One Confirmed and 2 Suspected Cases of Heartland Virus Disease.

Heartland virus is a tickborne phlebovirus first identified in Missouri in 2009; 11 human cases have been reported in the literature. Reported hallmarks of infection have included fever, malaise, anorexia, gastrointestinal complaints, thrombocytopenia, neutropenia, and aminotransferase elevations. We report 1 confirmed and 2 suspected cases and discuss implications for case-finding.

Heartland Virus in Lone Star Ticks, Alabama, USA.

We detected Heartland virus (HRTV) in lone star nymphs collected in 2018 in northern Alabama, USA. Real-time reverse transcription PCR selective for the small segment of the HRTV genome and confirmatory sequencing of positive samples showed high identity with HRTV strains sequenced from Tennessee and Missouri.

Targeted Metagenomics for Clinical Detection and Discovery of Bacterial Tick-Borne Pathogens.

Tick-borne diseases, due to a diversity of bacterial pathogens, represent a significant and increasing public health threat throughout the Northern Hemisphere. A high-throughput 16S V1-V2 rRNA gene-based metagenomics assay was developed and evaluated using >13,000 residual samples from patients suspected of having tick-borne illness and >1,000 controls. Taxonomic predictions for tick-borne bacteria were exceptionally accurate, as independently validated by secondary testing. Overall, 881 specimens were positive for bacterial tick-borne agents. Twelve tick-borne bacterial species were detected, including two novel pathogens, representing a 100% increase in the number of tick-borne bacteria identified compared to what was possible by initial PCR testing. In three blood specimens, two tick-borne bacteria were simultaneously detected. Seven bacteria, not known to be tick transmitted, were also confirmed to be unique to samples from persons suspected of having tick-borne illness. These results indicate that 16S V1-V2 metagenomics can greatly simplify diagnosis and accelerate the discovery of bacterial tick-borne pathogens.

A standardized method for the construction of a tick drag/flag sampling approach and evaluation of sampling efficacy.

Drag sampling and flagging are two of the most effective and widely applied techniques to monitor tick populations. Despite the importance of this sampling strategy, there is a lack of standardized protocols for the construction of an inexpensive tick drag/flag. To this end, we provide a step-by-step protocol that details the construction of a tick drag/flag. We provide evidence of efficacy by comparing results obtained over 3-months at 108 locations within the William B. Bankhead National Forest, Alabama, USA. Overall, our drag/flag sampling approach yielded 1127 larvae, 460 nymphs, and 53 adults for a total of 1640 ticks representing three species. We detected significant patterns in Amblyomma americanum abundance for nymphs and adults with greater counts in June (ß = 0.91 ± 0.36, 95% CI 0.55-1.27; ß = 2.44 ± 0.63, 95% CI 1.81-3.07, respectively) and July (ß = 0.73 ± 0.36, 95% CI 0.37-1.09; ß = 1.65 ± 0.66, 95% CI 0.99-2.31, respectively) as compared to August. We also detected a significant difference in tick captures by tick drag/flag fabric type with greater captures when muslin was used as compared to flannel (ß = 1.07 ± 0.06, 95% CI 1.01-1.13). Our goal is to provide instructions to assemble a highly effective tick drag/flag using minimal supplies. Evaluation and improvements of sampling techniques is essential to understand impacts of landscape management and larger stressors, such as climate change on tick populations but also for enhancing detection of invasive non-native species.

Chagas Disease Surveillance Activities - Seven States, 2017.

Chagas disease, a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi, has become a concern in the United States as a result of human emigration from Latin America where Chagas disease is endemic (1). It is estimated that as many as 8 million people living in Mexico, and Central and South America have Chagas disease.* Most cases of Chagas disease in the United States are chronic infections; however, rare cases of acute congenital infections and autochthonous vectorborne transmission have been reported (2). To understand how data are collected and used, a review of state-level public health surveillance for Chagas disease was conducted through semistructured interviews with health officials in six states (Arizona, Arkansas, Louisiana, Mississippi Tennessee, and Texas) where Chagas disease is reportable and one (Massachusetts) where it was previously reportable. States implemented surveillance in response to blood donor screening for Chagas disease and to identify the route of disease transmission. Many states reported primarily chronic cases and had limited ability to respond to local transmission because acute cases were infrequently reported. Surveillance remains important in states with large populations of immigrants or frequent travelers from countries with endemic disease and for states with a risk for local transmission. Surveillance efforts can also help increase awareness among providers and assist in linking patients with Chagas disease to treatment to help prevent cardiac and gastrointestinal complications.

Novel Clinical and Pathologic Findings in a Heartland Virus-Associated Death.

