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ABSTRACT: Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0.27 microm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0.74 and 0.22 mM, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.
Parasitology 05/2009; 136(6):595-602. · 2.96 Impact Factor
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ABSTRACT: Malaria is a common, life-threatening infection in endemic tropical areas and one that presents a diagnostic challenge to laboratories in most non-endemic countries. A rapid and accurate diagnosis is a prerequisite for effective treatment, especially for the potentially fatal cases of Plasmodium falciparum infection. In the present, multi-centre study, the performances of a rapid diagnostic test (NOW) Malaria) and several, commercial, PCR-based assays (AMS61, AMS42, AMS43, AMS4 and AMS45) were compared against the results of microscopical examination of bloodsmears (the current 'gold standard'). The subjects were either non-European immigrants (N=135) or international travellers (N=171). There was good concordance between the results of all the detection methods, with kappa values of >0.8. Although the NOW Malaria rapid test was both sensitive (100%) and specific (100%) in detecting P. falciparum infections, it was less specific (93.1%) and sensitive (90.7%) in identifying the other Plasmodium species. The results from the AMS61 assay, designed to detect any malarial infection, generally parallelled those of the microscopy (kappa = 0.89), giving a specificity of 98.2% and a sensitivity of 91.0%. Although the use of species-specific molecular primers to identify pure infections with P. falciparum and P. vivax gave results that were in good agreement with those of the microscopy, the subjects who had apparently pure infections with P. ovale or P. malariae were always found PCR-negative. Compared with the standard microscopy, both the NOW Malaria test and the PCR-based assays were therefore poor at identifying mixed infections. The NOW Malaria test and the PCR-based assays clearly need to be improved, particularly for the correct identification of infections with Plasmodium spp. other than P. falciparum, including mixed infections. For now, expert microscopy must remain the mainstay of the laboratory diagnosis of malaria.
Annals of Tropical Medicine and Parasitology 05/2007; 101(3):195-204. · 1.43 Impact Factor
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ABSTRACT: In the present study, 67 patients suspected to be cases of visceral leishmaniasis (VL) were each checked for leishmanial infection by the microscopical evaluation of various biological specimens, in-vitro culture, serology and an assay based on nested PCR. Most (35) of the subjects were immunocompetent (IC) but 32 were immunodeficient (ID) as the result of HIV infection (18 cases), treatment to prevent transplanted organs being rejected (six) or haematological malignancies (eight). Forty-one (61.2%) of the subjects (19 IC subjects, 12 HIV-positive patients, four transplant patients and six patients with malignancies) were considered true cases of VL. For the IC subjects, only the production and microscopical examination of leucocytoconcentrates and cultures of Buffy coats gave sensitivities of <80%, the results of the other methods showing higher sensitivities and almost perfect agreement with the 'gold-standard' diagnoses. For the ID subjects, however, only the serological tests and the PCR gave reasonable sensitivities (of >80%). For the initial diagnosis of leishmaniasis in ID patients, IFAT and western blots may be useful, as, among the present ID patients, they gave sensitivities (of 80.9% and 88.2%, respectively) that were almost as high as that for the PCR, and specificities of 100%. In the diagnosis of VL in either IC or ID patients, the assay based on a nested PCR appeared to be particularly reliable, with sensitivities of 88.9% and 95.2%, respectively, and a specificity of 100% in both groups of patients. The testing of bone-marrow aspirates by PCR revealed very few VL cases who were not found positive when samples of their peripheral blood were checked in the same assay. For both IC and ID subjects therefore, the use of the PCR-based method to test samples of peripheral blood (which can be collected much more easily than bone-marrow aspirates and with much less pain for the subject) is recommended.
