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 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.

Detection and prediction of Streptococcus pneumoniae serotypes directly from nasopharyngeal swabs using PCR

“…As previously demonstrated for this target, the lytA RT-PCR was specific and detected only S. pneumoniae isolates (Table S4) (Carvalho et al, 2010;Resti et al, 2010;Kim et al, 2012;Cvitkovic Spik et al, 2013;Grijalva et al, 2014;Pernica et al, 2014;Strålin et al, 2014). As for cpsA RT-PCR, the results showed it was no less specific than lytA.…”

“…Compared with culture, lytA-based PCR has previously been shown to be highly sensitive for the detection of S. pneumoniae (Carvalho et al, 2010;Resti et al, 2010;Kim et al, 2012;Cvitkovic Spik et al, 2013;Grijalva et al, 2014;Pernica et al, 2014;Strålin et al, 2014). In addition, the lytA gene target was shown to be specific for the detection of S. pneumoniae Abdeldaim et al, 2010;Greve & Møller, 2012;Strålin et al, 2014).…”

“…CAP and IPD surveillance often rely on a number of laboratory tests, including bacterial culture and antigen detection (Facklam & Washington, 1991;Spellerberg & Brandt, 2007;Harris et al, 2014). Increasingly, molecular methods such as PCR are being used for the detection of S. pneumoniae as they are highly sensitive compared with culture, can be applied to various specimen types and are not predicated on the growth of viable bacteria (Carvalho et al, 2010;Resti et al, 2010;Kim et al, 2012;Cvitkovic Spik et al, 2013;Grijalva et al, 2014;Pernica et al, 2014;Strålin et al, 2014). To assess S. pneumoniae serotype distribution, serotyping methods using Quellung reactions or latex agglutination are often used, but these require viable pneumococci and large panels of antisera (Austrian, 1976;Lovgren et al, 1998;Siira et al, 2012;Ortika et al, 2013).…”

Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection ofRhizoctonia solani

“…2010a;Nagdev et al 2011;Kim et al 2012), where reaction tubes are never opened, are suitable alternatives. Availability of fully portable and battery-operated devices e.g.…”

Methods of Rapid Diagnosis for the Etiology of Meningitis in Adults

“…LAMP use in separate studies detecting H. influenza and S. pneumoniae actually outperformed conventional PCR and specificity was 100% [113,114]. RT-PCR developed to combat the changing serotype epidemiology of H influenza showed proof of concept in detecting all known samples of H influenza nonserotype B [115] and has been used to detect L. monocytogenes [116].…”