Short Communication: Analysis of Minor Populations of Human Immunodeficiency Virus by Primer Identification and Insertion-Deletion and Carry Forward Correction Pipelines

Accurate analysis of minor populations of drug-resistant HIV requires analysis of a sufficient number of viral templates. We assessed the effect of experimental conditions on the analysis of HIV pol 454 pyrosequences generated from plasma using (1) the ‘‘Insertion-deletion (indel) and Carry Forward Correction’’ (ICC) pipeline, which clusters sequence reads using a nonsubstitution approach and can correct for indels and carry forward errors, and (2) the ‘‘Primer Identification (ID)’’ method, which facilitates construction of a consensus sequence to correct for sequencing errors and allelic skewing. The Primer ID and ICC methods produced similar estimates of viral diversity, but differed in the number of sequence variants generated. Sequence preparation for ICC was comparably simple, but was limited by an inability to assess the number of templates analyzed and allelic skewing. The more costly Primer ID method corrected for allelic skewing and provided the number of viral templates analyzed, which revealed that amplifiable HIV templates varied across specimens and did not correlate with clinical viral load. This latter observation highlights the value of the Primer ID method, which by determining the number of templates amplified, enables more accurate assessment of minority species in the virus population, which may be relevant to prescribing effective antiretroviral therapy

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories

Ethics of HIV research in children

No abstract available.http://www.thelancet.com/infection2016-06-30hb201

Emerging antiretroviral drug resistance in sub-Saharan Africa: novel affordable technologies are needed to provide resistance testing for individual and public health benefits

In industrialized countries, viral load monitoring and genotypic antiretroviral drug resistance testing (GART) play an important role in the selection of initial and subsequent combination antiretroviral therapy (cART) regimens. In contrast, resource constraints in Africa limit access to assays that could detect virologic failure, transmitted drug resistance (TDR) and acquired drug resistance to cART. This has adverse consequences for both individual and public health. Although the further roll-out of antiretrovirals for prevention, including preexposure prophylaxis (PrEP) and universal test and treat (UTT) strategies, could reduce HIV-1 incidence, these strategies may increase TDR [1,2]. Here, we present arguments that the scale up of antiretrovirals use should be accompanied by cost-effective assays for early detection of virologic failure, surveillance of TDR and GART for individual patient management

Implementation of HIV Drug Resistance Testing in Kenya

Background: Testing for HIV drug resistance prior to antiretroviral therapy (ART) is standard of care in the United States but is rarely performed in Africa due to its high cost. The prevalence of HIV drug resistance is increasing in resource-limited settings (RLS). An affordable and feasible strategy is needed in RLS to identify individuals with drug resistance and prescribe effective ART. Our group developed a low-cost oligonucleotide ligation assay (OLA) that detects mutations conferring resistance. Little data exists on implementing HIV drug resistance testing in RLS. Here, we describe the implementation of OLA in one laboratory in Kenya and discuss practical aspects of executing the OLA over two years. Methods: The OLA was transferred to a research laboratory at the Coptic Hope Center in Nairobi, Kenya, as part of a randomized trial testing use of the OLA in individuals starting first-line ART to improve virologic outcome. The Seattle Lab Manager (SLM) transported equipment needed for OLA to Nairobi, set up the laboratory, and trained two Kenyan lab technicians to perform OLA. Two additional technicians were later trained. Technicians had education either as a lab technologist or lab scientist, with limited training in molecular techniques. Results: OLA was successfully performed by Kenyan lab technicians on 565 blood samples. Each week, OLA was performed on approximately 7 samples, requiring an estimated 10 hours of technician labor, and 2 hours of remote technical support, review of test results and oversight from the SLM. Some sample results were delayed during two temporary, month-long pauses in testing of specimens, due to suboptimal performance of the OLA. This required trouble-shooting by the SLM in conjunction with lab personnel. Conclusion: OLA technology was successfully transferred to the Kenyan laboratory. However, it required time-intensive technician labor and substantial oversight by the SLM. The complexity of OLA, and a paucity of lab technicians and on-site supervisors trained in molecular techniques are potential bottlenecks for implementation of the current version of OLA at a larger population-level. Research is ongoing to develop OLA Simple, a simplified kit aimed to address these challenges and serve as a point-of-care assay

HIV-1 RNA Levels and Antiretroviral Drug Resistance in Blood and Non-Blood Compartments from HIV-1–Infected Men and Women enrolled in AIDS Clinical Trials Group Study A5077

Background: Detectable HIV-1 in body compartments can lead to transmission and antiretroviral resistance. Although sex differences in viral shedding have been demonstrated, mechanisms and magnitude are unclear. We compared RNA levels in blood, genital-secretions and saliva; and drug resistance in plasma and genital-secretions of men and women starting/changing antiretroviral therapy (ART) in the AIDS Clinical Trials Group (ACTG) 5077 study. Methods: Blood, saliva and genital-secretions (compartment fluids) were collected from HIV-infected adults (≥13 years) at 14 United-States sites, who were initiating or changing ART with plasma viral load (VL) ≥2,000 copies/mL. VL testing was performed on all compartment fluids and HIV resistance genotyping on plasma and genital-secretions. Spearman rank correlations were used to evaluate concordance and Fisher’s and McNemar’s exact tests to compare VL between sexes and among compartments. Results: Samples were available for 143 subjects; 36% treated (23 men, 29 women) and 64% ‘untreated’ (40 men, 51 women). RNA detection was significantly more frequent in plasma (100%) than genital-secretions (57%) and saliva (64%) (P<0.001). A higher proportion of men had genital shedding versus women (78% versus 41%), and RNA detection was more frequent in saliva versus genital-secretions in women when adjusted for censoring at the limit of assay detection. Inter-compartment fluid VL concordance was low in both sexes. In 22 (13 men, 9 women) paired plasma-genital-secretion genotypes from treated subjects, most had detectable resistance in both plasma (77%) and genital-secretions (68%). Resistance discordance was observed between compartments in 14% of subjects. Conclusions: HIV shedding and drug resistance detection prior to initiation/change of ART in ACTG 5077 subjects differed among tissues and between sexes, making the gold standard blood-plasma compartment assessment not fully representative of HIV at other tissue sites. Mechanisms of potential sex-dependent tissue compartmentalization should be further characterized to aid in optimizing treatment and prevention of HIV transmission. Trial Registration ClinicalTrials.gov NCT0000748

