Advancing HIV Drug Resistance Technologies and Strategies: Insights from South Africa's Experience and Future Directions for Resource-Limited Settings.

Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.

Clinical Presentation and Outcome of Acute Respiratory Illnesses in South African Children During the COVID-19 Pandemic.

BACKGROUND: Data from low- and middle-income countries (LMICs) show higher morbidity and mortality in children with acute respiratory illness (ARI) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, whether SARS-CoV-2 infection is distinct from other causes of ARI in this regard is unclear. We describe clinical characteristics and outcomes of South African children with SARS-CoV-2 and non-SARS-CoV-2 ARIs. METHODS: We performed a cross-sectional study including 0-13 years old children admitted to Tygerberg Hospital between May and December 2020 with an ARI. Routine clinical data were collected by the attending clinicians. All children underwent SARS-CoV-2 polymerase chain reaction testing. For severity of disease, the need for respiratory support and duration of support was considered. Multivariable logistic regression models were built to determine the factors associated with SARS-CoV-2 infection and severity. RESULTS: Data for 176 children were available, 38 (22%) children were SARS-CoV-2 polymerase chain reaction positive and 138 (78%) were negative. SARS-CoV-2 positive children were more likely to be female (OR: 2.68, 95% CI: 1.18-6.07), had lower weight-for-age Z score (OR: 0.76, 95% CI: 0.63-0.93), presented more frequently with fever (OR: 3.56, 95% CI: 1.54-8.24) and less often with cough (OR: 0.27, 95% CI: 0.11-0.66). SARS-CoV-2 infection was associated with significantly longer duration of oxygen treatment (median 8 vs. 3 days; OR: 1.1, 95% CI: 1.01-1.20). Overall, 66% of children had viral coinfection, with no significant difference between the groups. In total, 18% of SARS-CoV-2 positive children were readmitted within 3 months for a respiratory reason, compared with 15% SARS-CoV-2 negative children ( P = 0.64). CONCLUSIONS: Our data show that ARIs from SARS-CoV-2 cannot be easily differentiated, but were associated with a higher morbidity compared with ARIs from other causes. Overall outcomes were good. The long-term implications of severe SARS-CoV-2 pneumonia in young children in low- and middle-income countries require further study.

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.

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.

SARS-CoV-2 in children and their accompanying caregivers: Implications for testing strategies in resource limited hospitals.

Background: Identification of SARS-CoV-2 infected individuals is imperative to prevent hospital transmission, but symptom-based screening may fail to identify asymptomatic/mildly symptomatic infectious children and their caregivers. Methods: A COVID-19 period prevalence study was conducted between 13 and 26 August 2020 at Tygerberg Hospital, testing all children and their accompanying asymptomatic caregivers after initial symptom screening. One nasopharyngeal swab was submitted for SARS-CoV-2 using real-time reverse-transcription polymerase chain reaction (rRT-PCR). An additional Respiratory Viral 16-multiplex rRT-PCR test was simultaneously done in children presenting with symptoms compatible with possible SARS-CoV-2 infection. Results: SARS-Co-V 2 RT-PCR tests from 196 children and 116 caregivers were included in the analysis. The SARS-CoV-2 period prevalence in children was 5.6% (11/196) versus 15.5% (18/116) in asymptomatic caregivers (p<0.01). Presenting symptoms did not correlate with SARS-CoV-2 test positivity; children without typical symptoms of SARS-CoV-2 were more likely to be positive than those with typical symptoms (10.2% [10/99] vs 1% [1/97]; p<0.01). Children with typical symptoms (97/196; 49.5%) mainly presented with acute respiratory (68/97; 70.1%), fever (17/97; 17.5%), or gastro-intestinal complaints (12/97; 12.4%); Human Rhinovirus (23/81; 28.4%) and Respiratory Syncytial Virus (18/81; 22.2%) were frequently identified in this group. Children-caregiver pairs' SARS-CoV-2 tests were discordant in 83.3%; 15/18 infected caregivers' children tested negative. Symptom-based COVID-19 screening alone would have missed 90% of the positive children and 100% of asymptomatic but positive caregivers. Conclusion: Given the poor correlation between SARS-CoV-2 symptoms and RT-PCR test positivity, universal testing of children and their accompanying caregivers should be considered for emergency and inpatient paediatric admissions during high COVID-19 community transmission periods. Universal PPE and optimising ventilation is likely the most effective way to control transmission of respiratory viral infections, including SARS-CoV-2, where universal testing is not feasible. In these settings, repeated point prevalence studies may be useful to inform local testing and cohorting strategies.

