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Purpose: Overlapping clinical features of cutaneous leishmaniasis (CL) with ulcers caused by fungi and mycobacteria necessitate confirmatory diagnostic testing. We evaluated a handheld battery-operated device for detection of CL and common fungal and mycobacterial causes of ulcers. Methods: We validated Palm PCR™ for detection of common ulcerative skin pathogens using ATCC® reference and clinical strains of Leishmania, mycobacteria, and fungi in the lab and field. Amplified products were Sanger sequenced. Performance characteristics were calculated using conventional PCR as a reference standard. Results: Palm PCR™ detected 100% of ATCC® strains of Leishmania, fungi, and mycobacteria, with sensitivity and specificity of 90% and 91.7%, respectively. In the field, the sensitivity for detection of Leishmania in patients with suspected CL was 100%. In 61% of CL patients, co-colonization with genera such as Malassezia, Aspergillus, Candida, and Cladosporium was detected. In 50% of CL patients with an inflammatory (secondarily infected) phenotype, detected fungal species had known associations with human cutaneous disease. Conclusions: Palm PCR™ performs comparably to conventional PCR for detection of Leishmania, fungi, and mycobacteria. This work has implications for the diagnostic approach to tropical ulcers, and has the potential to improve field detection of ulcerative pathogens in resource constrained areas. [ABSTRACT FROM AUTHOR]

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America. Author Summary: Leishmaniasis is a neglected disease with more than two million new human infections annually worldwide. Tegumentary leishmaniasis, cutaneous and mucocutaneous, is mainly caused by five Leishmania species of the Viannia complex in South America. Different species can cause disease with similar symptoms but have dissimilar prognoses and may need different therapeutic regimens. Identification of Leishmania species traditionally relies on the multilocus enzyme electrophoresis (MLEE) assay, but it can only be applied to culture-positive samples and takes at least six weeks of intense laboratory work. A reliable and rapid assay for species identification can be a valuable tool. Molecular assays are the fastest and most accurate way to identify the etiological agents causing leishmaniasis. This paper describes a novel real-time PCR assay for identification of the five main species that cause tegumentary leishmaniasis in the New World. The assay correctly identified each of these five species of Leishmania directly from clinical samples. Because of its reliability, speed and simplicity, this assay could be used for species identification in routine laboratory diagnosis of leishmaniasis in endemic regions. [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]

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]

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]