Racial differences in seroprevalence of HAV and HEV in blood donors in the Western Cape, South Africa: a clue to the predominant HEV genotype?

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. This infection causes major water-borne outbreaks in low- and middle-income countries, whilst in industrialised countries this infection is zoonotic. These differences in epidemiology are related to different HEV genotypes. HEV genotype 3 is a zoonotic infection, whilst genotype 2 causes large outbreaks. This study determined the seroprevalence of HEV in blood donors from the Western Cape. Anti-hepatitis A virus (anti-HAV) antibody was detected in 184/300 (61%) donors. Antibody to HEV (anti-HEV) was detected in 78 of 300 donors (26%). It was highest in mixed race donors (62/100), followed by white donors (23/100) and lowest in black donors (19/100) P = 0.019. Since it is thought that genotypes 1 and 2 predominate both viruses would be acquired by the oro-faecal route, it is surprising that HEV seroprevalence does not mirror that of HAV. We postulate that this may reflect differences in socio-economic status and consumption of dietary meat. So the marked divergence between HEV and HAV seroprevalence may be the result of different routes of transmission. Further data are needed to explore the risk factors associated with HEV infection.

Clinical spectrum of disease and outcomes in children with Omicron SARS-COV-2 infection in Cape Town, South Africa.

INTRODUCTION: Children with underlying comorbidities and infants are most severely affected by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, including in low- and middle-income countries with a high prevalence of HIV and TB. We describe the clinical presentation of SARS-CoV-2 infection in children during the Omicron wave, in Cape Town, South Africa. METHODS: We analysed routine care data from a prospective cohort of children aged 0-13 years, with a positive SARS-CoV-2 real-time reverse-transcription polymerase chain reaction (rRT-PCR) or SARS-CoV-2 antigen test, admitted to Tygerberg Hospital between 1 November 2021 until 1 March 2022. Risk factors for severity of disease were assessed. RESULTS: Ninety-five children tested positive for SARS-CoV-2, of whom 87 (91.6%) were symptomatic. Clinical data were available for 86 children. The median age was 11 months (IQR 3.0-60.0), 37 (43.0%) were females, 21 (24.7%) were HIV-exposed and 7 (8.1%) were living with HIV (CLHIV). In total, 44 (51.2%) children had at least one underlying comorbidity. TB co-infection was seen in 11 children, 6 children were newly diagnosed and 5 children were already on TB treatment at the time of admission. CONCLUSION: There was no evidence of more severe disease in children living with HIV or TB.

INTRODUCTION: Les enfants et les nourrissons présentant des comorbidités sous-jacentes sont les plus gravement touchés par l'infection par le coronavirus-2 du syndrome respiratoire aigu sévère (SARS-CoV-2), y compris dans les pays à revenu faible ou intermédiaire où la prévalence du VIH et de la TB est élevée. Nous décrivons la présentation clinique de l'infection par le SARS-CoV-2 chez les enfants pendant la vague Omicron, au Cap, en Afrique du Sud. MÉTHODES: Nous avons analysé les données de soins de routine d'une cohorte prospective d'enfants âgés de 0 à 13 ans, avec un test positif de réaction en chaîne de la polymérase de transcription inverse en temps réel (rRT-PCR) ou d'antigène du SARS-CoV-2, admis à l'hôpital Tygerberg entre le 1er novembre 2021 et le 1er mars 2022. Les facteurs de risque de gravité de la maladie ont été évalués. RÉSULTATS: Quatre-vingt-quinze enfants ont été testés positifs au SARS-CoV-2, dont 87 (91,6%) étaient symptomatiques. Des données cliniques étaient disponibles pour 86 enfants. L'âge médian était de 11 mois (IQR 3,0­60,0), 37 (43,0%) étaient des filles, 21 (24,7%) étaient exposés au VIH et 7 (8,1%) vivaient avec le VIH (CLHIV). Au total, 44 (51,2%) enfants présentaient au moins une comorbidité sous-jacente. La co-infection par la TB a été observée chez 11 enfants, 6 enfants ont été nouvellement diagnostiqués et 5 enfants étaient déjà sous traitement antituberculeux au moment de l'admission. CONCLUSION: Il n'y a pas de preuve d'une maladie plus grave chez les enfants vivant avec le VIH ou la TB.

