Incompatible and sterile insect techniques combined eliminate mosquitoes.

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)-which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia-is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT-SIT) enables near elimination of field populations of the world's most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT-SIT for mosquito vector control.

Host-Guest Complexation-Induced Aggregation Based on Pyrene-Modified Cyclodextrins for Improved Electronic Circular Dichroism and Circularly Polarized Luminescence.

Several γ-cyclodextrin (CD) derivatives mono- or di-substituted by pyrenes at the primary rim of the CD were demonstrated to aggregate into nano-strips in aqueous solutions, with the pyrene moieties interpenetrating into γ-CD cavities. The hydrophobic complexation-induced aggregation provides a rigid chiral environment for the pyrenes and leads to significant electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) activities, giving unprecedently high gabs and glum values up to 4.3×10-2 and 5.3×10-2 , respectively. The aggregates lead to excimer emission with high quantum yields and show BCPL and BiCPL up to 338. 6 M-1 cm-1 and 169.3 M-1 cm-1 , respectively.

Wolbachia in Native Populations of Aedes albopictus (Diptera: Culicidae) From Yucatan Peninsula, Mexico.

This study reports the results of a molecular screening for Wolbachia (Wb) infection in Aedes albopictus (Skuse) populations recently established in the Yucatan Peninsula, Mexico. To do so, collections of free-flying adults with BG traps and emerged adults from eggs after ovitrap field collections were performed in three suburban localities of the city of Merida, Yucatan. Overall, local populations of Ae. albopictus present a natural Wb infection rate of ~40% (18 of 45). Wb infection was detected in both field-collected adults (76.5%, 13 of 17) and eggs reared (17.8%, 5 of 28) and in 37.9% (11/29) of females and 43.7% (7/16) of male Ae. albopictus mosquitoes. An initial screening for Wolbachia strain typing showed that native Ae. albopictus were naturally coinfected with both wAlbA and wAlbB strains. The knowledge of the prevalence and diversity of Wolbachia strains in local populations of Aedes mosquitoes is part of the baseline information required for current and future Wolbachia-based vector control approaches to be conducted in Mexico.

Controlled Drug Release from Cyclodextrin-Gated Mesoporous Silica Nanoparticles Based on Switchable Host-Guest Interactions.

With the development of materials science and pharmaceutics, the application of mesoporous silica nanoparticles with a gated switch in the field of drug delivery has attracted much attention in the past decades. Cyclodextrins (CD) as promising gated materials have become a new area of interest in recent years due to their properties of self-assembly and function of host-guest interaction. CD is one kind of extensively studied host molecules and the host-guest interactions with different guest molecules can respond to different signals, and thus can be applied as intelligent gated switches for smart drug carriers. Switchable gatekeepers based on CD hosts with different guest molecules (such as benzimidazole, azobenzene, and ferrocene) respond to different stimuli modes (such as pH, light, and redox) that change the host-guest interactions and trigger drug release. The different structural features, mechanisms of action, and potent applications of these switchable gatekeepers are discussed. In addition, some personal perspectives and challenge on this field are presented.

Homogeneity of the vaginal microbiome at the cervix, posterior fornix, and vaginal canal in pregnant Chinese women.

The vaginal microbiome is an emerging concern in prenatal health. Because the sampling process of vaginal microbiota may pose potential risks for pregnant women, the choice of sampling site should be carefully considered. However, whether the microbial diversity is different across various sampling sites has been controversial. In the present study, three repeated swabs were collected at the cervix (C), posterior fornix (P), and vaginal canal (V) from 34 Chinese women during different pregnancy stages, and vaginal species were determined using the Illumina sequencing of 16S rRNA tag sequences. The identified microbiomes were classified into four community state types (CSTs): CST I (dominated by L. crispatus), CST II (dominated by L. gasseri), CST III (dominated by L. iners), and CST IV-A (characterized by a low abundance of Lactobacillus, but with proportions of various species previously shown to be associated with bacterial vaginosis). All individuals had consistent CST at the three sampling sites regardless of pregnancy stage and CST group. In addition, there was little heterogeneity across community structures within each individual, as determined by LEfSe, indicating high vaginal microbiome homogeneity at the three sampling sites. The present study also revealed different beta diversity during pregnancy stages. The vaginal microbiome variation among women during trimester T1 (9 ± 2.6 weeks) is larger than that of non-pregnant women and women from other trimesters, as demonstrated by the UniFrac distance (P < 0.05). In particular, the present study is the first one that demonstrates the notably difference of vaginal microbiome of postpartum women compare to women in gestation. These results will be useful for future studies of the vaginal microbiota during pregnancy.