Prevalence of cardiotropic viruses in adults with clinically suspected myocarditis in South Africa.

OBJECTIVES: To determine the prevalence and types of viral pathogens in the myocardium of patients presenting with clinically suspected myocarditis in South Africa. METHOD: This is a prospective cross-sectional study. Consecutive adults presenting to a single tertiary centre in South Africa between August 2017 and January 2021 who fulfilled the European Society of Cardiology's diagnostic criteria for clinically suspected myocarditis and who had undergone the appropriate investigations, including cardiac MRI (CMR) and endomyocardial biopsy (EMB), were included. RESULTS: One hundred and two patients with clinically suspected myocarditis were enrolled. Acute myocarditis (AM) was confirmed by CMR or EMB in 82 (80.39%) patients. Viral genomes were detected by PCR in EMB specimens of 50 patients with AM. Parvovirus B19 (PVB19) was the most frequently detected virus, in 37 as monoinfection and 4 as coinfection. This was followed by Epstein-Barr virus (n=6), human herpesvirus 6 (n=2) and human bocavirus (n=1). PVB19 was also detected in 9 patients with no evidence of AM on CMR or EMB. CONCLUSION: Viral myocarditis is the most common form of myocarditis in South Africa. Local viral prevalence appears to be similar those of the developed world. The clinical significance and pathogenic role of PVB19 remains questioned, and its local background prevalence will have to be further investigated.

Xpert HPV as a Screening Tool for Anal Histologic High-Grade Squamous Intraepithelial Lesions in Women Living With HIV.

BACKGROUND: Women living with HIV (WLWH) experience high rates of anal cancer. Screening using anal cytology, high-resolution anoscopy (HRA) with biopsies, can histologically diagnose anal cancer precursors called high-grade squamous intraepithelial lesions (HSIL). The low specificity of screening using anal cytology results in HRA referral for many WLWH without HSIL. Screening using high-risk human papillomavirus (HR-HPV) may improve specificity. METHODS: Two hundred seven WLWH (63% non-Hispanic black) were screened for anal histologic HSIL (hHSIL) using cytology, HRA-guided biopsies, and Xpert HPV. Xpert performance for predicting anal hHSIL was compared with that of cytology. Usng Xpert 5 HPV genotypic results and accompanying cycle thresholds, receiver operator characteristic curve and recursive partitioning analyses were used to create predictive models for hHSIL. RESULTS: The performance of Xpert to predict hHSIL was not different from that of cytology with a sensitivity (Sn) of 89% and specificity (Sp) of 49%. Interpretation of Xpert was modified using genotypic results and receiver operator characteristic curve analysis, which produced a screen with an Sn and Sp of 75% and 84% for hHSIL, respectively. Another reinterpretation of Xpert was created using recursive partitioning and cycle thresholds, which predicted hHSIL with an Sn and Sp of 75% and 86%, respectively. The detection of HPV-16 was highly predictive of hHSIL in all analyses. These modified screening tests would reduce HRA referral in this population by almost half compared with anal cytology. CONCLUSIONS: Xpert HPV is an alternative to anal cytology to screen for anal HSIL and can be optimized to reduce the number of unnecessary HRAs performed in WLWH.

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.

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.

Resistance in patients failing integrase strand transfer inhibitors: a call to replace raltegravir with dolutegravir in third-line treatment in South Africa.

Data on integrase resistance patterns in low- and middle-income countries (LMICs) is scarce. We assessed genotypic drug resistance in 43 patients with virological failure on integrase strand transfer inhibitors (INSTIs) containing regimens as part of the third-line treatment program in South Africa. Of the raltegravir (RAL) exposed patients 20/34 (59%) had ≥1 major INSTI mutation, including two (6%) with dolutegravir (DTG) cross-resistance. DTG resistance was detected in one out of four DTG-exposed patients. Replacing RAL with DTG may reduce the risk of INSTI mutations. We recommend DTG drug resistance monitoring when DTG is introduced at a larger scale in LMICs.

