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Background: Cutaneous 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 Findings: We 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/Significance: Our 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. [ABSTRACT FROM AUTHOR]

Background. Traditional detection of Leishmania from ulcers involves collection of invasive specimens that cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared traditional diagnostic methods with a molecular noninvasive filter paper-based method for the diagnosis of cutaneous leishmaniasis. Methods. Consecutive patients presenting to the Leishmania Clinic at Hospital Nacional Cayetano Heredia were enrolled. Polymerase chain reaction (PCR) was performed on lesion scrapings, aspirates, and filter paper impressions. The reference standard was any 2 of 5 tests positive: smear, aspirate culture, invasive-specimen PCR (scrapings and aspirates), filter paper PCR, and leishmanin skin test. Outcome measures were sensitivity and specificity. Leishmania speciation was performed by PCR--restriction fragment length polymorphism (RFLP) of positive specimens. Results. Forty-five patients with 66 lesions were enrolled. Of 52 lesions diagnosed as cutaneous leishmaniasis, 50 were positive by PCR of invasive specimens versus 48 by PCR of filter papers (P = .930). Sensitivity and specificity of PCR on invasively obtained specimens were 94.2% (95% confidence interval [CI], 87.9%-100%) and 92.9% (95% CI, 79.4%-100%). Sensitivity and specificity of filter paper PCR were 92.3% (95% CI, 85.1%-99.5%) and 100%. Culture, smear, and leishmanin skin test all had inferior sensitivities, compared with PCR of invasive or noninvasive specimens (P < .001). Of 50 specimens positive by PCR, 19 had sufficient DNA for PCR-RFLP analysis. Conclusions. 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. [ABSTRACT FROM AUTHOR]

Mucocutaneous leishmaniasis (MCL) is an important health problem in many rural areas of Latin America, but there are few data on the results of programmatic approaches to control the disease. We report the results of a control programme in San Martin de Pangoa District, which reports one of the highest prevalences of MCL in Peru. For 2 years (2001–2002), the technicians at the health post were trained in patient case management, received medical support and were supplied with antimonials. An evaluation after 2 years showed the following main achievements: better diagnosis of patients, who were confirmed by microscopy in 34% (82/240) of the cases in 2001 and 60% of the cases (153/254) in 2002; improved follow-up during treatment: 237 of 263 (90%) patients who initiated an antimonial therapy ended the full treatment course; improved follow-up after treatment: 143 of 237 (60%) patients who ended their full treatment were correctly monitored during the required period of 6 (cutaneous cases) or 12 (mucosal cases) months after the end of treatment. These achievements were largely due to the human and logistical resources made available, the constant availability of medications and the close collaboration between the Ministry of Health, a national research institute and an international non-governmental organization. At the end of this period, the health authorities decided to register a generic brand of sodium stibogluconate, which is now in use. This should allow the treatment of a significant number of additional patients, while saving money to invest in other facets of the case management. [ABSTRACT FROM AUTHOR]

Accurate identification of Leishmania species is important for monitoring clinical outcome, adequately targeting treatment, and evaluation of epidemiological risk in tegumentary leishmaniasis. This is especially the case in regions where several species coexist and for travel medicine where the geographical source of infection is not always obvious. Species identification presently depends on parasite isolation, which is not very sensitive and not necessarily representative of parasites actually present in human tissues. We evaluated a polymerase chain reaction (PCR) assay combining amplification of the gp63 genes and restriction fragment length polymorphism (RFLP) analysis (gp63 PCR-RFLP) for direct Leishmania species-identification in tissues collected from Peruvian patients in 1999. By comparison with a kinetoplast DNA-based PCR, our PCR assay showed a detection sensitivity of 85%. Three species were engountered among patient samples, Leishmania (Viannia) braziliensis, L. (V.) peruviana and L. (V.) guyanensis, and their frequency and geographical distribution corresponded to earlier epidemiological studies of leishmaniasis in Peru. However, unexpected results raised questions about (i) the contribution of human migration to the emergence of new foci of given species, (ii) the pathogenicity of some species, and (iii) the frequency of mixed or hybrid infections. [Copyright &y& Elsevier]

In cutaneous leishmaniasis endemic areas it is a common practice for patients to manipulate their lesions with traditional treatments as a first therapeutic option. A case series study was conducted in order to describe the frequency and the variations of the patient manipulation of cutaneous leishmaniasis lesions at the Cayetano Heredia Hospital. The study included 124 patients with cutaneous leishmaniasis. From the patient population it was found that 54% (67/124) manipulated their lesions. Of this, 92.5% (62/67) did so with chemicals, and 43.3% (29/67) with plants. The most frequent local changes reported by patients were increased lesion size in 35.8% (24/67) and increased inflammation in 28.4% (19/67). Manipulation by patients decreased the positivity of the parasitological diagnosis in those patients with ulcerative lesions. [ABSTRACT FROM AUTHOR]

Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (K = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. Conclusions. The usefulness of this gene asa new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions. [ABSTRACT FROM AUTHOR]

Abstract: The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World. [Copyright &y& Elsevier]