Geographical distribution and epidemiological features of Old World cutaneous leishmaniasis foci, based on the isoenzyme analysis of 1048 strains

A series of 1048 Leishmania strains from Old World cutaneous leishmaniasis foci, isolated between 1981 and 2005, were studied by isoenzyme analysis. The strains were obtained from humans, rodents, dogs and sandflies from 33 countries. The four typically dermotropic species, Leishmania major , L. tropica , L. aethiopica and L. killicki , were found. The viscerotropic species L. donovani and L. infantum, which can occasionally be responsible for cutaneous leishmaniasis, are not considered in this paper. Leishmania major was the least polymorphic species (12 zymodemes, 638 strains). Leishmania tropica was characterized by a complex polymorphism varying according to focus (35 zymodemes, 329 strains). Leishmania aethiopica , a species restricted to East Africa, showed a high polymorphism, in spite of a limited number of strains (23 zymodemes, 40 strains). Leishmania killicki , mainly restricted to Tunisia had a single zymodeme for 39 strains. Recently a parasite close to L. killicki (one zymodeme, two strains) was isolated in Algeria, which lead us to revise the taxonomic status of this taxon.     

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.     

Identification of Leishmania strains from Jordan

The enzymatic profiles of 22 Jordanian Leishmania isolates obtained from humans, Psammomys obesus and Phlebotomus papatasi were determined using starch-gel electrophoresis and a 15-enzyme system. Thirteen of the isolates were typed as L. major and the other nine, all from Mediterranean or sub-Mediterranean regions, as L. tropica. The two zymodemes of L. major encountered, MON-26 and MON-103, differed in terms of purine nucleoside phosphorylase 2. The MON-26 isolates came from the Jordanian plateau whereas those of MON-103 were only collected from the Jordan valley. The four zymodemes of L. tropica observed (MON-7, MON-137, MON-200 and MON-265) were identical for only two of the 15 enzymes studied (i.e. isocitrate dehydrogenase and glucose phosphate isomerase), confirming the high level of enzymatic polymorphism of L. tropica. So far, MON-200 and MON-265 have only been found in Jordan.     

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

Objectives  To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil.
Methods  Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani . Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus.
Results  Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi . All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed.
Conclusions  Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi .     

Are cytochrome B gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina?

Recently, two techniques, polymerase chain reaction (PCR) amplification and sequencing of cytochrome b gene (cyt b gene sequencing) and polymorphism-specific PCR (PS-PCR) were recommended for Leishmania species identification. Before this study, however, the accuracy of these methods had not been tested against the multilocus enzyme electrophoresis, the current gold standard technique on this task. Therefore, a trial was done for the first time to compare the results obtained by these techniques, using 17 Argentinean Leishmania stocks in independent assays. For all the stocks examined, the same results at species level were obtained by the three techniques. Among them, 14 were assigned to L. (Viannia) braziliensis, and three to L. (V.) guyanensis. The two techniques, cyt b gene sequencing and PS-PCR, were able to distinguish between all the proven species responsible for leishmaniases in Argentina. Thus, both techniques were validated and could be used independently for the species designation of Leishmania parasites in the country.     

Epidemiologic and clinical features of cutaneous leishmaniasis in southeastern Tunisia

Species-specific diagnosis was performed in 66 patients with cutaneous leishmaniasis (CL) living in Tataouine focus in southeastern Tunisia. Leishmania DNA was extracted directly from dermal scrapings (n = 66) and from parasites obtained in culture (n = 12). Species were identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for internal transcribed spacer region 1 and isoenzyme analysis. Leishmania tropica and L. major were identified in 31 (47%) and 35 (53%) cases respectively. Leishmania tropica CL cases were geographically scattered, and L. major CL cases were clustered. Lesions caused by L. tropica were mostly single (83.8%) and face-localized (55.8%), and lesions caused by L. major were multiple (57.1%; P < 0.001) and situated on limbs (83.7%; P < 0.001). For both species, most lesion onsets were reported during June-January. However, lesions that emerged during February-May were mainly caused by L. tropica (83.3%; P < 0.01). Moreover, the delay before seeking medical advice was higher for L. tropica infections than for L. major infections (P < 0.05).     

