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.
The ability to detect and differentiate between Plasmodium falciparum and P. vivax is of great importance for the routine laboratory diagnosis of malaria, donor-blood screening and epidemiological studies. Most PCR-based methods for the discrimination of these two species require nested protocols or an additional hybridization reaction, leading to high labour costs and long turn-around times. A simple, time-effective and yet sensitive and specific technique, based on a multiplex PCR, has now been developed for the simultaneous detection and differentiation of P. falciparum and P. vivax in blood samples. Compared with the 'gold standard' of microscopy, this method had a sensitivity and specificity of 100%, with a detection limit of just one P. falciparum or three P. vivax parasites/microl blood.
Blastocystis is a polymorphic intestinal parasite that is common in humans. A total of 51 asymptomatic and symptomatic patients positive for Blastocystis only were included in the study. Symptoms were mainly nonspecific gastrointestinal symptoms. Blastocystis isolates were xenically cultured and subtyped. Blastocystis species subtype 3 was the predominant subtype. Intrasubtype differences (vacuolar/amoeboid presence) in subtype 3 morphotypes were observed in 32 asymptomatic and symptomatic subtype 3 cases and could possibly be related to Blastocystis pathogenic potential. Diverse morphologic features (vacuolar transiting to amoeboid), probably reflecting the progression from an asymptomatic to a symptomatic state, were observed in an asymptomatic subtype 3 carrier who later had symptoms. Searching for amoeboid forms might be helpful to presumptively screen symptomatic patients with subtype 3 or to follow up an asymptomatic subtype 3 carrier in case symptoms become evident before antiprotozoal treatment was attempted. Further studies on the roles of morphologic features and variation within Blastocystis species subtypes as predictors of symptoms are encouraged.
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