State‐wide survey of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Florida

Aedes aegypti and Aedes albopictus are invasive mosquito species with geographic ranges that have oscillated within Florida since their presence was first documented. Local transmission of dengue, chikungunya, and Zika viruses serves as evidence of the public health importance of these two species. It is important to have detailed knowledge of their distribution to aid in mosquito control efforts and understand the risk of arbovirus transmission to humans. Through a partnership involving the University of Florida Institute of Food and Agricultural Sciences Cooperative Extension Service and the Florida Medical Entomology Laboratory; the Florida Department of Health; and mosquito control agencies throughout Florida, a container mosquito surveillance program involving all life stages was launched in the summer of 2016 to detect the presence of Ae. aegypti and Ae. albopictus. Results from this survey were mapped to provide a picture of the current known distribution of Ae. aegypti and Ae. albopictus in Florida. Aedes aegypti and/or Ae. albopictus were detected in the 56 counties that were part of the survey. Only Aedes albopictus was detected in 26 counties, primarily in the panhandle region of Florida. The results of this work underscore the importance of maintaining container mosquito surveillance in a state where chikungunya, dengue, and Zika viruses are present and where there is continued risk for exotic arbovirus introductions.     

A decade of colonization: the spread of the Asian tiger mosquito in Pennsylvania and implications for disease risk

In recent decades, the Asian tiger mosquito expanded its geographic range throughout the northeastern United States, including Pennsylvania. The establishment of Aedes albopictus in novel areas raises significant public health concerns, since this species is a highly competent vector of several arboviruses, including chikungunya, West Nile, and dengue. In this study, we used geographic information systems (GIS) to examine a decade of colonization by Ae. albopictus throughout Pennsylvania between 2001 and 2010. We examined the spatial and temporal distribution of Ae. albopictus using spatial statistical analysis and examined the risk of dengue virus transmission using a model that captures the probability of transmission. Our findings show that since 2001, the Ae. albopictus population in Pennsylvania has increased, becoming established and expanding in range throughout much of the state. Since 2010, imported cases of dengue fever have been recorded in Pennsylvania. Imported cases of dengue, in combination with summer temperatures conducive for virus transmission, raise the risk of local disease transmission.     

A recent chikungunya outbreak associated with the occurrence of Aedes vectors (Diptera: Culicidae) in Kassala state,eastern Sudan in 2018

Aedes aegypti and Ae. albopictus (Diptera: Culicidae) are primary vectors of human arboviral diseases such as dengue, chikungunya, yellow fever, and Zika viruses. Aedes aegypti is well distributed in Sudan, except in Northern and Khartoum states, while Ae. albopictus has not been previously reported. Recently, Eastern Sudan witnessed an unprecedented large outbreak of chikungunya fever between 31 May 2018 and 30 March 2019. The outbreak was composed of four waves; one of them was in Kassala state. Aedes survey in localities of Kassala state (rather than Kassala city) is carried out to assess the possible expansion of the disease in the state. The results showed the presence of immature stages of Aedes spp. in four localities from ten localities. From the four localities that recorded Aedes spp., two localities (Rural Kassala and West Kassala) were reported with chikungunya cases. From this investigation, Ae. albopictus was reported for the first time in Sudan. Also, this investigation showed the importance of conducting entomological surveys with epidemiological surveys during outbreaks of arboviral diseases.     

The challenge of invasive mosquito vectors in the U.K. during 2016–2018: a summary of the surveillance and control of Aedes albopictus

Mosquito‐borne diseases resulting from the expansion of two key vectors, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), continue to challenge whole regions and continents around the globe. In recent years there have been human cases of disease associated with Chikungunya, dengue and Zika viruses. In Europe, the expansion of Ae. albopictus has resulted in local transmission of Chikungunya and dengue viruses. This paper considers the risk that Ae. aegypti and Ae. albopictus represent for the U.K. and details the results of mosquito surveillance activities. Surveillance was conducted at 34 points of entry, 12 sites serving vehicular traffic and two sites of used tyre importers. The most common native mosquito recorded was Culex pipiens s.l. (Diptera: Culicidae). The invasive mosquito Ae. albopictus was detected on three occasions in southern England (September 2016, July 2017 and July 2018) and subsequent control strategies were conducted. These latest surveillance results demonstrate ongoing incursions of Ae. albopictus into the U.K. via ground vehicular traffic, which can be expected to continue and increase as populations in nearby countries expand, particularly in France, which is the main source of ex‐continental traffic.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

Orally Co-Infected Aedes albopictus from La Reunion Island,Indian Ocean,Can Deliver Both Dengue and Chikungunya Infectious Viral Particles in Their Saliva

Background

First described in humans in 1964, reports of co-infections with dengue (DENV) and chikungunya (CHIKV) viruses are increasing, particularly after the emergence of chikungunya (CHIK) in the Indian Ocean in 2005–2006 due to a new variant highly transmitted by Aedes albopictus. In this geographic area, a dengue (DEN) outbreak transmitted by Ae. albopictus took place shortly before the emergence of CHIK and co-infections were reported in patients. A co-infection in humans can occur following the bite of two mosquitoes infected with one virus or to the bite of a mosquito infected with two viruses. Co-infections in mosquitoes have never been demonstrated in the field or in the laboratory. Thus, we question about the ability of a mosquito to deliver infectious particles of two different viruses through the female saliva.

Methodology/Principal Findings

We orally exposed Ae. albopictus from La Reunion Island with DENV-1 and CHIKV isolated respectively during the 2004–2005 and the 2005–2006 outbreaks on this same island. We were able to show that Ae. albopictus could disseminate both viruses and deliver both infectious viral particles concomitantly in its saliva. We also succeeded in inducing a secondary infection with CHIKV in mosquitoes previously inoculated with DENV-1.

Conclusions/Significance

In this study, we underline the ability of Ae. albopictus to be orally co-infected with two different arboviruses and furthermore, its capacity to deliver concomitantly infectious particles of CHIKV and DENV in saliva. This finding is of particular concern as Ae. albopictus is still expanding its geographical range in the tropical as well as in the temperate regions. Further studies are needed to try to elucidate the molecular/cellular basis of this phenomenon.     

Aedes (Stegomyia) albopictus (Skuse): A Potential Vector of Zika Virus in Singapore

Background

Zika virus (ZIKV) is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV.

Methodology/Principal Findings

To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80–85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi). Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose₅₀ assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious.

Conclusions/Significance

The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.     

Climatic and socio-economic factors supporting the co-circulation of dengue,Zika and chikungunya in three different ecosystems in Colombia

Dengue, Zika and chikungunya are diseases of global health significance caused by arboviruses and transmitted by the mosquito Aedes aegypti, which is of worldwide circulation. The arrival of the Zika and chikungunya viruses to South America increased the complexity of transmission and morbidity caused by these viruses co-circulating in the same vector mosquito species. Here we present an integrated analysis of the reported arbovirus cases between 2007 and 2017 and local climate and socio-economic profiles of three distinct Colombian municipalities (Bello, Cúcuta and Moniquirá). These locations were confirmed as three different ecosystems given their contrasted geographic, climatic and socio-economic profiles. Correlational analyses were conducted with both generalised linear models and generalised additive models for the geographical data. Average temperature, minimum temperature and wind speed were strongly correlated with disease incidence. The transmission of Zika during the 2016 epidemic appeared to decrease circulation of dengue in Cúcuta, an area of sustained high incidence of dengue. Socio-economic factors such as barriers to health and childhood services, inadequate sanitation and poor water supply suggested an unfavourable impact on the transmission of dengue, Zika and chikungunya in all three ecosystems. Socio-demographic influencers were also discussed including the influx of people to Cúcuta, fleeing political and economic instability from neighbouring Venezuela. Aedes aegypti is expanding its range and increasing the global threat of these diseases. It is therefore vital that we learn from the epidemiology of these arboviruses and translate it into an actionable local knowledge base. This is even more acute given the recent historical high of dengue cases in the Americas in 2019, preceding the COVID-19 pandemic, which is itself hampering mosquito control efforts.     

Population genomics of the Asian tiger mosquito,Aedes albopictus: insights into the recent worldwide invasion

Aedes albopictus, the “Asian tiger mosquito,” is an aggressive biting mosquito native to Asia that has colonized all continents except Antarctica during the last ~30–40 years. The species is of great public health concern as it can transmit at least 26 arboviruses, including dengue, chikungunya, and Zika viruses. In this study, using double‐digest Restriction site‐Associated DNA (ddRAD) sequencing, we developed a panel of ~58,000 single nucleotide polymorphisms (SNPs) based on 20 worldwide Ae. albopictus populations representing both the invasive and the native range. We used this genomic‐based approach to study the genetic structure and the differentiation of Ae. albopictus populations and to understand origin(s) and dynamics of the recent invasions. Our analyses indicated the existence of two major genetically differentiated population clusters, each one including both native and invasive populations. The detection of additional genetic structure within each major cluster supports that these SNPs can detect differentiation at a global and local scale, while the similar levels of genomic diversity between native and invasive range populations support the scenario of multiple invasions or colonization by a large number of propagules. Finally, our results revealed the possible source(s) of the recent invasion in Americas, Europe, and Africa, a finding with important implications for vector‐control strategies.     

