Hepatitis C virus 5' untranslated region variability correlates with treatment outcome.

Hepatitis C virus (HCV) variability affects viral-host interactions. We analysed HCV 5'untranslated region (5'UTR) in sera and peripheral blood mononuclear cells (PBMC) from chronic hepatitis C patients undergoing antiviral treatment. We studied 139 patients treated with pegylated interferon and ribavirin. The primary endpoint was a sustained virological response (SVR) defined as negative HCV RNA level 24 weeks after the end of therapy. 5'UTR was analysed by single-strand conformational polymorphism (SSCP) and sequencing. The pretreatment SSCP pattern in serum and PBMC differed in 26 (18.7%) patients. During therapy, the SSCP pattern remained stable in 65 (60.8%) patients, number of bands declined in 16 (15.0%), and in 18 (16.8%) patients, changes were qualified as 'shift' indicating change in band positions. In univariate analysis, there was a significant (P ≤ 0.05) positive association between SVR and pretreatment serum and PBMC dissimilarities, initial viral load <10(6) IU/mL, IL-28B CC genotype of the rs12979860 single nucleotide polymorphism and change in the SSCP band pattern (either 'shift' or decline) In multivariable analysis, only low initial viral load, IL-28B genotype, and changes in the SSCP band pattern were independent factors associated with SVR. In conclusion, stability of 5'UTR correlated with infection persistence, while changes correlated with SVR.

Seronegative hepatitis C virus infection in patients with lymphoproliferative disorders.

It has been reported that hepatitis C virus (HCV) RNA may be present in serum and/or lymphoid cells in the absence of specific circulating antibodies. The current study analysed seronegative HCV infection in patients with lymphoproliferative disorders. We studied 77 anti-HCV-negative patients (45 male and 32 female, mean age 54.8 ± 14.2 years) with various lymphoproliferative disorders. HCV-RNA was detected by RT-PCR in plasma, peripheral blood mononuclear cells (PBMC) and bone marrow. Furthermore, the presence of viral nonstructural protein 3 (NS3) was determined in PBMC and bone marrow by immunostaining. HCV-RNA was detectable in at least one compartment in 27 (35.1%) patients. Viral RNA was found in bone marrow in 22 patients (28.6%), in PBMC in 13 (16.9%) and in plasma in 10 (13%) patients. In nine patients, evidence of infection was confined to the bone marrow compartment. Viral load in HCV-RNA-positive plasma ranged from 15 to 1.17 × 10(3) IU/mL. NS3 was detected in all but two HCV-RNA-positive bone marrow samples and in all but one HCV-RNA-positive PBMC samples. All 27 HCV-RNA-positive patients remained anti-HCV-negative when tested again after 6-12 months, but only four remained HCV-RNA positive. In conclusion, among patients with lymphoproliferative disorders, HCV can be present in plasma, PBMC and bone marrow despite the lack of circulating specific antibodies. Further studies are required to analyse the phenomenon of seronegative infection and to determine whether such patients are infectious.

Tracing the HIV-1 subtype B mobility in Europe: a phylogeographic approach.

BACKGROUND: The prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced. RESULTS: In the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred. CONCLUSION: Subtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.

High prevalence of genotypic resistance to nucleoside reverse transcriptase inhibitors among therapy-naive individuals from the Warsaw cohort.

