West nile virus laboratory tests: Difference between revisions

Latest revision as of 19:28, 18 September 2017

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [2]; Associate Editor(s)-in-Chief: Rim Halaby, M.D. [3]

Overview

The front-line assay for laboratory diagnosis of WNV infection is the IgM assay. IgM and IgG ELISA tests can cross-react between flaviviruses; therefore, serum samples that are antibody-positive on initial screening should be evaluated by a more specific test. Currently the plaque reduction neutralization test (PRNT) is the recommended test for differentiating between flavivirus infections. Specimens submitted for WNV testing should also be tested by ELISA and PRNT against other arboviruses known to be active or be present in the area or in the region where the patient traveled. Numerous procedures have been developed for detecting viable WNV, WNV antigen or WNV RNA in human diagnostic samples. These procedures vary in their sensitivity, specificity, and time required to conduct the test. Among the most sensitive procedures for detecting WNV in samples are those using RT-PCR to detect WNV RNA in human CSF, serum, and other tissues. Confirmation of virus isolate identity can be accomplished by indirect immunofluorescence assay (IFA) using virus-specific monoclonal antibodies or nucleic acid detection. Immunohistochemistry (IHC) using virus-specific MAbs on brain tissue has been very useful in identifying cases of WNV infection.^[1]

Laboratory Tests

Appropriate selection of diagnostic procedures and accurate interpretation of findings requires information describing the patient and the diagnostic specimen. For human specimens, the following data must accompany sera, CSF, or tissue specimens for results to be properly interpreted and reported:^[1] 1) Symptom onset date (when known) 2) Date of sample collection 3) Unusual immunological status of patient (e.g., immunosuppression) 4) State and county of residence 5) Travel history (especially in flavivirus-endemic areas) 6) History of prior vaccination (e.g., yellow fever, Japanese encephalitis, or Tick-borne encephalitis viruses) 7) Brief clinical summary including clinical diagnosis (e.g., encephalitis, aseptic meningitis).

Minimally, onset and sample collection dates are required to perform and interpret initial screening tests. The remaining information is required to evaluate any specimens positive on initial screening. If possible, a convalescent serum sample taken at least 14 days following the acute sample should be obtained to enable confirmation by serological testing.^[1]

Serology

IgM and IgG ELISA tests

The front-line screening assay for laboratory diagnosis of human WNV infection is the IgM assay. WNV-specific IgM antibodies are usually detectable 3 to 8 days after onset of illness and persist for 30 to 90 days, but longer persistence has been documented. Therefore, positive IgM antibodies occasionally may reflect a past infection. If serum is collected within 8 days of illness onset, the absence of detectable virus-specific IgM does not rule out the diagnosis of WNV infection, and the test may need to be repeated on a later sample.

The presence of WNV-specific IgM in blood or CSF provides good evidence of recent infection but may also result from cross-reactive antibodies after infection with other flaviviruses or from non-specific reactivity. According to product inserts for commercially available WNV IgM assays, all positive results obtained with these assays should be confirmed by neutralizing antibody testing of acute- and convalescent-phase serum specimens at a state public health laboratory or CDC.

WNV IgG antibodies generally are detected shortly after IgM antibodies and persist for many years following a symptomatic or asymptomatic infection. Therefore, the presence of IgG antibodies alone is only evidence of previous infection and clinically compatible cases with the presence of IgG, but not IgM, should be evaluated for other etiologic agents.

Currently, the FDA has cleared four commercially-available test kits from different manufacturers, for detection of WNV IgM antibodies. These four kits are used in many commercial and public health laboratories in the United States. In addition the CDC-defined IgM and IgG ELISA can be used; protocols and reagents are available from the CDC DVBD Diagnostic Laboratory. There is also a microsphere-based immunoassay for the detection of IgM antibodies that can differentiate WNV from SLE.^[1]

Plaque reduction neutralization test

Because the IgM and IgG ELISA tests can cross-react between flaviviruses (e.g., SLE, dengue, yellow fever, WN), they should be viewed as screening tests only. For a case to be considered confirmed, serum samples that are antibody-positive on initial screening should be evaluated by a more specific test; currently the plaque reduction neutralization test (PRNT) is the recommended test for differentiating between flavivirus infections. Though WNV is the most common cause of arboviral encephalitis in the United States, there are several other arboviral encephalitides present in the country and in other regions of the world. Specimens submitted for WNV testing should also be tested by ELISA and PRNT against other arboviruses known to be active or be present in the area or in the region where the patient traveled.^[1]

