Reference strains used in this study.
Leishmaniasis is caused by Leishmania sp., which is transmitted to human beings and reservoirs by phlebotomine sand flies, with worldwide prevalence of approximately 12 million cases with population at risk of approximately 350 million. Cutaneous leishmaniasis (CL) is the most widespread form, causing localized skin lesions (LCL), mucocutaneous leishmaniasis (MCL), or nodular lesions in diffused cutaneous leishmaniasis (DCL). American CL includes LCL and DCL caused by Leishmania mexicana complex and MCL caused by the Leishmania braziliensis complex. In Mexico, CL is distributed in three endemic areas, Gulf of Mexico, Pacific of Mexico, and Central Mexico. In order to monitor clinical outcome and adequately target treatment as well as epidemiologic studies, diagnostic kinetoplast DNA (kDNA), polymerase chain reaction (PCR), Southern and dot blotting, and ITS1 PCR-RFLP of Leishmania DNA were evaluated in samples and Leishmania isolates from patients with cutaneous ulcers from several endemic areas. In Mexico, LCL can be caused by the L. mexicana, L. braziliensis, or both complexes. DCL is caused by L. (L.) mexicana or Leishmania (L.) amazonensis and visceral leishmaniasis (VL) by Leishmania (L.) chagasi and L. (L.) mexicana in immunocompromised patients. The geographic range in which CL is endemic has increased due to urbanization, new settlements, and ecological, social, and educative conditions, which favors its permanence and transmission.
- cutaneous leishmaniasis
Leishmaniasis is a group of clinical entities present in 79 countries at a rate of 400,000 cases per year. The World Health Organization estimates a worldwide prevalence of approximately 12 million cases with population at risk of approximately 350 million. It is caused by a parasitic protozoan, which belongs to the Leishmania genus that is transmitted to human beings and animal reservoirs by phlebotomine sand flies .
Cutaneous leishmaniasis (CL) is the most widespread form, causing primary localized skin lesions from which parasites can disseminate to the nasopharyngeal mucosa and cause mucocutaneous leishmaniasis (MCL) or disseminate to the entire body as nodular lesions in diffused cutaneous leishmaniasis (DCL). Visceral leishmaniasis (VL) is the most severe form of the disease; according to the WHO in areas endemic for VL, many people have asymptomatic infection and a concomitant HIV infection increases the risk of developing active VL by between 100 and 2320 times .
American cutaneous leishmaniasis is characterized by a spectrum of clinical presentations caused by Leishmania species grouped in complexes; these include LCL caused by Leishmania (L.) mexicana; DCL caused by Leishmania amazonensis, Leishmania venezuelensis, and Leishmania pifanoi, all of them belonging to the L. mexicana complex; and MCL caused by members of the L. braziliensis complex. VL is caused by L. (L.) chagasi belonging to the L. donovani complex. Symptomatic diagnosis confuses CL with unrelated disorders such as tropical ulcers, sporotrichosis, leprosy, and skin cancer, among others .
In Mexico, Seidelin first recorded LCL caused by L. (L.) mexicana in 1912, who called it “chiclero’s ulcer,” because he found the disease in rubber workers. CL is distributed in three main endemic areas: Gulf of Mexico, Pacific of Mexico, and Central Mexico. In these regions, multiple species of Leishmania may coexist and several species can cause both LCL and MCL [5–7]. Several methods of detection of Leishmania based on deoxyribonucleic acid (DNA) have been described. The polymerase chain reaction (PCR) has been employed for selective amplification of Leishmania DNA. Several molecular targets for a diagnostic PCR have been evaluated including the minicircle kinetoplast DNA (kDNA), the miniexon (spliced leader RNA) gene, and the internal transcribed spacer (ITS) [8–10], among others.
2. Materials and methods
In order to find a diagnostic method for leishmaniasis that combines high sensitivity with species differentiation in the field, rapid diagnosis, and low cost, several molecular targets for a diagnostic PCR were evaluated from patients with cutaneous ulcers suspected of having LC from several endemic areas. The target was minicircle kinetoplast DNA (kDNA) using specific primers or probes with the PCR and Southern or dot blotting  and PCR-RFLP of the internal transcribed spacer 1 (ITS1) [10, 12].
Distribution of CL or VL in social, educative, and ecological conditions was recorded. The patients diagnosed with CL were treated with meglumine antimoniate (Glucantime®).
