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

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.

Conjunctival Swab Real Time-PCR in Leishmania infantum Seropositive Dogs: Diagnostic and Prognostic Values

“…Recently, dogs were firstly found seropositive for L. tarentolae and L. infantum by IFAT in the same area of the present study [ 38 ], therefore suggesting a possible serological cross-reaction. Leishmania infantum DNA has been detected in PB samples of only a few seropositive dogs ( n = 4 in group HT in October 2020 and n = 1 in group HT in August 2021), confirming that PB is not the ideal tissue for molecular diagnosis of CanL [ 19 ]. However, a strong positive correlation between PB q PCR and clinical score was found in both time points ( Table 6 ), suggesting that PB q PCR may be a useful test for high positive predictive value based on clinical evaluation.…”

“…Moreover, factors related to the sampling technique and the molecular test performed (e.g., PCR method, target gene) may have influenced the obtained results. Nevertheless, it should be considered that q PCR targeting the parasite kDNA herein used represents to date one of the most sensitive methods for the molecular detection of L. infantum [ 19 ]. Finally, future studies involving the sampling and molecular analysis of CS at different time points during the transmission (e.g., beginning, middle, and end of sand fly season) as well as in the non-transmission periods are needed to support the hypothesis that CS q PCR results may be highly influenced by exposure time of dogs to L. infantum infected sand flies.…”

“…Conventional, nested, and real-time polymerase chain reactions (PCR) are sensitive and specific methods for the detection of Leishmania spp. infection both in clinically suspected and apparently healthy dogs, with the latter group being a potential source of the parasite to the phlebotomine vectors [ 4 , 18 , 19 ]. The diagnostic sensitivity of molecular assays relies considerably on the type of tissue evaluated, namely bone marrow, lymph node, and spleen as the most suitable samples for detecting Leishmania DNA [ 14 , 18 ], and on the PCR target being the kinetoplast DNA minicircle (kDNA) the most sensitive [ 20 ].…”

Liver- and Spleen-Specific Immune Responses in Experimental Leishmania martiniquensis Infection in BALB/c Mice

“…Lastly, our established qPCR in the present study, using L.ITS1.PCM2.4.6 primers, could differentiate L. martiniquensis (Tm = 84.9 • C) from L. orientalis (Tm = 81.0 • C), two major species endemic in Thailand (3), and from L. infantum (Tm = 82.6 • C), a species of sporadic occurrence (47). In the last two decades, the RT-qPCR application for the detection, quantification and genotyping of Leishmania species represents an advanced automation technique with high throughput, rapidity, and high sensitivity (48,49). qPCR has been used to quantify Leishmania loads in target organs of experimental animal models (23,50,51).…”

Nutritional Modulation of the Immune Response Mediated by Nucleotides in Canine Leishmaniosis

“…The diagnosis of CanL is complex, and it combines, besides compatible history and clinical signs, serological, molecular, and parasitological methods [7,29,[37][38][39]. Clinical staging, either following the LeishVet guidelines for the practical management of CanL [7] or the guidelines for the treatment of leishmaniasis in dogs [40], is also a very useful tool, especially during patient monitoring [41], as clinical evolution can have an impact on the prognosis and influence the efficacy of a particular treatment protocol.…”