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The preoperative contouring of plates decreases the duration of surgery and improves the quality of the reduction of pelvic fractures. Patient-tailored three-dimensionally printed pelvises might be an interesting tool for achieving that purpose. Currently, no study has evaluated the accuracy of measurements performed on three-dimensional printed models in comparison with computed tomography data for complex bones, such as the pelvis. This study examined whether the measurements obtained on pelvises printed using dual-material fused deposition modeling technology are not significantly different from those obtained on computed tomography images. The computed tomography images of the pelvic region from 10 dogs were used to produce three-dimensionally printed models with a dual-material fused deposition-modeling process. Four segments were measured on both three-dimensionally printed models and computed tomography images. The measurements were performed by three observers and repeated twice. Concordance correlation coefficients were used to assess the precision and accuracy of the measurements as well as evaluate the agreement between the methods. The accuracy of measurements between the methods was > 0.99 for all measurements. The precision was almost perfect for AE (0.996), substantial for BD and BC (0.963 and 0.958, respectively), and moderate for CD (0.912). These results indicate that, despite some minor variations, the measurements performed on printed models reproduced the computed tomography data reliably., Competing Interests: The authors declare no conflicts of interest., (© 2019 The Korean Society of Veterinary Science.)

The determination of body condition is a fundamental component of an evaluation during postmortem examinations of cetaceans. Three veterinarians experienced in beluga postmortem techniques subjectively evaluated 62 carcasses of beluga whales ( Delphinapterus leucas) in duplicate, using a visual analog scale. The result of evaluation and scoring was repeatable in cachectic animals and animals in very good condition but did not capture the continuum of body condition determinations between the obvious visual conditions.

Introduction: Presence of anti-E1E2 antibodies was previously associated with spontaneous cure of hepatitis C virus (HCV) and predictive before treatment of a sustained virological response (SVR) to bi- or tri-therapy in naïve or experienced patients, regardless of HCV genotype. We investigated the impact of anti-E1E2 seroprevalence at baseline on treatment response in patients receiving direct-acting antiviral (DAA) therapy., Material and Methods: We screened anti-E1E2 antibodies by ELISA in serum samples collected at treatment initiation for two groups of patients: 59 with SVR at the end of DAA treatment and 44 relapsers after DAA treatment. Nineteen patients received a combination of ribavirin (RBV) or PEG-interferon/ribavirin with sofosbuvir or daclatasvir and others received interferon-free treatment with DAA±RBV. HCV viral load was measured at different time points during treatment in a subgroup of patients., Results: A significant association was observed between presence of anti-E1E2 and HCV viral load<6log10 prior treatment. Among patients with anti-E1E2 at baseline, 70% achieved SVR whereas among patients without anti-E1E2, only 45% achieved SVR. Conversely, 66% of patients experiencing DAA-failure were anti-E1E2 negative at baseline. In the multivariate analysis, presence of anti-E1E2 was significantly associated with SVR after adjustment on potential cofounders such as age, sex, fibrosis stage, prior HCV treatment and alanine aminotransferase (ALT) level., Conclusions: The presence of anti-E1E2 at treatment initiation is a predictive factor of SVR among patients treated with DAA and more likely among patients with low initial HCV viral load (<6log10). Absence of anti-E1E2 at baseline could predict DAA-treatment failure., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The use of new Direct Acting Antivirals, specific of HCV, has greatly improved the HCV treatment. Most of the DAA are specific of HCV genotypes. Genotyping methods may target different regions of the HCV genome, though only the whole genome sequencing could confirm the correct genotype. The present study describes the virological investigation of a treatment failure due to the false identification of an unusual 2k/1b recombinant HCV form. It describes the sequencing methods, and the similarity analysis of the sequences to different genotype query sequences, to identify the recombination breakpoint., (© 2018 Wiley Periodicals, Inc.)

Direct-acting antiviral (DAA) drug performances depend on the viral genotype. So international recommendations give typing of the virus a prerequisite for treatment choice and patient management. Commercially available HCV genotyping kits are scarce and this analysis is often in-house using tedious PCRs and Sanger sequencing, leading to a lack of standardization. A newly commercialized HCV genotyping assay based on real-time PCR has been developed by Roche Diagnostics (Mannheim, Germany). We compared this new assay with our in-house PCRs -sequencing technique on 101 regular samples and 81 LiPA failures or low viral load samples. No genotype or 1a/1b subtype mismatch was observed. Two samples were misidentified at the subtype level without clinical impact. Three genotype 1b and two genotype 1a samples with low viral load could not be subtyped. Nevertheless, 13 (13%) samples from the regular panel and 35 (43%) from the more difficult-to-type panels failed to give results on first pass with the Roche kit. Failures were mostly associated with genotype 3 subtype a, with genotype 4 subtype non-a, or with viral loads <200 IU/mL (p = 0.0061). The workflow allowed a non-specialized technician to obtain results in less than 4 hours whereas 2 to 3 days and experienced staff were required with the in-house assay. In conclusion, the Roche cobas® HCV GT kit is easy and rapid to use and provides reliable results. The high rate of uninterpretable results particularly for low viral load samples and less frequent genotypes, and the absence of subtyping for non-genotype 1 could require sending complex samples to a specialized laboratory.

