Molecular characteristics of HIV-1 Subtype C viruses from KwaZulu-Natal, South Africa: implications for vaccine and antiretroviral control strategies

The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of Human Immunodeficiency Virus Type 1 (HIV-1) Subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or "lineages" of HIV-1 subtype C that segregated with other C viruses from Southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.This article was written by Prof Janse van Rensburg and Prof Cassol before they joined the University of Pretoria.Programme Grant 061238 from the Wellcome Trust, United Kingdom

Dried Blood Spots Collected on Filter Paper: An International Resource for the Diagnosis and Genetic Characterization of Human Immunodeficiency Virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large- scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia

Effect of age, polymicrobial disease,, and maternal HIV status on treatment response and cause of severe pneumonia in South African children: a prospective descriptive study

Background HIV-related pneumonia is the main cause of paediatric hospital admissions in southern Africa. We aimed to measure predictors of treatment failure and the cause of non-responsive pneumonia in children admitted to hospital with severe pneumonia in Durban, South Africa. Methods We investigated 358 children aged 1-59 months who presented with WHO-defined severe or very severe pneumonia. Children were recruited irrespective of HIV status and started on a standard antimicrobial regimen of benzylpenicillin and gentamicin. All infants also received high-dose trimethoprim-sulfamethoxazole. The primary outcome measure was treatment failure at 48 h. Findings 242 (68%) children were HIV infected, 41 (12%) HIV exposed, unindected, and 75 (21%) HIV uninfected. Failure to respond by 48 h was predicted by age under 1 year (adjusted odds ratio 6.38, 95% CI 2.72-14.91, p < 0.0001), very severe disease (2.47, 1.17-5.24, p=0.0181), HIV status (HIV infected 10.3, 3.26-32.51; HIV exposed, uninfected 6.02, 1.55-23.38; p=0.0003), and polymicrobial disease (one organism 2.06, 1.05-4.05; two organisms 10.75, 4.38-26.36; p < 0.0001) on logistic regression analysis. All children with three organisms failed treatment. 72/110 treatment failures had at least two organisms isolated. Three of nine HIV-exposed, uninfected infants, 29/74 HIV-infected, but no HIV-uninfected infants who failed study therapy had Pneumocystis jirovecii pneumonia. Interpretation For children younger than 1 year, the WHO guidelines are inadequate and need to be revised since both HIV-infected and HIV-exposed, uninfected infants had more treatment failures than did HIV-uninfected infants. Polymicrobial disease is an important reason for treatment failure, and we need to identify rapid low-cost diagnostic methods to assist clinicians

Persistence of HIV drug resistance among South African children given nevirapine to prevent mother-to-child-transmission

OBJECTIVES : We set out to examine the prevalence and persistence of mutations conferring high-level non-nucleoside-reverse-transcriptase (NNRTI)-resistance in a cohort of HIV-infected children who had failed prophylaxis to prevent mother-to-child-transmission (PMTCT). DESIGN : A prospective observational cohort study at the Pediatric HIV Clinic at Kalafong Provincial Tertiary Hospital in Pretoria, South Africa. METHODS : Children referred for initiation of antiretroviral therapy (ART) were enrolled from July 2010 through February 2013. HIV drug resistance testing was performed using the oligonucleotide ligation assay (OLA) on dried blood spots (DBS) collected at enrolment and monthly follow-up visits for 2 years. RESULTS : South African children who failed HIV-prophylaxis had a high prevalence of NNRTI-resistant HIV (46/88; 52%). Among children with NNRTI-resistance, the frequency of the predominant resistant variant in each child's HIV-quasispecies was high (median 96%) at study entry (median age 7.5 months), and in 26 out of 27 followed a median of 13 months persisted at a high frequency (median 89%). CONCLUSION : Our finding that infants who fail HIV-prophylaxis frequently have long-lived NNRTI-resistant HIV suggests that resistance will likely persist through 36 months of age, when children qualify for NNRTI-based ART. These children may benefit from HIV drug resistance testing to guide selection of their treatment.A grant from the Delegation of the European Union to South Africa: Drug Resistance Surveillance and Treatment Monitoring Network for the Public Sector HIV Antiretroviral Treatment Programme in the Free State-Sante 2007/147-790, National Research Council of South Africa, Unlocking the Future, 61509; NIH R01 AI058723; Clinical Research and Retrovirology Core of the Seattle Centers for AIDS Research (NIH P30 AI027757), NIH T32 HD007233-32, Seattle Children’s Research Institute Strategic Funds; NIH funding of the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) Virology Developmental Laboratory award (U01 AI068632).http://journals.lww.com/aidsonline2018-05-15hj2017Family Medicin

Multicenter Evaluation of Use of Dried Blood and Plasma Spot Specimens in Quantitative Assays for Human Immunodeficiency Virus RNA: Measurement, Precision, and RNA Stability

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at −70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy

Dried Blood Spots Collected on Filter Paper: An International Resource for the Diagnosis and Genetic Characterization of Human Immunodeficiency Virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large- scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia

An automated genotyping system for analysis of HIV-1 and other microbial sequences

The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entiret