Multicenter comparison of the new Cobas 6800 system with Cobas Ampliprep/Cobas TaqMan and Abbott RealTime for the quantification of HIV, HBV and HCV viral load.

BACKGROUND: The new Roche Cobas 6800 platform (C6800) has been recently introduced for viral load (VL) measurement. OBJECTIVES: Comparing C6800 to Cobas Ampliprep/Cobas TaqMan v2.0 (CAP/CTM) for the quantification of HIV, HBV and HCV viremia, and to the Abbott RealTime assay (ABB) for HCV quantification. STUDY DESIGN: We analysed 121 samples for HBV, and 139 for HIV-1 including 2 groupO and 137 group M viruses (36.5% subtype B, 27.0% CRF02_AG, 22.6% from other clades, and 14% subtype not available). For the 100 HCV samples compared with CAP/CTM, 42% were genotype 1 and 17% were genotype 4. For the 68 HCV samples compared with ABB, 52.9% were genotype 1 and 22.1% were genotype 4. RESULTS: C6800 results correlated well with those of CAP/CTM for all three viruses (R2: 0.97-0.99). However, C6800 can yield different viraemia results: higher for HIV (mean difference: +0.11 log10copies/mL, p<0.0001), and lower for HBV (mean difference:-0.11 log10 IU/mL, p<0.0001). Differences exceeded 0.5 log10 for 6.5% of HIV-1 samples and 7.4% of HBV samples. For HCV quantification, C6800 gave mostly lower values than the other assays towards the bottom of the range, and higher values in the upper part of the range, especially in comparisons with ABB, for which 28% of differences exceeded 0.5 log10 IU/mL. No particular HCV genotype was identified as responsible for these differences. CONCLUSION: Overall, the comparison tests between C6800 and CAP/CTM systems are satisfactory for the three viruses. Frequent discrepancies were observed between C6800 and ABB for HCV.

Improved detection of resistance at failure to a tenofovir, emtricitabine and efavirenz regimen by ultradeep sequencing.

OBJECTIVES: Resistant minority variants present before ART can be a source of virological failure. This has been shown for NRTIs, NNRTIs and CCR5 inhibitors. However, very few data are available for the detection of such minority resistant variants that could be selected at virological failure and not detected using classical Sanger sequencing. METHODS: We studied 26 patients treated with tenofovir, emtricitabine and efavirenz with their first virological failure (defined as two consecutive viral loads >50 copies/mL). We performed standard Sanger sequencing and ultradeep sequencing (UDS; Roche 454(®) Life Sciences) in plasma at failure. For UDS, mutations >1% were considered. We compared the presence of reverse transcriptase mutations between the two techniques, using the latest ANRS algorithm. RESULTS: UDS detected more resistance mutations in 38.5% of cases (10/26 patients) and the genotypic sensitivity score (GSS) was reduced for 6 of them (23.1%). The GSS was impacted more often for NRTIs than for NNRTIs, for which most mutations were already detected by Sanger sequencing. Resistant minority variants were detected even in patients with low viral load at failure. CONCLUSIONS: These results strongly argue for the use of next-generation sequencing in patients failing on an NRTI+NNRTI regimen, as UDS has the potential to modify the choice of the subsequent regimen.

Highly multidrug-resistant HIV: clonal analysis and therapeutic strategies.

