Infant mortality remains a major global concern. Sudden unexpected death in infancy (SUDI) is reported globally and an infant mortality rate of 23.129 per 1 000 live births has been reported in the Western Cape, South Africa, in 2024. Infections are often confirmed in SUDI cases admitted to the Tygerberg Medico-legal Mortuary in Cape Town, but molecular diversity in respiratory viruses is underreported. A total of 162 previously confirmed polymerase chain reaction (PCR)-positive trachea and / or lung samples from SUDI cases collected between 2015 and 2019 were retested for either rhinovirus or human respiratory syncytial virus (RSV). Sixty-four samples were positive for rhinovirus and 15 for RSV. Results from 5 of all positive samples were outside the PCR assay amplification limits determined by the cycle threshold (Ct) value and were excluded. Another 4 samples did not amplify, and the remaining 70 underwent subsequent sequencing, but successful sequences could only be obtained in 53 samples. All three rhinovirus (A, B and C) genotypes were identified, with RV-A most prevalent, followed by RV-C and RV-B. RSV-A and RSV-B were detected equally, and after amino acid alignment, 20 amino acid duplication and nine substitutions were found that confirmed two RSV-BA9 genotypes. This study describes the molecular and phylogenetic characterisation of specific respiratory viruses in SUDI cases in South Africa. However, the rapid decline in viral viability in post-mortem samples does not allow correlation between viral genotypes and cause of death or disease severity. Future prospective studies should therefore investigate temporality and associations between specific viral strains and clinical disease severity and mortality.

The mechanisms triggering the human immunodeficiency virus type I (HIV-1) to switch the coreceptor usage from CCR5 to CXCR4 during the course of infection are not entirely understood. While low CD4+ T cell counts are associated with CXCR4 usage, a predominance of CXCR4 usage with still high CD4+ T cell counts remains puzzling. Here, we explore the hypothesis that viral adaptation to the human leukocyte antigen (HLA) complex, especially to the HLA class II alleles, contributes to the coreceptor switch. To this end, we sequence the viral gag and env protein with corresponding HLA class I and II alleles of a new cohort of 312 treatment-naive, subtype C, chronically-infected HIV-1 patients from South Africa. To estimate HLA adaptation, we develop a novel computational approach using Bayesian generalized linear mixed models (GLMMs). Our model allows to consider the entire HLA repertoire without restricting the model to pre-learned HLA-polymorphisms. In addition, we correct for phylogenetic relatedness of the viruses within the model itself to account for founder effects. Using our model, we observe that CXCR4-using variants are more adapted than CCR5-using variants (p-value = 1.34e-2). Additionally, adapted CCR5-using variants have a significantly lower predicted false positive rate (FPR) by the geno2pheno[coreceptor] tool compared to the non-adapted CCR5-using variants (p-value = 2.21e-2), where a low FPR is associated with CXCR4 usage. Consequently, estimating HLA adaptation can be an asset in predicting not only coreceptor usage, but also an approaching coreceptor switch in CCR5-using variants. We propose the usage of Bayesian GLMMs for modeling virus-host adaptation in general.

Sudden and unexpected death in infancy (SUDI) may be triggered by an external risk or exposure. Intestinal infections with enteric viruses may disrupt the gut and enhance bacterial toxins present in SUDI cases. While diarrhoeal disease deaths have decreased worldwide, approximately half a million deaths still occur in children in Sub- Saharan Africa and South Asia. Furthermore, the role of viral enteropathogens in SUDI cases have not been investigated. The aim of this study was to describe specific viral pathogens in stool samples collected from SUDI cases and age-matched, apparently healthy infants in Cape Town, South Africa. Stool samples were collected from 176 SUDI cases between June 2017 and May 2018. In addition, stool samples were collected from the nappies of 30 age-matched, apparently healthy infants as a control group. Real-time polymerase chain reaction was performed on the stool samples for viral detection. A total of 111 SUDI cases were positive for viruses, with rotavirus (38.6%; 68/176) and norovirus GI and GII (30.0%; 53/176) were prevalent in SUDI cases. Adenovirus Type F was present in only 15.9% (28/176), astrovirus in 9.7% (17/176), and sapovirus in 0.6% (1/176) of cases. In the control samples, norovirus GII was detected most frequently (36.7%; 11/30), followed by rotavirus (33.3%; 10/30), and sapovirus in 6.7% (2/30). While there was no significant association between SUDI cases and enteric viruses, the majority of viruses were significantly associated with the seasons. The study confirms the importance of rotavirus vaccination and describes the significance of norovirus infection in children, post rotavirus vaccine introduction.

