Five-Antiretroviral Drug Class Resistant HIV-1

Five-Antiretroviral Drug Class Resistant HIV-1Five-Antiretroviral Drug Class Resistant HIV-1 In A Treatment Nave Patient Suppressed With Optimized Antiretroviral SelectionJoseph M Volpe, Douglas J Ward, Laura Na political leaderitano, Pham Phung, Jonathan Toma, Owen Solberg, Christos J Petropoulos, Charles M WalworthAbstractTransmission of HIV-1 exhibiting reduced susceptibility to protease and elevate transcriptase inhibitors is come up-documented, but is evolving for integrase inhibitors and is limited for the fusion inhibitor enfuvirtide. We describe here a case of contagious 5-drug class exemption involving protease, reverse transcriptase (nucleoside and non-nucleoside), integrase, and fusion inhibitors in an antiretroviral nave patient that subsequently was successfully treated ground on the optimized selection of an active antiretroviral drug regimen.KeywordsTransmitted Drug Resistance TDR Integrase Inhibitor Resistance Tropism creative activityDrug susceptibility can be a key determinant in choosing an initial antiretroviral (ARV)regimen for patients who are nave to ARV therapy.The selection of aregimen in which individual components be in possession of less than full susceptibility can result in virologic failure.Transmission of HIV-1 exhibiting electrical resistance to protease (PR) and reverse transcriptase (RT) inhibitors is intimately-documented1-3 andbecause of this,DHHS guidelines propose baseline genotypic resistance testing to guide drug selection in patients who are ARVnave4. However, given the relative newness of the integrase (IN)inhibitor class and the limited use of enfuvirtide (ENF), transmitted resistance for these drug classes is less well-defined5-7. To date, deuce cases of raltegravir disgusting HIV-1 transmission have been reported in the literature5-6.Although transmission of virus resistant to to a greater extent than one ARV class occurs less frequently than a unmarried class2-3, when it occurs, the selection of a base line regimen can bemore challenging. Such was the case in 2005 when a New York City man acquired a dual tropic, multidrug-resistant HIV-1 strain8, during a time when fewer therapeutic options were available. Here, we describe the first documented multidrug-resistant HIV-1 strain containing variants that exhibit resistance to five ARV classes. This reportnot only demonstrates one of the earliest cases of transmitted resistance to the integrase strand-transfer inhibitor (INSTI) class, but also exemplifies the need to develop a detailed resistance profile prior to initiating therapy.Case HistoryA man in his mid-forties was hospitalized in 2010 with severe flu-like illness. HIV-1 antibody testing during hospitalization was negative. HIV-1 RNA testing by PCR was not performed. Six months later, follow-up HIV-1 antibody testing was positiveand infection was corroborate by westbound blot. Initial CD4+ count and viral load were 376 cells/mm3 and 211,540 copies/mL, respectively. Baseline g enotypic resistance testing demonstrated extensive resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI, NNRTI) as well as protease inhibitors (PI) Figure 1. Confirmatory testing was performedto verify the initial genotypic resistance profile. Additional genotypic testing for INSTI resistanceand phenotypic testing for PI, NRTI, NNRTI and INSTIsusceptibility was conducted. phenotypic co-receptor tropism testing was also performed to evaluate additional ARV treatment options. Due to the complexity of the baseline resistance profile, ENF susceptibility was also assessed.MethodsResistance-associated mutations (RAMs) to inhibitors of HIV-1 PR, RT, and IN were identified by formal DNA sequencing(GenoSure MG, GenoSureIntegrase, Monogram Biosciences and LabCorp).Phenotypic susceptibility to PR, RT,and INinhibitors, ENF, and co-receptor tropism were also assessed using well-established pseudo-virus infectivity assays (PhenoSense, PhenoSense GT, PhenoSenseInteg rase , PhenoSense Entry, and Trofile, respectively Monogram Biosciences). Molecular clones of full-length envelope sequences were generated and evaluated for ENF susceptibility and co-receptor tropism (PhenoSense Entry, Trofile). The gp41 sequences of envelope clones were generated by conventional DNA sequencing. Phylogenetic synopsis was conducted on clonal gp41 sequences to rule out co-infection.PR and RT regions were also interrogated by massively parallel (deep) sequencing. A sequence library was generated using the IlluminaNextera XT library preparationkitand an IlluminaMiSeq2x250bp paired-end run resulted in 1,017,032 reads with an average read depth of 15,000X (after alignment). Reads were trimmed using cutadapt and fastx toolkits and aligned to the NL4-3 reference genome (accession number AX032749.1) using bowtie29.ResultsGenotypic resistance depth psychology of the baseline virus identified mutations associated with resistance to PI (L10Y, K20I, E35D, M36I, K43T, I62I/V, V 82K), NRTI (M41L, D67N, L74V, L118I), and NNRTI (K101E, Y181C, V189I, G190S) Figure 1. Repeat genotypic resistance analysis from a second draw confirmed the initial findings, as well as mutations associated with INSTIresistance (G140S, Q148H). Reductions in susceptibility to PI, NRTI, NNRTI, and INSTI were confirmed by phenotypic testing, which demonstrated large reductions in susceptibility to efavirenz, nevirapine, and raltegravir. Notably, the NRTI resistance mutation M184V was not identified bygenotypic legal opinion although a phenotypicassessment