A field survey for Wolbchia and phage WO infections of Aedes albopictus in Guangzhou City, China.

Wolbachia are maternal endosymbiotic bacterium, which infect a diverse range of arthropods, ranging from 20 to 76% in nature. They are capable of inducing a wide range of reproductive abnormalities to their hosts, such as cytoplasmic incompatibility (CI), which has been proposed to be used as a tool to modify mosquitoes that are resistant to the development of pathogen, as an alternative vector control strategy. Here, we evaluated the prevalence of Wolbachia and phage WO infections in the field population of Aedes albopictus in Guangzhou City via polymerase chain reaction (PCR) assay using the Wolbachia specific Wolbachia surface protein (wsp) and phage WO orf7 gene primers. Based on the results of PCR and phylogeny analysis, we found that A. albopictus in Guangzhou City were infected with two Wolbachia strains, wAlbA and wAlbB. Phage WO, the virus-infected Wolbachia, was also detected in A. albopictus. One hundred and ten female individuals were screened via PCR, with 109 super-infected with Wolbachia and one sample single-infected with wAlbB strain. And 104 of 113 male individuals were both infected with wAlbA and wAlbB, and nine male samples were found to be infected with wAlbA strain only. The infection rates of phage WO in female and male individuals were 82.73 and 46.02%, respectively. These results showed that the natural Wolbachia and phage WO infections in A. albopictus population in Guangzhou were at a higher frequency at present, indicating that Wolbachia appear to be a better candidate nature resource for biological control insect vectors to reduce vector-borne diseases.

MADS-box transcription factor SsMADS is involved in regulating growth and virulence in Sclerotinia sclerotiorum.

MADS-box proteins, a well-conserved family of transcription factors in eukaryotic organisms, specifically regulate a wide range of cellular functions, including primary metabolism, cell cycle, and cell identity. However, little is known about roles of the MADS-box protein family in the fungal pathogen Sclerotinia sclerotiorum. In this research, the S. sclerotiorum MADS-box gene SsMADS was cloned; it encodes a protein that is highly similar to Mcm1 orthologs from Saccharomyces cerevisiae and other fungi, and includes a highly conserved DNA-binding domain. MADS is a member of the MADS box protein SRF (serum response factor) lineage. SsMADS function was investigated using RNA interference. Silenced strains were obtained using genetic transformation of the RNA interference vectors pS1-SsMADS and pSD-SsMADS. SsMADS expression levels in silenced strains were analyzed using RT-PCR. The results showed that SsMADS mRNA expression in these silenced strains was reduced to different degrees, and growth rate in these silenced strains was significantly decreased. Infecting tomato leaflets with silenced strains indicated that SsMADS was required for leaf pathogenesis in a susceptible host. Our results suggest that the MADS-box transcription factor SsMADS is involved in S. sclerotiorum growth and virulence.

Biased virus transmission following sequential coinfection of Aedes aegypti with dengue and Zika viruses.

