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Recent proof-of-concept randomised controlled trials have shown a causal relation between herpes simplex virus (HSV) type 2 infection and HIV-1 replication in co-infected individuals. We explore the mechanisms that may operate to enhance reciprocal viral replication. Direct interactions could involve HIV-1-related immune deficiency, disruption of mucosal barrier by HSV infection/reactivation, HSV-induced mucosal cell recruitment, transactivation of HIV-1 replication by HSV proteins, and immune modulation by HSV decoys. Indirect interactions might coexist through disturbances of the vaginal flora during HSV shedding and systemic immune activation. In co-infected individuals, suppressive HSV treatment reduces HIV-1 genital and systemic excretion. This finding is a likely result of efficacious prevention of HSV2 reactivations, and perhaps of other herpesviruses. Strategies to control HSV2 and other herpesviruses deserve urgent attention and should become part of the HIV-1 prevention and care package.

Background: BK virus nephropathy (BKVN) is a severe complication of renal transplantation, resulting in graft loss in >50% of cases. Because patients with BKV viremia are at high risk for developing BKVN, the aim of our study was to analyze whether early reduction of immunosuppression (IS) could prevent BKVN in viremic patients., Methods: BKV viruria was prospectively screened every 3 months by real-time polymerase chain reaction during the first year after transplantation in 123 consecutive renal transplant recipients. Plasma viral load was measured by polymerase chain reaction whenever viruria was positive; in viremic patients a graft biopsy was systematically performed and IS was reduced., Results: Viruria, viremia, and BKVN occurred in 48.8%, 10.5%, and 2.4% of patients, respectively. In the 13 patients with positive viremia, initial graft biopsy showed BKVN in two. After reduction of IS in patients without BKVN, viremia disappeared in 8 of 11, decreased in 2 of 11, and increased in one patient who eventually developed BKVN. In contrast, viremia remained positive in one patient with BKVN and disappeared in the second but renal function deteriorated in both of them. Initial viral load was higher in patients who developed BKVN., Conclusion: Reduction of IS is probably an effective therapeutic option to clear viremia and prevent BKVN in viremic renal transplant patients.

Viral respiratory infections are very frequent diseases with variable degrees of severity. During the past few years, new respiratory viruses have been discovered by means of molecular biology techniques. In fact, these so far unidentified viruses have been present in humans for a long time but remained unidentified. These viruses are mainly represented by the human metapneumovirus (HMPV), the coronaviruses HCoV-NL63, HCoV-NH and HCoVHKU1, the human bocavirus (HBoV), and the newly described human polyomaviruses WU and KI. Beside these newly identified viruses, new respiratory viruses have emerged in humans within the last years. These viruses have been introduced from animal reservoirs and they generally present a high degree of pathogenicity with high level of mortality in humans. These emerging respiratory viruses are represented by the henipaviruses (Hendra and Nipah viruses), the New World hantaviruses associated with the Hantavirus Pulmonary Syndrome, the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS-CoV), and the avian influenza virus H5N1. This latter constitutes a major threat, with the risk of a murderous pandemic offering some parallels with the 1918-1919 Spanish flu which caused 20-50 millions deaths worldwide., (Copyright © 2007 Published by Elsevier Masson SAS.)

The newly identified human bocavirus is frequently detected by molecular techniques in respiratory samples from children with respiratory tract infections, but virions have not been observed so far. We report the electron microscopy observation of viral particles in nasopharyngeal aspirates previously found to be positive for human bocavirus DNA. The virions presented the expected structural characteristics of a Parvoviridae family member.

Background: Little is known about the semen shedding of human papillomaviruses (HPV) in African men., Objectives: To detect and identify HPV types in semen samples from men seeking fertility evaluation in Abidjan, Ivory Cost., Study Design: Sixty-three semen samples were tested by PCR using consensus primers and HPV types were determined by PCR product sequencing or by line probe assay (LiPA) when sequencing was inconclusive., Results: A total of 19 HPV isolates belonging to 11 HPV types were identified in semen samples from 18 (28.6%) men. Ten (55.5%) of the HPV-positive men shed oncogenic HPV types. An association of borderline significance (P=0.06) was observed between HPV semen shedding and a lower sperm count., Conclusion: HPV shedding in semen is common in African men. The relationship between HPV semen shedding and sperm quality merits to be further investigated.

The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women., ((c) 2007 Wiley-Liss, Inc.)

A new virus was recently discovered by molecular techniques in respiratory samples collected from young children with respiratory disease. This virus, which represents a new member of the Parvoviridae family is genetically related to the bovine parvovirus and the canine minute virus that belong to the Bocavirus genus. It has been classified in the Bocavirus genus and named human bocavirus (HBoV). Recent studies conducted in different countries have shown that HBoV is found in 3 to 18 %of children with respiratory disease worldwide. Genetic analysis indicate that this virus shows low genetic variability. The clinical signs observed in patients infected with HBoV are not different from those caused by other respiratory viruses.A frequent association of HBoV with other respiratory pathogens may be observed. Therefore, the exact role played by this virus in human diseases still remains unclear. Further studies including control populations are needed to ascertain the pathogenic potential of this virus.

