Laboratory Diagnosis and Testing

Laboratory testing for evidence of arboviral diseases typically involves serologic and molecular testing. For several viruses where humans are an amplification 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.

Eastern equine encephalitis (EEE) virus is a HHS Select Agent, and therefore, subject to strict regulations regarding its possession and use. Those intending to conduct EEE virus testing must be familiar with the complete information and specific guidance found at the Federal Select Agent Program website before conducting EEE virus testing.

Briefly, samples determined to be positive for EEE virus must be documented and reported to the Federal Select Agent Program via Form 4 (https://www.selectagents.gov/form4.html) within 7 calendar days of identification, and, if not diagnosed at a registered entity, they must then be transferred to a registered Select Agent facility or destroyed with documentation.

The front-line diagnostic assay for laboratory diagnosis of human EEE virus infection is the IgM antibody assay. Commercially available immunofluorescence assay (IFA) kits to detect IgM or IgG antibodies are often used in public health and other laboratories the United States. In addition, IgM and IgG assays developed at CDC are available in both ELISA and microsphere (IgM) immunoassay (MIA; Basile et al. 2013) formats; protocols and limited supplies of reagents are available from CDC’s DVBD Diagnostic Laboratory. CDC will provide positive controls and limited reagents considering commercial sources are available to state public health labs.

Because the IgM and IgG assays can be positive due to non-specific reactivity or rarely cross-reactivity (e.g., EEE virus is the only virus in the EEE antigenic complex in the United States, but low-level cross-reactivity might occur with other alphaviruses), they should be viewed as a presumptive positive. For a case to be considered confirmed, serum samples that are antibody-positive on initial testing should be evaluated by a more specific assay. Currently, the plaque reduction neutralization test (PRNT) is recommended for confirming IgM serological results. Although EEE virus is a rare cause of arboviral encephalitis in the United States, several other arboviral encephalitides are present in the United States and in other regions of the world. Specimens submitted for EEE virus testing should also be tested by ELISA and PRNT against other arboviruses known to be active or present in the area or in the region to where the patient traveled.

Numerous procedures have been developed for detecting viable EEE virus, EEE virus antigen, or EEE virus RNA in human diagnostic samples, many of which have been adapted to detecting EEE virus in other vertebrates and in mosquito samples. These procedures vary in their sensitivity, specificity, and time required to conduct the test. Among the most sensitive procedures for detecting EEE virus in samples are those using RT-PCR to detect EEE virus RNA in human CSF, serum, and other tissues. Real-time RT-PCR, standard RT-PCR, and nucleic acid sequence-based amplification (NASBA) amplification methods have been developed and validated for specific human diagnostic applications (Lambert et al. 2003); however, no commercially-produced or FDA-approved molecular EEE virus diagnostic tests are available.

EEE virus presence can be demonstrated by isolation of viable virus from samples taken from clinically ill patients. Appropriate samples include CSF, 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). 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). The IFA using well-defined murine MAbs is an efficient, economical, and rapid method to identify alphaviruses isolated in cell culture. 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 include real-time and standard RT-PCR methods.

Immunohistochemistry (IHC) using virus-specific MAbs on tissue has been useful in identifying both human and veterinary cases of EEE virus 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.

References

Basile AJ, et al. Multiplex microsphere immunoassays for the detection of IgM and IgG to arboviral diseases. PLoS One 2013;8:e75670.

Lambert AJ, et al. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays. J Clin Microbiol 2003; 41:379 –385.

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 (DVBD) in Fort Collins, CO. To obtain additional information about the proficiency testing program and about training in arbovirus diagnostic procedures, contact the DVBD 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/labs/BMBL.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.