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BACKGROUND We aimed to describe the prevalence of human respiratory syncytial virus (HRSV) and evaluate associations between HRSV subgroups and/or genotypes and epidemiologic characteristics and clinical outcomes in patients hospitalized with severe respiratory illness (SRI). METHODS Between January 2012 and December 2015, we enrolled patients of all ages admitted to two South African hospitals with SRI in prospective hospital-based syndromic surveillance. We collected respiratory specimens and clinical and epidemiological data. Unconditional random effect multivariable logistic regression was used to assess factors associated with HRSV infection. RESULTS HRSV was detected in 11.2% (772/6908) of enrolled patients of which 47.0% (363/772) were under the age of 6 months. There were no differences in clinical outcomes of HRSV subgroup A-infected patients compared with HRSV subgroup B-infected patients but among patients aged

Background: The SARS-CoV-2 Delta variant (B.1.617.2) has been associated with more severe disease, particularly when compared to the Alpha variant. Most of this data, however, is from high income countries and less is understood about the variant’s disease severity in other settings, particularly in an African context, and when compared to the Beta variant. Methods: A novel proxy marker, RNA-dependent RNA polymerase (RdRp) target delay in the Seegene AllplexTM 2019-nCoV (polymerase chain reaction) PCR assay, was used to identify suspected Delta variant infection in routine laboratory data. All cases diagnosed on this assay in the public sector in the Western Cape, South Africa, from 1 April to 31 July 2021, were included in the dataset provided by the Western Cape Provincial Health Data Centre (PHDC). The PHDC collates information on all COVID-19 related laboratory tests, hospital admissions and deaths for the province. Odds ratios for the association between the proxy marker and death were calculated, adjusted for prior diagnosed infection and vaccination status. Results: A total of 11,355 cases with 700 deaths were included in this study. RdRp target delay (suspected Delta variant) was associated with higher mortality (adjusted odds ratio [aOR] 1.45; 95% confidence interval [CI]: 1.13-1.86), compared to presumptive Beta infection. Prior diagnosed infection during the previous COVID-19 wave, which was driven by the Beta variant, was protective (aOR 0.32; 95%CI: 0.11-0.92) as was vaccination (aOR [95%CI] 0.15 [0.03-0.62] for complete vaccination [≥28 days post a single dose of Ad26.COV2.S or ≥14 days post second BNT162b2 dose]). Conclusion: RdRp target delay, a proxy for infection with the Delta variant, is associated with an increased risk of mortality amongst those who were tested for COVID-19 in our setting.

Background In June 2017, an outbreak of the highly pathogenic avian influenza A(H5N8) was detected in commercial poultry farms in South Africa, which rapidly spread to all nine South African provinces. Objectives We conducted active surveillance for the transmission of influenza A(H5N8) to humans working with infected birds during the South African outbreak. Methods Influenza A(H5N8)‐positive veterinary specimens were used to evaluate the ability of real‐time PCR‐based assays to detect contemporary avian influenza A(H5N8) strains. Whole genome sequences were generated from these specimens by next‐generation sequencing for phylogenetic characterization and screening for mammalian‐adaptive mutations. Results Human respiratory samples from 74 individuals meeting our case definition, all tested negative for avian influenza A(H5) by real‐time PCR, but 2 (3%) were positive for human influenza A(H3N2). 54% (40/74) reported wearing personal protective equipment including overalls, boots, gloves, masks, and goggles. 94% (59/63) of veterinary specimens positive for H5N8 were detected on an influenza A(H5) assay for human diagnostics. A commercial H5N8 assay detected H5 in only 6% (3/48) and N8 in 92% (44/48). Thirteen (13/25; 52%) A(H5N8) genomes generated from veterinary specimens clustered in a single monophyletic clade. These sequences contained the NS (P42S) and PB2 (L89V) mutations noted as markers of mammalian adaptation. Conclusions Diagnostic assays were able to detect and characterize influenza A(H5N8) viruses, but poor performance is reported for a commercial assay. Absence of influenza A(H5N8) in humans with occupational exposure and no clear impression of molecular adaptation for mammalian infection suggest that this avian pathogen continues to be low‐risk human pathogen.

