Fetal cerebral hemorrhage due to X-linked GATA1 gene mutation.

We report a multiplex family with a GATA1 gene mutation responsible for a massive fetal cerebral hemorrhage occurring at 36 weeks. Two other stillbirth cousins presented with fetal hydrops and congenital hemochromatosis' phenotype at 37 and 12 weeks of gestation. Molecular screening revealed the presence of a c.613G>A pathogenic allelic variation in exon 4 of GATA1 gene in the 3 male siblings and their carrier mothers. The diagnosis of a GATA1 gene mutation may be suspected in cases of male fetuses with intracerebral bleeding, particularly if a history of prior fetal loss(es) and mild maternal thrombocytopenia are also present.

Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer.

Inherited red blood cell (RBC) membrane disorders, such as hereditary spherocytosis, elliptocytosis and hereditary ovalocytosis, result from mutations in genes encoding various RBC membrane and skeletal proteins. The RBC membrane, a composite structure composed of a lipid bilayer linked to a spectrin/actin-based membrane skeleton, confers upon the RBC unique features of deformability and mechanical stability. The disease severity is primarily dependent on the extent of membrane surface area loss. RBC membrane disorders can be readily diagnosed by various laboratory approaches that include RBC cytology, flow cytometry, ektacytometry, electrophoresis of RBC membrane proteins and genetics. The reference technique for diagnosis of RBC membrane disorders is the osmotic gradient ektacytometry. However, in spite of its recognition as the reference technique, this technique is rarely used as a routine diagnosis tool for RBC membrane disorders due to its limited availability. This may soon change as a new generation of ektacytometer has been recently engineered. In this review, we describe the workflow of the samples shipped to our Hematology laboratory for RBC membrane disorder analysis and the data obtained for a large cohort of French patients presenting with RBC membrane disorders using a newly available version of the ektacytomer.

A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen.

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 µm drove flow into mechanical filtering units where RBCs flew either slowly through 5- to 2-µm-wide slits or rapidly along 10-µm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.

Low frequency of CXCR4-using viruses in patients at the time of primary non-subtype-B HIV-1 infection.

We used genotypic and phenotypic assays to estimate the frequency of X4/DM viruses in 131 patients infected with non-subtype-B viruses at the time of primary HIV-1 infection (PHI). All patients were enrolled in the French PRIMO Cohort from 1996 to 2007. Most strains belonged to CRF02_AG (51.1%) and subtype A (14.5%). Sixteen viruses (12.2%) were classified as CXCR4 tropic ("X4 strains") by the combined criteria of amino acids 11 and 25 of the V3 loop (11/25) and net charge rules and/or the SVMgeno2pheno(10%) algorithm: 6 strains by the combined genotypic rule, 7 by the SVMgeno2pheno(10%) algorithm, and 3, clustering in subtype D, by both algorithms. However, only one strain (0.8%), belonging to subtype A, was defined as a dual-tropic (DM) virus by the phenotypic assay. The 67 CRF02_AG strains included 2 classified as X4 strains by the combined genotypic rule (3%) and 2 others classified as X4 strains by SVMgeno2pheno(10%) (3%), but none of these 4 strains was an X4 or DM strain according to the phenotypic assay. These results suggest that the cellular virus reservoir was established with X4 strains in very few non-subtype-B-infected patients at the time of PHI. Genotypic predictions can overestimate the proportion of non-subtype-B X4 viruses at PHI.

Despite being highly diverse, immunovirological status strongly correlates with clinical symptoms during primary HIV-1 infection: a cross-sectional study based on 674 patients enrolled in the ANRS CO 06 PRIMO cohort.

