Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid

Kim, Dong Wook; Kilgore, Paul E.; Kim, Eun Jin; Kim, Soon Ae; Anh, Đặng Đức; Seki, Mitsuko

doi:10.1128/jcm.00515-11

Cited by 42 publications

(40 citation statements)

References 25 publications

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“…Our results showing higher clinical sensitivity of the Sp LAMP assay are consistent with results of the LAMP assay developed for detecting H. influenzae type b in CSF [19]. Previous studies demonstrated that the LAMP reaction is more tolerant of potentially perturbing biological substances than PCR [22].…”

Section: Resultssupporting

confidence: 89%

The Enhanced Pneumococcal LAMP Assay: A Clinical Tool for the Diagnosis of Meningitis Due to Streptococcus pneumoniae

et al. 2012

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Background Streptococcus pneumoniae is a leading cause of invasive bacterial disease in developed and developing countries. We studied the loop-mediated isothermal amplification (LAMP) technique to assess its suitability for detecting S. pneumoniae nucleic acid in cerebrospinal fluid (CSF).Methodology/Principal FindingsWe established an improved LAMP assay targeting the lytA gene (Streptococcus pneumoniae [Sp] LAMP). The analytical specificity of the primers was validated by using 32 reference strains (10 Streptococcus and seven non-Streptococcus species) plus 25 clinical alpha-hemolytic streptococcal strains, including four S. pneumoniae strains and 21 other strains (3 S. oralis, 17 S. mitis, and one Streptococcus species) harboring virulence factor-encoding genes (lytA or ply). Within 30 minutes, the assay could detect as few as 10 copies of both purified DNA and spiked CSF specimens with greater sensitivity than conventional polymerase chain reaction (PCR). The linear determination range for this assay is 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the clinical sensitivity and specificity of the Sp LAMP assay using 106 randomly selected CSF specimens from children with suspected meningitis in Korea, China and Vietnam. For comparison, CSF specimens were also tested against conventional PCR and culture tests. The detection rate of the LAMP method was substantially higher than the rates of PCR and culture tests. In this small sample, relative to the LAMP assay, the clinical sensitivity of PCR and culture tests was 54.5% and 33.3%, respectively, while clinical specificity of the two tests was 100%.Conclusions/SignificanceCompared to PCR, Sp LAMP detected S. pneumoniae with higher analytical and clinical sensitivity. This specific and sensitive LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

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Section: Resultssupporting

confidence: 89%

The Enhanced Pneumococcal LAMP Assay: A Clinical Tool for the Diagnosis of Meningitis Due to Streptococcus pneumoniae

et al. 2012

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“…The LAMP reaction mixture was prepared by following the manufacture’s protocol. Table S1 shows the LAMP primers purchased from Integrated DNA Technologies (Coralville, IA) for the target DNA sequences of the N. meningitidis ctrA gene, the S. pneumoniae lytA gene and the Hib capsulation locus region II (Kim et al 2012; Kim et al 2011; McKenna et al 2011). …”

Section: Methodsmentioning

confidence: 99%

“…The high strand displacement activity from the Bacillus stearothermophilus ( Bst ) DNA polymerase and identification of 6 distinct regions from 2 or 3 sets of primers in LAMP result in high specificity. There have been several reports on rapid diagnosis of bacterial meningitis (e.g., N. meningitidis , S. pneumoniae , Hib) by using conventional off-chip LAMP (Kim et al 2012; Kim et al 2011; McKenna et al 2011). However, none of these LAMP methods achieved multiplexed bacterial meningitis detection.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed instrument-free meningitis diagnosis on a polymer/paper hybrid microfluidic biochip

Dou

Sanjay

Domı́nguez

et al. 2017

Biosensors and Bioelectronics

121

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Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae), and Haemophilus influenzae type b (Hib) are three most common pathogens accounting for most bacterial meningitis, a serious global infectious disease with high fatality, especially in developing nations. Because the treatment and antibiotics differ among each type, the identification of the exact bacteria causing the disease is vital. Herein, we report a polymer/paper hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) for multiplexed instrument-free diagnosis of these three major types of bacterial meningitis, with high sensitivity and specificity. Results can be visually observed by the naked eye or imaged by a smartphone camera under a portable UV light source. Without using any specialized laboratory instrument, the limits of detection of a few DNA copies per LAMP zone for N. meningitidis, S. pneumoniae and Hib were achieved within 1 hour. In addition, these three types of microorganisms spiked in artificial cerebrospinal fluid (ACSF) were directly detected simultaneously, avoiding cumbersome sample preparation procedures in conventional methods. Compared with the paper-free non-hybrid microfluidic biochip over a period of three months, the hybrid microfluidic biochip was found to have a much longer shelf life. Hence, this rapid, instrument-free and highly sensitive microfluidic approach has great potential for point-of-care (POC) diagnosis of multiple infectious diseases simultaneously, especially in resource-limited settings.

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“…LAMP use in separate studies detecting H. influenza and S. pneumoniae actually outperformed conventional PCR and specificity was 100% [113,114]. RT-PCR developed to combat the changing serotype epidemiology of H influenza showed proof of concept in detecting all known samples of H influenza nonserotype B [115] and has been used to detect L. monocytogenes [116].…”

Section: Diagnostic Tests For Bacterial Meningitismentioning

confidence: 99%

Methods of Rapid Diagnosis for the Etiology of Meningitis in Adults

Bahr

Boulware

2014

Biomark. Med.

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Infectious meningitis may be due to bacterial, mycobacterial, fungal or viral agents. Diagnosis of meningitis must take into account numerous items of patient history and symptomatology along with regional epidemiology and basic cerebrospinal fluid testing (protein, etc.) to allow the clinician to stratify the likelihood of etiology possibilities and rationally select additional diagnostic tests. Culture is the mainstay for diagnosis in many cases, but technology is evolving to provide more rapid, reliable diagnosis. The cryptococcal antigen lateral flow assay (Immuno-Mycologics) has revolutionized diagnosis of cryptococcosis and automated nucleic acid amplification assays hold promise for improving diagnosis of bacterial and mycobacterial meningitis. This review will focus on a holistic approach to diagnosis of meningitis as well as recent technological advances.

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Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid

Cited by 42 publications

References 25 publications

The Enhanced Pneumococcal LAMP Assay: A Clinical Tool for the Diagnosis of Meningitis Due to Streptococcus pneumoniae

The Enhanced Pneumococcal LAMP Assay: A Clinical Tool for the Diagnosis of Meningitis Due to Streptococcus pneumoniae

Multiplexed instrument-free meningitis diagnosis on a polymer/paper hybrid microfluidic biochip

Methods of Rapid Diagnosis for the Etiology of Meningitis in Adults

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