Extent of the protection afforded by histo-blood group polymorphism against rotavirus gastroenteritis in metropolitan France and French Guiana.

Human rotaviruses attach to histo-blood group antigens glycans and null alleles of the ABO, FUT2 and FUT3 genes seem to confer diminished risk of gastroenteritis. Yet, the true extent of this protection remains poorly quantified. Here, we conducted a prospective study to evaluate the risk of consulting at the hospital in non-vaccinated pediatric patients according to the ABO, FUT2 (secretor) and FUT3 (Lewis) polymorphisms, in Metropolitan France and French Guiana. At both locations, P genotypes were largely dominated by P [8]-3, with P [6] cases exclusively found in French Guiana. The FUT2 null (nonsecretor) and FUT3 null (Lewis negative) phenotypes conferred near full protection against severe gastroenteritis due to P [8]-3 strains (OR 0.03, 95% CI [0.00-0.21] and 0.1, 95% CI [0.01-0.43], respectively in Metropolitan France; OR 0.08, 95% CI [0.01-0.52] and 0.14, 95%CI [0.01-0.99], respectively in French Guiana). Blood group O also appeared protective in Metropolitan France (OR 0.38, 95% CI [0.23-0.62]), but not in French Guiana. The discrepancy between the two locations was explained by a recruitment at the hospital of less severe cases in French Guiana than in Metropolitan France. Considering the frequencies of the null ABO, Secretor and Lewis phenotypes, the data indicate that in a Western European population, 34% (95% CI [29%; 39%]) of infants are genetically protected against rotavirus gastroenteritis of sufficient severity to lead to hospital visit.

Humoral immunity to SARS-CoV-2 and seasonal coronaviruses in children and adults in north-eastern France.

BACKGROUND: Children are underrepresented in the COVID-19 pandemic and often experience milder disease than adolescents and adults. Reduced severity is possibly due to recent and more frequent seasonal human coronaviruses (HCoV) infections. We assessed the seroprevalence of SARS-CoV-2 and seasonal HCoV specific antibodies in a large cohort in north-eastern France. METHODS: In this cross-sectional seroprevalence study, serum samples were collected from children and adults requiring hospital admission for non-COVID-19 between February and August 2020. Antibody responses to SARS-CoV-2 and seasonal HCoV (229E, HKU1, NL63, OC43) were assessed using a bead-based multiplex assay, Luciferase-Linked ImmunoSorbent Assay, and a pseudotype neutralisation assay. FINDINGS: In 2,408 individuals, seroprevalence of SARS-CoV-2-specific antibodies was 7-8% with three different immunoassays. Antibody levels to seasonal HCoV increased substantially up to the age of 10. Antibody responses in SARS-CoV-2 seropositive individuals were lowest in adults 18-30 years. In SARS-CoV-2 seronegative individuals, we observed cross-reactivity between antibodies to the four HCoV and SARS-CoV-2 Spike. In contrast to other antibodies to SARS-CoV-2, specific antibodies to sub-unit 2 of Spike (S2) in seronegative samples were highest in children. Upon infection with SARS-CoV-2, antibody levels to Spike of betacoronavirus OC43 increased across the whole age spectrum. No SARS-CoV-2 seropositive individuals with low levels of antibodies to seasonal HCoV were observed. INTERPRETATION: Our findings underline significant cross-reactivity between antibodies to SARS-CoV-2 and seasonal HCoV, but provide no significant evidence for cross-protective immunity to SARS-CoV-2 infection due to a recent seasonal HCoV infection. In particular, across all age groups we did not observe SARS-CoV-2 infected individuals with low levels of antibodies to seasonal HCoV. FUNDING: This work was supported by the « URGENCE COVID-19 ¼ fundraising campaign of Institut Pasteur, by the French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (Grant No. ANR-10-LABX-62-IBEID), and by the REACTing (Research & Action Emerging Infectious Diseases), and by the RECOVER project funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No. 101003589, and by a grant from LabEx IBEID (ANR-10-LABX-62-IBEID).

Association between Neu5Gc carbohydrate and serum antibodies against it provides the molecular link to cancer: French NutriNet-Santé study.

BACKGROUND: High consumption of red and processed meat is commonly associated with increased cancer risk, particularly colorectal cancer. Antibodies against the red meat-derived carbohydrate N-glycolylneuraminic acid (Neu5Gc) exacerbate cancer in "human-like" mice. Human anti-Neu5Gc IgG and red meat are both independently proposed to increase cancer risk, yet how diet affects these antibodies is largely unknown. METHODS: We used world global data to demonstrate that colorectal cancer incidence and mortality are associated with increased national meat consumption. In a well-defined large cohort, we used glycomics to measure daily Neu5Gc intake from red meat and dairy, and investigated serum as well as affinity-purified anti-Neu5Gc antibodies. Based on 24-h dietary records, daily Neu5Gc intake was calculated for 19,621 subjects aged ≥ 18 years of the NutriNet-Santé study. Serum and affinity-purified anti-Neu5Gc antibodies were evaluated by ELISA and glycan microarrays in representative 120 individuals, each with at least eighteen 24-h dietary records (aged 45-60, Q1-Q4; aged > 60, Q1 and Q4; 10 men/women per quartile). RESULTS: We found that high-Neu5Gc diet, gender, and age affect the specificity, levels, and repertoires of anti-Neu5Gc IgG immune responses, but not their affinity. Men consumed more Neu5Gc than women, mostly from red meat (p = 0.0015), and exhibited higher overall serum anti-Neu5Gc IgG levels by ELISA (3.94 ng/µl versus 2.22 ng/µl, respectively; p = 0.039). Detailed glycan microarray analysis against 56 different glycans revealed high Neu5Gc-specificity with increased anti-Neu5Gc IgG and altered repertoires, associated with higher consumption of Neu5Gc from red meat and cow dairy. Affinity purification of serum anti-Neu5Gc antibodies revealed increased levels and biased array repertoire patterns, without an increase in antibody affinity, in individuals consuming higher Neu5Gc levels. Furthermore, in a high-meat diet, antibody diversity patterns on glycan microarrays shifted towards Neu5Gcα3-linked glycans, increasing the α3/α6-glycans ratio score. CONCLUSIONS: We found a clear link between the levels and repertoire of serum anti-Neu5Gc IgG and Neu5Gc intake from red meat and dairy. These precise rational methodologies allowed to develop a Gcemic index to simplify the assessment of Neu5Gc in foods that could potentially be adapted for dietary recommendations to reduce cancer risk.

Host-Range Shift Between Emerging P[8]-4 Rotavirus and Common P[8] and P[4] Strains.

In Tunisia, we observed that rotavirus P[8]-3 and P[4] strains in young children with gastroenteritis associate with secretor histo-blood group phenotype. In contrast, the emerging P[8]-4 strain, representing 10% of cases, was exclusively found in nonsecretor patients. Unlike VP8* from P[8]-3 and P[4] strains, the P[8]-4 VP8* protein attached to glycans from saliva samples regardless of the donor's secretor status. Interestingly, a high frequency of FUT2 enzyme deficiency (nonsecretor phenotype) was observed in the population. This may allow cocirculation of P[8]-3 and P[8]-4 strains in secretor and nonsecretor children, respectively.

Impact of antiviral prophylaxis in adults Epstein-Barr Virus-seronegative kidney recipients on early and late post-transplantation lymphoproliferative disorder onset: a retrospective cohort study.

Post-transplantation lymphoproliferative disorder (PTLD) pathogenesis is related to EBV infection. Mismatch with the donor (EBV D+/R-) is the main risk factor for both early PTLD (<1 year post-transplantation) and late (>1 year). In these at-risk patients, the role of antiviral prophylaxis for preventing PTLD remains controversial. We analyzed the impact of antiviral drugs given to prevent CMV disease in a monocentric retrospective cohort of 73 adult kidney or kidney-pancreas EBV-seronegative recipients, transplanted between 01/01/2000 and 01/01/2016. Thirty-seven (50.7%, prophylaxis group) received (val-)aciclovir or (val-)ganciclovir for 3-6 months and 36 (49.3%, no-prophylaxis group) received no-prophylaxis. Mean follow-up was 69 ± 7.2 months in the prophylaxis group and 91 ± 10.3 months in the no-prophylaxis group. Monitoring of EBV PCR revealed that prophylaxis delayed primary infection at 100 days (43% vs. 84%, P = 0.02). Early PTLD incidence was not different between groups (4/37 vs. 4/36, P = 0.99). Concerning late events, EBV-related neoplasia incidence was significantly lower in treated patients among whom no cases were observed, while in the no-prophylaxis group 6 cases were reported (P = 0.02). Despite a weak level of evidence our study suggests that antiviral prophylaxis could prevent late onset PTLD.

