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

Abstract

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 Ϫ20ЊC (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 Ͼ 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.

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

“…Considering the 57 patients that had a lesion of up to two months, 54 (94.7%) had a positive PD. The positivity found in PD was similar to the described results in literature (33 a 77%) (Marques et al 2001).…”

“…In the 11-patient group where only the MST was positive, the PCR showed three positive results (27.3%), differing from the positivity (60%) found by Marques et al (2001). The low positivity found in this group can be explained by the fact that the MST can detect past infections (Oliveira et al 2005), besides the possibility of crossreactions with other infections, like sporotrichosis, as reported by Barros et al (2005).…”

“…Isaza et al (2002) have obtained 90.6% of PCR positivity in biopsies and 80.5% in lesion scrapping. Among the ATL (positive PD and/or MST), the results showed 91.1% of positivity, similar to the ones found by Lopez et al (1993), Rodriguez et al (1994, Pirmez et al (1999), Marques et al (2001), and Weigle et al (2002). In all cases where one of the conventional exams was positive, and the lesion PCR negative, another lesion material sample was extracted and amplified.…”

“…The MST is routinely used for the ATL diagnosis because it is a test of easy performance, however, it is not able to distinguish past from current infection (Marques et al 2001). The MST was positive in 51.9% of the patients and the association of PD and MST was useful to diagnose ATL in 57.7% of suspected patients (n = 156).…”

“…In the last years, molecular techniques have been used for the detection and identification of Leishmania in culture, biopsy, scrapes, blood, and phlebotomine (Marques et al 2001). The polymerase chain reaction (PCR) is able for detecting so little quantities as 1 phentogram (10 -15 g) of Leishmania DNA, equivalent to 1/10 of parasite (Gontijo & Carvalho 2003), offering an alternative way for the demonstration of parasites in clinical samples (Belli et al 1998, Medeiros et al 2002, Oliveira et al 2005.…”