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.

Persistent Severe Acute Respiratory Syndrome Coronavirus 2 Infection With accumulation of mutations in a patient with poorly controlled Human Immunodeficiency Virus infection.

A 22-year-old woman with uncontrolled advanced human immunodeficiency virus (HIV) infection was persistently infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beta variant for 9 months, the virus accumulating >20 additional mutations. Antiretroviral therapy suppressed HIV and cleared SARS-CoV-2 within 6 to 9 weeks. Increased vigilance is warranted to benefit affected individuals and prevent the emergence of novel SARS-CoV-2 variants.

Initial Supplementary Dose of Dolutegravir in Second-Line Antiretroviral Therapy: A Noncomparative, Double-Blind, Randomized Placebo-Controlled Trial.

BACKGROUND: Dolutegravir concentrations are reduced by efavirenz induction effect necessitating twice-daily dolutegravir dosing when coadministered. Efavirenz induction persists for several weeks after stopping, which could potentially select for dolutegravir resistance if switching occurred with unsuppressed human immunodeficiency virus type 1 (HIV-1) RNA levels and standard dolutegravir dosing. We evaluated the need for a lead-in supplementary dolutegravir dose in adults failing first-line tenofovir-emtricitabine-efavirenz (TEE). METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 2 trial in Khayelitsha, South Africa. Eligible patients had virologic failure (2 consecutive HIV-1 RNA ≥1000 copies/mL) on first-line TEE. Participants were randomly assigned (1:1) to switch to tenofovir-lamivudine-dolutegravir (TLD) with a supplementary 50 mg dolutegravir dose or placebo taken 12 hours later for 14 days. Primary outcome was proportion with HIV-1 RNA <50 copies/mL at week 24. This study was not powered to compare arms. RESULTS: One hundred thirty participants were randomized (65 to each arm). Median baseline HIV-1 RNA was 4.0 log10 copies/mL and 76% had baseline resistance to both tenofovir and lamivudine. One participant died and 2 were lost to follow-up. At week 24, 55 of 64 (86% [95% confidence interval {CI}: 75%-93%]) in the supplementary dolutegravir arm and 53 of 65 (82% [95% CI: 70%-90%]) in the placebo arm had HIV-1 RNA <50 copies/mL. Grade 3 or 4 adverse events were similar in frequency between arms. None of 6 participants (3 in each arm) eligible for resistance testing by 24 weeks developed dolutegravir resistance. CONCLUSIONS: Our findings do not support the need for initial dolutegravir dose adjustment in patients switching to TLD who failed first-line TEE. CLINICAL TRIALS REGISTRATION: NCT03991013.

Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Nano-RECall provides an integrated pipeline for HIV-1 drug resistance testing from Oxford Nanopore sequence data.

OBJECTIVES: Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing. METHODS: We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n = 46) and (b) homebrew integrase (IN) (n = 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only. RESULTS: The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both. CONCLUSIONS: The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings.

Perspectives of people living with HIV and health workers about a point-of-care adherence assay: a qualitative study on acceptability.

Current antiretroviral therapy (ART) adherence monitoring is premised on patients' self-reported adherence behaviour (prone to recall error) and verified by blood viral load measurement (which can delay results). A newly developed Urine Tenofovir Rapid Assay (UTRA) assesses tenofovir in urine at point-of-care and is a novel tool to test and immediately respond to adherence levels of people living with HIV (PLHIV). We explored PLHIV and health workers' initial perceptions about integrating the UTRA into routine medical care for adherence support. We conducted a series of once-off in-depth qualitative interviews with PLHIV (n = 25) and health workers (n = 5) at a primary care health facility in Cape Town, South Africa. Data analysis involved descriptive summaries of key emergent themes with illustrative case examples. We applied a deductive, outcomes-driven analytic approach to the summaries using the Implementation Outcomes Framework proffered by Proctor et al. (2011). The three relevant concepts from this framework that guided our evaluation were: acceptability, appropriateness, and feasibility. We found positive perceptions about the UTRA from many PLHIV and health worker participants. Many PLHIV reported that the immediate results offered by the UTRA could enable them to have constructive discussions with health workers on how to resolve adherence challenges in real-time. Few PLHIV reported concerns that drinking alcohol could affect their UTRA results. Many health workers reported that the UTRA could help them identify patients at risk of treatment failure and immediately intervene through counselling, though some relayed that they would support the UTRA's implementation if more staff members could be added in their busy facility. Overall, these findings show that the UTRA was widely perceived to be acceptable and actionable by many PLHIV and health workers in the study.

