ABSTRACT
Background: Lack of viral load monitoring of ART is known to be associated with slower switch from a failing regimen and thereby higher prevalence of MDR HIV-1. Many countries have continued to use thymidine analogue drugs despite recommendations to use tenofovir in combination with a cytosine analogue and NNRTI as first-line ART. The effect of accumulated thymidine analogue mutations (TAMs) on phenotypic resistance over time has been poorly characterized in the African setting. Patients and methods: A retrospective analysis of individuals with ongoing viral failure between weeks 48 and 96 in the NORA (Nevirapine OR Abacavir) study was conducted. We analysed 36 genotype pairs from weeks 48 and 96 of first-line ART (14 treated with zidovudine/lamivudine/nevirapine and 22 treated with zidovudine/lamivudine/abacavir). Phenotypic drug resistance was assessed using the Antivirogram assay (v. 2.5.01, Janssen Diagnostics). Results: At 96 weeks, extensive TAMs (≥3 mutations) were present in 50% and 73% of nevirapine- and abacavir-treated patients, respectively. The mean (SE) number of TAMs accumulating between week 48 and week 96 was 1.50 (0.37) in nevirapine-treated participants and 1.82 (0.26) in abacavir-treated participants. Overall, zidovudine susceptibility of viruses was reduced between week 48 [geometric mean fold change (FC) 1.3] and week 96 (3.4, P = 0.01). There was a small reduction in tenofovir susceptibility (FC 0.7 and 1.0, respectively, P = 0.18). Conclusions: Ongoing viral failure with zidovudine-containing first-line ART is associated with rapidly increasing drug resistance that could be mitigated with effective viral load monitoring.
Subject(s)
Anti-HIV Agents/therapeutic use , Drug Resistance, Viral/genetics , HIV-1/genetics , Mutation , Reverse Transcriptase Inhibitors/therapeutic use , Thymidine/analogs & derivatives , Zidovudine/therapeutic use , Adult , Africa South of the Sahara/epidemiology , Antiretroviral Therapy, Highly Active/adverse effects , CD4 Lymphocyte Count , Dideoxynucleosides/therapeutic use , Female , HIV Infections/drug therapy , HIV-1/drug effects , Humans , Lamivudine/therapeutic use , Male , Nevirapine/therapeutic use , Polymerase Chain Reaction , RNA, Viral/blood , Retrospective Studies , Reverse Transcriptase Inhibitors/administration & dosage , Thymidine/genetics , Treatment Failure , Viral Load/drug effects , Viral Load/methods , Zidovudine/administration & dosageABSTRACT
BACKGROUND: The 2013-2016 Ebola epidemic in West Africa resulted in accelerated development of rapid diagnostic tests for emergency outbreak preparedness. We describe the development and evaluation of the Idylla™ prototype Ebola virus test, a fully automated sample-to-result molecular diagnostic test for rapid detection of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV). METHODS: The Idylla™ prototype Ebola virus test can simultaneously detect EBOV and SUDV in 200 µL of whole blood. The sample is directly added to a disposable cartridge containing all reagents for sample preparation, RNA extraction, and amplification by reverse-transcription polymerase chain reaction analysis. The performance was evaluated with a variety of sample types, including synthetic constructs and whole blood samples from healthy volunteers spiked with viral RNA, inactivated virus, and infectious virus. RESULTS: The 95% limits of detection for EBOV and SUDV were 465 plaque-forming units (PFU)/mL (1010 copies/mL) and 324 PFU/mL (8204 copies/mL), respectively. In silico and in vitro analyses demonstrated 100% correct reactivity for EBOV and SUDV and no cross-reactivity with relevant pathogens. The diagnostic sensitivity was 97.4% (for EBOV) and 91.7% (for SUDV), the specificity was 100%, and the diagnostic accuracy was 95.9%. CONCLUSIONS: The Idylla™ prototype Ebola virus test is a fast, safe, easy-to-use, and near-patient test that meets the performance criteria to detect EBOV in patients with suspected Ebola.
