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BackgroundCharacterization of the respiratory tract bacterial microbiota is in its infancy when compared to the gut microbiota knowledge. As key methodological steps can directly affect the accuracy of the results, it is crucial to determine a robust methodology in order to limit bias.Two different pairs of primers 515F-806R targeting the V4 hypervariable region of the 16SrRNA gene, “S-V4” for “Stringent V4” primer pair and “R-V4” for “Relaxed V4” primer pair, are respectively used in two ongoing international projects “the Human microbiome project” and “the Earth microbiome project”. We compared two methods of sample processing using these two different primer pairs for bacterial microbiota analyses of respiratory samples from critically-ill ventilated patients. For the later, Illumina 250 paired-end sequencing was done on a MiSeq platform after DNA extraction using mechanical lysis (bead-beating) and NucliSENS easyMAG. The concordance with conventional microbiology is the criterion of choice to determine the optimal method.ResultsTwenty samples from seven patients and four controls were sequenced. The two primer pair provided highly different results. Only 54% of the samples had a similar microbial composition with both pairs of primers. “S-V4” gave the best agreement with the conventional microbiology for ETA: 89% as compared to 44% for the “R-V4” primer pair. The main difference related to Enterobacteriaceae, which were concordant between conventional cultures and microbiota analyses using “S-V4”. Enterobacteriaceae detection was poor for “R-V4”. Among patients with VAP, a decrease in alpha diversity in ETA was observed. The mean of pairwise Unifrac distance was higher inside this group of patients at the time of VAP diagnosis as compared to control patients.ConclusionAccuracy of the bacterial lung microbiota composition was highly correlated to the pair of primers used for amplification of the 16s rRNA hypervariable sequence. Comparison of microbiota results obtained by sequencing and conventional microbiology allowed us to select the best option for further studies. This work validates our methodology based on 16SrRNA gene amplification with 515F-806R “S-V4” primer pair associated to Illumina® MiSeq 250 paired-end sequencing.

We are grateful to the inhabitants and authorities in southeastern Cameroon for their warm welcome and their support of this study. We also acknowledge the invaluable assistance of the European zoo and its employees where we conducted the investigation. We also greatly appreciate the contributions of Olivia Cheny of the Center for Translational Science at the Institut Pasteur.; International audience; Comparisons of mammalian gut microbiota across different environmental conditions shed light on the diversity and composition of gut bacteriome and suggest consequences for human and animal health. Gut bacteriome comparisons across different environments diverge in their results, showing no generalizable patterns linking habitat and dietary degradation with bacterial diversity. The challenge in drawing general conclusions from such studies lies in the broad terms describing diverse habitats ("wild", "captive", "pristine"). We conducted 16S ribosomal RNA gene sequencing to characterize intestinal microbiota of free-ranging sympatric chimpanzees and gorillas in southeastern Cameroon and sympatric chimpanzees and gorillas in a European zoo. We conducted participant-observation and semi-structured interviews among people living near these great apes to understand better their feeding habits and habitats. Unexpectedly, bacterial diversity (ASV, Faith PD and Shannon) was higher among zoo gorillas than among those in the Cameroonian forest, but zoo and Cameroonian chimpanzees showed no difference. Phylogeny was a strong driver of species-specific microbial composition. Surprisingly, zoo gorilla microbiota more closely resembled that of zoo chimpanzees than of Cameroonian gorillas. Zoo living conditions and dietary similarities may explain these results. We encourage multidisciplinary approach integrating environmental sampling and anthropological evaluation to characterize better diverse environmental conditions of such investigations. Over the last decade, numerous studies have demonstrated the importance of environmental changes on the mammalian gut microbiome, which is strongly associated with host metabolic, immune, and neurological functions 1. Broad-ranging influences, including host genetics, living conditions, diet, stress, and antibiotic use can affect gut microbial diversity 2-5. Among these influences, diet and living conditions have been evaluated for human and animal populations, entailing significant effects on gut microbiota and consequences for human and animal health. Adverse microbial profile shifts, for instance, have been associated with dysbiosis and wide-ranging diseases among human beings, from obesity to pediatric environmental enteropathy, and from autism to asthma 2,6,7. Outside of laboratory conditions, disentangling the effects of living and dietary conditions on gut microbial composition from other influences remains a complex question. In humans, such questions have catalyzed multiple studies comparing environmental and gut microbiota between "westernized" and "rural" peoples 8-10. Among other mammalian populations, degradation in habitat quality affects the diversity of available flora and fauna for consumption, and in some cases, is associated with declines in microbial gut composition 11-14. Microbiome OPEN

Objectives Human adenovirus (HAdV) infections are associated with a high morbidity and mortality in transplant patients requiring the use of antiviral treatments. Brincidofovir (BCV), a cytidine analog, inhibits HAdV replication through viral DNA elongation termination and likely through other mechanisms. To elucidate if BCV regulates cellular antiviral pathways, we analyzed its impact on HAdV-infected and non-HAdV-infected lung epithelial cells. Methods We assessed the cellular and viral transcriptome of A549 cells infected and non-infected with HAdV C5 and treated or non-treated with BCV by RNAseq after 72 h. Results BCV treatment of HAdV infected cells resulted in a profound decrease of viral transcription associated with a relative overexpression of the early genes E1A and E4 and of the late gene L1. BCV had also a profound impact on A549 cells’ transcriptome. Ontologic analysis revealed an effect of BCV on several pathways known to interact with adenovirus replication as mTor signalling and Wnt pathways. A549 cells treated with BCV demonstrated a significant inhibition of the biological function of “viral replication” including 25 dysregulated genes involved in inflammation pathways. Conclusion We demonstrated that BCV alters viral gene expression and promotes the expression of antiviral cellular pathways in A549 cells. These results provide new insights how to interfere with cellular pathways to control HAdV infections.

Prior to transplantation of hematopoietic stem cells or solid organ, donor and recipient EBV serostatus has to be determined to assess risks of post-transplant lymphoproliferative disorders. Sensitivity of EBV Viral capsid antigens (VCA) IgG and EBV nuclear antigen-1 (EBNA-1) is critical to define past infection and a good specificity of VCA IgM is required to avoid any disqualification of cord blood (CB) units. Architect™ EBV antibody panel (Architect assay) providing a high throughput was compared to a semi-automated ELISA (Etimax assays Diasorin) to assess sensitivities and specificities of VCA and EBNA-1 IgG and VCA IgM on 419 sera collected from immunocompromised patients (n=184) and from pregnant women who agreed to give CB cells (n=235). Intra and inter-assay coefficient of variations ranged from 1.63 to 4.8 % for VCA IgM, VCA IgG and EBNA-1 IgG. Index of VCA IgG and IgM and EBNA IgG of the two assays were highly correlated. The concordance in the interpretation between the two assays was moderate for VCA IgM (kappa=0.5), substantial for VCA IgG (kappa =0.60) and good for EBNA-1 IgG (kappa=0.75). Using serial dilutions of positive controls and in accordance with clinical results VCA IgG and EBNA IgG were detected at lower dilutions with Architect than Etimax. Conversely, 96.1% (74/77) of samples negative with Architect and positive with Etimax for VCA IgM did not have any heterophile antibodies and had VCA IgG and EBNA IgG antibodies supporting past infections. Architect™ EBV serology panel provided good sensitivities and specificities for EBV serostatus determination prior to transplantation. This article is protected by copyright. All rights reserved

