A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasitesPlasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

Twenty-nine patients with chronic hepatitis C and 15 asymptomatic hepatitis C virus antibody-positive subjects who clinically recovered from hepatitis C virus infection were studied for their peripheral blood lymphomononuclear cell proliferative response to hepatitis C virus structural and nonstructural antigens (core, envelope, nonstructural 4 and nonstructural 5) expressed in yeast as superoxide dismutase fusion proteins, in an initial attempt to define some of the features of the virus-specific immune response. Hepatitis C virus core was the most immunogenic antigen for human leukocyte antigen class II-restricted T cells in both groups of patients studied, and the proliferative response to it was the most vigorous and the most frequently expressed in comparison with the other antigens tested. The specificity of the results was supported by the lack of response to hepatitis C virus antigens by healthy uninfected controls and confirmed by recognition of recombinant core proteins of different origin (yeast and baculovirus) by polyclonal T-cell lines produced by T-cell stimulation with yeast-derived core. Each of the antigens tested was able to induce significant although variable levels of proliferative response, indicating that all can be immunogenic at the T-cell level. Significant proliferative responses to core, nonstructural 4 and nonstructural 5 antigens were more frequently detected in subjects who were able to eradicate infection than in patients with chronic hepatitis C, although the difference was statistically not significant. No difference was observed between the two groups of patients with respect to the response to the putative envelope antigens.

Residues 11 to 27 of the hepatitis B virus nucleocapsid antigen contain a cytotoxic T-cell epitope that is recognized by cytotoxic T cells from virtually all HLA-A2-positive patients with acute hepatitis B virus infection. Using panels of truncated and overlapping peptides, we now show that the optimal amino acid sequence recognized by cytotoxic T cells is a lO-mer (residues 18 to 27) containing the predicted peptide-binding motif for HLA-A2 and that this peptide can stimulate cytotoxic T cells able to recognize endogenously synthesized hepatitis B core antigen. Since patients with chronic hepatitis B virus infection fail to mount an efficient cytotoxic T-cell response to it, this epitope might serve as the starting point for the design of synthetic * Corresponding author.

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