Health & Management / Managing for West Nile Virus Infection / List of hyperlinked Techniques & Protocols:
Ý ß  Human Surveillance for West Nile Virus:

Introduction and General Information
The main public health aim of surveillance for WN virus or other zoonotic arboviruses is to prevent human infection and disease. For this reason, surveillance of human cases is not generally appropriate as the primary/sole method of surveillance. However, it may be the most appropriate method in limited circumstances: where arbovirus activity is considered very unlikely or in regions where the resources required for bird-based and/or mosquito-based surveillance are not available. Human surveillance may also be used in conjunction with other forms of surveillance such as bird-based and mosquito-based surveillance. (D67, D147, J115.13.w2, P32.1.w21)

The goals of human surveillance in the USA are to: "1) assess the local, state and national public health impact of WNV disease and monitor national trends; 2) demonstrate the need for public health intervention programs; 3) allocate resources; 4) identify risk factors for infection and determine high-risk populations; 5) identify geographic areas in need of targeted interventions; and 6) identify geographical areas in which it may be appropriate to conduct analytic studies of important public health issues." (D147)

Timing and level of human surveillance:

Minimal human surveillance includes enhanced passive surveillance [see below] for individuals hospitalised with encephalitis of unknown origin and for patients with IgM antibodies to either WN virus or St Louis encephalitis virus (SLEV) in tests conducted in diagnostic or reference laboratories. (D147)

The appropriate level of surveillance will vary across the USA and with season. (D67):

  • The transmission season of WN virus is based on the activity of the insect vectors, thus the period of surveillance varies depending on the vector season in a given geographic area; in some areas a long period of surveillance is required during the year. (D147)
  • Northeastern and Midwestern USA: Enhanced passive surveillance for human cases, as well as active ecological surveillance,  should begin in early spring and continue through the fall (autumn) until mosquito activity ceases because of cold weather. Surveillance in urban and suburban areas should be emphasized. (D147)
    • During 2001-2002 onset of human illness onset in the northeastern states occurred as early as early July and as late as mid-November; in the same years cases in birds occurred as early as the first week of April and as late as the second week of December. (D147)
    • In New York State for 2001 it was suggested that: "Enhanced passive case surveillance is recommended for all counties from June through October. Active case surveillance is recommended for counties when there is evidence of WNV activity in their jurisdiction in 2001." (D72) 
  • Southern USA: Enhanced passive surveillance for human cases, as well as active ecological surveillance, should be conducted year round in these areas. (D67, D147)
    • N.B. in the Southern states, WN virus circulates throughout the year, with activity detected from January to December. In 2001-2004, onset of human illness was reported from as early as April to as late as mid-December; equine and avian infections were reported in all months of the year in these geographical areas. (D147, P39.3.w1, J84.10.w5, J84.11.w4)
  • Western USA:  Enhanced passive surveillance for human cases, as well as ecological surveillance, should be encouraged beginning in early spring and continuing through the fall until mosquito activity ceases due to cold weather. (D147)
    • In 2002, WNV activity was first reported among humans and animals in Rocky Mountain states and among animals in Pacific coast states. These events occurred relatively late in the year (mid-August). (D147)
  • Western Hemisphere outside the USA: Surveillance in other countries of the Western Hemisphere should be encouraged. (D67)
    • In Canada, in 2002 a WNV epidemic occurred in Ontario and Quebec provinces and an equine/avian epizootic occurred extending from the maritime provinces to Saskatchewan. By 2005, WV virus had been detected in Alberta, Manitoba, New Brunswick, Nova Scotia, Ontario, Quebec and Saskatchewan. (B526.21.w21, D147)
    • Development of surveillance systems capable of detecting WNV activity should be encouraged in the Caribbean and Central and South America. WNV surveillance should be integrated with dengue surveillance in these areas, and with yellow fever surveillance in areas where urban or peri-urban transmission of this virus occurs. (D147)
      • Wild bird and sentinel chicken surveillance has been recommended as part of surveillance for WNV in the Caribbean and in South America (Brazil). (J408.40.w1, W705.May08.w1)
      • By 2005, WV virus had been detected in equines and/or birds in Mexico, the Caribbean (the Bahamas, the Cayman Islands, Cuba, the Dominican Republic, Guadeloupe, Jamaica, Puerto Rico, Trinidad), Central America (Belize, Guatemala, El Salvador), and South America (Colombia); in 2006 it was detected in Venezuela and caused fatal illness in equines in Argentina; samples taken from birds in Argentina were first positive in 2005. (B526.21.w21, J84.12.w3, J84.13.w1, J84.14.w1, J270.45.w1, J491.19.w1)
  • Southern Africa: transmission of WNV increases in the early months of the year, following heavy rainfall in spring and summer. (J84.11.w4)

Record keeping:

Official data recording sheets should be used in order to standardise information and to facilitate entry of information into databases and comparison of data from different areas or collected by different personnel. Examples of data recording sheets for human viral encephalitis / meningitis cases and for human exposure to pesticides are given in the New York State West Nile Virus Response Plan - Guidance Document (D72) Appendix D: Surveillance Report Forms.

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Enhanced Passive Surveillance
Recognition of illness as a potential case of WNV infection is more likely to occur, and to occur rapidly, if health care professionals are aware that the disease occurs or may emerge in their local area and have been informed about the clinical signs and symptoms which may be seen in cases of WNV infection.

