezifo i-epidemiology nezindlela ezahlukile zokuhlolwa kwe-SARS-CoV-2 esimeni sokwesweleka kwezinsiza.
(2023) Duma, Zamathombeni.; Mkhize-Kwitshana, Zilungile Lynette.; Chuturgoon, Anil Amichund.; Ramsuran, Veron.
Background: In the context of the global battle to contain the rapidly mutating SARS-CoV-2, diagnostic testing for SARS-CoV-2 infection remains a challenge, particularly in low-middleincome countries (LMICs) due to low socioeconomic backgrounds. Concerningly, because less attention is paid to asymptomatic cases, particularly in LMICs with limited resources for SARSCoV- 2 testing, the virus is spreading silently in communities, and the majority of these individuals could be contributing to the resurgence of SARS-CoV-2 infection. This study aimed to determine the epidemiology and alternative approaches for SARS-CoV-2 testing within limited resources settings. Methods: A total sample size of 1335 residual patient samples from the Global Health Innovation (GHI) laboratory was used for the epidemiology study and methods comparison. Results and Discussion: Literature review showed that high income countries (HICs) test more frequently for SARS-CoV-2 infection, with a range of 113% to 146% higher than LMICs (1% to 43%). The present study demonstrated a higher proportion of asymptomatic cases (68%) among SARS-CoV-2 infected patients. Regarding the methods comparison for the detection of SARSCoV- 2, the evaluated alternative methods [three RNA extraction (Lucigen QuickExtract™ RNA Extraction Kit, Bosphore EX-Tract Dry Swab RNA Solution, Sonicator method and four commercial SARS-CoV-2 RT-PCR assay kits (Nucleic Acid COVID-19 Test Kit (SARS-CoV-2), abTESTM COVID-19 qPCR I Kit, PCL COVID19 Speedy RT-PCR Kit, and PCLMD nCoV One- Step RT-PCR Kit)] were found to be cheaper and faster. Conclusion: Notably LMICs are undertesting for SARS-CoV-2 infection compared to HICs, and there was a higher proportion of asymptomatic cases among SARS-CoV-2 infected patients in South Africa. This study suggests that using the above-mentioned cost-effective, quick, and accurate evaluated alternative methods for mass SARS-CoV-2 testing in routine diagnostic laboratories with limited resources can help to increase testing capacity for SARS-CoV-2 infection in LMICs. This means that the sooner SARSCoV- 2 infection control and prevention measures can be implemented to reduce community transmission. IQOQA Isendlalelo: Esimeni somzabalazo womhlaba jikelele wokuvimba i-SARS-CoV-2 eguquguquka ngokushesha, uvivinyo oluhlolayo lokutheleleka nge-SARS-CoV-2 luselokhu luyinselelo, ikakhulu emazweni anemiholo ephansi kuya kwephakathi nendawo (low-middle income countries - MICs) ngenxa yesizinda senhlalomnotho ephansi. Ngokuxakayo-ke, ngenxa yokunganakwa kokutheleleka okungenazimpawu, ikakhulu ama-LMIC anezinsiza zokuhlolela i-SARSCoV-2, igciwane liyanda buthule emiphakathini, futhi iningi lalaba bantu lingase libe nomthelela yokuvembuka kabusha kokuthelelana nge-SARSCoV-2. Lolu cwaningo lwaluhlose ukuthola imbangela nokusabalala kwezifo i-epidemiology nezinye izindlela zokuhlola i-SARSCoV-2 esimeni sokwesweleka kwezinsiza. Izindlela: Kwasetshenziswa isampula eliphelele lamasampula eziguli ezisilele eziyi-1332 avela emalaborethri akwaGlobal Health Innovation (GHI) ngokocwaningo lwe-ephidemiyoloji nokuqhathaniswa kwezindlela. Imiphumela nengxoxo: Imibhalo eyabuyekezwa yakhombisa ukuthi amazwe anemiholo ephezulu (high income countries - HICs) ahlolela ukutheleleka kwe-SARS-CoV-2 kaningana ngokwebanga eliyi-113% kuya kweliyi-146% ngaphezu kwama-LMICs (1% kuya kuma-43%). Lolu cwaningo lwakhombisa isibalo esiphezulu lotho olungenazimpawu (68%) phakathi kweziguli ezitheleleke nge-SARS-CoV-2. Mayelana nezindlela zoqhathaniso lokuhlolwa kwe-SARS-CoV-2, izindlela ezahlukile ezihloliwe zokukhishwa kokuthathu (Lucigen QuickExtract™ RNA Extraction Kit, Bosphore EX-Tract Dry Swab RNA Solution, nendlela iSonicator) nezinsiza ze-assayi i-SARS-CoV-2 RT-PCR (Nucleic Acid COVID-19 Test Kit (SARS-CoV-2), abTESTM COVID-19 qPCR I Kit, PCL COVID19 Speedy RT-PCR Kit, ne-PCLMD nCoV OneStep RT-PCR Kit)] okwatholwa kushibhile futhi kushesha. Isiphetho: Ngokuqaphelekayo ama-LMIC akuhlolela kancane ukutheleleka nge-SARS-CoV-2 uma kuqhathaniswa nama-HIC, futhi kwakunesibalo esiphezulu sotho olungenazimpawu phakathi kweziguli ezitheleleke nge-SARS-CoV-2 eSouth Africa. Lolu cwaningo lukhomba ukuthi ukusebenzisa izindlela ezibalwe ngenhla ezishibhile, ezisheshayo, futhi ezinembayo nezihloliwe futhi ezingezinye izindlela zokuhlola isisindo se-SARS-CoV-2 engukuhlola okwejwayelekile emalabhorethri anezinsiza ezingeziningi kungasiza ekukhuliseni ukukwazi ukuhlola kokutheleleka nge-SARS-CoV-2 ema-LMIC. Lokhu kuchaza ukuthi uma kungashesha ukulawulwa kokutheleleka nge-SARS-CoV-2 nezinyathelo zokuvikeleka kungenziwa ukunciphisa ukuthelelana emphakathini.
Fitness of multi-and extensively drug-resistant mycobacterium tuberculosis clinical strains.
(2015) Naidoo, Charissa Camille.; Pillay, Manormoney.
The biological fitness of a pathogen is defined as its ability to reproduce, survive, cause disease and be transmitted. Drug-resistant M. tuberculosis isolates often exhibit reduced competitive ability against susceptible isolates in the absence of drug selection. However, compensatory mutations may, to some extent, offset fitness costs associated with resistance-conferring mutations. Most studies to date have focused on the fitness of isogenic laboratory strains or globally-relevant strains. Fewer studies have addressed the fitness of endemic strains which propagate in multidrug and extensively drug-resistant forms (MDR and XDR, respectively). In this study, the fitness of four genotypes which drive the transmission of tuberculosis (TB) in KwaZulu-Natal (KZN), South Africa was explored. This included F15/LAM4/KZN genotype strains, some of which were involved in the notorious XDR-TB outbreak in Tugela Ferry, KZN as well as Beijing, F11 and F28 genotype strains. Drug-resistant F15/LAM4/KZN and F28 genotype strains demonstrated increased in vitro fitness, whilst Beijing and F11 MDR strains had markedly reduced fitness. These findings correlated with whole-genome and Sanger sequencing data which revealed the presence of low/no-cost resistance-conferring mutations as well as intra- and intergenic compensatory mutations in drug-resistant F15/LAM4/KZN and F28 strains, respectively. In contrast, high cost katG mutations and no accompanying compensatory mutations were identified in Beijing and F11 MDR strains. During co-culture experiments, a novel observation was made whereby susceptible and resistant strains exhibited synergistic growth compared with axenic cultures i.e. in vitro trans-complementation. Drug-resistant F15/LAM4/KZN strains did not undergo fitness costs in THP-1 macrophages and produced increasing levels of TNF-α which may enhance tissue destruction and dissemination to other hosts. Although demonstrating similar intracellular fitness, susceptible and MDR Beijing, F11 and F28 strains induced heterogeneous cytokine responses. Thus, the lack of a direct relationship between bacillary burden and cytokine responses indicates that this diversity results from strain heterogeneity. Relapse isolates, including those from F15/LAM4/KZN and Beijing genotypes, may reactivate without any changes in biological fitness, thereby retaining the potential to re-transmit. Taken together, the enhanced fitness of drug-resistant F15/LAM4/KZN and F28 genotype strains is due to the presence of beneficial mutations, while the reduced fitness of MDR Beijing and F11 strains is associated with high cost mutations.