Multifarious characterization of leishmania tropica from a Judean desert focus, exposing intraspecific diversity and incriminating phlebotomus sergenti as its vector

The predominant sand fly species collected inside houses in Kfar Adumim, an Israeli village in the Judean Desert that is a focus of cutaneous leishmaniasis, was Phlebotomus papatasi, which was also caught attempting to bite humans. Phlebotomus sergenti, which is rarely seen inside houses, constituted the predominant sand fly species in caves near the village. Leishmania isolates from Ph. sergenti and humans typed as Leishmania tropica. Sand fly and human isolates produced similar small nodular cutaneous lesions in hamsters. Isolates produced excreted factor (EF) of subserotypes A(9) or A(9)B(2), characteristic of L. tropica and reacted with L. tropica-specific monoclonal antibodies. Isoenzyme analysis consigned the strains to the L. tropica zymodemes MON-137 and MON-275. Molecular genetic analyses confirmed the strains were L. tropica and intraspecific microheterogeneity was observed. Genomic fingerprinting using a mini-satellite probe separated the L. tropica strains into two clusters that were not entirely congruent with geographic distribution. These results support the heterogeneous nature of L. tropica and incriminate Ph. sergenti as its vector in this Judean Desert focus.     

The PCR-based detection and identification of the parasites causing human cutaneous leishmaniasis in the Iranian city of Ahvaz

In Iran, Leishmania major or L. tropica cause almost all of the human cutaneous leishmaniasis (CL). Unfortunately, the detection methods frequently used for CL (the microscopical examination of direct smears or the culture of biopsies) are not very sensitive and the Leishmania species causing each case of CL in Iran is usually only tentatively identified from extrinsic factors, such as the case's clinical manifestations and region of residence. Recently, however, a nested PCR that targets the parasites' kinetoplast DNA has been used in the city of Ahvaz (the capital of the province of Khouzestan, in south-western Iran) to confirm the microscopical diagnosis of CL and to identify the causative parasites, to species level. Smears from the lesions on 100 suspected cases of CL were fixed, stained with Wright's eosin-methylene blue, and checked for amastigotes under a light microscope. Scrapings from the same smears were then tested for leishmanial DNA, using a nested PCR that allows the DNA from L. tropica to be identified and distinguished from that of L. major. The 100 smears investigated were all found amastigote-positive by microscopy and PCR-positive for either L. major DNA (97 smears) or L. tropica DNA (three smears). The predominant species causing CL in Ahvaz is therefore L. major.     

Identification and Characterization of Leishmania spp. in Impreession Smears of Patients with Cutaneous Leishmaniasis

Background: Monoclonal antibodies have been employed extensively for the identification of Leishmania species, development of diagnostic tests and in the characterization of defined leishmanial antigens. Objectives: Identification and characterization of  Leishmania spp. directly from cutaneous lesions of infected individuals. Methods: An immunoperoxidase test (Avidin-Biotin technique) using monoclonal antibodies was used for this purpose. One hundred and fifty individuals referring to Dermatology Clinic or Parasitology and Mycology Department of Shiraz University of Medical Sciences were chosen of whom a total of 28 individuals whose smears showed a large number of amastigotes after staining with Giemsa were included in this study. Five monoclonal antibodies designated: D2 (against L. donovani), A11 and T10 (against L. tropica), T1 (against L. major) and T7 (against L. tropica and L. major) were used. Amastigotes were identified by Labeled Avidin Biotin (LAB) method. Results: LAB method for identification of amastigotes in impression smears of patient lesions showed that 20 out of 28 cases (71%) were positive. Among these 12 (60%) and 7(35%) were identified as L. tropica and L. major respectively. Conclusion: The results showed that immunoperoxidase is suitable for in situ identification and characterization of Leishmania spp. at the species level.     

Cost effectiveness in the discrimination of leishmania species causing anthroponotic leishmaniases in Asia using selective enzymes

In this study, an attempt was made to evaluate the usefulness of selective enzymes in the identification of Leishmania spp causing anthroponotic leishmaniasis in Asia, especially from a cost effectiveness point of view. For this purpose cellulose acetate electrophoresis was carried out to identify the Leishmania species of the Old World. After analyzing 11 enzymes 6PGDH was found to be the most polymorphic enzyme which could distinguish the WHO reference strains of the Leishmania species endemic in Asian countries like L.(L.) donovani (DD8), L. (L.) infantum (IPT-I), L.(L.) major (5ASKH), and L.(L.) tropica (K-27). Addition of another enzyme G6PDH improved the quality of diagnosis. Cost could be reduced manifold to discriminate the Asian Leishmania parasites by analyzing these two enzymes.     

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan,Khyber Pakhtunkhwa,Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis(CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1(ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica(L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major(L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents(Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.     

Enzymatic polymorphism during Leishmania/HIV co-infection: a study of 381 Leishmania strains received between 1986 and 2000 at the international cryobank in Montpellier, France

Between 1986 and 2000, 381 Leishmania strains isolated from 288 HIV-positive patients were studied at the international cryobank in Montpellier, France. Most (95.1%) of the strains came from cases of visceral leishmaniasis but 4.9% were from HIV-positives with cutaneous leishmaniasis. The majority of the strains came from patients infected in the Mediterranean region, with a few originating in sub-Saharan Africa and South America. Isoenzymatic characterization revealed 28 zymodemes in four different species: L. infantum (which was predominant), L. donovani, L. major and L. guyanensis. The strains belonging to the L. infantum complex included 20 zymodemes, some of which have so far only been found in cases of Leishmania/HIV co-infection.     