Objective: To demonstrate a causal relationship between herpes simplex virus 2 (HSV-2) and increased genital HIV-1-RNA shedding in women on HAART., Design: A randomized, double-blind, placebo-controlled trial of herpes-suppressive therapy (valacyclovir 500 mg twice a day) in HIV-1/HSV-2-infected women taking HAART in Burkina Faso., Methods: Participants were followed for a total of 12 biweekly visits before and after randomization. The presence and frequency of genital and plasma HIV-1 RNA, and of genital HSV-2 were assessed using summary measures, adjusting for baseline values. Random effect linear regression models were used to assess the impact of treatment on genital and plasma viral loads among visits with detectable virus., Results: Sixty women were enrolled into the trial. Their median CD4 lymphocyte count was 228 cells/mul, and 83% had undetectable plasma HIV-1 RNA at baseline. Valacyclovir reduced the proportion of visits with detectable genital HSV-2 DNA [odds ratio (OR) 0.37, 95% confidence interval (CI) 0.13, 1.05], but had no significant impact on the frequency (OR 0.90, 95% CI 0.31, 2.62) or quantity (reduction of 0.33 log copies/ml, 95% CI -0.81, 0.16) of genital HIV-1 RNA. However, according to pre-defined secondary analyses restricted to women who shed HIV-1 at least once in the baseline phase, valacyclovir reduced both the proportion of visits with detectable HIV-1 shedding (OR 0.27, 95% CI 0.07, 0.99) and the quantity of genital HIV-1 RNA during these visits (-0.71 log10 copies/ml, 95% CI -1.27, -0.14)., Conclusion: HSV-2 facilitates residual genital HIV-1 replication among dually infected women taking HAART despite HIV-1 suppression at the systemic level.

A new virus has been recently identified by molecular biology technics in respiratory samples collected from young children with respiratory disease. This virus is a new member of the Parvoviridae family and has been classified in the Bocavirus genus. This virus named human bocavirus (HboV) is genetically related to the bovine parvovirus and the canine minus virus that belong to the Bocavirus genus. Recent studies conducted in different countries showed that this virus is found in 3 to 10 % of children with respiratory disease worldwide with no real seasonal distribution and that it is frequently associated with other respiratory pathogens. Genetic analysis indicated that this virus probably includes only one type. The clinical signs are not different from those caused by other respiratory viruses. The exact role played by this virus in human diseases needs to be further investigated., (Copyright © 2006 Published by Elsevier Masson SAS.)

Human bocavirus (HBoV), a new member of the genus Bocavirus in the family Parvoviridae, has been recently associated with respiratory tract infections. We report the epidemiologic and clinical features observed from a 1-year retrospective study of HBoV infection in young children hospitalized with a respiratory tract infection.

The performance characteristics of the INNO-LiPA Genotyping v2 test for human papillomavirus (HPV) identification were assessed by comparing results with those obtained by PCR product sequencing after subcloning, in genital samples from 20 highly sexually exposed African women. The INNO-LiPA HPV Genotyping v2 test identified more HPV types than subcloning/sequencing (56 versus 37, respectively). Overall, 86.5% (32/37) of the HPV types identified by subcloning/sequencing were identified by the INNO-LiPA HPV Genotyping v2 test, whereas 57.1% (32/56) of the HPV types identified by the INNO-LiPA HPV Genotyping v2 test were identified by subcloning/sequencing. Of the 20 clinical samples tested, 7 had identical types detected under both methods and a further 11 had more types detected under INNO-LiPA HPV Genotyping v2 than subcloning/sequencing. Of the remaining two samples, the same number of types were detected under both methods, but different types were detected. INNO-LiPA HPV Genotyping v2 test appears as a valid method for identifying HPV subtypes in women with multiple HPV infection.

The LCx human immunodeficiency virus (HIV) RNA Quantitative, RealTime HIV, and COBAS AmpliPrep-COBAS TaqMan assays for HIV type 1 (HIV-1) were compared for their abilities to quantitate HIV-1 RNA in plasma. High degrees of correlation and agreement were observed between the assays. Differences in HIV-1 RNA levels according to HIV-1 subtypes did not reach statistical significance.

Background: Human metapneumovirus (hMPV) is a newly recognized pathogen associated with respiratory tract disease (RTD)., Objectives: To evaluate the incidence of hMPV infection in children hospitalized with RTD and to analyze the virologic and clinical features of hMPV infection., Study Design: All children younger than 5 years of age hospitalized for RTD were included in this 1-year prospective study. hMPV was detected in nasopharyngeal secretions by reverse transcription polymerase chain reaction. The hMPV F gene amplification products were sequenced, and a phylogenetic tree was constructed. Samples were also tested for other respiratory viruses by both direct immunofluorescence assay and virus culture., Results: hMPV, detected in 50 of 589 (8.5%) children, represented the second leading cause of RTD after respiratory syncytial virus (RSV). Infections with hMPV occurred mainly between December and April. hMPV isolates clustered into the 4 subgroups (A1, A2, B1 and B2) currently recognized; the majority (72%) of hMPV isolates belonged to subgroup A1. Among the 35 children infected with hMPV alone, 23 (65.7%) had bronchiolitis, 5 (14.3%) had pneumonia, 2 (5.7%) had asthma exacerbation and 5 (14.3%) had a limited upper RTD. Fifteen (30%) of the hMPV-infected children were coinfected with RSV. As compared with children infected with hMPV or RSV alone, duration of hospitalization and requirement for supplemental oxygen were increased in the hMPV/RSV-coinfected children., Conclusions: hMPV is a frequent cause of RTD in young children. hMPV/RSV coinfection is frequent and could be more severe than a single hMPV or RSV infection.

HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

Forty-two HSV-2-seropositive women, of whom 22 were HIV-1 seropositive, were followed up weekly for 3 weeks for assessment of HIV-1 and HSV-2 genital shedding in Bobo Dioulasso, Burkina Faso. HSV-2 DNA and HIV-1 RNA were quantitated in cervicovaginal lavages enriched with a cervical swabbing (eCVL) using real-time polymerase chain reaction. HIV-1 RNA shedding was detected in eCVL from 19 of 22 (86.4%) of the HIV-1-seropositive women and HSV-2 DNA was detected in eCVL from 12 of 42 (28.6%) of the women. Compared with the HIV-1-seronegative women, the HIV-1-seropositive women showed more persistent HSV-2 genital shedding and higher HSV-2 DNA loads in eCVL. This study showed that eCVL is a reliable sampling method for detecting both viruses. Three measurement points (at weekly intervals in this study) seem to adequately allow detection of most HIV-1 or HSV-2 genital shedders.

The factors that determine the emergence of X4 isolates in some HIV-1-infected subjects are unknown. As the level of expression of CXCR4 could favor an R5 to X4 switch, quantitative flow cytometry was used to measure CXCR4 density on CD4 T cells in 200 HIV-1-positive adults, and this was compared with CD4 counts, interleukin-7 (IL-7), and RANTES (regulated on activation, normal T expressed and secreted) plasma levels and the R5/X4 virus phenotype. CD4 T-cell surface CXCR4 densities were increased in infected subjects and inversely correlated with CD4 T-cell count (r=-0.548, P<0.001). Yet, in vitro infection with either R5 or X4 strains and in vivo increases in viral load following interruption of antiretroviral treatment did not induce CXCR4 overexpression. The plasma levels of IL-7 and RANTES, 2 cytokines able to induce CXCR4 expression, did not correlate with CXCR4 density. Finally, higher CXCR4 densities were observed in patients harboring X4 strains (3300, 95% CI 2431-4169 CXCR4 molecules per cell) than in patients harboring only R5 strains (2406, 95% CI 2135-2677, P=0.027). These data suggest that CXCR4 overexpression during the course of the disease in some patients could favor the emergence of X4 strains.

The genotypic inhibitory quotient (GIQ) has been proposed as a way to integrate drug exposure and genotypic resistance to protease inhibitors and can be useful to enhance the predictivity of virologic response for boosted protease inhibitors. The aim of this study was to evaluate the predictivity of the GIQ in 116 protease inhibitor-experienced patients treated with lopinavir-ritonavir. The overall decrease in human immunodeficiency virus type 1 (HIV-1) RNA from baseline to month 6 was a median of -1.50 log(10) copies/ml and 40% of patients had plasma HIV-1 RNA below 400 copies/ml at month 6. The overall median lopinavir study-state C(min) concentration was 5,856 ng/ml. Using univariate linear regression analyses, both lopinavir GIQ and the number of baseline lopinavir mutations were highly associated with virologic response through 6 months. In the multivariate analysis, only lopinavir GIQ, baseline HIV RNA, and the number of prior protease inhibitors were significantly associated with response. When the analysis was limited to patients with more highly mutant viruses (three or more lopinavir mutations), only lopinavir GIQ remained significantly associated with virologic response. This study suggests that GIQ could be a better predictor of the virologic response than virological (genotype) or pharmacological (minimal plasma concentration) approaches used separately, especially among patients with at least three protease inhibitor resistance mutations. Therapeutic drug monitoring for patients treated by lopinavir-ritonavir would likely be most useful in patients with substantially resistant viruses.

Introduction: VZV virus-related peripheral neuropathies usually occur after shingles in adults and more rarely after chickenpox in childhood., Case Report: A 54-year-old patient presented with a right VIIth nerve palsy following a chickenpox rash and recovered after antiviral treatment. CSF analysis revealed lymphocytic meningitis and the virus was identified by PCR., Conclusions: Although previous chickenpox was not found in the patient's past history, the probability of reinfection is likely. The virus can be assumed to affect the nervous system directly; the axonal or demyelinating mechanism of the neuropathy may be discussed.

Introduction: Human metapneumovirus (hMPV) is a newly identified virus responsible for occasionally severe respiratory infections, mainly observed in children., Observation: A 59-year old man was hospitalised for respiratory problems and fever 3 days after his return from China during the epidemic of severe acute respiratory syndrome (SARS). Other than fever (>38 degrees C), the patient exhibited a dry cough, myalgias, arthralgias and mild lymphopenia and the chest radiography showed an opacity on the right lower lung. Conventional virological investigations remained negative. Detection of the novel SARS-associated coronavirus was negative but hMPV detection was positive., Discussion: The present observation indicates that hMPV may cause respiratory symptoms mimicking initial manifestations of SARS in adults visiting epidemic countries., Conclusion: hMPV testing has clinical utility in adult patients with acute respiratory tract infection.

