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Current status of Plasmodium knowlesi vectors: a public health concern?

Vythilingam I, Wong ML, Wan-Yussof WS

Parasitology, 2018 01;145(1):32-40.
PMID: 27222102 DOI: 10.1017/S0031182016000901

Plasmodium knowlesi a simian malaria parasite is currently affecting humans in Southeast Asia. Malaysia has reported the most number of cases and P. knowlesi is the predominant species occurring in humans. The vectors of P. knowlesi belong to the Leucosphyrus group of Anopheles mosquitoes. These are generally described as forest-dwelling mosquitoes. With deforestation and changes in land-use, some species have become predominant in farms and villages. However, knowledge on the distribution of these vectors in the country is sparse. From a public health point of view it is important to know the vectors, so that risk factors towards knowlesi malaria can be identified and control measures instituted where possible. Here, we review what is known about the knowlesi malaria vectors and ascertain the gaps in knowledge, so that future studies could concentrate on this paucity of data in-order to address this zoonotic problem.
Fulltext Zika virus and its potential re-emergence in Malaysia

Sam JI, Chan YF, Vythilingam I, Wan Sulaiman WY

Med J Malaysia, 2016 04;71(2):66-8.
PMID: 27326944 MyJurnal

Zika virus (ZIKV) has re-emerged to cause explosive epidemics in the Pacific and Latin America, and appears to be associated with severe neurological complications including microcephaly in babies. ZIKV is transmitted to humans by Aedes mosquitoes, principally Ae. aegypti, and there is historical evidence of ZIKV circulation in Southeast Asia. It is therefore clear that Malaysia is at risk of similar outbreaks. Local and international guidelines are available for surveillance, diagnostics, and management of exposed and infected individuals. ZIKV is the latest arbovirus to have spread globally beyond its initial restricted niche, and is unlikely to be the last. Innovative new methods for surveillance and control of vectors are needed to target mosquito-borne diseases as a whole.
Fulltext Community surveillance of Aedes albopictus associated with Wolbachia detection in low-rise residential areas in Selangor, Malaysia

Roslan MA, Ngui R, Vythilingam I, Wan Sulaiman WY

J Vector Ecol, 2022 Dec;47(2):142-152.
PMID: 36314668 DOI: 10.52707/1081-1710-47.2.142

The study assessed the distribution of Malaysian Ae. albopictus adults associated with Wolbachia detection in low-rise residential areas using a modified sticky ovitrap (MSO). The relationship between Ae. albopictus and climatological parameters were also determined. Fifty-two weeks of surveillance using 273 MSOs were conducted in four installation areas of eleven sampling sites. Specimens were subjected to PCR using wsp-specific primers for Wolbachia detection. The relationship between climatological parameters and Ae. albopictus captured were analyzed using Spearman rank correlation coefficient test. The majority of Ae. albopictus were captured in residential houses (87%), followed by playgrounds or parks (11.5%), guardhouses (1%), and community halls (0.5%). Most of the specimens (92%) were superinfected with wAlbA and wAlbB strains. A positive correlation with no significant association was found for rainfall (r = 0.015, P = 0.072), relative humidity (r = 0.005, P = 0.526), minimum temperature (r = 0.005, P = 0.516), and mean temperature (r = 0.003, P = 0.689). MSO effectively captured a high number of Ae. albopictus that was determined to be the predominant mosquito species found in low-rise residential areas. The adult collection is not only influenced by climatological parameters but also by other factors, including environmental conditions and general sanitation status.
Detection of Brugia malayi in mosquitoes by the polymerase chain reaction

Vythilingam I, Boaz L, Wa N

J Am Mosq Control Assoc, 1998 Sep;14(3):243-7.
PMID: 9813819

Accurate identification of filarial parasites in mosquitoes poses a major problem for the coordination of filariasis control programs. Traditional methods are tedious, and some are not specific enough to give satisfactory results. Amplification of specific gene sequences by primer-directed polymerase chain reaction (PCR) has been increasingly utilized as a diagnostic tool. However, current protocols for the extraction of parasite DNA from mosquito samples are tedious and could lead to failure of PCR amplification. We demonstrate that the use of Chelex is an efficient method for DNA extraction from mosquitoes and the parasite and that PCR amplification with primers specific for Brugia malayi yields a band of the expected size. The PCR products were transferred to a nylon membrane with Southern blotting, and a B. malayi-specific digoxigenin-labeled probe confirmed the sequence similarity of the PCR-amplified fragment and increased the sensitivity of the PCR assay. Use of this probe enabled us to detect PCR-amplified product from B. malayi even when a product was not visible on an ethidium bromide-stained agarose gel. This increased sensitivity allowed us to detect the parasite in the heads of mosquitoes.
Fulltext Plasmodium knowlesi and Wuchereria bancrofti: Their Vectors and Challenges for the Future