Genetic and microrna polymorphisms in young South African Indians with coronary artery disease.
(2015) Ramkaran, Prithiksha.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.; Khan, Sajidah.
The global burden of cardiovascular disease (CVD) is on the increase with coronary artery disease (CAD) estimated to become the leading cause of mortality worldwide by 2020. The age of onset of this chronic disorder is on the decline, particularly in the South African Indian population. Indians in South Africa (SA) have a higher prevalence of premature CAD compared to other ethnic groups in SA. Coronary artery disease is a lifestyle and genetic disease, and the inheritance of genetic variation from one or both parents plays an important role in the risk of an individual developing CAD. Genetic and epigenetic studies are being explored as potential tools for therapeutic interventions against CVDs. The role of single nucleotide polymorphisms (SNP) in microRNAs (miR, miRNA) and molecules regulating epigenetic pathways remains poorly understood. This study investigated SNPs in candidate genes; methylenetetrahydrofolate reductase (MTHFR), sirtuin (SIRT) 1, miR-499, and miR-146a; in young SA Indians with CAD. The study population included 106 SA Indian male CAD patients, 100 sex- and age-matched Indian, and 84 sex- and age-matched Black controls. The MTHFR, miR-146a, and miR-499 SNPs were investigated by PCR-RFLP, whilst a TaqMan SNP Genotyping assay assessed the SIRT1 SNPs. MiR-146a expression was measured by qPCR and western blot was used to assess the expression of NF-κB, IRAK-1, and TRAF-6. Interleukin (IL)-6 levels were analysed using an ELISA. All clinical parameters were obtained from pathology reports. Methylenetetrahydrofolate reductase is involved in folate metabolism and methylation pathways. The MTHFR rs1801133 has been associated with increased levels of homocysteine, a well known risk factor for CAD. Sirtuin 1, histone deacetylase, has been identified as a candidate molecule affecting the epigenetic mechanisms of CAD. Two common SNPs in the SIRT1 gene, rs7895833 and rs1467568, have been associated with several well-established risk factors for CAD. MicroRNAs (miRNAs) are small noncoding RNA molecules that inhibit messenger RNA (mRNA) translation or promoting mRNA degradation. MiR-499 and miR-146a are inflammatory-associated miRNAs. Two miRNA SNPs, miR-146a rs2910164 and miR-499 rs3746444, have been implicated in chronic inflammatory diseases. There was a significant association between the MTHFR variant (T) allele and CAD patients compared to Indian controls (p=0.0353, OR=2.105 95% CI 1.077–4.114). Indian controls presented with a higher frequency of the T allele compared to Black controls (7% vs. 2% respectively, p=0.0515 OR=3.086 95% CI 0.9958–9.564). The variant allele for the two SIRT1 SNPs occurred more frequently in the total Indian group compared to the total Black population (rs1467568: 41% vs. 18.5% respectively, p<0.0001, OR=3.190 95% CI 2.058-40943 and rs7895833: 41% vs. 22% respectively, p<0.0001, OR=2.466 95% CI 1.620–3.755). Indian controls presented with a higher frequency for both SNPs compared to Black controls (rs1467568: 40% vs. 18.5% respectively, p<0.0001, OR=2.996 95% CI 1.850–4.853 and rs7895833: 41% vs. 22% respectively, p<0.0001, OR=2.513 95% CI 1.578–4.004). No difference was seen in the distribution of both SNPs between CAD patients and either control group. The MTHFR and SIRT1 SNPs were not associated with any clinical parameters in CAD patients and controls. The miR-499 variant (G) allele was found at a higher frequency in the total Indian group (34%) compared to the total Black population (22%) (p=0.0070, OR=1.796 95% CI 1.182–2.730). Indian cases presented with higher frequency of the rs3746444 G allele compared to Indian controls (38% vs. 