Doctoral Degrees (Medical Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/9619

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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.
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.
Investigations into the morphometrics, uterine tissue adaptation, maternal fluid biochemistry, heavy metal offloading and early embryonic teeth development impacting the reproductive strategy of the female Ragged-tooth shark (Carcharias taurus)
(2018) Naidoo, Kristina.; Chuturgoon, Anil Amichund.; Gregory, Michael Alfred.
Vulnerable” status of ragged-tooth sharks (Carcharias taurus) in South Africa caused by overexploitation, late maturity and low fecundity suggests an intervention to increase the size of this population is needed. Achieving this will require an understanding of all aspects linked to this species maternal-embryonic relationship. Morphometric relationships, uterine histology and maternal fluid biochemistry were 8 assessed in C. taurus through all the respective reproductive stages (RS) from non-gravid (immature to mature-sexually active; RS1-3) to gravid (i.e. only capsules found; RS4 or capsules and pups found; RS5A-5E) females. Examination of metals in the maternal fluids and embryonic dentition were only examined in early-staged gravid females (i.e. RS5A). Haematoxylin/Eosin and Periodic Acid Schiff-Alcian Blue stains in conjunction with light microscopy was used to assess the uterine epithelium and wall while scanning electron microscopy further evaluated the epithelium. These techniques revealed an increase of the uterine lamellae (folds) protruding into the lumen lined with micro-ridges containing blood vessels. The close proximity of blood vessels to the lumen filled with uterine fluid and the decrease in wall thickness as pregnancy progressed suggests an adaption for the exchange of respiration and osmoregulation in the developing aplacental embryos. Although there is no evidence for uterine secretion through structural adaptations, the female supports the embryos nutritional requirement through embryonic tissue (intrauterine cannibalism) and yolk (oophagy) provisions. The pivotal interplay of the liver and ovary, during vitellogenesis, that impact on yolk formation was evident during the morphometric evaluation of hepatosomatic and gonadosomatic indices. Length, weight, uterine width, capsule production and migration trends of the females as well as length and weight relationship of the embryos were tabulated. Reproductive hormones, assessed in maternal fluids (i.e. plasma (in RS1-5D females), uterine fluid (in RS4-5D females) and intracapsular fluid (in RS5A females), showed that follicle-stimulating-, progesterone- and oestradiol hormones were responsible for promoting vitellogenesis and encapsulation which led to three main stages where the rate of ovulation increases in the female. Clinical biochemistry analysers confirmed the composition and concentration of biochemical analytes in same maternal fluids, which were found to be higher in the plasma. Finally, heavy metals were found to be present in all three fluids, but found highest in the plasma, using inductively coupled mass spectrophotometry. In addition, variable pressure (VP) SEM confirmed the dental composition of the embryonic teeth found in the jaws of some embryos that appeared to escape encapsulation earlier than previously documented. It would appear that the embryos are creating adaptive ways to survive the intracannibalistic stage (RS5C) by escaping encapsulation early. However, the presence of heavy metals in the maternal fluids that surround the embryos could compromise their development over time; creating concern for a species that is Vulnerable. This study, which serves as the first detailed analysis of the maternal-embryonic relationship, may serve as areas to model in forthcoming programmes aimed at increasing the numbers of this species.
Metformin alleviates neuronal and renal related stress signals in diabetic C57BL/6 mice.
(2019) Docrat, Taskeen Fathima.; Chuturgoon, Anil Amichund.
In recent years, diabetes has become more prevalent due to modern, demanding, and sedentary lifestyle patterns. This disease is characterised by insulin resistance and associated molecular complications. Metformin (MF) is a popular antidiabetic agent that has effects beyond glycaemic control such as regulation of metabolic molecular pathways. However, the exact mechanisms against hyperglycaemic induced end organ damage remain elusive. This study aimed to investigate the protective effects of MF in the brain and kidney in vivo, by exploring dysregulated pathways related to mitochondrial function, oxidative stress, ER stress, inflammation, and apoptosis. This study established a diabetic mouse (C57BL/6) model (Ethics no: AREC/057/016) through intraperitoneal multiple low-dose STZ (50 mg/kg BW) injections (10 days). Blood sugar levels of 7-16mmol/L were considered diabetic, and the 15 day treatment period (MF, 20 mg/kg BW per day, oral gavage) was inducted thereafter. Fasting (12 hr) plasma OGTT revealed MF significantly lowered blood glucose levels in diabetic mice. All mice experiments were performed by Dr. N. Naicker. Post-sacrifice (isoflurane), the investigator (author) of the work in the presented in this thesis assisted in harvesting Whole brain and kidney tissue and performed all downstream protein and mRNA analyses. Diabetic mice exhibited heightened oxidative stress by protein carbonylation, and diminished antioxidant responses in both the brain and kidney compared to normoglycaemic mice. Metformin significantly reduced protein carbonylation, increased GSTA4 expression in the brain; and Nrf2 and GPx mRNA levels in the kidney, alleviating oxidative stress. Further, MF improved mt activity, and decreased the HIF-1 expression in the kidney through upregulation of AMPK, and Sirt1 expression. In addition, MF induced epigenetic changes in mice brain through miR-148a repression and concomitant increases in PGC-1α, Sirt1, and Sirt3 protein and gene expressions, thus regulating mt biogenesis. Mitochondrial chaperone proteins HSP60, HSP70 and LonP1 in diabetic mice brain were upregulated through a MF-induced miR-132 repression mechanism. Regulation of the UPR by PERK-eIF2α inhibition after MF-treatment attenuated ER stress in diabetic mice brain and kidney tissue. Moreover, renal injury associated with diabetes was attenuated by MF through decreased CHOP expression, downstream to ER stress. This finding was supplemented by inhibition of Bax, cyt-c, and ultimately the intrinsic apoptotic pathway. MiR-141 modulates expression of PP2A, a phosphoesterase that regulates phosphorylation of tau protein. In hyperglycaemic mice there was increased miR-141 expression with concomitant PP2A downregulation in the brain. Treatment with MF exerted epigenetic regulation by downregulating miR-141 expression, concomitantly increasing PP2A and subsequent downregulation of tau protein phosphorylation at Ser396. Additionally, MF inhibited proinflammatory NLRP3 inflammasome and related components by regulation of the PP2A/ NF-κB cascade. Neuroplasticity was increased by increased BDNF overexpression by MF in diabetic mice. Herein we show that MF exerts protective mechanistic effects in the brain and kidney over an acute experimental period. We highlight that anti-oxidant and Sirt1 modulation are at the forefront of renal cell defence to metabolic stress. Neuroinflammatory and epigenetic therapeutic targets of MF are revealed through miRNA regulatory mechanisms, integrating the mechanisms of diabetic neuronal and renal damage.

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