Serological Responses in Patients Infected with Mayaro Virus and Evaluation of Cross-Protective Responses against Chikungunya Virus.

Mayaro virus (MAYV) is an alphavirus endemic to both Latin America and the Caribbean. Recent reports have questioned the ability of MAYV and its close relative, Chikungunya virus (CHIKV), to generate cross-reactive, neutralizing antibodies to one another. Since CHIKV was introduced to South America in 2013, discerning whether individuals have cross-reactive antibodies or whether they have had exposures to both viruses previously has been difficult. Using samples obtained from people infected with MAYV prior to the introduction of CHIKV in the Americas, we performed neutralizing assays and observed no discernable neutralization of CHIKV by sera from patients previously infected with MAYV. These data suggest that a positive CHIKV neutralization test cannot be attributed to prior exposure to MAYV and that previous exposure to MAYV may not be protective against a subsequent CHIKV infection.

The impact of insecticide treated curtains on dengue virus transmission: A cluster randomized trial in Iquitos, Peru.

Dengue is one of the most important vector-borne diseases, resulting in an estimated hundreds of millions of infections annually throughout the tropics. Control of dengue is heavily dependent upon control of its primary mosquito vector, Aedes aegypti. Innovative interventions that are effective at targeting the adult stage of the mosquito are needed to increase the options for effective control. The use of insecticide-treated curtains (ITCs) has previously been shown to significantly reduce the abundance of Ae. aegypti in and around homes, but the impact of ITCs on dengue virus (DENV) transmission has not been rigorously quantified. A parallel arm cluster-randomized controlled trial was conducted in Iquitos, Peru to quantify the impact of ITCs on DENV seroconversion as measured through plaque-reduction neutralization tests. Seroconversion data showed that individuals living in the clusters that received ITCs were at greater risk to seroconverting to DENV, with an average seroconversion rate of 50.6 per 100 person-years (PY) (CI: 29.9-71.9), while those in the control arm had an average seroconversion rate of 37.4 per 100 PY (CI: 15.2-51.7). ITCs lost their insecticidal efficacy within 6 months of deployment, necessitating re-treatment with insecticide. Entomological indicators did not show statistically significant differences between ITC and non-ITC clusters. It's unclear how the lack of protective efficacy reported here is attributable to simple failure of the intervention to protect against Ae. aegypti bites, or the presence of a faulty intervention during much of the follow-up period. The higher risk of dengue seroconversion that was detected in the ITC clusters may have arisen due to a false sense of security that inadvertently led to less routine protective behaviors on the part of households that received the ITCs. Our study provides important lessons learned for conducting cluster randomized trials for vector control interventions against Aedes-transmitted virus infections.

Estimating the impact of city-wide Aedes aegypti population control: An observational study in Iquitos, Peru.

During the last 50 years, the geographic range of the mosquito Aedes aegypti has increased dramatically, in parallel with a sharp increase in the disease burden from the viruses it transmits, including Zika, chikungunya, and dengue. There is a growing consensus that vector control is essential to prevent Aedes-borne diseases, even as effective vaccines become available. What remains unclear is how effective vector control is across broad operational scales because the data and the analytical tools necessary to isolate the effect of vector-oriented interventions have not been available. We developed a statistical framework to model Ae. aegypti abundance over space and time and applied it to explore the impact of citywide vector control conducted by the Ministry of Health (MoH) in Iquitos, Peru, over a 12-year period. Citywide interventions involved multiple rounds of intradomicile insecticide space spray over large portions of urban Iquitos (up to 40% of all residences) in response to dengue outbreaks. Our model captured significant levels of spatial, temporal, and spatio-temporal variation in Ae. aegypti abundance within and between years and across the city. We estimated the shape of the relationship between the coverage of neighborhood-level vector control and reductions in female Ae. aegypti abundance; i.e., the dose-response curve. The dose-response curve, with its associated uncertainties, can be used to gauge the necessary spraying effort required to achieve a desired effect and is a critical tool currently absent from vector control programs. We found that with complete neighborhood coverage MoH intra-domicile space spray would decrease Ae. aegypti abundance on average by 67% in the treated neighborhood. Our framework can be directly translated to other interventions in other locations with geolocated mosquito abundance data. Results from our analysis can be used to inform future vector-control applications in Ae. aegypti endemic areas globally.

