Recombination between two strains is a known phenomenon for enteroviruses replicating within a si... more Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence
JAIDS Journal of Acquired Immune Deficiency Syndromes, 2006
: The proportion of non-B HIV-1 variants is increasing in Western Europe. The impact of the high ... more : The proportion of non-B HIV-1 variants is increasing in Western Europe. The impact of the high polymorphism in the protease and reverse transcriptase genes, as recently described for CRF02-AG isolates of African origin, on antiretroviral resistance is still disputed. We first examined the polymorphism of these genes in CRF02-AG strains recovered from drug-naive patients followed at the University Hospital of Saint-Etienne in France, most of these of French origin and harboring a clonal strain as elicited by phylogenic analysis. The first plasma sample detected positive from 31 CRF02-AG and 23 B strains was used to compare sequences with their respective subtype consensus strain. The overall number of mutations was dramatically higher for CRF02-AG strains than for B strains in both protease and reverse transcriptase genes (P < 0.0001 and 0.009, respectively). In addition, no statistically significant difference in the number of therapeutic failures, mean CD4 cell count, and viral load was observed between 22 and 45 patients infected with CRF02-AG or B strains, respectively, during a mean treatment period of 25.5 months. Even if no striking antiretroviral failure linked to this polymorphism was observed during short-term follow-up, its impact on long-term therapy will have to be extensively evaluated in patients infected by non-B HIV-1 variants.
A RT-PCR approach for the direct detection and typing of human enteroviruses in the environment i... more A RT-PCR approach for the direct detection and typing of human enteroviruses in the environment is described in this study. A semi-nested RT-PCR using COnsensus-DEgenerated Hybrid Oligonucleotide Primers (CODEHOP) designed from the VP2 genome region has been developed for the direct typing of enteroviruses in clinical samples (Ibrahim et al., 2013). This CODEHOP/VP2 PCR strategy as well as the CODEHOP/VP1 technique described by Nix et al. (2006), were tested for the detection and typing of enteroviruses in wastewater samples. Virus particles were first extracted and concentrated from wastewater samples by using respectively beef extract and polyethylene glycol 6000, and the presence of enteroviruses was screened by a RT-PCR method using primers from the 5'-end non-coding region (5'NCR). Fifty-two of 172 samples (30.2%) were revealed positive by the 5'NCR method. From these 52 samples, only 19 samples (36.5%) were found positive by at least one of the two CODEHOP techniqu...
Immunosuppressive therapies used for treating ulcerative colitis are known to favor chronic and l... more Immunosuppressive therapies used for treating ulcerative colitis are known to favor chronic and latent viral diseases. This study aimed at evaluating prospectively the association between colonic cytomegalovirus (CMV) reactivation and anti-tumor necrosis factor (TNF) monoclonal antibodies (mabs) by comparison to azathioprine (AZA) in a series of flare-ups occurring in consecutive ulcerative colitis patients. A total of 109 flare-ups were recorded in 73 patients receiving a maintenance therapy by anti-TNF mabs (n = 69) or AZA (n = 40). The CMV DNA load in colonic tissue was determined by reverse transcription polymerase chain reaction on a pair of biopsies. The number of CMV reactivation was of 35% and 38% in patients receiving anti-TNF mabs and AZA, respectively. The median of CMV DNA load was 378 [10-29,800] and 8300 [10-3,25,000] copies/mg of tissue in patients treated by anti-TNF mabs and AZA, respectively (P = 0.11 by Mann-Whitney U test). In a subgroup of 45 patients under anti-TNF mabs requiring an optimized treatment by infliximab, clinical remission (partial Mayo score <3) was not significantly impacted by the presence of CMV reactivation at the time of flare-up (P = 0.52). Twenty of these patients underwent a second colonic biopsy 8 weeks after the initiation of flare-up therapy; except for 3 patients, the colonic CMV DNA load was stable or decreased. Patients under anti-TNF maintenance therapy are not at higher risk of CMV reactivation in case of flare-up. No reciprocal adverse influence was observed between anti-TNF mabs and CMV infection, suggesting that these drugs must be considered for treating flare-ups associated to CMV reactivation.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 2014
The management of children with community-acquired pneumonia (CAP) is largely influenced by the d... more The management of children with community-acquired pneumonia (CAP) is largely influenced by the development of new molecular diagnostic tests that allow the simultaneous detection of a wide range of pathogens. Evaluation of a diagnostic approach including multiplex PCR assays for revisiting the epidemiology and etiology of CAP in children at hospital. Children of all ages consulting at the Emergency Department of the University hospital of Saint-Etienne, France, during the 2012-2013 winter period were included. In addition to bacterial cultures, the following pathogens were detected using biplex commercially-available rt-PCR tests: adenovirus, respiratory syncytial virus, human metapneumovirus, bocavirus, rhinovirus/enterovirus, coronavirus, influenza viruses A and B, parainfluenza viruses, Mycoplasma pneumoniae and Chlamydophila pneumonia. From 85 patients with CAP, at least one pathogen was identified in 81 cases (95.3%), including 4 bacterial exclusive infections (4.7%), 53 viral...