Much attention has been focused on the role of the bacterial microbiome in human health, but the virome is understudied. Although previously investigated in individuals with inflammatory bowel diseases or solid-organ transplants, virome dynamics in allogeneic hematopoietic stem cell transplantation (HSCT) and enteric graft-versus-host disease (GVHD) remain unexplored. Here we characterize the longitudinal gut virome in 44 recipients of HSCT using metagenomics. A viral 'bloom' was identified, and significant increases were demonstrated in the overall proportion of vertebrate viral sequences following transplantation (P = 0.02). Increases in both the rates of detection (P < 0.0001) and number of sequences (P = 0.047) of persistent DNA viruses (anelloviruses, herpesviruses, papillomaviruses and polyomaviruses) over time were observed in individuals with enteric GVHD relative to those without, a finding accompanied by a reduced phage richness (P = 0.01). Picobirnaviruses were detected in 18 individuals (40.9%), more frequently before or within a week after transplant than at later time points (P = 0.008). In a time-dependent Cox proportional-hazards model, picobirnaviruses were predictive of the occurrence of severe enteric GVHD (hazard ratio, 2.66; 95% confidence interval (CI) = 1.46-4.86; P = 0.001), and correlated with higher fecal levels of two GVHD severity markers, calprotectin and α1-antitrypsin. These results reveal a progressive expansion of vertebrate viral infections over time following HSCT, and they suggest an unexpected association of picobirnaviruses with early post-transplant GVHD.

The virome has been recently studied in hematology and mostly in the setting of allogeneic hematopoietic stem cell transplantation. However, in hematology (as in the setting of nonhematological disorders) the study of the microbiome (that indeed includes the virome) is a growing field. The overall field is moving beyond species catalogue to the understanding of the complex ecological relationship that microbes have with each other and with their host. Here we review the existing literature on the virome in transplant recipients and in other settings, and discuss potential applications of the virome study in hematology.

Background Standardized and sensitive assays for Epstein Barr Virus (EBV) are needed to define universal cutoff for treatment initiation in allogeneic hematopoietic stem cells transplant recipients. In a context of accreditation and the availability of EBV international standard, we evaluated the Abbott RealTime EBV (RT) assay for EBV quantification in whole blood. Methods The RT assay was compared on 282 prospective clinical samples with the Artus EBV PCR Kit V1 assay (V1) and we analyzed the kinetics of EBV load in 11 patients receiving rituximab treatment. Results The estimated limit of detection was 88 IU/mL. The assay was linear (r2 = 0.9974) in the range of all samples tested (100 to 1,000,000 IU/mL). Intra-assay coefficients of variation (CV) ranged between 0.35 and 1.35%, and inter-assay CV between 3.40 and 4.5%. On samples above the limit of quantification, the two assays were strongly correlated. EBV RT values were on average 0.30 log10 IU/mL lower than those measured with the V1 assay. In patients treated with rituximab, the RT assay remained positive in 5 patients at the time it dropped below undetectable levels with the V1 assay. Conclusions In conclusion, the RT assay is a reliable assay for EBV load in whole blood. Its sensitivity will enable to estimate the kinetics of EBV load and the impact of treatments to control EBV reactivations.

Background Accurate quantification of Epstein-Barr virus (EBV) load in blood is essential for the management of post-transplant lymphoproliferative disorders. The automation of DNA extraction and amplification may improve accuracy and reproducibility. We evaluated the EBV PCR Kit V1 with fully automated DNA extraction and amplification on the m 2000 system ( Abbott assay ). Methodology Conversion factor between copies and international units (IU), lower limit of quantification, imprecision and linearity were determined in a whole blood (WB) matrix. Results from 339 clinical WB specimens were compared with a home-brew real-time PCR assay used in our laboratory ( in-house assay ). Results The conversion factor between copies and IU was 3.22 copies/IU. The lower limit of quantification (LLQ) was 1000 copies/mL. Intra- and inter-assay coefficients of variation were 3.1% and 7.9% respectively for samples with EBV load higher than the LLQ. The comparison between Abbott assay and in-house assay showed a good concordance (kappa = 0.77). Loads were higher with the Abbott assay (mean difference=0.62 log 10 copies/mL). Significance The EBV PCR Kit V1 assay on the m 2000 system provides a reliable and easy-to-use method for quantification of EBV DNA in WB.

Sensitive molecular assays have greatly improved the diagnosis of viral gastroenteritis. However, the proper preparation of stool samples for clinical testing remains an issue. bioMérieux has developed a stool preprocessing device (SPD) that includes a spoon for calibrated sampling and a vial containing buffer, glass beads, and two filters. The resulting stool filtrate is used for nucleic acid extraction. The purpose of this study was to evaluate the performance of the SPD for the quantification of human adenovirus (HAdV) DNA in stool samples collected from hematopoietic stem cell transplant (HSCT) recipients. HAdV DNA was quantified with the Adenovirus R-gene kit. The suitability of the device to reproducibly quantify HAdV DNA in stools using different extraction platforms (easyMAG and QIAsymphony) was determined using archived HAdV-positive stool samples. Coefficients of variation of HAdV DNA quantifications ranged from 1.79% to 1.83%, and no difference in quantification was observed between the two extraction systems. The HAdV DNA limit of quantification using the SPD was 3.75 log 10 copies/g of stool. HAdV DNA quantification using the SPD was then compared to that of the routine preprocessing technique on 75 fresh stool samples collected prospectively from pediatric HSCT recipients at risk for HAdV infections. Thirty-eight samples were HAdV DNA positive with both the SPD and routine preprocessing methods. HAdV DNA loads were on average 1.14-log 10 copies/g of stool higher with the SPD ( P < 0.0001) than with routine methods. This new device enabled a standardized preparation of stool samples in

Introduction The worldwide use of yellow fever (YF) live attenuated vaccines came recently under close scrutiny as rare but serious adverse events have been reported. The population identified at major risk for these safety issues were extreme ages and immunocompromised subjects. Study NCT01426243 conducted by the French National Agency for AIDS research is an ongoing interventional study to evaluate the safety of the vaccine and the specific immune responses in HIV-infected patients following 17D-204 vaccination. As a preliminary study, we characterized the molecular diversity from E gene of the single 17D-204 vaccine batch used in this clinical study. Materials and methods Eight vials of lyophilized 17D-204 vaccine (Stamaril®, Sanofi-Pasteur, Lyon, France) of the E5499 batch were reconstituted for viral quantification, cloning and sequencing of C/prM/E region. Results The average rate of virions per vial was 8.68 ± 0.07 log10 genome equivalents with a low coefficient of variation (0.81%). 246 sequences of the C/prM/E region (29–33 per vials) were generated and analyzed for the eight vials, 25 (10%) being defective and excluded from analyses. 95% of sequences had at least one nucleotide mutation. The mutations were observed on 662 variant sites distributed through all over the 1995 nucleotides sequence and were mainly non-synonymous (66%). Genome variability between vaccine vials was highly homogeneous with a nucleotide distance ranging from 0.29% to 0.41%. Average p-distances observed for each vial were also homogeneous, ranging from 0.15% to 0.31%. Conclusion This study showed a homogenous YF virus RNA quantity in vaccine vials within a single lot and a low clonal diversity inter and intra vaccine vials. These results are consistent with a recent study showing that the main mechanism of attenuation resulted in the loss of diversity in the YF virus quasi-species.