Lytic EBV infection investigated by detection of Soluble Epstein-Barr virus ZEBRA in the serum of patients with PTLD.

The ZEBRA protein (encoded by the BZLF1 gene), is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. Several studies highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders like post-transplant lymphoproliferative disease (PTLD). Here, we use an antigen-capture ELISA assay specifically designed to detecting the circulating soluble ZEBRA (sZEBRA) in serum samples (threshold value determined at 40ng/mL). We retrospectively investigated a population of 66 transplanted patients comprising 35 PTLD. All the samples from a control population (30 EBV-seronegative subjects and 25 immunocompetent individuals with EBV serological reactivation), classified as sZEBRA < 40ng/mL were assigned as negative. At PTLD diagnosis, EBV genome (quantified by qPCR with EBV DNA>200 copies/mL) and sZEBRA were detectable in 51% and 60% of cases, respectively. In the patients who developed a pathologically-confirmed PTLD, the mean sZEBRA value in cases, was 399 ng/mL +/- 141 versus 53ng/mL +/- 7 in patients who did not (p < 0,001). This is the first report relating to the detection of the circulating ZEBRA in serum specimens, as well as the first analysis dealing with the lytic cycle of EBV in PTLD patients with this new biomarker.

An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains.

The objective was to develop a method of HCV genome sequencing that allowed simultaneous genotyping and NS5A inhibitor resistance profiling. In order to validate the use of a unique RT-PCR for genotypes 1-5, 142 plasma samples from patients infected with HCV were analysed. The NS4B-NS5A partial region was successfully amplified and sequenced in all samples. In parallel, partial NS3 sequences were analyzed obtained for genotyping. Phylogenetic analysis showed concordance of genotypes and subtypes with a bootstrap >95% for each type cluster. NS5A resistance mutations were analyzed using the Geno2pheno [hcv] v0.92 tool and compared to the list of known Resistant Associated Substitutions recently published. In conclusion, this tool allows determination of HCV genotypes, subtypes and identification of NS5A resistance mutations. This single method can be used to detect pre-existing resistance mutations in NS5A before treatment and to check the emergence of resistant viruses while undergoing treatment in major HCV genotypes (G1-5) in the EU and the US.

Impact of stem cell graft on early viral infections and immune reconstitution after allogeneic transplantation in adults.

BACKGROUND: Viral infections are well-known complications after allogeneic stem cell transplant (allo-SCT). OBJECTIVES: We compared prospectively incidences of DNAemia and active infections (AI) for five opportunistic viruses (Human Herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), Cytomegalovirus (CMV) and Adenovirus (ADV)) and kinetics of immune reconstitution (IR) in adults receiving either double umbilical cord blood (dUCB group) or unrelated peripheral blood stem cell (uPBSC group) allo-SCT after a reduced-intensity conditioning (RIC) regimen. STUDY DESIGN: Whole blood samples were collected at transplant, every 15days during the first 3 months and at 4, 5 and 6 months post-transplant. RESULTS: Sixty-five patients were enrolled (uPBSC n=34; dUCB n=31). Incidences of HHV-6 and BKPyV DNAemia were significantly higher for dUCB (97% vs 23.5% and 58% vs 32%, respectively) while EBV DNAemia was more frequently detected in uPBSC (71% vs 26%). The incidence of CMV DNAemia was similar between both groups. ADV AI developed only in dUCB. HHV-6 AI were also higher in dUCB (84% vs 21%). In multivariate analysis, dUCB graft was the only independent factor associated with HHV-6 DNAemia (OR: 19.0; 95%CI: 5.2-69.1; p<0.0001) while EBV DNAemia were significantly associated with uPBSC (OR: 29.9; 95%CI: 5.68-158; p <0.0001). dUCB graft was also the only factor associated with HHV-6 AI. Finally, higher counts and faster recoveries of B lymphocytes (p<0.0001) and monocytes (p=0.0007) were observed in the dUCB group. CONCLUSION: We demonstrate a strong correlation between sources of graft and patterns of viral DNAemia and AI and IR after RIC allo-SCT.

Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses.

