Agnieszka Pawełczyk

Prevalence and abundance of Cryptosporidium parvum and Giardia spp. in wild rural rodents from the Mazury Lake District region of Poland

Prevalence and abundance of Cryptosporodium parvum and Giardia spp. were studied in 3 species of rodents from forests and abandoned agricultural fields in N.E. Poland (Clethrionomys glareolus n = 459; Microtus arvalis n = 274; Apodemus flavicollis n = 209). Overall prevalence was consistently higher in the voles compared with A. flavicollis (70.6, 73.0 and 27.8% respectively for C. parvum and 93.9, 96.3 and 48.3% respectively for Giardia spp.). Prevalence and abundance of infection also varied markedly across 3 years with 1998 being a year of higher prevalence and abundance with both species. Fewer older animals (especially C. glareolus and M. arvalis) carried infection with C. parvum and infections in these animals were relatively milder. Although seasonal differences were significant, no consistent pattern of changes was apparent. Host sex did not influence prevalence or abundance of infection with C. parvum, but made a small contribution to a 4-way interaction (in 5-way ANOVA) with other factors in the case of Giardia spp. The 2 species co-occurred significantly and in animals carrying both parasites there was a highly significant positive correlation between abundance of infection with each, even with between-year, seasonal, host age, sex and species differences taken into account. Quantitative associations were confined to the 2 vole species in the study. These results are discussed in relation to the importance of wild rodents as reservoir hosts and sources of infection for local human communities.

Evidence for viral persistence in patients who test positive for anti-hepatitis C virus antibodies and have normal alanine aminotransferase levels.

It is unclear whether patients who test positive for anti-hepatitis C virus (HCV) antibodies and have normal alanine aminotransferase (ALT) levels remain infected with the virus. Eleven patients who tested positive for anti-HCV antibodies, had persistently normal ALT levels, and tested negative for HCV RNA by commercial test were studied. Serum and peripheral blood mononuclear cells (PBMCs) were collected 2-3 times at 3-6-month intervals, and PBMCs were cultured with phytohemagglutinin and pokeweed mitogen. HCV RNA was detected in serum samples from 6 (55%) and in PBMCs from 11 (100%) patients. Our results suggest that, in asymptomatic patients who test positive for anti-HCV antibodies, small quantities of HCV RNA commonly persist, even in patients who test negative for HCV RNA in serum by commercial tests.

Factors affecting the component community structure of haemoparasites in common voles ( Microtus arvalis) from the Mazury Lake District region of Poland

The prevalence and abundance of infections with haemoparasites were studied over a 4-year period in Microtus arvalis (common vole, n =321) sampled from fallow grassland sites in north-eastern Poland. Total species richness was five (prevalence= Haemobartonella sp. 63.9%, Bartonella spp. 27.7%, Babesia microti 9.0%, Trypanosoma sp. 8.4% and and Hepatozoon lavieri 3.1%) with 76.9% of the voles carrying at least one species and a mean infracommunity species richness of 1.1. Variation in species richness was determined primarily by season and year, the interaction of these factors, and that of year with host age. The observed frequency distribution of infracommunity species richness did not differ from that predicted by a null model, suggesting that there were no marked associations between the species. Analyses of prevalence and abundance of infection with each species in turn, revealed that overall the principal causes of variation were temporal and seasonal, their interaction, and interactions with intrinsic factors (age and sex), the latter playing only a minor role in their own right. However, the relative importance of these combinations varied and was distinct for each of the species in the study. Prevalence data revealed eight sets of two- and three-way associations between species, mostly dependent to some extent on one of the intrinsic and extrinsic factors in the model. Analysis of quantitative associations suggested two sets of positive two-way interactions, none of which remained after controlling for the effect of extrinsic and intrinsic factors on the abundance of each species. These data are discussed in the context of the changing ecological profiles in this region of Eastern Europe and, in a wider context, in relation to current understanding of the factors that shape component community structures of haemoparasites in wild rodents.

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3+, CD14+ and CD19+.

BackgroundAlthough hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3+, CD14+ and CD19+. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.MethodsPBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3+, CD14+, CD19+ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.ResultsNegative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3+ cells then in 8/26 (30.8%) of CD14+ and CD19+ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3+ (2.4%), then in CD19+ (1.2%), and much lower in CD14+ (0.4%) cells.ConclusionsOur results indicate that CD3+ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

Ultradeep Pyrosequencing of Hepatitis C Virus Hypervariable Region 1 in Quasispecies Analysis

Genetic variability of hepatitis C virus (HCV) determines pathogenesis of infection, including viral persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR1) of a chronically infected patient by ultradeep 454 sequencing strategy. Three independent sequencing error correction methods were applied. First correction method (Method I) implemented cut-off for genetic variants present in less than 1%. In the second method (Method II), a condition to call a variant was bidirectional coverage of sequencing reads. Third method (Method III) used Short Read Assembly into Haplotypes (ShoRAH) program. After the application of these three different algorithms, HVR1 population consisted of 8, 40, and 186 genetic haplotypes. The most sensitive method was ShoRAH, allowing to reconstruct haplotypes constituting as little as 0.013% of the population. The most abundant genetic variant constituted only 10.5%. Seventeen haplotypes were present in a frequency above 1%, and there was wide dispersion of the population into very sparse haplotypes. Our results indicate that HCV HVR1 heterogeneity and quasispecies population structure may be reconstructed by ultradeep sequencing. However, credible analysis requires proper reconstruction methods, which would distinguish sequencing error from real variability in vivo.

