Characterisation of the cerebrospinal fluid of horses with West Nile virus neuroinvasive disease.

West Nile virus (WNV) is a zoonotic arbovirus transmitted by mosquitoes between wild birds (natural hosts) and other vertebrates. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Owing to the close contact of cerebrospinal fluid (CSF) with the extracellular fluid of the brain, the analysis of CSF composition can reflect central nervous system (CNS) impairments enabling the diagnosis and understanding of various neurodegenerative CNS disorders. Our objective was to compare the findings from the CSF samples of horses with neuroinvasive WNV infection with those of healthy controls. We compared findings from fifteen CSF samples of 13 horses with acute WNV encephalomyelitis with those of 20 healthy controls. Protein, particular enzymes and ions, glucose and lactate showed abnormal levels in a significant number of WNV cases. None of the six horses with elevated glucose concentrations survived. Rather neutrophilic than mononuclear pleocytosis was identified with WNV infection. Neutrophils probably play a role in the development of inflammatory response and brain damage. Although elevated glucose levels reliably predicted the outcome, they might be the consequence of increased plasma levels and reflect general stress rather than CNS pathophysiology. The CSF findings of WNV encephalomyelitis patients are non-specific and variable but facilitate the differential diagnosis.

Emergence of human West Nile Virus infection among pediatric population in Madhya Pradesh, India.

Human infections caused by West Nile virus (WNV) mostly remain subclinical and self-limited. However, nearly 20% infected people suffer from febrile illness and very few of them (<1%) may get neuroinvasive illness. Mortality has been reported among children. India somehow has reported very less number of WNV cases in the past. We collected cerebrospinal fluid (CSF) samples from 75 pediatric age group patients clinically suffering from acute encephalitis syndrome. Three of these samples were positive by reverse transcriptase polymerase chain reaction using pan flavivirus primers. On sequencing of the 212 bp long-amplified fragment, it was found to be WNV belonging to lineage 1. This is probably the first report of WNV causing encephalitis from this central part of India.

JC virus identified in a patient with persistent and severe West Nile virus disease.

West Nile virus is a notable cause of neuroinvasive disease, damage to the central nervous system, or even death. In this study, using metagenomics analysis and quantitative real-time PCR validation, we identified a JC virus infection in urine and cerebrospinal fluid samples of a West Nile virus patient with severe neurological symptoms and extended disease. JC virus is known to be involved in neurological complications, especially in immunocompromised individuals thus suggesting that the coinfection with JC virus is involved with the West Nile virus infection persistence and severe symptoms. JC virus was identified in urine samples from additional West Nile virus patients via quantitative real-time PCR, however, JC virus was not found in any cerebrospinal fluid samples of West Nile virus patients, suggesting that JC virus does not regularly infect the central nervous system of WNV patients. Overall, this study highlights the importance of identifying infection by opportunistic viruses in already-diagnosed patients and highlights the advantages of next-generation sequencing and metagenomics for viral diagnosis.

A probable case of West Nile virus transfusion transmission.

BACKGROUND: Transfusion-transmitted West Nile virus (WNV) infection is infrequent as a result of minipool (MP) and individual-donation (ID) nucleic acid testing (NAT) of blood donations. ID-NAT is triggered on the basis of local WNV activity identified by MP-NAT. STUDY DESIGN AND METHODS: A 78-year-old male patient who was treated for cardiac disease received 14 blood components from 30 donors in August 2016. He was discharged 7 days after aortic valve replacement and coronary bypass surgery, but was re-admitted on Day 12 with symptoms of viral infection, and eventually was diagnosed with aseptic meningitis. The patent died on Day 51. RESULTS: The patient was positive for WNV-immunoglobulin M (IgM) antibodies in his cerebrospinal fluid on Day 14 and was positive for WNV-IgM (on Days 14 and 16) and WNV-IgG antibodies (on Day 16) in his serum. All associated donations were nonreactive by MP-NAT or ID-NAT. However, one MP-NAT was noted to have an elevated (but negative) signal-to-cutoff ratio, and one donor from that MP was subsequently found positive for WNV-IgM and IgG antibodies; the donor was diagnosed with a WNV-like viral syndrome that had an onset 3 to 5 days postdonation. The donor's plasma was transfused 12 days before the patient's onset of WNV-meningoencephalitis. Conversion to ID-NAT was triggered for the region 7 days after the implicated donation, which was associated with the region's first human WNV case. CONCLUSION: Despite the possibility of mosquito-borne transmission, this was considered to be a case of transfusion-transmitted WNV infection from an MP-NAT-nonreactive donation collected just before triggering conversion to ID-NAT; a rare event recognized once in 84 million donations.