Primary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C+ NK cells, CD16+ Vδ2(-) γδ T cells, and conventional HCMV-specific CD8+ T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals.IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive immune responses with a deep HCMV imprint. The massive lymphocytosis could be the result of nonadapted or uncontrolled immune responses limiting the effectiveness of the specific responses mounted. Both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage. Furthermore, we cannot exclude a delayed immune response caused by immune escape established by HCMV strains.

Emergence of HCV resistance-associated variants in patients failing sofosbuvir-based regimens: an observational cohort.

BACKGROUND: Real-life effectiveness data of new hepatitis C direct-acting antivirals are now required. The present study aims to assess the rate of sustained viral response (SVR) and virological failure (VF) in patients infected with chronic HCV treated with sofosbuvir (SOF)-based regimens in routine medical practice. METHODS: This observational study included a total of 106 patients infected with HCV genotypes (G)1-4, who initiated SOF-based regimens in 2014. Viral load was followed at baseline, week (W)2, W4, W12 (or W24) and W12 post-treatment. For all VFs, resistance-associated variants (RAVs) were determined at baseline and failure by sequencing of NS5A, NS5B and/or NS3 genes, using the Sanger method. RESULTS: SVR rate was 85% for the whole cohort, 91% for the patients who underwent the full treatment course. The distribution of HCV genotypes was as follows: G1 n=66 (1a=33, 1b=29; 62%), G2 n=8 (8%), G3a n=20 (19%) and G4 n=12 (11%). The main regimens used were SOF+daclatasvir (37%), SOF+ribavirin (33%), SOF+simeprevir (26%) and SOF+ledipasvir (3%). Twenty-five (23%) patients were HIV-coinfected and 1 was HBV-coinfected. Seventy (65%) patients had a prior treatment experience. All VF were relapses (n=9): 3 G1a, 1 G2, 4 G3a and 1 G4, and mutations conferring resistance to NS5A inhibitors were found but none for NS5B polymerase inhibitors. CONCLUSIONS: In a real-life context, the rate of SVR in DAA-treated HCV-infected patients is close to clinical Phase III trial results. RAVs emerged for all patients treated by the anti-NS5A daclatasvir, and persisted several weeks after the end of treatment.

Decreased Frequency of Circulating Myelin Oligodendrocyte Glycoprotein B Lymphocytes in Patients with Relapsing-Remitting Multiple Sclerosis.

Although there is no evidence for a role of anti-MOG antibodies in adult MS, no information on B lymphocytes with MOG-committed BCR is available. We report here on the frequency of anti-MOG B cells forming rosettes with polystyrene beads (BBR) covalently bound to the extracellular domain of rhMOG in 38 relapsing-remitting patients (RRMS) and 50 healthy individuals (HI). We show a substantial proportion of circulating anti-MOG-BBR in both RRMS and HI. Strikingly, MOG-specific B cells frequencies were lower in MS than in HI. Anti-MOG antibodies measured by a cell-based assay were not different between MS patients and controls, suggesting a specific alteration of anti-MOG B cells in MS. Although anti-MOG-BBR were higher in CNS fluid than in blood, no difference was observed between MS and controls. Lower frequency of MOG-BBR in MS was not explained by an increased apoptosis, but a trend for lower proliferative capacity was noted. Despite an efficient B cell transmigration across brain derived endothelial cells, total and anti-MOG B cells transmigration was similar between MS and HI. The striking alteration in MOG-specific B cells, independent of anti-MOG antibody titers, challenges our view on the role of MOG-specific B cells in MS.

Human herpesvirus 6 reactivation before engraftment is strongly predictive of graft failure after double umbilical cord blood allogeneic stem cell transplantation in adults.