BACKGROUND: Mosquito-borne arboviruses are expanding their territory and elevating their infection prevalence due to the rapid climate change, urbanization, and increased international travel and global trade. Various significant arboviruses, including the dengue virus, Zika virus, Chikungunya virus, and yellow fever virus, are all reliant on the same primary vector, Aedes aegypti. Consequently, the occurrence of arbovirus coinfection in mosquitoes is anticipated. Arbovirus coinfection in mosquitoes has two patterns: simultaneous and sequential. Numerous studies have demonstrated that simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses. However, the viruses' interplay within a mosquito after the sequential coinfection seems intricated and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We conducted experiments aimed at examining the phenomenon of arbovirus sequential coinfection in both mosquito cell line (C6/36) and A. aegypti, specifically focusing on dengue virus (DENV, serotype 2) and Zika virus (ZIKV). We firstly observed that DENV and ZIKV can sequentially infect mosquito C6/36 cell line, but the replication level of the subsequently infected ZIKV was significantly suppressed. Similarly, A. aegypti mosquitoes can be sequentially coinfected by these two arboviruses, regardless of the order of virus exposure. However, the replication, dissemination, and the transmission potential of the secondary virus were significantly inhibited. We preliminarily explored the underlying mechanisms, revealing that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, leading to reduced susceptibility to the secondary arbovirus infections. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. However, it is important to note that more experimental investigations are needed to refine and expand upon this conclusion.

Mating harassment may boost the effectiveness of the sterile insect technique for Aedes mosquitoes.

The sterile insect technique is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. It has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development for Aedes mosquitoes. Here, we show that the release of sterile males at high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduce the longevity of female Aedes mosquitoes by reducing their feeding success. Under controlled conditions, blood uptake of females from an artificial host or from a mouse and biting rates on humans are also reduced. Finally, in a field trial conducted in a 1.17 ha area in China, the female biting rate is reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the sterile insect technique does not only suppress mosquito vector populations through the induction of sterility, but may also reduce disease transmission due to increased female mortality and lower host contact.

Laccase2 is required for sclerotization and pigmentation of Aedes albopictus eggshell.

Laccase (EC 1.10.3.2) is a member of multicopper oxidases that have been found in higher plants, fungus, bacterium, and insects. Two types of laccase genes have been detected in many species of insects: laccase1 and laccase2. It has been identified that laccase2 enzyme may play a key role in sclerotization and pigmentation of insect cuticle. But few attentions were given to the biological role of laccase2 in the synthesizing of similar structures, such as oothecae, eggshell, or silk cocoons. We cloned laccase2 gene from Aedes albopictus, one main mosquito vector of dengue virus in China. An upregulation of laccase2 gene was observed after a blood meal in female adult mosquitoes, suggesting that laccase2 gene may have an involvement in the development of ovary. RNA interference experiment was performed by using adult female mosquitoes. Female mosquitoes were injected with 20 ng of double-strain RNA into the thorax. Pigmentation of mosquito eggshell was blocked that these eggs never became dark. And the incomplete sclerotization of eggshell weakened the stability and flexibility of the eggs. These eggs without enough protection were deformed and died in water. These results demonstrate that laccase2 plays a critical role in the development of eggs of A. albopictus. Laccase2 may provide a novel target for mosquito control and management.

Molecular cloning, expression, and characterization of a putative activation-associated secreted protein from Angiostrongylus cantonensis.

Activation-associated secreted protein (ASP) had been found in many helminthes, which was associated with pathogenesis and stage transition. A complementary DNA (cDNA) sequence encoding a putative two-domain ASP was obtained from an Angiostrongylus cantonensis fourth-stage larvae cDNA library, which we designated as AgASP. The cDNA of AgASP contains an open reading frame encoding 424 amino acids, the first 19 residues being a putative secretion signal. The expression pattern of this protein was investigated by real-time polymerase chain reaction and Western blot. We found that this protein expressed most highly in the brain-stage larvae (Lbr) of this parasite and existed in the excretory/secretory products of this stage. Immunofluorescence showed it existed in the lumen of the Lbr. The recombinant protein can be recognized by the infection sera from mice (nonpermissive host), while it cannot be recognized by infection sera from rats (permissive host). The infiltration of neutrophils in infected nonpermissive host can be lessened by immunizing this host with this protein (immunized vs control group, 13.7 ± 10.2 vs 65.5 ± 19.2). These findings suggest that this protein plays a role in the pathogenesis of human angiostrongyliasis and is worthy of further study.