29%, p=0.059, respectively). No differences in genotypic frequency for rs2910164 was found (GG: 45 vs. 47 %, GC: 46 vs. 41 %, CC: 9 vs. 12 %) in controls and patients respectively (odds ratio=1.025; 95 % confidence interval 0.6782–1.550; p=0.9164). The lowest levels of NF-κB and C-reactive protein (hsCRP) were found in patients with the homozygous C allele compared to the heterozygous GC and wildtype variants. Higher levels of miR-499 targets, hsCRP and IL-6, were observed in CAD patients with the variant genotypes compared to those with the wild type genotypes (8.92±1.91 vs. 6.73±0.87 mg/L; p=0.299, 3.02±0.77 vs. 2.18±0.57 pg/mL; p=0.381 respectively). A 6.25-fold increase in miR-146a levels was observed in CAD patients with the CC genotype (relative to controls and patients with the wildtype variant, p<0.0001). These (CC genotype) patients had significantly lower levels of miR-146a targets, IRAK-1 (0.38±0.02; p=0.0072) and TRAF-6 (0.44±0.02; p=0.0146). Taken together, this study provides the frequency distribution of the SNPs in four candidate genes for CAD in young South African Indians compared to Indian and Black controls. The frequency of variant alleles of rs1801133, rs3746444, rs7895833 and rs1467568 was greater in the Indian population compared to Black South Africans. Although no difference in frequency was observed for rs2010164, our results suggest a role for miR-146a in toll-like receptor (TLR) signalling via a negative feedback mechanism involving the attenuation of NF-κB by downregulation of IRAK-1 and TRAF-6. Thus miR-146a can act as a target for therapy towards lowering inflammation in CAD patients.
Host induced microevolution of ESX secretion systems of M. Tuberculosis.
(2013) Sukkhu, Melisha.; Pym, Alexander S.
The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines.
Hydrogen sulfide (H2S) production by mycobacteria.
(2020) Kunota, Tafara Takunda Remigio.; Steyn, Andrie J. C.
The gasotransmitter hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. Many prokaryotic bacterial species endogenously generate H2S in their natural environments. However, to date, it is not known whether Mycobacterium tuberculosis (Mtb) is an endogenous producer of H2S. In this study, we tested the hypothesis that Mtb endogenously produces H2S to modulate respiration, central metabolism, oxidative stress, and drug susceptibility. We demonstrated that fast-growing non-pathogenic, slow-growing pathogenic mycobacterial species, as well as drug resistant clinical strains of Mtb species produce H2S. Here we demonstrate that fast-growing non-pathogenic M. smegmatis produces barely detectable quantities of H2S, whereas MDR Mtb produces large quantities of H2S. We have also developed a native PAGEbased assay for the rapid screening of H2S producing enzymes in the lysates of mycobacterial species. Using LC-MS/MS, we identified the protein, Rv3684 as an H2S-producing enzyme in Mtb. Disruption of rv3684, demonstrated using the genetic knock out of rv3684, reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. Whole Mtb cell-based and lysate assays showed reduced levels of H2S production in the Mtb knockout strain compared to the wild-type strain. Noticeably, we demonstrated that the Mtb mutant is resistant to oxidative stress and the anti-TB drugs clofazimine and rifampicin. We also found that endogenous H2S is an effector molecule that maintains bioenergetic homeostasis by regulating Mtb respiration, and that H2S also plays a key role in central metabolism by modulating the balance between oxidative phosphorylation (OXPHOS) and glycolysis. In summary, our findings reveal previously unknown concepts of Mtb physiology with respect to Mtb-derived H2S and energy metabolism which has significant implication for routine laboratory culturing, understanding susceptibility and TB disease.