Trypanosoma cruzi genotypes of insect vectors and patients with Chagas of Chile studied by means of Cytochrome b gene sequencing, minicircle hybridization, and nuclear gene polymorphisms

Fifty-six Trypanosoma cruzi stocks from Chile and neighboring countries and different hosts, humans, and Triatoma infestans and Mepraia sp., vectors of domiciliary and natural environments were characterized by using three molecular markers. These were cytochrome b (Cyt b) gene sequencing, minicircle DNA blotting, and hybridization with five genotype-specific DNA probes and nuclear analysis of 1f8 and gp72 by polymerase chain reaction-restriction fragment length polymorphism. The results with all three molecular markers are concordant, with minor limitations, grouping T. cruzi stocks into four discrete typing units (DTUs) (TcI, TcII, TcV, and TcVI). TcI and TcII stocks were heterogeneous. TcI and TcII stocks were clustered in two main subgroups determined by Cyt b gene sequencing and minicircle hybridization. However, TcV and TcVI stocks were homogeneous and not differentiated by Cyt b gene sequencing or minicircle DNA hybridization. The discriminatory power and limitations of the molecular markers are discussed, as well as the distribution of the four DTUs in the domiciliary and sylvatic transmission cycles of Chile and the limitations of each marker for molecular epidemiological studies performed with T. cruzi stocks rather than the analysis of direct T. cruzi samples from natural hosts.     

Genetic polymorphism within the leishmania donovani complex: correlation with geographic origin

Random amplified polymorphic DNA (RAPD) was used to detect intraspecific diversity for the Leishmania donovani complex. Fifty-two decameric to 21-meric primers of arbitrary sequence were applied to 15 strains that belong to nine zymodemes. Strains belonging to the species L. major and L. tropica were used as outgroups. A total of 902 amplicons generated by RAPD were scored. Most primers produced species-specific profiles, only 0.6% amplicons were shared by all species, while 4.3% amplicons were common for all 15 strains of the L. donovani complex. Well-supported trees have been constructed, which show a rather strong correlation between the genetic polymorphism of studied strains and their geographic origin. In all obtained trees, L. infantum was paraphyletic. The RAPD profiles suggest that MON-30 belongs to L. donovani. Moreover, the genetic distance between the L. archibaldi strain and other leishmanias does not warrant existence of a separate species.     

Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia

From 2009 to 2010, 3129 sandflies were caught in CDC light traps placed in various habitats in Ghomrassen, Tataouine governorate, southeast Tunisia, a mixed focus of human cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major. Species diversity was quantified in anthropogenic, semi-anthropogenic and semi-natural locations. Sandflies were identified according to morphological characters and also by the comparative sequence analysis of a fragment of the mitochondrial cytochrome b gene to distinguish between two putative local vectors of L. tropica, namely Phlebotomus chabaudi and Phlebotomus riouxi. The lowest sandfly diversities were found in L. major sites, where the incriminated vector P. papatasi predominated in the burrows of the rodent reservoir hosts (Meriones) as well as inside and outside houses of human cases. In L. tropica sites, the incriminated peri-domestic vector Phlebotomus sergenti was the most abundant species inside houses, whereas P. riouxi or P. chabaudi was the dominant species in the semi-natural rocky habitats favoured by the putative rodent reservoir, Ctenodactylus gundi. All specimens of P. chabaudi identified molecularly had the diagnostic cytochrome b characters of P. riouxi, indicating either that the latter represents only a geographical variant of P. chabaudi or that these two species may sometimes hybridize.     

Genetic analysis of leishmania parasites in Ecuador: are Leishmania (Viannia) panamensis and Leishmania (V.) Guyanensis distinct taxa?

In the course of an epidemiologic survey in Ecuador, the following collection of Leishmania stocks was isolated: 28 from patients with clinical signs of leishmaniasis, 2 from sloths, 1 from a dog, and 4 from sand flies. For genetic characterization of these stocks, multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) were used. Twenty six of the 35 stocks were identified as either Leishmania (V.) panamensis or L. (V.) guyanensis, 2 stocks were identified as L. (V.) braziliensis, the 2 stocks from sloths showed specific genotypes, and 5 stocks were characterized as hybrids between L. (V.) braziliensis and L. (V.) guyanensis. These data show that genetic diversity of Leishmania in Ecuador is high and that L. (V.) panamensis/guyanensis is the dominant group in this country. The genetic analysis questioned the distinctness between the two species L.(V.) panamensis and L. (V.) guyanensis, since MLEE and RAPD data did not indicate that L. (V.) panamensis and L. (V.) guyanensis correspond to distinct monophyletic lines. Population genetic analysis performed on the L. (V.) panamensis/guyanensis group favors the hypothesis of a basically clonal population structure.     

Recombinant kinetoplast DNA (kDNA) probe for identifying Leishmania tropica.

The paper reports on the construction of a kDNA library with DNA isolated from the WHO reference strain of Leishmania tropica IC-305 and subsequent identification and propagation of recombinant plasmids containing L. tropica kDNA sequences. It also shows that the cloned kDNA sequences can be used as genetic markers in restriction endonuclease, Southern blot transfer, and dot blot hybridisation analysis, to identify L. tropica parasites. When the pL 305-I kDNA probe was used in hybridisation experiments with DNAs from various Leishmania reference strains, species and isolates from different host species and from different geographical locations, hybridisation was detected only with L. tropica, thereby suggesting that the insert in recombinant plasmid 305-I was species-specific. The probe is sensitive to the level of 10(3) parasites in dot blot hybridisation. Additionally, orthogonal field alternation gel electrophoresis (OFAGE) and transverse alternating field electrophoresis (TAFE) were used to characterise Leishmania reference strains and Leishmania species. The molecular karyotypes resolved by these techniques showed significant differences in the profiles of chromosomal sized-DNA molecules among species of Leishmania. The DNA karyotypes of the two reference strains of L. tropica (IC-305 and NLB-067), while similar, were nevertheless distinct.     

Natural infection of North African gundi (Ctenodactylus gundi) by Leishmania tropica in the focus of cutaneous leishmaniasis, Southeast Tunisia

North African gundis (Ctenodactylus gundi) were trapped in the Leishmania (L.) tropica focus of cutaneous leishmaniasis, situated in southeast Tunisia and evaluated for Leishmania infection by real-time kinetoplast DNA polymerase chain reaction (PCR). Species identification was performed by internal transcribed spacer one (ITS1)-PCR-restriction fragment length polymorphism (RFLP) and high-resolution melting (HRM) analysis of the 7SL RNA gene. Real-time PCR on blood was positive in 6 of 13 (46.2%) tested gundis. Leishmania tropica was identified in five infected gundis and Leishmania major in one specimen. Alignments of the ITS-1 DNA sequences and 7S-HRM curves analysis indicated that similar genotypes were present in humans, a sandfly, and gundis from the same region suggesting a potential role of this rodent as reservoir host of L. tropica in southeast Tunisia.     

Genotypic characterization of cutaneous leishmaniasis at Al Baha and Al Qasim Provinces (Saudi Arabia)

Fifty samples of skin ulcers were collected from the western region of Saudi Arabia Kingdom (Al Baha and Al Qasim) to study genotypic characterization of Cutaneous Leishmaniasis in this area. Thirty-six samples were recorded as Leishmania isolates. The same isolates were subsequently tested with fingerprinting with single arbitrary primers. The primers used derived from the core sequence of the phage M13, and the repeat sequences (GTG)5 and (GACA)4. The 36 isolates were all identified as Leishmania major (n = 25 isolates) or Leishmania tropica (n = 11 isolates). All produced polymorphic patterns, which were grouped depending on the species they belonged to, next to the relevant well-characterized strains of the same species. Within the L. major and L. tropica group the subgroupings formed were mainly related to the geographical origin of the strains. A nested polymerase chain reaction-based schizodeme method for identifying Leishmania kinetoplast minicircle classes was used as a diagnostic tool for L. major and L. tropica.     

Re-emergence of visceral and cutaneous leishmaniasis in the Greek Island of Crete

Leishmaniases are vector-borne diseases transmitted by phlebotomine sand flies. Three species of Leishmania are found in the Mediterranean basin: Leishmania infantum, the most common species responsible for both visceral (VL) and cutaneous leishmaniasis (CL); Leishmania major, found in North Africa and Middle East causing CL; Leishmania tropica with a limited presence in Europe, causing CL. During the last 25 years, Crete has become an endemic zone for L. infantum with a high number of infected dogs and an increasing number of human cases every year; in the last 4 years, the incidence has reached an average of seven VL patients per year in a population of 600,000. At the same time, CL has re-emerged in Crete due to L. tropica, with an average of three CL cases per year in the last 4 years. Isolates were typed as L. infantum MON-1 and MON-98 and L. tropica MON-300, a zymodeme not reported before. Both VL and CL have spread to the whole of the island during the last 25 years, primarily in semi-urban and urban areas with altitudes of 0-50?m. The prevailing Phlebotomus species were Phlebotomus neglectus (proven vector of L. infantum) and Phlebotomus similis (suspected vector of L. tropica).