Annals of Tropical Medicine and Parasitology 11/2004; 98(7):667-76. · 1.43 Impact Factor
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ABSTRACT: The review summarizes the results in the main parasitological topics of our Lab: amoebic infections due to Entamoeba histolytica/Entamoeba dispar complex and to Acanthamoeba spp. respectively, and human infections caused by microsporidia. Different rapid and advanced techniques have been included in the standardized diagnostic protocols for each topic, and a critical comparison among them was made, in order to define the gold standard diagnostic method: a) E. histolytica/E. dispar: in vitro culture, zymodeme typization, biomolecular identification (PCR), immunoenzymatic assay (ELISA) for direct detection in stools of specific surface antigenic lectins; b) Acanthamoeba spp.: in vitro culture, light and ultrastructural characterization, species identification by immunofluorescence method with monoclonal antibodies, in vitro pharmacological studies; c) Microsporidia: ultrastructural (TEM), biomolecular (PCR), biochemical and immunological (SDS-PAGE, Immunoblotting) studies for species identification, use of advanced ultrastructural techniques ("freeze-etching", "deep-etching") in order to deepen the spore wall structure, to study the cytoskeletal function of actin and to define the mode of infection, in vitro pharmacological assays on some inhibitors of chitin-synthases.
Parassitologia 01/2002; 43 Suppl 1:37-43.
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ABSTRACT: Of 550 mentally retarded patients in an Italian institution, 125 (23%) were found to be infected with intestinal parasites. The infections were most frequent in young men, those with severe mental retardation, the chronically institutionalized and those living in older wards. Ninety-four (75.2%) of the parasitised subjects were infected only with protozoa, 25 (20%) only with helminths, and six (4.8%) with protozoa and helminths. Entamoeba histolytica and E. dispar infections were detected, but at low prevalences; in-vitro culture in Robinson's medium and isoenzyme electrophoresis of the cloned amoebic isolates indicated one infection with E. histolytica (zymodeme XII) and two infections with E. dispar (zymodemes I and III). All three Entamoeba-positive subjects were asymptomatic cyst-passers. Antibodies to E. histolytica were detected in seven (1%) of the sera from the 550 patients examined; only one of these was a carrier of an E. dispar strain at the time of investigation. The low prevalences of all the parasitic infections and of the amoebic infections in particular (compared with those observed previously in institutions for the mentally retarded) reflect relatively good facilities and sanitary conditions, an adequate number of well trained staff and good control of the more susceptible subjects.
Annals of Tropical Medicine and Parasitology 08/2000; 94(5):453-60. · 1.43 Impact Factor
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ABSTRACT: Four cases of amoebiasis are described: two symptomatic with intestinal and hepatic involvement and two asymptomatic, diagnosed in two, heterosexual, Italian couples. Infection was probably acquired first by the men, via an indirect faccal-oral route, and then transmitted to their partners in the same way. The two amoebic strains isolated, from the woman of one couple and the man of the other, were characterized by electrophoresis as zymodemes II alpha- and XIX of Entamoeba histolytica. These four cases emphasise once more the role of cyst-passers in the spread of infection and the importance of biochemical identification of the amoebic isolates, enabling more specific treatment.
Annals of Tropical Medicine and Parasitology 01/2000; 93(8):829-34. · 1.43 Impact Factor
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ABSTRACT: Cryptosporidium parvum is a coccidian protozoon that causes diarrhoeal enteritis in immunocompetent and immunocompromised humans and other mammals. Sometimes, chiefly in HIV-infected subjects, anatomical sites other than gastro-intestinal tract, such as the biliary and respiratory tree, are involved. We performed an experimental respiratory infection in immunosuppressed albino rats with a C. parvum human-derived isolate, to confirm the possibility of a primary infection at this site and to evaluate the protozoan damages by light and also by transmission electron microscopy (TEM). The animals were infected intratracheally with 1 x 10(6) C. parvum oocysts/ml and, from the 7th day post-infection, biological specimens of trachea, bronchi, lung and ileum were zoopsied. A sole cryptosporidial colonization of the respiratory tract, from the trachea to the median bronchi, without lung parenchyma infection, was observed. Moreover 13/33 (39.4%) rats also developed intestinal infection. TEM study of the respiratory tree specimens demonstrated that cryptosporidia infect either ciliated or goblet cells, and confirmed the role of microvilli in the parasite cell adhesion. The most relevant alterations involved the ciliated cells, with loss of cilia and nuclear and cytoplasmic damages.
