First finding of Echinococcus multilocularis DNA in soil: preliminary survey in Varmia-Masuria Province, northeast Poland.

The aim of this study was to estimate the presence of Echinococcus multilocularis DNA in the soil in rural areas of Varmia-Masuria Province, Poland, which is the region with the highest recorded number of human alveolar echinococcosis (AE) cases in this country. In total, 62 soil samples were analyzed. Samples were collected in forests near the lairs of foxes, as well as from arable fields, kitchen gardens and farmyards. The parasite DNA was detected in 11.3% of the samples. This study is the first environmental survey of the presence of E. multilocularis DNA in soil. The results demonstrate that the soil can be a direct source of human infections. These results should motivate the appropriate institutions to introduce informative and educational campaigns about the risk of infection, as well as developing methods for protecting the population, especially those living in at-risk areas.

The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples.

Toxoplasma gondii infections are prevalent in humans and animals all over the world. The aim of the study was to estimate the occurrence of T. gondii oocysts in fruits and vegetables and determine the genotype of the parasites. A total number of 216 fruits and vegetables samples were taken from shops and home gardens located in the area of northern Poland. Oocysts were recovered with the flocculation method. Then, real-time polymerase chain reaction (PCR) targeting the B1 gene was used for specific T. gondii detection and quantification. Toxoplasma DNA was found in 21 samples. Genotyping at the SAG2 locus showed SAG2 type I and SAG2 type II. This is the first investigation describing T. gondii DNA identification in a large number of fruits and vegetables samples with rapid molecular detection methods. The results showed that fruits and vegetables contaminated with T. gondii may play a role in the prevalence of toxoplasmosis in Poland.

Detection of Toxoplasma gondii oocysts in environmental soil samples using molecular methods.

Infections caused by Toxoplasma gondii are prevalent in humans and animals throughout the world. So far, there is no sufficient information concerning T. gondii oocysts prevalence in the environment, especially in soil. Therefore, the aim of this study was to estimate occurrence of T. gondii oocysts in soil and determine the genotype of detected parasites. A total of 101 soil samples were taken from different sites (sand-pits, "farming ground", areas around rubbish dumps) located in the Tri-City (Poland). Oocysts were recovered using the flotation method. Then, PCR reactions targeting the B1 gene were performed for specific T. gondii detection. The positive samples were further confirmed by PCR amplification of a repetitive element (REP) sequence [GenBank accession number AF146527]. Toxoplasma DNA was found in 18 samples. Among them, seven samples were successfully genotyped at the SAG2 locus. They were classified as SAG2 type I (5 samples) and SAG2 type II (2 samples). This is one of the first investigations describing T. gondii oocyst detection in environmental soil samples with rapid molecular detection methods and genotyping. The results of our findings showed that soil contaminated with T. gondii oocysts may play a role in the epidemiology of human toxoplasmosis in Poland.

Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis.

The precise diagnosis of an acute and recent Toxoplasma infection in pregnant women and the newborn child is important before treatment. This study describes a new Toxoplasma gondii IgG avidity test based on a combination of recombinant GRA1, GRA7 and SAG1 antigens and shows that this test is useful for diagnostic purposes and may replace the lysed, whole-cell antigens. Although more sera need to be tested, the results obtained here suggest that the IgG avidity test performed with rec-antigens correlated more with the stage of a T. gondii infection than the IgG avidity results obtained with the lysed, whole-cell antigen test, the VIDAS Toxo IgG avidity (bioMérieux).

Usefulness of Toxoplasma gondii-specific recombinant antigens in serodiagnosis of human toxoplasmosis.

Toxoplasma gondii SAG1, GRA1, and GRA7 recombinant antigens may be regarded as tools for the detection of T. gondii immunoglobulin G antibodies in persons with chronic and acute toxoplasmosis. GRA7 is more correlated with acute toxoplasmosis. A combination of these antigens will increase the sensitivity of enzyme-linked immunosorbent assays.

Human cystic and alveolar echinococcoses as indication to liver transplantation.