Vythilingam I

Front Physiol, 2012;3:115.
PMID: 22557977 DOI: 10.3389/fphys.2012.00115

Malaria and filariasis still continue to pose public health problems in developing countries of the tropics. Although plans are in progress for the elimination of both these parasitic vector borne diseases, we are now faced with a daunting challenge as we have a fifth species, Plasmodium knowlesi a simian malaria parasite affecting humans. Similarly in peninsular Malaysia, filariasis was mainly due to Brugia malayi. However, we now see cases of Wuchereria bancrofti in immigrant workers coming into the country. In order to successfully eliminate both these diseases we need to know the vectors involved and introduce appropriate control measures to prevent the diseases occurring in the future. As for knowlesi malaria it is still uncertain if human to human transmission through mosquito bites is occurring. However, P. knowlesi in human is not a rare occurrence anymore and has all the characteristics of a pathogen spreading due to changes in the ecosystem, international travel, and cross border migration. This has created a more complex situation. In order to overcome these challenges we need to revamp our control measures. This paper reviews the vectors of malaria and filariasis in Southeast Asia with special emphasis on P. knowlesi and W. bancrofti in Malaysia and their control strategies.
In vivo study of human Plasmodium knowlesi in Macaca fascicularis

Anderios F, Noorrain A, Vythilingam I

Exp Parasitol, 2010 Feb;124(2):181-9.
PMID: 19765587 DOI: 10.1016/j.exppara.2009.09.009

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per microL of blood for asexual stage and 88-264 parasites per microL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.
Fulltext Seasonal and Spatial Dynamics of the Primary Vector of Plasmodium knowlesi within a Major Transmission Focus in Sabah, Malaysia

Wong ML, Chua TH, Leong CS, Khaw LT, Fornace K, Wan-Sulaiman WY, et al.

PLoS Negl Trop Dis, 2015;9(10):e0004135.
PMID: 26448052 DOI: 10.1371/journal.pntd.0004135

BACKGROUND: The simian malaria parasite Plasmodium knowlesi is emerging as a public health problem in Southeast Asia, particularly in Malaysian Borneo where it now accounts for the greatest burden of malaria cases and deaths. Control is hindered by limited understanding of the ecology of potential vector species.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted a one year longitudinal study of P. knowlesi vectors in three sites within an endemic area of Sabah, Malaysia. All mosquitoes were captured using human landing catch. Anopheles mosquitoes were dissected to determine, oocyst, sporozoites and parous rate. Anopheles balabacensis is confirmed as the primary vector of. P. knowlesi (using nested PCR) in Sabah for the first time. Vector densities were significantly higher and more seasonally variable in the village than forest or small scale farming site. However An. balabacensis survival and P. knowlesi infection rates were highest in forest and small scale farm sites. Anopheles balabacensis mostly bites humans outdoors in the early evening between 1800 to 2000 hrs.
CONCLUSIONS/SIGNIFICANCE: This study indicates transmission is unlikely to be prevented by bednets. This combined with its high vectorial capacity poses a threat to malaria elimination programmes within the region.
Chikungunya Virus Infection of Aedes Mosquitoes

Wong HV, Chan YF, Sam IC, Sulaiman WY, Vythilingam I

Methods Mol Biol, 2016;1426:119-28.
PMID: 27233266 DOI: 10.1007/978-1-4939-3618-2_11

In vivo infection of mosquitoes is an important method to study and characterize arthropod-borne viruses. Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is transmitted primarily by Aedes mosquitoes. In this chapter, we describe a protocol for infection of CHIKV in two species of Aedes mosquitoes, Aedes aegypti and Aedes albopictus, together with the isolation of CHIKV in different parts of the infected mosquito such as midgut, legs, wings, salivary gland, head, and saliva. This allows the study of viral infection, replication and dissemination within the mosquito vector.
Fulltext A preliminary survey of Aedes aegypti in Selangor, Peninsular Malaysia

