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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.

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Faecal specimens were taken from 205 sheep and goats housed in five different localities in the west-central part of Poland. All faecal specimens were examined for Cryptosporidium by using microscopy screening of smears stained by modified Ziehl-Neelsen technique and commercial enzyme immunoassay. PCR technique using genus specific primers was additionally applied in the surveys of 10 faecal specimens collected from lambs. C. parvum infection was identified in 16 of 159 sheep (10.1%). Lambs were more often infected than adult sheep, and the intensity of infection was higher in lambs than in sheep, as a rule. Both lambs and sheep examined in the study were asymptomatically infected with Cryptosporidium. Both microscopy and enzyme immunoassay methods gave one false negative result. The examination of 10 faecal samples revealed 100% agreement among the results obtained by microscopic, immunologic and molecular methods. None of the goats raised on three farms were infected with Cryptosporidium.

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The present study was undertaken to investigate the prevalence of Cryptosporidium infection in horses used for recreational riding as well as in humans. A total of 106 faecal specimens from horses raised in 4 localities of western Poland and 6 stool samples from 3 persons who had constant or sporadic contact with horses were screened microscopically for oocysts using modified Ziehl-Neelsen staining. Enzyme immunoassay (EIA) was additionally used for the detection of coproantigen in human stool samples as well as in 43 randomly selected horse faecal samples. The overall infection rate of horses determined by microscopic examination was 9.4%. To our knowledge, this is the first report of cryptosporidial infection in horses in Poland. The infection was identified only in adult horses raised on 2 of 4 examined farms. The intensity of equine cryptosporidial infection was light, as a rule. None of the infected horses appeared clinically ill. The real overall infection rate in horses could be higher. Among 43 faecal specimens additionally processed by EIA, 5 samples were positive both for oocysts and coproantigen, whereas in 7 faecal samples only the parasite coproantigen was detected. The morphometric analysis of oocysts indicated that the horses were most probably infected with C. parvum. Of 3 examined persons, cryptosporidial infection was identified in a rider who had sporadic contact with horses.

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The application of biochemical and molecular techniques in parasitological studies has provided increasing evidences of genetic polymorphism among parasite populations. This review presents possible origins of genetic variation within populations of various protozoan species. Since the mode of reproduction has an important influence on genetic polymorphism within parasite populations these considerations refer mainly to some protozoan parasites which have various life cycles, e.g. Giardia, Trypanosoma, Cryptosporidium, Toxoplasma. Also other factors associated with parasites (such as: transmission and passage history in laboratory conditions; occurrence in different hosts or geographic regions; selective pressure of drugs; competitive interactions between populations) that affect parasite genetic diversity are discussed. However, the number of examined isolates of parasites and genetic markers, assortment of methods, probes, primers and reagents used is also of significance. The significance of genetic variability in parasite populations is still the subject of much interest and controversy. A simple interpretation of such variation is impossible because of the complexity of host-parasite interactions. The knowledge of parasite diversity at the nucleic acids level has continually increased, but a corect interpretation of this phenomenon requires at least the same knowledge of genetic variability in host populations. Nevertheless, genetic variability in protozoan parasites has many important implications, e.g. for taxonomy, epidemiology, control and evolution. Genetic differences within parasite populations might also be associated with phenotypic variability, e.g. virulence, antigenicity, infectivity, drug sensitivity, host preference etc.

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Cryptosporidium parvum is a coccidian parasite that affects millions of people worldwide. Clinical outcome of human cryptosporidiosis differs between immunocompetent and immnunodeficient individuals. C. parvum is responsible for causing protracted and life-threatening diarrhea, biliary, and pulmonary infections in immunocompromised persons, especially in patients with AIDS. Though no effective treatment has been found so far, early diagnosis may be useful in controlling the infection. Thirty-eight stool specimens obtained from 35 HIV-positive patients admitted to the Clinic of Infectious Diseases in Poznan, Poland, were examined for the detection of oocysts, coproantigen and DNA of Cryptosporidium using standard microscopic, immunologic and molecular diagnostic methods. The presence of Cryptosporidium was detected in 10 HIV-positive patients. Oocysts, coproantigen and DNA of this parasite were identified solely in one specimen while Cryptosporidium DNA was detected in 8 specimens. Cryptosporidium coproantigen was found only in one sample. Although, the PCR was the most useful technique in the detection of Cryptosporidium in HIV-positive patients it should be noted that PCR has many pitfalls and needs to be carefully controlled to avoid both false positive and false negative results.

