Prevalence and Distribution of HPV Genotypes in Immunosuppressed Patients in Lorraine Region.

BACKGROUND: The primary objective of this work was to assess the prevalence and distribution of HPV genotypes in immunosuppressed patients, and to compare them with the French Monsonego cohort. Secondary objectives were to evaluate whether the risk of HPV infection was correlated with HIV viral load, CD4 cell count in HIV-infected patients and the type, number of immunosuppressive therapies or type of pathology (transplant vs. autoimmune diseases) in patients undergoing long-term immunosuppressive therapy. METHODS: An observational, monocentric and historical study was conducted including all immunosuppressed patients having received an HPV testing, in the Laboratory of Virology, Nancy Regional Teaching Hospital Center, between 2014 and 2020. Immunosuppressed patients were either HIV-infected or received long-term immunosuppressive therapy. RESULTS: In our cohort, the prevalence of HPV infection (75.6% vs. 16.1% p < 0.05), the proportion of patients with high-risk HPV infection (48.9% vs. 15.1% p < 0.05) and with multiple HPV infection (41.1% vs. 5.7% p < 0.05) were significantly higher than in the Monsonego cohort. HPV 52 (13%), 53 (13%) and 16 (10%) were the most common in the immunosuppressed population, while it was HPV 16, 42 and 51 in the Monsonego cohort. CONCLUSIONS: This study supports that a particular attention must be given to all the immunosuppressed patients for the screening and care of HPV-related diseases because of major modifications of HPV epidemiology compared with the overall population.

Full-length genome sequencing of RNA viruses-How the approach can enlighten us on hepatitis C and hepatitis E viruses.

Among the five main viruses responsible for human hepatitis, hepatitis C virus (HCV) and hepatitis E virus (HEV) are different while sharing similarities. Both viruses can be transmitted by blood or derivatives whereas HEV can also follow environmental or zoonotic routes. These highly variable RNA viruses can cause chronic hepatitis potentially leading to hepatocarcinoma. HCV and HEV can develop new structures and functions under selective pressure to adapt to host immunity, human tissues, treatments or even various animal reservoirs. Elsewhere, with directly acting antiviral treatments, HCV can be eradicated whereas HEV is an emerging pathogen against which specific treatments have to be improved. As a unique molecular tool able to explore viral genomic plasticity, full-length genome (FLG) sequencing has become easier, faster and cheaper. The present review will show how FLG sequencing can explore these RNA viruses with the aim to investigate key genomics data to improve basic knowledge, patients' healthcare and preventive tools.

SARS-Cov-2 fulminant myocarditis: an autopsy and histopathological case study.

The coronavirus disease 2019 (COVID-19), due to SARS-CoV-2, is primarily a respiratory disease, causing in most severe cases life-threatening acute respiratory distress syndrome (ARDS). Cardiovascular involvement can also occur, such as thrombosis or myocarditis, generally associated with pulmonary lesions. Little is known about SARS-CoV-2-induced myocarditis. We report the case of a 69-year-old man suffering from a refractory cardiogenic shock, without significant lung involvement. Prior to death, several nasopharyngeal swabs and distal bronchoalveolar lavage were sampled in order to perform RT-PCR analyses for SARS-CoV-2-RNA, which all gave negative results. Autopsy showed coronary atherosclerosis, without acute complication. Microscopic examination of the heart revealed the existence of an intense multifocal inflammatory infiltration, in both ventricles and septum, composed in its majority of macrophages and CD8+ cytotoxic T lymphocytes (CD4/CD8 ratio: 0.11). Immunohistochemistry for anti-SARS nucleocapsid protein antibody was strongly positive in myocardial cells, but not in lung tissue. RT-PCR was realized on formalin-fixed paraffin-embedded lung and heart tissue blocks: only heart tissue was positive for SARS-CoV-2 RNA. In conclusion, this exhaustive post-mortem pathological case study of fulminant myocarditis demonstrates the presence of SARS-CoV-2 RNA in heart tissue, without significant lung involvement. Immunohistochemistry showed that the virus was specifically localized in cardiomyocytes and induced a strong cytotoxic T cells inflammatory response. This case report thus gives new insight in the pathogenesis of SARS-CoV-2-induced myocarditis and emphasizes on the importance and reliability of post-mortem analyses in order to better understand the physiopathology of this worldwide spreading new viral disease.

Curative or pre-emptive adenovirus-specific T cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: a successful phase I/II multicenter clinical trial.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT), the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD) and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST) infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB). METHODS: Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN)-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9%) or a third party haploidentical donor (57.1%). One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. RESULTS: One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 103 CD3+IFN-γ+ cells/kg) up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%). Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. CONCLUSIONS: Adoptive transfer of rapidly isolated ADV-VST is an effective therapeutic option for achieving in vivo expansion of specific T cells and clearance of viral load, even as a pre-emptive treatment. Our study highlights that third party haploidentical donors are of great interest for ADV-VST generation in the context of UCB transplantation. (N° Clinical trial.gov: NCT02851576, retrospectively registered).

Near Full-Length Characterization and Population Dynamics of the Human Immunodeficiency Virus Type I Circulating Recombinant Form 42 (CRF42_BF) in Luxembourg.

A new recombinant form representing a mosaic of HIV-1 subtype B and F1 and designated as CRF42_BF was identified in Luxembourg. We confirmed the inedited nature of CRF42_BF by near full-length genome characterization and retrieved a possible ancestor originating from Brazil. The demographic history of CRF42_BF in Luxembourg using Bayesian coalescent-based methods was investigated. The exponential phase of the logistic growth happened in a very short time period of approximately 5 months associated with a high mean rate of population growth of 15.02 new infections per year. However, CRF42_BF was not characterized by either a higher ex vivo replication capacity in peripheral blood mononuclear cells (PBMCs) or a higher ex vivo transmission efficiency from monocyte-derived dendritic cells to PBMCs as compared to B and F1 viruses. These data do not support a high pathogenic potential of CFR42_BF but rather an initial bursting spread of the recombinant probably due to a more favorable transmission route.

Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy.

BACKGROUND: Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen. METHODS: Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool. RESULTS: After immunoselection, a mean of 0.53 ± 0.25 × 106 cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection. CONCLUSIONS: Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.

Human herpesvirus 6 infection after allogeneic stem cell transplantation: incidence, outcome, and factors associated with HHV-6 reactivation.

BACKGROUND: Human herpesvirus 6 (HHV-6) is susceptible to latency and reactivation in hematopoietic stem cell transplant (HSCT) recipients. We investigated the incidence of HHV-6 DNAemia and factors related to HHV-6 DNAemia and death after allogeneic stem cell transplantations. We also explored the relationship between HHV-6 viral load and the presence of clinical signs. METHODS: Data concerning age, sex, transplantation conditions, graft-versus-host disease (GVHD), treatments, clinical signs, outcome, HHV-6, and other infections were collected for a historical cohort of 390 HSCT performed between 1999 and 2008 in the Transplant Unit of Nancy University Hospital Center. Univariate analysis was used to evaluate influences between the different parameters. RESULTS: The study included 220 of the 390 allogeneic HSCTs. For the analyzed period, 44 patients (n=44/220, 20%) presented HHV-6 DNAemia in whole blood, including three integrated forms. Fifteen percent (7/41) of HHV-6-positive patients presented clinical signs not related to higher viral load (P=0.164). The factors associated with HHV-6 DNAemia were as follows: cord blood transplantation (P<0.001), conditioning regimen (P=0.030), acute GVHD (P=0.003), and the type of prophylactic treatment for GVHD (P=0.001). HHV-6 DNAemia was not significantly associated with cytomegalovirus infection (P=0.937). HHV-6 DNAemia was not associated with death (P=0.151). CONCLUSIONS: HHV-6 DNAemia was not so frequent after allogeneic transplantation. Factors associated with HHV-6 DNAemia were similar to those for other infections. No abnormally high death rate was observed in the HHV-6 positive population. The presence of clinical signs did not appear to be statistically related to HHV-6 viral load.

Varicella arthritis in childhood: a case report and review of the literature.