International audience; Background: Human Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. Methods: We assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. Results: We identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (+/- 6.2) and 5.2 (+/- 2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. Conclusions: Our study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments.

Objectives Interleukin-2 (IL-2) therapy increased CD4 cell counts and delayed antiretroviral therapy (ART) initiation in HIV-infected patients in the Agence Nationale de Recherche sur le SIDA et les Hepatites Virales (ANRS) 119 trial. However, four cases of lymphoma were reported. Epstein−Barr virus (EBV) replication is associated with an increased risk of lymphoma in immunocompromised patients. We assessed whether IL-2 had an impact on EBV replication and the development of lymphoma. Methods A total of 130 ART-naive patients were randomized to receive IL-2 therapy (n = 66) or no treatment (n = 64). Clinical data for patients with lymphomas were reviewed and tumours assessed for evidence of EBV infection and CD25 (the IL-2 receptor) expression. EBV DNA levels were measured in whole blood and plasma in both arms using real-time polymerase chain reaction (PCR), up to 48 weeks after baseline (BL). Results Four lymphomas occurred, a median of 61 weeks [range 40−94 weeks] after randomization at a median CD4 cell count of 396 cells/μL (IQR 234–536 cells/μL). In the IL-2 arm, two patients developed EBV-positive Hodgkin's lymphoma, and one developed EBV-negative Burkitt-type lymphoma. One patient in the control group developed EBV-positive non-Hodgkin's lymphoma. CD25 was negative in all cases. Among the 41 of 55 (control arm) and 44 of 58 (IL-2 arm) patients with detectable EBV DNA in whole blood at both BL and week 48, the median change in EBV DNA between BL and week 48 was +0.04 log10 copies/ml in both arms (P = 0.7). In plasma, EBV was detected at least once in 22 of 52 controls and 21 of 54 IL-2-treated patients (P = 0.8). Conclusions IL-2 therapy had no significant effect on EBV replication over 48 weeks in these ART-naive patients. The occurrence of lymphomas did not seem to be associated with IL-2 therapy.

We report the near-full-length genome sequence of a hepatitis C virus (HCV) isolate from a man originating from Democratic Republic of Congo, the genotype of which could not be determined by the routinely used sequencing technique. The near-complete genome sequence of this variant BAK1 was obtained by the association of two next-generation sequencing technologies. Evolutionary analysis indicates that this isolate, BAK1, could be the first reported strain belonging to a new HCV-7b subtype. This new subtype has been incorrectly identified as genotype 2 by the Versant HCV Genotype 2.0 assay (LiPA). The requirement of three independent isolates has been filled, and a new subtype can be assigned. More examples of HCV-7 are required to better understand its origin, its pathogenicity and its relationship with genotype 2.

Skin rash are common in immunocompromised patients, particularly after bone marrow transplantation. Human herpes virus 6 (HHV6) reactivation is often suspected, but its clinical presentation and the routine laboratory tests may be unspecific, thus leading to late diagnosis. In this case, we report specific intralymphocytic cytopathic inclusions on skin biopsy as a sign of systemic HHV6 reactivation. A 56-year-old patient presented progressive erythroderma and fever occurring after autologous hematopoietic stem-cell transplantation for mantle cell lymphoma. The skin biopsy showed a perivascular infiltrate of medium-to-large lymphocytes with irregular nuclei containing a large central basophilic inclusion surrounded by a clear halo. High levels of HHV-6 genomic in skin biopsy confirm HHV-6-induced cytopathic effect. The clinical course improved with intravenous foscavir. The specific histopathological findings encountered in this case are exceptional but recognizable, and along with HHV-6 DNA detection allow a prompt recognition of HHV6 skin rash.

OBJECTIVES: Few studies have examined the frequency and duration of genital herpes simplex virus (HSV) shedding in sub-Saharan Africa. This study describes HSV shedding patterns among a sample of HSV-2-seropositive women enrolled in a placebo-controlled trial of HSV suppressive therapy (acyclovir 400 mg twice a day) in Tanzania. METHODS: Trial participants were invited to participate in a substudy involving 12 clinic visits over 4 weeks. At each visit, cervical, vaginal and external skin swabs were taken and analysed for HSV DNA using inhouse real-time PCR. RESULTS: HSV shedding was mainly subclinical (90%; 57/63 shedding days in the placebo arm). The most frequent shedding site was the external skin, but HSV DNA was detected from all three sites on 42% (27/63) of shedding days. In HIV-negative women, HSV DNA was detected on 3% (9/275) of days in the acyclovir versus 11% (33/309) in the placebo arm, while in HIV-positive women, detection was on 14% (23/160) versus 19% (30/155) of days, respectively. CONCLUSIONS: HSV shedding was common, varying greatly by individual. Shedding rates were similar to studies in African and non-African settings. Among HIV-negative women, shedding rates were lower in the acyclovir arm; however, acyclovir did not substantially impact on HSV shedding in HIV-positive women.

Herpes simplex virus (HSV) suppressive therapy reduces genital and plasma human immunodeficiency virus type 1 (HIV-1) RNA over periods up to 3 months, but the long-term effect is unknown.A total of 484 HIV-1 and HSV type 2 seropositive Tanzanian women aged 16-35 years were enrolled in a randomized placebo-controlled trial of acyclovir administered at a dosage of 400 mg twice daily. Cervico-vaginal lavage and blood samples were collected at 6 months, 12 months, and 24 months for quantification of genital and plasma HIV-1 RNA and genital HSV DNA. Primary outcomes were detection and quantity of cervico-vaginal HIV-1 RNA at 6 months.At 6 months, there was little difference between the acyclovir and placebo arms for cervico-vaginal HIV-1 RNA detection (88 [ 41 .3%] of 213 vs 84 [ 44 .0%] of 191; odds ratio [OR], 0.90; 95% confidence interval [CI], 0.60-1.33), HSV DNA detection (20 [ 9 .4%] of 213 vs 22 [ 11 .5%] of 191; OR, 0.80; 95% CI, 0.42-1.51), genital HIV or HSV loads, or plasma HIV-1 RNA load. Estimated median adherence was 91%. There was a suggestion of an impact on cervico-vaginal HIV-1 RNA detection among women with estimated adherence 90% (OR, 0.74; 95% CI, 0.50-1.09) when data from all 3 visits were included.Acyclovir administered at a dosage of 400 mg twice daily is unlikely to be a useful long-term intervention to reduce HIV transmission. The lack of effect on HIV may be attributable to suboptimal adherence or treatment regimen.