We analysed the genetic changes in gp41 in a population of 30 heavily pretreated HIV-1-infected patients failing on a T-20-containing salvage therapy. This study confirms the key role of the number of drugs presumed to be still effective associated with T-20 in the magnitude of the virological response. Our results suggest that only HR-1 sequencing is necessary to characterize resistance to T-20, and that HR-2 domain sequencing is probably not required.

Objective: To compare the prevalence of mutations associated with resistance to antiretroviral drugs and their interpretation in patients infected with non-B HIV-1 variants versus HIV-1subtype B-infected patients with similar treatment regimens., Methods: The reverse transcriptase (RT) and protease genes of HIV-1 were sequenced, and subtypes were determined by phylogenetic analysis. Each sequence belonging to a non-B variant was matched with a sequence belonging to subtype B. Patterns of resistance mutations were interpreted in terms of drug resistance using the HIV db algorithm., Results: RT mutations M41L, L210W, and, to a lesser extent, T215Y were less prevalent in patients infected with non-B variants. This lower prevalence was associated with subtypes A (A1/A2), C, F (F1/F2), and CRF06&#95;cpx. A lower prevalence of high-level resistance to zidovudine was also observed in patients infected with these HIV-1 variants. In the protease gene, differences between patients infected with B or non-B strains were mainly observed for mutations playing a minor role in drug resistance and known to occur mainly as a natural polymorphism in non-B strains: K20R/M/I, M36I, L63P, A71V/T, and V77I. Interpretation of genotypes using the HIV db algorithm indicated that resistance to saquinavir, ritonavir, indinavir, and amprenavir was more frequently a high-level resistance for subtype B and an intermediate-level resistance for non-B variants, but this difference was only significant for amprenavir., Conclusion: Our results suggest that the genetic diversity of HIV-1 does not play a major role in the development of resistance to antiretroviral drugs.

Human cytomegalovirus (HCMV) resistance to ganciclovir results from mutations in viral phosphotransferase (UL97) and/or DNA polymerase (UL54) genes. The HCMV isolates from the blood of immunocompromised patients with persisting presence of the pp65 antigen in the blood in spite of ganciclovir therapy were tested for ganciclovir susceptibility by an immediate-early antigen plaque reduction assay, and the UL54 and UL97 genes were sequenced. Nine isolates from eight patients (six patients with acquired immune deficiency syndrome (AIDS), one liver transplant recipient and one renal transplant recipient) showed phenotypic resistance to ganciclovir. All these ganciclovir-resistant HCMV isolates harbored one or more of the following UL97 mutations: M460V, A594V, A594T, L595S, C603W, and M615V. Two isolates harbored the P522S mutation in the UL54 gene. The M615V mutation in the UL97 gene has not been reported earlier and its role in ganciclovir resistance remains to be elucidated. In ganciclovir-resistant HCMV isolates the UL54 gene was less frequently mutated than the UL97 gene. The P522S mutation was relatively frequent in UL54-mutated HCMV isolates.

Objectives: As spontaneous anti-HIV-1 antibody and IgG secretion by peripheral blood mononuclear cells (PBMC) reflect immune system activation by HIV-1 antigens, we evaluated the impact of antiretroviral therapies on HIV-1 specific and non-specific B cell responses., Methods: Anti-HIV-1 antibody and non-specific IgG were measured by ELISA in supernatants of PBMC cultured during 7 day from 30 patients initiating an antiretroviral therapy at baseline, 8, 16, 24, 36 and 48 weeks., Results: An early and sustained fall in plasma viral load to below the detection limit (20 copies/ml) was observed in 17 sustained responder patients (SR), whereas HIV-1 RNA remained detectable in 13 others incomplete responders. In both groups, HIV-1 specific antibody secretion decreased significantly in parallel with plasma viral load and polyclonal immunoglobulin production became similar to that of PBMC controls. However, HIV-1 specific antibody production became negative in only six SR, exhibiting a greater increase of CD4 T-cell counts and higher levels of the spontaneous HIV-1 specific IgA secretion at baseline than the other SR., Conclusions: Antiretroviral therapy induced a rapid and dramatic decrease of spontaneous HIV-1 specific and non-specific B cell responses. These results pointed out that HIV-1 specific antibody secretion persisted in 11 out of 17 SR patients, suggesting persistent immune system activation by residual HIV-1 antigens., (Copyright 2002 Lippincott Williams & Wilkins)

The factors governing interindividual variability in disease progression among children vertically infected with human immunodeficiency virus type 1 (HIV-1) remain unclear. Because it has recently been shown in infected adults that the density of CC chemokine receptor 5 (CCR5) molecules at the surface of nonactivated (human leukocyte antigen [HLA]-DR(-)) CD4+ T cells correlates with disease progression, the same correlation was sought in children. HLA-DR(-)CD4+ T cell surface CCR5 density was constant over time and correlated with the bioclinical stage and with the CD4 cell slope observed before antiretroviral treatment. In addition, CCR5 density was negatively correlated with the intensity of the decrease in viremia during antiretroviral therapy and was positively correlated with CD4 cell slope since birth. These results are compatible with the hypothesis that CCR5 density is a key factor governing disease progression in pediatric HIV-1 infection and, thereby, an indicator of prognosis. Moreover, they suggest that therapies aimed at reducing CCR5 accessibility should slow down HIV disease evolution in children.