2.1. Patient population
In these studies, we evaluated samples from patients with clinical symptoms and skin lesions suggestive of CL, MCL, and DCL from several endemic areas of Mexico—Campeche, Tabasco, Veracruz, Nayarit and Chiapas, and Quintana Roo—or samples from VL patients from Chiapas and Tabasco states. The clinical samples were taken on filter papers or smears, needle aspirates, and tissue biopsy samples (1–2 mm) from the edge of cutaneous or bone marrow aspirates (Figure 1).
2.2. Ethical considerations
For bleeding human beings for diagnosis and therapeutics, informed consent was obtained from all the adults who participated in the study. Consent for including young children was obtained from their parents or guardians. The ethics committee of the corresponding health authorities, in agreement with International Ethical Guidelines for Biomedical Research involving human subjects (Norma Oficial Mexicana de Salud: NOM-003-SSA 2-1993), reviewed and approved the protocols of the present studies.
2.3. Leishmania reference strains and Mexican isolate culture conditions
Reference Leishmania strains (Table 1), used as control and Mexican isolates of Leishmania from Tabasco, Veracruz, Campeche, and Quintana Roo states (Table 2 and Figure 1), were cultured in Roswell Park Memorial Institute medium 1640 (RPMI medium 1640) supplemented with 10% fetal calf serum at 26°C. DNAs of Trypanosoma cruzi and Mycobacterium tuberculosis were used as negative controls.
|1||MHOM/BZ/82/BEL21||BEL21||L. (L.) mexicana|
|2||MHOM/BZ/62/M379||M379||L. (L.) mexicana|
|3||IFLA/BR/67/PH8||PH8||L. (L.) amazonensis|
|4||MHOM/BR/73/M2269||M2269||L. (L.) amazonensis|
|5||MHOM/PE/84/LC53||LC53||L. (V.) braziliensis|
|6||MHOM/BR/84/LTB300||LTB300||L. (V.) braziliensis|
|7||MHOM/BR/75/M2903||M2903||L. (V.) braziliensis|
|8||MHOM/BR/75/M2904||M2904||L. (V.) braziliensis|
|9||MHOM/BR/75/M4147||M4147||L. (V.) guyanensis|
|10||MHOM/PE/84/LC26||LC26||L. (V.) peruviana|
|11||MHOM/CR/87/NEL3||NEL3||L. (V.) panamensis|
|12||MHOM/PA/72/LS94||LS94||L. (V.) panamensis|
|13||MHOM/IN/80/DD8||DD8||L. (L.) donovani|
|14||MHOM/BR/74/PP75||PP75||L. (L.) infantum/chagasi|
|Number||Code||Origin||Clinical expression||Leishmania species|
|1||MHOM/MX/88/HRC JS||Tabasco||DCL||L. am + L. mex|
|2||MHOM/MX/88/HRC MC||Tabasco||LCL||L. (L.) mexicana|
|3||MHOM/MX/84/ISET GS||Tabasco||DCL||L. am + L. mex|
|4||MHOM:MX:83:UAVY CV||Yucatan||LCL||L. (L.) mexicana|
|5||MHOM/MX/85/ISET HF||Veracruz||DCL||L. am + L. mex|
|6||LVER||Veracruz||DCL||L. am + L. mex|
|7||REP||Campeche||LCL||L. am + L. mex|
|8||MHM/MX/06/ENCB/MIC||Campeche||LCL||L. (L.) mexicana|
|9||MHM/MX/06/ENCB CDL||Campeche||LCL||L. (L.) mexicana|
|10||MHM/MX/06/ENCB FDL||Campeche||LCL||L. (L.) mexicana|
|11||CR||Campeche||LCL||L. (V.) braziliensis|
|12||PVS||Campeche||LCL||Mx. L. mexicana|
|13||RGL||Campeche||LCL||L. b + L. mx|
|14||FJJ||Campeche||LCL||L. b + L. mx|
|15||ESP||Campeche||LCL||Mx. L. mexicana|
2.4. Isolation of DNA
Clinical specimens cut from the filter paper or eluted from the smear, bone marrow aspirates, skin aspirates, and tissue biopsy samples (1–2 mm) were incubated in 250 μL of cell lysis buffer for 1 h at 56°C. DNA from Leishmania cultures was prepared by centrifuging 109 parasites in the exponential phase of growth at 2000 × g for 10 min at 4°C. The DNA was extracted from the pellet using the High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer’s instructions. The DNA was stored at −20°C until used.