Methods: We compared the performance of 3 collection methods for cervicovaginal secretions [cervicovaginal lavage (CVL), CVL enriched with a cervical swab (eCVL), and vaginal tampon (VT)] to identify the most reliable method for detection of cervicovaginal HIV-1 and herpes simplex virus type 2 (HSV-2). HIV-1 RNA (Nuclisens EasyQ; BioMerieux, Marcy-l'Etoile, France), HSV-2 DNA (real-time polymerase chain reaction), and microscopic blood and semen traces were detected in samples from 19 HIV-1-HSV-2-coinfected women seen at 4 weekly visits. Results: HIV-1 RNA was detected in 49 (79%) of 62 eCVLs, 41 (61%) of 67 CVLs, and 27 (57%) of 47 VTs. Detection of HIV-1 RNA was higher in eCVL compared with CVL [45/58 (78%) vs. 32/58 (55%); risk ratio 1.41, 95% confidence interval 1.05 to 1.88]. Conclusions: Although more eCVLs were contaminated with microscopic blood (29%) than CVLs (22%) or VTs (7%), detection of HIV-1 RNA remained higher using eCVL compared with CVL (risk ratio 1.43, 95% confidence interval 1.02 to 2.02) in uncontaminated samples. HSV-2 DNA was detected in less than 10% of samples by each method but in 7 (37%) of 19 women overall by 1 or more methods.

The performances of commercial enzyme-linked immunosorbent assays (ELISAs) in detecting herpes simplex virus type 2 (HSV-2) antibodies have been inconsistent for African and human immunodeficiency virus (HIV)-positive populations. We compared the performances of the HerpeSelect and Kalon glycoprotein G2 ELISAs for patients with genital ulcer disease in Ghana and the Central African Republic. Sera from 434 women were tested with the HerpeSelect assay, and a subsample ( n = 199) was tested by the Kalon assay. Ulcer swabs and cervicovaginal lavage samples were tested for HSV-2 DNA by PCR. HSV-2-seronegative women with detectable genital HSV-2 DNA were retested for HSV-2 antibodies 14 and 28 days later by the two ELISAs. A total of 346 (80%) women were positive by HerpeSelect at baseline, and 225 (54%) had detectable genital (lesional or cervicovaginal) HSV-2 DNA. Sixty-six (19%) HerpeSelect-positive samples had low-positive index values (1.1 to 3.5), and 58% of these samples had detectable genital HSV-2 DNA. Global agreement between the two serological assays was 86%. Concordance was high (99%) for sera that were negative by HerpeSelect or had high index values (>3.5). Defining infection detected by HSV-2 DNA PCR and/or Kalon assay as true infection, 71% of sera with low-positive index values were associated with true HSV-2 infection. Twenty-five women were identified as having nonprimary first-episode genital HSV-2 infection. Rates of HSV-2 seroconversion at day 14 were 77% (10/13 patients) by HerpeSelect assay and 23% (3/13 patients) by Kalon assay, with four additional seroconversions detected by Kalon assay at day 28. HIV serostatus did not influence assay performance. Low index values obtained with the HerpeSelect assay may correspond to true HSV-2 infection, in particular to nonprimary first episodes of genital HSV-2 infection, and need to be interpreted in the context of clinical history.

Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.

In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4+ T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1BaL (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1NDK (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN+ CR3− cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV.

BACKGROUND Sequence variations in HR-1 gp41 env gene region encoding the target for T-20 have previously been reported among patients naive to inhibitory fusion. OBJECTIVE To evaluate whether a previous therapeutic history of patients could have an impact on a differential evolution of the gp41 polymorphism. METHODS We assessed the genetic polymorphism within the critical HR-1 gp41 env gene region in HIV-1 variants from 108 T-20-naive patients (Groups I-III) and 12 patients receiving T-20 as part of a salvage regimen (Group IV). T-20-naive patients included 50 patients exhibiting variants harboring resistance mutations to NRTIs, NNRTIs, and PIs (Group I), 24 patients with variants harboring resistance mutations for NRTIs and/or NNRTIs (Group II), and 34 antiretroviral drug-naive patients (Group III). RESULTS In T-20-naive patients whose HIV harbored resistance mutations to NRTIs, NNRTIs, and/or PIs, the mean number of synonymous mutations (ds) per patient was decreased and the mean number of nonsynonymous (da) mutations per patient was increased, resulting in a significant decrease in the mean Sigmads/Sigmada ratio as compared with antiretroviral drug-naive patients (Group III; 4.1 vs. 11.6; P < 0.0001). The mean number of polymorphic mutations in HR-1 gp41 per patient was two-fold higher in patients exhibiting antiretroviral drug resistance mutations (Groups I and II) than in antiretroviral drug-naive patients (Group III; 0.41 vs. 0.20; P < 0.05). CONCLUSION Our observations indicate that the HR-1 gp41 T-20 target is subjected to high genetic variability, including intrinsic polymorphism and selection of T-20 resistance mutations under T-20 intake, that is increased by the presence of resistance mutations to NRTIs, NNRTIs, and/or PIs. Our data provide a basis for a potential impact of previous antiretroviral drug history on the therapeutic efficacy of T-20.

The accuracy and usefulness of laboratory-developed real-time PCR procedures using a Light Cycler instrument (Roche Diagnostics) for detecting and quantifying human immunodeficiency virus type 1 (HIV-1) RNA and DNA as well as herpes simplex virus type 1 (HSV-1)/HSV-2 DNA in cervicovaginal secretions from women coinfected with HIV and HSV were evaluated. For HIV-1, the use of the NEC152 and NEC131 primer set and the NEC-LTR probe in the long terminal repeat gene allowed us to detect accurately the majority of HIV-1 subtypes of group M circulating in sub-Saharan Africa, including subtypes A, B, C, D, and G as well as circulating recombinant forms 02 and 11. The detection threshold of real-time PCR for HIV in cervicovaginal lavage samples was 5 copies per assay for both RNA and DNA; the intra- and interassay coefficients of variation of C T values were 1.30% and 0.69% (HIV-1 RNA) and 1.84% and 0.67% (HIV-1 DNA), respectively. Real-time PCR for HSV using primers and probe targeting the HSV DNA polymerase gene allowed both detection and quantification of HSV DNA and also differentiation between HSV-1 and HSV-2 genotypes. The detection threshold of real-time PCR for HSV was 5 copies per assay; the intra- and interassay coefficients of variation of C T values were 0.96% and 1.49%, respectively. Both manual and automated silica-based procedures were appropriate for combined extraction of HIV and HSV genomes from female genital secretions. Taken together, these findings indicate that real-time PCR may be used as a unique nucleic acid amplification procedure to detect and quantify HIV and HSV genomes in cervicovaginal secretions and thus to assess at reduced costs the genital shedding of both viruses in women included in intervention studies.