We describe an investigation into a Heartland virus (HRTV)-associated death in Tennessee with novel clinical and pathologic findings. HRTV can cause rapidly fatal, widely disseminated infection with multisystem organ failure in patients without substantial comorbidities. We identified viral antigen in multiple organ tissues where it was not detected previously.

Comparative sequence analyses of La Crosse virus strain isolated from patient with fatal encephalitis, Tennessee, USA.

We characterized a La Crosse virus (LACV) isolate from the brain of a child who died of encephalitis-associated complications in eastern Tennessee, USA, during summer 2012. We compared the isolate with LACV sequences from mosquitoes collected near the child's home just after his postmortem diagnosis. In addition, we conducted phylogenetic analyses of these and other sequences derived from LACV strains representing varied temporal, geographic, and ecologic origins. Consistent with historical findings, results of these analyses indicate that a limited range of LACV lineage I genotypes is associated with severe clinical outcomes.

La Crosse Encephalitis Virus Infection in Field-Collected Aedes albopictus, Aedes japonicus, and Aedes triseriatus in Tennessee.

La Crosse virus (LACV) is a mosquito-borne virus and a major cause of pediatric encephalitis in the USA. La Crosse virus emerged in Tennessee and other states in the Appalachian region in 1997. We investigated LACV infection rates and seasonal abundances of the native mosquito vector, Aedes triseriatus, and 2 recently introduced mosquito species, Ae. albopictus and Ae. japonicus, in an emerging disease focus in Tennessee. Mosquitoes were collected using multiple trapping methods specific for Aedes mosquitoes at recent human case sites. Mosquito pools were tested via reverse transcriptase-polymerase chain reaction (RT-PCR) of the S segment to detect multiple Bunyamwera and California serogroup viruses, including LACV, as well as real-time RT-PCR of the M segment. A total of 54 mosquito pools were positive, including wild-caught adult females and laboratory-reared adults, demonstrating transovarial transmission in all 3 species. Maximum likelihood estimates (per 1,000 mosquitoes) were 2.72 for Ae. triseriatus, 3.01 for Ae. albopictus, and 0.63 for Ae. japonicus. We conclude that Ae. triseriatus and Ae. albopictus are important LACV vectors and that Ae. japonicus also may be involved in virus maintenance and transmission.

Seroepidemiologic study of human infections with spotted fever group Rickettsiae in North Carolina.

Increasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsii are responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was conducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickettsia parkeri, and Rickettsia amblyommii antigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more antigens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsii are associated with illness among North Carolina residents and that serologic testing using R. rickettsii antigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

An outbreak of bed bug infestation in an office building.

Since 2000, resurgence in bed bugs has occurred in the U.S. Reports of infestations of homes, hospitals, hotels, and offices have been described. On September 1, 2011, complaints of itching and bites among workers in an office were reported to the Tennessee Department of Health. A retrospective cohort study and environmental assessments were performed in response to the complaints. Canines certified to detect live bed bugs were used to inspect the office and arthropod samples were collected. Of 76 office workers, 61 (80%) were interviewed; 39 (64%) met the case definition. Pruritic maculopapular lesions were consistent with arthropod bites. One collected arthropod sample was identified as a bed bug by three entomologists. Exposures associated with symptoms included working in a cubicle in which a canine identified bed bugs (risk ratio [RR]: 1.8; 95% confidence interval [CI]: 1.3-3.6), and self-reported seasonal allergies (RR: 1.6, 95% CI: 1.0-2.4). Bed bugs represent a reemerging and challenging environmental problem with clinical, psychological, and financial impacts.

Persistent spatial clustering and predictors of pediatric La Crosse virus neuroinvasive disease risk in eastern Tennessee and western North Carolina, 2003-2020.

The combined region of eastern Tennessee and western North Carolina has a persistently high risk of pediatric La Crosse virus neuroinvasive disease (LACV-ND). To guide public health intervention in this region, the objectives of this retrospective ecological study were to investigate the geographic clustering and predictors of pediatric LACV-ND risk at the ZIP code tabulation area (ZCTA) level. Data on pediatric cases of LACV-ND reported between 2003 and 2020 were obtained from Tennessee Department of Health and North Carolina Department of Health and Human Services. Purely spatial and space-time scan statistics were used to identify ZCTA-level clusters of confirmed and probable pediatric LACV-ND cases from 2003-2020, and a combination of global and local (i.e., geographically weighted) negative binomial regression models were used to investigate potential predictors of disease risk from 2015-2020. The cluster investigation revealed spatially persistent high-risk and low-risk clusters of LACV-ND, with most cases consistently reported from a few high-risk clusters throughout the entire study period. Temperature and precipitation had positive but antagonistic associations with disease risk from 2015-2020, but the strength of those relationships varied substantially across the study area. Because LACV-ND risk clustering in this region is focally persistent, retroactive case surveillance can be used to guide the implementation of targeted public health intervention to reduce the disease burden in high-risk areas. Additional research on the role of climate in LACV transmission is warranted to support the development of predictive transmission models to guide proactive public health interventions.