Frequency, kinetics and determinants of viable SARS-CoV-2 in bioaerosols from ambulatory COVID-19 patients infected with the Beta, Delta or Omicron variants.

Airborne transmission of SARS-CoV-2 aerosol remains contentious. Importantly, whether cough or breath-generated bioaerosols can harbor viable and replicating virus remains largely unclarified. We performed size-fractionated aerosol sampling (Andersen cascade impactor) and evaluated viral culturability in human cell lines (infectiousness), viral genetics, and host immunity in ambulatory participants with COVID-19. Sixty-one percent (27/44) and 50% (22/44) of participants emitted variant-specific culture-positive aerosols <10µm and <5µm, respectively, for up to 9 days after symptom onset. Aerosol culturability is significantly associated with lower neutralizing antibody titers, and suppression of transcriptomic pathways related to innate immunity and the humoral response. A nasopharyngeal Ct <17 rules-in ~40% of aerosol culture-positives and identifies those who are probably highly infectious. A parsimonious three transcript blood-based biosignature is highly predictive of infectious aerosol generation (PPV > 95%). There is considerable heterogeneity in potential infectiousness i.e., only 29% of participants were probably highly infectious (produced culture-positive aerosols <5µm at ~6 days after symptom onset). These data, which comprehensively confirm variant-specific culturable SARS-CoV-2 in aerosol, inform the targeting of transmission-related interventions and public health containment strategies emphasizing improved ventilation.

Pooling strategies to reduce the cost of HIV-1 RNA load monitoring in a resource-limited setting.

BACKGROUND: Quantitative human immunodeficiency virus (HIV) RNA load testing surpasses CD4 cell count and clinical monitoring in detecting antiretroviral therapy (ART) failure; however, its cost can be prohibitive. Recently, the use of pooling strategies with a clinically appropriate viral load threshold was shown to be accurate and efficient for monitoring when the prevalence of virologic failure is low. METHODS: We used laboratory request form information to identify specimens with a low pretest probability of virologic failure. Patients aged ≥15 years who were receiving first-line ART had individual viral load results available were eligible. Blood plasma, dried blood spots, and dried plasma spots were evaluated. Two pooling strategies were compared: minipools of 5 samples and a 10 ×10 matrix platform (liquid plasma specimens only). A deconvolution algorithm was used to identify specimens(s) with detectable viral loads. RESULTS: The virologic failure rate in the study sample was <10%. Specimens included were liquid plasma specimens tested in minipools(n = 400), of which 300 were available for testing by matrix, and specimens tested with minipools only: dried blood spots (n = 100) and dried plasma spots (n = 185). Pooling methods resulted in 30.5%-60% fewer HIV RNA tests required to screen the study sample. For plasma pooling, the matrix strategy had the better efficiency, but minipools of 5 dried blood spots had the best efficiency overall and were accurate at a >95% negative predictive value with minimal technical requirements. CONCLUSIONS: In resource-constrained settings, a combination of preselection of patients with low pretest probability of virologic failure and pooled testing can reduce the cost of virologic monitoring without compromising accuracy.

A position statement and practical guide to the use of particulate filtering facepiece respirators (N95, FFP2, or equivalent) for South African health workers exposed to respiratory pathogens including Mycobacterium tuberculosis and SARS-CoV-2.