Ho TM, Vythilingam I

Med J Malaysia, 1980 Jun;34(4):409-14.
PMID: 7219273

A preliminary survey of Aedes aegypti was carried out in 6 areas in the state of Selangor, Malaysia. The densities of Ae. aegypti and Ae. albopictus in the areas were discussed. Results indicated that the distribution of larval habitats varied with the housing type. The most ommon indoor larval habitat in urban areas is the bathroom tank. In both urban and rural areas, outdoor preference is for the earthenware jars. Anttraps have decreased in importance as larvae breeding habitats. The accuracy of house searches can be increased by increasing the number of larvae examined per container to 5 or more. Further study is required to determine whether the findings of this survey is peculiar to the areas surveyed or is representative of the whole country, and whether there is a seasonal fluctuation in the types of preferred larval habitats.
Fulltext Gravid oviposition sticky trap and dengue non-structural 1 antigen test for early surveillance of dengue in multi-storey dwellings: study protocol of a cluster randomized controlled trial

Liew JWK, Selvarajoo S, Tan W, Ahmad Zaki R, Vythilingam I

Infect Dis Poverty, 2019 Sep 03;8(1):71.
PMID: 31477185 DOI: 10.1186/s40249-019-0584-y

BACKGROUND: Dengue is a global disease, transmitted by the Aedes vectors. In 2018, there were 80 615 dengue cases with 147 deaths in Malaysia. Currently, the nationwide surveillance programs are dependent on Aedes larval surveys and notifications of lab-confirmed human infections. The existing, reactive programs appear to lack sensitivity and proactivity. More efficient dengue vector surveillance/control methods are needed.
METHODS: A parallel, cluster, randomized controlled, interventional trial is being conducted for 18 months in Damansara Damai, Selangor, Malaysia, to determine the efficacy of using gravid oviposition sticky (GOS) trap and dengue non-structural 1 (NS1) antigen test for early surveillance of dengue among Aedes mosquitoes to reduce dengue outbreaks. Eight residential apartments were randomly assigned into intervention and control arms. GOS traps are set at the apartments to collect Aedes weekly, following which dengue NS1 antigen is detected in these mosquitoes. When a dengue-positive mosquito is detected, the community will be advised to execute vector search-and-destroy and protective measures. The primary outcome concerns the the percentage change in the (i) number of dengue cases and (ii) durations of dengue outbreaks. Whereas other outcome measures include the change in density threshold of Aedes and changes in dengue-related knowledge, attitude and practice among cluster inhabitants.
DISCUSSION: This is a proactive and early dengue surveillance in the mosquito vector that does not rely on notification of dengue cases. Surveillance using the GOS traps should be able to efficiently provide sufficient coverage for multistorey dwellings where population per unit area is likely to be higher. Furthermore, trapping dengue-infected mosquitoes using the GOS trap, helps to halt the dengue transmission carried by the mosquito. It is envisaged that the results of this randomized controlled trial will provide a new proactive, cheap and targeted surveillance tool for the prevention and control of dengue outbreaks.
TRIAL REGISTRATION: This is a parallel-cluster, randomized controlled, interventional trial, registered at ClinicalTrials.gov (ID: NCT03799237), on 8th January 2019 (retrospectively registered).
Gender beyond male and female: Occurrence of a gynandromorph in the Japanese encephalitis vector Culex sitiens (Diptera: Culicidae)

Low VL, Wong ML, Liew JWK, Pramasivan S, Jeyaprakasam NK, Vythilingam I

Acta Trop, 2020 Jan;201:105207.
PMID: 31586449 DOI: 10.1016/j.actatropica.2019.105207

A gynandromorph of Culex sitiens Wiedemann (Diptera: Culicidae) was attracted to a human during a mosquito surveillance programme conducted in Kuala Lipis, Pahang, Malaysia on July 20, 2019. Gynandromorphism was observed in antennae, maxillary palps, legs and wings of the specimen, with distinct male characters on the left and female characters on the right, though the left maxillary palp is slightly shorter than the proboscis of a typical male. The abdomen, however, displays well-developed male genitalia. This study represents the first report of oblique gynandromorphism in Cx. sitiens, one of the vectors of Japanese encephalitis in Asia.
Genetic diversity of zoonotic malaria parasites from mosquito vector and vertebrate hosts

Wong ML, Ahmed MA, Sulaiman WYW, Manin BO, Leong CS, Quan FS, et al.