Enhanced passive surveillance is defined as passive surveillance enhanced by general alerts to key health personnel such as primary care providers, infectious disease physicians, neurologists, hospital infection control personnel, and diagnostic laboratories. (D67, D147)

"In the absence of known WN virus activity in an area, enhanced passive surveillance for hospitalized cases of encephalitis of unknown etiology**, and for patients who test positive for antibodies to either WN or SLE virus in commercial or government laboratories, should be employed. A high index of suspicion for arboviral encephalitis should be encouraged. When in doubt, appropriate clinical specimens should be submitted to CDC or another laboratory capable of reliably diagnosing arboviral infections. It is important that paired acute- and convalescent-phase serum samples be submitted to ensure accurate interpretation of serologic results." (D67)

"(** While human infections with neurotropic arboviruses are usually clinically inapparent, most clinically apparent infections are febrile illnesses associated with a wide range of neurologic manifestations. These range from mild aseptic meningitis to fulminant and fatal encephalitis. Signs and symptoms may include fever, headache, stiff neck, confusion or other mental status changes, nausea, vomiting, meningismus, cranial nerve abnormalities, paresis or paralysis, sensory deficits, altered reflexes, abnormal movements, convulsions, and coma of varying severity. Arboviral meningitis or encephalitis cannot be clinically distinguished from other central nervous system infections. Notably, of the cases of WN viral encephalitis diagnosed in NYC in 1999, approximately 40% of laboratory positive cases had severe muscle weakness; of these, 10 % developed flaccid paralysis with electromyographic findings consistent with an axonal neuropathy. This profound muscle weakness initially raised the possibility of botulism or Guillain-Barré syndrome.)"

Clinical syndromes suitable for surveillance:

In the USA in general, monitoring of encephalitis cases is the highest priority. Monitoring of milder illnesses such as aseptic meningitis, Guillain-Barré syndrome, acute flaccid paralysis, brachial plexopathy and fever with rash illness is resource-dependent and are considered to be of lower priority. (D67, D147)

  • Following a number of reported cases of acute flaccid paralysis in human patients during 2002 enhanced surveillance for this condition associated with WNV infection, even in the absence of fever or other neurological signs, has been suggested. (J84.9.w13, P39.4.w3, P48.1.w7)

The following Recommended Criteria for Suspected Cases of West Nile Virus Infection adopted in New York State has been taken directly from the New York State West Nile Virus Response Plan - Guidance Document (D72): [Text copied directly]

Recommended Criteria for Suspected Cases of West Nile Virus Infection

· Any adult or pediatric patient with suspected viral encephalitis (criteria a, b, and c below) with or without associated muscle weakness (criteria d):

a) Fever > 38 ° C or 100 ° F, and 

b) Altered mental status (altered level of consciousness, agitation, lethargy) and/or other evidence of cortical involvement (e.g., focal neurologic findings, seizures), and

c) Cerebrospinal fluid (CSF) pleocytosis with predominant lymphocytes and/or elevated protein and a negative gram stain and culture, with or without

d) Muscle weakness (especially flaccid paralysis) confirmed by neurologic exam or electromyography (EMG).

· Any adult or pediatric (aged 2 years or older) patient with suspected viral (aseptic) meningitis (criteria a, b, and c below):

a) Fever > 38 ° C or 100 ° F, and

b) Headache, stiff neck and/or other meningeal signs, and

c) CSF pleocytosis with predominant lymphocytes and/or elevated protein and a negative gram stain and culture.

· Any adult or pediatric patient with Guillain-Barré syndrome (particularly individuals with atypical features such as fever, altered mental status and a CSF pleocytosis).
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Active Surveillance
Active surveillance should be considered in areas with known WN virus activity. 

It has been recommended that, in general, one or both of the following approaches to active surveillance should be taken: 

1) Identify physicians in appropriate specialties (such as. infectious diseases, neurology, and intensive care medicine) and hospital infection control personnel and contact them on a regular basis to inquire about patients with potential arboviral infections; 

2) Implement laboratory-based surveillance for specimens of cerebrospinal fluid (CSF) meeting sensitive but non-specific criteria for arboviral infections (e.g. mild to moderate pleocytosis together with negative tests for the presence of non-arboviral agents such as bacteria, fungi, herpesviruses, and enteroviruses) and test these for evidence of WN virus infection.

(D67, D147)

An additional benefit has been recognised if special surveillance projects are integrated into active surveillance:

"Certain special projects may be used to enhance arboviral disease surveillance. Such projects include the Infectious Diseases Society of America Emerging Infections Network (IDSA EIN), Emergency Department Sentinel Network for Emerging Infections (EMERGEncy ID NET), Emerging Infections Programs (EIP) Unexplained Deaths and Critical Illnesses Surveillance, and the Global Emerging Infections Sentinel Network of the International Society of Travel Medicine (GeoSentinel). In some areas, syndromic surveillance systems are in place or being developed. The "piggy-backing" of surveillance for WN meningoencephalitis and milder clinical forms of WN fever, such as fever with rash or lymphadenopathy, onto existing syndromic surveillance systems, including those involving large health maintenance organizations, should be encouraged. Real-time computerized syndromic surveillance in emergency departments, and special surveillance projects to identify WN virus disease in pediatric populations, may be useful." (D67)

The following example of an Active Case Surveillance program has been taken directly from the New York State West Nile Virus Response Plan - Guidance Document (D72): [Text copied directly]

Active Case Surveillance (One or more of the following options should be conducted by counties if current WNV activity is detected in their jurisdiction.)

1. Active surveillance conducted at sentinel acute care hospitals.

a) Weekly contact with key medical staff (e.g., infectious disease, neurology or intensive care subspecialists) and/or the infection control practitioner (ICP) regarding hospitalized case-patients with suspected viral encephalitis or viral meningitis.

b) LHUs [Local Health Units] should ensure that appropriate laboratory specimens are obtained on all suspected cases and tested for WNV.

2. Laboratory-based surveillance conducted at sentinel hospitals. This system will provide back up to ensure that suspected viral encephalitis and viral meningitis cases that are not reported by clinicians are tested for arthropod-borne diseases.

a) LHUs should ask laboratory staff to store all spinal fluid samples that have parameters suggestive of a viral etiology (e.g. increased protein, pleocytosis and negative gram stain and culture).

b) Samples should be collected weekly and submitted to NYSDOH [New York State Department of Health] or NYCDOH [New York City Department of Health] and screened for arboviruses by IgM-capture enzyme linked immunoassays (ELISA) and/or reverse transcriptase polymerase chain reaction (RT-PCR) testing.

3. Retrospective surveillance conducted for patients discharged with a diagnosis of encephalitis/aseptic meningitis of unknown etiology.

a) LHUs should ask sentinel hospitals to search databases for patients discharged with select ICD-9 codes.

b) If additional patients are identified, hospital laboratory directors should be contacted to determine if serum or CSF is available on identified suspect patients.

  • If specimens are available, arrangements should be made for testing at NYSDOH or NYCDOH.
  • If specimens are not available, patients should be contacted to obtain convalescent sera.

Since the discovery in 2002 that WNV infection may be transmitted through the donation of blood, blood products and solid organs, it has been recommended that :

  • When an individual is diagnosed with WNV infection with an onset within four weeks after receiving a blood transfusion or organ(s), the case should be reported, via local public health authorities, to CDC. For the purpose of later studies serum or tissue samples from such patients should be retained. (W380.Dec02.wnv1)
  • When an individual is diagnosed with WNV infection, the date of onset having been within two weeks of donation of blood or organ(s), the case should be reported via state and local public health departments to CDC.  (W380.Dec02.wnv1)
  • For asymptomatic potential donors with a previous diagnosis of WNV infection or illness, the time since the diagnosis of WNV infection and the resolution of symptoms should be considered in deciding whether donation should be accepted or deferred (standard blood collection procedures already include deferral of a donor who is not in good health or is not feeling well at the time of intended donation). (W380.Dec02.wnv1)
    • For donors definitively diagnosed with WNV infection donation it is recommended that should be deferred "until 14 days after the condition is considered to be resolved and at least 28 days from onset of symptoms or diagnosis whichever is the later date." However "in the absence of current or recent symptoms, an IgM positive antibody test result alone should not be grounds for deferral."  (W380.Dec02.wnv1)
  • Blood collection centres should actively encourage donors to report illness which starts post-donation and which could be associated with WNV infection.  (W380.Dec02.wnv1)
  • Laboratory screening tests may be required for routine screening to detect acute donor WNV infections.  (W380.Dec02.wnv1)
  • If WNV infection or illness is diagnosed in a donor, in-date components from current, prior and subsequent collections should be quarantined and retrieved promptly for products collected back to 14 days prior to the onset of illness and for 28 says after the onset of illness.  (W380.Dec02.wnv1)
  • If a blood recipient develops post-donational illness potentially associated with WNV infection, within 28 days of receiving a donation, all donations received during this period should be considered "suspect". Product quarantine and retrieval should be applied promptly to in-date components which were collected "in the period from 28 days prior to the suspect donation to 28 days after the suspect donation".  (W380.Dec02.wnv1)
  • If a medical diagnosis of WNV infection is made for a donor and that information is received by a blood establishment, it is recommended that the establishment considers "tracing records and notifying transfusion services so that they, in turn, may notify treating physicians of prior recipients of blood and blood components collected from that donor." Additionally, "If an epidemiological infection suggests that a specific donor is the likely source of transmission of WNV to a transfusion recipient, we recommend that blood establishments consider tracing records and notifying transfusion services so that they, in turn, may notify treating physicians of prior recipients of blood and blood components collected from that donor." In such situations "relevant units" are considered to be those dating 28 days prior to the date of suspect donation to 28 days after the suspect donation.  (W380.Dec02.wnv1)
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The Role of Pathology
Studies which may be carried out on tissues taken at autopsy, or by brain biopsy, include gross pathological examination, histopathology, RT-PCR, virus isolation and immunohistochemistry. (D147)

Pathological examination, particularly histopathology and immunohistochemistry, was important in the initial diagnosis of West Nile Virus Infection in New York in 1999. (J84.6.w4)

  • "At autopsy, particular attention should be paid to complete neurological examination, including the spinal cord and nerve roots. If autopsy is limited for any reason, the brainstem, particularly the medulla, should be sampled. Gross and histological examinations of the pancreas, liver and heart are also important." (J123.31.w1)

It is important to ensure that the correct samples are taken and that the correct storage conditions are used. Information provided by the appropriate laboratories, to which samples are to be sent, should be consulted prior to sample collection. 