It has been reported that a new pattern of mutations, E44D/A and/or V118I, in the reverse transcriptase (RT) gene of HIV-1 confers a moderate level of resistance to lamivudine in the absence of the M184V mutation. The prevalence of this mutational pattern was studied in HIV-1 isolates obtained from 280 patients. These mutations were not identified in the RT sequences from 23 antiretroviral-naive patients but were detected in 82 (31.9%) of the 257 RT sequences obtained from nucleoside reverse transcriptase inhibitors (NRTI)-experienced patients. Mutation at codon 44 was identified in 41 patients (7 mutations E44A and 34 mutations E44D), mutation V118I was identified in 73 patients and a combination of mutations at codons 44 and 118 was found in 32 patients. Multivariate analysis showed an association between the E44D/A and/or V118I mutational pattern and the RT mutations D67N, T69D, L210W, and T215Y/F. No relationship was observed between this mutational pattern and the lamivudine-specific resistance mutation M184V. The prevalence of these mutations increased significantly with the number of drug regimens experienced and a prevalence of 42.4% was observed in patients who had received >or= 4 antiretroviral regimens. A relationship was found between the E44D/A and/or V118I mutational pattern and experience with didanosine or stavudine but not with lamivudine. The results suggest that the development of the E44D/A and/or V118I mutational pattern is frequent in patients treated with NRTIs. Thymidine analogues and didanosine, but not lamivudine, could promote the development of these mutations., (Copyright 2002 Wiley-Liss, Inc.)

Objective and Design: We have recently shown that the number of CCR5 molecules at the surface of peripheral blood CD4 T cells (CCR5 density) correlates with the viral RNA plasma level in HIV-1-infected individuals. As viral load is a strong predictor of outcome in HIV infection, the present study examines the correlation between CCR5 density and HIV-1 disease progression., Methods: Using a quantitative flow cytometry assay, we measured CCR5 density in HIV-1-infected adults and control healthy volunteers. The CCR5 genotype (presence of a Delta 32 allele) was also determined., Results: CCR5 density was stable over time on non-activated, HLA-DR(-)CD4 T cells of infected individuals. In a study cohort of 25 patients, asymptomatic and non-treated, we observed a correlation between CCR5 density on HLA-DR(-)CD4 T cells and the CD4 T cell slope (P = 0.026), which was independent of the presence or absence of the Delta 32CCR5 deletion. In particular, slow progressors expressed lower CCR5 densities than non-slow progressors (P = 0.004) and non-infected control subjects (P = 0.002)., Conclusion: These results are compatible with the hypothesis that CCR5 density, which is a key factor of HIV-1 infectability, determines in-vivo HIV production, and thereby the rate of CD4 cell decline. Consequently, CCR5 density quantitation could be a new valuable prognostic tool in HIV-1 infection. Moreover, these data emphasize the therapeutic potential of treatments that reduce functional CCR5 density.

Background: Primary cutaneous CD30+ anaplastic large cell lymphoma (ALCL) is a rare subset of cutaneous lymphoma, with a much better prognosis than its nodal counterpart. The pathogenesis of both nodal and primary cutaneous CD30+ ALCL is largely unknown but experimental data support the hypothesis that the Epstein-Barr virus could play a role in the nodal subset., Objective: To evaluate the involvement of Epstein-Barr Virus (EBV) in primary cutaneous CD30+ ALCL by searching for both nucleic acids and EBV proteins in cutaneous lesions., Setting: Two University Hospitals in Southern France (secondary referral hospitals)., Patients: Eight consecutive patients with typical primary cutaneous CD30+ anaplastic large cell lymphoma were studied., Methods: Search for the presence of DNA, RNA and EBV proteins in cutaneous lesions by PCR, in situ hybridization and immunohistochemistry., Results: EBV DNA and RNA was identified in only one lesion of primary cutaneous CD30+ ALCL and in none of the normal adjacent skin samples. In situ hybridization and immunohistological studies were consistently negative in all samples., Conclusion: These results do not support an early role of EBV in the oncogenetic pathogenesis of primary cutaneous CD30+ ALCL.