Effects of radiation on the fitness, sterility and arbovirus susceptibility of a Wolbachia-free Aedes albopictus strain for use in the sterile insect technique.

BACKGROUND: The sterile insect technique (SIT) is a green and species-specific insect pest control technique that suppresses target populations by releasing factory-reared, radiosterilized males into the wild. Once released, it is important to be able to distinguish the released males from the wild males for monitoring purposes. Several methods to mark the sterile males exist. However, most have limitations due to monetary, process efficiency, or insect quality. Aedes albopictus is naturally infected with Wolbachia at a high prevalence, therefore the elimination of Wolbachia can serve as a biomarker to distinguish factory-reared male mosquitoes from wild conspecifics. RESULTS: In this study, a Wolbachia-free Ae. albopictus GT strain was developed and its fitness evaluated, which was found to be comparable to the wild GUA strain. In addition, GT male mosquitoes were irradiated at the adult stage and a dose of 20 Gy or more induced over 99% sterility. Moreover, a dose of 30 Gy (almost completely sterilizing male and female mosquitoes) had limited effects on the mating competitiveness of GT males and the vector competence of GT females, respectively. However, radiation reduced mosquito longevity, regardless of sex. CONCLUSION: Our results indicate that the Ae. albopictus GT strain can be distinguished from wild mosquitoes based on Wolbachia status and shows similar fitness, radio-sensitivity and arbovirus susceptibility to the GUA strain, indicating that it is feasible to use the GT strain to suppress Ae. albopictus populations for SIT programmes. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Current Status of Mosquito Handling, Transporting and Releasing in Frame of the Sterile Insect Technique.

The sterile insect technique (SIT) and its related technologies are considered to be a powerful weapon for fighting against mosquitoes. As an important part of the area-wide integrated pest management (AW-IPM) programs, SIT can help reduce the use of chemical pesticides for mosquito control, and consequently, the occurrence of insecticide resistance. The mosquito SIT involves several important steps, including mass rearing, sex separation, irradiation, packing, transportation, release and monitoring. To enable the application of SIT against mosquitoes to reduce vector populations, the Joint Food and Agriculture Organization of the United Nations (FAO) and the International Atomic Energy Agency (IAEA) Centre (previously called Division) of Nuclear Techniques in Food and Agriculture (hereinafter called Joint FAO/IAEA Centre) and its Insects Pest Control sub-program promoted a coordinated research project (CRP) entitled "Mosquito handling, transport, release and male trapping methods" to enhance the success of SIT. This article summarizes the existing explorations that are critical to the handling and transporting of male mosquitoes, offers an overview of detailed steps in SIT and discusses new emerging methods for mosquito releases, covering most processes of SIT.

Biocompatible Polyelectrolyte Complex Nanoparticles for Lycopene Encapsulation Attenuate Oxidative Stress-Induced Cell Damage.

In this study, lycopene was successfully encapsulated in polyelectrolyte complex nanoparticles (PEC NPs) fabricated with a negatively charged polysaccharide, TLH-3, and a positively charged sodium caseinate (SC) via electrostatic interactions. Results showed that the lycopene-loaded PEC NPs were spherical in shape, have a particle size of 241 nm, have a zeta potential of -23.6 mV, and have encapsulation efficiency of 93.6%. Thus, lycopene-loaded PEC NPs could serve as effective lycopene carriers which affected the physicochemical characteristics of the encapsulated lycopene and improved its water dispersibility, storage stability, antioxidant capacity, and sustained release ability in aqueous environments when compared with the free lycopene. Moreover, encapsulated lycopene could enhance the cells' viability, prevent cell apoptosis, and protect cells from oxidative damage through the Nrf2/HO-1/AKT signalling pathway, via upregulation of antioxidase activities and downregulation of MDA and ROS levels. Therefore, the biocompatible lycopene-loaded PEC NPs have considerable potential use for the encapsulation of hydrophobic nutraceuticals in the food and pharmaceutical industries.

Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence.

BACKGROUND: Anopheles stephensi Listen (1901) is a major vector of malaria in Asia and has recently been found in some regions of Africa. The An. stepehnsi species complex is suspected to have three sibling species: type, intermediate, and mysorensis, each with its own vector competence to the malaria parasite and ecology. To identify the members of the species complex in our An. stephensi insectary colony, we used the morphological features of eggs and genetic markers such as AnsteObp1 (Anopheles stephensi odorant binding protein 1), mitochondrial oxidases subunit 1 and 2 (COI and COII), and nuclear internal transcribed spacer 2 locus (ITS2). METHODS: Eggs were collected from individual mosquitoes (n = 50) and counted for the number of ridges under stereomicroscope. Genomic DNA was extracted from female mosquitoes. After the amplification of partial fragments of AnsteObp1, COI, COII and ITS2 genes, the PCR products were purified and sequenced. Phylogenetic analysis was performed after aligning query sequences against the submitted sequences in GenBank using MEGA 7. RESULTS: The range of ridges number on each egg float was 12-13 that corresponds to the mysorensis form of An. stephensi. The generated COI, COII and ITS2 sequences showed 100%, 99.46% and 99.29% similarity with the sequences deposited for Chinese, Indian and Iranian strains of An. stephensi, respectively. All the generated AnsteObp1 intron I region sequences matched 100% with the sequences deposited for An. stephensi sibling species C (mysorensis form) from Iran and Afghanistan. CONCLUSIONS: This manuscript precisely describes the morphological and molecular details of the 'var mysorensis' form of An. stephensi that could be exploited in elucidating its classification as well as in differentiation from other biotypes of the same or other anopheline species. Based on our findings, we recommend AnsteObp1 as a robust genetic marker for rapid and accurate discrimination (taxonomic identification) of the An. stephensi species complex, rather than the COI, COII, and ITS2 marker, which could only be utilized for interspecies (Anopheles) differentiation.

The Protective Effect of Polysaccharide SAFP from Sarcodon aspratus on Water Immersion and Restraint Stress-Induced Gastric Ulcer and Modulatory Effects on Gut Microbiota Dysbiosis.

Sarcodon aspratus is a popular edible fungus for its tasty flavour and can be used as a dietary supplement for its functional substances. This study was conducted to evaluate the potential health benefits of Sarcodon aspratus polysaccharides (SAFP) on water immersion and restraint stress (WIRS)-induced gastric ulcer in rats. The results indicated that SAFP could decrease myeloperoxidase (MPO) activity and plasma corticosterone levels, as well as enhance Prostaglandin E2 (PGE2) and Nitrate/nitrite (NOx) concentration in rats. Furthermore, SAFP significantly attenuated the stress damage, inflammation, pathological changes and gastric mucosal lesion in rats. Moreover, high-throughput pyrosequencing of 16S rRNA suggested that SAFP modulated the dysbiosis of gut microbiota by enhancing the relative abundance of probiotics, decreasing WIRS-triggered bacteria proliferation. In summary, these results provided the evidence that SAFP exerted a beneficial effect on a WIRS-induced gastric ulcer via blocking the TLR4 signaling pathway and activating the Nrf2 signaling pathway. Notably, SAFP could modulate the WIRS-induced dysbiosis of gut microbiota. Thus, SAFP might be explored as a natural gastric mucosal protective agent in the prevention of gastric ulcers and other related diseases in the food and pharmaceutical industries.

Catalytic Chiral Photochemistry Sensitized by Chiral Hosts-Grafted Upconverted Nanoparticles.

