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West Nile virus (WNV) IgM antibodies typically indicate a recent infection. However, WNV IgM antibodies can remain detectable for months to years following illness onset. We found that 23% (11/47) of samples tested with a WNV ELISA and 43% (20/47) of samples tested with WNV microsphere immunoassay (MIA) at 16-19 months following WNV illness onset were positive for IgM antibodies. The proportion of samples testing positive for WNV IgM by ELISA decreased over time, but 5% (2/44) of individuals remained positive at 60-63 months after their acute illness and 4% (2/50) were WNV IgM equivocal at 72-81 months. Testing by MIA showed the same general trend of decreased proportion positive over time though the rates of positivity were higher at most time points compared with the ELISA, including 6% (3/50) of participant's samples identified as IgM positive by MIA at 72-81 months post their acute illness. With the MIA, there also was a high proportion of samples with nonspecific results at each time point; average of 23% across all time points. Clinicians and public health officials should consider these findings along with clinical and epidemiologic data when interpreting WNV IgM antibody test results.

West Nile virus (WNV) is the most common domestic arbovirus in the United States. During 2018, WNV was transmitted through solid organ transplantation to 2 recipients who had neuroinvasive disease develop. Because of increased illness and death in transplant recipients, organ procurement organizations should consider screening during region-specific WNV transmission months.

Eastern equine encephalitis virus (EEEV) is an arbovirus in the family Togaviridae, genus Alphavirus, found in North America and associated with freshwater/hardwood swamps in the Atlantic, Gulf Coast, and Great Lakes regions. EEEV disease in humans is rare but causes substantial illness and death. To investigate the molecular epidemiology and microevolution of EEEV from a fatal case in Alabama, USA, in 2019, we used next-generation sequencing of serum and cerebrospinal fluid (CSF). Phylogenetic inference indicated that the infecting strain may be closely related to isolates from Florida detected during 2010-2014, suggesting potential seeding from Florida. EEEV detected in serum displayed a higher degree of variability with more single-nucleotide variants than that detected in the CSF. These data refine our knowledge of EEEV molecular epidemiologic dynamics in the Gulf Coast region and demonstrate potential quasispecies bottlenecking within the central nervous system of a human host.

Background: Heartland virus (HRTV), a recently reclassified member of the genus Bandavirus, family Phenuiviridae, was first isolated in 2009 from a Missouri farmer exhibiting leukopenia and thrombocytopenia with suspected ehrlichiosis. Since then, more HRTV cases have been diagnosed, and firstline laboratory diagnostic assays are needed to identify future infections Objectives. We sought to develop rapid and reliable IgM and IgG microsphere immunoassays (MIAs) to test sera of patients suspected of having HRTV infection, and to distinguish between recent and past infections., Study Design: Heartland virus antigen was captured by an anti-HRTV monoclonal antibody covalently bound to microspheres. Antibodies in human sera from confirmed HRTV-positive and negative cases were reacted with the microsphere complexes and detected using a BioPlex® 200 instrument. Assay cutoffs were determined by receiver operator characteristic analysis of the normalized test output values, equivocal zones for each assay were defined, and sensitivities, specificities, accuracies, and imprecision values were calculated., Results: Sensitivities, specificities and accuracies of the IgM and IgG MIAs were all >95 %. Both tests were precise within and between assay plates, and cross-reactivity with other arboviruses was not observed., Conclusions: HRTV IgM and IgG MIAs are accurate and rapid first-line methods to serologically identify recent and past HRTV infections., (Published by Elsevier B.V.)