First report of two asymptomatic cases of human infection with Babesia microti (Franca, 1910) in Poland

Human infection by Babesia microti has been recognized as an emerging zoonosis with important public health implications worldwide. In Europe the reported cases of human babesiosis have been attributed mostly to B. divergens infection, with only sporadic cases of the disease caused by B. microti or B. venatorum. This study, based on molecular methods (PCR, R-T PCR, DNA sequencing and phylogenetic analysis), reveals for the first time in Poland, asymptomatic infection with . microti in immunocompetent healthy individuals working in forest ecosystems. Of the 58 professional foresters examined, two (3.4%) were identified as B. microti-positive by specific PCR. The results of this study also provide strong evidence that in eastern Poland, where tick-borne diseases (TBDs) are endemic, there is a potential risk of acquiring human babesiosis due to zoonotic B. microti parasites commonly found in rodents and I. ricinus ticks. The potential public health importance of this finding is discussed.

Differential display analysis of gene expression in brains from hepatitis C-infected patients.

Objectives:Hepatitis C virus (HCV) infection is often associated with cognitive dysfunction, fatigue and depression. The current study was undertaken to determine whether HCV infection affects gene expression in brain tissue. Design:We analysed the gene expression pattern in brain tissue in a group of HCV-infected patients compared with HCV-negative controls. Methods:Brain tissue samples were obtained at autopsy from three HCV-positive patients and three HCV-negative control patients. The analysis of gene expression was conducted using differential display and reverse Northern hybridization. Only those genes that were up or downregulated more than 1.8 times were considered to be differentially expressed. Results:Altogether, 29 differentially expressed genes were identified by differential display and subsequently confirmed by reverse Northern hybridization. A prominent finding was the downregulation of mitochondrial oxidative phosphorylation genes in HCV-infected patients. The impairment of brain oxidative/energy metabolism has previously been suggested to be the proximate cause of many disorders that impair mentation. Another finding was the downregulation of some ribosomal protein genes and several genes involved in transcription regulation, perhaps reflecting reduced metabolic activities. Conclusion:Our findings suggest for the first time that there may be a biological basis for the neuropsychiatric symptoms and cognitive impairment associated with HCV infection.

A Cluster of Fatal Tick-borne Encephalitis Virus Infection in Organ Transplant Setting.

word count: 200 Text word count: 2,640Background Tick-borne encephalitis virus (TBEV) infection has become a major health problem in Europe and is currently a common cause of viral brain infection in many countries. Encephalitis in transplant recipients, althrough rare, is becoming a recognized complication. Our study provides the first description of transmission of TBEV through transplantation of solid organs. Methods Three patients who received solid organ transplants from a single donor (2 received kidney, and 1 received liver) developed encephalitis 17-49 days after transplantation and subsequently died. Blood and autopsy tissue samples were tested by next-generation sequencing (NGS) and reverse transcription polymerase chain reaction (RT-PCR). Results All 3 recipients were first analyzed in autopsy brain tissue samples and/or cerebrospinal fluid by NGS, which yielded 24-52 million sequences per sample and 9-988 matched TBEV sequences in each patient. The presence of TBEV was confirmed by RT-PCR in all recipients and in the donor, and direct sequencing of amplification products corroborated the presence of the same viral strain. Conclusions We demonstrated transmission of TBEV by transplantation of solid organs. In such a setting, TBEV infection may be fatal, probably due to pharmacological immunosuppression. Organ donors should be screened for TBEV when coming from or visiting endemic areas.

Evidence for immune activation in patients with residual hepatitis C virus RNA long after successful treatment with IFN and ribavirin.

Low-level hepatitis C virus (HCV) RNA may persist in PBMCs after successful treatment of chronic hepatitis C, but the consequences of this phenomenon are unclear. Forty-nine patients who achieved a sustained virological response (SVR) after pegylated IFN and ribavirin therapy were analysed 52-66 months after the SVR. HCV RNA was detected in PBMCs from 18 patients (47.4 %), and PBMCs in two patients stained positive for non-structural protein 3 (NS3). Quantification of various cytokine and chemokine transcripts in PBMCs revealed that levels of IL-6, IL-8, IL-12, TNF-α and macrophage inflammatory protein 1β were significantly higher in HCV-positive patients than in HCV-negative individuals. In conclusion, persistence of HCV RNA in PBMCs of patients with a SVR appears to be associated with immune activation.

Deep sequencing of hepatitis C virus hypervariable region 1 reveals no correlation between genetic heterogeneity and antiviral treatment outcome

BackgroundHypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable part of HCV genome and its translation product is a major target for the host immune response. Variability within HVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study was to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of PEG-IFN-α (pegylated interferon α) and ribavirin treatment.MethodsHVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype 1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche). Reads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using three different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mann–Whitney and Fisher’s exact tests.ResultsComplexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic substitutions were not significantly different between responders and non-responders, when analyzing viral populations at any of the three frequencies (≥1%, ≥2% and ≥5%). When clonal sample was used to determine pyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a frequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous variant present at frequency of 0.5%.ConclusionsWhile deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients, there was no correlation between treatment outcome and any of the analyzed quasispecies parameters.