Real-time PCR assay has many advantages over conventional PCR methods, including rapidity, quantitative measurement, low risk of contamination, high sensitivity, high specificity, and ease of standardization (Mackay et al., Nucleic Acids Res 30:1292-1305, 2002). The real-time PCR system relies upon the measurement of a fluorescent reporter during PCR, in which the amount of emitted fluorescence is directly proportional to the amount of the PCR product in a reaction (Gibsons et al., Genome Res 6:995-1001, 1996). Here, we describe the use of SYBR Green I-based and TaqMan(®) real-time reverse transcription polymerase chain reaction (RT-PCR) for the detection and quantification of Chikungunya virus (CHIKV).
The Filariasis Control Program was established more than 30 years ago in the country and the disease is still a public health problem in some states. Since 1983, a total of 17 filariasis control teams were formed throughout the country to carry out filariasis control work. The teams conduct house and population censuses, nocturnal mass blood surveys and treatment of microscopically confirmed cases. Individual case follow-up is being carried out after 3-5 months while the locality is resurveyed after about 2-3 years. During the years 1988 to 1990, there appeared to be a decreasing trend in the number of filariasis cases detected countrywide. In 1991, brugian filariasis accounted for 92% of the cases detected. The microfilaria rate (MFR) also showed a decreasing trend countrywide for the years 1988 (0.57%) to 1990 (0.35%) but there was an increase in 1991 although it remained well below the 5% MFR targeted in the program objective, In 1991, the filariasis control teams and the district multi-purpose teams collected a total of 167, 151 blood slides out of which 871 were found to be positive for microfilaria. To determine the true endemicity of filariasis in the country, the malaria district multi-purpose teams are also utilized to assist in probe surveys in new areas of the district. Two species of filarial worms, namely Brugia malayi and Wuchereria bancrofti, and the mosquito vectors belonging to the Anopheles and Mansonia genera are involved in the transmission of filariasis in Malaysia. Monkeys and domestic cats are the reservoir hosts for the subperiodic strain of B. malayi.
In Armigeres subalbatus, 60% and 3% of the ingested Brugia pahangi microfilariae (mf) respectively migrated into the haemocoel and the thorax within 5 minutes post ingestion (p.i.). Most of the mf had migrated from the gut into the haemocoel within the first 10 minutes p.i. There was no correlation between the number of mf ingested and the migration rate though those in mosquitoes with a low mf burden tend to migrate earlier. At 24 hours p.i., 5-30% of the mf were still in the gut; 19% of these mf were immobile. At 48 hours p.i. only 2% of the mf were mobile. B. pahangi mf isolated from blood meals at 24 hours p.i., failed to develop when inoculated into Armigeres subalbatus. 54% and 73% of the mf isolated from a 24 hour old clotted blood of a B. pahangi-infected cat and fresh peripheral cat blood respectively developed into stage-1 larva. Probably mf left in the midgut at 24 hours p.i. are the young and immature worms and are physiologically incapable of penetrating the gut.
The efficacy of two formulations, wettable powder and emulsifiable concentrate, of cyfluthrin sprayed on plywood [10 mg (ai)/m2] was assessed against six species of mosquitos. The bioassay followed the WHO standard method, with some modification for the bioassay of insecticidal deposits on wall surfaces. The results indicated that these two formulations of cyfluthrin were effective against Anopheles dirus and Mansonia uniformis, moderately toxic to Aedes aegypti and Ae. albopictus in decreasing mortality through out the study period. It was least effective against Culex quinquefasciatus and An. maculatus, respectively. There was no statistically significant difference between these two formulations.
Dengue viruses pose a considerable global public health problem with an estimated 100 million cases of illness every year. This illustrates the need for rapid and reliable diagnostic methods for proper patient management and disease control. Currently, laboratory diagnosis depends on serology or virus isolation, with both methods having certain drawbacks. Alternatively, reverse transcription and polymerase chain reaction (RT-PCR) offers the potential for the rapid, highly sensitive and specific detection of dengue viruses. Since we occasionally encounter the problem of insufficient amounts of patient serum for the direct detection of dengue viruses, a method was developed for the extraction of viral RNA after biological amplification in mosquito larvae. Using this method, 15 of 19 clinical samples tested were correctly identified using RT-PCR.