Background: Recent data have suggested that failure to achieve sustained virological response with direct-acting antiviral therapy is usually due to relapse and is primarily associated with the emergence of resistance-associated substitutions. The aim of this study was to investigate the prevalence and characterization of non-structural-5A resistance-associated substitutions in patients infected with HCV genotypes 1, 3 and 4 treated by direct-acting antiviral therapy, including anti-non-structural-5A, and to characterize the pre-existing resistance-associated substitutions in subjects treated with anti-non-structural-5A inhibitors., Methods: From January 2014 to March 2016, 2,995 patients infected with HCV genotypes 1, 3 and 4 were exposed to non-structural-5A inhibitors. Sequencing results at the time of virological failure were available for 61 patients; sequencing at baseline was available for 35 of these patients., Results: Among the 35 patients with sequencing results available at baseline, 15 had no resistance-associated substitution, 16 had only one resistance-associated substitution, and 4 had more than one resistance-associated substitution. Resistance-associated substitutions were harbored in 57% of the sequences in the non-structural-5A region. Among the 61 patients sequenced at virological failure, 50 (82%) patients presented at least one resistance-associated substitutions inducing a high level of resistance to non-structural-5A inhibitors (>10-fold resistance)., Conclusions: This pooled analysis suggests that non-structural-5A resistance-associated substitutions screening should be recommended when considering retreatment with a non-structural-5A inhibitor regimen in patients who have previously experienced failed non-structural-5A treatment.

Background: Ultradeep pyrosequencing technologies permit an assessment of the genetic diversity and the presence and frequency of minority variants in a viral population. The effect of these parameters on the outcome of highly active antiretroviral therapy (HAART) in HIV-infected patients is poorly understood., Objectives: The present study used the pyrosequencing Roche 454 prototype assay to determine whether antiretroviral efficacy is correlated with viral diversity and minority drug resistance mutations in HIV-infected treatment-naive patients and to compare assay performance in B and non-B subtypes., Study Design: The study included 30 HIV-1 infected naive patients (20 with subtype non-B and 10 with subtype B). Ultradeep pyrosequencing of protease and reverse transcriptase genes was performed at baseline and 1 month after HAART initiation. Plasma HIV VL was measured at 0 and after 1, 3, and 6 months of HAART., Results: Pre-HAART minority drug resistance mutations were observed to NRTI in 4 patients, to NNRTI in 6 patients, and to PI in 1 patient; there was no difference in HAART-induced VL decay between patients. Pre-HAART diversity was significantly correlated with the time elapsed since HIV-1 infection diagnosis, but not with the subtype, VL, or CD4 count. Patients with an undetectable VL after 3 months of HAART had a higher pre-HAART diversity. Pre- and post-HAART diversities were not statistically different. There was no difference in assay performance between subtype B and non-B., Conclusions: A high pre-HAART viral diversity might have a positive effect on the outcome of HAART. Pre-therapeutic minority drug resistance mutations are uncommon in naive patients., (Copyright © 2017. Published by Elsevier B.V.)

Fish and aquatic invertebrates deserve evidence-based medicine. Pharmacologic information is available; most pharmacokinetic studies are derived from the aquaculture industry and extrapolated to ornamental fish. Conversely, advanced diagnostics and information regarding diseases affecting only ornamental fish and invertebrates require more peer-reviewed experimental studies; the examples of carp edema virus, sea star wasting disease, seahorse nutrition, and gas bubble disease of fish under human care are discussed. Antinociception is also a controversial topic of growing interest in aquatic animal medicine. This article summarizes information regarding new topics of interest in companion fish and invertebrates and highlights some future avenues for research., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines., Methods: First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments., Results: Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism)., Conclusions: These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

To evaluate the toxicity of short-term high doses of meloxicam in American kestrels ( Falco sparverius ), 32 male captive-born, 1- to 4-year-old American kestrels were randomly assigned to 4 groups: 3 groups treated with meloxicam (n = 9 per group) and a control group (n = 5). Meloxicam was administered orally via feeding tube in the proventriculus at 2, 10, and 20 mg/kg every 12 hours for 7 days for the treatment groups, while the control group received saline solution. The birds were evaluated for the presence of clinical signs, abnormalities in the complete blood cell count and in the plasma biochemical panel for the 20-mg/kg group, and gross and histopathologic lesions. No clinical signs or mortality were observed in any group. No significant differences of clinical relevance were found in results of the packed cell volume, total solids, and biochemical panel, and no evidence of renal toxicity was found in the treatment or control groups. A significant correlation was found between hepatic lipidosis and meloxicam dose (P = .02). Two of 9 birds in the 20-mg/kg group developed gastric ulcers, although this result was not significant. None of the birds in the 2- and 10-mg/kg groups had similar lesions. Finally, meloxicam dosages up to 20 mg/kg did not result in nephrotoxicity in American kestrels. Further toxicologic studies to evaluate hepatotoxicity and gastrotoxicity of meloxicam in avian species are needed.

Background: The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotype homologous recombination. Non-homologous recombinations may be deleterious and are rarely detected. In previous studies, we identified HCV-1b strains bearing two tandemly repeated V3 regions in the NS5A gene without ORF disruption. This polymorphism may be associated with an unfavorable course of liver disease and possibly involved in liver carcinogenesis. Here we aimed at characterizing the origin of these mutant strains and identifying the evolutionary mechanism on which the V3 duplication relies., Methods: Direct sequencing of the entire NS5A and E1 genes was performed on 27 mutant strains. Quasispecies analyses in consecutive samples were also performed by cloning and sequencing the NS5A gene for all mutant and wild strains. We analyzed the mutant and wild-type sequence polymorphisms using Bayesian methods to infer the evolutionary history of and the molecular mechanism leading to the duplication-like event., Results: Quasispecies were entirely composed of exclusively mutant or wild-type strains respectively. Mutant quasispecies were found to have been present since contamination and had persisted for at least 10 years. This V3 duplication-like event appears to have resulted from non-homologous recombination between HCV-1b wild-type strains around 100 years ago. The association between increased liver disease severity and these HCV-1b mutants may explain their persistence in chronically infected patients., Conclusions: These results emphasize the possible consequences of non-homologous recombination in the emergence and severity of new viral diseases.

Therapy for hepatitis C is currently undergoing a revolution. The arrival of new antiviral agents targeting viral proteins reinforces the need for a better knowledge of the viral strains infecting each patient. Hepatitis C virus (HCV) whole genome sequencing provides essential information for precise typing, study of the viral natural history or identification of resistance-associated variants. First performed with Sanger sequencing, the arrival of next-generation sequencing (NGS) has simplified the technical process and provided more detailed data on the nature and evolution of viral quasi-species. We will review the different techniques used for HCV complete genome sequencing and their applications, both before and after the apparition of NGS. The progress brought by new and future technologies will also be discussed, as well as the remaining difficulties, largely due to the genomic variability.

Background and Aim: The impact of basal core promoter (BCP) and precore (PC) mutants of the hepatitis B virus (HBV) on liver disease severity remains controversial. The aim of the present study was to screen BCP and PC mutations in 252 HBV surface antigen (HBsAg) positive carriers in France and to assess relationships between these mutations and severe fibrosis., Methods: Direct sequencing of the precore/core gene was used to detect A1762T/G1764A and G1757A mutations in the BCP and G1896A and G1899A mutations in the PC region., Results: The prevalences of A1762T/G1764A, G1757A, G1896A, and G1899A mutations were 34.1%, 38.7%, 54.9%, and 29.3% (P < 0.001), respectively. The independent predictors of severe fibrosis (≥F3 Metavir) were older age (P < 0.001), male gender (P = 0.012), elevated alanine aminotransferase (P < 0.001), and the double A1762T/G1764A mutant with no other mutations (P = 0.011). Interestingly, the association of the G1899A mutation with the double A1762T/G1764A mutant significantly counteracted the deleterious effect of the sole double A1762T/G1764A mutant (odds ratio [OR] = 0.28 vs. OR = 3.55, respectively, P = 0.028)., Conclusions: Patients with the A1762T/G1764A mutation have a higher risk of severe fibrosis. The G1899A mutation is a protective factor against severe fibrosis that counteracted the deleterious effect of the A1762T/G1764A mutation. Finally, host phenotypic and HBV genotypic markers independently predict fibrosis severity., (© 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Veterinarians caring for companion animals may encounter microsporidia in various host species, and diagnosis and treatment of these fungal organisms can be particularly challenging. Fourteen microsporidial species have been reported to infect humans and some of them are zoonotic; however, to date, direct zoonotic transmission is difficult to document versus transit through the digestive tract. In this context, summarizing information available about microsporidiosis of companion exotic animals is relevant due to the proximity of these animals to their owners. Diagnostic modalities and therapeutic challenges are reviewed by taxa. Further studies are needed to better assess risks associated with animal microsporidia for immunosuppressed owners and to improve detection and treatment of infected companion animals.

