Expression of Antimicrobial Peptides in Human Monocytic Cells and Neutrophils in Response to Dengue Virus Type 2.

BACKGROUND/AIMS: The innate immune response is remarkably important for controlling infections. Information about the participation of antimicrobial peptides (AMPs) in response to dengue virus (DENV) is scarce. The aim of this study was to examine the AMP response to DENV-2 in human THP-1 cells and neutrophils. METHODS: Protein and mRNA levels of two AMPs - hBD-1 and cathelicidin LL-37 - were assessed in DENV-infected macrophage-like THP-1 cells using qRT-PCR and indirect immunofluorescence. Also, mRNA levels of α-defensins (hDEFAs) and LL-37 were examined by qRT-PCR in human neutrophils taken from peripheral blood and treated with DENV-2. RESULTS: mRNA expression of hBD-1 rose in THP-1 cells at 24-72 h, while protein expression increased later, from 48 to 72 h after infection. Cathelicidin LL-37 mRNA expression of DENV-infected THP-1 cells was observed at 6-48 h after infection, while protein levels increased importantly up to 72 h after infection. Regarding neutrophils, the mRNA expression of hDEFAs and LL-37 increased slightly at 2 and 5 h after the contact with DENV-2. CONCLUSION: THP-1 cells and human neutrophils strongly respond to DENV by producing AMPs: hBD-1 and LL-37 for the THP-1 cells and hDEFAs and LL-37 for neutrophils. However, the direct effect of these molecules on DENV particles remains unclear.

Serum levels of IFN-ß are associated with days of evolution but not with severity of dengue.

The serum levels of beta interferon (IFN-ß) were evaluated in clinical samples taken in the acute phase of dengue fever for 107 patients during the 2011 dengue outbreak in Yucatán, México. Dengue diagnoses were confirmed by NS1 or IgM/IgG serology in all patients. Average serum IFN-ß levels in patients with dengue fever without warning signs (n = 53) were 140 pg/ml, and 105 pg/ml for patients with warning signs (n = 54). There was no statistically significant difference between IFN-ß levels for the two groups (Mann-Whitney U Test P > 0.05) and no association with warning or mild signs (OR: 0.57; 95%CI: 0.26-1.22) was indicated. Thrombocytopenia was the most prevalent warning sign (91%) in one group of patients (49) with mean 113 pg/ml IFN-ß levels. In contrast, the patients without thrombocytopenia (50) had 126 pg/ml of IFN-ß, but this level was not statistically significant (Mann-Whitney U Test P > 0.05). The average levels of IFN-ß were also found to have statistically similar results, using the 1997 WHO classification system. The amount of IFN-ß at 1-3, 4-6, and 7-9 days after onset of illness, however, did show significant differences (ANOVA P = 0.038) between patients for the 1-3 and 4-6 days pair (Scheffe post-hoc P = 0.043). These results suggest that serum levels of IFN-ß do not correlate well with the severity of dengue illness, but there is a clear association between changes in IFN-ß levels and the days of evolution during the acute phase of the disease.

Local evolution of pyrethroid resistance offsets gene flow among Aedes aegypti collections in Yucatan State, Mexico.

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.

Urban Mosquito Fauna in Mérida City, México: Immatures Collected from Containers and Storm-water Drains/Catch Basins.

We examined the species composition and temporal occurrence of immature mosquitoes in containers and storm-water drains/catch basins from November 2011 to June 2013 in Mérida City, México. A wide range of urban settings were examined, including residential premises, vacant lots, parking lots, and streets or sidewalks with storm-water drains/catch basins. In total, 111,776 specimens of 15 species were recorded. The most commonly collected species were Aedes (Stegomyia) aegypti (L.) (n = 60,961) and Culex quinquefasciatus Say (45,702), which together accounted for 95.4% of the immatures collected. These species were commonly encountered during both rainy and dry seasons, whereas most other mosquito species were collected primarily during the rainy season. Other species collected were Aedes (Howardina) cozumelensis Diaz Najera, Aedes (Ochlerotatus) taeniorhynchus (Wiedemann), Aedes (Ochlerotatus) trivittatus (Coquillett), Culex coronator Dyar and Knab, Culex interrogator Dyar and Knab, Culex lactator Dyar and Knab, Culex nigripalpus Theobald, Culex salinarius Coquillett, Culex tarsalis Coquillett, Culex thriambus Dyar, Haemagogus equinus Theobald, Limatus durhamii Theobald, and Toxorhynchites rutilus (Coquillett). The greatest number of species was recorded from vacant lots (n = 11), followed by storm-water drains/catch basins (nine) and residential premises (six). Our study demonstrated that the heterogeneous urban environment in Mérida City supports a wide range of mosquito species, many of which are nuisance biters of humans and/or capable of serving as vectors of pathogens affecting humans or domestic animals. We also briefly reviewed the medical importance of the encountered mosquito species.

