Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity.

Germano Castelli,Fabrizio Vitale,Federica Bruno,Stefano Reale,Simone Catanzaro,Viviana Valenza

doi:10.3390/pathogens10070865

Germano Castelli, Fabrizio Vitale + Show 4 more

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https://doi.org/10.3390/pathogens10070865

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Abstract

Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.

Highlights

Leishmania is a parasitic protozoa belonging to the family Trypanosomatidae, which can infect both humans and other mammals [1]
The present study aims to evaluate the potential use of the conserved motif of kinetoplast DNA (kDNA) minicircles in a Taqman-based real-time PCR approach, to discriminate between L. infantum from the other species and identify correlation with the clinical status of the subjects, allowing us to discriminate between symptomatic patients, cured patients, and asymptomatic carriers
The sensitivity of the quantitative PCR (qPCR) reaction was tested by using serial dilutions of L. infantum p2a.r1a.sSiteenDsitNivAityeaxntrdacStpeedcififrcoitmy oaf tkhneoRweanl-nTuimmebPeCr RofApsasraaysites

Summary

Introduction

Leishmania is a parasitic protozoa belonging to the family Trypanosomatidae, which can infect both humans and other mammals [1]. Dogs are considered the main reservoir of Leishmania infantum (L. infantum), developing canine Leishmaniosis (CanL) but there is clear evidence that some wild and synanthropic mammals and domestic cats can infect sand flies and they play a variable role in a reservoir system according to local and ecological peculiarities [3]. Cats develop clinical disease, feline leishmaniasis (FeL) due to L. infantum infection and may have dermal as well as visceral manifestations of this infection [4,5]. They can play a role as additional reservoir hosts of L. infantum and in a “One Health” perspective; preventative measures should be taken in this species based on epidemiological data [6]. Other assays were developed to estimate the parasite load through absolute or relative quantification

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