Marie-Hélène Schrive

HIV type 1 isolates from 200 untreated individuals in Ho Chi Minh City (Vietnam): ANRS 1257 Study. Large predominance of CRF01_AE and presence of major resistance mutations to antiretroviral drugs.

HIV-1 isolates from 200 untreated patients recruited in 2001 and 2002 in the south part of Vietnam and particularly in Ho Chi Minh City were sequenced in the RT, protease, and env genes. Out of 200 isolates 198 belonged to CRF01_AE while only one subtype B and one intersubtype (B-CRF01_AE) recombinant could be observed. Of the isolates 6.5% had major resistance mutations to antiretroviral drugs.

New Molecular Assays to Predict Occurrence of Cytomegalovirus Disease in Renal Transplant Recipients

Thirty renal transplant recipients, after transplantation, were tested weekly with the following assays: cytomegalovirus (CMV) antigenemia (pp65 Ag), plasma qualitative Amplicor CMV (P-AMP), plasma and peripheral blood leukocyte quantitative Amplicor CMV monitor (P- and PBL-CMM), peripheral blood leukocyte (PBL) quantitative Quantiplex bDNA CMV, version 2.0 (bDNA), and whole-blood Nuclisens pp67 CMV (pp67). Eleven patients developed symptomatic CMV disease, and 7 developed asymptomatic CMV infection. For prediction of CMV disease, the sensitivity, specificity, and positive and negative predictive values, respectively, were as follows: 100%, 63%, 61%, and 100% for pp65 Ag; 100%, 42%, 50%, and 100% for bDNA; 91%, 47%, 50%, and 90% for PBL-CMM; 55%, 74%, 55%, and 74% for P-AMP; 55%, 74%, 55%, and 74% for P-CMM; and 64%, 79%, 64%, and 79% for pp67. First positive results in PBL were obtained 9-10 days before symptoms of CMV disease, compared with 5-6 days in plasma and 0 days in whole blood. PBL assays appear to be more appropriate than plasma assays when pre-emptive therapy is required to prevent the rapid progression from the first detection of the virus to CMV disease.

Cell-associated HIV-1-DNA quantitation after highly active antiretroviral therapy-treated primary infection in patients with persistently undetectable plasma HIV-1 RNA.

ObjectiveTo determine the usefulness of cell-associated HIV-1-DNA quantification during the follow-up of highly active antiretroviral therapy (HAART)-treated primary-infected patients with persistently undetectable plasma RNA loads. Patients and methodsIn 27 patients given HAART within a median of 24 days after symptomatic primary HIV infection, plasma and peripheral blood mononuclear cell (PBMC) HIV-1 RNA were less than 50 copies/ml and less than 50 copies/106 cells after 18 months of treatment. HIV-1 RNA and DNA were quantified every 6 months in PBMC in these 27 patients, 14 of whom accepted excision lymph node biopsy after month 18 for HIV-1-RNA and -DNA quantification in lymph node mononuclear cells (LNMC). ResultsThe median decreases in plasma HIV-1 RNA, PBMC HIV-1 RNA and DNA over the 18 months of follow-up were 3.6 log (P < 0.005), 1.1 log (P < 0.05), and 1.0 log (P < 0.001), respectively. HIV-1 DNA was detected in 92.3% of PBMC samples at baseline and at month 18. In LNMC, 100% of samples were detectable for HIV-1 DNA. ConclusionIn this highly selected population of patients with excellent plasma virological response under HAART, HIV-1 DNA showed a progressive decrease but was still detectable in 92.3% of samples at month 18, whereas all LNMC samples tested scored positive for HIV-1 DNA. The utility of proviral HIV-1-DNA monitoring was not clearly demonstrated in this 18-month follow-up of HAART-treated primary-infected patients. However, this finding could be reconsidered when using other therapeutic strategies such as structured treatment interruptions, reinforced treatment or additive immunotherapy.

Quantitation of HIV-1 RNA in dried blood and plasma spots

Dried blood spots (DBSs) and dried plasma spots (DPSs) are an attractive method for serological and molecular diagnosis of HIV infection. Recently, Youngpairoj et al. [Youngpairoj, A.S., Masciotra, S., Garrido, C., Zahonero, N., de Mendoza, C., Garcia-Lerma, J.G., 2008. HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C. J. Antimicrob. Chemother. 61, 1217-1220] showed that HIV-1 can be genotyped efficiently from DBS specimens stored at 4 degrees C for 1 year. The viral load obtained from DBS and DPS samples was compared with that obtained from plasma samples. A total of 86 samples was prepared from people infected with HIV subtype B or non-B and spotted on 903 filter papers stored with desiccant at 4 degrees C. RNA was extracted using the QIAamp Viral RNA mini kit (Qiagen, Courtaboeuf, France). RNA from DBS or DPS samples was quantified in accordance with the Agence Nationale de Recherche sur le SIDA (ANRS, AC11, Paris, France) assay for HIV-1 quantitation. When the mean viral load of plasma samples and DPS samples or plasma samples and DBS samples were compared, there were no significant differences. The overall data showed that although the sensitivity threshold of the assays was different, there was a correlation between the three specimen types and that DBS and DPS samples can be routinely used for viral load quantification particularly in resource-limited settings.

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

Analysis of HIV-1 expression in vivo with in situ hybridization and the polymerase chain reaction

The objective of the present study was to compare the data of in situ hybridization (ISH), RNA polymerase chain reaction (PCR/RNA) and p24 core antigen (p24 Ag) enzyme immunoassay (EIA) for the detection of HIV-1 expression in peripheral blood mononuclear cells (PBMCs) and in plasma of infected patients at various CDC stages. PBMCs of 24 patients mostly of CDC stage II were obtained from heparinized blood samples, cytocentrifuged and hybridized with a (35S) labelled single-stranded RNA probe specific for gag-pol of LAVBru HIV-1 allowing the detection of genomic and/or messenger RNA. The corresponding plasma samples were used for the determination of p24 Ag by EIA and detection of HIV-1 genomic RNA by RT-PCR using specific primers in the LTR, gag and env regions. Whereas p24 was detected in only six out of 24 patients, both ISH and PCR/RNA enabled the detection of viral RNAs in more than 60% of the patients; cumulation of positive results of ISH and RT-PCR showed that 100% of patients at stage IV and 83% of patients at stages II/III have molecular signs of HIV expression therefore indicating that transcription of the provirus is a highly frequent event, even in the early stages of the disease, and, pleading for undertaking a very early antiviral chemotherapy.

Pol Bootscanning Analysis of HIV Type 1 Can Exhibit Unexpected Recombinations

In France the recommendation is to sequence the RT gene of HIV-1 isolates prior to initiation of antiretroviral therapy. The data are routinely used for molecular characterization of the viruses yielding the subtype or CRF of the isolates investigated together with the absence or presence of drug resistance mutations. In this study, we performed bootscanning analysis on the whole pol gene, in which in vitro and in vivo intersubtype recombination has been reported to occur frequently. We showed that out of 15 HIV-1 isolates, two exhibited a recombination unexpected by this routine sequencing method.