Immunopathogenesis of vulvo-vaginal candidiasis in human immunodeficiency virus infected women.
(2014) Apalata, Teke.; Moodley, Prashini.
Vulvovaginal candidiasis (VVC) is an important cause of lower genital tract infections in women. There are currently numerous clinical observations linking increased cases of symptomatic VVC to the progression of HIV epidemic. While the pathogenesis of other commonly encountered mucosal candidiasis (oral and oesophageal) in the context of HIV infection has been well studied, gaps in our knowledge remain regarding candida vaginitis. With increasing degree of immunosuppression, symptomatic VVC in HIV infected women is frequent, severe, recurrent and less responsive to conventional anti-fungal therapy. The quality of life is greatly diminished for women who experience recurrent episodes of symptomatic VVC. Furthermore, the high prevalence of HIV infected women adds to the burden of healthcare. In this study, we sought to further understand the pathogenesis of symptomatic VVC and the associated host defense mechanisms in HIV infected women. The results of this study are presented as a collection of 5 papers, 4 of which are published in peer reviewed journals, 1 is still under review. The initial chapter of this thesis, ‘Introduction and Background’, is followed by the 5 manuscripts which are grouped into 3 consecutive result chapters as follows: I. Impact of HIV on symptomatic VVC. II. Impact of symptomatic VVC on HIV RNA levels in plasma and genital secretions. III. Plasma and vaginal-associated immune responses in women with symptomatic VVC. The final chapter, ‘Discussion and Conclusions’, rounds up the thesis.
The impact of semen exposure on the immune and microbial environments of the female genital tract.
(2021) Jewanraj, Janine.; Liebenberg, Lenine Julie.; Ngcapu, Sinaye.
Background: Semen is an immunomodulatory fluid that induces mucosal changes at the female genital tract (FGT) for sperm survival and conception. Semen-induced alterations necessary for reproduction may also modulate the inflammatory environment related to HIV risk in women. This thesis investigated the impact of semen exposure on biomarkers of female genital inflammation (GI) and the persistence of these associations over time. Methods: Stored genital specimens were assessed from HIV-negative women participating in the CAPRISA 008 trial. Cervicovaginal lavage (CVL) samples were screened for Y-chromosome DNA (YcDNA) by real-time PCR as a biomarker of semen exposure within 15 days of genital sampling. Prostate-specific antigen (PSA) detection by ELISA stratified CVLs into semen exposure within 48 hours (PSA+YcDNA+) and between 3-15 days (PSA-YcDNA+). Vaginal cytokine concentrations, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were assessed in CVLs using multiplexed ELISA. Endocervical T-cell frequencies were measured in cytobrushes by flow-cytometry. Vaginal microbes and sexually transmitted infections (STIs) were detected in vulvovaginal swabs by PCR. Results: Self-reported condom use as a measure of semen exposure was not associated with changes in the FGT microenvironments. Conversely, YcDNA detection predicted significant increases in several cytokines, barrier-related proteins, and Prevotella bivia detection (p=0.001). Since YcDNA detection alone was not associated with the immune environment linked to HIV risk, this thesis further investigated the contribution of more recent sex to female GI. PSA detection (semen exposure within 48 hours) was associated with higher YcDNA concentrations (p<0.0001), suggesting a relationship between the timing of semen exposure and vaginal YcDNA concentrations after condomless sex. In support of this, both PSA detection and higher YcDNA concentrations predicted significant increases in several cytokines, barrier-related proteins (MMP-2, TIMP-1, TIMP-4), and higher frequencies of activated CD4+HLA-DR+ T-cells (p=0.032) and CD4+CCR5+HLA-DR+ HIV targets (p=0.046). PSA detection was also associated with increased detection of several bacterial vaginosis (BV)-associated microbes and reduced Lactobacillus jensenii detection. Conclusion: Recent semen exposure contributes to the inflammatory environment associated with HIV risk in women. These studies highlight the need for clinical and immunological studies of STIs and their biomedical interventions to consider semen’s contribution to the immune and microbial microenvironments of the FGT.
