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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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