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The authors present the actual review on several publications concerning the molecular characterizations of the viruses found in parasitic protozoa such as Giardia, Trichomonas, Leishmania and Entamoeba histolytica. All of the RNA viruses observed in parasitic protozoa showed several similarities and did not considerably differ from the viruses found in simple eukaryotic cells; they closely correspond to dsRNA viruses of yeast. The supposition that the protozoan symbionts detected in laboratories transfer to their hosts in natural conditions seemed to be rational, though, there are no evidences that these symbionts are potential pathogens. However, the opinion reiterates that intestinal protozoa (e.g. Entamoeba histolytica) may serve as vectors for HIV or cofactors of HIV infection. The authors point out that irrespective of the potential role of viruses as vectors in the transfection system for parasitic protozoa, the observed viral system constitutes an unusual experimental system to solve the problems of gene expression.

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The article reviews the problems on epidemiology of cryptosporidiosis and gives a detailed description of the massive waterborne outbreak in Milwaukee (Wisconsin, USA) associated with a break in filtration capacity of a public water supply. The authors emphasize the need for high-quality diagnostic procedures as current coprodiagnostic microscopical techniques seemed to fail to detect Cryptosporidium oocysts.

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The authors present the actual informations on waterborne outbreaks of giardiasis as well as the evidences of Giardia cyst transmission by municipal water supply. In the article a recommendations is given to molecular biology techniques which contributed to the armentarium applied in the modern diagnostics of giardiasis.

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The article comprises a critical review on practical applications of molecular technology in parasitological diagnostics in a broad sense, also as a diagnosis of species and a method of epidemiological analysis. Techniques of genome analysis at different levels, as specific nucleic acid probes, DNA restriction profiles (RFLP), hybridization techniques, pulse-field gel electrophoresis, in vitro nucleic acid amplification, and DNA fingerprint technique used in studies on Giardia and Cryptosporidium were discussed. The essential reservation as far as this technology is concerned refers to its usefulness in parasitological diagnostics; there is no sense in working out methods for recognizing parasites which could otherwise be identified by well trained parasitologists and simple microscopic methods. The improved diagnosis of parasites resulting from the application of molecular technology significantly contributed to the armarium of parasitologists. Application of recent molecular technology in diagnosis of giardiasis and cryptosporidiosis may basically support clinical diagnosis which provides possibilities of early and selective treatment and makes possible epidemiological studies. These assays will permit not only a rapid diagnosis and exact differentiation but will also enable a better recognition of Giardia and Cryptosporidium genome organization. However, in spite of the wide availability of this new techniques they have not been fully applied--as yet--in diagnosis and in epidemiological studies on these parasites. The authors share the opinion of Busch (1991) on the need of proper recognition of high-quality and rigorous work in employing new molecular assays, because their wide availability and high sensitivity could cause "false-positive" results by contamination with amplified DNA sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

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A human volunteer and Mongolian gerbils were shown to be susceptible to infection with Giardia from a Gambian giant pouched rat. The course of infection and the pattern of cyst excretion, as well as the number of cysts in gerbils which were given the same inocula of Giardia, differed from the course of infection in humans. The latent period in gerbils was longer and the gerbils continually excreted numerous cysts, whereas excretion of cysts, in smaller numbers, by the volunteer was intermittent. Moderate clinical symptoms were noted in the volunteer, and the infection was confirmed by the development of anti-Giardia antibodies. The study demonstrated the zoonotic character of giardiasis and has important implications for the epidemiology of Giardia.

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In man, as well as in many species of vertebrates there exist several populations of Giardia duodenalis group which, though morphologically indistinguishable, show different level of heterogenicity in several characteristics, a.o. in surface antigens, isoenzyme make-up, RFLP, invasiveness for different hosts or pattern of experimental infection. Also the clinical observations in man distinctly suggested that G. intestinalis comprises several different populations. In the course of giardiasis apparent variability in clinical manifestations can be observed. In many patients the infection is symptomless and resolves spontaneously and in some others--variable intensity of symptoms is observed. Most likely both the parasite's characteristics and the host's feature will determine the clinical character of infection. It is well known that in some cases it is the host that responsible for the symptoms of the infection. Thus, for instance, clinical giardiasis links with immunodeficiency, malnutrition or young age. There are also evidences that some Giardia isolates have enhanced potency to provoke the disease of the host. It is considered that the variable pattern of the infection may be related to three different factors in host-parasite relationship: extra- and intrapopulation variability of Giardia isolates, the microenvironmental factors of the host's intestine, and the variable immune response of the host. The author describes data on the two first factors.

