Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease

被引:12
作者
David de Paz, Hector [1 ]
Brotons, Pedro [1 ,2 ,3 ]
Esteva, Cristina [1 ,2 ]
Munoz-Almagro, Carmen [1 ,2 ,3 ]
机构
[1] Hosp St Joan de Deu, Dept Mol Microbiol, Inst Recerca Pediat, Barcelona, Spain
[2] CIBERESFP, CIBER Epidemiol & Publ Hlth, Madrid, Spain
[3] Univ Int Catalunya, Dept Med, Barcelona, Spain
来源
FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY | 2020年 / 10卷
关键词
Streptococcus pneumoniae; loop-mediated isothermal amplification; invasive pneumococcal disease; rapid diagnosis; diagnostic accuracy; STREPTOCOCCUS-PNEUMONIAE; YOUNG-CHILDREN; IDENTIFICATION; PCR; DNA; PATHOGENS; BLOOD;
D O I
10.3389/fcimb.2020.00115
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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 10(8)-10(4) copies/mL; limit of detection, 5 center dot 10(3) 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.
引用
收藏
页数:7
相关论文
共 34 条
[1]  
[Anonymous], 2001, FDA GUIDANCE IND BIO
[2]   PCR Using Blood for Diagnosis of Invasive Pneumococcal Disease: Systematic Review and Meta-Analysis [J].
Avni, Tomer ;
Mansur, Nariman ;
Leibovici, Leonard ;
Paul, Mical .
JOURNAL OF CLINICAL MICROBIOLOGY, 2010, 48 (02) :489-496
[3]   Real-Time PCR Measurement of Persistence of Bordetella pertussis DNA in Nasopharyngeal Secretions during Antibiotic Treatment of Young Children with Pertussis [J].
Bidet, Philippe ;
Liguori, Sandrine ;
De Lauzanne, Agathe ;
Caro, Valerie ;
Lorrot, Mathie ;
Carol, Agnes ;
Faye, Albert ;
Guiso, Nicole ;
Bingen, Edouard ;
Bonacorsi, Stephane .
JOURNAL OF CLINICAL MICROBIOLOGY, 2008, 46 (11) :3636-3638
[4]   Streptococcus pneumoniae colonisation:: the key to pneumococcal disease [J].
Bogaert, D ;
de Groot, R ;
Hermans, PWM .
LANCET INFECTIOUS DISEASES, 2004, 4 (03) :144-154
[5]   Validation of a loop-mediated isothermal amplification assay for rapid diagnosis of pertussis infection in nasopharyngeal samples [J].
Brotons, Pedro ;
de Paz, Hector D. ;
Esteva, Cristina ;
Latorre, Irene ;
Munoz-Almagro, Carmen .
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2016, 16 (01) :125-130
[6]  
Centers for Disease Control Prevention (CDC), 2015, 10 PCR DETECTION CHA
[7]   Comparison of reverse transcription loop-mediated isothermal amplification, conventional PCR and real-time PCR assays for Japanese encephalitis virus [J].
Chen, Zhiyong ;
Liao, Yuxue ;
Ke, Xuemei ;
Zhou, Jie ;
Chen, Yixiong ;
Gao, Lulu ;
Chen, Qing ;
Yu, Shouyi .
MOLECULAR BIOLOGY REPORTS, 2011, 38 (06) :4063-4070
[8]   Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics [J].
David de Paz, Hector ;
Brotons, Pedro ;
Munoz-Almagro, Carmen .
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2014, 14 (07) :827-843
[9]  
DELLINGER RP, 2013, CRIT CARE MED, V41, P580, DOI DOI 10.1097/CCM.0b013e31827e83af
[10]  
Fierz Walter, 2004, Methods Mol Med, V94, P393