SUMMARY: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is transmitted mainly by aerosol in particles <10 µm that can remain suspended for hours before being inhaled. Because particulate filtering facepiece respirators ('respirators'; e.g. N95 masks) are more effective than surgical masks against bio-aerosols, many international organisations now recommend that health workers (HWs) wear a respirator when caring for individuals who may have COVID-19. In South Africa (SA), however, surgical masks are still recommended for the routine care of individuals with possible or confirmed COVID-19, with respirators reserved for so-called aerosol-generating procedures. In contrast, SA guidelines do recommend respirators for routine care of individuals with possible or confirmed tuberculosis (TB), which is also transmitted via aerosol. In health facilities in SA, distinguishing between TB and COVID-19 is challenging without examination and investigation, both of which may expose HWs to potentially infectious individuals. Symptom-based triage has limited utility in defining risk. Indeed, significant proportions of individuals with COVID-19 and/or pulmonary TB may not have symptoms and/or test negative. The prevalence of undiagnosed respiratory disease is therefore likely significant in many general clinical areas (e.g. waiting areas). Moreover, a proportion of HWs are HIV-positive and are at increased risk of severe COVID-19 and death. RECOMMENDATIONS: Sustained improvements in infection prevention and control (IPC) require reorganisation of systems to prioritise HW and patient safety. While this will take time, it is unacceptable to leave HWs exposed until such changes are made. We propose that the SA health system adopts a target of 'zero harm', aiming to eliminate transmission of respiratory pathogens to all individuals in every healthcare setting. Accordingly, we recommend: the use of respirators by all staff (clinical and non-clinical) during activities that involve contact or sharing air in indoor spaces with individuals who: (i) have not yet been clinically evaluated; or (ii) are thought or known to have TB and/or COVID-19 or other potentially harmful respiratory infections;the use of respirators that meet national and international manufacturing standards;evaluation of all respirators, at the least, by qualitative fit testing; andthe use of respirators as part of a 'package of care' in line with international IPC recommendations. We recognise that this will be challenging, not least due to global and national shortages of personal protective equipment (PPE). SA national policy around respiratory protective equipment enables a robust framework for manufacture and quality control and has been supported by local manufacturers and the Department of Trade, Industry and Competition. Respirator manufacturers should explore adaptations to improve comfort and reduce barriers to communication. Structural changes are needed urgently to improve the safety of health facilities: persistent advocacy and research around potential systems change remain essential.

Diagnosis of COVID-19: Considerations, controversies and challenges.

Coronavirus disease 2019 (COVID-19) due to a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that has resulted in over 1.5 million confirmed cases and close to 100 000 deaths. In the majority of symptomatic cases, COVID-19 results in a mild disease predominantly characterised by upper respiratory tract symptoms. Reverse transcription polymerase chain reaction (RT-PCR) using a nasopharyngeal sample is the mainstay of diagnosis, but there is an ~30% false negative rate early in the disease and in patients with mild disease, and therefore repeat testing may be required. RT-PCR positivity can persist for several days after resolution of symptoms. IgM and IgG antibody responses become positive several days after the onset of symptoms, and robust antibody responses are detectable in the second week of illness. Antibody-based immunoassays have a limited role in the diagnosis of early symptomatic disease. However, their incremental benefit over RT-PCR in the first 2 weeks of illness is currently being clarified in ongoing studies. Such assays may be useful for surveillance purposes. However, their role in potentially selecting individuals who may benefit from vaccination, or as a biomarker identifying persons who could be redeployed into essential employment roles, is being investigated. Rapid antibody-based immunoassays that detect viral antigen in nasopharyngeal samples are being developed and evaluated.

The role of serological testing in the SARS-CoV-2 outbreak.

Antibody tests for the novel coronavirus, SARS-CoV2, have been developed both as rapid diagnostic assays and for high-throughput formal serology platforms. Although these tests may be a useful adjunct to a diagnostic strategy, they have a number of limitations. Because of the antibody and viral dynamics of the coronavirus, their sensitivity can be variable, especially at early time points after symptom onset. Additional data are required on the performance of the tests in the South African population, especially with regard to development and persistence of antibody responses and whether antibodies are protective against reinfection. These tests may, however, be useful in guiding the public health response, providing data for research (including seroprevalence surveys and vaccine initiatives) and development of therapeutic strategies.