Peptide insertions in codons 67–71 of the reverse transcriptase (RT) pol gene were detected in 11 (2.7%) of 414 genotypic analyses performed in a hospital cohort of 2900 outpatients with human immunodeficiency virus type 1 (HIV-1) infection. The duration of antiretroviral treatment (bi- or tri-therapy) before the detection of insertions ranged from 12 to 60 months. Dipeptide insertions were detected in ten patients, of which the most frequent was serine–serine. A monopeptide insertion was diagnosed once. The amino-acid composition patterns of insertions varied with time in five of the 11 patients. Peptide insertions were always associated with various patterns of pre-existing or appearing resistance mutations in the RT pol gene to different antiretroviral drugs. Genotypic-guided treatment resulted in virological and immunological improvement in two patients. In contrast, the remaining patients did not respond to any of the various antiretroviral regimens prescribed. Furthermore, various patterns of resistance mutations developed to the prescribed antiretroviral drugs, with AIDS-related conditions leading to death in two patients. It was concluded that peptide insertion in this region of the HIV-1 RT pol gene constitutes a rare cause of persistent therapeutic failure, and that management of such patients remains challenging despite successive genotypic analyses aimed at detecting mutations conferring antiretroviral drug resistance.

Background: Lack of mucosal hepatitis C virus (HCV) transmission may be due to fairly low infectivity of body fluids in HCV-infected individuals in association with yet unknown innate or acquired resistance factors in individuals exposed to the virus. Objective: To evaluate HCV excretion patterns in cervicovaginal secretions obtained from chronically HCV-infected women. Study design: Fifteen chronically HCV-infected women of childbearing age hospitalized for chronic hepatitis were prospectively recruited. Cervicovaginal secretions were obtained by vaginal washing with 3 ml phosphate-buffered saline (PBS). All cervicovaginal secretions were free of hemoglobin traces and also free of semen traces. Free HCV-RNA and cell-associated HCV-RNA were examined in acellular part and cellular part of the cervicovaginal secretions, respectively, by in-house qualitative PCR for 5′-HCV-non-coding region (NCR). Negative strand HCV-RNA, a marker of HCV replication, was searched by using tag-RT-nested PCR (tag-RT-NPCR). Results: HCV-RNA could not be detected in the acellular fractions of the 15 evaluated cervicovaginal secretions. In contrast, HCV-RNA could be detected in the cellular fractions of four of 15 (27%) cervicovaginal secretions. None of the cervicovaginal secretions, including the four positive cell-associated HCV-RNA, contained negative strand, replicating HCV-RNA. Conclusions: Our results suggest that positive strand HCV-RNA may be present outside the menstruation periods as cell-associated virus in the cervicovaginal secretions of a minority of untreated HCV-seropositive, HCV-RNA-viremic women, and that the lower female genital tract does not constitute a reservoir where HCV replicates. These observations thus provide the basis for the low risk of female-to-male sexual transmission of HCV infection.

Background/Aims : We herein focused on identifying biological factors possibly involved in non-parenteral transmission of hepatitis C virus (HCV), such as HCV excretion patterns and antibody-based immunity to the virus in saliva and/or cervicovaginal secretions (CVS). Methods : Paired blood, saliva and cervicovaginal lavage samples were obtained from HCV-RNA plasma-positive hemoglobin (Hb) antigen and HIV-seronegative, HCV-seropositive males ( n =13) and females ( n =21). HCV-specific antibodies were detected by ELISA in paired samples, and HCV-RNA was detected in cell-free and cell-associated body fluids. Results : Antibodies to E1 HCV surface glycoprotein of the IgG and IgA isotypes showed similar, but less pronounced, profiles as IgG and IgA to E2. HCV-specific IgG and IgA in mucosal fluids likely originated predominantly from the systemic compartment, because HCV-specific mucosal immunoglobulins involved primarily monomeric antibodies, including monomeric IgA, and because their specific activities for HCV antigens in corporeal fluids were similar to those in paired serum (Se). Viral shedding in saliva or CVS was restricted to cell-associated, non-replicating strand (+) HCV-RNA in 42% (12 out of 28) of saliva and in 19% (four out of 21) of cervicovaginal fluids. Conclusions : The association in body fluids of HCV-specific IgG, and to a lesser extent IgA, directed to E1/E2 surface glycoproteins (which may block critical steps of virus–cell interactions), of undetectable free viral RNA, and of occasional non-replicating cell-associated HCV, suggests a resulting poor infectivity of saliva or cervicovaginal fluid in chronically HCV-infected individuals. Taken together, these observations provide the basis for the low risk of non-parenteral transmission of HCV infection.

The levels of cervicovaginal HIV-1 proviral DNA in contrast to HIV-1 RNA were not associated with those of HSV-2 DNA even in women shedding HSV-2 DNA in their genital secretions. These observations suggest that HSV-2 genital replication may influence the genital production of cell-free HIV-1 RNA rather than that of cell-integrated HIV-1 DNA. The existence of a reservoir of productively infected cells with rapid turnover within the female genital tract has been previously hypothesized based on the marked and rapid effect of antiretroviral treatment in decreasing the quantity of HIV-1 RNA in CVSs. We can in turn hypothesize that HSV-2 genital replication positively influences this genital reservoir of rapid turnover cells. Conversely the lack of association between cervicovaginal HSV-2 DNA and HIV-1 DNA shedding suggests the existence of a cellular genital reservoir of HIV-infected cells with low turnover similar to that previously recognized in the systemic compartment. (excerpt)

International audience; Detection of viral and bacterial DNA in nasopharyngeal aspirates (NPAs) is now a routine practice in emergency cases of febrile pneumonia. We investigated whether Pneumocystis jirovecii DNA could also be detected in these cases by conducting retrospective screening of 324 consecutive NPAs from 324 adult patients (198 or 61% were immunocompromised) admitted with suspected pulmonary infections during the 2012 influenza epidemic season, using a real-time quantitative polymerase chain reaction (PCR) assay (PjqPCR), which targets the P. jirovecii mitochondrial large subunit ribosomal RNA gene. These NPAs had already been tested for 22 respiratory pathogens (18 viruses and 4 bacteria), but we found that 16 NPAs (4.9%) were PjqPCR-positive, making P. jirovecii the fourth most prevalent of the 23 microorganisms in the screen. Eleven of the 16 PjqPCR-positive patients were immunocompromised, and five had underlying pulmonary conditions. Nine NPAs were also positive for another respiratory pathogen. Six had PjqPCR-positive induced sputa less than 3 days after the NPA procedure, and five were diagnosed with pneumocystis pneumonia (four with chronic lymphoproliferative disorders and one AIDS patient). In all six available pairs quantification of P. jirovecii DNA showed fewer copies in NPA than in induced sputum and three PjqPCR-negative NPAs corresponded to PjqPCR-positive bronchoalveolar lavage fluids, underscoring the fact that a negative PjqPCR screen does not exclude a diagnosis of pneumocystosis. Including P. jirovecii DNA detection to the panel of microorganisms included in screening tests used for febrile pneumonia may encourage additional investigations or support use of anti-pneumocystis pneumonia prophylaxis in immunocompromised patients.