The occurrence of rotavirus (RV) infection among vaccinated children in high-burden settings poses a threat to further disease burden reduction. Genetically altered viruses have the potential to evade both natural infection and vaccine-induced immune responses, leading to diarrheal diseases among vaccinated children. Studies characterizing RV strains responsible for breakthrough infections in resource-limited countries where RV-associated diarrheal diseases are endemic are limited. We aimed to characterize RV strains detected in fully vaccinated children residing in Zambia using next-generation sequencing. We conducted whole genome sequencing on Illumina MiSeq. Whole genome assembly was performed using Geneious Prime 2023.1.2. A total of 76 diarrheal stool specimens were screened for RV, and 4/76 (5.2%) were RV-positive. Whole genome analysis revealed RVA/Human-wt/ZMB/CIDRZ-RV2088/2020/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 mono and multiple reassortant (exchanged genes in bold) respectively, whilst 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 and VP4 antigenic epitope of breakthrough strains and Rotarix strain revealed several amino acid differences. Variations in amino acids in antigenic epitope suggested they played a role in immune evasion of neutralizing antibodies elicited by vaccination. Findings from this study have the potential to inform national RV vaccination strategies and the design of highly efficacious universal RV vaccines.

Child mortality is a major health concern worldwide with over 4.2 million infants dying before reaching the age of one year in 2016 alone. Several international intervention initiatives have resulted in a decrease in the number of infant deaths; however, the incidence of sudden unexpected death in infancy (SUDI) and sudden infant death syndrome (SIDS) remain unacceptably high. SIDS still accounts for approximately 50–80% of SUDI cases, followed by infection. The aim of this study was to investigate a selection of immune biomarkers that are associated with an immune response in an effort to support the diagnosis of an infectious cause ("Infection") e.g. bronchopneumonia, interstitial pneumonitis, etc., instead of SIDS in SUDI cases. C-reactive protein and 18 different cytokines were retrospectively quantified in serum collected during post-mortem investigations of SUDI cases admitted to the Tygerberg Medico-legal Mortuary in the Western Cape Province of South Africa between 2015 and 2017. Statistical comparison was done between infants with a final cause of death (COD) of Infection and SIDS to investigate any correlations between the immune markers and sociodemographic information of the groups. A p-value of < 0.0026, after Bonferroni correction for multiple comparisons, was considered as statistically significant. A total of 169 cases were included, of which 65 (38.5%) were assigned a cause of death of Infection and 104 (61.5%) SIDS by forensic pathologists. The male to female ratio of the entire group was 1:0.97 and the median age at the time of death was 9 (interquartile range [IQR] 10.9) weeks. The majority (56.8%) of deaths occurred during the colder seasons (autumn and winter) and the median post-mortem interval was 4 (IQR 3) days. No statistically significant differences were demonstrated for gender, season, sleeping position or bed-sharing between the Infection and SIDS groups. Age and interleukin-1α were identified as predictors of a COD of Infection before adjusting for the multiple comparisons problem. C-reactive protein was a statistically significant predictor of a COD of Infection even after adjusting for the effect of multiple comparisons. The COD is primarily based on histopathology of the lungs, where other causes of interstitial inflammation have been ruled out, and where there are morphological changes present suggestive of infection, but not enough evidence to assign a final COD of Infection, the cases are concluded as SIDS. These biomarkers can therefore be valuable in the investigation protocol of SUDI cases to increase the number Infection cases where the histopathology of the lungs is suggestive of, but does not support conclusive evidence of infection.

