Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka
The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by <i>Leishmania donovani</i>) nested Internal Transcribed Spacer-1 (ITS1) PCR-...
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MDPI AG
2022-05-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/5/990 |
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| author | Nirmitha Lalindi De Silva Viraji Nefertiti Hiromel De Silva Arachchige Theja Hemapala Deerasinghe Upeksha Lakmini Rathnapala Makoto Itoh Hidekazu Takagi Mirani Vasanthamala Weerasooriya Hirotomo Kato Thishan Channa Yahathugoda |
| author_facet | Nirmitha Lalindi De Silva Viraji Nefertiti Hiromel De Silva Arachchige Theja Hemapala Deerasinghe Upeksha Lakmini Rathnapala Makoto Itoh Hidekazu Takagi Mirani Vasanthamala Weerasooriya Hirotomo Kato Thishan Channa Yahathugoda |
| author_sort | Nirmitha Lalindi De Silva |
| collection | DOAJ |
| description | The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by <i>Leishmania donovani</i>) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (10<sup>3</sup> to 10<sup>−2</sup>) of the DNA extract of a cultured <i>L. donovani</i> DD8 strain. Patients (<i>n</i> = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; <i>p</i> < 0.01). The RFLP pattern was <i>L. donovani</i> in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; <i>p</i> < 0.01), ulcerated nodules (91% vs. 71.8%; <i>p</i> < 0.01) and plaques (100% vs. 66.7%; <i>p</i> < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy. |
| first_indexed | 2024-03-10T03:22:03Z |
| format | Article |
| id | doaj.art-62a809f91c91494a95d041aa10abdc85 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T03:22:03Z |
| publishDate | 2022-05-01 |
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| series | Microorganisms |
| spelling | doaj.art-62a809f91c91494a95d041aa10abdc852023-11-23T12:16:08ZengMDPI AGMicroorganisms2076-26072022-05-0110599010.3390/microorganisms10050990Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri LankaNirmitha Lalindi De Silva0Viraji Nefertiti Hiromel De Silva1Arachchige Theja Hemapala Deerasinghe2Upeksha Lakmini Rathnapala3Makoto Itoh4Hidekazu Takagi5Mirani Vasanthamala Weerasooriya6Hirotomo Kato7Thishan Channa Yahathugoda8Department of Parasitology, Faculty of Medicine, University of Ruhuna, Galle 80000, Sri LankaBase Hospital Tangalle, Tangalle 82200, Sri LankaDistrict General Hospital Hambantota, Hambantota 82000, Sri LankaSchool of Biosciences, University of Melbourne, Melbourne, VIC 3010, AustraliaDepartment of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi 480-1195, JapanDepartment of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi 480-1195, JapanDepartment of Parasitology, Faculty of Medicine, University of Ruhuna, Galle 80000, Sri LankaDivision of Medical Zoology, Department of Infection and Immunity, Jichi Medical University, Tochigi 329-0498, JapanDepartment of Parasitology, Faculty of Medicine, University of Ruhuna, Galle 80000, Sri LankaThe recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by <i>Leishmania donovani</i>) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (10<sup>3</sup> to 10<sup>−2</sup>) of the DNA extract of a cultured <i>L. donovani</i> DD8 strain. Patients (<i>n</i> = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; <i>p</i> < 0.01). The RFLP pattern was <i>L. donovani</i> in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; <i>p</i> < 0.01), ulcerated nodules (91% vs. 71.8%; <i>p</i> < 0.01) and plaques (100% vs. 66.7%; <i>p</i> < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy.https://www.mdpi.com/2076-2607/10/5/990cutaneous leishmaniasisITSPCR-RFLPSri Lanka |
| spellingShingle | Nirmitha Lalindi De Silva Viraji Nefertiti Hiromel De Silva Arachchige Theja Hemapala Deerasinghe Upeksha Lakmini Rathnapala Makoto Itoh Hidekazu Takagi Mirani Vasanthamala Weerasooriya Hirotomo Kato Thishan Channa Yahathugoda Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka Microorganisms cutaneous leishmaniasis ITS PCR-RFLP Sri Lanka |
| title | Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka |
| title_full | Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka |
| title_fullStr | Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka |
| title_full_unstemmed | Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka |
| title_short | Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka |
| title_sort | development of a highly sensitive nested pcr and its application for the diagnosis of cutaneous leishmaniasis in sri lanka |
| topic | cutaneous leishmaniasis ITS PCR-RFLP Sri Lanka |
| url | https://www.mdpi.com/2076-2607/10/5/990 |
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