OBJECTIVES: There are today HIV-infected patients in therapeutic impasses because of highly multidrug-resistant (HMDR) viruses. We studied the distribution of resistance mutations at clonal level, and we analysed the therapeutic strategies used in such cases to achieve undetectable viraemia. METHODS: The HMDR profile was defined as a genotypic sensitivity score (GSS) ≤ 1.5 for etravirine and raltegravir with full resistance to darunavir. About 30 clones per gene and per patient were sequenced. Virtual phenotypes were determined. Efficacy of therapeutic strategies was evaluated by follow-up of viral loads, CD4 cell counts and trough concentrations of drugs. RESULTS: Among 1310 patients on treatment and with genotypic resistance testing, 25 (2%) were resistant to darunavir and 11 (0.8%) had an HMDR profile. Five-hundred clones could be analysed for four of them. HMDR profiles were harboured by the great majority of clones and all resistance mutations were located on the same strains for all genes. Despite this and a regimen with a GSS <2.0 in three patients, they achieved a viraemia <20 copies/mL. These results were obtained using different strategies: high doses of drugs; combination of antiretrovirals with full or intermediate susceptibility, such as tipranavir, etravirine or maraviroc; and use of alternative compounds, such as foscarnet or interferon. CONCLUSION: Patients with HMDR HIV were uncommon, but, in such cases, all resistance mutations were borne on the same majority strains. In this study, tipranavir was the only protease inhibitor with full or intermediate susceptibility. Despite very limited therapeutic options, an undetectable viraemia can be achieved by combining different strategies.

Historical HIV-RNA resistance test results are more informative than proviral DNA genotyping in cases of suppressed or residual viraemia.

BACKGROUND: Resistance genotyping is often requested due to residual HIV viraemia or for treatment optimization, but may be unsuccessful if plasma RNA levels are too low or undetectable. Analyses of proviral HIV-DNA can provide information about the viral reservoir, because integrated DNA reflects both actively and latently infected cells. OBJECTIVES: To determine whether proviral DNA is a potential relevant alternative to HIV-RNA for resistance genotyping in this context. METHODS: The resistance mutations harboured by the proviral DNA were compared with the cumulative data for all plasma RNA genotypes previously obtained for the patient concerned. We also investigated whether various parameters, such as CD4 count, level of viraemia or drug pressure, affected the results. RESULTS: We collected 134 and 141 DNA genotypes with 443 and 462 corresponding RNA sequences for the reverse transcriptase and protease genes, respectively. The mean rates of concordance between DNA and RNA genotypes were 46.7% for nucleoside reverse transcriptase inhibitors (NRTIs), 26.3% for non-NRTIs and 43.7% for protease inhibitors (PIs). Mixtures were detected for most DNA mutations. The rate of concordant PI mutations was significantly higher for patients taking PIs at the time of DNA genotyping (48% versus 26%; P=0.004). The other factors studied had no impact. CONCLUSIONS: In the context of low or undetectable viraemia, it is difficult to reach the archived mutated DNA. Classical RNA genotyping during previous periods of virological failure remains the gold standard for documenting resistance mutations and for the monitoring of future treatments.

Role of HIV-1 DNA levels as clinical marker of HIV-1-associated nephropathies.

BACKGROUND: HIV-associated nephropathy (HIVAN) is usually diagnosed in virologically uncontrolled infected patients. However, HIVAN can occur in some cases with undetectable HIV-1 RNA levels. We suggested that intracellular HIV-1 proviral DNA load can be used as a marker in this particular patient population. METHODS: Renal tissue HIV-1 proviral DNA, peripheral blood mononuclear cells (PBMCs) HIV-1 proviral DNA, plasma HIV-1 RNA and peripheral blood CD4-positive lymphocyte counts were quantified and assessed in 100 HIV-1-infected individuals (49 with undetectable HIV-1 RNA level viraemia) who underwent a kidney biopsy for renal abnormalities in a prospective study. RESULTS: A proviral DNA load was detected in patients with HIV-1-associated nephropathy with a negative plasma viral RNA load. None of the patients with a proviral DNA load of <10 copies/1.5×10(5) PBMCs had HIVAN. In renal tissue, proviral DNA load was detected in all patients with HIVAN. CONCLUSIONS: PBMC HIV-1 proviral DNA load is potentially the optimal clinical marker to exclude the diagnosis of HIVAN. On the other hand, cellular incorporation of HIV-1 within the kidney is a requisite for the development of this disorder.

