María Alma Bracho

Enterovirus co-infections and onychomadesis after hand, foot, and mouth disease, Spain, 2008.

Mixed infection of enteroviruses may explain the rare complication of nail shedding. Onychomadesis after HFMD

Onychomadesis Outbreak in Valencia, Spain Associated with Hand, Foot, and Mouth Disease Caused by Enteroviruses

Abstract:  This report evaluates the June 2008 onychomadesis outbreak in Valencia, Spain. The study sample consisted of 221 onychomadesis cases and 77 nonaffected individuals who lived close to those affected. We collected data on dietary variables, hygiene products, and individual pathological histories. Feces and blood specimens were collected from 44 cases and 24 controls to evaluate exposure to infectious agents. Pathological background data revealed a high frequency (61%) of hand, foot, and mouth disease among the onychomadesis cases. Coxsackievirus A10 was the most commonly detected enterovirus in both case and control groups (49%). Other enteroviruses such as coxsackieviruses A5, A6, A16, B1, and B3; echoviruses 3, 4, and 9; and enterovirus 71 were present in low frequencies in the case and control groups (3–9%). The 2008 onychomadesis outbreak in the metropolitan area of Valencia was associated with an outbreak of hand, foot, and mouth disease primarily caused by coxsackievirus A10.

Evidence of recombination in intrapatient populations of hepatitis C virus.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare in vivo. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naïve), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially relevant mechanism generating genetic variation in HCV and with important implications for the treatment of this infection.

Recombination in Hepatitis C Virus

Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

Molecular Epidemiology of a Hepatitis C Virus Outbreak in a Hemodialysis Unit

ABSTRACT We analyzed a hepatitis C virus (HCV) transmission case in the hemodialysis unit of a private clinic by sequencing two genome regions of virus isolates from a number of patients attending this unit and some external controls. The analysis of 337 nucleotides (nt) in the NS5B region did not provide enough resolution to ascertain which patients were actually involved in the outbreak and the potential source. Nevertheless, this region allowed the exclusion of several patients as putative sources of the transmission case based on their genotypes and phylogenetic relationships. On the other hand, the analysis of several 472-nt-long clone sequences per sample in a more rapidly evolving region of the HCV genome, coding for the envelope proteins and encompassing hypervariable region 1, allowed us to establish the existence of at least two independent transmission events involving two different source patients and three recipients. The direction of the transmissions was further corroborated by different measures of genetic variability within and among samples.

Molecular evolution in court: analysis of a large hepatitis C virus outbreak from an evolving source

BackgroundMolecular phylogenetic analyses are used increasingly in the epidemiological investigation of outbreaks and transmission cases involving rapidly evolving RNA viruses. Here, we present the results of such an analysis that contributed to the conviction of an anesthetist as being responsible for the infection of 275 of his patients with hepatitis C virus.ResultsWe obtained sequences of the NS5B and E1-E2 regions in the viral genome for 322 patients suspected to have been infected by the doctor, and for 44 local, unrelated controls. The analysis of 4,184 cloned sequences of the E1-E2 region allowed us to exclude 47 patients from the outbreak. A subset of patients had known dates of infection. We used these data to calibrate a relaxed molecular clock and to determine a rough estimate of the time of infection for each patient. A similar analysis led to an estimate for the time of infection of the source. The date turned out to be 10 years before the detection of the outbreak. The number of patients infected was small at first, but it increased substantially in the months before the detection of the outbreak.ConclusionsWe have developed a procedure to integrate molecular phylogenetic reconstructions of rapidly evolving viral populations into a forensic setting adequate for molecular epidemiological analysis of outbreaks and transmission events. We applied this procedure to a large outbreak of hepatitis C virus caused by a single source and the results obtained played a key role in the trial that led to the conviction of the suspected source.

Genetic variability in hepatitis C virus and its role in antiviral treatment response

Summary.  Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. The high genetic variability of HCV contributes to the chronicity of hepatitis C. Here, we report results from a large‐scale sequence analysis of 67 patients infected with HCV genotype 1, 23 with subtype 1a and 44 with subtype 1b. Two regions of the HCV genome were analysed in samples prior to combined therapy with alpha interferon plus ribavirin, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the E2 glycoprotein and another one including the interferon‐sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Genetic diversity measures showed a clear tendency to higher genetic variability levels in nonresponder patients to antiviral treatment than in responder patients, although highly disperse values were present within each response group for both subtypes. A more detailed analysis of amino acid composition revealed the presence of several subtype‐specific variants in a few positions, but no discriminating positions between responder and nonresponder patients were detected. Our results also revealed that most amino acid positions were highly conserved, especially for subtype 1a. We conclude that the outcome of the antiviral treatment might depend not only on the nature of one or a few independent positions, but more likely on the combination of several positions along the HCV genome. Moreover, the own host’s ability to generate an appropriate systemic response, in combination with the action of antivirals, is also likely to be essential for treatment outcome.