Infect Genet Evol, 2019 09;73:26-32.
PMID: 30999059 DOI: 10.1016/j.meegid.2019.04.010

We explored and constructed haplotype network for simian malaria species: Plasmodium knowlesi, P. cynomolgi and P. inui aiming to understand the transmission dynamics between mosquitoes, humans and macaques. Mosquitoes were collected from villages in an area where zoonotic malaria is prevalent. PCR analysis confirmed Anopheles balabacensis as the main vector for macaque parasites, moreover nearly 60% of the mosquitoes harboured more than one Plasmodium species. Fragments of the A-type small subunit ribosomal RNA (SS rRNA) amplified from salivary gland sporozoites, and equivalent sequences obtained from GenBank were used to construct haplotype networks. The patterns were consistent with the presence of geographically distinct populations for P. inui and P. cynomolgi, and with three discrete P. knowlesi populations. This study provides a preliminary snapshot of the structure of these populations, that was insufficient to answer our aim. Thus, collection of parasites from their various hosts and over time, associated with a systematic analysis of a set of genetical loci is strongly advocated in order to obtain a clear picture of the parasite population and the flow between different hosts. This is important to devise measures that will minimise the risk of transmission to humans, because zoonotic malaria impedes malaria elimination.
Fulltext Plasmodium knowlesi infecting humans in Southeast Asia: What's next?

Jeyaprakasam NK, Liew JWK, Low VL, Wan-Sulaiman WY, Vythilingam I

PLoS Negl Trop Dis, 2020 12;14(12):e0008900.
PMID: 33382697 DOI: 10.1371/journal.pntd.0008900

Plasmodium knowlesi, a simian malaria parasite, has been in the limelight since a large focus of human P. knowlesi infection was reported from Sarawak (Malaysian Borneo) in 2004. Although this infection is transmitted across Southeast Asia, the largest number of cases has been reported from Malaysia. The increasing number of knowlesi malaria cases has been attributed to the use of molecular tools for detection, but environmental changes including deforestation likely play a major role by increasing human exposure to vector mosquitoes, which coexist with the macaque host. In addition, with the reduction in human malaria transmission in Southeast Asia, it is possible that human populations are at a greater risk of P. knowlesi infection due to diminishing cross-species immunity. Furthermore, the possibility of increasing exposure of humans to other simian Plasmodium parasites such as Plasmodium cynomolgi and Plasmodium inui should not be ignored. We here review the current status of these parasites in humans, macaques, and mosquitoes to support necessary reorientation of malaria control and elimination in the affected areas.
Improved Aedes/dengue field surveillance using Gravid Oviposition Sticky trap and dengue NS1 tests: Epidemiological, entomological outcomes and community acceptance

Liew JWK, Selvarajoo S, Phang WK, Mah Hassan M, Redzuan MS, Selva Kumar S, et al.

Acta Trop, 2021 Apr;216:105829.
PMID: 33465350 DOI: 10.1016/j.actatropica.2021.105829

The aim of this study is to investigate the feasibility and outcomes of using Gravid Oviposition Sticky (GOS) trap and dengue NS1 antigen tests for indoor and outdoor dengue/Aedes surveillance in the field. A one-year community-based study was carried out at Sungai Buloh Hospital Quarters, Selangor, Malaysia. GOS traps were first placed outdoors in three apartment blocks (Anggerik, Bunga Raya and Mawar). Beginning 29th week of the study, indoor traps were set in two apartment units on every floor in Anggerik. All female Aedes mosquitoes caught were tested for the presence of dengue NS1 antigen. Dengue seroprevalence and knowledge, attitude and practices on dengue prevention of the community and their reception to the surveillance approach were also assessed. Dengue-positive mosquitoes were detected at least 1 week before a dengue onset. More mosquitoes were caught indoors than outdoors in block Anggerik, but the total number of mosquitoes caught in all 3 blocks were similar. There was a significant difference in distribution of Ae. aegypti and Ae. albopictus between the 3 blocks. 66.1% and 3.4% of the community were positive for dengue IgG and IgM, respectively. Most respondents think that this surveillance method is Good (89%) and support its use nationwide. Dengue case ratio in the study apartment blocks decreased from year 2018 to 2019. This study demonstrated the practicality of performing proactive dengue/Aedes surveillance inside apartment units using the GOS traps. This surveillance method can be performed with immediate result output in the field.
Fulltext Plasmodium knowlesi in humans: a review on the role of its vectors in Malaysia

