American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous, diffuse cutaneous, and mucocutaneous leishmaniasis which can be caused by different strains of Leishmania belonging to the L. mexicana or L. braziliensis complexes which may coexist in the same endemic area. We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania. In relation to the Mexican isolates of Leishmania 52% displayed a pattern similar to the L. (L.) mexicana, 5% showed a mixed pattern compatible with L. (L.) mexicana and L. (V.) braziliensis, eight with L. (L.) amazonensis and L. (L.) mexicana, and one to L. (V.) braziliensis. Most of the clinical samples, 109/116 (94%), gave a pattern similar to that of the L. mexicana, two clinical samples gave similar patterns to that of Leishmania braziliensis, and 5 samples gave patterns that suggest a coinfection of L. (L.) mexicana and L. (V.) braziliensis or L. (L.) mexicana and L. (L.) amazonensis. The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enables determination of the infecting species of New World Leishmania in the field in relatively short time and low cost.

An epidemiological study was carried out in the northernUntil 1994 only four cases of MCL were recorded in Mexico. These cases were confined to the states of Oaxaca, Veracruz and Tabasco and the causal agent was considered to be L. (L.) mexicana. In the State of Campeche, LCL was found to be caused by members of L. mexicana complex and by members of the L. braziliensis complex (Velasco et al. 1989a,b, OPS/OMSSecretaria de Salud 1994, Perez-Motul et al. 1994, Hernandez-Montes et al. 1998.Between 1987 and 1994, 326 cases of LCL have been recorded in Nayarit. These cases were attributed to infection with L. (L.) mexicana. Diagnosis of LCL was carried out by serology and skin testing, however Leishmania species were not identified. The presence of L. (V.) braziliensis has, until now, not been recorded in Nayarit (OPS/OMS 1994). Calera de Cofrados, district of Nayarit endemic for LCL, is near to the Pacific coast where the climate is hot with high humidity woods with deep hills surrounded by small brooks.

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Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR 1 Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR 1 Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

Objective. To study cutaneous leishmaniasis (CL), in the Calakmul municipality of the Campeche State, during two years. Materials and methods. Individuals with skin lesions were evaluated. Aspirates taken from the lesions were cultured, PCR was performed to diagnose the Leishmania species. Results. The culture detected 42% of the samples. PCR diagnosed CL in 76% of the samples; of those 38% were from children and 62% from adults. 89% of the patients were infected with L. mexicana; 14.4% with Mexican strains of L.mexicana; 7% with L. braziliensis; 3.6% with L. mexicana and L. braziliensis. The most affected villages with CL were Dos Lagunas Sur with 12.3%, La Mancolona with 6.5% and La Guadalupe with 2.2% of prevalence, respectively. After the treatment with Glucantime, 96% of the patients were healed. Conclusion. CL is an important public health concern in Calakmul, and the parasite causing it belongs to Leishmania mexicana and Leishmania braziliensis complexes.