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Background Babesia infection is caused by intraerythrocytic tick-borne parasites. Cases of transfusion-transmitted babesiosis have been increasingly recognized. To date, no Babesia test has been licensed for screening US blood donors. We conducted a longitudinal study to assess the course and markers of Babesia infection among seropositive donors identified in a seroprevalence study. Study Design and Methods Eligible donors had B. microti indirect fluorescent antibody (IFA) titers of 64 or greater. Enrollees were monitored up to 3 years, by IFA and three methods for evidence of parasitemia: B. microti nested polymerase chain reaction (PCR) analysis (at two laboratories), hamster inoculation, and blood-smear examination. Results Among 115 eligible donors, 84 (73%) enrolled. Eighteen enrollees (21%) had evidence of parasitemia for 30 total specimens (17% of 181), which were collected in 9 different months and tested positive by various approaches: PCR (25 specimens/16 persons), hamster inoculation (13 specimens/8 persons), and blood smear (one specimen positive by all three approaches). Overall, 14 persons had one or more specimen with positive PCR results at both laboratories (12 persons) and/or had parasitologically confirmed infection (eight persons). Three of nine persons who had more than one specimen with evidence of parasitemia had nonconsecutive positives. Several enrollees likely had been infected at least 1 year when their last positive specimen was collected. The final three specimens for seven persons tested negative by all study methods, including IFA. Conclusion Seropositive blood donors can have protracted low-level parasitemia that is variably and intermittently detected by parasitologic and molecular methods. Donor-screening algorithms should include serologic testing and not solely rely on molecular testing.

Resumen Las tecnicas de diagnostico molecular por PCR permiten distinguir entre las diferentes especies de Cryptosporidium morfologicamente identicas capaces de infectar a humanos. De las 23 especies actualmente reconocidas en el genero, al menos 9 son capaces de infectar a humanos. Por ello, y debido a que la intensidad de las manifestaciones clinicas, la patogenicidad, la excrecion de ooquistes y la incidencia varian entre ellas, la realizacion de estudios moleculares es crucial para una mejor comprension de la epidemiologia de la criptosporidiosis humana. En el presente trabajo se analizan muestras procedentes de 2 estudios independientes: uno formado por 23 muestras procedentes de Madrid y otro compuesto por 72 muestras procedentes de La Coruna, todas ellas positivas para Cryptosporidium spp. por metodos microscopicos y pertenecientes a casos aislados de criptosporidiosis. Para la identificacion a nivel de especie se utilizaron las regiones de diagnostico descritas para el ADNr 18S y las regiones de diagnostico del gen de la COWP. De las 95 muestras analizadas, se consiguio extraer y amplificar ADN en 77 casos, en los que las especies causantes de la infeccion fueron: C. parvum (40 casos: 2 Madrid y 38 La Coruna), C. hominis (30 casos: 10 Madrid y 20 La Coruna) y C. meleagridis (2 casos: uno Madrid y uno La Coruna). En otros 5 casos fue imposible detectar la especie responsable de la infeccion, aunque se confirmara su positividad por PCR (4 Madrid y uno La Coruna). Los genotipos aislados en estos pacientes se correlacionaron con los hallados en animales de las mismas regiones.

BACKGROUND: Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions. CASE REPORT: The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donora 67-year-old California residenthad a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008. CONCLUSIONS: This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.

The Phylum Microsporidia comprises >1,200 species, only 15 of which are known to infect humans, including the genera Trachipleistophora, Pleistophora, and Brachiola. We report an infection by Tubulinosema sp. in an immunosuppressed patient.

Isoenzyme analysis of cultured parasites is the conventional approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed PCR generic primers to amplify a segment of the rRNA internal transcribed spacer 2 (ITS2) from multiple Leishmania species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR followed by DNA sequencing analysis) with those from the parasitologic approach ( in vitro culture followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of eight Leishmania species were identified. The species results were concordant for all but two patients: for one patient, the results were Leishmania ( Viannia ) guyanensis by the molecular approach versus L. (V.) braziliensis by the parasitologic approach; for the other patient, the results were L. ( Leishmania ) tropica versus L. (L.) major , respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among Leishmania species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens.

Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia, designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri. The isolate grew at 33 degrees C, 37 degrees C, 40 degrees C, and 42 degrees C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli, the usual food source of Paravahlkampfia ustiana, the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae.

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5%); seven (64%) acquired HIV through male-to-male sexual contact, two (18%) through male-to-male sexual contact and injection drug use, and one (9%) through heterosexual contact; one (9%) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/µL (range 3 to 319 cells/µL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16%. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts. Para determinar a prevalência de microsporidiose intestinal em pacientes infectados pelo HIV foi realizado um estudo prospectivo em três hospitais dos Estados Unidos da América do Norte (EUA) e analizada uma base de dados nacional composta de dados coletados de pacientes infectados pelo HIV em 10 cidades dos EUA. De um total de 737 amostras de fezes de pacientes infectados pelo HIV que apresentavam diarréia, amostras de 11 pacientes (prevalência de 1,5%) foram positivas para microsporídios. Todos os positivos eram do sexo masculino e, entre eles, sete (64%) pacientes adquiriram a infecção pelo HIV através de relação homossexual, dois (18%) através de relação sexual e drogas injetáveis e um (9%) através de contato heterosexual, enquanto que em um paciente o modo de transmissão do HIV não foi determinado. A contagem média de linfócitos CD4 realizada até seis meses do diagnóstico de microsporidiose foi de 33 células/microlitro (3 a 319 células/microlitro). A análise da base de dados nacional (n = 24.098) mostrou uma prevalência de microsporidiose de 0,16%. A prevalência de microsporidiose em pacientes HIV-positivos com diarréia é baixa. Entretando, como a microsporidiose é mais frequentemente diagnosticada em pacientes com contagens de CD4 muito baixas, a indicação de pesquisa de microsporídios é justificada, especialmente para estes pacientes.

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.

Most reported U.S. zoonotic cases of babesiosis have occurred in the Northeast and been caused by Babesia microti. In Washington State, three cases of babesiosis have been reported previously, which were caused by WA1 (for “Washington 1”)-type parasites. We investigated a case of babesiosis in Washington in an 82–year-old man whose spleen had been removed and whose parasitemia level was 41.4%. The complete 18S ribosomal RNA gene of the parasite was amplified from specimens of his whole blood by polymerase chain reaction. Phylogenetic analysis showed the parasite is most closely related, but not identical, to B. divergens (similarity score, 99.5%), a bovine parasite in Europe. By indirect fluorescent-antibody testing, his serum reacted to B. divergens but not to B. microti or WA1 antigens. This case demonstrates that babesiosis can be caused by novel parasites detectable by manual examination of blood smears but not by serologic or molecular testing for B. microti or WA1-type parasites.

This study reports the molecular and morphologic characterization of a Cryptosporidium sp., identified in stools of captive lemurs Propithecus verreauxi coquereli. Stool samples were collected from seven animals (n=7) presenting episodes of diarrhea. Bright-field light microscopy of stool smears stained with modified acid-fast technique revealed the presence of Cryptosporidium sp. oocysts in four of the stool samples analyzed. All microscopically positive samples were confirmed by PCR using primers designed to amplify DNA fragments from two independent loci, i.e. the Cryptosporidium oocyst wall protein (COWP) gene and the small subunit ribosomal RNA (ssrRNA) gene. Phylogenetic analysis based on the full-length ssrRNA gene placed this isolate within a clade that contains all currently known C. parvum species/genotypes, closely related to the C. parvum pig genotype. Comparison with partial ssrRNA sequences available in the GenBank™ revealed 100% sequence identity with the genotype previously identified in Canadian patients. This finding was confirmed further by comparison of the COWP gene partial sequences.

Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum = 80% and G. lamblia = 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITC-conjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of human-infectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.

Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.

