Molecular Serotype-Specific Identification of Non-type b Haemophilus influenzae by Loop-Mediated Isothermal Amplification

Abstract

Over the past four decades, the incidence of meningitis caused by Haemophilus influenzae in children has decreased due to widespread vaccination against H. influenzae type b (Hib). The incidence of invasive diseases due to H. influenzae types not included in the vaccines, however, has increased. At present, there are a limited number of diagnostics available to detect non-type b H. influenzae. To address this issue, we developed a rapid, simple, and cost-effective method for detecting serotypes of H. influenzae. We designed LAMP primer sets based on published sequences for H. influenzae capsular types a, c, d, e, and f. The assay was evaluated to determine test reactivity, specificity, and sensitivity. To support its use in patients with suspected meningitis, we evaluated the detection limit of the non-Hib serotype specific LAMP assay using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) specimens. The reactivity and specificity of the LAMP assays were confirmed using six serotypes and non-typeable H. influenzae strains, plus eight strains of other Haemophilus species and non-Haemophilus genera. The detection limits of the LAMP assay for capsular types a, c, d, e, and f were 102, 102, 102, 103, and 10 copies per reaction, while those of the PCR assay were 104, 104, 103, 103, and 104 genome copies per reaction, respectively. Using DNA-spiked CSF specimens, the detection limit of the LAMP assay was equivalent to that using purified DNA as the template. However, the detection limit of the PCR was reduced from 103 to 104 genome copies per reaction for serotype d and from 103 to 105 genome copies per reaction for serotype e. To the best of our knowledge, this is the first report of a serotype-specific identification assay for H. influenzae using the LAMP method. Our results suggest the potential of LAMP methods for patients with suspected meningitis in resource-limited laboratories or public health surveillance systems.

Loop-Mediated Isothermal Amplification Methods for Diagnosis of Bacterial Meningitis

“…Loop-mediated isothermal amplification methods for serogrouping meningococcus and serotyping Haemophilus influenzae have been established ( 9 , 14 , 15 ). However, LAMP methods for pneumococcus serotyping have not yet been available, and some studies have reported the incidence of invasive diseases due to pneumococcal strains that were not included in the vaccines ( 2 , 40 , 41 ).…”

“…LAMP-based diagnostic assays for S. pneumoniae, H. influenzae , and N. meningitidis have been established ( 9 – 13 ). The typing assays using LAMP for the detection of N. meningitidis serogroups [A, B, C, W, X, and Y ( 14 )] and H. influenzae serotypes [a, b, c, d, e, and f ( 15 )] were released.…”

Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification

“…LAMP reactions were not inhibited, or were inhibited only slightly, when using DNA-spiked CSF and blood. PCR is inhibited by biological substances, particularly heparin 30 and other blood components, including haem, leukocyte DNA, and immunoglobulin G 22,31,32 . The LAMP assay can be performed using simple DNA preparation methods because the LAMP reaction more readily tolerates potentially disturbing biological elements (i.e.…”

“…LAMP-based diagnostic assays for S. pneumoniae , Haemophilus influenzae , and Neisseria meningitidis in cerebrospinal fluid (CSF) specimens have been established 17–20 . LAMP-based methods for meningococcal typing to detect meningococcal serogroups A, B, C, X, Y, and W 21 , as well as H. influenzae serotypes a, b, c, d, e, and f, have been developed 17,22 .…”

Combination of Loop-Mediated Isothermal Amplification and AuNP-Oligoprobe Colourimetric Assay for Pork Authentication in Processed Meat Products

“…The most frequently used DNA detection techniques for meat identification are based on polymerase chain reaction (PCR) such as randomly amplified polymorphic DNA-PCR (RAPD-PCR), restriction fragment length polymorphism-PCR (RFLP-PCR), PCR with species-specific primers, real-time PCR/quantitative PCR (RT-PCR/qPCR), and digital PCR (dPCR) (Alikord et al 2017;Ballin et al 2009;Farag et al 2015). Despite their high sensitivity and specificity, PCR methods have some limitations including their complicated experimentation, the need of a thermal cycler or an additional detection machine, their relatively high cost of reagents and time consumption, and the need of skilled operators (Ali et al 2012;Ckumdee et al 2016;Seetang-Nun et al 2013;Takano et al 2017;Tomita et al 2008). This thus presents an opportunity for developing novel and more efficient approaches for authenticating meat species.…”