Field study to determine the reliability of HIV viral load results shows minimal impact of delayed testing in South Africa.

Background: Understanding factors that impact HIV viral load (VL) accuracy in resource-limited settings is key to quality improvement. Objective: We evaluated whether testing delay and specimen storage between 25 °C and 30 °C before testing affected results. Methods: Between November 2019 and June 2023, 249 individuals on antiretroviral therapy, or with newly diagnosed HIV, were recruited from clinics in Cape Town and Gqeberha, South Africa, and three plasma preparation tubes were collected. One tube was tested within 24 h, while the others were stored uncentrifuged at ambient temperatures before testing. Centrifugation and testing of matched samples were performed on Day 4 and Day 7 after collection. Results: Time delay and ambient storage had minimal impact in specimens with a Day 1 VL of > 100 copies/mL. When grouped by Day 1 VL range, 96% - 100% of specimens at Day 4 and 93% - 100% at Day 7 had VLs within 0.5 log copies/mL of the first result. The greatest variability at Days 4 and 7 was observed when the Day 1 VL was < 100 copies/mL. However, there was no trend of increasing difference over time. Of Day 1 specimens with undetectable VL, or VL < 50 copies/mL, 80% had concordant results at Day 4 and 78% at Day 7. Conclusion: These results show that VL is stable in plasma preparation tubes for 7 days when stored at room temperature. There is significant variability in specimens with low VL, but variability is not affected by testing delay. What this study adds: Ideal HIV VL testing conditions are frequently unachievable in resource-limited settings. Data are needed on whether this impacts on the validity of test results. Our results provide reassurance that storage at ambient temperature for up to 7 days before testing does not substantially affect the VL result.

HIV Viral Load Testing in the South African Public Health Setting in the Context of Evolving ART Guidelines and Advances in Technology, 2013-2022.

HIV viral load (VL) testing plays a key role in the clinical management of HIV as a marker of adherence and antiretroviral efficacy. To date, national and international antiretroviral treatment recommendations have evolved to endorse routine VL testing. South Africa (SA) has recommended routine VL testing since 2004. Progressively, the centralised HIV VL program managed by its National Health Laboratory Service (NHLS) has undergone expansive growth. Retrospective de-identified VL data from 2013 to 2022 were evaluated to review program performance. Test volumes increased from 1,961,720 performed in 2013 to 45,334,864 in 2022. The median total in-laboratory turnaround time (TAT) ranged from 94 h (2015) to 51 h (2022). Implementation of two new assays improved median TATs in all laboratories. Samples of VL greater than 1000 copies/mL declined steadily. Despite initial increases, samples of fewer than 50 copies/mL stagnated at about 70% from 2019 and declined to 68% in 2022. Some variations between assays were observed. Overall, the SA VL program is successful. The scale of the VL program, the largest of its kind in the world by some margin, provides lessons for future public health programs dependent on laboratories for patient outcome and program performance monitoring.

Human Parainfluenza Virus (HPIV) Detection in Hospitalized Children with Acute Respiratory Tract Infection in the Western Cape, South Africa during 2014-2022 Reveals a Shift in Dominance of HPIV 3 and 4 Infections.

The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2-10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study.

A case for investment in clinical metagenomics in low-income and middle-income countries.

Clinical metagenomics is the diagnostic approach with the broadest capacity to detect both known and novel pathogens. Clinical metagenomics is costly to run and requires infrastructure, but the use of next-generation sequencing for SARS-CoV-2 molecular epidemiology in low-income and middle-income countries (LMICs) offers an opportunity to direct this infrastructure to the establishment of clinical metagenomics programmes. Local implementation of clinical metagenomics is important to create relevant systems and evaluate cost-effective methodologies for its use, as well as to ensure that reference databases and result interpretation tools are appropriate to local epidemiology. Rational implementation, based on the needs of LMICs and the available resources, could ultimately improve individual patient care in instances in which available diagnostics are inadequate and supplement emerging infectious disease surveillance systems to ensure the next pandemic pathogen is quickly identified.

Epidemiology and outcomes of SARS-CoV-2 infection associated with anti-nucleocapsid seropositivity in Cape Town, South Africa.

Background: In low- and middle-income countries where SARS-CoV-2 testing is limited, seroprevalence studies can characterise the scale and determinants of the pandemic, as well as elucidate protection conferred by prior exposure. Methods: We conducted repeated cross-sectional serosurveys (July 2020 - November 2021) using residual plasma from routine convenient blood samples from patients with non-COVID-19 conditions from Cape Town, South Africa. SARS-CoV-2 anti-nucleocapsid antibodies and linked clinical information were used to investigate: (1) seroprevalence over time and risk factors associated with seropositivity, (2) ecological comparison of seroprevalence between subdistricts, (3) case ascertainment rates, and (4) the relative protection against COVID-19 associated with seropositivity and vaccination statuses, to estimate variant disease severity. Findings: Among the subset sampled, seroprevalence of SARS-CoV-2 in Cape Town increased from 39.2% in July 2020 to 67.8% in November 2021. Poorer communities had both higher seroprevalence and COVID-19 mortality. Only 10% of seropositive individuals had a recorded positive SARS-CoV-2 test. Antibody positivity before the start of the Omicron BA.1 wave (28 November 2021) was strongly protective for severe disease (adjusted odds ratio [aOR] 0.15; 95%CI 0.05-0.46), with additional benefit in those who were also vaccinated (aOR 0.07, 95%CI 0.01-0.35). Interpretation: The high population seroprevalence in Cape Town was attained at the cost of substantial COVID-19 mortality. At the individual level, seropositivity was highly protective against subsequent infections and severe COVID-19. Funding: Wellcome Trust, National Health Laboratory Service, the Division of Intramural Research, NIAID, NIH (ADR) and Western Cape Government Health.

