Babesia microti and Borrelia bissettii transmission by Ixodes spinipalpis ticks among prairie voles, Microtus ochrogaster, in Colorado.

An endemic transmission cycle of Babesia microti was discovered in Colorado in the foothills of the Rocky Mountains. B. microti were found by PCR in 4 of 25 Ixodes spinipalpis tick pools tested (a 3.2 % minimum infection rate) and in 87% (13 of 15) of Microtus ochrogaster (the prairie vole) spleen and blood samples. Using naturally infected I. spinipalpis collected from wild-caught M. ochrogaster as vectors, B. microti and Borrelia bissettii were successfully transmitted to laboratory-born M. ochrogaster. Neither I. spinipalpis, nor M. ochrogaster (the prairie vole) have been previously reported as a vector or a reservoir host of B. microti. Unlike the east coast of the United States where Peromyscus leucopus is an important reservoir for B. microti, evidence for Peromyscus spp. (neither P. maniculatus nor P. difficilis) as B. microti reservoirs was not found in this study.

New cryptosporidium genotypes in HIV-infected persons.

Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes.

Fast and reliable extraction of protozoan parasite DNA from fecal specimens.

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.

Ultrastructure, immunofluorescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS.

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.

Diagnosis of Enterocytozoon bieneusi (microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for small-subunit ribosomal RNA.

OBJECTIVE: Enterocytozoon bieneusi is the most prevalent microsporidian causing chronic diarrhea in patients with acquired immunodeficiency syndrome. The current methods used for routine diagnosis of infections caused by microsporidia are based on microscopic detection of the microorganism spores in stained smears. We evaluated the usefulness of the polymerase chain reaction (PCR) technique as a tool to diagnose Enterocytozoon bieneusi infections, using the species-specific diagnostic primer pair EBIEF1/EBIER1 on stool samples that were also analyzed by optical microscopy. DESIGN: To perform PCR in such samples, we developed a novel protocol to obtain DNA free of PCR inhibitors. This protocol was based on disruption of spores using glass beads and overnight digestion with proteinase K; final purification was accomplished with the RapidPrep Micro Genomic DNA isolation Kit for Cells and Tissues (Pharmacia Biotech Inc, Piscataway, NJ). We also evaluated this approach on aliquots of a sample fixed in formalin from 1 to 10 days. PATIENTS AND SAMPLES: We evaluated the PCR technique on 64 stool samples obtained from patients with acquired immunodeficiency syndrome who had persistent chronic diarrhea. Patients were from Spain, Brazil, Germany, and the United States. RESULTS: Using this approach, we could confirm the presence of E bieneusi in all 17 positive samples; no false-positive results were observed. We could also amplify E bieneusi DNA in 10 aliquots of one sample fixed up to 10 days in 10% formalin. CONCLUSION: We conclude that PCR technology is very suitable for species identification of microsporidia in stool samples and may have a potential application in prospective studies in formalin-fixed samples.

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.

Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for small-subunit rRNA.

Enterocytozoon bieneusi is the most common microsporidian infecting patients with AIDS. We have developed a PCR primer pair, named EBIEF1/EBIER1, based on the small-subunit rRNA sequence of this microsporidian. Compared with other PCR-based methods, this primer pair shows a higher efficiency of detection in diagnostic applications than does another previously described primer pair, V1/EB450.

Short-term in vitro culture and molecular analysis of the microsporidian, Enterocytozoon bieneusi.

The microsporidium, Enterocytozoon bieneusi, causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon, is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.

Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in a patient with AIDS: successful therapy with albendazole.

Infections due to microsporidia are being recognized increasingly, especially in AIDS patients. A patient with disseminated microsporidiosis with advanced renal failure due to Encephalitozoon cuniculi (confirmed by culture and polymerase chain reaction [PCR]) is described. The organism from urine and sputum was characterized by culture, Weber's chromotrope-based staining, transmission electron microscopy, and indirect immunofluorescence (IIF) tests. PCR was done on DNA extracted from the infected cell cultures. Treatment with albendazole resulted in improvement in serum creatinine levels, complete disappearance of spores from sputum, a negative urine culture, and a 3-log decline in the number of spores in the urine, as evidenced by chromotrope-based staining. IIF and PCR were used to confirm E. cuniculi as the etiologic agent. Our findings indicate that disseminated microsporidiosis with renal failure in AIDS is treatable.

In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS.

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).

Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection.