Singlet chiral photocatalysis is highly challenging. Herein, we report fluorescence resonance energy transfer (FRET)-based chiral photocatalysis with γ-cyclodextrin (CD)-grafted lanthanide-doped upconverted nanoparticles (UCNP). The CD-modified UCNP strongly emits in the UV wavelength region upon excitation with a 980 nm laser, which selectively sensitizes the photosubstrates complexed by CD on the surface of UCNP through FRET. Therefore, enantiodifferentiating photocyclodimerization of anthracene or naphthalene derivatives sensitized by the CD-modified UCNP gives photoproducts in good enantioselectivity even in the presence of a catalytic amount of CD-modified UCNP. Moreover, the photocatalysts are readily separated and could be reused for at least six cycles without decreasing the enantioselectivity.

Predicting the Impact of Climate Change on the Distribution of a Neglected Arboviruses Vector (Armigeres subalbatus) in China

The geographic boundaries of arboviruses continue to expand, posing a major health threat to millions of people around the world. This expansion is related to the availability of effective vectors and suitable habitats. Armigeres subalbatus (Coquillett, 1898), a common and neglected species, is of increasing interest given its potential vector capacity for Zika virus. However, potential distribution patterns and the underlying driving factors of Ar. subalbatus remain unknown. In the current study, detailed maps of their potential distributions were developed under both the current as well as future climate change scenarios (SSP126 and SSP585) based on CMIP6 data, employing the MaxEnt model. The results showed that the distribution of the Ar. subalbatus was mainly affected by temperature. Mean diurnal range was the strongest predictor in shaping the distribution of Ar. subalbatus, with an 85.2% contribution rate. By the 2050s and 2070s, Ar. subalbatus will have a broader potential distribution across China. There are two suitable expansion types under climate change in the 2050s and 2070s. The first type is continuous distribution expansion, and the second type is sporadic distribution expansion. Our comprehensive analysis of Ar. subalbatus's suitable distribution areas shifts under climate change and provides useful and insightful information for developing management strategies for future arboviruses.

A cross-sectional study to assess the epidemiological situation and associated risk factors of dengue fever; knowledge, attitudes, and practices about dengue prevention in Khyber Pakhtunkhwa Province, Pakistan.

Background: Dengue fever has been responsible for around 12 countrywide large outbreaks in Pakistan, resulting in 286,262 morbidities and 1,108 deaths. Khyber Pakhtunkhwa (KP) is the most recently impacted province. This study aimed to investigate the molecular, epidemiological, and potential elements that contribute to increasing dengue transmission patterns, and knowledge, attitude, and practice (KAP) toward dengue in KP province. Method: This cross-sectional community-based study was conducted (June-December, 2021) in two phases. Phase I involved the epidemiological (n = 5,242) and molecular analysis of DENV in 500 randomly collected blood samples of the 2021 dengue outbreak in KP. Phase II focused on assessing dengue-KAP levels in healthy communities (n = 14,745, aged >18 years), adopting a cross-sectional clustered multistage sampling in eight districts (dengue-hotspot vs. non-hotspot) of KP. Chi-square tests and logistic regression analysis were applied. Results: Peshawar district had the highest dengue cases (60.0%) associated with the predominant co-circulation of DENV-2 (45.8%) and DENV-3 (50.4%) serotypes. A rise in cases was reported in October (41.8%) followed by September (27.9%) and August (14.4%; p < 0.001). Males (63.7%, p < 0.001) and individuals aged 16-30 years (37.0%, p < 0.001) were highly affected. General workers (18.0%), families with a monthly income of 10,000-20,000 Pak rupees (50.5%), unmarried (71.0%), uneducated (31%), families with higher human density (>10 individuals per household), and those (29.0%) who faced power outages for more than 7/24 h were the most affected. Moreover, co-morbidities like renal failure and bronchial asthma were associated with disease severity. A community survey on KAP revealed that an average of 74, 60, and 43% of the participants demonstrated good knowledge, attitudes, and dengue preventive practices, respectively. Conclusion: Multiple poor socioeconomic elements are influencing dengue fever transmission in the province. Higher KAP levels may explain the low frequency of dengue in non-hotspot districts. Our study emphasizes the need for effective and long-term public health education, strengthened vector surveillance, and expanded laboratory capacity for better diagnosis and management of dengue cases to better predict the burden and seasonality of disease in the country.