Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100% and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was investigated. Diluting crude extracts of skin biopsies was sufficient to eliminate sample inhibition yet maintain required sensitivity. Leishmania braziliensis was also detected by PCR in PBMCs of individuals with active cutaneous leishmaniasis. The simplifications described here significantly improve the feasibility of using the PCR in endemic countries as the primary method for detection and preliminary characterization of Leishmania in clinical specimens of cutaneous leishmaniasis.

A simplified polymerase chain reaction (PCR)-based assay was used for detection and typing of Leishmania parasites in clinical specimens from patients suspected of cutaneous leishmaniasis. Using cultures as the reference standard, our PCR detection method was more sensitive (92%) than classical diagnostic techniques, including microscopy (42% sensitivity), histologic staining (33%), and IgG enzyme-linked immunosorbent (20%). The PCR assay was also 100% specific. Parasites in both lesion biopsies and isolates cultured from lesion aspirates were identified as Leishmania braziliensis by PCR. In this study, we have demonstrated the suitability of simplified PCR assays for the simultaneous diagnosis and typing of parasites causing cutaneous leishmaniasis in a developing country where leishmaniasis is endemic.

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, andLeishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to theLeishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi,Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmaniaparasites, particularly where different Leishmaniacomplexes are found in the same geographical area.

As part of a survey of human leishmaniasis in Nicaragua we examined phenotypic and genotypic characteristics of 40 Leishmania isolates. We identified 3 distinct parasites associated with cutaneous disease in this area; Leishmania panamensis (40% of cases), Leishmania braziliensis (33%), and a strain which exhibits the heterozygous isoenzyme and DNA fingerprinting patterns expected of a L. panamensis/L. braziliensis hybrid (27%). There was complete correlation between the isoenzyme and DNA data for each of the putative hybrids examined. All of the 'hybrids' were obtained from foci in the northern region of the country where L. panamensis and L. braziliensis occur sympatrically. These observations provide strong evidence for sexual reproduction in New World Leishmania populations and suggest that it is of taxonomic and epidemiological significance.

Abstract. Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recently been associated with atypical cutaneous leishmaniasis (ACL) in Central America; however, little comprehensive information about this disease is available. Clinical, epidemiologic, and parasitologic characteristics of 252 ACL cases and 44 VL cases in Nicaragua were analyzed. Visceral leishmaniasis is primarily associated with malnourished children less than five years of age, whereas ACL is found predominantly in children greater than five years of age and young adults. Genetically similar parasites are associated with both disease manifestations. The sand fly Lutzomyia evansi, in addition to Lu. longipalpis, may be involved in transmission of L. chagasi to humans. Our results indicate that ACL is more prevalent than previously thought, affecting up to 10% of a local population. The fact that the same parasite appears to cause both ACL and the potentially fatal visceral disease suggests that the host immune response is critical in determining the outcome of L. chagasi infection. The public health implications of the widespread presence of L. chagasi are discussed.Parasitic protozoa of the genus Leishmania are responsible for a group of diseases that constitute an important public health problem in tropical and subtropical regions worldwide. Leishmaniasis presents a wide spectrum of clinical manifestations, depending on the species of the parasite, the host immune response, and possibly factors in the saliva of the sand fly vector.