OBJECTIVES: To analyse immunovirological status during primary HIV-1 infection (PHI) according to contemporary clinical status and time since infection. METHODS: Plasma HIV-RNA and peripheral blood mononuclear cell (PBMC) HIV-DNA levels and CD4 cell counts were determined at enrolment in the ANRS PRIMO cohort. Time since infection was estimated based on both the number of antibodies on western blot at enrolment (0-1, 2-4 or > or =5 specific antibodies) and the estimated interval between infection and enrolment based on clinical and epidemiological features. Patients were classified according to the presence or absence of clinical symptoms at enrolment. RESULTS: Between 1996 and 2006, 674 patients were enrolled an estimated median of 47 days after infection. Median marker values were as follows: HIV-RNA 5.10 log(10) copies/mL (range <1.70-8.33); HIV-DNA 3.30 log(10) copies/10(6) PBMCs (<1.84-4.93); and 506 CD4 cells/mm(3) (40-1542). Median HIV-RNA and PBMC HIV-DNA levels were significantly higher in patients with 0 or 1 specific antibody (n = 71) than in patients with 2-4 (n = 228) or > or =5 antibodies (n = 375). Symptomatic patients had significantly higher HIV-RNA and PBMC HIV-DNA levels and lower CD4 cell counts. However, 10% of symptomatic patients recruited shortly after infection had favourable immunovirological status. CONCLUSIONS: Plasma HIV-RNA, PBMC HIV-DNA and CD4 cell count values were highly diverse and correlated strongly with clinical status during PHI. Early diagnosis was not always associated with severe PHI. Combining PBMC HIV-DNA with HIV-RNA, CD4 cell count and clinical symptoms would have allowed identification of 179 patients (26.5%) at high risk of rapid disease progression who did not meet current guidelines for early treatment initiation.

Undetectable HIV-1 RNA load with the Cobas TaqMan v1.0 in a patient diagnosed at the time of primary HIV-1 infection.

Diagnosis of primary HIV-1 infection is challenging due to the presence of a serological window; thus, HIV-1-RNA quantitation and/or measurement of p24 antigenemia are recommended in such cases. A patient was diagnosed at the time of primary HIV-1 infection, he harbored a CFR02_AG subtype virus; quantitation of plasma HIV-1-RNA yielded an undetectable result according to one commercial assay, while HIV-1-RNA was detectable when measured with three other assays.

Red Blood Cell AE1/Band 3 Transports in Dominant Distal Renal Tubular Acidosis Patients.

INTRODUCTION: Anion exchanger 1 (AE1) (SLC4A1 gene product) is a membrane protein expressed in both kidney and red blood cells (RBCs): it exchanges extracellular bicarbonate (HCO3 -) for intracellular chloride (Cl-) and participates in acid-base homeostasis. AE1 mutations in kidney α-intercalated cells can lead to distal renal tubular acidosis (dRTA). In RBC, AE1 (known as band 3) is also implicated in membrane stability: deletions can cause South Asian ovalocytosis (SAO). METHODS: We retrospectively collected clinical and biological data from patients harboring dRTA due to a SLC4A1 mutation and analyzed HCO3 - and Cl- transports (by stopped-flow spectrophotometry) and expression (by flow cytometry, fluorescence activated cell sorting, and Coomassie blue staining) in RBCs, as well as RBC membrane stability (ektacytometry). RESULTS: Fifteen patients were included. All experience nephrolithiasis and/or nephrocalcinosis, 2 had SAO and dRTA (dRTA SAO+), 13 dominant dRTA (dRTA SAO-). The latter did not exert specific RBC membrane anomalies. Both HCO3 - and Cl- transports were lower in patients with dRTA SAO+ than in those with dRTA SAO- or controls. Using 3 different extracellular probes, we report a decreased expression (by 52%, P < 0.05) in dRTA SAO+ patients by fluorescence activated cell sorting, whereas total amount of protein was not affected. CONCLUSION: Band 3 transport function and expression in RBCs from dRTA SAO- patients is normal. However, in SAO RBCs, impaired conformation of AE1/band 3 corresponds to an impaired function. Thus, the driver of acid-base defect during dominant dRTA is probably an impaired membrane expression.

High frequency of X4/DM-tropic viruses in PBMC samples from patients with primary HIV-1 subtype-B infection in 1996-2007: the French ANRS CO06 PRIMO Cohort Study.

