Single-Step Multiplex PCR Assay for Characterization of New World Leishmania Complexes

Abstract

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.

Diagnostic approach and significance of inducible nitric oxide positivity in human cutaneous leishmaniasis caused by leishmania tropica

“…PCR has the added advantage of identifying the subtypes of leishmania parasites. [7][8][9]22,23 In recent studies, Brujin MHL et al . reported that positive detection rates were 38.5% by direct visualization of smear and 84.6% by PCR.…”

“…Sometimes biopsy material had been badly handled or fixed, making interpretation of the pathology and identification of the parasites impossible; however, even in such tissues, detection of parasite DNA is possible by PCR. [7][8][9]22,23,24 Although direct microscopy is a simple and a rapid technique, it may not detect parasites. Culture may be contaminated and thus take a long time to obtain a result.…”

“…Culture may be contaminated and thus take a long time to obtain a result. [7][8][9]22,23,24 WHO has designated leishmaniasis as one of six major diseases, with the possibility of becoming more widespread in the near future. 27 The current treatment of leishmaniasis is expensive and toxic.…”

Sustainable Transfer of Biotechnology to Developing Countries

“…The collaboration with the Department of Parasitology began in 1988, first with support of biochemical and immunological diagnostic and typing methods and then with the introduction of simplified PCR techniques for the diagnosis and characterization of leishmaniasis, which have been used routinely for the last 17 years. 12 New molecular typing methods were also developed, 13 and use of these assays in-country led to the discovery of a new form of disease caused by Leishmania chagasi infection in Nicaragua (atypical cutaneous leishmaniasis [ACL]). 14 Further studies examining the parasitological, immunological, epidemiological, and entomological characteristics of ACL were conducted, and the Ministry of Health incorporated information about recognition, diagnosis, and treatment of ACL into posters that were distributed throughout the nation's health centers.…”

A rapid assay for simultaneous detection of Spiroplasma eriocheiris and white spot syndrome virus in Procambarus clarkii by multiplex PCR

“…It is a widely utilized assay for specifically amplifying small fragments of nucleic acid. In the field of infectious diseases, multiplex PCR has been proven to be a valuable tool for identification of viruses (Stockton et al 1998), bacteria (Kong et al 2002) and parasites (Harris et al 1998).…”