Masters Degrees (Medical Virology)

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An anti-SARS-CoV-2 quantitative study to determine the effect of time on the antibody titre, post-vaccination
(Stellenbosch : Stellenbosch University, 2024-01) Meyer, Burnet Adriaan; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: In 2019, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) outbreak emerged in Wuhan, China, rapidly evolving to a pandemic. Vaccines such as messenger ribonucleic acid (mRNA)- based and Adenovirus-based, have been developed to counter severe Coronavirus Disease 2019 (COVID-19). However, many factors, for example, immunosuppression and prior infection can affect the antibody titre. This study evaluated the effectiveness in Anti-SARS-CoV-2 immunoglobulin (Ig) G production and decline over time, indicating the potential requirement for additional boosters. Furthermore, this study focused on how Adenovirus-based vaccine boosters and breakthrough infections affected the antibody titre. A total of 184 samples were collected from two cohorts which consisted of 67 participants. Samples were collected at baseline, week 3, and week 6 post-booster for Cohort A (laboratory workers at the Western Cape Blood Service). Cohort B (staff and students at the Faculty of Medicine and Health Sciences, Stellenbosch University) had samples collected at baseline, week 2, week 4 and week 8 post-booster. The samples were tested for the presence of the SARS-CoV-2 Anti-nucleocapsid (N) as well as Anti-spike (S) antibodies. The Anti-N antibodies indicated that a participant had a natural infection to SARS-CoV-2 while the Anti-S antibodies were utilised to examine the influence of age, gender and time on vaccine-induced antibodies. This findings of this study demonstrated that vaccine-induced antibodies were still detectable six months following the initial vaccination. A significant increase was observed for both cohorts, when comparing the baseline titre with the different timepoints, post-booster. Multiple samples were found to be Anti-N positive, suggesting that these participants were potential asymptomatic cases. Variations were found in the Anti-S antibody titre based on different age groups, although these variations were not statistically significant. Moreover, the results have indicated that gender does not affect the Anti-S antibody titre. This research enhances the understanding of antibody titre dynamics, contributing to a better grasp of immune longevity. Further studies are recommended to determine the impact of combining different boosters on the antibody titre, and assess the difference in antibody titre between monovalent boosters with bivalent boosters.
Characterisation of rotavirus strains responsible for breakthrough diarrhoeal disease among Zambian children using whole genome sequencing
(Stellenbosch : Stellenbosch University, 2024-01) Mwape, Innocent; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background Genetically altered viruses or variants have the potential to increase their virulence, pathogenicity, transmission, and ability to evade both natural and vaccine-induced immune responses leading to diarrhoeal disease in under five-year-old children who have received all recommended doses of Rotavirus (RV) vaccines, also known as breakthrough infections. Studies characterising RV strains responsible for breakthrough infections are rare in resource-limited countries like Zambia where RV-associated diarrhoeal disease is endemic. We aimed to characterise RV strains detected in fully vaccinated under five-year-old children residing in Zambia using next generation sequencing. Methods This was a case study nested under an open label randomised controlled RV vaccine clinical trial that evaluated safety and immune boosting effects of a third dose of Rotarix compared to a two-dose schedule. The Rotaclone kit was used to screen for RV in stool. We performed VP7 and VP4 genotyping on RV positive stool using Sanger sequencing. Whole genome sequencing was done on the Illumina Miseq platform. Genome assembly was done using Geneious software and multiple sequence alignment using Muscle in MEGA version 6. Results A total of 76 diarrhoeal stool specimens were collected and screened for RV of which 4/76 (5.2%) were positive. Genotypes of three of the four cases were identified as G1P[4], G12P[4] and G12P[8] using Sanger sequencing. Whole genome analysis revealed that the RVA/Human-wt/ZMB/CIDRZRV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and RVA/Human-wt/ZMB/CIDRZ-RV2106/ 2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 strains were mostly DS-1-like with mono and multiple reassortant respectively, whilst the RVA/Human-wt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1- M1-A1-N1-T1-E1-H1 was a typical Wa-like strain. Comparison of VP7 antigenic epitope of strains causing breakthrough infections and Rotarix vaccine strains revealed several amino acid differences like G96P and M217E. Comparison of P[4] strains with VP4 of the Rotarix vaccine strain demonstrated two amino acids differences (P114Q and V115T) were P114Q is an immune escape mutation. Discussion and Conclusion Differences observed in amino acids in antigenic epitope suggested its role in the immune evasion of neutralising antibodies elicited by the vaccine. Findings from this study have potential to inform national RV vaccination strategies and the design of highly efficacious universal RV vaccines. Furthermore, there might be need to monitor strains that have escaped vaccine-induced immunity to prevent diarrheal diseases in children under five years of age.