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.

Molecular and antigenic characterization of group C orthobunyaviruses isolated in Peru.

Group C orthobunyaviruses (GRCVs) are a complex of viruses in the genus Orthobunyavirus and are associated with human febrile disease in tropical and subtropical areas of South and Central America. While numerous GRCVs have been isolated from mosquitoes, animals, and humans, genetic analysis of these viruses is limited. In this study, we characterized 65 GRCV isolates from febrile patients identified through clinic-based surveillance in the northern and southern Peruvian Amazon. A 500 base pair region of the S segment and 750 base pair regions of the M and L segments were sequenced. Pairwise sequence analysis of the clinical isolates showed nucleotide identities ranging from 68% to 100% and deduced amino acid sequence identities ranging from 72% to 100%. Sequences were compared with reference strains of the following GRCVs: Caraparu virus (CARV), Murutucu virus (MURV), Oriboca virus (ORIV), Marituba virus (MTBV), Itaqui virus (ITQV), Apeu virus (APEUV), and Madrid virus (MADV). Sequence comparison of clinical isolates with the prototype strains based on the S and L segments identified two clades; clade I included isolates with high genetic association with CARV-MADV, and clade II included isolates with high genetic association with MURV, ORIV, APEUV, and MTBV. Genetic relationships based on the M segment were at time inconsistent with those based on the S and L segments. However, clade groupings based on the M segment were highly consistent with relationships based on microneutralization assays. These results advance our understanding of the genetic and serologic relationships of GRCVs circulating in the Peruvian Amazon.

Isolation of Complete Equine Encephalitis Virus Genome from Human Swab Specimen, Peru.

While studying respiratory infections in Peru, we identified Venezuelan equine encephalitis virus (VEEV) in a nasopharyngeal swab, indicating that this alphavirus can be present in human respiratory secretions. Because VEEV may be infectious when aerosolized, our finding is relevant for the management of VEEV-infected patients and for VEEV transmission studies.

Malaria surveys using rapid diagnostic tests and validation of results using post hoc quantification of Plasmodium falciparum histidine-rich protein 2.

BACKGROUND: Rapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys. METHODS: Individuals' blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey. RESULTS: There was a sigmoidal dose-response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD. CONCLUSIONS: Measures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration.

Serologic Evidence of Scrub Typhus in the Peruvian Amazon.

Using a large, passive, febrile surveillance program in Iquitos, Peru, we retrospectively tested human blood specimens for scrub typhus group orientiae by ELISA, immunofluorescence assay, and PCR. Of 1,124 participants, 60 (5.3%) were seropositive, and 1 showed evidence of recent active infection. Our serologic data indicate that scrub typhus is present in the Peruvian Amazon.

Genome Sequence of Bellavista Virus, a Novel Orthobunyavirus Isolated from a Pool of Mosquitoes Captured near Iquitos, Peru.

A novel orthobunyavirus, Bellavista virus, was isolated from Culex (Melanoconion) portesi mosquitoes in the Bellavista neighborhood of Iquitos, Peru, in 2009. The assembled segment L, M, and S sequences of strain PRD0552 are 6,950, 4,469, and 1,256 bases in length, respectively, comprising complete protein-coding sequences and partial terminal untranslated sequences.

Identification of Blood Meals from Potential Arbovirus Mosquito Vectors in the Peruvian Amazon Basin.

The transmission dynamics of many arboviruses in the Amazon Basin region have not been fully elucidated, including the vectors and natural reservoir hosts. Identification of blood meal sources in field-caught mosquitoes could yield information for identifying potential arbovirus vertebrate hosts. We identified blood meal sources in 131 mosquitoes collected from areas endemic for arboviruses in the Peruvian Department of Loreto by sequencing polymerase chain reaction amplicons of the cytochrome b gene. Psorophora (Janthinosoma) albigenu, Psorophora (Grabhamia) cingulata, Mansonia humeralis, Anopheles oswaldoi s.l., and Anopheles benarrochi s.l. had mainly anthropophilic feeding preferences; Aedes (Ochlerotatus) serratus, and Aedes (Ochlerotatus) fulvus had feeding preferences for peridomestic animals; and Culex (Melanoconion) spp. fed on a variety of vertebrates, mainly rodents (spiny rats), birds, and amphibians. On the basis of these feeding preferences, many mosquitoes could be considered as potential enzootic and bridge arbovirus vectors in the Amazon Basin of Peru.

