Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR.
Control of human leishmaniases relies on appropriate diagnosis and reliable methods for monitoring chemotherapy. The current method used for estimation of parasite burden during chemotherapy patient follow-up as well as in pharmacological studies performed in experimental models involves PCR-based assays. Compared to time-consuming conventional methods, this type of Leishmania DNA detection-based method is extremely sensitive, but could fail in distinguishing viable Leishmania from slowly degenerating ones. We have used an in vitro model to monitor the duration of Leishmania DNA persistence in mouse macrophages following exposure to l-leucine ester, a molecule otherwise known to rapidly kill intracellular Leishmania amazonensis amastigotes. At 1h of post l-leucine ester exposure, more than 98% of amastigote-loaded macrophages harbored killed parasites and parasite remnants, as assessed by microscopy. This dramatic decrease in parasite load and the microscopic parasite follow-up over the 120 h time period studied were correlated with Leishmania DNA as quantified by real-time PCR. Our results indicate that kinetoplast and nuclear parasite DNA degradation occurs very rapidly after amastigote death. These data add further weight to the argument that PCR assays represent not only a robust method for diagnosis but can also be reliable for monitoring parasite size reduction rate post any intervention (Leishmania-targeting molecules, immunomodulators...).
Prina
Eric
E
Unité d'Immunophysiologie et Parasitisme Intracellulaire, Département de Parasitologie et Mycologie, Institut Pasteur 25-28 rue du Dr Roux, 75724 Paris cédex 15, France. eprina@pasteur.fr
Roux
Emeric
E
Mattei
Denise
D
Milon
Geneviève
G
D000818:Animals; D000982:Antipruritics / Q000494:pharmacology; D016923:Cell Death; D016054:DNA, Protozoan / Q000378:metabolism*; D005260:Female; D007891:Leishmania / Q000166:cytology / Q000235:genetics*; D007930:Leucine / Q000494:pharmacology; D008264:Macrophages / Q000737:chemistry / Q000469:parasitology; D051379:Mice; D008853:Microscopy; D016133:Polymerase Chain Reaction
D000982:Antipruritics; D016054:DNA, Protozoan; D007930:Leucine