PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

Abstract

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Detection of Leishmania (V) guyanensis in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) collected from Pecari tajacu

“…y la HRM-PCR para identificar la especie del parásito, se emplearon en el intento de amplificar el citocromo b para su posterior secuenciación, aunque sin éxito. El citocromo b se localiza en el maxicírculo del cinetoplasto con un menor número de copias que el minicírculo, también localizado en este (22,23), y se empleó como sección de molde para la amplificación mediante los dos tipos de PCR. El fallido intento de secuenciación usando el citocromo b se explicaría por el bajo número de parásitos en R. microplus.…”

Phylogenetic study of treatment failure clinical isolates of Leishmania major

“…They are considered one of the most useful genes for molecular and phylogenetic studies [19,20]. The COXII has been sown a higher evolutionary rate in some other studies [6,21]. Because of its multicopy nature, it can be used for clinical material and environmental samples, such as sandflies.…”

“…Because of its multicopy nature, it can be used for clinical material and environmental samples, such as sandflies. Nevertheless, only a few phylogenetic studies of the genus Leishmania have included this marker and its discriminative power has not been established in detail [6,19,21]. Pilot studies have shown it to be useful for the discrimination of subgenera or species complexes, but its applicability for species delimitation has to be further studied.…”

“…Molecular techniques also help to nd out the genetic characterization of the parasite, which can be considered to result in precise and appropriate planning and control, as well as e cient vaccine and drug development [5]. Various target genes used in this eld include a variety of mitochondrial and nucleus barcodes [6,7].…”

<b>Aspectos clínicos e moleculares no diagnóstico da leishmaniose visceral canina</b>

“…Esses marcadores possuem uma característica única que possibilita a discriminação de complexos, espécies, subespécies, estirpes e até isolados de Leismania spp., dependendo da região genômica a ser pesquisada. Além disto mostram-se sensíveis em detectar casos incipientes de leishmaniose (LOPES, 2010) Lopes et al (2016),. em seu estudo para determinar a performance de primers utilizados em cPCR direcionados a amplificar gene kDNA e DNA nuclear de leishmanias encontrou uma alta sensibilidade dos que amplificam kDNA, como os primers 13A/13B(RODGERS et al, 1990), pois como já descrito, o locus gênico que eles amplificam está presente em milhares de cópias no genoma celular do parasita, explicando assim a maior sensibilidade analítica da cPCR baseada em primers de kDNA.…”