4. Discussion

3. Results

Quintana Roo (Tables 1 and 2, Figure 1) [17].

from Nayarit and several skin samples from Campeche state.

Campeche and some from Nayarit states (Figure 1) [20].

tuberculosis, did not amplify [17].

158 The Epidemiology and Ecology of Leishmaniasis

did not hybridize.

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

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.

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

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

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

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 In Mexico since 1985, cases of LCL, DCL, MCL, and VL clinical expressions were reported in 15 states; the species involved were L. (L.) mexicana, L. (V.) braziliensis, and L. (L.) chagasi. LCL was the most common, and all cases were considered caused by L. (L.) mexicana [6, 22]. The five major foci of Leishmania transmission were in rain forest of southern Campeche, La Chontalpa (the cocoa-producing district of Tabasco), and the southern coffee producing of Nayarit, southern Quintana Roo, and Chiapas (Figure 1).

In Nayarit, state of the Pacific endemic region, LCL was recorded in Caleras de Cofrados since 1987 [22], a district near Tepic, the state capital city (Figure 1). The etiological agent was thought to be L. (L.) mexicana. In our studies using kDNA PCR and hybridization techniques, we have demonstrated that the L. braziliensis complex is present in Nayarit, and we were able to distinguish between two variants or two different species of L. (V.) braziliensis. We believe this was the first report of L. (V.) braziliensis in Nayarit, Mexico [20]. The population affected with skin lesion were 5–65 years old; males were the most affected and their main activity was the harvesting and/or growing coffee. The possible vectors are Lutzomyia cruciata, Lutzomyia diabolica, and Lutzomyia shannoni, which were captured and identified at the plantation. In relation with the animal reservoirs, no studies have been reported [20].

Biopsies, clinical samples, and isolates from LCL patients from several districts of Campeche state, mainly from Calakmul, were PCR amplified with specific primers for kDNA of L. braziliensis and L. mexicana complex members and primers specific for Mexican strains of L. mexicana [19] and also were analyzed by ITS1 PCR-RFLP [12]. We detected in Northern Calakmul 43% of cases infected with L. mexicana, 25% of cases with L. braziliensis complex members, 62% of mixed infection of Mx L. mexicana + L. (L.) mexicana, and 25% of cases infected with L. braziliensis complex + L. (L.) mexicana. The most affected community of this area was La Mancolona, with a 6.5% of prevalence; this village is located 3–4 km away from the crops and is more urbanized due to deforestation (Figure 3a). The most affected population in this village were adult males (66%) [19].

In central Calakmul 15% of the cases were infected with L. (L.) mexicana, 25% of the cases infected with L. braziliensis complex members, and 37% of the cases infected with Mx L. mexicana L. (L.) mexicana. La Guadalupe village had the highest prevalence rate (2.2%) and children were the most affected (67%) [19].

In southern Calakmul 25% of the cases were infected with L. (L.) mexicana, 62% with L. braziliensis complex members, and 75% with both L. (L.) mexicana and L. braziliensis complex members. Dos Lagunas Sur was the most affected community, located close to the border with Belize, with 12% prevalence (Figure 2c). People in this village farm chili crops around their houses, which are located very close to the forest, and the population affected were children (50%), women, and men (50%) (Figures 2a–c and 3a–c) [19]. In relation to the vectors, L. mexicana infections in two sand fly species, Lu. shannoni and Lutzomyia ylephiletor, were found in Dos Lagunas Sur, whereas in La Mancolona, L. (L.) mexicana infections were found in Lu. shannoni, Lu. cruciata, Lu. o. olmeca, and Lu. Panamensis [23].

Regarding to the animal reservoirs, L. (L.) mexicana was identified in four species of wild rodents: the black-eared rice rat, Oryzomys melanotis; the hispid cotton-rat, Sigmodon hispidus; the big-eared climbing rat, Ototylomys phyllotis; and the Yucatan deer mouse, Peromyscus yucatanicus [24].

We found most of the cases of DCL in the states of Tabasco and Veracruz (Figure 1). These states have a common border in the endemic region of the Gulf of Mexico and are

Figure 2. Patients from the endemic Gulf of Mexico region, with skin lesions suffering from cutaneous leishmaniasis.

