RNA and DNA Sanger sequencing versus next-generation sequencing for HIV-1 drug resistance testing in treatment-naive patients.

Background: Sanger sequencing of plasma RNA is the standard method for HIV-1 drug resistance testing in treatment-naive patients, but is limited by the non-detection of resistance-associated mutations (RAMs) with prevalence below approximately 20%. Objectives: We compared RNA and DNA Sanger sequencing (RSS and DSS) with RNA next-generation sequencing (NGS) for RAM detection in HIV-1 reverse transcriptase (RT), protease (PR) and integrase (IN) genes. Methods: Sanger sequencing was performed on RNA and DNA, following the recommendations of the French Agency for AIDS Research (ANRS). NGS was performed on RNA using the HIV-1 Drug Resistance Assay, v. 3.0 (Roche) on the 454 GS Junior sequencer. The IAS-USA list was used to identify RAMs. ANRS, Rega and Stanford algorithms were used for drug resistance interpretation. Results: The study included 48 ART-naive patients. The number of patients with at least one major RAM was 3, 3, 4 and 8 when using RSS, DSS, NGS 20% and NGS 5%, respectively. Numerous minor mutations were detected in patients, especially in the protease gene. None of the methods detected any major mutation in the integrase gene. Overall, the mutation detection rate was similar between RSS and DSS, and higher with NGS 20%. Differences in drug resistance interpretation were found between algorithms. No impact of the minority RAMs detected by NGS was found on the short-term treatment outcome. Conclusions: DSS does not clearly improve the detection of RAMs in ART-naive patients, as compared with RSS. NGS allows detection of additional minority RAMs; however, their clinical relevance requires further investigation.

Counter-intuitive plasma vitamin D and zinc status in HIV-1-infected adults with persistent low-level viraemia after treatment initiation: a pilot case-control study.

Determinants of persistent low-level viraemia [PLLV, a viral load (VL) of between 50 and 500 copies/mL] have not been elucidated. In a case-control study, we evaluated the influence of micronutrients on PLLV in a population of 454 HIV-1 adults having initiated antiretroviral therapy (ART) between January 2007 and December 2011. Plasma levels of retinol (vitamin A), 25-OH vitamin D2 + D3, vitamin E and zinc were measured at ART initiation in cases (PLLV after 6 months of ART) and in controls (VL <50 copies/mL after 6 months). Cases and controls were matched for the CD4 cell count (±50/mm3) and ethnic origin. Intergroup differences in demographic, biological and treatment parameters and sunshine intensity at ART initiation were adjusted using a propensity score. A receiver operating characteristic (ROC) curve was used to assess intergroup differences in plasma micronutrient levels. Thirty-three of the 454 patients (7.3%) displayed PLLV (median VL: 92 copies/mL). Patients were predominantly male (89%), Caucasian (64%) and CDC stage C (25%). The median age was 38 years, the median initial VL was 5.2 log10 copies/mL and the median CD4 count was 74/mm3. The 22 cases and matched controls were balanced in these respects, and had similar vitamin A/E levels. Two cases (9%) and 9 controls (41%) had a vitamin D level <10.3 ng/mL (p = 0.0015), and 2 cases (9%) and 10 controls (48%) had a zinc level <74.6 µg/dL (p = 0.04). Our results support in vitro studies suggesting that vitamin D favours HIV-1 replication and that HIV-1 is zinc-dependent. Wide-scale, prospective studies are required.

Factors associated with virological response to a switch regimen containing maraviroc for antiretroviral-experienced HIV-1-infected patients.

