Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology.

Linfu Zhou,Ying He,Dongyun Feng,Wen Dai,Rui Wu,Xiaodan Shi,Tingting Liu,Ting Bian,Ming Shi,Guodong Feng,Gang Zhao,Yining Yang

doi:10.3390/ijerph13020193

Abstract

Early diagnosis and treatment are crucial for the outcome of central nervous system (CNS) infections. In this study, we developed a multiplex PCR-Luminex assay for the simultaneous detection of five major pathogens, including Mycobacterium tuberculosis, Cryptococcus neoformans, Streptococcus pneumoniae, and herpes simplex virus types 1 and 2, which frequently cause CNS infections. Through the hybridization reaction between multiplex PCR-amplified targets and oligonucleotide “anti-TAG” sequences, we found that the PCR-Luminex assay could detect as low as 101–102 copies of synthetic pathogen DNAs. Furthermore, 163 cerebrospinal fluid (CSF) specimens from patients with suspected CNS infections were used to evaluate the efficiency of this multiplex PCR-Luminex method. Compared with Ziehl-Neelsen stain, this assay showed a high diagnostic accuracy for tuberculosis meningitis (sensitivity, 90.7% and specificity, 99.1%). For cryptococcal meningitis, the sensitivity and specificity were 92% and 97.1%, respectively, compared with the May Grunwald Giemsa (MGG) stain. For herpes simplex virus types 1 and 2 encephalitis, the sensitivities were 80.8% and 100%, and the specificities were 94.2% and 99%, respectively, compared with Enzyme Linked Immunosorbent Assay (ELISA) assays. Taken together, this multiplex PCR-Luminex assay showed potential efficiency for the simultaneous detection of five pathogens and may be a promising supplement to conventional methods for diagnosing CNS infections.

Highlights

central nervous system (CNS) infections caused by viruses, bacteria, fungi, parasites and prions still remain diseases with significant morbidity and mortality [1,2]
We developed a rapid, high-throughput diagnostic method integrated multiplex polymerase chain reaction (PCR) and Luminex technology for detecting five pathogens, including M. tuberculosis, C. neoformans, S. pneumoniae, herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which account for more than 70% of CNS infections in Northwest China
The results showed that the limit of detection was 102 plasmid copies for M. tuberculosis, C. neoformans, HSV-2 and S. pneumoniae and 101 plasmid copies for HSV-1 (Table 2)

Summary

Introduction

CNS infections caused by viruses, bacteria, fungi, parasites and prions still remain diseases with significant morbidity and mortality [1,2]. In many developing countries affected by endemic diseases such as tuberculosis, HIV/AIDS, and cryptococcal meningitis, delayed diagnosis, treatment and high economic burden often lead to avoidable deaths or severe sequelae [3]. To reduce the mortality and improve the cure rates, it is vital to screen and identify pathogens in clinical specimens from patients as early as possible [4,5]. And accurate diagnosis will support physicians with the selection of the appropriate antimicrobial agents [6,7]. Most laboratory methods for diagnosing CNS infections. Res. Public Health 2016, 13, 193; doi:10.3390/ijerph13020193 www.mdpi.com/journal/ijerph

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Conclusion