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Next-Generation Sequencing Pipeline Finds Cause of Critically Ill Boy's Disease

NEW YORK (GenomeWeb) — After conventional diagnostic approaches came up dry, researchers turned to a next-generation sequencing pipeline to pinpoint the cause of a teenaged boy's encephalitis.

The boy, who also has severe combined immunodeficiency, was hospitalized for six weeks after being ill on and off for nearly a year while clinicians and researchers scrambled to determine what was causing his escalating symptoms.

Unbiased sequencing of cerebrospinal fluid from the boy revealed a leptospira infection that the doctors then treated with penicillin, as the researchers led by Charles Chiu, a professor of laboratory medicine at the University of California, San Francisco, reported in a case study in the online, early edition of the New England Journal of Medicine yesterday.

"This case was a test run for how you would validate and deploy a clinically-approved test," Chiu said in a statement.

While next-gen sequencing holds promise for diagnosing disease and affecting clinical care, whether it can move from a patient sample to an actionable result within a clinically relevant timeframe remains unclear.

Chiu and his colleagues relied on a bioinformatics pipeline they developed called SURPI — for sequence-based ultra-rapid pathogen identification — to quickly sequence and identify pathogen DNA within their samples. They described this approach in a related paper that appeared online in Genome Research yesterday, and said that it could process between 7 million and 50 million reads in 10 minutes to 30 minutes in "fast" mode and in one hour to five hours in "comprehensive" mode.

"From the perspective of cost and turnaround time, this is a very powerful technology that has become practical to implement routinely in clinical laboratories," Chiu said.

The 14-year-old boy presented with a number of symptoms, including fever and headache, beginning in September 2012, as Chiu and his colleagues recounted in their NEJM case study. Then in 2013, he was hospitalized three times for fever, photophobia, and headache.

An infectious disease workup at his first admission in April 2013 did not find anything.

When the boy was readmitted in July, he was treated with steroids as clinicians suspected an autoimmune cause of his encephalitis because of his immunodeficiency disorder. However, he failed to improve and developed uncontrollable seizures that led clinicians to place him in a medically induced coma.

After obtaining consent from his parents, the study authors sequenced cerebrospinal fluid and serum from the boy alongside negative control samples on the Illumina MiSeqDx platform.

Chiu and his colleagues fed the sequencing reads into their SURPI pipeline. SURPI, a cloud-compatible bioinformatics tool, relies on a combination of two alignment tools, SNAP and RAPSearch, to quickly compare DNA from the samples — after human host DNA has been subtracted — to what's housed in GenBank, as they reported in Genome Research.

In this case, the researchers reported a sample-to-answer turnaround time of 48 hours, and 97 minutes to analyze the resulting reads.

In the CSF samples, the majority of the bacterial reads — some 80 percent — were from the Leptospiraceae family. The closest match in the NCBI database was L. borgpetersenii, with 3.7 percent coverage of chromosome 1 and 2.2 percent coverage for chromosome 2, Chiu and his colleagues said.

No other convincing bacterial or viral hits were found in the CSF samples, the researchers added, nor were any found in the serum or in the negative control samples.

To confirm their Leptospiraceae result, the researchers amplified the bug's ompA and secY genes and Sanger sequenced them.

They also amplified and sequenced the lipL32, lipL41, rposB, and secY genes to perform a phylogenetic analysis of the Leptospiraceae species in the sample.

From that, they uncovered that the boy's CSF sample harbored L. santarosai, which the researchers noted has been sequenced previously but was not deposited in the NCBI database when SUPRI searched it in August 2013.

A separate confirmation of L. santarosai in the CSF sample was made a few months later by the US Centers for Disease Control and Prevention using a leptospira PCR assay targeting lipL34. The CDC subsequently detected L. santarosai in a serum sample from the boy using a newly approved qualitative IgM agglutination assay.

The boy's doctors decided to begin treating him with penicillin even before these confirmatory tests were finished.

"The concern that the finding was spurious was considered to be negligible given that a large number of reads spanning the leptospira genome were detected in the patient's CSF, leptospira reads were absent in the patient's serum and in a control sample from an unrelated patient processed concomitantly, and leptospira sequences have hitherto never been detected in the next-generation sequencing laboratory," Chiu and his colleagues said in their case study. They also noted that the boy had been on vacation to Puerto Rico where he may have contracted the spirochete.

The boy slowly recovered upon receiving penicillin, was discharged to an in-patient rehabilitation facility two weeks later, and returned home at the end of September, some 76 days after he was admitted. Tests at that time were negative for leptospira.

"[U]nbiased next-generation sequencing coupled with a rapid bioinformatics pipeline provided a clinically actionable diagnosis of a specific infectious disease from an uncommon pathogen that eluded conventional testing for months after the initial presentation," the study authors added. "This approach thus facilitated the use of targeted and efficacious antimicrobial therapy."