BACKGROUND: The purpose of this study was to recognize nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV-1 strains among a group of therapy-naive patients, and subsequently to guide the choice of antiretroviral therapy. PATIENTS AND METHODS: HIV strains present in sera of 128 antiretroviral therapy-naive patients were tested for NRTI resistance-associated mutations. The RT-PCR amplified HIV rt regions were analyzed with an INNI-LiPA-HIV-1 RT assay. The number and pattern of resistance-associated mutated codons were determined and interpreted as resistance to particular drugs. RESULTS: Mutated codons were detected in 66 (51.5%) out of 128 tested samples. In 54 (81.8%) out of 66 samples, a K70R mutation was identified, which was followed by an M184V mutation in 22 cases (33.3%). For a majority of these samples, mixed wild/mutant populations were recognized in 44 (66.6%) cases. The interpretation of hybridization data revealed drug-resistant strains in 37 (28.9%) out of all samples tested. The determined resistance to particular drugs was as follows: 20 strains were resistant to zidovudine (ZDV), ten to lamivudine (3TC) and six to didanosine (ddI)/zalcitabine (ddC)/abacavir (ABC). In one case, a ZDV/3TC-resistant HIV strain was found. CONCLUSION: The prevalence of mutations associated with NRTI resistance was high in the tested cohort. The key reasons for that were most probably needle and drug sharing within the group of intravenous drug users (IVDUs) and the use of ZDV in monotherapeutic regimes in the early 1990s.

Serological and molecular evidence of human granulocytic ehrlichiosis focus in the Bialowieza Primeval Forest (Puszcza Bialowieska), northeastern Poland.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne zoonosis. First described in the USA, it is being increasingly reported from several European countries. This study was undertaken to provide serological and molecular evidence of the occurrence of the HGE focus in the Bialowieza Primeval Forest, located in northeastern Poland. To this end, the seroprevalence of HGE in this area, where Lyme borreliosis and tickborne encephalitis are highly endemic, was determined by means of an indirect immunofluorescence antibody assay. In addition, the frequency of granulocytic Ehrlichia spp. infection in Ixodes ricinus ticks from the same area was estimated using a polymerase chain reaction method with EHR 521 and EHR 747 primers, which amplified a fragment of 16S rDNA. The rate of seropositivity for HGE was 6.2% (8/130 subjects). Individuals seropositive for Lyme borreliosis were more likely to have anti-HGE antibodies than seronegative ones (P<0.05; OR=6.34, 95%CI=1.12-36.98). There was no association between self-reported frequency of tick bites or forestry employment and HGE seropositivity. Sixty of 376 (16%) Ixodes ricinus ticks tested were positive for the Ehrlichia phagocytophila genogroup by polymerase chain reaction. Ehrlichial DNA was present in 59 of 302 (19.5%) adult ticks and in 1 of 74 nymphs (1.4%). There was a significantly higher infection rate among female ticks (32.9%; 49/149) than among male ticks (6.5%; 10/153) (P<0.05). Dual infection with Ehrlichia spp. and Borrelia burgdorferi sensu lato was detected in 10 samples that were positive for ehrlichiae. The results obtained confirm the perpetuation of the HGE agent in the primeval forest ecosystem of northeastern Poland.

[Usefulness of the molecular techniques for detecting and/or identifing of parasites and fungi in humans and animals or pathogens transmitted by ticks (Part I)]. / Zastosowanie technik molekularnych do wykrywania i/lub identyfikacji pasozytów i grzybów u ludzi i zwierzat oraz pa- togenów przenoszonych przez kleszcze. Czesc.

After a long period of using basic microscopic, immunological and biochemical methods for diagnosis, rapid development of nucleic acids investigation enabled introduction of specific and sensitive methods of detection of pathogenic agents on the molecular level. Among others, polymerase chain reaction (PCR), discovered in mid of 80'ies and then automatized, offered an attractive alternative to conventional testing systems. In this paper we describe reliable diagnostic tests widely used in the world, including Poland, and capable of detecting different disease agents as parasites and fungi in clinical specimens and pathogens of emerging zoonotic diseases in ticks. The possibilities of using molecular methods for determination of Plasmodium falciparum drug resistance is also discussed. Moreover, the report offers information concerning kinds of molecular tests and institutions in which there are executed.

Detection of three genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different regions of Poland.

