Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method.

The aim of this study was to develop a simple, low-cost method for the detection and species differentiation of Leishmania directly from clinical samples, for routine use in a parasitology laboratory. A total of 87 samples was used, including 60 peripheral blood, seven bone marrow and 17 skin lesion material samples, derived from Greek patients with visceral or cutaneous leishmaniasis, and three reference strains. PCR was performed using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene. Identification of the Leishmania species studied was achieved by digestion with a single restriction endonuclease (RFLP), single-strand conformational polymorphism (SSCP) and DNA sequencing of the PCR-generated fragments. Typing identified all visceral and one cutaneous leishmaniasis strains as L. infantum, twelve of the cutaneous leishmaniasis strains as L. tropica and four as L. major. The described PCR method proved efficient for the detection of pathogenic Leishmania species in various clinical samples, most importantly in peripheral blood samples. Furthermore, PCR followed by a simple RFLP using a single restriction endonuclease was capable of identifying all Leishmania species commonly encountered in Greece.

Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing.

Blastocystis is an anaerobic parasitic microorganism, which has been found in the intestinal tract of many vertebrates including humans. Recently, members of Blastocystis sp. were classified into nine subtypes, based on phylogenetic trees derived from sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene. The role of Blastocystis in human disease remains uncertain and the existence of pathogenic and non-pathogenic subtypes is under investigation. We report the development of a polymerase chain reaction (PCR)-based assay that is able to detect Blastocystis directly from human faeces. Furthermore, combined with single strand conformational polymorphism (SSCP) analysis and/or sequencing of the respective PCR product, the protocol can classify Blastocystis among the nine established subtypes. The method was applied to 45-positive and 30-negative faecal samples and proved to be highly sensitive and specific. Genotyping using SSCP analysis and sequencing revealed that subtype 3 is the most frequent in Greece, while subtypes 1, 2, 4, 6 and 7 are also present but in lower frequencies. Hopefully, the simplicity of the proposed method will contribute toward large-scale epidemiological studies for prompt clarification of the role of the parasite.

Association study of human VN1R1 pheromone receptor gene alleles and gender.

Pheromones are water-soluble chemicals that elicit neuroendocrine and physiological changes, while they also provide information about gender within individuals of the same species. VN1R1 is the only functional pheromone receptor in humans. We have undertaken a large mutation screening approach in 425 adult individuals from the Hellenic population to investigate whether the allelic differences, namely alleles 1a and 1b present in the human VN1R1 gene, are gender specific. Here we show that both VN1R1 1a and 1b alleles are found in chromosomes of both male and female subjects at frequency of 26.35% and 73.65%, respectively. Given the fact that those allelic differences potentially cause minor changes in the protein conformation and its transmembrane domains, as simulated by the TMHMM software, our data suggest that the allelic differences in the human VN1R1 gene are unlikely to be associated with gender and hence to contribute to distinct gender-specific behavior.

Increased gamma-globin gene expression in beta-thalassemia intermedia patients correlates with a mutation in 3'HS1.

We report a novel set of genetic markers in the DNaseI hypersensitive sites comprising the human beta-globin locus chromatin hub (CH), namely HS-111 and 3'HS1. The HS-111 (-21 G>A) and 3'HS1 (+179 C>T) transitions form CH haplotypes, which occur at different frequencies in beta-thalassemia intermedia and major patients and normal (nonthalassemic) individuals. We also show that the 3'HS1 (+179 C>T) variation results in a GATA-1 binding site and correlates with increased fetal hemoglobin production in beta-thalassemia intermedia patients. In contrast, the HS-111 (+126 G>A) transition, found in three normal chromosomes, is simply a rare polymorphism. We conclude that the CH haplotypes are useful genetic determinants for beta-thalassemia major and intermedia patients, while the 3'HS1 (+179 C>T) mutation may have functional consequences in gamma-globin genes expression.

Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis.

The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.