Improved oral detection is a characteristic of Omicron infection and has implications for clinical sampling and tissue tropism.

BACKGROUND: The Omicron variant of concern is characterised by more than 50 distinct mutations, most in the spike protein. The implications of these for disease transmission, tissue tropism and diagnostic testing needs study. OBJECTIVES: We evaluated the performance of RT-PCR on saliva (SA) swabs and antigen testing on mid-turbinate MT samples relative to RT-PCR on MT swabs. Patients (n = 453) presenting for outpatient testing at the Groote Schuur Hospital COVID-19 testing centre in Cape Town South Africa were recruited. Participants were recruited during the Delta (n = 304) and Omicron (n = 149) waves. RESULTS: In 30 confirmed Delta infections, positive percent agreement (PPA) of RT-PCR on saliva was only 73% compared to a composite standard of either MT or SA RT-PCR positivity, with rapid decay by day 3 after symptom onset. In contrast, in the 70 Omicron infections, SA performed as well as, or better than, MT samples up to day 5, with an overall PPA of SA swabs of 96% and MT of 93%. A change in antigen test performance was noted, with PPA of 93% in Delta, but only 68% for Omicron. CONCLUSIONS: Altered shedding kinetics appear to be present in Omicron-infected patients with more viral RNA detectable in saliva. Saliva swabs are a promising alternative to nasal samples, especially early in infection when sampling of both sites could improve detection. Lower sensitivity of antigen tests in Omicron is a concern and requires further study.

Assessing the clinical severity of the Omicron variant in the Western Cape Province, South Africa, using the diagnostic PCR proxy marker of RdRp target delay to distinguish between Omicron and Delta infections - a survival analysis.

BACKGROUND: At present, it is unclear whether the extent of reduced risk of severe disease seen with SARS-Cov-2 Omicron variant infection is caused by a decrease in variant virulence or by higher levels of population immunity. METHODS: RdRp target delay (RTD) in the Seegene AllplexTM 2019-nCoV PCR assay is a proxy marker for the Delta variant. The absence of this proxy marker in the transition period was used to identify suspected Omicron infections. Cox regression was performed for the outcome of hospital admission in those who tested positive for SARS-CoV-2 on the Seegene AllplexTM assay from November 1 to December 14, 2021 in the Western Cape Province, South Africa, in the public sector. Adjustments were made for vaccination status and prior diagnosis of infection. RESULTS: A total of 150 cases with RTD and 1486 cases without RTD were included. Cases without RTD had a lower hazard of admission (adjusted hazard ratio [aHR], 0.56; 95% confidence interval [CI], 0.34-0.91). Complete vaccination was protective against admission, with an aHR of 0.45 (95% CI, 0.26-0.77). CONCLUSION: Omicron has resulted in a lower risk of hospital admission compared with contemporaneous Delta infection, when using the proxy marker of RTD. Under-ascertainment of reinfections with an immune escape variant remains a challenge to accurately assessing variant virulence.

Varicella-zoster virus reactivation is frequently detected in HIV-infected individuals presenting with stroke.

Infections are an underappreciated cause of stroke, particularly in young and immunocompromised individuals. Varicella-zoster virus (VZV) reactivation, particularly ophthalmic zoster, has been linked to increased risk of stroke but diagnosing VZV-associated cerebral vasculopathy is challenging as neither a recent zoster rash, nor detectable levels of VZV DNA are universally present at stroke presentation. Detection of VZV IgG in cerebrospinal fluid (CSF-VZVG) presents a promising alternative, but requires evaluation of individual blood-CSF dynamics, particularly in the setting of chronic inflammatory states such as HIV infection. Consequently, its use has not been broadly adopted as simple diagnostic algorithms are not available. In this study looking at young adults presenting with acute stroke, we used an algorithm that includes testing for both VZV nucleic acids and CSF-VZVG which was corrected for blood-CSF barrier dynamics and poly-specific immune activation. We found that 13 of 35 (37%), including 7 with a positive CSF VZV PCR, young HIV-infected adults presenting with stroke, 3 of 34 (9%) young HIV-uninfected adults presenting with stroke, and 1 of 18 (6%) HIV-infected nonstroke controls demonstrated evidence of central nervous system reactivation of VZV.

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa.

