Elsevier

Experimental Parasitology

Volume 148, January 2015, Pages 81-85
Experimental Parasitology

Full length article
The Jordanian Mid Jordan Valley is a classic focus of Leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples

https://doi.org/10.1016/j.exppara.2014.11.006Get rights and content

Highlights

  • A molecular diagnostic analysis on high number of CL patients.

  • A better epidemiological assurance of CL distribution and causative species in JMidJV.

  • ITS-1 PCR is a good technique for diagnosis of typical and atypical lesions of CL.

  • RFLP is confirmed useful for species identification of Leishmania.

  • The JMidJV is a classic focus of L. major.

Abstract

The identity of the causative species of cutaneous leishmaniasis (CL) in the endemic Jordanian Mid Jordan Valley (JMidJV) was investigated using the polymerase chain reaction (PCR) amplifying the ribosomal internal transcribed spacer 1 (ITS-1) followed by the restriction fragment length polymorphism (RFLP). The geographical distribution of CL and the usefulness of ITS1 PCR in diagnosis of suspected CL in the study area were also addressed. Over the period from 2004 to 2009, 56 clinical isolates of Leishmania promastigotes and 185 lesion scrapings spotted on filter papers were obtained from suspected CL patients living in the JMidJV, which is divided into northern and southern districts. The majority (67.1%) of patients occurred in the populated eastern part of the southern district. Of the 185 suspected CL patients, 173 (93.5%) were confirmed positive using PCR. Leishmanial DNA was detected in 27 (90%) of 30 patients having clinically atypical lesions of CL and in 60 (92%) of 65 smear- and culture-negative cases having typical lesions of CL. The parasites in all of the 56 isolates and the 173 PCR-positive scrapings were classified as Leishmania major. In conclusion, PCR is useful in diagnosis of CL especially when smear and culture are negative. It is also recommended as a differential diagnostic tool of atypical lesions when CL is endemic. The identification of L. major as the causative species in such a considerable number of CL cases, representative of all mini foci of CL in the study area, shows that the JMidJV is a classic focus of L. major.

Introduction

The Jordanian Mid Jordan Valley (JMidJV, Fig 1) is endemic for cutaneous leishmaniasis (Arbaji et al, 1993, Khoury et al, 1996) and the disease is underreported (Mosleh et al., 2008a). The actual annual incidence of the disease is, however, known to be quite high (Khoury et al, 1996, Mosleh et al, 2008a, Oumeish, 1991). Khoury et al. (1996), for example, reported 987 cases from the Jordanian side of the Jordan Valley between 1983 and 1992. In the same region, the highest incidence, of more than 313/100 000 cases per year, was reported in 2004/2005 (Mosleh et al., 2008a). The desert rodent (Psammomys obesus) was confirmed as the main reservoir host (Saliba et al., 1994) and Phlebotomus papatasi as the vector of the disease caused by L. major (Janini et al., 1995b). In Jordan, the disease is diagnosed clinically, and when parasitological investigation is performed, it is restricted, in most of the cases, to the smears of lesion scrapings stained with Giemsa.

Species identification of the causative agent is vital to determine the best ways of control measures and treatment regimens (Schönian et al., 2003). A well-established PCR method, targeting the internal transcribed spacer 1 (ITS1) region between the SSU and 5.8S rRNA genes, is useful for the direct diagnosis of different forms of leishmaniasis. The method is highly specific and sensitive, able to detect approximately 0.2 parasites per sample (Schönian et al., 2003) and identify all medically relevant Leishmania species which are distinguished by DNA sequencing or restriction fragment length polymorphism (RFLP) of the PCR product (Dávila, Momen, 2000, Schönian et al, 2000, Schönian et al, 2001, Schönian et al, 2003).

Few studies have investigated the identity of the causative species of cutaneous leishmaniasis (CL) in the endemic JMidJV (Mosleh et al, 2008b, Mosleh et al, 2009, Nimri et al, 2002, Saliba et al, 1988, Saliba et al, 2004). These studies either performed on a limited number of cases, not designed to cover all of the foci in the region, or do not precisely record the origin of the reported Leishmania species. The present study is a wide scale investigation of the identity and the geographical distribution of Leishmania along the JMidJV. It also investigates the usefulness of ITS1 PCR in diagnosis of Leishmania in lesion scrapings of Jordanian patients.

Section snippets

Study area

The JMidJV is divided into two districts (Fig. 1) with a total population of around 87 000 (Department of Statistics, Jordan). The majority of the population is distributed in 24 towns and villages, which are mainly located in the eastern part of the two districts. The strip of the valley that is located along the eastern bank of the Jordan River (‘X's’ in Fig. 1) is a poorly inhabited military zone. There are 28 governmental health centers distributed in the villages and towns of the two

PCR results and geographical distribution of CL patients

DNA extracts from the lesion scrapings of the suspected CL patients (n = 185, one lesion/patient), the cultured promastigotes of the clinical isolates (n = 56), and the reference strains were investigated by PCR. All the (56) clinical isolates and 173 out of the 185 lesion scrapings gave an amplicon similar to that shown in Fig. 3a.

PCR results of the 185 patients, categorized into groups 1–5, are shown in Table 1. PCR detected leishmanial DNA in at least 90% of the patients in all of the five

Discussion

ITS1 PCR followed by RFLP was used to identify Leishmania species found in the JMidJV. This technique worked well regardless of the type of the clinical sample investigated. The investigation on a considerable number of cultured promastigotes and lesion scrapings spotted on filter papers revealed that only one species, L. major, is the causative species of the endemic CL in the JMidJV. Earlier studies performed in the study area using the isoenzyme characterization (Nimri et al, 2002, Saliba et

Acknowledgments

We thank the Director of South Shuneh Hospital, Dr. Saed Kharabsheh and Major Mohammad Shgeerat and Major Ahmad Khateeb of the Jordanian Army who facilitated access to the patients during the course of this work. The senior author (I.M.M.) was supported by the Deanship of Scientific Research, University of Jordan, and the Deutsche Forschungsgemeinschaft (DFG), Germany.

References (28)

  • BensoussanE. et al.

    Comparison of PCR assays for diagnosis of cutaneous leishmaniasis

    J. Clin. Microbiol

    (2006)
  • BrycesonA.D.M.

    Leishmaniasis

  • DávilaA.M. et al.

    Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania

    Ann. Trop. Med. Parasitol

    (2000)
  • DweikA. et al.

    Evaluation of PCR-RFLP (based on ITS-1 and HaeIII) for the detection of Leishmania species, using Greek canine isolates and Jordanian clinical material

    Ann. Trop. Med. Parasitol

    (2007)
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