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

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 findings: 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%.

Conclusions/significance: 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.

Figure 1. Nucleotide sequence of the lytA gene used to design the Sp LAMP primer.

The sequences used for Sp LAMP primers are indicated by arrows (A). Structure and sequence of the primers used in the Sp LAMP reaction (B).

Figure 2. Real-time sensitivity of Sp LAMP, as monitored by the measurement of turbidity.

Shown from left to right in the figure are the curves of decreasing concentration (1,000,000 to 1) of bacteria. The detection limit was 10 copies (A). The relationship between the threshold time (Tt) of each sample and the log of the amount of initial template DNA (B).

Figure 3. Electrophoretic analysis of Sp LAMP-amplified products.

Lane M, 100-bp ladder (New England Biolabs, Beverly, MA, USA) used as a size marker; lane 1, Sp LAMP product from lane 2 after digestion with TasI (Fermentas, Inc.). The digested fragments were 107 and 108 bp; lane 2, 10⁶ copies of the genomic DNA of S. pneumoniae ATCC 6305; lane 3, no template.