First Evidence for the Disease-Stage, Cell-Type, and Virus Specificity of microRNAs during Human Immunodeficiency Virus Type-1 Infection.

The potential involvement of host microRNAs (miRNAs) in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+) individuals (viremic and aviremic individuals while receiving highly active antiretroviral therapy (HAART), therapy-naïve long-term non-progressors (LTNP), and HIV-negative (HIV-) healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV- samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted p-value < 0.05. The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV- controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs.

Varicella zoster virus quantitation in blood from symptomatic and asymptomatic individuals.

Primary infection with varicella zoster virus (VZV) occurs in immunocompromised and immunocompetent individuals. Clinical and asymptomatic reactivation with shedding of infectious virus and viremia may occur. The prevalence of VZV viremia is unknown. The aim of this study was to detect VZV viremia and quantify VZV DNA using quantitative polymerase chain reaction (qPCR) in blood from different populations. A qPCR-based method using EvaGreen® was used to quantify VZV DNA in 491 samples, including whole blood, plasma and buffy-coat, from patients hospitalized with varicella-associated disease (Group 1, n=10) and three groups with no VZV disease: individuals with a first clinical diagnosis of central nervous system demyelination (Group 2, n=213) with their age and sex-matched controls (Group 3, n=218); and HIV-infected individuals (Group 4, n=50). VZV-specific IgG antibody titres were measured in Group 3. The proportion positive for viremia and mean detectable VZV DNA load (copies/ml) were: Group 1: 100% (10/10) and 4.6 × 10(6) ± 1.4 × 10(7) ; Group 2: 4% (9/213) and 1.5 × 10(3) ± 1.8 × 10(4) ; Group 3: 8% (17/218) and 1.1 × 10(3) ± 7.8 × 10(3) ; Group 4: 12% (6/50) and 7.7 × 10(1) ± 2.8 × 10(2) . VZV DNA load and IgG titres were not significantly correlated (Group 3 only). VZV load in Group 1 was significantly elevated compared to Groups 2-4 (P<0.001); the latter were not significantly different from each other (P=0.05). VZV genotypes from clades 1-5 were identified in Group 1. VZV DNA was detected but at low frequency and viral load in both immunocompetent and immunocompromised individuals asymptomatic for VZV infection, compared to individuals with active VZV infection.

Sensitive detection of the K103N non-nucleoside reverse transcriptase inhibitor resistance mutation in treatment-naïve HIV-1 infected individuals by rolling circle amplification.

Primary or transmitted antiretroviral drug resistance mutations pose a significant obstacle for optimizing antiviral treatment. When present at low-levels, resistance mutations are less likely to be detected by standard genotyping assays. This study utilizes a novel rolling circle amplification (RCA) method using padlock probes to achieve the sensitive, specific and low-level detection of the NNRTI resistance K103N from 59 HIV+ treatment-naïve patients from Beijing, China. Using standard genotyping methods, primary drug resistance mutations to either protease or RT inhibitors were found in 25% (15/59) of patients attending hospital clinics in Beijing. Among these 15 patients with antiretroviral (ARV) resistance mutations, standard sequence-based genotyping revealed that most (10/15) had the 103N. Using a highly sensitive RCA assay, 5 more patients among the 59 treatment-naïve cohort were found to have the 103N, but at low-levels, leading to an overall rate of 103N at 25.4% (15/59) in this population. The high prevalence of the 103N suggests that baseline resistance testing should be performed before treatment in this population. Importantly, the new RCA technology allows large-scale, sensitive detection of drug resistance mutations, including detection of minority populations with minimal equipment requirement.

External quality assessment of HLA-B*5701 reporting: an international multicentre survey.

OBJECTIVES: HLA-B*5701 strongly predicts abacavir hypersensitivity (HSR), but implementation of effective routine screening into clinical practice requires testing be practical and accurate. We tested the proficiency of HLA-B*5701 typing among laboratories using sequence-specific primer PCR. DESIGN AND METHODS: DNA panels (1 and 2) were distributed to seven laboratories (A to G) for blinded typing of the HLA-B*5701 allele. Panel 1 (n = 10 samples; n = 7 laboratories) included 3 positives and other closely related B17 subtypes (B*5702, B*5703, B*5704 and B*5801). Panel 2 (n = 96 samples; n = 4 laboratories) included 36 positives among a broad spectrum of other B alleles. Two laboratories (A and B) also submitted 96 routine samples, typed by the same methodology, to the reference centre for additional analysis by sequence-based typing. RESULTS: All laboratories correctly typed panel 1 for HLA-B*5701 carriage. Laboratories A, B and C identified HLA-B*5701 alleles in panel 2 with 100% sensitivity and 100% specificity. Laboratory D reported one false negative, reportedly due to a sampling error. The results obtained for routine samples typed by laboratories A and B and those generated by the reference laboratory using sequencing were fully concordant. CONCLUSIONS: Detection of HLA-B*5701 alleles among laboratories was 100% specific and 99.4% sensitive, indicating that participating HIV testing laboratories were currently offering effective primary screening to identify individuals at high risk of abacavir HSR. Accurate reporting of HLA-B*5701 status is critical for the safe administration of this drug and participation in quality assurance programmes by all sites who report HLA-B*5701 status should be promoted.

