Seroprevalence of Heartland Virus Antibodies in Blood Donors, Northwestern Missouri, USA.

We estimated the seroprevalence of Heartland virus antibodies to be 0.9% (95% CI 0.4%-4.2%) in a convenience sample of blood donors from northwestern Missouri, USA, where human cases and infected ticks have been identified. Although these findings suggest that some past human infections were undetected, the estimated prevalence is low.

Ability To Serologically Confirm Recent Zika Virus Infection in Areas with Varying Past Incidence of Dengue Virus Infection in the United States and U.S. Territories in 2016.

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.

Measuring Haitian children's exposure to chikungunya, dengue and malaria.

OBJECTIVE: To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti. METHODS: We used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinant Plasmodium falciparum antigens. Most (217) of the blood samples investigated were collected longitudinally, from each of 61 children, between 2011 and 2014 but another 127 were collected from a cross-sectional sample of children in 2014. FINDINGS: Of the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Léogâne and towards the ocean. CONCLUSION: Serological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015.

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Methods for detection of West Nile virus antibodies in mosquito blood meals.

We describe and compare 2 qualitative serologic techniques for detecting West Nile virus (WNV)-specific antibodies in mosquito blood meals. The techniques are the biotin microsphere immunoassay (b-MIA) and the inhibition platform of the VectorTest™ WNV antigen assay (VecTest-inhibition). To demonstrate the ability of these tests to detect WNV-neutralizing antibodies, we experimentally exposed feeding mosquitoes to blood containing 5 concentrations of 6B6C-1, a flavivirus-neutralizing monoclonal antibody. Antibody concentrations were quantified using the 90% plaque-reduction neutralization test (PRNT90). After 24 h of blood-meal digestion at 22.5°C, the threshold PRNT90 titer of detection was ≤18 for b-MIA and ≤50 for VecTest-inhibition. Both tests reliably detected antibodies in 3 of 3 blood meals that had been digested for up to 30 h, or were about 25% digested. The b-MIA was also applied to mosquitoes that had engorged on avian blood in Arizona following a WNV epidemic in 2010. There was no significant difference in the WNV antibody prevalence determined by b-MIA (52% of 71 avian blood meals) compared to the WNV-neutralizing antibody prevalence in birds determined by direct sampling (49% of 234 birds). VecTest-inhibition requires fewer resources and may be used in the field without a laboratory, but consumes the entire blood meal and relies on subjective interpretation of results. The b-MIA requires a laboratory and sophisticated equipment and reagents. Results for b-MIA are analyzed objectively and can be applied to mosquito blood meals with greater confidence than the VecTest-inhibition method and thus can contribute substantially to research and surveillance programs that would benefit from the detection of specific WNV antibodies in mosquito blood meals.

Duration of West Nile Virus Immunoglobulin M Antibodies up to 81 Months Following West Nile Virus Disease Onset.

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.

Laboratory evaluation of RealStar Yellow Fever Virus RT-PCR kit 1.0 for potential use in the global yellow fever laboratory network.

BACKGROUND: Early detection of human yellow fever (YF) infection in YF-endemic regions is critical to timely outbreak mitigation. African National Laboratories chiefly rely on serological assays that require confirmation at Regional Reference Laboratories, thus delaying results, which themselves are not always definitive often due to antibody cross-reactivity. A positive molecular test result is confirmatory for YF; therefore, a standardized YF molecular assay would facilitate immediate confirmation at National Laboratories. The WHO-coordinated global Eliminate Yellow Fever Epidemics Laboratory Technical Working Group sought to independently evaluate the quality and performance of commercial YF molecular assays relevant to use in countries with endemic YF, in the absence of stringent premarket assessments. This report details a limited laboratory WHO-coordinated evaluation of the altona Diagnostics RealStar Yellow Fever Virus RT-PCR kit 1.0. METHODOLOGY AND PRINCIPAL FINDINGS: Specific objectives were to assess the assay's ability to detect YF virus strains in human serum from YF-endemic regions, determine the potential for interference and cross-reactions, verify the performance claims as stated by the manufacturer, and assess usability. RNA extracted from normal human serum spiked with YF virus showed the assay to be precise with minimal lot-to-lot variation. The 95% limit of detection calculated was approximately 1,245 RNA copies/ml [95% confidence interval 497 to 1,640 copies/ml]. Positive results were obtained with spatially and temporally diverse YF strains. The assay was specific for YF virus, was not subject to endogenous or exogenous interferents, and was clinically sensitive and specific. A review of operational characteristics revealed that a positivity cutoff was not defined in the instructions for use, but otherwise the assay was user-friendly. CONCLUSIONS AND SIGNIFICANCE: The RealStar Yellow Fever Virus RT-PCR kit 1.0 has performance characteristics consistent with the manufacturer's claims and is suitable for use in YF-endemic regions. Its use is expected to decrease YF outbreak detection times and be instrumental in saving lives.

Seroprevalence and Symptomatic Attack Rate of Chikungunya Virus Infection, United States Virgin Islands, 2014-2015.

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.

Serological Survey for Antibodies to Mosquito-Borne Bunyaviruses Among US National Park Service and US Forest Service Employees.

Serum samples from 295 employees of Great Smoky Mountains National Park (GRSM), Rocky Mountain National Park (ROMO), and Grand Teton National Park with adjacent Bridger-Teton National Forest (GRTE-BTNF) were subjected to serological analysis for mosquito-borne bunyaviruses. The sera were analyzed for neutralizing antibodies against six orthobunyaviruses: La Crosse virus (LACV), Jamestown Canyon virus (JCV), snowshoe hare virus (SSHV), California encephalitis virus, and Trivittatus virus (TVTV) belonging to the California serogroup and Cache Valley virus (CVV) belonging to the Bunyamwera serogroup. Sera were also tested for immunoglobulin (Ig) G antibodies against LACV and JCV by enzyme-linked immunosorbent assay (ELISA). The proportion of employees with neutralizing antibodies to any California serogroup bunyavirus was similar in all three sites, with the prevalence ranging from 28% to 36%. The study demonstrated a seroprevalence of 3% to CVV across the three parks. However, proportions of persons with antibodies to specific viruses differed between parks. Participants residing in the eastern regions had a higher seroprevalence to LACV, with 24% (18/75) GRSM employees being seropositive. In contrast, SSHV seroprevalence was limited to employees from the western sites, with 1.7% (1/60) ROMO and 3.8% (6/160) GRTE-BTNF employees being positive. Seroprevalence to JCV was noted in employees from all sites at rates of 6.7% in GRSM, 21.7% in ROMO, and 15.6% in GRTE-BTNF. One employee each from ROMO (1.7%) and GRTE-BTNF (1.9%) were positive for TVTV. This study also has illustrated the greater sensitivity and specificity of plaque reduction neutralization test compared to IgG ELISA in conducting serosurveys for LACV and JCV.

Multiplex microsphere immunoassays for the detection of IgM and IgG to arboviral diseases.

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.