SARS-CoV-2 variants that escape neutralization and potentially affect vaccine efficacy have emerged. T cell responses play a role in protection from reinfection and severe disease, but the potential for spike mutations to affect T cell immunity is incompletely understood. We assessed neutralizing antibody and T cell responses in 44 South African COVID-19 patients either infected with the Beta variant (dominant from November 2020 to May 2021) or infected before its emergence (first wave, Wuhan strain) to provide an overall measure of immune evasion. We show that robust spike-specific CD4 and CD8 T cell responses were detectable in Beta-infected patients, similar to first-wave patients. Using peptides spanning the Beta-mutated regions, we identified CD4 T cell responses targeting the wild-type peptides in 12 of 22 first-wave patients, all of whom failed to recognize corresponding Beta-mutated peptides. However, responses to mutated regions formed only a small proportion (15.7%) of the overall CD4 response, and few patients (3 of 44) mounted CD8 responses that targeted the mutated regions. Among the spike epitopes tested, we identified three epitopes containing the D215, L18, or D80 residues that were specifically recognized by CD4 T cells, and their mutated versions were associated with a loss of response. This study shows that despite loss of recognition of immunogenic CD4 epitopes, CD4 and CD8 T cell responses to Beta are preserved overall. These observations may explain why several vaccines have retained the ability to protect against severe COVID-19 even with substantial loss of neutralizing antibody activity against Beta.

A novel proxy for the Delta variant, RNA-dependent RNA polymerase target delay in the Seegene Allplex™ 2019-nCoV PCR assay, was associated with higher mortality (adjusted Odds Ratio 1.45 [95%CI 1.13-1.86]), compared to presumptive Beta infection, in the Western Cape, South Africa (April-July 2021). Prior diagnosed infection and vaccination were protective.

Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced PCR amplification efficiency of the RdRp gene target of the Seegene, Allplex 2019-nCoV diagnostic assay when detecting the Delta variant. We propose that this can be used as a surrogate for variant detection.

HLA-DQB1*05:272 has a single transversion compared to the most similar variant, HLA-DQB1*05:01:01 in IPD-IMGT/HLA database. This article is protected by copyright. All rights reserved.

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced RT-PCR amplification efficiency of the RdRp-gene target of the Seegene, Allplex 2019-nCoV diagnostic assay from June 2021 when detecting the Delta variant. We investigated whether the reduced amplification efficiency denoted by an increased RT-PCR cycle threshold value (RΔE) can be used as an indirect measure of SARS-CoV-2 Delta variant prevalence. We found a significant increase in the median RΔE for patient samples tested from June 2021, which coincided with the emergence of the SARS-CoV-2 Delta variant within our sample set. Whole genome sequencing on a subset of patient samples identified a highly conserved G15451A, non-synonymous mutation exclusively within the RdRp gene of Delta variants, which may cause reduced RT-PCR amplification efficiency. While whole genome sequencing plays an important in identifying novel SARS-CoV-2 variants, monitoring RΔE value can serve as a useful surrogate for rapid tracking of Delta variant prevalence.

Trivalent seasonal influenza vaccine effectiveness during the 2015 season in South Africa was assessed using a test-negative case control study design. Influenza A(H1N1)pdm09 was the dominant circulating strain. Overall influenza vaccine coverage was 3.2% (29/899). The vaccine effectiveness estimate, against any influenza virus infection, adjusted for age, underlying conditions and timing within season was 46.2% (95% CI: -23.5 to 76.5), and 53.6% (95% CI: -62.6 to 80.3) against influenza A(H1N1)pdm09.

Background We estimated the household secondary infection risk (SIR) and serial interval (SI) for influenza transmission from HIV-infected and HIV-uninfected index cases. Methods Index cases were the first symptomatic person in a household with influenza-like illness, testing influenza positive on real-time reverse transcription polymerase chain reaction (rRT-PCR). Nasopharyngeal swabs collected from household contacts every 4 days were tested by rRT-PCR. Factors associated with SIR were evaluated using logistic regression. Results We enrolled 28 HIV-infected and 57 HIV-uninfected index cases. On multivariable analysis, HIV-infected index cases were less likely to transmit influenza to household contacts (odds ratio [OR] 0.2; 95% confidence interval [CI], 0.1-0.6; SIR 16%, 18/113 vs 27%, 59/220). Factors associated with increased SIR included index age group 1-4 years (OR 3.6; 95% CI, 1.2-11.3) and 25-44 years (OR 8.0; 95% CI, 1.8-36.7), and contact age group 1-4 years (OR 3.5; 95% CI, 1.2-10.3) compared to 5-14 years, and sleeping with index case (OR 2.7; 95% CI, 1.3-5.5). HIV infection of index case was not associated with SI. Conclusions HIV-infection was not associated with SI. Increased infectiousness of HIV-infected individuals is likely not an important driver of community influenza transmission.

