Use of Staged Molecular Analysis to Determine Causes of Unexplained Central Nervous System Infections - Vol. 19 No. 9 - September 2013 - Emerging Infectious Disease journal - CDC
Table of Contents
Volume 19, Number 9–September 2013
Volume 19, Number 9—September 2013
Use of Staged Molecular Analysis to Determine Causes of Unexplained Central Nervous System Infections
Central nervous system (CNS) infections pose a diagnostic challenge because clinical manifestations are not typically pathognomonic for specific pathogens, and a wide range of agents can be causative. An infectious cause of encephalitis is determined for < 40%–70% of cases worldwide (1–5). Culture is of limited use, particularly for viral infections. In recent studies, only 1.9% of cerebrospinal fluid (CSF) viral cultures were positive (3), and < 0.1% of CSF cultures recovered viruses other than enteroviruses or herpesviruses (6).
AbstractNo agent is implicated in most central nervous system (CNS) infections. To investigate cerebrospinal fluid samples from patients with CNS infections of unknown cause in 1 hospital in Taiwan, we used a staged molecular approach, incorporating techniques including multiplex MassTag PCR, 16S rRNA PCR, DNA microarray, and high-throughput pyrosequencing. We determined the infectious agent for 31 (24%) of 131 previously negative samples. Candidate pathogens were identified for 25 (27%) of 94 unexplained meningitis cases and 6 (16%) of 37 unexplained encephalitis cases. Epstein-Barr virus (18 infections) accounted for most of the identified agents in unexplained meningitis cases, followed by Escherichia coli (5), enterovirus (2), human herpesvirus 2 (1), and Mycobacterium tuberculosis. Herpesviruses were identified in samples from patients with unexplained encephalitis cases, including varicella-zoster virus (3 infections), human herpesvirus 1 (2), and cytomegalovirus (1). Our study confirms the power of multiplex MassTag PCR as a rapid diagnostic tool for identifying pathogens causing unexplained CNS infections.
PCR enables sensitive detection of microbial nucleic acids in clinical samples, which may be useful for identifying pathogens that are nonviable, uncultivable, or fastidious. MassTag PCR is a multiplex platform that enables inexpensive, sensitive, and simultaneous detection of multiple pathogens (7–10). Originally implemented for differential diagnosis of respiratory tract infections (7), MassTag PCR has been expanded to several syndrome-based panels for differential diagnosis of hemorrhagic fever and for detection of tick-borne pathogens (9,11).
Amplification and sequencing of the 16S ribosomal RNA (rRNA) gene is a well-established technique for identifying bacterial pathogens (12). Broad-range 16S rRNA PCR with subsequent sequencing is superior to bacterial culture for diagnosing bacterial meningitis, with a sensitivity of 86% and a specificity of 97% (13). It is particularly useful for slow-growing bacteria (e.g., Mycobacterium tuberculosis) and for diagnosis of cases that remain culture-negative as a result of antimicrobial drug treatment before lumbar puncture.
Microarray technology also has been applied to the detection and identification of infectious diseases (10,14) and has the potential to test for virtually all known viruses, bacteria, fungi, and parasites (15).The advent of high-throughput sequencing affords unique opportunities for pathogen surveillance and discovery with regard to CNS infections. We have successfully used high-throughput sequencing to identify causative agents of disease for patients with transplant-associated encephalopathy (16) and encephalitis associated with agammaglobulinemia (17).
Our staged molecular approach used complementary tools for pathogen detection and discovery that include syndrome-based multiplex PCRs, DNA microarray, and high-throughput sequencing (18). We report the results of our investigation of CNS infections of unknown cause in 1 hospital in Taiwan, which we conducted by using MassTag PCR, 16S rRNA PCR, DNA microarray, and high-throughput sequencing.