Ethical Approval

This study was approved by the ethics committee of the Stefan S. Nicolau Institute of Virology (Bucharest, Romania). Although this was a retrospective study, informed consent was obtained from each patient included in the study, as part of routine hospital activity.

Patients

We conducted a retrospective study of 7 patients given a diagnosis of influenza in whom neurologic complications developed. These patients were hospitalized in a tertiary care facility (Dr. Victor Babes Clinical Hospital of Infectious and Tropical Diseases, Bucharest, Romania) during the 2014–15 influenza season.

We used the case definition for encephalitis from the 2013 Consensus Statement of the International Encephalitis Consortium. Major criterion was altered mental status (defined as decreased or altered level of consciousness, lethargy, or personality change) lasting >24 hours with no alternative cause identified. Minor criteria were fever (temperature >38°C), generalized or partial seizures, new onset of focal neurologic findings, CSF leukocyte count >5 cells/mm³, and abnormality of brain parenchyma on neuroimaging suggestive of encephalitis (13).

We collected nasopharyngeal swab specimens from all patients with ILI and sent these specimens to the National Reference Center for Influenza (Cantacuzino Institute, Bucharest, Romania) for antigenic and genetic characterization. Specimens were examined by using real-time reverse transcription PCRs (RT-PCRs) with the Superscript III Platinum One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) for influenza type A and type B viruses. Samples positive for influenza A viruses were tested by using a second real-time RT-PCR that discriminated between influenza A(H1N1)pdm09 and A(H3N2) virus subtypes. For samples positive for influenza B viruses, we used a second real-time RT-PCR and specific minor-groove binder probes to determine lineage (14).

Positive specimens were inoculated into an MDCK line, and virus isolates were characterized antigenically by using a hemagglutination inhibition assay and turkey/guinea pig erythrocytes. We used the conventional Sanger sequencing technique to monitor influenza virus evolution for the complete hemagglutinin (HA) gene. The PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used to sequence DNA templates on a PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). We determined genotypes of all virus sequences from patients by alignment with sequences found in Romania with World Health Organization reference viruses. We performed phylogenic analysis by using maximum-likelihood inference and a generalized time-reversible model of nucleotide substitution and a Γ model of rate heterogeneity with RAxML version 8.00 software (15).

Patients

Patient 1 was a 28-year-old woman who had uncontrolled hyperthyroidism, a 3-day history of high fever (temperature >39°C), headache, sleepiness, left upper limb motor deficit, and a Glasgow Coma Scale (GCS) score of 3−4/15. Results of cerebrospinal fluid (CSF) testing were unremarkable. The patient was intubated and mechanically ventilated after 4 hours of hospitalization.

Figure 1

Figure 1. Magnetic resonance imaging of the brain of a 28-year-old woman (patient 1) who had neurologic complications of influenza B virus infection, Romania. A) Axial T2 image showing multiple areas of T2-associated...

Magnetic resonance imaging (MRI) of the brain (Figure 1) showed multiple areas of T2-associated hyperintensities associated with restricted diffusion with involvement of the genu corpus callosum bilateral internal capsule and several areas of white matter in the right frontal lobe. Hyperintensities were visible at the limit between the right parietal and occipital lobe (axial T2-associated and diffusion-weighted imaging), and multiple high-signal lesions associated with restricted diffusion were present in the right caudate nucleus head and the subcortical and deep white matter of the frontal lobes (coronal fluid–attenuated inversion recovery and diffusion-weighted imaging). Despite intensive antimicrobial drug treatment with oseltamivir, acyclovir, meropenem, mannitol, corticosteroids, and thyrozol, the patient died 7 days after admission.

Patient 2 was a previously healthy 37-year-old woman who was hospitalized after 2 days of fever, rhinorrhea, myalgia that progressed to a headache, sleepiness, photophobia, vertigo, stiff neck, and a positive Romberg sign. Results of computed tomography (CT) and analysis of CSF were unremarkable. Complete resolution occurred after 9 days of treatment with oseltamivir and mannitol.

Figure 2

Figure 2. Magnetic resonance imaging of the brain of a 55-year-old woman (patient 3) who had neurologic complications of influenza B virus infection, Romania. Axial diffusion-weighted image showing restricted diffusion in the bilateral...

Patient 3 was a previously healthy 55-year-old woman with a history of influenza A(H3N2) virus encephalitis (in 2012). The patient was hospitalized after transfer from another clinic 10 days after onset of illness with signs and symptoms that included fever, confusion, photophobia, dizziness, right facial paralysis, aphasia, stiff neck, and coma (GCS score 8). Onset of neurologic signs occurred on day 5. Results of CT on day 7 were unremarkable. We found increased levels of proteins and cells in the CSF. Brain MRI showed symmetric diffusion restriction in the bilateral anterior frontal cortex (Figure 2). The patient was intubated and mechanically ventilated for 24 hours. She showed good progression after being given mannitol, dexamethasone, oseltamivir, and acyclovir. The patient was discharged with complete resolution after 18 days of hospitalization.

Patient 4 was a 20-year-old woman who had Russell−Silver syndrome and was being given prednisone and levothyroxine. She was admitted to an intensive care unit (ICU) already mechanically ventilated. Onset of illness was 5 days before admission and included fever, cough, and agitation. After 3 days in the hospital, nystagmus, stiff neck, and a GCS score of 9−10 were observed. Results of CSF testing and cerebral CT and MRI were unremarkable. She was given mannitol, methylprednisolone, and oseltamivir. Her condition improved after 17 days of hospitalization and was followed by complete recovery.