Purpose. This study aimed to determine the prevalence of human respiratory syncytial virus (HRSV) acute respiratory infection (ARI) in children under the age of 5 years at the Provincial General Hospital of Bukavu (PGHB), and to analyse factors associated with the risk of ARI being diagnosed as lower respiratory tract infection (LRTI). Methodology. A total of 146 children under 5 years visiting the PGHB for ARI between August and December 2016 were recruited, and socio-demographic information, clinical data and nasopharyngeal swabs were collected. The samples were analysed by a multiplex reverse transcriptase polymerase chain reaction targeting 15 different viruses. Results. 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 fewer cases. There were only 11 (7.5%) cases of co-infection. HRSV infection, malnutrition, younger age, rural settings, low income and mother illiteracy were associated with the risk of ARI being diagnosed as LRTI in bivariate analyses but, after adjusting for the confounding factors, only HRSV infection and younger age were independently associated with LRTI. Conclusion. The prevalence of HRSV is high among children visiting the PGHB for ARI. HRSV infection and lower age are independently associated with the risk of ARI being diagnosed as LRTI.

Background: Mucins are large O-linked glycosylated proteins which give mucus their gel-forming properties. There are indications that mucus and mucins in saliva, breast milk and in the cervical plug inhibit the human immunodeficiency virus (HIV-1) in an in vitro assay.Main body of abstract: Crude mucus gels form continuous layers on the epithelial surfaces of the major internal tracts of the body and protect these epithelial surfaces against aggressive luminal factors such as hydrochloric acid and pepsin proteolysis in the stomach lumen, the movement of hard faecal pellets in the colon at high pressure, the effects of shear against the vaginal epithelium during intercourse and the presence of foreign substances in the respiratory airways. Tumour-associated epitopes on mucins make them suitable as immune-targets on malignant epithelial cells, rendering mucins important as diagnostic and prognostic markers for various diseases, even influencing the design of mucin-based vaccines.Sub-Saharan Africa has the highest prevalence of HIV-AIDS in the world. The main points of viral transmission are via the vaginal epithelium during sexual intercourse and mother-to-child transmission during breast-feeding. There have been many studies showing that several body fluids have components that prevent the transmission of HIV-1 from infected to non-infected persons through various forms of contact.Crude saliva and its purified mucins, MUC5B and MUC7, and the purified mucins from breast milk, MUC1 and MUC4 and pregnancy plug cervical mucus (MUC2, MUC5AC, MUC5B and MUC6), inhibit HIV-1 in an in vitro assay. There are conflicting reports of whether crude breast-milk inhibits HIV-1 in an in vitro assay. However studies with a humanised BLT mouse show that breast-milk does inhibit HIV and that breast-feeding is still advisable even amongst HIV-positive women in under-resourced areas, preferably in conjunction with anti-retroviral treatment.Conclusion: These findings raise questions of how such a naturally occurring biological substance such as mucus, with remarkable protective properties of epithelial surfaces against aggressive luminal factors in delicate locations, could be used as a tool in the fight against HIV-AIDS, which has reached epidemic proportions in sub-Saharan Africa.

