In this article, we report the case of a 16-month-old German boy who was admitted to the Children's Hospital of Stuttgart with a 4-week history of intermittent fever, decreased appetite, weakness, fatigue, and difficulty sleeping. He was healthy at birth and remained so for the first 15 months of his life. On admission, physical examination showed enlarged cervical, axillary, and inguinal lymph nodes, as well as hepatosplenomegaly. Laboratory data revealed pancytopenia, elevated liver function tests, and hypergammaglobulinemia. Blood, stool, and urine culture results were negative. Viral infections and rheumatologic and autoimmune disorders were ruled out, but a positive titer for Leishmania antibodies was noted. In a liver and bone marrow biopsy, the amastigote form of the parasite could not be seen in cells. The promastigote form of Leishmania was found and the diagnosis of visceral leishmaniasis was made by combining the cultures of both the liver and the bone marrow biopsy material in 5 mL 0.9% saline on brain heart infusion agar, supplemented with defibrinated rabbit blood and incubated at 25 to 26 degrees C for 5 days. The parasite was identified by Southern blot analysis as Leishmania infantum. Specific therapy with the antimonial compound sodium stibogluconate with a dose of 20 mg/kg body weight was begun immediately. Within 4 days, the patient became afebrile. The side effects of treatment, including erosive gastritis, cholelithiasis, worsening hepatosplenomegaly, elevation of liver enzymes, pancreatitis, and electrocardiogram abnormalities, necessitated the discontinuation of treatment after 17 days. On discharge 4 weeks later, the patient was stabilized and afebrile with a normal spleen, normal complete blood count, normal gammaglobulins, and decreasing antibody titers to Leishmania. During the next 24 months, the patient experienced intermittent episodes of abdominal pain, decreased appetite, recurrent arthralgia, and myalgia. But at his last examination in January 1998, he was well; all symptoms mentioned above had disappeared. Because the child had never left Germany, nonvector transmission was suspected and household contacts were examined. His mother was the only one who had a positive antibody titer against Leishmania donovani complex. She had traveled several times to endemic Mediterranean areas (Portugal, Malta, and Corse) before giving birth to the boy. But she had never been symptomatic for visceral leishmaniasis. Her bone marrow, spleen, and liver biopsy results were within normal limits. Culture results and polymerase chain reaction of this material were negative. A Montenegro skin test result was positive, indicating a previous infection with Leishmania. Western blot analysis showed specific recognition by maternal antibodies of antigens of Leishmania cultured from the boy's tissue. Visceral leishmaniasis is endemic to several tropical and subtropical countries, but also to the Mediterranean region. It is transmitted by the sand fly (Phlebotomus, Lutzomyia). Occasional nonvector transmissi...

The prevalence of intestinal microsporidiosis among human immunodefiency virus (HIV)-infected persons with chronic diarrhea varies from 7% to 50%; thus, microsporidia are a significant source of morbidity and, occasionally, mortality among these patients. Anecdotal reports suggest that intestinal microsporidiosis is also an important infection in patients with AIDS in Germany. To determine the prevalence of microsporidiosis among HIV-infected patients in Germany, we performed a prospective coprodiagnostic study of 97 consecutive HIV-infected patients. Microsporidia were the most common enteropathogen identified in 18 (36.0%) of 50 patients with diarrhea and 2 (4.3%) of 47 patients without diarrhea (P < .001; chi2 test). Microsporidia were present in 60% of patients with chronic diarrhea and 5.9% of patients with acute diarrhea. The etiologic agent was Enterocytozoon bieneusi in 18 patients and Encephalitozoon intestinalis in two patients. The prevalence of intestinal microsporidiosis in this cohort of German patients with AIDS and diarrhea is one of the highest to be reported anywhere in the world. Microsporidiosis seems to represent one of the most important causes of diarrhea in HIV-infected patients in Germany and thus must be considered in the differential diagnosis for all AIDS patients presenting with diarrhea.

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In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for species-specific detection of Encephalitozoon cuniculi. Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Enterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected with Enterocytozoon bieneusi (n = 9), Encephalitozoon spp. (n = 2), and Encephalitozoon intestinalis (n = 1) as well as stool spiked with spores of Encephalitozoon cuniculi and Encephalitozoon hellem and tissue cultures of Encephalitozoon cuniculi and Encephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected with Encephalitozoon cuniculi and Encephalitozoon hellem. Moreover, identification of Encephalitozoon spp. could be specified as Encephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates species-specific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.

Enterocytozoon bieneusi is emerging as an important cause of chronic diarrhoea in AIDS patients. Its reservoirs and transmission patterns are unknown. In this study, we have examined E. bieneusi sequences from four Rhesus macaques of different origin, which were kept at one animal facility. The sequences were identical in all animals, which suggested that infection had occurred within the facility. Full sequence agreement of E. bieneusi from macaques was found with an E. bieneusi genotype that occurs frequently in humans. To clarify, the relevance of possible inter-species transmission from man to macaque, a phylogenetic analysis was conducted including all sequences of E. bieneusi deposited in GenBank. The hitherto used system of diverse nomenclatures could be reduced to an outlier group and three main lineages, one of which could be further sub-divided into five subgroups. Based in this phylogeny, an association of parasites and host species could be observed for main lineages 2 and 3, as well as for most of the subgroups of main lineage 1. For confirmation, the phylogeny of main lineage 1 was reconstructed with an alternative method of distance estimation, yielding essentially the same parasite-host associations. Zoonotic potential of E. bieneusi is thus supported on a phylogenetic basis.

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. helleri, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Examination of 54 Triatoma infestans from a village near the European Southern Observatory La Silla in Chile and of 9 Triatoma spinolai from the territory of the observatory showed that 10 T. infestans were infected with trypanosomatids. Mice were infected with in vitro cultures initiated with five different trypanosomatid isolates and treated with the immunosuppressive drug cyclophosphamide to increase the parasitemia of the flagellates. Evidence of the presence of T. cruzi was provided by a comparative biometrical analysis of blood trypomastigotes and the occurrence of intracellular amastigotes. Three methods for further identification were used: examination of kDNA ultrastructure, disc electrophoresis of soluble proteins and the Aaptos papillata II lectin induced agglutination. We obtained the following results for all isolates: (1) presence of a central band of the kDNA; (2) T. cruzi specific double bands of the protein patterns; (3) positive reaction with Aaptos papillata II. No differences between the five isolates from Chile and T. cruzi or T. cruzi-like strains from other countries could be observed. Based on these results an infection of the bugs with T. rangeli and T. conorhini could be excluded.