Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease
Current molecular PCR-based techniques used for detecting Streptococcus pneumoniae, the causative pathogen of invasive pneumococcal disease (IPD), are accurate but have a run time of several hours. We aimed to develop and validate a novel real-time loop mediated amplification (LAMP) assay for rapid...
Main Authors: | , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Frontiers Media S.A.
2020-03-01
|
Series: | Frontiers in Cellular and Infection Microbiology |
Subjects: | |
Online Access: | https://www.frontiersin.org/article/10.3389/fcimb.2020.00115/full |
id |
doaj-7a075f54c14040bba9ce44d351ff9e54 |
---|---|
record_format |
Article |
spelling |
doaj-7a075f54c14040bba9ce44d351ff9e542020-11-25T02:05:08ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882020-03-011010.3389/fcimb.2020.00115460713Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal DiseaseHéctor David de Paz0Pedro Brotons1Pedro Brotons2Pedro Brotons3Cristina Esteva4Cristina Esteva5Carmen Muñoz-Almagro6Carmen Muñoz-Almagro7Carmen Muñoz-Almagro8Department of Molecular Microbiology, Institut de Recerca Pediatrica, Hospital Sant Joan de Déu, Barcelona, SpainDepartment of Molecular Microbiology, Institut de Recerca Pediatrica, Hospital Sant Joan de Déu, Barcelona, SpainCIBER of Epidemiology and Public Health, CIBERESP, Madrid, SpainDepartment of Medicine, Universitat Internacional de Catalunya, Barcelona, SpainDepartment of Molecular Microbiology, Institut de Recerca Pediatrica, Hospital Sant Joan de Déu, Barcelona, SpainCIBER of Epidemiology and Public Health, CIBERESP, Madrid, SpainDepartment of Molecular Microbiology, Institut de Recerca Pediatrica, Hospital Sant Joan de Déu, Barcelona, SpainCIBER of Epidemiology and Public Health, CIBERESP, Madrid, SpainDepartment of Medicine, Universitat Internacional de Catalunya, Barcelona, SpainCurrent molecular PCR-based techniques used for detecting Streptococcus pneumoniae, the causative pathogen of invasive pneumococcal disease (IPD), are accurate but have a run time of several hours. We aimed to develop and validate a novel real-time loop mediated amplification (LAMP) assay for rapid detection of pneumococcus in normally sterile samples with accuracy comparable to a gold standard real-time PCR. Conserved regions of lytA were used for the design of the LAMP test. Analytical validation included assessment of linearity, limit of detection (LOD), intra-assay and inter-assay precision and analytical specificity, which was evaluated by using reference strain S. pneumoniae R6 and a quality control panel. Clinical performance was assessed on all samples collected from children with suspicion of IPD attended in Hospital Sant Joan de Deu (Barcelona, Spain) during the period April-September 2015. Fresh samples were analyzed after DNA extraction. The following values of analytical parameters were determined: linearity within the range 108-104 copies/mL; limit of detection, 5·103 copies/mL; intra- and inter-assay precision measured by mean coefficient of variance, 3.61 and 6.59%; analytical specificity, 9/9 pathogens similar to S. pneumoniae and 14/14 strains of different S. pneumoniae serotypes correctly identified as negative and positive results, respectively. Diagnostic sensitivity and specificity values were 100.0 and 99.3%. Median time of DNA amplification was 15 min. The new LAMP assay showed to have similar accuracy as PCR while being 5-fold faster and could become a useful diagnostic tool for early diagnosis of IPD.https://www.frontiersin.org/article/10.3389/fcimb.2020.00115/fullStreptococcus pneumoniaeloop-mediated isothermal amplificationinvasive pneumococcal diseaserapid diagnosisdiagnostic accuracy |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Héctor David de Paz Pedro Brotons Pedro Brotons Pedro Brotons Cristina Esteva Cristina Esteva Carmen Muñoz-Almagro Carmen Muñoz-Almagro Carmen Muñoz-Almagro |
spellingShingle |
Héctor David de Paz Pedro Brotons Pedro Brotons Pedro Brotons Cristina Esteva Cristina Esteva Carmen Muñoz-Almagro Carmen Muñoz-Almagro Carmen Muñoz-Almagro Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease Frontiers in Cellular and Infection Microbiology Streptococcus pneumoniae loop-mediated isothermal amplification invasive pneumococcal disease rapid diagnosis diagnostic accuracy |
author_facet |
Héctor David de Paz Pedro Brotons Pedro Brotons Pedro Brotons Cristina Esteva Cristina Esteva Carmen Muñoz-Almagro Carmen Muñoz-Almagro Carmen Muñoz-Almagro |
author_sort |
Héctor David de Paz |
title |
Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease |
title_short |
Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease |
title_full |
Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease |
title_fullStr |
Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease |
title_full_unstemmed |
Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease |
title_sort |
validation of a loop-mediated isothermal amplification assay for rapid diagnosis of invasive pneumococcal disease |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cellular and Infection Microbiology |
issn |
2235-2988 |
publishDate |
2020-03-01 |
description |
Current molecular PCR-based techniques used for detecting Streptococcus pneumoniae, the causative pathogen of invasive pneumococcal disease (IPD), are accurate but have a run time of several hours. We aimed to develop and validate a novel real-time loop mediated amplification (LAMP) assay for rapid detection of pneumococcus in normally sterile samples with accuracy comparable to a gold standard real-time PCR. Conserved regions of lytA were used for the design of the LAMP test. Analytical validation included assessment of linearity, limit of detection (LOD), intra-assay and inter-assay precision and analytical specificity, which was evaluated by using reference strain S. pneumoniae R6 and a quality control panel. Clinical performance was assessed on all samples collected from children with suspicion of IPD attended in Hospital Sant Joan de Deu (Barcelona, Spain) during the period April-September 2015. Fresh samples were analyzed after DNA extraction. The following values of analytical parameters were determined: linearity within the range 108-104 copies/mL; limit of detection, 5·103 copies/mL; intra- and inter-assay precision measured by mean coefficient of variance, 3.61 and 6.59%; analytical specificity, 9/9 pathogens similar to S. pneumoniae and 14/14 strains of different S. pneumoniae serotypes correctly identified as negative and positive results, respectively. Diagnostic sensitivity and specificity values were 100.0 and 99.3%. Median time of DNA amplification was 15 min. The new LAMP assay showed to have similar accuracy as PCR while being 5-fold faster and could become a useful diagnostic tool for early diagnosis of IPD. |
topic |
Streptococcus pneumoniae loop-mediated isothermal amplification invasive pneumococcal disease rapid diagnosis diagnostic accuracy |
url |
https://www.frontiersin.org/article/10.3389/fcimb.2020.00115/full |
work_keys_str_mv |
AT hectordaviddepaz validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT pedrobrotons validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT pedrobrotons validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT pedrobrotons validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT cristinaesteva validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT cristinaesteva validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT carmenmunozalmagro validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT carmenmunozalmagro validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease AT carmenmunozalmagro validationofaloopmediatedisothermalamplificationassayforrapiddiagnosisofinvasivepneumococcaldisease |
_version_ |
1724939716818632704 |