Moderate levels of pre-therapy drug resistance (PDR) in a generalised epidemic: time for better first-line ART?

BACKGROUND: The WHO-recommended first-line antiretroviral therapy (ART) as a fixed dose combination (FDC) of efavirenz (EFV) and tenofovir disoproxil fumarate (TDF) with lamivudine (3TC) or emtricitabine (FTC) has been preferred in the large scale unprecedented ART roll out in Southern Africa. Models and recent reports suggest that pre-ART HIV drug resistance (PDR) is increasing with high treatment coverage. METHOD: We therefore investigated PDR and any local transmission clusters in a setting where high treatment coverage was further enhanced by universal test and treat (UTT). Surveillance drug resistance mutations (SDRMs) were identified with an in-house PCR and population sequencing method and calibrated population resistance (CPR) tool. RESULTS: Of 60 patients, six (10%) had an SDRM mutation: five (8.3%) had nonnucleoside reverse transcriptase (NNRT) mutations, one had an nucleos(t)ide reverse transcriptase inhibitor mutation and none had protease inhibitor (PI) mutations. Phylogenetic analysis revealed no large transmission clusters. CONCLUSION: An increase to the current moderate PDR levels and the better tolerability and durability, may support a recent drive to avail FDC integrase strand transfer inhibitor (ISTI)-based regimens as the new preferred first-line ART in the Southern African region for individual benefit and to contribute to limiting transmission of infection and drug resistant virus.

Mutational Heterogeneity in p6 Gag Late Assembly (L) Domains in HIV-1 Subtype C Viruses from South Africa.

Contradictory results have been reported on the impact of duplications/insertions in the HIV-1 gag-p6 late assembly domains [TSG101-binding P(T/S)APP motif and ALIX-binding LYPxnLxxL motif] heterogeneity following therapy failure. However, most studies are limited to small numbers of patients and do not include samples from South Africa, which has the largest number of HIV-1C-infected patients (HIV-1CZA). In this study we compared the gag-p6 variability among HIV-1CZA-infected patients from a South African clinical cohort who experienced antiretroviral therapy (ART) failure (n = 845) with ART-naive HIV-1CZA sequences (n = 706) downloaded from the Los Alamos database. Partial (PTA/PTV/APP) or complete P(T/S)APP duplications were less frequent in HIV-1CZA with ART failure compared to therapy-naive ones (14% vs. 30%; p < 0.001). In contrast, the tetrapeptide PYxE insertion, recently described by us, occurred more frequently (5-fold) in therapy-failure patients (p < 0.001) and was associated with a higher number of reverse transcriptase inhibitor (RTI) mutations (p = 0.04) among patients failing ART.

Trends in Genotypic HIV-1 Antiretroviral Resistance between 2006 and 2012 in South African Patients Receiving First- and Second-Line Antiretroviral Treatment Regimens.

OBJECTIVES: South Africa's national antiretroviral (ARV) treatment program expanded in 2010 to include the nucleoside reverse transcriptase (RT) inhibitors (NRTI) tenofovir (TDF) for adults and abacavir (ABC) for children. We investigated the associated changes in genotypic drug resistance patterns in patients with first-line ARV treatment failure since the introduction of these drugs, and protease inhibitor (PI) resistance patterns in patients who received ritonavir-boosted lopinavir (LPV/r)-containing therapy. METHODS: We analysed ARV treatment histories and HIV-1 RT and protease mutations in plasma samples submitted to the Tygerberg Academic Hospital National Health Service Laboratory. RESULTS: Between 2006 and 2012, 1,667 plasma samples from 1,416 ARV-treated patients, including 588 children and infants, were submitted for genotypic resistance testing. Compared with 720 recipients of a d4T or AZT-containing first-line regimen, the 153 recipients of a TDF-containing first-line regimen were more likely to have the RT mutations K65R (46% vs 4.0%; p<0.001), Y115F (10% vs. 0.6%; p<0.001), L74VI (8.5% vs. 1.8%; p<0.001), and K70EGQ (7.8% vs. 0.4%) and recipients of an ABC-containing first-line regimen were more likely to have K65R (17% vs 4.0%; p<0.001), Y115F (30% vs 0.6%; p<0.001), and L74VI (56% vs 1.8%; p<0.001). Among the 490 LPV/r recipients, 55 (11%) had ≥1 LPV-resistance mutations including 45 (9.6%) with intermediate or high-level LPV resistance. Low (20 patients) and intermediate (3 patients) darunavir (DRV) cross resistance was present in 23 (4.6%) patients. CONCLUSIONS: Among patients experiencing virological failure on a first-line regimen containing two NRTI plus one NNRTI, the use of TDF in adults and ABC in children was associated with an increase in four major non- thymidine analogue mutations. In a minority of patients, LPV/r-use was associated with intermediate or high-level LPV resistance with predominantly low-level DRV cross-resistance.

