scholarly journals Sequencing of Cytomegalovirus UL97 Gene for Genotypic Antiviral Resistance Testing

2001 ◽  
Vol 45 (10) ◽  
pp. 2775-2780 ◽  
Author(s):  
Nell S. Lurain ◽  
Adriana Weinberg ◽  
Clyde S. Crumpacker ◽  
Sunwen Chou
Keyword(s):  
Drug Resistance ◽  
Clinical Isolates ◽  
Resistance Testing ◽  
Virus Infectivity ◽  
Resistance Mutations ◽  
Sequence Identity ◽  
Complete Sequences ◽  
Immunosuppressed Patients ◽  
Ul97 Gene ◽  
Genotypic Assay

ABSTRACT The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CMV) infections in immunosuppressed patients has led to the development of drug resistance. Phenotypic assays for CMV drug resistance are presently too time-consuming to be therapeutically useful. To support the development of genotypic assays for GCV resistance, the complete sequences of the UL97 phosphotransferase genes in 28 phenotypically GCV-sensitive CMV clinical isolates were determined. The gene was found to be highly conserved, with nucleotide sequence identity among strains ranging from 98.6 to 100% and amino acid sequence identity of >99%. Primers for a genotypic assay were designed to amplify codons 400 to 707, because all known UL97 mutations conferring drug resistance occur at three sites within this region. This part of the UL97 gene was amplified from over 50 clinical isolates, and two sequencing reactions for the coding strand were successfully used to identify GCV resistance mutations. This genotypic assay can be performed in 48 h using genomic DNA extracted from cell monolayers at very low levels of virus infectivity, thus rapidly providing therapeutically useful results.

Impact of genotypic diversity on selection of subtype-specific drug resistance profiles during raltegravir-based therapy in individuals infected with B and BF recombinant HIV-1 strains

2020 ◽  
Vol 75 (6) ◽  
pp. 1567-1574
Author(s):  
Daniela Sánchez ◽  
Solange Arazi Caillaud ◽  
Ines Zapiola ◽  
Silvina Fernandez Giuliano ◽  
Rosa Bologna ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Current Knowledge ◽  
Phylogenetic Analyses ◽  
Genotypic Diversity ◽  
Resistance Testing ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Middle Income ◽  
Subtype B ◽  
Hiv 1

Abstract Background Current knowledge on HIV-1 resistance to integrase inhibitors (INIs) is based mostly on subtype B strains. This contrasts with the increasing use of INIs in low- and middle-income countries, where non-B subtypes predominate. Materials and methods HIV-1 drug resistance genotyping was performed in 30 HIV-1-infected individuals undergoing virological failure to raltegravir. Drug resistance mutations (DRMs) and HIV-1 subtype were characterized using Stanford HIVdb and phylogenetic analyses. Results Of the 30 integrase (IN) sequences, 14 were characterized as subtype F (47%), 8 as subtype B (27%), 7 as BF recombinants (23%) and 1 as a putative CRF05_DF (3%). In 25 cases (83%), protease and reverse transcriptase (PR-RT) sequences from the same individuals confirmed the presence of different BF recombinants. Stanford HIVdb genotyping was concordant with phylogenetic inference in 70% of IN and 60% of PR-RT sequences. INI DRMs differed between B and F IN subtypes, with Q148K/R/H, G140S and E138K/A being more prevalent in subtype B (63% versus 0%, P = 0.0021; 50% versus 0%, P = 0.0096; and 50% versus 0%, P = 0.0096, respectively). These differences were independent of the time on raltegravir therapy or viral load at the time of genotyping. INI DRMs in subtype F IN genomes predicted a lower level of resistance to raltegravir and no cross-resistance to second-generation INIs. Conclusions Alternative resistance pathways to raltegravir develop in subtypes B and F IN genomes, with implications for clinical practice. Evaluating the role of HIV-1 subtype in development and persistence of mutations that confer resistance to INIs will be important to improve algorithms for resistance testing and optimize the use of INIs.

scholarly journals Monitoring of HIV Drug Resistance Mutations in Newly Diagnosed Patients in Cyprus (2010–2012)

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S424-S424
Author(s):  
Ioannis Demetriades
Keyword(s):  
Drug Resistance ◽  
Phylogenetic Trees ◽  
Resistance Mutation ◽  
Drug Resistance Mutation ◽  
High Rate ◽  
Resistance Testing ◽  
Newly Diagnosed ◽  
Resistance Mutations ◽  
Subtype B ◽  
Hiv 1

Abstract Background A molecular epidemiology study of HIV-1 infection was conducted in 100 HIV-1 diagnosed and untreated patients in Cyprus representing 65.4 percent of all the reported HIV-1 infections in Cyprus between 2010 and 2012. Methods Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and 18 patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Results Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other CRFs (7.0%) and unknown recombinant forms, URFs (12%). Most of newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33–48) reporting having sex with other men, MSM (51%). Conclusion A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM. Twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are subtype A1 and three subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with subtype A2 strain, both with the PI drug resistance mutation M46L and one patient from Greece with subtype A1 strain with the NNRTI drug resistance mutation K103N. Disclosures All authors: No reported disclosures.

scholarly journals Drug Resistance Mutations in HIV-Infected Patients in the Spanish Drug Resistance Database Failing Tipranavir and Darunavir Therapy

2010 ◽  
Vol 54 (7) ◽  
pp. 3018-3020 ◽  
Author(s):  
Eva Poveda ◽  
Lourdes Anta ◽  
José Luis Blanco ◽  
José Luis Casado ◽  
Félix Gutiérrez ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Research Network ◽  
Resistance Testing ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
National Drug ◽  
Aids Research

ABSTRACT The presence of resistance mutations in patients failing tipranavir or darunavir was examined at the national drug resistance database of the Spanish AIDS Research Network. Although mutations emerging during tipranavir and darunavir failures differed considerably, cross-resistance was found in up to half of the patients tested. Interestingly, mutation 54L, which is associated with tipranavir hypersusceptibility, was selected in half of the darunavir failures. Thus, resistance testing seems mandatory to ensure the benefit of the sequential use of these drugs.

scholarly journals Evaluation of the HIV-1 drug resistance to elsulfavirine and the effectiveness of it among Russian treatment-naïve patients

2021 ◽  
Vol 12 (5) ◽  
pp. 29-39
Author(s):  
A. A. Kirichenko ◽  
D. E. Kireev ◽  
A. V. Kravchenko ◽  
A. V. Pokrovskaya ◽  
U. A. Kuimova ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Antiretroviral Therapy ◽  
Virological Failure ◽  
Resistance Testing ◽  
Study Group ◽  
Resistance Mutations ◽  
Drug Resistance Testing ◽  
The Third ◽  
Treatment Naïve ◽  
Hiv 1

The aim of the study: to analyze the prevalence of resistance mutations to elsulfavirine and to evaluate the effectiveness of it among HIV-infected treatment-naïve patients in real clinical practice.Materials and methods. The study included 578 patients with HIV infection, which divided into 3 groups. The first group is 354 HIV-infected treatment-naïve patients for whom HIV-1 nucleotide sequences were obtained as part of routine drug resistance testing. The second study group included 111 HIV-infected treatment-naïve patients, tested for drug resistance before the antiretroviral therapy containing elsulfavirine. The third study group included 113 HIV-infected treatment-naïve patients, each of whom was assigned a treatment regimen containing elsulfavirine without prior drug resistance testing. The observation period for patients of the second and third groups who received treatment was 24 weeks. To assess the effectiveness of antiretroviral therapy in patients, viral load, CD4+ T-cell counts, and adherence to therapy were assessed. HIV-1 subtypes and drug resistance mutations were determined using the Stanford HIV Resistance Database (v. 8.9-1). To clarify the results of subtyping, phylogenetic analysis of nucleotide sequences was carried out using the MEGA program (v. 6.0).Results. The prevalence of mutations associated with decreased susceptibility to elsulfavirine among HIV-infected treatment-naïve patients was 1.7% and 4.5% for the first and second groups of patients, respectively. All of the patients have only single resistance mutations which, according to the results of preclinical studies, cannot cause drug resistance. The use of elsulfavirine in real clinical practice among treatment-naïve patients has demonstrated good virological and immunological efficacy of the drug. As a result of 24 weeks of therapy in patients of the second group, no treatment ineffectiveness, and the development of drug resistance were observed. Among the patients of the third group, 6 patients (5.3%) have the virological failure of therapy associated with the resistance to the used drugs. All patients with virological failure had a resistance mutation profile associated with a high level of drug resistance to one of the drugs in the treatment regimen, lamivudine. Additionally, 1 patient had a combination of mutations that reduce susceptibility to elsulfavirine, and 4 patients had mutations that can reduce susceptibility to elsulfavirine in combination with other mutations.Conclusion. The low prevalence of mutations associated with a decrease in susceptibility to elsulfavirine and the absence of combinations of mutations make it possible to predict the successful use of this drug for Russian treatment-naïve patients. Reported cases of virological failure of antiretroviral therapy are difficult to interpret in the context of elsulfavirine due to the lack of an exact list of mutations and their combinations, and associations with the degree of resistance to it. This study describes for the first time the mutation profiles in patients with virological failure of therapy containing elsulfavirine and demonstrates the necessity of the further study of drug resistance profile to drug in vitro and in vivo.

scholarly journals Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

2018 ◽  
Author(s):  
Ronit Dalmat ◽  
Negar Makhsous ◽  
Gregory Pepper ◽  
Amalia Magaret ◽  
Keith R. Jerome ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Virological Failure ◽  
Sanger Sequencing ◽  
Low Frequency ◽  
Resistance Testing ◽  
Antiviral Resistance ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Next Generation Sequencing Ngs

AbstractHIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.ImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

scholarly journals Detectable viral load among HIV -1 positive pregnant women on ART (Option B +) in northern Tanzania: Baseline results from the HIVDR study

2021 ◽  
Vol 3 (1) ◽  
pp. 44-50
Author(s):  
Nicholaus Steven Mazuguni ◽  
Festo Mazuguni ◽  
Eva Prosper Muro
Keyword(s):  
Drug Resistance ◽  
Viral Load ◽  
Virological Failure ◽  
Virologic Failure ◽  
Resistance Mutation ◽  
Resistance Testing ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Hiv 1 ◽  
Level Resistance

Introduction: In Tanzania, the Ministry of Health, Community Development, Gender, Elderly and Children (MoHCDEC) has implemented the Option B+ as one of the strategies to facilitate achievement of elimination of mother to child transmission of HIV. To prevent emergence of drug resistance mutations early identification of option B+ failure is critical. The emergence of drug resistance mutation and subsequent treatment failure poses a major concern for HIV program in low- and middle-income resource settings where treatment options are limited. Methodology: We recruited treatment naïve, treatment experienced HIV-1 positive pregnant women and those who had prophylaxis in their previous pregnancy in Kilimanjaro, northern Tanzania August 2016 to February 2017. Whole blood (2ml) for biochemistry, viral load and drug resistance testing were taken at baseline. ARV drug resistance testing was done on women with VL ≥ 1000 copies/ml. We used descriptive statistic and logistic regression to determine the strength of association between virologic outcome (virologic failure) and independent predictors. Results: One hundred and forty eight (148) pregnant HIV-positive women were enrolled in the study with mean age of 29.82 years (SD=6.17) from August, 2016 to February, 2017. Virologic failure was demonstrated in 34 (23%) with viral load   ≥ 1,000 copies/ml. Genotyping results were available from 26 women, mutations associated with ARV resistance were detected in 23.1% (n = 6/26). Among the six women with ARV resistance mutation 4(66.7%) had high level resistance and 2(33.3%) had low level resistance. Among the 26 samples genotyped 15(58%) viruses were subtype A, while eight were subtype C (31%) and three subtypes D (11%). The most dominant drug resistance mutations against the reverse transcriptase inhibitors for the women with high level resistance were K103N, Y188L, D67N, K70R, M184V, T215F, K219EQ, and the low-level resistance was E138A. The older age was associated with virological failure compared to those who were < 20 year of age. Conclusion: Viral load testing should be done on women who were already on antiretroviral treatment on their first antenatal visit to ensure early detection of virological failure and enable clinicians to take an appropriate course of action on their management. Educational intervention on adherence should be targeted at an early stage to women with virological failure during pregnancy to reduce the emergence of HIV-1 drug resistance mutations.

scholarly journals Are We Ready for NGS HIV Drug Resistance Testing? The Second “Winnipeg Consensus” Symposium

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 586 ◽  
Author(s):  
Hezhao Ji ◽  
Paul Sandstrom ◽  
Roger Paredes ◽  
P. Richard Harrigan ◽  
Chanson J. Brumme ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Care ◽  
Resistance Testing ◽  
Quantitative Detection ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Global Challenge ◽  
Symposium Series ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

HIV drug resistance is a major global challenge to successful and sustainable antiretroviral therapy. Next-generation sequencing (NGS)-based HIV drug resistance (HIVDR) assays enable more sensitive and quantitative detection of drug-resistance-associated mutations (DRMs) and outperform Sanger sequencing approaches in detecting lower abundance resistance mutations. While NGS is likely to become the new standard for routine HIVDR testing, many technical and knowledge gaps remain to be resolved before its generalized adoption in regular clinical care, public health, and research. Recognizing this, we conceived and launched an international symposium series on NGS HIVDR, to bring together leading experts in the field to address these issues through in-depth discussions and brainstorming. Following the first symposium in 2018 (Winnipeg, MB Canada, 21–22 February, 2018), a second “Winnipeg Consensus” symposium was held in September 2019 in Winnipeg, Canada, and was focused on external quality assurance strategies for NGS HIVDR assays. In this paper, we summarize this second symposium’s goals and highlights.

scholarly journals Zero prevalence of primary drug resistance-associated mutations to protease inhibitors in HIV-1 drug-naive patients in and around Aligarh, India