Subject(s)
Disease Outbreaks , Ebolavirus/isolation & purification , Hemorrhagic Fever, Ebola/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Africa, Western/epidemiology , Ebolavirus/genetics , Hemorrhagic Fever, Ebola/epidemiology , Hemorrhagic Fever, Ebola/virology , Humans , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Sensitivity and SpecificityABSTRACT
INTRODUCTION: Large-scale vaccination campaigns can benefit from using digital health tools, particularly in low- and middle-income countries (LMICs). Selecting the best tool to fit into a pre-existing digital landscape can be challenging. AREAS COVERED: We conducted a narrative review in PubMed and the grey literature for data available within 5 years to provide an overview of digital health tools used in large-scale vaccination campaigns for outbreak response in LMICs. We discuss tools used along the typical steps of a vaccination process. Digital tool functionalities and technical specifications, open-source options, data privacy and security concerns, and lessons learned from the use of these tools are discussed. EXPERT OPINION: The landscape of digital health tools for large-scale vaccination processes in LMICs is growing. For efficient implementation, countries should prioritize the appropriate tool(s) depending on their needs and available resources, develop a robust framework around data privacy and security, and select sustainable features. Improving internet connectivity and digital literacy in LMICs will facilitate adoption. This review may aid LMICs still needing to prepare large-scale vaccination campaigns in the selection of supporting digital health tools. Further research on impact and cost-effectiveness is needed.
Subject(s)
Developing Countries , Pandemics , Humans , Pandemics/prevention & control , Vaccination , Immunization ProgramsABSTRACT
The failure of raltegravir (RAL) is generally associated with the selection of mutations at integrase position Y143, Q148, or N155. However, a relatively high proportion of failures occurs in the absence of these changes. Here, we report the phenotypic susceptibilities to RAL and elvitegravir (EVG) for a large group of HIV-infected patients failing on RAL-containing regimens. Plasma from HIV-infected individuals failing on RAL-containing regimens underwent genotypic and phenotypic resistance testing (Antivirogram v2.5.01; Virco). A control group of patients failing on other regimens was similarly tested. Sixty-one samples were analyzed, 40 of which belonged to patients failing on RAL-containing regimens. Full RAL susceptibility was found in 20/21 controls, while susceptibility to EVG was diminished in 8 subjects, with a median fold change (FC) of 2.5 (interquartile range [IQR], 2.1 to 3.1). Fourteen samples from patients with RAL failures showed diminished RAL susceptibility, with a median FC of 38.5 (IQR, 10.8 to 103.2). Primary integrase resistance mutations were found in 11 of these samples, displaying a median FC of 68.5 (IQR, 23.5 to 134.3). The remaining 3 samples showed a median FC of 2.5 (IQR, 2 to 2.7). EVG susceptibility was diminished in 19/40 samples from patients with RAL failures (median FC, 7.71 [IQR, 2.48 to 99.93]). Cross-resistance between RAL and EVG was high (R(2) = 0.8; P < 0.001), with drug susceptibility being more frequently reduced for EVG than for RAL (44.3% versus 24.6%; P = 0.035). Susceptibility to RAL and EVG is rarely affected in the absence of primary integrase resistance mutations. There is broad cross-resistance between RAL and EVG, which should preclude their sequential use. Resistance to EVG seems to be more frequent and might be more influenced by integrase variability.