Background: Heartland virus (HRTV) was first described as a human pathogen in 2012. From 2013 to 2017, the Centers for Disease Control and Prevention (CDC) implemented a national protocol to evaluate patients for HRTV disease, better define its geographic distribution, epidemiology, and clinical characteristics, and develop diagnostic assays for this novel virus., Methods: Individuals aged ≥12 years whose clinicians contacted state health departments or the CDC about testing for HRTV infections were screened for recent onset of fever with leukopenia and thrombocytopenia. A questionnaire was administered to collect data on demographics, risk factors, and signs and symptoms; blood samples were tested for the presence of HRTV RNA and neutralizing antibodies., Results: Of 85 individuals enrolled and tested, 16 (19%) had evidence of acute HRTV infection, 1 (1%) had past infection, and 68 (80%) had no infection. Patients with acute HRTV disease were residents of 7 states, 12 (75%) were male, and the median age (range) was 71 (43-80) years. Illness onset occurred from April to September. The majority reported fatigue, anorexia, nausea, headache, confusion, arthralgia, or myalgia. Fourteen (88%) cases were hospitalized; 2 (13%) died. Fourteen (88%) participants reported finding a tick on themselves in the 2 weeks before illness onset. HRTV-infected individuals were significantly older ( P <  .001) and more likely to report an attached tick ( P =  .03) than uninfected individuals., Conclusions: Health care providers should consider HRTV disease testing in patients with an acute febrile illness with either leukopenia or thrombocytopenia not explained by another condition or who were suspected to have a tickborne disease but did not improve following appropriate treatment., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2020.)

Background: In fall 2017, 3 solid organ transplant (SOT) recipients from a common donor developed encephalitis within 1 week of transplantation, prompting suspicion of transplant-transmitted infection. Eastern equine encephalitis virus (EEEV) infection was identified during testing of endomyocardial tissue from the heart recipient., Methods: We reviewed medical records of the organ donor and transplant recipients and tested serum, whole blood, cerebrospinal fluid, and tissue from the donor and recipients for evidence of EEEV infection by multiple assays. We investigated blood transfusion as a possible source of organ donor infection by testing remaining components and serum specimens from blood donors. We reviewed data from the pretransplant organ donor evaluation and local EEEV surveillance., Results: We found laboratory evidence of recent EEEV infection in all organ recipients and the common donor. Serum collected from the organ donor upon hospital admission tested negative, but subsequent samples obtained prior to organ recovery were positive for EEEV RNA. There was no evidence of EEEV infection among donors of the 8 blood products transfused into the organ donor or in products derived from these donations. Veterinary and mosquito surveillance showed recent EEEV activity in counties nearby the organ donor's county of residence. Neuroinvasive EEEV infection directly contributed to the death of 1 organ recipient and likely contributed to death in another., Conclusions: Our investigation demonstrated EEEV transmission through SOT. Mosquito-borne transmission of EEEV to the organ donor was the likely source of infection. Clinicians should be aware of EEEV as a cause of transplant-associated encephalitis., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

When introduced into a naïve population, chikungunya virus generally spreads rapidly, causing large outbreaks of fever and severe polyarthralgia. We randomly selected households in the U.S. Virgin Islands (USVI) to estimate seroprevalence and symptomatic attack rate for chikungunya virus infection at approximately 1 year following the introduction of the virus. Eligible household members were administered a questionnaire and tested for chikungunya virus antibodies. Estimated proportions were calibrated to age and gender of the population. We enrolled 509 participants. The weighted infection rate was 31% (95% confidence interval [CI]: 26-36%). Among those with evidence of chikungunya virus infection, 72% (95% CI: 65-80%) reported symptomatic illness and 31% (95% CI: 23-38%) reported joint pain at least once per week approximately 1 year following the introduction of the virus to USVI. Comparing rates from infected and noninfected study participants, 70% (95% CI: 62-79%) of fever and polyarthralgia and 23% (95% CI: 9-37%) of continuing joint pain in patients infected with chikungunya virus were due to their infection. Overall, an estimated 43% (95% CI: 33-52%) of the febrile illness and polyarthralgia in the USVI population during the outbreak was attributable to chikungunya virus and only 12% (95% CI: 7-17%) of longer term joint pains were attributed to chikungunya virus. Although the rates of infection, symptomatic disease, and longer term joint symptoms identified in USVI are similar to other outbreaks of the disease, a lower proportion of acute fever and joint pain was found to be attributable to chikungunya virus.

