Next-Generation Sequencing Nabs Culprit in Brain Infection

Janis C. Kelly

June 09, 2014

Next-generation DNA sequencing leapt convincingly from bench to bedside when clinicians used the new tool to identify treatable bacterial meningoencephalitis in a 14-year old boy with severe combined immunodeficiency, fever, headache, hydrocephalus, and status epilepticus. The dramatic case provided proof of principle that sequencing can provide actionable data in a clinically relevant time frame.

Michael R. Wilson, MD, from the Departments of Biochemistry, Biophysics, and Neurology, University of California, San Francisco, and colleagues used the experimental technique after the patient's infection had stubbornly eluded diagnosis over the course of 4 months of intensive clinical investigation using standard approaches. Within 48 hours after specimens were received for analysis, unbiased next-generation sequencing of DNA from the patient's cerebrospinal fluid (CSF) had identified 475 (of 3,063,784 reads) sequences that corresponded to leptospira infection, the authors report in an article published online June 4 in New England Journal of Medicine.

By that point, the previously healthy patient had been hospitalized repeatedly and was in a drug-induced coma to control new-onset status epilepticus. Although clinical assays for leptospirosis were negative, the University of California, San Francisco, team decided to begin treatment for neuroleptospirosis before confirmatory testing had been completed because the patient's condition was so serious. High-dose intravenous penicillin G (13 million units daily for 7 days) brought the infection under control, with resolution of status epilepticus, normalization of CSF, and resolution of leptomeningitis lesions that had persisted on serial magnetic resonance imaging scans.

At 14 days, the patient was discharged to inpatient rehabilitation with occasional right facial twitching from simple partial seizures. After 11 days of rehab (76 days after hospital admission), the patient was discharged home "close to his premorbid functional status," the authors write.

"Due to the patient's underlying [severe combined immunodeficiency] and prior of use of intravenous immunoglobulin G, conventional diagnosis by serological antibody testing was not possible, and next-generation sequencing was required to diagnose the leptospira infection," coauthor Charles Y. Chiu, MD, PhD, told Medscape Medical News. Dr. Chiu is assistant professor, laboratory medicine and medicine/infectious diseases, director of the University of California, San Francisco-Abbott Viral Diagnostics and Discovery Center, and associate director of the University of California, San Francisco, Clinical Microbiology Laboratory, San Francisco.

How Next-Generation Sequencing Found the Cause of Meningoencephalitis

The patient's severe combined immunodeficiency was caused by adenosine deaminase deficiency, and his immune function had been partially restored by 2 haploidentical bone marrow transplants. He used monthly intravenous immune globulin for hypogammaglobulinemia and trimoprim-sulfamethoxazole or atovaquone for prophylaxis against Pneumocystis jirovecii pneumonia.

One month after a trip to Puerto Rico (during which the patient swam in a river and the ocean and a fellow traveler developed fever and hematuria suggestive of leptospira-associated nephritis), the patient developed fever, headache, and bilateral conjunctivitis that resolved spontaneously. The next month, he developed photophobia and pain with movement of his left eye and was treated for uveitis. Two months after the trip, he developed thrombocytopenia, which was treated with rituximab for presumed immune thrombocytopenic purpura.

Eight months after the Puerto Rico trip, the patient was admitted to the hospital with fever, photophobia, daily frontotemporal headaches, increasing fatigue, abdominal pain, weight loss, and evidence of loss of control of adenosine deaminase deficiency. Extensive laboratory analysis of blood and CSF turned up nothing abnormal, and head MRI was unremarkable. Extensive infectious disease workup was unrevealing.

The patient was admitted again 3 months later with fever, headache, diffuse weakness, myalgias, nausea, and vomiting. He was treated with 2 doses of rituximab for possible autoimmune involvement, with no effect on symptoms. Magnetic resonance imaging showed patchy lesions in the basal ganglia, and repeat imaging 13 days later showed persistent basal ganglia lesions and development of basilar leptomeningitis extending into the cerebral hemispheres. Biopsy of the frontal lobe showed inflamed leptomeninges with a granulomatous infiltrate, but neither immunohistochemical testing nor electron microscopy could identify fungi, bacteria, or viruses.

The patient was treated with intravenous glucocorticoids for possible neurosarcoidosis and with polyethylene glycol-modified adenosine deaminase in an attempt to boost his immune system, but his condition continued to decline. Hydrocephalus worsened, and new-onset status epilepticus required initiation of a medically induced coma. At that point, the patient's family consented to his enrolment in a research study for pathogen detection using unbiased next-generation sequencing.

Next-generation sequencing involves miniaturization of individual sequencing chemical reactions, random amplification of clinical samples, and parallel sequencing of thousands or millions of the amplified DNA sequences at the same time. The sequencing reads are then analyzed using sequence-based ultrarapid pathogen identification, which subtracts human host sequences and checks the remaining sequences against the National Center for Biotechnology Information databases for bacteria, viruses, fungi, and parasites.

In this case, next-generation sequencing produced more than 10 million reads, including 2 million from control samples, and the full set was analyzed using sequence-based ultrarapid pathogen identification in approximately 100 minutes. The researchers found 589 bacterial reads, 80% of which corresponded to the Leptospiraceae family.

Five months later, Leptospira santarosai was confirmed at the Centers for Disease Control and Prevention by polymerase-chain reaction and serologic testing.

Routine Clinical Use Expected in 3 to 5 Years

"Although currently a research tool, I feel that [next-generation] sequencing will likely be used in regular clinical practice in 3 to 5 years in fields ranging from genetic testing to cancer to infectious diseases. Since encephalitis is undiagnosed up to 60% of the time, these emerging diagnostic tools are urgently needed in hospitals and clinics. The technology and bioinformatics have evolved to where it is feasible to perform the test routinely in clinical laboratories, although there are still several hurdles to overcome," Dr. Chiu said.

Dr. Chiu predicted that commercial development of this assay will require partnership with industry, academic research laboratories, and clinicians. Major challenges include compliance with regulatory requirements for such a broad detection assay, cost and turnaround time, reimbursement issues, and clinical interpretation of the genomics data in a form usable by physicians and other healthcare personnel, Dr. Chiu said.

The study was supported by the American Brain Foundation Clinical Research Training Fellowship, a National Human Genome Research Institute Intramural Research Program appointment, a Howard Hughes Medical Institute appointment, a grant from the National Institutes of Health, a University of California Discovery Grant, an Amazon Web Services in Education Research Grant, and an Abbott Viral Discovery Award. Dr. Chiu has received research grants from Abbott Laboratories and from, advisory board fees from Rubicon Genomics, and speakers bureau fees from Cubist Pharmaceuticals. The other authors have disclosed no relevant financial relationships.

N Engl J Med. Published online June 4, 2014. Full text


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