Subject(s)
HIV Infections/drug therapy , HIV Infections/genetics , Pyrrolidinones/therapeutic use , Drug Resistance, Viral/genetics , Genotype , HIV Integrase Inhibitors/therapeutic use , Humans , Molecular Sequence Data , Quinolones/therapeutic use , Raltegravir PotassiumABSTRACT
OBJECTIVES: To investigate the best conditions of raltegravir use to avoid the selection of resistance mutations in the three main genetic pathways: 148, 155 and 143. METHODS: A total of 161 patients failing on raltegravir with two consecutive HIV-1 viral loads >20 copies/mL were studied. Ten parameters [HIV-1 RNA and CD4 at baseline and failure, genotypic sensitivity score (GSS) of treatment associated with raltegravir, protease inhibitors used, time spent on raltegravir, subtype, sex and age] were tested in univariate and multivariate logistic regression analyses and compared with the emergence of resistance mutations to raltegravir at failure. Phenotypic susceptibility to raltegravir was studied in 16 patients without the main resistance mutations to raltegravir at failure. RESULTS: At raltegravir failure, 46/161 patients (28.6%) had integrase resistance mutations, whereas 115/161 (71.4%) had no resistance mutations. High HIV-1 viral load level at failure (ORâ=â2.81, 95% CI 1.8-4.6, Pâ<â0.001) and low GSS of treatment associated with raltegravir (ORâ=â11.6, 95% CI 4.5-36.4, Pâ<â0.001) were independently associated with the selection of raltegravir mutations. The percentages of patients with integrase resistance mutations were 7.7% (6/78) versus 48.1% (40/83) when viral load is ≤200 or >200 copies/mL and 47.5% (39/82) versus 8.9% (7/79) when GSS is <2 or ≥2. Among patients without main resistance mutations, two patients showed raltegravir phenotypic resistance, one naturally with F121Y at baseline and the other acquiring G118R at failure. CONCLUSIONS: Our results show that to avoid the selection of raltegravir resistance mutations, patients have to be treated with at least two active drugs in combination with raltegravir and to maintain a viral load ≤200 copies/mL.
Subject(s)
Anti-HIV Agents/administration & dosage , Antiretroviral Therapy, Highly Active/methods , Drug Resistance, Viral , HIV-1/drug effects , Pyrrolidinones/administration & dosage , Adult , Aged , Anti-HIV Agents/pharmacology , CD4 Lymphocyte Count , Female , HIV-1/isolation & purification , Humans , Male , Microbial Sensitivity Tests , Middle Aged , Pyrrolidinones/pharmacology , Raltegravir Potassium , Risk Factors , Viral Load , Young AdultABSTRACT
OBJECTIVES: HIV-1 genotyping is widely accepted as a diagnostic tool to optimize therapy changes in patients whose antiretroviral regimen is failing. Phenotyping can substantially complement the information obtained from genotyping, especially in the presence of complex mutational patterns. However, drug susceptibility tests are laborious and require biosafety facilities. We describe the molecular mechanism of a non-infectious HIV-1 protease phenotypic assay in eukaryotic cells and validate its applicability as a tool for monitoring drug resistance. METHODS: A cloning vector containing the fusion protein green fluorescent protein-HIV-1 protease (GFP-PR) was modified to facilitate the insertion of HIV-1 protease from infected subjects. Real-time quantitative PCR and western blot analysis were used to establish the molecular mechanism of the new phenotypic assay. The method was validated by analysing HIV-1 protease from 46 clinical isolates. Statistical comparisons were made between values obtained using our assay and those reported from alternative standardized phenotypic assays. RESULTS: The capacity of HIV-1 protease to cleave cellular translation factors, such as the eukaryotic translation initiation factor 4 (eIF4GI) and the poly(A)-binding protein (PABP), led to cyclical accumulation of GFP that varied with the dose of protease inhibitors. Validation and comparison revealed a significant correlation with the Virco TYPE HIV-1 test (P < 0.0001, Spearman's ρ = 0.60), the Antivirogram test (P = 0.0001, Spearman's ρ = 0.60) and the Stanford HIVdb (P < 0.0001, Spearman's ρ = 0.69). CONCLUSIONS: This cell-based non-infectious phenotypic method with a well-understood molecular mechanism was highly reliable and comparable to other widely used assays. The method can be used for both phenotyping of HIV-1 viral isolates resistant to protease inhibitors and screening of new protease inhibitors.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Blotting, Western , Cells, Cultured , Cloning, Molecular , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HIV Infections/virology , HIV Protease/genetics , HIV Protease/metabolism , HIV-1/isolation & purification , Humans , Microbial Sensitivity Tests/methods , Real-Time Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
