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 [11] 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).

protozoan, which belongs to the Leishmania genus that is transmitted to human beings and

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 [1]. VL is characterized by irregular fever, weight loss, swelling of the liver and spleen, and

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, sporotri-

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)

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 [11] and PCR-RFLP of the

Distribution of CL or VL in social, educative, and ecological conditions was recorded. The

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,

patients diagnosed with CL were treated with meglumine antimoniate (Glucantime®).

animal reservoirs by phlebotomine sand flies [1].

154 The Epidemiology and Ecology of Leishmaniasis

chosis, leprosy, and skin cancer, among others [4].

2. Materials and methods

2.1. Patient population

internal transcribed spacer 1 (ITS1) [10, 12].

anemia. After recovery, patients sometime develop chronic DCL [2, 3].

gene, and the internal transcribed spacer (ITS) [8–10], among others.

Figure 1. Map of Mexico, showing the Ocean Pacific, Gulf of Mexico, and Central Leishmaniasis endemic regions.

#### 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.


Table 1. Reference strains used in this study.

#### 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 [13]. PCR of the L. mexicana complex was carried out using the M1 and M2 primers [14] and the LMO1 and LMO2 primers specific for minicircles of Mexican L. (L.) mexicana strains [15]). PCR of the L. braziliensis complex was done with the B1 and B2 primers [8]. PCR for L. donovani complex was done with the D1 and D2 primers [16]. PCR amplification conditions were performed as described previously [8, 13, 14, 16, 17].