Coreceptor expression and T lymphocyte subset distribution in HIV-infected and TB co-infected South African patients on anti-retroviral therapy
(Stellenbosch : University of Stellenbosch, 2009-12) Ngandu, Jean Pierre Kabue; De Beer, Corena; Glashoff, R. H.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: In 2007, AIDS caused an estimated 2.1 millions deaths worldwide; about 70% in sub-Saharan Africa. HIV preferentially targets activated CD4 T cells, expressing the major HIV receptor CD4, as well as the major chemokine coreceptors CCR5 and CXCR4. These coreceptors play a prominent role during HIV cell entrance phase, HIV transmission and also disease progression. They have been found to be differentially expressed by CD4 T cell subsets. Tuberculosis coinfection may enhance immune activation in vivo thus accelerating HIV disease progression and has become a major challenge in the control of TB in Africa. Introduction of HAART has reduced disease progression to AIDS, as well as risk of further morbidity and mortality. HAART results in a rapid decline of viral load and an initial increase of peripheral CD4 count, however little is known on the effect of HAART in regulation of coreceptor expression, immune activation status and CD4 T cell subset distribution in HIV infection and HIV/TB coinfection. This study is a cross-sectional analysis of coreceptor expression, immune activation status and CD4 T cell subpopulation distribution in South African HIV and HIV/TB coinfected patients before and after ARV. A total of 137 South African individuals were investigated, comprising 15 healthy normal donors (healthy subgroup), 10 patients with active pulmonary tuberculosis (PTB subgroup), 33 HIV-1 positive patients without active PTB (HIV subgroup), 23 positive patients with active PTB (HIV/PTB subgroup), 36 HIV-1 positive patients on ARV (HIV on ARV subgroup) and 20 HIV-1 positive patients with active PTB on ARV (HIV/PTB on ARV subgroup). CD4 absolute count and plasma viral load were determined for all donors. Freshly isolated PBMC were classified by flow cytometry into the following CD4+ T lymphocyte subsets: naïve (CD45+, CD27+), effector memory (CD45-, CD27-), central memory (CD45-, CD27+), and effector (CD45+, CD27-). Coreceptor expression and activation status was assessed by CCR5, CXCR4 and CD38 expression on CD4 T cell subsets. HIV, TB and HIV/TB coinfection was associated with a decrease in percentage CCR5+ T cells as compared to healthy controls, with the HIV/TB group showing the most extensive decrease. In treatment naive patients, CD4 T cells showed elevated surface expression of CCR5 and CD38 as determined by mean fluorescence intensity in HIV/TB co-infection compared to HIV infection alone. The percentage of antigen-experienced cells was higher in the HIV/TB co-infected group compared to the HIV group. The percentage of naïve T cells was decreased in both the HIV infected and the HIV/TB co-infected groups compared to healthy controls. HIV patients with more than 6 months of ARV showed decreased CCR5 and CD38 surface level expression in the HIV and the HIV/ TB co-infected subgroups. An increased percentage of naïve T cells was observed in the HIV infected subgroup, but not in the HIV/TB subgroup, similarly, a decreased percentage of antigen-experienced cells was observed in the HIV subgroup, but not in the HIV/TB co-infected subgroup. A positive correlation was found between CCR5 and CD38 expression, and CXCR4 and CD38 expression (Spearman coefficient of correlation respectively: r=0.59, p<0.001 and r=0.55, p<0.001). Furthermore we found plasma viral load positively associated with CD38 expression (r=0.31, p<0.001) and percentage activated CCR5+ expressing CD4 T cells positively related to viral load (r=0.31, p<0.001). Percentage naïve CD4 T cells was positively associated with CD4 count (r=0.60, p<0.001) and negatively correlated to viral load (r=-0.42, p<0.001). These results indicate that TB coinfection exacerbates certain aspects of dysregulation of CD4 T cell homeostasis and activation caused by HIV infection. In addition, ARV-associated decrease in coreceptor expression, immune activation status and a normalisation of CD4 T cell subset distribution was observed in HIV infected individuals, but not in HIV/TB coinfection. Despite viral suppression after ARV treatment, the decline in the immune activation marker CD38 and coreceptor CCR5 expression, increase in percentage naïve CD4 T cells and decrease of antigen-experienced cells did not reach the levels displayed in the healthy control group. This may indicate that ongoing (albeit reduced) T cell immune activation may occur in the presence of ARV. Further longitudinal studies are needed to closely monitor immune activation during ARV treatment. This study highlighted an association of TB disease with immune activation in HIV infection, the importance of T-cell activation in HIV pathogenesis and its impact on ARV treatment. Further studies are needed to identify causative factors that may lead to a persistent immune activation status during ARV treatment, and how TB coinfection confounds normal responses to ARV.
Genotyping 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.