Primary genotypic resistance of HIV-1 to the maturation inhibitor PA-457 in protease inhibitor-experienced patients.

Sequences from 82 protease inhibitors (PI)-experienced patients were analysed for the presence of previously described in-vitro resistance mutations to PA-457 located in the C-terminal capside (H226Y, L231F, L231M) and in the N-terminal SP1 (A1V, A3T, A3V) within the CA-SP1 boundary domain. Overall, the CA-SP1 cleavage site was highly conserved in PI pre-treated patients and only one patient showed an L231M mutation. The impact of this mutation should be further addressed in vivo.

Association of Gag cleavage sites to protease mutations and to virological response in HIV-1 treated patients.

OBJECTIVES: The sequence variability in the protease and in the 5 Gag cleavage sites (CS) were explored to look for eventual associations between the mutations. Moreover, we have evaluated associations between the Gag region sequence and the virological response to Protease Inhibitors (PI). METHODS: The protease and the 5 Gag CS sequences from 98 PI-experienced patients were sequenced and compared to the HXB2 reference sequence. Sixty patients, treated by Saquinavir plus Ritonavir, were studied to evaluate the clinical impact of the Gag region variability. RESULTS: The relationship between 63 protease mutations and 21 Gag CS mutations were explored. Two patterns of mutations in the protease were identified: (M46I/L, I54V, V82A/T/F) was associated to the A431V and (K20I/R/M, L89M/I) to the S373Q and L449P. None of the Gag CS mutations resulting from PI treatment was associated to the virological response to SQV/r. On the other hand, the S373P mutation had a negative impact on the virological response that remained statistically significant in a multivariate analysis after adjustment on the number of PI resistance mutations. CONCLUSIONS: These results evoke the pertinence to introduce some mutations found in the Gag CS in the algorithms used for the interpretation of resistance testing.

Foscarnet salvage therapy for patients with late-stage HIV disease and multiple drug resistance.

OBJECTIVE: To evaluate the efficacy of foscarnet on HIV infection in patients with late-stage HIV disease and multiple drug resistance. METHODS: Three drugs experienced patients with plasma viral load (pVL) > 50,000 copies/ml and CD4+ T-cell counts < 100/mm3 were eligible for this open-label, single-arm, add-on pilot study. Foscarnet induction therapy consisted of 5 g intravenously twice daily for 6 weeks, in addition to a stable antiretroviral regimen. Patients with at least 1 log10 decrease in pVL at week 6 (W6), were given foscarnet 5 g intravenously twice daily on two consecutive days each week. Primary endpoint was the virological response rate at W6. RESULTS: Eleven patients were enrolled with a median baseline pVL at 5.16 log10 copies/ml, median CD4+ T-cell count at 10/mm3 and median number of mutations of 9, 2 and 12 associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-NRTIs and protease inhibitors, respectively. One patient discontinued foscarnet at W2 because of renal toxicity. In an intent-to-treat analysis, the median change in pVL from baseline was -1.99 log10 copies/ml at W2 and -1.79 log10 copies/ml at W6. Eight out of eleven patients had a fall in pVL of at least 1 log10 at W6, and six started maintenance therapy. The median fall in pVL after 12 weeks of maintenance therapy was -0.85 log10 copies/ml in the four patients who reached W12, and the median increase of CD4+ T-cell count was 60/mm3. CONCLUSION: In patients with HIV mutations conferring resistance to all antiretroviral drug classes, foscarnet markedly reduced plasma HIV load and improved immunological status.

Role of HIV-1 minority populations on resistance mutational pattern evolution and susceptibility to protease inhibitors.

We investigated, in the protease and GAG cleavage sites, the role of minority populations on the evolution of resistance to a subsequent boosted protease inhibitor (PI) regimen after a first failure to nelfinavir. Two pathways were observed: the addition of mutations to a currently dominant genotype when minority variants are not shown, or the emergence of a minority variant as a dominant strain. These minor species, not detected by standard genotype, may influence PI susceptibility.