Virus detection assay

Numerous procedures have been developed for detecting viable WNV, WNV antigen or WNV RNA in human diagnostic samples. These procedures vary in their sensitivity, specificity, and time required to conduct the test.^[1]

RT-PCR

Among the most sensitive procedures for detecting WNV in samples are those using RT-PCR to detect WNV RNA in human CSF, serum and other tissues. Fluorogenic 5' nuclease techniques (real-time PCR) and nucleic acid sequence-based amplification (NASBA) methods have been developed and validated for specific human diagnostic applications and for detecting WNV RNA in blood donations.^[1]

WNV presence can be demonstrated by isolation of viable virus from samples taken from clinically ill humans. Appropriate samples include CSF (serum samples may be useful very early in infection) and brain tissue (taken at biopsy or postmortem). Virus isolation should be performed in known susceptible mammalian (e.g., Vero) or mosquito cell lines (e.g., C6/36). Mosquito origin cells may not show obvious cytopathic effect and must be screened by immunofluorescence or RT-PCR. Appropriate samples for virus isolation from clinically ill humans include CSF (serum samples may be useful very early in infection) and brain tissue (taken at biopsy or postmortem).^[1]

Indirect immunofluorescence assay

Confirmation of virus isolate identity can be accomplished by indirect immunofluorescence assay (IFA) using virus-specific monoclonal antibodies or nucleic acid detection. The IFA using well-defined murine monoclonal antibodies (MAbs) is an efficient, economical, and rapid method to identify flaviviruses. MAbs are available that can differentiate WNV and SLE virus from each other and from other flaviviruses. Incorporating MAbs specific for other arboviruses known to circulate in various regions will increase the rapid diagnostic capacities of state and local laboratories. Nucleic acid detection methods including RT-PCR, TaqMan and nucleic acid sequence based amplification (NASBA) methods may be used to confirm virus isolates as WNV.^[1]

While these tests can be quite sensitive, virus isolation and RT-PCR to detect WNV RNA in sera or CSF of clinically ill patients have limited utility in diagnosing human WNV neuroinvasive disease due to the low level viremia present in most cases at the time of clinical presentation. However, one study demonstrated that combining detection of IgM with detection of WNV RNA in plasma significantly increased the number of WNV non-neuroinvasive (i.e., fever) cases detected. Virus isolation or RT-PCR on serum may be helpful in confirming human WNV infection in immunocompromised patients when antibody development is delayed or absent.^[1]

Immunohistochemistry

Immunohistochemistry (IHC) using virus-specific MAbs on brain tissue has been very useful in identifying both human and avian cases of WNV infection. In suspected fatal cases, IHC should be performed on formalin fixed autopsy, biopsy, and necropsy material, ideally collected from multiple anatomic regions of the brain, including the brainstem, midbrain, and cortex.^[1]

References

↑ ^1.00 ^1.01 ^1.02 ^1.03 ^1.04 ^1.05 ^1.06 ^1.07 ^1.08 ^1.09 ^1.10 <West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [1]

Template:WS

[CDC-1] 1.00 ^1.01 ^1.02 ^1.03 ^1.04 ^1.05 ^1.06 ^1.07 ^1.08 ^1.09 ^1.10 <West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [1]

[1]