Background: Bacille Calmette-Guerin (BCG) is routinely given at birth in tuberculosis-endemic settings due to its protective effect against disseminated tuberculosis in infants. BCG is however contraindicated in HIV-infected infants. We investigated whether delaying BCG vaccination to 14 weeks of age affected vaccine-induced antibody responses to Haemophilus influenzae type b (Hib)-conjugate, pertussis, tetanus and Hepatitis B (HBV) vaccines, in HIV-exposed uninfected (HEU) and -unexposed uninfected (HUU) infants.Methods: Infants were randomized to receive BCG at birth or at 14 weeks of age. Blood was taken at 14, 24, and 52 weeks of age and analyzed for Hib, pertussis, tetanus and HBV specific antibodies.Results: BCG was given either at birth (106 infants, 51 HEU) or at 14 weeks of age (74 infants, 50 HEU). The timing of BCG vaccination did not influence the antibody response to any antigen studied. However, in a non-randomized comparison, HEU infants had higher Hib antibody concentrations at weeks 14 and 24 (p=0.001 and <0.001, respectively) and pertussis at week 24 (p = 0.003). Conversely, HEU infants had lower antibody concentrations to HBV at 14 and 52 weeks (p = 0.032 and p = 0.031) with no differences in tetanus titres.Conclusions: HIV exposure, but not the timing of BCG vaccination, was associated with antibody concentrations to Hib, pertussis, HBV and tetanus primary immunization. (C) 2016 Elsevier Ltd. All rights reserved.

Background: The HIV-AIDS pandemic is prevalent in sub-Saharan Africa. Breastfeeding is a risk factor, with transmission from mother to child being as high as 40%. Objectives: To determine the antiviral activity of crude breast milk and its purified mucins MUC1 and MUC4 against HIV-1 in patients who were HIV positive compared to those who were not. Methods: Twenty-one human milk samples were taken from both groups. Breast milk mucins were purified by density-gradient ultracentrifugation in caesium chloride and analyzed by SDS-PAGE, Western blotting and amino acid content. The inhibition of the virus by crude milk and purified mucin was assayed by an in vitro HIV-1 p24 assay. Results: SDS-PAGE for purified mucin showed several high-molecular-weight bands for the HIV-negative group and prominently stained single bands on the stacking gel with faintly periodic acid Schiff-positive glycoprotein bands observed in some cases in the running gel for the HIV-positive mucins. Western blot analysis identified the mucins in both groups to be MUC1 and MUC4. Both mucins showed more intensity on Western blotting for the HIV-positive group. There was no difference in the content of serine, threonine and proline of purified mucins for both groups. HIV-1 was not inhibited by crude breast milk from normal (13/14 samples) and infected individuals (19/19 samples). Fifteen of 20 and 16/18 samples of purified mucin from the uninfected and HIV-positive groups, respectively, inhibited the virus. Conclusions: Crude breast milk does not inhibit HIV-1, whilst purified mucins do in an in vitro assay.

Currently in South Africa research into sudden unexpected death in infancy (SUDI) is limited. The causes of sudden infant death syndrome (SIDS) remain obscure despite full medico-legal investigations inclusive of autopsy, scene visit and ancillary studies. Viral infections play an important role as a multitude of respiratory viruses have been detected in autopsy specimens and are implicated in these deaths. The specific contribution of viruses in the events preceding SIDS still warrants deciphering. Infancy is characterised by marked vulnerability to infections due to immaturities of the immune system that may only resolve by the age of 24 months. Routine viral screening of all SUDI cases at Tygerberg Forensic Pathology Service (FPS) Mortuary in Cape Town focuses on only a portion of respiratory viruses from lung and liver tissue. This review highlights important virological and immunological aspects regarding investigations into the infectious nature of SUDI, including the lack of national standardised guidelines for appropriate specimen collection at autopsy and subsequent laboratory analysis.