BACKGROUND: Prolonged shedding of influenza viruses may be associated with increased transmissibility and resistance mutation acquisition due to therapy. We compared duration and magnitude of influenza shedding between human immunodeficiency virus (HlV)-infected and -uninfected individuals. METHODS: A prospective cohort study during 3 influenza seasons enrolled patients with influenza-like illness and a positive influenza rapid test. Influenza viruses were detected by real-time reverse transcription polymerase chain reaction. Weibull accelerated failure time regression models were used to describe influenza virus shedding. Mann-Whitney U tests explored initial influenza viral loads (VL). RESULTS: Influenza virus shedding duration was similar in 65 HIV-infected (6 days; interquartile range [IQR] 3–10) and 176 HIV-uninfected individuals (7 days; IQR 4–11; P = .97), as was initial influenza VL (HIV-uninfected 5.28 ± 1.33 log(10) copies/mL, HIV-infected 4.73 ± 1.68 log(10) copies/mL; P = .08). Adjusted for age, HIV-infected individuals with low CD4 counts shed influenza virus for longer than those with higher counts (adjusted hazard ratio 3.55; 95% confidence interval, 1.05–12.08). DISCUSSION: A longer duration of influenza virus shedding in HIV-infected individuals with low CD4 counts may suggest a possible increased risk for transmission or viral evolution in severely immunocompromised individuals. HIV-infected individuals should be prioritized for annual influenza immunization.

Summary Objectives The household is important in influenza transmission due to intensity of contact. Previous studies reported secondary attack rates (SAR) of 4–10% for laboratory-confirmed influenza in the household. Few have been conducted in middle-income countries. Methods We performed a case-ascertained household transmission study during May–October 2013. Index cases were patients with influenza-like-illness (cough and self-reported or measured fever (≥38 °C)) with onset in the last 3 days and no sick household contacts, at clinics in South Africa. Household contacts of index cases with laboratory-confirmed influenza were followed for 12 days. Results Thirty index cases in 30 households and 107/110 (97%) eligible household contacts were enrolled. Assuming those not enrolled were influenza negative, 21/110 household contacts had laboratory-confirmed influenza (SAR 19%); the mean serial interval was 2.1 days (SD = 0.35, range 2–3 days). Most (62/82; 76%) household contacts who completed the risk factor questionnaire never avoided contact and 43/82 (52%) continued to share a bed with the index case after illness onset. Conclusion SAR for laboratory-confirmed influenza in South Africa was higher than previously reported SARs. Household contacts did not report changing behaviors to prevent transmission. These results can be used to understand and predict influenza transmission in similar middle-income settings.

Background Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children in both the community and hospital setting. Methods The clinical presentation, patient and phylogenetic characteristicsof laboratory-confirmed cases of RSV, as well as risk factors for nosocomial infectionat Red Cross War Memorial Children’s Hospital in Cape Town were analysed. A multiplex PCR assay that detects 7 respiratory viruses was used to identify RSV nucleic acid on respiratory specimens. Results A total of 226 children were studied, ages ranging between 1 week and 92.5 months (median: 2.8 months, IQR: 1.3–6.3 months) and 51.8 % were males. The median duration of symptoms prior to diagnosis was 2 days (IQR: 1–4 days). Nosocomial infections wereidentified in 22 (9.7 %) children. There were pre-existing medical conditions in 113 (50.0 %) excluding HIV, most commonly prematurity (n = 58, 50.0 %) and congenital heart disease (n = 34, 29.3 %). The commonest presenting symptoms were cough (196, 86.7 %), difficulty in breathing (115, 50.9 %) and fever (91, 41.6 %).A case fatality rate of 0.9 % was recorded. RSV group A predominated (n = 181, 80.1 %) while group B accounted for only 45 (19.9 %) of the infections. The prevalent genotypes were NA1 (n = 127,70.1 %), ON1 (n = 45,24.9 %) and NA2 (n = 9,5.0 %) for group A while the only circulating RSV B genotype was BA4. There was no significant difference in the genotype distribution between the nosocomial and community-acquired RSV infections. Age ≥ 6 months was independently associated with nosocomial infection. Conclusions A large percentage of children with RSV infection had pre-existing conditions. Approximately one tenth of the infections were nosocomial with age 6 months or older being a risk factor. Though both RSV groups co-circulated during the season, group A was predominant and included the novel ON1 genotype. Continued surveillance is necessary to identify prevalent and newly emerging genotypes ahead of vaccine development and efficacy studies.

