Parasitology
Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World,☆☆,

https://doi.org/10.1016/j.diagmicrobio.2012.06.010Get rights and content

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.

Introduction

Leishmaniasis is the collective term for several disease manifestations caused by the trypanosomatid genus Leishmania and is endemic in 88 countries on 4 continents. It has been categorized as one of the “most neglected diseases” (Hotez et al., 2004). In the Americas, it occurs in 3 clinical variations: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and diffuse CL (DCL) (Marzochi, 1992). American tegumentary leishmaniasis (ATL), as it is collectively called, is a zoonosis encountered from the south of the United States to the north of Argentina (Marzochi, 1992). As the severity of the disease varies according to the infecting Leishmania species, the presence of multiple Leishmania species with overlapping clinical features and geographical distribution requires not only the specific detection of Leishmania parasites, but also accurate species identification for evaluating prognosis and choosing appropriate therapy (Arévalo et al., 2007). The traditional diagnosis of ATL is performed using clinical and epidemiologic features, immunologic methods, and parasitologic tests such as direct examination of smears after Giemsa staining, culture, and histopathology (Grimaldi and Tesh, 1993), but none of these identifies the infecting parasite species.

Molecular methods are increasingly employed for diagnosis, clinical, and epidemiologic studies on leishmaniasis in an effort to detect infection and categorize Leishmania at the genus, species, or strain level (Schönian et al., 2008). Ideal assays should be easy to perform and interpret, rapid, sensitive, specific, and able to detect and identify parasites accurately in reservoirs and vectors to allow studying transmission dynamics (García et al., 2007b, Talmi-Frank et al., 2010). Many different polymerase chain reaction (PCR)–based methods have been described for the detection of Leishmania spp. in different skin lesion specimens (Boggild et al., 2010, Deborggraeve et al., 2008, López et al., 1993, Marfurt et al., 2003, Rotureau et al., 2006, Schönian et al., 2001, Simon et al., 2010, Victoir et al., 2003).With multilocus enzyme electrophoresis (MLEE) (Rioux et al., 1990) being increasingly replaced by genetic characterization for species identification (Bañuls et al., 2007, Rotureau et al., 2006), several techniques and targets have been developed (Nasereddin et al., 2008, Reithinger and Dujardin, 2007, Schönian et al., 2001). Unfortunately, many assays are homemade and therefore difficult to compare. For that reason, standardized tests that are validated and applicable globally on biological, clinical, and environmental samples are of crucial importance.

The cytoplasmic heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World using PCR, followed by sequencing or restriction fragment length polymorphism (RFLP) analysis (García et al., 2004, García et al., 2007a, García et al., 2007b, Alves da Silva et al., 2010, Fraga et al., 2010, Montalvo et al., 2010a, Montalvo et al., 2010b, Montalvo et al., 2011, Veland et al., 2012). Recently, 3 improved PCRs were developed for this target, with increased sensitivity and specificity, which facilitates their use for both Leishmania detection and identification (Montalvo et al., 2012). The aim of this work was to evaluate the performance of the original PCR of García et al. (2004) and the 2 improved hsp70 PCRs most useful in the New World on ATL samples from Peru, where ATL is a major health problem with more than 10,000 cases per year (PAHO, 2010). In Peru, the disease is caused predominantly by 3 Leishmania (Viannia) species: L. (V.) peruviana, L. (V.) guyanensis, and L. (V.) braziliensis. Other species occasionally found are L. (Leishmania) amazonensis and L. (V.) lainsoni (Arévalo et al., 2007, Lucas et al., 1998). CL is endemic in 70% of the country, resulting in high morbidity, lifelong scarring, and cumbersome treatment in many resource-deprived communities (Llanos-Cuentas et al., 2008).

Section snippets

Reference strains and clinical isolates

Table 1 lists all 5 Leishmania reference strains and 20 clinical isolates used in this study. The reference strains were previously classified according to MLEE, and DNA was isolated from cultures at the Institute of Tropical Medicine Antwerp. DNA from 20 clinical isolates was obtained from cultured parasites from Peruvian patients consulting the Institute of Tropical Medicine Alexander von Humboldt (IMTAvH, Lima, Peru), following international ethical guidelines after review and approval by

Reference panel

The analytical sensitivity of PCR-G, PCR-F, and PCR-N was determined using 1 Peruvian reference isolate from each genotype (Fig. 2). The detection limit in each case was between 100 and 10 fg of DNA. PCR-N showed the highest product yield and was slightly (up to 10-fold) more sensitive than PCR-G, with PCR-F holding an intermediate position.

With the use of the algorithm in Fig. 1, the reference strains and isolates listed in Table 1 were characterized with the 3 hsp70 RFLPs. PCR amplicons were

Discussion

Of the 3 PCRs evaluated in this study, PCR-N was diagnostically the most sensitive as assayed in various clinical samples. PCR-F was found less sensitive, and, finally, PCR-G showed the lowest sensitivity. In line with these findings, the typing efficiency, i.e., the number of samples that could be successfully typed with the corresponding RFLP analyses RFLP-N, RFLP-F, and RFLP-G, followed the same order. This seems logical as the PCRs generate fragments of 1422 (PCR-G), 1286 (PCR-F), and 593

Acknowledgments

The authors are grateful to the Infectious Disease Research Institute for inclusion of samples from the study IDRI-LCVTC-202, funded by the Bill and Melinda Gates Foundation under grant no. 39129. The authors also would like to thank Ms Milena Alba (IMTAvH, UPCH) for technical support.

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    Work performed at the Institute of Tropical Medicine Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Urb. Ingeniería, Lima, Peru.

    ☆☆

    Presented in part at the MEEGID X Conference: Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases (3–5 November 2010, Amsterdam, The Netherlands, poster P2.6.02).

    This work was supported by the third framework program of the Belgian Development Cooperation with the Institute of Tropical Medicine Antwerp and by the European Commission's FP6 INCO-DEV program “Control Strategies for Visceral Leishmaniasis (VL) and Mucocutaneous Leishmaniasis (MCL) in South America: Applications of Molecular Epidemiology” (grant INCO-CT2005-015407).

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