Resistance-mutation patterns in the reverse transcriptase (RT) and protease genes of HIV-1 were analyzed in 22 patients who had been extensively pretreated and who failed to respond to highly active antiretroviral therapy (HAART). The number of mutations ranged from 8 to 19 (median, 13): 4 to 12 (median, 6) mutations in the RT gene, and 4 to 8 (median, 7) mutations in the protease gene. In the RT gene, the most frequent resistance mutations were found at codons 215 (100%), 41 (95%), 67 (91%), and 210 (77%). Multidrug-resistant mutation patterns including Q151M and insertion mutations at codon 69, which confer cross-resistance to the different nucleoside analogue RT inhibitors were detected in 1 and 3 patients, respectively; 1 patient with insertion mutation displayed a NGQGV [corrected] sequence at codons 67 to 70. In the protease gene, the most frequent mutations were found at codons 63 (95%), 10 (86%), 90(86%), 71(77%), 46 (50%), 36 (45%), and 84 (45%). Genotypic resistance to zidovudine, saquinavir, and indinavir was found in 100% of the patients. All patients showed also resistance or possible resistance to stavudine, abacavir, ritonavir, and nelfinavir. Mutations conferring genotypic resistance to nonnucleoside analogue RT inhibitors (NNRTIs) were found in 12 (80%) of the NNRTI-experienced patients and 1 of 7 NNRTI-naive patients. Our results indicate that failure of HAART in the patients extensively pretreated results from the multiplicity of RT and protease mutations that confer genotypic resistance to almost all available antiretroviral drugs. In these patients, genotypic resistance tests confirm the lack of alternative salvage therapy strategies based on the currently available antiretroviral drugs.

In the present study, we show that IL-2, IL-4, IL-7, and IL-15 are able to induce functional CXCR4 surface expression on resting in vitro-generated CD4+ CXCR4- CCR7+ memory T cells. Cytokine-mediated induction of CXCR4 expression was associated with an increase in CXCR4 transcription, enhanced stromal-derived factor-1-induced T cell migration in vitro, and increased susceptibility of these cells to infection with X4 strains of HIV-1. CXCR4 expression could also be induced through an alternative pathway, following coculture of these cells with CD40-activated, autologous, CD34+ progenitor-derived dendritic cells. Although these dendritic cells express transcripts for IL-7 and IL-15, addition of neutralizing anti-IL-7R and IL-15 mAbs did not block induction of CXCR4 expression. Indeed, dendritic cell-mediated up-regulation of CXCR4 expression was found to depend on CD40/CD154 and CD134/CD134L interactions. Whereas activated autologous dendritic cells induced the expression of both CXCR4 and CD25 on a portion of CCR7+ memory T cells, concomitant CD3-mediated activation of these cells further enhanced CD25 expression, but, in contrast, prevented induction of CXCR4 expression. This observation suggests that triggering of the CD134 and CD154 molecules, in contrast to TCR/CD3 complex-mediated stimulation, results in simultaneous T cell activation and CXCR4 expression. Taken together, these results show that common gamma-chain-interacting cytokines as well as signals mediated via noncognate interactions between activated dendritic cells and memory T cells are involved in the up-regulation of CXCR4 expression.

We report a successful treatment of a B19 parvovirus-induced fetal hydrops diagnosed at 16 weeks of gestation. This disease could be corrected by means of a unique intraperitoneal blood transfusion performed at 18 weeks, once diagnosis was established. The delivery occurred at 36 weeks, leading to the birth of a healthy baby. This case suggests that transfusion should be attempted, as the spontaneous fetal recovery remains uncertain and shows that intraperitoneal blood transfusion is an effective therapeutic option of the B19 parvovirus-induced anemia, in the absence of a viral myocarditis., (Copyright 2000 S. Karger AG, Basel.)

The intensity of expression of the chemokine receptor CCR5 is involved in in vitro cell infectability by human immunodeficiency virus (HIV)-1 R5 isolates. Because CCR5 expression varies among individuals, the hypothesis that this expression could determine virus load in HIV-1-infected persons was tested. The mean number of CCR5 molecules per cell was measured on peripheral blood CD4+ T lymphocytes (CCR5 density) from HIV-1-infected, asymptomatic, nontreated adults. There was a strong correlation between HIV RNA plasma level and CCR5 density (P=.009) that was independent of cell activation and was not due to an HIV-induced CCR5 up-regulation. These data are compatible with the hypothesis that CCR5 density is a key factor governing cell infectability and in vivo virus production and explain the protective effect of the Delta32CCR5 deletion, which results in low CCR5 expression. CCR5 density might be of critical predictive value in HIV infection.

Objective: Assessment of genotypic changes in the reverse transcriptase gene of HIV-1 occurring in antiretroviral naive patients treated by stavudine plus didanosine combination therapy., Methods: Sequence analysis (codons 1-230) was performed after amplification of the reverse transcriptase gene from plasma samples collected at baseline and at the end of treatment from 39 previously treatment-naive patients treated for 24-48 weeks., Results: At baseline, mutations associated with zidovudine resistance were detected in plasma from two patients: Asp67Asn/Lys219Gln and Leu210Trp. Among the 39 subjects, 18 (46%) developed mutations: one developed the Val75Thr/Ala mutation, four (10%) developed a Gln151Met multidrug-resistance mutation (MDR), associated in one of them with the Phe77Leu and the Phe116Tyr MDR mutations and 14 (36%) developed one or more zidovudine-specific mutations (Met41Leu, Asp67Asn, Lys70Arg, Leu210Trp, Thr215Tyr/Phe). The development of a Met41Leu zidovudine-specific mutation was associated with the development of a Gln151Met mutation in one patient. Other reverse transcriptase mutations known to confer resistance to nucleoside analogues were not detected. At inclusion, there was no statistical difference in HIV-1 load between patients who developed resistance mutations and those who did not. RNA HIV-1 load decrease was higher (P = 0.05) in patients who maintained a wild-type reverse transcriptase genotype (-2.22 log10 copies/ml) than in patients who developed resistance mutations (-1.14 log10 copies/ml)., Conclusion: Stavudine/didanosine combination therapy is associated with emergence of zidovudine-related resistance or MDR mutations in naive patients. These findings should be considered when optimizing salvage therapy for patients who have received a treatment including stavudine/didanosine combination.