revealedmodest reductions in susceptibility to emtricitabine and lamivudine (IC50fold change (FC) 7.16 and 5.25 respectively)that exceeded the biological cutoff (FC 3.5).Further analysis of the sample using deep sequencing failed touncover electric razor variants harboring an M184V substitution. Neither deep sequencingnor the phylogenetic analysis of envelope clone sequences yielded evidence for dual infection.Initial phenotypic anal ysis for ENFsusceptibility (FC 6.31)fell within 0.2 log10of the biological cutoff(FC 6.48). This cutoff is based on a reference population of ENF-nave baseline isolates from the TORO clinical trials 10. Given the proximity of the measuredENF susceptibility of the patient sample to the biological cutoff and considering both the large-minded distribution of the susceptibility of the reference population and the inherent variability of the phenotypic assay (3-fold), further analysis was warranted. Consequently, envelope gp41 sequencing was performed on 44 molecular clones generated from the virus population. Phenotypic analysis to determine ENF susceptibility was performed on 20 of the 44 clones that had unique gp41 sequences relative to the consensus sequence of the virus population. Two clones, (10 and 25), exhibitedreducedENF susceptibility(FC 46 and MAX, respectively), well above the biological cutoff. The gp41 sequence of these two clonesrevealed novel substitutions (Q40R, N43S) at amino acid positions previously associated with ENFresistance 11. Co-receptor tropism testing indicated that 19 of the 20 selected clones exhibited R5 tropism, consistent with the R5 tropism determination for the boilers suit virus population.Based on resistance and co-receptor tropism testing, the patient was placed on a regimen of ritonavir-boosted darunavir, tenofovir/emtricitabine, and maraviroc, which successfully suppressed viral replication to 3 at diagnosis to 614 cells/mm3 at one year of treatment. At three years post-initiation of treatment, the patients virus remains suppressed with a CD4 count of 865 cells/mm3 (Table 1). intelligenceTo our knowledge, this case representsthe first confirmed report of the transmission of HIV-1 containing variants exhibiting resistance to five antiretroviral drug classes,as well as the third confirmed report of transmitted INSTI resistant HIV-1.The selection of tenofovir/emtricitabinein the treatment regimen was based upon an assumption that anM184V variant might have been present below the limit of detection for population sequencing. Often, lamivudine or emtricitabineis incorporated intoARV treatment regimens toexploit the impaired replication of M184Vvariants, despitelimited evidence to support this approach. Detectable reductions in phenotypic susceptibility due to M184V variantsrequire a viral subpopulation approximating 40% of the good viral population12. Here, the absence of an M184V-containing subpopulation below the limit of detection of genotypic assays was confirmed by deep sequencing.Thus, the observed reduction in phenotypic susceptibility to emtricitabine and lamivudine waslikely due to the combination of L74V and V118I substitutions along with the thymidine analog substitutions M41L and D67N.This case further demonstrates the clinical utility of co-receptor tropism testing to guide maraviroc prescription.ARV treatment experienced patients have a lower prevalence of R5 tropic virus compared with ARV treatment nave patients. Laboratory studies demonstrate preferential transmission of R5 virus13 and data from clinical cohorts demonstrate that over 70% of ARV nave patients harbor R5 tropic virus14. In ARV treatment nave patients, there is no genetic linkage data to suggest that ARV resistant profiles in pol influence envelope co-receptor tropism. Despite the extensive ARV resistance profile identified within the pol gene, the case patient was treatment nave and thus more likely to harbor R5 tropic virus.Envelope substitutions associated with ENFresistance include Q40H and the N43D.Clonal analysis of this case virus led to the identification of two novel substitutions Q40Rand N43S that were demonstrated to confer highschool level phenotypic resistance to ENF.The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report. Current DHHS guidelines recommend supplemental genotypic INSTI resistance testing when transmitted INS TI resistance may be a concern4. There is evidence that transmitted INSTI resistance is followingthe same lay coursepreviously observed for NRTI, NNRTI and PI15. With the recent approval of a third INSTI, more widespread INSTI use, overlapping INSTI cross resistance profilesand documentation of this third case of transmitted INSTI resistance, baseline testing for INSTI resistance may become prudent.Figure 1Results of both the genotypic and phenotypic drug resistance analyses are listed here. The net assessment column considers both the genotype and phenotype test results and provides a final resistance call based on the cumulative data. Single values represent biological cutoffs, and ranges indicate lower and upper clinical cutoffs. Fold change is defined as the ratio of the measured IC50 for the patient-derived virus to that of the NL4-3reference virus.Table 1 Patient clinical ParametersViral loads, CD4 and CD8 counts, and CD4/CD8 ratiosfor this patient are listed over the treatme nt period. The initial viral load measurements were obtained using the Roche COBAS TaqMan 2.0until 7/12. Subsequent values were obtained using the Siemens versant HIV-1 Branched DNA assay.