Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis
To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DN...
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MDPI AG
2020-06-01
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| Series: | Tropical Medicine and Infectious Disease |
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| Online Access: | https://www.mdpi.com/2414-6366/5/2/95 |
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| author | Rajashree Chowdhury Prakash Ghosh Md. Anik Ashfaq Khan Faria Hossain Khaledul Faisal Rupen Nath James Baker Ahmed Abd El Wahed Shomik Maruf Proggananda Nath Debashis Ghosh Md. Masud-Ur-Rashid Md. Utba Bin Rashid Malcolm S. Duthie Dinesh Mondal |
| author_facet | Rajashree Chowdhury Prakash Ghosh Md. Anik Ashfaq Khan Faria Hossain Khaledul Faisal Rupen Nath James Baker Ahmed Abd El Wahed Shomik Maruf Proggananda Nath Debashis Ghosh Md. Masud-Ur-Rashid Md. Utba Bin Rashid Malcolm S. Duthie Dinesh Mondal |
| author_sort | Rajashree Chowdhury |
| collection | DOAJ |
| description | To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments. |
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| series | Tropical Medicine and Infectious Disease |
| spelling | doaj.art-e40d2041d1504b93a5ae9fb5fd2ab74d2023-11-20T03:02:06ZengMDPI AGTropical Medicine and Infectious Disease2414-63662020-06-01529510.3390/tropicalmed5020095Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal LeishmaniasisRajashree Chowdhury0Prakash Ghosh1Md. Anik Ashfaq Khan2Faria Hossain3Khaledul Faisal4Rupen Nath5James Baker6Ahmed Abd El Wahed7Shomik Maruf8Proggananda Nath9Debashis Ghosh10Md. Masud-Ur-Rashid11Md. Utba Bin Rashid12Malcolm S. Duthie13Dinesh Mondal14Nutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshInstitute of Animal Hygiene and Veterinary Public Health, University of Leipzig, D-04103 Leipzig, GermanyNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshInfection and Tropical Medicine, Mymensingh Medical College and Hospital (MMCH), Mymensingh 2200, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshDepartment of Cardiovascular and Thoracic Surgery, National Heart Foundation Hospital and Research Institute, Dhaka 1216, BangladeshNutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshHost Directed Therapeutics (HDT) Bio Corp, Seattle, WA 98102, USANutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, BangladeshTo detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.https://www.mdpi.com/2414-6366/5/2/95post-kala-azar dermal leishmaniasis (PKDL)point-of-need diagnosisDNA extractionrecombinase polymerase amplification (RPA)real-time PCR |
| spellingShingle | Rajashree Chowdhury Prakash Ghosh Md. Anik Ashfaq Khan Faria Hossain Khaledul Faisal Rupen Nath James Baker Ahmed Abd El Wahed Shomik Maruf Proggananda Nath Debashis Ghosh Md. Masud-Ur-Rashid Md. Utba Bin Rashid Malcolm S. Duthie Dinesh Mondal Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis Tropical Medicine and Infectious Disease post-kala-azar dermal leishmaniasis (PKDL) point-of-need diagnosis DNA extraction recombinase polymerase amplification (RPA) real-time PCR |
| title | Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis |
| title_full | Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis |
| title_fullStr | Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis |
| title_full_unstemmed | Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis |
| title_short | Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis |
| title_sort | evaluation of rapid extraction methods coupled with a recombinase polymerase amplification assay for point of need diagnosis of post kala azar dermal leishmaniasis |
| topic | post-kala-azar dermal leishmaniasis (PKDL) point-of-need diagnosis DNA extraction recombinase polymerase amplification (RPA) real-time PCR |
| url | https://www.mdpi.com/2414-6366/5/2/95 |
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