Nonbacterial arthritis is a rare but well-recognized complication of acute varicella in children. Reported cases usually described monoarthritis of the knee that occurs at the onset of the rash or shortly after. Herein, we describe a case of arthritis of the hip that occurred in a 4-year-old girl 6 days before the onset of rash. The presence of varicella-zoster virus DNA in synovial fluid was confirmed by real-time polymerase chain reaction. A review of the literature reported 26 previous cases of aseptic arthritis due to varicella infection among children.

The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Monitoring of EBV DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary, but not sufficient, to identify patients at risk of EBV-induced post-transplantation lymphoproliferative disorders (PTLD). Combining this with quantifying EBV-specific cellular immunity was shown to be helpful. In this study, we evaluated the value of IFNγ-Elispot assay in monitoring EBV DNAemia after HSCT. METHODS: EBV-DNA load in whole blood was monitored at least weekly using real-time PCR in 40 recipients of HSCT. Quantitative and qualitative T-cell recoveries, including EBV-specific T-cell quantification by Elispot assay, were studied 60, 100, 180 and 360 days after HSCT. RESULTS: Among the 35 evaluable patients, 14 (35%) presented EBV DNAemia, only 2/14 (14%) needing pre-emptive treatment with rituximab. The greatest risk factor for EBV DNAemia was the presence of anti-thymocyte globulin (ATG) (p=0.005). EBV-specific cellular immune recovery was monitored by IFNγ-Elispot assay. Using multivariate analysis, four factors were found to significantly influence IFNγ-Elispot results at defined times post-HSCT: EBV DNAemia, young age, global T-cell recovery and severe acute GVHD. In those cases where EBV DNAemia occurred and cleared spontaneously, Elispot results gave more than 1000 spot-forming cells (SFC)/10(6)PBMC. CONCLUSION: Elispot assay may be usefully combined with EBV-DNA load monitoring to determine when a patient should receive pre-emptive treatment, or when the clinician should avoid Rituximab use which severely immunocompromises patients.

Analyses of the effects of collection and processing time on the results of serology testing of cadaveric cornea donors.

Serologic testing results are one of the major reasons for discarding donor corneas. This study assessed the prevalence of serologic markers and the times of preanalytic steps. The prevalence of serologic markers in 705 cornea donors of the Lorraine region was studied. One hundred forty-one (20%) corneas were discarded on the basis of serologic testing. In a subsample of 180 consecutive cornea donors, data concerning the time of death, sampling, and serology testing were collected. When the time between death and sampling did not exceed 8 h (n = 122), immediate decantation (separation of serum from clot) led to less discarding of corneas than delayed decantation (2/46 [4.3%] discarded in the group with immediate decantation versus 13/76 [17.1%] discarded in the group with delayed decantation [P = 0.038]). For blood samples collected within 8 h of death, the rate of reactive results is significantly lower if samples are immediately decanted.

Comparison of in-house real-time quantitative PCR to the Adenovirus R-Gene kit for determination of adenovirus load in clinical samples.

In the context of hematopoietic stem cell transplantation, adenovirus infections are associated with relevant mortality and morbidity. Detection of adenovirus DNA by quantitative PCR is the "gold standard" for these patients. A total of 150 samples, namely, 78 whole-blood, 22 cerebrospinal fluid, 24 digestive biopsy, and 26 stool samples, from 29 patients, including 24 hematopoietic stem cell transplant recipients, were tested for the detection of adenovirus using an in-house real-time quantitative PCR assay (A. Heim, C. Ebnet, G. Harste, and P. Pring-Akerblom, J. Med. Virol. 70:228-239, 2003) and the commercially available Adenovirus R-Gene kit. Adenovirus DNA was automatically isolated from whole-blood samples (Magna Pure LC system; Roche) or was manually extracted from other specimens (QIAamp; Qiagen) using the appropriate kit. The intra- and interassay reproducibilities and sensitivities were evaluated with cell culture supernatant dilutions. Of the 150 samples tested, 86 were found to be positive and 55 were found to be negative using both techniques. Nine (6%) discordant results were obtained. In most cases, discrepant results concerned samples with low viral loads. Quantitative results for all concordant positive samples were analyzed using the Spearman correlation test. A good correlation between the results of the in-house assay and those of the kit assay was obtained (r = 0.95; P < 0.001). Regarding the threshold cycle value for internal control spiked samples, none of the 150 samples tested contained a PCR inhibitor. In conclusion, a relevant correlation of results between the in-house assay and the kit assay, as well as the high-quality reproducibility and sensitivity of the kit assay, warranted its use for follow-up of hematopoietic stem cell transplantation recipients.