SARS-CoV-2 infection and pulmonary tuberculosis in children and adolescents: a case-control study.

BACKGROUND: The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic has had an impact on the global tuberculosis (TB) epidemic but evidence on the possible interaction between SARS-CoV-2 and TB, especially in children and adolescents, remains limited. We aimed to evaluate the relationship between previous infection with SARS-CoV-2 and the risk of TB in children and adolescents. METHODS: An unmatched case-control study was conducted using SARS-CoV-2 unvaccinated children and adolescents recruited into two observational TB studies (Teen TB and Umoya), between November 2020 and November 2021, in Cape Town, South Africa. Sixty-four individuals with pulmonary TB (aged < 20 years) and 99 individuals without pulmonary TB (aged < 20 years) were included. Demographics and clinical data were obtained. Serum samples collected at enrolment underwent quantitative SARS-CoV-2 anti-spike immunoglobulin G (IgG) testing using the Abbott SARS-CoV-2 IgG II Quant assay. Odds ratios (ORs) for TB were estimated using unconditional logistic regression. RESULTS: There was no statistically significant difference in the odds of having pulmonary TB between those who were SARS-CoV-2 IgG seropositive and those who were seronegative (adjusted OR 0.51; 95% CI: 0.23-1.11; n = 163; p = 0.09). Of those with positive SARS-CoV-2 serology indicating prior infection, baseline IgG titres were higher in individuals with TB compared to those without TB (p = 0.04) and individuals with IgG titres in the highest tertile were more likely to have pulmonary TB compared to those with IgG levels in the lowest tertile (OR: 4.00; 95%CI: 1.13- 14.21; p = 0.03). CONCLUSIONS: Our study did not find convincing evidence that SARS-CoV-2 seropositivity was associated with subsequent pulmonary TB disease; however, the association between magnitude of SARS-CoV-2 IgG response and pulmonary TB warrants further investigation. Future prospective studies, evaluating the effects of sex, age and puberty on host immune responses to M. tuberculosis and SARS-CoV-2, will also provide more clarity on the interplay between these two infections.

UMOYA: a prospective longitudinal cohort study to evaluate novel diagnostic tools and to assess long-term impact on lung health in South African children with presumptive pulmonary TB-a study protocol.

BACKGROUND: Despite a high paediatric tuberculosis (TB) burden globally, sensitive and specific diagnostic tools are lacking. In addition, no data exist on the impact of pulmonary TB on long-term child lung health in low- and middle-income countries. The prospective observational UMOYA study aims (1) to build a state-of-the-art clinical, radiological, and biological repository of well-characterised children with presumptive pulmonary TB as a platform for future studies to explore new emerging diagnostic tools and biomarkers for early diagnosis and treatment response; and (2) to investigate the short and long-term impact of pulmonary TB on lung health and quality of life in children. METHODS: We will recruit up to 600 children (0-13 years) with presumptive pulmonary TB and 100 healthy controls. Recruitment started in November 2017 and is expected to continue until May 2023. Sputum and non-sputum-based samples are collected at enrolment and during follow-up in TB cases and symptomatic controls. TB treatment is started by routine care services. Intensive follow-up for 6 months will allow for TB cases to retrospectively be classified according to international consensus clinical case definitions for TB. Long-term follow-up, including imaging, comprehensive assessment of lung function and quality of life questionnaires, are done yearly up to 4 years after recruitment. DISCUSSION: The UMOYA study will provide a unique platform to evaluate new emerging diagnostic tools and biomarkers for early diagnosis and treatment response and to investigate long-term outcomes of pulmonary TB and other respiratory events on lung health in children.

Prevalence and patterns of HIV drug resistance in patients with suspected virological failure in North-Western Tanzania.

