Abstract
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.
Highlights
Streptococcus pneumoniae is associated with invasive and noninvasive diseases, such as meningitis, bacteremia, septicemia, community-acquired pneumonia, and otitis media [1]
S. mitis and S. oralis are most closely related to pneumococcus on this ecological basis, and their 16S rRNA sequences share over 99% identity with pneumococcus, which presents a challenge for clinical laboratories to differentiate pneumococcus from other alpha-hemolytic oral streptococci [4]
The supernatant of a pooled pneumococcus-negative cerebrospinal fluid (CSF) specimen [16] was used for a spiking assay in which serial ten-fold dilutions of genomic DNA were amplified, and the results were compared between Sp loop-mediated isothermal amplification (LAMP) and conventional Sp polymerase chain reaction (PCR)
Summary
Streptococcus pneumoniae is associated with invasive and noninvasive diseases, such as meningitis, bacteremia, septicemia, community-acquired pneumonia, and otitis media [1]. We established an improved LAMP assay by adding a loop primer targeting the lytA gene (Sp LAMP), and we compared the detection performance of the Sp LAMP assay with that of Sp PCR by using cerebrospinal fluid (CSF) specimens from patients with suspected meningitis in Korea, China and Vietnam [16]. To our knowledge, this is the first report of using a LAMP assay for detecting S. pneumoniae in clinical CSF specimens
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