Investigating meningitis in cases of sudden and unexpected death in infancy (SUDI) at the Tygerberg Medico-legal Mortuary in the Western Cape Metropole
(Stellenbosch : Stellenbosch University, 2023-02) Nortjé, Simoné-Odette; De Beer, Corena; Verster, Janette; Nel, Pieter; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: The infant mortality rate in South Africa is currently 24.3 per 1 000 live births, with infections accounting for the majority of infant deaths. All unexplained deaths between the ages of seven days and one year that occur before any investigation has taken place are classified as Sudden Unexpected Death in Infancy (SUDI). Sudden Infant Death Syndrome (SIDS) is the classification assigned to cases where no cause of death could be found, after conclusion of the investigation. An interlinking of several factors is widely accepted to contribute to the occurrence of unexplained infant death, but one possible cause, infective meningitis, has largely remained unexplored and underreported despite the regular detection of viral and bacterial pathogens in SUDI post-mortem specimens. The investigators postulate that these pathogens could contribute as an exogenous trigger to SUDI in a susceptible infant during a particular developmental phase and set out to investigate the exact contribution of meningeal pathogens in SUDI cases. The primary aim of this study was to investigate the presence of viral and bacterial pathogens, commonly associated with meningitis, isolated from cerebrospinal fluid (CSF) as either single or co-infections, over the course of one year from all SUDI cases admitted to Tygerberg Medico-legal Mortuary. The secondary aim was to investigate the prevalence of neonatal meningitis in the Eastern Metropole of Cape Town in the Western Cape by performing a retrospective review on all infants under the age of one year admitted to Tygerberg Hospital that were diagnosed with suspected meningitis due to viral, bacterial and tuberculosis causes over a five-year period (2016 to 2021). Additionally, an ancillary investigation was conducted to determine if the modified CSF extraction technique has a lower risk of contamination. Methods Post-mortem samples were collected from 133 SUDI cases that were admitted to the Tygerberg Medico-Legal Mortuary between March 2021 and March 2022. CSF collected during the autopsies was investigated for the presence of certain viruses and bacteria that are commonly associated with meningitis. Two multiplex assays specific for the various pathogens in question were used to analyse extracted deoxyribonucleic acid (DNA). The one-step multiplex real-time polymerase chain reaction (PCR) assay, Seegene AllplexTM Meningitis-V1 Assay was used for the detection of seven double-stranded DNA human herpes viruses (HHV-1 to HHV-7). The one-step multiplex real-time PCR assay, Seegene AllplexTM Meningitis-B Assay was used for the detection of six bacterial pathogens including Group B Streptococcus, Escherichia coli, Haemophilus influenzae type b, Listeria monocytogenes, Neisseria meningitidis and Streptococcus pneumoniae. Fresh CSF samples were analysed with the Xpert® MTB/RIF Ultra assay in order to detect Mycobacterium tuberculosis. Data were stored securely in an encrypted Microsoft® Office® Excel® database. Results Multiple well-known demographic risk factors for SUDI were observed, such as age 11.3 ± 9.9 weeks, majority males, cold season, prematurity, low birthweight, bed-sharing, prone sleeping position, little to no ventilation, exposure to smoke and young maternal age. With the AllplexTM Meningitis-V1 and Meningitis-B real-time PCR, 24% (31/131) of cases had viral pathogens detected and 9% (12/131) had bacterial pathogens detected. HHV-6 (47%) and cytomegalovirus (23%) were the most commonly detected viruses. A significant association was observed between viral PCR-positive results and age, with a peak incidence between 10 and 14 weeks. Neisseria meningitidis (42%) and Haemophilus influenzae type b (25%) were the most frequently detected bacteria. Most PCR-positive cases were identified during the cold season. With the Xpert® MTB/RIF Ultra assay, no tuberculosis meningitis was detected over a six-month period. Infection was the most frequent cause of death (COD) in cases, with a significance observed between both PCR-positive viral (p=0.017) and bacterial (p=0.002) results and a COD of Infection. Five percent (7/131) of the cases were signed out as meningitis as final diagnosis, of which Neisseria meningitidis was detected in two of those cases. The retrospective audit showed PCR-positive results for any of the six HHVs in 21% (213/1 037) of cases. HHV-6 was positive most often in 43% (91/213) of samples, followed by CMV in 27% (58/213), EBV in 17% (37/213), VZV in 5% (11/213), HHV-2 in 4% (8/213) and HHV-1 in 4% (8/213). No HHV-7 was detected over the five-year period. In total 6 535 CSF samples were cultured and of those 1% (65/6 535) were positive for one or more bacteria. Thirty-five percent (23/65) were positive for natural skin flora, 20% (13/65) each for E. coli and K. pneumoniae, 15% (10/65) for Staphylococcus aureus and 9% (6/65) for mixed growth. Conclusion In some cases that were assigned a COD of SIDS, the PCR results cannot be ignored. If performed routinely, the PCR results could have favoured a final COD of Infection instead of SIDS in some of these cases. Further research is needed to elucidate the significance of detecting pathogens from SUDI cases where no other ancillary investigations yield evidence of infection. Although only five cases of Neisseria meningitidis were detected, the severity of the pathogen cannot be ignored since two of those cases were assigned a final COD of meningitis. This finding supports the need for further investigations on the prevalence of Neisseria meningitidis in South Africa and whether or not meningococcal vaccines need to be reinstated in the Expanded Immunisation Program of South Africa.
Investigation of the molecular epidemiology of HIV-1 in Khayelitsha, Cape Town, using serotyping and genotyping techniques
(Stellenbosch : University of Stellenbosch, 2005-12) Jacobs, Graeme Brendon; Engelbrecht, Susan; De Beer, Corena; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
There are currently an estimated 5.3 million people infected with human immunodeficiency virus / acquired immunodeficiency syndrome (HIV/AIDS) in South Africa. HIV-1 group M Subtype C is currently responsible for the majority of HIV infections in sub-Saharan Africa (56% worldwide). The Khayelitsha informal settlement, located 30 km outside Cape Town, has one of the highest HIV prevalence rates in the Western Cape. The objective of this study was to investigate the molecular epidemiology of HIV-1 in Khayelitsha using serotyping and genotyping techniques. Patient samples were received from the Matthew Goniwe general health clinic located at site C in Khayelitsha. Serotyping was performed through a competitive enzymelinked immunosorbent assay (cPEIA). RNA was isolated from patient plasma and a two step RT-PCR amplification of the gag p24, env gp41 IDR, env gp120 V3 and pol genome regions performed. Sequences obtained were used for detailed sequence and phylogenetic analysis. Neighbour-joining and maximum likelihood phylogenetic trees were drawn to assess the relationship between the Khayelitsha sequences obtained and a set of reference sequences obtained from the Los Alamos National Library (LANL) HIV database (http://www.hiv.lanl.gov/). Through serotyping and genotyping the majority of HIV strains were characterised as HIV-1 group M subtype C. One sample (1154) was characterised as a possible C / D recombinant strain. In 9 other samples HIV-1 recombination cannot be excluded, as only one of the gene regions investigated could be amplified and characterised in these samples. The gag p24 genome region was found to be more conserved than the env gp41 IDR, with the env gp41 IDR more conserved than the env gp120 V3. The variability of the env gp120 V3 region indicates that patients might be dually infected with variant HIV-1 subtype C strains or quasispecies. Conserved regions identified in the Khayelitsha sequences can induce CD4+ T-cell responses and are important antibody recognition target sites. These conserved regions can play a key role in the development of an effective HIV-1 immunogen reactive against all HIV-1 subtypes. The majority of subtype C viruses were predicted to use CCR5 as their major chemokine co-receptor. The pol sequences analysed indicate that mutations associated with minor resistance to Protease Inhibitors (PIs) might be present in the Khayelitsha community. The identification of resistant mutations is vital for people receiving antiretroviral treatment (ART). It can influence the success of their treatment and delay the onset of AIDS. Serotyping is a quick characterisation method, but not always accurate. With genotyping detailed molecular analysis can be performed. However, with genotyping the success of amplification often depends on viral load. In Southern Africa a subtype C candidate vaccine appears to be the best option for future vaccine considerations. The sporadic detection of non-subtype C and recombinant subtype C viruses remains a concern and will thus have to be closely monitored. Phylogenetic analysis can help to classify and monitor the spread and evolution of these viruses.
