Assessment of a Multiplex LAMP Assay (Eazyplex® CSF Direct M) for Rapid Molecular Diagnosis of Bacterial Meningitis: Accuracy and Pitfalls

Abstract

Background: Automated molecular panels are attractive tools for improving early meningitis diagnosis. This study assessed the Eazyplex^® CSF direct M panel (EP), a multiplex real-time Loop-Mediated Isothermal Amplification assay. Methods: From December 2016 to December 2019, cerebrospinal fluid (CSF) samples were routinely tested with the EP V1.0. CSF parameters and microbiological and clinical data were retrospectively collected. Results: Out of 230 CSF samples, the EP yielded positive, negative, and invalid results for 32 (13.9%) (16 N. meningitidis, nine S. pneumoniae, two S. agalactiae, two E. coli, two H. influenzae, one L. monocytogenes), 182 (79.1%), and 16 (7%) samples, respectively. Among the positive samples, 14 (44%) remained negative in culture (antibiotic therapy before lumbar puncture (n = 11), meningococcal meningitis (n = 3)). High CSF protein concentrations and cellularity were associated with LAMP inhibition, counteracted by centrifugation. The automated software yielded 13 false positive and five false negative results. Amplification curve analysis was necessary and enabled the attainment of positive (PPA) and negative percentage agreement and positive and negative predictive values of 91.4%, 100%, 100%, and 98.3%. Three false negative results remained (two E. coli and one N. meningitidis). E. coli presented the poorest PPA (50%). Conclusion: This work confirms the strong performance of the EP, of particular interest in cases of antibiotic therapy before lumbar puncture.

Keywords: Eazyplex® CSF direct M panel; Loop-Mediated Isothermal Amplification (LAMP); amplification curves analysis; bacterial meningitis; performance; rapid diagnosis.

Figure 1

Study selection flow diagram. Abbreviations: CSF, cerebrospinal fluid; LAMP, Loop-mediated isothermal amplification.

Figure 2

Impact of CSF protein concentration, WBC and RBC count on LAMP inhibition. Abbreviations: CSF, cerebrospinal fluid; IQR, interquartile range; WBC, white blood cell; RBC, red blood cell; IC, inhibition control. ^a missing data: valid IC group (n = 9) and invalid IC group (n = 4); ^b missing data: valid IC group (n = 10) and invalid IC group (n = 4); ^c missing data: valid IC group (n = 63) and invalid IC group (n = 15). * p < 0.05; **** p < 0.0001; n.s: not significant.

Figure 3

Amplification curves and rates of true positive (A,B) and false positive (C,D) results.

Figure 4

Impact of CSF centrifugation on amplification curves. CSF protein and WBC in valid inhibition control group versus in CSF needed centrifugation before analysis (A); Amplification curves and rates before (B,C) and after (B’,C’) centrifugation. Abbreviations: CSF, cerebrospinal fluid; WBC, white blood cells. ^a missing data: valid IC group (n = 9) and centrifugation needed group (n = 1); ^b missing data: valid IC group (n = 10) and invalid IC group (n=1).