Hospital-based enhanced surveillance for West Nile virus neuroinvasive disease.

Accurate data on the incidence of West Nile virus (WNV) disease are important for directing public health education and control activities. The objective of this project was to assess the underdiagnosis of WNV neuroinvasive disease through laboratory testing of patients with suspected viral meningitis or encephalitis at selected hospitals serving WNV-endemic regions in three states. Of the 279 patients with cerebrospinal fluid (CSF) specimens tested for WNV immunoglobulin M (IgM) antibodies, 258 (92%) were negative, 19 (7%) were positive, and two (1%) had equivocal results. Overall, 63% (12/19) of patients with WNV IgM-positive CSF had WNV IgM testing ordered by their attending physician. Seven (37%) cases would not have been identified as probable WNV infections without the further testing conducted through this project. These findings indicate that over a third of WNV infections in patients with clinically compatible neurological illness might be undiagnosed due to either lack of testing or inappropriate testing, leading to substantial underestimates of WNV neuroinvasive disease burden. Efforts should be made to educate healthcare providers and laboratorians about the local epidemiology of arboviral diseases and the optimal tests to be used in different clinical situations.

Plasma cell cerebrospinal fluid pleocytosis does not predict West Nile virus infection.

PURPOSE: Diagnosis of WNV (WNV) relies upon serologic testing which may take several days after the onset of clinical symptoms to turn positive. Anecdotal reports suggest the presence of plasma cells or plasmacytoid lymphocytes in the cerebrospinal fluid (CSF) may be an early indicator of WNV infection. METHODS: The CSFs of 89 patients (12 with WNV, 12 with other viral illness {OVI}, and 65 with nonviral illness{NVI}) were compared for the presence of either plasma cells or plasmacytoid lymphocytes. RESULTS: Plasma cells were rarely seen in any of the patients. Plasmacytoid lymphocytes were more commonly seen in WNV (58%) and OVI (50%) than NVI (11%). The differences were significant for WNV versus NVI, but not WNV versus OVI (P < 0.001 and P = 0.58, resp.). CONCLUSIONS: A CSF pleocytosis with plasma cells or plasmacytoid lymphocytes was neither sensitive nor specific for the diagnosis of WNV infection.

Detection of West Nile virus genome and specific antibodies in Iranian encephalitis patients.

West Nile virus (WNV) is a mosquito-borne flavivirus which circulates in birds, horses and humans. An estimated 80% of WNV infections are asymptomatic. Fewer than 1% of infected persons develop neuroinvasive disease, which typically presents as encephalitis, meningitis, or acute flaccid paralysis. This study was conducted from January 2008 to June 2009 in Isfahan, Iran. Patients attending the emergency department with fever and loss of consciousness were consecutively included. Cerebrospinal fluids (CSF) were initially analysed through bacteriology and biochemistry examinations, resulting in those with evidence of meningitis being excluded. Patients' CSF and serum were diagnosed by serological and molecular assays. A total of 632 patients with fever and loss of consciousness were tested by CSF analyses. Samples of the remaining patients (39·4%) were referred for WNV investigation. Three (1·2%) of the patients were positive for both serum and CSF by RT-PCR, and six (2·4%) were positive only for IgG antibodies. History of insect bite, and blood transfusion and transplantation were risk factors for being positive by RT-PCR (P=0·048) and being IgG positive (P=0·024), respectively. The results of this study showed that the prevalence of West Nile fever is low in patients with encephalitis.