Prevalence and risk factors of acute respiratory infection by human respiratory syncytial virus in children at Provincial General Hospital of Bukavu, Democratic Republic of the Congo
(Stellenbosch : Stellenbosch University, 2017-12) Cihambanya, Landry Kabego; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT : Human Respiratory Syncytial Virus (HRSV) is the major cause of acute respiratory infection in children (ARI) and it is responsible for substantial morbidity and mortality, especially in younger children. The present study had two main objectives. The first one was to determine the prevalence of HRSV and non-HRSV ARI in children under the age of 5 years at the Provincial General Hospital of Bukavu (PGHB). The second objective was to analyse factors associated with the risk of ARI to be diagnosed as lower respiratory tract infection (LRTI). A total of 146 children under 5 years visiting the PGHB for ARI between August and December 2016 were recruited. A clinical examination was made and a questionnaire was completed by the parent or the guardian after which a nasopharyngeal swab was performed to collect respiratory fluid. The sample was analysed by a multiplex reverse transcriptase polymerase chain reaction for the detection of 15 different viruses, among which HRSV A and B, Influenza A and B, human Rhinovirus (HRV) A/B/C, Parainfluenza (PIV) viruses 1, 2, 3 and 4, Adenovirus (ADV), Bocavirus, Coronavirus OC43 and 229E/NL63, Enterovirus and human Metapneumovirus. Of 146 samples collected, 84 (57.5%) displayed a positive result of at least one of the 15 viruses. The overall prevalence of HRSV was 21.2%. HRSV A (30, 20.5%) was the virus the most detected, followed by HRV (24, 16.4%), PIV3 (20, 16.6) and ADV (7, 4.79%). The other viruses were detected in three or less cases. There were only 11 (7.5%) of co-infection. In bivariate analyses, HRSV infection, malnutrition, younger age, rural settings, low income and mother illiteracy were associated with the risk of ARI to be diagnosed as LRTI. However, in multivariate analyses, only HRSV infection and younger age predicted LRTI. Children with HRSV infection had 6.45 times higher odds to exhibit LRTI when compared to children without HRSV infection. Older children (by one month) had 6% lower odds of LRTI than younger children (adjusted odds ratio = 0.94, 95% CI: 0.90 – 0.97, p-value = 0.004).
Profiling cardiovascular viral pathogens in cases of sudden and unexpected death in infants (SUDI) at the Tygerberg Medico-legal Mortuary and the role of myocarditis as a possible cause of death
(Stellenbosch : Stellenbosch University., 2020-03) Venter, Michaela Lucienne; De Beer, Corena; Dempers, Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Introduction: Sudden unexpected death in infancy (SUDI) is a heterogeneous group in which pathological changes, when observed, can either be an adequate or non-conclusive cause of death (COD). A non-conclusive COD is referred to as Sudden Infant Death Syndrome (SIDS), which is postulated to be the result of multiple risk factors including infection. Viral heart infections, resulting in myocarditis, reportedly contribute to SUDI cases. Numerous viruses have been associated with myocarditis, with few ever having been investigated in a South African context. Aim: The study aimed to investigate the presence of three specific viruses in the heart of SUDI cases admitted to the Tygerberg Medico-legal Mortuary over a one-year period. Methodology: Swab samples of the left ventricle of the heart were collected from SUDI cases admitted to the Tygerberg Medico-legal Mortuary over the period of one year. Concurrently, swabs and tissue were retrieved for microbiological and histological analysis, respectively. Conventional qualitative polymerase chain reaction assays were used to detect three deoxyribonucleic acid viruses, namely human adenovirus (HAdV), human bocavirus (HBoV) and parvovirus B19 (PVB19), possibly linked to myocarditis. Clinical history, sociodemographic information and the final COD were collected from case files. All viral results were compared to the histology of the tissue. Associations were investigated between sociodemographic information and viral presence through statistical analysis in order to identify significant risk factors. Results: Heart swabs were collected from 173 SUDI case, consisting of 93 males and 80 females and a mean age of 12.1 ± 9.8 weeks. Over half of the SUDI cases occurred in the cold seasons. The majority of the cases were assigned Infection as a COD, with just under half assigned as SIDS. Only one virus, HBoV, was detected in the heart tissue with implications of myocarditis histologically observed in one of the viral positive SUDI cases. Bacterial presence was also confirmed in only one case. All viral infections were observed in the cold seasons. Risk factors were highlighted between variable associations. Significant associations were observed between prematurity, room ventilation, birthweight and the COD. Significant associations were also observed between microbiology results, histology and the temperature on the day of death. Conclusion: The study expanded the knowledge regarding myocardial infections contributing to SUDI in the study population, as well as significant risk factors. Viral detection in the myocardium, supported by histological evidence, provided an improved way of classifying COD as infection.
