Cerebrospinal Fluid Cytology in Seasonal Epidemic West Nile Virus Meningo-Encephalitis Ajay Rawal,
1,2 M.D.,
Patrick J. Gavin,
M.D.,
3,4
and Charles D. Sturgis, M.D.4,5*
The incidence of West Nile Virus (WNV) infection has progressively increased in North America since the first epidemic in 1999. Formal scholarly documentation of cerebrospinal fluid (CSF) cytology changes in patients with WNV infection is limited. We report our experience with CSF cytospins from a population of consecutive patients with documented CSF WNV-specific IgM. Thirty-two patients (12 male, 20 female) with a median age of 52 yr (range, 19–88) diagnosed with WNV meningo-encephalitis were studied. Symptoms were present for a mean of 5 days (range, 1– 14) prior to lumbar puncture. CSF proteins were elevated in 94% of patients (30/32) with a mean value of 79 mg/dl (range, 36–185). CSF glucose was normal to elevated in all cases. All cytomorphologically adequate samples demonstrated a pleocytosis with a mean of 156 cells/mm3 (range, 13–683). Nearly, all (26/28) patients showed increased CSF neutrophils- mean 43% (range, 1– 83). Mean lymphocyte and monocyte fractions were 44% (range, 8–85) and 14% (range, 2–27), respectively. Three cases showed 1–4% plasma cells. Mean total leukocyte counts (TLC) (197 cells/ mm3) and mean neutrophil fractions (50%) were greater in patients sampled within the first 3 days of symptoms than in those sampled beyond day 3 (mean TLC, 126 cells/mm3; mean neutrophil fraction, 37%). Relative lymphocyte proportions increased from a mean of 39 to 48% after 3 days of illness. WNV should be considered as a potential etiology of infectious CSF pleocytosis in the North American late summer and early fall seasons. Diagn. Cytopathol. 2006;34:127–129. ' 2006 Wiley-Liss, Inc. Key Words: pleocytosis
West Nile virus; cytology; cerebrospinal fluid;
1 Department of Anatomic and Clinical Pathology, Evanston Northwestern Healthcare, Evanston, Illinois 2 Department of Anatomic and Clinical Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 3 Department of Microbiology, Evanston Northwestern Healthcare, Evanston, Illinois 4 Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 5 Department of Cytopathology, Evanston Northwestern Healthcare, Evanston, Illinois *Correspondence to: Charles D. Sturgis, M.D., Department of Cytopathology, Evanston Hospital, 2650 Ridge Avenue, Evanston, IL 60201. E-mail:
[email protected] Received 31 May 2005; Accepted 4 August 2005 DOI 10.1002/dc.20410 Published online in Wiley InterScience (www.interscience.wiley.com).
'
2006 WILEY-LISS, INC.
In recent years, West Nile virus (WNV) has emerged as a mosquito-transmitted threat to public health in North America. The incidence of WNV infections in the United States has progressively increased since the first epidemic in 1999. While only 149 cases were reported to the Center for Disease Control from 1999 through 2001, the number of reported cases has since escalated sharply, with 9,862 cases confirmed in 2003 alone, with a mortality rate of 2.6%.1 Most WNV infections are sub-clinical or manifest only as a mild febrile illness. In 1 out every 150 (0.7%) cases, however, the infection manifests with severe neurological symptoms such as encephalitis, meningitis, or acute flaccid paralysis.2 The diagnosis of WNV infection is based on a strong clinical index of suspicion, supported by appropriate serological testing of the involved patient’s serum or cerebrospinal fluid (CSF). Limited documentation of CSF cytology exists in patients infected with WNV.3–7 This study was performed to systematically evaluate cytological features and other routine CSF biochemical parameters in a cohort of patients with documented WNV meningo-encephalitis.