First record of Stegomyia albopicta (= Aedes albopictus) in Morocco: a major threat to public health in North Africa?

The Asian tiger mosquito Stegomyia albopicta (= Aedes albopictus) (Diptera: Culicidae), native to Asian forests, is a nuisance mosquito and is responsible for the transmission of arboviruses of public health importance, such as dengue, chikungunya and Zika viruses. It has colonized parts of all continents, except Antarctica, over the past 30–40 years. However, to date, the only records of S. albopicta in North Africa refer to occasional collections in 2010 and 2014 in Algeria. In early September 2015, S. albopicta larvae and adults were collected in a district of Rabat, Morocco. Morphological identification was confirmed by molecular analysis. This is the first record of this invasive mosquito in Morocco. A national surveillance programme will be implemented in 2016 to establish its geographical distribution in Morocco and to instigate control measures to prevent the establishment of new populations and the transmission of arboviruses.     

Aedes hensilli as a Potential Vector of Chikungunya and Zika Viruses

An epidemic of Zika virus (ZIKV) illness that occurred in July 2007 on Yap Island in the Federated States of Micronesia prompted entomological studies to identify both the primary vector(s) involved in transmission and the ecological parameters contributing to the outbreak. Larval and pupal surveys were performed to identify the major containers serving as oviposition habitat for the likely vector(s). Adult mosquitoes were also collected by backpack aspiration, light trap, and gravid traps at select sites around the capital city. The predominant species found on the island was Aedes (Stegomyia) hensilli. No virus isolates were obtained from the adult field material collected, nor did any of the immature mosquitoes that were allowed to emerge to adulthood contain viable virus or nucleic acid. Therefore, laboratory studies of the probable vector, Ae. hensilli, were undertaken to determine the likelihood of this species serving as a vector for Zika virus and other arboviruses. Infection rates of up to 86%, 62%, and 20% and dissemination rates of 23%, 80%, and 17% for Zika, chikungunya, and dengue-2 viruses respectively, were found supporting the possibility that this species served as a vector during the Zika outbreak and that it could play a role in transmitting other medically important arboviruses.     

High Efficiency of Temperate Aedes albopictus to Transmit Chikungunya and Dengue Viruses in the Southeast of France

Background

Since 2005, cases of chikungunya (CHIK) were caused by an unusual vector, Aedes albopictus. This mosquito, present in Europe since 1979, has gained importance since its involvement in the first CHIK outbreak in Italy in 2007. The species is capable of transmitting experimentally 26 arboviruses. However, the vectorial status of its temperate populations has remained little investigated. In 2010, autochthonous cases of CHIK and dengue (DEN) were reported in southeastern France. We evaluated the potential of a French population of Ae. albopictus in the transmission of both viruses.

Methodology and Principal Findings

We used two strains of each virus, CHIK and DEN: one strain was isolated from an imported case, and one from an autochthonous case. We used as controls Aedes aegypti from India and Martinique, the source of the imported cases of CHIK and DEN, respectively. We showed that Ae. albopictus from Cagnes-sur-Mer (AL-CSM) was as efficient as the typical tropical vector Ae. aegypti from India to experimentally transmit both CHIK strains isolated from patients in Fréjus, with around 35–67% of mosquitoes delivering up to 14 viral particles at day 3 post-infection (pi). The unexpected finding came from the high efficiency of AL-CSM to transmit both strains of DENV-1 isolated from patients in Nice. Almost 67% of Ae. albopictus AL-CSM which have ensured viral dissemination were able to transmit at day 9 pi when less than 21% of the typical DEN vector Ae. aegypti from Martinique could achieve transmission.

Conclusions/Significance

Temperate Ae. albopictus behaves differently compared to its counterpart from tropical regions, where recurrent epidemic outbreaks occur. Its potential responsibility for outbreaks in Europe should not be minimized.     

Comparative Host Feeding Patterns of the Asian Tiger Mosquito,Aedes albopictus,in Urban and Suburban Northeastern USA and Implications for Disease Transmission

Background

Aedes albopictus is an invasive species which continues expanding its geographic range and involvement in mosquito-borne diseases such as chikungunya and dengue. Host selection patterns by invasive mosquitoes are critically important because they increase endemic disease transmission and drive outbreaks of exotic pathogens. Traditionally, Ae. albopictus has been characterized as an opportunistic feeder, primarily feeding on mammalian hosts but occasionally acquiring blood from avian sources as well. However, limited information is available on their feeding patterns in temperate regions of their expanded range. Because of the increasing expansion and abundance of Ae. albopictus and the escalating diagnoses of exotic pathogens in travelers returning from endemic areas, we investigated the host feeding patterns of this species in newly invaded areas to further shed light on its role in disease ecology and assess the public health threat of an exotic arbovirus outbreak.

Methodology/Principal Findings

We identified the vertebrate source of 165 blood meals in Ae. albopictus collected between 2008 and 2011 from urban and suburban areas in northeastern USA. We used a network of Biogents Sentinel traps, which enhance Ae. albopictus capture counts, to conduct our collections of blooded mosquitoes. We also analyzed blooded Culex mosquitoes collected alongside Ae. albopictus in order to examine the composition of the community of blood sources. We found no evidence of bias since as expected Culex blood meals were predominantly from birds (n = 149, 93.7%) with only a small proportion feeding on mammals (n = 10, 6.3%). In contrast, Aedes albopictus fed exclusively on mammalian hosts with over 90% of their blood meals derived from humans (n = 96, 58.2%) and domesticated pets (n = 38, 23.0% cats; and n = 24, 14.6% dogs). Aedes albopictus fed from humans significantly more often in suburban than in urban areas (χ², p = 0.004) and cat-derived blood meals were greater in urban habitats (χ², p = 0.022). Avian-derived blood meals were not detected in any of the Ae. albopictus tested.

Conclusions/Significance

The high mammalian affinity of Ae. albopictus suggests that this species will be an efficient vector of mammal- and human-driven zoonoses such as La Crosse, dengue, and chikungunya viruses. The lack of blood meals obtained from birds by Ae. albopictus suggest that this species may have limited exposure to endemic avian zoonoses such as St. Louis encephalitis and West Nile virus, which already circulate in the USA. However, growing populations of Ae. albopictus in major metropolitan urban and suburban centers, make a large autochthonous outbreak of an arbovirus such as chikungunya or dengue viruses a clear and present danger. Given the difficulties of Ae. albopictus suppression, we recommend that public health practitioners and policy makers install proactive measures for the imminent mitigation of an exotic pathogen outbreak.     

Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector

Mosquitoes transmit numerous arboviruses including dengue and chikungunya virus (CHIKV). In recent years, mosquito species Aedes albopictus has expanded in the Indian Ocean region and was the principal vector of chikungunya outbreaks in La Reunion and neighbouring islands in 2005 and 2006. Vector‐associated bacteria have recently been found to interact with transmitted pathogens. For instance, Wolbachia modulates the replication of viruses or parasites. However, there has been no systematic evaluation of the diversity of the entire bacterial populations within mosquito individuals particularly in relation to virus invasion. Here, we investigated the effect of CHIKV infection on the whole bacterial community of Ae. albopictus. Taxonomic microarrays and quantitative PCR showed that members of Alpha‐ and Gammaproteobacteria phyla, as well as Bacteroidetes, responded to CHIKV infection. The abundance of bacteria from the Enterobacteriaceae family increased with CHIKV infection, whereas the abundance of known insect endosymbionts like Wolbachia and Blattabacterium decreased. Our results clearly link the pathogen propagation with changes in the dynamics of the bacterial community, suggesting that cooperation or competition occurs within the host, which may in turn affect the mosquito traits like vector competence.     

Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico

Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue‐1 during 2009, 2010, and 2013 in Florida and dengue‐1 and −2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler‐imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV‐1 and −2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV‐1 (6–14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV‐2 than Ae. albopictus (Ae. aegypti, ∼28%; Ae. albopictus, ∼9%). The dissemination rate with dengue‐2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV‐2 isolate from Puerto Rico. These results are discussed in light of Florida's vulnerability to DENV transmission.     

Genetic structure of the tiger mosquito, Aedes albopictus, in Cameroon (Central Africa)

Background

Aedes albopictus (Skuse, 1884) (Diptera: Culicidae), a mosquito native to Asia, has recently invaded all five continents. In Central Africa it was first reported in the early 2000s, and has since been implicated in the emergence of arboviruses such as dengue and chikungunya in this region. Recent genetic studies of invasive species have shown that multiple introductions are a key factor for successful expansion in new areas. As a result, phenotypic characters such as vector competence and insecticide susceptibility may vary within invasive pest species, potentially affecting vector efficiency and pest management. Here we assessed the genetic variability and population genetics of Ae. albopictus isolates in Cameroon (Central Africa), thereby deducing their likely geographic origin.