Extraction buffer contaminated bacterially as a cause of invalid HIV-1 viral load results on the NucliSens EasyQ system.

Shortly after starting to use the NucliSens EasyQ HIV-1 V1.1 system for HIV-1 RNA load testing, the number of invalid tests per assay run gradually increased. Within five days, approximately 50% of tests showed a total lack of amplification of the calibrator and in most cases also of the HIV-1 template. According to the manufacturer's specifications, the lysis buffer and three extraction buffers remain on the automated NucliSens easyMAG extraction system between assay runs. Therefore possible microbial contamination of these buffers was investigated, after they had been on the automated system for approximately one week. The NucliSens easyMAG extraction buffer 2 yielded bacterial growth identified as Acinetobacter baumannii. After regular decontamination of the machine's tubing system with 70% alcohol and storage of the buffers at 4 degrees C between assay runs were commenced, invalid results due to failed internal calibrator signal occurred no longer. It is likely that bacterial contamination of the buffer was the cause of assay failure, probably due to ribonuclease (RNase) activity. Bacterial contamination of PCR systems should be added to the list of potential hazards in diagnostic virology. This experience underlines the necessity of state-of-the-art assay design incorporating adequate internal controls and calibrators.

Pitfalls with rapid HIV antibody testing in HIV-infected children in the Western Cape, South Africa.

BACKGROUND: Rapid HIV antibody tests are commonly used for HIV diagnosis in the developing world. These tests are generally reported as sensitive, despite paucity of evaluations in paediatric populations. OBJECTIVES: We tested specimens of paediatric patients, known to be HIV-infected, to detect any false negative tests and determine associations with such an outcome. STUDY DESIGN: One hundred and fifty-three specimens, from 109 patients, recorded to be HIV-infected by standard testing, were tested on the Capillustrade mark HIV-1/HIV-2 test (Trinity Biotech, Ireland); 150 specimens also had sufficient volume to be tested on Abbott Determinetrade mark HIV1/2 assay (Abbott GmbH, Wiesbaden, Germany). Treatment information, CD4 counts and HIV-1 viral load measurements were obtained from patient files and laboratory databases. RESULTS: Twenty-one of 153 specimens tested negative on the Capillus (sensitivity 86.3%). False negative results by Capillus were associated with antiretroviral treatment (ART) (p=0.0018) and lower HIV-1 viral load (p=0.013). Serial dilutions of some of the specimens indicated that both rapid tests, and the Capillus in particular, became negative at lower dilutions than an HIV enzyme immunoassay (EIA). CONCLUSIONS: The Capillus test had an unexpectedly low sensitivity in a South African population of HIV-infected children that had access to antiretroviral treatment, posing a risk of false negative HIV testing.

Variety of interpretation systems for human immunodeficiency virus type 1 genotyping: confirmatory information or additional confusion?

The emergence of drug resistance remains a major problem during antiretroviral treatment of patients infected with human immunodeficiency virus type 1 (HIV-1). As phenotypic drug resistance is laborious and expensive to determine, and because numerous specific mutations are known to be correlated with different resistance patterns, genotyping of the reverse transcriptase and protease genes of HIV is fast becoming an integral part of HIV management in industrialized countries. A number of software-based interpretation systems have been developed for the interpretation of the resulting complex nucleotide sequences. These programs either employ rule-based algorithms or are based on a genotype-phenotype database. This paper reviews recent publications that compare different such systems, trying to identify the degree of discordance between different systems and the reasons underlying such discrepant interpretations. The highest discordance rate was observed for nucleoside reverse transcriptase inhibitors (NRTIs) followed by protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). For the NRTIs, it is the role of nucleoside analogue associated mutations, for the PIs and for the NNRTIs, that of secondary mutations that causes most discrepancies. As the complexity of the mutation pattern is likely to increase further with new drugs becoming available, rule-based genotype interpretation algorithms need to be updated frequently. Whilst not recommending one particular system, the authors believe that the correlation of genotypic with clinical data is probably the best way to develop an optimal algorithm.

