DIAGNOSIS OF WEST NILE NEUROINVASIVE DISEASE IN HUMANS

West Nile virus (WNV) is arbovirus distributed all around the world. In humans, 80% of infection cases are asymptomatic. In 20% of infected people, a febrile self-limiting illness is reported. WNV has the potential for fatal neuroinvasive disease. In 1% of cases, the infection may result in neuroinvasive disease with permanent neurological consequences or death outcome. Neurological forms may vary presenting with encephalitis, meningitis, meningoencephalitis or acute flaccid paralysis. Outbreaks with neurological forms of WNV infection were recorded in different areas of Greece, Italy, Romania, Hungary and Serbia. During the period from 2013 to 2016, 114 samples of cerebrospinal fluid and 107 serum samples were taken from 114 patients suspected of WNV neuroinvasive disease (WNND). The presence of specific anti-WNV IgM and IgG antibodies in cerebrospinal fluid (CSF) and sera samples were tested by WNV IgM and IgG ELISA (Euroimmun, Germany). In addition, 48 samples of CSF or/and serum of people with suspected WNV infection were examined by commercial molecular tests real time RT-PCR (WNV Real-TM, Sacace biotechnologies, Italy). The IgM antibodies against WNV were present in 25.4% (29/114) of CSF samples, and in 31.8% (34/107) of serum samples tested from 114 patients suspected of WNND. The IgG antibodies against WNV were detected in 3.5% (4/114) of CSF samples, and in 11.2% (12/107) of serum samples. The WNV RNA was detected by real time RT-PCR test in 7 out of 48 (14.6%) 1* Corresponding author: ivana.hrnjakovic-cvjetkovic@mf.uns.ac.rs Arhiv veterinarske medicine, Vol. 11, No. 1, 79 89, 2018 Hrnjaković Cvetković I. ... et al.: Diagnosis of West Nile neuroinvasive disease... 80 CSF or/and serum samples. In this study, detection of IgM antibodies in CSF is more frequent than detection of WNV RNA in CSF or serum samples. WNV RNA detection in CSF is confirmatory diagnostic test but has limited utility in the diagnosis of WNV neuroinvasive disease due to low viremia level at the time of clinical presentation of the disease. The limitations in the use of ELISA IgM test are linked to cross reactivity among flaviviruses and long persistence of IgM antibodies in the serum and CSF.


Abstract
West Nile virus (WNV) is arbovirus distributed all around the world. In humans, 80% of infection cases are asymptomatic. In 20% of infected people, a febrile self-limiting illness is reported. WNV has the potential for fatal neuroinvasive disease. In 1% of cases, the infection may result in neuroinvasive disease with permanent neurological consequences or death outcome. Neurological forms may vary presenting with encephalitis, meningitis, meningoencephalitis or acute flaccid paralysis. Outbreaks with neurological forms of WNV infection were recorded in different areas of Greece, Italy, Romania, Hungary and Serbia. During the period from 2013 to 2016, 114 samples of cerebrospinal fluid and 107 serum samples were taken from 114 patients suspected of WNV neuroinvasive disease (WNND). The presence of specific anti-WNV IgM and IgG antibodies in cerebrospinal fluid (CSF) and sera samples were tested by WNV IgM and IgG ELISA (Euroimmun, Germany). In addition, 48 samples of CSF or/and serum of people with suspected WNV infection were examined by commercial molecular tests -real time RT-PCR (WNV Real-TM, Sacace biotechnologies, Italy). The IgM antibodies against WNV were present in 25.4% (29/114) of CSF samples, and in 31.8% (34/107) of serum samples tested from 114 patients suspected of WNND. The IgG antibodies against WNV were detected in 3.5% (4/114) of CSF samples, and in 11.2% (12/

