h Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r ‫؍‬ 0.87; P < 0.0001), but the former showed higher sensitivity (P ‫؍‬ 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P ‫؍‬ 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P ‫؍‬ 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.

Filter paper PCR constitutes a sensitive and specific alternative to traditional diagnostic assays. This novel, rapid, well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is most difficult to perform, and can potentially be used for rapid species identification.

BackgroundCutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion.Methodology/Principal FindingsWe applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4).Conclusion/SignificanceOur results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.

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Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific.

* LST = leishmanin skin test; NNN = Novy-MacNeal-Nicolle; kDNA = kinetoplast DNA; PCR = polymerase chain reaction.† Four persons did not undergo leishmanin skin testing. ‡ Per patient analysis.

We compared traditional cutaneous leishmaniasis diagnostic methods to filter paper lesion impression (FPLI) PCR for secondarily infected ulcers and nonulcerative lesions. The sensitivity and specificity of FPLI PCR for secondarily infected lesions (n ‫؍‬ 8) were 100%. In primarily nonulcerative lesions (n ‫؍‬ 15), the sensitivity of FPLI PCR was inferior to that of pooled-invasive-specimen PCR (72.7% versus 100%) (P ‫؍‬ 0.10). FPLI PCR is sensitive, specific, and unlike invasive procedures, can be used in secondarily infected ulcers. Invasive specimen collection is superior in nonulcerative lesions.

Background Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus identified in 20–25% of Viannia —species endemic to Latin America, and is believed to accelerate cutaneous to mucosal leishmaniasis over time. Our objective was to quantify known virulence factor (VF) RNA transcript expression according to LRV1 status, causative species, and isolate source. Methods Eight cultured isolates of Leishmania were used, four of which were LRV1-positive ( Leishmania Viannia braziliensis [ n = 1], L . ( V .) guyanensis [ n = 1], L . ( V .) panamensis [ n = 2]), and four were LRV1-negative ( L . ( V .) panamensis [ n = 3], L . ( V .) braziliensis [ n = 1]). Promastigotes were inoculated into macrophage cultures, and harvested at 24 and 48 h. RNA transcript expression of hsp23 , hsp70 , hsp90 , hsp100 , mpi , cpb , and gp63 were quantified by qPCR. Results RNA transcript expression of hsp100 ( p = 0.012), cpb ( p = 0.016), and mpi ( p = 0.022) showed significant increases from baseline pure culture expression to 24- and 48-h post-macrophage infection, whereas hsp70 ( p = 0.004) was significantly decreased. A trend toward increased transcript expression of hsp100 at baseline in isolates of L . ( V .) panamensis was noted. Pooled VF RNA transcript expression by L . ( V .) panamensis isolates was lower than that of L . ( V .) braziliensis and L . ( V .) guyananesis at 24 h ( p = 0.03). VF RNA transcript expression did not differ by LRV1 status, or source of cultured isolate at baseline, 24, or 48 h; however, a trend toward increased VF RNA transcript expression of 2.71- and 1.93-fold change of mpi ( ...