Vythilingam I

Trop Biomed, 2010 Apr;27(1):1-12.
PMID: 20562807 MyJurnal

Plasmodium knowlesi in humans is life threatening, is on the increase and has been reported from most states in Malaysia. Anopheles latens and Anopheles cracens have been incriminated as vectors. Malaria is now a zoonoses and is occurring in malaria free areas of Malaysia. It is also a threat to eco-tourism. The importance of the vectors and possible control measures is reviewed here.
Fulltext A comparative study of adult mosquito trapping using dry ice and yeast generated carbon dioxide

Oli K, Jeffery J, Vythilingam I

Trop Biomed, 2005 Dec;22(2):249-51.
PMID: 16883295 MyJurnal

Adult mosquito collections were conducted for 12 weeks in two residential areas in Kuala Lumpur. The CDC light traps were compared using dry ice and yeast as sources of carbon dioxide attractants for mosquitoes. The efficacy of the dry ice baited trap was significant over yeast generated CO2 trap. The predominant species obtained were Culex quinquefasciatus, Stegomyia albopicta and Armigeres subalbatus.
Fulltext Prevalence of intestinal protozoa in an aborigine community in Pahang, Malaysia

Noor Azian MY, San YM, Gan CC, Yusri MY, Nurulsyamzawaty Y, Zuhaizam AH, et al.

Trop Biomed, 2007 Jun;24(1):55-62.
PMID: 17568378 MyJurnal

The objective was to estimate the prevalence of intestinal protozoa among the aborigines and to determine the problems regarding the infection. The study was carried out in January 2006 in Pos Senderut, Pahang, Malaysia. Samples of faeces were collected from children and adults and these were fixed in PVA and trichrome staining was carried out. From the 130 individuals studied, 94 (72.3%) were positive with at least one intestinal protozoa. Nine intestinal protozoa namely Blastocystis hominis, Giardia lamblia, Entamoeba histolytica, Entamoeba coli, Endolimax nana, Entamoeba hartmani, Entamoeba polecki, Iodamoeba butschlii and Chilomastix mesnili were detected. The prevalent species were B. hominis (52.3%), followed by G. lamblia (29.2%), E. coli (26.2%) and E. histolytica (18.5%). The other species ranged from 1.5 to 10.8%. Among the positive samples, mixed infection with E. histolytica and G. lamblia was 3.8%, E. histolytica and B. hominis was 15.4%, G. lamblia and B. hominis was 17.7%. Triple infection of E. histolytica, G. lamblia and B. hominis was 3.1%. The infection was more prevalent in children below 10 years age group (45.4%) and lowest in the age above 60 years (3.8%). The high prevalence was attributable to poor environmental management, poor personal hygiene and lack of health education.
Histopathology of Brugia pahangi and Plasmodium berghei ANKA co-infection in the Gerbil (Meriones unguiculatus)

Junaid OQ, Wong KT, Khaw LT, Mahmud R, Vythilingam I

Trop Biomed, 2018 Dec 01;35(4):981-998.
PMID: 33601846

Co-infection with multiple different parasites is a common phenomenon in both human and animals. Among parasites that frequently co-infect the same hosts, are the filarial worms and malaria parasites. Despite this, the mechanisms underlying the interactions between these parasites is still relatively unexplored with very few studies available on the resulting pathologies due to co-infection by filarial nematodes and malaria parasites. Hence, this study investigated the histopathological effect of Brugia pahangi and Plasmodium berghei ANKA (PbA) infections in gerbil host. Gerbils grouped into B. pahangi-infected, PbA-infected, B. pahangi and PbA-coinfected, and uninfected control, were necropsied at different time points of post PbA infections. Brugia pahangi infections in the gerbils were first initiated by subcutaneous inoculation of 50 infective larvae, while PbA infections were done by intraperitoneal injection of 106 parasitized red blood cells after 70 days patent period of B. pahangi. Organs such as the lungs, kidneys, spleen, heart and liver were harvested aseptically at the point of necropsy. There was significant hepatosplenomegaly observed in both PbA-infected only and coinfected gerbils. The spleen, liver and lungs were heavily pigmented. Both B. pahangi and PbA infections (mono and coinfections) resulted in pulmonary edema, while glomerulonephritis was associated with PbA infections. The presence of both parasites induced extramedullary hematopoiesis in the spleen and liver. These findings suggest that the pathologies associated with coinfected gerbils were synergistically induced by both B. pahangi and PbA infections.
Fulltext A new paradigm for Aedes spp. surveillance using gravid ovipositing sticky trap and NS1 antigen test kit

