A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey
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...
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| creator | Toz, Seray Ozensoy Culha, Gulnaz Zeyrek, Fadile Yıldız Ertabaklar, Hatice Alkan, M Ziya Vardarlı, Aslı Tetik Gunduz, Cumhur Ozbel, Yusuf |
| description | 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. |
| doi_str_mv | 10.1371/journal.pntd.0002205 |
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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.</description><identifier>ISSN: 1935-2735</identifier><identifier>ISSN: 1935-2727</identifier><identifier>EISSN: 1935-2735</identifier><identifier>DOI: 10.1371/journal.pntd.0002205</identifier><identifier>PMID: 23675543</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Diagnosis ; Diseases ; DNA, Intergenic - chemistry ; DNA, Intergenic - genetics ; DNA, Protozoan - chemistry ; DNA, Protozoan - genetics ; Dog Diseases - diagnosis ; Dog Diseases - parasitology ; Dogs ; Genotype ; Humans ; Leishmania - classification ; Leishmania - genetics ; Leishmania - isolation & purification ; Leishmaniasis ; Leishmaniasis - diagnosis ; Leishmaniasis - parasitology ; Leishmaniasis - veterinary ; Medicine ; Methods ; Microbiology ; Molecular Diagnostic Techniques - methods ; Molecular Sequence Data ; Parasites ; Parasitic diseases ; Parasitology - methods ; Polymerase chain reaction ; Real-Time Polymerase Chain Reaction - methods ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Studies ; Transition Temperature ; Turkey</subject><ispartof>PLoS neglected tropical diseases, 2013-05, Vol.7 (5), p.e2205-e2205</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Ozensoy Toz et al 2013 Ozensoy Toz et al</rights><rights>2013 Ozensoy Toz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited: Toz SO, Culha G, Zeyrek FY, Ertabaklar H, Alkan MZ, et al. (2013) A Real-Time ITS1-PCR Based Method in the Diagnosis and Species Identification of Leishmania Parasite from Human and Dog Clinical Samples in Turkey. 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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.</description><subject>Animals</subject><subject>Diagnosis</subject><subject>Diseases</subject><subject>DNA, Intergenic - chemistry</subject><subject>DNA, Intergenic - genetics</subject><subject>DNA, Protozoan - chemistry</subject><subject>DNA, Protozoan - genetics</subject><subject>Dog Diseases - diagnosis</subject><subject>Dog Diseases - parasitology</subject><subject>Dogs</subject><subject>Genotype</subject><subject>Humans</subject><subject>Leishmania - classification</subject><subject>Leishmania - genetics</subject><subject>Leishmania - isolation & purification</subject><subject>Leishmaniasis</subject><subject>Leishmaniasis - diagnosis</subject><subject>Leishmaniasis - parasitology</subject><subject>Leishmaniasis - veterinary</subject><subject>Medicine</subject><subject>Methods</subject><subject>Microbiology</subject><subject>Molecular Diagnostic Techniques - methods</subject><subject>Molecular Sequence Data</subject><subject>Parasites</subject><subject>Parasitic diseases</subject><subject>Parasitology - methods</subject><subject>Polymerase chain reaction</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Sensitivity and Specificity</subject><subject>Sequence Analysis, DNA</subject><subject>Studies</subject><subject>Transition Temperature</subject><subject>Turkey</subject><issn>1935-2735</issn><issn>1935-2727</issn><issn>1935-2735</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>DOA</sourceid><recordid>eNptUluLEzEYHURx1-o_EA0I4svU3GameRFK8VIoKFqfQyaXTtZMMiYzwv4Nf7GZbXdpQfKQ8OWcky_nO0XxEsElIg16fxOm6IVbDn5USwghxrB6VFwjRqoSN6R6fHa-Kp6ldANhxaoVelpcYVI3VUXJdfF3DaIWrhxtr8F2_wOV3zbfQSuSVqDXYxcUsB6MnQbKioMPySYgvAJp0NLqBKzSfrTGSjHa4EEwYKdt6nrhrQCDiCLZUQMTQw-6KVfvyCocgHTWZ5YDSfSDm5U82E_xl759XjwxwiX94rQvip-fPu43X8rd18_bzXpXyrrGY4kMhkJiw1pJG6EbWjeohbqCECloEKpbpShCLcZSysbUiDFBWqNaygQjkpJF8fqoO7iQ-MnOxBGpVzUhKzwjtkeECuKGD9H2It7yICy_K4R44CKOVjrNjcJt1bKWGKEpJaZFjWYy90DRijFqstaH02tT22sls21RuAvRyxtvO34IfzipKWMVywLvTgIx_J50Gnlvk9TOCa_DNPddYdywVZ73onhzhB5Ebs16E7KinOF8TQiFFBNIMmr5H1ReSvdWBq-NzfULwtszQpdjM3YpuGmefLoE0iNQxpBS1ObhmwjyObz3bvM5vPwU3kx7dW7RA-k-reQfUgDtvw</recordid><startdate>20130501</startdate><enddate>20130501</enddate><creator>Toz, Seray