The impact of sexually transmitted infections (STI) and genital tract inflammation on HIV-1 acquisition and rate of disease progression in subtype C infected women.
(2014) Mlisana, Koleka Patience.; Kharsany, Ayesha Bibi Mahomed.; Passmore, Jo-Ann Shelley.
Introduction: Women carry more than half the burden of HIV disease globally and this burden is even higher in sub-Saharan Africa (SSA). Young women, in particular, are at disproportionate risk of HIV infection in South Africa. Understanding risk behaviours and factors associated with ability to negotiate safe sex and condom use is one of the key elements in curbing the spread of HIV. Sexually transmitted infections (STI) and bacterial vaginosis (BV), which cause female genital tract inflammation, have been identified as key drivers of the HIV epidemic. This inflammation, which is also present in the absence of symptoms, is associated with increased susceptibility to HIV infection. Although syndromic management of symptomatic STIs or BV at the first encounter with a health care provider is an important public health measure, its effectiveness is minimised because a substantial proportion of individuals have either asymptomatic infections or fail to recognise signs and symptoms of STI and are therefore excluded. Most new HIV infections in SSA occur among young people and particularly among young girls. Prompt diagnosis of acute HIV infection (AHI) is critical and benefits the individual as well as providing opportunities for public health intervention. In South Africa, the majority of HIV infections are due to infection with HIV-1 subtype C for which there is limited data compared to subtype-B HIV-1 infections. The overall aim of this study was to assess the impact of BV, STIs, and associated genital tract inflammation on acquisition of HIV-1 subtype C infections; and evaluate the rate of subsequent disease progression in women. The objectives were: i. to investigate STIs and genital tract inflammation as risk factors for HIV infection in high risk women and adequacy of syndromic management; ii. to evaluate the challenges associated with diagnosing recently acquired (acute) HIV infection in a subtype C prevalent population; iii. and to evaluate the relationship between clinical disease progression and genital and or systemic inflammation in high-risk women who became infected with HIV. We assessed the adequacy of syndromic diagnosis of STIs, compared with laboratory diagnosis of STIs, and evaluated the association between STI diagnosis and the risk of HIV acquisition in a cohort of high-risk women. Genital cytokine profiles and the degree of inflammation associated with common STIs and bacterial vaginosis were assessed. The most common signs and symptoms of acute HIV infection (AHI) were described and a clinical algorithm to identify acute HIV cases was developed. We investigated rates of HIV disease progression of subtype C–infected South African women. Methods: The CAPRISA 002 study was a prospective cohort study established to examine the pathogenesis and natural history of HIV-1 subtype C infection and to describe the immunologic, virologic and clinical characteristics of acute and early infection in KwaZulu Natal, South Africa. A total of 775 high-risk women were screened for HIV infection, and 245 HIV-uninfected women were enrolled into the study. At each monthly visit for a total of 24 months behavioural and clinical data were collected. Cervico-vaginal lavage (CVL) samples were collected at enrolment and at each six month follow-up visits and were tested for STI pathogens (including Chlamydia trachomatis, Neisseria gonorrhoeae, herpes simplex virus type 2 (HSV-2) and Trichomonas vaginalis) and bacterial vaginosis. Forty-two cytokines were measured from the CVL and 13 from the plasma samples at enrolment. All women received monthly HIV-1 antibody and RNA testing and were assessed for AHI. Signs and symptoms at the visit with HIV-1 antibody or HIV-1 RNA positive test were compared to HIV negative visits. Logistic regression identified clinical predictors of AHI and a model-based score