Our main objective was to determine new factors associated with engraftment and single-unit predominance after double umbilical cord blood (UCB) allogeneic stem-cell transplantation. Engraftment occurred in 78% of cases in this retrospective study including 77 adult patients. Three-year overall survival, disease-free survival, relapse incidence, and nonrelapse mortality were 55 ± 6%, 44 ± 6%, 33 ± 5%, and 23 ± 4%, respectively. In multivariate analysis, Human herpesvirus 6 reactivation during aplasia (hazard ratio [HR] = 2.63; 95% confidence interval [CI]: 1.64-4.17; p < 0.001), younger recipient age (<53 years) (HR = 1.97; 95% CI: 1.16-3.35; p = 0.012), and lower human leukocyte antigen matching between the two units (3 of 6 or 4 of 6) (HR = 2.09; 95% confidence interval: 1.22-3.59; p = 0.013) were the three factors independently associated with graft failure. Also, factors independently predicting the losing UCB unit were younger age of the UCB unit (odds ratio [OR] = 1.01; 95% CI: 1-1.02; p = 0.035), lower CD34(+) cell dose contained in the UCB unit (≤ 0.8 × 10(5)/kg) (OR = 2.55; 95% CI: 1.05-6.16; p = 0.04), and presence of an ABO incompatibility between the UCB unit and the recipient (OR = 2.53; 95% CI: 1.15-5.53; p = 0.02). Thus, Human herpesvirus 6 reactivation during aplasia, lower unit-unit human leukocyte antigen matching, and younger UCB unit age, as new unfavorable predictive factors, may represent new parameters to take into account after double UCB allogeneic stem-cell transplantation in adults. These results need to be confirmed prospectively, as they may influence unit selections and patient outcomes.

Long-lasting HHV-6 reactivation in long-term adult survivors after double umbilical cord blood allogeneic stem cell transplantation.

Higher incidence of human herpesvirus 6 (HHV-6) infection has been documented after umbilical cord blood allo-transplant in adults. Here we demonstrate that HHV-6 reactivation persists for a very long time in half of the patients after this type of graft. Long-term immune reconstitution does not explain this event, which remains to be explained.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection.

BACKGROUND: Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. OBJECTIVES: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. STUDY-DESIGN: Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. RESULTS: Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood. CONCLUSION: The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.

A FUT2 gene common polymorphism determines resistance to rotavirus A of the P[8] genotype.

Attachment to carbohydrates of the histo-blood group type of several human Rotavirus strains (RVA) has recently been described. Synthesis of these ligands requires a functional FUT2 enzyme, suggesting that FUT2 null homozygote (ie, nonsecretor) individuals may not be recognized by most human RVA strains. Whereas such individuals represent 20% of the control population, this retrospective study determined that none of 51 patients infected by P[8] rotavirus strains were nonsecretors. The lack of α1,2fucosylated carbohydrate motifs in the gut surface mucosa is thus associated with resistance to symptomatic infection and virus attachment to such motifs is essential to the infection process.

Amplified NKG2C+ NK cells in cytomegalovirus (CMV) infection preferentially express killer cell Ig-like receptor 2DL: functional impact in controlling CMV-infected dendritic cells.

CMV infection represents a major complication in hematopoietic stem cell transplantation, which compromises graft outcome. Downregulation of HLA class I expression is one mechanism by which CMV evades T cell-mediated immune detection, rendering infected cells vulnerable to killer cell Ig-like receptor (KIR)(+) NK cells. In this study, we observed that the amplified NKG2C(+) NK cell population observed specifically in CMV seropositive individuals mainly expressed KIR2DL receptors. We have shown that HLA class I expression was downregulated on CMV-infected immature dendritic cells (iDCs), which escape to HLA-A2-pp65-specific T lymphocytes but strongly trigger the degranulation of KIR2D(+) NK cells. CMV infection conferred a vulnerability of C2C2(+) iDCs to educated KIR2DL1(+) and KIR2DL3(+) NK cell subsets. Alloreactivity of KIR2DL1(+) NK cell subsets against C1C1(+) iDCs was maintained independently of CMV infection. Unexpectedly, CMV-infected C1C1(+) iDCs did not activate KIR2DL3(+) NK cell reactivity, suggesting a potential CMV evasion to KIR2DL3 NK cell recognition. Altogether, the coexpression of KIR and NKG2C on expanded NK cell subsets could be related to a functional contribution of KIR in CMV infection and should be investigated in hematopoietic stem cell transplantation, in which the beneficial impact of CMV infection has been reported on the graft-versus-leukemia effect.

HHV-6 cell receptor CD46 expression on various cell subsets of six blood and graft sources: a prospective series.

BACKGROUND: Cord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpesvirus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT. Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference. OBJECTIVES: To prospectively compare the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand. STUDY DESIGN: 52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n = 10), G-CSF mobilised PB (GCSF-PB, n = 10), cord blood (CB, n = 10), unmanipulated bone marrow (uBM, n = 5), leukapheresis product (LP, n = 10) and thawed CB graft (n = 7). CD46 expression was assessed by FACS analysis on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells. RESULTS: As all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison between the three blood sources and the three graft sources. The most impressive result observed was that HHV-6 cell receptor CD46 expression was significantly reduced in almost all cell components of thawed CB graft compared to other graft sources. CONCLUSIONS: This original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults.