Background: Treatment of cytomegalovirus (CMV) diseases with protracted administration of ganciclovir can promote the development of resistant CMV that is associated with a poor response to therapy. It has been shown that the majority of ganciclovir-resistant CMV isolates carry mutations in the UL97 phosphotransferase gene., Objectives: To evaluate the frequency of CMV resistance to ganciclovir in patients with AIDS treated with ganciclovir and to identify the UL97 gene mutations associated with ganciclovir resistance., Study Design: Analysis of CMV blood isolates obtained over 1 year from patients treated with ganciclovir. CMV susceptibility to ganciclovir was determined by an immediate early antigen plaque reduction assay; UL97 gene mutations were identified by restriction enzyme digest analysis and sequencing., Results: Twenty-nine patients were followed-up; 17 CMV blood isolates were obtained from 10 ganciclovir-experienced patients. Thirteen (76%) of these isolates, obtained from seven (24%) patients after a median treatment duration of 5.5 months, were resistant to ganciclovir. Five of the seven patients who had a ganciclovir-resistant CMV in blood showed retinitis progression. UL97 gene mutations were identified in nine CMV isolates at codons 460 (M --> V), 594 (A --> V and A --> T), and 595(L --> S). Three patients developed a ganciclovir-resistant virus after a ganciclovir treatment shorter than 60 days (28-58 days). In another patient, we observed that ganciclovir resistance persisted 4 months after discontinuation of ganciclovir therapy., Conclusion: Our results indicate that ganciclovir resistance due to UL97 gene mutations is common in subjects with AIDS-related CMV diseases treated with ganciclovir. Detection of these mutations represents a tool for the management of patients with ganciclovir therapy.

The enzyme-linked immunospot (ELISPOT) assay was adapted to detect and enumerate HIV-1-producing cells at the single cell level. With CEM cells or peripheral blood mononuclear cells (PBMC) infected in vitro with HIV-1, the ELISPOT assay detected cells that produced HIV-1 antigens and showed that between 5.4% and 9.5% of the p24 antigen-positive CEM cells and 11.1% to 23.6% of the p24 antigen-positive PBMC were productively infected. In HIV-1-infected patients in early stage of the disease and without antiretroviral therapy, up to 4.54 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in peripheral blood and up to 277.75 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in splenic lymphoid tissue. Our results indicate that the ELISPOT assay could represent a new tool to study HIV-1 replication in vivo.

Objective: To analyse retrospectively the clinical, biological and epidemiological features of cytomegalovirus (CMV) infection in the immunocompetent host., Patients and Methods: A retrospective study was conducted in 116 cases of CMV infection (74 inpatients, 42 out-patients) collected from 1981 to 1997 in a university hospital. Diagnostic was established on serological criteria in all cases., Results: Fever was observed in all cases but one (mean duration: 21 days). The most frequent symptoms were headache (51%) and myalgia (46%). Splenomegaly was the most frequent sign (36%). Pulmonary interstitial opacities on chest x-ray were found in 8.5% of patients. Pulmonary (1 case) and neurological (2 cases) complications occurred. Concomitant HIV primary infection was observed in 2 patients. Mononucleosis and ALAT and LDH elevations were observed in 95%, 85% and 95% of cases respectively. Viremia was positive in 79% (30/38). pp65 antigenemia was useful to establish the diagnosis in 9 patients. Antibiotics were prescribed in 46% before diagnosis was established., Conclusion: CMV infection in the immunocompetent host is a frequent and rarely complicated disease. pp65 antigenemia should be evaluated more in this setting. Most hospitalizations due to this affection could be avoided.

Nuclisens HIV-1 QT is a new version of the NASBA HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma. The specificity of this assay was 100% in one laboratory and 99%-with nonrepeatability of the initial false positive-in another. The test was linear between 2.0 and 6.0 log RNA copies per ml. According to the input HIV-1 RNA concentration, accuracy varied from -0.11 to +0.10 log RNA copy per ml and precision varied from 0.66 to 0.14 log RNA copy per ml. Reproducibility decreased when the HIV-1 RNA level was near the lower limit of quantitation of the test. HIV-1 RNA could be quantitated by Nuclisens HIV-1 QT in 36% (laboratory 1) and 24% (laboratory 2) of clinical samples with HIV-1 RNA levels lower than the lower limit of quantitation by NASBA HIV-1 QT. Nuclisens HIV-1 QT was not suitable for measurement of RNA from clade G and group O HIV-1 strains.