Compartmentalization of HIV-1 within the Female Genital Tract Is Due to Monotypic and Low-Diversity Variants Not Distinct Viral Populations

BACKGROUND:Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. METHODOLOGY/PRINCIPAL FINDINGS:Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. CONCLUSIONS/SIGNIFICANCE:In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood

The elusive source of HIV-1 rebound after treatment interruption

Identifying the source of viral rebound during a monitored analytical treatment interruption (ATI) would reveal potential targets for cure strategies. Therefore, we examined the genetic composition of proviral DNA in different subsets from participants on antiretroviral therapy and compared this to rebounding virus after an ATI. Eleven participants underwent a monitored ATI and were sampled from different anatomical sites prior to and after the ATI. From the peripheral blood, Naïve (TNA), central (TCM), transitional (TTM) and effector (TEM) memory CD4+ T cells were sorted as were CD45 cells from gut-associated lymphoid tissue (GALT). Using single-genome sequencing (SGS) the env region of HIV DNA and plasma-derived RNA was sequenced. In an ongoing study, Full-Length Individual Proviral Sequencing (FLIPS) and Integration Site Loop Amplification (ISLA) assays were performed on the T cell subsets from 2 participants. For participant STAR10, 87 integration sites (IS) and 113 proviral genomes were sequenced while only 3 unique intact proviruses (3%) were identified. A cluster of 17 identical defective proviruses were linked to an IS (9% of all IS) in STAT5B located in TCM, TNA, TEM and TTM. When comparing the FLIPS to SGS env sequences a 100% match was found between one defective provirus and one plasma HIV RNA sequence after rebound. For participant STAR11, 37 IS and 105 proviral genomes were sequenced yielding 14 intact proviruses (13%) with the highest proportion found predominantly in the TEM subset (n=13, 45%). Four different clusters of identical sequences could be identified of which 2 (n=3 and n=9) consisted of intact TEM sequences with the smaller cluster linked to an IS in ZNF274. A 99% match between 2 env from rebounding plasma RNA and this smaller cluster of intact proviral genomes was identified. Comparing proviral sequences and their IS to plasma-derived RNA sequences after an ATI reveals additional information in terms of the source of viral rebound. However, this comparison is complicated by multiple factors. For example, we found a plasma-derived RNA sequence obtained during viral rebound matched a defective proviral sequence which highlights the problem of using one HIV RNA subgenomic region for identifying replication-competent virus. In addition, ongoing viral replication during rebound may prevent a 100% match with genetically intact proviral sequences making it challenging to determine the absolute source of rebound

Oligonucleotide ligation assay detects HIV drug resistance associated with virologic failure among antiretroviral-naive adults in Kenya

Background: Transmitted drug resistance (TDR) is increasing in some areas of Africa. Detection of TDR may predict virologic failure of first-line non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). We evaluated the utility of a relatively inexpensive oligonucleotide ligation assay (OLA) to detect clinically relevant TDR at time of ART initiation. Methods: Pre-ART plasmas from ART-naive Kenyans initiating an NNRTI-based fixed-dose combination ART in a randomized adherence trial conducted in 2006 were retrospectively analyzed by OLA for mutations conferring resistance to NNRTI (K103N, Y181C, and G190A) and lamivudine (M184V). Post-ART plasmas were analyzed for virologic failure (≥1,000 copies/mL) at 6 month intervals over 18-month follow-up. Pre-ART plasmas of those with virologic failure were evaluated for drug resistance by consensus and 454-pyrosequencing. Results: Among 386 participants, TDR was detected by OLA in 3.89% [95% Confidence Interval (CI), 2.19-6.33], and was associated with a 10-fold higher rate of virologic failure [Hazard Ratio (HR), 10.39; 95% CI, 3.23-32.41; p Conclusions: Detection of TDR by a point mutation assay may prevent use of sub-optimal ART

Classification of hyperbolic Dynkin diagrams, root lengths and Weyl group orbits

We give a criterion for a Dynkin diagram, equivalently a generalized Cartan matrix, to be symmetrizable. This criterion is easily checked on the Dynkin diagram. We obtain a simple proof that the maximal rank of a Dynkin diagram of compact hyperbolic type is 5, while the maximal rank of a symmetrizable Dynkin diagram of compact hyperbolic type is 4. Building on earlier classification results of Kac, Kobayashi-Morita, Li and Sa\c{c}lio\~{g}lu, we present the 238 hyperbolic Dynkin diagrams in ranks 3-10, 142 of which are symmetrizable. For each symmetrizable hyperbolic generalized Cartan matrix, we give a symmetrization and hence the distinct lengths of real roots in the corresponding root system. For each such hyperbolic root system we determine the disjoint orbits of the action of the Weyl group on real roots. It follows that the maximal number of disjoint Weyl group orbits on real roots in a hyperbolic root system is 4.Comment: J. Phys. A: Math. Theor (to appear