@@ Line 1: / Line 1: @@
+__NOTOC__
 {{West nile virus}}
-{{CMG}}
+{{CMG}}; {{AE}} {{Rim}}
 ==Overview==
-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
+The front-line assay for laboratory diagnosis of WNV infection is the [[IgM]] assay. IgM and IgG ELISA tests can cross-react between [[flavivirus]]es; therefore, serum samples that are antibody-positive on initial screening should be evaluated by a more specific test. Currently the plaque reduction neutralization test (PRNT) is the recommended test for differentiating between flavivirus infections. Specimens submitted for WNV testing should also be tested by ELISA and PRNT against other arboviruses known to be active or be present in the area or in the region where the patient traveled. Numerous procedures have been developed for detecting viable WNV, WNV antigen or WNV RNA in human diagnostic samples. These procedures vary in their [[sensitivity]], [[specificity]], and time required to conduct the test. Among the most sensitive procedures for detecting WNV in samples are those using RT-[[PCR]] to detect WNV RNA in human CSF, serum, and other tissues. Confirmation of virus isolate identity can be accomplished by indirect [[immunofluorescence assay]] (IFA) using virus-specific monoclonal antibodies or nucleic acid detection.  [[Immunohistochemistry]] (IHC) using virus-specific MAbs on brain tissue has been very useful in identifying cases of WNV infection.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-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.
-A high clinical suspicion for arboviral encephalitis should be encouraged among health care providers. When the diagnosis is in doubt, appropriate clinical specimens should be submitted to CDC or another laboratory capable of performing reliable serologic testing for antibodies to domestic arboviruses. Testing of CSF and paired acute- and convalescent-phase serum samples should be strongly encouraged to maximize the accuracy of serologic results.
+==Laboratory Tests==
+Appropriate selection of diagnostic procedures and accurate interpretation of findings requires information describing the patient and the diagnostic specimen. For human specimens, the following data must accompany sera, [[CSF]], or tissue specimens for results to be properly interpreted and reported:<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
+) Symptom onset date (when known)
+) Date of sample collection
+) Unusual immunological status of patient (e.g., [[immunosuppression]])
+) State and county of residence
+) Travel history (especially in flavivirus-endemic areas)
+) History of prior [[vaccination]] (e.g., [[yellow fever]], Japanese encephalitis, or Tick-borne encephalitis viruses)
+) Brief clinical summary including clinical diagnosis (e.g., [[encephalitis]], [[aseptic meningitis]]).
-==Laboratory Findings==
+Minimally, onset and sample collection dates are required to perform and interpret initial screening tests. The remaining information is required to evaluate any specimens positive on initial screening. If possible, a convalescent [[serum]] sample taken at least 14 days following the acute sample should be obtained to enable confirmation by serological testing.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-The basic laboratory diagnostic tests—and how they should be used at the national, state, and local level—are outlined below. The initial designation of reference and regional laboratories that can do all testing will be based on the availability of biosafety level 3 (BSL3) containment facilities.
-===Serologic Laboratory Diagnosis===
+===Serology===
+====IgM and IgG ELISA tests====
+The front-line screening assay for laboratory diagnosis of human WNV infection is the [[IgM]] assay.  WNV-specific IgM antibodies are usually detectable 3 to 8 days after onset of illness and persist for 30 to 90 days, but longer persistence has been documented. Therefore, positive IgM antibodies occasionally may reflect a past infection. If serum is collected within 8 days of illness onset, the absence of detectable virus-specific IgM does not rule out the diagnosis of WNV infection, and the test may need to be repeated on a later sample.
-Accurate interpretation of serologic findings requires knowledge of the specimen. For human specimens the following data must accompany specimens submitted for serology before testing can proceed or results can be properly interpreted and reported:
+The presence of WNV-specific IgM in [[blood]] or [[CSF]] provides good evidence of recent infection but may also result from cross-reactive [[antibodies]] after infection with other [[flavivirus]]es or from non-specific reactivity. According to product inserts for commercially available WNV IgM assays, all positive results obtained with these assays should be confirmed by neutralizing antibody testing of acute- and convalescent-phase serum specimens at a state public health laboratory or CDC.
-) symptom onset date (when known);
+WNV IgG antibodies generally are detected shortly after [[IgM]] antibodies and persist for many years following a symptomatic or asymptomatic infection. Therefore, the presence of IgG antibodies alone is only evidence of previous infection and clinically compatible cases with the presence of IgG, but not IgM, should be evaluated for other etiologic agents.
-) date of sample collection;
+Currently, the [[FDA]] has cleared four commercially-available test kits from different manufacturers, for detection of WNV IgM [[antibodies]]. These four kits are used in many commercial and public health laboratories in the United States. In addition the CDC-defined [[IgM]] and [[IgG]] ELISA can be used; protocols and reagents are available from the CDC DVBD Diagnostic Laboratory.  There is also a microsphere-based immunoassay for the detection of IgM antibodies that can differentiate WNV from SLE.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-) unusual immunological status of patient (e.g., immunosuppression);
+====Plaque reduction neutralization test====