The consequences of zidovudine (ZDV) replacement by other nucleoside reverse transcriptase inhibitors on the expression of resistance mutations at codons 215 and 41 of the reverse transcriptase (RT) gene was investigated prospectively in 66 patients harboring mutant genotypes who were changed to an effective two- or three-drug combination antiretroviral regimen. Quantitation of mutant (MUT) viral populations at codon 215 by means of RT-PCR with differential hybridization of amplicons specific for MUT and wild (WT) variants revealed no difference in the proportion of 215 MUT variants prior to (93.5 ± 2.4%) and 12 to 20 months after (96.9 ± 1.9%) ZDV replacement, independently of a therapeutic change for stavudine. The fitness of the variants harboring the ZDV-resistant MUT 215 genotype following drug withdrawal was calculated to be 96 to 99% of that of the variants harboring the WT 215 genotype. The apparent stability of ZDV-resistant variants in the study population may have two main complementary explanations: persistent selective pressure secondary to partial cross-resistance due to the new regimens given after the therapeutic alteration and suppression of viral replication after the therapeutic alteration that could have hampered the replacement of less fit variants by fitter variants. These findings indicate that, at least within 15 months following discontinuation of ZDV, an effective antiretroviral therapy is insufficient to allow for ZDV-resistant strains to disappear, and thus to allow for the safe re-introduction of the drug. J. Med. Virol. 68:1–6, 2002. © 2002 Wiley-Liss, Inc.

The interplay between immune recovery, cytomegalovirus (CMV)-reactivation, CMV-driven immunity and graft-versus-leukaemia effect (GVL) was analysed in 108 children (median age: 8 years) who underwent haematopoietic-stem cell transplantation (HSCT) for acute leukaemia. Follow-up was 2 years unless death or relapse occurred. CMV-polymerase chain reaction (PCR) was programmed weekly until month +3 post-HSCT. Immunomonitoring consisted of sequential lymphocyte subset enumerations and analyses of T-cell proliferative and γ-interferon responses to CMV and to adenovirus. In the 108 recipients, the 2-year relapse rate (RR) was 25% (median time to onset 4·5 months; range: 24 d-17 months). CMV reactivation occurrence was 31% (median time to onset 26 d). Donor/recipient CMV serostatus did not influence RR. Among the 89 recipients disease-free after day +120, i) early CMV-reactivation before day +30 was more frequent (P = 0·01) in the relapse recipient group opposed to the non-relapse group. ii) CD8(+) /CD28(-) and CD4(+) CD45RA(-) T-cell expansions induced by CMV did not influence RR, iii) Recovery of anti-CMV and also anti-adenovirus immunity and of naive CD4(+) T-cells was faster in the non-relapse group (P = 0·008; 0·009 and 0·002 respectively). In contrast to adult acute myeloid leukaemia, CMV reactivation was associated with increased RR in this paediatric series. Accelerated overall immune recovery rather than CMV-driven immunity had a favourable impact on RR.

International audience; Since the type of herpes simplex virus (HSV) infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is always recommended. Although PCR has been the diagnostic standard method for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, could replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, antigen detection-an immunofluorescence test or enzyme immunoassay from samples from symptomatic patients--could be employed, but HSV type determination is of importance. Type-specific serology based on glycoprotein G should be used for detecting asymptomatic individuals but widespread screening for HSV antibodies is not recommended. In conclusion, rapid and accurate laboratory diagnosis of HSV is now become a necessity, given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy.

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [ n = 13], parvovirus B19 [ n = 2], and adenovirus [ n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays ( P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections.

Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples ( n = 92) and 78% from stool samples ( n = 100). No adenovirus was detected in samples from noninfected patients ( n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml ( r = 0.82 and 0.78, respectively) were correlated up to 6 log 10 copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus ( n = 41), CMV ( n = 30), EBV ( n = 26), JC virus ( n = 9), and HSV-1 ( n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.

The usefulness of a point-of-care immunochromatographic dual test for the simultaneous detection of both nontreponemal and Treponema pallidum-specific antibodies (Chembio Diagnostics Systems Inc., Medford, NY, USA) was assessed in various situations related to syphilis, by reference to conventional syphilis serology. Thawed sera were obtained from 100 adults including 36 primary syphilis, 6 secondary syphilis, 6 re-infection, 9 recently-treated syphilis, and 43 old syphilis. Doubtful reactivities for the treponemal line were considered positive; doubtful reactivities for the nontreponemal line were considered positive only when the treponemal line was present. The sensitivity, the specificity, and its concordance to gold standard serology of treponemal line were high, around 90%. The sensitivity of nontreponemal line was 96.3%, its specificity 76.7%, and its concordance 83.4%. In conclusion, the dual rapid test from Chembio Diagnostics Systems Inc. is useful for rapid point-of-care diagnosis in the various situations encountered with patients suffering from syphilis.

IgG and IgA antibodies with specificities to a latent and a lytic antigen of human herpes virus 8 (HHV-8) were detectable in the saliva and serum of eight patients whose Kaposi's sarcoma had regressed, seven of whom were HIV-1 infected. The measurement of antibody-specific activity and secretion rate, and the detection of secretory IgA all indicate anti-HHV-8 antibody activity in saliva. The specific humoral responses possibly influence mucosal replication of HHV-8, and in turn, that of HIV.

Asymptomatic genital shedding of herpes simplex virus type 2 (HSV-2) DNA was evidenced by real-time PCR in 25 (13.2%) of 188 cervicovaginal lavage samples and in only 13 (6.9%) paired cervicovaginal samples from 188 HSV-2-seropositive, nonpregnant childbearing-aged human immunodeficiency virus-seronegative women living in Gabon. These observations demonstrate that cervicovaginal washing is more suitable than endocervicovaginal swabbing for detecting and quantifying HSV-2 DNA by PCR in female genital secretions.

The BioPlex platform was evaluated for the detection of herpes simplex virus 2 (HSV-2) antibodies in sub-Saharan Africa individuals in comparison to clinicovirological standards and compared to HerpeSelect. The sensitivities and specificities were, respectively, 88.9% and 93.5% for BioPlex and 89.9% and 92.7% for HerpeSelect. The agreement between both assays was 95.7%.

The human polyomavirus JC virus (JCV) is the agent of progressive multifocal leukoencephalopathy (PML). It has also recently been involved in cerebellar atrophy. Factors involved in this entity are elusive. We present a case of a human immunodeficiency virus (HIV)-infected patient with PML and cerebellar atrophy. In addition to a compartmentalization of JCV strains between urine, cerebrospinal fluid, and cerebellum, specific rearrangements in the JCV regulatory region were observed in the cerebellum, resulting in alterations of transcription factor binding sites. Our data underline the importance of searching for JCV in HIV-infected patients with cerebellar disorders and suggest that mutations in the regulatory region may be involved in cerebellar degeneration.

