Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients.

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species.     

Detection of the microsporidian parasite Enterocytozoon bieneusi in specimens from patients with AIDS by PCR.

Microsporidia are protozoa parasites responsible for significant gastrointestinal disease in patients infected with human immunodeficiency virus. We evaluated a PCR assay of stool samples, duodenal aspirates, and biopsy specimens from patients with Enterocytozoon bieneusi infection. A 210-bp DNA fragment of the unique rRNA intergenic spacer could be amplified from all samples infected with E. bieneusi, but no amplification was seen by using DNA purified from samples with Septata intestinalis or other parasites and from negative control human cells. These results suggest that the PCR in stool samples may be a useful tool for the diagnosis of intestinal microsporidiosis in patients with AIDS.     

Identification of genotypes of Enterocytozoon bieneusi from stool samples from human immunodeficiency virus-infected patients in Thailand

We identified genotypes of Enterocytozoon bieneusi from 33 stool samples of Thai human immunodeficiency virus (HIV)-infected adult patients. Genotype D was identified at the highest frequency (36.4%), while genotype E was the second most common (15.1%). Genotypes O and PigEBITS 7, previously found only in pigs, were observed in Thai HIV-infected patients. Phylogenetic analysis supported a zoonotic nature for E. bieneusi.     

Quantitative light microscopic detection of Enterocytozoon bieneusi in stool specimens: a longitudinal study of human immunodeficiency virus-infected microsporidiosis patients.

The clinical course of microsporidiosis caused by Enterocytozoon bieneusi and the pattern of intestinal shedding of spores have not been correlated, at least in part because detection of E. bieneusi in stools is more difficult than detection of other protozoa because of its smaller size and less intense staining. We examined with a modified trichrome stain 124 stool specimens collected over a 2-year follow-up period from 23 human immunodeficiency virus-infected patients with electron microscopic-proven E. bieneusi infection and correlated the results with electron microscopic observations from duodenal biopsy specimens taken at the beginning of the study period. E. bieneusi was detected in the stool at least once in 74% (17 of 23) of all patients, in 100% (9 of 9) of patients in whose tissue moderate or abundant numbers of parasites were seen, and in 57% (8 of 14) of patients in whose tissue few parasites were seen. In two patients with abundant tissue parasites, many microsporidia were detected in every stool specimen (13 of 13) during the follow-up period, whereas among the patients with few tissue parasites, only 23% (15 of 64) of stool specimens were positive. Furthermore, if spore stages as well as plasmodial stages were detected in tissue, stool specimens were more likely to be positive. Although most of the heavily infected stools were from patients with chronic diarrhea, microsporidia were detected in 33, 28, and 42% of stool specimens from patients with nil, intermittent, and chronic diarrhea patterns, respectively. Although quantitation of E. bieneusi spores in stool specimens was closely correlated with quantitation in tissue, it was not correlated with reported patterns of diarrhea.     

Development and application of genetic probes for detection of Enterocytozoon bieneusi in formalin-fixed stools and in intestinal biopsy specimens from infected patients.

The microsporidium Enterocytozoon bieneusi is closely linked to wasting and diarrhea in a high proportion of individuals with AIDS. However, its relative contribution to disease is uncertain because diagnosis until recently depended on procedures involving endoscopy. A sensitive PCR technique which amplifies a fragment of the small-subunit rRNA gene of E. bieneusi from formalin-fixed stool samples was developed. Of 80 formalin-fixed stool samples collected from 74 Zimbabweans and 6 U.S. patients who were human immunodeficiency virus positive, 50% tested positive for E. bieneusi by PCR, whereas 24% tested positive for E. bieneusi by light microscopy of trichrome-stained fecal smears. In addition, we describe an in situ hybridization technique which detected and identified E. bieneusi as the causative agent in all six intestinal biopsy specimens tested. Both the PCR and in situ hybridization procedures are sensitive diagnostic tools which will complement currently available techniques and enable the differentiation of E. bieneusi from other microsporidia to be made.     

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.     

Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta)

CONTEXT: Enterocytozoon bieneusi is the most frequent microsporidian parasite of human patients with acquired immunodeficiency syndrome and is a significant cause of diarrhea and wasting. Recently, this organism has also been recognized as a spontaneous infection of several species of captive macaques. As in humans, E bieneusi frequently causes enteropathy and cholangiohepatitis in immunodeficient simian immunodeficiency virus (SIV)-infected macaques. OBJECTIVE: To examine E bieneusi as an etiologic agent of nonsuppurative proliferative serositis in immunodeficient rhesus macaques (Macaca mulatta). DESIGN: Retrospective analysis of necropsy material obtained from immunodeficient SIV-infected rhesus macaques. RESULTS: Examination of SIV-infected rhesus macaques (n = 225) revealed E bieneusi proliferative serositis in 7 of 16 cases of peritonitis of unknown origin. The organism could be identified by in situ hybridization and polymerase chain reaction in sections of pleura and peritoneum obtained at necropsy. Serositis was always accompanied by moderate-to-severe infection of the alimentary tract, and morphologic evidence suggested dissemination through efferent lymphatics. Colabeling experiments revealed most infected cells to be cytokeratin positive and less frequently positive for the macrophage marker CD68. Sequencing of a 607-base pair segment of the small subunit ribosomal gene revealed 100% identity to sequences obtained from rhesus macaques (Genbank accession AF023245) and human patients (Genbank accession AF024657 and L16868). CONCLUSIONS: These findings indicate that E bieneusi disseminates in immunodeficient macaques and may be a cause of peritonitis in the immunocompromised host.     

Electron microscopy identification of microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis from stool samples of HIV infected patients

Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis have been reported worldwide causing diarrhoea in AIDS patients. Stool samples from HIV infected patients were subjected to routine examination for parasites, followed by special staining techniques to detect microsporidia and Cyclospora cayetanensis. Confirmed positive cases of these parasites were further processed for electron microscopy identity of the parasites and characteristic details. Scanning and transmission electron microscopy showed better morphological and structural details of the parasites.     

Detection and identification of Enterocytozoon bieneusi and Encephalitozoon species in stool and urine specimens by PCR and differential hybridization

Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. Enterocytozoon bieneusi and Encephalitozoon intestinalis are most commonly associated with chronic diarrhea. All Encephalitozoon species, including E. intestinalis, E. hellem, and E. cuniculi, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and Vittaforma corneae as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three Encephalitozoon species and V. corneae. An analytical sensitivity of 3.5 x 10(2) to 3.5 x 10(3) spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different Encephalitozoon species.     

Evidence of different Enterocytozoon bieneusi genotypes in patients with and without human immunodeficiency virus infection.

We classified 100 Enterocytozoon bieneusi isolates into five genotypes by a PCR-restriction fragment length polymorphism method. Type I strains were encountered only in human immunodeficiency virus (HIV)-infected patients, whereas type II strains were more frequently found in non-HIV-infected patients (75 versus 10%, respectively; P < 10(-4)), suggesting differences in the epidemiology of E. bieneusi among these patients.     

Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques

A three-step method for the purification of Enterocytozoon bieneusi spores from stool specimens was developed. The primary process of purification of the spores from bacterial contaminants involved Percoll gradient centrifugation followed by additional separation using cesium chloride density gradient centrifugation. The cesium chloride-isolated spores were further purified using a flow cytometer with cell sorting capabilities. Sorting was performed without the use of antibodies, fluorochromes, or dyes, leaving the sorted spores in their native state, which appears to be less destructive for spores. When quantified by flow cytometry using tubes with known numbers of highly fluorescent polystyrene beads, the sorted material showed a slight decrease in light scatter characteristics compared with the slightly larger Encephalitozoon species spores. Although the overall recovery of the E. bieneusi spores was low, calcofluor and Gram chromotrope staining, indirect immunofluorescence assay, and transmission electron microscopy revealed that the sorted material was highly purified and contained large numbers of E. bieneusi spores and relatively few bacteria and other debris. The sorted material appeared to be sufficiently pure and could be used for in vitro culture and for the development of a variety of diagnostic reagents as well as in studying the genome of E. bieneusi and host-parasite interactions.     

Diagnosis of Enterocytozoon bieneusi by PCR in stool samples eluted from filter paper disks

We report a PCR-based assay for the detection of Enterocytozoon bieneusi. We extracted DNA from feces which had been applied to filter paper disks and evaluated four preserving solutions. Infected specimens were identified by electrophoresis of amplicons from concentrated formalin-fixed samples and unconcentrated fresh feces. Our findings demonstrate that this methodology is effective for sample collection, mailing, and diagnosis of this pathogen.     

Diagnosis of Enterocytozoon bieneusi by PCR in Stool Samples Eluted from Filter Paper Disks

We report a PCR-based assay for the detection of Enterocytozoon bieneusi. We extracted DNA from feces which had been applied to filter paper disks and evaluated four preserving solutions. Infected specimens were identified by electrophoresis of amplicons from concentrated formalin-fixed samples and unconcentrated fresh feces. Our findings demonstrate that this methodology is effective for sample collection, mailing, and diagnosis of this pathogen.     

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

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 speciesspecific detection ofEncephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, andEnterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected withEnterocytozoon bieneusi (n=9),Encephalitozoon spp. (n=2), andEncephalitozoon intestinalis (n=1) as well as stool spiked with spores ofEncephalitozoon cuniculi andEncephalitozoon hellem and tissue cultures ofEncephalitozoon cuniculi andEncephalitozoon 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 ofEnterocytozoon bieneusi andEncephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected withEncephalitozoon cuniculi andEncephalitozoon hellem. Moreover, identification ofEncephalitozoon spp. could be specified asEncephalitozoon 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 speciesspecific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.     