OBJECTIVES: A wide array of monitoring tests is commercially available to gauge HIV-1 disease progression and the overall health status of an HIV-1-infected patient. Viral load tests provide a picture of viral activity, while CD4 cell counts shed light on the immune status and can help physicians to prevent the development of opportunistic infections in patients. On the other hand, genotypic and phenotypic resistance testing and therapeutic drug monitoring help to optimize HIV-1 antiretroviral therapy. Resistance testing is currently recommended within the standard of care guidelines to aid the choice of new drug regimens following treatment failure(s). METHODS: Genotypic testing described here is based on the amplification and sequencing of an HIV-1 protease (PR) and reverse transcriptase (RT) region from a patient sample to identify resistance mutations associated with PR and RT inhibitor resistance. A genotypic test takes a week to perform and the results are reported as a list of detected mutations. The virco®TYPE HIV-1 report uses genotypic data to predict phenotypic susceptibility by linear regression modeling that uses a large correlative database of genotype-phenotype pairs. Phenotypic testing measures the ability of the virus to replicate in the presence of a drug and provides a direct measurement of drug susceptibility in vitro. Since phenotypic analysis is laborious and time consuming (28 days), genotypic resistance testing is currently the standard reference method used for HIV-1 resistance testing. However, a phenotypic test is important when a patient harbors virus with complex genetic patterns, or when the mutational resistance profile for a particular drug is not well-characterized. RESULTS AND CONCLUSIONS: Some of the currently used resistance tests are partially automated enabling laboratories to increase overall efficiency. However, maximum automation and standardization of the process, instruments and software that we have described here can overcome many of the problems encountered with current tests and aims at having a compliant, high-throughput, diagnostic laboratory, which can guarantee sample integrity from sample reception to result reporting. We also describe in detail the development and performance of virco®TYPE HIV-1 (genotype) and Antivirogram® (phenotype) assay on PR and RT genes to evaluate antiretroviral resistance.
Subject(s)
Anti-HIV Agents/pharmacology , Drug Monitoring/methods , Drug Resistance, Viral , HIV Infections/virology , HIV-1/drug effects , HIV-1/genetics , Molecular Typing/methods , Genotype , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV-1/enzymology , HIV-1/isolation & purification , Humans , Microbial Sensitivity Tests/methods , Mutation, Missense , PhenotypeABSTRACT
Background: The National TB Elimination Programme (NTEP) has quite successfully involved private sector for referral of presumptive drug resistant TB (DR-TB) patients for molecular testing and referral for DR-TB management. There was a challenge as all the referred patients were not reaching to the facilities. A "DOST" intervention model was implemented to strengthen the patient care pathway. We conducted this study to describe the patient care cascade, the clinico-demographic characteristics of patients linked to the treatment and to estimate the mean turn-around time for drug resistant TB care services. Methods: It is a cross-sectional study conducted at New Delhi during the period July 2019-December 2020 under programmatic settings. Results: A total of 9,331 patients were subjected to CB-NAAT test and 382 (4%) were found to be resistant for rifampicin and 231 (76%) were initiated on treatment in the public sector under NTEP. Conclusion: The DOST intervention model developed to link the DR-TB patients from private sector to the public sector DR-TB centers is found to be efficient and effective.
Subject(s)
Private Sector , Tuberculosis, Multidrug-Resistant , Cross-Sectional Studies , Humans , India , Public Sector , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country's testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.