OBJECTIVES: There are few data on clinical and virological factors associated with maraviroc virological response (VR) in clinical practice. This study aimed to identify factors associated with VR in 94 treatment-experienced, but CCR5 inhibitor-naive, HIV-1 patients switched to maraviroc-containing regimens. METHODS: Patients with HIV-1 RNA viral load (VL) <50 copies/mL switching to an antiretroviral treatment containing maraviroc were followed. VR was defined at month 3 as VL <50 copies/mL. The impact of age, baseline tropism, zenith VL, nadir CD4 cell count and CD4 cell count, HIV subtype (B versus non-B), genotypic susceptibility score of treatment, once- or twice-daily treatment and presence of raltegravir in optimized background therapy on VR was investigated. RESULTS: Baseline characteristics were: median age 49 years (range 25-73 years), median CD4 cell count 481 cells/mm(3) (range 57-1830 cells/mm(3)) and median nadir CD4 cell count 99 cells/mm(3) (range 3-585). Maraviroc was administered twice daily in 88 of 94 patients and once daily in 6 of 94 patients (300 mg/day for 4 of 6 and 150 mg/day for 2 of 6). At month 3, 89.4% of patients were responders. A better VR to a switch regimen containing maraviroc was associated with the B subtype (P = 0.0216) and a lower zenith VL (median of 5.24 and 5.70 log10 copies/mL for patients in success or in failure, respectively) in univariate analysis. Only B subtype was associated with a better VR in multivariate analysis. CONCLUSIONS: This study evidenced the efficacy of a switch regimen containing maraviroc in clinical practice. VR was better for patients with a lower zenith VL and B subtype.

Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes.

A 2-log drop in viral load at 1 month is the best predictor of sustained response in HCV patients with normal ALT: a kinetic prospective study.

The most reliable predictor of a sustained virological response in patients with persistently normal ALT has not been identified. We analysed 17 patients with genotype 1 chronic HCV who underwent therapy with pegylated interferon alfa 2b and ribavirin for 48 weeks. Two patients discontinued therapy within 28 days because of side effects and the remaining 15 patients were analysed in detail. An analysis of on treatment virological response using area under the receiver operating characteristic analyses showed that a 2 log drop in HCV RNA at day 28 was the best predictor of a sustained virological response and a failure to reduce viral load by 2 logs correctly identified patients with a low (<15%) probability of achieving a sustained virological response. Introduction of this early discontinuation rule in patients with normal ALT would allow nearly half of the patients to discontinue futile therapy at an early stage.

Clinically validated mutation scores for HIV-1 resistance to fosamprenavir/ritonavir.

BACKGROUND: We developed clinically relevant genotypic scores for resistance to fosamprenavir/ritonavir in HIV-1 protease inhibitor (PI)-experienced patients. METHODS: PI-experienced patients with virological failure receiving fosamprenavir/ritonavir as the sole PI for at least 3 months and with detectable fosamprenavir plasma levels were included. The impact of baseline protease mutations on virological response (VR, i.e. decrease in plasma HIV-1 RNA between baseline and month 3) was analysed using the Mann-Whitney test. Mutations with prevalence >10% and P value <0.10 were retained. The Jonckheere-Terpstra test was used to select the combination of mutations most strongly associated with VR. The association between score and VR was assessed by multivariate backward regression. RESULTS: In the 73 patients included, the median baseline HIV-1 RNA was 4.6 log(10) copies/mL (range: 2.7-6.9) and the mean decrease at month 3 was -1.07 +/- 1.40 log(10) copies/mL. Ninety per cent of the patients were infected by HIV-1 subtype B variants. Two fosamprenavir/ritonavir mutation scores were constructed: score A (L10F/I/V + L33F + M36I + I54L/M/V/A/T/S + I62V + V82A/F/C/G + I84V + L90M) was based only on mutations associated with a worse VR, whereas score B (L10FIV + L33F + M36I + I54L/M/V/A/T/S + A71V - V77I - N88S + L90M) also took into account favourable mutations. Both scores were independent predictors of VR, however, co-administration of tenofovir was associated with a worse VR and the presence of the N88S protease mutation and co-administration of enfuvirtide with a better VR. CONCLUSIONS: These clinically validated mutation scores should be of interest for the clinical management of PI-experienced patients. The fosamprenavir/ritonavir score A was introduced in the 2006 ANRS algorithm along with isolated mutations I50V and V32I + I47V.

ABCB1 allele polymorphism is associated with virological efficacy in naïve HIV-infected patients on HAART containing nonboosted PIs but not boosted PIs.