INTRODUCTION
The West Nile virus (WNV) is an arbovirus (arthropod-borne virus), the member of the family Flaviviridae, genus Flavivirus. WNV is widely distributed across Africa, Europe, Asia, Australia, and America among mosquitoes and vertebrates including humans and horses. Birds, as main reservoir, and mosquitoes (primarily Culex species) as vectors play a central role in transmission cycle of WNV in nature. Humans, equines and other animals are generally considered the "dead end" hosts because they have low viremia levels, insufficient to sustain transmission from vertebrate to feeding mosquitoes. Most WNV infections in humans remain asymptomatic. In one of four cases of WNV infection, WNV fever is developed (Petersen et al., 2013). In 1% of cases, infection may result in West Nile neuroinvasive diseases (WNND) such as meningitis, encephalitis, poliomyelitis-as a condition with flaccid paralysis, with permanent neurological consequences or lethal outcome. Estimated percentages of WNND are as following: meningitis 35 -40%, encephalitis 55-60% and poliomyelitis 5-10% (Sejvar et al., 2007). Clinically, WNV meningitis is indistinguishable from aseptic meningitis caused by other neurotropic viruses.
WNV encephalitis is usually associated with seizures, mental status changes, focal neurologic deficits or movement disorders.
Until 1996, West Nile virus was not considered significant human pathogen because most reported infections were mild febrile disease in rural populations and a few cases of WNND were reported. WNV acquired neurotropism during the second half of the 20th century and two events occurred: outbreaks of WNND in Europe and introduction of WNV in The United States. In 1996, first explosive epidemic of WNND occurred in Europe. It was an epidemic in Romania in Bucharest, with 393 patients with chronologically confirmed or probable WNV infection, of whom 352 had acute central-nervous-system infections. Seventeen patients (5%) older than 50 years died (Tsai et al., 1998). WNV had never been detected in the Western Hemisphere of America until August 1999, when the first cases of WNV infection appeared in New York City. In the outbreak in New York City, 59 people were infected and 63% of them had clinical signs of encephalitis, and seven patients died (12 %) (Nash et al., 2001). In 1999, explosive epidemic of WNV occurred in Volgograd City, Russia, on the bank of Volga River, where WNV infection has never been detected before. 826 patients were admitted to hospitals with invasive forms of the infection (acute aseptic encephalitis, meningitis) or fever. Case fatality rate was high. Out of total 84 cases of acute aseptic meningoencephalitis, 40 (48%) were fatal (Platonov et al., 2001). At the beginning of 21 st century, outbreaks of WNND continued to occur in Europe. Outbreaks of WNND were recorded in different areas of Greece, Italy, Romania, Hungary, Serbia (ECDC, 2017). Sporadic cases were recorded in Spain, Portugal, Austria and Croatia (Vilibić-Čavlek et al., 2013). In Asia and Africa where WNV has been endemic for many years, WNV encephalitis occurs in children and has benign course and good prognosis. In Europe and USA, where WNV emerged only recently, WNV encephalitis is a severe disease and many of the survivors are left with severe neurologic deficits and cognitive dysfunctions. During the 2015 season, testing for WNV infection was performed on 21 patients with suspected WNND. In total, 21 samples of CSF and 21 serum samples were tested applying serological and molecular tests. In CSF samples testing WNV IgM ELISA was found to be positive for 4 patients out of 21 (19.0%). All these patients also reacted positive for WNV IgM antibodies in serum samples. The WNV IgM ELISA was positive for 38.1% (8/21) serum samples of the patients with suspected WNND. The WNV IgG ELISA were found negative for all CSF samples, however, two serum samples (2/21) were tested positive. The WNV RNA was detected in sample of CSF of only one patient.

MATERIAL AND METHODS
During the season 2016, in total 21samples of CSF and 20 serum samples were collected from 21 patients with suspected WNND. The WNV IgM antibodies were detected in CSF of 6 patients (28.6%). Out of 20 patients' serum samples tested with WNV IgM ELISA, 7 (35%) were found to be IgM antibody positive. None of the samples of CSF tested positive for WNV IgG antibodies, but those antibodies were detected in 1 out of 20 (5.0%) tested serum samples.

