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A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey.

Toz SO, Culha G, Zeyrek FY, Ertabaklar H, Alkan MZ, Vardarlı AT, Gunduz C, Ozbel Y - PLoS Negl Trop Dis (2013)

Bottom Line: However, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification.The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples.Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.

View Article: PubMed Central - PubMed

Affiliation: Ege University, Medical School Department of Parasitology, Bornova, Izmir, Turkey.

ABSTRACT
Human visceral leishmaniasis (VL) caused by L. infantum and cutaneous leishmaniasis (CL) caused by L. tropica and L. infantum have been reported in Turkey. L. infantum is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish Leishmania isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as L.infantum and 6.52% as L.tropica; 52.46% of CL samples as L. infantum and 26.90% as L. tropica. In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. However, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.

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Related in: MedlinePlus

The working chart of the present study.1: Optimization; 2: Diagnosis; 3: Species identification.
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pntd-0002205-g004: The working chart of the present study.1: Optimization; 2: Diagnosis; 3: Species identification.

Mentions: The ITS1 real time PCR results of clinical samples were classified into three groups which were clearly distinguishable as (a) 191 samples diagnosed as L. infantum including 29 visceral, 45 dog and 117 cutaneous samples; (b) 66 samples of L. tropica including 60 cutaneous and four visceral and two dog samples and (c) 58 samples of two peaks including 46 cutaneous, seven visceral and five dog samples. The working process and the results were summarized in Figure 4. L. infantum and L. tropica were found to be causative agents of both clinical forms of human leishmaniasis as well as canine leishmaniasis in Turkey.


A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey.

Toz SO, Culha G, Zeyrek FY, Ertabaklar H, Alkan MZ, Vardarlı AT, Gunduz C, Ozbel Y - PLoS Negl Trop Dis (2013)

The working chart of the present study.1: Optimization; 2: Diagnosis; 3: Species identification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649959&req=5

pntd-0002205-g004: The working chart of the present study.1: Optimization; 2: Diagnosis; 3: Species identification.
Mentions: The ITS1 real time PCR results of clinical samples were classified into three groups which were clearly distinguishable as (a) 191 samples diagnosed as L. infantum including 29 visceral, 45 dog and 117 cutaneous samples; (b) 66 samples of L. tropica including 60 cutaneous and four visceral and two dog samples and (c) 58 samples of two peaks including 46 cutaneous, seven visceral and five dog samples. The working process and the results were summarized in Figure 4. L. infantum and L. tropica were found to be causative agents of both clinical forms of human leishmaniasis as well as canine leishmaniasis in Turkey.

Bottom Line: However, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification.The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples.Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.

View Article: PubMed Central - PubMed

Affiliation: Ege University, Medical School Department of Parasitology, Bornova, Izmir, Turkey.

ABSTRACT
Human visceral leishmaniasis (VL) caused by L. infantum and cutaneous leishmaniasis (CL) caused by L. tropica and L. infantum have been reported in Turkey. L. infantum is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish Leishmania isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as L.infantum and 6.52% as L.tropica; 52.46% of CL samples as L. infantum and 26.90% as L. tropica. In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. However, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.

Show MeSH
Related in: MedlinePlus