BACKGROUND : Babesiosis is a tick-borne zoonosis caused by intraerythrocytic protozoa. More than 40 US cases of Babesia microti infection acquired by blood transfusion have been reported. This report describes the identification of a transfusion-associated case of babesiosis and the subsequent identification of the infected blood donor and three other infected recipients of cellular blood components from three other donations by this donor. STUDY DESIGN AND METHODS : Serum specimens from the donors of blood that had been made into cellular components received by the index recipient and from other recipients of such components from the implicated donor were tested by the indirect fluorescent antibody (IFA) assay for antibodies to B. microti. Whole blood from IFA-positive persons was tested by PCR for B. microti DNA. RESULTS : IFA testing of serum from 31 of 36 donors implicated a 45-year-old man (titer, 1 in 256), whose donation had been used for RBCs. He likely became infected when bitten by ticks while camping in Minnesota in June 1999 and had donated blood four times thereafter. As demonstrated by PCR, he remained parasitemic for at least 10 months. Of the five other surviving recipients of cellular blood components from the implicated donor, three recipients (one for each of the three other donations) had become infected through either RBC or platelet transfusions. CONCLUSIONS : Babesiosis should be included in the differential diagnosis of posttransfusion febrile illness, and effective means for preventing transmission by blood transfusion are needed.

Summary Background Investigators have reported that patients infected with Pneumocystis carinii containing mutations in the DHPS (dihydropteroate synthase) gene have a worse outcome than those infected with P carinii containing wild-type DHPS . We investigated patients with HIV-1 infection and P carinii pneumonia to determine if DHPS mutations were associated with poor outcomes in these patients. Methods We compared presence of mutations at the DHPS locus with survival and response of patients to co-trimoxazole or other drugs. Findings For patients initially given co-trimoxazole, nine (14%) of 66 with DHPS mutant died, compared with nine (25%) of 36 with wild type (risk ratio=0·55 [95% CI=0·24–1·25]; p=0·15). Ten (15%) of 66 patients with a DHPS mutant did not respond to treatment, compared with 13 (36%) of 36 patients with the wild type (0·42 [0·20–0·86]; p=0·02). For patients aged 40 years or older, four (14%) of 29 with the mutant and nine (56%) of 16 with the wild type died (0·25 [0·09–0·67]; p=0·005). Interpretation These results, by contrast with those of previous studies, suggest that patients with wild-type P carinii do not have a better outcome than patients with the mutant when given co-trimoxazole. Our results suggest that presence of a DHPS mutation should be only one of several criteria guiding the choice of initial drug treatment of P carinii pneumonia in patients with HIV-1 infection.

SUMMARYInvestigations of queen, worker and male bumble bees (Bombus terrestris) showed that all individuals became infected with Nosema bombi. Infections were found in Malpighian tubules, thorax muscles, fat body tissue and nerve tissue, including the brain. Ultrastructural studies revealed thin walled emptied spores in host cell cytoplasm interpreted as autoinfective spores, besides normal spores (environmental spores) intended for parasite transmission between hosts. The nucleotide sequence of the gene coding for the small subunit rRNA (SSU-rRNA) from Microsporidia isolated from B. terrestris, B. lucorum, and B. hortorum were identical, providing evidence that N. bombi infects multiple hosts. The sequence presented here (GenBank Accession no AY008373) is different from an earlier submission to GenBank (Accession no U26158) of a partial sequence of the same gene based on material collected from B. terrestris. It still remains to be investigated if there is species diversity among Microsporidia found in b...

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.

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 μm; range, 3.5 to 5.0 μm), 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.

We demonstrate, based on the light, electron microscopic, and immunofluorescence studies carried out on two isolates of Encephalitozoon cuniculi established in culture, that E. cuniculi exhibits di-, tri-, tetra- and octosporous sporogony. We therefore propose that the generic characters of Encephalitozoon should be amended to include tetra-sporous sporogony as generic features. Additionally, the molecular phylogenetic analysis indicates that E. cuniculi, E. hellem, and E. (Septata) intestinalis form a cohesive group.

Background : Polymerase chain reaction (PCR) detection of intestinal protozoa in fecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this study we describe a novel method for DNA extraction from such specimens containing spores and oocysts of Enterocytozoon bieneusi and Cryptosporidium parvum , respectively. Methods and Results : Extraction was done using commercial kits modified to maximize the recovery and purity of extracted DNA. In comparison with a procedure we previously reported, we estimate that this method may increase the sensitivity of parasite DNA detection in fecal specimens up to tenfold. An additional advantage of this method is that up to 12 samples may be processed simultaneously within 2 hours. Conclusions : By using this method, we were able to increase reproducibility of PCR amplification on fecal specimens and significantly reduce the hands-on time required to process the samples.

Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species. This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using "quick-hot" Gram-chromotrope stain. None of the 10 samples reacted with anti-E. hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.

Intestinal microsporidiosis has been associated traditionally with severely immunocompromised patients with AIDS. We describe two new cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in human immunodeficiency virus-negative adults. Both patients presented with chronic nonbloody diarrhea, and one had intestinal lymphangiectasia as well. Intestinal microsporidiosis was diagnosed by evaluation of stool samples, and the specific species was determined by use of polymerase chain reaction (PCR) in duodenal biopsy specimens. To our knowledge, this is the first report of confirmation of E. bieneusi in the intestinal epithelium of HIV-negative individuals by use of PCR in duodenal biopsy specimens. Cases of intestinal microsporidiosis in HIV-negative individuals reported in the English-language literature are reviewed. These two new cases along with those described previously corroborate the need to evaluate for microsporidia in HIV-negative individuals with unexplained diarrhea.

Microsporidia are ancient, intracellular, eukaryotic protozoan parasites that form spores and that lack mitochondria. Currently, as many as eight species included under six genera are known to infect humans, mostly patients with AIDS. Among these, Enterocytozoon bieneusi , the agent of gastrointestinal (GI) disease, is the most frequently identified microsporidian in clinical laboratories in the United States. Encephalitozoon ( Septata ) intestinalis , the agent that causes a disseminated infection including infection of the GI tract, is the second most frequently identified microsporidian parasite. In spite of this, not many isolates of E. intestinalis have been established in culture. We describe here the continuous cultivation of eight isolates of E. intestinalis obtained from different samples including the urine, sputum, and duodenal aspirate or biopsy specimens from five AIDS patients originating from California, Colorado, and Georgia. The specific identification was made on the bases of ultrastructural, antigenic, and PCR analyses.

We compared a nested PCR assay and microscopic examination of Giemsa-stained blood films for detection and identification of Plasmodium spp. in blood specimens. PCR was more sensitive than microscopy and capable of identifying malaria parasites at the species level when microscopy was equivocal.

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.

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.

Background: The microsporidian Septata intestinalis, recently suggested to be reclassified as Encephalitozoon intestinalis, is probably the second most common microsporidian isolated from AIDS patients after Enterocytozoon bieneusi. S. intestinalis causes a disseminated disease, including infections of the gastointestinal tract, whereas E. bieneusi is confined strictly to the gastrointestinal tract. It is important to differentiate between these two microsporidians, as only infections caused by S. intestinalis can, at this time, be effectively treated. Currently, diagnosis of infections caused by S. intestinalis can be achieved only by transmission electron microscopy. Methods and Results: In this study are described specific polymerase chain reaction primers for diagnosis of S. intestinalis infections based on the region coding for the small subunit ribosomal RNA cloned from a S. intestinalis isolate. These primers were tested for specificity on cloned ribosomal RNA sequences of different species of microsporidia, as well as on cultured samples of E. bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem and Vittaforma corneae (Nosema corneum), without showing any cross-amplification. By use of these polymerase chain reaction primers, eight different microsporidian isolates grown in culture and one diagnostic sample, collected as an ethanol-fixed duodenal-jejunal segment, were identified as S. intestinalis. Conclusion: These primers are powerful diagnostic tools and can enhance or replace traditional methods used to diagnose this microsporidian.

