Lionel F. Schnur

Comparison of PCR Assays for Diagnosis of Cutaneous Leishmaniasis

ABSTRACT Three PCR assays for diagnosing leishmaniasis were compared and validated against parasite cultures and microscopic evaluation of stained tissue smears using 92 specimens from suspected cases of cutaneous leishmaniasis (CL) in Israel and the West Bank. Samples from imported and locally acquired disease were examined. The kinetoplast DNA (kDNA) PCR showed the highest sensitivity (98.7%) of any assay, correctly diagnosing 77/78 of the confirmed positive samples, followed by the rRNA gene internal transcribed spacer 1 (ITS1) PCR (71/78 positive, 91.0% sensitivity) and then the spliced leader mini-exon PCR (42/78 positive, 53.8% sensitivity). Either parasite culture or microscopy alone detected 62.8% (49/78) or 74.4% (58/78) of the positive specimens, respectively, while culture and microscopy together improved overall sensitivity to 83.3% (65/78). Except for the kDNA PCR that had six false positives, all other assays were 100% specific. Further, restriction enzyme analysis of the ITS1 PCR product enabled identification of 74.6% of the positive samples, which included strains of Leishmania major (50.9%), Leishmania tropica (47.2%), and the Leishmania braziliensis complex (1.9%). This suggests that a PCR using kDNA should be used for the diagnosis of CL and that an ITS1 PCR can be reliably used for the diagnosis of CL when rapid species identification is needed.

An experimental model for canine visceral leishmaniasis.

Summary Seven mixed‐breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post‐infection. Anti‐parasite specific antibody levels were measured by enzyme‐linked immunosorbence, immunofluorescence, crossed‐immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70‐2. was also done. Mitogenic and antigen‐specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen‐specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 15 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen.

Outbreak of Cutaneous Leishmaniasis in Northern Israel

This study describes a new focus of cutaneous leishmaniasis (CL) due to Leishmania tropica, in the Galilee region of northern Israel. Thirty-three cases from 4 villages (northern part) and from the city of Tiberias (southern part) have been clinically diagnosed since 1996. Parasites from 13 patients and from 6 sand flies were characterized by isoenzyme electrophoresis, 2 immunological methods, and 3 polymerase chain reaction (PCR)-based methods. Isolates from the northern part were antigenically similar to Leishmania major and were different from other L. tropica isolates, including those from the southern part of the focus. They belonged to a newly reported zymodeme and were separable from all known Israeli L. tropica isolates, by use of 2 different PCR-based methods. Five (5.2%) of 97 Phlebotomus (Adlerius) arabicus and 2 (1.2%) of 162 Phlebotomus (Paraphlebotomus) sergenti females from the northern part of the focus were found to be infected with L. tropica. Three of 29 hyraxes (Procavia capensis) were positive for Leishmania ribosomal DNA. Thus, the northern part of this emerging focus of CL in Israel is distinct from all known L. tropica foci. P. arabicus is the main vector, and it transmits parasites that are different from other L. tropica isolates, with respect to antigenic, molecular, and biochemical parameters.

Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, Northern Israel.

TOC summary for table of contents: Infection with Leishmania tropica is emerging because of encroachment of rock hyraxes and transmission by multiple vector species.

Leishmaniasis in Israel: reservoir hosts, sandfly vectors and leishmanial strains in the Negev, Central Arava and along the Dead Sea

The reservoir animals, sandfly vectors and strains of Leishmania from foci in the southern region of Israel were studied. The rodent host species are: Psammomys obesus, Meriones crassus and probably Nesokia indica. The vector species are Phlebotomus papatasi, which were caught at all collecting sites and Ph. sergenti, which were collected in the area of the Dead Sea and in the Central Arava. Strains of Leishmania major isolated from rodents, vectors and man were serologically and enzymologically identical with regard to their excreted factor (EF) serotypes, their malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI) and glucose-6-phosphate dehydrogenase (G6PDH) enzyme variant types, but exhibited three variant subtypes of 6-phosphogluconate dehydrogenase (6PGDH). The distribution of the 6PGDH subtypes correlates with three different geographical locations. Scarcity of water is the main factor limiting the biotopes of the sandflies and the spread of leishmaniasis. The subjects discussed are the dependence of sandfly distribution on rodent-burrow depth in arid areas and the inter-relationship between the leishmanial subtypes, vectors and hosts.

