Human Laboratory Diagnosis and Testing

Laboratory testing for evidence of arboviral diseases typically involves serologic and molecular testing. For several viruses where humans are an amplifying host, molecular testing is more specific and can be used to confirm the diagnosis in the first week of illness. For viruses that typically are neuroinvasive, serology is more likely to be used to determine if someone was recently infected.

In most patients, infection with an arbovirus that can cause encephalitis is clinically inapparent or causes a nonspecific viral syndrome. Numerous pathogens cause encephalitis, aseptic meningitis, and febrile disease with similar clinical symptoms and presentations and should be considered in the differential diagnosis. Definitive diagnosis can only be made by laboratory testing using specific reagents. Selection of diagnostic test procedures should take into consideration patient factors (e.g., age, immune status, vaccination history), timing of infection, the range of pathogens in the differential diagnosis, the criteria for classifying a case as confirmed or probable, as well as the capability of the primary and confirming diagnostic laboratories.

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) 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); and 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 test results from 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.

Serology. The front-line screening assay for laboratory diagnosis of human WNV infection is the IgM assay. Currently, the FDA has cleared three commercially available test kits from different manufacturers, for detection of WNV IgM antibodies. These kits are used in many commercial and public health laboratories in the United States. In addition, the CDC-defined IgM and IgG EIA [i.e., ELISA or microsphere-based immunoassay (MIA)] can be used (Martin et al. 2000; Johnson et al. 2000; Johnson et al. 2005). The CDC MIA can differentiate WNV from St. Louis encephalitis (SLE). Protocols are available for the CDC-developed assays from CDC’s DVBD Diagnostic Laboratory (Martin et al. 2000; Johnson et al. 2000). CDC also will provide positive controls and limited reagents as commercial sources are available to state public health labs.

Because the IgM and IgG antibody tests can cross-react between flaviviruses (e.g., SLE, dengue, yellow fever, WNV, Powassan), 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 against other arboviruses known to be active or be present in the area or in the region where the patient traveled.

Virus Detection Assays. Numerous procedures have been developed for detecting viable WNV, WNV antigen, or WNV RNA in human diagnostic samples, many of which have been adapted to detecting WNV in other vertebrates and in mosquito samples. These procedures vary in their sensitivity, specificity, and time required to conduct the test (Table).

WNV presence can be demonstrated by isolation of viable virus from samples taken from clinically ill humans. Appropriate samples include CSF and serum samples obtained 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). However, viremia is almost always absent by the time a patient presents with neuroinvasive illness and thus viral isolation is generally not recommended as part of a testing algorithm in immune competent patients. Mosquito origin cells may not show obvious cytopathic effect and must be screened by immunofluorescence or RT-PCR. Confirmation of virus isolate identity can be accomplished by indirect immunofluorescence assay (IFA) using virus-specific monoclonal antibodies (MAbs) or nucleic acid detection (e.g. RT-PCR, real-time RT-PCR or sequencing). IFA using well-defined murine MAbs is an efficient, economical, and rapid method to identify flaviviruses isolated in cell culture. 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, real-time and nucleic acid sequence-based amplification (NASBA) methods may be used to confirm virus isolates (Briese et al. 2000; Shi et al. 2001; Lanciotti et al. 2000).

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. Virus isolation or RT-PCR on serum may be helpful in confirming WNV infection in immunocompromised patients when antibody development is delayed or absent.

Immunohistochemistry (IHC) using virus-specific MAbs on brain tissue has been very useful in identifying human 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 (Bhatnagar et al. 2007).

Clinical Laboratory Improvements Amendments (CLIA) certification: To maintain certification, CLIA recommendations for performing and interpreting human diagnostic tests should be followed. Laboratories performing arboviral serology or RNA-detection testing are invited to participate in the annual proficiency testing that is available from CDC’s Division of Vector-Borne Diseases in Fort Collins, CO. To obtain additional information about the proficiency testing program and about training in arbovirus diagnostic procedures, contact the Division of Vector-Borne Diseases by phone: 970-261-6400 or email: dvbid2@cdc.gov.

Biocontainment: Containment specifications are available in the CDC/National Institutes of Health publication Biosafety in Microbiological and Biomedical Laboratories (BMBL 6). This document can be found online at: https://www.cdc.gov/csels/dls/locs/2020/cdc_releases_6th_edition_of_biosafety_in_microbiology_and_biomedial_labs.html

Shipping of diagnostic samples and agents. Shipping and transport of clinical specimens should follow current International Air Transport Association (IATA) and Department of Commerce recommendations. For more information, visit the IATA dangerous goods Web site at:

http://www.iata.org/publications/dgr/Pages/index.aspx, and the USDA Animal and Plant Health Inspection Service (APHIS), National Center for Imports and Exports website: https://www.aphis.usda.gov/aphis/ourfocus/importexport

References

Bhatnagar J, Guarner J, Paddock CD, Shieh WJ, Lanciotti RS, Marfin AA, Campbell GL, Zaki SR. 2007. Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: a useful adjunct to conventional tissue-based diagnostic methods. J Clin Virol. 38(2):106-11. Epub 2006 Dec 8. 48

Briese T, Glass WG, Lipkin WI.2000. Detection of West Nile virus sequences in cerebrospinal fluid. Lancet. 355:1614-5.

Johnson AJ, Martin DA, Karabatsos N, Roehrig JT. 2000. Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay. J Clin Microbiol. 38:1827-31.

Johnson AJ, Noga AJ, Kosoy O, Lanciotti RS, Johnson AA, Biggerstaff BJ. 2005. Duplex microsphere-based immunoassay for detection of anti-West Nile virus and anti-St. Louis encephalitis virus immunoglobulin m antibodies. Clin Diagn Lab Immunol. 12(5):566-74.

Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, et al. 2000. Rapid detection of West Nile virus from human clinical specimens, field- collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol. 38:4066-71.

Martin DA, Muth DA, Brown T, Johnson AJ, Karabatsos N, Roehrig JT. 2000 Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J Clin Microbiol. 38:1823-6.

Shi PY, Kauffman EB, Ren P, Felton A, Tai JH, Dupuis AP. et al. 2001. High-throughput detection of West Nile virus RNA. J Clin Microbiol. 39:1264-71.