Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus.

BACKGROUND: The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources. METHODS: We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively. RESULTS: These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/µl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83). CONCLUSIONS: The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas.

Molecular identification of sand flies (Diptera: Psychodidae) in eastern North America by using PCR-RFLP.

Sand flies (Diptera: Psychodidae) are small blood-feeding dipterans that are primary vectors of numerous human and livestock pathogens. Effective surveillance programs with accurate identification tools are critical in development and implementation of modern integrated pest management programs. Although morphological keys are available for North American species, identification can still be challenging owing to the nature of sample preparation and incompatibility with molecular or biochemical-based pathology assays. Further, the potential for introduction of Old World or other exotic species is not accounted for by current keys. Herein, we present the development and validation of a restriction fragment-length polymorphism-based molecular identification method. Specifically, cytochrome c oxidase subunit I, a mitochondrial DNA marker, was used to distinguish two species of adult sand flies indigenous to eastern North America with two exotic species not yet known to occur in the United States.

Risk factors for infection with chikungunya and Zika viruses in southern Puerto Rico: A community-based cross-sectional seroprevalence survey.

Chikungunya virus (CHIKV) caused a large outbreak in Puerto Rico in 2014, followed by a Zika virus (ZIKV) outbreak in 2016. Communities Organized for the Prevention of Arboviruses (COPA) is a cohort study in southern Puerto Rico, initiated in 2018 to measure arboviral disease risk and provide a platform to evaluate interventions. To identify risk factors for infection, we assessed prevalence of previous CHIKV infection and recent ZIKV and DENV infection in a cross-sectional study among COPA participants. Participants aged 1-50 years (y) were recruited from randomly selected households in study clusters. Each participant completed an interview and provided a blood specimen, which was tested by anti-CHIKV IgG ELISA assay and anti-ZIKV and anti-DENV IgM MAC-ELISA assays. We assessed individual, household, and community factors associated with a positive result for CHIKV or ZIKV after adjusting for confounders. During 2018-2019, 4,090 participants were enrolled; 61% were female and median age was 28y (interquartile range [IQR]: 16-41). Among 4,035 participants tested for CHIKV, 1,268 (31.4%) had evidence of previous infection. CHIKV infection prevalence was lower among children 1-10 years old compared to people 11 and older (adjusted odds ratio [aOR] 2.30; 95% CI 1.71-3.08). Lower CHIKV infection prevalence was associated with home screens (aOR 0.51; 95% CI 0.42-0.61) and air conditioning (aOR 0.64; 95% CI 0.54-0.77). CHIKV infection prevalence also varied by study cluster of residence and insurance type. Few participants (16; 0.4%) had evidence of recent DENV infection by IgM. Among 4,035 participants tested for ZIKV, 651 (16%) had evidence of recent infection. Infection prevalence increased with older age, from 7% among 1-10y olds up to 19% among 41-50y olds (aOR 3.23; 95% CI 2.16-4.84). Males had an increased risk of Zika infection prevalence compared with females (aOR 1.31; 95% CI 1.09-1.57). ZIKV infection prevalence also decreased with the presence of home screens (aOR 0.66; 95% CI 0.54-0.82) and air conditioning (aOR 0.69; 95% CI 0.57-0.84). Similar infection patterns were observed for recent ZIKV infection prevalence and previous CHIKV infection prevalence by age, and the presence of screens and air conditioners in the home decreased infection risk from both viruses by as much as 50%.

Investigation of habitat effects on the spatial distribution of Lutzomyia shannoni across heterogeneous environments, with note of respective mosquito species compositions.

Sand flies are small blood feeding dipterans that are primary vectors of numerous human and livestock pathogens. Control efforts are often complex and multidimensional. A fundamental step in the development and implementation of any integrated pest management (IPM) program is the refining of effective surveillance and identification techniques. Before this study, the presence of two species, Lutzomyia shannoni (Dyar) and Lutzomyia vexator (Coquillett) became known in Kentucky and surrounding states. To understand the spatial distribution of these species across heterogeneous landscapes in this region, trapping was conducted at random locations in previously designated habitats. Although a lack of clear data prevented any conclusions to be drawn for the preferences of L. vexator, the species of greater medical and veterinary importance, L. shannoni, was documented to show a strong preference for habitats along the wooded edges of pasture lands. In the region of study, these lands are frequently used in the production of beef cattle and other livestock, reinforcing the veterinary significance of the findings. Because of their familiarity to vector control officials and operators, mosquito species compositions were compared with the recorded abundance of L. shannoni across the varied habitats. The linking of this information should prove useful to management plans for the phlebotomine, if warranted in the future.

Assessment of community support for Wolbachia-mediated population suppression as a control method for Aedes aegypti mosquitoes in a community cohort in Puerto Rico.