Ongoing outbreak of West Nile virus infection in humans, Greece, July to August 2011.

Between 16 July and 21 August 2011, 31 cases of West Nile neuroinvasive disease were reported from four regions in Greece. Of these, 17 occurred in districts that had not been affected in 2010. The reoccurrence of human cases in two consecutive years (following the large 2010 outbreak) and the spread of the virus in new areas suggest that West Nile virus is established in Greece, and its transmission may continue to occur in the future.

Neuronal and glial cerebrospinal fluid protein biomarkers are elevated after West Nile virus infection.

Neurotrophic West Nile virus (WNV) disease is a severe arbovirus infection in which neuronal loss is the likely anatomical substrate for the high morbidity and mortality. We investigated whether cerebrospinal fluid (CSF) protein biomarkers were elevated in vivo and related to disease severity in patients with WNV infection. This exploratory study included 114 patients (24 acute WNV, 77 noninflammatory controls, six peripheral neuropathies, seven aseptic meningoencephalitis). CSF levels of neuronal (neurofilaments, NfH-SMI35) and glial (glial fibrillary acidic protein, GFAP, S100B) biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). Immunocytochemistry was performed in two fatal WNV cases. A significant proportion of patients with WNV had pathological CSF levels for NfH-SMI35 (58%, median concentration 1.01 ng/mL), GFAP (58%, 10 pg/mL), and S100B (90%, 1.29 ng/mL). The results were consistent with postmortem evidence for neuronal death and astrogliosis. Surprisingly, CSF protein biomarker levels were also found to be pathological in a considerable proportion of patients who presented with WNV fever only (100% for GFAP and S100B and 43% for NfH-SMI35). Elevated CSF protein biomarker levels are suggestive of neuronal death and glial pathology in human WNV infection. The results indicate the presence of neuroinvasive disease across the spectrum of WNV disease, including WNV fever.

Arboviral Surveillance among Pediatric Patients with Acute Febrile Illness in Houston, Texas.

We instituted active surveillance among febrile patients presenting to the largest Houston-area pediatric emergency department to identify acute infections of dengue virus (DENV), West Nile virus (WNV), and chikungunya virus (CHIKV). In 2014, 1,063 children were enrolled, and 1,015 (95%) had blood and/or cerebrospinal fluid specimens available for DENV, WNV, and CHIKV testing. Almost half (49%) reported recent mosquito bites, and 6% (N = 60) reported either recent international travel or contact with an international traveler. None were positive for acute WNV; three had false-positive CHIKV results; and two had evidence of DENV. One DENV-positive case was an acute infection associated with international travel, whereas the other was identified as a potential secondary acute infection, also likely travel-associated. Neither of the DENV-positive cases were clinically recognized, highlighting the need for education and awareness. Health-care professionals should consider the possibility of arboviral disease among children who have traveled to or from endemic areas.

West Nile virus immunoglobulin A (WNV IgA) detection in cerebrospinal fluid in relation to WNV IgG and IgM reactivity.

BACKGROUND: Diagnostic criteria for neurologic involvement in WNV infection include WNV IgM detection in CSF; however, WNV IgM can persist in CSF >6 months. CSF IgA characterizes other flavivirus infections, but WNV IgA in CSF has not been evaluated. WNV IgM in CSF correlates with IgM in serum but the presence of WNV IgA in CSF compared to serum is unknown. OBJECTIVES: Evaluate WNV IgA detection in CSF as a marker of WNV neuroinvasive infection, initially with samples pre-selected based on WNV IgG and IgM reactivity and subsequently with all available CSF samples submitted for WNV antibody testing over an entire WNV season. STUDY DESIGN: Selected CSF samples and CSF/serum pairs previously tested for WNV IgG and IgM were assayed for WNV IgA. Subsequently, all available CSF samples tested for WNV antibodies during the 2005 season were tested for WNV IgA, including those where paired sera were available and tested for IgA, IgG and IgM. RESULTS: For most samples, including paired CSF and serum, the IgA result qualitatively agreed with the IgM result, regardless of the IgG result. CONCLUSION: IgA detection is equivalent to IgM detection as a marker of WNV infection in CSF.