2014 ◽  
Vol 8 (01) ◽  
pp. 079-085 ◽  
Author(s):  
Mohd Azam ◽  
Abida Malik ◽  
Meher Rizvi ◽  
Arvind Rai
Keyword(s):  
Drug Resistance ◽  
Protease Inhibitors ◽  
Resistance Testing ◽  
Protease Gene ◽  
Resistance Mutations ◽  
Subtype C ◽  
Primary Resistance ◽  
Primary Drug Resistance ◽  
Treatment Naïve ◽  
Hiv 1

Introduction: This study aimed to evaluate the prevalence of resistance mutations in the protease gene of HIV-1 strains isolated from north Indian antiretroviral (ARV) treatment-naive patients and to assess the phylogenetic relatedness of these strains with known HIV-1 strains. Methodology: Fifty-four HIV-1 strains isolated from treatment-naive patients (n = 54) were included in this study. Resistance genotyping for the protease gene was performed using semi-nested PCR and DNA sequencing. The sequences were aligned (ClustalW) and a phylogenetic tree was built (MEGA 4 software). Drug resistance (DR) pattern was analyzed using the Stanford HIV-DR database and the IAS-USA mutation list. For subtyping purposes, all the nucleotide sequences were submitted to the REGA HIV-1 subtyping tool version 2.0l. Results: All the strains (100%) were found to belong to the C subtype and to harbor at least two secondary mutations in the protease gene. The most frequent mutations were H69K and I93L (52 of 52 strains), followed by I15V (80.7%), L19I (69.2%), M36I (67.3%), R41K (94.2%), L63P (61.5%), and L89M (82.7%). Conclusion: This study confirms that HIV-1 subtype C predominates in northern India. Protease secondary mutations associated with drug resistance to protease inhibitors (PIs) were present with high frequency in the HIV-1 C subtype strains isolated from north Indian ARV treatment-naive patients, but no primary resistance mutations were found in this region. We suggest that resistance testing in HIV-1 infected patients should ideally be performed before the initiation of therapy to tailor the treatment for the individual to achieve the optimal therapeutic outcome

scholarly journals Increase in HIV-1 transmitted drug resistance among untreated 16~25-y-old youths in the China-Myanmar border areas during 2009~2017

2020 ◽  
Author(s):  
Yibo DING ◽  
Min CHEN ◽  
Jibao WANG ◽  
Yuecheng YANG ◽  
Yi FENG ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Phylogenetic Trees ◽  
Cd4 Count ◽  
Molecular Networks ◽  
Resistance Testing ◽  
Transmitted Drug Resistance ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
The Mean ◽  
Hiv 1

Abstract Background Transmitted drug resistance (TDR) can affect antiretroviral therapy (ART) efficacy. Surveillance drug resistance mutations in untreated youths newly reported with HIV-1 are highly representative of local TDR. We investigated HIV-1 TDR, TDR transmission based on molecular networks, and the effect of TDR mutations (TDRMs) on the CD4 count among youths in the China-Myanmar border area near the "Golden Triangle" to better understand TDR and guide ART.Methods From 2009 to 2017, 573 ART-naïve youths (16~25 y) newly reported with HIV-1 were enrolled. CD4 counts were obtained from whole blood. HIV pol gene sequences were amplified from RNA extracted from plasma. The Stanford REGA program and phylogenetic trees were used to determine genotypes. TDRMs were analyzed using the Stanford Calibrated Population Resistance tool. TDR transmission was evaluated from molecular networks of HIV-1 pol genes.Results The average prevalence of TDR was 6.3%, and the resistance to NNRTIs, NRTIs, and PIs was 3.49%, 2.62%, and 0.52%, respectively. TDR prevalence increased significantly during the period 2009~2017 (3.92%~9.48%, p<0.05). The mean CD4 count was significantly lower among individuals with TDRMs (373/mm3 vs. 496/mm3, p=0.013). The rate of network entry of youths harboring TDRMs (63.89%) was significantly higher than that of youths without TDRMs (44.9%).Conclusions The HIV-1 TDR increase and low CD4 count of patients with TDRMs in Dehong at the China-Myanmar border suggest the need for early ART and completion of resistance testing before initiating ART in HIV hotspots. Youths with TDRMs are likely to have links to others, necessitating intervention in onward transmission.

scholarly journals HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

2015 ◽  
Vol 59 (11) ◽  
pp. 6824-6833 ◽  
Author(s):  
H. R. Lapointe ◽  
W. Dong ◽  
G. Q. Lee ◽  
D. R. Bangsberg ◽  
J. N. Martin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Sanger Sequencing ◽  
Illumina Miseq ◽  
Quality Data ◽  
Resistance Testing ◽  
Study Cohort ◽  
Miseq Sequencing ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing

ABSTRACTLimited access to HIV drug resistance testing in low- and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n= 759) and from a Ugandan study cohort (n= 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log10HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex “wide” sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.

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