Materials and Methods In the calendar year 2002, a total of 4,156 WNV infections were reported to the Centers for Disease Control. Of these cases, 884 came from Illinois. Eighty of these patients were originally diagnosed at Evanston Northwestern Healthcare, more than at any other hospital in the state. Thirty-two patients had WNV-specific IgM antibodies detected in CSF. These individuals are the subject of this study. Diagnostic serological testing was performed at the Illinois Department of Public Health laboratory using an IgM antibody capture enzyme-linked immunosorbent assay. Wright-Giemsa stained CSF cytospin preparations were cytologically evaluated at Evanston Northwestern Healthcare by two of the authors (AR and CDS). Results of CSF biochemistry (protein and glucose levels) and total leukocyte counts (TLC) were retrieved from laboratory information systems, and clinical symptoms and their durations were gleaned from the medical record. (All Diagnostic Cytopathology, Vol 34, No 2
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Diagnostic Cytopathology DOI 10.1002/dc
RAWAL ET AL. Table I. Presenting Symptoms in Patients with Documented CSF WNV IgM Clinical symptom
Prevalence %
Fever Headache Neck stiffness Myalgia Nausea Weakness Rash Photophobia Altered sensorium Diplopia
91 81 60 41 41 38 34 16 9 9
(29/32) (26/32) (19/32) (13/32) (13/32) (12/32) (11/32) (5/32) (3/32) (3/32)
Table II. Spinal Fluid Biochemical and Cellular Parameters in Patients with Documented CSF WNV IgM (N ¼ 32) Parameter
Mean 6 SD
Range
Reference range
Proteins Glucose (mg/dl) TLC (cells mm3) Neutrophils (%) Lymphocytes (%) Monocytes (%)
79 66 156 43 44 14
6 6 6 6 6 6
36–185 43–121 13–683 1–83 8–85 2–27
15–45 40–80 0–8 0–6 40–80 15–45
33 20 156 24 22 2
Fig. 1. CSF cytospin photomicrograph with intense neutrophil-rich pleocytosis from patient on day three of clinical central nervous system symptomatology (Wright-Giemsa stain, original magnification 4003).
electronic and paper chart record reviews was performed with ENH Institutional Review Board Approval, E04-003, to ensure patient privacy and confidentiality).
Results Twelve of the 32 patients were males and 20 were females. The median age was 52 yr (range, 19–88). The median duration of symptoms prior to lumbar puncture was 5 days (range, 1–14). Fever, headache, and neck stiffness were the most commonly reported clinical symptoms (Table I). Spinal fluid protein was elevated in 94% (30/ 32) of the CSF IgM-confirmed cases (Table II). CSF glucose was normal in majority of the patients (26/32, 81%). Four cytology samples were excluded from the morphologic review because of gross red cell contamination. Spinal fluid pleocytoses were present in 100% (28/28) of the patients, and neutrophil fractions were increased in 93% (26/28) of cases (Table II and Fig. 1). Identified lymphocytes were cytomorphologically reactive and demonstrated a heterogeneous range of appearances, varying from small mature-appearing forms to larger transformed lymphocytes with less condensed nuclear chromatin and cytoplasmic basophilia. Plasma cells were identified in 11% (3/28) of the cases, where they comprised a minority (1–4%) of the cellular infiltrate (Fig. 2). Mean TLC and mean neutrophil fractions were noted to be consistently higher in the initial 3 days of illness and tended to decrease after 3 days (Table III).
Discussion Accurate diagnosis of WNV-induced meningo-encephalitis is based on both clinical and laboratory data. Confir128
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Fig. 2. CSF cytospin photomicrograph with plasmacytoid lymphocyte (arrow), showing cytoplasmic basophilia, nuclear eccentricity, and perinuclear cytoplasmic clearing (Wright-Geimsa stain, original magnification 4003). Table III. Relationship between Symptom Duration, CSF Total Leukocyte Count, Neutrophilic Fraction, and Lymphocyte Fraction in Patients with Documented CSF WNV IgM (N ¼ 32)
Parameter Mean total leukocyte count (mm3) Mean neutrophilic fraction (%) Mean lymphocytic fraction (%)
Symptom duration 3 days
197 50 39
126 37 48
mation of clinical suspicion of this disease process is based on the demonstration of WNV specific IgM proteins in either spinal fluid or serum of infected patients.
Diagnostic Cytopathology DOI 10.1002/dc
CEREBROSPINAL FLUID CYTOLOGY IN MENINGO-ENCEPHALITIS Table IV. Summary of CSF Biochemical and Cytological Parameters from Literature on WNV Infections of the Nervous System Protein (mg/dl)a
Reference 3
Weiss et al Nash et al4 Sejvar et al5 Pepperell et al6 Sejvar et al7
111 104 84 90 126
Glucose (mg/dl)a
(56–555) (38–899) (40–234) (30–200) (37–234)
67 80 65 X 82
(48–95) (46–143) (37–119) (X) (54–119)
Cell count (mm3)a 308 38 76 X 566
(0–1782) (0–525) (0–2317) (0–1179) (3–2600)
Predominating cell type Neutrophils Lymphocytes Lymphocytes Lymphocytes (Neutrophils in early infection) X
X, Values not reported. a Values in parentheses indicate mean (range) values.