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

Leishmania in the Old World: 1. The geographical and hostal distribution of L. major zymodemes

135 stocks of Leishmania major from man, reservoir hosts and sandflies were characterized using thin-layer starch-gel electrophoresis of 13 enzymes: MDH, 6PGD, GD, SOD, ASAT, ALAT, PK, PGM, ES, NH, PEPD, MPI, GPI. Homogeneity in this species was demonstrated by identical electrophoretic mobilities in nine enzymes. Polymorphism in four enzymes: 6PGD, GPI, PEPD, ES, gave six zymodemes among the collection. Stocks from sandflies and several species of burrowing rodents were indistinguishable from those from man in the same areas. Stocks of Leishmania from North-West India were identified as L. major. In some foci the distribution of zymodemes has some correlation with the presence of particular rodent reservoir hosts. The enzymic homogeneity of L. major throughout its geographical and host range appears to be correlated with the close association between L. major and sandflies of the subgenus Phlebotomus. The status of L. major as a distinct species is supported.

Detection and identification of Old World Leishmania by high resolution melt analysis.

Background Three major forms of human disease, cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis, are caused by several leishmanial species whose geographic distribution frequently overlaps. These Leishmania species have diverse reservoir hosts, sand fly vectors and transmission patterns. In the Old World, the main parasite species responsible for leishmaniasis are Leishmania infantum, L. donovani, L. tropica, L. aethiopica and L. major. Accurate, rapid and sensitive diagnostic and identification procedures are crucial for the detection of infection and characterization of the causative leishmanial species, in order to provide accurate treatment, precise prognosis and appropriate public health control measures. Methods/Principal Findings High resolution melt analysis of a real time PCR product from the Internal Transcribed Spacer-1 rRNA region was used to identify and quantify Old World Leishmania in 300 samples from human patients, reservoir hosts and sand flies. Different characteristic high resolution melt analysis patterns were exhibited by L. major, L. tropica, L. aethiopica, and L. infantum. Genotyping by high resolution melt analysis was verified by DNA sequencing or restriction fragment length polymorphism. This new assay was able to detect as little as 2-4 ITS1 gene copies in a 5 µl DNA sample, i.e., less than a single parasite per reaction. Conclusions/Significance This new technique is useful for rapid diagnosis of leishmaniasis and simultaneous identification and quantification of the infecting Leishmania species. It can be used for diagnostic purposes directly from clinical samples, as well as epidemiological studies, reservoir host investigations and vector surveys.

Leishmaniasis in the Jordan Valley II. Sandflies and transmission in the central endemic area

Cutaneous leishmaniasis in the Jordan Valley is maintained within the close association of the rodent Psammomys obesus and sandfly Phlebotomus papatasi, which appear to be the exclusive host and vector species. The incidence of the disease was similar to the distribution of Psammomys colonies in the region, i.e., the plains of light stoneless soil. An infection rate of 93% was recorded in the very common P. obesus. Other potential host species, except for Mus musculus, were scarce and no infection with Leishmania was found in them. The only Phlebotomus species caught in significant numbers was Ph. papatasi and this was also the only species harbouring leishmanial parasites, up to 56% in one sample. All Leishmania isolates from Psammomys and from Ph. papatasi were identical to those from local human cases. The density of Ph. papatasi populations in uncultivated areas was correlated with soil conditions favouring high humidity in Psammomys burrows. A very low rate of engorged females among the Sergentomyia species collected suggests that the common species, S. antennata and S. africana asiatica, are highly autogenous.

The recent emergence of Leishmania tropica in Jericho (A'riha) and its environs, a classical focus of L. major.

Between 1997 and 2002, 49 strains of Leishmania were isolated from the cutaneous lesions of Palestinians living in and around Jericho. A polymerase chain reaction (PCR) amplifying the ribosomal internal transcribed spacer 1 (ITS1‐PCR) was applied to their cultured promastigotes and to 207 individuals’ skin scrapings spotted on filter‐papers, 107 of which proved positive for leishmanial DNA. Species identification was performed by restricting the ITS1‐PCR amplification products from the cultured promastigotes and the amastigotes in the scrapings with the endonuclease HaeIII. Of the 49 cultures, 28 (57%) were L. major and 21 (43%) were L. tropica. Of the 107 dermal samples tested directly, 53 (49.5%) were infected with L. major, 52 (48.5%) with L. tropica and two remained unidentified. This is the first time L. tropica has been exposed in the population of the Jericho area and on such a large scale. The itinerant behaviour of some of this population precludes categorically declaring that L. tropica has recently become established in this classical focus of L. major. For this and although 88.2% of the cases of L. tropica claimed not to have travelled out of the vicinity of Jericho, local infected sand fly vectors of L. tropica must be caught, identified and, if possible, shown to harbour infections, and, if one exists, an animal reservoir host should also be exposed to endorse whether the cases caused by L. tropica were imported or autochthonous.