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Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort.Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens.Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks.Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Background Whether young adults who are infected with SARS-CoV-2 are at risk of subsequent infection is uncertain. We investigated the risk of subsequent SARS-CoV-2 infection among young adults seropositive for a previous infection. Methods This analysis was performed as part of the prospective COVID-19 Health Action Response for Marines study (CHARM). CHARM included predominantly male US Marine recruits, aged 18–20 years, following a 2-week unsupervised quarantine at home. After the home quarantine period, upon arrival at a Marine-supervised 2-week quarantine facility (college campus or hotel), participants were enrolled and were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a dilution of 1:150 or more on receptor-binding domain and full-length spike protein ELISA. Participants also completed a questionnaire consisting of demographic information, risk factors, reporting of 14 specific COVID-19-related symptoms or any other unspecified symptom, and brief medical history. SARS-CoV-2 infection was assessed by PCR at weeks 0, 1, and 2 of quarantine and participants completed a follow-up questionnaire, which included questions about the same COVID-19-related symptoms since the last study visit. Participants were excluded at this stage if they had a positive PCR test during quarantine. Participants who had three negative swab PCR results during quarantine and a baseline serum serology test at the beginning of the supervised quarantine that identified them as seronegative or seropositive for SARS-CoV-2 then went on to basic training at Marine Corps Recruit Depot—Parris Island. Three PCR tests were done at weeks 2, 4, and 6 in both seropositive and seronegative groups, along with the follow-up symptom questionnaire and baseline neutralising antibody titres on all subsequently infected seropositive and selected seropositive uninfected participants (prospective study period). Findings Between May 11, 2020, and Nov 2, 2020, we enrolled 3249 participants, of whom 3168 (98%) continued into the 2-week quarantine period. 3076 (95%) participants, 2825 (92%) of whom were men, were then followed up during the prospective study period after quarantine for 6 weeks. Among 189 seropositive participants, 19 (10%) had at least one positive PCR test for SARS-CoV-2 during the 6-week follow-up (1·1 cases per person-year). In contrast, 1079 (48%) of 2247 seronegative participants tested positive (6·2 cases per person-year). The incidence rate ratio was 0·18 (95% CI 0·11–0·28; p

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic’s Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92–2.41) and 10:1 (CT increase of 3.03–3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer’s setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic’s SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.

Simple Summary The influence of environmental and physiological factors on various aspects of normal mosquito behavior is unclear. The efficacy of repellents against mosquitoes depends on the vector species as well as the concentration and formulation of the repellent. In this study, we observed the behavioral responses of two night-biting malaria vectors in Thailand, Anopheles minimus and Anopheles dirus, during daytime and nighttime. We demonstrated that time of observation has a considerable impact on behavioral responses for both species. When optimizing an excito-repellency assay system, time of observation-based testing should be considered in order to prevent an under- or overestimation of behavioral responses. Abstract Information on factors influencing the behavioral responses of mosquitoes to repellents is lacking and poorly understood, especially in the Anopheles species, night-biting mosquitoes. Our goal was to investigate the impact of different time periods on circadian activity and behavioral responses of two malaria vectors, Anopheles minimus and An. dirus, to 5% DEET using an excito-repellency test system. Each mosquito species was exposed to the repellent during the daytime (06.00–18.00) and nighttime (18.00–06.00), and time of observation was further divided into four 3-h intervals. Significant escape responses were observed between daytime and nighttime for An. minimus in both noncontact and contact tests. An. dirus showed statistical differences in contact irritancy escape response, whereas no significant difference was found in noncontact repellency tests. Both mosquito species showed more significantly higher escape responses when exposed to DEET during the afternoon and late in the night. This finding indicates that the time of testing may affect the behavioral responses of mosquitoes to repellents, especially in An. minimus and An. dirus. A better understanding of nocturnally active mosquito behavioral responses spanning from dusk to dawn would assist in optimizing product development, screening, and effective evaluation.

Whether young adults who are infected with SARS-CoV-2 are at risk of subsequent infection is uncertain. We investigated the risk of subsequent SARS-CoV-2 infection among young adults seropositive for a previous infection.This analysis was performed as part of the prospective COVID-19 Health Action Response for Marines study (CHARM). CHARM included predominantly male US Marine recruits, aged 18-20 years, following a 2-week unsupervised quarantine at home. After the home quarantine period, upon arrival at a Marine-supervised 2-week quarantine facility (college campus or hotel), participants were enrolled and were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a dilution of 1:150 or more on receptor-binding domain and full-length spike protein ELISA. Participants also completed a questionnaire consisting of demographic information, risk factors, reporting of 14 specific COVID-19-related symptoms or any other unspecified symptom, and brief medical history. SARS-CoV-2 infection was assessed by PCR at weeks 0, 1, and 2 of quarantine and participants completed a follow-up questionnaire, which included questions about the same COVID-19-related symptoms since the last study visit. Participants were excluded at this stage if they had a positive PCR test during quarantine. Participants who had three negative swab PCR results during quarantine and a baseline serum serology test at the beginning of the supervised quarantine that identified them as seronegative or seropositive for SARS-CoV-2 then went on to basic training at Marine Corps Recruit Depot-Parris Island. Three PCR tests were done at weeks 2, 4, and 6 in both seropositive and seronegative groups, along with the follow-up symptom questionnaire and baseline neutralising antibody titres on all subsequently infected seropositive and selected seropositive uninfected participants (prospective study period).Between May 11, 2020, and Nov 2, 2020, we enrolled 3249 participants, of whom 3168 (98%) continued into the 2-week quarantine period. 3076 (95%) participants, 2825 (92%) of whom were men, were then followed up during the prospective study period after quarantine for 6 weeks. Among 189 seropositive participants, 19 (10%) had at least one positive PCR test for SARS-CoV-2 during the 6-week follow-up (1·1 cases per person-year). In contrast, 1079 (48%) of 2247 seronegative participants tested positive (6·2 cases per person-year). The incidence rate ratio was 0·18 (95% CI 0·11-0·28; p0·001). Among seropositive recruits, infection was more likely with lower baseline full-length spike protein IgG titres than in those with higher baseline full-length spike protein IgG titres (hazard ratio 0·45 [95% CI 0·32-0·65]; p0·001). Infected seropositive participants had viral loads that were about 10-times lower than those of infected seronegative participants (ORF1ab gene cycle threshold difference 3·95 [95% CI 1·23-6·67]; p=0·004). Among seropositive participants, baseline neutralising titres were detected in 45 (83%) of 54 uninfected and in six (32%) of 19 infected participants during the 6 weeks of observation (ID50 difference p0·0001).Seropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies.Defense Health Agency and Defense Advanced Research Projects Agency.

