3. Results

Primers DeB8 and AJS1, specific for the Leishmania (L.) subgenus [13], amplified the kDNA of L. (L.) mexicana Bel 21, L. (L.) mexicana M379, L. (L.) amazonensis PH8, L. (L.) amazonensis M2269, L. (L.) donovani DD8, L. (L.) infantum/chagasi PP75, 10 Mexican strains of Leishmania, and many clinical samples from patients with skin lesion from Campeche, Tabasco, Veracruz, and Quintana Roo (Tables 1 and 2, Figure 1) [17].

PCR with the primers M1 and M2 specific for the L. mexicana complex [14] resulted in the amplification of kDNA of L. (L.) amazonensis PH8 and M2269 with a band size of 700 bp and L. (L.) mexicana BEL21 with a band size of 800–820 bp. This difference can be used diagnostically to distinguish between L. (L.) amazonensis and L. (L.) mexicana isolates. The size of the kDNA amplicons of the Mexican strains is more similar to the size of the amplicons of L. (L.) amazonensis group than the amplicons of L. (L.) mexicana. Negative controls, T. cruzi and M. tuberculosis, did not amplify [17].

PCR specific for the L. braziliensis complex carried out with B1 and B2 primers [8] produced a kDNA amplification band of 750 bp of L. (V.) braziliensis LTB300, LC53, L. (V.) braziliensis M2903, L. (V.) braziliensis M2904, L. (V.) braziliensis reference strains, and some skin biopsies from Nayarit and several skin samples from Campeche state.

In order to have a more accurate identification of the Leishmania species in Nayarit, the skin biopsies were PCR analyzed with primers 3J1 and 3J2 specific for DNA genomic of L. (V.) braziliensis. Most of the samples amplified giving a band of 617 bp. The PCR products hybridized positively with the LbJ38 probe, which is species specific for L. braziliensis complex [18, 20].

PCR with specific primers D1 and D2 for the L. donovani complex resulted in the amplification of kDNA of the L. (L.) donovani DD8 and L. (L.) infantum/chagasi PP75 reference strains, and bone marrow and liver biopsy from a patient from Chiapas with VL were amplified [16, 21].

PCR products of the kDNA of Mexican strains of Leishmania mexicana and clinical samples amplified with primers AJS1 and DeB8, specific for the subgenus Leishmania, were dot blotted and tested with probe 9.2, specific for the L. mexicana complex. The probe hybridized with high affinity to L. (L.) mexicana BEL21, the 10 Mexican strains of Leishmania mexicana, several samples and biopsies from Campeche state, and DNA from a bone marrow aspirate, from a patient from Tabasco, with VL; kDNA from the reference strains other than L. mexicana that did not hybridize.

PCR products amplified with primers B1 and B2, specific for the L. braziliensis complex, were Southern blotted and tested with probe B18, specific for the L. braziliensis complex. This probe hybridized to L. (V.) braziliensis LTB300 and to DNA from skin biopsies from patients from Campeche and some from Nayarit states (Figure 1) [20].

PCR with specific primers for ITS1 resulted in the amplification of the Leishmania reference strains, the Mexican strains and isolates of L. mexicana, and the clinical samples from Campeche giving 300–350 bp amplification bands. Restriction of the ITS1 gene amplicons of L. (V.) panamensis, L. (V.) guyanensis, and L. (L.) braziliensis reference strains with the endonuclease HaeIII generated patterns with two bands of 170 and 150 bp; L. (L.) amazonensis generated two bands of 220 and 140 bp; and L. mexicana generated three bands of 200, 80, and 40 bp.

Most of the Mexican strains and isolates of Leishmania displayed a restriction pattern similar to that of L. (L.) mexicana reference strain; nine of these were obtained from LCL patients from Campeche. Some showed a mixed pattern compatible with L. (L.) mexicana and L. (V.) braziliensis; some others showed a mixed pattern compatible with L. (L.) amazonensis and L. (L.) mexicana (Table 2) [11].

In relation to the clinical samples from Campeche, most of them amplified a restriction pattern similar to the L. (L.) mexicana reference strain. In some samples, extra bands of 50 and 25 bp were observed, suggesting a coinfection, as it was found in a previous study with kDNA PCR analysis of clinical samples that DNA from both L. (L.) mexicana and L. (V.) braziliensis was identified (Table 2) [11, 15].