ABSTRACT
Concordance between the conventional HIV-1 phenotypic drug resistance assay, PhenoSense (PS), and vircoTYPE HIV-1 (vT), a drug resistance assay based on prediction of the phenotype, was investigated in a data set from the Stanford HIV Resistance database (hivdb). Depending on the drug, between 287 and 902 genotype-phenotype data pairs were available for comparisons. Test results (fold-change values) in the two assays were highly correlated, with an overall mean correlation coefficient of 0.90 using single PS measurements. This coefficient rose to 0.94 when the vT results were compared to the mean of repeat PS measurements. These results are comparable with the corresponding correlation coefficients of 0.87 and 0.95, calculated using single measurements, and the mean of repeat measurements, respectively, as obtained in the Antivirogram assay, the conventional HIV-1 phenotypic drug resistance test on which vT is based. The proportion of resistance calls resulting in a "major" discordance (fully susceptible or maximal response by one assay but fully resistant or minimal response by the other) ranged from 0% to 8.1% for drugs for which two clinical test cut-offs were available in both assays (didanosine, abacavir, tenofovir, saquinavir/r, fosamprenavir/r, and lopinavir/r), from 2.4% to 8.1% for the drugs for which two clinical test cut-offs were available in the vT assay and one clinical test cut-off in the PS assay (lamivudine, stavudine, indinavir/r, and atazanavir/r) and from 3.1% to 10.3% for drugs for which biological test cut-offs were used (zidovudine, nevirapine, delavirdine, efavirenz, indinavir, ritonavir, nelfinavir, saquinavir, and fosamprenavir). Our analyses suggest that these assays provide comparable resistance information, which will be of value to physicians who may be presented with either or both types of test report in their practice.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , HIV-1/genetics , Microbial Sensitivity Tests/methods , Mutation, Missense , Genotype , Humans , Phenotype , Statistics as TopicABSTRACT
The objective of this study was to compare the performance of the Idylla™ Respiratory (IFV-RSV) panel to the GeneXpert Xpert® Flu/RSV assay and establish the performance of a midturbinate swab compared to nasopharyngeal sampling. Considering GeneXpert® assay as imperfect reference standard, a positive percentage agreement between both assays of 98-100% for influenza A and 96-99% for influenza B could be calculated when 354 nasopharyngeal and 325 midturbinate swabs were retrospectively analyzed. Comparing midturbinate samples to nasopharyngeal specimens of 321 subjects, positive percentage agreement varied from 42% to 94% depending on both target virus and assay used. Negative percentage agreements ranged from 98% to 100% for both methods and sample type comparison. The Idylla™ assay showed excellent performance compared to the GeneXpert® assay for the detection of influenza virus. The study also showed a slightly better performance for nasopharyngeal sampling compared to the use of a midturbinate swab.
Subject(s)
Influenza, Human/diagnosis , Molecular Diagnostic Techniques/methods , Nasal Cavity/virology , Nasopharynx/virology , Orthomyxoviridae/isolation & purification , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus, Human/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Retrospective Studies , Specimen Handling/methods , Young AdultABSTRACT
Mutation proI47A has recently been associated with lopinavir/ritonavir (LPV/r) resistance. Only four out of 1859 specimens (0.2%) sent for drug resistance testing (219 drug-naive and 1650 antiretroviral-experienced) showed I47A. All belonged to patients failing LPV/r. The prevalence among protease inhibitor-experienced patients was 0.6%. Phenotypic testing showed that proI47A caused high-level lopinavir resistance (> 100-fold) and cross-resistance to amprenavir, whereas it caused hypersusceptibility to saquinavir. ProI47A should thus be considered the primary lopinavir resistance mutation.
Subject(s)
HIV Infections/genetics , HIV Protease Inhibitors/therapeutic use , HIV-1/genetics , Pyrimidinones/therapeutic use , Carbamates/therapeutic use , Codon/genetics , Drug Resistance, Viral/genetics , Furans , Genotype , HIV Infections/drug therapy , HIV Protease Inhibitors/blood , HIV-1/drug effects , Humans , Indinavir/therapeutic use , Lopinavir , Mutation , Nelfinavir/therapeutic use , Phenotype , Pyrimidinones/blood , Ritonavir/therapeutic use , Saquinavir/therapeutic use , Sulfonamides/therapeutic useABSTRACT
BACKGROUND: The K103N mutation in HIV-1 reverse transcriptase (RT) confers high-level resistance to current non-nucleoside reverse transcriptase inhibitors (NNRTI). The prevalence and resistance profile of HIV-1 with other substitutions at RT codon 103 is less well documented. METHODS: K103 substitutions among over 70,000 clinical samples submitted for routine antiretroviral resistance testing at two independent centres were examined. Phenotypic resistance profiles of isolates harboring rare K103 variants in the absence of known NNRTI-associated resistance mutations were retrieved from Virco's correlative genotype/phenotype database. Genotyped samples with known treatment histories were retrieved from the British Columbia Centre for Excellence in HIV/AIDS database. Site-directed mutants containing K103 variants were constructed and phenotyped. RESULTS: K103N, R and S were observed in 29, 1.8, and 0.9% of Virco isolates and in 16, 1.5 and 0.4% of British Columbia isolates. K103T/Q/H substitutions were observed only rarely (<0.2%). The prevalence of unusual codon 103 substitutions remained stable over 5 years, except K103S, which increased over fourfold in both datasets. K103R/Q-containing clinical isolates remained phenotypically susceptible to NNRTI, whereas K103S/T/H-containing isolates showed over 10-fold decreased NNRTI susceptibility. Among patients with a known treatment history, K103S/T/H were observed primarily in individuals failing NNRTI-containing regimens. Site-directed mutants confirmed decreased susceptibility to NNRTI in K103S/T/H-containing recombinants. CONCLUSION: Variants at HIV RT codon 103 other than K103N are observed relatively rarely in clinical isolates, but K103 S, T and H confer decreased susceptibility to NNRTI. These data are relevant for interpretive genotype algorithms and in the design of assays specific to RT codon 103 mutations.
