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...

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Abstract. Quantification of Leishmania infantum DNA in blood samples by an ultrasensitive quantitative polymerase chain reaction (QPCR) detected parasitemias in different clinical presentations. We observed a large range of parasitemias, more than 9 log values, and could determine the threshold between asymptomatic carriage and disease in the Mediterranean area (approximately one parasite/mL of blood. Based on kinetoplast DNA amplification, this assay had a sensitivity of 0.001 parasite DNA equivalents/mL and detected asymptomatic carriage of Leishmania. It detected parasite DNA in 58% of healthy subjects, while an immunoblot detected specific antibodies in only 16%. For initial diagnosis of disease, this quantitative PCR with blood samples constitutes a non-invasive alternative to bone marrow aspiration. Its main applications are monitoring of drug therapy and follow-up of immunodeficient patients for biologic confirmation of relapses.

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Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.