Abstract. A fluorescent DNA probe (LEIS.P1) specific for a conserved region of the small-subunit ribosomal RNA gene of Leishmania and a pair of flanking primers (LEIS.U1 and LEIS.L1) were designed for use in a fluorogenic polymerase chain reaction. Optimal assay conditions with zero background were established to detect low levels of Leishmania from clinical samples. By use of this assay, we amplified DNA from 27 strains of cultured Leishmania (both Old and New World strains) and selectively amplified Leishmania DNA from 12 paraffin-embedded human biopsy samples and 3 fresh human skin biopsy specimens. For the fresh human tissue biopsies, the turnaround time from biopsy to test result was Ͻ 24 hr. No amplification was detected in negative control samples (including the kinetoplastid protozoa Trypanosoma rangelli and Crithidia fasiculata). This assay provides a specific and rapid diagnostic modality to detect infection with Leishmania.
Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzymelinked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity.
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