BACKGROUND: More than 15 million people in sub-Saharan Africa receive ART. Treatment failure is common, but the role of HIV drug resistance in treatment failure is largely unknown because drug resistance testing is not routinely done. This study determined the prevalence and patterns of HIV drug resistance in patients with suspected virological failure. MATERIALS AND METHODS: A single high viral load of >1000 viral RNA copies/mL of plasma at any point during ART was considered as suspected virological failure. HIV-1 RNA was extracted from plasma samples of these patients using the QIAamp Viral RNA kit. The protease and part of the RT regions of the HIV pol gene were characterized. RESULTS: Viral load was determined in 317 patients; 64 (20.2%) had suspected virological failure. We successfully genotyped 56 samples; 48 (85.7%) had at least one major resistance-associated mutation (RAM). Common mutations in RT were M184V (75%), T215Y (41.1%), K103N (39.3%), M41L (32.1%), D67DN (30.3%), G190A (28.6%) and A98G (26.8%). No RAMs were detected in ART regimens based on a ritonavir-boosted PI. CONCLUSIONS: The Tanzanian national guidelines define 'virological failure' as two consecutive viral load measurement results, at 3 month intervals, above the WHO threshold (1000 copies/mL). Here, we show that a single viral load above the WHO threshold is associated with high rates of RAMs. This suggests that a single high viral load measurement could be used to predict virological failure and avoid delays in switching patients from first-line to higher genetic barrier second-line regimens.

Primary resistance against integrase strand transfer inhibitors in integrase strand transfer inhibitor-naive patients failing first- and second-line ART in Tanzania.

INTRODUCTION: Sub-Saharan African countries are introducing integrase strand transfer inhibitors (INSTIs) in their ART programmes as the preferred first-line regimen, and dolutegravir is the INSTI of choice due to its potency, tolerability and high genetic barrier to resistance. Dolutegravir was introduced into the first-line ART regimen in Tanzania in 2019. However, there is a paucity of data on the occurrence of mutations in HIV lineages circulating in Tanzania. This study aimed to determine the prevalence of INSTI primary resistance mutations in Tanzanian patients exposed to ART but not INSTIs. METHODS: Plasma samples from 50 INSTI-naive patients failing first- or second-line ART [median (IQR) age: 40 (21.93-46.41) years; 68% women] were subjected to Sanger sequencing of the HIV integrase gene. Participants had been on ART for a median (IQR) duration of 7.32 (4.73-9.29) years, with 80% and 20% failing first- and second-line ART, respectively. RESULTS: No major INSTI mutations were found, but 2 (4%) participants had the accessory mutation T97A. Using the REGA HIV-1 subtyping tool, HIV subtype A1 (53.1%) was found to be dominant, followed by subtypes C (30.6%) and D (16.3%). CONCLUSIONS: This study found no current evidence for transmitted resistance against INSTIs among unexposed patients failing ART and supports the scale-up of INSTI-based regimens. However, the presence of accessory mutations calls for the surveillance of INSTI resistance mutations to ensure that the anticipated long-term desired outcomes are achieved.

Routine use of fluoroscopic and real-time transthoracic echocardiographic guidance to ensure safety of right ventricular endomyocardial biopsy in a low-volume center.

To determine whether the routine use of real-time transthoracic echocardiographic (TTE) guidance in addition to fluoroscopy would ensure the safety of right ventricular endomyocardial biopsy (RV EMB) in a low-volume center. RV EMB is a valuable tool and plays an important role in the diagnosis and management of patients with myocardial diseases. However, it has yet to gain widespread acceptance due to its perceived low diagnostic yield and concerns regarding its invasive nature and potential complications. Although the safety of EMB when performed by experienced operators in high-volume centers is well established, the complication rate in low-volume centers is less well defined but appears to be higher. This is a retrospective single-center cross-sectional study. Consecutive adult patients who underwent RV EMB procedures at Tygerberg Hospital (Cape Town, South Africa) between August 2017 and December 2020 were included. RV EMB was successfully performed in 85 patients. No major complications were reported. Five (5.88%) patients experienced minor complications: three transient right bundle branch blocks and two hemodynamically stable ventricular tachycardia. A definitive biopsy diagnosis was made in 37 (43.54%) patients. The average procedural time was 27.06 min, which equated to 4.09 min per specimen taken. The routine use of real-time TTE guidance in addition to fluoroscopy ensured the safety of RV EMB in a low-volume center without unnecessarily prolonging procedural time.

Comparing mutational pathways to lopinavir resistance in HIV-1 subtypes B versus C.

