A validated in-house assay for HIV drug resistance mutation surveillance from dried blood spot specimens.

Despite increasing scale-up of antiretroviral therapy (ART) coverage, challenges related to adherence and HIV drug resistance (HIVDR) remain. The high cost of HIVDR surveillance is a persistent challenge with implementation in resource-constrained settings. Dried blood spot (DBS) specimens have been demonstrated to be a feasible alternative to plasma or serum for HIVDR genotyping and are more suitable for lower resource settings.

HIV acquisition prior to entry into formal sex work: inference from next-generation viral sequencing.

Objective: To infer the timing of HIV acquisition in relation to self-reported events in the sexual life course of adolescent girls and young women (AGYW) who self-identify as female sex workers (FSW) in Mombasa, Kenya.

Design: Next-generation viral sequencing of samples of AGYW living with HIV in the Transitions study, a cross-sectional bio-behavioural survey of AGYW aged 14-24 years in Mombasa, Kenya.

Method: Dried blood spot specimens were collected from study participants ( n  = 37, all FSW).

Point-of-Care Tests for HIV Drug Resistance Monitoring: Advances and Potentials.

HIV/AIDS is a global public health crisis that is yet to be contained. Effective management of HIV drug resistance (HIVDR) supported by close resistance monitoring is essential in achieving the WHO 95-95-95 targets, aiming to end the AIDS epidemic by 2030. Point-of-care tests (POCT) enable decentralized HIVDR testing with a short turnaround time and minimal instrumental requirement, allowing timely initiation of effective antiretroviral therapy (ART) and regimen adjustment as needed.

Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing.

The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS.

Quality Control of Next-Generation Sequencing-Based HIV-1 Drug Resistance Data in Clinical Laboratory Information Systems Framework.

Next-generation sequencing (NGS) in HIV drug resistance (HIVDR) testing has the potential to improve both clinical and public health settings, however it challenges the normal operations of quality management systems to be more flexible due to its complexity, massive data generation, and rapidly evolving protocols. While guidelines for quality management in NGS data have previously been outlined, little guidance has been implemented for NGS-based HIVDR testing. This document summarizes quality control procedures for NGS-based HIVDR testing laboratories using a laboratory information systems (LIS) framework.

Performance comparison of next generation sequencing analysis pipelines for HIV-1 drug resistance testing.

Next generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Many NGS HIVDR data analysis pipelines have been independently developed, each with variable outputs and data management protocols. Standardization of such analytical methods and comparison of available pipelines are lacking, yet may impact subsequent HIVDR interpretation and other downstream applications.

A MiSeq-HyDRA platform for enhanced HIV drug resistance genotyping and surveillance.

Conventional HIV drug resistance (HIVDR) genotyping utilizes Sanger sequencing (SS) methods, which are limited by low data throughput and the inability of detecting low abundant drug resistant variants (LADRVs). Here we present a next generation sequencing (NGS)-based HIVDR typing platform that leverages the advantages of Illumina MiSeq and HyDRA Web. The platform consists of a fully validated sample processing protocol and HyDRA web, an open web portal that allows automated customizable NGS-based HIVDR data processing.

Bioinformatic data processing pipelines in support of next-generation sequencing-based HIV drug resistance testing: the Winnipeg Consensus.

Introduction: Next-generation sequencing (NGS) has several advantages over conventional Sanger sequencing for HIV drug resistance (HIVDR) genotyping, including detection and quantitation of low-abundance variants bearing drug resistance mutations (DRMs). However, the high HIV genomic diversity, unprecedented large volume of data, complexity of analysis and potential for error pose significant challenges for data processing. Several NGS analysis pipelines have been developed and used in HIVDR research; however, the absence of uniformity in data processing strategies results in lack of consistency and comparability of outputs from different pipelines.

Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs.

Background: Mosquito-borne diseases threaten over half the world's human population, making the need for environmentally-safe mosquito population control tools critical. The sterile insect technique (SIT) is a biological control method that can reduce pest insect populations by releasing a large number of sterile males to compete with wild males for female mates to reduce the number of progeny produced. Typically, males are sterilized using radiation, but such methods can reduce their mating competitiveness.

Immunogenicity of sequences around HIV-1 protease cleavage sites: potential targets and population coverage analysis for a HIV vaccine targeting protease cleavage sites.

Developing an effective preventative vaccine against HIV-1 has proved to be a great challenge. The classical and proven vaccine approach has failed so far or produced a modest effect, new approaches are needed. In this study we evaluated the immunogenicity of the sequences around the protease cleavage sites (PCS) and the population coverage of a vaccine targeting HIV-1 PCS.

HIV-1 clade D is associated with increased rates of CD4 decline in a Kenyan cohort.

HIV-1 is grouped phylogenetically into clades, which may impact rates of HIV-1 disease progression. Clade D infection in particular has been shown to be more pathogenic. Here we confirm in a Nairobi-based prospective female sex worker cohort (1985-2004) that Clade D (n = 54) is associated with a more rapid CD4 decline than clade A1 (n = 150, 20.

For protection from HIV-1 infection, more might not be better: a systematic analysis of HIV Gag epitopes of two alleles associated with different outcomes of HIV-1 infection.

A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce.

A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1.

Background: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution.

Methodology/principal Findings: HIV-1 gag gene was amplified from 96 patients using nested PCR.

Identification and characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a novel approach.

Identification of CTL epitopes correlated to immune protection is important for the development of vaccines that enhance T-cell mediated immune responses. The correlation of positively selected amino acids (PS) of HIV-1 with host HLA alleles can identify regions containing potential T-cell epitopes. However, the specific epitopes have to be identified and characterized using overlapping peptides through T-cell functional assays.

Clade-specific evolution mediated by HLA-B*57/5801 in human immunodeficiency virus type 1 clade A1 p24.

HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate.

An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani Sex Worker Cohort.

Human immunodeficiency virus type 1 (HIV-1) is able to evade the host cytotoxic T-lymphocyte (CTL) response through a variety of escape avenues. Epitopes that are presented to CTLs are first processed in the presenting cell in several steps, including proteasomal cleavage, transport to the endoplasmic reticulum, binding by the HLA molecule, and finally presentation to the T-cell receptor. An understanding of the potential of the virus to escape CTL responses can aid in designing an effective vaccine.