Diagnosis of visceral leishmaniasis: the sensitivities and specificities of traditional methods and a nested PCR assay.

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.

Multicentre study, in patients with imported malaria, on the sensitivity and specificity of a dipstick test (ICT Malaria P.f./P.v.) compared with expert microscopy.

A prospective, multicentre study was carried out in Italy to assess the sensitivity and specificity of a rapid dipstick test (ICT Malaria P.f./P.v.) in the diagnosis of imported malaria caused by Plasmodium falciparum and other Plasmodium spp. The test is based on the detection of histidine-rich protein-2 (HRP-2) from P. falciparum and 'panmalarial' antigen in peripheral blood. The 241 subjects were international travellers or immigrants from areas where malaria is endemic. When compared with the microscopical examination of bloodsmears (used as the 'gold standard'), the dipsticks were found to be 94.4% sensitive and 94.5% specific for pure infections with P. falciparum. The performance of the tests when used on patients infected with species other than P. falciparum or more than one Plasmodium spp. showed a high degree of variability. Although the dipsticks represent a very simple, rapid, and valuable diagnostic aid, they should not be considered a complete substitute for direct microscopical diagnosis using stained bloodsmears.

Reappearance of human diphyllobothriasis in a limited area of Lake Como, Italy.

BACKGROUND: Very few sporadic cases of autochthonous diphyllobothriasis have been reported in Italy during the past 20 years, probably due to the drastic decrease or disappearance of some fish species, mainly Perca fluviatilis L., known as the intermediate host of the infective larval stage of this tapeworm. However, increasing consumption of raw or undercooked fish during the last years as influenced by Oriental cuisine, played an important role in a number of new single cases of the infection or microepidemics. PATIENTS: We describe six cases which were diagnosed within a period of 4 months, which all originated from the area of Lake Como (Lombardy region). All subjects had previously eaten raw perch fillets. Four individuals complained of gastrointestinal signs and symptoms, whereas two were asymptomatic. DISCUSSION: Parasitological diagnosis was achieved in four cases by recovery of typical operculated eggs in stool specimens; in two others by morphological features of proglottids recovered from feces. Treatment with niclosamide, as a single oral dose, cured the infection in all subjects. This is the first report of this infection in Italy in the last 10 years.

[Pathogenic protozoans in man: differential characterization of Entamoeba histolytica/Entamoeba dispar complex, Acanthamoeba spp., Microsporidia]. / Protozoi patogeni per l'uomo: protocolli di caratterizzazione differenziale di genere e di specie nell'ambito del complesso Entamoeba histolytica/Entamoeba dispar, Acanthamoeba spp., Microsporidi.

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.

In vitro efficacy of nikkomycin Z against the human isolate of the microsporidian species Encephalitozoon hellem.

Since 1985 microsporidia have been recognized as a cause of emerging infections in humans, mainly in immunocompromised human immunodeficiency virus-positive subjects. As chitin is a basic component of the microsporidian infective stage, the spore, we evaluated in vitro the susceptibility of a human-derived strain of Encephalitozoon hellem to nikkomycin Z, a peptide-nucleoside antibiotic known as a competitive inhibitor of chitin synthase enzymes. Transmission electron microscopy showed that this drug, at 25 microgram/ml, reduced the number of parasitic foci by about 35% +/- standard deviation after 7 days of culture (P < 0.0001) and induced cell damage of both mature and immature spores and also other sporogonic and merogonic stages. In particular, an irregular outline of the cell shape and an abnormally condensed cytoplasm in meronts and sporonts were documented. Also, the polar tubule and the polaroplast membranes appeared disarrayed in the sporoblast stage. The spore wall showed an enlarged endospore and delaminated exospore. Mature spores had a complete cytoplasmic disorganization and a swollen and delaminated cell wall. No ultrastructural cell damage was observed in uninfected control cultures treated with the drug.

Intestinal parasitic infections in an institution for the mentally retarded.

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.

Symptomatic and asymptomatic amoebiasis in two heterosexual couples.

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.

