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Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species.

ABSTRACT: The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10^-4-1 × 10^-6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

SUBMITTER: Ceccarelli M

PROVIDER: S-EPMC7765608 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species.

Ceccarelli Marcello M Buffi Gloria G Diotallevi Aurora A Andreoni Francesca F Bencardino Daniela D Vitale Fabrizio F Castelli Germano G Bruno Federica F Magnani Mauro M Galluzzi Luca L

Microorganisms 20201216 12

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that ...[more]

PMID: 33339158

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Project description:The most abundant surface macromolecule on the promastigote stage of leishmanial parasites is a polymorphic lipophosphoglycan (LPG). We have elucidated the structures of two new LPGs, from Leishmania tropica (LRC-L36) and L. aethiopica (LRC-L495), and investigated the nature of intra-specific polymorphism in the previously characterized LPG of L. major (LRC-L456 and -L580). These molecules contain a phosphoglycan chain, made up of repeating PO4-6Gal beta 1-4Man units and a conserved hexaglycosyl-phosphatidylinositol membrane anchor. Extensive polymorphism occurs in the extent to which the LPG repeat units are substituted with different glycan side chains. The L. tropica LPG is the most complex LPG characterized to date, as most of the repeat units are substituted with more than 19 different glycan side chains. All of these side chains, including the novel major glycans, Arap beta 1-3Glc beta 1- and +/- Arap beta 1-2Glc beta 1-4[+/- Arap beta 1-2]Glc beta 1-, are linked to the C-3 position of the backbone disaccharide galactose. In contrast, the L. aethiopica LPG repeat units are partially substituted (35%) with single alpha-mannose residues that are linked, unusually, to the C-2 position of the mannose in the backbone disaccharide. Polymorphism is also evident in the spectrum of alpha-mannose-containing oligosaccharides that cap the non-reducing terminus of the phosphoglycan chains of these LPGs. Finally, analysis of the L. major LPGs showed that, while some strains contain LPGs which are highly substituted with side chains of beta Gal, Gal beta 1-3Gal beta 1- and Arap beta 1-2Gal beta 1-3Gal beta 1-, the LPGs of other strains (i.e L. major LRC-L456) are essentially unsubstituted. Recent studies have shown that the LPG side chains and cap structures can mediate promastigote attachment to a number of different receptors along the midgut of the sandfly vector. The possible significance of LPG polymorphism on the ability of these parasites to infect a number of different sandfly vectors is discussed.

Project description:BackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.MethodsField-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.ResultsThe qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionsThis study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Project description:BACKGROUND:A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. RESULTS:The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. CONCLUSIONS:This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

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Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species.

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Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species.

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