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Development of a TaqMan Array card to target 21 purulent meningitis-related pathogens.

ABSTRACT: BACKGROUND:Purulent meningitis (PM) is a serious life-threatening infection of the central nervous system (CNS) by bacteria or fungi and associated with high mortality and high incidence of CNS sequelae in children. However, the conventional cerebrospinal fluid (CSF) culture method is time-consuming and has a low sensitivity. METHODS:Our study developed a real-time PCR-based purulent meningitis-TaqMan array card (PM-TAC) that targeted 21?PM-related pathogens and could produce results within 3?h. Primers and probes were adapted from published sources possibly. The performance of them were evaluated and optimized and then they were spotted on TAC. RESULTS:The PM-TAC showed a sensitivity and specificity of 95 and 96%, respectively. For all of the 21 targeted pathogens, the PM-TAC assay had a LOD ranging from 5 copies/reaction to 100 copies/reaction, an intra-assay variation of 0.07-4.45%, and an inter-assay variation of 0.11-6.81%. Of the 15 CSF samples collected from patients with PM after empiric antibiotic therapies, the positive rate was 53.3% (8/15) for our PM-TAC assay but was only 13.3% (2/15) for the CSF culture method. Of the 17 CSF samples showing negative CSF culture, the PM-TAC assay identified a case of Neisseria meningitidis infection. Furthermore, all of the 10 CSF samples from patients without CNS infection showed negative for the PM-TAC assay. CONCLUSIONS:Our PM-TAC assay also demonstrated that the pathogen loads in the CSF samples correlated with the severity of PM. Thus, the PM-TAC may be helpful to improve the prognosis of PM and clinical outcomes from antibiotic therapies.

SUBMITTER: Zhao C

PROVIDER: S-EPMC6438039 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.

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Development of a TaqMan Array card to target 21 purulent meningitis-related pathogens.

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