Antibodies to S2 domain of SARS-CoV-2 spike protein in Moderna mRNA vaccinated subjects sustain antibody-dependent NK cell-mediated cell cytotoxicity against Omicron BA.1.

Vaccination with the primary two-dose series of SARS-CoV-2 mRNA protects against infection with the ancestral strain, and limits the presentation of severe disease after re-infection by multiple variants of concern (VOC), including Omicron, despite the lack of a strong neutralizing response to these variants. We compared antibody responses in serum samples collected from mRNA-1273 (Moderna) vaccinated subjects to identify mechanisms of immune escape and cross-protection. Using pseudovirus constructs containing domain-specific amino acid changes representative of Omicron BA.1, combined with domain competition and RBD-antibody depletion, we showed that RBD antibodies were primarily responsible for virus neutralization and variant escape. Antibodies to NTD played a less significant role in antibody neutralization but acted along with RBD to enhance neutralization. S2 of Omicron BA.1 had no impact on neutralization escape, suggesting it is a less critical domain for antibody neutralization; however, it was as capable as S1 at eliciting IgG3 responses and NK-cell mediated, antibody-dependent cell cytotoxicity (ADCC). Antibody neutralization and ADCC activities to RBD, NTD, and S1 were all prone to BA.1 escape. In contrast, ADCC activities to S2 resisted BA.1 escape. In conclusion, S2 antibodies showed potent ADCC function and resisted Omicron BA.1 escape, suggesting that S2 contributes to cross-protection against Omicron BA.1. In line with its conserved nature, S2 may hold promise as a vaccine target against future variants of SARS-CoV-2.

Direct mosquito feedings on dengue-2 virus-infected people reveal dynamics of human infectiousness.

Dengue virus (DENV) transmission from humans to mosquitoes is a poorly documented, but critical component of DENV epidemiology. Magnitude of viremia is the primary determinant of successful human-to-mosquito DENV transmission. People with the same level of viremia, however, can vary in their infectiousness to mosquitoes as a function of other factors that remain to be elucidated. Here, we report on a field-based study in the city of Iquitos, Peru, where we conducted direct mosquito feedings on people naturally infected with DENV and that experienced mild illness. We also enrolled people naturally infected with Zika virus (ZIKV) after the introduction of ZIKV in Iquitos during the study period. Of the 54 study participants involved in direct mosquito feedings, 43 were infected with DENV-2, two with DENV-3, and nine with ZIKV. Our analysis excluded participants whose viremia was detectable at enrollment but undetectable at the time of mosquito feeding, which was the case for all participants with DENV-3 and ZIKV infections. We analyzed the probability of onward transmission during 50 feeding events involving 27 participants infected with DENV-2 based on the presence of infectious virus in mosquito saliva 7-16 days post blood meal. Transmission probability was positively associated with the level of viremia and duration of extrinsic incubation in the mosquito. In addition, transmission probability was influenced by the day of illness in a non-monotonic fashion; i.e., transmission probability increased until 2 days after symptom onset and decreased thereafter. We conclude that mildly ill DENV-infected humans with similar levels of viremia during the first two days after symptom onset will be most infectious to mosquitoes on the second day of their illness. Quantifying variation within and between people in their contribution to DENV transmission is essential to better understand the biological determinants of human infectiousness, parametrize epidemiological models, and improve disease surveillance and prevention strategies.

MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016-2018: results from the MERCURIAL multiyear prospective cohort study.

Prospective cohort study to investigate the potential exposure to the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) following Hajj pilgrims is still very limited. Here, we report the antibody seroconversion study results obtained from successive three years cohort studies (2016-2018) involving the Malaysian Hajj pilgrims returning from the Middle East. A cohort study of Hajj pilgrims from Malaysia enrolled 2,863 participants from 2016-2018, all of whom consented to provide paired blood samples for both pre- and post-Hajj travel to the Middle East. ELISAs and micro-neutralization assays were performed to detect the presence of MERS-CoV IgG antibodies. Sociodemographic data, symptoms experienced during Hajj, and history of exposure to camels or camel products were recorded using structured pre- and post-Hajj questionnaires. A 4-fold increase in anti-MERS-CoV IgG between paired pre-Hajj and post-Hajj serum samples in twelve participants was observed. None of the twelve ELISA-positive sera had detectable levels of virus-neutralizing antibodies. All reportedly had mild symptoms of respiratory symptoms at a certain point during the pilgrimage, implying mild or asymptomatic infections. No association between post-Hajj serum positivity and a history of exposure to camels or camel products was obtained. Findings from the study suggest that serologic conversion to MERS-CoV occurred in at least 0.6% of the Hajj pilgrims returning from the Middle East. Since all the seroconvertants had mild to no symptoms during the sampling period, it highlights the likelihood of occurrence of only low infectivity spillover infections among the Hajj pilgrims.

Etiology and risk factors for diarrheal disease amongst rural and peri-urban populations in Cambodia, 2012-2018.

Diarrheal diseases are a leading cause of mortality and morbidity, disproportionally affecting persons residing in low and middle-income countries. Accessing high-resolution surveillance data to understand community-level etiology and risk remains challenging, particularly in remote and resource limited populations. A multi-year prospective cohort study was conducted in two rural and two peri-urban villages in Cambodia from 2012 to 2018 to describe the epidemiology and etiology of acute diarrheal diseases within the population. Suspected diarrheal episodes among participants were self-reported or detected via routine weekly household visits. Fresh stool and fecal swabs were tested, and acute-illness and follow-up participant questionnaires collected. Of 5027 enrolled participants, 1450 (28.8%) reported at least one diarrheal incident. A total of 4266 individual diarrhea case events were recorded. Diarrhea incidence rate was calculated to be 281.5 persons per 1000 population per year, with an event rate of 664.3 individual diarrhea events occurring per 1000 population per year. Pathogenic Escherichia coli, Aeromonas spp., and Plesiomonas shigelloides were the most prevalent bacterial infections identified. Hookworm and Strongyloides stercoralis were the predominant helminth species, while Blastocystis hominis and Giardia lamblia were the predominant protozoan species found. Norovirus genotype 2 was the predominant virus identified. Mixed infections of two or more pathogens were detected in 36.2% of positive cases. Risk analyses identified unemployed status increased diarrhea risk by 63% (HR = 1.63 [95% CI 1.46, 1.83]). Individuals without access to protected water sources or sanitation facilities were 59% (HR = 1.59 [95% CI 1.49, 1.69]) and 19% (HR = 1.19 [95% CI 1.12, 1.28]) greater risk of contracting diarrhea, respectively. Patient-level surveillance data captured in this long-term study has generated a unique spatiotemporal profile of diarrheal disease in Cambodia. Understanding etiologies, together with associated epidemiological and community-level risk, provides valuable public health insight to support effective planning and delivery of appropriate local population-targeted interventions.

Antibody Responses to the SARS-CoV-2 Ancestral Strain and Omicron Variants in Moderna mRNA-1273 Vaccinated Active-Duty US Navy Sailors and Marines.

Omicron and its subvariants have steadily gained greater capability of immune escape compared to other variants of concern, resulting in an increased incidence of reinfections even among vaccinated individuals. We evaluated the antibody response to Omicron BA.1, BA.2, and BA.4/5 in US military members vaccinated with the primary 2-dose series of Moderna mRNA-1273 in a cross-sectional study. While nearly all vaccinated participants had sustained spike (S) IgG and neutralizing antibodies (ND50) to the ancestral strain, only 7.7% participants had detectable ND50 to Omicron BA.1 at 8 months postvaccination. The neutralizing antibody response to BA.2 and BA.5 was similarly reduced. The reduced antibody neutralization of Omicron correlated with the decreased antibody binding to the receptor-binding domain. The participants' seropositivity to the nuclear protein positively correlated with ND50. Our data emphasizes the need for continuous vigilance in monitoring for emerging variants and the need to identify potential alternative targets for vaccine design.

Multi-omics of NET formation and correlations with CNDP1, PSPB, and L-cystine levels in severe and mild COVID-19 infections.

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics and correlative analysis of an immunologically naïve SARS-CoV-2 clinical cohort from blood plasma of uninfected controls, mild, and severe infections. Consistent with previous observations, severe patient populations showed an elevation of pulmonary surfactant levels. Intriguingly, mild patients showed a statistically significant elevation in the carnosine dipeptidase modifying enzyme (CNDP1). Mild and severe patient populations showed a strong elevation in the metabolite L-cystine (oxidized form of the amino acid cysteine) and enzymes with roles in glutathione metabolism. Neutrophil extracellular traps (NETs) were observed in both mild and severe populations, and NET formation was higher in severe vs. mild samples. Our correlative analysis suggests a potential protective role for CNDP1 in suppressing PSPB release from the pulmonary space whereas NET formation correlates with increased PSPB levels and disease severity. In our discussion we put forward a possible model where NET formation drives pulmonary occlusions and CNDP1 promotes antioxidation, pleiotropic immune responses, and vasodilation by accelerating histamine synthesis.

Quantifying heterogeneities in arbovirus transmission: Description of the rationale and methodology for a prospective longitudinal study of dengue and Zika virus transmission in Iquitos, Peru (2014-2019).

Current knowledge of dengue virus (DENV) transmission provides only a partial understanding of a complex and dynamic system yielding a public health track record that has more failures than successes. An important part of the problem is that the foundation for contemporary interventions includes a series of longstanding, but untested, assumptions based on a relatively small portion of the human population; i.e., people who are convenient to study because they manifest clinically apparent disease. Approaching dengue from the perspective of people with overt illness has produced an extensive body of useful literature. It has not, however, fully embraced heterogeneities in virus transmission dynamics that are increasingly recognized as key information still missing in the struggle to control the most important insect-transmitted viral infection of humans. Only in the last 20 years have there been significant efforts to carry out comprehensive longitudinal dengue studies. This manuscript provides the rationale and comprehensive, integrated description of the methodology for a five-year longitudinal cohort study based in the tropical city of Iquitos, in the heart of the Peruvian Amazon. Primary data collection for this study was completed in 2019. Although some manuscripts have been published to date, our principal objective here is to support subsequent publications by describing in detail the structure, methodology, and significance of a specific research program. Our project was designed to study people across the entire continuum of disease, with the ultimate goal of quantifying heterogeneities in human variables that affect DENV transmission dynamics and prevention. Because our study design is applicable to other Aedes transmitted viruses, we used it to gain insights into Zika virus (ZIKV) transmission when during the project period ZIKV was introduced and circulated in Iquitos. Our prospective contact cluster investigation design was initiated by detecttion of a person with a symptomatic DENV infection and then followed that person's immediate contacts. This allowed us to monitor individuals at high risk of DENV infection, including people with clinically inapparent and mild infections that are otherwise difficult to detect. We aimed to fill knowledge gaps by defining the contribution to DENV transmission dynamics of (1) the understudied majority of DENV-infected people with inapparent and mild infections and (2) epidemiological, entomological, and socio-behavioral sources of heterogeneity. By accounting for factors underlying variation in each person's contribution to transmission we sought to better determine the type and extent of effort needed to better prevent virus transmission and disease.

Clinical and epidemiologic evaluation of a 2020 chikungunya outbreak in Cambodia.

BACKGROUND: In 2020, the Kingdom of Cambodia experienced a nationwide outbreak of chikungunya virus (CHIKV). Despite an increase in the frequency of outbreaks and expanding geographic range of CHIKV, diagnostic challenges remain, and limited surveillance data of sufficient granularity are available to characterize epidemiological profiles and disease dynamics of the virus. METHODS: An ongoing and long-standing cross-sectional study of acute undifferentiated febrile illness (AUFI) in Cambodia was leveraged to describe the disease epidemiology and characterize the clinical presentation of patients diagnosed with CHIKV during the 2020 outbreak. Participants presenting with AUFI symptoms at ten study locations provided acute and convalescent blood samples and were tested for CHIKV using a reverse transcription-polymerase chain reaction (RT-PCR) and serological diagnostic methods including IgM and IgG. Acute and follow-up clinical data were also collected. RESULTS: From 1194 participant blood samples tested, 331 (27.7%) positive CHIKV cases were detected. Most CHIKV positive individuals (280, 84.6%) reported having a fever 3 to 4 days prior to visiting a health facility. Symptoms including chills, joint pain, nausea, vomiting, and lesions were all statistically significant among CHIKV positive participants compared to CHIKV negative AUFI participants. Cough was negatively associated with CHIKV positive participants. Positivity proportions were significantly higher among adults compared to children. No significant difference was found in positivity proportion between rainy and dry seasons during the outbreak. Positive CHIKV cases were detected in all study site provinces, with the highest test positivity proportion recorded in the rural northeast province of Kratie. CONCLUSIONS: Surveillance data captured in this study provided a clinical and epidemiological characterization of positive CHIKV patients presenting at selected health facilities in Cambodia in 2020, and highlighted the widespread distribution of the outbreak, impacting both urban and rural locations. Findings also illustrated the importance of utilizing both RT-PCR and serological testing for effective CHIKV surveillance.

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos, Peru.

Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

A case-control study of the causes of acute respiratory infection among hospitalized patients in Northeastern Laos.

With the advent of highly sensitive real-time PCR, multiple pathogens have been identified from nasopharyngeal swabs of patients with acute respiratory infections (ARIs). However, the detection of microorganisms in the upper respiratory tract does not necessarily indicate disease causation. We conducted a matched case-control study, nested within a broader fever aetiology project, to facilitate determination of the aetiology of ARIs in hospitalised patients in Northeastern Laos. Consenting febrile patients of any age admitted to Xiengkhuang Provincial Hospital were included if they met the inclusion criteria for ARI presentation (at least one of the following: cough, rhinorrhoea, nasal congestion, sore throat, difficulty breathing, and/or abnormal chest auscultation). One healthy control for each patient, matched by sex, age, and village of residence, was recruited for the study. Nasopharyngeal swabs were collected from participants and tested for 33 pathogens by probe-based multiplex real-time RT-PCR (FastTrack Diagnostics Respiratory pathogen 33 kit). Attributable fraction of illness for a given microorganism was calculated by comparing results between patients and controls (= 100 * [OR - 1]/OR) (OR = odds ratio). Between 24th June 2019 and 24th June 2020, 205 consenting ARI patients and 205 matching controls were recruited. After excluding eight pairs due to age mismatch, 197 pairs were included in the analysis. Males were predominant with sex ratio 1.2:1 and children < 5 years old accounted for 59% of participants. At least one potential pathogen was detected in 173 (88%) patients and 175 (89%) controls. ARI in admitted patients were attributed to influenza B virus, influenza A virus, human metapneumovirus (HMPV), and respiratory syncytial virus (RSV) in 17.8%, 17.2%, 7.5%, and 6.5% of participants, respectively. SARS-CoV-2 was not detected in any cases or controls. Determining ARI aetiology in individual patients remains challenging. Among hospitalised patients with ARI symptoms presenting to a provincial hospital in Northeastern Laos, half were determined to be caused by one of several respiratory viruses, in particular influenza A virus, influenza B virus, HMPV, and RSV.

Contribution of Viral Respiratory Infections to Dengue-Like Illness Presentation at a Community Clinic in Southern Malaysia.

This study explored the contribution of viral respiratory infections (VRIs) in dengue-like illness (DLI) patients and their distinguishing clinicolaboratory parameters. Two hundred DLI patients were prospectively recruited (July 1- October 1, 2019) from a community clinic in Southern Malaysia. Patients ≥ 18 years with acute fever and fulfilling the WHO criteria of probable dengue were recruited. They underwent blood testing: blood counts, rapid dengue tests (nonstructural antigen-1/IgM) and polymerase chain reaction (PCR) for dengue, Zika, chikungunya, and Leptospira. Nasopharyngeal swabs (NPSs) were collected for FilmArray®RP2plus testing. From the 200 NPSs, 58 respiratory viruses (RVs) were detected in 54 patients. Of the 96 dengue-confirmed cases, 86 had dengue mono-infection, and 10 were coinfected with RVs. Of the 104 nondengue, 44 were RV positive and 4 Leptospira positive. Zika and chikungunya virus were not detected. Overall, the etiological diagnosis was confirmed for 72% of patients. Clinicolaboratory parameters were compared between dengue mono-infection and VRI mono-infection. Patients with coinfections were excluded. Multiple logistic regression showed that recent household/neighborhood history of dengue (adjusted odds ratio [aOR]: 5.9, 95% CI = 1.7-20.7), leukopenia (aOR: 12.5, 95% CI = 2.6-61.4) and thrombocytopenia (aOR: 5.5, 95% CI = 1.3-23.0) predicted dengue. Inversely, rhinorrhoea (aOR: 0.1, 95% CI = 0.01-0.3) and cough (aOR: 0.3, 95% CI = 0.1-0.9) favored VRI. Thus, VRIs comprise many infections diagnosed initially as DLIs. Early clinicolaboratory parameters can guide physicians screen patients for further testing.

Multiyear prospective cohort study to evaluate the risk potential of MERS-CoV infection among Malaysian Hajj pilgrims (MERCURIAL): a study protocol.

INTRODUCTION: Middle East respiratory syndrome (MERS) is a viral respiratory infection caused by the MERS-CoV. MERS was first reported in the Kingdom of Saudi Arabia in 2012. Every year, the Hajj pilgrimage to Mecca attracts more than two million pilgrims from 184 countries, making it one of the largest annual religious mass gatherings (MGs) worldwide. MGs in confined areas with a high number of pilgrims' movements worldwide continues to elicit significant global public health concerns. MERCURIAL was designed by adopting a seroconversion surveillance approach to provide multiyear evidence of MG-associated MERS-CoV seroconversion among the Malaysian Hajj pilgrims. METHODS AND ANALYSIS: MERCURIAL is an ongoing multiyear prospective cohort study. Every year, for the next 5 years, a cohort of 1000 Hajj pilgrims was enrolled beginning in the 2016 Hajj pilgrimage season. Pre-Hajj and post-Hajj serum samples were obtained and serologically analysed for evidence of MERS-CoV seroconversion. Sociodemographic data, underlying medical conditions, symptoms experienced during Hajj pilgrimage, and exposure to camel and untreated camel products were recorded using structured pre-Hajj and post-Hajj questionnaires. The possible risk factors associated with the seroconversion data were analysed using univariate and multivariate logistic regression. The primary outcome of this study is to better enhance our understanding of the potential threat of MERS-CoV spreading through MG beyond the Middle East. ETHICS AND DISSEMINATION: This study has obtained ethical approval from the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia. Results from the study will be submitted for publication in peer-reviewed journals and presented in conferences and scientific meetings. TRIAL REGISTRATION NUMBER: NMRR-15-1640-25391.

Rapid testing requires clinical evaluation for accurate diagnosis of dengue disease: A passive surveillance study in Southern Malaysia.

BACKGROUND: Dengue fever is the most common mosquito-borne infection worldwide where an expanding surveillance and characterization of this infection are needed to better inform the healthcare system. In this surveillance-based study, we explored the prevalence and distinguishing features of dengue fever amongst febrile patients in a large community-based health facility in southern peninsular Malaysia. METHODS: Over six months in 2018, we recruited 368 adults who met the WHO 2009 criteria for probable dengue infection. They underwent the following blood tests: full blood count, dengue virus (DENV) rapid diagnostic test (RDT), ELISA (dengue IgM and IgG), nested RT-PCR for dengue, multiplex qRT-PCR for Zika, Chikungunya and dengue as well as PCR tests for Leptopspira spp., Japanese encephalitis and West Nile virus. RESULTS: Laboratory-confirmed dengue infections (defined by positive tests in NS1, IgM, high-titre IgG or nested RT-PCR) were found in 167 (45.4%) patients. Of these 167 dengue patients, only 104 (62.3%) were positive on rapid diagnostic testing. Dengue infection was significantly associated with the following features: family or neighbours with dengue in the past week (AOR: 3.59, 95% CI:2.14-6.00, p<0.001), cutaneous rash (AOR: 3.58, 95% CI:1.77-7.23, p<0.001), increased temperature (AOR: 1.33, 95% CI:1.04-1.70, p = 0.021), leucopenia (white cell count < 4,000/µL) (AOR: 3.44, 95% CI:1.72-6.89, p<0.001) and thrombocytopenia (platelet count <150,000/µL)(AOR: 4.63, 95% CI:2.33-9.21, p<0.001). Dengue infection was negatively associated with runny nose (AOR: 0.47, 95% CI:0.29-0.78, p = 0.003) and arthralgia (AOR: 0.42, 95% CI:0.24-0.75, p = 0.004). Serotyping by nested RT-PCR revealed mostly mono-infections with DENV-2 (n = 64), DENV-1 (n = 32) and DENV-3 (n = 17); 14 co-infections occurred with DENV-1/DENV-2 (n = 13) and DENV-1/DENV-4 (n = 1). Besides dengue, none of the pathogens above were found in patients' serum. CONCLUSIONS: Acute undifferentiated febrile infections are a diagnostic challenge for community-based clinicians. Rapid diagnostic tests are increasingly used to diagnose dengue infection but negative tests should be interpreted with caution as they fail to detect a considerable proportion of dengue infection. Certain clinical features and haematological parameters are important in the clinical diagnosis of dengue infection.

Potential for community based surveillance of febrile diseases: Feasibility of self-administered rapid diagnostic tests in Iquitos, Peru and Phnom Penh, Cambodia.

Rapid diagnostic tests (RDTs) have the potential to identify infectious diseases quickly, minimize disease transmission, and could complement and improve surveillance and control of infectious and vector-borne diseases during outbreaks. The U.S. Defense Threat Reduction Agency's Joint Science and Technology Office (DTRA-JSTO) program set out to develop novel point-of-need RDTs for infectious diseases and deploy them for home use with no training. The aim of this formative study was to address two questions: 1) could community members in Iquitos, Peru and Phnom Penh, Cambodia competently use RDTs of different levels of complexity at home with visually based instructions provided, and 2) if an RDT were provided at no cost, would it be used at home if family members displayed febrile symptoms? Test kits with written and video (Peru only) instructions were provided to community members (Peru [n = 202]; Cambodia [n = 50]) or community health workers (Cambodia [n = 45]), and trained observers evaluated the competency level for each of the several steps required to successfully operate one of two multiplex RDTs on themselves or other consenting participant (i.e., family member). In Iquitos, >80% of residents were able to perform 11/12 steps and 7/15 steps for the two- and five-pathogen test, respectively. Competency in Phnom Penh never reached 80% for any of the 12 or 15 steps for either test; the percentage of participants able to perform a step ranged from 26-76% and 23-72%, for the two- and five-pathogen tests, respectively. Commercially available NS1 dengue rapid tests were distributed, at no cost, to households with confirmed exposure to dengue or Zika virus; of 14 febrile cases reported, six used the provided RDT. Our findings support the need for further implementation research on the appropriate level of instructions or training needed for diverse devices in different settings, as well as how to best integrate RDTs into existing local public health and disease surveillance programs at a large scale.

Heterogeneity of Dengue Illness in Community-Based Prospective Study, Iquitos, Peru.

Measuring heterogeneity of dengue illness is necessary to define suitable endpoints in dengue vaccine and therapeutic trials and will help clarify behavioral responses to illness. To quantify heterogeneity in dengue illness, including milder cases, we developed the Dengue Illness Perceptions Response (IPR) survey, which captured detailed symptom data, including intensity, duration, and character, and change in routine activities caused by illness. During 2016-2019, we collected IPR data daily during the acute phase of illness for 79 persons with a positive reverse transcription PCR result for dengue virus RNA. Most participants had mild ambulatory disease. However, we measured substantial heterogeneity in illness experience, symptom duration, and maximum reported intensity of individual symptoms. Symptom intensity was a more valuable predicter of major activity change during dengue illness than symptom presence or absence alone. These data suggest that the IPR measures clinically useful heterogeneity in dengue illness experience and its relation to altered human behavior.

Guaroa Virus and Plasmodium vivax Co-Infections, Peruvian Amazon.

During April-June 2014 in a malaria-endemic rural community close to the city of Iquitos in Peru, we detected evidence of Guaroa virus (GROV) infection in 14 febrile persons, of whom 6 also had evidence of Plasmodium vivax malaria. Cases were discovered through a long-term febrile illness surveillance network at local participating health facilities. GROV cases were identified by using a combination of seroconversion and virus isolation, and malaria was diagnosed by thick smear and PCR. GROV mono-infections manifested as nonspecific febrile illness and were clinically indistinguishable from GROV and P. vivax co-infections. This cluster of cases highlights the potential for GROV transmission in the rural Peruvian Amazon, particularly in areas where malaria is endemic. Further study of similar areas of the Amazon may provide insights into the extent of GROV transmission in the Amazon basin.

Potential Use of Community-Based Rapid Diagnostic Tests for Febrile Illnesses: Formative Research in Peru and Cambodia.

In 2012, the U.S. Defense Threat Reduction Agency Joint Science and Technology Office initiated a program to develop novel point-of-need diagnostic devices for surveillance of emerging infectious diseases including dengue, malaria, plague, and melioidosis. Prior to distribution of devices to observe their correct use among community members in Iquitos, Peru, and Phnom Penh, Cambodia, research was conducted to: 1) assess acceptability of use, including the motivation to use a rapid diagnostic test (RDT) before or instead of seeking care at a health facility, 2) explore comprehension of RDT use instructions, and 3) examine possible strategies for large scale RDT distribution and use at each site. In February 2014, 9 focus group discussions (FGD) with community members and 5 FGD with health professionals were conducted in Iquitos, and 9 FGD with community members and 9 in-depth interviews with health professionals in Phnom Penh. In both places, participants agreed to use the device themselves (involving finger prick) or could identify someone who could do so in their home or neighborhood. The main incentive to RDT use in both sites was the ability for device results to be used for care facilitation (post confirmatory tests), specifically reduced wait times to be seen or obtain a diagnosis. Comprehension of RDT use instructions was assessed in Iquitos by asking some participants to apply the device to research team members; after watching a short video, most steps were done correctly. In Phnom Penh, participants were asked to describe each step after reading the instructions; they struggled with comprehension. Health professionals' main concerns in both sites were their community's ability to accurately use the test, handle complicated instructions, and safety (i.e., disposal of lancets). Health system structure and ability to use home diagnostic devices varied in the two disease endemic sites, with substantial challenges in each, suggesting the need for different strategies for RDT large scale community use, and illustrating the value of formative research before deployment of novel technologies.

Acceptability of Aedes aegypti blood feeding on dengue virus-infected human volunteers for vector competence studies in Iquitos, Peru.

As part of a study to investigate drivers of dengue virus (DENV) transmission dynamics, this qualitative study explored whether DENV-infected residents of Iquitos, Peru, considered it acceptable (1) to participate in direct mosquito feeding experiments (lab-reared Aedes aegypti mosquitoes fed directly on human volunteers) and (2) to provide blood meals indirectly (Ae. aegypti fed on blood drawn from participants by venipuncture). Twelve focus group discussions (FGDs; 94 participants: 82 females and 12 males) were conducted in January 2014 to explore six themes: (1) concerns and preferences regarding direct mosquito feeds and blood draws, (2) comprehension of and misconceptions about study procedures, (3) motivating factors for participation, (4) acceptability of children's participation, (5) willingness to provide multiple samples over several days, and (6) preference for direct feedings in homes versus the study laboratory. Results of FGDs, including one with 5 of 53 past direct mosquito feed participants, indicated that mosquito feeding procedures are acceptable to Iquitos residents when they are provided with information and a few key messages are properly reinforced. FGD participants' concerns focused primarily on safety issues rather than discomfort associated with mosquito bites. A video explaining the study dramatically increased comprehension of the study procedures. The majority of participants expressed a preference for mosquito feeding over venipuncture. Adults supported child participation if the children themselves assented. For most participants, home feedings were preferred over those in a laboratory. A major impetus for participation was the idea that results would contribute to an improved understanding of DENV transmission in Iquitos. Findings from our study will support future large-scale studies that employ direct mosquito feeding, a low-risk, non-invasive procedure that is experimentally superior to artificial mosquito feeding methods.

Feasibility of feeding Aedes aegypti mosquitoes on dengue virus-infected human volunteers for vector competence studies in Iquitos, Peru.

BACKGROUND: Transmission of dengue virus (DENV) from humans to mosquitoes represents a critical component of dengue epidemiology. Examinations of this process have generally been hampered by a lack of methods that adequately represent natural acquisition of DENV by mosquitoes from humans. In this study, we assessed artificial and natural blood feeding methods based on rates of DENV infection and dissemination within mosquitoes for use in a field-based epidemiological cohort study in Iquitos, Peru. METHODOLOGY/PRINCIPAL FINDINGS: Our study was implemented, stepwise, between 2011 and 2015. Participants who were 5 years and older with 5 or fewer days of fever were enrolled from ongoing clinic- and neighborhood-based studies on dengue in Iquitos. Wild type, laboratory-reared Aedes aegypti were fed directly on febrile individuals or on blood collected from participants that was either untreated or treated with EDTA. Mosquitoes were tested after approximately 14 days of extrinsic incubation for DENV infection and dissemination. A total of 58 participants, with viremias ranging from 1.3 × 10(2) to 2.9 × 10(6) focus-forming units per mL of serum, participated in one or more feeding methods. DENV infection and dissemination rates were not significantly different following direct and indirect-EDTA feeding; however, they were significantly lower for mosquitoes that fed indirectly on blood with no additive. Relative to direct feeding, infection rates showed greater variation following indirect-EDTA than indirect-no additive feeding. Dissemination rates were similar across all feeding methods. No differences were detected in DENV infection or dissemination rates in mosquitoes fed directly on participants with different dengue illness severity. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates the feasibility of using direct and indirect feeding methods for field-based studies on vector competence. Direct mosquito feeding is preferable in terms of logistical ease, biosecurity, and reliability.

Human Antibody Responses to Emerging Mayaro Virus and Cocirculating Alphavirus Infections Examined by Using Structural Proteins from Nine New and Old World Lineages.

Mayaro virus (MAYV), Venezuelan equine encephalitis virus (VEEV), and chikungunya virus (CHIKV) are vector-borne alphaviruses that cocirculate in South America. Human infections by these viruses are frequently underdiagnosed or misdiagnosed, especially in areas with high dengue virus endemicity. Disease may progress to debilitating arthralgia (MAYV, CHIKV), encephalitis (VEEV), and death. Few standardized serological assays exist for specific human alphavirus infection detection, and antigen cross-reactivity can be problematic. Therefore, serological platforms that aid in the specific detection of multiple alphavirus infections will greatly expand disease surveillance for these emerging infections. In this study, serum samples from South American patients with PCR- and/or isolation-confirmed infections caused by MAYV, VEEV, and CHIKV were examined by using a protein microarray assembled with recombinant capsid, envelope protein 1 (E1), and E2 from nine New and Old World alphaviruses. Notably, specific antibody recognition of E1 was observed only with MAYV infections, whereas E2 was specifically targeted by antibodies from all of the alphavirus infections investigated, with evidence of cross-reactivity to E2 of o'nyong-nyong virus only in CHIKV-infected patient serum samples. Our findings suggest that alphavirus structural protein microarrays can distinguish infections caused by MAYV, VEEV, and CHIKV and that this multiplexed serological platform could be useful for high-throughput disease surveillance. IMPORTANCE Mayaro, chikungunya, and Venezuelan equine encephalitis viruses are closely related alphaviruses that are spread by mosquitos, causing diseases that produce similar influenza-like symptoms or more severe illnesses. Moreover, alphavirus infection symptoms can be similar to those of dengue or Zika disease, leading to underreporting of cases and potential misdiagnoses. New methods that can be used to detect antibody responses to multiple alphaviruses within the same assay would greatly aid disease surveillance efforts. However, possible antibody cross-reactivity between viruses can reduce the quality of laboratory results. Our results demonstrate that antibody responses to multiple alphaviruses can be specifically quantified within the same assay by using selected recombinant protein antigens and further show that Mayaro virus infections result in unique responses to viral envelope proteins.