Survey of Cutaneous Leishmaniasis in Mexico: *Leishmania* Species, Clinical Expressions and Risk Factors http://dx.doi.org/10.5772/65501 161

In central Calakmul 15% of the cases were infected with L. (L.) mexicana, 25% of the cases infected with L. braziliensis complex members, and 37% of the cases infected with Mx L. mexicana L. (L.) mexicana. La Guadalupe village had the highest prevalence rate (2.2%) and

In southern Calakmul 25% of the cases were infected with L. (L.) mexicana, 62% with L. braziliensis complex members, and 75% with both L. (L.) mexicana and L. braziliensis complex members. Dos Lagunas Sur was the most affected community, located close to the border with Belize, with 12% prevalence (Figure 2c). People in this village farm chili crops around their houses, which are located very close to the forest, and the population affected were children (50%), women, and men (50%) (Figures 2a–c and 3a–c) [19]. In relation to the vectors, L. mexicana infections in two sand fly species, Lu. shannoni and Lutzomyia ylephiletor, were found in Dos Lagunas Sur, whereas in La Mancolona, L. (L.) mexicana infections were found in Lu.

Regarding to the animal reservoirs, L. (L.) mexicana was identified in four species of wild rodents: the black-eared rice rat, Oryzomys melanotis; the hispid cotton-rat, Sigmodon hispidus; the big-eared climbing rat, Ototylomys phyllotis; and the Yucatan deer mouse, Peromyscus

We found most of the cases of DCL in the states of Tabasco and Veracruz (Figure 1). These states have a common border in the endemic region of the Gulf of Mexico and are

Figure 2. Patients from the endemic Gulf of Mexico region, with skin lesions suffering from cutaneous leishmaniasis.

children were the most affected (67%) [19].

160 The Epidemiology and Ecology of Leishmaniasis

yucatanicus [24].

shannoni, Lu. cruciata, Lu. o. olmeca, and Lu. Panamensis [23].

Figure 3. Communities situated in the leishmaniasis endemic region of Gulf of Mexico. People in these villages farm chili crops around their houses, located very near the forest close to the border of Belize and Guatemala.

characteristically tropical rain forest, with considerable rainfall and important agricultural activities, including the production of cocoa, sugar cane, and rubber. We collected isolates from patients with DCL or LCL in these states and some from Campeche. Their DNA was amplified with primers M1 and M2 [17] specific for kDNA of L. mexicana complex. The size of PCR products (680–720 bp) of the Mexican isolates is more similar to the size of the PCR products (700 bp) of L. (L.) amazonensis group than the PCR products (800–820 bp) of L. (L.) mexicana BEL21. The isolate PCR products hybridized with probe 9.2 specific for the L. mexicana complex. Their DNA was also analyzed using ITS1 PCR-RFLP, and we confirmed the presence of both DNA of L. (L.) amazonensis and L. (L.) mexicana in the same isolate (Table 2) [12, 17].

In Mexico, it has been reported that VL was caused by L. (L.) chagasi and confined to Central endemic region [22]. Subsequently, in the Pacific endemic region states of Chiapas, Guerrero VL was detected. In Tabasco, only cases of LCL and DCL caused by L. (L.) mexicana have previously been reported [25]. In our studies by kDNA analysis, we have found VL cases in Tabasco (a 6-month-old immunosuppressed male) [21] and in Chiapas (a 36-year-old male coinfected with HIV and Pneumocystis carinii) to be caused by L. (L.) mexicana [26]. These findings are important because it indicates that these species, typically cutaneous, can visceralize in immunocompromised patient, and in Mexico, MCL, LCL, and VL coexist in some endemic areas. This is the first case reported in Mexico of coinfection by L. (L.) mexicana and HIV, which was manifested as VL. Our results agree with those found in Hernandez [26], who reported in Venezuelan patient displaying the symptoms of VL, a coinfection with HIV and a Leishmania variant strain sharing kDNA sequences with L. braziliensis and L. mexicana [27].

Treatment of CL patients with Glucantime® was successful in 96% of cases, regardless of the number and location of lesions. To obtain complete healing of lesions, the doses needed were in children from 2 to 20 and in adults from 2 to 67 ampules, although some patients cure spontaneously [19].

In the endemic areas evaluated in the present studies, the risk factors associated with CL were identified as the human colonization of large areas of previously untouched rain forests, where CL is endemic. The urbanization and deforestation are important factors because the Leishmania transmission cycles are adapting to peridomestic environments and are spreading to previously no endemic areas with domestic animals as potential reservoirs and spending nocturnal periods in the forest for cultivation of agricultural crops (e.g., chili and coffee) (Figure 3a–d) [11, 19, 20].