+Because the IgM and IgG ELISA tests can cross-react between [[flavivirus]]es (e.g., [[SLE]], [[dengue]], [[yellow fever]], WN), they should be viewed as screening tests only. For a case to be considered confirmed, serum samples that are [[antibody]]-positive on initial screening should be evaluated by a more specific test; currently the plaque reduction neutralization test (PRNT) is the recommended test for differentiating between [[flavivirus]] infections.  Though WNV is the most common cause of arboviral [[encephalitis]] in the United States, there are several other arboviral encephalitides present in the country and in other regions of the world.  Specimens submitted for WNV testing should also be tested by ELISA and PRNT against other [[arbovirus]]es known to be active or be present in the area or in the region where the patient traveled.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-) state and county of residence;
+===Virus detection assay===
+Numerous procedures have been developed for detecting viable WNV, WNV antigen or WNV RNA in human diagnostic samples.  These procedures vary in their [[sensitivity]], [[specificity]], and time required to conduct the test.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-) travel history in flavivirus-endemic areas;
+====RT-PCR====
+Among the most sensitive procedures for detecting WNV in samples are those using RT-[[PCR]] to detect WNV RNA in human CSF, serum and other tissues. Fluorogenic 5' nuclease techniques (real-time PCR) and nucleic acid sequence-based amplification (NASBA) methods have been developed and validated for specific human diagnostic applications and for detecting WNV RNA in blood donations.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-) history of prior vaccination against flavivirus disease (e.g., yellow fever, Japanese encephalitis, or Central European encephalitis); and
+WNV presence can be demonstrated by isolation of viable [[virus]] from samples taken from clinically ill humans. Appropriate samples include [[CSF]] (serum samples may be useful very early in infection) and brain tissue (taken at biopsy or postmortem). Virus isolation should be performed in known susceptible mammalian (e.g., Vero) or mosquito cell lines (e.g., C6/36). Mosquito origin cells may not show obvious cytopathic effect and must be screened by immunofluorescence or RT-PCR. Appropriate samples for virus isolation from clinically ill humans include CSF (serum samples may be useful very early in infection) and brain tissue (taken at biopsy or postmortem).<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-) brief clinical summary including clinical diagnosis (e.g., encephalitis, aseptic meningitis).
+====Indirect immunofluorescence assay====
+Confirmation of virus isolate identity can be accomplished by indirect [[immunofluorescence assay]] (IFA) using virus-specific monoclonal antibodies or nucleic acid detection. The IFA using well-defined murine monoclonal [[antibodies]] (MAbs) is an efficient, economical, and rapid method to identify flaviviruses. MAbs are available that can differentiate WNV and [[SLE]] virus from each other and from other [[flavivirus]]es. Incorporating MAbs specific for other arboviruses known to circulate in various regions will increase the rapid diagnostic capacities of state and local laboratories. Nucleic acid detection methods including RT-PCR, TaqMan and nucleic acid sequence based amplification (NASBA) methods may be used to confirm virus isolates as WNV.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-''1. Human''
+While these tests can be quite sensitive, virus isolation and RT-PCR to detect WNV RNA in sera or CSF of clinically ill patients have limited utility in diagnosing human WNV neuroinvasive disease due to the low level [[viremia]] present in most cases at the time of clinical presentation. However, one study demonstrated that combining detection of IgM with detection of WNV RNA in plasma significantly increased the number of WNV non-neuroinvasive (i.e., fever) cases detected. Virus isolation or RT-PCR on serum may be helpful in confirming human WNV infection in [[immunocompromised]] patients when antibody development is delayed or absent.<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-a) Commercial kits for human serologic diagnosis of WNV infection are currently in development. Until these kits are available, the CDC-defined IgM and IgG ELISA should be the front-line tests for serum and CSF. These ELISA tests are the
+====Immunohistochemistry====
-most sensitive screening assays available. The HI and indirect immunofluorescent antibody (IFA) test may also be used to screen samples for flavivirus antibodies. Laboratories performing HI assays need be aware that the recombinant WNV
+[[Immunohistochemistry]] (IHC) using virus-specific MAbs on brain tissue has been very useful in identifying both human and avian cases of WNV infection. In suspected fatal cases, IHC should be performed on formalin fixed autopsy, biopsy, and necropsy material, ideally collected from multiple anatomic regions of the brain, including the [[brainstem]], [[midbrain]], and [[cortex]].<ref name=CDC><West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. 4th revision, 2013. [http://www.cdc.gov/westnile/resources/pdfs/wnvGuidelines.pdf] </ref>
-antigens produced to date are not useful in the HI test; mouse brain source antigen (available from CDC) must be used in HI tests. The recombinant WNV antigen is available from commercial sources.
-b) To date, the prototype WNV strains Eg101 or NY99 strains have performed equally well as antigens in diagnostic tests for WNV in North America.
-c) To maintain Clinical Laboratory Improvements Amendments (CLIA) certification, CLIA recommendations for positive and negative ranges should be followed, and laboratories doing WNV testing should participate in a proficiency testing program
-through experienced reference laboratories; CDC’s Division of Vector-Borne Infectious Diseases in Fort Collins, Colorado and the National Veterinary Services Laboratories in Ames, Iowa both offer this type of program.
-d) 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 ubmitted 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).
 ==References==
@@ Line 49: / Line 54: @@
 [[Category:Disease]]
-[[Category:Infectious disease]]
 [[Category:Neurology]]