Complex patterns of the HIV-1 epidemic in Kuala Lumpur, Malaysia: evidence for expansion of circulating recombinant form CRF33_01B and detection of multiple other recombinants.

The HIV protease-reverse transcriptase (PR-RT) (1047 bp), gp120-env (891 bp) and gp41-env (547 bp) regions from the plasma of 115 HIV-1-infected patients in Kuala Lumpur (KL), Malaysia were sequenced. Detailed phylogenetic and bootscanning analyses were performed to determine the mosaic structure of the HIV-1 strains and their recombination breakpoint(s). Among the 50 patient samples in which all three regions could be amplified, the HIV-1 CRF01_AE subtype (46%) was predominant followed by subtypes B (10%) and B' (6%). A total of 9/50 (18%) patients were infected with a CRF01_AE/B inter-subtype recombinant, displaying a recombinant form (RF)(PR-RT), CRF01_AE(gp120-env) and CRF01_AE(gp41-env). This RF was derived from the Thai variants of CRF01_AE and B' subtype, with two distinct B' subtype segments in the backbone of CRF01_AE, similar to the newly identified CRF33_01B. In addition, one sample demonstrated a close structural relationship with the new CRF33_01B in the PR-RT region but displayed B' segment in part of the env region (RF(PR-RT), CRF01_AE/B'(gp120-env) and B'(gp41-env)) indicating continuing evolution of CRF33_01B. The remaining 18% of samples were identified as unique recombinant forms (URFs).

Drug resistance and viral evolution in plasma and peripheral blood cells during structured treatment interruption (STI) and non-interrupted HAART.

BACKGROUND: Highly active antiretroviral therapy (HAART) can successfully reduce plasma and tissue levels of HIV-1 RNA and results in reductions in HIV-related morbidity and mortality, but the slow viral evolution during therapy in cellular reservoirs is a continuing problem. In addition, little remains known how viral evolutionary process may differ between cell-free and cell-associated compartments, over time, in vivo in patients receiving HAART or STI. OBJECTIVES: The main objectives of this study were to assess viral replication kinetics, drug resistance and viral evolution during HAART and STI. STUDY DESIGN: We have conducted a longitudinal study of virus culture kinetics in vitro, molecular analysis of uncultured HIV-1 variants from plasma and PBMC of 6 patients on HAART, 4 patients on STI, and 6 from treatment-naïve patients. RESULTS: Our data suggest that drug resistance mutations remained compartmentalized between plasma and PBMC. The divergent distribution of resistance mutations between plasma and PBMC coincided with divergent env gene evolution in these compartments. In contrast, the HIV strains from therapy-naive patients showed tight genetic and phylogenetic concordance between plasma and PBMC. Both STI and non-STI groups showed the presence of resistance mutations to both RT and protease inhibitors, which correlated with inadequate suppression of viremia and partially with the virus culture isolation in vitro. CONCLUSIONS: Overall, STI for HIV patients has no added advantage over regular HAART at the virologic level and in the diminution of resistance mutations that result in therapy failure. Under both forms of anti-retroviral therapies, virus could be isolated in vitro from the PBMC showing continuing low-level viral replication under suppressive therapy. Overall, these data may be useful in predicting the late emergence of drug resistance mutations via the latent integrated provirus.

Genetic analyses reveal structured HIV-1 populations in serially sampled T lymphocytes of patients receiving HAART.