Background: Few studies have assessed the duration of humoral immunity following yellow fever (YF) vaccination in a non-endemic population. We evaluated seropositivity among US resident travellers based on time post-vaccination., Methods: We identified serum samples from US travellers with YF virus-specific plaque reduction neutralization testing (PRNT) performed at CDC from 1988 to 2016. Analyses were conducted to assess the effect of time since vaccination on neutralizing antibody titer counts., Results: Among 234 travellers who had neutralizing antibody testing performed on a specimen obtained ≥1 month after vaccination, 13 received multiple YF vaccinations and 221 had one dose of YF vaccine reported. All 13 who received more than one dose of YF vaccine had a positive PRNT regardless of the amount time since most recent vaccination. Among the 221 travellers with one reported dose of YF vaccine, 155 (70%) were vaccinated within 10 years (range 1 month-9 years) and 66 (30%) were vaccinated ≥10 years (range 10-53 years) prior to serum collection. Among the 155 individuals vaccinated, <10 years prior to serum collection, 146 (94%) had a positive PRNT compared with 82% (54/66) of individuals vaccinated ≥10 years prior to serum collection (P = 0.01). Post-vaccination PRNT titers showed a time-dependent decrease. Individuals with immunocompromising conditions were less likely to have a positive PRNT (77%) compared with those who were not immunocompromised (92%; P = 0.04)., Conclusion: Although the percentage of vaccinees with a positive PRNT and antibody titers decreased over time, a single dose of YF vaccine provided long-lasting protection in the majority of US travellers. A booster dose could be considered for certain travellers who are planning travel to a high risk area based on immune competence and time since vaccination.

Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate the serological diagnosis of Zika virus (ZIKAV) infection. We assessed the utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive immunoglobulin M (IgM) results in areas with various levels of past dengue virus (DENV) infection incidence. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from 1 January through 31 August 2016 with positive ZIKAV IgM results, and ZIKAV and DENV PRNTs were performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false positive for samples interpreted as a DENV infection or negative. In U.S. states, 208 (27%) of 759 IgM-positive results were confirmed to be ZIKAV compared to 11 (21%) of 52 in the U.S. Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM-positive results were unspecified flavivirus infections. The false-positivity rate was 27% in U.S. states, 18% in the USVI, 2% in American Samoa, and 6% in Puerto Rico. In U.S. states, the PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM-positive samples. Almost a third of ZIKAV IgM-positive results were not confirmed; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher rates of DENV transmission, the PRNT usually could not differentiate between ZIKAV and DENV infections., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

In 2016, Zika virus disease developed in a man (patient A) who had no known risk factors beyond caring for a relative who died of this disease (index patient). We investigated the source of infection for patient A by surveying other family contacts, healthcare personnel, and community members, and testing samples for Zika virus. We identified 19 family contacts who had similar exposures to the index patient; 86 healthcare personnel had contact with the index patient, including 57 (66%) who had contact with body fluids. Of 218 community members interviewed, 28 (13%) reported signs/symptoms and 132 (61%) provided a sample. Except for patient A, no other persons tested had laboratory evidence of recent Zika virus infection. Of 5,875 mosquitoes collected, none were known vectors of Zika virus and all were negative for Zika virus. The mechanism of transmission to patient A remains unknown but was likely person-to-person contact with the index patient.

Zika virus is an emerging mosquito-borne flavivirus that typically causes a mild febrile illness with rash, arthralgia, or conjunctivitis. Zika virus has recently caused large outbreaks of disease in southeast Asia, Pacific Ocean Islands, and the Americas. We identified all positive Zika virus test results performed at U.S. Centers for Disease Control and Prevention from 2010 to 2014. For persons with test results indicating a recent infection with Zika virus, we collected information on demographics, travel history, and clinical features. Eleven Zika virus disease cases were identified among travelers returning to the United States. The median age of cases was 50 years (range: 29-74 years) and six (55%) were male. Nine (82%) cases had their illness onset from January to April. All cases reported a travel history to islands in the Pacific Ocean during the days preceding illness onset, and all cases were potentially viremic while in the United States. Public health prevention messages about decreasing mosquito exposure, preventing sexual exposure, and preventing infection in pregnant women should be targeted to individuals traveling to or living in areas with Zika virus activity. Health-care providers and public health officials should be educated about the recognition, diagnosis, and prevention of Zika virus disease., (© The American Society of Tropical Medicine and Hygiene.)