  • Necropsy samples which should be submitted (formalin-fixed in the case of tissues other than blood) for routine histological examination and special studies include:
    • Pancreas
    • Liver
    • Heart
    • Lung
    • Spleen
    • Kidney
    • Brain: including several sections from the brainstem, particularly the medulla
    • Spinal cord: including the dorsal and ventral roots.
    • Blood
  • Necropsy samples which should be submitted on dry ice (not formalin-fixed) include:

    (J123.31.w1, J133.951.w20)

CDC-recommended samples to be taken at autopsy include brain tissue, particularly samples of the cortex, midbrain and brainstem, heart blood or venous blood, and buffy coat. Tissue samples should be divided and preserved in two ways: half the sample fixed in formalin and the other half frozen at -70°C. These samples should be submitted to CDC or to another appropriate specialised laboratory. (D147)

It is important to recognise that pathological findings, including histopathology, do not allow a definitive aetiological diagnosis; they cannot definitely distinguish between arboviral encephalitides. WNV infection does not cause any characteristic viral inclusions or other distinctive pathology. (B253.6.w6, B245.29.w29, P48.4.w11)

  • Immunohistochemical (IHC) staining, RT-PCR and virus isolation from tissues, if positive, may be used to confirm cases of WNV infection. However, negative results do not necessarily rule out WNV infection.  (J84.6.w3, J84.6.w4, D72)
  • IHC can now be carried out using monoclonal antibodies that are highly specific for WN virus rather than polyclonal antibodies that do not distinguish between WN and related viruses such as St Louis encephalitis virus. (P48.4.w11)
  • At least SIX tissue samples from the brain stem and spinal cord should be submitted to allow for confidence in excluding WNV infection as the cause if samples are negative. (P48.4.w8)
  • It has been noted that some seronegative individuals, particularly immunosuppressed and diabetic patients, are IHC positive, while some seropositive individuals are IHC-negative. (P48.4.w11)
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Laboratory Diagnosis
It is not possible to distinguish a patient with WNV encephalitis from one with viral encephalitis due to another virus solely on clinical grounds (B245.29.w29, J84.7.w14). Additionally, many cases of WNV infection in humans are clinically inapparent or cause only a mild, non-specific illness. 

For further information see:  

Clinical pathology findings in WNV infection are generally not useful for diagnosis; findings are non-specific.

For further information see: 

Definitive diagnosis requires laboratory testing using specific reagents; adequate laboratory support is vital for the success of  active surveillance. (D67)

Samples for testing:

Cerebrospinal fluid: this may be tested for WN virus-specific IgM antibody, which is commonly present in the CSF on the day of onset of illness and can be detected using antibody-capture ELISA. Additionally it may rarely be possible to isolate virus from the CSF and in up to 60% of cases the presence of WN virus may be detected in acute-phase samples using RT-PCR. (D147)

Serum: paired serum samples, one acute-phase (collected 0-8 days after onset of illness) and one convalescent-phase (collected 14-21 days after the acute specimen)  are useful for demonstration of seroconversion to WN virus or other arboviruses by ELISA or neutralization tests. Evidence of recent WN virus infection may be provided by tests on a single acute-phase serum specimen may provide evidence of a recent WNV infection, but recent infection cannot be ruled out if the acute-phase sample is negative, therefore collection of paired samples is important. It must be remembered also that development of antibodies may be delayed or absent in immunocompromised individuals. (D147)

Tissues: see section above on "The Role of Pathology".

In 2003 for human serum or CSF first choice tests ELISA and PRNT, other possibilities TaqMan, NASBA and virus isolation; TaqMan may detect in 57% of acute CSF but less than 10% of serum samples. (P39.4.w9)

For humans, the following advice, quoted directly from New York State West Nile Virus Response Plan - Guidance Document [2001] (D72) for the most appropriate specimens to test for viral encephalitides, has been provided through the Emerging Infections Encephalitis Project, funded by the Centers for Disease Control and Prevention (CDC): [Text copied directly]

This project includes testing CSF specimens on patients with encephalitis of unknown etiology for 13 different viruses.

Appropriate specimens for testing include:

  1. CSF - can be tested by IgM capture ELISA and RT-PCR

    If there is insufficient quantity of CSF for both ELISA and RT-PCR, testing priority should be determined by the ordering physician.

    1. Capture ELISA is more sensitive than RT-PCR for WNV testing and should be considered when there is stronger suspicion of WNV than other etiologic agents.
    2. RT-PCR is less sensitive for WNV, but provides testing for 13 different viruses. This test should be considered if suspicion of the etiologic agent is stronger than for viruses other than WNV.
  2. Serum - Acute and convalescent sera can be tested by IgM Capture and IgG ELISA testing, with indeterminate and positive specimens confirmed by PRNT.
  3. Brain Tissue - Can be tested by RT-PCR and viral culture.

It was further noted that if acute specimens (obtained within eight days of the onset of illness) are negative by RT-PCR  (reverse transcriptase polymerase chain reaction) or negative or indeterminate by ELISA  testing, a follow-up (convalescent) serum collected at least 3 weeks after the acute specimen should be obtained. (D72)

Details of samples required, shipping and handling conditions should be obtained prior to sending specimens to a laboratory for testing. Details of requirements for human samples sent to CDC are provided in: West Nile Virus - Detection and Identification Techniques (Viral Reports) - Specimen Sampling & Shipping

Interpretation of serological results:

It is important to recognise that  accurate interpretation of serologic findings requires knowledge of the specimen submitted. For human specimens, it is important that the following data accompany specimens submitted for serology before testing can proceed or the results can be properly interpreted and reported: 1) the date of the onset of symptoms, when known; 2) date of sample collection; 3) any unusual immunological status of patient (e.g. immunosuppression); 4) current address of the patient and any travel history in flavivirus-endemic areas; 5) any history of prior vaccination against flavivirus disease (e.g. yellow fever, Japanese encephalitis, or Central European encephalitis); and 6) a brief clinical summary including the suspected diagnosis (e.g. encephalitis, aseptic meningitis). (D67, D147)