Ixodes ricinus ticks, collected in 1996-1998 in different Polish woodlands, were examined to assess the frequency of the occurrence of Lyme borreliosis-associated genospecies. A total of 568 samples of individual adults and 162 samples of individual (n =48) and pooled (of 2 to 7) samples of nymphs were analysed by the polymerase chain reaction (PCR) for Borrelia burgdorferi sensu lato. Spirochetes were detected in 130 adult ticks (22.9%) and in a minimum of 32 (5.3%) nymphs. Further identification of 153 B. burgdorferi s.l.-positive samples by nested PCR using three species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto and B. garinii. Both single-species and mixed infections were noted. Single-species infections were observed in the majority of samples (n = 83/153; 54.2%). Within this group B. afzelii was found in 38/153 samples (24.9%), followed by B. burgdorferi sensu stricto (n = 23/153; 15.0%) and B. garinii (n = 22/153; 14.4%). Dual infections with B. burgdorferi s.s. and B. afzelii were detected in 17/121 (14.0%) adults, while both B. burgdorferi s. s./B. garinii and B. afzelii/B. garinii coinfected 11/121 (9.1%) adult ticks. Triple infection with B. burgdorferi s.s., B. afzelii and B. garinii was noted twice (1.6%). In general, B. afzelii was found in 72/153 (47.1%) tick samples and was the predominant species. B. burgdorferi s. s. and B. garinii were detected in a total of 60/153 (39.2%) and 51/153 (33.3%) samples, respectively. Although, 21 (13.7%) samples were infected by B. burgdorferi s.l. genospecies undetectable by the primers used, results of our study confirm that Lyme borreliosis pathogenic genospecies are well established in tick populations throughout Poland.

[Use of PCR for evaluating detection of coa and nuc genes in methicillin-resistant, coagulase-negative strains of Staphylococcus aureus (MRSA-CN)]. / Zastosowanie PCR do oceny wystepowania genów coa i nuc u metycylino-opornych, koagulazo-ujemnych szczepów Staphylococcus aureus (MRSA-CN).

Strains showing a negative reaction in tube test for coagulase constitute 10 to 20% of all Staphylococcus aureus isolated from patients of Hospital of Infant Jesus in Warsaw. Most of them are MRSA. In 42 MRSA strains showing negative reaction for coagulase, the presence of coagulase (coa) and nuclease (nucA) genes was checked. Determination of whole cell DNA with PCR reaction was performed. The obtained results revealed that all 42 strains possessed gene nucA, but only 39 strains possessed the coa gene.

Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari, Ixodidae) in different Polish woodlands.

In 1996-1998, a total of 2285 Ixodes ricinus ticks (1063 nymphs, 637 males, 585 females) were collected from vegetation from 25 different localities in the 8 Polish provinces throughout the country. Ticks inhabited all 25 collection sites. The average number of ticks per collection site was 91.4 +/- 13.7. All 2285 ticks were examined for Borrelia burgdorferi sensu lato (s.l.) presence, of which 1333 specimens from 3 provinces were tested by routine indirect immunofluorescence assay (IFA) using polyclonal antibody PAB 1B29. The remaining 952 specimens from 5 provinces were examined by polymerase chain reaction (PCR), using FL6 and FL7 primers. The overall infection rate in ticks estimated by these 2 methods was 10. 2%. Nymphs showed lower positivity rate (6.2%) as compared to adult ticks (14.9% in females and 12.4% in males). The highest percentage of infected I. ricinus ticks (37.5%) was noted in the Katowice province while the lowest (4.1%) in the Bia ystok province. In particular collection sites, infection rates varied from 0-37.5%. The obtained results confirmed that B. burgdorferi s.l. is present throughout the distributional areas of I. ricinus in Poland and that a prevalence of spirochete-infected ticks may be high in some locations.

[Genetic diagnosis of hepatitis C virus infection]. / Diagnostyka genetyczna zakazen wirusem zapalenia watroby typu C.

Hepatitis C virus, a small enveloped RNA member of Flaviviridae, is the major causative agent of non-A, non-B hepatitis. The discovery and genetic characterization of HCV is described. Modern genetic procedures and methods involved in the studies of HCV biology, genetic heterogenity and diagnostics of HCV infections are presented. Interferon-alfa treatment is the only licensed therapy for chronic hepatitis C until now, but an efficacy of such treatment is highly variable. Viral factors predicting the long-term outcome of interferon alfa therapy, such as genotype, baseline viral load, quasispecies or ISDR mutations, and current concepts of their predictive values are discussed. Our own results are presented and compared with those present in the latest publications.

