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In this study we have developed a modified CLARK and DIAMOND (1992, 1993) method using the polymerase chain reaction to amplify amoebic ribosomal RNA genes that allow either specific detection of Entamoeba histolytica s. str. or amoeba species identification. DNA was isolated from cultured protozoa or from stool samples (cysts andlor trophozoites). Cultures of xenie or axenic material (also stool samples) were treated with Easy Genomic DNA Prep or Genomic DNA Prep Plus (A&A Biotechnology, Poland) for DNA isolation. With these new procedures, DNA can be obtained effectively and with high degree of purity. 13 amoeba strains were investigated. Three strains produced an 876 bp fragment with Psp5 and Psp3 primers (specific product for E. histolytica s. str). Three strains gave a specific 876 bp product for E. dispar with NPsp5 and NPsp3 primers. Two strains were E. moshkovskii as identified by KpnI digestion of PCR products obtained with RD5 and RD3 primers. Four strains (one of them was cultured in two laboratories) were E. invadens as identified by HinfI or HhaI digestion of PCR products obtained with RD5 and RD3 primers. Characteristic RFLP patterns with these enzymes was also obtained for E. terrapinae and Blastocystis hominis. This RFLP analysis show significant promise as a medical diagnostic tool particularly useful for species identification.
The identified mutations in the pfcrt, dhfr and dhps genes of Plasmodium falciparum show a very high correlation with resistance to chloroquine, pyrimethamine and sulfadoxine, the drugs that are still used as malaria chemoprophylaxis or treatment. We undertook a molecular screening of 82 Polish P. falciparum isolates, mainly imported from different countries of sub-Saharan Africa to assess their molecular drug-resistance profiles. Only 4 isolates showed no mutations in the three analyzed gene fragments. In the remaining isolates from one to six mutations in one or more examined genes were found. Different mutations in the pfcrt, dhfr and dhps genes were found in ca. 76%, 80% and 70% of P. falciparum isolates, respectively. About forty our patients used chloroquine or pyrimethamine + sulfadoxine as malaria chemoprophylaxis and/or antimalarial treatment, but without success. In all but 5 of the P. falciparum isolates obtained from these persons, mutations associated to resistance of the parasite to chloroquine and the antifolate drugs were found.
Nematodes from the superfamily Ascaridoidea (families Anisakidae and Raphidascarididae) are worldwide distributed parasites. Their live cycles include many species of water invertebrates and teleostean fish as intermediate hosts, and fish, sea mammals or fish-eating birds being definitive hosts. Humans can be infected with some of these parasites after consumption of raw or wrongly processed fish. The parasitological investigations of fish (herring, cod and flatfish) from southern Baltic (ICES 24-26) provided in the years 80 and 90 showed their infection with larvae of several anisakid species: Anisakis simplex s. str., Contracaecum osculatum C and Hysterothylacium auctum. Sporadically Pseudoterranova decipiens and Raphidascaris acus were also found. Larvae of Anisakis simplex were noted mainly in herrings, C. osculatum primarily in cods and H. auctum in flounders. Additionally, preserved herrings (marinated, smoked) were also investigated and sporadically live larvae of A. simplex were found. The main etiological agent of human anisakidosis worldwide is A. simplex. Although the live cycle of this nematode cannot be completed in the Baltic Sea - this nematode is brought to the Baltic by infected herring migrating from the North Sea for spawning in coastal waters of the Southern Baltic - the prevalence and intensity of infection with larvae of this nematode species were the highest in fish investigated by us. The results obtained suggest the possibility of the human infection with A. simplex larvae in Poland.
Parasitological examinations comprised above 20000 fish which were searched for parasitic nematoda of Anisakidae. It was evidenced that herrings were infected with anisakid larvae in 8%, cods and flatfish in 4% and the eelpouts in as many as in 52%. The species that prevails in these fish were: Anisakis simplex, Contracaecum osculatum C, Hysterothylacium auctum and Pseudoterranova decipiens, respectively. In direction of the bacteria pathogenic to man 765 fish were examined; in 38% there were found pathogenic strains such as coagulaso-positive staphylococci, even Salmonella and Shigella. Besides there were cultivated 109 strains of bacteria pathogenic to fish belonging to the genera: Aeromonas, Pseudomonas, Acinetobacter and Moraxella. Virological evaluation comprised 527 fish, in that number Enteroviruses (Coxsackie, ECHO) were identified in 31 % fish and viral agent, likely to be pathogenic to fish in 8% specimens. Not a one case of infection with Ichthyophonus hoferi fungus in the herrings hitherto examined was noted.
The description of very efficient system for production and purification of Toxoplasma gondii recombinant antigens, GRA6, p35 and SAG2 is given in this study. The usefulness of these antigens for diagnostic of human infections was tested in an ELISA using 99 sera obtained during routine diagnostics. The sera for testing were selected from either acute or chronically infected patients. Both r-GRA6 and r-p35 antigens detected antibodies more frequently (p<0.01) from acute (93.9 and 87.9%) rather than chronic (60.6 and 53.0%) infections. The r-SAG2 gave a similar sensitivity in both groups of patients (93.9 and 95.5%).
Leishmania parasites are the etiological agents of leishmaniosis, with severe course and often fatal prognosis, and the global number of cases has increased in recent decades. The gold standards for the diagnosis of leishmaniosis are microscopic examinations and culture in vitro of the different clinical specimens. The sensitivity of these methods is insufficient. Recent development in specific and sensitive molecular methods (PCR) allows for detection as well as identification of the parasite species (subspecies). The aim of the study was to estimate the usefulness of molecular methods (PCR) for detection of Leishmania species and consequently for the implementation of such methods in routine diagnostics of leishmaniosis in Polish patients returning from endemic areas of the disease. In our investigations we used 54 known Leishmania positive DNA templates (from culture and clinical specimens) received from the CDC (Atlanta, GA, USA). Moreover, 25 samples of bone marrow, blood or other tissues obtained from 18 Polish individuals suspected of leishmaniosis were also examined. In PCR we used two pairs of primers specific to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircle (13A/13B and F/R). Using these primers we obtained amplicons in all DNA templates from the CDC and in three Polish patients suspected for Leishmania infection. In one sample from among these cases we also obtained positive results with DNA isolated from a blood specimen which was previously negative in microscopic examinations.
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