was assigned to each predictor to create a risk score for every woman. All women who seroconverted were followed up for more than five years and monitored for HIV disease progression. Rapid disease progression was defined as CD4+ T cell count decline to <350 cells/μl within two years post-infection. Serial clinical and laboratory assessments were compared using survival analysis and logistic regression models. CAPRISA had established several cohorts of HIV negative women to determine the feasibility of establishing cohorts and sites for HIV biomedical prevention trials. Women seroconverting in these studies were referred to the CAPRISA 002 study for longitudinal follow-up for HIV post seroconversion. Results: In this study, the HIV-1 prevalence at screening was 59.6% (95% CI: 55.9% to 62.8%). During a total of 390 person-years of follow-up, 28 new infections occurred, yielding a seroincidence rate of 7.2 (95% CI: 4.5 to 9.8) per 100 person-years. A total of 62 participants, including seroconvertors from other CAPRISA cohorts, were enrolled into the acute HIV infection Women from the HIV-1 negative cohort generally demonstrated a high level of knowledge on HIV/AIDS, and 60.3% reported use of condoms. Reported condom use at last sexual encounter varied slightly by partner type (57.0% with steady versus 64.4% with casual partners; p = 0.36), whilst self-perceived ability to choose to use a condom was significantly lower with steady partners compared to casual partners (20.8% versus 53.9%; p=0.01). An important finding was that vaginal discharge was a poor predictor of laboratory-diagnosed STIs, as only 12.3% of women (25/204) who had a laboratory-confirmed discharge causing pathogen had clinical evidence of a discharge (yielding a sensitivity of 12.3% and a specificity of 93.8%). CVL cytokine concentrations did not differ between women with asymptomatic or symptomatic STIs; and were elevated in women with either asymptomatic or symptomatic STIs compared to women with no STIs or BV. Women with chlamydia or gonorrhoea had the highest genital cytokine concentrations, with 17/42 and 14/42 of the cytokines measured in CVL being up-regulated compared with women with no infections, respectively. While BV was associated with elevated pro-inflammatory cytokine concentrations in CVL, women with BV had lower levels of chemokines and haematopoietic cytokines than women with no infections or BV. HSV-2 reactivation was inflammatory, but yielded a comparatively lower level of inflammation than Chlamydia or gonnorhoea. Trichomoniasis, despite being relatively common in this cohort, did not cause significant changes in genital tract cytokine concentrations compared to women with no infections or BV. Genital infections did not influence plasma cytokine concentrations, suggesting that the compartments were not linked with respect to cytokine responses. Although laboratorydiagnosed STIs were associated with increased risk of HIV infection [hazard ratio, 3.3 (95% confidence interval, 1.5 – 7.2)], clinical symptoms were not. Of the women who became infected, factors predictive of AHI included age, 25 years (OR = 3.2; 1.4 – 7.1), rash (OR = 6.1; 2.4 –15.4), sore throat (OR = 2.7; 1.0 – 7.6), weight loss (OR = 4.4; 1.5 – 13.4), genital ulcers (OR = 8.0; 1.6 – 39.5) and vaginal discharge (OR = 5.4; 1.6 – 18.4). A risk score of 2 correctly predicted AHI in 50.0% of cases. The number of signs and symptoms correlated with higher HIV-1 RNA at diagnosis (r = 0.63; p = 0.001). The 62 acutely infected women were identified at a median of 42 days post-infection (IQR = 34 – 59). Mean CD4 count dropped by 39.6% at 3 months and 46.7% at 6 months post-infection in women with pre-infection measurements. CD4 decline to <350 cells/μL occurred in 31%, 44%, and 55% of women at 1, 2, and 3 years post-infection, respectively, and to <500 cells/μL in 69%, 79%, and 81% at equivalent time-points. Predictors of rapid progression were CD4 count at 3 months post-infection (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.31–3.28; P = .002), setpoint viral load (HR, 3.82; 95% CI, 