Comparative analysis of cytokine gene expression in cerebrospinal fluid of horses without neurologic signs or with selected neurologic disorders.

OBJECTIVE: To determine gene transcription for cytokines in nucleated cells in CSF of horses without neurologic signs or with cervical stenotic myelopathy (CSM), West Nile virus (WNV) encephalitis, equine protozoal myeloencephalitis (EPM), or spinal cord trauma. ANIMALS: 41 horses (no neurologic signs [n = 12], CSM [8], WNV encephalitis [9], EPM [6], and spinal cord trauma [6]). PROCEDURES: Total RNA was extracted from nucleated cells and converted into cDNA. Gene expression was measured by use of real-time PCR assay and final quantitation via the comparative threshold cycle method. RESULTS: Cytokine genes expressed by nucleated cells of horses without neurologic signs comprised a balance between proinflammatory tumor necrosis factor-alpha (TNF-alpha), anti-inflammatory cytokines (interleukin [IL]-10 and transforming growth factor [TGF]-beta), and Th1 mediators (interferon [IFN]-gamma). Cells of horses with CSM mainly expressed genes for TNF-alpha, TGF-beta, and IL-10. Cells of horses with WNV encephalitis mainly expressed genes for IL-6 and TGF-beta. Cells of horses with EPM mainly had expression of genes for IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, and TGF-beta. Cells from horses with spinal cord trauma had expression mainly for IL-6; IFN-gamma; TGF-beta; and less frequently, IL-2, IL-10, and TNF-alpha. Interleukin-8 gene expression was only detected in CSF of horses with infectious diseases. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the small number of CSF samples for each group, results suggest distinct gene signatures expressed by nucleated cells in the CSF of horses without neurologic signs versus horses with inflammatory or traumatic neurologic disorders.

Focal neurological injury caused by West Nile virus infection may occur independent of patient age and premorbid health.

INTRODUCTION: Limited evidence suggests that focal neurological injury (e.g., acute flaccid paralysis) caused by infection with the West Nile virus (WNV) is more common in older patients. We re-evaluate this association in a series of patients who were infected with the WNV during the 2002 epidemic. METHODS: We performed a retrospective chart review of 34 patients who were hospitalized for treatment of serologically confirmed WNV infection. Measurements included the patient's demographic characteristics, baseline medical diagnoses, the occurrence of symptoms and exam findings, the results of various diagnostic tests, and the patient's clinical outcome. RESULTS: Patients infected with the WNV who developed focal neurological injury were found to be comparable to patients who did not develop focal neurological injury both in terms of patient age and the number of medical conditions the patient had prior to infection. This is in contrast to WNV-infected patients who developed an encephalitis-like clinical course, or who died or were institutionalized after their hospitalization; such patients tended to be older and-in cases with a poor outcome-have more medical conditions prior to WNV infection. CONCLUSIONS: In our patient group, focal neurological injury caused by WNV infection was not related to advanced patient age or to the number of medical conditions the patient had prior to infection. Our findings bring into question commonly held views about the development of focal neurological injury caused by WNV infection, and they raise concerns about the management of future WNV epidemics and the testing and use of potential antiviral treatments against this infection.

Plasma cell pleocytosis in cerebrospinal fluid in patients with West Nile virus encephalitis.

