Cerebrospinal Fluid Examination in Survivors of Ebola Virus Disease

Abstract

This cohort study assesses the role of the central nervous system as a potential reservoir for Ebola virus after initial clinical recovery in a population of patients who survived Ebola virus disease.

With the extent of the recent Ebola virus disease (EVD) epidemic in West Africa (including >28 000 cases and ≤17 000 survivors), many aspects of Ebola virus have become clearer, including acute manifestations, sequelae, and the possibility of relapse and persistence. Neurologic complications are becoming more commonly recognized in EVD during the acute phase, with long-term sequelae. More remarkable, however, is the potential for persistence and relapse of EVD in the central nervous system (CNS), as in the case of a nurse who developed meningoencephalitis with seizures, cranial nerve involvement, and radiculitis 9 months after EVD recovery. Ebola virus was recovered from the cerebrospinal fluid (CSF) at higher levels than from the blood, indicating viral replication in the CNS. This case reveals the need to determine the role of the CNS as a potential reservoir for Ebola virus after initial clinical recovery.

Methods

To investigate potential Ebola virus persistence in the CNS, we undertook a systematic study of CSF samples from patients who survived EVD (hereinafter referred to as EVD survivors). Through partnership between the National Institutes of Health and Liberia’s Ministry of Health, the Partnership for Research on Ebola Virus in Liberia (PREVAIL) III Ebola natural history study is ongoing at John F. Kennedy Medical Center in Monrovia, Liberia. This study was approved by the institutional review boards of the National Institute of Allergy and Infectious Diseases and the Liberian National Research Ethics Board. All patients included in the analysis provided written informed consent.

A subset of EVD survivors (n = 165) underwent evaluation by neurologists as part of this study. Survivors who reported coma, delirium, or meningitis during acute EVD, who reported new neurologic symptoms since recovery, and who had neurologic abnormalities on examination were selected as candidates for lumbar puncture (LP). These survivors (n = 22) were informed about LP using illustrated flip charts; those who decided to proceed were screened for eligibility (n = 18). Eligibility criteria included age 18 to 60 years, positive for Ebola virus antibody, no new neurologic findings at time of LP, negative for human immunodeficiency virus antibody, no recent seizure or head trauma, no use of nonsteroidal anti-inflammatory drugs, no papilledema, and normal results of coagulation studies.

For patients who met criteria and signed informed consent (n = 8), LPs were performed in standard fashion by operators in full personal protective equipment (B.J.B., J.D., and B.R.S.). Samples were collected from December 15 to 18, 2015. Each LP sample collected included 15 to 20 mL of CSF. Fluid was analyzed for Ebola viral RNA by reverse transcriptase–polymerase chain reaction (RT-PCR) at the Liberian Institute for Biomedical Research laboratory. The RT-PCR systems used to detect nucleoprotein and glycoprotein gene targets included ABI 7500 (Applied Biosystems) and GeneXpert IV (Cepheid); the level of detection for the GeneXpert assay is 232 copies/mL. The CSF cell count was assessed using a hemocytometer. Glucose and protein levels were measured using a semiautomated clinical chemistry analyzer (Excel; Stanbio Laboratory).

Results

We collected CSF samples from 7 participants (5 men and 2 women; mean [SD] age, 34.9 [12.0] years). Median time from discharge from the Ebola treatment unit was 414 days (range, 364-459 days). No Ebola viral RNA was detected in the 7 CSF samples by either RT-PCR assay. Furthermore, all samples had protein and glucose levels and white blood cell counts within reference range, although 1 patient had borderline elevation of protein levels. Another patient had red blood cells in the CSF due to a traumatic LP (Table).

Table. Patient Demographic and Clinical Characteristics and Lumbar Puncture Findings^a.

Patient No./Sex/Age, y	Length of ETU Stay, d	Time From ETU Discharge to LP, d	Neurologic Symptoms		Abnormal Neurologic Examination Findings	CSF Measurement
			During ETU Stay	Since EVD Discharge		WBCs/hpf	RBCs/hpf	Protein Level, mg/dL	Glucose Level, mg/dL
1/Female/28	21	364	Hallucinations, confusion, headache, vision change	Paresthesias, hallucinations, headache, memory loss, confusion	Frontal release signs (glabellar, grasp), choppy pursuits, dysmetric saccades, increased reflexes	0	0	25.5	47.2
2/Male/23	8	459	Headache, AMS, hallucinations, vertigo, paresthesias	New headache	Cerebellar findings (dysmetria, dysdiadochokinesia), slowed saccades to left and left extinguishing horizontal nystagmus	0	0	18.2	58.7
3/Male/50	32	414	Headache, hallucinations, AMS, tremor, vertigo, tinnitus, paresthesias	Memory loss, tinnitus	Mild action tremor, peripheral neuropathy	0	0	17.9	53.6
4/Female/22	19	418	Headache, confusion, hallucinations, bilateral LE weakness and numbness	Headache, memory loss, depression	Choppy smooth pursuits, slowed saccades, decreased hearing, increased reflexes (3+ at knees)	0	0	42.5	61.5
5/Male/44	26	401	Headache, neck stiffness, AMS, hallucinations, paresthesias, tremor, hearing loss, vertigo	New headache, memory loss, paresthesias	Loss of sensation in V2 and V3, facial weakness, decreased hearing, mild postural tremor, peripheral neuropathy	0	0	30.7	44.5
6/Male/25	23	404	Headache, hallucinations, tremor, paresthesias, vertigo	New headache, paresthesias	Choppy pursuits, slowed saccades, tongue deviation, mild postural tremor, sensory neuropathy	0	0	31.8	69.9
7/Male/46	13	415	Headache, neck stiffness, double vision, hearing loss, vertigo, tinnitus, trouble swallowing, LOC, confusion	Paresthesias, new headache, memory loss	Parkinsonism with bradykinesia, mild cogwheeling and retropulsion; slowed saccades	3	2850	40.2	64.1

Abbreviations: AMS, altered mental state; CSF, cerebrospinal fluid; ETU, Ebola treatment unit; EVD, Ebola virus disease; hpf, high-powered field; LE, lower extremity; LOC, loss of consciousness; RBCs, red blood cells; WBCs, white blood cells.

SI conversion factors: To convert glucose to millimoles per liter, multiply by 0.0555; protein to grams per liter, multiply by 0.01.

^aEbola virus was not detected on reverse transcriptase–polymerase chain reaction results using the GeneXpert (Cepheid) or ABI 7500 (Applied Biosciences) device in any patient.

Discussion

Few reports exist of CSF studies during or after Ebola virus infection. This study represents the first systematic evaluation of CSF in EVD survivors. The CSF from all 7 patients undergoing analysis was negative for Ebola viral RNA and showed no signs of inflammation; however, this finding could be related to the relatively long period from resolution of acute EVD to performance of LP. Alternately, if Ebola virus is dormant in the CNS of EVD survivors and is cell associated, it may not be released into CSF. Any release of virus from reservoirs into the CSF would be expected to cause acute meningoencephalitis. Thus, EVD survivors should be monitored for neurologic symptoms suggestive of EVD relapse in the CNS because of the potential for Ebola virus transmission during relapse.