Lau SM, Chua TH, Sulaiman WY, Joanne S, Lim YA, Sekaran SD, et al.

Parasit Vectors, 2017 Mar 21;10(1):151.
PMID: 28327173 DOI: 10.1186/s13071-017-2091-y

BACKGROUND: Dengue remains a serious public health problem in Southeast Asia and has increased 37-fold in Malaysia compared to decades ago. New strategies are urgently needed for early detection and control of dengue epidemics.
METHODS: We conducted a two year study in a high human density dengue-endemic urban area in Selangor, where Gravid Ovipositing Sticky (GOS) traps were set up to capture adult Aedes spp. mosquitoes. All Aedes mosquitoes were tested using the NS1 dengue antigen test kit. All dengue cases from the study site notified to the State Health Department were recorded. Weekly microclimatic temperature, relative humidity (RH) and rainfall were monitored.
RESULTS: Aedes aegypti was the predominant mosquito (95.6%) caught in GOS traps and 23% (43/187 pools of 5 mosquitoes each) were found to be positive for dengue using the NS1 antigen kit. Confirmed cases of dengue were observed with a lag of one week after positive Ae. aegypti were detected. Aedes aegypti density as analysed by distributed lag non-linear models, will increase lag of 2-3 weeks for temperature increase from 28 to 30 °C; and lag of three weeks for increased rainfall.
CONCLUSION: Proactive strategy is needed for dengue vector surveillance programme. One method would be to use the GOS trap which is simple to setup, cost effective (below USD 1 per trap) and environmental friendly (i.e. use recyclable plastic materials) to capture Ae. aegypti followed by a rapid method of detecting of dengue virus using the NS1 dengue antigen kit. Control measures should be initiated when positive mosquitoes are detected.
Inapparent dengue in a community living among dengue-positive Aedes mosquitoes and in a hospital in Klang Valley, Malaysia

Tan W, Liew JWK, Selvarajoo S, Lim XY, Foo CJ, Refai WF, et al.

Acta Trop, 2020 Apr;204:105330.
PMID: 31917959 DOI: 10.1016/j.actatropica.2020.105330

The public health burden of dengue is most likely under reported. Current dengue control measures only considered symptomatic dengue transmission. Hence, there is a paucity of information on the epidemiology of inapparent dengue. This study reports that many people have been unknowingly exposed to dengue infection. Almost 10% and 70% of individuals without any history of dengue infection and living in a dengue hotspot, in Selangor, Malaysia, were dengue IgM and IgG positive respectively. When dengue-positive mosquitoes were detected in the hotspot, 11 (6.3%) of the 174 individuals tested were found to have dengue viremia, of which 10 were asymptomatic. Besides, upon detection of a dengue-infected mosquito, transmission was already widespread. In a clinical setting, it appears that people living with dengue patients have been exposed to dengue, whether asymptomatic or symptomatic. They can either have circulating viral RNA and/or presence of NS1 antigen. It is also possible that they are dengue seropositive. Collectively, the results indicate that actions taken to control dengue transmission after the first report of dengue cases may be already too late. The current study also revealed challenges in diagnosing clinically inapparent dengue in hyperendemic settings. There is no one best method for diagnosing inapparent dengue. This study demonstrates empirical evidence of inapparent dengue in different settings. Early dengue surveillance in the mosquito population and active serological/virological surveillance in humans can go hand in hand. More studies are required to investigate the epidemiology, seroprevalence, diagnostics, and control of inapparent dengue. It is also crucial to educate the public, health staff and medical professionals on asymptomatic dengue and to propagate awareness, which is important for controlling transmission.

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