Asymptomatic respiratory tract microsporidiosis due to Encephalitozoon hellem in three patients with AIDS.

Microsporidia of the genera Enterocytozoon and Encephalitozoon have been identified as frequent causes of intestinal and disseminated infections, respectively, in patients with AIDS. Even though most subjects infected with these protozoa develop overt disease, simple colonization without illness may occur, as we observed in three severely immunosuppressed patients with AIDS. The parasites, recognized in and isolated from bronchoalveolar lavage sediment specimens, were characterized as Encephalitozoon hellem. Colonization of the bronchial tree was temporary, and treatment with albendazole was not needed to clear the infection.

The relationship of bacterial vaginosis, Candida and Trichomonas infection to symptomatic vaginitis in postmenopausal women attending a vaginitis clinic.

OBJECTIVE: To estimate the prevalence of bacterial vaginosis, Candida albicans, and Trichomonas vaginalis infections in a population of postmenopausal women with symptoms of vaginitis seen at a vaginitis clinic either as self-referred or clinician referred patients. METHODS: A cross-sectional study of 148 postmenopausal women (cases) and 1564 controls of reproductive age attending a vaginitis clinic. C. albicans and T. vaginalis infections were diagnosed by culture techniques. Bacterial vaginosis was diagnosed on the basis of clinical findings. RESULTS: Fifty-six (37.8%) postmenopausal women and 834 (53.3%) controls were diagnosed with T. vaginalis or C. albicans infection, or bacterial vaginosis, or mixed infection (odds ratio (OR) 0.53, 95% confidence interval (CI) 0.37-0.75). C. albicans and T. vaginalis infection were diagnosed in 34.1% (534/1564) and 1.92% (30/1564) of women of childbearing age and in 13.5% (20/148) and 10.8% of postmenopausal women, respectively. (P < 0.05 for both comparisons). The prevalence of bacterial vaginosis was similar between the two groups (14/148 in postmenopausal patients and 210/1564 in controls of reproductive age; P = 0.22). CONCLUSIONS: Among postmenopausal women attending a vaginitis clinic, a defined diagnosis of bacterial vaginosis, C. albicans or T. vaginalis infection can be made in about one third of such patients. Concerning the two thirds of symptomatic women lacking such a microbiologic diagnosis, alternative causes (e.g., estrogen deficiency, nonanaerobic bacterial infections, local irritants or allergenes, and dermatologic conditions) need to be considered.

Pulmonary microsporidiosis due to Encephalitozoon hellem in a patient with AIDS.

The microsporidian Encephalitozoon hellem is being reported with increasing frequency in HIV-positive subjects, as an agent of disseminated microsporidiosis without involving the gastrointestinal tract. We describe a case of pulmonary microsporidiosis in a 27-year-old Italian man with AIDS who developed fever, cough, and dyspnea. A chest X-ray showed multiple bilateral pulmonary opacities and mediastinal lymph-node enlargement. Stained smears of bronchoalveolar lavage sediment showed oval structures consistent with microsporidian spores. Viral, bacterial and fungal cultures were repeatedly negative, whereas microsporidia were successfully cultured in human and bovine fibroblast cell lines. Analysis of electron micrographs indicated that the isolate belonged to the genus Encephalitozoon. Based on further immunological, biochemical and molecular studies it was characterized as E. hellem. Even though a temporary improvement with albendazole therapy was noticed, the patient deteriorated clinically and died of severe respiratory distress.

Isolation and identification of Encephalitozoon hellem from an Italian AIDS patient with disseminated microsporidiosis.

Microsporidia are primitive mitochondria-lacking spore-forming eukaryotic protozoa that infect a wide variety of animals and also humans. Of the five genera (Encephalitozoon, Enterocytozoon, Septata, Nosema and Pleistophora) that cause infections in humans, Enterocytozoon bieneusi, Septata intestinalis, and Encephalitozoon hellem are being increasingly identified in patients with acquired immunodeficiency syndrome (AIDS). E. bieneusi causes gastrointestinal disease, S. intestinalis causes gastrointestinal and disseminated disease, and E. hellem causes ocular as well as disseminated disease. We have established in continuous culture a strain of microsporidia isolated from the urine and throat washings of an Italian AIDS patient and identified it as Encephalitozoon hellem, based on its ultrastructural morphology, antigenic pattern, and polymerase chain reaction-amplified small subunit ribosomal RNA. We believe that this is the first time that a strain of microsporidia has been isolated from the throat washings of a patient with microsporidiosis.

