New primers for the detection Leishmania species by multiplex polymerase chain reaction

Abstract

Leishmaniasis is caused by protozoa of the Leishmania genus, which is divided into subgenus Viannia and Leishmania. In humans, the course of infection largely depends on the host-parasite relationship and primarily of the infective species. The objective of the present study was to design specific primers to the identification of Leishmania species using multiplex PCR. Four primers were designed, based on the GenBank sequences of the kDNA minicircle, amplifying 127 bp for subgenus Viannia, 100 bp for L. amazonensis, and 60 bp for Leishmania donovani complex and L. major. None of the primers amplified Trypanosoma cruzi or L. mexicana. The limit of detection of multiplex PCR was 2 × 10 parasites for L. braziliensis, 2 x 10 parasites for L. amazonensis, and 1.4 × 10 parasites for L. infantum. The high sensitivity of multiplex PCR was confirmed by the detection of parasites in different biological samples, including lesion scrapings, spleen imprinting of a hamster, sandflies, and blood. The multiplex PCR that was developed herein presented good performance with regard to detecting and identifying the parasite in different biological samples and may thus be useful for diagnosis, decision making with regard to the proper therapeutic approach, and determining the geographic distribution of Leishmania species.

Overcoming the Negligence in Laboratory Diagnosis of Mucosal Leishmaniasis

“…Whole-blood samples were collected from 14 (15.9%) ML patients with a positive result for kDNA PCR. DNA extracted from these samples was analyzed using a standardized qPCR hsp 70 protocol [ 35 ]. The time of appearance of mucosal lesions was variable in these patients, ranging from recent lesions (0.25 months) to old lesions (72 months) ( Table 6 ).…”

“…Although in this study, standardized protocol was employed for sample collection and slide preparation, and the same professional examined all the slides, 206 patients would not have been diagnosed if only DP was performed. Molecular tests are more sensitive for TL than for DP, with sensitivity for TL of up to 100% [ 35 , 68 , 69 , 70 ]. As expected, higher sensitivity was observed for molecular tests (PCR) employed in this work, and only 10 samples positive for DP were negative for both molecular tests.…”

“…PCR targeting minicircle kDNA is considered the most sensitive molecular target for both CL and ML diagnosis, with reports of up to 97% sensitivity [ 71 , 72 , 73 ]. High sensitivity is also reported for PCR hsp 70, although, this approach showed lower sensitivity than PCR kDNA [ 35 , 74 , 75 ]. Our results are in agreement with the sensitivity reported for the different tests.…”

“…There are several PCR-based methods for diagnosis of TL, targeting kinetoplast DNA (kDNA), transcribed internal spacer (ITS), and heat shock protein 70 gene ( hsp 70), among others [ 31 , 34 , 35 , 36 , 37 , 38 , 39 ]. Each marker has both advantages and disadvantages, thus, validation with clinical samples is necessary.…”

In silico and in vitro evaluation of primers for molecular differentiation of Leishmania species

“…Several authors have used PCR-based techniques to differentiate Leishmania species (Akhoundi et al, 2013;Morais et al, 2016;Conter et al, 2018). These molecular techniques, which include real-time quantitative polymerase chain reaction (qPCR), have provided an alternative to multilocus enzyme electrophoresis (MLEE) (Galluzzi et al, 2018), which has been considered the "gold standard" technique for the differentiation of Leishmania species (Schönian et al, 2011), and enables testing of a wide range of biological samples (blood, lymph node, bone marrow, skin, etc.).…”