Helminth parasites in faecal samples from the endangered Iberian lynx (Lynx pardinus).

The Iberian lynx is the most endangered felid in the world. Enteropathogens may threaten its survival, and therefore we analysed faecal samples from 66 different individuals (37 males and 29 females), the largest population representation studied to date. The samples were obtained from November 2005 to October 2008 in the two areas where the Iberian lynx survives: Sierra Morena and Doñana (Andalusia, southern Spain). A total of 56.1% samples were parasitized with at least 6 species of helminths, including two cestodes (Hymenolepis spp. and Taenia spp.) and four Nematodes (Ancylostoma spp., Toxocara spp., Toxascaris leonina, and Capillaria sp.). In this work, the presence of Hymenolepis is reported for the first time in Lynx pardinus. The relevance of our findings is discussed focussed on the conservation of this endangered felid.

Comparative in vitro antifungal activity of amphotericin B lipid complex, amphotericin B and fluconazole.

Amphotericin B (AMB) is considered the gold standard in the treatment of serious systemic mycoses in spite of its nephrotoxicity and adverse effects. Association with lipids enables larger doses of AMB to be given with a longer t((1/2)) and C(max), without the toxic effects at lower concentrations. Liposome-encapsulated AMB shows a lower affinity for mammalian cells and improves V(d), thus decreasing toxicity. Amphotericin B lipid complex (ABLC) is an AMB formulation associated with a biodegradable phospholipid matrix (5% molar) from which the drug is released by cell phospholipases. ABLC is recommended for serious mycoses refractory to conventional antifungal therapy or when AMB is contraindicated. We compared the in vitro antifungal activity of ABLC, AMB and fluconazole (FLZ) against 328 strains of clinically significant opportunistic fungi using a microdilution method (NCCLS, M-27A). 64.9% of the yeasts were inhibited by MIC of ABLC

[Determination of the in vitro antifungal susceptibility of clinically important yeasts using the Sensititre system]. / Determinación mediante el sistema Sensititre de la sensibilidad in vitro a los antifúngicos de levaduras de interés médico.

Using Sensititre (AccuMed, USA) we studied the in vitro antifungal activity of amphotericin B, fluconazole, itraconazole, ketoconazole and 5-fluorocytosine against 250 clinical yeast isolates taken from different hospitals, including Candida (151 C. albicans, 15 C. krusei, 14 C. parapsilosis, 11 C. tropicalis, 10 C. glabrata, 4 C. guilliermondii, 3 C. rugosa, 2 C. viswanathii, 2 C. famata and 2 C. kefyr), Cryptococcus (32 C. neoformans and 1 C. laurentii), Trichosporon (2 isolates) and Rhodotorula rubra (1 isolate). All the strains were susceptible to amphotericin B and showed an MIC <1 mg/l. The susceptibility of C. albicans (MIC(90) <256 mg/l), C. krusei (MIC(90) <64 mg/l), C. glabrata (MIC(90) <64 mg/l) and C. neoformans (MIC(90) 32 mg/l) to fluconazole was lower (14% isolates being resistant and 16.8% susceptible depending on the dose). The largest number of strains resistant to itraconazole was observed in C. albicans and C. glabrata (17.2% resistant and 24% susceptible and susceptible depending on the dose, respectively). Ketoconazole and 5-fluorocytosine were not effective in vitro against 12.8% and 2%, respectively, of all the isolates studied. Nine C. krusei and seven C. neoformans (12.9%) showed dose-dependent susceptibility to 5-fluorocytosine.

Comparative study of the in vitro antifungal activity of bifonazole, naftifine and sertaconazole against yeasts.

The in vitro activity of three antifungal agents was tested and compared against 151 yeast strains, including ten Candida species, Cryptococcus neoformans, Rhodotorula rubra, and Trichosporon cutaneum. Minimum inhibitory concentrations (MICs) were determined by a microdilution technique in Shadomy modified liquid medium. The mean MICs of sertaconazole (0.34 mg/L) were lower than those of naftifine (16.3 mg/L) and bifonazole (13.2 mg/L). These results suggest that sertaconazole is more active against Candida spp than other topical agents such as bifonazole and naftifine.

Detection by an immunofluorescence test of Encephalitozoon intestinalis spores in routinely formalin-fixed stool samples stored at room temperature.

Of the several microsporidia that infect humans, Enterocytozoon bieneusi is known to cause a gastrointestinal disease whereas Encephalitozoon intestinalis causes both a disseminated and an intestinal disease. Although several different staining techniques, including the chromotrope technique and its modifications, Uvitex 2B, and the quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples, they do not identify the species of microsporidia. A need for an easily performed test therefore exists. We reevaluated 120 stool samples that had been found positive for microsporidia previously, using the quick-hot Gram-chromotrope technique, and segregated them into two groups on the basis of spore size. We also screened the smears by immunofluorescence microscopy, using a polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity with bacteria, yeasts, or other structures in the stool samples was seen. Additionally, the numbers of spores that fluoresced in seven of these samples were substantially smaller than the numbers of spores that were present in the stained smears, indicating that these samples were probably derived from patients with mixed infections of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae or with Enterocytozoon bieneusi spores in feces, we concluded that an immunofluorescence test using this serum is a good alternative for the specific identification of E. intestinalis infections.

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.

Identification of Cryptosporidium felis in a cow by morphologic and molecular methods.

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 micrometer; range, 3.5 to 5.0 micrometer), 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.

Subconjunctival infection with Dirofilaria repens: serological confirmation of cure following surgery.

Cases of zoonotic dirofilariasis infection, caused by Dirofilaria repens, occur widely throughout European, African, Middle Eastern, and Asian countries. The reports of this infection in humans in Spain are limited, and we herein report the case of a 43-year-old man from Elche (Alicante), Spain, who was seen with acute hyperemic reactivity of the temporal limbus of the right eye. A large nematode was visualized on examination and the intact worm was surgically removed. The parasite was identified as a mature but infertile female D repens. The level of serum antibodies against D repens was monitored for 6 months after surgery using immunoenzymatic assays. Serological results confirmed, as expected, the presence of a single worm and the parasitological cure after the surgical removal of the parasite. To our knowledge, this is the fourth autochthonous case of D repens infecting humans in Spain and also the first autochthonous case of subconjunctival localization.

Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans.

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.

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.