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1 Supplementary Online Content Van Haren K, Ayscue P, Waubant E, et al. Acute flaccid myelitis of unknown etiology in California, JAMA. doi: /jama emethods eresults efigure 1. Age distribution of 59 patients with acute flaccid myelitis of unknown etiology in California, June 2012 July 2015 efigure 2. Administrative region of residence for 59 reported cases of acute flaccid myelitis in California with onset June 1, 2012 July 31, 2015 efigure 3. Based on abstraction of MRI reports from all 59 patients with acute flaccid myelitis in California, June 2012 July 2015 efigure 4. Cerebral spinal fluid (CSF) studies from patients with acute flaccid myelitis in California, June 2012 July 2015 etable 1. Neurophysiology findings from all available electromyography reports were consistent with a spinal motor neuron localization etable 2. Specimen testing results for non-enterovirus pathogens*, acute flaccid myelitis cases of unknown etiology in California, June 2012 July 2015 ereferences This supplementary material has been provided by the authors to give readers additional information about their work.

2 emethods Assessment of Muscle Tone for Case Inclusion If the available clinical records for a case did not adequately specify the patients tone as flaccid/low or spastic/increased, we used hyporeflexia as a proxy for flaccidity and/or hyperreflexia as proxy for spasticity. If neither muscle tone nor reflexes specifications were available, the case was excluded. Infectious Disease Testing Methodologies The Viral and Rickettsial Disease Laboratory at the California Department of Public Health (CDPH) tested available specimens for adenoviruses, respiratory syncytial virus, rhinoviruses, human metapneumovirus, parainfluenza viruses, Mycoplasma pneumoniae, herpes viruses, enteroviruses, and West Nile virus by real-time PCR and serologic methods, if testing had not previously been performed at a commercial laboratory. Because of cross-reactivity, distinguishing enteroviruses from rhinoviruses can present a diagnostic challenge 1 ; CDPH tested specimens for the presence of enterovirus by an enterovirus-specific PCR assay. Typing of enterovirus species was performed by sequencing of the VP1 gene coding region 2. Additional diagnostic testing by CDPH was directed by clinical and epidemiologic findings and included tests for rabies and arboviruses other than non-west Nile virus. Enterovirus/rhinovirus testing on clinical specimens was also independently performed by the Abbot Viral Diagnostics and Discovery Center at University of California, San Francisco using an enterovirus D68 specific PCR assay targeting the VP1 gene coding region 3 and a panenterovirus/rhinovirus assay targeting the 5 -UTR 4. All throat and nasopharyngeal swab samples testing enterovirus D68 negative were also analyzed for other viruses by unbiased metagenomic next-generation sequencing 5.

3 eresults Clinical Findings Of the 26 patients with sensory deficits documented on at least one exam, 8 had deficits only in pain or temperature, 3 had deficits only in fine touch or vibration, 10 had deficits in both pain/temperature and fine touch/vibration, and 5 had unspecified deficits. A sensory level was specified or implied in 6 patients. Sensory deficits, including sensory levels, have also been reported in association with paralytic poliovirus 6-8 and enterovirus MRI & Clinical Findings A total 77 MRI reports available for review and abstraction; all 59 patients had at least 1 spinal MRI report available. Three patients had reports from 3 studies available, 12 patients had reports from 2 studies available, and the remaining forty-five patients had only 1 MRI report available. Timing of spinal MRI relative to onset of weakness was as follows: 18, 30, 12, 10, and 7 patients received a spinal MRI study at 0-48 hours, 2-4 days, 5-10 days, days, and >20 days, respectively. Radiologic signs of denervation 10, including nerve root enhancement, nerve root thickening or clumping, and paraspinal muscle enhancement were infrequently reported but were absent among the 18 patients who had an MRI within 48 hours of onset. In contrast, nerve root thickening/clumping and paraspinal muscle edema were reported in 4 of the 7 patients with studies completed 20 or more days from onset of weakness, suggesting these radiologic findings may manifest later in the course of disease. Of the 20 patients requiring intubation, 16 had T2 signal abnormalities involving some portion of the cervical cord between C2 and C5. Among the 48 patients who had reports available for brain MRI studies, supratentorial lesions were reported among less than one-third of patients; these supratentorial lesions often localized to either cortical or subcortical gray matter structures (Table 1), a pattern reminiscent of ADEM 11.

