Detection of microsporidia (Enterocytozoon bieneusi) in intestinal biopsy specimens from human immunodeficiency virus-infected patients by PCR.

Intestinal microsporidiosis has been implicated as a major cause of chronic diarrhea in human immunodeficiency virus (HIV)-infected patients. So far diagnosis depends on direct visualization of the parasites by light and transmission electron microscopy. We evaluated the diagnostic value of microsporidian DNA amplification by PCR on duodenal biopsy specimens obtained from patients with and without intestinal microsporidiosis caused by Enterocytozoon bieneusi. Thirteen HIV-infected patients (all CDC stage C3) were studied. Eight patients had intestinal microsporidiosis caused by E. bieneusi (n = 6), Septata intestinalis (n = 1), and Encephalitozoon cuniculi (n = 1); microsporidioses were diagnosed by light microscopy of stool samples and confirmed by light and electron microscopy of intestinal biopsy specimens. Five patients had no microsporidia in their stool samples or in their intestinal biopsy specimens, as examined by light and electron microscopy. Additionally, DNA prepared from Toxoplasma gondii derived from mouse ascites was used as a further control. A 353-bp DNA fragment of the small-subunit rRNA gene could be amplified from all six biopsy specimens infected with E. bieneusi, and the nature of the PCR products was confirmed by Southern blot hybridization. No amplification of DNA fragments was seen by using DNA extracted from biopsy specimens with S. intestinalis or E. cuniculi infection or without microsporidian infection and with template DNA extracted from T. gondii. The results suggest that PCR testing of intestinal biopsy specimens may be a useful approach to diagnosing microsporidiosis in HIV-infected patients.     

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.     

Purification of Enterocytozoon bieneusi from stools and production of specific antibodies

Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 microm) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.     

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.     

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.     

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.     

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.     

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.     

Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens.

The human amyloidoses represent a heterogeneous group of disorders characterized by the deposition of fibrillar protein in vital organs. Given the fact that at least 20 different molecules can form fibrils, the unambiguous identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these disorders. Heretofore, this information has been inferred from particular clinical features of the disease, ancillary laboratory tests, and results of immunohistochemical analyses. However, to establish unequivocally the kind of protein that is deposited as amyloid, it is necessary to determine its chemical composition through amino acid sequencing or mass spectroscopy of material extracted from fibrillar deposits. We have developed a micromethod whereby such studies can be performed readily using sections of formalin-fixed, paraffin-embedded biopsy specimens. The ability to identify precisely the nature of the tissue deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disorders.     

Culture of intestinal biopsy specimens and stool culture for detection of bacterial enteropathogens in patients infected with human immunodeficiency virus. The Berlin Diarrhea/Wasting Syndrome Study Group.

The diagnostic yields of stool cultures and biopsy specimens for the detection of enteric bacterial pathogens in 213 human immunodeficiency virus-infected patients were compared. Forty-five percent (19 of 42) of the pathogens were detected exclusively by stool culture, 2% (1 of 42) of the isolates were detected exclusively by culture of biopsy specimens, and 53% (22 of 42) were detected by both methods. Repeated stool cultures remain the most important means of diagnosing enteric bacterial pathogens, which were encountered in 20% (40 of 213) of all patients. The additional culture of biopsy specimens should be reserved for patients with suspected mycobacteriosis.     

Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis.

Enterocytozoon bieneusi, a microsporidian parasite, has been recognized since 1985 as an agent of intestinal microsporidiosis leading to malabsorption syndrome, diarrhea, and weight loss in AIDS patients. Recently, however, we have identified E. bieneusi spores in the sputum, bronchoalveolar lavage, and stool samples of an AIDS patient with a 2-year history of intestinal microsporidiosis. The spores were characterized by Weber's chromotrope-based staining, immunofluorescence tests, and PCR. No microsporidia were detected in urine samples by the same techniques. PCR was performed with DNAs purified from specimens with E. bieneusi-, Encephalitozoon cuniculi-, Encephalitozoon hellem-, and Encephalitozoon (Septata) intestinalis-specific primers. Treatment with albendazole and loperamide resulted in an improvement of intestinal symptoms, without eradication of the parasite. To our knowledge, this is the second report of the identification of E. bieneusi spores in respiratory and enteric samples obtained from an AIDS patient. Although no pulmonary pathology could be established in either of these cases, it is now clear that E. bieneusi is capable of colonizing the respiratory tract and it is suggested that investigators should be aware of the possibility of finding E. bieneusi spores in respiratory secretions.     

