Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

Abstract

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.