DISCUSSION
Various methods are available for detecting viable WNV, WNV antigen or WNV RNA in human diagnostic samples. The viable WNV can be detected by isolation on cell culture (e.g. Vero cell) or in the brain tissue samples from the laboratory infected suckling mice. Disadvantages of virus isolation are that the method is time-consuming and it requires the use of cell cultures and handling with live, infectious virus and BSL-3 (Biosafety level 3) facility. For virus genome detection and amplification molecular techniques such as real time reverse transcription polymerase chain reactions (real time RT-PCR) and nucleic acid sequence based amplification (NASBA) methods are applied. The WNV genome RNA can be detected in human CSF, serum and other tissues. Molecular methods have great sensitivity but limited utility in diagnosing human WNV neuroinvasive disease, which is due to the low level of viremia present in most cases at the time of clinical presentation. Isolation of the virus has similarly limited significance in the diagnosis of WNND. Molecular methods and virus isolation are useful only at the beginning of the infection. The best choice at the later stages of illness are serologic methods. The serological screening method for laboratory diagnosis of human WNND is the ELISA IgM antibody assay. ELISA is the most widely used serological method because it is sensitive and easy to perform in routine practice in a diagnostic virology laboratories. In this study, IgM antibodies against WNV were detected in 31.8% of tested serum samples from suspected WNND patients. According to the results of many studies, IgM antibodies against WNV can persist in serum for months (Papa et al., 2015). Murray detected immunoglobulin M antibodies up to 8 years after infection with WNV in a study encompassing 163 participants (Murray et al., 2013). In the study of Roehrig at al. (2003), 60% of patients with laboratory -confirmed WNV encephalitis had WNV IgM antibodies approximately 500 days after onset of the disease. Because of that, detection of WNV IgM antibodies in blood sera should not be considered a marker of acute West Nile virus infection and determination of avidity of WNV IgG is necessary to ensure the accurate and reliable diagnosis (Hrnjaković et al., 2017a). The presence of IgM antibodies in CSF establishes the diagnosis of WNND since IgM antibody does not cross the blood-brain barrier. The IgM antibodies appear in CSF within 8 days of the beginning of disease in at least 90% of patients with encephalitis or meningitis (Petersen et al., 2013). co-circulate in humans in Vojvodina and because of that, misinterpretation of serological results is possible. The issue of cross-reactivity may be overcome by performing the plaque reduction neutralization test (PRNT). PRNT is the gold standard for the serological diagnosis of flaviviral infections. Disadvantages of this test are that it requires facilities that are available only in specialized research institutions (BSL-3 laboratories). In this study, PRNT was not used as confirmatory assay and it was the limitation of this study.
In 2010, WNV RNA genome was detected in mosquito Culex pipiens collected in the city of Novi Sad applying real time RT PCR (Petrić et al., 2012). Circulation of WNV in wild birds in Vojvodina was also detected by using molecular methods and serological tests. The WNV isolate from wild birds belonged to the lineage 2 and clustered with WNV strains isolated in Hungary 2004, Greece 2010 and Italy 2012 (Petrović et al., 2013). Circulation of WNV in pigs, boars and roe deer was also detected in Serbia using molecular methods and serological tests (Escribano-Romero et al., 2015). The first human cases of WNND on the territory of South Bačka district in Vojvodina Province of Serbia were registered in 2012. It was the first epidemic in South Bačka district with 32 patients in which neuroinvasive form of WNV infection was diagnosed. All patients were hospitalized at the Clinic for Infectious Disease of the Clinical Center of Vojvodina in Novi Sad. The largest number of patients has recovered completely. In 6.25% of patients, sequelaes were developed. Lethal outcome was registered in 3.13% cases (Sević et al, 2015). Since 2013, human WNV infections on the territory of South Bačka district have been diagnosed every year from July to October at the Institute of Public Health of Vojvodina. Complete genome sequences of the WNV isolate from a human patient with neuroinvasive disease, resident of Novi Sad, were described (Jovanović Galović et al., 2017). It was the first whole genome sequencing of the isolate from human sample in Serbia. The isolate belonged to lineage 2 and similarities with strains circulating in Greece, Hungary, Austria, and Italy were demonstrated (Jovanović Galović et al., 2017).

CONCLUSION
WNV has the ability to cause neuroinvasive disease (WNND) that can result in debilitating morbidities and long-term sequelaes. WNV RNA detection in CSF or serum is confirmatory diagnostic test but has limited utility in the diagnosis of WNV neuroinvasive disease due to low viremia level at the time of clinical presentation of disease. Diagnosis of WNND usually relies on detection of IgM antibodies in serum or cerebrospinal fluid. Special emphasis in diagnosing of WNV infection should be put on CSF analyses (IgM ELISA and RT-PCR).

ACKNOWLEDGMENT
This work was financed by the Ministry of Education, Science and Technological Development, Republic of Serbia -projects TR31084 and III43007