Antiretroviral resistance patterns and factors associated with resistance in adult patients failing NNRTI-based regimens in the Western Cape, South Africa.

Antiretroviral drug resistance in patients failing non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line combination antiretroviral treatment (ART) is influenced by: regimen choice, HIV-1 subtype, detection of and response to therapy failure. In order to describe resistance patterns by genotypic testing, at the time of first-line ART failure and to describe associations with having M184I/V, K65R, three or more thymidine analog mutations (TAMs) and etravirine (ETV) resistance, the prevalence of antiretroviral drug resistance associated mutations in a cross-sectional study, at two South African public health clinic settings, at the time of virologic failure (HIV-1 RNA load >400 copies/ml) are described. Also reported are associations of therapy choice, prolonged virologic failure, and concurrent HIV viral load and CD4 count with the presence of M184I/V, TAMs, K65R, and resistance to ETV. Of 167 adult patients with virologic failure on first-line ART, 28 (17%) had no resistance, 137 (82%) had NNRTI resistance, 101 (60%) M184I/V, 20 (12%) TAMs, of which 4 had 3 or more TAMs, and 7 (4%) had K65R, of which 6 were on D4T and one on AZT. A prolonged estimated period of failure was associated with having ≥3 TAMs. Patients treated with nevirapine (NVP) were more likely to have ETV resistance than those treated with efavirenz (EFV). Major protease inhibitor mutations were not detected. A delayed response to ART failure may risk accumulation of TAMs in patients on an NNRTI-based regimen. The use of NVP rather than EFV was associated with ETV resistance.

Surveillance of transmitted resistance to antiretroviral drug classes among young children in the Western Cape Province of South Africa.

There are limited data on transmitted antiretroviral resistance in young children who require antiretroviral therapy. We adapted the World Health Organization surveillance strategy, testing antiretroviral naive infants (<18 months) in the Western Cape Province of South Africa, and detecting only 3 non-nucleoside reverse transcriptase inhibitors (NNRTI) and no NRTI or protease inhibitor surveillance mutations in 49 patients. The estimated NRTI and protease inhibitor transmitted antiretroviral resistance prevalence is low (<5%), predicting good therapeutic response in Western Cape infants.

Protease inhibitor resistance in South African children with virologic failure.

BACKGROUND: In South Africa, first-line antiretroviral therapy for children younger than 3 years of age combines a protease inhibitor (PI) with 2 nucleoside reverse transcription inhibitors. In our study, some pediatric patients received ritonavir (RTV) as single PI (RTV-sPI) and others ritonavir-boosted lopinavir (LPV/r), which has a higher resistance barrier. We explored antiretroviral resistance mutations in pediatric patients failing PI-based antiretroviral therapy and the predictors of major PI resistance mutations (MPIRM) in these patients. MATERIALS AND METHODS: We studied pediatric HIV patients at Tygerberg Academic Hospital experiencing virologic failure on a PI regimen. Mixed-effects linear- and mixed-effect logistic regression modeling, were used to explore predictors of MPIRM. RESULTS: MPIRM were found in 12 of 17 patients exposed to RTV-sPI compared with 1 of 13 patients treated with LPV/r. Exposure to RTV-sPI was significantly associated with MPIRM, with both exposure time and estimated failing time on RTV-sPI being significant positive predictors of MPIRM. Neither CD4 count, viral load, age at first visit nor receiving rifampin predicted MPIRM. CONCLUSIONS: RTV-sPI in infants and children poses a significant risk of MPIRM which is dependent on the exposure time and time failing while receiving the regimen.