Human herpesvirus 6 (HHV6) is an emerging cause of interstitial pneumonia in immunocompromised hosts. However, the clinical significance of a positive PCR test for HHV6 in respiratory samples from patients with hematological malignancies remains unclear.We retrospectively studied the features and outcomes of 29 critically ill hematology patients with acute respiratory failure and lung pulmonary infiltrates visible on a chest radiograph, who tested positive for a qualitative PCR for HHV6 in bronchoalveolar lavage fluid.Of the 29 patients, 18 (62%) were stem cell transplant recipients and 11 (38%) had received chemotherapy. All patients had a fever. Clinical manifestations consistent with extra-pulmonary HHV6 disease were noted in 17 (59%) patients. One or more co-pathogens were found in 25 (86%) patients. The four remaining patients diagnosed with HHV6 pneumonia and subsequently recovered with foscarnet therapy. Antiviral therapy was also given to seven patients with co-infections, of whom two ultimately died.In most cases, HHV6 recovered from BAL fluid is a co-pathogen whose clinical relevance remains undetermined. However, in some cases, HHV6 is the only pathogen, along with disseminated systemic viral disease, and the patient is likely to benefit from foscarnet therapy.

Several studies suggest that herpes simplex virus type 2 (HSV-2) may enhance HIV-1 transmission and disease progression.We conducted a randomized, double-blind, placebo-controlled trial of aciclovir 400 mg twice daily for 3 months in 300 HSV-2/HIV-1 co-infected women not yet on highly active antiretroviral therapy (HAART). Participants were evaluated prerandomization and at monthly visits for 3 months. Primary outcomes were the detection and quantity of genital HIV-1 RNA at the month 3 (M3) visit. Analyses were also undertaken using data from all visits. The treatment effects on plasma HIV-1 RNA, CD4 cell count and genital HSV-2 DNA were also assessed.At M3 fewer women had detectable genital HIV in the aciclovir group compared to placebo, but this was not significant [61/132 (46%) vs. 71/137 (52%), risk ratio (RR) 0.89, 95% confidence interval (CI) 0.70-1.14; P = 0.36]. There was also little difference in quantity of HIV-1 RNA among shedders (+0.13 log10 copies/ml, 95% CI -0.14 to 0.39) at M3. However, aciclovir significantly decreased the frequency of HIV-1 shedding over all visits [adjusted odds ratio (OR) 0.57, 95% CI 0.36-0.89]. Significant reductions in M3 plasma HIV-1 RNA (-0.34 log10 copies/ml, 95% CI 0.15-0.54), genital HSV-2 DNA (8 vs. 20%, RR 0.37, 95% CI 0.19-0.73) and genital ulceration (8 vs. 18%, RR 0.43, 95% CI 0.22-0.84) were observed in the aciclovir group.HSV-2 suppressive therapy, by reducing HIV-1 plasma viral load and altering the pattern of genital HIV-1 shedding, may contribute to the reduction in sexual transmission of HIV-1 and may delay the requirement for HAART initiation.

We evaluated the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for the detection of influenza A and influenza B viruses and respiratory syncytial viruses from 353 pediatric nasopharyngeal aspirates. As assessed by comparison with the results of immunofluorescence testing and cell culture, the specificity and the sensitivity of the ProFlu-1 assay ranged from 97% to 100%. In addition, the ProFlu-1 assay amplified 9% of samples not detected by conventional methods.

OBJECTIVE:: To investigate correlates of herpes simplex virus type 2 (HSV-2) DNA and HIV-1 RNA among women with genital ulcer disease (GUD). DESIGN:: Baseline data from a randomized placebo-controlled trial of episodic herpes treatment in Ghana and the Central African Republic. METHODS:: GUD aetiology was determined by polymerase chain reaction (PCR) from a lesional swab. Real-time PCR was used to quantify HIV-1 RNA, and HSV-2 DNA in cervicovaginal lavages (CVL) and HIV-1 RNA in plasma. Genital infection was defined as the presence of virus in the lesion or CVL. RESULTS:: Of 441 women enrolled, 79.0% were HSV-2 seropositive, 46.6% were HIV-1 seropositive, and 50.0% had an HSV-2 ulcer. Among 180 HSV-2/HIV-1 co-infected women, cervicovaginal HIV-1 RNA was detected more frequently in women with HSV-2 ulcers (67.9%) or cervicovaginal HSV-2 DNA only (72.3%) compared with women without genital HSV-2 infection (42.4%) (P = 0.004). Women with genital HSV-2 infection had higher median cervicovaginal HIV-1-RNA loads (3.14 log10 copies/mL versus 2.10 log10 copies/mL; P = 0.003), higher plasma HIV-1-RNA loads (median 5.10 versus 4.65 log10 copies/mL; P = 0.07), and lower median CD4 cell counts) (198 versus 409 cells/mm, P = 0.03). Cervicovaginal HIV-1 RNA and HSV-2 DNA were significantly correlated after adjusting for plasma HIV-1 RNA and CD4 cell counts (P < 0.001) and a 10-fold increase in cervicovaginal HSV-2 DNA was associated with a 1.7-fold increase in plasma HIV-1 RNA (P = 0.003). CONCLUSION:: Genital HSV-2 infection is associated with increased cervicovaginal and plasma HIV-1 RNA among co-infected women with genital ulcers, independently of the level of immunodeficiency, highlighting the close interaction between these two viruses and the role of HSV-2 as a co-factor for the sexual transmission of HIV-1.

Clinical and subclinical genital herpes simplex virus type 2 (HSV-2) reactivations have been associated with increases in human immunodeficiency virus (HIV)-1 genital shedding. Whether HSV-2 shedding contributes to the selection of specific genital HIV-1 variants remains unknown. We evaluated the genetic diversity of genital and blood HIV-1 RNA and DNA in 14 HIV-1/HSV-2-co-infected women, including seven with HSV-2 genital reactivation, and seven without as controls. HIV-1 DNA and HIV-1 RNA env V1–V3 sequences in paired blood and genital samples were compared. The HSV-2 selection pressure on HIV was estimated according to the number of synonymous substitutions (dS), the number of non-synonymous substitutions (dN) and the dS/dN ratio within HIV quasi-species. HIV-1 RNA levels in cervicovaginal secretions were higher in women with HSV-2 replication than in controls (p0.02). Plasma HIV-1 RNA and genital HIV-1 RNA and DNA were genetically compartmentalized. No differences in dS, dN and the dS/dN ratio were observed between the study groups for either genital HIV-1 RNA or plasma HIV-1 RNA. In contrast, dS and dN in genital HIV-1 DNA were significantly higher in patients with HSV-2 genital reactivation (p

Variability in the handling of samples of genital secretions prior to quantitation of HIV-1 RNA and HSV DNA may profoundly affect both the detection and quantitation of these nucleic acids. Over 144 h, we evaluated, the influence of storage temperature (4 degrees C, 20 degrees C, 30 degrees C) on the quantity of HIV-1 RNA and HSV-2 DNA in HIV and HSV negative cervicovaginal lavage pools spiked with known amounts of HIV-1 and HSV-2 and in HIV-1 and HSV-2 co-infected cervicovaginal lavage pools. The level of viral nucleic acids remained stable at 4 degrees C for 24h but decreased significantly when cervicovaginal lavages were stored at 20 degrees C and 30 degrees C, demonstrating that, cervicovaginal lavages to be quantified for viral RNA or DNA require, at minimum, immediate storage at 4 degrees C.

