Epidemiology and molecular investigation of hepatitis C infection following holiday haemodialysis.

BACKGROUND: Hepatitis C virus infection (HCV) is not infrequent among haemodialysis patients. Most published reports suggest that patient-to-patient spread, either directly or indirectly, is the most common mode of transmission in renal units. AIM: To investigate the source of an outbreak, and the route of transmission, of acute HCV infection in two Scottish patients occurring within eight weeks of receiving haemodialysis in the same unit while on holiday in Majorca. METHODS: This was an international epidemiological and molecular investigation of HCV infection among a cohort of haemodialysis patients from nine countries. FINDINGS: No further HCV-positive infections were observed among residents and holidaymakers receiving haemodialysis at the unit in Majorca. Molecular investigations confirmed that a Spanish healthcare worker (HCW) was the source of infection for the two Scottish patients. The investigators were unable to determine the route of transmission. CONCLUSIONS: This outbreak is the first reported case of HCW-to-patient transmission of HCV in a renal unit, and the third reported case of transmission involving a HCW who had not performed invasive procedures. The issue of whether renal units are an exceptional case with regards to the risk of transmission associated with non-invasive procedures should be considered, in conjunction with the need to improve surveillance of blood-borne virus transmissions in renal units in the UK and abroad.

Refined analysis of genetic variability parameters in hepatitis C virus and the ability to predict antiviral treatment response.

Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres-Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large-scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha-interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region.

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

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.

Genetic similarity of hepatitis C virus and fibrosis progression in chronic and recurrent infection after liver transplantation.

The effect of hepatitis C virus (HCV) genetic heterogeneity on clinical features of post-transplantation hepatitis C is controversial. Different regions of the HCV genome have been associated with apoptosis, fibrosis, and other pathways leading to liver damage in chronic HCV infection. Besides, differences in immunodominant regions, such as NS3, may influence HCV-specific immune responses and disease outcome. In the liver transplant setting, a recent study has reported a positive association between HCV-1b Core region genetic relatedness 5-year post-transplantation and histological severity of recurrent hepatitis C. We have compared nucleotide sequences of HCV Core, NS3 and NS5b regions in HCV-1b-infected patients 3 years post-transplantation (n = 22). A cohort of nontransplanted patients (n = 22) was used as control of natural chronic HCV-1b infection. Histological evaluation was used to define the rate of fibrosis progression. Molecular variance analysis did not show significant differences in HCV sequences between transplanted and nontransplanted patients, or between those with fast or slow fibrosis progression. The same results were obtained when analysing phylogenetic trees for Core, NS3 and NS5b regions. A more appropriate clustering method (using minimum spanning networks) revealed a significant positive relationship between HCV genetic similarity in Core (r = 0.550, P < 0.01) and NS5b regions (r = 0.847, P < 0.01) and the yearly rate of fibrosis progression in nontransplanted patients which, in contrast, was not observed in transplanted patients. Our results indicate that some strains of HCV-1b might be more pathogenic in the natural course of chronic infection by this virus subtype. In the liver transplant setting, when the immune response is severely compromised, other mechanisms are probably more important in determining hepatitis C progression.

Efecto de la alimentación sobre la condición fisiológica del mejillón Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN / Food effect on the physiological condition of the Perna viridis mussel (Bivalvia: Mytilidae), using the RNA/DNA quotient

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.

Efecto de la temperatura de aclimatación sobre el crecimiento instantáneo de Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN / Effects of acclimation temperature on the instant growth of Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA quotation

Temperature affects growth rate in aquatic organisms. This can be evaluated in short term using biochemical indexes (RNA/DNA and Protein/DNA). The effect of acclimatization temperature on the instantaneous growth and physiological condition of Perna viridis was studied in organisms collected in La Esmeralda, Sucre State (Venezuela) and taken to the laboratory, where groups of 100 organisms (size 3.0 - 3.5 cm, anteroposterior measurement) were acclimatized at 15, 20, 26 or 28 degrees C during four weeks. Later they were kept in a 60 liters aquarium for another six weeks under the same conditions. Each week, ten organisms per group were extracted to measure concentrations of RNA, DNA (by a fluorometric method with ethidium bromide) and proteins (by a colorimetric method), in tissues (digestive gland, adductor muscle and gills). Protein concentration was greater and highly significant at 15 degrees C for all studied tissues. The opposite was obtained with the RNA/DNA and Protein/DNA ratios: the greatest increase was observed at the highest temperature (28 degrees C) for all tissues. At the lowest temperature there was a tendency to reduce both indexes with time. Greater instantaneous growth can be expected at higher temperatures and 28 degrees C was optimal for growth in these specimens.

[Effects of acclimation temperature on the growth of Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio]. / Efecto de la temperatura de aclimatación sobre el crecimiento instantáneo de Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN.

Temperature affects growth rate in aquatic organisms. This can be evaluated in short term using biochemical indexes (RNA/DNA and Protein/DNA). The effect of acclimatization temperature on the instantaneous growth and physiological condition of Perna viridis was studied in organisms collected in La Esmeralda, Sucre State (Venezuela) and taken to the laboratory, where groups of 100 organisms (size 3.0 - 3.5 cm, anteroposterior measurement) were acclimatized at 15, 20, 26 or 28 degrees C during four weeks. Later they were kept in a 60 liters aquarium for another six weeks under the same conditions. Each week, ten organisms per group were extracted to measure concentrations of RNA, DNA (by a fluorometric method with ethidium bromide) and proteins (by a colorimetric method), in tissues (digestive gland, adductor muscle and gills). Protein concentration was greater and highly significant at 15 degrees C for all studied tissues. The opposite was obtained with the RNA/DNA and Protein/DNA ratios: the greatest increase was observed at the highest temperature (28 degrees C) for all tissues. At the lowest temperature there was a tendency to reduce both indexes with time. Greater instantaneous growth can be expected at higher temperatures and 28 degrees C was optimal for growth in these specimens.

[Food effect on the physiological condition of the mussel Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio]. / Efecto de la alimentación sobre la condición fisiológica del mejillón Perna viridis (Bivalvia: Mytilidae), según el cociente ARN/ADN.

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.

Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity.

The genetic diversity of a vesicular stomatitis virus population was analysed by RT-PCR, cloning and sequencing of two approximately 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with both Taq and Pfu DNA polymerases, and important differences were observed. Between 10 and 22 mutations were detected when virus populations were analysed by Taq amplification (20 clones from each region), whereas amplification of the same samples with Pfu revealed between 0 and 5 mutations. PCR fidelity assays, performed under the same PCR conditions as those used in the population analysis, showed that the Taq error-rate estimate of 0.27 x 10(-4) misincorporations per bp per cycle was within the range estimated elsewhere from PCR amplification of recombinant plasmids (0.27-0.85 x 10(-4) errors per bp per cycle) or from functional assays (0.2-2 x 10(-4) errors per bp per cycle). The error rate of Taq was found to be 9.3 times higher than the error rate of Pfu with DNA as a template, and about 10 times higher with cDNAs obtained by reverse transcription of viral RNA templates from natural populations. In the present study, we discuss (i) the implications of Taq errors on the analysis of genetic variability, based on both the frequency and nature (replacement vs synonymous) of the observed substitutions and (ii) the sample size required to assess the genetic variability in a virus population generated by a single infection.