The role of viral load determination for the management of human immunodeficiency virus, hepatitis B virus and hepatitis C virus infection.

During the last 5 years, considerable scientific and financial efforts have been made in the development of quantitative nucleic acid detection technology. For detection of human immunodeficiency virus (HIV), quantitative culture is time consuming, cumbersome and requires appropriate laboratory safety equipment. Quantitative determination of p24 antigen by enzyme immunoassay is of limited value due to its relatively poor sensitivity. Therefore, quantitative determination of viral load using nucleic acid amplification techniques is the most accurate, prognostic marker for HIV type 1 infection, independently of the CD4+ cell count. Hepatitis B virus (HBV) is not cultivable in vitro. Serological assays allow an accurate diagnosis and follow-up of acute or chronic infection. Quantification of HBV DNA is used for the monitoring of antiviral therapy, determination of infectivity and for resolution of unclear serological profiles, e.g. isolated anti-HBc reactivity, as well as for patients in which HBV mutants are suspected. Hepatitis C virus (HCV) can only be detected by molecular based assays because no cell culture system, which permits a reliable isolation of clinical specimens, is currently available. Furthermore, early diagnosis and follow-up of infection cannot be achieved with antibody serology. The prognostic relevance of quantitative HCV RNA determination is of limited value for the long-term prognosis of chronic hepatitis C. However, viral load may predict the outcome of antiviral therapy. Genetic diversity is another challenge for HCV RNA quantification.

Severe acute respiratory syndrome (SARS)--paradigm of an emerging viral infection.

An acute and often severe respiratory illness emerged in southern China in late 2002 and rapidly spread to different areas of the Far East as well as several countries around the globe. When the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (SARS) came to an end in July 2003, it had caused over 8000 probable cases worldwide and more than 700 deaths. Starting in March 2003, the World Health Organization (WHO) organised an unprecedented international effort by leading laboratories working together to find the causative agent. Little more than one week later, three research groups from this WHO-coordinated network simultaneously found evidence of a hitherto unknown coronavirus in SARS patients, using different approaches. After Koch's postulates had been fulfilled, WHO officially declared on 16 April 2003 that this virus never before seen in humans is the cause of SARS. Ever since, progress around SARS-associated coronavirus (SARS-CoV) has been swift. Within weeks of the first isolate being obtained, its complete genome was sequenced. Diagnostic tests based on the detection of SARS-CoV RNA were developed and made available freely and widely; nevertheless the SARS case definition still remains based on clinical and epidemiological criteria. The agent's environmental stability, methods suitable for inactivation and disinfection, and potential antiviral compounds have been studied, and development of vaccines and immunotherapeutics is ongoing. Despite its grave consequences in humanitarian, political and economic terms, SARS may serve as an example of how much can be achieved through a well-coordinated international approach, combining the latest technological advances of molecular virology with more "traditional" techniques carried out to an excellent standard.

No apparent effect of cidofovir in epidermodysplasia verruciformis.

This paper reports the failure of a patient suffering from Epidermodysplasia verruciformis, characterised by widespread infection of the skin with human papillomaviruses, to respond to topical and systemic treatment with the antiviral agent, Cidofovir, despite its previously demonstrated effectiveness against a range of different papillomavirus-associated conditions.

Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients of allogeneic stem cell transplants.