Objectives: MDR Candida strains are emerging. Next-generation sequencing (NGS), which enables extensive and deep genome analysis, was used to investigate echinocandin and azole resistance in clinical Candida isolates., Methods: Six genes commonly involved in antifungal resistance (ERG11, ERG3, TAC1, CgPDR1, FKS1 and FKS2) were analysed using NGS in 40 Candida isolates (18 Candida albicans, 15 Candida glabrata and 7 Candida parapsilosis). The strategy was validated using strains with known sequences. Then, 8 clinical strains displaying antifungal resistance and 23 sequential isolates collected from 10 patients receiving antifungal therapy were analysed., Results: A total of 391 SNPs were detected, among which 6 coding SNPs were reported for the first time. Novel genetic alterations were detected in both azole and echinocandin resistance genes. A C. glabrata strain, which was resistant to echinocandins but highly susceptible to azoles, harboured an FKS2 S663P mutation plus a novel presumed loss-of-function CgPDR1 mutation. This isolate was from a patient with deep-seated and urinary candidiasis. Another C. glabrata isolate, with an MDR phenotype, carried a new FKS2 S663A mutation and a new putative gain-of-function CgPDR1 mutation (T370I); this isolate showed mutated (80%) and WT (20%) populations and was collected after 75 days of exposure to caspofungin from a patient who underwent complicated abdominal surgery., Conclusions: This study shows that NGS can be used for extensive assessment of genetic mutations involved in antifungal resistance. This type of wide genome approach will become very valuable for detecting mechanisms of resistance in clinical strains subjected to multidrug pressure., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Hepatitis E virus infection - mainly genotype 3 - is increasingly common in industrialized countries. Infection is usually asymptomatic, but cases of central or peripheral neurological symptoms with hepatitis E have been described. The most frequent is Guillain-Barre but somes cases of neuralgic amyotrophy have been described. In our center, since 2010, we have identified five cases of neuralgic amyotrophy associated with acute hepatitis E in immunocompetent patients. For all these patients, neuralgic amyotrophy was diagnosed with electromyogram and positive IgM for hepatitis E, and detectable HEV RNA in 4 of the cases. Including our patients, we count 26 cases in literature. The mean age of the patients was 44 years old, with a large predominance of males (88%). The disorder is bilateral and asymmetric in 69% of cases. Peripheral nerves other than the brachial plexus were affected in 6 patients (23%). In industrialized countries, any neuralgic amyotrophy, particularly if there is bilateral, asymmetric associated with involvement of nerves outside the brachial plexus, should lead physicians to consider a diagnosis of acute hepatitis E., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila., Methods: We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples., Findings: We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment., Interpretation: In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Despite the gain in sustained virological responses (SVR) provided by protease inhibitors (PIs), failures still occur. The aim of this study was to determine if a baseline analysis of the NS3 region using ultradeep pyrosequencing (UDPS) can help to predict an SVR. Serum samples from 40 patients with previously nonresponding genotype 1 chronic hepatitis C who were retreated with triple therapy, including a PI, were analyzed. Baseline UDPS of the NS3 gene was performed on plasma and peripheral blood mononuclear cells (PBMC). Mutations conferring resistance to PIs were sought. The overall diversity of the quasispecies was evaluated by calculating the Shannon entropy (SE). Resistance mutations were found in plasma and PBMC but were not discriminating enough to predict an SVR. NS3 quasispecies heterogeneity was significantly lower at baseline in patients achieving an SVR than in those not achieving an SVR (SE of 26.98 ± 16.64 × 10(-3) versus 44.93 ± 19.58 × 10(-3), P = 0.0047). With multivariate analysis, the independent predictors of an SVR were fibrosis of stage F ≤2 (odds ratio [OR], 13.3; 95% confidence interval [CI], 1.25 to 141.096; P < 0.03) and SE below the median (OR, 5.4; 95% CI, 1.22 to 23.87; P < 0.03). More than the presence of minor mutations at the baseline in plasma or in PBMC, the NS3 viral heterogeneity determined by UDPS is an independent factor for an SVR in previously treated patients receiving triple therapy that includes a PI., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Introduction: When diagnosing Pneumocystis jirovecii pneumonia (PJP), the clinical suspicion must be confirmed by laboratory tests. PJP is rarely described in patients with idiopathic CD4 + lymphocytopenia (ICL), a rare T-cell deficiency of unknown origin with persistently low levels of CD4 + T-cells (<300 µl -1 or <20 % of total lymphocytes) but repeated negative human immunodeficiency virus (HIV) tests. We retrospectively analysed a case of an ICL patient with severe PJP associated with multiple opportunistic infections (OIs). We also reviewed the literature since 1986., Case Presentation: A laboratory-confirmed case of PJP associated with invasive candidiasis and cytomegalovirus infection was reported in an ICL patient. Despite early treatment, the patient died of respiratory failure under polymicrobial pneumonia. According to the literature, the mortality rate of ICL patients is 10.4 % (33/316). In ICL patients, the risk of OI is 83.2 % (263/316), with viral infections being the most prevalent (58.2 %, 184/316), followed by fungal infections (52.2 %, 165/316) and mycobacterial infections (15.5 %, 49/316). Dysimmunity is reported in 15.5 % (49/316) of ICL patients. Among the fungal infections, cryptococcal infections are the most prevalent (24.1 %, 76/316), followed by candidiasis (15.5 %, 49/316) and PJP (7.9 %, 25/316)., Conclusions: The high risk of OIs underlines the importance of more vigorous preventative actions in hospitals. The response to therapy and the detection of early relapse of PJP may be monitored by several laboratory tests including quantitative PCR. It is essential to treat the ICL and to follow the guidelines concerning therapy and prophylaxis of OIs as given to HIV patients.

This study compared blood glucose concentrations measured with a portable blood glucometer and a validated laboratory analyzer in venous blood samples of 20 pet ferrets (Mustela putorius furo). Correlation and agreement were evaluated with a Bland-Altman plot method and Lin's concordance correlation coefficient. Blood glucose concentrations measured with the laboratory analyzer and the glucometer ranged from 1.9 to 8.6 mmol/L and from 0.9 to 9.2 mmol/L, respectively. The glucometer had a poor agreement and correlation with the laboratory analyzer (bias, -0.13 mmol/L; level of agreement, -2.0 to 3.6 mmol/L, concordance correlation coefficient 0.665). The relative sensitivity and specificity of the portable blood glucometer for detection of hypoglycemia were 100% (95% CI: 66% to 100%) and 50% (95% CI: 20% to 80%), respectively. Positive and negative predictive values were 67% (95% CI: 39% to 87%) and 100% (95% CI: 46% to 100%), respectively. Based on these results, clinicians are advised to be cautious when considering the results from this handheld glucometer in pet ferrets, and blood glucose concentrations should be determined with a laboratory analyzer validated for this species.

Tetralogy of Fallot associated with bidirectional shunting across a large ventricular septal defect, was found in a 6-year-old ferret. The prognosis associated with tetralogy of Fallot is usually poor. This case is interesting given the advanced age of the ferret. The bidirectional shunting, responsible for an acyanotic disease, may explain the unexpected prolonged survival in this ferret.

