Protease Reverse Transcriptase Regions Research Articles

Shall I trust the report? Variable performance of Sanger sequencing revealed by deep sequencing on HIV drug resistance mutation detection

BackgroundThe clinical utilisation of deep sequencing in HIV treatment has been hindered due to its unknown correlation with standard Sanger genotyping and the undetermined value of minority drug resistance mutation (DRM) detection. ObjectivesTo compare deep sequencing performance to standard Sanger genotyping with clinical samples, in an effort to delineate the correlation between the results from the two methods and to find the optimal deep sequencing threshold for clinical utilisation. MethodsWe conducted a retrospective study using stored plasma collected from August 2014 to March 2018 for HIV genotyping with the commercial Sanger genotyping kit. Samples with available Sanger genotyping reports were further deep sequenced. Drug resistance was interpreted according to the Stanford HIV drug resistance database algorithm. ResultsAt 15–25% minority detection thresholds, 9–15% cases had underestimated DRMs by Sanger sequencing. The concordance between the Sanger and deep sequencing reports was 68–82% in protease-reverse transcriptase region and 88–97% in integrase region at 5–25% thresholds. The undetected drug resistant minority variants by Sanger sequencing contributed to the lower negative predictive value of Sanger genotyping in cases harbouring DRMs. ConclusionsUse of deep sequencing improved detection of antiretroviral resistance mutations especially in cases with virological failure or previous treatment interruption. Deep sequencing with 10–15% detection thresholds may be considered a suitable substitute for Sanger sequencing on antiretroviral DRM detection.

Исследование частоты встречаемости двойной ВИЧ-инфекции в России

Objective. To develop and validate the method of dual HIV infection detection based on the results of Sanger sequencing and to evaluate of dual HIV infection frequency in Russia. Materials and methods. We carried out the sequencing of HIV-1 pol gene fragment in model samples of dual HIV infection, the calculation of degeneracy bases index (DB-index), and the index of synonymity (SM-index) and determination of their threshold values for dual HIV infection detection. We analyzed nucleotide sequences of the pol gene fragment in HIV-1 samples (n = 1561) taken from Russian patients. Results. The threshold values of 34 for the DB-index and 0.05 for the SM-index allow detecting effectively the dual HIV infection using reverse transcriptase (RT) and protease-reverse transcriptase (PR-RT) fragments of the pol gene correspondingly. At total 21 (1.35%) dual HIV-infected samples (18 of which were collected in 2014 and later) were revealed in the collection studied; 12 of them were obtained from patients residing in the Central Federal District. An inverse correlation between the likelihood of dual HIV infection detecting and the duration of the patient's infection was found. In total, 11 out of 21 samples with dual HIV infection had mutations associated with drug resistance; 7 of them (33.3%) had substitutions that were not associated with natural polymorphism of HIV-1. Conclusion. The problem of dual HIV infection in Russia is actual and requires further study. Key words: HIV, dual HIV infection, drug resistance, nucleotide sequence, DB-index, SM-index, PR-RT region

High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

Discrepant amplification results during the development of an assay leads to reclassification of two AIDS reagent repository HIV-2 isolates as HIV-1.

The development and verification of HIV-2 assays depends in part on the availability of well-characterized samples, including those from reagent repositories. During the development of an HIV-2 RNA quantification assay, two HIV-2 viral isolates (CDC 301340 and CDC 301342) obtained from the NIAID AIDS Reagent and Reference Repository were not detected leading to an investigation. Two HIV-2 primers/probe sets of known performance in real-time viral RNA quantification assays, targeting different regions of the virus, also failed to generate RT-PCR products for these two isolates. These isolates were tested in the HIV-1 specific COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (Roche Molecular Diagnostics) and were quantified at high copy number. Other HIV-2 isolates tested were not amplified in the COBAS HIV-1 TaqMan assay. Furthermore, the discrepant isolates were highly reactive in an HIV-1 p24 antigen test while the other HIV-2 isolates showed very weak, if any, cross-reactivity with the HIV-1 p24 assay. Phylogenetic tree analysis of sequences from the protease-reverse transcriptase regions of the discrepant HIV-2 isolates mapped with HIV-1 Group M, Subtype CRF02_AG confirming these isolates were of HIV-1 origin and had been misclassified as HIV-2. The use of misclassified isolates in the verification of molecular and immunological assays can lead to misinterpretation of test results, misdirection of efforts into assay redesign and increased development costs. The results of this study were shared with the NIAID AIDS Reagent Program, leading to the reclassification of the two discrepant isolates as HIV-1.

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).