Rickettsial Disease in the Peruvian Amazon Basin.

Using a large, passive, clinic-based surveillance program in Iquitos, Peru, we characterized the prevalence of rickettsial infections among undifferentiated febrile cases and obtained evidence of pathogen transmission in potential domestic reservoir contacts and their ectoparasites. Blood specimens from humans and animals were assayed for spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) by ELISA and/or PCR; ectoparasites were screened by PCR. Logistic regression was used to determine associations between patient history, demographic characteristics of participants and symptoms, clinical findings and outcome of rickettsial infection. Of the 2,054 enrolled participants, almost 2% showed evidence of seroconversion or a 4-fold rise in antibody titers specific for rickettsiae between acute and convalescent blood samples. Of 190 fleas (Ctenocephalides felis) and 60 ticks (Rhipicephalus sanguineus) tested, 185 (97.4%) and 3 (5%), respectively, were positive for Rickettsia spp. Candidatus Rickettsia asemboensis was identified in 100% and 33% of the fleas and ticks tested, respectively. Collectively, our serologic data indicates that human pathogenic SFGR are present in the Peruvian Amazon and pose a significant risk of infection to individuals exposed to wild, domestic and peri-domestic animals and their ectoparasites.

Calling in sick: impacts of fever on intra-urban human mobility.

Pathogens inflict a wide variety of disease manifestations on their hosts, yet the impacts of disease on the behaviour of infected hosts are rarely studied empirically and are seldom accounted for in mathematical models of transmission dynamics. We explored the potential impacts of one of the most common disease manifestations, fever, on a key determinant of pathogen transmission, host mobility, in residents of the Amazonian city of Iquitos, Peru. We did so by comparing two groups of febrile individuals (dengue-positive and dengue-negative) with an afebrile control group. A retrospective, semi-structured interview allowed us to quantify multiple aspects of mobility during the two-week period preceding each interview. We fitted nested models of each aspect of mobility to data from interviews and compared models using likelihood ratio tests to determine whether there were statistically distinguishable differences in mobility attributable to fever or its aetiology. Compared with afebrile individuals, febrile study participants spent more time at home, visited fewer locations, and, in some cases, visited locations closer to home and spent less time at certain types of locations. These multifaceted impacts are consistent with the possibility that disease-mediated changes in host mobility generate dynamic and complex changes in host contact network structure.

Symptoms and Immune Markers in Plasmodium/Dengue Virus Co-infection Compared with Mono-infection with Either in Peru.

BACKGROUND: Malaria and dengue are two of the most common vector-borne diseases in the world, but co-infection is rarely described, and immunologic comparisons of co-infection with mono-infection are lacking. METHODOLOGY AND PRINCIPAL FINDINGS: We collected symptom histories and blood specimens from subjects in a febrile illness surveillance study conducted in Iquitos and Puerto Maldonado, Peru, between 2002-2011. Nineteen symptoms and 18 immune markers at presentation were compared among those with co-infection with Plasmodium/dengue virus (DENV), Plasmodium mono-infection, and DENV mono-infection. Seventeen subjects were identified as having Plasmodium/DENV co-infection and were retrospectively matched with 51 DENV mono-infected and 44 Plasmodium mono-infected subjects. Those with Plasmodium mono-infection had higher levels of IL-4, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, and MIP1-α/CCL3 compared with DENV mono-infection or co-infection; those with Plasmodium mono-infection had more cough than those with DENV mono-infection. Subjects with DENV mono-infection had higher levels of TGF-ß1 and more myalgia than those with Plasmodium mono-infection. No symptom was more common and no immune marker level was higher in the co-infected group, which had similar findings to the DENV mono-infected subjects. CONCLUSIONS/SIGNIFICANCE: Compared with mono-infection with either pathogen, Plasmodium/DENV co-infection was not associated with worse disease and resembled DENV mono-infection in both symptom frequency and immune marker level.

Factors Associated with Correct and Consistent Insecticide Treated Curtain Use in Iquitos, Peru.

Dengue is an arthropod-borne virus of great public health importance, and control of its mosquito vectors is currently the only available method for prevention. Previous research has suggested that insecticide treated curtains (ITCs) can lower dengue vector infestations in houses. This observational study investigated individual and household-level socio-demographic factors associated with correct and consistent use of ITCs in Iquitos, Peru. A baseline knowledge, attitudes, and practices (KAP) survey was administered to 1,333 study participants, and ITCs were then distributed to 593 households as part of a cluster-randomized trial. Follow up KAP surveys and ITC-monitoring checklists were conducted at 9, 18, and 27 months post-ITC distribution. At 9 months post-distribution, almost 70% of ITCs were hanging properly (e.g. hanging fully extended or tied up), particularly those hung on walls compared to other locations. Proper ITC hanging dropped at 18 months to 45.7%. The odds of hanging ITCs correctly and consistently were significantly greater among those participants who were housewives, knew three or more correct symptoms of dengue and at least one correct treatment for dengue, knew a relative or close friend who had had dengue, had children sleeping under a mosquito net, or perceived a change in the amount of mosquitoes in the home. Additionally, the odds of recommending ITCs in the future were significantly greater among those who perceived a change in the amount of mosquitoes in the home (e.g. perceived the ITCs to be effective). Despite various challenges associated with the sustained effectiveness of the selected ITCs, almost half of the ITCs were still hanging at 18 months, suggesting a feasible vector control strategy for sustained community use.

Incomplete Protection against Dengue Virus Type 2 Re-infection in Peru.

BACKGROUND: Nearly half of the world's population is at risk for dengue, yet no licensed vaccine or anti-viral drug is currently available. Dengue is caused by any of four dengue virus serotypes (DENV-1 through DENV-4), and infection by a DENV serotype is assumed to provide life-long protection against re-infection by that serotype. We investigated the validity of this fundamental assumption during a large dengue epidemic caused by DENV-2 in Iquitos, Peru, in 2010-2011, 15 years after the first outbreak of DENV-2 in the region. METHODOLOGY/PRINCIPAL FINDINGS: We estimated the age-dependent prevalence of serotype-specific DENV antibodies from longitudinal cohort studies conducted between 1993 and 2010. During the 2010-2011 epidemic, active dengue cases were identified through active community- and clinic-based febrile surveillance studies, and acute inapparent DENV infections were identified through contact tracing studies. Based on the age-specific prevalence of DENV-2 neutralizing antibodies, the age distribution of DENV-2 cases was markedly older than expected. Homologous protection was estimated at 35.1% (95% confidence interval: 0%-65.2%). At the individual level, pre-existing DENV-2 antibodies were associated with an incomplete reduction in the frequency of symptoms. Among dengue cases, 43% (26/66) exhibited elevated DENV-2 neutralizing antibody titers for years prior to infection, compared with 76% (13/17) of inapparent infections (age-adjusted odds ratio: 4.2; 95% confidence interval: 1.1-17.7). CONCLUSIONS/SIGNIFICANCE: Our data indicate that protection from homologous DENV re-infection may be incomplete in some circumstances, which provides context for the limited vaccine efficacy against DENV-2 in recent trials. Further studies are warranted to confirm this phenomenon and to evaluate the potential role of incomplete homologous protection in DENV transmission dynamics.

Long-Term Arthralgia after Mayaro Virus Infection Correlates with Sustained Pro-inflammatory Cytokine Response.

Mayaro virus (MAYV), an alphavirus similar to chikungunya virus (CHIKV), causes an acute debilitating disease which results in the development of long-term arthralgia in more than 50% of infected individuals. Currently, the immune response and its role in the development of MAYV-induced persistent arthralgia remain unknown. In this study, we evaluated the immune response of individuals with confirmed MAYV infection in a one-year longitudinal study carried out in Loreto, Peru. We report that MAYV infection elicits robust immune responses that result in the development of a strong neutralizing antibody response and the secretion of pro-inflammatory immune mediators. The composition of these inflammatory mediators, in some cases, differed to those previously observed for CHIKV. Key mediators such as IL-13, IL-7 and VEGF were strongly induced following MAYV infection and were significantly increased in subjects that eventually developed persistent arthralgia. Although a strong neutralizing antibody response was observed in all subjects, it was not sufficient to prevent the long-term outcomes of MAYV infection. This study provides initial immunologic insight that may eventually contribute to prognostic tools and therapeutic treatments against this emerging pathogen.

Dengue Knowledge and Preventive Practices in Iquitos, Peru.

As part of a cluster-randomized trial to evaluate insecticide-treated curtains for dengue prevention in Iquitos, Peru, we surveyed 1,333 study participants to examine knowledge and reported practices associated with dengue and its prevention. Entomological data from 1,133 of these households were linked to the survey. Most participants knew that dengue was transmitted by mosquito bite (85.6%), but only few (18.6%) knew that dengue vectors bite during daytime. Most commonly recognized dengue symptoms were fever (86.6%), headache (76.4%), and muscle/joint pain (67.9%). Most commonly reported correct practices for mosquito control were cleaning homes (61.6%), using insecticide sprays (23%), and avoiding having standing water at home (12.3%). Higher education was associated with higher knowledge about dengue, including transmission and vector control. Higher socioeconomic status was associated with increased reported use of preventive practices requiring money expenditure. We were less likely to find Aedes aegypti eggs, larvae, or pupae in households that had < 5-year-old children at home. Although dengue has been transmitted in Iquitos since the 1990s and the Regional Health Authority routinely fumigates households, treats domestic water containers with larvicide, and issues health education messages through mass media, knowledge of dengue transmission and household practices for prevention could be improved.

Evolutionary and Ecological Characterization of Mayaro Virus Strains Isolated during an Outbreak, Venezuela, 2010.

In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions.

Weather Regulates Location, Timing, and Intensity of Dengue Virus Transmission between Humans and Mosquitoes.

BACKGROUND: Dengue is one of the most aggressively expanding mosquito-transmitted viruses. The human burden approaches 400 million infections annually. Complex transmission dynamics pose challenges for predicting location, timing, and magnitude of risk; thus, models are needed to guide prevention strategies and policy development locally and globally. Weather regulates transmission-potential via its effects on vector dynamics. An important gap in understanding risk and roadblock in model development is an empirical perspective clarifying how weather impacts transmission in diverse ecological settings. We sought to determine if location, timing, and potential-intensity of transmission are systematically defined by weather. METHODOLOGY/PRINCIPAL FINDINGS: We developed a high-resolution empirical profile of the local weather-disease connection across Peru, a country with considerable ecological diversity. Applying 2-dimensional weather-space that pairs temperature versus humidity, we mapped local transmission-potential in weather-space by week during 1994-2012. A binary classification-tree was developed to test whether weather data could classify 1828 Peruvian districts as positive/negative for transmission and into ranks of transmission-potential with respect to observed disease. We show that transmission-potential is regulated by temperature-humidity coupling, enabling epidemics in a limited area of weather-space. Duration within a specific temperature range defines transmission-potential that is amplified exponentially in higher humidity. Dengue-positive districts were identified by mean temperature >22°C for 7+ weeks and minimum temperature >14°C for 33+ weeks annually with 95% sensitivity and specificity. In elevated-risk locations, seasonal peak-incidence occurred when mean temperature was 26-29°C, coincident with humidity at its local maximum; highest incidence when humidity >80%. We profile transmission-potential in weather-space for temperature-humidity ranging 0-38°C and 5-100% at 1°C x 2% resolution. CONCLUSIONS/SIGNIFICANCE: Local duration in limited areas of temperature-humidity weather-space identifies potential locations, timing, and magnitude of transmission. The weather-space profile of transmission-potential provides needed data that define a systematic and highly-sensitive weather-disease connection, demonstrating separate but coupled roles of temperature and humidity. New insights regarding natural regulation of human-mosquito transmission across diverse ecological settings advance our understanding of risk locally and globally for dengue and other mosquito-borne diseases and support advances in public health policy/operations, providing an evidence-base for modeling, predicting risk, and surveillance-prevention planning.