Background: The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subsequent infection among seropositive young adults was studied prospectively. Methods: The study population comprised 3,249 predominantly male, 18-20-year-old Marine recruits. Upon arrival at a Marine-supervised two-week quarantine, participants were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a 1:150 dilution or greater on receptor binding domain and full-length spike protein enzyme-linked immunosorbent (ELISA) assays. SARS-CoV-2 infection was assessed by PCR at initiation, middle and end of the quarantine. After appropriate exclusions, including participants with a positive PCR during quarantine, we performed three biweekly PCR tests in both seropositive and in seronegative groups once recruits left quarantine and entered basic training and baseline neutralizing antibody titers on all subsequently infected seropositive and selected seropositive uninfected participants. Findings: Among 189 seropositive participants, 19 (10.1%) had at least one positive PCR test for SARS-CoV-2 during the six-week follow-up (1.1 cases per person-year). In contrast, 1,079 (48.0%) of the 2,247 seronegative participants tested positive (6.2 cases per person-year). The incidence rate ratio was 0.18 (95% CI 0.11-0.28, p

The Naval Infectious Diseases Diagnostic Laboratory (NIDDL) serves as a reference clinical laboratory that supports Department of Defense (DoD) military treatment facilities worldwide in the detection and identification of high-risk and emerging infectious diseases. Since the emergence of Zika virus (ZIKV) in the Western Hemisphere in 2016, the NIDDL has been a central hub for ZIKV testing for DoD personnel and beneficiaries. Samples collected during patients' clinical evaluations were screened for evidence of possible exposure to ZIKV using molecular and serological methods. An in-house ZIKV plaque reduction neutralization test was used to confirm the presence of ZIKV immunoglobulin M antibody. Of 1,420 individuals tested, ZIKV infection was confirmed by quantitative real-time polymerase chain reaction (PCR) assay in 11 (0.8%); an additional 26 recent flaviviral infections (possibly ZIKV) were identified based on serology (1.8%). These findings contribute to the understanding of the burden of ZIKV infections among DoD personnel and beneficiaries and highlight the role of the NIDDL in clinical diagnosis during emerging infectious disease outbreaks.

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Mosquito behavior is heavily influenced by the chemical molecules in the environment. This knowledge can be used to modify insect behaviors; particularly to reduce vector-host contact as a powerful method for disease prevention. N,N-Diethyl-meta-toluamide (DEET) is the most widely used insect repellent in the market and an excellent example of a chemical that has been used to modify insect behavior for disease prevention. However, genetic insensitivity and habituation in Aedes aegypti (L.) mosquitoes after preexposure to DEET have been reported. In this study, we investigated the effect of preexposure to DEET on the downstream blood-feeding behavior of Ae. aegypti mosquitoes and the duration of the effect. We exposed mosquitoes to four different DEET concentrations: 0.10, 0.12, 0.14, and 0.16% for 10 min then allowed the mosquitoes to blood-feed on an artificial blood-feeding system either immediately or after being held for 1, 3, 6, or 24 h following DEET exposure. We found that preexposing Ae. aegypti mosquitoes to 0.14 or 0.16% DEET lowered their blood engorgement level, but did not alter their landing and probing behavior when compared to the control test populations. The reduction in complete blood-feeding was observed at all time periods tested, but was only statistically significant at 3 and 6 h after the preexposure process. Because reduction in blood meal has been associated with increased refeeding, future studies analyzing the effect of this behavior using arbovirus-infected mosquitoes are needed to address the concern of potentially increased vectorial capacity.

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

No licensed vaccine or antiviral drug against dengue virus (DENV) is available; therefore, most of the effort to prevent this disease is focused on reducing vector-host interactions. One of the most widely accepted methods of blocking vector-human contact is to use insect repellents to interfere with mosquito host-seeking behavior. Some arboviruses can replicate in the nervous system of the vector, raising the concern that arboviral infection may alter the insect behavioral response toward chemical stimuli. Three different Aedes aegypti (L.) mosquito cohorts: DENV-1-injected, diluent-injected, and uninjected were subjected to behavioral tests using a high-throughput screening system with 2.5% DEET and 0.14% DEET on 1, 4, 7, 10, 14, and 17 d postinjection. All test cohorts exhibited significant contact irritant or escape responses when they were exposed to 2.5% or 0.14% DEET. However, we found no biologically relevant irritancy response change in DENV-1-infected Ae. aegypti mosquitoes when they were exposed to DEET. Further studies evaluating the effects of other arboviral infections on insect repellents activity are necessary in order to provide better recommendation on the prevention of vector-borne disease transmission.