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.

DRB polymerase chain reaction fingerprinting, DQA oligotyping and mixed lymphocyte culture results in related bone marrow donors and recipients.

Donor and recipient are usually regarded as well matched for bone marrow transplantation when they are compatible in HLA class I and II antigens and in mixed lymphocyte culture (MLC). However, results of serological typing of class II antigens may be unreliable. Hence, polymerase chain reaction fingerprinting of HLA DRB (PCR FP) was introduced for the screening of related donors for 29 patients awaiting bone marrow transplantation. In addition, the sequence-specific oligonucleotide (SSO) typing of DQA alleles was performed. In 18 pairs the results of DNA analysis methods were compared with the results of MLC. 72% of pairs were HLA DQA compatible and 59% showed compatibility in PCR FP. MLC compatibility was found in 61%. A higher correlation of PCR FP and MLC results was observed.

Experimental parameters of vanadium determination by atomic-absorption spectroscopy with graphite-furnace atomization.

It was found that impregnation of a graphite cuvette (HGA-72) with salts of elements which form stable carbides (Ta, Si Nb, Zr, W, La) decreases the absorbance signal for vanadium. The slope of the atomization curves indicates that formation of vanadium atoms is inhibited, probably by formation of a ternary compound between the impregnating element, vanadium and graphite. On the contrary bigger signals and better repeatability of results may be achieved when the cuvette is coated with pyrolytic graphite and methane is added to the sheath gas. The presence of methane increases the atomization efficiency and compensates for the disadvantageous influence of any air present in the sheath gas.

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.

[Identification of toxigenic Clostridium difficile strains isolated from alimentary tract of dogs by PCR]. / Identyfikacja metoda PCR toksynotwórczych szczepów Clostridium difficile wyhodowanych z przewodu pokarmowego psów.

Twenty eight Clostridium difficile strains were investigated. Eleven strains were toxin A and toxin B gene positive. Four strains were negative for both genes. These results correlate with results of cytotoxicity assay. We identified 13 Clostridium difficile strains toxin A gene positive and toxin B gene negative.

[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.

[The role of ticks in the epidemiology of Lyme borreliosis]. / Rola kleszczy w epidemiologii boreliozy z Lyme.

In this paper the current knowledge of the natural circulation of Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, and its relationships both to hard ticks (Ixodidae) serving as vectors, and to certain vertebrates which function as tick hosts and pathogen reservoirs is reviewed. The modes of transmission of borreliae (by salivation, regurgitation, defecation) by ticks to their animal hosts and men as well as prophylactic measures against tick-bites are also discussed.

[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.

[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.

[Experimental studies of the control of mosquitoes of the genus Anopheles using selected insecticides]. / Badania doswiadczalne nad redukcja komarów z rodzaju Anopheles przy zastosowaniu wybranych insektycydów.

In the research 4 compounds: Iodofenphos, Propetamphos, Dioxacarb and Permethrin and two commercial insecticides: Pibutoks Super (Permethrin) and Safrotin 10 WP (Propetamphos) were used to control anophelini females. The chemicals were tested in the laboratory on mosquitoes caught in the nature and the contact method was employed. Propetamphos and Permethrin at concentrations 0.05% and 0.1%, after 2 hours of 1 hour exposure reduced the mosquitoes in 96% and 99.9% respectively. Iodofenphos and Dioxacarb, even at concentration 0.1%, after 24 hours of 1 hour exposure caused barely 78.3% and 83.9% mortality of these insects. Both insecticides tested, in the 7 days period after the spraying, caused more than 97% reduction of the Anopheles population in the cow-sheds.

[Mosquitoes (Diptera, Culicidae) found in the coastal region from Wejherowo to Darlowo]. / Komary (Diptera, Culicidae) znalezione w pasie przymorskim od Wejherowa do Darlowa.

Species composition Anophelini were investigated in the years 1986/87 and Culicini in 1987/88 at the part of the coastal region. The mosquitoes were caught from April till October, every week, at 47 stations (water reservoirs, free nature and cattle-sheds). Total 16,194 mosquitoes (2452 Anophelini and 13,742 Culicini) were caught. They belong to 26 species of 5 genera: Anopheles (4 spp.), Aedes (15 spp.), Coquillettidia (1 sp.), Culex (1 sp.) and Culiseta (5 spp.). The highest numbers of Anophelini were observed in July (1986) and in September (1987). The aggressiveness toward man showed by Culicini was found to be highest in the second half of May and at the end of July.

[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.

[The polymerase chain reaction (PCR) and its application in transfusion medicine and hematology]. / Reakcja lancuchowa polimerazy (PCR) i jej zastosowanie w transfuzjologii i hematologii.

The new technique, polymerase chain reaction, used for in vitro DNA fragments amplification is described. The conditions of the reaction and methods of data analysis are discussed as well as the usefulness of this technique in research and medical practice.

Occurrence of pediculosis capitis in a population of children from Gdansk, Sopot, Gdynia and the vicinities.

Elementary schools in Gdansk, Sopot, Gdynia and the neighbourhood, were studied during 1990-1992 to determine the actual extent of head louse infestation in school children as well as to see if there was correlation between incidence of head lice (Pediculus humanus capitis) and some environmental factors as: the sex and the age of a child, the parents education, family situation and living condition of a child etc.--In the examined sample of normal population of 27,800 school children (aged 6-15 years) 881 (3.2%) pupils were infested with head lice, with the incidence being higher in girls. As many as 382 cases (1.3%) were qualified as a focal type, i.e. with stubborn, repeated reinfestations after following treatments. In the group with focal pediculosis 48.9% and 79.4% of children originated from the families of 4 or more children and from the families where parents had only elementary or trade education. Later, over 50% (53.3-56.6%) of village elementary school children focally infested with head lice lived in old houses mostly supplied only with cold water or even without running water at all. These data confirm the association between head louse infestation and familial, social and hygienic conditions of an infested child.

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.