Background Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF).Methodology/Principal FindingsWe developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively.Conclusions/SignificanceCompared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.
We have developed a novel Neisseria meningitidis serogroup-specific loop-mediated isothermal amplification (LAMP) assay for six of the most common meningococcal serogroups (A, B, C, W, X, and Y). The assay was evaluated using a set of 31 meningococcal LAMP assay positive cerebrospinal fluid (CSF) specimens from 1574 children with suspected meningitis identified in prospective surveillance between 1998 and 2002 in Vietnam, China, and Korea. Primer specificity was validated using 15 N. meningitidis strains (including serogroups A, B, C, E, W, X, Y, and Z) and 19 non-N. meningitidis species. The N. meningitidis serogroup LAMP detected down to ten copies and 100 colony-forming units per reaction. Twenty-nine CSF had N. meningitidis serogroup identified by LAMP compared with two CSF in which N. meningitidis serogroup was identified by culture and multi-locus sequence typing. This is the first report of a serogroup-specific identification assay for N. meningitidis using the LAMP method. Our results suggest that this assay will be a rapid, sensitive, and uniquely serogroup-specific assay with potential for application in clinical laboratories and public health surveillance systems.
Background The clinical impact of infection with Mycobacterium ( M. ) abscessus complex (MABC), a group of emerging non-tuberculosis mycobacteria (NTM), is increasing. M. abscessus subsp. abscessus / bolletii frequently shows natural resistance to macrolide antibiotics, whereas M. abscessus subsp. massiliense is generally susceptible. Therefore, rapid and accurate discrimination of macrolide-susceptible MABC subgroups is required for effective clinical decisions about macrolide treatments for MABC infection. We aimed to develop a simple and rapid diagnostic that can identify MABC isolates showing macrolide susceptibility. Methods Whole genome sequencing (WGS) was performed for 148 clinical or environmental MABC isolates from Japan to identify genetic markers that can discriminate three MABC subspecies and the macrolide-susceptible erm (41) T28C sequevar. Using the identified genetic markers, we established PCR based- or DNA chromatography-based assays. Validation testing was performed using MABC isolates from Taiwan. Finding We identified unique sequence regions that could be used to differentiate the three subspecies. Our WGS-based phylogenetic analysis indicated that M. abscessus carrying the macrolide-susceptible erm (41) T28C sequevar were tightly clustered, and identified 11 genes that were significantly associated with the lineage for use as genetic markers. To detect these genetic markers and the erm (41) locus, we developed a DNA chromatography method that identified three subspecies, the erm (41) T28C sequevar and intact erm (41) for MABC in a single assay within one hour. The agreement rate between the DNA chromatography-based and WGS-based identification was 99·7%. Interpretation We developed a novel, rapid and simple DNA chromatography method for identification of MABC macrolide susceptibility with high accuracy. Funding AMED, JSPS KAKENHI
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