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

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.

[1]  D. Deforce,et al.  Current and New Approaches in GMO Detection: Challenges and Solutions , 2015, BioMed research international.

[2]  M. Gottschalk,et al.  Simultaneous detection of 33 Streptococcus suis serotypes using the luminex xTAG® assay™. , 2015, Journal of microbiological methods.

[3]  A. Morita.  [Clinical and Laboratory Diagnosis of Central Nervous System Infections]. , 2015, Brain and nerve = Shinkei kenkyu no shinpo.

[4]  T. William,et al.  Diagnosis of tuberculous meningitis: challenges and promises. , 2015, The Malaysian journal of pathology.

[5]  J. Nemeth,et al.  Bacteriostatic versus bactericidal antibiotics for patients with serious bacterial infections: systematic review and meta-analysis. , 2015, The Journal of antimicrobial chemotherapy.

[6]  Kwang Sik Kim,et al.  Treatment strategies for central nervous system infections: an update , 2015, Expert opinion on pharmacotherapy.

[7]  Heather E. Lynch,et al.  Development and implementation of a proficiency testing program for Luminex bead-based cytokine assays. , 2014, Journal of immunological methods.

[8]  Min Zhang,et al.  Diagnostic accuracy of intracellular mycobacterium tuberculosis detection for tuberculous meningitis. , 2014, American journal of respiratory and critical care medicine.

[9]  Cynthia Bautista.  Central nervous system infections. , 2013, Critical care nursing clinics of North America.

[10]  R. Peeling,et al.  Rapid diagnostic tests for neurological infections in central Africa. , 2013, The Lancet. Infectious diseases.

[11]  M. Otto,et al.  Aktuelle Liquordiagnostik bei erregerbedingten Krankheiten , 2013, Der Nervenarzt.

[12]  E. Auvinen,et al.  Performance of the Luminex xTAG Respiratory Viral Panel Fast in a clinical laboratory setting , 2012, Journal of Virological Methods.

[13]  Nicole Wolter,et al.  Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens , 2011, Journal of Clinical Microbiology.

[14]  E. Houpt,et al.  Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples. , 2011, Diagnostic microbiology and infectious disease.

[15]  S. Aman,et al.  Multiplex PCR for rapid diagnosis of tuberculous meningitis , 2011, Journal of Neurology.

[16]  K. Pabbaraju,et al.  Comparison of the Luminex xTAG Respiratory Viral Panel with xTAG Respiratory Viral Panel Fast for Diagnosis of Respiratory Virus Infections , 2011, Journal of Clinical Microbiology.

[17]  Shihab U. Sobuz,et al.  High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites. , 2011, The American journal of tropical medicine and hygiene.

[18]  Guy Thwaites,et al.  Tuberculous meningitis: a uniform case definition for use in clinical research. , 2010, The Lancet. Infectious diseases.

[19]  S. Cui,et al.  A mouse model to study infection against porcine circovirus type 2: viral distribution and lesions in mouse , 2010, Virology Journal.

[20]  Z. Duan,et al.  [Application of Luminex xMAP technology in infectious diseases]. , 2010, Bing du xue bao = Chinese journal of virology.

[21]  R. Holdsworth,et al.  Review article: Luminex technology for HLA antibody detection in organ transplantation , 2009, Nephrology.

[22]  J. Møller,et al.  Eight-Plex PCR and Liquid-Array Detection of Bacterial and Viral Pathogens in Cerebrospinal Fluid from Patients with Suspected Meningitis , 2009, Journal of Clinical Microbiology.

[23]  A. Vaheri,et al.  Improved multiplex-PCR and microarray for herpesvirus detection from CSF. , 2008, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[24]  David J. Marshall,et al.  High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses , 2007, Journal of Clinical Microbiology.

[25]  S. Dunbar.  Applications of Luminex® xMAP™ technology for rapid, high-throughput multiplexed nucleic acid detection , 2005, Clinica Chimica Acta.

[26]  J. Schrenzel,et al.  Use of Diagnostic Microarrays for Determination of Virulence Gene Patterns of Escherichia coli K1, a Major Cause of Neonatal Meningitis , 2005, Journal of Clinical Microbiology.

[27]  S. Kamei.  [Bacterial meningitis: determination of pathogens and therapeutic management]. , 2004, Rinshō shinkeigaku Clinical neurology.

[28]  Stephan Preibisch,et al.  Specific and nonspecific hybridization of oligonucleotide probes on microarrays. , 2004, Biophysical journal.

[29]  Paula M. Imbro,et al.  A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents. , 2004, Molecular and cellular probes.

[30]  R. Booy,et al.  Diagnosis and treatment of bacterial meningitis , 2003, Archives of disease in childhood.

[31]  S. Dunbar,et al.  Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system. , 2003, Journal of microbiological methods.

[32]  M. Drebot,et al.  Design of oligonucleotide arrays to detect point mutations: molecular typing of antibiotic resistant strains of Neisseria gonorrhoeae and hantavirus infected deer mice. , 2003, Molecular and cellular probes.

[33]  R. Thomson,,et al.  Laboratory diagnosis of central nervous system infections. , 2001, Infectious Disease Clinics of North America.

[34]  M. Biros,et al.  Presentation, time to antibiotics, and mortality of patients with bacterial meningitis at an urban county medical center. , 2001, The Journal of emergency medicine.

[35]  A. Steel,et al.  Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly. , 2000, Biophysical journal.

[36]  K. Zhang.  The expectation of life without disability in England and Wales. , 1993, Social science & medicine.

[37]  R. V. D. van der Vlugt,et al.  Multiplex detection of plant pathogens through the Luminex MagPlex bead system. , 2015, Methods in molecular biology.

[38]  S. Süssmuth,et al.  [Current cerebrospinal fluid diagnostics for pathogen-related diseases]. , 2013, Der Nervenarzt.

[39]  M. Bissell.  A Highly Efficient Ziehl-Neelsen stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid , 2013 .

[40]  J. Møller.  Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology. , 2012, Methods in molecular biology.

[41]  Z. Lei.  Advances in research on multiple biological detecting technologies , 2012 .

[42]  A. Ramsay,et al.  Investing time in microscopy: an opportunity to optimise smear-based case detection of tuberculosis. , 2007, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[43]  K. Peck,et al.  Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression. , 2004, Nucleic acids research.