Clonal analyses of HIV quasispecies in patients harbouring plasma genotype with K65R mutation associated with thymidine analogue mutations or L74V substitution.

We analysed the quasispecies at a clonal level in patients whose plasma genotypic test harboured K65R with L74V or thymidine analogue mutations (TAM). We showed that the K65R and TAM such as M41L, D67N, T215Y/D, L210W and K219E can be borne by the same virus. We found no clone bearing both K65R and L74V substitutions. Moreover, the S68G and V75I mutations are not necessarily linked with K65R, and could thus have their own resistance effect.

French national sentinel survey of antiretroviral drug resistance in patients with HIV-1 primary infection and in antiretroviral-naive chronically infected patients in 2001-2002.

OBJECTIVE: To survey the frequency of genotypic antiretroviral resistance and the spread of non-B subtypes in patients with primary HIV-1 infection (2001-2002) and in treatment-naive chronically HIV-1-infected patients (2001). METHODS: Plasma samples from 303 patients with acute HIV-1 infection (Primo study) and 363 treatment-naive patients with chronic HIV-1 infection (Odyssee study) were tested for genotypic resistance. Resistance mutations were identified from the International AIDS Society Resistance Testing-USA panel and resistant viruses were defined according to the French Agence Nationale de Recherches sur le SIDA (ANRS) resistance algorithm. RESULTS: In the Primo study, 14% of the patients had viruses with resistance mutations and 12% of patients had viruses with mutations conferring resistance to least 1 antiretroviral drug. Thirty patients had viruses with mutations to at least 1 antiretroviral drug in a single pharmacologic class. Six patients were infected by viruses resistant to 2 or 3 classes of drugs. In the Odyssee study, the prevalence of reverse transcript (RT) associated and major protease inhibitor-associated mutations was 6.1% (95% CI: 3.6-8.6). Six patients had viruses resistant to at least 1 antiretroviral drug and 3 patients had viruses resistant to 2 classes of antiretroviral drugs. Twenty-four percent of acutely infected patients harbored non-B subtype strains (19% in 1999-2000) and 33.2% of chronically infected patients (10% in 1998; P < 0.0001). CONCLUSION: In France, the frequency of HIV-1 resistance in untreated patients was not significantly higher in 2001-2002 than in previous surveys while the prevalence of non-B subtypes is increasing.

Resistance profiles observed in virological failures after 24 weeks of amprenavir/ritonavir containing regimen in protease inhibitor experienced patients.

Amprenavir (APV) is an HIV protease inhibitor (PI) used for the treatment of either naive or PI-experienced HIV-infected patients. Several genotypic resistance pathways in protease gene have been described to be associated to unboosted APV failure (I50V, V32I + I47V, I54L/M, or less commonly I84V, which may be accompanied by one ore more accessory mutations such as L10F, L33F, M46I/L). The aims of this study were to investigate the efficacy up to week 24 of an APV plus ritonavir containing regimen in PI experienced patients and to determine the genotypic resistance profiles emerging in patients failing to this therapy. Forty-nine, PI experienced but APV naïve patients were treated with APV (600 mg bid) plus ritonavir (100 mg bid). By intent-to-treat analysis, the median decrease in viral load (VL) was -1.32 log10 (min +0.6; max -2.8) and -1.46 log10 (min +0.5; max -2.8) 12 and 24 weeks after initiating APV plus ritonavir regimen, respectively. Twelve patients harboured a VL >200 copies/ml at week 24. Among these patients, the selection of mutations previously described with the use of APV as first PI (V32I, L33F, M46I/L, I50V, 54M/L, and I84V) was observed. However, in some cases, mutations classically described after the use of other PIs (V82F and L90M) were selected but always with APV-specific mutations. There was no relation between the resistance pathways selected with either APV or ritonavir plasma minimal concentration, but higher APV plasma minimal concentration were associated with a lower rate of resistance mutations selection.