BACKGROUND: Although several diagnostic methods are available for the surveillance of patients at risk of human cytomegalovirus (CMV) infection and disease, little data is available on their comparative performances in the diagnostic setting. OBJECTIVES: To compare different assays for CMV detection, especially assays based on (quantitative) DNA and mRNA detection. STUDY DESIGN: Eight allogeneic bone marrow and stem cell transplant recipients at high risk for developing CMV disease (donor CMV-negative, recipient positive) were regularly tested for 7-20 weeks post-transplant by spin-amplification rapid culture from urine (viruria), antigenemia (pp65 assay), pp67 mRNA in whole blood (NASBA), and CMV DNA both qualitatively (in-house PCR, whole blood) and quantitatively (in-house PCR, plasma; Cobas Amplicor CMV Monitor Test, plasma and whole blood; Hybrid Capture, whole blood). RESULTS: Four patients (50%) suffered CMV reactivation during follow-up. Out of 104 sample dates, 41 (39.4%) yielded a positive CMV result in at least one assay. Out of the 28 samples tested by all assays, the highest percentage of positive results was obtained with the in-house quantitative PCR (60.7%), followed by the Hybrid Capture system (39.3%), the Cobas Amplicor CMV Monitor Test, plasma version (35.7%), the Cobas Amplicor CMV Monitor Test, whole blood version (32.1%), in-house qualitative PCR (28.6%), and the mRNA assay (21.4%). Viruria was positive in one sample and pp65 antigenemia was found in two samples. CONCLUSIONS: Despite a considerable incidence of CMV reactivations, pre-emptive anti-CMV chemotherapy prevented the development of CMV disease with the exception of one case. The molecular assays had superior sensitivity to conventional ones. The antigenemia assay proved unsuitable for the surveillance of hematological transplant patients. However, none of the tests recognized all timepoints with CMV reactivation. Further comparative studies are needed to determine their respective diagnostic values.

In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from subjects coinfected with human immunodeficiency virus and Leishmania infantum.

To explore the type 1 and type 2 cytokine profile in cases coinfected with human immunodeficiency virus (HIV) and Leishmania infantum, production of interleukin-4 (IL-4), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) was investigated in mitogen-stimulated and unstimulated peripheral blood mononuclear cell cultures from eight HIV/Leishmania coinfected subjects matched with eight anti-HIV-positive subjects with no evidence of Leishmania coinfection. Levels of IL-4 and IL-2R increased significantly from the baseline levels in the peripheral blood mononuclear cell supernatants of HIV/Leishmania coinfected subjects following stimulation with phytohemoagglutin, whereas the postchallenge concentration of IFN-gamma was significantly increased in the HIV-infected group. The levels of IL-4 and IL-10 were significantly higher in the HIV/Leishmania group throughout evaluation. Post-stimulation IFN-gamma production was significantly higher in the HIV-positive group in comparison with that of the HIV-Leishmania coinfected subjects. These observations support the notion that a Th2 cytokine response is present during a Leishmania infection, even among HIV-coinfected individuals.

Evaluation of 11 enzyme immunoassays for the detection of immunoglobulin M antibodies to Epstein-Barr virus.

Laboratory diagnosis of Epstein-Barr virus (EBV) infection is based mainly on serological tests. The analysis of the pattern of class-specific immunoglobulin response against defined viral antigens, i.e. virus capsid antigen (VCA), early antigen (EA) and Epstein-Barr nuclear antigen (EBNA), permits the differentiation between primary, latent and secondary (reactivated) EBV infection. In recent months, numerous test kits for the detection of VCA specific IgM antibody have been introduced on the international market. With a panel of well defined sera, the sensitivity and specificity of eleven different commercially available IgM ELISAs was evaluated. A well established, commercially available, indirect immunofluorescence assay (IFA) served as the reference test. Compared to the IFA, the Biotest and Sigma Diagnostic assays had the highest sensitivity for the detection of IgM antibody to VCA or EA. A variable number of false positive results (n = 0-9) was obtained with the EBV-IgM assays by testing potentially cross-reactive serum samples. Up to 19 serum samples from immunocompromised organ transplant recipients were found positive with the Sigma Diagnostics assay. The results of this study show that there are great differences in quality of current EBV-IgM-ELISA test kits. Depending on the clinical setting, it may be important to use a test kit which detects immunoglobulin M reactivity to EA in order to warrant an optimal sensitivity for the serological diagnosis of EBV reactivation in immunocompromised patients. However, since most immunosuppressed patients have serological reactivations, and in most cases these are asymptomatic, the clinical relevance of the detection of EA-IgM is very low.