  • IgM may be detectable in serum by ELISA more than a year after acute infection, therefore although a single high serum IgM titre is suggestive of infection, the presence of IgM in serum alone is not sufficient for confirmation of WNV infection as the cause of illness. (J84.9.w3)
  • N.B. "Because the ELISA can cross-react between flaviviruses (e.g., SLE, dengue, yellow fever, WN), it should be viewed as a screening test only. Initial serologically positive samples should be confirmed by neutralization test. Specimens submitted for arboviral serology should also be tested against other arboviruses known to be active or be present in the given area (e.g., test against SLE, WN and EEE viruses in Florida)."(D67)
  • It may be epidemiologically important to use cross-neutralization tests with an appropriate battery of closely related viruses. (D147)

In immunosuppressed patients:

  • Some immunosuppressed or diabetic patients may be seronegative but are later confirmed positive by IHC. (P48.4.w11)
  • In immunosuppressed individuals development of antibodies may be delayed or may not occur. (D147)

Appropriate serological tests:

The CDC-defined IgM and IgG ELISA should be the front-line tests for serum and CSF; no commercial kit is yet available for human serologic diagnosis of WNV infection. These ELISA tests are the most sensitive screening assays available. The HI test may also be used to screen samples for flavivirus antibodies however laboratories performing HI assays need be aware that mouse brain source antigen (available from CDC) must be used in HI test as the recombinant WN virus antigens produced to date are not useful in this test. (D67, D147)

Testing for the presence of virus:

  • Virus is most likely to be detected (rarely isolated, more commonly detected by RT-PCR on the first days of illness in serum or more often in acute-phase samples of CSF. (B244.w1, J129.42.w1, D147)
    • In immunosuppressed individuals virus or viral RNA may be detected in the serum or CSF for longer periods than normal. (D147)
  • Attempts at virus isolation should be performed in known susceptible mammalian or mosquito cell lines. (D147)
    • Cells of mosquito origin may not show a cytopathic effect; immunofluorescence should be used for screening. (D147)
    • If virus is isolated its identity should be confirmed using indirect immunofluorescence assay (IFA) with virus-specific monoclonal antibodies, detection of WN viral nucleic acid or virus neutralization.(D147)
    • Detection of fourfold or greater titre differences between paired acute- and convalescent-phase serum samples  in virus neutralization assays may also be used to differentiate between viruses. (D147)
  • Virus may be detected in tissues using detection of viral antigen with immunohistochemistry on fixed tissues, using virus-specific monoclonal antibodies. (D147)

Further information on virus identification is provided in:

CDC definitions for "confirmed" and "probable" cases of arboviral encephalitis or meningitis, and of West Nile Fever, are provided in: West Nile Virus Disease (Viral Disease) - Diagnostic Criteria

  • The following "Case definition for human WNV infection" is quoted directly from the CDC Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention and Control (D67) Appendix B [2001]: [Text copied directly]
CASE DEFINITION FOR HUMAN WEST NILE VIRUS INFECTION:

Confirmed case: A confirmed case of WN encephalitis is defined as a febrile illness associated with neurologic manifestations ranging from headache to aseptic meningitis or encephalitis, plus at least one of the following:

  • Isolation of WN virus from, or demonstration of WN viral antigen or genomic sequences in, tissue, blood, CSF, or other body fluid;1
  • Demonstration of IgM antibody to WN virus in CSF by IgM-capture EIA;2-4
  • A >4-fold serial change in plaque-reduction neutralizing (PRNT) antibody titer to WN virus in paired, appropriately timed serum or CSF samples; 2, 3, 5
  • Demonstration of both WN virus-specific IgM (by EIA) and IgG (screened by EIA or HI and confirmed by PRNT) antibody in a single serum specimen.2, 4-6

Probable case: A probable case is defined as a compatible illness (as above) that does not meet any of the above laboratory criteria, plus at least one of the following:

  • Demonstration of serum IgM antibody against WN virus (by EIA);3, 4
  • Demonstration of an elevated titer of WN virus-specific IgG antibody in convalescent-phase serum (screened by EIA or HI and confirmed by PRNT).3-6

Non-Case: A non-case is defined as an illness that does not meet any of the above laboratory criteria, plus:

  • A negative test for IgM antibody to WN virus (by EIA) in serum or CSF collected 8-21days after onset of illness;3, 4 

and/or

  • A negative test for IgG antibody to WN virus (by EIA, HI, or PRNT) in serum collected >22 days after onset of illness.3-5

Notes:

1. Although tests of tissues or fluids by PCR, antigen detection, or virus isolation can be used to confirm WN encephalitis cases, they cannot be used to rule-out cases because the negative predictive values of these test methods in this disease are unknown.

2. See the above discussion concerning serologic cross-reactivity between WN and SLE viruses. Prior to a more definitive demonstration of WN virus as the cause of an epidemic or a sporadic viral encephalitis case, this serologic criterion should be used to classify human cases as probable only, pending definitive identification of the circulating flavivirus type (see discussion above).