Ozensoy</creator><creator>Culha, Gulnaz</creator><creator>Zeyrek, Fadile Yıldız</creator><creator>Ertabaklar, Hatice</creator><creator>Alkan, M Ziya</creator><creator>Vardarlı, Aslı Tetik</creator><creator>Gunduz, Cumhur</creator><creator>Ozbel, Yusuf</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130501</creationdate><title>A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey</title><author>Toz, Seray Ozensoy ; Culha, Gulnaz ; Zeyrek, Fadile Yıldız ; Ertabaklar, Hatice ; Alkan, M Ziya ; Vardarlı, Aslı Tetik ; Gunduz, Cumhur ; Ozbel, Yusuf</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c662t-1f20ac2f9bc47ae74671b0e5001d0f116bdd411b22ccc7f6199a3bfdb49a93c43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Animals</topic><topic>Diagnosis</topic><topic>Diseases</topic><topic>DNA, Intergenic - chemistry</topic><topic>DNA, Intergenic - genetics</topic><topic>DNA, Protozoan - chemistry</topic><topic>DNA, Protozoan - genetics</topic><topic>Dog Diseases - diagnosis</topic><topic>Dog Diseases - parasitology</topic><topic>Dogs</topic><topic>Genotype</topic><topic>Humans</topic><topic>Leishmania - classification</topic><topic>Leishmania - genetics</topic><topic>Leishmania - isolation & purification</topic><topic>Leishmaniasis</topic><topic>Leishmaniasis - diagnosis</topic><topic>Leishmaniasis - parasitology</topic><topic>Leishmaniasis - veterinary</topic><topic>Medicine</topic><topic>Methods</topic><topic>Microbiology</topic><topic>Molecular Diagnostic Techniques - methods</topic><topic>Molecular Sequence Data</topic><topic>Parasites</topic><topic>Parasitic diseases</topic><topic>Parasitology - methods</topic><topic>Polymerase chain reaction</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>Sensitivity and Specificity</topic><topic>Sequence Analysis, DNA</topic><topic>Studies</topic><topic>Transition Temperature</topic><topic>Turkey</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Toz, Seray Ozensoy</creatorcontrib><creatorcontrib>Culha, Gulnaz</creatorcontrib><creatorcontrib>Zeyrek, Fadile Yıldız</creatorcontrib><creatorcontrib>Ertabaklar, Hatice</creatorcontrib><creatorcontrib>Alkan, M Ziya</creatorcontrib><creatorcontrib>Vardarlı, Aslı Tetik</creatorcontrib><creatorcontrib>Gunduz, Cumhur</creatorcontrib><creatorcontrib>Ozbel, Yusuf</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PLoS neglected tropical diseases</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Toz, Seray Ozensoy</au><au>Culha, Gulnaz</au><au>Zeyrek, Fadile Yıldız</au><au>Ertabaklar, Hatice</au><au>Alkan, M Ziya</au><au>Vardarlı, Aslı Tetik</au><au>Gunduz, Cumhur</au><au>Ozbel, Yusuf</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey</atitle><jtitle>PLoS neglected tropical diseases</jtitle><addtitle>PLoS Negl Trop Dis</addtitle><date>2013-05-01</date><risdate>2013</risdate><volume>7</volume><issue>5</issue><spage>e2205</spage><epage>e2205</epage><pages>e2205-e2205</pages><issn>1935-2735</issn><issn>1935-2727</issn><eissn>1935-2735</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23675543</pmid><doi>10.1371/journal.pntd.0002205</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Diagnosis Diseases DNA, Intergenic - chemistry DNA, Intergenic - genetics DNA, Protozoan - chemistry DNA, Protozoan - genetics Dog Diseases - diagnosis Dog Diseases - parasitology Dogs Genotype Humans Leishmania - classification Leishmania - genetics Leishmania - isolation & purification Leishmaniasis Leishmaniasis - diagnosis Leishmaniasis - parasitology Leishmaniasis - veterinary Medicine Methods Microbiology Molecular Diagnostic Techniques - methods Molecular Sequence Data Parasites Parasitic diseases Parasitology - methods Polymerase chain reaction Real-Time Polymerase Chain Reaction - methods Sensitivity and Specificity Sequence Analysis, DNA Studies Transition Temperature Turkey |
| title | A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey |
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