Evaluation of 4 polymerase chain reaction protocols for cultured Leishmania spp. typing

Abstract

Leishmaniasis is a disease caused by the protozoan Leishmania resulting in a variety of clinical manifestations, from self-healing skin lesions to fatal visceral disease. The development of polymerase chain reaction (PCR)-based techniques has made species identification easier, faster, and less labor intensive. The main targets for PCR amplification include kinetoplastid DNA (kDNA), miniexon, and conserved regions such as the internal transcribed spacer. The objective of this work was to evaluate 4 different PCR techniques designed to type Leishmania using laboratory strains. Parasites were subjected to 4 PCR procedures using specific Leishmania primers for miniexon (designated A1 and A2) and kDNA (designated B1 and B2, C1 and C2, and D1, D2 and D3). Discrimination between some species and the 2 main subgenera Leishmania and Viannia was achieved. Unweighted pair group method analysis resulted in the expected clustering of the 2 species from the subgenus Leishmania. However, some species in the subgenus Viannia could not be distinguished, representing a continued challenge for PCR-based protocols. Results are discussed in terms of advantages, limitations, and reproducibility of these 4 PCR-based techniques in the taxonomy of Leishmania.

Species Typing in Dermal Leishmaniasis

“…Only the specific primers of Meredith et al (123) discriminated 4 Old World species, but that assay was validated on one strain of each species only. Several authors have observed cross-reactions between different species (104,147), which is probably caused by the use of slightly different reaction conditions across labs and the variability of the minicircle population in each species. De Bruijn and Barker (148) reported a widely used Leishmania (Viannia)-specific PCR, validated on one strain each of 10 species, but cross-reaction of one primer with human and mouse DNAs was found (149), which could lead to false-positive results.…”

“…Even though some species groups can be discriminated based on size only (60,132,147), RFLP analysis is needed for discriminating species. The PCR (Fig.…”

Intraspecies Variation in Trypanosoma cruzi GPI-Mucins: Biological Activities and Differential Expression of α-Galactosyl Residues

“…26 To evaluate if such discrimination would be observed in this work, a taxon/character matrix was constructed based on the presence/absence of bands from FACE analysis and biological techniques performed. 35 The relationship among the strains was determined after a phenogram construction using the Dice similarity coefficient 36 from the NTSYSpc software package (version 2.02).…”

Identificación molecular de aislamientos clínicos de Leishmania spp. procedentes de Colombia con base en el gen hsp70

“…Esta identificación puede hacerse de manera directa con una PCR específica para la especie (12), o mediante procedimientos posteriores a la amplificación molecular, como el análisis del tamaño de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP) (13-15). En este contexto, la variante PCR-hsp70/RFLP basada en el gen hsp70, propuesta por García, et al (11), y modificada posteriormente, detecta 14 especies de importancia médica usando un solo par de cebadores y, a partir de un único producto de amplificación, logra identificar individualmente la mayoría de dichas especies mediante una o dos restricciones enzimáticas (15).…”