Actionable diagnosis of neuroleptospirosis by next-generation sequencing

Abstract

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Figure 1. Clinical Course of the 14-Year-Old Patient with Fulminant Meningoencephalitis

Panel A shows a timeline beginning with the patient’s trip to Puerto Rico in August 2012 and ending after his recovery in October 2013. Major events during the course of the patient’s illness are indicated by arrows. Panel B shows laboratory values obtained and pertinent medications administered during the patient’s third hospitalization. The upper graph shows the body-temperature curve (red line) and peripheral-blood leukocyte counts (purple bars). The lower graph shows the leukocyte count and differential (bars) and the glucose (dashed line) and protein (solid line) levels in serially collected cerebrospinal fluid samples. The horizontal thick gray lines show the medications administered. The asterisk denotes the first course of cefuroxime (CFX) given to the patient, which did not result in clinical improvement. CFPM denotes cefepime, EVD extraventricular drain, MEP methylprednisolone, MRI magnetic resonance imaging, PCN G penicillin G, PEG-ADA polyethylene glycol–modified adenosine deaminase, and VANC vancomycin.

Figure 2. Neuroradiologic MRI and Brain-Biopsy Findings

The images shown in Panels A, B, and C were acquired on day 13 of the patient’s third hospitalization, whereas the image shown in Panel D was acquired on day 55, after the patient had completed a 7-day course of intravenous penicillin G. An axial T₂-weighted image of the head revealed persistent hyperintensities in the basal ganglia and deep frontal white matter (Panel A, arrows). Sagittal and axial T₂-weighted fluid-attenuated inversion recovery (FLAIR) images (Panels B and C, respectively) showed thickening in and around the basilar meninges (arrows). An axial T₂-weighted FLAIR image (Panel D) depicts near resolution of the previously seen (Panels B and C) basilar meningitis. Results of a biopsy of the right frontal lobe performed 2 weeks after the third hospital admission showed infiltration by lymphocytes and epithelioid histiocytes in the subarachnoid space with a perivascular predilection (Panel E, hematoxylin and eosin). T lymphocytes were visualized by means of immunolabeling with anti-CD3 antibody (Panel F). Various stains also showed the absence of mycobacteria (Panel G, acid-fast), fungi (Panel H, Gomori methenamine silver), and leptospira or other spirochetes (Panel I, Warthin–Starry silver). Electron microscopy revealed the presence of an inflammatory infiltrate and the absence of inclusion bodies, viral particles, or other evidence of microorganisms (Panel J).

Figure 3. Diagnosis of Leptospira Infection by Means of Unbiased Next-Generation Sequencing (NGS)

A clinical laboratory workflow for a comprehensive pathogen-detection assay with the use of unbiased NGS is shown (Panel A). The total time required for the bioinformatics analysis of NGS data, 97 minutes (pie chart, inset), was only 3.4% of the 48-hour turnaround time for the assay. Sequence-based ultra-rapid pathogen identification (SURPI) is a bioinformatics pipeline for pathogen identification from data obtained by means of next-generation sequencing of clinical samples. In de novo assembly, overlapping sequence reads are joined together in order to create longer contiguous sequences. The overhead time refers to computational time necessary for file-format conversion, sequence retrieval of identified matches in the database, and taxonomic classification of aligned reads. A total of 475 sequence reads derived from the patient’s cerebrospinal fluid (CSF) sample were mapped to the closest matched leptospira genome in the reference database, Leptospira borgpetersenii (Panel B). The distribution of bacterial and viral sequences identified in the patient’s CSF included propionibacteria, anelloviruses, and bacteriophages, which are generally considered to be nonpathogenic body flora (Panel C). Phylogenetic analysis of the full-length gene rpoB showed sequences from representative serovars, strains, and species of leptospira, highlighting the strain identified in the patient with fulminant meningoencephalitis (provisionally named L. santarosai strain UW [University of Wisconsin]) (Panel D). The scale bar denotes the number of nucleotide substitutions per site. GenBank accession numbers are provided in the Supplementary Appendix.