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Prevalence of hepatitis B and C and risk factors for nonvaccination in inflammatory bowel disease patients in Northeast France.

BACKGROUND: Data regarding the prevalence of hepatitis C (HCV) and hepatitis B (HBV) in inflammatory bowel disease (IBD) patients are conflicting. METHODS: In all, 315 IBD (252 Crohn's disease [CD] and 63 ulcerative colitis [UC]) patients were consecutively recruited between June 2005 and May 2009. RESULTS: The median age was 33 years (interquartile range [IQR]: 24-43) and median disease duration was 5 years (IQR: 2-11). Present and/or past HBV and HCV infection was found in 2.86% of 315 patients (CD: HBsAg 0.79%, anti-HBc 2.78%, anti-HCV 0.79%; UC: HBsAg 1.59%, anti-HBc 1.59%, anti-HCV 1.59%). Effective vaccination (anti-HBs without anti-HBc) was present in 48.9% of 315 patients. In multivariate analysis, age at diagnosis over 31 years (odds ratio [OR] 0.29; 95% confidence interval [CI] 0.15-0.58; P = 0.005), disease duration over 7 years (OR 0.43; 95% CI 0.23-0.83; P = 0.005), age at inclusion over 33 years (OR 0.44; 95% CI 0.20-0.94; P = 0.005), and CD (OR 0.29; 95% CI 0.15-0.58; P = 0.005) were associated with the lack of effective vaccination. Two HBsAg-positive patients, including 1 under curative nucleoside/nucleotide analog treatment, had received 6 and 7 infliximab infusions, and 1 HCV RNA-positive subject had been receiving corticosteroid and azathioprine therapies for 12 and 33 months, respectively. No viral reactivation occurred in these patients. CONCLUSIONS: The prevalence of HBV and HCV infection in French IBD patients is similar to that of the general population. While the ECCO recommends an effective HBV vaccination in IBD, half of the patients were not vaccinated. The nonvaccination risk factors identified in our study may allow targeted vaccination coverage.

Contribution of human herpesvirus 6 (HHV-6) viral load in whole blood and serum to investigate integrated HHV-6 transmission after haematopoietic stem cell transplantation.

BACKGROUND: Human herpesvirus 6 (HHV-6) is susceptible to latency and recurrence. A less-frequent form of HHV-6 persistence is the integration of viral DNA into host chromosomes. OBJECTIVES: To investigate HHV-6 viral load after haematopoietic stem cell transplantation (HSCT) in whole blood (WB) and serum with regard to integrated HHV-6 transmission diagnosis. STUDY DESIGN: HHV-6 DNA quantitation in serum and WB was performed using quantitative polymerase chain reaction for the follow-up of a 16-year-old girl after HSCT. In whole blood, results were expressed as HHV-6 genomic equivalent copies (gec) per milliliter of WB or per million cells. RESULTS: HHV-6 viral load (undetectable before HSCT) increased up to 3.05 x 10(7)gec/10(6)cells. HHV-6 viral load in the donor sample (3.44 x 10(6)gec/10(6)cells) was in favor of viral transmission through HSCT. The correlation between viral load in WB and serum was significant (p=0.0005). Viral load results expressed as gec/10(6)cells in WB was more reliable than results expressed as gec/ml of whole blood. CONCLUSION: These findings indicate that HHV-6 may be transmitted during HSCT as integrated virus contained in the graft. This reiterates that in the setting of HSCT, HHV-6 viral load must be correctly interpreted. Using HHV-6 viral load expressed as gec/10(6) cells may be more suitable for the follow-up of patients with integrated HHV-6.

