Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.

Intramammary infections with the contagious Staphylococcus aureus genotype B in Swiss dairy cows are associated with low prevalence of coagulase-negative staphylococci and Streptococcus spp

The association between the contagious Staphylococcus aureus genotype B (GTB) and the presence of coagulase-negative staphylococci (CNS) and Streptococcus spp. (non-agalactiae streptococci), was investigated, and the identification of problem herds without genotyping was evaluated. Milk samples from 10 herds with Staph. aureus GTB herd problems (PH cases) were compared with samples from 19 herds with at least one Staph. aureus isolate of non-B genotype (CH cases). All samples were bacteriologically analysed and Staph. aureus genotyping carried out using a ribosomal spacer-PCR. Cow and quarter prevalences of Staph. aureus, CNS and Streptococcus spp. differed significantly between PH and CH groups. PH cases were highly associated with decreased cow prevalences of CNS and Streptococcus spp. These altered prevalences also contributed significantly to the identification of problem herds without resorting to genotyping. Common herd-level risk factors did not explain the difference between the prevalences in PH and CH cases.

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C. Methodology/Principal Findings Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome. Conclusions/Significance Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.

Serum ferritin levels are associated with a distinct phenotype of chronic hepatitis C poorly responding to pegylated interferon-alpha and ribavirin therapy

Elevated serum ferritin levels may reflect a systemic inflammatory state as well as increased iron storage, both of which may contribute to an unfavorable outcome of chronic hepatitis C (CHC). We therefore performed a comprehensive analysis of the role of serum ferritin and its genetic determinants in the pathogenesis and treatment of CHC. To this end, serum ferritin levels at baseline of therapy with pegylated interferon-alpha and ribavirin or before biopsy were correlated with clinical and histological features of chronic hepatitis C virus (HCV) infection, including necroinflammatory activity (N = 970), fibrosis (N = 980), steatosis (N = 886), and response to treatment (N = 876). The association between high serum ferritin levels (> median) and the endpoints was assessed by logistic regression. Moreover, a candidate gene as well as a genome-wide association study of serum ferritin were performed. We found that serum ferritin ≥ the sex-specific median was one of the strongest pretreatment predictors of treatment failure (univariate P < 0.0001, odds ratio [OR] = 0.45, 95% confidence interval [CI] = 0.34-0.60). This association remained highly significant in a multivariate analysis (P = 0.0002, OR = 0.35, 95% CI = 0.20-0.61), with an OR comparable to that of interleukin (IL)28B genotype. When patients with the unfavorable IL28B genotypes were stratified according to high versus low ferritin levels, SVR rates differed by > 30% in both HCV genotype 1- and genotype 3-infected patients (P < 0.001). Serum ferritin levels were also independently associated with severe liver fibrosis (P < 0.0001, OR = 2.67, 95% CI = 1.68-4.25) and steatosis (P = 0.002, OR = 2.29, 95% CI = 1.35-3.91), but not with necroinflammatory activity (P = 0.3). Genetic variations had only a limited impact on serum ferritin levels. Conclusion: In patients with CHC, elevated serum ferritin levels are independently associated with advanced liver fibrosis, hepatic steatosis, and poor response to interferon-alpha-based therapy.

Liver kidney microsomal type 1 antibodies reduce the CYP2D6 activity in patients with chronic hepatitis C virus infection

Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease the CYP2D6 activity in vitro and are present in a minority of patients with chronic hepatitis C infection. We investigated whether LKM-1 antibodies might reduce the CYP2D6 activity in vivo. All patients enrolled in the Swiss Hepatitis C Cohort Study and tested for LKM-1 antibodies were assessed (n = 1723): 10 eligible patients were matched with patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specific substrate using the dextromethorphan/dextrorphan metabolic ratio to classify patients into four activity phenotypes. All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with the CYP2D6 genotype in most LKM-negative patients, whereas only three LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was sixfold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, P = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies. In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies.