Previous studies have suggested an association between cytomegalovirus (CMV) infection and steroi... more Previous studies have suggested an association between cytomegalovirus (CMV) infection and steroid-refractory inflammatory bowel disease. In this study, the use of CMV DNA load during acute flare-ups of ulcerative colitis (UC) to predict resistance to immunosuppressive therapy was evaluated in intestinal tissue. Forty-two consecutive patients (sex ratio M/F: 0.9, mean age: 43.6 years) hospitalized for moderate to severe UC and treated with IV steroids were included prospectively. A colonoscopy was performed for each patient at inclusion; colonic biopsy samples of the pathological tissue, and if possible, of the healthy mucosa, were tested for histological analysis and determination of CMV DNA load by real-time polymerase chain reaction assay. Patients were treated as recommended by the current guidelines. Sixteen patients were found positive for CMV DNA in inflamed intestinal tissue but negative in endoscopically healthy tissue; all of these patients were positive for anti-CMV IgG, three exhibited CMV DNA in blood, and none was positive for intestinal CMV antigen by immunohistochemistry detection. In the 26 remaining patients, no stigmata of recent CMV infection were recorded by any technique. By multivariate analysis, the only factor associated with CMV DNA in inflammatory tissue was the resistance to steroids or to three lines of treatment (risk ratio: 4.7; 95% confidence interval: 1.2-22.5). A CMV DNA load above 250 copies/mg in tissue was predictive of resistance to three successive regimens (likelihood ratio+=4.33; area under the receiver-operating characteristic curve=0.85). Eight UC patients with CMV DNA in inflamed tissue and therapeutic failure received ganciclovir; a clinical remission was observed in seven cases, with a sustained response in five of them. The CMV DNA load determined in inflamed intestinal tissue predicts resistance to steroid treatment and to three drug regimens in UC. Initiation of an early antiviral treatment in these patients might delay the occurrence of resistance to current treatments.
A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) an... more A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.
Human papillomavirus (HPV) infection, especially by HPV 16, is frequently detected in oropharynge... more Human papillomavirus (HPV) infection, especially by HPV 16, is frequently detected in oropharyngeal squamous cell carcinoma (OSCC). The expression of viral oncoproteins in tumoral tissues of OSCCs suggests the implication of HPV in tumorogenesis. It should now be systematically detected and considered in each patient's treatment and outcome. To investigate the prevalence of HPV infection, the oncogenic role of HPV in patients from the South-East of France with head and neck squamous cell carcinoma (HNSCC), and the resulting clinical implications. Biopsy samples from 200 patients with HNSCC were analyzed. For each patient, one or two biopsies of tumoral tissue were analyzed simultaneously with a biopsy of healthy tissue. Fresh frozen tissues were tested by molecular techniques for HPV DNA detection and genotyping as well as mRNA expression of oncoproteins E6 and E7. Expression of p16 was also analyzed by immunohistochemical staining. HPV DNA tested positive in 11.5% of biopsy samples. The HPV prevalence was higher in OSCCs (91.3 vs 27.3, p < 0.0001) and in patients not consuming tobacco (65.2% vs 95.4%, p < 0.0001). The estimated 3-year overall survival rates were 67.0% for HPV-infected patients versus 39.9% for non-infected patients. The high-risk HPV 16 was the most common type detected (65.2%). In 12 of 18 patients exhibiting DNA of high-risk HPV in their tumor tissue, the same viral genome was also present in normal tissue. E6 and E7 expression was found in 9 of 14 tumoral biopsies tested for these markers.
Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to... more Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.
