PCR AND IN-VITRO CULTIVATION FOR DETECTION OF LEISHMANIA SPP IN DIAGNOSTIC SAMPLES FROM HUMANS AND DOGS

A PCR assay for the diagnosis of leishmaniosis was developed by using primers that were selected from the sequence of the small-subunit rRNA gene. The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (lysis of erythrocytes in Tris-EDTA buffer, digestion with proteinase K directly in PCR buffer, and no further purification steps). Furthermore, an internal control i s included in every specimen in order to detect the presence of PCR in hibitors. The PCR was compared with diagnostic in vitro cultivation of promastigote stages for the detection of Leishmania spp. in clinical specimens from humans and dogs with a tentative diagnosis of leishmani osis. PCR and cultivation gave identical results with all but 1 of the 95 specimens from humans. The PCR result in this case was false negat ive, possibly because of unequal apportionment of this sample. With 10 skin biopsies from fix patients with cutaneous Leishmaniosis, the sen sitivity nas 60%. For six human immunodeficiency virus-positive patien ts with visceral leishmaniosis, all bone marrow biopsies and 7 of 11 w hole blood samples (after isolation of leukocytes by Ficoll-Paque) wer e positive in both tests. PCR detected one more case dth the use of 50 0 mu l of whole blood with direct lysis of the erythrocytes in Tris-ED TA buffer. With dog lymph node aspirates, the sensitivity was 100% (16 of 16 samples) for both methods; furthermore, PCR was positive for 5 of 13 whole blood samples from dogs with leishmaniosis. The specificit y of the PCR was 100% (70 specimens from patients without leishmaniosi s). This PCR assay proved to be feasible as a routine diagnostic test, being reliable and faster than in vitro cultivation.