Utilization of a zoo for mosquito (Diptera: Culicidae) diversity analysis, arboviral surveillance, and blood feeding patterns.

Zoos provide a unique opportunity to study mosquito feeding ecology as they represent areas where exotic animals, free-roaming native animals, humans, and mosquito habitats overlap. Therefore, these locations are a concern for arbovirus transmission to both valuable zoo animals and human visitors. We sampled mosquitoes in and around The Nashville Zoo at Grassmere in Tennessee, USA, over 4 months in 2020 using 4 mosquito trap methods and 12 sampling locations. Mosquitoes were identified to species, Culex mosquitoes were analyzed for arboviruses, and all engorged mosquitoes were preserved for host usage analysis. We captured over 9,000 mosquitoes representing 27 different species, including a new species record for Davidson County, TN (Culex nigripalpus Theobald). Minimum infection rates for West Nile virus (WNV) (Flaviviridae: Flavivirus), St. Louis encephalitis virus (Flaviviridae: Flavivirus), and Flanders virus (Hapavirus: Rhabdoviridae) were 0.79, 0, and 4.17, respectively. The collection of 100 engorged mosquitoes was dominated by Culex pipiens pipiens Linnaeus (38%), Culex erraticus Dyar and Knab (23%), and Culex pipiens pipiens-Culex pipiens quinquefasciatus hybrids (10%). Host DNA from 84 engorged mosquitoes was successfully matched to a variety of host species (n = 23), with just 8 species belonging to the zoo. Wild birds were the most frequently fed upon host, in particular northern cardinals (Cardinalis cardinalis L. Passeriformes: Cardinalidae), which are competent WNV reservoirs. Taken together, our results demonstrate the utility of zoos as sentinels for emerging pathogens, for studying wildlife and human risk of zoonotic diseases, and for assessing vector diversity.

West Nile Virus and Epizootic Hemorrhagic Disease Virus Co-Infection in a Novel Host at the Nashville Zoo.

On August 30, 2017, one of five bontebok in a mixed-species exhibit at the Nashville Zoo at Grassmere exhibited acute hind-limb ataxia and altered demeanor. Pathological examination demonstrated meningoencephalitis and spinal myelitis. Coinfection of West Nile virus (WNV) and epizootic hemorrhagic disease virus (EHDV) was revealed by quantitative real-time and traditional reverse transcription-polymerase chain reaction assays and virus isolation/whole genome sequencing from brain tissue, respectively. Whole genome sequencing was conducted for EHDV. Mosquito testing from September 19 to October 13, 2017, demonstrated a higher WNV infection rate in mosquitoes at the zoo compared with the rest of Nashville-Davidson County. EHDV is endemic in wild white-tailed deer (family Cervidae) in Tennessee, and the prevalence in wildlife depends on environmental influences. This case illustrates the potential susceptibility of exotic zoo animals to endemic domestic arthropod-borne viruses (arboviruses) and reinforces the importance of cooperative antemortem and postmortem surveillance strategies among human, wildlife, and domestic animal health agencies.

Eastern equine encephalitis in Tennessee: 2002-2008.

Human and equine outbreaks caused by eastern equine encephalomyelitis virus (EEEV) typically occur in North America adjacent to coastal wetlands associated with the presence of Culiseta melanura (Coquillet) mosquitoes. Eastern equine encephalomyelitis (EEE) is an emerging disease in Tennessee, as the first records of equine disease began in 2002. In 2006 we trapped and tested mosquitoes for EEEV at hardwood swamps in western Tennessee that were at the epicenter of a multi-equine outbreak in fall of 2005. Additionally, the Tennessee Valley Authority tested mosquito pools collected in Tennessee swamps from 2000 to 2007 for the presence of arboviruses. Two pools of EEEV positive Culex erraticus (Dyer and Knab) mosquitoes were found (one each in 2003 and 2004) in a county adjacent to where the 2005 outbreak occurred. In 2008, another EEE outbreak involving multiple horses occurred in West Tennessee. A brain specimen was collected from a horse during this outbreak and the first isolate of EEEV from Tennessee was obtained. In total, 74,531 mosquitoes collected from 2000 to 2008 were tested via polymerase chain reaction and VecTest for EEEV. The traditional enzootic vector, Cs. melanura, was found in low numbers at all collection sites. Cx. erraticus, however, was consistently found in high numbers and was the only mosquito species in which EEEV was detected. We suggest that EEE transmission may be maintained by Cx. erraticus in a nontraditional cycle. We discuss the importance of a nontraditional cycle from the perspective of EEEV adaptation and emergence.

La Crosse virus spread within the mosquito population in Knox County, TN.