Parasite (Paris, France) 10/1999; 6(3):217-22. · 1.00 Impact Factor
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New England Journal of Medicine 04/1999; 340(9):738. · 53.30 Impact Factor
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ABSTRACT: Proteinases play an important role in survival of microorganisms and in pathogenicity of diseases. By using a modified SDS-gelatin-polyacrylamide gel system, proteinases of rat-P.carinii were detected as bands of proteolytic digestion after electrophoresis. P.carinii organisms obtained from dexamethasone immunosuppressed transtracheally infected rats were cultured in spinner flask suspension cultures to minimize host cell contamination. At pH 8.3, seven Pc-specific proteolytic bands were detected in three clusters of different molecular weights clearly different from host cell patterns. By using a range of pH, various preparations of organisms and both infected and uninfected culture media, proteolytic activities have been partially characterized. Elastase secretion has been assessed based on elastin digestion model. Proteinase inhibitors have been tested for their ability to inhibit P.carinii growth in HEL299 short-term monolayer cultures. Results indicate that proteolytic activities are involved in the proliferation of microorganisms since leupeptin exerted in vitro antipneumocystis activity while aprotinin enhanced P.carinii growth.
Parasite (Paris, France) 04/1999; 6(1):9-16. · 1.00 Impact Factor
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ABSTRACT: Acanthamoeba keratitis is a severe ocular infection secondary to accidental macro- or microscopic trauma of the cornea. Starting in 1985, a dramatic increase of this infection was recorded along with the spread of contact lens use. This protozoal disease is difficult to treat because of the scarcity of efficacious topical and systemic drugs. We evaluated the in vitro effectiveness of povidone-iodine (PVP-I [Betadine]), an agent with broad antibacterial and antiviral activity, compared to that of chlorhexidine (CXD), a cationic antiseptic, on Acanthamoeba isolates from patients with amebic keratitis. The results showed that PVP-I solution from 0.5 to 2.5% has a better antiamebic activity both on trophic and cystic stages of Acanthamoeba spp. than does CXD.
Antimicrobial Agents and Chemotherapy 10/1998; 42(9):2232-4. · 4.84 Impact Factor
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ABSTRACT: An epidemiological survey to characterize Entamoeba histolytica/E dispar isolates from 123 human subjects was carried out in the Wonji area of Central Ethiopia, where an increased incidence of amoebic infection has been reported. In a randomized, coproparasitological study, 93 (75.6%) of the subjects were found positive for at least one species of intestinal parasite: 14 (15.1%) harboured only one species and 79 (84.9%) were infected with at least two species. In-vitro culture in Robinson's medium revealed amoebic parasites in 52 (82.5%) of the 63 cases tested. Of the 29 amoebic isolates successfully stabilized, cloned and characterized by Sargeaunt's electrophoretic technique, 27 (93.1%) were of E. dispar zymodemes (19 of zymodeme I, two each of zymodemes III, V and XI, and one each of zymodemes X and XV) and two (6.9%) were of E. histolytica (zymodeme XIII).
Annals of Tropical Medicine and Parasitology 04/1998; 92(2):173-9. · 1.43 Impact Factor
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ABSTRACT: Isoenzyme analysis by starch-gel electrophoresis has proved to be a useful method for the biochemical differentiation of pathogenic Entamoeba histolytica and non-pathogenic E. dispar isolates. Of the known 24 zymodemes, 3 are laboratory-made and have not previously been identified in humans. Parasitology screening was carried out in a psychiatric institution. Two amebic stocks were isolated and characterized that had never previously been found in humans and that have protein patterns identical to that of the laboratory-made zymodeme XX.
Parasitology Research 02/1997; 83(7):716-8. · 2.15 Impact Factor
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ABSTRACT: Pneumocystis carinii pneumonia (PCP) is a highly frequent cause of morbidity and mortality in immunocompromised subjects, particularly in HIV-infected ones. The biology of P. Carinii is poorly understood because of the lack of reliable synthetic media or adequate cell lines to grow this opportunistic pathogen in continuous culture. We reported the suitability of the MDCK (Madin Darby Canine Kidney, ATTC CCL 34) cell line to support the temporary microorganism's growth in vitro and the experimental pharmacological trials, in comparison with the HEL 299 cell line, used as reference standard.