OBJECTIVE: To assess the effect of the multidrug resistance-1 single nucleotide polymorphism (ABCB1 SNP) C3435T in exon 26 on the virological responses to first-line protease inhibitor (PI)-containing HAART regimens. METHOD: A cohort of 182 HIV-infected patients with a PI-containing HAART regimen initiated from 1997 to 2004 was enrolled. Time to the first indetectable viral load (VL) was determined in patients with the CC, CT, or TT genotype. RESULTS: There were 37%, 44%, and 19% of patients who had the CC, CT and TT genotypes, respectively. The median estimated times to VL indetectability in the CC, CT, and TT groups were respectively 5.9, 3.9, and 4.8 months (p= .06). In patients on a non-boosted PI regimen, ABCB1 genotype was associated with time to VL indetectability that was shorter in patients with the CT than CC genotype (CT vs. CC, hazard ratio [HR]=0.62, p= .02; TT vs. CC, HR= 0.72, p= .21). This association was not found in patients with first-generation boosted PI-containing regimens and especially not with second-generation boosted PI-containing regimens. CONCLUSION: Our results show that the ABCB1 SNP in exon 26 is associated with virological efficacy in HIV-infected patients treated with non-boosted PI-containing regimens but not with those containing boosted PIs, particularly of the second generation.

Fatal hemophagocytic syndrome related to human herpesvirus-6 reinfection following liver transplantation: a case report.

Human herpesvirus-6 (HHV-6) has been identified as the causal agent of exanthema subitum in early childhood (also called sixth disease or roseola), a mononucleosis-like disease in adults, and as an opportunistic pathogen in transplant recipients. In the latter setting, most infections are caused by reactivation of the latent virus in the recipient and generally have a paucisymptomatic course. Only limited data on HHV-6 infection are available for liver transplant recipients. Herein we have reported a case of fatal hemophagocytic syndrome related to HHV-6 reactivation 2 weeks after liver transplantation (LT). This case suggests that this virus may be a serious and potentially life-threatening pathogen following LT.

Sanger sequencing versus INNO-LiPA® HBV PreCore assay for routine detection of precore and basal core promoter mutations in hepatitis virus B chronically infected patients.

We compared the Sanger sequencing and the commercial INNO-LiPA® HBV assay for the routine detection of precore (PC) and basal core promoter (BCP) mutations of hepatitis B virus in chronically infected patients. The overall agreement rate between assays was 94.2% and 98.8% for the detection of PC and BCP mutations, respectively.

[Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins]. / Rôle des N-glycanes dans les fonctions des glycoprotéines d'enveloppe du virus de l'hépatite C.

Hepatitis C virus (HCV) is an enveloped virus and encodes two envelope glycoproteins, E1 and E2. E1 and E2 are transmembrane type I proteins with a N-terminal ectodomain and C-terminal anchor. During their synthesis, E1 and E2 ectodomains are targeted in the endoplasmic reticulum lumen where they are modified by N-linked glycosylation. After their synthesis, E1 and E2 assemble as a non-covalent heterodimer. The N-linked glycosylation is based on the recognition of specific asparagine residue in the context of the consensus sequence Asn-X-Ser/Thr. E1 contains potentially 4 or 5 N-linked glycosylation sites and E2 up to 11. Recent data indicated that some glycans of glycoproteins E1 and E2 play a major role in protein folding and heterodimer formation. Some N-linked glycans of E2 were involved in interactions with CD81, a putative cellular receptor for HCV. It appeared that N-linked glycans of E1 and E2 played an important role of in the viral entry.

[Simultaneous detection of IgM anti-Epstein-Barr virus and IgM anti-hepatitis A during an acute hepatitis]. / Détection simultanée d'IgM anti-Epstein-Barr virus et d'IgM anti-hépatite A lors d'une hépatite aiguë.

We report a case of a 15-year-old young man who was admitted for an acute hepatitis. Virological assessment showed both IgM anti-EBV and IgM anti-hepatitis A. IgG anti-EBNA and clinical history allowed to rule out the hypothesis of a recent EBV infection and confirmed the diagnosis of acute hepatitis A infection.

[Emergent viruses: SARS-associate coronavirus and H5N1 influenza virus]. / Virus respiratoires émergents : virus du Sras et virus influenza A/H5N1 hautement pathogène.

