LQCL2611 - CMLE - Diagnosing a Culture-Negative Infection with Targeted Next-Generation Sequencing-LQCL2611

LQCL2611 - Educational Activity

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This document reviews the diagnostic workup of cerebrospinal fluid (CSF) infections and highlights how next-generation sequencing (NGS) can resolve culture-negative meningitis/ventriculitis cases. Standard CSF evaluation includes cell count/differential and chemistry (protein, glucose) alongside microbiology testing: Gram stain, culture on routine media (eg, sheep blood, chocolate, MacConkey, enrichment broth) with prolonged incubation up to 14 days, followed by organism identification (biochemical tests or MALDI-TOF) and antimicrobial susceptibility testing.<br /><br />Multiplex meningitis/encephalitis PCR panels can rapidly detect common bacterial, viral, and fungal pathogens directly from CSF within hours and improve sensitivity, especially when patients received prior antibiotics or organisms are fastidious. However, these panels are limited to “on-panel” targets; they may yield “no targets detected” despite a positive Gram stain if the causative organism is not included (and many panels also exclude organisms such as Mycobacterium tuberculosis and parasites).<br /><br />The text compares three NGS strategies: (1) whole-genome sequencing (WGS), which requires a cultured isolate and is best for outbreak investigation, strain typing, and resistance gene surveillance; (2) targeted amplicon-based sequencing (eg, broad-range 16S rRNA), which can be performed directly on specimens, is more sensitive with less background DNA, and is well-suited for culture-negative infections when there is an a priori suspicion (eg, Gram-stain morphology); and (3) metagenomic sequencing, a hypothesis-free approach that sequences all nucleic acids but is costlier, more complex, and challenged by host DNA and incidental findings.<br /><br />In the presented case, a young woman with neutrophilic CSF, low glucose, brain abscesses, and gram-positive cocci in chains had negative culture and multiplex PCR. Broad-range 16S targeted sequencing identified Streptococcus intermedius (Streptococcus anginosus group), a pathogen strongly associated with abscess formation. Therapy was adjusted to ceftriaxone plus metronidazole for six weeks with clinical improvement.

Keywords

cerebrospinal fluid infection

meningitis

ventriculitis

culture-negative meningitis

multiplex meningitis encephalitis PCR panel

next-generation sequencing NGS

16S rRNA broad-range sequencing

metagenomic sequencing

whole-genome sequencing WGS

Streptococcus intermedius (Streptococcus anginosus group)