Parasite (Paris, France) 07/1996; 3(2):183-5. · 1.00 Impact Factor
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ABSTRACT: Since 1993, strains of Entamoeba histolytica sensu lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1.7% nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.
Parasitology 05/1996; 112 ( Pt 4):363-9. · 2.96 Impact Factor
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Parassitologia 01/1994;
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ABSTRACT: A sample of 300 sexually-active adults was selected at random from patients, from the rural area of Malenga Makali, Tanzania, who were attending a dispensary because they had diarrhoea of at least 2 weeks' duration. The potential associations between the patient's health (in terms of the World Health Organization's clinical definition of AIDS), HIV-1 seroprevalence and malaria and other parasitic infections were then investigated. Although, HIV-1 seroprevalence was 20.6% overall, the level of seroprevalence was directly correlated with the distance between the patients' home villages and the nearest main road. Strict application of the clinical definition of AIDS gave 98.7% specificity, 46% sensitivity and a predictive value of 90.6% when validated by HIV-1 seropositivity. Although malaria infection was more common in HIV-1 seropositives than in the seronegatives, the intensity of the Plasmodium falciparum infections, intestinal amoebiasis and giardiasis did not appear to be correlated with HIV-1 infection. In contrast, intestinal infections with Cryptosporidium parvum and Isospora belli were virtually restricted to HIV-1 seropositive individuals who had had diarrhoea for a relatively long time.
Annals of Tropical Medicine and Parasitology 01/1994; 87(6):585-93. · 1.43 Impact Factor
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ABSTRACT: The sesquiterpene bilobalide, extracted from Ginkgo biloba leaves, was tested in vitro and in vivo for the ability to inhibit Pneumocystis carinii growth. Bilobalide was inhibitory to trophozoites cultured on human embryonic lung fibroblasts (HEL 299) at approximately the same concentration as trimethoprim plus sulfamethoxazole (lowest effective concentration, 50 micrograms of bilobalide per ml versus 9/45 microgram of trimethoprim-sulfamethoxazole per ml), inducing microscopically detectable morphological changes in the cytoplasm of the parasite. In pharmacologically immunosuppressed Sprague-Dawley rats transtracheally infected with a suspension of about 5 x 10(6) P. carinii trophozoites per ml, the daily intraperitoneal administration of bilobalide (10 mg/kg of body weight for 8 days) lowered the number of organisms by approximately 2 logs (that is, about 99%). There was no apparent toxicity either in uninfected HEL 299 feeder cells or in infected and uninfected animals. These studies suggest that the sesquiterpene bilobalide might be useful for therapy of and prophylaxis against P. carinii infections in humans.
Antimicrobial Agents and Chemotherapy 08/1993; 37(7):1492-6. · 4.84 Impact Factor
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Clinical Infectious Diseases 03/1993; 16(2):344. · 9.15 Impact Factor
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ABSTRACT: We report a case of intestinal capillariasis in a 32-year-old Italian man. After he made a trip to Indonesia that lasted approximately one month, he developed heartburn, abdominal pain, irregular bowel movements, headache, fatigue, weight loss, low-grade fever, and severe itching. The diagnosis was provided by the recovery of Capillaria philippinensis eggs in the stool. Treatment with oral albendazole, 200 mg twice a day for 21 days, resulted in clinical and parasitologic cure. This is the first report of C. philippinensis infection acquired in Indonesia.
The American journal of tropical medicine and hygiene 08/1992; 47(1):10-2. · 2.59 Impact Factor
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ABSTRACT: Three autochthonous cases of Entamoeba histolytica infection in institutionalized mentally-retarded patients are reported. Isoenzyme analysis by starch-gel electrophoresis shows the pathogenicity of the three isolates: two belong to zymodeme II, and one to zymodeme XIX. The study shows that invasive E. histolytica strains occur in Italy and can be isolated from institutionalized oligophrenic patients.
Annals of Tropical Medicine and Parasitology 11/1991; 85(5):509-13. · 1.43 Impact Factor