BackgroundSudden unexpected death in infancy is one of the main contributory factors to high infant mortality rates world-wide. Several risk factors, including viral infection, have been implicated in SUDI cases, but no single factor has been confirmed as the main cause of death. At the Tygerberg Medico-legal Laboratory, Cape Town, South Africa, investigation of lung tissue for viral infection forms part of an institutional protocol for the examination of cases of sudden unexpected death in infancy.MethodsLung tissue from 82 cases of sudden unexpected death in infancy was collected over a 10 month period. Routine shell vial cultures and histological examination of the tissue were performed according to the standard institutional protocol on fresh and formalin-fixed tissue, respectively. In addition, real-time polymerase chain reactions and immunohistochemical staining for adenovirus, cytomegalovirus and respiratory syncytial virus were done on fresh and formalin-fixed lung tissue, respectively.ResultsHuge variation was found in the number of positive cases confirmed by shell vial culture, real-time polymerase chain reaction and immunohistochemistry (0, 2 and 0 for adenovirus; 3, 29 and 2 for cytomegalovirus; and 0, 0 and 4 for respiratory syncytial virus, respectively).ConclusionsIn the absence of a National Protocol for investigation of sudden unexpected death in infancy, we conclude that the selection of viruses and routine diagnostic technique included in the institutional investigation protocol might be suboptimal and should be re-evaluated.

Background: Sudden infant death syndrome (SIDS) is considered the second most frequent cause of infant mortality worldwide. Identifiable causes remain challenging despite medico-legal investigations. Testing for viral infections can significantly contribute to some of these deaths, especially since a multitude of viruses have been detected from autopsy specimens. In South Africa there is a lack of a standard protocol for investigations into causes of SIDS. In this prospective descriptive study we aimed to investigate the prevalence of viruses in SIDS cases at Tygerberg Forensic Pathology Service (FPS) Mortuary. We also assessed signs of infection from lung histology. Methods & Materials: Between May 2012 and May 2013 tracheal and lung swabs were collected from 148 SUDI cases. The Seeplex® RV15 Ace detection polymerase chain reaction (PCR) assay was used to investigate 13 ribonucleic acid (RNA) viruses (influenza A and B, PIV 1-4, HCoV [OC43, 229E/NL63], HRV A, B and C, RSV A and B, human enterovirus and HMPV). Results: During our study we confirm multiple known demographic risk factors for SIDS and we detected between 1 and 3 viruses 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 2-3 viruses. Microscopic signs of infection on lung tissue were confirmed in 59.5% (88/148) of cases. Most of the viruses are included in the routine shell vial culture (SVC) test that forms part of the medico-legal investigation for all SIDS cases at Tygerberg. However, SVCs were positive in 9.5% of cases only and thus seems to miss most of the 13 repisratory viruses we investigated. Conclusion: In some cases the PCR viruses detected may be important 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 cases. The histological picture requires further characterisation to determine if it accurately predicts infections and can support virology findings. Without determining the true prevalence of viruses and the viral-specific immune response, the contribution of virus-specific infections to this syndrome will remain largely undetermined.

Background:Early in life, HIV-exposed uninfected (HEU) infants are at an increased risk of morbidity and mortality from infectious disease compared with HIV-unexposed (UE) infants. To improve our understanding of the mechanisms underlying their increased risk, we contrasted innate immune development between HEU and UE infants in a developing world setting, where early life infectious disease risk is exceptionally high.Methods:A prospective longitudinal cohort of HEU and UE newborns was established, and the most detailed characterization to date of HEU infant immune development was performed. Single-cell cytokine production was analyzed by flow cytometry after stimulation of whole blood with pathogen-associated molecular patterns (PAMPs).Results:Monocyte, classical dendritic cell, and plasmacytoid dendritic cell composition was similar between HEU and UE infants throughout the first year of life. However, HEU mononuclear cells mounted an enhanced pro-inflammatory response to PAMP stimulation, both in quantity of cytokine produced per cell and in proportion of responder cells. Significant differences in cytokine production were detected on the single-cell level in a PAMP-specific pattern, but only at 2 and 6 weeks of age; all differences normalized by 12 months of age.Conclusions:This time course of innate immune deviation early in life corresponds to the clinical window of vulnerability to infections in HEU infants and may be at least partially responsible for their increased morbidity and mortality from infectious disease.

