Masters Degrees (Medical Virology)
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- ItemGenotyping respiratory viruses in sudden unexpected death in infancy cases (SUDI) at Tygerberg Medico-legal Mortuary(Stellenbosch : Stellenbosch University, 2023-03) Vanmali, Hameer Deepak; De Beer, Corena; Claassen, Mathilda; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.ENGLISH SUMMARY: Background: Infant mortality remains a major global concern. Sudden unexpected death in infancy (SUDI) is reported globally and accounted for 40% of infant deaths between 2012 and 2016 in the Western Cape. No standardised investigation protocol exists for SUDI cases in South Africa, and research is poorly funded. Previous research highlight the burden of respiratory viruses in infants and SUDI cases, however molecular typing of respiratory viruses in SUDI cases is lacking. Methods: HRV and RSV polymerase chain reaction (PCR)-positive trachea and lung swab samples of SUDI cases admitted to Tygerberg Medico-legal Mortuary between 2015 and 2019 were identified and included in this study. The Allplex™ RV Essential Assay was repeated on these samples to confirm that only PCR-positive samples were selected and included in this study. Positive samples underwent automated nucleic acid extraction, one-step, nested RT-PCR, and confirmed for amplification by gel electrophoresis before sequencing. Sequencing results were aligned and underwent phylogenetic analysis. Results: A total of 116 SUDI cases were HRV and/or RSV PCR-positive and included in the study, with a median age of SUDI of 10.9 weeks. More cases were female (53.4%), of African ethnicity (51.7%), and from informal housing (54.3%). Of the infants that were included in this study, 35.4% died during the autumn and winter, compared to summer and spring. According to the information provided by parents or caregivers, most infants bed-shared (94.8%) and when compared to prone and supine positions, most infants were placed to sleep on their side (49.14%) and were found in this position (40.05%). Human rhinovirus (HRV) detected in trachea and respiratory syncytial virus (RSV) detected in the lung were significantly associated with sex and all four seasons (p<0.05). HRV samples fell within three distinct species , HRV-A (n=28), followed by -C (n=11), and -B (n=4). In total, eight HRV-A (A28, A80, A10, A82, A43, A56, A11, and A63), one -B (B84), and seven -C (C22, C19, C24, C35, C29, C38, and C2) genotypes were identified. Two RSV groups were identified, RSV-A (n=5) and -B (n=5). No RSV-A sequences were assigned as ON1, and two samples were assigned BA9 after amino acid alignment indicating characteristic of 20 amino acid duplication, as well as K218T, L223P, K225I, V251T, D253N, S267P, H287Y, S288L, and E292G substitutions. Conclusion: These study results were comparable to SUDI trends within the East Metropole of Cape Town, as well as globally. Respiratory viruses, especially in combination with sociodemographic and meteorological factors, remain key contributors to SUDI. The phylogenetic analysis describes the first molecular characterisation of respiratory viruses in SUDI cases in Africa. Without prospective sampling the current burden and characterisation of circulating HRV and RSV types and associations with clinical disease severity are unknown.
- ItemImplementation, evaluation and use of methods to identify SARS-CoV-2 genetic variants of significance(Stellenbosch : Stellenbosch University, 2023-03) Karabo, Phadu; Wolfgang, Preiser; Tongai, Maponga; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.ENGLISH SUMMARY: Background: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants, designated as variants of concern (VOCs) by the World Health Organization (WHO) continue to threaten measures in place to contain the spread of the virus. Consequently, efforts have been made to intensify genomic surveillance to track and monitor the evolution and spread of SARS-CoV-2 VOCs to help inform public health interventions in a timely manner. However, whole genome sequencing (WGS) is expensive, has a longer turnaround time and requires expert bioinformatics analysis, making it unfeasible for near real-time monitoring and reporting of SARS-CoV-2 variants. There is a need for affordable variant screening methods for the rapid detection and differentiation of SARS-CoV-2 VOCs. Methods: Ten SARS-CoV-2 variant screening assays targeting SARS-CoV-2 Alpha, Beta, Delta and Omicron VOCs were used. The analytical sensitivity of the assays was assessed using RNA extracted from cell culture supernatants of isolates of the Beta and Delta VOCs and the limit of detection (LOD) and linearity of the assays were determined. Furthermore, the assays were used to screen for SARS-CoV-2 VOCs in 898 patient samples diagnosed with infection between November 2020 and January 2022. Screening results were validated against WGS to determine the clinical sensitivity, and specificity of variant screening assays, and the agreement between assays (using WGS as the gold standard) was reported as the kappa value. Lastly, the TaqMan SNP genotyping panel, TaqPath COVID-19, and ModularDx assays were implemented to estimate the proportion of SARS-CoV-2 VOCs that circulated between November 2020 and January 2022. Results: The SARS-COV-2 variant screening assay had a correlation coefficient between 0.98 and 0.99, indicating good linearity between the dilution and the corresponding average cycle threshold values (Ct-values) at each standard dilution. Of the 10 screening assays, the Smartchek B.1.351 assay had the lowest clinical sensitivity (86.4%) followed by the Allplex Variants I assay (95.5%). The sensitivity of the ModularDx assay and the TaqPath COVID-19 assays was 98.6% and 99.7%, respectively. Furthermore, the TaqMan mutation panel, Allplex Variants II, IV and Master assays were 100% sensitive. All assays demonstrated 100% specificity and moderate to high concordance in comparison with WGS. The predominant circulating variant before and during May 2021 was the Beta VOC, constituting 86.7%, 100% and 79.4% of samples screened in November and December 2020 and May 2021 respectively. The Delta VOC rapidly spread, displacing the Beta VOC in May 2021 and dominating from June until October 2021. In November, December 2021, and January 2022 the Omicron VOC predominated, with the proportion of samples resulting in failure of the S gene target to amplify, SGTF rising from 87.7% in November to 99.5% in December. Conclusion: This study demonstrated that SARS-CoV-2 variant screening assays can be used as rapid and affordable tools to monitor signature mutations in SARS-CoV-2 VOCs. In addition, they can be useful tools for scaling up SARS-CoV-2 genomic surveillance. However, they need to be regularly updated and cannot replace traditional sequencing methods, but rather serve as tools to complement sequencing in monitoring circulating SARS-CoV-2 VOCs.