Profiling Enterovirus and Parvovirus B19 in sudden and unexpected death in infancy (SUDI) at the Tygerberg Medico-legal Mortuary and the role of myocarditis as a possible cause of death
(Stellenbosch : Stellenbosch University, 2018-03) Saayman, Jamie Ester; De Beer, Corena; Dempers, Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH SUMMARY: Background: Sudden infant death syndrome (SIDS) remains one of the leading causes of death among infants. The Triple-Risk Model has contributed to identifying modifiable risk factors that may lead to a reduction of SIDS occurrences. Cardiovascular infection contributes significantly to mortality and morbidity in children and adults. Acute myocarditis affects infants more severely than adults and has a known association with Coxsackie-B, Adeno-, parvo- B19, Epstein Barr-, Cytomegalo-, Human herpes-6 viruses. These viruses have been explored in sudden unexpected death in infancy (SUDI) and shown some association between SIDS and myocardial infection. Aim: This study aimed to describe two cardiovascular viruses in SUDI cases and to determine the association of myocarditis to these deaths. Methodology: Heart swab and tissue samples were prospectively collected from SUDI cases at the Tygerberg Medico-legal Mortuary over a one year period. The samples were collected in parallel with routine heart swabs for microbiology analysis and peripheral blood for HIV screening. The SUDI samples were additionally screened for enterovirus and parvovirus B19 by polymerase chain reaction assays. The heart tissue was processed for histological analysis. Sociodemographic information, medical history and final cause of death were obtained during the initial interview with family / caregiver(s) and from case files respectively, and potential risk factors in the SUDI population were identified from the data by statistical analysis. Results: Heart swab and tissue samples were collected from 168 and 161 SUDI cases respectively. The SUDI population consisted of 81 males and 87 females. Majority of deaths (64%) were in infants younger than 14 weeks and 67% occurred during the colder months of the year. In more than half of the cases an infectious cause of death was confirmed, while in 40% death was attributed to SIDS. There was a higher frequency of death among black infants, which is consistent with the literature, however it is not clinically relevant as it is not a representation of the general population profile in the Western Cape. The heart tissue for histology was within normal limits in all but 10 of 161 SUDI cases examined for morphological change associated with viral myocarditis, and 1 of these 10 cases was diagnosed as myocarditis as the final cause of death. The only significant risk factor identified in this population was ethnicity, but the finding was not clinically relevant. Conclusion: The results obtained from this study support the Triple-Risk Model of SIDS. The high proportion of deaths that remained unexplained (i.e. SIDS) emphasizes the need to introduce additional testing, such as molecular based tests which provide significant value when establishing a final cause of death. SIDS research in South Africa is limited and would be valuable in the forensic environment.
Profiling the approach to the investigation of viral infections in cases of Sudden Unexpected Death in Infancy (SUDI) in the Western Cape Province
(Stellenbosch : University of Stellenbosch, 2011-03) Burger, Marilize Cornelle; De Beer, Corena; Dempers, Johan; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Sudden Unexpected Death in Infancy (SUDI) refers to any such sudden demise in a child. If the child dies while asleep within the first year of life, and if no conclusive cause of death can be ascertained by means of complete autopsy and investigation into the circumstances surrounding death, including visit of the death scene, such a case is classified as one of Sudden Infant Death Syndrome (SIDS). By South African law, a full medico-legal autopsy is mandated in cases where the cause of death is not evident – including cases of possible SIDS. There can be little doubt that viral infection can be a cause of death in cases of supposed SUDI. At the Tygerberg medico-legal (forensic) laboratory, the evaluation of lung tissue for the presence of fatal viral lung infections forms part of the institutional protocol for the examination of SUDI cases. Lung samples of these SUDI cases are routinely tested for the presence of Cytomegalovirus (CMV), adenovirus and respiratory syncytial virus (RSV) by means of shell vial cultures. In a retrospective pilot study of 366 SUDI case files from Tygerberg Hospital, Western Cape, from 2004 – 2006, it was evident that in only 13.9% of possible SIDS cases, positive results for one or more of the aforementioned viruses were obtained. We hypothesise that the current method of virus detection, together with other factors such as the interval between death and post mortem examination, transport time of the specimens to the laboratory etc. might not be optimal to give a realistic picture of death in infancy caused by viral pulmonary infection. As other test modalities exist for the diagnosis of pulmonary viral infections, these methods were compared in terms of positive yield and association with viral pneumonitis, keeping the cost and time needed for each assay in mind. A total of 82 samples were collected over an 8 month period and routine shell vial cultures were done, followed by real-time Polymerase Chain Reaction (PCR) and immunohistochemical (IHC) staining of the lung sections with consensus pathology opinion. As expected, the real-time PCR method was much more better suited for identifying positive samples than shell vials (35% vs. 3.7% respectively). IHC staining also aided the pathologist in diagnosing viral infections microscopically. We expect the findings to be instrumental in streamlining not only our institutional SIDS investigation protocol, but also the development of a standardised national SIDS investigation protocol.