2.5. Polymerase chain reaction
PCR analysis of kDNA for subgenus Leishmania was carried out by using the AJS1 and DeB8 primers . PCR of the L. mexicana complex was carried out using the M1 and M2 primers  and the LMO1 and LMO2 primers specific for minicircles of Mexican L. (L.) mexicana strains ). PCR of the L. braziliensis complex was done with the B1 and B2 primers . PCR for L. donovani complex was done with the D1 and D2 primers . PCR amplification conditions were performed as described previously [8, 13, 14, 16, 17].
2.6. PCR analysis of genomic DNA of L. (V.) braziliensis
PCR species specific for nuclear DNA from variants of L. (V.) braziliensis was carried out by using the primers 3J1 and 3J2. Amplification conditions were as described elsewhere .
2.7. PCR analysis of the internal transcribed spacer 1 (ITS1)
Some samples were analyzed for ITS1 PCR using the primers: LITSR and L5.8S . Amplification conditions were as described . PCR products were digested with HaeIII enzyme, according to the manufacturer’s instructions. The amplicons and restriction products were analyzed as described elsewhere .
2.8. Southern or dot blot hybridization of kDNA PCR products of biopsies, isolates and Leishmania reference strains
The kDNA PCR products of clinical samples, Mexican isolates and reference strains, were Southern or dot blotted onto nylon membranes and were hybridized with the cloned fragments of kDNA used as probes: B4Rsa, which hybridizes specifically to members of the L. donovani complex; 9.2 and 9.3, specific for the L. mexicana complex; and B18, specific for members of the L. braziliensis complex. The probes were labeled with DIG Random Primer DNA labeling kit (Boehringer Mannheim) and either visualized colorimetrically with NBT and BCIP (Boehringer Mannheim) or labeled with [32P]d ATP, using the Prime-it™ Random Primer DNA labeling kit (Stratagene). The hybridization conditions were described elsewhere [14, 17].
2.9. Administration of meglumine antimoniate (Glucantime®)
Patients diagnosed with CL accepted treatment with meglumine antimoniate (Glucantime®). Glucantime is marketed in 5 mL ampules containing 1.5 g of N-methyl-glucamine antimoniate, which corresponds to 425 mg of Sb51. Treatment consisted in one ampule by intramuscular injection per day until healing .
Primers DeB8 and AJS1, specific for the Leishmania (L.) subgenus , 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) .
PCR with the primers M1 and M2 specific for the L. mexicana complex  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 .
PCR specific for the L. braziliensis complex carried out with B1 and B2 primers  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) .
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) .
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].
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 , 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 . 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 .
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  and also were analyzed by ITS1 PCR-RFLP . 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%) .
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%) .
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) . 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 .
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 .
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 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  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 . 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 . In our studies by kDNA analysis, we have found VL cases in Tabasco (a 6-month-old immunosuppressed male)  and in Chiapas (a 36-year-old male coinfected with HIV and Pneumocystis carinii) to be caused by L. (L.) mexicana . 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 , 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 .
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 .
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].
In conclusion, our findings are interesting because we have shown that in the typical endemic regions of Gulf of Mexico and Ocean Pacific of Mexico, CL can be caused by several species of the L. mexicana and L. braziliensis complexes and in some clinical samples, we found DNA of both complexes. Furthermore, we found DCL caused by a mix infection with strains of L. (L.) amazonensis and L. (L.) mexicana , both belonging to the L. mexicana complex. VL can be caused by L. (L.) chagasi and in immunocompromised patients by L. (L.) mexicana. Diagnosis of leishmaniasis by PCR and hybridization of kDNA and ITS1 PCR-RFLP analysis of Leishmania DNA must be combined for the reliable characterization of Leishmania species mainly in endemic areas where the presence of multiple species of Leishmania overlap clinical pictures demands simultaneous species identification . In Mexico, the geographic range in which CL is endemic has increased in size due to urbanization, new settlements, and ecological, social, and educative conditions, which favors its permanence and transmission, as it has occurred in Calakmul.
Financial support for this research was provided by Secretaría de Investigacion y Posgrado, Instituto Politecnico Nacional, Mexico, and Conacyt. Amalia Monroy-Ostria is a fellow of COFAA, Instituto Politecnico Nacional, Mexico. We thank Erik Fabila-Monroy, MBA, for reviewing the English of the manuscript.