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.     

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.     

Evaluation of initial and deeper sections of esophageal biopsy specimens for detection of intestinal metaplasia

There are wide variations in the preparation of histologic sections from endoscopic esophageal biopsy specimens. We evaluated serial step sections from 261 esophageal or gastroesophageal junction biopsies at 4 levels to determine the first level at which goblet cell metaplasia (GCM) was detected. Deeper step sections of 152 paraffin blocks also were obtained to determine whether additional sections are useful in detecting GCM not seen in initial levels. GCM was identified in 95.3% of blocks in 3 levels. GCM was seen at level 4 in 12 blocks (4.7%). In the blocks that did not reveal intestinal metaplasia in the initial 4 levels, deeper sections disclosed GCM in only 1 (0.8%) of 120 blocks. However, deeper sections revealed initially undetected GCM in 4 of 32 blocks from patients with a history of documented Barrett esophagus. We conclude that 4 levels of step sections are adequate in routine processing of esophageal biopsy specimens for demonstration of GCM. Deeper sections may be obtained for patients with known Barrett esophagus to better evaluate for dysplasia or find additional foci of GCM.     

Genotyping of Helicobacter pylori strains in formalin-fixed or Formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens.

Different genotypes of Helicobacter pylori can play a role in the development of atrophic gastritis, peptic ulcer disease, and gastric carcinomas. In this study the authors developed polymerase chain reaction assays for the detection and identification of vacA (s- and m-regions), cagA, and iceA genotypes of H. pylori in formalin-fixed or formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens. Polymerase chain reaction products were analyzed by reverse hybridization on a line-probe assay. Complete genotyping was achieved in 26 of 28 samples (93%), and multiple genotypes, indicating the presence of multiple strains, were detected in nine samples (32%). This genotyping method offers the possibility for long-term retrospective studies on H. pylori genotypes and gastric pathology in the same archival gastric tissue specimens.     

Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens

An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.     

Transmission and Serial Propagation of Enterocytozoon bieneusi from Humans and Rhesus Macaques in Gnotobiotic Piglets

For over a decade Enterocytozoon bieneusi infections in people with AIDS have been linked with chronic diarrhea and wasting. The slow scientific progress in treating these infections is attributed to the inability of investigators to cultivate the parasite, which has also precluded evaluation of effective therapies. We report here successful serial transmissions of E. bieneusi from patients with AIDS and from macaques with AIDS to immunosuppressed gnotobiotic piglets. One infected piglet was still excreting spores at necropsy 50 days after an oral challenge. Spores in feces were detected microscopically by trichrome stain and by PCR and within enterocytes by in situ hybridization and immunohistochemistry. E. bieneusi infection induced no symptoms. The development of an animal model for E. bieneusi will open up new opportunities for investigating this parasite.     

Use of Probemix and OmniProbe biotinylated cDNA probes for detecting HPV infection in biopsy specimens from the genital tract.

AIMS--To compare two commercially available pan probes for the identification of human papillomavirus (HPV) DNA expression in histological sections and to type the HPV positive cases. METHODS--97 formalin fixed, paraffin wax embedded biopsy specimens from the genital tract were tested for HPV positivity with in situ hybridisation using biotinylated cDNA pan probes--Probemix (Enzo) and OmniProbe (Digene). The HPV positive cases were further tested with HPV types 6/11, 16/18, and 31/33/35/51, and the HPV type was related to the histological diagnosis. Formalin fixed, HeLa cells (10-50 HPV 18 copies per cell) and SiHa cells (1-2 HPV 16 copies per cell) were used as reference cell lines. RESULTS--32% of the specimens gave positive nucleic signals with both Probemix and OmniProbe. Of these, 84% could be further characterised with regard to HPV types 6/11, 16/18, and 31/33/35/51; 4% of all cases were positive with either Probemix or OmniProbe. The concordance of these probes was high, 96% altogether. HeLa cells stained positive but SiHa cells did not. CONCLUSION--There is no difference between Probemix and OmniProbe for the general detection of HPV. The mean detection limit of these probes is about 20 copies a cell.     

Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsy specimens

In this work, a fluorescence in situ hybridization (FISH) method for the rapid detection of Helicobacter pylori using a novel peptide nucleic acid (PNA) probe is reported. Laboratory testing with several different bacterial species, including other Helicobacter spp., has shown that this probe is highly specific for H. pylori strains. In addition, the PNA FISH method has been successfully adapted for detection of the pathogen in paraffin-embedded gastric biopsy specimens.