Detection of Entamoeba histolytica DNA in the saliva of amoebic liver abscess patients who received prior treatment with metronidazole.

Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR.

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.

Bromo-deoxyuridine based assay for detection of parasite and drug sensitivity in Plasmodium falciparum in vitro.

A non-radioactive, thymidine analogue-bromodeoxyuridine (Brdu), derivative of uridine has been used for incorporation in DNA in culture of P. falciparum at various dosages and at different time period. Parasite growth rate and effect of chloroquine in culture were monitored by microscopic observation of stained smears and incorporation of Brdu molecules were visualized by immunofluorescence and measured by enzyme immuno assay using anti-Brdu. Uptaking of Brdu in parasite is slower unlike tumour cells. A positive correlation between parasite growth and Brdu uptake measurement by ELISA has been observed.

Isolation of virulence genes directing GPI synthesis by functional complementation of Leishmania

Recent advances in molecular genetics of Leishmania parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant LPG, an abundant GPI-anchored polysaccharide. LPG1, the gene product identified by complementation of our R2D2 LPG- mutant, may be a glycosyltransferase responsible for the addition of galactofuranose to the nascent chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism