Time-varying, serotype-specific force of infection of dengue virus.

Infectious disease models play a key role in public health planning. These models rely on accurate estimates of key transmission parameters such as the force of infection (FoI), which is the per-capita risk of a susceptible person being infected. The FoI captures the fundamental dynamics of transmission and is crucial for gauging control efforts, such as identifying vaccination targets. Dengue virus (DENV) is a mosquito-borne, multiserotype pathogen that currently infects ∼390 million people a year. Existing estimates of the DENV FoI are inaccurate because they rely on the unrealistic assumption that risk is constant over time. Dengue models are thus unreliable for designing vaccine deployment strategies. Here, we present to our knowledge the first time-varying (daily), serotype-specific estimates of DENV FoIs using a spline-based fitting procedure designed to examine a 12-y, longitudinal DENV serological dataset from Iquitos, Peru (11,703 individuals, 38,416 samples, and 22,301 serotype-specific DENV infections from 1999 to 2010). The yearly DENV FoI varied markedly across time and serotypes (0-0.33), as did daily basic reproductive numbers (0.49-4.72). During specific time periods, the FoI fluctuations correlated across serotypes, indicating that different DENV serotypes shared common transmission drivers. The marked variation in transmission intensity that we detected indicates that intervention targets based on one-time estimates of the FoI could underestimate the level of effort needed to prevent disease. Our description of dengue virus transmission dynamics is unprecedented in detail, providing a basis for understanding the persistence of this rapidly emerging pathogen and improving disease prevention programs.

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.

Experiences with insecticide-treated curtains: a qualitative study in Iquitos, Peru.

BACKGROUND: Dengue is an arthropod-borne viral disease responsible for approximately 400 million infections annually; the only available method of prevention is vector control. It has been previously demonstrated that insecticide treated curtains (ITCs) can lower dengue vector infestations in and around houses. As part of a larger trial examining whether ITCs could reduce dengue transmission in Iquitos, Peru, the objective of this study was to characterize the participants' experience with the ITCs using qualitative methods. METHODS: Knowledge, attitudes, and practices (KAP) surveys (at baseline, and 9 and 27 months post-ITC distribution, with n = 593, 595 and 511, respectively), focus group discussions (at 6 and 12 months post-ITC distribution, with n = 18 and 33, respectively), and 11 one-on-one interviews (at 12 months post-distribution) were conducted with 605 participants who received ITCs as part of a cluster-randomized trial. RESULTS: Focus groups at 6 months post-ITC distribution revealed that individuals had observed their ITCs to function for approximately 3 months, after which they reported the ITCs were no longer working. Follow up revealed that the ITCs required re-treatment with insecticide at approximately 1 year post-distribution. Over half (55.3 %, n = 329) of participants at 9 months post-ITC distribution and over a third (34.8 %, n = 177) at 27 months post-ITC distribution reported perceiving a decrease in the number of mosquitoes in their home. The percentage of participants who would recommend ITCs to their family or friends in the future remained high throughout the study (94.3 %, n = 561 at 9 months and 94.6 %, n = 488 at 27 months post-distribution). When asked why, participants reported that ITCs were effective at reducing mosquitoes (81.6 and 37.8 %, at 9 and 27 months respectively), that they prevent dengue (5.7 and 51.2 %, at 9 and 27 months), that they are "beautiful" (5.9 and 3.1 %), as well as other reasons (6.9 and 2.5 %). CONCLUSION: ITCs have substantial potential for long term dengue vector control because they are liked by users, both for their perceived effectiveness and for aesthetic reasons, and because they require little proactive behavioral effort on the part of the users. Our results highlight the importance of gathering process (as opposed to outcome) data during vector control studies, without which researchers would not have become aware that the ITCs had lost effectiveness early in the trial.

House-to-house human movement drives dengue virus transmission.

Dengue is a mosquito-borne disease of growing global health importance. Prevention efforts focus on mosquito control, with limited success. New insights into the spatiotemporal drivers of dengue dynamics are needed to design improved disease-prevention strategies. Given the restricted range of movement of the primary mosquito vector, Aedes aegypti, local human movements may be an important driver of dengue virus (DENV) amplification and spread. Using contact-site cluster investigations in a case-control design, we demonstrate that, at an individual level, risk for human infection is defined by visits to places where contact with infected mosquitoes is likely, independent of distance from the home. Our data indicate that house-to-house human movements underlie spatial patterns of DENV incidence, causing marked heterogeneity in transmission rates. At a collective level, transmission appears to be shaped by social connections because routine movements among the same places, such as the homes of family and friends, are often similar for the infected individual and their contacts. Thus, routine, house-to-house human movements do play a key role in spread of this vector-borne pathogen at fine spatial scales. This finding has important implications for dengue prevention, challenging the appropriateness of current approaches to vector control. We argue that reexamination of existing paradigms regarding the spatiotemporal dynamics of DENV and other vector-borne pathogens, especially the importance of human movement, will lead to improvements in disease prevention.

Spatiotemporal analysis of Guaroa virus diversity, evolution, and spread in South America.

We conducted phylogeographic modeling to determine the introduction and spread of Guaroa virus in South America. The results suggest a recent introduction of this virus into regions of Peru and Bolivia over the past 60-70 years and emphasize the need for increased surveillance in surrounding areas.

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.

Itaya virus, a Novel Orthobunyavirus Associated with Human Febrile Illness, Peru.

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.

Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks.

Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru.

Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.

Reduced risk of disease during postsecondary dengue virus infections.

BACKGROUND: Antibodies induced by infection with any 1 of 4 dengue virus (DENV) serotypes (DENV-1-4) may influence the clinical outcome of subsequent heterologous infections. To quantify potential cross-protective effects, we estimated disease risk as a function of DENV infection, using data from longitudinal studies performed from September 2006 through February 2011 in Iquitos, Peru, during periods of DENV-3 and DENV-4 transmission. METHODS: DENV infections before and during the study period were determined by analysis of serial serum samples with virus neutralization tests. Third and fourth infections were classified as postsecondary infections. Dengue fever cases were detected by door-to-door surveillance for acute febrile illness. RESULTS: Among susceptible participants, 39% (420/1077) and 53% (1595/2997) seroconverted to DENV-3 and DENV-4, respectively. Disease was detected in 7% of DENV-3 infections and 10% of DENV-4 infections. Disease during postsecondary infections was reduced by 93% for DENV-3 and 64% for DENV-4, compared with primary and secondary infections. Despite lower disease rates, postsecondary infections constituted a significant proportion of apparent infections (14% [for DENV-3 infections], 45% [for DENV-4 infections]). CONCLUSIONS: Preexisting heterotypic antibodies markedly reduced but did not eliminate the risk of disease in this study population. These results improve understanding of how preinfection history can be associated with dengue outcomes and DENV transmission dynamics.

Characterization of a novel flavivirus isolated from Culex (Melanoconion) ocossa mosquitoes from Iquitos, Peru.

We describe the isolation and characterization of a novel flavivirus, isolated from a pool of Culex (Melanoconion) ocossa Dyar and Knab mosquitoes collected in 2009 in an urban area of the Amazon basin city of Iquitos, Peru. Flavivirus infection was detected by indirect immunofluorescent assay of inoculated C6/36 cells using polyclonal flavivirus antibodies (St. Louis encephalitis virus, yellow fever virus and dengue virus type 1) and confirmed by RT-PCR. Based on partial sequencing of the E and NS5 gene regions, the virus isolate was most closely related to the mosquito-borne flaviviruses but divergent from known species, with less than 45 and 71 % pairwise amino acid identity in the E and NS5 gene products, respectively. Phylogenetic analysis of E and NS5 amino acid sequences demonstrated that this flavivirus grouped with mosquito-borne flaviviruses, forming a clade with Nounané virus (NOUV). Like NOUV, no replication was detected in a variety of mammalian cells (Vero-76, Vero-E6, BHK, LLCMK, MDCK, A549 and RD) or in intracerebrally inoculated newborn mice. We tentatively designate this genetically distinct flavivirus as representing a novel species, Nanay virus, after the river near where it was first detected.

Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites.

Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC(50) in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.

In Peru, reporting male sex partners imparts significant risk of incident HIV/sexually transmitted infection: all men Engaging in same-sex behavior need prevention services.

BACKGROUND: Detailed information on the sexual behavior of bisexual, non-gay-identified men and the relationship between same-sex behavior and HIV/sexually transmitted infection (STI) incidence is limited. This study provides information on the sexual behavior with male partners of non-gay-identified men in urban, coastal Peru and the relationship of this behavior with HIV/STI incidence. METHODS: We analyzed data from 2146 non-gay-identified men with a baseline and then 2 years of annual follow-up, including detailed information on sexual behavior with up to 5 sex partners, to determine the characteristics associated with bisexual behavior. Discrete time proportional hazards models were used to determine the effect of self-reported sex with men on subsequent HIV/STI incidence. RESULTS: Over the 3 study visits, sex with a man was reported by 18.9% of men, 90% of whom also reported sex with a female partner. At baseline, reported bisexual behavior was associated with other sexual risk behaviors such as exchanging sex for money and increased risk of HIV, herpes simplex virus type 2, and gonorrhea. The number of study visits in which recent sex with men was reported was positively correlated with risk of other sexual risk behaviors and incident HIV, herpes simplex virus type 2, and gonorrhea. Recent sex with a man was associated with increased HIV/STI incidence (hazard ratio, 1.79; confidence interval, 1.19-2.70), after adjusting for sociodemographics and other sexual risk behaviors. CONCLUSIONS: Given the prevalence of recent sex with men and the relationship of this behavior with HIV/STI incidence, interventions with non-gay-identified men who have sex with men and their partners are warranted.

Human rhinoviruses and enteroviruses in influenza-like illness in Latin America.

BACKGROUND: Human rhinoviruses (HRVs) belong to the Picornaviridae family with high similarity to human enteroviruses (HEVs). Limited data is available from Latin America regarding the clinical presentation and strains of these viruses in respiratory disease. METHODS: We collected nasopharyngeal swabs at clinics located in eight Latin American countries from 3,375 subjects aged 25 years or younger who presented with influenza-like illness. RESULTS: Our subjects had a median age of 3 years and a 1.2:1.0 male:female ratio. HRV was identified in 16% and HEV was identified in 3%. HRVs accounted for a higher frequency of isolates in those of younger age, in particular children < 1 years old. HRV-C accounted for 38% of all HRVs detected. Phylogenetic analysis revealed a high proportion of recombinant strains between HRV-A/HRV-C and between HEV-A/HEV-B. In addition, both EV-D68 and EV-A71 were identified. CONCLUSIONS: In Latin America as in other regions, HRVs and HEVs account for a substantial proportion of respiratory viruses identified in young people with ILI, a finding that provides additional support for the development of pharmaceuticals and vaccines targeting these pathogens.

Detection of human leptospirosis as a cause of acute fever by capture ELISA using a Leptospira interrogans serovar Copenhageni (M20) derived antigen.

BACKGROUND: Leptospirosis is a potentially lethal zoonosis mainly affecting low-resource tropical countries, including Peru and its neighbouring countries. Timely diagnosis of leptospirosis is critical but may be challenging in the regions where it is most prevalent. The serodiagnostic gold standard microagglutination test (MAT) may be technically prohibitive. Our objective in this study was to assess the sensitivity, specificity, and predictive value of an IgM antibody capture enzyme-linked immunoassay (MAC-ELISA) derived from the M20 strain of Leptospira interrogans serovar Copenhageni (M20) by comparison to MAT, which was used as the gold standard method of diagnosis. METHODS: Acute and convalescent sera from participants participating in a passive febrile surveillance study in multiple regions of Peru were tested by both IgM MAC-ELISA and MAT. The sensitivity, specificity, positive and negative predictive value (PPV, NPV) of the MAC-ELISA assay for acute, convalescent and paired sera by comparison to MAT were calculated. RESULTS: The sensitivity, specificity, PPV and NPV of the MAC-ELISA assay for acute sera were 92.3%, 56.0%, 35.3% and 96.6% respectively. For convalescent sera, the sensitivity, specificity, PPV and NPV of the MAC-ELISA assay were 93.3%, 51.5%, 63.6% and 89.5% respectively. For paired sera, the sensitivity, specificity, PPV and NPV of the MAC-ELISA assay were 93.6%, 37.5%, 59.2%, 85.7% respectively. CONCLUSIONS: The M20 MAC-ELISA assay performed with a high sensitivity and low specificity in the acute phase of illness. Sensitivity was similar as compared with MAT in the convalescent phase and specificity remained low. Paired sera were the most sensitive but least specific by comparison to MAT serodiagnosis. NPV for acute, convalescent and paired sera was high. The limited specificity and high sensitivity of the MAC-ELISA IgM suggests that it would be most valuable to exclude leptospirosis in low-resource regions that lack immediate access to definitive reference laboratory techniques such as MAT.

The battle between infection and host immune responses of dengue virus and its implication in dengue disease pathogenesis.

Dengue virus (DENV) is a mosquito-transmitted single stranded RNA virus belonging to genus Flavivirus. The virus is endemic in the tropical and subtropical countries of the world, causing diseases classified according to symptoms and severity (from mild to severe) as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Among a variety of human cell types targeted by DENV, monocytes, macrophages, and dendritic cells are members of innate immunity, capable of mounting rapid inflammatory responses. These cells are also major antigen presenting cells, responsible for activating the adaptive immunity for long-term memory. This paper is an overview of the current understanding of the following mutually affected aspects: DENV structure, viral infectivity, cellular receptors, innate immune response, and adaptive immunity.

Novel strain of Andes virus associated with fatal human infection, central Bolivia.

To better describe the genetic diversity of hantaviruses associated with human illness in South America, we screened blood samples from febrile patients in Chapare Province in central Bolivia during 2008-2009 for recent hantavirus infection. Hantavirus RNA was detected in 3 patients, including 1 who died. Partial RNA sequences of small and medium segments from the 3 patients were most closely related to Andes virus lineages but distinct (<90% nt identity) from reported strains. A survey for IgG against hantaviruses among residents of Chapare Province indicated that 12.2% of the population had past exposure to >1 hantaviruses; the highest prevalence was among agricultural workers. Because of the high level of human exposure to hantavirus strains and the severity of resulting disease, additional studies are warranted to determine the reservoirs, ecologic range, and public health effect of this novel strain of hantavirus.

Susceptibility and lack of evidence for a viremic state of rabies in the night owl monkey, Aotus nancymaae.

BACKGROUND: Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World non-human Primates (NHP). To investigate the suitability of the owl monkey Aotus nancymaae asissue sections examined were unremarkable for inflammation or other histologic signs of rabies a viable animal model for rabies virus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus. METHODS: Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed. RESULTS: Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p = 0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies within the asymptomatic animal. Similarly none of the brain sections exhibited immunoreactivity for rabies virus antibody. DISCUSSION: This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain. CONCLUSIONS: Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.

Venezuelan equine encephalitis and 2 human deaths, Peru.

Studies have suggested that enzootic strains of Venezuelan equine encephalitis (VEE) subtype ID in the Amazon region, Peru, may be less pathogenic to humans than are epizootic variants. Deaths of 2 persons with evidence of acute VEE virus infection indicate that fatal VEEV infection in Peru is likely. Cases may remain underreported.

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.