The development and/or improvement of diagnostic tools remain a priority in the research field of leishmaniasis. In particular, much work has to be done for increasing the sensitivity and specificity of the diagnostic tests for ATL in endemic areas, such as NW Argentina . In this work, we assessed both the impact of adding a PS-PCR analysis to the diagnosis-methods currently used in Argentina, and its usefulness to the identification of the causal-agent of the disease at the species level.
Indeed, by performing PS-PCR directly on clinical samples, we enhanced significantly the efficacy of the ATL diagnosis, especially for MCL. Appling this method to patients with negative results for the microscopic search of parasites in dermal smears, allowed us to increase substantially the diagnostic sensitivity from 70.5% for smears alone to 97.6% for the combination (Table 3). Thus, these results confirm that the PCR analysis is a valuable and applicable strategy for these cases of suspected ATL with negative microscopic examination. The relevance of this strategy becomes apparent when considering the availability of effective treatments or the tendency of the disease to evolve into severe clinical forms if left untreated. These factors indicate that the sensitivity of the diagnostic method should have the priority for maximization, rather than its specificity . Previous PCR analyses for the diagnosis of leishmaniases displayed sensitivities ranging from 51.7% to 100% [18–20]. This wide variability in the sensitivity may very well be due to the methods employed, but also attributable to differences in the sampling techniques, or the clinical and laboratory criteria applied for defining the presence or absence of the disease . The sensitivity of the PS-PCR analysis estimated in this study (81%) is in close agreement with those reported by other investigators when the method was performed on samples from cutaneous lesions of patients with ATL from endemic areas of Ecuador [21, 22]. Among the reasons for false negative PS-PCR results, the low parasite burden observed in the borders of the lesions might be of importance. Therefore, improving the sampling method, for example, using a syringe to aspirate the sample or collecting the exudates after scraping the lesions, in addition to efficient DNA extraction, would appear to substantially increase sensitivity and diagnostic efficacy .
In the present work, three non-ATL cases were PS-PCR positive, lowering the specificity to 84.2%. Since these patients presented atypical lesions, and were negative for the microscopic parasitological examination and the MST test, we think that these results were due to contamination of the DNA sample.
An identification of Leishmania spp. with PS-PCR applied directly on material scrapped from the lesions was achieved in 70.5% of ATL cases. Leishmania (V.) braziliensis was the most prevalent species (90.3%) among ATL patients, and this is consistent with previous studies aimed to characterize Leishmania stocks isolated in NW Argentina [5, 23]. Since we performed the analysis on samples collected directly from the lesions, the indicators of species prevalence reported here might be more accurate than those published before. Moreover, the method has additional advantages, as it gives the Leishmania spp. identification without further procedures, such as digestion analysis of the PCR products or gene sequencing, and it is applicable in relatively low-resource laboratories.
Leishmania (V.) braziliensis was involved in two cases DSCL reported for the first time in Argentina (Figure 1). Local ATL cases due to L. (V.) guyanensis were also found, confirming our previous report on the presence of this species in this area . These findings are of clinical relevance for the patients, because they require a different treatment, such as a protocol with pentamidine rather than the pentavalent antimonials commonly prescribed in this area [19, 24]. Interestingly L. (V.) panamensis was identified in one sample (Figure 2C). This is the first case reported in Argentina. Although parasites belonging to the Leishmania mexicana or donovani complexes were found involved in cases of leishmaniasis in this area [4, 25], we could not prove their involvement in the present series (Table 4).