Baseline Prediction of Combination Therapy Outcome in Hepatitis C Virus 1b Infected Patients by Discriminant Analysis Using Viral and Host Factors

Background Current treatment of chronic hepatitis C virus (HCV) infection has limited efficacy −especially among genotype 1 infected patients−, is costly, and involves severe side effects. Thus, predicting non-response is of major interest for both patient wellbeing and health care expense. At present, treatment cannot be individualized on the basis of any baseline predictor of response. We aimed to identify pre-treatment clinical and virological parameters associated with treatment failure, as well as to assess whether therapy outcome could be predicted at baseline. Methodology Forty-three HCV subtype 1b (HCV-1b) chronically infected patients treated with pegylated-interferon alpha plus ribavirin were retrospectively studied (21 responders and 22 non-responders). Host (gender, age, weight, transaminase levels, fibrosis stage, and source of infection) and viral-related factors (viral load, and genetic variability in the E1–E2 and Core regions) were assessed. Logistic regression and discriminant analyses were used to develop predictive models. A “leave-one-out” cross-validation method was used to assess the reliability of the discriminant models. Principal Findings Lower alanine transaminase levels (ALT, p = 0.009), a higher number of quasispecies variants in the E1–E2 region (number of haplotypes, nHap_E1–E2) (p = 0.003), and the absence of both amino acid arginine at position 70 and leucine at position 91 in the Core region (p = 0.039) were significantly associated with treatment failure. Therapy outcome was most accurately predicted by discriminant analysis (90.5% sensitivity and 95.5% specificity, 85.7% sensitivity and 81.8% specificity after cross-validation); the most significant variables included in the predictive model were the Core amino acid pattern, the nHap_E1–E2, and gamma-glutamyl transferase and ALT levels. Conclusions and Significance Discriminant analysis has been shown as a useful tool to predict treatment outcome using baseline HCV genetic variability and host characteristics. The discriminant models obtained in this study led to accurate predictions in our population of Spanish HCV-1b treatment naïve patients.

Molecular Epidemiology and Forensic Genetics: Application to a Hepatitis C Virus Transmission Event at a Hemodialysis Unit

Molecular phylogenetic analyses are frequently used in epidemiologic testing, although only occasionally in forensics. Their acceptability is hampered by a lack of statistical confidence in the conclusions. However, maximum likelihood testing provides a sound statistical framework for the testing of phylogenetic hypotheses relevant for forensic analysis. We present the results of applying this method to a small hepatitis C outbreak produced in a hospital hemodialysis unit that involved 6 patients. Polymerase chain reaction products from a 472-nt fragment of the E1-E2 region, including the hypervariable region, HVR-1, of the hepatitis C virus genome were cloned, and an average of 10 clones/patient and from 11 additional control patients were sequenced. The method allows a statistical evaluation that the likelihood of each sample belonging or not to a given group, a question of relevance in many forensic and epidemiological analyses of molecular sequences.

Patient-to-patient transmission of hepatitis C virus (HCV) during colonoscopy diagnosis.

BackgroundNo recognized risk factors can be identified in 10-40% of hepatitis C virus (HCV)-infected patients suggesting that the modes of transmission involved could be underestimated or unidentified. Invasive diagnostic procedures, such as endoscopy, have been considered as a potential HCV transmission route; although the actual extent of transmission in endoscopy procedures remains controversial. Most reported HCV outbreaks related to nosocomial acquisition have been attributed to unsafe injection practices and use of multi-dose vials. Only a few cases of likely patient-to-patient HCV transmission via a contaminated colonoscope have been reported to date. Nosocomial HCV infection may have important medical and legal implications and, therefore, possible transmission routes should be investigated. In this study, a case of nosocomial transmission of HCV from a common source to two patients who underwent colonoscopy in an endoscopy unit is reported.ResultsA retrospective epidemiological search after detection of index cases revealed several potentially infective procedures: sample blood collection, use of a peripheral catheter, anesthesia and colonoscopy procedures. The epidemiological investigation showed breaches in colonoscope reprocessing and deficiencies in the recording of valuable tracing data. Direct sequences from the NS5B region were obtained to determine the extent of the outbreak and cloned sequences from the E1-E2 region were used to establish the relationships among intrapatient viral populations. Phylogenetic analyses of individual sequences from viral populations infecting the three patients involved in the outbreak confirmed the patient pointed out by the epidemiological search as the source of the outbreak. Furthermore, the sequential order in which the patients underwent colonoscopy correlates with viral genetic variability estimates.ConclusionsPatient-to-patient transmission of HCV could be demonstrated although the precise route of transmission remained unclear. Viral genetic variability is proposed as a useful tool for tracing HCV transmission, especially in recent transmissions.