HIV-1 infection and compartmentalization in diverse leukocyte targets significantly contribute to viral persistence during suppressive highly active antiretroviral therapy (HAART). Longitudinal analyses were performed on envelope sequences of HIV-1 populations from plasma, CD4+ and CD8+ T lymphocytes in 14 patients receiving HAART and 1 therapy-naive individual. Phylogenetic reconstructions and analysis of molecular variance revealed that HIV-1 populations in CD4+ and CD8+ T cells remained compartmentalized over time in most individuals. Analyses of viral genetic variation demonstrated that, despite compartmentalization remaining over time, viral subpopulations tended not to persist and evolve but instead broke down and became reconstituted by new founder viruses. Due to the profound impact of HAART on viral evolution, it was difficult to discern whether these dynamics were ongoing during treatment or predominantly established prior to the commencement of therapy. The genetic structure and viral founder effects observed in serially sampled T lymphocyte populations supported a scenario of metapopulation dynamics in the tissue(s) where different leukocytes become infected, a factor likely to contribute to the highly variable way that drug resistance evolves in different individuals during HAART.

Assessment of drug resistance mutations in plasma and peripheral blood mononuclear cells at different plasma viral loads in patients receiving HAART.

BACKGROUND: HIV drug resistance mutations both in peripheral blood mononuclear cells (PBMCs) and plasma have the ability to influence the outcome of highly active antiretroviral therapy for HIV patients. PBMCs harbor archival proviral DNA, are a major source of HIV and also underdo latent infection during suppressive HAART. OBJECTIVES: The main objectives of this study were to assess whether specific viral load groups are better predictors of drug resistance and to examine the utility of PBMCs for drug resistance testing during HAART. STUDY DESIGN: Patients were grouped into a plasma panel comprising of 100 patients and a PBMC/plasma panel of 45 patients. These two groups were further divided according to plasma viral load (low, medium and high). Therapy naive patients were also included. Resistance to protease and reverse transcriptase inhibitors was assessed in each group over different viral load categories. RESULTS: Our data indicated that in addition to plasma, PBMCs also are a reliable predictor of drug resistance. Drug resistance mutations analyzed from each panel demonstrated that intermediate and high viral loads were strong indicators of drug resistance in both the plasma and PBMC compartments. Despite this, a significant portion of patients with high viral loads showed reduced levels of drug resistance indicating that factors including poor compliance, drug pharmacokinetics and host genetic factors are also likely to contribute to therapy failure. A significant degree of resistance to NRTI and PI resistance was found in treatment-naive individuals, demonstrating the transmission of circulating drug resistant HIV-1 variants. CONCLUSIONS: Our data emphasize the need for stronger pharmacokinetic evaluation during HAART, especially for patients with intermediate or high plasma viremia. The utility of PBMCs as an alternative source of resistance profiling was also demonstrated, and this approach may benefit the assessment of future drug regimens for HIV-infected patients.

Obstacles to successful antiretroviral treatment of HIV-1 infection: problems & perspectives.

Mutations in human immunodeficiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretroviral therapy (HAART) and the design of anti-HIV vaccines. Although HAART has made long-term suppression of HIV a reality, drug resistance, drug toxicity, drug penetration, adherence to therapy, low levels of continued viral replication in cellular reservoirs and augmentation of host immune responses are some of the most important challenges that remain to be sorted out. Continuing viral replication in the face of HAART leads to the accumulation of drug resistance mutations, increase in viral loads and eventual disease progression. Patients who fail therapy have minimal options for their clinical management. Therefore, a clear understanding of the pathogenesis of drug-resistant HIV-1, and all of the issues that influence the success of HAART is urgently needed. In the present article, we discuss various obstacles to HIV therapy, and provide perspectives relating to these issues that are critical in determining the success or failure of HAART.

HIV-1 compartmentalization in diverse leukocyte populations during antiretroviral therapy.

CD4+ T lymphocytes are the primary target of human immunodeficiency virus type 1 (HIV-1), but there is increasing evidence that other immune cells in the blood, including CD8+ T lymphocytes and monocytes, are also productively infected. The extent to which these additional cellular reservoirs contribute to ongoing immunodeficiency and viral persistence during therapy remains unclear. In this study, we conducted a detailed investigation of HIV-1 diversity and genetic structure in CD4+ T cells, CD8+ T cells, and monocytes of 13 patients receiving highly active antiretroviral therapy (HAART). Analysis of molecular variance and nonparametric tests performed on HIV-1 envelope sequences provided statistically significant evidence of viral compartmentalization in different leukocyte populations. Signature pattern analysis and predictions of coreceptor use provided no evidence that selection arising from viral tropism was responsible for the genetic structure observed. Analysis of viral genetic variation in different leukocyte populations demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. In the absence of evidence for leukocyte-specific selection, these features were supportive of a metapopulation model of HIV-1 replication as described previously among HIV-1 populations in the spleen. Compartmentalization of the virus in different leukocytes may have significant implications for current models of HIV-1 population genetics and contribute to the highly variable way in which drug resistance evolves in different individuals during HAART.