Subject(s)
Amino Acid Substitution/genetics , Drug Resistance, Viral/genetics , HIV Infections/genetics , HIV Reverse Transcriptase/genetics , Mutation/genetics , Codon/genetics , HIV Infections/drug therapy , Humans , Phenotype , Polymorphism, Genetic , Reverse Transcriptase Inhibitors/therapeutic useABSTRACT
The rhizobacterium Pseudomonas aeruginosa 7NSK2 produces secondary metabolites such as pyochelin (Pch), its precursor salicylic acid (SA), and the phenazine compound pyocyanin. Both 7NSK2 and mutant KMPCH (Pch-negative, SA-positive) induced resistance to Botrytis cinerea in wild-type but not in transgenic NahG tomato. SA-negative mutants of both strains lost the capacity to induce resistance. On tomato roots, KMPCH produced SA and induced phenylalanine ammonia lyase activity, while this was not the case for 7NSK2. In 7NSK2, SA is probably very efficiently converted to Pch. However, Pch alone appeared not to be sufficient to induce resistance. In mammalian cells, Fe-Pch and pyocyanin can act synergistically to generate highly reactive hydroxyl radicals that cause cell damage. Reactive oxygen species are known to play an important role in plant defense. To study the role of pyocyanin in induced resistance, a pyocyanin-negative mutant of 7NSK2, PHZ1, was generated. PHZ1 is mutated in the phzM gene encoding an O-methyltransferase. PHZ1 was unable to induce resistance to B. cinerea, whereas complementation for pyocyanin production or co-inoculation with mutant 7NSK2-562 (Pch-negative, SA-negative, pyocyanin-positive) restored induced resistance. These results suggest that pyocyanin and Pch, rather than SA, are the determinants for induced resistance in wild-type P. aeruginosa 7NSK2.
Subject(s)
Botrytis/growth & development , Plant Diseases/microbiology , Pseudomonas aeruginosa/metabolism , Solanum lycopersicum/microbiology , Thiazoles , Cysteine/pharmacology , Genetic Complementation Test , Immunity, Innate/physiology , Mutation , Phenols/metabolism , Plant Roots/metabolism , Plant Roots/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Pyocyanine/metabolism , Salicylic Acid/metabolism , Siderophores/metabolismABSTRACT
Genotypic testing based on subtype-specific amplification and population Sanger sequencing for two nonstructural (NS) protein-coding regions, the NS3/4A protease and the NS5B polymerase, of the hepatitis C virus (HCV) genome is described here. The protocols include the molecular steps for RNA extraction, one-step RT-PCR followed by inner PCR and population Sanger sequencing, to obtain the sequence information of the target regions from the clinical isolates of HCV subtypes 1a and 1b, which can be used to detect any sequence change in the viral genome as for example caused by the development of drug resistance in these two common viral targets.