The SARS-CoV-2 epidemic in southern Africa has been characterized by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, while the second and third waves were driven by the Beta (B.1.351) and Delta (B.1.617.2) variants, respectively1-3. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron, B.1.1.529) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, which are predicted to influence antibody neutralization and spike function4. Here we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity.

Reduced amplification efficiency of the RNA-dependent-RNA-polymerase target enables tracking of the Delta SARS-CoV-2 variant using routine diagnostic tests.

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.

Higher mortality associated with the SARS-CoV-2 Delta variant in the Western Cape, South Africa, using RdRp target delay as a proxy: a cross-sectional study.

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 Allplex TM 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.

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain.

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.

Molecular characterisation and epidemiology of enterovirus-associated aseptic meningitis in the Western and Eastern Cape Provinces, South Africa 2018-2019.

BACKGROUND: Enteroviruses are amongst the most common causes of aseptic meningitis. Between November 2018 and May 2019, an outbreak of enterovirus-associated aseptic meningitis cases was noted in the Western and Eastern Cape Provinces, South Africa. OBJECTIVES: To describe the epidemiology and phylogeography of enterovirus infections during an aseptic meningitis outbreak in the Western and Eastern Cape Provinces of South Africa. METHODS: Cerebrospinal fluid samples from suspected cases were screened using a polymerase chain reaction targeting the 5'UTR. Confirmed enterovirus-associated meningitis samples underwent molecular typing through species-specific VP1/VP2 primers and pan-species VP1 primers. RESULTS: Between November 2018 and May 2019, 3497 suspected cases of aseptic meningitis were documented in the Western and Eastern Cape Provinces. Median age was 8 years (range 0-61), interquartile range (IQR=4-13 years), 405/735 (55%) male. 742/3497 (21%) cases were laboratory - confirmed enterovirus positive by routine diagnostic PCR targeting the 5'UTR. 128/742 (17%) underwent molecular typing by VP1 gene sequencing. Echovirus 4 (E4) was detected in 102/128 (80%) cases. Echovirus 9 was found in 7%, Coxsackievirus A13 in 3%. 10 genotypes contributed to the remaining 10% of cases. Synonymous mutations were found in most cases, with sporadic amino acid changes in 13 (12.7%) cases. CONCLUSION: The aseptic meningitis outbreak was associated with echovirus 4. Stool samples are valuable for molecular typing in CSF confirmed EV-associated aseptic meningitis.

Detection of a SARS-CoV-2 variant of concern in South Africa.

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus1,2. Here we describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by eight mutations in the spike protein, including three substitutions (K417N, E484K and N501Y) at residues in its receptor-binding domain that may have functional importance3-5. This lineage was identified in South Africa after the first wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be determined, the genomic data-which show rapid expansion and displacement of other lineages in several regions-suggest that this lineage is associated with a selection advantage that most plausibly results from increased transmissibility or immune escape6-8.

Sixteen novel lineages of SARS-CoV-2 in South Africa.

The first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in South Africa was identified on 5 March 2020, and by 26 March the country was in full lockdown (Oxford stringency index of 90)1. Despite the early response, by November 2020, over 785,000 people in South Africa were infected, which accounted for approximately 50% of all known African infections2. In this study, we analyzed 1,365 near whole genomes and report the identification of 16 new lineages of SARS-CoV-2 isolated between 6 March and 26 August 2020. Most of these lineages have unique mutations that have not been identified elsewhere. We also show that three lineages (B.1.1.54, B.1.1.56 and C.1) spread widely in South Africa during the first wave, comprising ~42% of all infections in the country at the time. The newly identified C lineage of SARS-CoV-2, C.1, which has 16 nucleotide mutations as compared with the original Wuhan sequence, including one amino acid change on the spike protein, D614G (ref. 3), was the most geographically widespread lineage in South Africa by the end of August 2020. An early South African-specific lineage, B.1.106, which was identified in April 2020 (ref. 4), became extinct after nosocomial outbreaks were controlled in KwaZulu-Natal Province. Our findings show that genomic surveillance can be implemented on a large scale in Africa to identify new lineages and inform measures to control the spread of SARS-CoV-2. Such genomic surveillance presented in this study has been shown to be crucial in the identification of the 501Y.V2 variant in South Africa in December 2020 (ref. 5).

HIV-1 viral load testing in resource-limited settings: Challenges and solutions for specimen integrity.

HIV-1 viral load (VL) testing is a crucial element in providing an antiretroviral treatment monitoring program. The success of these programs depends on the availability and quality of the VL testing services. There are several pre-analytic factors which can affect the quality of VL testing. Many of the challenges faced by resource-limited countries result in a compromise of specimen integrity, thus limiting widespread access to VL monitoring. The various logistic and financial challenges that exist are not insurmountable and several innovative solutions currently exist to overcome these barriers to providing widespread VL testing. This review summarizes the VL testing challenges in resource-limited settings and provides an overview of potential solutions including testing dried blood spots, dried plasma spots, plasma separation cards and the use of point of care tests.