Objective: To confirm the interest of the reverse transcription-coupled polymerase chain reaction (RT-PCR) in prenatal diagnosis of rubella., Patients and Methods: Maternal blood, amniotic fluid and foetal blood specimens collected at 21 to 24 weeks of amenorrhea from two pregnant women with serologically proven rubella infection were tested by RT-PCR and cell culture for detection of rubella virus. Rubella virus-specific IgM antibodies were also assayed in foetal blood., Results: In both cases, rubella virus was detected in foetal blood by RT-PCR and cell culture, whereas it was not detected in maternal blood. Rubella virus-specific IgM were also detected in foetal blood. In amniotic fluid specimens, rubella virus was detected by RT-PCR in one case and by cell culture in the other one. A termination of pregnancy was carried out in both cases., Comments: RT-PCR is a rapid test. It could be useful to confirm foetal infection and could represent a criterion for considering a pregnancy termination. However, this technique which is not yet standardized for detection of rubella virus, needs further validation by additional studies.

We compared the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma in the Stadi trail, which evaluated a stavudine plus didanosine combination therapy in 52 patients. HIV-1 RNA baseline values measured with AMPLICOR HIV-1 MONITOR 1.0 were significantly higher than those measured with QUANTIPLEX HIV-1 RNA 2.0, and decreases in HIV-1 RNA levels from baseline were also found to be significantly higher when measured with the AMPLICOR HIV-1 MONITOR 1.0 assay. The frequency of HIV-1 RNA levels below the lower limit of quantitation was significantly higher with QUANTIPLEX HIV-1 RNA 2.0 than with AMPLICOR HIV-1 MONITOR 1.0. Reanalysis of these results by an ultrasensitive procedure of AMPLICOR HIV-1 MONITOR 1.0 or by a modified version of the test that included additional primers adapted for non-B HIV-1 clades yielded greater differences between the QUANTIPLEX HIV-1 RNA 2.0 assay and the AMPLICOR HIV-1 MONITOR 1.0 assay. Our results indicate that a valid comparison of the virological efficacies obtained with different antiretroviral drug regimens requires the use of the same viral load quantitation procedure; further standardization between the different HIV-1 RNA quantitation kits is therefore needed.

Objective: This study was performed to investigate the hyporeactivity of purified B lymphocytes from HIV-1-infected patients., Design: Given the importance of the B-cell Ag receptor (BCR) and CD40 in B-lymphocyte activation, we assessed the capacity of purified peripheral blood B lymphocytes from HIV-1-infected patients to differentiate into Ig-secreting cells in a T-cell- and accessory-cell-independent system of BCR and CD40 costimulation., Methods: B lymphocytes from 21 HIV-1-infected patients were purified by immunomagnetic cell separation and costimulated with immobilized anti-CD40 monoclonal antibodies and Staphylococcus aureus Cowan I particles in the presence of interleukin (IL)-2 and IL-10. Homotypic aggregate formation, apoptosis, cell cycle entrance, proliferation and Ig secretion of B cells were analysed., Results: Costimulation by the BCR and CD40 induced proliferation and differentiation of B lymphocytes into Ig-secreting cells in 13 patients (group I) but not in eight patients (group II). For three patients in group II, the dual triggering induced apoptosis of B cells. The unexpected inability of these cells to differentiate was associated with a high CD38 expression and a weak spontaneous production of Ig or anti-HIV-1 antibodies in patients with a high viral load and a low CD4+ lymphocyte count. Despite this anomaly, the B cells from group II were able to progress through the cell cycle after stimulation with a combination of phorbol ester and ionomycin in complete medium, suggesting an impairment in BCR and CD40 early signal transduction., Conclusion: Intrinsic in vitro hyporeactivity of B lymphocytes to dual triggering of BCR and CD40 was observed in advanced HIV-1 disease and appeared to be related to in vivo hyperactivation of B cells.

An acute wheezing episode is the most common feature of severe lower respiratory tract infection during infancy. Respiratory syncytial virus (RSV) is the major causative agent. In order to study inflammation during acute wheezing episodes in infants, we wanted to assess the feasibility and contribution of induction of pharyngeal secretions. We therefore compared inflammatory markers in the pharyngeal secretions of 27 infants suffering from acute wheezing episodes with an RSV infection (RSV+) and 18 infants suffering with acute wheezing episodes without RSV infection (RSV-). Pharyngeal secretions were recovered by physiotherapy using isotonic saline. The safety of the procedure was carefully checked. Pharyngeal secretions were homogenized with dithiothreitol. Total cells and eosinophils were counted and levels of eosinophil cationic protein (ECP) and histamine were measured. Induction of pharyngeal secretion was always well tolerated. Eosinophils were present in five RSV+ and seven RSV- patients. ECP levels were not significantly different between the groups. Histamine levels after protein adjustment were significantly increased in RSV+ patients (p<0.01) in comparison to RSV- patients. In this study, we have shown, that pharyngeal secretion can be safely recovered from infants suffering from acute wheezing episodes, and that it can be analysed for enumeration of inflammatory cells and measurement of inflammatory mediators.