Characterization of various blood and graft sources: a prospective series.

BACKGROUND: Studies comparing cell components of blood and graft sources are very scarce. We present here a thorough study examining the cellular content of various sources of blood and cell therapy products. STUDY DESIGN AND METHODS: We have prospectively compared by fluorescence-activated cell sorting analyses the cellular composition of three blood sources on the one hand--peripheral blood (PB; n = 10) versus granulocyte-colony-stimulating factor (G-CSF)-mobilized PB (GCSF-PB, n = 10) versus cord blood (CB, n = 10)--and of three graft sources on the other hand--unmanipulated bone marrow (uBM, n = 5) versus leukapheresis product (LP, n = 10) versus thawed CB graft (n = 7). RESULTS: All median absolute numbers of cell subsets were found significantly higher in GCSF-PB and LP, except for monocytoid dendritic cells (mDCs) in CB and uBM. The most impressive results were the median quantities of memory T and B lymphocytes but also of plasmacytoid DCs (pDCs) contained in LP compared to thawed CB graft, with ratios of 375, 318, and 247, respectively. The proportions of naive and CD4+/CD8- T cells, transitional B cells, and CD5+ and naive B lymphocytes were found significantly higher in CB samples while the proportions of mDCs and pDCs were found significantly lower. CONCLUSION: Our study shows strong differences in terms of quantitative and qualitative cellular composition between several blood or graft sources, possibly explaining the differences observed in terms of outcomes after transplant.

Genotyping and resistance profile of hepatitis C (HCV) genotypes 1-6 by sequencing the NS3 protease region using a single optimized sensitive method.

The objective was to develop a method of NS3 gene sequencing that allowed simultaneous genotyping and protease inhibitor (PI) resistance profiling of HCV genotypes 1-6. To validate the use of a unique RT-PCR for genotypes 1-6 and evaluate its sensitivity, the NS3 protease region was amplified from 140 plasma samples from patients infected with HCV without previous PI therapy. In parallel, NS5b sequences were obtained. Amplification of NS3 was successful in 139/140 samples (99%). For the 135 samples with both NS5b and NS3 sequencing results, phylogenetic analysis showed concordance of genotypes with a bootstrap >90% for each cluster. PI resistance mutations were analyzed using the Geno2pheno [hcv] v1.0 tool. For the 63 genotype 1 (G1) Nantes clinical strains, 12 (19%) presented a natural resistance mutation. This proportion was higher (p<0.05) than that observed in a sample of 374 G1 reference sequences. This significant difference was observed only in subtype 1b (n=7; 25% against n=19; 8%). In conclusion, this tool allows determination of both HCV genotype and identification of PI-resistance mutations. It can be used to detect pre-existing resistance mutations in NS3 before treatment and follow the emergence of resistant viruses during therapy.

Detection of herpes simplex virus (1 and 2), varicella-zoster virus, cytomegalovirus, human herpesvirus 6 and enterovirus in immunocompetent Tunisian patients with acute neuromeningeal disorder.

Enteroviruses (EVs) and human herpesviruses (HHVs) are involved frequently in acute neurological disorders of viral etiology. This study aimed to investigate the incidence of herpes simplex virus types-1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human enteroviruses (EVs) in cerebrospinal fluid (CSF) samples of Tunisian immunocompetent patients with neuromeningeal disorders. The patients had been hospitalized at the Fattouma Bourguiba University Hospital (Monastir, Tunisia) between September 2007 and June 2009. At least one viral genome was detected in 58 (46%) out of 126 CSF samples collected. Enterovirus was detected in 31 of the positive samples (53.4%), CMV in 20 (34.5%), HSV-1 in 3 (5.2%), HSV-2 in 6 (10.3%), VZV in 4 (6.9%), HHV-6 in 2 (3.4%). More than one viral genome was detected in seven CSF samples, including CMV DNA in six of the samples. The high frequency of enteroviral infections in aseptic meningitis was confirmed. The detection of CMV DNA only suggests a direct role of this virus in the etiology of acute neuromeningeal disorder.