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A total of 13 new Giardia isolates were established in axenic culture. All of the new isolates were obtained by excystation of Giardia cysts from the feces of patients in Dutch hospitals. These isolates were subjected to isoenzyme and DNA analysis together with isolates from Poland, Belgium, and various other parts of the world. Isoenzyme analysis revealed that nearly all of the newly established isolates exhibited unique zymodemes. Isolates obtained from individuals from Belgium and Poland, on the other hand, displayed single zymodemes. Genomic DNA libraries were constructed from isolates belonging to the latter two zymodemes; specific and common recombinant DNA clones were selected from these libraries. Differential screening revealed that the two isolates had only 80% of the clones in common. Restriction-fragment-length polymorphism analysis using three different probes together with two synthetic probes that are complementary to Giardia structural protein genes led to the separation of all isolates into two major groups; within these groups, a further division could be made by application of other techniques or probes. The results of DNA analysis and zymodeme classification were in general agreement; in the present report they are compared with the data in the literature and discussed.

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The sensitivity in vitro to metronidazole and ornidazole of 7 stocks and of the cloned lines of 5 stocks of Giardia isolated from humans, rodents and monkeys was studied by the growth inhibition test. All 7 stocks of Giardia, irrespective of the host, differed in their sensitivity to these drugs, commonly used in therapy of human giardiasis. The differences were greater with ornidazole than with metronidazole. The 5 Giardia stocks from which clones were prepared were found to consist of populations with significantly (P less than 0.05) differing sensitivities to ornidazole and metronidazole. There was a positive correlation between high resistance in vitro to both drugs of all clones of one parent stock and treatment failures of giardiasis in the patient from which the parasite stock had been isolated. The spectra of sensitivity of Giardia to anti-giardial drugs may have implications concerning the suspected zoonotic character of human giardiasis.

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Five Giardia isolates from primates and rodents were grown axenically and compared by different electrophoretic techniques. One isolate from a lemur (slow loris) contained a dsRNA virus also found in some of the Giardia of human origin. Using ethidium bromide stained gels and also Southern blots hybridized with a rDNA probe, two profiles of restriction fragment length polymorphism were found in the animal Giardia, which are identical to two profiles found previously in strains of human origin. Isoenzyme and total protein patterns obtained with agarose isoelectric focusing divided the strains in the same two groups. With pulsed field gradient gel electrophoresis, the isolates showed 6-8 chromosomal bands but none of the band patterns were identical. The size of the chromosomes varied from 0.8 to over 3.0 Mb. A ribosomal DNA probe hybridized with different bands.

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During the examination of animals at the Poznan Zoological Gardens, attempts were made to isolate Giardia strains. Using an in vitro excystation procedure, eight samples of cysts from animals with asymptomatic giardiasis were inoculated on BI-S-33 medium. The ease of isolation and axenization of Giardia was surprising; five axenic isolates of Giardia, belonging to the G. duodenalis morphological group, were established from primates (slow loris, lesser slow loris and siamang) and from rodents (Gambian giant pouched rat and cuis). The growth of all isolates was abundant and similar; the peak number of trophozoites on the seventh day (depending on the Giardia isolate) was 2.3 X 10(6)-3.2 X 10(6) and generation times were 8.2-19.3 h. The easy establishment of these isolates confirmed that they belong to the G. duodenalis morphological group. The recent hypothesis that Giardia may be introduced to a human population from an animal source implies the necessity to isolate and differentiate parasite strains from various hosts. In this respect, the first isolation of Giardia strains from non-human primates and from rodents is of particular importance.

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Restriction enzyme digestion of bulk DNA from Giardia intestinalis reveals the presence of repeated sequences. A prominent 1.8 kb band in the Alu I profile was cloned into the pUC8 plasmid (pGI7) and used for comparing strains. When blots of DNA of 34 isolates from different geographic areas are probed with pGI7, hybridization with identical intensities can be detected. However, some strains give different hybridization patterns with several restriction enzymes. No hybridization of pGI7 can be detected with DNA from Trypanosoma brucei, Naegleria fowleri, Entamoeba histolytica and Trichomonas vaginalis. Therefore probe pGI7 may be useful in comparing different isolates as well as in screening for G. intestinalis infection.

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