The human cystic and alveolar echinococcoses are zoonotic diseases caused by larval stages of the tapeworms Echinococcus granulosus and E multilocularis. In man the liver form of the diseases develops most frequently. Recent epidemiological data indicate that the distribution of E multilocularis in the central Europe is wider than was previously anticipated; more cases of human alveococcosis during the last years have also been noted in Poland. In the present paper we analyzed several cases of human echinococcoses from Poland with respect to diagnosis and treatment as well as the indications for liver transplantation.

Identification of anisakid nematodes from the Southern Baltic Sea using PCR-based methods.

Four common species of Baltic anisakids Anisakis simplex, Hysterothylacium auctum, Contracaecum osculatum from fish (the last one also from the grey seal) and C. rudolphii from cormorants were examined using PCR-RFLP technique for working out the method allowing their identification and differentiation. A fragment of nuclear DNA containing intergenic regions (ITS-1 and ITS-2) of ribosomal DNA together with adjacent sequences of genes coding 18S and 28S rRNA (in total; approximately 1500 bp) was amplified and the products were digested using three endonucleases Hin fI, Hae III and Pvu II. Digestion with Hin fI endonuclease enabled species identification and differentiation between all nematode species studied. Digestion with Hae III endonuclease enabled identification of A. simplex and H. auctum while electrophoretic patterns obtained for both Contracaecum species were similar (differentiation between A. simplex, H. auctum and the genus Contracaecum). Pvu II endonuclease was not suitable to this purpose. On the basis of nucleotide sequences obtained, the pairs of species-specific primers were designed for each species studied. By the use of these pairs of primers the species-specific PCR reactions were carried out. The sizes of specific products were: 486 bp for A. simplex, 491 bp for C. osculatum, 505 bp for C. rudolphii and 663 bp for H. auctum. The methods presented herein can be useful in diagnostics of human anisakidosis, especially when parasites are disrupted.

Usefulness of PCR for diagnosis of imported malaria in Poland.

The aim of the present study was to use the polymerase chain reaction (PCR) to detect and identify Plasmodium spp. in diagnostic specimens, especially in those from patients diagnosed by microscopy as having possible mixed infections, and in those demonstrating low parasitemia or those that were parasite-negative. For most of the specimens, the PCR results were in accordance with microscopic findings, and in 16.2% of the cases with low parasitemia PCR enhanced the results by identifying the parasite species. This method detected one additional case of Plasmodium falciparum malaria among the patients with fever of unknown origin. The sensitivity of PCR for detecting Plasmodium DNA was found to correspond to 1.35-0.38 and 0.12 for Plasmodium falciparum and Plasmodium vivax parasites per microliter of blood, respectively. Follow-up examinations demonstrated that most of the patients became negative for Plasmodium DNA from 1 to 4 days after the disappearance of parasitemia, as determined by examination of blood films. In conclusion, PCR performed by the reference laboratory significantly enhanced the microscopic diagnosis of malaria and proved very helpful in cases of low parasitemia and in cases of mixed infection.

[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.

Liver injury in the course of malaria.

121 malaria cases treated in the Ward of Tropical Diseases of the Clinic of the Institute of Maritime and Tropical Medicine in Gdynia in the years 1993-1999 were analysed. About 37% of the patients showed symptoms of parenchymal liver injury manifested by increased bilirubin concentration, elevated transaminase, alkaline phosphatase and GGTP levels. Histopathological examination of the liver revealed the activation of cells of mononuclear phagocyte system, Kupffer's cells in particular, with the presence of granules of browny-black ,,malarial" pigment and iron deposits. In one fatal tropical malaria case, symptoms of disseminated intravascular coagulation were found along with a few lymphocytic infiltrations in portal spaces, and focal necrosis of hepatocytes. In some patients with the so-called ,,untypical" secondary liver lesions present in the histopathological examination, the toxic effect of antimalarial drugs should be taken into account. A liver biopsy is justified in patients manifesting multiple courses of malaria. It is indispensable in cases of suspected polyetiological changes in the liver and in cases of recompensation payment claims.

