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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 Leishmania donovani) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragmen...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9145043/ https://www.ncbi.nlm.nih.gov/pubmed/35630433 http://dx.doi.org/10.3390/microorganisms10050990 |
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author | De Silva, Nirmitha Lalindi De Silva, Viraji Nefertiti Hiromel Deerasinghe, Arachchige Theja Hemapala Rathnapala, Upeksha Lakmini Itoh, Makoto Takagi, Hidekazu Weerasooriya, Mirani Vasanthamala Kato, Hirotomo Yahathugoda, Thishan Channa |
author_facet | De Silva, Nirmitha Lalindi De Silva, Viraji Nefertiti Hiromel Deerasinghe, Arachchige Theja Hemapala Rathnapala, Upeksha Lakmini Itoh, Makoto Takagi, Hidekazu Weerasooriya, Mirani Vasanthamala Kato, Hirotomo Yahathugoda, Thishan Channa |
author_sort | De Silva, Nirmitha Lalindi |
collection | PubMed |
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 Leishmania donovani) 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(3) to 10(−2)) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 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%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 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. |
format | Online Article Text |
id | pubmed-9145043 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91450432022-05-29 Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka De Silva, Nirmitha Lalindi De Silva, Viraji Nefertiti Hiromel Deerasinghe, Arachchige Theja Hemapala Rathnapala, Upeksha Lakmini Itoh, Makoto Takagi, Hidekazu Weerasooriya, Mirani Vasanthamala Kato, Hirotomo Yahathugoda, Thishan Channa Microorganisms Article 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 Leishmania donovani) 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(3) to 10(−2)) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 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%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 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. MDPI 2022-05-09 /pmc/articles/PMC9145043/ /pubmed/35630433 http://dx.doi.org/10.3390/microorganisms10050990 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article De Silva, Nirmitha Lalindi De Silva, Viraji Nefertiti Hiromel Deerasinghe, Arachchige Theja Hemapala Rathnapala, Upeksha Lakmini Itoh, Makoto Takagi, Hidekazu Weerasooriya, Mirani Vasanthamala Kato, Hirotomo Yahathugoda, Thishan Channa Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in 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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9145043/ https://www.ncbi.nlm.nih.gov/pubmed/35630433 http://dx.doi.org/10.3390/microorganisms10050990 |
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