Two viral agents with RNA genome are responsible for emerging illnesses: influenza virus A/H5N1 and Severe Acute Respiratory Syndrome virus (SARS). For the diagnosis of SARS virus infection, an epidemiological investigation is necessary to know whether the patient has been exposed to a risk in a country where the SARS virus is circulating or whether the patient had worked in a laboratory handling SARS virus. The detection of SARS virus is possible in various clinical samples (including urine) by viral culture or RT-PCR. The handling of those samples and RNA extraction must be performed in a BSL3 laboratory. The SARS virus RT-PCR is poorly sensitive, therefore the test should be performed on samples collected consecutively for several days. In front of a suspicion of A/H5N1, similar procedures are recommended. An epidemiologic investigation is necessary to specify whether the patient stayed in a country where A/H5N1 virus was circulating. Clinical samples needed for a specific diagnosis are: nasopharyngeal, throat-swab or fecal samples, cerebrospinal fluid and blood. The presence of A/H5N1 virus is confirmed by viral isolation or RNA detection by RT-PCR. RNA extraction must be performed in a BSL3 laboratory. For diagnosis of A/H5N1 virus infection, RT-PCR test amplifies specifically a fragment of H5 gene (Hemagglutinin). In french laboratories of medical virology, procedures are ready to diagnose the first case of A/H5N1 virus infection and cases of reemerging SARS virus infection.

A patient with fever, abdominal pain and bicytopenia: Trouble once again with these IgM antibodies!

We here report the case of a 30-year old man with a history of ulcerative colitis, who presented clinical and biological features compatible with a viral hepatitis. Initial serological results revealed the presence of IgM antibodies against many viruses, and the most likely diagnosis was viral hepatitis A. However, further investigations were performed and concluded to cytomegalovirus primary infection.

[Various approaches of therapeutic vaccination for the treatment of HIV type 1 infection]. / Différentes approches de vaccination thérapeutique dans le traitement de l'infection par le VIH-1.

HIV-1 infection is a major pandemic situation. With the advent of highly active antiretroviral therapy (HAART), morbidity and mortality associated with HIV-1 infection have been dramatically reduced. However, HAART does not enable eradication of the virus. The efficacy of these new regimens is limited by problems over long-term use such as toxicity and resistance. Therapeutic vaccination is an alternative approach to HIV-1 infection. The main aim is to boost and reinforce virus-specific host immune responses. Several immunogens and schedules of immunization have been tested. In this review, various strategies designed for therapeutic vaccines for HIV-1 infection are presented.

Human polyomavirus JC latency and reactivation status in blood of HIV-1-positive immunocompromised patients with and without progressive multifocal leukoencephalopathy.

BACKGROUND: Human polyomavirus JC (JCV) induces human progressive multifocal leukoencephalopathy (PML) in patients with AIDS. Peripheral blood mononuclear cells (PBMC) of HIV-1-positive immunocompetent and immunocompromised patients can harbour JCV genome, but their precise role in JCV latency or reactivation status before the onset of PML remains hypothetical. OBJECTIVES: To assess JCV latency or reactivation status in PBMC of HIV-1-positive immunocompromised patients without PML. DESIGN: A group of 82 HIV-1-positive immunocompromised patients who did not have PML were compared with 10 patients with AIDS and PML and with 69 HIV-1-positive immunocompetent patients without PML. METHODS: DNA and total RNA were extracted from PBMC. The presence of JCV DNA was demonstrated by a semi-nested polymerase chain reaction (PCR). By using primer pairs specific for an early gene,T, and a late gene, VP1, the expression of both early and late gene mRNA in PBMC could be identified using reverse transcriptase (RT) PCR. RESULTS: JCV DNA was detected by PCR in 17.4% of 69 HIV-1-positive immunocompetent patients, in 23.2% of 82 HIV-1-positive immunocompromised patients, and in 60% of 10 patients with AIDS and PML. No correlation could be drawn between the detection of JCV DNA in the PBMC and the clinical or biological status of the HIV-1-positive patients. By using RT-PCR procedures, no expression of JCV early and late mRNA in PBMC was found in any patients. CONCLUSIONS: JCV DNA is detectable in the PBMC of 20.5% of 151 HIV-1-infected patients independently of the CDC (Centers for Disease Control and Prevention) stages of the infection. Moreover, our results suggest that active replication of JCV in PBMC appears to be absent or at least a very rare event in HIV-1-positive immunocompromised patients with and without PML.

Soluble tumor necrosis factor receptor type II (sTNFRII) in HIV-infected patients: relationship with the plasma level of HIV-1 RNA.