OBJECTIVES: To estimate the frequency of viruses with X4 or dual-X4/DM tropism from peripheral blood mononuclear cells (PBMCs) of 390 human immunodeficiency virus type 1 (HIV-1) subtype-B patients diagnosed at the time of primary HIV-1 infection (PHI) between 1996 and 2007 and enrolled in the PRIMO Cohort. METHODS: V3 loop sequences were amplified from HIV-1-DNA and analysed with a combination of five genotypic rules to predict tropism: (i) the '11/25 rule'; (ii) the net charge rule; (iii) the PSSM(X4/DM) algorithm; (iv) the PSSM(SI/NSI) algorithm; and (v) the SVM(Geno2pheno) algorithm. RESULTS: A high proportion (62/390, 15.9%) of patients harboured X4/DM-tropic viruses. This prevalence was stable over time: 18.1% before 2003 versus 14.8% since 2003. No difference according to HIV tropism was noted in HIV-RNA levels, CD4 cell count, time between infection and enrolment, and HIV infection risk factor. The frequency of X4/DM-tropic virus was similar among patients infected with a resistant virus (12/62, 19.4%) compared with patients harbouring wild-type strains (50/328, 15.2%). CONCLUSIONS: This large French epidemiological study evidenced a high proportion of patients (15.9%) harbouring X4/DM-tropic viruses in PBMCs at the time of PHI, suggesting the existence of a cellular X4/DM viral reservoir that could persist for lengthy period of time. Several reports identified that HIV-1 CXCR4 usage was more frequent among patients who developed AIDS and was a powerful predictor of the response to antiretrovirals. Further studies are needed to evaluate the impact of such strains on the outcome of HIV disease, when they are detected at the time of primary infection.

New and old complex recombinant HIV-1 strains among patients with primary infection in 1996-2006 in France: the French ANRS CO06 primo cohort study.

BACKGROUND: Prevalence of HIV-1 non-B subtypes has increased overtime in patients diagnosed at the time of primary infection (PHI) in France. Our objective was to characterize in detail non-B strains which could not be genetically classified into the known subtypes/Circulating Recombinant Forms (CRFs). METHODS: Among 744 patients enrolled in the ANRS PRIMO Cohort since 1996, 176 (23.7%) were infected with HIV-1 non-B strains. The subtype/CRF could not be identified in RT for 15 (2%). The V3-V5 env region was sequenced and 3 strains (04FR-KZS, 06FR-CRN, 04FR-AUK) were full-length sequenced. Phylogenetic and bootscan analyses were used to characterize the mosaic structures. RESULTS: Among V3-V5 sequences, 6 were divergent A, 2 distantly related to E or D, 2 C, 1 B and 2 remained unclassified. 04FR-KZS, isolated in a Congolese woman infected in France, clustered with 2 previously described viruses from the Democratic Republic of Congo. They represent CRF27_cpx involving A/E/G/H/J/K/U subtypes. 06FR-CRN, isolated in a homosexual Caucasian patient, was a B/C/U recombinant involving a Brazilian C strain. 04FR-AUK, isolated in a Congolese patient infected in France, was a A/K/CRF09/U recombinant clustering from gag to vif with HIV-1 MAL. Others PHI were further observed in 2006-2007 with 1 KZS and 5 CRN-like viruses, suggesting their spread in France. CONCLUSION: This study illustrates the increasing HIV-1 diversity in France associating new (06FR-CRN) and old (CRF27_cpx and "MAL-like" 04FR-AUK) strains, which are rare in their region of origin but may have a possible founder effect in France. Our results strengthen the French guidelines recommending viro-epidemiological surveillance of HIV-1 diversity.

Absence of HIV-1 shedding in male genital tract after 1 year of first-line lopinavir/ritonavir alone or in combination with zidovudine/lamivudine.

BACKGROUND: New strategies such as boosted-protease inhibitor (PI) monotherapy are being investigated. However, a concern remains regarding the efficacy of this strategy in viral sanctuaries such as the male genital tract. More than 80% of untreated HIV-infected men have detectable HIV-RNA in semen and such a strategy could favour local selection of resistant variants, given the poor penetration of most PIs in semen. OBJECTIVES: To evaluate the impact of a first-line lopinavir/ritonavir alone or standard triple combination on HIV-1 shedding in the genital tract. METHODS: HIV-1-infected men enrolled in the Monark randomized trial were eligible for the present study after 48 weeks of a first-line lopinavir/ritonavir alone or in combination with zidovudine and lamivudine. Single-paired samples of blood and semen were collected at week 48. Blood plasma HIV-RNA and seminal plasma HIV-RNA were measured at week 48. Lopinavir and ritonavir concentrations were measured in blood and in semen at week 48 by high-performance liquid chromatography. RESULTS: Ten patients were included: five of them received lopinavir/ritonavir monotherapy and five received a triple combination. At week 48, all patients had blood plasma HIV-RNA <1.7 log(10) copies/mL. Median lopinavir and ritonavir concentrations were within the expected therapeutic target range in blood plasma (4896 and 130.5 ng/mL, respectively), whereas both lopinavir and ritonavir were undetectable in all seminal plasma samples (<30 ng/mL). All 10 patients had undetectable seminal plasma HIV-RNA at week 48 (<2.3 log(10) copies/mL). CONCLUSIONS: No local viral production was evident in semen, despite the local absence of therapeutic antiretroviral drug concentrations in the five patients receiving lopinavir/ritonavir alone.