Subject(s)
Drug Resistance, Viral/genetics , Genotype , Hepacivirus/drug effects , Hepacivirus/genetics , Microbial Sensitivity Tests/methods , Viral Nonstructural Proteins/genetics , Cell Line , Humans , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Major advances in antiretroviral (ARV) therapy during the last decade have made HIV-1 infections a chronic, manageable disease. In spite of these significant advancements, ARV drug resistance remains a hurdle for HIV-infected patients who are committed to lifelong treatments. Several commercially marketed and/or laboratory-developed tests (LDT) are available to detect resistance-associated mutations (RAMs) in HIV-1, by genotyping. These genotyping tests mainly comprise polymerase chain reaction (PCR)-amplification and population, nucleotide sequencing (Sanger methodology) of a large part of the protease (PR), reverse transcriptase (RT), and integrase (IN) genes. In this chapter, we describe HIV-1 PR, RT, and IN genotyping on clinical samples (plasma), using the LDT methodology performed at Janssen Diagnostics BVBA, Belgium (JDx), where the PR-RT genotyping is used as input, to generate a CE-marked vircoTYPE™ HIV-1 report while the IN genotyping is performed as a research-use-only (RUO) assay. The complete HIV-1 PR gene (297 bp; 99 amino acids) and a large part of the RT gene (the first 1,200 bp; 400 amino acids) are amplified and sequenced as a single 1,497 bp fragment. Genotyping of the IN gene is performed by amplification and sequencing of the RT-IN region (the last 459 bp; 153 amino acids of RT with the complete 867 bp; 289 amino acids of IN). This methodology allows identification of nucleoside/-nucleotide reverse transcriptase, non-nucleoside reverse transcriptase, protease, and integrase inhibitor (NRTI, NtRTI, NNRTI, PI, INI) RAMs in the PR-RT and IN genes, which allows to predict viral response against current ARV regimens.
Subject(s)
Drug Resistance, Viral/genetics , Genotyping Techniques , HIV Integrase/genetics , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , HIV-1/genetics , Computational Biology/methods , Genotype , Genotyping Techniques/methods , HIV Integrase/metabolism , HIV Protease/metabolism , HIV Reverse Transcriptase/metabolism , Humans , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
In recent years, increasing numbers of patients infected with HIV-1 non-B subtypes have been treated with modern antiretroviral regimens. Therefore, a better knowledge of HIV drug resistance in non-B strains is crucial. Thus, we compared the mutational pathways involved in drug resistance among the most common non-B subtypes in Italy (F, C, and CRF02_AG) and the B subtype. In total, 2234 pol sequences from 1231 virologically failing patients from Central Italy were analyzed. The prevalence of resistance mutations in protease and reverse transcriptase between non-B and B subtypes has been evaluated. Among patients treated with nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and with thymidine analogues (TA) experience, TAMs1 M41L and L210W were less prevalent in CRF02_AG, while TAMs2 T215F and K219E were more prevalent in the F subtype. In NRTI-treated patients having experience with abacavir, didanosine, tenofovir, or stavudine the K65R mutation was mostly prevalent in the C subtype. In non-NRTI (NNRTI)-treated patients infected by the C subtype the prevalence of K103N was lower than in patients infected with other subtypes, while the prevalence of Y181C and Y188L was higher compared to subtype B. The prevalence of Y181C was higher also in subtype F as compared to subtype B. In patients treated with protease inhibitors, L89V was predominantly found in CRF02_AG, while the TPV resistance mutation T74P was predominantly found in the C subtype. Some differences in the genotypic drug resistance have been found among patients infected with B, C, F, and CRF02_AG subtypes in relationship to treatment. These results may be useful for the therapeutic management of individuals infected with HIV-1 non-B strains.