HIV-exposed but uninfected (HEU) infants born to HIV-infected mothers from areas in the world with a high burden of infectious disease suffer higher infectious morbidity and mortality than their HIV unexposed uninfected (HUU) peers. Vaccination provides protection from infection. The possibility exists that altered response to vaccination contributes to the higher rate of infection in HEU than in HUU infants. While short-term, cross-sectional studies support this notion, it is unclear whether or not HEU infants develop long-term protective immune responses following the WHO extended program on immunization (EPI). Vaccine-specific antibody responses were compared between HEU and HUU infants from 2 weeks until 2 years of age in a longitudinal South African cohort. Total IgG and antibodies specific for Bordetella pertussis, Haemophilus influenzae type b (Hib), tetanus toxoid, hepatitis B virus (HepB), and measles virus were measured at multiple time points throughout the first 2 years of life. Prevaccine antibodies (maternal antibodies passively acquired) specific for tetanus were lower in HEU than in HUU infants, while prevaccine antibodies to HepB were higher in HEU than in HUU infants. Both groups responded similarly to tetanus, Hib, and HepB vaccination. HEU demonstrated stronger pertussis vaccine responses, developing protective titers 1 year earlier than HUU patients, and maintained higher anti-tetanus titers at 24 months of age. Vaccine-induced antibodies to measles virus were similar in both groups at all time points. Our results suggest that the current EPI vaccination program as practiced in South Africa leads to the development of vaccine-specific antibody responses that are equivalent in HEU and HUU infants. However, our data also suggest that a large fraction of both HEU and HUU South African infants have antibody titers for several infectious threats that remain below the level of protection for much of their first 2 years of life.

Pertussis is a highly contagious respiratory disease that can occur in all age groups. Over the past 15 years, an increase in the incidence of reported cases has been observed globally. Local surveillance data have also shown an increase in the number of laboratory-confrmed cases. This raises concerns about waning immunity in adolescents and adults. The aim of this study was to screen for seropositivity against pertussis infection by measuring immunoglobulin G (IgG) antibody titers of Bordetella pertussis in an adolescent population in the Western Cape, South Africa. Serum samples were collected from 182 adolescents aged 15-18 years from different racial groups. The SERION ELISA® classic B. pertussis IgG assay was used to detect human antibodies in serum. Twenty-seven subjects (15%) were seronegative and 135 (74%) seropositive for IgG antibodies. Racial breakdown showed that 84% of the black, 74% of the coloured, and 69% of the white subjects were seropositive, while the largest percentage (24%) of sero-negative individuals was in the white population group. This study demonstrated a high percentage of individuals in the adolescent age group with sero-positive antibody levels. However, it is unknown if these antibody levels are functionally protective. A more rigourous surveillance system would assist in defning and understanding the epidemiology of pertussis in South Africa, and could provide evidence of the need for booster vaccinations in adolescence and adulthood.

The first year of life represents a time of marked susceptibility to infections; this is particularly true for regions in sub-Saharan Africa. As innate immunity directs the adaptive immune response, the observed increased risk for infection as well as a suboptimal response to vaccination in early life may be due to less effective innate immune function. In this study, we followed a longitudinal cohort of infants born and raised in South Africa over the first year of life, employing the most comprehensive analysis of innate immune response to stimulation published to date. Our findings reveal rapid changes in innate immune development over the first year of life. This is the first report depicting dramatic differences in innate immune ontogeny between different populations in the world, with important implications for global vaccination strategies.

HIV-exposed uninfected (HEU) infants have higher infectious morbidity than HIV-unexposed uninfected (HUU) infants. We present the clinical outcomes from a pilot cohort study of 27 HEU and 28 HUU infants. In the absence of infant malnutrition or advanced maternal HIV, HEU infants experienced a 2.74 (0.85-8.78) times greater risk of hospitalization in the first year.