Phenotype-genotype correlation in eight Polish patients with inherited Factor XIII deficiency: identification of three novel mutations

Inherited factor XIII (FXIII) deficiency is known as one of the most rare blood coagulation disorder in humans. In the present study, phenotype and genotype of eight FXIII deficient Polish patients from five unrelated families were compared. The patients presented with a severe phenotype demonstrated by a high incidence of intracerebral haemorrhages (seven of eight patients), haemarthrosis (six patients) and bleeding due to trauma (five patients). Introduction of regular substitution with FXIII concentrate prevented spontaneous bleeding in seven patients. In all patients, mutations within the F13A gene have been identified revealing four missense mutations (Arg77Cys, Arg260Cys, Ala378Pro, Gly420Ser), one nonsense mutation (Arg661X), one splice site mutation (IVS5-1 G>A) and one small deletion (c.499-512del). One homozygous large deletion involving exon 15 was detected by failure of PCR product. The corresponding mutations resulted in severely reduced FXIII activity and FXIII A-subunit antigen concentration, while FXIII B-subunit antigen remained normal or mildly decreased. Structural analysis demonstrated that the novel Ala378Pro mutation may cause a disruption of the FXIII catalytic triad leading to a non-functional protein which presumably undergoes premature degradation. In conclusion, the severe phenotype with high incidence of intracranial bleeding and haemarthrosis was in accordance with laboratory findings on FXIII and with severe molecular defects of the F13A gene.

Topical caspofungin for treatment of keratitis caused by Candida albicans in a rabbit model

Candida albicans is the most frequent cause of fungal keratitis in temperate regions. Caspofungin has potent activity against Candida spp. in a variety of clinical settings. Little is known, however, about its activity against fungal keratitis. We compared the efficacy of topical caspofungin with that of topical amphotericin B (AMB) in a rabbit model of experimental keratomycosis. Keratitis was induced with a standardized inoculum of Candida albicans (SC 5314) placed on the debrided cornea. Twenty-four hours after infection, animals were randomly assigned to treatment with 0.15% caspofungin, 0.5% caspofungin, 0.15% AMB, and a saline control (n = 12 rabbits in each group). For the first 12 h, treatment was repeated every 30 min and, after a 12-h pause, was resumed at hourly intervals for another 12 h. The animals were examined and killed 12 h after administration of the last dose. Treatment effects were evaluated by clinical assessment, fungal culture, and histopathology. Drug treatment significantly reduced corneal fungal recovery from 3.78 log10 CFU in saline-treated animals to 2.97, 1.76, and 1.18 log10 CFU in animals treated with 0.15% caspofungin, 0.5% caspofungin, and 0.15% AMB, respectively. By histopathology, the mean hyphal density was significantly lower in the corneas of treated animals than in those of the controls; there was no difference in hyphal densities between the different treatment groups. The depth of corneal invasion was not significantly reduced by the antifungal treatments. By clinical assessment, keratitis progressed in animals treated with saline, whereas disease progression was inhibited by all drug treatment regimens. In our rabbit model, 0.5% caspofungin was as effective as 0.15% AMB for the topical treatment of Candida keratitis. The potential clinical efficacy of caspofungin awaits further investigation.

Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C

BACKGROUND: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)-related steatosis. The MTP -493G/T polymorphism may promote liver fat accumulation, but its role in HCV-related steatosis is still unclear. METHODS: Two hundred ninety-eight CHC patients were studied and genotyped for MTP -493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. RESULTS: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV-3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2-3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non-3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22-3.86; P=0.008). Moreover, TT carriers had higher high-density lipoprotein (65.6+/-14.6 vs 56.1+/-16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80+/-0.3 vs 1.60+/-0.3 g/L; P=0.005) levels than G allele carriers. CONCLUSIONS: Chronic hepatitis C patients with the MTP -493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non-3 patients.

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.

Pegylated interferon-alpha2a/ribavirin treatment of recurrent hepatitis C after liver transplantation

Hepatitis C virus (HCV) infection invariably recurs after liver transplantation (LT), leading to significant morbidity and mortality. Although the combination of pegylated interferon-alpha (IFN-alpha)/ribavirin is the preferred treatment for these patients, the optimal schedule remains undetermined. In an uncontrolled trial, 19 patients with HCV infection recurring after LT received pegylated IFN-alpha(2a), 180 mug weekly, and ribavirin, 10 mg/kg body weight daily, for 48 weeks. The proportion of patients with undetectable HCV RNA in their serum after 12 weeks of treatment was 53%. Five patients (26%) dropped out of the study due to intolerance (in 2 cases), depression (in 1), or infectious complications (in 2). A sustained virological response (SVR), defined as undetectable serum HCV RNA 24 weeks after the end of treatment, was observed in 9/19 patients (47%). SVR was associated with an early virological response after 12 weeks of therapy (P<0.001) and a treatment duration >80% (P=0.02), but not with baseline HCV RNA level or a cumulative dose of pegylated IFN-alpha(2a) or ribavirin >80% of the scheduled dose. All 4 patients with genotype 2 or 3 reached SVR, as compared with 33% of patients with genotype 1 or 4 (P=0.03). A 48-week course of pegylated IFN-alpha(2a)/ribavirin therapy is effective in patients with recurrent HCV infection after LT.

Little evidence that hepatitis C virus leads to a higher risk of mortality in the absence of cirrhosis and excess alcohol intake: the Swiss Hepatitis C Cohort Study

The objective of this study was to describe the all-cause mortality of participants in the Swiss Hepatitis C Cohort compared to the Swiss general population. Patients with hepatitis C virus (HCV) infection attending secondary and tertiary care centres in Switzerland. One thousand six hundred and forty-five patients with HCV infection were followed up for a mean of over 2 years. We calculated all-cause standardized mortality ratios (SMR) and 95% confidence intervals (CI) using age, sex and calendar year-specific Swiss all-cause mortality rates. Multivariable Poisson regression was used to model the variability of SMR by cirrhotic status, HCV genotype, infection with hepatitis B virus or HIV, injection drug use and alcohol intake. Sixty-one deaths were recorded out of 1645 participants. The crude all-cause SMR was 4.5 (95% CI: 3.5-5.8). Patients co-infected with HIV had a crude SMR of 20 (95% CI: 11.1-36.1). The SMR of 1.1 (95% CI: 0.63-2.03) for patients who were not cirrhotic, not infected with HBV or HIV, did not inject drugs, were not heavy alcohol consumers (genotype 3, indicated no strong evidence of excess mortality. We found little evidence of excess mortality in hepatitis C infected patients who were not cirrhotic, in the absence of selected risk factors. Our findings emphasize the importance of providing appropriate preventive advice, such as counselling to avoid alcohol intake, in those infected with HCV.

Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy

The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P ce sites. CONCLUSION: Drug and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host "immune resistance" in the STAT-C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C.

Frequency of PRRS live vaccine virus (European and North American genotype) in vaccinated and non-vaccinated pigs submitted for respiratory tract diagnostics in North-Western Germany

The frequency of PRRSV corresponding to live vaccines and wild-type was determined in 902 pigs from North-Western Germany submitted for post-mortem examination. Overall, 18.5% of the samples were positive for the EU wild-type virus. EU genotype vaccine virus was detected in 1.3% and the NA genotype vaccine virus in 8.9% of all samples. The detection of the EU vaccine was significantly higher in pigs vaccinated with the corresponding vaccine (OR=9.4). Pigs vaccinated with NA genotype had significantly higher detection chances for the corresponding vaccine virus when compared to non-vaccinated animals (OR=3.34) animals, however, NA vaccine was also frequently detected in non-vaccinated pigs. Concluding, the dynamics of NA genotype vaccine and EU wild-type virus corresponds with studies on PRRSV spread in endemically infected herds. The potential of spontaneous spread of the NA genotype vaccine should be considered in the planning of eradication programs.