HIV-1 DNA for the measurement of the HIV reservoir is predictive of disease progression in seroconverters whatever the mode of result expression is.

BACKGROUND: HIV-1 DNA levels, reported as copies/10(6) peripheral blood mononuclear cells (PBMC), are very predictive of disease progression in seroconverters, independently of CD4(+)T cell count and HIV-RNA. Previously, HIV-DNA levels have sometimes been reported by other means: copies/10(6) CD4(+)T cells, reflecting the proportion of infected cells; or copies/mL whole blood, reflecting the global blood reservoir size. OBJECTIVES: We investigated if the predictive value over the natural course of the disease depends on how the results are reported. STUDY DESIGN: Results reported as HIV-DNA copies/10(6) PBMC were converted to copies/10(6) CD4(+)T cells or to copies/mL whole blood for 422 seroconverters included in the French SEROCO cohort (ANRS). RESULTS: The three methods for reporting HIV-DNA levels yielded different ranges, but these values were highly correlated. The level of HIV-DNA during the seroconversion period was strongly associated with disease progression in all three reporting methods. CONCLUSIONS: This reinforces the value of HIV-DNA quantification in physiopathological and therapeutical studies, particularly in an era of research aimed at diminishing the HIV reservoir. Even if blood represents a small part of this reservoir, HIV-DNA in blood is a simple marker that provides an informative picture of the global reservoir and is strongly predictive of disease progression.

Response to HAART in French patients with resistant HIV-1 treated at primary infection: ANRS Resistance Network.

OBJECTIVE: The aim of the study was to analyse the response to highly active antiretroviral therapy (HAART) initiated at the time of primary HIV infection (PHI) in patients infected with a virus resistant to > or = 1 drug of their treatment compared with patients infected with a wild-type virus. METHODS: We analysed data from 350 patients who were enrolled from 1996-2004 in the French ANRS PRIMO Cohort or in the ANRS Resistance Group and treated with HAART during PHI. During the study period, HAART was initiated before the result of the genotypic resistance test was available. We compared patients infected with a virus resistant to > or = 1 drug of their regimen (GR group, n = 46) with patients harbouring a wild-type virus (WT group, n = 304). Virological and immunological response to treatment according to drug-resistance profile was analysed 3 months and 6 months after HAART initiation. RESULTS: In GR and WT groups, HIV RNA level was < 400 copies/ml in 68% and 83% (P = 0.02) and < 50 copies/ml in 23% and 40% (P = 0.08) 3 months after HAART initiation. In multivariable logistic regression taking into account gender, age, boosted PI regimen, plasma HIV RNA and CD4+ T-cell count at HAART initiation, patients with virus resistant to > or = 1 drug of their regimen were significantly less likely to achieve undetectable viral load at month 3 (odds ratio 0.32, 95% confidence interval 0.15-0.72) than the others. This difference was sustained up to month 6. CONCLUSION: In this large cohort of HAART-treated PHI-patients, the presence of drug resistance mutations led to suboptimal response to early therapy.

HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time.

