Molecular phylogenetic analysis of Enterobius vermicularis and development of an 18S ribosomal DNA-targeted diagnostic PCR.

We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed.

Specific inhibitors of mitogen-activated protein kinase and PI3-K pathways impair immune responses by hemocytes of trematode intermediate host snails.

To characterize molecular mechanisms regulating snail cellular immune responses, the contributions of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3-K) were examined in hemocytes of the trematode intermediate host snails Biomphalaria glabrata and Lymnaea stagnalis. Simultaneous measurement of phagocytosis/encapsulation and H2O2 production by hemocytes in the presence or absence of specific signal transduction inhibitors was used to assess the role of extracellular-signal regulated kinases 1 and 2 (ERK1/2), p38, JNK and PI3-K. Hemocyte spreading was significantly reduced in a dose-dependent manner by the ERK inhibitor, PD098059, and by wortmannin, a potent PI3-K inhibitor. The JNK inhibitor, SP600125, and the p38 kinase inhibitor, SB203580, had no effect on hemocyte spreading. Sheep red blood cell phagocytosis was significantly impaired by PD098059, SP600125, and SB203580. Hydrogen peroxide production during phagocytosis was severely inhibited by PD098059. Additionally, PD098059, but not the other inhibitors, significantly impaired the cellular encapsulation of trematode larvae and H2O2 production during encapsulation. These results suggest that MAPK and PI3-K signal transduction pathways play a pivotal role in the immune responses of snail hemocytes. PI3-K and ERK appear to strongly regulate cell motility. ERK, JNK and p38 contribute to phagocytosis-mediated signal transduction. ERK also play a major role in oxidative burst activation and the encapsulation of trematode larvae by snail hemocytes.

Coccidioidomycosis and blastomycosis: advances in molecular diagnosis.

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.

Comparison of autofluorescence and iodine staining for detection of Isospora belli in feces.

To evaluate the sensitivity of autofluorescence for detection of Isospora oocysts, wet preparations of 192 stool samples from patients with chronic diarrhea were examined by fluorescence microscopy and by light microscopy after iodine staining used for routine screening for ova and parasites. Silicon-chambered glass coverslips were used for fluorescence microscopy. Isospora oocysts were detected in 46 iodine-stained concentrated stool samples; 91 samples were positive by autofluorescence. According to the maximum likelihood estimates, examination by fluorescence (95.7%; 95% confidence interval [CI], 85.2-99.5) was significantly more sensitive than iodine staining (48.4%; 95% CI, 37.7-59.1). Examination for autofluorescence is a simple, highly sensitive, inexpensive, and easily applicable method to detect Isospora spp. oocysts in feces.

Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvum in fecal specimens.

To optimize routine screening for cryptosporidiosis, 198 stool samples from patients at risk and from calves were examined by enzyme immunoassay (EIA), a direct fluorescent-antibody (DFA) and a modified immunofluorescence assay. Ninety-nine samples were positive in at least one assay, whereas 99 were negative in all three assays. Sensitivity of antigen EIA and DFA were similar (94%, 95% CI: 88-98%, and 91%, 95% CI: 84-95%). The modified immunofluorescence was significantly less sensitive (64%, 95% CI: 55-74%). 149 samples were also examined by two nested PCR assays targeting either the 18S rRNA or Cryptosporidium outer wall protein (COWP) gene. A PCR product was amplified from 86 out of 89 samples being positive in at least one other assay (sensitivity 97%, 95% CI: 91-99%). None was obtained from 60 samples negative in the three other assays. PCR assays did not increase the detection rate. Antigen EIA or DFA appear sufficient for routine Cryptosporidium screening of fecal samples.

A comparative study of the organic acid content of the hemolymph of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata

The freshwater snail Biomphalaria glabrata is an intermediate host of the trematode Schistosoma mansoni. However, some strains of B. glabrata are resistant to successful infection by S. mansoni larvae. The present work examines the profile of organic acids present in S. mansoni-resistant and susceptible strains of B. glabrata, in order to determine whether the type of organic acid present is related to susceptibility. The organic acids were extracted from the hemolymph of two susceptible B. glabrata strains (PR, Puerto Rico and Ba, Jacobina-Bahia from Brazil), and from the resistant strains 13-16-R1 and 10R2, using solid phase extraction procedures followed by high performance liquid chromatography. The organic acids obtained were analyzed and identified by comparison with known standards. Pyruvate, lactate, succinate, malate, fumarate, acetate, propionate, ß-hydroxybutyrate and acetoacetate were detected in all hemolymph samples. Under standard conditions, the concentration of each of these substances varied among the strains tested and appeared to be specific for each strain. An interesting variation was the low concentration of pyruvate in the hemolymph of PR-snails. Only the concentration of fumarate was consistently different (pó0.05) between resistant and susceptible strains.