Baseline PI susceptibility by HIV-1 Gag-protease phenotyping and subsequent virological suppression with PI-based second-line ART in Nigeria.

OBJECTIVES: Previous work showed that gag-protease-derived phenotypic susceptibility to PIs differed between HIV-1 subtype CRF02_AG/subtype G-infected patients who went on to successfully suppress viral replication versus those who experienced virological failure of lopinavir/ritonavir monotherapy as first-line treatment in a clinical trial. We analysed the relationship between PI susceptibility and outcome of second-line ART in Nigeria, where subtypes CRF02_AG/G dominate the epidemic. METHODS: Individuals who experienced second-line failure with ritonavir-boosted PI-based ART were matched (by subtype, sex, age, viral load, duration of treatment and baseline CD4 count) to those who achieved virological response ('successes'). Successes were defined by viral load <400 copies of HIV-1 RNA/mL by week 48. Full-length Gag-protease was amplified from patient samples for in vitro phenotypic susceptibility testing, with PI susceptibility expressed as IC50 fold change (FC) relative to a subtype B reference strain. RESULTS: The median (IQR) lopinavir IC50 FC was 4.04 (2.49-7.89) for virological failures and 4.13 (3.14-8.17) for virological successes (P = 0.94). One patient had an FC >10 for lopinavir at baseline and experienced subsequent virological failure with ritonavir-boosted lopinavir as the PI. There was no statistically significant difference in single-round replication efficiency between the two groups (P = 0.93). There was a moderate correlation between single-round replication efficiency and FC for lopinavir (correlation coefficient 0.32). CONCLUSIONS: We found no impact of baseline HIV-1 Gag-protease-derived phenotypic susceptibility on outcomes of PI-based second-line ART in Nigeria.

Graphene oxide-based biosensing platform for rapid and sensitive detection of HIV-1 protease.

HIV-1 protease is essential for the life cycle of the human immunodeficiency virus (HIV), and is one of the most important clinical targets for antiretroviral therapies. In this work, we developed a graphene oxide (GO)-based fluorescence biosensing platform for the rapid, sensitive, and accurate detection of HIV-1 protease, in which fluorescent-labeled HIV-1 protease substrate peptide molecules were covalently linked to GO. In the absence of HIV-1 protease, fluorescein was effectively quenched by GO. In contrast, in the presence of HIV-1 protease, it would cleave the substrate peptide into short fragments, thus producing fluorescence. Based on this sensing strategy, HIV-1 protease could be detected at as low as 1.18 ng/mL. More importantly, the sensor could successfully detect HIV-1 protease in human serum. Such GO-based fluorescent sensors may find useful applications in many fields, including diagnosis of protease-related diseases, as well as sensitive and high-throughput screening of drug candidates. Graphical abstract ᅟ.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients.

Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.

Recent progress in prostate-specific antigen and HIV proteases detection.

Proteases mediate a wide variety of biological events and have a critical role in the development of many diseases. Protease detection methods can be hindered by the limitation of assay safety, sensitivity, specificity, time constraints and ease of on-site analysis. Notably, the implementation of various detection methods on biosensing platforms translates them into practical biosensing applications. Currently, the detection of prostate cancer and AIDS at the earliest occasion is one of the major research obstacles. Therefore, recent advances focus on the development of portable detection systems toward point-of-care testing. These detection systems should be highly sensitive and specific for the detection of their prognostic biomarkers, such as the prostate-specific antigen and HIV load assay for prostate cancer and AIDS, respectively. These methods will also facilitate decision-making on a treatment regimen.

Genetic diversity of HIV type 1 in Montenegro.

Human immunodeficiency virus type 1 (HIV-1) is characterized by high genetic variability due to its high replication rate and the lack of proofreading activity of the reverse transcriptase enzyme. On the basis of phylogenetic analysis performed on numerous isolates from all over the world, HIV-1 is subdivided into types, subtypes, subsubtypes, circulating recombinant forms, and unique recombinant forms. No data are currently available about the circulation of HIV-1 types in Montenegro. Here, we describe the genetic variability of HIV-1 strains identified in plasma samples of patients from Montenegro. Phylogenetic analysis on 32 HIV-1 sequences was carried out. The prevalent circulating HIV-1 subtype is B. The strains were interspersed within the tree. Two main clades (I and II) may suggest independent introductions of HIV-1 subtype B into Montenegro, although other epidemiological evidence will be needed to assume a small number of introductions. No obvious evidence of clustering by residence, age, or sex was found (data not shown). Nelfinavir resistance was found, though lopinavir is the only PI administered. Continuous monitoring of HIV-1-infected individuals is crucial to a better understand of the epidemiology of the B subtype in Montenegro.

Genetic polymorphisms and resistance mutations of HIV type 2 in antiretroviral-naive patients in Burkina Faso.

Natural polymorphisms in the pol gene of HIV-2 may influence the susceptibility to antiretroviral drugs and the choice of treatment. We collected samples in centers for anonymous HIV testing in Ouagadougou, Burkina Faso, in patients supposedly naive for any antiretroviral treatment. Eighty-four samples were first tested as HIV-2 positive in Burkina Faso and then shipped to Brussels, Belgium, for confirmation of the serological status and plasma viral load. Fifty-two samples were confirmed as HIV-2 positive in Belgium. Twelve others were HIV-1 positive and 20 were dually reactive. Twenty-one of HIV-2 confirmed samples had an HIV-2 plasma viral load higher than 1000 copies/ml. These viruses were sequenced in the protease and reverse trancriptase genes and 17 sequences of the pol gene were obtained. Highly polymorphic positions were identified in protease and RT genes. Two samples harbored known resistance mutations: M184V RT mutation in one and Q151M with M184V in the other. Phylogenetic analysis showed that viruses in Burkina Faso did not cluster separately from published sequences from neighboring countries. The two resistant strains were unrelated. Our findings imply either that resistant viruses are circulating in Burkina Faso or that some individuals take unsupervised treatment. Both hypotheses present problems.

Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.

High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients.

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

Adaptation of the plasma renin radioimmunoassay for use with HIV-1 protease.

We have demonstrated the use of a radioimmunoassay to quantitate the peptidolytic activity of human immunodeficiency virus, type 1 (HIV-1) protease using a tetradecapeptide substrate of porcine renin, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser. HIV-1 protease catalyzes cleavage of this substrate at the same Leu-Leu bond as does porcine renin, resulting in the formation of authentic angiotensin-I. The angiotensin-I product is then detected by use of a commercially available renin plasma assay kit, which constitutes the basis of the RIA. The radioimmunoassay provides detection of the protease-catalyzed formation of angiotensin-I at picomolar concentrations in vitro. We demonstrate the use of this assay in determining IC50 values for two HIV-1 protease inhibitors present in cell culture media and in standard assay buffer. An example of the potential development of this assay for the quantitation of these inhibitors present in ex vivo plasma samples is also presented.