Methods and Results

Mosquitoes were sampled in 2007 in 12 localities in southern Cameroon and analyzed for polymorphism at six microsatellite loci and in two mitochondrial DNA regions (ND5 and COI). All the microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structureamong geographic populations (F_ST = 0.068, P<0.0001). Analysis of mtDNA sequences revealed four haplotypes each for the COI and ND5 genes, with a dominant haplotype shared by all Cameroonian samples. The weak genetic variation estimated from the mtDNA genes is consistent with the recent arrival of Ae. albopictus in Cameroon. Phylogeographic analysis based on COI polymorphism indicated that Ae. albopictus populations from Cameroon are related to tropical rather than temperate or subtropical outgroups.

Conclusion

The moderate genetic diversity observed among Cameroonian Ae. albopictus isolates is in keeping with recent introduction and spread in this country. The genetic structure of natural populations points to multiple introductions from tropical regions.     

The Effects of Climate Change and Globalization on Mosquito Vectors: Evidence from Jeju Island,South Korea on the Potential for Asian Tiger Mosquito (Aedes albopictus) Influxes and Survival from Vietnam Rather Than Japan

Background

Climate change affects the survival and transmission of arthropod vectors as well as the development rates of vector-borne pathogens. Increased international travel is also an important factor in the spread of vector-borne diseases (VBDs) such as dengue, West Nile, yellow fever, chikungunya, and malaria. Dengue is the most important vector-borne viral disease. An estimated 2.5 billion people are at risk of infection in the world and there are approximately 50 million dengue infections and an estimated 500,000 individuals are hospitalized with dengue haemorrhagic fever annually. The Asian tiger mosquito (Aedes albopictus) is one of the vectors of dengue virus, and populations already exist on Jeju Island, South Korea. Currently, colder winter temperatures kill off Asian tiger mosquito populations and there is no evidence of the mosquitos being vectors for the dengue virus in this location. However, dengue virus-bearing mosquito vectors can inflow to Jeju Island from endemic area such as Vietnam by increased international travel, and this mosquito vector''s survival during colder winter months will likely occur due to the effects of climate change.

Methods and Results

In this section, we show the geographical distribution of medically important mosquito vectors such as Ae. albopictus, a vector of both dengue and chikungunya viruses; Culex pipiens, a vector of West Nile virus; and Anopheles sinensis, a vector of Plasmodium vivax, within Jeju Island, South Korea. We found a significant association between the mean temperature, amount of precipitation, and density of mosquitoes. The phylogenetic analyses show that an Ae. albopictus, collected in southern area of Jeju Island, was identical to specimens found in Ho Chi Minh, Vietnam, and not Nagasaki, Japan.

Conclusion

Our results suggest that mosquito vectors or virus-bearing vectors can transmit from epidemic regions of Southeast Asia to Jeju Island and can survive during colder winter months. Therefore, Jeju Island is no longer safe from vector borne diseases (VBDs) due to the effects of globalization and climate change, and we should immediately monitor regional climate change to identify newly emerging VBDs.     

Impact of the introduction of chikungunya and zika viruses on the incidence of dengue in endemic zones of Mexico

BackgroundWith the arrival of chikungunya (CHIKV) and zika (ZIKV) viruses in Mexico, there was a decrease in diagnosed dengue virus (DENV) cases. During the first years of cocirculation (2015–2017), the algorithms established by epidemiological surveillance systems and the installed capacity limited us to one diagnostic test per sample, so there was an underestimation of cases until September 2017, when a multiplex algorithm was implemented. Therefore, the objective of this study was determine the impact of the introduction of CHIKV and ZIKV on the incidence of diagnosed DENV in endemic areas of Mexico, when performing the rediagnosis, using the multiplex algorithm, in samples from the first three years of co-circulation of these arboviruses.Methodology and principal findingsFor this, 1038 samples received by the Central Laboratory of Epidemiology between 2015 and 2017 were selected for this work. Viruses were identified by multiplex RT-qPCR, and the χ² test was used to compare categorical variables. With the new multiplex algorithm, we identified 2.4 times the rate of arbovirosis as originally reported, evidencing an underestimation of the incidence of the three viruses. Even so, significantly less dengue was observed than in previous years. The high incidence rates of chikungunya and Zika coincided with periods of dengue decline. The endemic channel showed that the cases caused by DENV rose again after the circulation of CHIKV and ZIKV decreased. In addition, 23 cases of coinfection were identified, with combinations between all viruses.Conclusions and significanceThe results obtained in this study show for the first time the real impact on the detected incidence of dengue after the introduction of CHIKV and ZIKV in Mexico, the degree of underestimation of these arboviruses in the country, as well as the co-infections between these viruses, whose importance clinical and epidemiological are still unknown.