3. Although the antibody response to human infection with WN virus has not been thoroughly or systematically studied, the following are reasonable assumptions, based on extensive experience with other flaviviruses, or preliminary conclusions based on empirical observations made during the 1999 and 2000 New York epidemic of WN encephalitis:

  • IgM antibody in serum: By the eighth day of illness, a large majority of infected persons will have detectable serum IgM antibody to WN virus; in most cases it will be detectable for at least 1-2 months after illness onset; in some cases it will reach undetectable levels prior to 1 month after illness onset; in some cases it will be detectable for 12 months or longer.
  • IgG antibody in serum: By 3 weeks post-infection (and often earlier), virtually all infected persons should demonstrate long-lived serum IgG antibody to WN virus by EIA, HI, and PRNT.
  • IgM antibody in CSF: In WN encephalitis cases, IgM antibody will virtually always be detectable in CSF by the eighth day of illness and sometimes as early as the day of onset; the duration of WN virus-specific IgM antibody in CSF has not been studied.
  • IgG antibody in CSF: IgG antibody in CSF often does not reach detectable levels and thus is a relatively insensitive indicator of infection.
  • Specificity of IgM-capture EIA: Serum (and CSF) from recently WN virus-infected persons will cross-react in IgM-capture EIAs when either WN virus or any closely related flavivirus is used as antigen. The homologous (infecting) serotype should be determined by cross-neutralization.
  • Specificity of IgG EIA: WN viral IgG antibody detectable by EIA (or HI) is broadly cross-reactive with all closely related flaviviruses, and this usually cannot be resolved with comparative EIAs (or HIs) using various flavivirus antigens. The homologous serotype should be determined by cross-neutralization.
  • Specificity of PRNT: In previously WN virus-infected persons without an antecedent history of infection with another flavivirus (e.g., yellow fever vaccine virus or dengue), serum cross-neutralization tests against a battery of flaviviruses will usually implicate WN virus as the homologous virus. Serum from previously WN virus-infected persons with an antecedent history of infection with another flavivirus is often broadly cross-reactive by PRNT against a variety of other flaviviruses, and comparative titers are often insufficiently different to implicate the homologous virus.

Based on these assumptions or preliminary conclusions:

  • Persons whose acute-phase serum or CSF specimens (collected 0-7 days after illness onset) test negative for IgM antibody to WN virus should have convalescent-phase serum specimens submitted for testing. Generally, convalescent-phase specimens should be drawn at least 2 weeks after acute-phase specimens. These intervals are arbitrary and not part of the national arboviral encephalitis surveillance case definition. In some cases, for example, seroconversion to WN virus can be demonstrated in specimens collected only a few days apart during the late acute or early convalescent phase of the illness.
  • Negative tests for IgM antibody to WN virus in serum specimens collected more than 3 weeks after illness onset could be due to rapid waning of antibody; these results should be considered as potential false-negatives, pending IgG antibody testing.
  • The EIA (or HI) for serum IgG antibody is a sensitive but relatively nonspecific test for previous WN virus infection. Positive results should be confirmed by PRNT.
  • CSF should generally not be tested by WN viral IgG EIA (or HI). Instead, it should usually be reserved for testing by IgM-capture EIA and possibly by other means, including virus isolation, PCR, and neutralization.

4. At CDC, EIA results are based on "P/N ratios", which are optical density (OD) ratios or signal-to-noise ratios, not titers. A P/N ratio is calculated by dividing the OD of the test sample, P, by the OD of a normal, N, human antibody control. At CDC, serum specimens are routinely tested at a dilution of 1:400 and CSF specimens are tested undiluted. Empirically, CSF P/N ratios of >3 are considered positive for flavivirus IgM antibody at CDC, and serum IgM P/N ratios of 2.00-2.99 are considered to be equivocal pending further serologic testing (e.g., EIA endpoint titration), and ratios <2 are is considered uninterpretable if the OD of the test sample with viral antigen is <2 times the OD of the test serum with normal mouse brain antigen. Because of the potential for interlaboratory variability in P/N ratios generated for identical serum samples, appropriate positive, negative, and equivocal ranges of P/N ratios must be empirically determined by each laboratory.

5. At CDC, a serum PRNT titer of 10 (i.e., a 1:10 dilution of serum neutralizes at least 90% of the test virus dose) or greater is considered positive.

6. Longitudinal studies of WN encephalitis cases have shown that WN virus-specific IgM antibody can persist in serum for 12 months or longer. Thus, the presence of serum anti-WN viral IgM antibody is not necessarily diagnostic of acute WN viral infection. For this reason, especially in areas where WN virus is known to have circulated previously, suspected cases of acute WN encephalitis or meningitis should be confirmed by the demonstration of WN virus-specific IgM antibody in CSF, the development of WN virus-specific IgG antibody in convalescent-phase serum, or both.
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History and Recent Experience Regarding Human Surveillance
History:

In the past, with West Nile virus perceived as a disease causing mainly mild, non-specific illness, with serious neurological or other complications seen only rarely, it has been suggested that human surveillance would be the most practical form of surveillance.

  •  "The most practical form of surveillance is human case detection based on an awareness of the disease, and the use of virological tests (serum IgM detection) in cases of undifferentiated fever and encephalitis occurring during the summer/rainy months in areas endemic for the virus. Virus activity may also be monitored by vector surveillance and isolation attempts, particularly where Cx. univittatus serves as a vector. WNV isolation rates and seroconversion rates in wild birds are too low to be of use as a surveillance tool. However, the use of sentinel chickens and pigeons have been reported to correlate with virus activity". (B244.w1)

In the USA recently dead bird surveillance has been the most sensitive and practical surveillance tool and has provided the earliest warning of WN virus activity in a locality or state. (V.w42)

Recent Experience in the USA:

The following comments summarising recent experience with human surveillance in the USA has been quoted directly from the CDC Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control 3rd Revision (D147): [Text copied directly]

a) In the U.S. during 1999-2002, the peak human risk for WN viral infection occurred in August and September, although in 2002 human illness onset was reported as early as mid-May and as late as mid-December. In many regions, the peak minimum infection rates in mosquitoes and a rapid increase in the number of reported avian and equine WN viral infections occurred just prior to the period of maximal human risk.

b) In 1999-2002, the majority of reported, confirmed, or probable cases of human WN viral disease were among persons with meningoencephalitis. Testing of patients with aseptic meningitis or unexplained febrile illnesses for evidence of WN viral infection may be beneficial, but can also overwhelm laboratory testing capacity and appears to be of relatively low yield for surveillance purposes since the majority of these cases will not be due to WNV infection.

c) Most patients with WN encephalitis or meningitis (WNME) are older adults, generally over 50 years old. In the U.S. in 1999-2001, the median age among the 142 reported WNME cases was 68 years. In 2002, among 2,942 reported cases of WN meningoencephalitis, the median age was 59 years. Although 21% of reported cases were in persons younger than 40, only 4% of reported cases were in persons younger than 18.

d) When WN viral infections were first identified in the U.S., WN encephalitis was associated with a Guillain-Barré syndrome with generalized muscle weakness. In 1999-2000, generalized muscle weakness was reported in 29% of WN encephalitis cases. In 2002, at least 2 new neurologic syndromes associated with WN viral infection were identified: acute flaccid paralysis (“WN poliomyelitis-like syndrome”) and brachial plexopathy.

e) Using CDC-recommended test methods in public health laboratories, WNV-specific IgM antibody was detected in acute-phase (i.e., those collected 8 or less days after illness onset) serum or CSF specimens, or both, in the large majority of confirmed cases. In contrast, only a small minority of suspected cases were subsequently confirmed in which specific IgM antibody reactivity in acute-phase serum or CSF was in the equivocal or low-positive range.

f) Longitudinal studies of WNME cases have shown that WNV-specific IgM antibody can persist in serum for 12 months or longer. Thus, the presence of WNV-specific IgM antibody in a single serum sample is not necessarily diagnostic of acute WN viral infection. For this reason, especially in areas where WNV is known to have circulated previously, suspected, acute WN viral disease cases should be confirmed by observing a fourfold or more change in titer of WNV-specific antibody in serum and the presence of WNV-specific IgM antibody in CSF, when available.

g) In 1999 in the U.S., the sensitivity of polymerase chain reaction (PCR) tests of CSF for the diagnosis of human WN encephalitis cases was only 57%; more recent statistics are currently unavailable. Thus, PCR for the diagnosis of WN viral infections of the human central nervous system (CNS) continues to be experimental and should not replace tests for the detection of WNV-specific antibody in CSF and serum, tests that are far more sensitive.

h) During 1999-2001, 7 cases of uncomplicated WN fever (WNF) were reported in the U.S., which represents 5% of the total number of WNV disease cases reported. In 2002, over 1,100 WNF cases were reported (30% of total). Contributing factors likely include the intensive media attention paid to the 2002 epidemic that may have led to increased consumer demand for WNV diagnostic testing by patients and physicians, and the greater availability of commercial testing. Nevertheless, during 1999-2002, WNF was probably significantly underdiagnosed in the U.S. It has been estimated that approximately 20 WNF cases occur for every WNME case.

i) For suspected WNV disease cases in immunocompromised patients, WNV-specific antibody may not be present. Since longer viremias may be observed in these patients, testing serum and CSF samples for the presence of virus or viral RNA may be useful.
  • The peak human risk for WN viral infection in the USA during 1999-2000 was mid- to late August appearing to decline rapidly after the first week in September. The highest measured minimum infection rates in mosquitoes, and the majority of equine cases of WN viral infection, occurred after the apparent decrease in human risk. (D67)
  • In 1999-2000 in the USA most confirmed cases of human illness due to WN virus infection were in individuals with encephalitis. Few cases were identified by testing of patients with aseptic meningitis or Guillain-Barré syndrome for evidence of WN viral infection. (D67)
  • Most patients with WNV encephalitis or meningitis are older adults, generally more than 50 years old. In the USA in 1999 the median age of hospitalized patients was 71 years and in 2000 it was 63 years. Cases of WNV infection with encephalitis or meningitis are unusual in children. (D67)
  • In the USA, WNV encephalitis has been associated with a Guillain-Barrè-like syndrome with generalized muscle weakness; generalized muscle weakness was seen in 34% of WN encephalitis cases in 199 and in 16% of WNV encephalitis in 2000. (D67)
  • In the USA WNV-positive dead birds (particularly American crows (Corvus brachyrhynchos - American Crow)) usually have been detected from several to many weeks prior to the onset on human cases.(V.w42)
  • In 2001 in the USA in 2/359 counties (0.6%) the earliest detection of WN virus activity was by detection of human cases. (P39.3.w7)
  • Totals of humans in the USA with WNV infection reported to CDC were 21 in 2000 and 66 in 2001. (P39.3.w7)
  • In Chicago based on data in 2002 it was considered that surveillance for fever and aseptic meningitis was of value. (P39.4.w6)

NOTE: Data on surveillance in humans in the USA to 2007 is available summarised in map form. See: Map0001 - Spread of West Nile Virus in the USA (2000-2007) - Human maps