Babesia infection is caused by intraerythrocytic tick-borne parasites. Cases of transfusion-transmitted babesiosis have been increasingly recognized. To date, no Babesia test has been licensed for screening US blood donors. We conducted a longitudinal study to assess the course and markers of Babesia infection among seropositive donors identified in a seroprevalence study.Eligible donors had B. microti indirect fluorescent antibody (IFA) titers of 64 or greater. Enrollees were monitored up to 3 years, by IFA and three methods for evidence of parasitemia: B. microti nested polymerase chain reaction (PCR) analysis (at two laboratories), hamster inoculation, and blood-smear examination.Among 115 eligible donors, 84 (73%) enrolled. Eighteen enrollees (21%) had evidence of parasitemia for 30 total specimens (17% of 181), which were collected in 9 different months and tested positive by various approaches: PCR (25 specimens/16 persons), hamster inoculation (13 specimens/8 persons), and blood smear (one specimen positive by all three approaches). Overall, 14 persons had one or more specimen with positive PCR results at both laboratories (12 persons) and/or had parasitologically confirmed infection (eight persons). Three of nine persons who had more than one specimen with evidence of parasitemia had nonconsecutive positives. Several enrollees likely had been infected at least 1 year when their last positive specimen was collected. The final three specimens for seven persons tested negative by all study methods, including IFA.Seropositive blood donors can have protracted low-level parasitemia that is variably and intermittently detected by parasitologic and molecular methods. Donor-screening algorithms should include serologic testing and not solely rely on molecular testing.

Summary A phylogenetic analysis of the 16S-like ribosomal RNA coding regions from Labyrinthuloides haliotidis, Developayella elegans, Proteromonas lacertae and other organisms corroborates morphological evidence that proteromonads and other eukaryotes with tripartite tubular hairs form a monophyletic group of organisms, the stramenopiles. Within the stramenopiles, the heterotrophic groups (proteromonads, Labyrinthulida, bicosoecids, Developayella and oomycetes) diverge before the radiation of the “heterokont algae”, the autotrophic stramenopiles. The stramenopiles were initially “protozoan” but their ecological success is largely attributable to the late symbiotic acquisition of chloroplasts. The stramenopiles and other taxa with chlorophyll a+c containing chloroplasts (cryptomonads, dinoflagellates, and haptophytes) do not share a common autotrophic ancestor. These photosynthetic assemblages acquired their plastids independently.

Summary Based on light microscopic and ultrastructural characteristics as well as on the nucleotide sequence of the small subunit ribosomal RNA coding region, the microsporidium Nosema ceranae n. sp., a parasite of the Asian honey bee Apis cerana is described. Merogonial stages and sporonts are diplokaryotic. Merozoites are mostly formed by cytoplasmic fission in quadrinucleate meronts and the number of merogonial cycles may vary. The sporogony is disporoblastic. The living mature spore is ovocylindrical, straight to slightly curved and measures 4.7 × 2.7 μm whereas fixed and stained spores measure 3.6 × 1.7 μm. The polar filament is isofilar with a diameter of 96–102 nm and is arranged in 20–23 coils in the posterior and mid-part of the spore. In the anterior part of the polaroplast there are closely packed approximately 11 nm thick lamellae. The lamellae of the posterior polaroplast are thicker and less regular. In the posterior part of the mature spore a well fixed posterior body interpreted as a posterosome was often observed. Phylogenetic analysis, based on the sequence of the small subunit ribosomal RNA, places Nosema ceranae in the Nosema clade, as defined by Nosema bombycis , the type species of the Nosema genus.

We analyzed the small subunit ribosomal RNA (SSUrRNA) gene sequences from 13 malaria species parasitic to humans, chimpanzees/gorillas, Old World monkeys, rodents, birds, and lizards in order to reconstruct the phylogenetic relationships among the Plasmodium species. The SSUrRNA genes of Plasmodium vivax and P. ovale were sequenced by the dideoxy method in our laboratory; other sequences were retrived from GenBank. These sequences were aligned with the SSUrRNA gene sequence of outgroup species, Paramecium and Toxoplasma. After gaps and ambiguous regions were deleted, the aligned sequences were used for phylogenetic analysis by maximum likelihood and distance methods. The tree defines two major clades, the first with the bird and reptile parasites, the second with the rest of the species. The two bird parasites, P. gallinaceum and P. lophurae, do not closely cluster with human, chimpanzee/gorilla, Old World monkey, or rodent parasites, but cluster with the lizard parasites. P. vivax clusters with three Old World monkey parasites, P. cynomolgi, P. fragile, and P. knowlesi in decreasing order of closeness. P. ovale, while in a separate clade, is more closely related to P. vivax than to P. malarie or P. falciparum. P. malariae and P. berghei do not closely cluster with any of the other clades or with each other. Statistical analysis proves that the placement of P. falciparum in the bird malaria clade is less likely than in the mammalian malaria clade. Our analysis reveals that: (1) human malaria parasites have an evolutionary independent origin; (2) P. falciparum is most closely related to P. reichenowi and did not arise from lateral transfer of a bird parasite, as was previously suggested; and (3) the lizard malaria parasites are true members of the genus Plasmodium.