A prospective study investigating the role of respiratory viral infections in Sudden Unexpected Death in Infancy (SUDI) at Tygerberg Medico-legal Mortuary
(Stellenbosch : Stellenbosch University., 2020-03) Ferreira, Janca; De Beer, Corena; Dempers, Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background: The current South African infant mortality rate is 22.1 per 1 000 live births with respiratory infections, pneumonia, influenza and interstitial lung infections being responsible for most infant deaths. Sudden Unexpected Death in Infancy (SUDI) includes all infant deaths between the age of 7 days and 1 year without an apparent cause before any investigation has occurred. However, cases that remain unexplained following thorough investigation are classified as Sudden Infant Death Syndrome (SIDS). SIDS is regarded as a disease in search of a cause with several interlinking risk factors. Numerous respiratory viruses have been detected from SUDI autopsy specimens, therefore, viral infections could contribute to some SUDI cases as an exogenous trigger on a vulnerable infant during a specific developmental stage. This might be due to the infants’ vulnerability to infections due to immaturities of their immune systems. Nonetheless, the exact contribution of respiratory viruses preceding death still needs further investigation. Objectives: The primary aim of this study was to investigate the role of major respiratory viruses, found in the lungs and trachea as either single or co-infections of all SUDI cases admitted to Tygerberg Medico-legal Mortuary (MLM) over a 1-year period. The secondary aim entailed collecting all the epidemiological information and other relevant laboratory data from the retrospective cases from the Tygerberg MLM (2015-2016) to assess any trends or differences between the 2 studies and to evaluate how risk factors associated with SUDI cases at the Tygerberg MLM might have differed or remained constant over 2 study periods. Finally, laboratory results from all infants aged between 7 days and 1 year admitted to Tygerberg Hospital (TBH) due to respiratory infections during the study period were retrieved in order to identify if similar single and multiple viruses were circulating in both populations. Methods: Between March 2018 and March 2019 samples were collected from 173 SUDI cases admitted to Tygerberg MLM. As part of the mandatory routine investigations into SUDI cases bacterial culture swabs were collected from the lower left and right lung lobes at autopsy to investigate the role of single and co-infections of viruses associated with SUDI. The Seegene AllplexTM RV Essential Assay one-step multiplex, real-time Polymerase Chain reaction (PCR) assay was used for the detection of 6 ribonucleic acid (RNA) respiratory viruses, Influenza A (Flu A), Influenza B (Flu B), Human Metapneumovirus (HMPV), Human Parainfluenza virus (HPIV), Respiratory syncytial virus A and B (RSVA/B) and Human Rhinovirus (HRV) from RNA extracted from lower left and right lung lobe and tracheal swabs. Tissue sections from the lower left and right lung lobes were also assessed for histology signs of infection. TIBCO Statistica® version 13.5.0 was used to identify any similarities or differences between the current prospective study and retrospective study, as well as the comparison group of infants admitted to TBH. Results: During this study multiple known demographic risk factors for SUDI, such as age (12.1 ± 9.8 weeks), male predominance, prematurity, low birthweight, cold season, bedsharing, prone sleeping position and ventilation were observed. With the AllplexTM RV Essential real-time PCR assay between 1 and 5 viruses were detected in 90.2% (156/173) of cases. RSV A/B (31.7%) and HRV (24.8%) were the most commonly detected viruses. The majority of PCR-positive cases were detected in the cold season, with a statistical significance observed for Flu A (p = 0.04), Flu B (p = 0.04), HPIV (p = 0.03) and RSV (p = 0.02) and cold season. The most frequently detected co-infection was between RSVA/B and HRV. Thirty-three cases had 2 viruses detected, 5 had 3 viruses and 1 case had 5 different viruses (Flu A - Flu B - HMPV - RSV A/B - HRV). The majority of cases had a cause of death (COD) of Infection. Furthermore, Flu A was significantly associated with the COD Infection and Flu B with SIDS. In 4 SIDS cases with positive histology, positive viral PCR results were observed leading to a change in COD to Infection. Major differences between the prospective and retrospective studies included female predominance, COD of SIDS, HRV being the most frequently detected virus and co-infection only being observed in 3 cases (Flu A - HRV, Flu B - HRV; HPIV - HRV - RSV A/B). The same viruses were circulating in SUDI cases and the comparison group. Conclusion: In cases that had a COD of SIDS, the PCR viruses detected cannot be ignored, especially when it is supported by histological signs of infection as seen during this study Therefore, the use of real-time PCR could alter a COD Classification from SIDS to Infection. However, the role of single or co-infection with respiratory viruses in SUDI cases wherein no histological sign of infection was observed requires further investigation. Future research is needed to determine the exact role of co-infections in those who succumb to SUDI, more specifically how viral interactions play a role in disease progression and severity in a vulnerable infant. Finally, research should be aimed at determining the effect of PCR-positive viral results in the absence of histology to identify the true cause of vulnerability in infants.
A 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.