Temporal correlations between mosquito-based dengue virus surveillance measures or indoor mosquito abundance and dengue case numbers in Mérida City, México.

Surveillance of dengue virus (DENV) in Aedes (Stegomyia) aegypti (L.) females is of potential interest because human DENV infections are commonly asymptomatic, which decreases the effectiveness of dengue case surveillance to provide early warning of building outbreaks. Our primary aim was to examine if mosquito-based virological measures--monthly percentages of examined Ae. aegypti females infected with DENV or examined homes from which at least one DENV-infected Ae. aegypti female was collected--are correlated with reported dengue cases in the same or subsequent months within study neighborhoods in Méida City, México. The study encompassed approximately 30 neighborhoods in the southern and eastern parts of the city. Mosquitoes were collected monthly over a 15-mo period within study homes (average of 145 homes examined per month); this produced approximately 5,800 Ae. aegypti females subsequently examined for DENV RNA. Although monthly dengue case numbers in the study neighborhoods varied > 100-fold during the study period, we did not find statistically significant positive correlations between monthly data for mosquito-based DENV surveillance measures and reported dengue cases in the same or subsequent months. Monthly average temperature, rainfall, and indoor abundance of Ae. aegypti females were positively correlated (P < or = 0.001) with dengue case numbers in subsequent months with lag times of 3-5, 2, and 1-2 mo, respectively. However, because dengue outbreak risk is strongly influenced by serotype-specific susceptibility of the human population to DENV, the value of weather conditions and entomological indices to predict outbreaks is very limited. Potential ways to improve the sensitivity of mosquito-based DENV surveillance are discussed.

Household use of insecticide consumer products in a dengue-endemic area in México.

OBJECTIVES: To evaluate the household use of insecticide consumer products to kill mosquitoes and other insect pests, as well as the expenditures for using these products, in a dengue-endemic area of México. METHODS: A questionnaire was administered to 441 households in Mérida City and other communities in Yucatán to assess household use of insecticide consumer products. RESULTS: A total of 86.6% of surveyed households took action to kill insect pests with consumer products. The most commonly used product types were insecticide aerosol spray cans (73.6%), electric plug-in insecticide emitters (37.4%) and mosquito coils (28.3%). Mosquitoes were targeted by 89.7% of households using insecticide aerosol spray cans and >99% of households using electric plug-in insecticide emitters or mosquito coils. Products were used daily or every 2 days in most of the households for insecticide aerosol spray cans (61.4%), electric plug-in insecticide emitters (76.2%) and mosquito coils (82.1%). For all products used to kill insect pests, the median annual estimated expenditure per household that took action was 408 Mexican pesos ($MXN), which corresponded to approximately 31 $US. These numbers are suggestive of an annual market in excess of 75 million $MXN (>5.7 million $US) for Mérida City alone. CONCLUSION: Mosquitoes threaten human health and are major nuisances in homes in the study area in México. Households were found to have taken vigorous action to kill mosquitoes and other insect pests and spent substantial amounts of money on insecticide consumer products.

Vacant lots: productive sites for Aedes (Stegomyia) aegypti (Diptera: Culicidae) in Mérida City, México.

We assessed the potential for vacant lots and other nonresidential settings to serve as source environments for Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) in Mérida City, México. Mosquito immatures were collected, during November 2011-June 2013, from residential premises (n = 156 site visits) and nonresidential settings represented by vacant lots (50), parking lots (18), and streets or sidewalks (28). Collections totaled 46,025 mosquito immatures of 13 species. Ae. aegypti was the most commonly encountered species accounting for 81.0% of total immatures, followed by Culex quinquefasciatus Say (12.1%). Site visits to vacant lots (74.0%) were more likely to result in collection of Ae. aegypti immatures than residential premises (35.9%). Tires accounted for 75.5% of Ae. aegypti immatures collected from vacant lots. Our data suggest that vacant lots should be considered for inclusion in mosquito surveillance and control efforts in Mérida City, as they often are located near homes, commonly have abundant vegetation, and frequently harbor accumulations of small and large discarded water-holding containers that we now have demonstrated to serve as development sites for immature mosquitoes. In addition, we present data for associations of immature production with various container characteristics, such as storage capacity, water quality, and physical location in the environment.

Stormwater drains and catch basins as sources for production of Aedes aegypti and Culex quinquefasciatus.

We present data showing that structures serving as drains and catch basins for stormwater are important sources for production of the mosquito arbovirus vectors Aedes aegypti and Culex quinquefasciatus in Mérida City, México. We examined 1761 stormwater drains - located in 45 different neighborhoods spread across the city - over dry and wet seasons from March 2012 to March 2013. Of the examined stormwater drains, 262 (14.9%) held water at the time they were examined and 123 yielded mosquito immatures. In total, we collected 64,560 immatures representing nine species. The most commonly encountered species were Cx. quinquefasciatus (n=39,269) and Ae. aegypti (n=23,313). Ae. aegypti and Cx. quinquefasciatus were collected during all 11 months when we found water-filled stormwater drains, and both were found in stormwater drains located throughout Mérida City. We also present data for associations between structural characteristics of stormwater drains or water-related characteristics and the abundance of mosquito immatures. In conclusion, stormwater drains produce massive numbers of Ae. aegypti and Cx. quinquefasciatus across Mérida City, both in the wet and dry seasons, and represent non-residential development sites that should be strongly considered for inclusion in the local mosquito surveillance and control program.

[Immune responses and immunopathogenesis in infections with dengue virus]. / Respuesta inmune e inmunopatogénesis en las infecciones con el virus del dengue.

We still have an incomplete understanding of both immunoprotection and immunopathogenesis mechanisms in dengue. Proper balance among the components of the immune response plays an important role in protection as well as in pathogenesis because these impact clinical outcomes and severity of dengue cases. In this article, we review the elements of the immune response that participate in DENV infections, and we contrast the levels of immune effectors in both classic dengue fever and the severe dengue fever cases. We also emphasize the components frequently related to the immunopathogenesis in dengue. It is clear that several effectors are increased or dysregulated in the severe cases. Finally, the global mechanism that contributes to the subversion of the immune system in dengue hemorrhagic fever or dengue shock syndrome still requires complete elucidation.

Towards a Casa Segura: a consumer product study of the effect of insecticide-treated curtains on Aedes aegypti and dengue virus infections in the home.

The home, or domicile, is the principal environment for transmission of dengue virus (DENV) between humans and mosquito vectors. Community-wide distribution of insecticide-treated curtains (ITCs), mimicking vector control program-driven interventions, has shown promise to reduce DENV infections. We conducted a Casa Segura consumer product intervention study in Mérida, Mexico to determine the potential to reduce intradomicillary DENV transmission through ITC use in individual homes. Dengue virus infections in mosquitoes and in humans were reduced in homes with ITCs in one of two study subareas. Overall, ITCs reduced intradomicillary DENV transmission; ITC homes were significantly less likely to experience multiple DENV infections in humans than NTC homes. Dengue virus-infected Aedes aegypti females were reduced within the ITC homes where curtain use was highest. Some homes yielded up to nine infected Ae. aegypti females. This study provides insights regarding best practices for Casa Segura interventions to protect homes from intradomicillary DENV transmission.

Antibodies to West Nile virus in wild and farmed crocodiles in southeastern Mexico.