The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.

Microsporidian spores were identified, on the basis of Weber's staining, in urine, stool, nasal, and saliva samples of an AIDS patient with diarrhea, hematuria, dysuria, and dementia. Urine and stool samples contained numerous spores, whereas few spores were seen in the nasal and saliva samples. Spores were concentrated from urine samples and inoculated into monkey kidney cell (E6) monolayers. After 6 to 8 weeks of culture, infected E6 cells filled with spores as well as spores free in the culture supernatants were seen daily. Transmission electron microscopy revealed that all stages of the parasite (CDC:V297) developed within septated, honeycomb-shaped parasitophorous vacuoles. Indirect immunofluorescence and immunoblotting studies using rabbit anti-Encephalitozoon cuniculi, anti-Encephalitozoon hellem, and anti-CDC:V297 sera revealed that CDC:V297 reacted intensely with the homologous serum but minimally with the heterologous sera. DNA isolated from CDC:V297, when PCR amplified with E. hellem and E. cuniculi primers, did not produce the diagnostic bands of approximately 547 and approximately 549 bp characteristic of E. hellem and E. cuniculi, respectively. On the basis of these studies, we concluded that CDC:V297 fits the description of Septata intestinalis (A. Cali, D. P. Kotler, and J. M. Orenstein, J. Eukaryot, Microbiol. 40:101-112, 1993).

Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.

SUMMARYNosema apis has been found on all continents where there is beekeeping using Apis mellifera. However, there are few data on the prevalence and impact of Nosema apis in honey bee colonies in tropical climates and it may be uncertain if all records of microsporidia in honey bees actually are records of the same parasite. Also, the development of N. apis has not been documented in tropical races of honey bees. We sampled honey bees from five different colonies in two apiaries on a weekly basis for a full year in Zimbabwe and investigated the samples for N. apis. The development of the parasite was monitored by infection experiments. The gene sequence of the 16S small subunit ribosomal RNA gene of a Zimbabwean isolate of microsporidia from honey bees was sequenced and compared to sequences for N. apis registered in GenBank. The molecular results demonstrate that the Zimbabwean isolates of microsporidia are N. apis. Sampling results show that N. apis may occur at high levels of prevalence at colony leve...

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions.

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.

Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions.The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008.This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.

There is a need for the development of methods for the detection of Cryptosporidium in environmental samples. Two assays which detect the DNA of this parasite were developed and compared to the traditional method of detection, immunofluorescence assay (IFA). Oligonucleotide primers hybridizing to small subunit ribosomal RNA coding sequences were used in the polymerase chain reaction to develop highly specific detection of Cryptosporidium species. The PCR assay could detect about 20-100 oocyst equivalents in clean samples, but the sensitivity was reduced in some environmental samples. Detection based on direct hybridization of repetitive DNA probes from C. parvum was also accomplished, but the assay was not as sensitive as PCR or IFA and yielded false positive signals with environmental samples.

For behavioral research and due to growing ecotourism, some populations of free-ranging mountain gorillas (Gorilla gorilla beringei) have become habituated to humans. Molecular analysis of two Cryptosporidium sp. oocyst isolates originating from two human-habituated gorilla groups and two oocyst isolates from non-habituated gorillas yielded positive identification of C. parvum Genotype 2 (G2; i.e., "cattle", "animal-adapted", or "zoonotic"). As G2 is cross-transmissible between humans and animals, C. parvum infections can be propagated in the habitats of human-habituated, free-ranging gorillas through both zoonotic and anthroponotic transmission cycles.