聚合酶链反应酶谱分型检测宫颈癌中人乳头瘤病毒 …

目的 探讨人乳头瘤病毒（ＨＰＶ）和单纯疱疹病毒（ＨＳＶ）等对宫颈癌的病因学作用。方法 应用聚合酶链反应（ＰＣＲ）－核酸内切酶分型检测宫颈癌活检组织中ＨＰＶ－ＤＮＡ和ＨＳＶ－ＤＮＡ基因,以正常宫颈组织作对照。结果 在宫颈癌活检细胞中ＨＰＶ－１６,１８型和ＨＳＶ－２型阳性率分别为３８．９％和３４．６％,与正常妇女宫颈组织阳性率均为３．２％比较,差异均有非常显著意义（Ｐ〈０．００１）。结论 ＨＰＶ－１６     

Limited value of PCR for detection of Toxoplasma gondii in blood from human immunodeficiency virus-infected patients.

Cerebral toxoplasmosis is a common, opportunistic, and often life-threatening disease in HIV-infected patients. Diagnosis is supported mainly by clinical evidence and computerized tomography or magnetic resonance imaging scans, but brain images may share features with other brain diseases occurring in HIV-infected patients. To determine the diagnostic value of PCR for the detection of Toxoplasma gondii in blood from HIV-infected patients, we examined 89 blood samples from 59 HIV-infected patients. PCR and Southern blot hybridization were done with DNA extracted from blood samples from 20 patients with confirmed cerebral toxoplasmosis and from 10 patients with suspected but not confirmed cerebral toxoplasmosis. The samples were taken before and 7 to 10 days after the beginning of antiparasitic therapy. For 9 patients who suffered from cerebral toxoplasmosis more than 6 months prior to the study and for 20 patients without any evidence for toxoplasmosis only one blood sample per patient was examined. PCR gave positive results with 5 of the 20 blood samples from patients who suffered from cerebral toxoplasmosis. After 7 to 10 days of therapy PCR results became negative in all these five cases. No amplification was seen with DNA from blood samples from the other 54 patients as the target. The results presented here show that PCR testing of blood samples from HIV-infected patients is of limited value for the diagnosis of cerebral toxoplasmosis. The sensitivity was only 25%, but the specificity was very high (100%), so this technique may be useful for discriminating between cerebral toxoplasmosis and other brain diseases which may be mistaken for toxoplasmosis.     

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients.

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.     

Molecular study of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among human immunodeficiency virus-infected patients from two geographical areas: Niamey, Niger, and Hanoi, Vietnam

Microsporidiosis cases due to Enterocytozoon bieneusi and Encephalitozoon intestinalis are emerging opportunistic infections associated with a wide range of clinical syndromes in humans. The aim of this study was to specify microsporidial epidemiology in two different geographical areas. From November 2004 to August 2005, 228 and 42 stool samples were collected in Niamey, Niger, and Hanoi, Vietnam, respectively. Screening for microsporidia was performed using UV-light microscopy. Detection was confirmed by molecular biology using two methods specific for E. bieneusi and E. intestinalis. All samples positive for E. bieneusi were subjected to genotyping. In this study, we found high prevalences of microsporidiosis among human immunodeficiency virus-infected patients, 10.5% and 9.5%, respectively, in Niamey and Hanoi. These levels of prevalence are similar to those recorded in European countries before highly active antiretroviral therapy was introduced. In the samples positive for E. bieneusi, we found seven distinct genotypes, including two genotypes not previously described. The E. bieneusi genotype distributions in the two geographical areas suggest different routes of infection transmission, person-to-person in Niger and zoonotic in Vietnam.     

One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.     

Sensitivity and specificity of a monoclonal antibody-based fluorescence assay for detecting Enterocytozoon bieneusi spores in feces of simian immunodeficiency virus-infected macaques

Enterocytozoon bieneusi is clinically the most significant among the microsporidia causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. Microscopy with either calcofluor or modified trichrome stains is the standard diagnostic test for microsporidiosis and does not allow species identification. Detection of E. bieneusi infection based on PCR is limited to a few reference laboratories, and thus it is not the standard diagnostic assay. We have recently reported the development and characterization of a panel of monoclonal antibodies against E. bieneusi, and in this publication we evaluated the specificity and sensitivity of an immunofluorescence assay (IFA), compared with PCR, in simian immunodeficiency virus-infected macaques. The IFA, which correlated with the primary PCR method, with a detection limit of 1.5 x 10(5) spores per gram of feces, will simplify considerably the detection of E. bieneusi spores in clinical and environmental specimens and in laboratory and epidemiological investigations.