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Surveillance of Possible Health Effects from Pesticide Exposure
Evaluation of pesticide exposure of humans has been recognised as a priority area for further research. Appropriate larvicidal and adulticidal agents must be chosen for use in mosquito control measures for the prevention of  human WNV infection. Clear and unambiguous information must be collected on the negative effects that the application of such substances may have on human health, providing scientific data which can then be used for scientific support of the selection of mosquito control methods and for providing information to the public. (D67)

The following suggestions for "Surveillance of Possible Health Effects from Pesticide Exposure" have been taken directly from the New York State West Nile Virus Response Plan - Guidance Document (D72): [Text copied directly]

Surveillance of Possible Health Effects from Pesticide Exposure

I. Introduction

The plan for controlling the transmission of WNV is to use a tiered approach, starting with education, mosquito habitat reduction, larvicides, and adulticides in that order. There are limited opportunities for human exposure to larvicides. If adulticides are used, either by ground or aerial application, people may be exposed to them. Also, the threat of viral transmission may result in greater-than-average use of repellents or individually applied pesticides. Pesticide exposure carries some inherent risk to people. This risk can result from the active ingredients or the carrier agents. If the pesticides are applied properly, it is expected that most people would not experience health symptoms. However, some people may be particularly sensitive to certain pesticide products or their carriers, which may include petroleum solvents. Such individuals could experience short-term, temporary effects such as skin, eye or mucous membrane irritation, as well as exacerbation of conditions such as asthma. Therefore, monitoring of possible health effects is important.

Many signs or symptoms that might be related to pesticide exposure are non-specific and could be confused with other conditions or caused by other exposures. Also, individuals with symptoms may not seek medical care, and the provider may not associate symptoms or conditions with pesticide exposure. Thus, surveillance of possible health effects cannot provide a comprehensive accounting of all outcomes. However, it is prudent to monitor the nature and extent of concerns and common outcomes to verify that no severe, unanticipated health effects are occurring.

II. Objectives

· To review data as it becomes available to help identify any potential acute health effects associated with pesticide use that might require immediate investigation and response.

· To retrospectively assess collected data to help identify possible health effects of pesticide exposure.

· To assess year 2000 and 2001 surveillance results to determine if ongoing surveillance activities should be continued in the future and, if so, at what level.

· To determine the feasibility and usefulness of evaluating possible asthma exacerbations around the time pesticides are applied. The New York State and New York City Departments of Health are analyzing asthma reports during the 1999 outbreak to evaluate this activity.

III. Activities

During the mosquito control season, the NYSDOH [New York State Department of Health] should collect information on calls received reporting health symptoms possibly related to WNV-related adulticide applications. In addition, the NYSDOH should contact agencies (Local Health Units and Poison Control Centers) receiving similar reports to collect those data and maintain communication to encourage sharing of information. By monitoring reports of health effects possibly related to pesticide use, the NYSDOH may identify:

· Serious, unusual, or repeated acute health effects that show a pattern of association with local or aerial spraying that might need further evaluation.

· Unexpected avenues of exposure that might require investigation and referral to the appropriate agency.

· Problems in responding to concerns and inquiries about pesticide health effects, including knowledge gaps or problems with educational materials.

The NYSDOH should use a standard form (see Appendix D [D72 - Appendix D]) and data collection processes for reports of potential pesticide-related health effects from individuals and health care providers. The data collection form may be used by local health units. This form should be compatible with those already in use by NYS Poison Control Centers. At the end of the spraying season, the NYSDOH should summarize information on all calls received including the volume of calls and the nature of the complaints. NYSDOH should monitor all reports received by the Pesticide Poisoning Registry. By law, physicians, health facilities and clinical laboratories are required to report confirmed or suspected pesticide poisonings to the NYSDOH, via a toll-free telephone number. Reports received through the WNV surveillance activity should be reviewed to identify potential registry cases. These reports should be followed up as necessary. The NYSDOH has prepared a physician fact sheet for the commonly used pesticides and has distributed it to hospital emergency rooms and physicians. In addition, this fact sheet is available on the NYSDOH web site.
  • Symptoms associated with exposure to adulticides do not appear to be common. In New York City 157 people called the New York City Poison Control Center in 2000 to report symptoms which they perceived to be due to exposure to pesticides. Symptoms reported included "exacerbation of existing respiratory illness or allergies; shortness of breath; coughing; congestion; skin, eye and throat irritation; nausea and light-headedness, among others." The preliminary determination was that 11 people "possibly or probably" had symptoms which were related to pesticide exposure. (W214.Nov01.WNV6)
  • Data collected by New York City Department of Health, based on reports of visits to physicians and emergency departments, reports to the Poison Control center, number of calls to the emergency services on 911 related to asthma attacks and a population-level ecological study (asthma care regression analysis) showed no apparent reports of adverse effects in the five days following pesticide use. (P39.3.w12)
  • Levels of metabolised pesticides in urine in people from two regions with permethrin application were compared with levels in people in regions not applying pesticides; no differences were found. However higher levels of permethrin metabolites were found in individuals using home-formulations of mosquito control chemicals. (P48.1.w2)
  • Personnel involved in use of pesticides should be monitored, for example workers involved with mixing, loading or applying any organophosphorus insecticides should have their personal baseline level of serum and red blood cell cholinesterase activities established prior to the start of the mosquito season and the levels should be tested periodically thereafter, with reassignment to tasks away from pesticide exposure if blood enzyme levels drop 25% or more below their initial baseline.  (P32.1.w8)
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Authors & Referees
Authors Debra Bourne (V.w5)
Referee Suzanne I. Boardman (V.w6); Becki Lawson (V.w26); Dr Robert G. McLean (V.w42)

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