Patient 5 was a 57-year-old man who came to our clinic after 3 days of fever, chills, and cough. In the preceding 24 hours, headache, dysarthria, right side motor deficit, and vomiting developed. Results of CSF testing and cranial CT and MRI were unremarkable. The patient was given oseltamivir and mannitol. After 10 days, the patient was discharged, and he showed complete resolution.

Patient 6 was a 31-year-old woman who had recent breast implants. The patient was hospitalized after 4 days of fever, headache, vomiting, vertigo, photophobia, and movement and balance disorders. Results of CSF testing were unremarkable. She was given oseltamivir and mannitol, and her condition improved after 5 days.

Patient 7 was a 27-year-old woman who had influenza, fever, myalgia, headache, and vomiting 3 days before hospitalization. After 2 days, she became lethargic, had aphasia and seizures, and entered a coma; GCS score decreased to 3. Results of initial CSF testing were unremarkable. She was intubated, mechanically ventilated, and transferred to our clinic. Results of CT were unremarkable. However, MRI showed an abnormal result (multiple areas of hyperintensities).

At admission, patient 7 was comatose and intubated. She had unreactive fixed mydriasis, upward deviation of the eyes, and no corneal reflex and plantar cutaneous reflexes. Results of additional CSF testing showed pleocytosis and a high level of albumin (3.906 g/L). Renal failure and ventricular tachyarrhythmia developed. Despite treatment with oseltamivir, acyclovir, mannitol, methylprednisolone, and meropenem, the patient continued to show signs of brain death and died 3 days after admission.

Observations

The peak of the influenza season in Romania was during February−March 2015. A total of 110 patients with ILI (90 female patients and 20 male patients) were hospitalized at the tertiary care facility during January−April 2015. The median age of patients was 43 years (range 4−93 years); only 3 patients were <18 years of age. None of the patients had been vaccinated against influenza for the current season. There were 57 patients with ILI laboratory-confirmed influenza infection: 32 patients (56.2%) were infected with an influenza B strain, 14 (24.5%) with an influenza A/H3 strain, and 11 (19.3%) with the A(H1N1)pdm09 strain. These 3 strains were present in Romania at similar prevalences throughout the influenza season.

A complicated form of influenza was diagnosed for 28 (49.1%) patients, of whom 19 (33.3%) had respiratory complications (8 infected with the A(H1N1)pdm09 strain, 6 with the A/H3 strain, and 5 with the B strain), and 9 (15.7%) had neurologic complications (8 with the B strain and 1 with the (A/H1N1)pdm09 strain). Two patients who had febrile seizures as the only neurologic manifestation were excluded from analysis (1 child with a history of febrile seizures and 1 adult with epilepsy). Eight of the 9 patients with neurologic complications were female patients. Of those infected with an influenza B strain, 7 were adults (median age 31 years, range 20−57 years), and 1 was a 4-year-old girl. Six of the 7 adult patients infected with an influenza B strain in whom neurologic manifestations developed were women.

Antiviral treatment with a neuraminidase inhibitor (oseltamivir, 75 mg 2×/d for adults and 37.5 mg 2×/d for the child) was administered to all patients upon presentation. However, most patients presented late after onset of disease, and only 1 received oseltamivir within the first 48 hours of disease onset; the other patients received antiviral treatment 72 hours after disease onset.

Demographic, clinical, imaging and laboratory data for the 7 adult patients with neurologic manifestations and laboratory-confirmed influenza B virus infection are shown in the Table. None of the patients had preexisting neurologic diseases, and the average time from disease onset to hospitalization was 4.28 days. On the basis of the case definition, 4 patients were given a diagnosis of confirmed encephalitis (major criteria and >3 minor criteria and laboratory-confirmed influenza), 2 were given a diagnosis of possible encephalitis (major criteria and 2 minor criteria), and 1 was given a diagnosis of cerebellar ataxia (no major criteria but with neurologic manifestations).

Cerebral MRI was performed for 6 patients and CT was performed for 2 patients. Abnormal brain imaging results were observed for 3 patients: changes consistent with multiple areas of hyperintensities visible in T2-associated with restricted diffusion for patients 1 and 7 (patient 1; Figure 1) and cerebral edema and diffusion restriction for patient 3 (Figure 2). Six patients were admitted to the ICU; 4 of these patients required intubation and mechanical ventilation because of neurologic complications. Two patients died, 3 and 7 days after admission. The other 5 patients showed a good outcome with complete resolution. At a follow-up 1 month after discharge, results of neurologic examinations for the 5 patients were unremarkable without any signs or symptoms during this period.

Lumbar puncture was performed for all 7 patients. Results of CSF analysis were abnormal for 3 patients (pleocytosis [>5 cells/mm³] and increased protein levels). All CSF samples were negative for influenza virus nucleic acids, enteroviruses, and herpes viruses.

Genetic analysis of the HA sequences of influenza B viruses isolated from nasopharyngeal swab specimens was successful for 4 patients (patients 1, 2, 5, and 7) (Table). HA phylogenetic analysis showed that all strains belonged to Yamagata-lineage clade 3, representative strain B/Phuket/3073/2013, which is distinct from the World Health Organization recommended strain included in the 2014–2015 vaccine (B/Massachusetts/2/2012–like virus).