Although combination antiretroviral therapies seem to be effective at controlling HIV-1 infections regardless of the viral subtype, there is increasing evidence for subtype-specific drug resistance mutations. The order and rates at which resistance mutations accumulate in different subtypes also remain poorly understood. Most of this knowledge is derived from studies of subtype B genotypes, despite not being the most abundant subtype worldwide. Here, we present a methodology for the comparison of mutational networks in different HIV-1 subtypes, based on Hidden Conjunctive Bayesian Networks (H-CBN), a probabilistic model for inferring mutational networks from cross-sectional genotype data. We introduce a Monte Carlo sampling scheme for learning H-CBN models for a larger number of resistance mutations and develop a statistical test to assess differences in the inferred mutational networks between two groups. We apply this method to infer the temporal progression of mutations conferring resistance to the protease inhibitor lopinavir in a large cross-sectional cohort of HIV-1 subtype C genotypes from South Africa, as well as to a data set of subtype B genotypes obtained from the Stanford HIV Drug Resistance Database and the Swiss HIV Cohort Study. We find strong support for different initial mutational events in the protease, namely at residue 46 in subtype B and at residue 82 in subtype C. The inferred mutational networks for subtype B versus C are significantly different sharing only five constraints on the order of accumulating mutations with mutation at residue 54 as the parental event. The results also suggest that mutations can accumulate along various alternative paths within subtypes, as opposed to a unique total temporal ordering. Beyond HIV drug resistance, the statistical methodology is applicable more generally for the comparison of inferred mutational networks between any two groups.

Comparing the findings and diagnostic sensitivity of cardiovascular magnetic resonance in biopsy confirmed acute myocarditis with infarct-like vs. heart failure presentation.

BACKGROUND: Cardiovascular magnetic resonance (CMR) is considered the reference imaging modality in providing a non-invasive diagnosis of acute myocarditis (AM), as it allows for the detection of myocardial injury associated with AM. However, the diagnostic sensitivity and pattern of CMR findings appear to differ according to clinical presentation. METHODS: This is a retrospective cross-sectional study. Consecutive adult patients presenting to a single tertiary centre in South Africa between August 2017 and January 2022 with AM confirmed on endomyocardial biopsy (EMB) were enrolled. Patients with infarct-like symptoms, defined as those presenting primarily with chest pain syndrome with associated ST-T wave changes on electrocardiogram, or heart failure (HF) symptoms, defined as clinical signs and symptoms of HF without significant chest discomfort, were compared using contrasted CMR and parametric techniques with EMB confirmation of AM as diagnostic gold standard. RESULTS: Forty-one patients were identified including 23 (56%) with infarct-like symptoms and 18 (44%) with HF symptoms. On CMR, the infarct-like group had significantly higher ejection fractions of both ventricles (LVEF 55.3 ± 15.3% vs. 34.4 ± 13.5%, p < 0.001; RVEF 57.3 ± 10.9% vs. 42.9 ± 18.2%, p = 0.008), without significant differences in end diastolic volumes (LVEDVI 82.7 ± 30.3 ml/m2 vs. 103.4 ± 35.9 ml/m2, p = 0.06; RVEDVI 73.7 ± 22.1 ml/m2 vs. 83.9 ± 29.9 ml/m2, p = 0.25). Myocardial oedema was detected more frequently on T2-weighted imaging (91.3% vs. 61.1%, p = 0.03) and in more myocardial segments [3.0 (IQR 2.0-4.0) vs. 1.0 (IQR 0-1.0), p = 0.003] in the infarct-like group. Despite the absence of a significant statistical difference in the prevalence of late gadolinium enhancement (LGE) between the two groups (95.7% vs. 72.2%, p = 0.07), the infarct-like group had LGE detectable in significantly more ventricular segments [4.5 (IQR 2.3-6.0) vs. 2.0 (IQR 0-3.3), p = 0.02] and in a different distribution. The sensitivity of the original Lake Louise Criteria (LLC) was 91.3% in infarct-like patients and 55.6% in HF patients. When the updated LLC, which included the use of parametric myocardial mapping techniques, were applied, the sensitivity improved to 95.7% and 72.2% respectively. CONCLUSION: The pattern of CMR findings and its diagnostic sensitivity appears to differ in AM patients presenting with infarct-like and HF symptoms. Although the sensitivity of the LLC improved with the addition of parametric mapping in the HF group, it remained lower than that of the infarct-like group, and suggests that EMB should be considered earlier in the course of patients with clinically suspected AM presenting with HF.

Novel Criteria for Diagnosing Acute and Early Human Immunodeficiency Virus Infection in a Multinational Study of Early Antiretroviral Therapy Initiation.

