Evidence for leishmania (viannia) parasites in the skin and blood of patients before and after treatment.

BACKGROUND: American cutaneous leishmaniasis is considered to be a zoonotic disease transmitted by sand flies that feed on infected sylvatic mammals. However, the "domestication" of transmission and the increase in treatment failure with antimonial drugs have raised the suspicion of anthroponotic transmission of American cutaneous leishmaniasis. METHODS: The objective of the present study was to explore the potential of humans as a source of infection for sand flies. Biological (xenodiagnosis and culture) and molecular (polymerase chain reaction/Southern blot) detection methods were used to evaluate peripheral-blood monocytes and tissue fluids from sites accessible to sand flies from 59 adult patients with parasitologically confirmed American cutaneous leishmaniasis. RESULTS: Overall, 44.1% of patients (26/59) presented biological and/or molecular evidence of Leishmania parasites in normal skin, peripheral-blood monocytes, lesion scars, or lesion border (by xenodiagnosis) before (18/59 [30.5%]) or after (10/27 [37.0%]) treatment. Leishmania parasites were cultured from the unaffected skin of 2 (3.6%) of 55 patients, and xenodiagnosis gave positive results for 5 (8.8%) of 57 patients before treatment. CONCLUSIONS: The presence of Leishmania parasites in the unaffected skin and peripheral-blood monocytes of a high proportion of patients even after treatment and the acquisition of infection by sand flies support the plausibility of anthroponotic transmission of American cutaneous leishmaniasis.

Amplification of human DNA by primers targeted to Leishmania kinetoplast DNA and post-genome considerations in the detection of parasites by a polymerase chain reaction.

We evaluated the Leishmania Viannia-specific primers B1-B2 to detect Leishmania in normal skin and peripheral blood monocytes of patients with active cutaneous leishmaniasis. Southern blotting and sequencing of polymerase chain reaction (PCR) products confirmed the specificity of kinetoplast DNA (kDNA) amplification from tissue fluid from healthy skin, whereas the PCR with monocytes also amplified a human sequence of a size similar (718 basepairs) to the expected kDNA product (750 basepairs), resulting in false-positive results. Although B1 was not homologous to any human DNA sequence, B2 showed homology to a human chromosome 2 intergenic region (AC010878) at positions 35,881-36,599, which are spaced 718 nucleotides apart. Amplification of the human artifact from monocyte DNA was confirmed using the primer B2 alone. Examination of other primers reported for the PCR of kDNA from various species of Leishmania showed that six of seven were homologous to human DNA sequences. These findings underscore the importance of exploiting sequencing, bioinformatics, and DNA probes to refine molecular amplification techniques and to validate the performance of primers when used for new applications.