In Appalachia, La Crosse virus (LACV) is a leading pediatric arbovirus and public health concern for children under 16 years. LACV is transmitted via the bite of an infected Aedes mosquito. Thus, it is imperative to understand the dynamics of the local vector population in order to assess risk and transmission. Using entomological data collected from Knox County, Tennessee in 2013, we formulate an environmentally-driven system of ordinary differential equations to model mosquito population dynamics over a single season. Further, we include infected compartments to represent LACV transmission within the mosquito population. Findings suggest that the model, with dependence on degree days and accumulated precipitation, can closely describe field data. This model confirms the need to include these environmental variables when planning control strategies.

Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus.

The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/µL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Assessment of Risk Factors and Outcomes of Severe Ehrlichiosis Infection.

Importance: Ehrlichiosis cases in the US have increased more than 8-fold since 2000. Up to 57% of patients with ehrlichiosis require hospitalization and 11% develop a life-threatening complication; however, risk factors for serious disease are not well documented. Objective: To examine risk factors associated with severe ehrlichiosis. Design, Setting, and Participants: An analytic cross-sectional study of patients diagnosed with ehrlichiosis by polymerase chain reaction (PCR) between January 1, 2007, and December 31, 2017, was conducted in a single tertiary-care center in a region endemic for ehrlichiosis. Analysis was performed from February 27, 2018, to September 9, 2020. A total of 407 positive Ehrlichia PCR results were identified from 383 unique patients, with 155 unique patients meeting study criteria. Patients hospitalized at other institutions who had a positive Ehrlichia PCR performed as a reference test (n = 222) were excluded as no clinical data were available. Electronic medical record review was performed to collect demographic, clinical, laboratory, treatment, and outcomes data. Cases were excluded when there were insufficient clinical data to assess the severity of illness (n = 3) and when the clinical illness did not meet the case definition for ehrlichiosis (n = 3). Exposures: Date of presentation, onset of symptoms, date of PCR testing, date of treatment initiation, site of care, age, birth sex, race/ethnicity, Charlson Comorbidity Index, trimethoprim with sulfamethoxazole use within the prior 2 weeks, and immunosuppression. Main Outcomes and Measures: Requirement for intensive care unit (ICU) admission. Results: Of the 155 patients who met inclusion criteria, 99 patients (63.9%) were men, and 145 patients (93.5%) identified as non-Hispanic White; median age was 50 years (interquartile range, 23-64 years). Intensive care unit admission was indicated in 43 patients (27.7%), 94 patients (60.6%) were hospitalized on general medical floors, and 18 patients (11.6%) received care as outpatients. In adjusted analysis, time to treatment initiation was independently associated with an increased risk for ICU admission (adjusted prevalence ratio [aPR], 1.09; 95% CI, 1.04-1.14; P < .001). Documentation of tick exposure was independently associated with a decreased risk for ICU admission (aPR, 0.54; 95% CI, 0.34-0.86; P = .01). There appeared to be a nonsignificant change toward a decreased need for ICU care among immunosuppressed persons (aPR, 0.51; 95% CI, 0.26-1.00; P = .05). Conclusions and Relevance: This study suggests that delay in initiation of doxycycline therapy is a significant factor associated with severe ehrlichiosis. Increased recognition of infection by front-line clinicians to promote early treatment may improve outcomes associated with this increasingly common and life-threatening infection.

Rickettsia parkeri in Amblyomma americanum ticks, Tennessee and Georgia, USA.

To determine the geographic distribution of the newly recognized human pathogen Rickettsia parkeri, we looked for this organism in ticks from Tennessee and Georgia, USA. Using PCR and sequence analysis, we identified R. parkeri in 2 Amblyomma americanum ticks. This rickettsiosis may be underdiagnosed in the eastern United States.

Use of hamster-baited no. 17 Trinidad mosquito traps at an eastern equine encephalomyelitis focus in Tennessee.

We set out no. 17 Trinidad traps baited with hamsters at a swamp in Tennessee, where recent eastern equine encephalomyelitis (EEE) equine outbreaks had occurred, to determine which mosquito species at this site were attracted to these traps. We also set out CO2-baited CDC light traps for comparison. Of 11 species present in CO2-baited CDC light traps, only Culex (Melanoconion) erraticus (Dyar and Knab) blood fed on hamsters in the Trinidad traps. Significantly less Cx. erraticus mosquitoes entered Trinidad traps per trap night (mean = 17.6, median = 6.0) compared with CDC traps (mean = 38.7, median = 35.0). Advantages and disadvantages in using hamster-baited no. 17 Trinidad traps compared with CO2-baited CDC light traps to capture Cx. erraticus mosquitoes are discussed. Further understanding of the feeding behavior and ecology of this potential vector is warranted.