Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity

Abstract

A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 10 6 nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.Leishmania infantum is the causative agent of visceral leishmaniasis in the Mediterranean area. Host-parasite relationships may vary from asymptomatic carriage of the parasite to the typical presentation of the disease.The opportunistic character of this parasite is well demonstrated in immunosuppressed or immunocompromised patients (8); in cases of coinfection by human immunodeficiency virus and Leishmania, relapses are common even when a correct therapeutic scheme has been applied, and there is a need for biological indicators of evolution of the parasitosis in order to start earlier treatment of relapses or to prevent relapses by use of maintenance therapy (7). Asymptomatic carriage was recognized as the most common status in host-parasite relationships (25). In healthy humans, asymptomatic carriage of Leishmania infantum is diagnosed only by immunological tests because parasitic loads seem to be very low compared to those observed during the acute phase of the disease or in dog leishmaniasis (2).In addition to the conventional microscopic, cultural, and serological methods, numerous DNA-based tests have been described, particularly the use of PCR technology (1,4,6,9,23). The sensitivities of such methods seem to be variable, depen...

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

“…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. In all asymptomatic carriers of L. infantum, parasitemia was less than one parasite/mL of blood.…”

“…These results agree with the sensitivity limits of classic techniques previously described. 7 Since the interval of time between the first episode of VL and the relapse is variable, the frequency of biologic tests should be adapted to the clinical presentation of the patients. In a patient coinfected with HIV and Leishmania whose leishmaniasis was previously treated with antimony, a long-term survey showed efficacy of liposomal amphotericin B treatment of the first relapses.…”

“…7 DNA was extracted by using the mini Qia-amp kit (Qiagen, Courtaboueuf, France) according to the instructions of the manufacturer. To optimize the extraction yield, proteinase K digestion of the nucleated cells (equivalent to 2 mL of blood) was performed overnight at 56°C.…”

“…5,6 A quantitative PCR reached such sensitivity that quantification of parasites over an interval of 9 log units, starting at a 1:10,000 single cell equivalent amounts of DNA per reaction tube. 7 The aim of this study was to obtain reference values and thresholds for parasitemias in cases with asymptomatic infections and those with acute-phase infections during and after treatment of Mediterranean VL and to illustrate the usefulness of parasite quantification in blood for therapy monitoring and follow-up of patients at risk of relapse.…”

Diagnosis of Leishmania (Leishmania) chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas, state of Tocantins, Brazil

“…The technique has been consistently validated as the fastest and most sensitive and specific one as compared to other diagnosis protocols -apart from its suitability when used in leishmaniasis surveillance programs 2,[8][9][10][11] . Concerning molecular diagnosis, kinetoplast DNA minicircles (KDNA) are good targets in the Leishmania genome, as these present up to 10,000 copies per cell, which increases sensitivity of detection procedures 2,12 .…”