Methylenetetrahydrofolate reductase 677 T allele protects against persistent HBV infection in West Africa.

BACKGROUND/AIMS: Homocysteine metabolism is linked to DNA methylation, a mechanism potentially involved in the course of hepatitis B virus (HBV) infection. We evaluated the association of determinants of homocysteine metabolism with the outcome of HBV infection. METHODS: Four hundred and fifty-five healthy adults from Togo and Benin were tested for HBV serologic markers, HLA DR alleles, folate, vitamin B12, methylenetetrahydrofolate reductase (MTHFR) 677 C-->T, 1298 A-->C and methionine synthase 2756 A-->G polymorphisms. RESULTS: Seventy-eight percent of the study population was anti-HBc positive. Among them, 202 (56.9%) were anti-HBs positive and 58 (16.3%) were HBsAg positive. After stepwise logistic regression, the MTHFR 677 T allele was independently associated with persistence of detectable anti-HBs antibodies (OR: 2.47; 95% CI: 1.29-4.71; p=0.006). The mean HBV DNA level was significantly lower in HBsAg positive subjects carrying the 677 T allele than in those with the 677 CC genotype (1000+/-1406 vs. 2,400,000+/-214,000 copies/ml, p=0.005). Beninese origin and HLA-DRB1*09 allele were the other determinants independently associated with favorable outcome of HBV infection. CONCLUSIONS: The methylenetetrahydrofolate reductase 677 T allele seems to protect against chronic HBV infection in young African adults.

Central African Republic is part of the West-African hepatitis B virus genotype E crescent.

BACKGROUND: Recent studies have shown that Hepatitis B virus (HBV) genotype E predominates in a vast crescent in West-Africa spanning from Senegal to Angola. OBJECTIVES: To determine whether HBV strains in the Central African Republic (CAR) belong predominately to the homogeneous West-African genotype E or whether they are more closely related to genotypes found in East Africa. STUDY DESIGN: Serum samples were randomly collected from 196 patients admitted with symptoms of acute or chronic hepatitis to the Central Hospital in Bangui. Thirty complete and 36 partial sequences of HBV strains were obtained. RESULTS: Ninety-four percent (62/66) of the strains belonged to genotype E, while genotype A1, most closely related to a strain from Tanzania and genotype D were detected in only one and three samples, respectively. One strain presented a recombination between the S and X gene of a genotype E precursor and a partial PreC/C gene of a genotype D precursor. CONCLUSIONS: Genotype E is predominant in CAR with little overlap with genotypes from Eastern Africa, extending the West-African HBV genotype E crescent further to the East.

Phylogenetic analysis of the precore/core gene of hepatitis B virus genotypes E and A in West Africa: new subtypes, mixed infections and recombinations.

One hundred and twenty-two new hepatitis B virus (HBV) preC/C sequences and three complete genomes from three major countries in West Africa were analysed. The majority of sequences were of genotype E and the only other genotype found was genotype A. Although for genotype E sequences, the genetic diversity of the preC/C gene was about two to three times higher than that of the preS/S gene, it was still considerably lower than that for genotype A sequences. The HBV/E preC/C gene was related most closely to subgenotype D1 and D2 sequences. Evidence of recombination was found in two strains that were of genotype A in the preS/S gene and of genotype E in the preC/C gene. The genotype A strains from Cameroon, Mali and Nigeria could be divided phylogenetically into three subtypes, A3 and two new subtypes, tentatively designated A4 and A5. Each subtype presented a genetic diversity of 2.19-3.85 % and intersubtype distances of 4.47-5.97 %. Interestingly, one sample from Nigeria showed evidence of a triple recombination of genotypes E/D and A, separated by a genotype G-specific insert of 36 bp. Of 110 patients, 19 (17.3 %) showed a coinfection of genotypes A and E, mostly in human immunodeficiency virus-positive children from Cameroon. Thus, in Cameroon, where both genotypes coexist, 37 % of all individuals tested had mixed infections. The low genetic variability in the preC/C gene of genotype E supports our previous speculation about a relatively short evolutionary history of this genotype, in contrast to the subtype-rich African genotype A strains.