- ItemA real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples(Stellenbosch : Stellenbosch University, 2023-03) Mpazi, Nothukela; Nkosi, Nokwazi; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.ENGLISH SUMMARY: Hepatitis A virus (HAV) is a growing public health concern worldwide due to its contribution to acute viral hepatitis. In South Africa, HAV data has been accounted for in clinical and environmental studies separately. Simultaneous detection of the HAV in clinical and environmental samples in the Western Cape province has not yet been explored. The current serological method of HAV detection has been linked to cross-reactivity between antibodies resulting in false positive results. Supplemental use of real-time reverse transcription (RT) polymerase chain reaction (PCR) may have the benefit of limiting cross-reactivity and serving as an early detection method in outbreak settings as it detects HAV ribonucleic acid (RNA) two weeks prior to seroconversion. The aim was to assess the presence of HAV (RNA) in various clinical and environmental wastewater samples using an in-house established real-time RT-PCR assay and to describe circulating HAV genotype(s). The objectives were to identify serologically tested HAV residual serum, plasma and stool samples referred to the National Health Laboratory Service (NHLS) Medical Virology Laboratories at Tygerberg Hospital (TBH), Groote Schuur Hospitals (GSH); and Western Cape Blood Service (WCBS); to screen untreated wastewater environmental samples for HAV presence from the South African Medical Research Council (SAMRC); to analyse data by age, sex, liver function enzyme parameters and location of retrieval using Microsoft Excel and to assess circulating genotype (s) through Sanger sequencing. Two different primers and probes were evaluated for HAV RNA detection in 353 samples comprising of 179 clinical (serum, stool, and plasma) and 174 environmental (untreated wastewater RNA eluates) samples obtained through convenience sampling from TBH; GSH NHLS serology laboratories; WCBS and SAMRC (from five Stellenbosch University residences and two Cape Town areas). BioEdit software; Genome detectives Krisp genotyping tool version 2.43 and Mega 11 software were used to assess HAV genotype(s) on selected clinical samples only. HAV RNA was positive in 43.6% (78/179) clinical samples and 32.7% (57/174) environmental samples. In the ages younger 15 years HAV RNA positive samples were higher in males in compared to females. In contrast age groups >16 years HAV RNA positive samples were higher in females compared to males. The City of Cape Town areas had a higher detection of HAV RNA positive environmental samples (>40%), while a 28% detection was recorded from the Stellenbosch University student residence samples. The identified circulating HAV genotype was HAV IB. Using the two primer and probe sets this assay successfully detected HAV RNA in various clinical and environmental untreated wastewater samples offering a 92% and 100% sensitivity for the first and second primer set, respectively. Specificity was 100% and 75% respectively. Performance characteristics of second primer set evaluated limited by sample number evaluated. Using assays with 100% specificity might be used in diagnostic settings as a supplemental confirmatory technique. This study was semi-qualitative in nature, future studies may assess the quantitative aspect and cost related to the implementation of the assay in diagnostic settings.
