Development and validation of an advanced fragment analysis‐based assay for the detection of 22 pathogens in the cerebrospinal fluid of patients with meningitis and encephalitis

Abstract

BackgroundMeningitis and encephalitis (ME) are central nervous system (CNS) infections mainly caused by bacteria, mycobacteria, fungi, viruses, and parasites that result in high morbidity and mortality. The early, accurate diagnosis of pathogens in the cerebrospinal fluid (CSF) and timely medication are associated with better prognosis. Conventional methods, such as culture, microscopic examination, serological detection, CSF routine analysis, and radiological findings, either are time‐consuming or lack sensitivity and specificity.MethodsTo address these clinical needs, we developed an advanced fragment analysis (AFA)‐based assay for the multiplex detection of 22 common ME pathogens, including eight viruses, 11 bacteria, and three fungi. The detection sensitivity of each target was evaluated with a recombinant plasmid. The limits of detection of the 22 pathogens ranged from 15 to 120 copies/reaction. We performed a retrospective study to analyze the pathogens from the CSF specimens of 170 clinically diagnosed ME patients using an AFA‐based assay and compared the results with culture (bacteria and fungi), microscopic examination (fungi), polymerase chain reaction (PCR) (Mycobacterium tuberculosis), and Sanger sequencing (virus) results.ResultsThe sensitivity of the AFA assay was 100% for 10 analytes. For Cryptococcus neoformans, the sensitivity was 63.6%. The overall specificity was 98.2%. The turnaround time was reduced to 4‐6 hours from the 3‐7 days required using conventional methods.ConclusionsIn conclusion, the AFA‐based assay provides a rapid, sensitive, and accurate method for pathogen detection from CSF samples.

Multiplex detection of meningitis and encephalitis pathogens: A study from laboratory to clinic

“…Among them, MME-18 was positive in 51.8% of cases, and in two cases with only positive Xpert MTB/RIF results. According to other studies in China, the identification of ME etiologies varies from 7.5 to 81.8% (14,15,27). In mono infections, the most are MTB (23.7%, 33/139), which also represents one of the most important etiological agents for ME.…”

“…The PCR product was subjected to capillary electrophoresis and fragment analysis using a 3500Dx Genetic Analyzer (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's protocol (Supplementary Figure S1). The procedure details had been reported previously (27). The panel was designed to detect 18 pathogens within 4 h.…”

“…Each positive viral result sample with sequencing comparator assays (Sangon Biotech, Shanghai, China) was conducted. Information for all of the primers has been listed in the previous report (27). The sequencing data were compared with the data provided by NCBI to confirm the pathogen.…”

“…Multiplex PCR is a PCR amplification technique that can simultaneously and quickly detect various pathogens in a PCR reaction system. The procedure details have been reported previously (27), but its utilization for MME-18 has not been investigated. Therefore, this study investigated the epidemiological characteristics of ME pathogens in Southwestern China using the novel detection method.…”

Diagnostic Yield of Multiplex PCR Method in Cerebrospinal Fluid for the Diagnosis of Purulent Meningitis in Children

“…Nucleic acid was extracted from each well-stored CSF specimen using the QIAamp DNA mini kit (QIAGEN; Hilden, Germany) on an automated operating platform (Smart LabAssist-32; Taiwan Advanced Nanotech, Taiwan, China). The extracted DNA samples were amplified by PCR, using primers designed by Long et al 11 with the following touch-down PCR program: (1) 25°C for 5 minutes, 50°C for 30 minutes, and 95°C for 15 minutes; (2) 6 cycles of denaturation at 94°C for 30 seconds, annealing at the temperature from 65°C to 60°C (1°C reduction per cycle) for 30 seconds and extension at 72°C for 60 seconds; (3) 29 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 60 seconds; and (4) a final extension at 72°C for 15 minutes and hold at 4°C. PCR products were analyzed by capillary electrophoresis and transferred to ABI 3500Dx genetic analyzer for separation and detection.…”