The value of soluble receptor for tumor necrosis factor type II (sTNFRII) as a strong and early predictor of HIV disease progression was suggested. Recently it has been reported that sTNFRII may provide an indication of the HIV load. In this work we focused on the relationship between sTNFRII and HIV burden in 95 HIV-1+ patients without AIDS grouped according to the 1993 classification of the CDC as group A, n = 55, and group B, n = 40. Compared with healthy controls, higher values of sTNFRII were obtained in all groups of HIV-1 infected patients (P < 0.001), but we found no inverse correlation between sTNFRII and CD4+ lymphocyte counts in CDC group A and B of the disease, and no correlation with log RNA copy number in patients with CD4 T-cell counts > 499/microl. A correlation was obtained between sTNFRII and the viral load in patients with CD4 T-cell counts ranging from 200 to 499/microl, but only in CDC group B patients (P < 0.01, n = 26). There was no correlation between the variations of sTNFRII and HIV-1 RNA levels in 19 CDC group A and 15 CDC group B clinically stable patients in the course of a short follow up. The plasma level of sTNFRII do not appear as a valuable surrogate marker of the plasma level of HIV-1 RNA in patients. Further investigations are needed to define the mechanism of the raised level of sTNFRII in HIV-1 infected patients.

In HIV-1-infected patients, plasma levels of HIV-1 RNA are inversely correlated with IFN-alpha responsiveness of whole-blood cultures to sendai virus.

BACKGROUND: A diminished or totally blocked IFN-alpha production in cells from HIV-1-infected patients has been reported. OBJECTIVE: To investigate the relationship between the decreased in vitro production of IFN-alpha and the plasma level of HIV-1 RNA. STUDY DESIGN: Whole blood samples of 39 healthy subjects and 44 HIV-1-infected patients were incubated in the presence of Sendai virus for 24 h. IFN-alpha contained in supernatants was assayed by using an immunochemical method (DELFIA) and by using an antiviral assay. Plasma HIV-1 RNA was measured by the Amplicor HIV-1 monitor test. RESULTS: The levels of IFN-alpha obtained were significantly lower in cultures from HIV-1 infected patients than in control subjects (P<0.0001). The antiviral activity in supernatants of Sendai virus-activated whole-blood cultures, assayed by protection of MDBK cells against vesicular stomatitis virus (VSV), was significantly lower in cultures from HIV-1 infected patients than in corresponding controls (P<0.0001). IFN-alpha values determined by DELFIA and those determined by bioassay were significantly correlated. In vitro production of IFN-alpha by whole-blood cultures correlated well with the plasma levels of HIV-1 RNA (P<0.001). CONCLUSIONS: In HIV-infected patients an increased rate of HIV-1 replication is associated with reduced responsiveness to induction of IFN-alpha by indicator virus, suggesting that HIV-1 replication causes impaired production of IFN-alpha by blood cells or vice-versa.

Combination of whole blood culture and a rapid and sensitive cell assay for the determination of the cytopathogenicity of human immunodeficiency virus type-1 isolates.

It has been reported that in vitro biological properties of human immunodeficiency virus type 1 (HIV-1) isolates from patients are correlated with the prognosis of HIV-1 infection. A rapid assay was developed to study the phenotype of HIV-1 isolates. The P4 cell line is a HIV-1 infectible Hela CD4 cell carrying the bacterial LacZ gene under the control of the HIV-1 LTR (long terminal repeat). Conventional peripheral blood mononuclear cells (PBMCs) co-culture and heparinized whole blood (HWB) co-culture with normal PBMCs were used for HIV-1 isolated strains from 17 HIV-1-infected patients. The sensitivity of P4 cells was higher than that of MT-2 cells for detecting syncytia induced by HIV-1LAI (lymphadenopathy-associated virus). Like MT-2 cells, P4 cells enable the detection of syncytium inducing strains isolated in peripheral blood mononuclear cells (PBMCs) and HWB cultures. HIV-1 isolates with both culture methods from certain patients induced cytolysis without syncytium in P4 cells but had no cytopathic effect on MT-2 cells. The experiments are in favour of the direct effect of HIV-1 isolates of these patients in the lysis of P4 cells but its mechanism has not been elucidated. It was shown that the combination of whole blood culture for HIV-1 isolation and phenotype study with P4 cell assay is rapid and sensitive and could be used to monitor HIV-1-infected patients.