HIV-1 and parasitic infections in rural Tanzania.

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.

Intestinal capillariasis (Capillaria philippinensis) acquired in Indonesia: a case report.

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.

Naegleria australiensis ssp. italica: experimental study in mice.

A subspecies of Naegleria australiensis, N. australiensis italica, pathogenic for mice, was recently isolated and identified from an Italian thermal spa. We describe the histopathological changes of the central nervous system with experimental infection of albino mice. The histopathological patterns are intermediate to those seen with infection caused by N. fowleri and N. australiensis or Acanthamoeba spp. An acute inflammatory reaction was present within the choroid plexus, ependyma, midbrain, cerebellum, and basal ganglia. Occasional single amebic trophozoites were found within some microabscesses. Cysts were not identified. Involvement of the olfactory neuroepithelium and of the nasal mucosa was not detected.

An in vitro comparative study on the effect of amphotericin B, econazole, and 5-fluorocytosine on Naegleria fowleri, Naegleria australiensis, and Naegleria australiensis s.sp. italica.

We tested in vitro amphotericin B (AMP-B), econazole (ECO), and 5-fluorocytosine (5-FC) on pathogenic Naegleria fowleri (KUL strain), Naegleria australiensis s.sp. italica (AB-T-F3, original strain) to assess their sensitivity to chemotherapeutic compounds. Previous reports have shown the polyene antibiotic AMP-B to be the most active agent. It was, however, much more active on N. fowleri than on N. australiensis and N. australiensis s.sp italica. 5-FC and ECO gave rise to non appreciable effect at non-toxic corresponding dosages in vivo. The results of these in vitro tests are discussed.

Pathogenic and non-pathogenic Naegleria and Acanthamoeba spp.: a new autochthonous isolate from an Italian thermal area.

We performed an epidemiological survey of 17 thermal baths and the same number of mud-basins. This study aimed to ascertain the presence and incidence of small free-living amoebae, particularly species and/or strains of Naegleria and Acanthamoeba spp., occasional etiological agents of fatal meningoencephalitis and/or ocular infections in man. Over 51 samples of water and mud incubated at 37 degrees C and at 45 degrees C, 34 (66.7%) became positive at 37 degrees C and 33 (64.7%) at 45 degrees C. We isolated 7 (6%) strains of Naegleria spp., 6 (5.2%) of Acanthamoeba spp., 39 (33.6%) of Vahlkampfia spp., 28 (24.1%) of Hartmannella spp. and 36 (31.1%) strains of other species of free-living amoebae. 4 strains of Naegleria spp. and 6 of Acanthamoeba spp. proved pathogenic both in vivo, after experimental infection (meningoencephalitis) in the albino mouse, and in vitro, having previously contaminated monolayers of Vero cell line (cytopathic effect). Within the isolated pathogenic strains of Naegleria spp., a new strain of N. australiensis s.sp. italica was typified from an immunochemical point of view. This should be added to previous isolations reported by us.

[First isolation in Italy of Naegleria australiensis (De Jonckheere, 1981)]. / Primo isolamento in Italia di Naegleria australiensis (De Jonckheere, 1981).

An epidemiological investigation was carried out in Northern Italian spa to detect presence and incidence of free-living amoebae, mostly belonging to amphizoic species Acanthamoeba and Naegleria. Seven pathogenic strains of Naegleria spp. were isolated. Morpho-biological characterization and indirect immunofluorescence assays showed that all these strains belong to N. australiensis, a new pathogenic species. Such a species could induce, along with N. fowleri and Acanthamoeba spp., fatal meningo-encephalitis in man and other mammals.