OBJECTIVE: Characterization of the early establishment of the viral reservoir in patients acquiring resistant strains at primary HIV-1 infection (PHI), and longitudinal analysis of resistance mutations in circulating virions and intracellular HIV strains. PATIENTS AND METHODS: Drug-resistance was compared between HIV RNA and peripheral blood mononuclear cell (PBMC)-HIV DNA at the time of PHI in 44 patients enrolled in the Primo Cohort and harbouring plasma HIV-1 resistant to at least one antiretroviral drug. Longitudinal monitoring of viral load and resistance genotype was performed in plasma-HIV RNA and PBMC HIV DNA for at least 24 months in a subset of 10 patients. Phylogenetic analysis of HIV DNA protease gene clones was used to explore the diversity of quasi-species at baseline. RESULTS: Baseline resistance profile was identical in paired HIV RNA and PBMC HIV DNA for all 44 patients. All resistance-associated mutations persisted in plasma and PBMC over 2 years in the five untreated patients. Of the five patients started on empirical HAART, two achieved undetectable HIV RNA at month 6, with long-term persistence of archived drug-resistance mutations in PBMC HIV DNA. Virological failure was observed in the other three patients, resulting in the accumulation of additional drug-resistance mutations in HIV RNA and HIV DNA for two of them. Phylogenetic analysis of HIV DNA clones showed highly homogenous and exclusively resistant quasi-species in the cellular reservoir at baseline. CONCLUSION: HIV resistant strains acquired at the time of PHI massively fuel the cellular reservoir, and their prolonged persistence is supported by the early expansion of a dominant homogenous and resistant viral population. Results in treated patients showed that classical empirical triple-combination may be suboptimal.

CD4 cell count and HIV DNA level are independent predictors of disease progression after primary HIV type 1 infection in untreated patients.

BACKGROUND: Treatment initiation at the time of primary human immunodeficiency virus (HIV) type 1 (HIV-1) infection has become less frequent in recent years. METHODS: In the French prospective PRIMO Cohort, in which patients are enrolled at the time of primary HIV-1 infection, 30% of the 552 patients recruited during 1996-2004 did not start receiving antiretroviral treatment during the first 3 months after diagnosis. We analyzed the patients' clinical and immunological outcomes and examined potential predictors of disease progression. Progression was defined as the occurrence of an acquired immunodeficiency syndrome (AIDS)-related clinical event or a CD4 cell count <350 cells/mm3. RESULTS: Fifty-six (34%) of the untreated patients experienced immunological progression during a median duration of follow-up of 24 months, and 1 of these patients had an AIDS-related event. The estimated risks of progression were 25%, 34%, and 42% at 1, 2, and 3 years after enrollment, respectively. Compared with patients who did not have progression, those with progression had significantly lower CD4 cell counts at diagnosis (455 vs. 738 cells/mm3), higher plasma HIV RNA levels (4.9 vs. 4.5 log10 copies/mL), and higher HIV DNA levels (3.3 vs. 3.0 log(10) copies/10(6) peripheral blood mononuclear cells [PBMCs]). All 3 parameters were significantly associated with progression in univariate analysis. In multivariate analysis, only the CD4 cell count and HIV DNA level were independently predictive of disease progression (relative hazard for CD4 cell count, 1.84 per decrease of 100 cells/mm3; relative hazard for HIV DNA level, 2.73 per increase of 1 log(10) copies/10(6) PBMCs). CONCLUSIONS: Both a low initial CD4 cell count and a high HIV DNA level are predictive of rapid progression of untreated primary HIV-1 infection. Affected patients may therefore benefit from close clinical and laboratory monitoring and/or early administration of treatment.

A virologic survey of patients admitted to a critical care unit for acute cardiorespiratory failure.

OBJECTIVE: To document the prevalence of respiratory virus infections in patients with chronic cardiac or pulmonary disorders admitted to a critical care unit for acute cardiorespiratory failure. DESIGN, SETTING, PATIENTS: The study took place in a critical care unit during two consecutive winters. All patients admitted to the unit for acute respiratory or cardiac failure were enrolled. A nasal swab was taken for polymerase chain reaction (PCR) detection of influenza virus, respiratory syncytial virus (RSV), metapneumovirus, rhinovirus, and coronavirus. RESULTS: One hundred twenty-two patients were enrolled. Their mean age was 69 years; 42% of the patients were female; the new simplified acute physiology score (SAPS II) score on admission was 35.6; 94% of patients had acute respiratory failure, 14% reported "influenza-like" illness before admission, and 11% of patients died in the unit. Twenty-one patients (17%) tested positive for a respiratory virus. The per-1,000 positivity rates were influenza virus 66, RSV 49, rhinovirus 33, metapneumovirus 16, and coronavirus 8. No link was found between virologic results and clinical outcome. A strong link was found between the rate of influenza virus positivity and the incidence of flu-like illness in the community (p=0.017). CONCLUSION: These results show that respiratory virus infection-particularly influenza virus infection during epidemic periods-is common among patients hospitalized for acute cardiorespiratory failure.