For optimal antiviral therapy, the hepatitis C virus (HCV) genotype needs to be determined, as it remains a strong predictor of sustained viral response. In this study, we assessed the number of HCV genotyping results that could not be determined using the commercially available line probe assay (LiPA) (Versant hepatitis C virus genotype 2.0 assay) in a large international panel of samples from 9,874 HCV-positive patients. In-house sequencing assays targeting the 5' untranslated region (UTR), core region, NS3 region, and NS5B region of the HCV genome and phylogenetic analyses were used to resolve these LiPA failures. Among all cases, the genotypes of 51 samples (0.52%) could not be determined with the LiPA. These undetermined results were observed more frequently among samples from non-European regions (mainly the Arabian Peninsula). The use of sequencing assays coupled with phylogenetic analysis provided reliable genotype results for 86% of the LiPA failures, which exhibited higher rates of genotypes 4, 5, and 6 than did LiPA-resolved genotypes. As expected, the 5' UTR was not sufficiently variable for clear discrimination between genotypes 1 and 6, but it also resulted in errors in classification of some genotype 3 and 4 cases using well-known Web-based BLAST programs. This study demonstrates the low frequency of genotyping failures with the Versant hepatitis C virus genotype 2.0 assay (LiPA) and also underlines the need for a complex combination of sequences and phylogenetic analyses in order to genotype these particular HCV strains correctly.

The pathogenesis of the recently recognized "red vent syndrome" in wild Atlantic salmon (Salmo salar) is not fully understood. Pathologic observations indicate that this syndrome is associated with the presence of nonencapsulated larvae of the nematode Anisakis simplex in the body wall, the lower intestinal wall, and the visceral cavity surrounding the vent region. We evaluated the relationship between the occurrence of red vent syndrome and intensity of infection with Anisakis sp. larvae in naturally infected fish. Salmon caught by sport anglers were opportunistically evaluated to detect red vent syndrome. We included 106 salmon with red vent syndrome and 98 without red vent syndrome in this study. Intensity of infection was established by counting the total number of perivisceral larvae and by determining the number of larvae per gram in 10 g of pepsin-digested perianal tissue. The severity of inflammatory changes was also evaluated in standard histologic sections of the perianal area using a semiquantitative scale. Salmon with red vent syndrome had significantly higher intensity of inflammation than salmon without red vent syndrome (P=0.008). The odds of having red vent syndrome increased with the number of perianal larvae per gram of perianal tissue (P=0.002; odds ratio [OR]=1.12; 95% confidence interval: [1.05; 1.22]) but not with the number of perivisceral larvae, fish length, or gender. Although these results support the association between this syndrome and intensity of infection by A. simplex, the relationship is not strong (OR near 1), suggesting that the clinical expression of red vent syndrome at an individual level, and the emergence of this disease on a global scale, must be determined by other factors, such as timing of infection.

HBV reactivations are observed frequently in patients with past hepatitis B infection receiving cytotoxic and/or immunosuppressive chemotherapy for hemato-oncological malignancies or autoimmune diseases. Recent ischemic stroke was shown to induce immunodepression by misunderstood mechanisms. To our knowledge, the association between HBV reactivation and ischemic stroke has not been reported before. This study reports the case of an anti-HBs- and anti-HBc-positive patient who presented HBV reactivation in a context of recent ischemic stroke, with no other intercurrent iatrogenic phenomenon or usual immunosuppressive pathology., (Copyright © 2012 Wiley Periodicals, Inc.)