Microsporidia are primitive, spore-forming, mitochondria-lacking, eukaryotic protozoa that are obligate intracellular parasites. They are known to parasitize almost every group of animals including humans. Recently, microsporidia have increasingly been found to infect patients with AIDS. Five genera (Encephalitozoon, Enterocytozoon, Nosema, Septata, and Pleistophora) of microsporidia are known to infect humans. Enterocytozoon organisms cause gastrointestinal disease in a majority of AIDS patients with microsporidiosis. However, a smaller, but an expanding, number of patients with AIDS are being diagnosed with ocular and disseminated infection with Encephalitozoon hellem. Although microsporidial spores can be identified in clinical samples by a staining technique such as one with Weber's chromotrope stain, identification to the species level is dependent on cumbersome and time-consuming electron microscopy. We have recently isolated and established in continuous culture several strains of E. hellem from urine, bronchoalveolar lavage, and sputum samples from AIDS patients with disseminated microsporidiosis. We developed polyclonal and monoclonal antibodies and PCR primers to a strain of E. hellem that can be used successfully to identify E. hellem from other species of microsporidia either in clinical specimens or in cultures established from clinical specimens. Since patients infected with Encephalitozoon spp. are known to respond favorably to albendazole, identification of the parasite to the species level would be invaluable in the treatment of disseminated microsporidiosis.

Isotypic analysis of humoral immune responses in rhesus monkeys to an adult microsomal antigen of Schistosoma mansoni: an indicator of successful treatment.

A study was undertaken to examine the potential role of immunodiagnostic methods in determining successful chemotherapy in schistosomiasis. Fifteen rhesus monkeys were infected with 1,500 Schistosoma mansoni (Puerto Rico strain) cercariae, and 10 of the monkeys were then treated with a curative dose of praziquantel 13 weeks after infection. Five monkeys remained untreated. One monkey was not successfully cured, as confirmed by the presence of both male and female worms at the time of perfusion. Serum samples were longitudinally collected and specific Ig isotypes were quantified with an adult microsomal antigen of S. mansoni using the FAST-ELISA. Specific isotypes were detected with monoclonal antibodies specific for each human Ig isotype, followed by a peroxidase-conjugated anti-mouse Ig. Longitudinally, all monkeys showed similar isotype patterns. Isotypes increased for the first nine weeks following infection, and then began to decrease. Ten to 14 days following treatment, all isotypes increased. The Ig isotype responses of all monkeys followed classic patterns of isotype expression. A ratio of pretreatment (week 13) IgG1 absorbance values to post-treatment IgG1 absorbance values was generated for each monkey. All successfully treated monkeys, determined to be worm-free by perfusion, had IgG1 ratios at week 53 greater than 2.4 (range 2.4-181). The untreated monkeys and the single monkey that was a treatment failure had IgG1 ratios less than 2.1 (range 0.09-2.05) for the same time period.

A specific diagnostic antigen of Echinococcus granulosus with an apparent molecular weight of 8 kDA.

An echinococcus antigen with an apparent molecular weight of 8 kDa was identified as diagnostically important. An immunoblot assay using this antigen was 91% sensitive for surgically confirmed Echinococcus granulosus hydatid disease of the liver. Specificity was 100% for echinococcosis. Marked cross-reactivity was observed with serum specimens from patients with E. multilocularis and E. vogeli infections. The 8 kDa component was not related to the widely recognized echinococcus antigen 5.

Diagnosis of paragonimiasis by immunoblot.

A sensitive and specific immunoblot assay was used to rapidly and accurately diagnose paragonimiasis. The immunoreactivity of a complex Paragonimus westermani Chaffee antigen was evaluated by SDS-PAGE and Western blot analysis. Initial probing with pooled human serum from proven Paragonimus infections revealed many bands, including a significant antibody response to an approximately 8,000 molecular weight (8 kDa) protein. Forty-three of 45 proven paragonimiasis serum specimens had antibodies to this diagnostic band. Of 29 normal serum specimens and 210 serum specimens from patients with other parasitic and nonparasitic infections, only 1 serum, from a schistosomiasis haematobium patient, reacted positively. These results indicate that our immunoblot for paragonimiasis, which uses a comparatively crude antigen, is highly sensitive (96%) and specific (99%).

Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool.

A standardized microtest plate enzyme-linked immunosorbent assay was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. The standard reference serum pool was prepared from acutely and chronically infected rhesus monkeys and was shown to be appropriate as a standard for measuring the levels of reactivity of the unknowns. The standard serum pool was arbitrarily designated as having 100 activity units per microliter. The levels of reactivity of the unknowns were expressed as activity units per microliter. Serum specimens were obtained from 190 patients infected with S. mansoni in the Caribbean, South America, and Africa. Serum was obtained from small numbers of patients infected with S. haematobium, S. japonicum, or S. mekongi. Controls were 136 patients with other helminthic infections, 142 patients with protozoal or other diseases with liver involvement, and 81 healthy serum donors. The J index (or predictability) of the assay was calculated to determine the significant level of reactivity. The assay has a predictability of 95% for both patients with S. mansoni infections and those with other infections. The sensitivity of the assay for S. mansoni infections was 96%, and the specificity (in terms of cross-reactions with infections with other parasite genera or with other liver diseases) was 99%. The heterologous Schistosoma species showed a markedly lower level of reactivity, with an overall sensitivity of 55%. This is in accord with the species-specificity previously recognized in MAMA, and emphasizes the need for standard reference pools of human sera prepared from patients infected with single species of each of the Schistosoma. Use of these pools in assays with antigens of the respective schistosome species would allow optimum serologic evaluation.