The enhanced pneumococcal LAMP assay: a clinical tool for the diagnosis of meningitis due to Streptococcus pneumoniae

• Published in: PloS one Vol. 7; no. 8; p. e42954
• Main Authors: Kim, Dong Wook, Kilgore, Paul E, Kim, Eun Jin, Kim, Soon Ae, Anh, Dang Duc, Dong, Bai Qing, Kim, Jung Soo, Seki, Mitsuko
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.08.2012; Public Library of Science (PLoS)
• Subjects: Antimicrobial agents; Assaying; Bacterial infections; Bacteriology; Biology; Cerebrospinal fluid; Child, Preschool; Children; Culture; Dentistry; Deoxyribonucleic acid; Developing countries; Diagnosis; Disease; DNA; DNA, Bacterial - chemistry; Epidemiology; Genes; Genes, Bacterial; Health sciences; Hospitals; Humans; Infant; Laboratories; LDCs; Medical diagnosis; Medicine; Meningitis; Meningitis, Pneumococcal - diagnosis; Methods; Microorganisms; N-Acetylmuramoyl-L-alanine Amidase - chemistry; N-Acetylmuramoyl-L-alanine Amidase - genetics; Nucleic Acid Amplification Techniques - methods; Nucleic acids; Pharmacy; Pneumonia; Polymerase chain reaction; Primers; Process controls; Reproducibility of Results; Sensitivity; Sensitivity analysis; Sensitivity and Specificity; Strains (organisms); Streptococcus infections; Streptococcus pneumoniae; Streptococcus pneumoniae - genetics; Surveillance; Turbidimetry; Vaccines; Virulence; Virulence (Microbiology); Virulence factors; South Korea; Vietnam; United States > US; China; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0042954

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). We 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%. Compared 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.