Subject(s)
Anti-HIV Agents/pharmacology , Drug Resistance, Viral , HIV Protease Inhibitors/pharmacology , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV Seropositivity/genetics , HIV-1/genetics , Reverse Transcriptase Inhibitors/pharmacology , Adult , CD4 Lymphocyte Count , Female , Genotype , HIV Protease/drug effects , HIV Reverse Transcriptase/drug effects , HIV Seropositivity/drug therapy , HIV Seropositivity/epidemiology , HIV-1/drug effects , Humans , Italy/epidemiology , Male , Middle Aged , Mutation, Missense/genetics , Phylogeny , Prevalence , Treatment Failure , Viral LoadABSTRACT
HIV-1 Protease (PR) and Reverse Transcriptase (RT) genotyping is well established for the management of antiretroviral (ARV) drug therapy, as it is able to detect gene mutations encoding resistance to ARV compounds or drug classes, that are associated with reduced drug susceptibility (i.e. phenotype). A correct phenotypic interpretation from the derived PR-RT genotype (i.e. virtual phenotype), requires a well characterized geno-phenotype correlative database and appropriate statistical predictive models. The applicability of the virtual phenotype for the patient, will, however, not only depend on the accuracy of the statistical models and the database they rely on, but also depend largely on the sequence information that is provided. Since HIV-1 evolves as a complex of closely related but non-identical viral genomes (i.e. quasispecies) it is crucial that the sequencing method used, is able to characterize most of the genetic mixtures that make up the different quasispecies within a single patient. US regulatory agencies require that developers of HIV-1 genotyping assays, determine and report the HIV-1 mixture detection level of their assay. Hence, the mixture scoring sensitivity of the population-based Sanger sequencing method, along with the defined mixture scoring rules, used to drive the virco(®)TYPE HIV-1 virtual phenotype, was investigated by comparing it to the 454 pyrosequencing technique, which is able to generate the complete viral population sequence. To this end the PR-RT coding sequence of 20 clinical isolates was determined by both sequencing methodologies. The genotyping assay which feeds the virco(®)TYPE HIV-1 virtual phenotype was able to call automatically 97.5% (i.e. 268 mixtures) and 95.3% (i.e. 326 mixtures) of the mixtures that were present between 25 and 75% and between 20 and 80% in the viral population, as detected by 454. From the not called mixtures, all but one did present a mixture sequence in the Sanger DNA chromatograms, however, with a peak surface area for the second peak that was below the threshold setting for automatic mixture calling in the basecaller software (i.e. 25%). Viral loads ranged from 470 to 629,000 copies/mL and exerted no effect on the mixture calling relationship between both sequencing methodologies (R(2)=0.92). In some occasions (i.e. 55 mixtures) the genotyping assay would detect automatically mixtures that were present below 20% in the viral population, when measured by 454. Hence, the mixture scoring sensitivity of the automated high throughput virco(®)TYPE HIV-1 genotyping assay is currently set at 97.5% and 95.3%, for mixtures present at 25 and 20% in the viral population and may identify occasionally mutations that are present at lower frequencies. These findings were not influenced by the viral load of the examined samples.
Subject(s)
HIV-1/genetics , Sequence Analysis, RNA/methods , Drug Resistance, Viral , Genotype , HIV Infections/drug therapy , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , Humans , MutationABSTRACT
We investigated the phenotypic impact of a number of uncommon amino acid substitutions at HIV-1 reverse transcriptase positions 103 and 138, which are not part of the etravirine resistance score and were found in combination with the high-impact mutation K101P. Etravirine phenotypic fold changes were 380-1400 for K101P + E138A/G/Q + K103N/S/T + V179I and 12-130 for K101P + (K103S +/- V179I) in the absence of E138A/G/Q. Although the effect of K103S is unclear, additional position 138 substitutions seem important for etravirine susceptibility.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/genetics , Mutation , Pyridazines/pharmacology , Drug Resistance, Viral/genetics , Genotype , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , Humans , Nitriles , Phenotype , PyrimidinesABSTRACT
The phenotypic impact on etravirine susceptibility was examined in plasma specimens from 40 nonnucleoside reverse transcriptase inhibitor-experienced HIV patients with distinct nonnucleoside reverse transcriptase inhibitor resistance-associated mutations. Some etravirine resistance-associated mutations produced larger reductions in fold change than others. Y181C in combination with at least one etravirine resistance-associated mutation caused, on average, a 12.6-fold reduced susceptibility to etravirine. Two novel changes, K101H and E399D, significantly diminished etravirine susceptibility. Thus, the original etravirine resistance-associated mutation list should be updated and weighted for a suitable genotypic resistance interpretation.