4 Neurophysiologic Features Twelve complete nerve conduction/emg reports from 9 patients were available for review and abstraction (etable 1). All EMGs were interpreted as diagnostic of or compatible with spinal motor neuron injury. Infectious Agent Testing Testing was limited by incomplete and late collection of specimens. Among 42 patients with a respiratory prodrome prior to acute flaccid paralysis onset, 9 had respiratory specimens collected within 7 days of respiratory symptom onset and an additional 14 had respiratory specimens collected within 7 days of acute flaccid paralysis onset date. Specimens meeting U.S. Centers for Disease Control and Prevention (CDC) guidelines for poliovirus detection (e.g., 2 stool specimens collected >24 hours apart and <14 days after symptom onset) were submitted for 7 patients, of which 3 yielded non-polio enteroviruses. Of the serum samples from 32 patients submitted to the state lab, 13 were collected after the patient had received IVIG, precluding interpretation of serologic testing. The non-enterovirus infectious agents detected in our sample (etable 2), which occur more commonly in the general population, have a more ambiguous relationship with our cases. These agents are more likely to represent incidental findings, although we cannot exclude the possibility that one or more of these may trigger or worsen infection in the setting of a co-infection with a more traditional neurotropic agent 12.

5 efigure 1. Age distribution of 59 patients with acute flaccid myelitis of unknown etiology in California, June 2012 July 2015 Median age at onset of weakness was 9 years (blue arrow) with an interquartile range from 4 years to 14 years. Fifty of 59 patients were 21 years or younger at onset.

6 efigure 2. Administrative region of residence for 59 reported cases of acute flaccid myelitis in California with onset June 1, 2012 July 31, 2015

7 efigure 3. Based on abstraction of MRI reports from all 59 patients with acute flaccid myelitis in California, June 2012 July 2015 pons medulla C1 C2 C3 C4 C5 C6 C7 C8 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 conus Cases with T2 lesions described on spine MRI report (n=59) T2 lesions were most commonly reported in the cervical cord.

8 efigure 4. Cerebrospinal fluid (CSF) white blood cell (WBC) differential among patients with pleocytosis during course of acute flaccid myelitis in California, June 2012 July 2015 Among 41 patients with pleocytosis and a differential available at the time of first lumbar puncture (median of 2 days after onset of weakness, range 6 days prior to 90 days after), CSF profile was significantly more lymphocytic by ANOVA, p<0.0001; no patient had an eosinophilic differential exceeding 4%. Each green pixel represents 1 patient; bar demarcates median; hashed line demarcates 50% threshold. Four of the 9 patients with neutrophilic pleocytosis had persistent pleocytosis on subsequent lumbar punctures at which time 3 had converted to lymphocytic predominance and 1 to monocytic predominance (data not shown).

9 etable 1. Neurophysiology findings from all available electromyography reports were consistent with a spinal motor neuron localization 0-7 days after onset 8-21 days after onset 3-20 weeks after onset >20 wks after onset Case # CMAPs Yes No Yes No Presence of No No No No fibrillations Not Yes MUPs specified Yes Yes CMAPs Yes No No Presence of Yes Yes No fibrillations MUPs Yes Yes Yes CMAPs Yes Yes Yes Presence of Yes Yes No fibrillations MUPs Yes Yes Yes CMAPs Yes Yes Presence of Yes Yes fibrillations MUPs Yes Yes Conduction velocities (not shown) were normal or reduced in accordance with low CMAPs. CMAP = compound motor action potential. MUP = motor unit potential

10 etable 2. Specimen testing results for non-enterovirus pathogens*, acute flaccid myelitis cases of unknown etiology in California, June 2012 July 2015 Number of patients tested Number of patients testing positive Non-enterovirus testing results from nasopharyngeal samples Rhinovirus A65 PCR 41 2 Respiratory syncytial virus PCR 33 2 Human metapneumovirus PCR 33 1 Adenovirus PCR 32 1 Parainfluenza 1-4 PCR 32 0 Influenza A PCR 32 0 Influenza B PCR 32 0 Mycoplasma PCR 19 0 Parechovirus PCR 7 0 Non-enterovirus testing in serum & CSF Mycoplasma IgM, serum 13 2 Rabies virus indirect fluorescent antibody, serum 11 0 Herpes virus PCR, CSF 12 0 *If more than one positive test result for the same pathogen on more than one respiratory specimen on the same patient, only one result reported. One patient was co-positive for enterovirus D68 and respiratory syncytial virus, both from nasopharynx

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