Recombination between two strains is a known phenomenon for enteroviruses replicating within a si... more Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence
JAIDS Journal of Acquired Immune Deficiency Syndromes, 2006
: The proportion of non-B HIV-1 variants is increasing in Western Europe. The impact of the high ... more : The proportion of non-B HIV-1 variants is increasing in Western Europe. The impact of the high polymorphism in the protease and reverse transcriptase genes, as recently described for CRF02-AG isolates of African origin, on antiretroviral resistance is still disputed. We first examined the polymorphism of these genes in CRF02-AG strains recovered from drug-naive patients followed at the University Hospital of Saint-Etienne in France, most of these of French origin and harboring a clonal strain as elicited by phylogenic analysis. The first plasma sample detected positive from 31 CRF02-AG and 23 B strains was used to compare sequences with their respective subtype consensus strain. The overall number of mutations was dramatically higher for CRF02-AG strains than for B strains in both protease and reverse transcriptase genes (P < 0.0001 and 0.009, respectively). In addition, no statistically significant difference in the number of therapeutic failures, mean CD4 cell count, and viral load was observed between 22 and 45 patients infected with CRF02-AG or B strains, respectively, during a mean treatment period of 25.5 months. Even if no striking antiretroviral failure linked to this polymorphism was observed during short-term follow-up, its impact on long-term therapy will have to be extensively evaluated in patients infected by non-B HIV-1 variants.
A RT-PCR approach for the direct detection and typing of human enteroviruses in the environment i... more A RT-PCR approach for the direct detection and typing of human enteroviruses in the environment is described in this study. A semi-nested RT-PCR using COnsensus-DEgenerated Hybrid Oligonucleotide Primers (CODEHOP) designed from the VP2 genome region has been developed for the direct typing of enteroviruses in clinical samples (Ibrahim et al., 2013). This CODEHOP/VP2 PCR strategy as well as the CODEHOP/VP1 technique described by Nix et al. (2006), were tested for the detection and typing of enteroviruses in wastewater samples. Virus particles were first extracted and concentrated from wastewater samples by using respectively beef extract and polyethylene glycol 6000, and the presence of enteroviruses was screened by a RT-PCR method using primers from the 5'-end non-coding region (5'NCR). Fifty-two of 172 samples (30.2%) were revealed positive by the 5'NCR method. From these 52 samples, only 19 samples (36.5%) were found positive by at least one of the two CODEHOP techniqu...
Immunosuppressive therapies used for treating ulcerative colitis are known to favor chronic and l... more Immunosuppressive therapies used for treating ulcerative colitis are known to favor chronic and latent viral diseases. This study aimed at evaluating prospectively the association between colonic cytomegalovirus (CMV) reactivation and anti-tumor necrosis factor (TNF) monoclonal antibodies (mabs) by comparison to azathioprine (AZA) in a series of flare-ups occurring in consecutive ulcerative colitis patients. A total of 109 flare-ups were recorded in 73 patients receiving a maintenance therapy by anti-TNF mabs (n = 69) or AZA (n = 40). The CMV DNA load in colonic tissue was determined by reverse transcription polymerase chain reaction on a pair of biopsies. The number of CMV reactivation was of 35% and 38% in patients receiving anti-TNF mabs and AZA, respectively. The median of CMV DNA load was 378 [10-29,800] and 8300 [10-3,25,000] copies/mg of tissue in patients treated by anti-TNF mabs and AZA, respectively (P = 0.11 by Mann-Whitney U test). In a subgroup of 45 patients under anti-TNF mabs requiring an optimized treatment by infliximab, clinical remission (partial Mayo score <3) was not significantly impacted by the presence of CMV reactivation at the time of flare-up (P = 0.52). Twenty of these patients underwent a second colonic biopsy 8 weeks after the initiation of flare-up therapy; except for 3 patients, the colonic CMV DNA load was stable or decreased. Patients under anti-TNF maintenance therapy are not at higher risk of CMV reactivation in case of flare-up. No reciprocal adverse influence was observed between anti-TNF mabs and CMV infection, suggesting that these drugs must be considered for treating flare-ups associated to CMV reactivation.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 2014
The management of children with community-acquired pneumonia (CAP) is largely influenced by the d... more The management of children with community-acquired pneumonia (CAP) is largely influenced by the development of new molecular diagnostic tests that allow the simultaneous detection of a wide range of pathogens. Evaluation of a diagnostic approach including multiplex PCR assays for revisiting the epidemiology and etiology of CAP in children at hospital. Children of all ages consulting at the Emergency Department of the University hospital of Saint-Etienne, France, during the 2012-2013 winter period were included. In addition to bacterial cultures, the following pathogens were detected using biplex commercially-available rt-PCR tests: adenovirus, respiratory syncytial virus, human metapneumovirus, bocavirus, rhinovirus/enterovirus, coronavirus, influenza viruses A and B, parainfluenza viruses, Mycoplasma pneumoniae and Chlamydophila pneumonia. From 85 patients with CAP, at least one pathogen was identified in 81 cases (95.3%), including 4 bacterial exclusive infections (4.7%), 53 viral...