Traditional modes of investigating influenza nosocomial transmission have entailed a combination of confirmatory molecular diagnostic testing and epidemiological investigation. Common hospital-acquired infections like influenza require a discerning ability to distinguish between viral isolates to accurately identify patient transmission chains. We assessed whether influenza hemagglutinin sequence phylogenies can be used to enrich epidemiological data when investigating the extent of nosocomial transmission over a four-month period within a paediatric Hospital in Cape Town South Africa. Possible transmission chains/channels were initially determined through basic patient admission data combined with Maximum likelihood and time-scaled Bayesian phylogenetic analyses. These analyses suggested that most instances of potential hospital-acquired infections resulted from multiple introductions of Influenza A into the hospital, which included instances where virus hemagglutinin sequences were identical between different patients. Furthermore, a general inability to establish epidemiological transmission linkage of patients/viral isolates implied that identified isolates could have originated from asymptomatic hospital patients, visitors or hospital staff. In contrast, a traditional epidemiological investigation that used no viral phylogenetic analyses, based on patient co-admission into specific wards during a particular time-frame, suggested that multiple hospital acquired infection instances may have stemmed from a limited number of identifiable index viral isolates/patients. This traditional epidemiological analysis by itself could incorrectly suggest linkage between unrelated cases, underestimate the number of unique infections and may overlook the possible diffuse nature of hospital transmission, which was suggested by sequencing data to be caused by multiple unique introductions of influenza A isolates into individual hospital wards. We have demonstrated a functional role for viral sequence data in nosocomial transmission investigation through its ability to enrich traditional, non-molecular observational epidemiological investigation by teasing out possible transmission pathways and working toward more accurately enumerating the number of possible transmission events.

The epidemiology and impact of multiple concurrent Human papillomavirus (HPV) infections on the natural history of cervical disease is uncertain, but could have significant implications for cervical cancer prevention and HPV vaccination strategies.A cross-sectional prevalence study was conducted to determine the overall prevalence of HPV and the rate of multiple concurrent HPV infections, in a cohort of sexually active HIV-uninfected South African adolescents. HPV genotyping was performed using the polymerase chain reaction.Overall prevalence of HPV was 64.1%. Multiple concurrent HPV infections were found in 43.6% of participants and 68% of HPV-infected participants. Non-vaccine high-risk HPV (HR-HPV) genotypes were found much more often than vaccine types (HPV16 and HPV18).Our cohort of young South African females was found to have a high overall prevalence of HPV and multiple concurrent HPV infections. Most HR-HPV infections found were genotypes other than HPV16 or HPV18.

It is well established that there is a severe genetic bottleneck associated with human immunodeficiency virus (HIV) transmission, resulting in low genetic diversity in early infection. Recently, larger studies that sequenced single HIV genomes from mainly heterosexual cohorts were able to more accurately quantitate this bottleneck [1–4]. It is estimated that approximately 80% (141 of 175) of sexually transmitted HIV infections are the result of only a single variant crossing the mucosal barrier to establish clinical infection. However, HIV and simian immunodeficiency virus studies have shown that the virus transmission bottleneck is affected by both the dose and the route of infection [4–7]. In macaques, a lower-dose inoculum resulted in a reduced number of transmitted variants [6]. Although in both macaques and humans injection drug use (IDU) resulted in an increased frequency of multiple-variant infections [5, 7], a separate study from Russia found that IDU did not relax the genetic bottleneck [8]. This conflict in findings about IDU could be due to differences in cohort characteristics, including risk behavious. Men who have sex with men are also at higher risk of infection than heterosexuals [5] and were found to be twice as likely to acquire multiple-variant HIV infections, possibly due to anatomical and histological differences between the 2 modes of transmission [4]. Similarly, inflammatory genital infections have also been shown to be associated with both increased risk of transmission and multivariant infection [2]. Together these studies suggest that breaching or bypassing the mucosal barrier can mitigate the viral transmission bottleneck and that characterizing the diversity following transmission can provide a surrogate marker of increased transmission risk. Microbicides for women aim to reinforce the vaginal epithelial barrier against infection, although some microbicides, such as nonoxyl-9, compromise the mucosal barrier and increase the risk of transmission [9]. The recent pivotal CAPRISA 004 microbicide trial showed that women in urban and rural KwaZulu-Natal, South Africa, who used 1% tenofovir-based gel had a significantly lower incidence of HIV acquisition (5.6 per 100 women-years) than women on the placebo arm (9.1 per 100 women-years Africa) [10], with an overall 39% protective effect. These results demonstrate the utility of having tenofovir at the site of transmission to inhibit the early rounds of viral replication [10]. We studied intrapatient genetic diversity of HIV infection in the CAPRISA 004 trial participants with breakthrough infections to determine whether the use of the 1% tenofovir gel affected the genetic bottleneck.

Background HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.