We describe 4 patients with West Nile virus encephalitis who all displayed previously unreported plasma cell pleocytosis of the cerebrospinal fluid (CSF). Three patients recovered but had varying degrees of mild neurologic disability on discharge from the hospital, and 1 patient died. The finding of significant numbers of plasma cells in CSF may serve as a useful early diagnostic clue for West Nile virus encephalitis.

Persistence of West Nile Virus (WNV) IgM antibodies in cerebrospinal fluid from patients with CNS disease.

The Michigan Department of Community Health (MDCH) reported 644 laboratory positive human cases of West Nile Virus (WNV) in the 2002 outbreak in the US, of which 559 cases presented with either meningitis or encephalitis. The first line test utilized for diagnosis of WNV infection was the immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (MAC-ELISA). We continued testing for WNV even during winter months of the year 2002-2003 due to the awareness of other modes of WNV transmission (blood transfusion, organ transplantation, transplacental, breast milk, and occupational) as well as concern for people traveling to endemic areas. As a result of year-round testing for WNV infections during 2002-2003, we detected WNV IgM-specific antibodies in cerebrospinal fluid (CSF) specimens from three patients persisting for 110, 141, and 199 days post acute phase infection in patients with central nervous system (CNS) disease. This is a new observation and there is no published data on the persistence of WNV IgM antibodies in CSF specimens beyond 47 days. Thus, it is important to note that the presence of WNV IgM class antibodies may not always reflect acute phase infection with this virus.

The West Nile virus encephalitis outbreak in the United States (1999-2000): from Flushing, New York, to beyond its borders.

Viruses cause most forms of encephalitis. The two main types responsible for epidemic encephalitis are enteroviruses and arboviruses. The City of New York reports about 10 cases of encephalitis yearly. Establishing a diagnosis is often difficult. In August 1999, a cluster of five patients with fever, confusion, and weakness were admitted to a community hospital in Flushing, New York. Flaccid paralysis developed in four of the five patients, and they required ventilatory support. Three, less severe, cases presented later in the same month. An investigation was conducted by the NewYork City (NYC) and New York State (NYS) health departments and the national Centers for Disease Control and Prevention (CDC). The West Nile virus (WNV) was identified as the etiologic agent. WNV is an arthropod-borne flavivirus, with a geographic distribution in Africa, the Middle East, and southwestern Asia. It has also been isolated in Australia and sporadically in Europe but never in the Americas. The majority of people infected have no symptoms. Fever, severe myalgias, headache, conjunctivitis, lymphadenopathy, and a roseolar rash can occur. Rarely, encephalitis or meningitis is seen. The NYC outbreak resulted in the first cases of WNV infection in the Western Hemisphere and the first arboviral infection in NYC since yellow fever in the nineteenth century. The WNV is now a public health concern in the United States.

Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes.

Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic were conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, including 5 of 13 ducks (38%), 1 of 1 goose, 19 of 52 chickens (37%), 1 of 1 peahen, and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 26) from the urban Bucharest site was not significantly different (P = 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46). Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted for 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obtained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97-50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained from a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenetic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest the existence of two large lineages of viruses. Bootstrap analysis of the nucleotide alignment indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Algeria, and west, central, and east Africa, all of the European isolates, those from France and Romania, an Israeli isolate, and an isolate of KUN virus from Australia. The nucleotide sequence of RO97-50 was identical to the sequence of a WN virus isolate obtained from Cx. neavei mosquitoes from Senegal and Cx. univittatus mosquitoes from Kenya. The phylogenetic analyses were compatible with the introduction of virus into Romania by birds migrating from sub-Saharan Africa, to northern Africa, and into southern Europe.

West Nile virus: case report with MR imaging findings.

We report a case of serologically proved West Nile virus encephalitis. Striking bilateral hyperintensities were noted in the basal ganglia and thalami on T2-weighted and fluid-attenuated inversion recovery MR images, similar to previous accounts of the imaging findings in cases of Japanese encephalitis. Recognition of the MR imaging appearance of this entity is important because of the expanding epidemic.