BACKGROUND: Antiretroviral therapy (ART) initiation during acute and early human immunodeficiency virus infection (AEHI) limits HIV reservoir formation and may facilitate post-ART control but is logistically challenging. We evaluated the performance of AEHI diagnostic criteria from a prospective study of early ART initiation. METHODS: AIDS Clinical Trials Group A 5354 enrolled adults at 30 sites in the Americas, Africa, and Asia who met any 1 of 6 criteria based on combinations of results of HIV RNA, HIV antibody, Western blot or Geenius assay, and/or the signal-to-cutoff (S/CO) ratio of the ARCHITECT HIV Ag/Ab Combo or GS HIV Combo Ag/Ab EIA. HIV status and Fiebig stage were confirmed by centralized testing. RESULTS: From 2017 through 2019, 195 participants were enrolled with median age of 27 years (interquartile range, 23-39). Thirty (15.4%) were female. ART was started by 171 (87.7%) on the day of enrollment and 24 (12.3%) the next day. AEHI was confirmed in 188 (96.4%) participants after centralized testing, 4 (2.0%) participants were found to have chronic infection, and 3 (1.5%) found not to have HIV discontinued ART and were withdrawn. Retrospectively, a nonreactive or indeterminate HIV antibody on the Geenius assay combined with ARCHITECT S/CO ≥10 correctly identified 99 of 122 (81.2%) Fiebig II-IV AEHI cases with no false-positive results. CONCLUSIONS: Novel AEHI criteria that incorporate ARCHITECT S/CO facilitated rapid and efficient ART initiation without waiting for an HIV RNA result. These criteria may facilitate AEHI diagnosis, staging, and immediate ART initiation in future research studies and clinical practice. CLINICAL TRIALS REGISTRATION: NCT02859558.

Clinical Experience With Severe Acute Respiratory Syndrome Coronavirus 2-Related Illness in Children: Hospital Experience in Cape Town, South Africa.

BACKGROUND: Children seem relatively protected from serious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related disease, but little is known about children living in settings with high tuberculosis and human immunodeficiency virus (HIV) burden. This study reflects clinical data on South African children with SARS-CoV-2. METHODS: We collected clinical data of children aged <13 years with laboratory-confirmed SARS-CoV-2 presenting to Tygerberg Hospital, Cape Town, between 17 April and 24 July 2020. RESULTS: One hundred fifty-nine children (median age, 48.0 months [interquartile range {IQR}, 12.0-106.0 months]) were included. Hospitalized children (n = 62), with a median age of 13.5 months (IQR, 1.8-43.5 months) were younger than children not admitted (n = 97; median age, 81.0 months [IQR, 34.5-120.5 months]; P < .01.). Thirty-three of 159 (20.8%) children had preexisting medical conditions. Fifty-one of 62 (82.3%) hospitalized children were symptomatic; lower respiratory tract infection was diagnosed in 21 of 51 (41.2%) children, and in 11 of 16 (68.8%) children <3 months of age. Respiratory support was required in 25 of 51 (49.0%) children; 13 of these (52.0%) were <3 months of age. One child was HIV infected and 11 of 51 (21.2%) were HIV exposed but uninfected, and 7 of 51 (13.7%) children had a recent or new diagnosis of tuberculosis. CONCLUSIONS: Children <1 year of age hospitalized with SARS-CoV-2 in Cape Town frequently required respiratory support. Access to oxygen may be limited in some low- and middle-income countries, which could potentially drive morbidity and mortality. HIV infection was uncommon but a relationship between HIV exposure, tuberculosis, and SARS-CoV-2 should be explored.

HIVIntact: a python-based tool for HIV-1 genome intactness inference.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

Intact HIV Proviruses Persist in Children Seven to Nine Years after Initiation of Antiretroviral Therapy in the First Year of Life.

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation.IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower (P < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited.

An assessment of hepatitis B virus prevalence in South African young blood donors born after the implementation of the infant hepatitis B virus immunization program: Implications for transfusion safety.