Previous studies have suggested an association between cytomegalovirus (CMV) infection and steroi... more Previous studies have suggested an association between cytomegalovirus (CMV) infection and steroid-refractory inflammatory bowel disease. In this study, the use of CMV DNA load during acute flare-ups of ulcerative colitis (UC) to predict resistance to immunosuppressive therapy was evaluated in intestinal tissue. Forty-two consecutive patients (sex ratio M/F: 0.9, mean age: 43.6 years) hospitalized for moderate to severe UC and treated with IV steroids were included prospectively. A colonoscopy was performed for each patient at inclusion; colonic biopsy samples of the pathological tissue, and if possible, of the healthy mucosa, were tested for histological analysis and determination of CMV DNA load by real-time polymerase chain reaction assay. Patients were treated as recommended by the current guidelines. Sixteen patients were found positive for CMV DNA in inflamed intestinal tissue but negative in endoscopically healthy tissue; all of these patients were positive for anti-CMV IgG, three exhibited CMV DNA in blood, and none was positive for intestinal CMV antigen by immunohistochemistry detection. In the 26 remaining patients, no stigmata of recent CMV infection were recorded by any technique. By multivariate analysis, the only factor associated with CMV DNA in inflammatory tissue was the resistance to steroids or to three lines of treatment (risk ratio: 4.7; 95% confidence interval: 1.2-22.5). A CMV DNA load above 250 copies/mg in tissue was predictive of resistance to three successive regimens (likelihood ratio+=4.33; area under the receiver-operating characteristic curve=0.85). Eight UC patients with CMV DNA in inflamed tissue and therapeutic failure received ganciclovir; a clinical remission was observed in seven cases, with a sustained response in five of them. The CMV DNA load determined in inflamed intestinal tissue predicts resistance to steroid treatment and to three drug regimens in UC. Initiation of an early antiviral treatment in these patients might delay the occurrence of resistance to current treatments.
A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) an... more A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.
Human papillomavirus (HPV) infection, especially by HPV 16, is frequently detected in oropharynge... more Human papillomavirus (HPV) infection, especially by HPV 16, is frequently detected in oropharyngeal squamous cell carcinoma (OSCC). The expression of viral oncoproteins in tumoral tissues of OSCCs suggests the implication of HPV in tumorogenesis. It should now be systematically detected and considered in each patient's treatment and outcome. To investigate the prevalence of HPV infection, the oncogenic role of HPV in patients from the South-East of France with head and neck squamous cell carcinoma (HNSCC), and the resulting clinical implications. Biopsy samples from 200 patients with HNSCC were analyzed. For each patient, one or two biopsies of tumoral tissue were analyzed simultaneously with a biopsy of healthy tissue. Fresh frozen tissues were tested by molecular techniques for HPV DNA detection and genotyping as well as mRNA expression of oncoproteins E6 and E7. Expression of p16 was also analyzed by immunohistochemical staining. HPV DNA tested positive in 11.5% of biopsy samples. The HPV prevalence was higher in OSCCs (91.3 vs 27.3, p < 0.0001) and in patients not consuming tobacco (65.2% vs 95.4%, p < 0.0001). The estimated 3-year overall survival rates were 67.0% for HPV-infected patients versus 39.9% for non-infected patients. The high-risk HPV 16 was the most common type detected (65.2%). In 12 of 18 patients exhibiting DNA of high-risk HPV in their tumor tissue, the same viral genome was also present in normal tissue. E6 and E7 expression was found in 9 of 14 tumoral biopsies tested for these markers.
Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to... more Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.
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