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

Chika Takano,Tomonori Hoshino,Dong Wook Kim,Eun Jin Kim,Yoko Kuramochi,Daisuke Omagari,Kazumasa Fuwa,Makoto Ohnishi,Paul E Kilgore,Mari Sasano,Satoshi Hayakawa,Mitsuko Seki,Bin Chang

doi:10.1038/s41598-019-56225-0

Abstract

In children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). However, since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. However, invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. To develop a rapid, simple, and cost-effective assay to detect serotypes of Streptococcus pneumoniae, we developed a novel loop-mediated isothermal amplification (LAMP) assay based on the sequences available for the 13 capsular types that are included in PCV13: 1, 3, 4, 5, 6 A, 6B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, and 23 F. We evaluated test reactivity, specificity, sensitivity and performance, and compared the results between established LAMP and conventional PCR assays. To support its clinical use, the detection limits of the LAMP assay were evaluated using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) and blood specimens. We confirmed the specificity of the LAMP assay using 41 serotypes of pneumococcal strains. The sensitivity of the LAMP assay was 10 to 100 copies per reaction, compared to 10 to 104 copies per reaction for PCR assays. The detection limits of the LAMP assay were comparable when using DNA-spiked CSF and blood specimens, as compared to using purified DNA as the template. In conclusion, a rapid and simple LAMP-based pneumococcal serotyping method has been developed. This is the first report of a LAMP method for a PCV13 serotype-specific identification assay, which could be a promising step to facilitate epidemiological studies of pneumococcal serotyping.

Highlights

In children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13)
Since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13), the incidence of meningitis caused by S. pneumoniae in children has decreased[4,5]
The analytical reactivity and specificity of loop-mediated isothermal amplification (LAMP)-based pneumococcal serotyping (Table 1) were evaluated using 55 pneumococcal strains belonging to 41 pneumococcal serotypes (Table 2)

Summary

Introduction

The incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). Since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. Invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. A rapid and simple LAMP-based pneumococcal serotyping method has been developed. Non-PCV13 serotypes have become an emerging problem since the introduction of the vaccine. Serological typing of pneumococci is performed using the Quellung reaction with type-specific pneumococcal antisera In this method, serum is added to the bacteria obtained from the medium, and swelling of the capsule is scored under a microscope. Accurately determining pneumococcal serotypes remains challenging due to the limited availability of routine microbiology laboratory services and facilities in developing countries[10]

Methods

Results

Conclusion