Identification of Cryptosporidium felis in a cow by morphologic and molecular methods.

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 +/- 0.4 micrometer; range, 3.5 to 5.0 micrometer), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.

Usefulness of new DNA extraction procedure for PCR technique in species identification of Entamoeba isolates.

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 and/or trophozoites). Cultures of xenic 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 Hinfl 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.

Malaria as an Occupational Disease in Polish Citizens.

Background: Since 1963, malaria has been exclusively recognized in Poland as an imported disease, which has become increasingly frequent in recent years. When associated with performing duties in malaria-endemic areas, malaria is acknowledged as an occupational disease. Methods: Ninety five cases of malaria recognized as an occupational disease in Polish citizens in the years 1984-1993 were studied retrospectively in terms of working conditions, epidemiologic and clinical features, and permanent disability sequelae. Results: Among patients examined, young and professionally active persons who called at tropical ports or who worked in rural areas or in the regions of military conflict were predominant. Most infections were contracted in Africa during the first few months of the stay. The observed morbidity and recrudescence rates were mainly attributable to Plasmodium falciparum. Disregard of prevention and chemoprophylaxis of malaria were key risk factors. In 33% of the patients, severe symptoms and complications occurred and these required long-term therapy. In seven cases, disturbances of the central nervous system, hepatic, renal, or cardiac system occurred 6 months after termination of treatment, and these occurrences resulted in permanent disability and an inability to work. Conclusions: The disability and inability to work and its legal consequences, such as indemnity, demonstrate that malaria may present a serious socioeconomic problem, even in the country where it is an imported disease.

[Occurrence of parasites, bacteria, viruses and fungi in fish that are pathogenic to men and fish]. / Wystepowanie u ryb baltyckich pasozytów, bakterii, wirusów i grzybów patogennych dla ludzi i ryb.

Parasitological examinations comprised above 20,000 fish which were searched for parasitic nematoda of Anisakidae. It was evidenced that herring were infected with anisakid larvae in 8%, cod 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 coagulase-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 herring hitherto examined was noted.

[Anemia and thrombocytopenia in malaria. II. Mean erythrocyte and thrombocyte count in 45 patients with malaria observed in the Clinic of the Institute of Maritime and Tropical Medicine in Gdynia]. / Niedokrwistosc i maloplytkowosc w malarii. II. Srednia liczba erytrocytów i plytek krwi u 45 chorych z malaria obserwowanych w Klinice Instytutu Medycyny Morskiej i Tropikalnej w Gdyni.

In the years 1987-92, 45 patients with malaria were hospitalized in our clinic. All the patients were before staying in tropical countries. 30 patients were infected with PI. falciparum while the rest - with other species of this parasite. From among numerous different symptoms of malaria, in the majority of the patients profound anaemia and thrombocytopenia occurred. The simultaneously observed drop in the erythrocyte and blood platelet levels confirms that the destruction pathways of them in patients with malaria may be similar, as are the reasons for ineffective erythro- and thrombocytopoiesis. Anaemia and thrombocytopenia gradually subsided in the course of the therapy, as the parasitemia decreased and the state of the patients improved.

Malaria as an occupational disease.

In Poland, cases of imported malaria are noted at present. In Polish citizens who work abroad, malaria is considered as an occupational disease. 95 cases of malaria in Polish citizens treated at the Clinic of the Institute of Maritime and Tropical Medicine in 1984-1993 were subjected to retrospective analysis, considering their occupational, epidemiological and clinical aspects as well as permanent sequelae of the disease. The most numerous groups among the patients examined were seafarers, fishermen, priests and missionaries. They were infected mostly in West Africa, during the first year of work. The majority of patients neglected chemoprophylaxis of malaria. In about 1/3 of patients, the resulting incapacity for work exceeded 1 month and more. In 7% of patients, 6 months after the treatment, permanent sequelae of the disease were observed: damage to the central nervous system, liver, kidneys and heart. The social and material costs of this invasive occupational disease were high. Malaria is an important health problem in Poles who work in endemic areas. Malaria prevention should be intensified.