Fluorogenic detection of viable Toxoplasma gondii.

In order to easily assess growth and destruction of Toxoplasma gondii in vitro, this report describes two double staining assays that both visualize live and dead organisms: acridine orange--ethidium bromide (AO-EB) and bisbenzimide (Hoechst 33258)--propidium iodide (B-PI). EB and PI were chosen for dead organisms staining while AO and B stain viable organisms. Thus, both double staining assays seem more informative than Giemsa staining or indirect immunofluorescence. They offer methods to study internal structure of the parasite as well as information on host-parasite relationships. Moreover, detection in culture are sensitive, easier, and less time consuming than previous methods. So, they should to be useful in strains behaviour analysis.

Heterogeneity in cellular and humoral immune responses against Toxoplasma gondii antigen in humans.

Protection against Toxoplasma gondii in infected patients is mainly attributed to cellular immunity. We here attempt to improve the characterization of the proteins that induce cellular immunity in naturally infected patients. Cellular immunity was evaluated by flow cytometry after 7 days of blood culture from 31 chronically T. gondii infected and 8 noninfected pregnant women, in the presence of soluble T. gondii antigen (ST-Ag) or fractionated proteins from ST-Ag, separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Blood cultures from infected patients with ST-Ag induced 39.5 +/- 12.7% of activated (CD25+) CD4+ T cells using flow cytometry. This contrasts with the absence of activated CD4+ T cells after either culture with PBS or in blood cultures from noninfected women. The protein fraction between 21 and 41.9 kD induced the highest response (14.7 +/- 10.0%). Blood samples from 20 infected and 5 uninfected women were cultured in presence of 12 protein subfractions of 2-208 kD. The highest frequencies of response among infected patients were seen with fractions (Fr) 26-31.9 kD (C.I. 85-100%) and Fr 32-36.9 kD (C.I. 77-100%). Although we note a good concordance between cellular and humoral response, Western blot analysis of ST-Ag does not completely predict the panel of proteins recognized by cellular immunity. Two-dimensional separation of the ST-Ag revealed more than 200 protein spots in these fractions. However, only two proteins in the 20-40 kD range induced a significant humoral response. Further studies are necessary to determine which proteins in the Fr 26-31.9 kD and 32-36.9 kD are superior immunogens for cellular responses.

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR.

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR.

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.