Arboviral diseases transmitted by Aedes species mosquitoes pose an increasing public health challenge in tropical regions. Wolbachia-mediated population suppression (Wolbachia suppression) is a vector control method used to reduce Aedes mosquito populations by introducing male mosquitoes infected with Wolbachia, a naturally occurring endosymbiotic bacterium. When Wolbachia-infected male mosquitoes mate with female wild mosquitoes, the resulting eggs will not hatch. Public support is vital to the successful implementation and sustainability of vector control interventions. Communities Organized to Prevent Arboviruses (COPA) is a cohort study to determine the incidence of arboviral disease in Ponce, Puerto Rico and evaluate vector control methods. Focus groups were conducted with residents of COPA communities to gather their opinion on vector control methods; during 2018-2019, adult COPA participants were interviewed regarding their views on Wolbachia suppression; and a follow-up questionnaire was conducted among a subset of participants and non-participants residing in COPA communities. We analyzed factors associated with support for this method. Among 1,528 participants in the baseline survey, median age was 37 years and 63% were female. A total of 1,032 (68%) respondents supported Wolbachia suppression. Respondents with an income of $40,000 or more were 1.34 times as likely [95% CI: 1.03, 1.37] to support Wolbachia suppression than those who earned less than $40,000 annually. Respondents who reported repellant use were 1.19 times as likely to support Wolbachia suppression [95% CI: 1.03, 1.37]. A follow-up survey in 2020 showed that most COPA participants (86%) and non-participants living in COPA communities (84%) supported Wolbachia suppression during and after an educational campaign. The most frequent questions regarding this method were related to its impact on human and animal health, and the environment. Continuous community engagement and education efforts before and during the implementation of novel vector control interventions are necessary to increase and maintain community support.

Circadian activity of Lutzomyia shannoni (Diptera: Psychodidae) during late season population peaks.

The phlebotomine sand fly, Lutzomyia shannoni (Dyar), is considered both zoophillic and anthropophillic and may be a potential vector of human and livestock pathogens. While the known range of this species in North America has recently been expanded, many aspects of its natural history, which are fundamental components for assessments of disease risk or development of integrated pest management programs for the vector, remain unclear. One is the time of day when individuals are active and searching for blood meals. Through the use of a rotating trap mechanism, male L. shannoni were found to be active earlier in the evening (1-4 h after sunset), whereas the majority of female activity occurred after midnight.

Species diversity and relative abundance of phlebotomine sand flies (Diptera: Psychodidae) on three Army installations in the southern United States and susceptibility of a domestic sand fly to infection with Old World Leishmania major.

Leishmania infections in American veterans of Iraq and Afghanistan have raised concern that veterans could serve as reservoirs of Old World parasites for domestic vector populations. A survey of sand flies on three U.S. Army facilities in the southern United States was conducted to identify potential vectors. Five species, including two new state records, are reported for Fort Hood, TX. Very few flies were detected in Fort Bragg, NC. Large numbers of a man-biting species, Lutzomyia shannoni, were trapped on Fort Campbell, KY. Weekly activity patterns for dominant species are presented. In addition, an infection experiment was conducted to determine if a domestic sand fly is susceptible to infection with Old World Leishmania major. Lu. shannoni became infected and supported Le. major up to 6 days postprandial. Metacyclogenesis and actual transmission of Le. major to an uninfected mouse did not occur because infected flies did not take subsequent blood meals.

Quantitative Analysis of Aedes albopictus Movement Behavior Following Sublethal Exposure To Prallethrin.

The pyrethroid prallethrin, an AI in DUET™ (Clarke Mosquito Control, St. Charles, IL), is widely marketed ultra-low volume (ULV) mosquito adulticide. Volatilized prallethrin is intended to stimulate mosquito flight, increasing its adulticide effectiveness. However, field tests using volatilized prallethrin have not produced significant differences in mosquito trap catches, leading to questions regarding prallethrin's behavioral impact efficacy. Thus, we conducted laboratory tests of prallethrin's effect on flight behavior of adult female Asian tiger mosquitoes, Aedes albopictus. Mosquitoes were divided into 3 groups: untreated control, exposed to volatilized prallethrin, and exposed to a liquid spray calibrated to simulate a ULV application at label rates. After exposure, mosquito behavior in an airstream of 0.5 m/sec was recorded and analyzed using motion-tracking software. No significant differences in flight behavior were found between the control and treated mosquitoes exposed to volatilized prallethrin. The ULV-sprayed mosquitoes exhibited a significant increase in the number of flight events, the turning frequency, overall movement speed, and flight speed compared to the control-a significant difference in locomotor stimulation response that would increase exposure to a ULV spray cloud. However, our results showed that volatilization alone was insufficient to increase ULV efficacy in the field and suggested that incorporating a more volatile flight stimulant into ULV adulticides would provide a measurable improvement in mosquito control.

Ochlerotatus japonicus japonicus in the state of Kentucky.

Ochlerotatus j. japonicus (Theobald) was first collected and identified in 2003 from CDC gravid trap collections in Boone, Campbell, and Wolfe counties, Kentucky. Subsequent collections during 2004 and 2005 indicate that the species has become established in Boone, Campbell, Fayette, Fleming, Grant, Jefferson, and Kenton counties.

Wax structures of Scymnus louisianae attenuate aggression from aphid-tending ants.

The cuticular wax structures of Scymnus louisianae J. Chapin larvae were investigated as a defense against ant aggression by Lasius neoniger Emery. The presence of wax structures provided significant defense against ant aggression compared with denuded larvae in that these structures attenuated the aggressive behavior of foraging ants. Furthermore, reapplication of wax dissolved in hexane partially restored defenses associated with intact structures, showing an attenuation of aggression based in part on cuticular wax components rather than solely on physical obstruction to ant mouthparts.