Background & Aims: Hepatitis B virus (HBV) modulates the immune system to escape clearance. Plasmacytoid dendritic cells (pDCs) initiate antiviral immunity and might determine outcomes of HBV infections. Functional defects in pDCs and natural killer (NK) cells have been reported in patients with chronic HBV infection. However, the mechanisms of these immune dysfunctions and the interactions between pDCs and NK cells have not been determined. We investigated features of pDCs from patients with chronic HBV infection and their interactions with NK cells., Methods: We used flow cytometry and cytokine assays to analyze pDCs from patients with chronic HBV infection (118 aviremic and 67 viremic) and compared them with pDCs from uninfected individuals (controls). We performed coculture assays to analyze the ability of pDCs to activate heterologous NK cells., Results: Circulating and hepatic pDCs from patients with chronic HBV infection had higher levels of activation than pDCs from controls and defective responses to stimulation with Toll-like receptor 9 ligand (TLR9-L), regardless of the patient's viral load. TLR9-L-activated pDCs from viremic patients with HBV did not induce cytolytic activity of NK cells. This altered function of pDCs was associated with reduced expression of OX40L and could be reproduced by incubating control pDCs with plasma from viremic patients with HBV. A high level of interferon-induced protein 10 (IP-10 or CXCL10) and hepatitis B surface and e antigens might induce these defective pDC functions., Conclusions: HBV escapes antiviral immunity by altering pDC functions, to disrupt interactions between pDC and NK cells. This could reduce immune control of HBV and lead to chronic infection., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Unlabelled: The immune control of hepatitis B virus (HBV) infection is essential for viral clearance. Therefore, restoring functional anti-HBV immunity is a promising immunotherapeutic approach to treatment of chronic infection. Plasmacytoid dendritic cells (pDCs) play a crucial role in triggering antiviral immunity through their ability to capture and process viral antigens and subsequently induce adaptive immune responses. We investigated the potential of pDCs to trigger antiviral cellular immunity against HBV. We used a human leukocyte antigen A (HLA-A)*0201(+) pDC line loaded with HLA-A*0201-restricted peptides derived from hepatitis B core/hepatitis B surface (HBc/HBs) antigens to amplify specific CD8 T cells ex vivo from chronic HBV patients and established a Hepato-HuPBL mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMCs or liver-infiltrating lymphocytes from HLA-A*0201(+) chronic HBV patients by HBc peptide-loaded pDCs elicited up to 23.1% and 76.1% HBV-specific CD8 T cells in 45.8% of cases. The specific T cells from the "responder" group secreted interferon-γ, expressed CD107 upon restimulation, and efficiently lysed HBV antigen-expressing hepatocytes. Circulating hepatitis B e antigen (HBeAg) was found to distinguish the group of patients not responding to the pDC stimulation. The therapeutic efficacy of the pDC vaccine was evaluated in immunodeficient NOD-SCID β(2) m(-/-) mice reconstituted with HBV patients' PBMCs and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of Hepato-HuPBL mice with the HBc/HBs peptide-loaded pDCs elicited HBV-specific T cells able to specifically lyse the transfected hepatocytes and reduce the systemic viral load., Conclusion: pDCs loaded with HBV-derived peptides can elicit functional virus-specific T cells. HBeAg appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. A pDC-based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background: Hepatitis C virus (HCV) is underdiagnosed and therefore increasing the opportunities for HCV testing without venipuncture may be useful., Objectives: We evaluated the analytical performance of a modified, commercially available, combined HCV antigen-antibody assay (cEIA) (Monolisa(®) HCV-Ag-Ab-ULTRA) and a commercially available point-of-care (POC) device (OraQuick(®) HCV) on fingerstick blood (FSB) and oral mucosal transudate (OMT)., Study Design: FSB, OMT and serum samples were collected from 113 cases of HCV-antibody-positive patients and 88 HCV-antibody-negative controls. The HCV-antibody-positive group included 63 patients with quantifiable HCV-RNA (56%) and 17 HIV/HCV co-infected patients (15%). FSB and OMT specimens were collected as dried blood spots (DBSs) or with the OraSure collection system, before testing with cEIA., Results: With FSB specimens, the cEIA and the POC device exhibited 100% specificity and 98.2% and 97.4% sensitivity, respectively. The specificity of the cEIA in FSB sharply decreased if stored 3days at room temperature. With OMT specimens, the cEIA sensitivity (71.7%) and specificity (94.3%) were significantly lower than the performance of OraQuick(®) HCV (sensitivity, 94.6%; specificity, 100%). The optical densities obtained with the cEIA in FSB and OMT were lower in HIV/HCV co-infected patients compared with HCV monoinfected patients., Conclusion: The cEIA using FSB specimens collected on DBSs preserved in appropriate storage conditions was a reliable alternative, equivalent to the POC assay, for HCV testing without venipuncture. The cEIA was not adapted for HCV testing on OMT., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Multiple control methods have been used in North America to manage the spread of rabies caused by the raccoon (Procyon lotor) rabies virus variant (RRVV). Recently, oral vaccination with ONRAB(®) vaccine baits, which contain an adenovirus rabies glycoprotein recombinant, has been made available as an additional tool for rabies control. Our objectives were to estimate rabies antibody prevalence in wild-caught raccoons and striped skunks (Mephitis mephitis), and identify factors influencing the probability of being antibody positive at the individual level in these species, following oral rabies vaccination (ORV) campaigns in which ONRAB was distributed aerially in 2007-2009 in southern Québec, Canada. Following the aerial distribution of 43-155 ONRAB baits/km(2), the annual percentages of antibody-positive raccoons and skunks varied between 35% and 56% and 11% and 17%, respectively. In raccoons, the probability of being antibody positive was positively associated with age and density of ONRAB distributed, and influenced by the number of previous ORV campaigns conducted. Conversely, this probability was negatively associated with estimated abundance of raccoons in the trapping cell and proportion of residential areas near the raccoon capture location. None of the variables examined explained variation in the probability of being antibody positive in skunks. Our results indicate that the ONRAB density applied during ORV campaigns should be adjusted to account for variations in raccoon population density and presence of residential areas to increase the likelihood of creating an effective immunological barrier against RRVV. The high percentage of juvenile raccoons (annual mean =45 ± 3 [SE]%) and skunks (66 ± 2%) captured during post-ORV monitoring suggests that ORV campaigns should be conducted at least annually to account for the recruitment of naïve individuals into the populations. In Québec, the increasing use of ONRAB coincided with the elimination of rabies caused by RRVV. Nonetheless, our results indicate that improvements to this vaccine bait and/or the distribution techniques are required to increase its efficacy, especially in striped skunks.

