Vici Varghese

HIV-1 integrase inhibitor resistance and its clinical implications.

With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development—elvitegravir and S/GSK1349572—may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Minority Variants Associated with Transmitted and Acquired HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor Resistance: Implications for the Use of Second-Generation Nonnucleoside Reverse Transcriptase Inhibitors

Objectives:K103N, the most common nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutation in patients with transmitted resistance and in patients receiving a failing NNRTI-containing regimen, is fully susceptible to the new NNRTI, etravirine. Therefore, we sought to determine how often NNRTI-resistant mutations other than K103N occur as minority variants in plasma samples for which standard genotypic resistance testing detects K103N alone. Methods:We performed ultradeep pyrosequencing (UDPS; 454 Life Sciences a Roche Company, Branford, CT) of plasma virus samples from 13 treatment-naive and 20 NNRTI-experienced patients in whom standard genotypic resistance testing revealed K103N but no other major NNRTI-resistance mutations. Results:Samples from 0 of 13 treatment-naive patients vs. 7 of 20 patients failing an NNRTI-containing regimen had minority variants with major etravirine-associated NNRTI-resistant mutations (P = 0.03, Fisher exact test): Y181C (7.0%), Y181C (3.6%) + G190A (3.2%), L100I (14%), L100I (32%) + 190A (5.4%), K101E (3.8%) + G190A (4.9%), K101E (4.0%) + G190S (4.8%), and G190S (3.1%). Conclusions:In treatment-naive patients, UDPS did not detect additional major NNRTI-resistant mutations suggesting that etravirine may be effective in patients with transmitted K103N. In NNRTI-experienced patients, UDPS often detected additional major NNRTI-resistant mutations suggesting that etravirine may not be fully active in patients with acquired K103N.

Emergence of Novel Human Group A Rotavirus G12 Strains in India

ABSTRACT Three rare human G12 strains were detected from diarrheic clinical samples of children (<8 months of age) in Calcutta during a routine surveillance study of rotaviral diarrhea in India. The VP7 genes of G12 strains and their products showed maximum homology (97 to 99% at the nucleotide level and 98% at the amino acid level, respectively) with those of two recently reported G12 strains (from the United States and Thailand) but lesser homology with those of prototype G12 strain L26.

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations

ABSTRACT T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants.

Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4

Summary.Long electropherotype with Subgroup I specificity is a common feature of animal rotaviruses. In an epidemic of infantile gastroenteritis in Manipur, India, long but SG I strains predominated in the outbreak in the year 1987–88. One such strain isolated from that region, following the outbreak had G9P [19] specificity. As this is a rare combination, the gene sequences encoding VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 of this strain were analyzed. All these genes except VP7 were closely related to porcine rotaviruses (95–99% identity at amino acid level) and clustered with the porcine strains in phylogenetic analysis. In addition, it had subgroup I nature and belonged to NSP4 genotype B which is characteristic of animal rotaviruses. This is the first report of a rotavirus with VP6 and NSP4, two crucial proteins thought to be involved in host range restriction and pathogenicity, were of porcine origin and caused diarrhoea in a human host. Among the genes of this strain sequenced so far, only VP7 had highest identity to human strains at amino acid level. This study suggests reassortment may be occurring between human and other animal strains and some of the reassortant viruses may be virulent to humans.

Molecular characterization of a porcine Group A rotavirus strain with G12 genotype specificity

Summary.A porcine Group A rotavirus strain (RU172) was detected and molecularly characterized during a surveillance study conducted for rotavirus infection in a pig farm located in a suburban area of Kolkata City, India. The G12 genotype specificity of RU172 was revealed by PCR-based genotyping assays following addition of a G12 type-specific primer (designed in our laboratory to pick up G12 isolates from field samples) and was confirmed by sequence analysis of the VP7-encoding gene. The RU172 strain exhibited maximum VP7 identities of 93.6% to 94.5% with human G12 strains at the deduced amino acid level. In spite of its G12 genotype nature, RU172 appeared to be distinct from human G12 rotaviruses and, on phylogenetic analysis, formed a separate lineage with human G12 strains. Among the other gene segments analyzed, RU172 belonged to NSP4 genotype B, had a NSP5 and VP6 of porcine origin, and shared maximum VP4 identities with porcine P[7] rotaviruses (94.3%–95.4% at the deduced amino acid level). Therefore, to the best of our knowledge, this is the first report of detection of an animal rotavirus strain with G12 genotype specificity. Detection of strains like RU172 provides vital insights into the genomic diversity of Group A rotaviruses of man and animals.

Genomic Diversity of Group A Rotavirus Strains Infecting Humans in Eastern India

ABSTRACT Between 1998 and 2000, a total of 266 samples were found positive for group A rotaviruses by RNA electrophoresis. Samples were collected from patients admitted to two leading hospitals in Calcutta. Serotyping could be done only with 22% of the positive samples, leaving 78% untypeable. The G (VP7 genotypes) and P (VP4 genotypes) types were determined for 159 samples by reverse transcription and multiplex PCR. The predominant genotype was G1P[8] (20%), followed by G2P[4] (15%) and G4P[8] (6%). A number of uncommon genotypes, G1P[4] (4%), G2P[8] (2.5%), G2P[6] (0.6%), G4P[4] (2.5%), and G4P[6] (1.25%), were also detected during this study period. Twenty two percent of specimens showed mixed infections, 38 (24%) of the total samples remained untypeable for either VP7 or VP4, while only 4 (2.5%) of the samples were untypeable for both genes. Eleven specimens collected from Manipur were also genotyped and revealed a very high degree of genomic reassortment.

Nucleic acid template and the risk of a PCR-Induced HIV-1 drug resistance mutation.

Background The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses. Methods and Findings We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses. Conclusions This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.

Non-nucleoside reverse transcriptase inhibitor (NNRTI) cross-resistance: implications for preclinical evaluation of novel NNRTIs and clinical genotypic resistance testing

OBJECTIVES The introduction of two new non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the past 5 years and the identification of novel NNRTI-associated mutations have made it necessary to reassess the extent of phenotypic NNRTI cross-resistance. METHODS We analysed a dataset containing 1975, 1967, 519 and 187 genotype-phenotype correlations for nevirapine, efavirenz, etravirine and rilpivirine, respectively. We used linear regression to estimate the effects of RT mutations on susceptibility to each of these NNRTIs. RESULTS Sixteen mutations at 10 positions were significantly associated with the greatest contribution to reduced phenotypic susceptibility (≥10-fold) to one or more NNRTIs, including: 14 mutations at six positions for nevirapine (K101P, K103N/S, V106A/M, Y181C/I/V, Y188C/L and G190A/E/Q/S); 10 mutations at six positions for efavirenz (L100I, K101P, K103N, V106M, Y188C/L and G190A/E/Q/S); 5 mutations at four positions for etravirine (K101P, Y181I/V, G190E and F227C); and 6 mutations at five positions for rilpivirine (L100I, K101P, Y181I/V, G190E and F227C). G190E, a mutation that causes high-level nevirapine and efavirenz resistance, also markedly reduced susceptibility to etravirine and rilpivirine. K101H, E138G, V179F and M230L mutations, associated with reduced susceptibility to etravirine and rilpivirine, were also associated with reduced susceptibility to nevirapine and/or efavirenz. CONCLUSIONS The identification of novel cross-resistance patterns among approved NNRTIs illustrates the need for a systematic approach for testing novel NNRTIs against clinical virus isolates with major NNRTI-resistance mutations and for testing older NNRTIs against virus isolates with mutations identified during the evaluation of a novel NNRTI.