The etiological agent of Lyme disease, Borrelia burgdorferi, in ticks (Acari:Ixodidae) from eastern Poland.

In 1994, 4204 out of 4656 Ixodes ricinus collected both from vegetation in three different areas and from hunter-killed deer and wild boar in the Bialystok province (eastern Poland) were examined individually for the presence of Borrelia burgdorferi s.I. Detection of spirochetes was carried out by the routine indirect immunofluorescence assay (IFA) using polyclonal antibody (anti-B. burgdorferi, strain 1 B 29). B. burgdorferi spirochetes were evident in 349 (8.8%) nymphal and adult I. ricinus collected from vegetation (n = 3958) and in 8 (3.2%) of those removed from hosts (n = 246). Among the ticks collected from vegetation, infection rates in nymphs (5.8-6.4%) in particular areas were about 2-4 times lower than in adults (8.1-24.6%). The calculated minimal and maximal infection rates of ticks collected from different sites were 1.6% and 15.4%, respectively. Prevalence of B. burgdorferi in I. ricinus was determined with respect to the abundance and seasonal activity of the ticks.

[The prevalence of Ixodes ricinus ticks (Acari, Ixodidae) in the forested areas of Gdansk, Sopot, and Gdynia and their infection rate with Borrelia burgdorferi spirochetes]. / Wystepowanie kleszczy Ixodes ricinus (Acari, Ixodidae) na zalesionych obszarach Trojmiasta i ich zakazenie kretkami Borrelia burgdorferi.

Data are presented on the variable patterns on the seasonal activity of Ixodes ricinus questing on vegetation in 6 study sites in the forested areas of Gdansk, Sopot and Gdynia in 1993-1995. A total of 8992 specimens collected there show that ticks frequently occupy habitats closely associated with man. Out of them 5775 (4328 nymphs, 713 females and 680 males) collected in 1994 and 1995 were examined individually for Borrelia burgdorferi sensu lato-the etiologic agent of Lyme borreliosis-using in-direct immunofluorescence assay (IFA). Spirochetes were detected in 577 (10.3%) of the ticks tested. The overall infection rate was 8.2% for nymphs (n = 353), 14.9% for females (n = 102) and 18.9% for males (n = 122). The infection rates in particular study sites varied between 1.4% and 16.4% in 1994 and between 3.6% and 26.9% in 1995. The highest prevalence of B. burgdorferi was observed in June (10%) and October (11.9%) in 1994 and in August (16.3%) and October (25%) in 1995. Detection of B. burgdorferi in ticks derived from the same area in the two following years shows that the infection of the I. ricinus population with this pathogen in the forested areas of Gdansk, Sopot and Gdynia is permanent.

[Selected diagnostic and clinical aspects of chronic viral hepatitis type C]. / Wybrane zagadnienia diagnostyczne i kliniczne przewleklego wirusowego zapalenia watroby typu C.