Objectives: During the 1980s, walrus-meat consumption caused infections with the parasite Trichinella nativa in Nunavik inhabitants. In response to these events, stakeholders set up the community-based Nunavik Trichinellosis Prevention Program (NTPP). The objectives of the present communication are to review the NTPP, describe how science and action were interwoven in its development and identify its assets and limitations. Study design. Descriptive study., Methods: The NTPP relies on a pooled digestion assay of tongue samples taken from each harvested walrus. The public health recommendations depend on the results of the analyses: infected walrus meat should be destroyed; parasite-free meat may be eaten raw or cooked., Results: All communities involved in the walrus hunt participate in the NTPP and a high percentage of harvested walruses are included in the NTPP. Infected animals account for 2.9% of the walruses tested (20/694) since 1992. The NTPP permitted the early management of a trichinellosis event in 1997. Since then, it prevented the new occurrence of outbreaks related to walruses hunted by Nunavimmiut., Conclusions: The absence of recent major outbreaks of trichinellosis in Nunavik may reasonably be attributed to the NTPP. The success of the program stands on many facilitating factors such as the nature of the disease and its source, the existence of an efficient analytic method, the strong involvement of the different partners including direct resource users, as well as the comprehensive bidirectional science-to-action approach that has been followed.

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.

The objectives of this study were twofold: to determine the sensitivity of gill biopsies used for the screening of Pseudodactylogyrus bini in subclinically infested American eels (Anguilla rostrata) and to evaluate the effect of bath anesthesia on the proportion of infested eels. Fifty-four eels were euthanized following either intracoelomic injection of T61 or deep anesthesia with tricaine methanesulfonate. Proportions of eels infested by P. bini diagnosed by gill biopsies taken through the gill slit and by microscopic examination of the entire gill arches were compared. The euthanasia protocol had no significant effect on parasite abundance. The prevalence of monogeneans detected by examination of nonterminal antemortem gill biopsies and by complete gill examinations was 20% and 70%, respectively. The sensitivity of gill biopsies (29%) to detect P. bini in subclinically infested eels is therefore low, and should be used for group screening rather than individual screening.

Microfilariae are considered non-pathogenic in wild birds. The objective of the current communication is to report host reactions to microfilarial infection of unusual intensity in emaciated boreal owls (Aegolius funereus). An unusually large number of boreal owls (n = 21) were submitted to the Canadian Cooperative Wildlife Health Center-Quebec Region for post-mortem examination during the winter of 2009. Nineteen out of 21 birds were considered emaciated based on atrophy of adipose tissue and pectoral muscles and suboptimal weight. A microscopic examination of a subset of nine owls revealed the presence of microfilariae in six owls. Three of the birds with a heavy parasite burden had masses of larval nematodes obstructing large vessels of the lungs. The emaciated owls are believed to have died from starvation due to a cyclic decrease in prey abundance in the boreal forest. This cycle also drives winter movements of boreal owls to urbanized areas of southern Quebec, presumably accounting for the large number of birds submitted in 2009. In the most severely infected owls, the extreme microfilarial burden might have caused an alteration in circulatory dynamics, gaseous exchanges and also probably some metabolic cost. Consequently, microfilariae could have significantly contributed to the death of some of these owls.

The prevalence of occult hepatitis B, defined by absence of HBsAg and HBV DNA, ranges widely in patients with hepatitis C. This may influence the treatment of hepatitis C and the severity of liver disease. Sensitive and specific real-time PCR techniques are available commercially and can detect more reliably low HBV DNA levels. The aim of this study was to determine the prevalence of occult hepatitis B virus infection using the COBAS Taqman assay (Roche Diagnostics, Meylan, France) in the serum and liver of HBsAg negative patients with chronic hepatitis C and to evaluate its clinical consequences on liver pathology and its impact on the response to treatment with peg-IFNalpha and Ribavirin. HBV DNA detection was assessed retrospectively on 140 sera and 113 liver biopsies of HCV positive/HBsAg negative patients before treatment. A 4.4% (5/113) prevalence of occult hepatitis B was recorded in liver samples and in none of the sera. Anti-HBc was not detected in one, three of whom were sustained virological responders to treatment, one was relapsed responder and one was non-responder. Furthermore, in this cohort composed of 12% anti-HBs negative/anti-HBc positive and 20% anti-HBs positive/anti-HBc positive patients, anti-HBc was not associated with pre-therapeutic viral load, ALT serum levels, and histological activity or fibrosis. Using a commercial real-time PCR assay, we observed a low prevalence of occult B hepatitis. This, just as anti-HBC status, had no clinical impact in a large cohort of hepatitis C patients. It therefore does not appear useful to screen for occult hepatitis B in these patients with this test before beginning HCV treatment., ((c) 2010 Wiley-Liss, Inc.)

A selective real-time PCR (sPCR) assay has been developed to detect the rtM204V/I and rtN236T mutations of hepatitis B virus (HBV) associated with resistance to lamivudine and adefovir. Using mixtures of mutant and wild-type plasmids, this sPCR was able to detect 0.1% of mutated strain in a total plasmid population of 10(5) copies and was more sensitive in detecting resistant strains than the line probe INNO-LiPA-DR-v2 assay and a direct sequencing assay. The comparison of these methods on 20 clinical specimens from treated patients confirmed the plasmid results: the three methods were concordant for the detection of the mutant strains in 72% of the cases and the discrepant results were caused mainly by the sequencing assay's lack of sensitivity. The line probe assay was more sensitive for detecting mutations than sPCR when the viral load was less than 10(4) copies/ml; conversely, the sPCR provided a more sensitive detection when the viral load was greater than 10(4) copies/ml. Although difficult to perform in clinical practice, sPCR appears to be a reliable technique for detecting and quantifying quasi-species resistant to lamivudine (LAM) and adefovir (ADV) and can be useful to gain a better understanding of the natural history of antiviral resistance during the treatment of chronic hepatitis B (CHB).

