Real-Time PCR for Detection and Quantitation of Leishmania in Mouse Tissues

Abstract

Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 10 4 parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of Leishmania spp .Leishmania spp. are intracellular protozoa that affect humans and dogs worldwide and are transmitted by the bite of hematophagous sand flies. They cause a large spectrum of diseases, ranging from spontaneously healing skin lesions to fatal visceral symptoms, if left untreated. Two million new human cases arise every year, and at least 350 million people are exposed to the risk of the Leishmania parasite infection (2, 9). Experimental hosts, such as laboratory mice, are largely used to study the immunobiology of these parasites and to screen the efficacy of newly developed drugs and vaccines (4,11,20,27). Most of those studies require detection and quantitation of the Leishmania burdens in different mouse tissues. This is still routinely performed by culture-based techniques (6, 28), which have several limitations, in particular, the amount of time required and the putative presence of viable but noncultivable parasites, such as persistent parasites (1). PCR-based methods for detecting Leishmania species have been developed to amplify rRNA genes, miniexon genes, kinetoplast DNA (kDNA), and repetitive nuclear sequences (15,18,24,26). Recently, we have developed a PCR-based assay with which to quantify the parasite load in mice infected with Leishmania major (23) by using primers from the conserved sequences of kDNA. However, this technique is still cumbersome as it requests agarose gel image analysis.A more rapid alternative is real-time quantitative PCR, which quantifies DNA...

Reference Values for Leishmania Infantum Parasitemia in Different Clinical Presentations: Quantitative Polymerase Chain Reaction for Therapeutic Monitoring and Patient Follow-Up

“…8 Realtime PCR assays targeted to nuclear genome sequences were designed for quantification of parasites in experimental studies in mice, but their sensitivity was only approximately one genome equivalent per reaction tube. 9 Only a newly developed quantitative PCR assay that targeted a consensus sequence of kinetoplast DNA showed a sensitivity of less than one parasite/mL of blood. 7 This report gives the order of magnitude of parasitemias for different clinical presentations of Leishmania infection in the Mediterranean area.…”

Therapeutic Efficacy Evaluation of Metronidazole and Some Antifungal Agents with Meglumine Antimoniate on Visceral Leishmaniasis by Real-Time Light-Cycler (LC) PCR in BALB/c Mice

“…PCR-based methods for detecting Leishmania species have been developed to amplify rRNA genes, miniexon genes, kinetoplast DNA (kDNA), and repetitive nuclear sequences [7]. Recently, a PCR-based assay to quantify the parasite load in mice infected with L. major using primers from the conserved sequences of kDNA [8] was reported. However, this technique is still cumbersome as it requires agarose gel image analysis.…”

Transplacental Transmission of Cutaneous Leishmania mexicana Strain in BALB/c Mice

“…Finally, the precipitates were rehydrated with 100 μL ultrapure water and stored at −20 C. The PCR mixture was prepared using the Taq PCR Master Mix from QIAGEN (1.5 mM MgCl 2 , 0.5 U/μL Taq DNA polymerase, 200 μM 2-deoxyribonucleoside 5-triphosphates (DNTPs) and 10 + PCR Buffer QIAGEN), 10 pM each JW11 and JW12 primers, 18,19 and genomic DNA. Assay samples, at a final volume of 25 μL/vial, were generated for the healthy and infected mice, the positive controls (i.e., L. mexicana cultures), and the negative controls (i.e., samples without DNA)…”