- ItemScreening for viruses associated with myocarditis and genotyping human parvovirus B19 in cases of sudden and unexpected death in Infancy (SUDI) at Tygerberg Medico-Legal Mortuary(Stellenbosch : Stellenbosch University, 2023-03) Ngobeni, Trevor; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.ENGLISH SUMMARY: Introduction: The sudden infant death syndrome (SIDS) is one of the primary contributors of infant mortality. The Triple Risk model has identified risk factors that are modifiable and can help in reducing the incidence of SIDS. Sudden unexpected death in infancy (SUDI) is diverse and can be either an appropriate or indeterminate cause of death when pathological changes are observed. Cardiovascular infections are a major contributor to morbidity and mortality in both infants and adults. Aim: The aim of this study was to investigate the presence of specific viruses associated with myocarditis and to genotype Parvovirus B19 in the heart specimens of SUDI cases admitted to the Tygerberg Medico-legal Mortuary. Methodology: Heart samples were collected during the autopsy of the SUDI cases admitted to the Tygerberg Medico-legal Mortuary. Samples for histology and microbiological analysis were also collected. The SUDI samples were further screened for Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Herpes Simplex Virus (HSV) 1 & 2, Human Herpes Virus (HHV) 6 & 7, Varicella-zoster Virus (VZV), Human Adenovirus (HAdV), Human Enteroviruses (HEV), Human parechovirus (HPeV), Mumps virus and Parvovirus B19 (PVB19). Specimens that tested positive for PVB19 were genotyped. Ten residual adult heart specimens positive for PVB19 were also genotyped for comparison. Results: Heart swabs from 40 infants were tested for viruses associated with myocarditis. The study consisted of 18 male and 22 female infants. The mean age of death in weeks was 12.8 weeks. Most deaths occurred during winter (47.5%). More than 50% of infants were sleeping on their side. The following viruses were detected in the infant heart swabs: CMV, EBV, HSV-1, HHV-6, HHV-7, VZV, HAdV, HEV, HPeV and PVB19. All the three PVB19 genotypes were identified, 8 of the adult residual samples were genotype 2 and the other 2 adult specimens were genotype 3 and 1A. The 2 infant samples were both genotype 1A. Conclusion: This study has contributed previously unknown knowledge about myocardial infections and specific PVB19 genotypes found in SUDI cases that could have contributed to myocarditis and sudden death in infants. SUDI research in South Africa is limited and may be of value in the forensic environment when formulating a cause of death in SUDI cases.
- ItemThe role of underdiagnosed viruses in neonatal sepsis(Stellenbosch : Stellenbosch University, 2022-12) Mahlangu, Tshegofatso Perseverance; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.ENGLISH ABSTRACT: Background Neonatal sepsis is a clinical syndrome that is difficult to diagnose due to the unspecific symptoms that patients present with. Several different diagnostic techniques are used to diagnose neonatal sepsis; but currently, blood cultures are regarded as the gold standard for diagnosis of bacterial sepsis. Broad spectrum antibiotics are administered to neonates with suspected sepsis and the therapy is adjusted accordingly after diagnosis. This is, however, inappropriate therapy if sepsis is caused by viral and not bacterial agents. Numerous studies have found certain viruses to cause sepsis or a sepsis-like syndrome, but because viruses require virus specific assays, they may be underdiagnosed. Although the incidence of viral neonatal sepsis has been low, the effect of its underdiagnosis has detrimental effects. Therefore, in this study, a rapid detection multiplex polymerase chain reaction (PCR) was developed to potentially assist with the diagnosis of viral neonatal sepsis. A third-generation sequencing (TGS) technology was used for species specific identification of the agents detected by the multiplex PCR. TGS technology is increasingly utilised in medical research and appears promising for future rapid infectious agent diagnosis due to its real-time and long read sequencing. Methods Various sample types were included: residual patient laboratory samples with known positive diagnoses (none of which were from neonatal sepsis patients), external quality assurance panel samples and culture isolates from known agents. These samples were used to first optimise a singleplex assay followed by the multiplex assay. The assay was developed to have two rounds of PCR to ensure increased sensitivity. Two different extraction methods were compared to determine the method best suited for the assay. In addition, a simple sensitivity test was conducted using serially diluted samples for both the singleplex, and multiplex assays. As a result of the presence of an unknown fragment in the second-round multiplex PCR non-template controls (NTCs), several troubleshooting experiments were performed. Products with the incorrect band size or present in NTCs, which were observed during assay optimisation, were initially investigated with Sanger sequencing. Thereafter Oxford Nanopore Technology (ONT) sequencing was used to confirm the PCR amplified products. In addition, ONT sequencing was used to attempt to identify the unknown fragment in the NTCs. Results The singleplex assay was successfully optimised, however, during the multiplex optimisation an unknown fragment was discovered in the second round PCR NTCs. Troubleshooting excluded all other sources and we concluded that the “contaminant” was possibly an impurity of bacterial origin carried over from recombinant technology used in the production of the enzyme system. A new enzyme system was introduced to continue the multiplex optimisation, but the sensitivity was lower than with the previous enzyme system. As a result, the previous enzyme system was utilised for all targets except bacterial targets, for which a separate reaction including the 16s rRNA primers were used. Thereafter, the multiplex assay was further optimised. ONT sequencing confirmed that the correct agents were amplified, rendering the entire workflow successful. The unknown fragment of bacterial origin discovered in the NTCs was sequenced with ONT as well as Sanger sequencing. However, both platforms failed to confirm the specific bacterial species present. Conclusion The multiplex assay developed in this study can successfully amplify the intended agents but only at high concentrations. The comparison of sensitivity of the singleplex versus the multiplex versions of the assay revealed that the multiplex assay was significantly less sensitive. ONT sequencing could rapidly confirm all the amplified agents, and this is beneficial for diagnostic purposes. Currently, this study serves as a proof of concept because the multiplex assay, in its current form, does not appear to be suitable for primary (uncultured) diagnostic samples due to inadequate sensitivity for a low target concentration input.