Hepatitis C virus in the semen of men coinfected with HIV-1: prevalence and origin.

OBJECTIVE: To compare the prevalence of hepatitis C (HCV) RNA in semen from men infected with HCV and those coinfected with HIV-1/HCV and to study the origin of HCV shed in semen. DESIGN: Two prospective studies (HC EP09 and BINECO) included 120 HCV-positive men, 82 coinfected with HIV-1; all had positive HCV RNA detection in blood. METHODS: Paired blood and semen samples were collected for HCV RNA detection and quantification in seminal plasma and in blood serum; repeated semen samples were obtained for 45 men. HCV RNA was sought in spermatozoa and non-sperm cells. Phylogenetic analysis of the HVR-1 region of HCV compared the quasispecies in blood serum and seminal plasma of two men. RESULTS: HCV RNA was more frequently found in the semen of men coinfected with HIV-1 (37.8%) than in those with only HCV infection (18.4%) (P = 0.033). HCV RNA detection in semen was intermittent and was positive in at least one semen sample of 42.8% of HIV-1/HCV-coinfected men who provided repeated samples. Men with HCV-positive semen had significantly higher HCV load in blood than men with HCV-negative semen (P = 0.038). Phylogenetic comparison of HCV quasispecies in blood and in semen showed no evidence of HCV replication in genital leukocytes; however, a phenetic structure was observed between compartments (P < 0.001). CONCLUSIONS: HCV particles in semen originate from passive passage from blood, with preferential transfer of some variants. Nearly half of HIV-1/HCV-coinfected men may intermittently harbour HCV in their semen. Recommendations of protected sex for HIV-infected individuals should be reinforced.

Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.

Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane, a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry, electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane proteins.

X4 tropic multi-drug resistant quasi-species detected at the time of primary HIV-1 infection remain exclusive or at least dominant far from PHI.

Our objective was to analyze the evolution of resistance mutations (RM) and viral tropism of multi-drug-resistant (MDR) strains detected at primary HIV-1 infection (PHI). MDR HIV strain was defined as the presence of genotypic resistance to at least 1 antiretroviral of the 3 classes. Tropism determinations (CCR5 or CXCR4) were performed on baseline plasma HIV-RNA and/or PBMC-HIV-DNA samples, then during follow-up using population-based sequencing of V3 loop and phenotypic tests. Clonal analysis was performed at baseline for env, RT and protease genes, and for HIV-DNA env gene during follow-up. Five patients were eligible. At baseline, RT, protease and env clones from HIV-RNA and HIV-DNA were highly homogenous for each patient; genotypic tropism was R5 in 3 (A,B,C) and X4 in 2 patients (D,E). MDR strains persisted in HIV-DNA throughout follow-up in all patients. For patient A, tropism remained R5 with concordance between phenotypic and genotypic tests. Clonal analysis on Month (M) 78 HIV-DNA evidenced exclusively R5 (21/21) variants. In patient B, clonal analysis at M36 showed exclusively R5 variants (19/19) using both genotypic and phenotypic tests. In patient C, baseline tropism was R5 by genotypic test and R5/X4 by phenotypic test. An expansion of these X4 clones was evidenced by clonal analysis on M72 HIV-DNA (12/14 X4 and 2/14 R5 variants). In patient D, baseline tropism was X4 with concordance between both techniques and HIV-RNA and HIV-DNA remained X4-tropic up to M72, confirmed by the clonal analysis. Patient E harboured highly homogenous X4-using population at baseline; tropism was unchanged at M1 and M18. In all patients, the initial MDR population was highly homogenous initially, supporting the early expansion of a monoclonal population and its long-term persistence. X4-tropic variants present at baseline were still exclusive (patients D and E) or dominant (at least one time point, patient C) far from PHI.

Polymorphism in Gag gene cleavage sites of HIV-1 non-B subtype and virological outcome of a first-line lopinavir/ritonavir single drug regimen.

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial.