Diagnosis of Fatal Human Case of St. Louis Encephalitis Virus Infection by Metagenomic Sequencing, California, 2016

We used unbiased metagenomic next-generation sequencing to diagnose a fatal case of meningoencephalitis caused by St. Louis encephalitis virus in a patient from California in September 2016. This case is associated with the recent 2015–2016 reemergence of this virus in the southwestern United States.

of the predicted 10,936-bp virus genome. Subsequent mNGS testing of the patient's CSF from hospitalization day 3 also was positive for SLEV.
The patient's SLEV genome sequence was >99% identical with previously sequenced 2014-2015 SLEV isolates from mosquitoes in California and Arizona (4). Phylogenetic analysis placed the patient's strain in a cluster containing these isolates and viruses sequenced from mosquitoes in Argentina in 1978 and 2005 (9) (Figure). The patient's SLEV was genetically distinct from the 2003 Imperial Valley strain that had been circulating in California before an 11-year absence (12), suggesting that he was infected by the 2015-2016 reemergent genotype currently circulating in the southwestern United States (3,4). Furthermore, the patient's SLEV genome was closely related to a strain sequenced from a mosquito pool collected in June 2016 from Kern County ( Figure, panel A), with 99.9% pairwise nucleotide identity and only 5 single-nucleotide variants across the genome.
After extensive discussion with his wife regarding the patient's SLEV diagnosis and poor prognosis, the patient was transitioned to comfort care on hospitalization day 23 and died the following day. Autopsy revealed residual mantle cell lymphoma and bronchopneumonia consistent with infection or chemical pneumonitis from aspiration. The diagnosis of SLEV meningoencephalitis was subsequently confirmed by positive reverse transcription PCR and virus culture testing from multiple laboratories (Table  2). However, follow-up testing for SLEV from the patient's CSF and serum was negative.

Conclusions
We present a case of SLEV infection in an elderly immunocompromised patient hospitalized with fever and AMS  and who experienced critical respiratory failure. Most SLEV infections are asymptomatic; when infections are symptomatic, clinical features include fever, lethargy, and confusion (1), with potential complications including sepsis, gastrointestinal hemorrhage, pulmonary embolism, and bronchopneumonia. In hindsight, SLEV infection is consistent with the patient's clinical presentation, with pneumonitis and respiratory decompensation possibly resulting from aspiration during the patient's AMS from viral meningoencephalitis. Deaths from SLEV infection during the first 2 weeks are generally from encephalitis, whereas later deaths are more often caused by complications of hospitalization, such as this patient's bronchopneumonia. Routine diagnosis of SLEV is challenging because serologic testing is only performed by specialized reference laboratories, the period of viremia is brief, and molecular testing by reverse transcription PCR is not widely used. Clinicians in California might fail to consider SLEV when examining a patient with nonspecific febrile illness, especially given the lack of virus or disease activity in the state during 2004-2015. Antibody testing can be complicated by the absence of seroreactivity in elderly and immunocompromised patients, as observed in the case of this patient (Table 1), as well as potential cross-reactivity with other flavivirus infections, such as dengue, Zika virus, and West Nile virus (3).
The identification of SLEV infection in CSF by using a panpathogen metagenomic sequencing assay is another demonstration of the power of an unbiased approach to infectious disease testing (5-7), although challenges remain with respect to test availability, interpretation, and validation (8). No antiviral therapy for SLEV has been proven to be efficacious, although interferon-α has been tried (13). With a laboratory sample-to-reporting time of 4 days, earlier sample submission might have spared our patient from the side effects of antimicrobial drug therapy, costly laboratory testing, and invasive procedures. Importantly, the family obtained reassurance and closure from communication of an established diagnosis.
During summer 2016, SLEV was reported in mosquitoes from 7 counties in California, including Kern County, where the patient resided (4). According to his wife, the patient often sat outdoors during the few weeks before hospitalization, although she did not recall his reporting any mosquito bites. Nevertheless, we believe he most likely contracted SLEV in California, because his history of travel to Arizona 5 months prior was not consistent with the incubation period of the disease (4-21 days); mosquitoes are less prevalent at the higher altitudes of Payson, Arizona; and the patient's SLEV sequence was most closely related to a strain from a June 2016 Kern County mosquito pool. Given the reemergence of SLEV in the southwestern United States, clinicians from affected areas will need to maintain a high index of suspicion for this disease, particularly during local community outbreaks or high SLEV activity detected through mosquito surveillance efforts.  (3,9) CDPH Positive SLEV, RT-PCR (3,9) CDC Positive Viral culture (3,9) CDC Positive, confirmed as SLEV by RT-PCR SLEV, IgG/IgM antibody (3,9) Quest Diagnostics Negative, <1:10 SLEV, PRNT for neutralizing antibodies (9) CDPH Negative, <1:10 WNV, IgM WNV, PRNT for neutralizing antibodies (9 C.Y.C. is the director of the UCSF-Abbott Viral Diagnostics and Discovery Center and receives research support from Abbott Laboratories, Inc. C.Y.C., S.N.N., and S.M. are co-inventors of the sequence-based ultra-rapid pathogen identification computational pipeline (known as SURPI+) and associated algorithms; a patent pending for SURPI+ has been filed by the University of California, San Francisco.
Dr. Chiu is an associate professor at the University of California, San Francisco, who heads a translational research laboratory focused on clinical metagenomic assay development for infectious diseases and characterization of emerging outbreak viruses.