1.51–9.67; P = .005), and hepatitis B coinfection (HR, 4.54; 95% CI, 1.31–15.69; P = .017). Conversely, presence of any of HLAB*1302, B*27, B*57, B*5801, or B*8101 alleles predicted non–rapid progression (HR, 0.19; 95% CI, .05–.74; P = .016). Discussion/Conclusion: This study showed that syndromic STI diagnosis, which is dependent on clinical signs of vaginal discharge, was poorly predictive of laboratory-diagnosed STIs or BV and missed a significant proportion of women with asymptomatic infections. However, the level of genital inflammation, as measured by cytokine concentrations in CVL, was similar in women with symptomatic and asymptomatic infections and therefore place women at increased risk for HIV infection. While laboratory-diagnosed STIs and the presence of inflammatory cytokines in the genital tract were associated with increased susceptibility to HIV acquisition, vaginal discharge was not. Chlamydial infection was associated with the highest genital cytokine concentrations, followed by gonorrhoea, HSV-2, trichomoniasis, and BV. In regions where HIV is prevalent and STIs are managed syndromically, targeted screening of populations at risk for STIs is critical and urgently needed. Recognition of signs and symptoms of AHI is important for early diagnosis of HIV infection. The proposed algorithm of risk-stratifying individuals for AHI provides a useful clinical tool especially in resource-limited settings where there are limited or no routinely available tests for AHI. However, validation of the algorithm on another cohort is needed to assess its utility further. Point-of-care HIV antigen or viral load testing is needed, to detect asymptomatic, antibody negative cases enabling early interventions and prevention of transmission. This cohort showed high rates of rapid disease progression, with nearly half of these subtype C–infected women progressing to a CD4+ T cell count of below 350 cells/μL within 2 years of infection. Implementing 2013 World Health Organization treatment guidelines (CD4+ T cell counts less than 500cells/μL) would require most individuals to start antiretroviral therapy within 1 year of HIV infection. The economic and health systems planning implicated by these findings need to be explored and addressed to guide policy makers as countries adopt the current WHO guidelines.
Improving the efficacy of bacillus calmette guerin vaccine by concomitant inhibition of T regulatory and T helper 2 cells.
(2015) Kumar, Santosh.; Moodley, Prashini.; Das, Gobardhan.
Tuberculosis (TB) remains a major threat to human population as currently Mycobacterium tuberculosis (MTB) infects nearly 33% of the global population. Annually, about one and a half million deaths are caused by tuberculosis (TB). Current reports suggest that approximately nine million new TB cases are reported every year. There is an available therapy for TB, however it is quite lengthily and consists of numerous antibiotics leading to treatment dropout. This treatment incompletion has been linked to the major cause for the appearance of drug-resistant species of MTB. Consequently, alternate therapies to treat TB are needed. Bacillus Calmette-Guerin (BCG) remains the only vaccine of choice since its inception in 1921. Although BCG mounts host protective T helper1 (Th1) cell activation, which plays a pivotal role in host protection against TB, its efficacy is inadequate, suggesting that additional methods to enhance protective immune responses are needed. We have also shown that simultaneous inhibition of Th2 cells and Tregs by using pharmacological inhibitors (suplatasttosylate and D4476, respectively) dramatically enhance MTB clearance and induces a superior Th1 response. Here we show that treatment with these two immuno-modulators during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in T cell memory development, towards central memory T (Tcm) cell responses. Collectively, our findings provide evidence that concurrent inhibition of T helper cells type 2 and Tregs during BCG vaccination promotes vaccine efficacy.

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