Abstract:
Visceral leishmaniasis is caused by parasites of L. donovani species complex that can spread to
internal organs and cause death. The diagnostic methods of Leishmaniasis are based on clinical
sign and symptoms, microscopy, serological, and molecular techniques. Because of a broad
spectrum of diverse clinical manifestations and the similarities of the responses to different
species, identification to the species level is often difficult. Therefore, the objective of this study
was to evaluate the PCR-RFLP assay of the ITS1 region for direct identification of Leishmania
donovani species from clinically confirmed smear positive and negative slide samples. Ninety
smears positive (47) and negative (43) slide samples of visceral leishmaniasis were taken from
Leishmaniasis Research and Treatment Center, at the University of Gondar Comprehensive
Specialized Hospital. The DNA was extracted from 85, clinical samples using the phenol -
chloroform method, whereas the 5 slides were wasted during the extraction. Tenfold serial
dilution was performed from Leishmania donovani reference strain DNA, for determination of
detection limits of the ITS1-PCR method. The statistical analysis of the present study was
performed by using chi-square and kappa index. The ITS1 region was amplified at 320bp using
LITSR/L5.8S genus specific primers, then the ITS1-PCR products were subjected to RFLP assay
using HaeIII restriction enzyme. Of the 85 smear positive (44) and negative (41) clinical
samples, 39 (45.9%) true positive, 5 (5.9%) false positive, 6(7.1%) false negative and 35 (41.2%)
true negative results were obtained. According to this result and considering microscopy as the
gold standard, the sensitivity, specificity, positive predictive values and negative predictive
values of the ITS1- PCR technique were 86.7%, 87.5%, 88.6%, and 85.4% respectively. All ITS1-
PCR positive clinical samples were identified as belonging to the Leihmania donovani species by
PCR-RFLP patterns and the digestion revealed three different bands (190bp, 80bp and 50bp). In
conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of
Leishmania donovani species in the clinical samples of visceral leishmaniasis patients than the
microscopic method, depending on statistical analysis of the present finding there is also
significant association and substantial levels of degree of agreement between the two methods.
Direct identification of visceral leishmaniasis from clinical samples irrespective of species and
genus level, ITS1-RFLP is recommendable than a microscope.