Surveillance for evidence of West Nile virus (WNV) infection in Morelet's crocodiles (Crocodylus moreletii) was conducted in Campeche State, Mexico, in 2007. Sera from 62 crocodiles (32 free-ranging and 30 captive) were assayed for antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay. Antibodies to WNV were detected in 13 (41%) wild and nine (30%) captive crocodiles, and the overall antibody prevalence was 35%. Although evidence of WNV infection in captive crocodiles has been reported in Mexico, we provide the first evidence of WNV exposure in wild crocodiles in Mexico.

Serologic evidence of flavivirus infection in bats in the Yucatan Peninsula of Mexico.

We captured 140 bats of seven species in Merida City in the Yucatan Peninsula of Mexico in 2010. Serum was collected from each bat and assayed by plaque reduction neutralization test (PRNT) using six flaviviruses: West Nile virus, St. Louis encephalitis virus, and dengue viruses 1-4. Flavivirus-specific antibodies were detected in 26 bats (19%). The antibody-positive bats belonged to three species: the Pallas's long-tongued bat (Glossophaga soricina), Jamaican fruit bat (Artibeus jamaicensis), and great fruit-eating bat (Artibeus lituratus), and their flavivirus antibody prevalences were 33%, 24%, and 9%, respectively. The PRNT titers were usually highest for dengue virus 2 or dengue virus 4, but none of the titers exceeded 80. These data could indicate that most of the antibody-positive bats had been infected with dengue virus. However, because all titers were low, it is possible that the bats had been infected with another (perhaps unrecognized) flavivirus not included in the PRNT analysis, possibly a virus more closely related to dengue virus than to other flaviviruses. Each serum sample was assayed for flavivirus RNA by reverse transcription PCR, but all were negative.

Aedes (Stegomyia) aegypti and Aedes (Howardina) cozumelensis in Yucatán State, México, with a summary of published collection records for Ae. cozumelensis.

We collected mosquito immatures from artificial containers during 2010-2011 from 26 communities, ranging in size from small rural communities to large urban centers, located in different parts of Yucatán State in southeastern México. The arbovirus vector Aedes (Stegomyia) aegypti was collected from all 26 examined communities, and nine of the communities also yielded another container-inhabiting Aedes mosquito: Aedes (Howardina) cozumelensis. The communities from which Ae. cozumelensis were collected were all small rural communities (<6,000 inhabitants) in the north-central part of Yucatán State. These new collection records for Ae. cozumelensis demonstrate that this mosquito has a far broader geographic range in the Yucatán Peninsula than previously known. Ae. cozumelensis immatures were collected from both residential premises and cemeteries, with specimens recovered from rock holes as well as various artificial containers including metal cans, flower vases, buckets, tires, and a water storage tank. The co-occurrence with Ae. aegypti in small rural communities poses intriguing questions regarding linkages between these mosquitoes, including the potential for direct competition for larval development sites. Additional studies are needed to determine how commonly Ae. cozumelensis feeds on human blood and whether it is naturally infected with arboviruses or other pathogens of medical or veterinary importance. We also summarize the published records for Ae. cozumelensis, which are restricted to collections from México's Yucatán Peninsula and Belize, and uniformly represent geographic locations where Ae. aegypti can be expected to occur.

Productive container types for Aedes aegypti immatures in Mérida, México.

During 2007-2010, we examined which container types in Mérida, México, are most productive for Aedes aegypti (L.) immatures. Surveys for mosquito immatures followed routine surveillance methodology and container type classifications used by Servicios de Salud de Yucatán. Our main findings were that (1) small and larger discarded containers that serve no particular purpose and therefore can be removed from the environment contribute strongly to larval and pupal production in Mérida, and (2) the importance of different container types can vary among sets of residential premises as well as between dry and wet periods. These results may help to guide future implementation in Mérida of control efforts that target the most productive container types for Ae. aegypti immatures. Furthermore, if the Patio Limpio cleanup campaign that currently is ongoing in Mérida proves successful in removing discarded containers as important immature development sites, then we should see dramatic changes in the most productive container types in the future as the mosquito is forced to switch to other container types, which perhaps also will be easier to include in highly targeted mosquito control interventions.

Mosquito infestation and dengue virus infection in Aedes aegypti females in schools in Merida, Mexico.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from schools in Mérida, México, during 2008 and 2009. Backpack aspiration from 24 schools produced 468 females of Ae. aegypti and 1,676 females of another human biter, Culex quinquefasciatus. Ae. aegypti females were collected most commonly from classrooms followed by offices and bathrooms. Of these females, 24.7% were freshly fed. Examination of 118 pools of Ae. aegypti females (total of 415 females) for presence of DENV RNA produced 19 positive pools (16.1%). DENV-infected pools were detected from 11 (45.8%) of 24 schools and came from different room types, including classrooms, offices, and bathrooms. The overall rate of DENV infection per 100 Ae. aegypti females was 4.8. We conclude that schools in Mérida present a risk environment for students, teachers, and other personnel to be exposed to mosquitoes and bites of DENV-infected Ae. aegypti females.

Dengue virus-infected Aedes aegypti in the home environment.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from premises of laboratory-confirmed dengue patients over a 12-month period (March 2007 to February 2008) in Merida, Mexico. Backpack aspiration from 880 premises produced 1,836 females and 1,292 males indoors (predominantly from bedrooms) and 102 females and 108 males from patios/backyards. The mean weekly indoor catch rate per home peaked at 7.8 females in late August. Outdoor abundances of larvae or pupae were not predictive of female abundance inside the home. DENV-infected Ae. aegypti females were recovered from 34 premises. Collection of DENV-infected females from homes of dengue patients up to 27 days after the onset of symptoms (median, 14 days) shows the usefulness of indoor insecticide application in homes of suspected dengue patients to prevent their homes from becoming sources for dispersal of DENV by persons visiting and being bitten by infected mosquitoes.

Use of Google Earth to strengthen public health capacity and facilitate management of vector-borne diseases in resource-poor environments.

OBJECTIVE: Novel, inexpensive solutions are needed for improved management of vector-borne and other diseases in resource-poor environments. Emerging free software providing access to satellite imagery and simple editing tools (e.g. Google Earth) complement existing geographic information system (GIS) software and provide new opportunities for: (i) strengthening overall public health capacity through development of information for city infrastructures; and (ii) display of public health data directly on an image of the physical environment. METHODS: We used freely accessible satellite imagery and a set of feature-making tools included in the software (allowing for production of polygons, lines and points) to generate information for city infrastructure and to display disease data in a dengue decision support system (DDSS) framework. FINDINGS: Two cities in Mexico (Chetumal and Merida) were used to demonstrate that a basic representation of city infrastructure useful as a spatial backbone in a DDSS can be rapidly developed at minimal cost. Data layers generated included labelled polygons representing city blocks, lines representing streets, and points showing the locations of schools and health clinics. City blocks were colour-coded to show presence of dengue cases. The data layers were successfully imported in a format known as shapefile into a GIS software. CONCLUSION: The combination of Google Earth and free GIS software (e.g. HealthMapper, developed by WHO, and SIGEpi, developed by PAHO) has tremendous potential to strengthen overall public health capacity and facilitate decision support system approaches to prevention and control of vector-borne diseases in resource-poor environments.

Epidemic dengue 4 in the Yucatán, México, 1984

An outbreak of dengue 4 occurred in the Yucatán, México in 1984. During the course of the outbreak, 538 of 5486 reported cases of dengue-like illness were studied; 200 were confirmed as dengue serologically and/or virologically. Dengue 4 virus was isolated from 34 patients and dengue 1 from one. Severe haemorrhagic symptoms were observed in 9 laboratory confirmed patients, including four deaths. Thus, the outbreak in Yucatán is the second dengue epidemic in the Americas after the Cuban epidemic in 1981 in which a number of patients suffered from haemorrhagic complications. It was notable that 5 of 9 hospitalized, severe cases were young adults and that only one met the WHO criteria of DHF, in contrast to primary pediatric nature of DHF in Southeast Asia. In this paper we describe clinical, serologic, and virologic studies conducted during the outbreak.