Respiratory infections and immune biomarkers of infection and inflammation in cases of Sudden Unexpected Death in Infancy (SUDI) at the Tygerberg Medico-legal Mortuary
(Stellenbosch : Stellenbosch University, 2018-03) Matshazi, Don Makwakiwe; De Beer, Corena; Dempers, Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH SUMMARY: infant death syndrome (SIDS) is the leading cause of infant death in the post-neonatal stage in developing and developed countries. A diagnosis of SIDS is made after medico-legal investigations fail to demonstrate an adequate cause of death in sudden unexpected death in infancy (SUDI) cases and SIDS is diagnosed in over 60% of SUDI cases. The peak incidence of SUDI is observed when infants are aged between two to four months. It is also at this stage that infants lose their maternally acquired immunity against infections. During medico-legal investigations in SUDI cases, viruses and bacteria have been confirmed, although none of them was consistently and exclusively associated with SUDI to date. Furthermore, viral respiratory symptoms are reported in 44% of SUDI cases, but the exact role they play in the events leading up to the death of the infant remains unknown. Viral infections can either facilitate easier bacterial colonisation of the respiratory system or induce an unregulated immune response through the release of cytokines and chemokines (immune biomarkers) that lead to formation of immune complexes in lungs and other respiratory organs. This could result in the loss of functionality and ultimately death of the infant. The role played by viruses and the interaction of the immune system in the events leading to SIDI therefore require further investigation to get a clearer understanding. Objectives: The first aim of this cross-sectional, descriptive study was to characterise the respiratory viruses observed in SUDI cases investigations at the Tygerberg Medico-Legal Mortuary using the Seeplex RV15 ACE detection multiplex PCR kit. The multiplex PCR viral detection results were compared to routine shell vial culture results to determine the superior viral detection method. The second aim was to assess 16 target immune biomarkers as indicators of infection or inflammation prior to or at the time of death of an infant. Methods: Samples were collected from 183 SUDI cases admitted to the Tygerberg Medico-Legal Mortuary between July 2015 and June 2016. Swabs collected from the trachea and left and right lungs were collected for multiplex PCR detection of 15 respiratory viruses. These viral targets were human adenovirus, human bocavirus, human coronavirus 229E/NL63, OC43, human enterovirus, influenza A and B, human metapneumovirus, human parainfluenza 1-4, respiratory syncytial virus A and B and human rhinovirus A/B/C. Serum was also collected for immune biomarker testing. Tissue from both lungs were collected for shell vial culture and blood was collected for HIV 1/2 antibody testing. Microbiology routine testing included culture of heart, left and right lung swab samples. Results: The gender distribution of infants in this study was not consistent with literature. There were 93 (50.8%) females and 90 (49.2%) males, although males have been identified as being at a greater risk of SUDI than females. However, other socio-demographic risk factors for SUDI, such as the greatest risk of death being at age two to four months were consistent with literature. The detection of viruses by multiplex PCR proved to be superior to SVC as the former detected viruses in more cases than the latter. The most commonly detected virus by multiplex PCR was human Rhinovirus A/B/C, which was detected in 65 (35.5%) of the 183 cases tested. Adenovirus was the second most frequently detected virus as it was present in 18 (12.6%) of the cases tested. Parainfluenza 3, Enterovirus and RSV B were detected in 10 (5.5%), 9 (4.9%) and 7 (3.8%) cases respectively. Human metapneumovirus was not detected at all by either assay. The serum concentrations of CRP and IL-6 were significantly elevated in the serum of SUDI cases where infection was the cause death compared to cases that were diagnosed as SIDS. However, levels of IL-18 were significantly reduced in the serum of SUDI cases where infection was the cause of death compared to cases with a final cause of death classification of SIDS. Conclusion: It is possible to change the cause of death from SIDS to infection if serum immune biomarker results and multiplex PCR are used in addition to tissue histology. The current study showed that serum CRP, IL-6 and IL-18 levels can possibly be regarded as candidates for use as indicators of infectious death and these findings need to be further investigated in larger study cohorts and over longer study periods.
Respiratory pathogens in cases of Sudden Unexpected Death in Infancy (SUDI) at Tygerberg forensic pathology service mortuary
(Stellenbosch : Stellenbosch University, 2014-04) La Grange, Heleen; De Beer, Corena; Dempers, Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Medical Virology.
ENGLISH ABSTRACT: Background: Sudden infant death syndrome (SIDS) is considered the second most frequent cause of infant mortality worldwide. Research specifically pertaining to SIDS is limited in the South African setting. Identifiable causes for sudden infant death remain challenging despite full medico-legal investigations inclusive of autopsy, scene visit and ancillary studies. Viral infections could contribute to some sudden unexpected death in infancy (SUDI) cases, especially since a multitude of respiratory viruses have been detected from autopsy specimens. The specific contribution of viruses in the events preceding death, including the subsequent involvement of the immature immune response in infants, still warrants deciphering. Infancy is characterised by marked vulnerability to infections due to immaturities of their immune systems that may only resolve as infants grow older when these sudden deaths rarely still occur. In South Africa there is a lack of a standard protocol for investigations into the causes of SIDS, including the lack of standard guidelines as to which specimens should be taken, which viruses should be investigated and which laboratory assays should be utilised. Objectives: In this prospective descriptive study we aimed to investigate the prevalence of viruses in SUDI and SIDS cases at Tygerberg Forensic Pathology Service (FPS) Mortuary over a one year period. The primary aim was to explore possible respiratory viral infections in SUDI and SIDS cases and to determine the usefulness of molecular techniques to detect viruses from SUDI cases. To determine the significance of viruses, we assessed signs of infection from lung histology. The secondary objectives included collecting demographic data to investigate possible risk factors for SUDI and to look for possible similarities between viruses confirmed in living