Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.
Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene ( hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L. ) amazonensis, L. (L. ) mexicana, L. (Viannia) lainsoni, L. (V. ) braziliensis, L. (V. ) guyanensis, L. (V. ) naiffi and L. (V. ) shawi, and three species found in Eurasia and Africa, including L. (L. ) tropica, L. (L. ) donovani and L. (L. ) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.
HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.
The different clinical forms of the Leishmaniases range from cutaneous to visceral infections and are caused by organisms belonging to the genus Leishmania. Controversy over the validity of different molecular methods to correctly identify a species hinders the association of a given species with different clinical forms, complicating the prognosis and the development of suitable treatment protocols. A correct identification leads to a better understanding of the action and consequent development of new drugs and immunological reactions. It also provides important information about the relationship of each species with its hosts (humans, animal reservoirs and sandflies) in different geographical areas and ecological situations, helping to design control strategies. Today, PCR is the most commonly used method for Leishmania identification, but even though several targets have been described, no simple and direct protocol has emerged. In this paper, we coupled hsp70 real-time PCR with the determination of amplicon melting profiles in order to explore polymorphic regions by HRM analysis. This methodology yielded discriminatory melting temperature (Tm) values for Brazilian and Eurasian/African Leishmania species. The protocol has proven to be 100% reliable with both clinical and experimental samples. The major advantage of the presently described method is that it is simple, less expensive, highly sensitive and easily automated.