The aim of this study was to evaluate selected diagnostic and clinical aspects of chronic hepatitis C (CH-C) in the group of 80 patients: 68 males aged 24-65 (mean 39.8 +/- 10,5) and 12 females aged 35-66 (mean 48.7 +/- 12.6). The epidemiological data allowed to divide the basic group into 3 subgroups: patients with transfusion-associated CH-C (subgroup I: 12 males, mean age 38 +/- 6.7 and 2 females aged 40 and 46), CH-C patients with parenteral hepatitis C virus exposure-other than blood transfusion (subgroup II: 25 males, mean age 40.6 +/- 8.2 and 5 females aged 43 +/- 15.1) and sporadic cases with unknown HCV exposure (subgroup III: 31 males, mean age 38.2 +/- 11.2 and 5 females, mean age 50.5 +/- 10.3). The duration of the disease (CH-C) was calculated from the incident of acute viral hepatitis or the first signs of liver damage caused by HCV to the confirmation of CH-C by liver biopsy. The following data were analyzed: a frequency of acute viral hepatitis with jaundice at the beginning of the disease, ALT flare-ups, mean highest activities of ALP and GGT, frequency of hypergammaglobulinaemia and sings of fatty liver in ultrasonographic finding (USG). In all patients but one anti-HCV antibodies (ELISA 2nd generation test by Abbott) were detected. In 64/80 subjects antibodies to HCV antigens: 5-1-1, C 100-3, C 33c and C 22 were determined by RIBA-2 test (Ortho). In 62/80 patients HCV-RNA in serum was determined by RT PCR. Liver biopsy was performed in 71/80 patients. Other co-existent liver diseases were excluded. The similarity between 3 subgroups was shown: similar percentage of males and females, similar patients mean age and the duration of the disease. It was shown that the acute beginning of the disease with jaundice has been observed twice as frequent in subgroups I and II compared with subgroup III. The same frequency of ALT flare-ups in all subgroups was observed (25-28.6%). No differences in mean highest ALP and GGT activities in 3 subgroups were observed. It was shown, however, that hypergammaglobulinaemia was detected more frequently in subgroup III (30.5%) compared with subgroup I (7.1%) and II (16.7%). The signs of fatty liver in ultrasonographic findings were also observed more frequently in subgroup III (30.5%) than in subgroup I (14.3%) or II (16.7%). In all patient but one, in which anti-HCV antibodies by ELISA test were detected, anti-C 33c and anti-C 22 antibodies by RIBA were present. HCV-RNA in serum was detected in 77.8% subjects from subgroup I. 73.9%-from subgroup II and 66.7%-from subgroup III. In all HCV-RNA positive patients anti-HCV antibodies were detected. The evidence of chronic active hepatitis confirmed by liver biopsy was shown in 63.6%, 67.8% and 71.8% of patients from subgroup I, II and III, respectively. In no case normal liver morphology was present. Authors concluded the distressing fact of the high incidence of chronic active hepatitis in patients unaware of HCV infection, without the incident of acute hepatitis at the beginning of the disease (over 1/3 of all described subjects). The differences of the clinical course of the disease between subgroups 1 + II and subgroup III suggest two different routes of HCV infection or the presence of two different HCV mutants in Polish population. Authors emphasise the necessity of HCV gene typing in CH-C patients, which might explain the surprisingly high incidence of chronic active hepatitis in the reported group. The use of the presented data for the general practitcioner making the diagnosis of crytogenetic liver disease is also accentuated.

Sulfate-activating enzymes in normal and brachymorphic mice: evidence for a channeling defect.

The severe reduction in the amount of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in cartilage from homozygous brachymorphic mice results from a decrease in the activities of both ATP sulfurylase (50%) and adenosine 5'-phosphosulfate (APS) kinase (14% of normal). In order to better understand the etiology of this double enzyme defect, a dual approach to elucidating the nature of the enzyme complex as well as its mechanistic properties was undertaken. Antibody reagents that react with both activities provide evidence for a single, bifunctional protein in both normal and mutant cartilage. Quantitative Western blot analysis indicates that a normal amount of a dysfunctional protein is produced in mutant cartilage. Kinetic studies show that the Vmax for mutant kinase is significantly reduced and that mutant sulfurylase and kinase appear to have lower KmAPS values than normal. Interestingly, the mutation appears to disrupt the channeling mechanism that has recently been demonstrated for this pathway [Lyle et al. (1994) Biochemistry 33, 6822-6827]. APS kinase from normal mouse cartilage utilizes APS supplied by ATP sulfurylase much more efficiently than APS which is added exogenously; i.e., channeling efficiency is > 90%. In contrast, the mutant enzymes exhibit only 54% channeling efficiency. Lastly, isotope dilution and enrichment experiments show directly that the APS binding sites of the mutant enzymes are more accessible to free APS than are those of the normal enzymes. These data suggest that the mutation primarily affects the catalytic properties of the PAPS activation system by altering the function of the novel coupling mechanism between the two activities, causing a decrease in the ability to channel APS and produce PAPS efficiently.