BACKGROUND: The prevalence of hepatitis B surface antigen is estimated to be 6.7% in the South African population and in April 1995 the nation introduced universal hepatitis B virus (HBV) vaccination for newborns and infants. We studied the temporal association of this program with HBV prevalence in young blood donors and the contemporary HBV incidence and residual risk of transfusion-transmitted HBV infection (TT-HBV). METHODS: We used blood donation data from January 2011 to December 2019. Estimation of HBV prevalence donations made by first-time blood donors were analyzed by birth cohort and covariates. To estimate the incidence and residual risk of TT-HBV, mathematical models used data from both first time and repeat donors. RESULTS: HBV prevalence in first-time donors decreased from 0.84% (95% confidence interval [CI] 0.78-0.90) in 2011 to 0.66% (95% CI 0.61-0.70) in 2019. The post-1995 birth cohort had a significantly lower HBV prevalence of 0.14% (95% CI 0.13-0.15) than the pre-1985 birth cohort of 1.29% (95% CI 1.25-1.33) and the odds of HBV infection were reduced in a multivariable model (odds ratio [OR] = 0.28, 95% CI 0.24-0.34). The residual risk of TT-HBV occurring from window-period, occult, and possible vaccine breakthrough infections were estimated at 36.9, 5.8, and 2.2 per million red blood cell transfusions, respectively. CONCLUSION: Donors born after the start of routine HBV immunization had significantly lower prevalence of HBV infection, supporting the effectiveness of the vaccination program. The contemporary residual risk of TT-HBV has decreased and should decline further as more vaccinated young people join the donor pool.

HIV incidence in South African blood donors from 2012 to 2016: a comparison of estimation methods.

BACKGROUND: Measuring incidence is important for monitoring and maintaining the safety of the blood supply. Blood collected from repeat-donors has provided the opportunity to follow blood donors over time and has been used to estimate the incidence of viral infections. These incidence estimates have been extrapolated to first-time donors using the ratio of NAT yield cases in first-time versus repeat-donors. We describe a model to estimate incidence in first-time donors using the limiting antigen (LAg) avidity assay and compare its results with those from established models. METHODS: HIV-positive first-time donations were tested for recency using the LAg assay. Three models were compared; incidence estimated for (1) first-time donors using LAg avidity, (2) first-time and repeat-donors separately using the NAT yield window period (WP) model and (3) repeat-donors using the incidence/WP model. RESULTS: HIV incidence in first-time donors was estimated at 3·32 (CI 3·11, 3·55) and 3·81 (CI 3·07, 4·73) per 1000 PY using the LAg assay and NAT yield WP models, respectively. Incidence in repeat-donors was between 2·0- and 2·5-fold lower than in first-time donors estimated at 1·56 (CI 1·37, 1·77) and 1·94 (CI 1·86-2·01) per 1000 PY using the NAT yield/WP and incidence/WP models, respectively. CONCLUSION: Testing HIV-positive donations using the LAg assay provides a reliable method to estimate incidence in first-time donors for countries that collect the majority of blood from first-time donors and do not screen with NAT.

Better Virological Outcomes Among People Living With Human Immunodeficiency Virus (HIV) Initiating Early Antiretroviral Treatment (CD4 Counts ≥500 Cells/µL) in the HIV Prevention Trials Network 071 (PopART) Trial in South Africa.

BACKGROUND: There have been concerns about reduced adherence and human immunodeficiency virus (HIV) virological suppression (VS) among clinically well people initiating antiretroviral therapy (ART) with high pre-ART CD4 cell counts. We compared virological outcomes by pre-ART CD4 count, where universal ART initiation was provided in the HIV Prevention Trials Network 071 (PopART) trial in South Africa prior to routine national and international implementation. METHODS: This prospective cohort study included adults initiating ART at facilities providing universal ART since January 2014. VS (<400 copies/mL), confirmed virological failure (VF) (2 consecutive viral loads >1000 copies/mL), and viral rebound were compared between participants in strata of baseline CD4 cell count. RESULTS: The sample included 1901 participants. VS was ≥94% among participants with baseline CD4 count ≥500 cells/µL at all 6-month intervals to 30 months. The risk of an elevated viral load (≥400 copies/mL) was independently lower among participants with baseline CD4 count ≥500 cells/µL (3.3%) compared to those with CD4 count 200-499 cells/µL (9.2%) between months 18 and 30 (adjusted relative risk, 0.30 [95% confidence interval, .12-.74]; P = .010). The incidence rate of VF was 7.0, 2.0, and 0.5 per 100 person-years among participants with baseline CD4 count <200, 200-499, and ≥500 cells/µL, respectively (P < .0001). VF was independently lower among participants with baseline CD4 count ≥500 cells/µL (adjusted hazard ratio [aHR], 0.23; P = .045) and 3-fold higher among those with baseline CD4 count <200 cells/µL (aHR, 3.49; P < .0001). CONCLUSIONS: Despite previous concerns, participants initiating ART with CD4 counts ≥500 cells/µL had very good virological outcomes, being better than those with CD4 counts 200-499 cells/µL. CLINICAL TRIALS REGISTRATION: NCT01900977.