Background: The Epstein-Barr virus (EBV) protease (PR), coded by the BVRF2 gene, is essential for the maturation of the viral capsid and viral DNA packaging during the late stage of the EBV lytic cycle. Like the other herpesvirus serine PRs, EBV PR could be a target for the inhibition of EBV replication. To date, no data have been reported on the inhibition of EBV PR messenger RNA (mRNA) by small interfering RNA (siRNA)., Methods: In this study, siRNAs targeting EBV PR were delivered to the epithelial 293 cell line stably transfected with the complete B95-8 EBV episome. EBV DNA and PR mRNA were quantified by real-time PCR in cells and supernatant, protein expression was assessed by immunoblotting, and production of EBV infectious particles in the culture medium was measured by Raji cell superinfection., Results: The EBV PR mRNA within the cells was reduced by 73%, the PR protein by 35% and the amount of virus in the cell supernatant was drastically decreased by 86% or 95%, depending on the method., Conclusions: The strong effect of the siRNA targeting EBV PR on EBV replication attests to the crucial role played by EBV PR in the production of infectious particles and suggests that targeting this enzyme can be a new strategy against EBV-associated diseases where virus replication occurs.

Background/aims: It has been suggested that, in HIV-HCV co-infected patients, co-infections with other viruses may affect the response to HCV therapy. We aimed to assess the prevalence of GBV-C, SEN-V and occult HBV infections, their impact on HCV and HIV infections and on the response to HCV therapy in HIV-HCV co-infected patients., Methods: Three-hundred and sixty eight patients were tested before starting interferon-ribavirin for the presence of occult hepatitis B DNA, GBV-C RNA and SEN-V DNA by using real time PCR. Clinical, immunological, virological, histological characteristics and response to HCV therapy were compared according to the presence or not of each viral co-infection., Results: HBV DNA, GBV-C RNA and SEN-V DNA were found in 5 (1.4%, CI95%: 0.2-2.4%), 104 (29.9%, CI95%: 25.1-34.7%) and 209 patients (57.9%, CI95%: 52.8-63.0%), respectively. GBV-C positive patients had significantly higher CD4 count at baseline, during and after HCV therapy, even after stratification on antiretroviral treatment. No other significant difference was observed according to the presence or not of GBV-C or SEN-V co-infection, in particular regarding virological responses to HCV combination therapy., Conclusions: There is no reason to withhold HCV therapy in HIV infected patients who have access to HAART, because of occult HBV, GBV-C or SEN-V co-infections.

Background: Sequential monotherapies of nucleotide analogs used in chronic hepatitis B treatment can lead to the selection of a resistance mutation to each antiviral drug., Case Presentation: A patient with chronic hepatitis B was successively treated with lamivudine monotherapy, lamivudine-adefovir dual therapy, adefovir monotherapy and again with an adefovir-lamivudine dual therapy. Lamivudine-associated mutations (rtL180M and rtM204V/I) followed by adefovir-associated mutations (rtN236T and rtA181V) emerged during the two monotherapy regimens. Despite the presence of rtM204V/I, rtA181V, and rtN236T mutations at the beginning of the second dual therapy, sustained biochemical and virological responses have been observed thus far after 23 months., Conclusion: This case illustrates that rtM204V/I, rtA181V, and rtN236T resistance mutations can coexist in a patient but do not preclude the recycling of lamivudine and adefovir in combination therapy, when no other therapeutic choices are available.

Background: Concerns about the potential use of smallpox in bioterrorism have stimulated interest in the development of novel antiviral treatments. Currently, there are no effective therapies against smallpox and new treatment strategies are greatly needed., Methods: In this study, specifically designed small interfering RNAs (siRNAs), targeting five proteins essential for orthopoxvirus replication, were investigated for their ability to inhibit vaccinia virus strain Western Reserve (VACVWR) replication., Results: Among these siRNAs, 100 nM siD5R-2, an siRNA targeting the D5 protein, decreased VACVWR replication up to 90% when used either prophylactically or therapeutically in human lung carcinoma A549 cells. This siRNA induced a striking concentration-dependent inhibition of VACVWR replication and a prolonged prophylactic antiviral effect that lasted for 72 h, at a concentration of 100 nM. Confocal microscopy of Alexa-siD5R-2-treated VACVWR-infected cells confirmed a decrease in viral replication. Furthermore, siD5R-2 was shown to specifically reduce the D5R mRNA and protein expression using real-time reverse transcriptase-PCR and western blotting analysis, without inducing interferon-13 in A549 cells. We also demonstrated the antiviral potency of siD5R-2 against different pathogenic orthopoxviruses, such as cowpox and monkeypox viruses, which were inhibited up to 70% at the lowest concentration (1 nM) tested. Finally, siD5R-2 showed antiviral effects in VACVWR-infected human keratinocyte and fibroblast cell cultures., Conclusions: These results suggest that siD5R-2 could be a potential candidate to treat poxvirus infections.

A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log(10) copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the Cobas TaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log(10) copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C(18)-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4 degrees C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 +/- 1.06 micro g/ml (n = 24) at week 12 of treatment (W12) and 3.24 +/- 1.35 micro g/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.