Development of a PCR based diagnostic system for kala azar

Abstract

Kala-azar or black fever is a severe form of Leishmaniasis, a disease caused by protozoan parasite, Leishmania spp. one of the largest parasites-borne illnesses, this disease could be fatal, if untreated, as a consequence of infection to different internal organs such as liver, spleen, and bone marrow resulting in death. The conventional approach for the diagnosis of the disease relies on collecting samples from bone marrow and splenic tissues from suspected patients, a method which is not only painful, stressful and risky for patients, but also cumbersome. This prompted us to develop a simple, rapid PCR-based diagnostic assay for the detection of Leishmania using patient‟s venous blood sample. A total of thirty five clinically suspected febrile patients who had sign and symptoms for the disease, and produced positive reaction for rk39 immunochromatographic test were taken for the diagnosis. In addition to the blood samples, specimens from bone marrow/ splenic aspirations were collected from those individuals. While presence of Leishmania spp. was confirmed in twenty six patients either by microscopy and by culture on Nicolle Novy McNeal (NNN) culture media from the samples collected from bone marrow/spleen aspirations of patients with Kala-Azar, however their buffy coat preparations of blood samples could not produce the same, indicating the unsuitability of the blood samples to be used as a specimen for diagnosis by conventional methods. In an attempt to address whether those blood samples could be used for diagnosis, a pair of oligonucleotides was designed using conserved sequences of kinetoplast DNA minicircles of Leishmania spp and was used as primers to detect the presence of Leishmania genome in a polymerase chain reaction. In addition, blood samples collected from sixteen and twenty five healthy individuals from the endemic and non-endemic regions respectively, and twenty five patients with other similar diseases (TB, malaria and dengue) were used as controls. All the twenty six blood samples from patients with Kala-azar yielded specific amplifications in PCR, while blood samples taken as controls produced no amplicon, producing the sensitivity and specificity records as 98% and 100% respectively. Such an analysis was found superior when compared with two other sets of primers reported in the literature. The identity of the amplicon was subsequently confirmed as Leishmania by DNA sequencing and BLAST search. Therefore, the primers designed for this study could be used to score the presence of Leishmania in a PCR-based detection system.