Yellow fever virus (YFV) is endemic in tropical and sub-tropical regions of the world, with around 180,000 human infections a year occurring in Africa. Serologic testing is the chief laboratory diagnostic means of identifying an outbreak and to inform the decision to commence a vaccination campaign. The World Health Organization disseminates the reagents for YFV testing to African reference laboratories, and the US Centers for Disease Control and Prevention (CDC) is charged with producing and providing these reagents. The CDC M-antibody capture ELISA is a 2-day test, requiring titration of reagents when new lots are received, which leads to inconsistency in testing and wastage of material. Here we describe the development of a kit-based assay (YF MAC-HD) based upon the CDC method, that is completed in approximately 3.5h, with equivocal samples being reflexed to an overnight protocol. The kit exhibits >90% accuracy when compared to the 2-day test. The kits were designed for use with a minimum of equipment and are stored at 4°C, removing the need for freezing capacity. This kit is capable of tolerating temporary sub-optimal storage conditions which will ease shipping or power outage concerns, and a shelf life of >6 months was demonstrated with no deterioration in accuracy. All reagents necessary to run the YF MAC-HD are included in the kit and are single-use, with 8 or 24 sample options per kit. Field trials are envisioned for the near future, which will enable refinement of the method. The use of the YF MAC-HD is anticipated to reduce materials wastage, and improve the quality and consistency of YFV serologic testing in endemic areas., (Published by Elsevier B.V.)

Heartland virus is a newly identified phlebovirus that was first isolated from two northwestern Missouri farmers hospitalized with fever, leukopenia, and thrombocytopenia in 2009. Based on the patients' clinical findings and their reported exposures, the virus was suspected to be transmitted by ticks. After this discovery, CDC worked with state and local partners to define the ecology and modes of transmission of Heartland virus, develop diagnostic assays, and identify additional cases to describe the epidemiology and clinical disease. From this work, it was learned that Heartland virus is found in the Lone Star tick (Amblyomma americanum). Six additional cases of Heartland virus disease were identified during 2012-2013; four of those patients were hospitalized, including one with comorbidities who died.

Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic cross-reactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression "Logitboost" as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests.

The duration of anti-yellow fever (YF) virus immunoglobulin M (IgM) antibodies following YF vaccination is unknown, making it difficult to interpret positive IgM antibody results in previously vaccinated travelers. We evaluated the frequency and predictors of YF IgM antibody positivity 3-4 years following YF vaccination. Twenty-nine (73%) of 40 participants had YF IgM antibodies 3-4 years postvaccination. No demographic or exposure variables were predictive of YF IgM positivity. However, persons who were YF IgM positive at 3-4 years postvaccination had earlier onset viremia and higher neutralizing antibody geometric mean titers at 1 month and 3-4 years postvaccination compared with persons who were YF IgM negative. Detection of YF IgM antibodies several years postvaccination might reflect remote YF vaccination rather than recent YF vaccination or YF virus infection.

We surveyed state public health and commercial diagnostic reference laboratories regarding current testing practices for West Nile virus (WNV). The majority of WNV testing is now performed in commercial diagnostic reference laboratories using commercially available Food and Drug Administration-cleared kits labeled for the presumptive diagnosis of WNV. However, only 25% of surveyed state public health or commercial diagnostic reference laboratories currently have the capacity to perform the recommended confirmatory testing. These findings indicate the need for both manufacturers and laboratories to monitor the performance of these WNV test kits. Further, clinicians should be aware of the limitations of these kits and the need for additional testing to confirm a diagnosis of WNV disease.

From September through early December 2005, an outbreak of yellow fever (YF) occurred in South Kordofan, Sudan, resulting in a mass YF vaccination campaign. In late December 2005, we conducted a serosurvey to assess YF vaccine coverage and to better define the epidemiology of the outbreak in an index village. Of 552 persons enrolled, 95% reported recent YF vaccination, and 25% reported febrile illness during the outbreak period: 13% reported YF-like illness, 4% reported severe YF-like illness, and 12% reported chikungunya-like illness. Of 87 persons who provided blood samples, all had positive YF serologic results, including three who had never been vaccinated. There was also serologic evidence of recent or prior chikungunya virus, dengue virus, West Nile virus, and Sindbis virus infections. These results indicate that YF virus and chikungunya virus contributed to the outbreak. The high prevalence of YF antibody among vaccinees indicates that vaccination was effectively implemented in this remotely located population.

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, is endemic in Africa and Asia. In 2005-2006, CHIKV epidemics were reported in islands in the Indian Ocean and in southern India. We present data on laboratory-confirmed CHIKV infections among travelers returning from India to the United States during 2006.