CSF biochemical and cytological examinations are an integral part of front-line testing in patients with symptoms or signs of central nervous system inflammatory processes and provide vital corroborative evidence of disease. The current study is the first formal review focusing on CSF cytological parameters from a cohort of patients with confirmed WNV meningo-encephalitis. Findings of elevations in CSF protein levels, normal to slight elevations in CSF glucose measurements, and cellular pleocytoses were uniformly noted in the patient set (Table II). These findings parallel WNV-related spinal fluid changes reported in the clinical literature (Table IV).3–7 Two previous clinical reviews of patients with WNV infection have reported predominant early neutrophilic responses in the CSF of WNV meningo-encephalitis patients, as seen in the current data set.3,6 In the study by Weiss et al., no association was found between the intensity of neutrophilic response and the severity of presentation of encephalitis. Interestingly, from the cytopathology perspective, six of the 56 patients in a series by Pepperell et al. demonstrated atypical lymphocytes, prompting work-ups for possible central nervous system lymphoproliferative disorders, which were ultimately found to be non-clonal pleocytoses.6 In the current study, the lymphocyte morphology ranged from small, mature-appearing cells to those exhibiting conspicuous reactive features, including the presence of plasma cells in 3 cases (Fig. 2). All cases, however, demonstrated a spectrum of alterations in lymphocyte morphology, suggesting a reactive etiology, and no case was thought to be cytomorphologically suspicious enough to prompt exclusionary work-up for a neoplastic lymphoproliferative disorder. Viral infections of the central nervous system are known to be associated with cytomorphologic alterations in lymphocyte populations, and some cells may assume bizarre immunoblastic morphologies.8 From an interpretive cytopathologic perspective, recognition of a true heterogeneity in leukocyte response is crucial in arriving at an appropriate morphologic interpretation. In those cases in which clinical his-
tory and cytomorphology combined do not exclude the possibility of central nervous system lymphoma, immunophenotyping by flow cytometry may be a valuable adjunctive test. In a study by Sejvar et al., CSF protein levels, leukocyte counts, and lymphocyte percentages trended to higher values in the subset of patients presenting with acute flaccid paralysis.5 In another study of 7 patients with acute flaccid paralysis due to WNV, the presence of both pleocytosis and elevated proteins in the CSF helped substantiate the clinical impression of an acute poliomyelitis-like illness as the cause for paralysis, in contrast to Guillian– Barre´ syndrome, which is characterized only by elevated proteins and no pleocytosis.7 Recognition of patterns of CSF inflammatory responses in real time clinical cohorts of appropriate patient samples seen within a given geographic population / laboratory may be valuable in pinpointing WNV outbreaks early in their progression. WNV should be considered as a potential etiology of infectious CSF pleocytosis in the North American late summer and early fall seasons.
References 1. http://www.cdc.gov. 2. Peterson LR, Marfin AA. West Niles virus: a primer for the clinician. Ann Int Med 2002;137:173–179. 3. Weiss D, Carr D, Kellachan J, et al. Clinical findings of West Nile virus infection in hospitalized patients, New York and New Jersey, 2000. Emerg Infect Dis 2001;7:654–658. 4. Nash D, Mostashari F, Fine A, et al. The outbreak of West Nile virus infection in the New York City area in 1999. N Engl J Med 2001; 344:1807–1814. 5. Sejvar JJ, Haddad MB, Tierney BC, et al. Neurologic manifestations and outcome of West Nile virus infection. JAMA 2003;290: 511–515. 6. Pepperell C, Rau N, Krajden S, et al. West Nile virus infection in 2002: morbidity and mortality among patients admitted to hospital in southcentral Ontario. CMAJ 2003;168:1399–1405. 7. Sejvar JJ, Leis AA, Stokic DS, et al. Acute flaccid paralysis and West Nile virus infection. Emerg Infect Dis 2003;9:788–793. 8. Bigner SH. Cerebrospinal fluid (CSF) cytology: current status and diagnostic applications. J Neuropathol Exp Neurol 1992;3:235–245.
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