Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR

Abstract

To cite this version:Eric Prina, Emeric Roux, Denise Mattei, Geneviève Milon. Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR.. Microbes and Infection, Elsevier, 2007, 9 (11) Abstract 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 one hour 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 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 (Leishmaniatargeting molecules, immunomodulators…).

Molecular detection, identification and phylogenetic inference of Leishmania spp. in some desert lizards from Northwest China by using internal transcribed spacer 1 (ITS1) sequences

“…In this study, we found Leishmania DNA in all of the detected desert lizard species with exception to H42 H41 H40 H39 H38 H37 H36 H35 HG512925 H8 H4 H6 / HG 512919 H14 H3 H13 H5 H1 H2 H10 H11 HG512927 H23 H22 H21 H20 H19 H18 H17 H16 H15 H12 H7 HG512922 H9 HG512908 HG512948 T. przewalskii. Because DNA persists in the serum for approximately 24 h (Prina et al, 2007), positive results of PCR become a good indicator of current or recent infection. Our results demonstratethat the occurrence of Leishmania species varies among lizard species, with E. velox roborowskii showing lower prevalence value (56%) than do others.…”

Continual renewal and replication of persistentLeishmania majorparasites in concomitantly immune hosts

“…Parasite genome integrity was assessed by the TUNEL assay (35). PIPs visualized in iNOS + cells showed no TUNEL + nuclei, but 26 ± 15% of PIPs showed TUNEL + kinetoplast (mitochondrial) DNA (Fig.…”

SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples

“…The result of the present study showed comparable efficiency of the real time-PCR assay to the nested PCR, which is being used widely for the diagnosis of leishmaniasis. The existence of one (4.3 %) false-negative could have resulted from factors such as the technique employed for sample extraction, the presence of inhibitors, or the degradation of DNA, particularly kDNA, which is more readily degraded than nuclear DNA (Prina et al 2007). The kDNA nested-PCR and real-time PCR presented the same analytical sensitivity level (1 parasite/ml) and specificity.…”