hospitalised infants at Tygerberg, during the study period compared to viruses detected from SUDI cases. Methods: Between May 2012 and May 2013 samples were collected from 148 SUDI cases presenting at Tygerberg FPS Mortuary. As part of the mandatory routine investigations into SUDI, shell vial culture (SVC) results were collected from lung and liver tissue specimens and bacterial culture results were collected from left and right lung and heart swabs at autopsy. To investigate the possibility of viruses implicated in some of the infant deaths we used the Seeplex® RV15 Ace detection multiplex polymerase chain reaction (PCR) assay to establish the frequency of 13 ribonucleic acid (RNA) respiratory viruses (influenza A and B, human parainfluenza 1-4, human coronavirus [OC43, 229E/NL63], human rhinovirus A, B and C, respiratory syncytial virus A and B, human enterovirus and human metapneumovirus) from RNA extracted from tracheal and lower left and right lung lobe swabs. Tissue from the lower left and right lung lobes were also assessed for histology signs of infection. Results: During our study we confirmed multiple known demographic risk factors for SIDS, such as the age peak around 1-3 months, the male predominance, bed-sharing, sleeping in the prone position, heavy wrapping in warm blankets, prenatal smoke exposure, and socio-economic factors. With the Seeplex® RV15 Ace detection assay between one and three viruses were detected in 59.5% (88/148) of cases. Of the 88 cases that had viruses detected, 75% (66/88) had one virus and 25% (22/88) had co-detections of two to three viruses. The most common viruses detected were HRV in 77% (68/88) of cases, RSV in 18% (16/88) of cases and HCoV in 14% (12/88) of cases. Many of the viruses we detected from our cases are included in the SVC test that forms part of the medico-legal laboratory investigation for all SUDI cases at Tygerberg FPS Mortuary. SVCs were positive in 9.5% (14/148) of all cases only. We showed that the SVC method is potentially missing most of the 13 respiratory viruses we investigated that could contribute to death in some of the SUDI cases. Conclusion: In some cases that had a Cause of Death Classification - SIDS, the PCR viruses detected cannot be ignored, especially when it is supported by histological evidence of infection. We thus propose that the use of PCR could alter a Cause of Death Classification from SIDS to Infection in some of these cases. Further research is needed to determine the significance of detecting viruses from SUDI cases wherein no significant histological evidence of infection was observed. This questions whether PCR may be too sensitive and is detecting past and latent viral infections that do not play any role in the cause of death. The histological picture also requires further characterisation to determine if it accurately predicts infections or lethal events and can truly support virology findings, especially in young infants whose immune systems are still maturing. Without determining the true prevalence of viruses in SUDI cases and the viral-specific immune response, the contribution of virus-specific infections to this syndrome will remain largely undetermined.
Screening 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.
T lymphocyte inhibitory/exhaustion marker expression in chronic HIV-1 infection and the impact of TB co-infection
(Stellenbosch : Stellenbosch University, 2016-03) Golden, Maxine; Glashoff, Richard; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Introduction Chronic HIV-1 infection is driven by inflammation and immune activation which ultimately induces T lymphocyte exhaustion. Co-infection with TB is problematic as the HIV-impaired immune system is unable to effectively contain the TB, and in turn the TB disease promotes further immune activation and exhaustion through additional antigenic burden. In the current two component study, a total of 101 HIV-1 infected, 25 HIV-1-TB co-infected and 52 uninfected control individuals were included from the Cape Town peri-urban region. A cross sectional investigation of expression of markers of immune activation (CD38 and HLA-DR), exhaustion/inhibition (PD-1, Tim-3, LAG-3, 2B4) and apoptosis (CD95) was investigated on both CD4+ and CD8+ T cells. Functional proliferative responsiveness of T cells was also assessed. Materials and Methods Flow cytometry (both 4–colour and 10-colour) was used to determine expression of phenotypic markers using both fresh whole blood (pilot study) and PBMC (main study). CD4+ and CD8+ T lymphocyte populations of all study groups were stained for all the markers and evaluated for positive expression or co-expression. A functional proliferation assay (αCD3 and αCD28 stimulation) was conducted using CFSE-staining on the HIV-1+ samples. Impact of blocking Tim-3 and PD-1 pathways was evaluated. Results Chronic HIV-1 infection was accompanied by a significant increase in CD38 (p<0.0001), CD95 (p<0.01), PD-1 (p<0.01) and Tim-3 (p<0.01) expression on CD8+ T cells (pilot study). TB co-infection led to significantly elevated expression of CD38, CD95 and Tim-3, but not PD-1 (all p<0.05). CD4+ T cells displayed decreased expression of all of these markers in the infected groups, except for Tim-3, which was consistently <5%. In the main study CD8+ T cell-associated CD38, CD95, and PD-1 displayed a similar trend, with significant higher expression in the infected groups (p<0.0001, p<0.05, and p<0.0001, respectively), In contrast to the pilot study, Tim-3 expression was consistently <10%, with no difference between the groups. The novel marker 2B4 showed high level baseline expression (median 59.2%) which was significantly increased in the HIV and HIV/TB groups (69.6% and 75.1% respectively, p=0.025). LAG-3 was poorly expressed. Co-expression of PD-1 and 2B4 as well as CD95 and CD38 was also significantly increased (p<0.0001 for both). Discussion Increased immune activation and exhaustion was evident in the infected groups in both studies. PD-1 and 2B4 were both strongly expressed on CD8+ T cells and were significantly up-regulated in infected groups. PD-1 correlated positively with Tim-3 and LAG-3, and 2B4 with LAG-3, (both p<0.01) indicating that they are useful as biomarkers of exhaustion. Although significantly elevated exhaustion markers were observed in the TB co-infected setting in the pilot study, this did not reflect in the main study (except for CD95, 2B4). This suggests that immune dysfunction is mainly driven by HIV-1 infection alone. Short-term anti-TB therapy may also have had a restorative impact on the exhaustive marker expression. Differences in expression patterns using fresh whole blood vs. PBMC (especially Tim-3) warrant further investigation. Blocking Tim-3 and PD-1 expression on CD4+ and CD8+ T cells did not appear to have beneficial effects on T cell proliferation.

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