Immunodeficiency related to HIV may increase the incidence of EBV-associated lymphomas, by altering EBV-specific immune control and consequently favoring EBV reactivation. The aim of the present study was to assess the relationship between the decrease of EBV-specific cellular immunity and the increase of EBV reactivation in a prospective cohort of 72 unselected HIV-infected individuals. EBV-specific immunity was evaluated by a highly sensitive IFN-gamma ELISPOT assay using 22 peptides mimicking latent and lytic antigens, and circulating mononuclear (PBMC) EBV DNA load was quantified by real-time quantitative PCR. The mean circulating cell-associated EBV DNA load was higher in HIV-infected patients (639 copies/10(6) PBMC) than in healthy controls (21, n = 14) ( P = 0.005) and was higher in patients with CD4(+) T-cell count below 350/microL than that in patients harboring higher CD4(+) T-cell count (1112 vs. 389, P = 0.003). The mean intensity of EBV-specific cellular responses was lower in HIV-infected patients than in controls ( P = 0.001), even in patients with CD4(+) T-cell count above 350/-microL ( P = 0.007). The number of EBV peptides recognized was lower in HIV-infected patients than in controls (frequency: 0.44 vs. 0.67; P = 0.02), indicating reduced polyclonality in HIV-infected patients. The polyclonality was 1.5-fold lower in HIV-infected patients with CD4(+) T-cell count below 350/-microL ( P =0.007). For EBV load >1000 copies/10(6) PBMC, the levels of cell-associated EBV DNA and those of EBV-specific cellular immunity, either in intensity or in polyclonality, or both, were inversely correlated. These findings demonstrate early impairment of the EBV-specific cellular immune control with progressive increase of EBV reactivation in the course of HIV infection. These observations likely provide a basis for appreciating the risk to develop non-Hodgkin's lymphomas in HIV-infected individuals.

Antibodies to herpes simplex virus type 1 (HSV-1) and HSV-2 of the immunoglobulin G (IgG) and IgA isotypes were detected in the cervicovaginal secretions (CVS) of 77 HSV-1- and HSV-2-seropositive but clinically asymptomatic African women by type-specific enhanced chemiluminescence Western blotting (ECL-WB). Of the 77 subjects, 34 were HIV negative, shedding HSV-2 DNA in their genital secretions; 20 were HIV positive, shedding HSV-2 DNA; and 23 were HIV negative, not shedding HSV-2 DNA. HSV-specific IgG was detected in CVS of nearly 70% of the women studied. HSV-specific IgA was found in CVS of 50% of the women studied. The distribution of CVS HSV-specific antibodies to each HSV type was highly heterogeneous, with a slight predominance of detectable IgG to HSV-1 (59%) over IgG to HSV-2 (41%), whereas the frequency of detectable IgA to HSV-1 (39%) was similar to that of IgA to HSV-2 (36%). The presence of detectable HSV-specific antibodies was inversely associated with HSV-2 DNA genital asymptomatic shedding but was not affected by HIV seropositivity. In addition, 13 of 77 (17%) CVS samples showed neutralizing activity against HSV-2, as assessed by an HSV-2 in vitro infectivity reduction assay. Neutralizing activity in CVS was associated with the presence of IgG and/or IgA antibodies to HSV-1 and/or to HSV-2 by ECL-WB. Among women whose CVS showed HSV-2-neutralizing activity, the specific activity of HSV-specific neutralizing antibodies was substantially (fivefold) higher in HSV-2 DNA shedders than in nonshedders. In conclusion, HSV-specific antibodies are frequently detected in CVS of asymptomatic African women seropositive for HSV-1 and HSV-2. A subset of these women had functional neutralizing activity against HSV-2 in their CVS. The origin of these antibodies and their role in HSV-2 disease of the female genital tract remain to be determined.

Using ultrasensitive polymerase chain reaction–based techniques we assessed levels of cell-free and cellassociated human immunodeficiency virus (HIV)–1 in paired blood and genital samples of 30 clinically asymptomatic treatment-naive women. Levels of HIV-1 RNA in cervicovaginal-lavage samples were positively correlated with those in plasma samples (r=.50; P=.008) whereas levels of HIV-1 DNA in genital samples were loosely correlated with those in blood samples (r=.31; P=.041). In plasma of peripheral blood levels of HIV-1 DNA were positively correlated with those of HIV-1 RNA (r=.64; P < .001) whereas no correlation between HIV-1 DNA and HIV-1 RNA was evident in genital secretions. Our results indicate that levels of HIV-1 RNA and HIV-1 DNA are unrelated in the genital tracts of treatment-naive women and suggest that the level of genital HIV-1 RNA is influenced by systemic viral replication—in contrast to genital HIV-1 provirus which may be influenced as well by local cofactors triggering the migration of HIV-infected cells originating from the cervicovaginal submucosa. These features may be relevant for an understanding of HIV-1 transmission in heterosexual individuals. (authors)

Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.

BackgroundResistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility.MethodsThe NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0-15) and zanamivir (days 15-25 and 70-80).ResultsCompared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6-69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin.ConclusionsReduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised.

Background A randomized controlled trial in South Africa found a beneficial effect of acyclovir on genital ulcer healing, but no effect was seen in trials in Ghana, Central African Republic and Malawi. The aim of this paper is to assess whether the variation in impact of acyclovir on ulcer healing in these trials can be explained by differences in the characteristics of the study populations. Methodology/Principal Findings Pooled data were analysed to estimate the impact of acyclovir on the proportion of ulcers healed seven days after randomisation by HIV/CD4 status, ulcer aetiology, size and duration before presentation; and impact on lesional HIV-1. Risk ratios (RR) were estimated using Poisson regression with robust standard errors. Of 1478 patients with genital ulcer, most (63%) had herpetic ulcers (16% first episode HSV-2 ulcers), and a further 3% chancroid, 2% syphilis, 0.7% lymphogranuloma venereum and 31% undetermined aetiology. Over half (58%) of patients were HIV-1 seropositive. The median duration of symptoms before presentation was 6 days. Patients on acyclovir were more likely to have a healed ulcer on day 7 (63% vs 57%, RR = 1.08, 95% CI 0.98–1.18), shorter time to healing (p = 0.04) and less lesional HIV-1 RNA (p = 0.03). Small ulcers (