Norman J. Pieniazek

Fast and reliable extraction of protozoan parasite DNA from fecal specimens2

BACKGROUNDnPolymerase 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.nnnMETHODS AND RESULTSnExtraction 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.nnnCONCLUSIONSnBy 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.

Detection of Septata intestinalis (microsporidia) cali et al. 1993 using polymerase Chain reaction primers targeting the small subunit ribosomal RNA coding region

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.

Molecular and morphologic characterization of a Cryptosporidium genotype identified in lemurs

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.

Short‐Term In Vitro Culture and Molecular Analysis of the Microsporidian, Enterocytozoon bieneusi

ABSTRACT. 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.

Molecular characterization of Nosema bombi (Microsporidia : Nosematidae) and a note on its sites of infection in Bombus terrestris (Hymenoptera : Apoidea)

SUMMARY Investigations 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 bumble bees.

Identification of Leishmania spp. by Molecular Amplification and DNA Sequencing Analysis of a Fragment of rRNA Internal Transcribed Spacer 2

ABSTRACT 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.

Asymptomatic Respiratory Tract Microsporidiosis Due to Encephalitozoon hellem in Three Patients with AIDS

Microsporidia of the genera Enterocytozoon and Encephalitozoon have been identified as frequent causes of intestinal and disseminated infections, respectively, in patients with AIDS. Even though most subjects infected with these protozoa develop overt disease, simple colonization without illness may occur, as we observed in three severely immunosuppressed patients with AIDS. The parasites, recognized in and isolated from bronchoalveolar lavage sediment specimens, were characterized as Encephalitozoon hellem. Colonization of the bronchial tree was temporary, and treatment with albendazole was not needed to clear the infection.

Morphological and molecular characterization of Antonospora scoticae n. gen., n. sp. (Protozoa, microsporidia) a parasite of the communal bee, Andrena scotica Perkins, 1916 (Hymenoptera, andrenidae)

Summary The new microsporidium Antonospora scoticae n. gen., n. sp., a parasite of the communal bee Andrena scotica , is described based on light microscopy, ultrastructural characteristics and the nucleotide sequence of the small subunit ribosomal RNA coding region. The parasite is apansporoblastic and develops in close contact with the host cytoplasm. All developmental stages are diplokaryotic. Cytoplasmic fission was not observed, but the sporogony is believed to be disporoblastic. Live spores are ovocylindrical, straight to slightly curved, and measure 6.8 × 2.7 μn whereas spores fixed and stained for TEM measure 5.0 × 1.8 μm. The exospore is four-layered, with an internal single layer followed by a thicker, more electron dense layer, then another single layer followed by a thin external double layer. The polar filament is isofilar and arranged in 15–22 coils in the posterior and mid-part of the spore. The polaroplast has tightly packed lamellae that become less densely packed in the posterior region. The coding region of the small subunit ribosomal RNA is 1371 base pairs long. Its GC content (62%) is significantly higher than previously reported for this group of organisms. The systematic position of the described microsporidium was found to be ambiguous and is discussed in the context of inconsistencies between the molecular and morphological taxonomy of microsporidia. A new genus is proposed for A. scoticae without defining superior taxa because the current developmental and morphological evidence is limited and partly contradictory to the molecular data. Current taxonomies of microsporidia are based on characters that are most likely polyphyletic in nature. Traditional systems of microsporidian taxonomy may need to be extensively revised, as molecular data become available.

Blood and sporozoite stage-specific small subunit ribosomal RNA-encoding genes of the human malaria parasite Plasmodium vivax

Malaria parasites, unlike other eukaryotes, have developmentally controlled distinct small subunit ribosomal RNA (SSUrRNA)-encoding genes (SSUrDNA), sporozoite stage-specific C and blood stage-specific A genes. This report describes characterization of the C and A forms of SSUrDNA from the human malaria parasite Plasmodium vivax. We have aligned and compared these sequences with the reported SSUrDNA sequences of other human malaria parasites to identify the regions with potential for diagnostic probes. The comparison revealed the presence of seven conserved regions (> or = 90% similarity), four highly variable regions (< 60% similarity) and three semiconserved regions. The analysis also revealed that the A and C genes of P. vivax share more similarity with each other, as compared to the A and C genes of P. falciparum. Comparison of the SSUrDNA of human, monkey and rodent malaria parasites revealed that the A genes share more similarity with each other than the C genes share with each other.

Isolation and identification of Encephalitozoon hellem from an Italian AIDS patient with disseminated microsporidiosis.

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, Septula, Nosema and Pleistophora) that cause infections in humans, Enterocytozoon bieneusi. Septula intestinulis, and Encephulitozoon hellem are being increasingly identified in patients with acquired immunodeficiency syndrome (AIDS). E. bieneusi causes gastrointestinal disease, S. intestinulis 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.