We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.The leishmaniases are a group of illnesses of the skin, oral and respiratory mucosae and the reticuloendothelium caused by protozoa of the genus Leishmania. Of these, the cutaneous form is the most widespread, afflicting primarily rural and periurban populations exposed to the infected sand fly vector. Cutaneous leishmaniasis (CL) is most frequently diagnosed by clinical evaluation, either alone or in combination with the leishmanin skin test. Clinical evaluation usually suffers from lack of standardization (13,38) and is hampered by the fact that even fairly typical acute lesions can be confused with other dermatological problems, such as sporotrichosis (9). The leishmanin skin test is highly sensitive but lacks specificity when employed in areas of endemicity because it cannot distinguish acute lesions from past infection. A definitive, laboratory diagnosis of mucosal or cutaneous leishmaniasis traditionally requires either the visualization of amastigotes or the isolation of replicative Leishmania from lesions (24). The most widely employed laboratory methods for CL are microscopic examination of lesion scrapings, biopsy impression smears, and histopathology. The most sensitive conventional diagnostic methods, culture of lesion biopsies and aspirates, are available only in reference laboratories. Even these less available, more sensitive methods ...

Phenotypic characterization of 511 strains of Leishmania, subgenus Viannia, isolated from Colombian patients was conducted based on electrophoretic polymorphisms of 13 isoenzymes. Ninety-one Colombian strains of L. braziliensis were the most heterogeneous, constituting seven zymodemes while 397 L. panamensis and 22 L. guyanensis strains yielded five and three zymodemes, respectively. Phosphogluconate dehydrogenase, nucleoside hydrolase, and superoxide dismutase were the most polymorphic enzymes in this collection of strains, and together with glucose-6-phosphate dehydrogenase, allowed the discrimination of the three aforementioned species. Hierarchical cluster analysis of the zymodemes using Jaccard's coefficient of similarities revealed two clusters, one constituted by L. braziliensis zymodemes, and another by three subgroups consisting of zymodemes of L. panamensis closely related to the species reference strain, another consisting of L. guyanensis zymodemes, and a third group distinguished by new electromorphs of proline iminopeptidase and aspartate aminotransferase that reacted with the L. panamensisspecific monoclonal antibody B-11. Multiple zymodemes of L. panamensis and L. guyanensis were found to be sympatrically transmitted in foci along the Pacific coast. Leishmania braziliensis variants were ubiquitous throughout the territory of Colombia; L. panamensis was prevalent in the western region and L. guyansis was prevalent in the Orinoco and Amazon river basins in the eastern half of the country. Distinct zymodemes of L. panamensis predominated in the northern and southern regions of the Pacific coast. Nine zymodemes of all three species were isolated from mucosal lesions. Zymodeme 1.1 of L. braziliensis had the highest frequency of mucosal involvement (10% of the cases), and disease caused by this zymodeme had the longest mean time of evolution (31 months; P ϭ 0.002). In addition to being useful in describing epidemiologic relationships, the intraspecific heterogeneity of strains of the Viannia subgenus within and among foci can be used to understand such fundamental questions as the pathogenicity of different populations of parasites, and the induction of cross-protection against related parasites.

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This prospective study measured the incidence of Leishmania infection, by Leishmanin skin test (LST) conversion, and leishmaniasis, by new acquisition of lesions, in a Leishmania braziliensis endemic area of Colombia, during 7243 person-years. The incidence rate of infection and leishmaniasis varied greatly by village, ranging from 2.8 to 23.0/100 person-years and 0.0 to 20.4/1000 person-years, respectively. Adult males experienced greater rates of both infection and leishmaniasis. Most primary infections (91%) were subclinical initially. Typical scars were predictive of subsequent leishmaniases both for persons initially LST-reactive (risk ratio = 11.3, P = .003) and for those initially nonreactive (risk ratio = 3.2, P = .02). Only one-third of the diagnosed leishmaniasis cases (24/77) were due to newly acquired infections in naive hosts. The relative contribution of existing lesions, recurrences, and new infections to the burden of disease should be considered in the planning of leishmaniasis control programs.