Serum specimens were collected from 6 species of animals living in 9 states of Malaysia including Sabah, North Borneo in 1993. Antibodies against Japanese encephalitis (JE) virus in these sera were detected by means of hemagglutination-inhibition (HI) and neutralization (NT) tests. By HI test, 702 of 2,152 (32.6%) sera showed positive results. Higher positive rates were obtained by the NT test, in which 1,787 of 1,927 (92.7%) sera had antibodies against JE virus. All serum specimens with positive HI were confirmed as positive by the NT. Swine sera showed especially higher rates of antibody positive and higher antibody titers compared with other animals. These results suggest that JE infections are widely distributed among many animals of Malaysia, and pig is the most susceptible amplifier host for JE virus.
A total of 37 species of mosquitoes from seven genera were collected in six villages in the Bengkoka Peninsula, Sabah State, during two visits in 1981 in connection with studies on malaria and filariasis. Fifty-five per cent of the total mosquitoes collected were Mansonia. An. collessi constituted a new record of the species from Sabah. An. balabacensis was found to be naturally infected with sporozoites. Ma. bonneae was found to be naturally infected with Brugia, probably B. malayi. Parous rates of An. balabacensis and Ma. bonneae were very high with consequent high probability of survival ideally suiting transmission of malaria and filariasis.
A total of 82 persons have been found to be positive for microfilaria a sub-periodic Brugia malayi out of 1,613 examined in seven villages in Serian District. This represents an average microfilaria infection rate of 5.1% with a range of zero to 10.7%. It is found that males are more predominantly affected than females with a ratio of 3:1. It is also confirmed that Mansonia dives and M. bonneae are possible vectors for the transmission of the disease although Anopheles species cannot be ruled out owing to the small number of specimens examined. The study is confined to areas where indoor DDT spraying has been done since 1960 and only in two of the areas it has been terminated in 1966. Yet to be published data shows higher filaria infection rate than any of these villages.
Japanese encephalitis virus (JEV) and West Nile virus (WNV) provide some of the most important examples of emerging zoonotic viral encephalitides. For these flaviviruses, only a small proportion of those infected develop clinical features, and these may range from a non-specific flu-like illness to a severe fatal meningoencephalitis, often with Parkinsonian features, or a poliomyelitis-like flaccid paralysis. The factors governing the clinical presentations, and outcome of flavivirus infections are poorly understood, but studies have looked at viral virulence determinants and the host immune response. Previous studies on JEV have suggested that the distribution of the four genotypes across Asia may relate to the differing clinical epidemiology (epidemic disease in the north, endemic disease in the south). However, new data based on the complete nucleotide sequence of a virus representing one of the oldest lineages, and phylogenetic analyses of all JEV strains for which genetic data are available, suggest that the distribution is best explained in terms of the virus' origin in the Indonesia-Malaysia region (where all genotypes have been found), and the spread of the more recent genotypes to new geographical areas. Clinical studies have shown that innate immunity, as manifested by interferon alpha levels, is important in JEV and other flaviviruses, but treatment with interferon alpha did not improve the outcome. A failure of the humoral immune response, is associated with death from encephalitis caused by JEV and WNV. Cellular immunity has been less well characterized, but CD8+ and CD4+ T cells are thought to be important.
Eleven viral isolates derived mostly in albopictus C6/36 cells from mosquito pools collected in Southeast Asia and the Americas between 1966 and 2014 contained particles with electron microscopy morphology typical of reoviruses. Metagenomics analysis yielded the near complete genomes of three novel reoviruses, Big Cypress orbivirus, Ninarumi virus, and High Island virus and a new tetravirus, Sarawak virus. Strains of previously characterized Sathuvarachi, Yunnan, Banna and Parry's Lagoon viruses (Reoviridae), Bontang virus (Mesoniviridae), and Culex theileri flavivirus (Flaviviridae) were also characterized. The availability of these mosquito virus genomes will facilitate their detection by metagenomics or PCR to better determine their geographic range, extent of host tropism, and possible association with arthropod or vertebrate disease.
Clostridium bifermentans serovar malaysia (C.b.m.) is highly toxic to mosquito larvae. In this study, the following aquatic nontarget invertebrates were treated with high C.b.m. concentrations (up to 1,600-fold the toxic concentration for Anopheles stephensi) to study their susceptibility towards the bacterial toxin: Planorbis planorbis (Pulmonata); Asellus aquaticus (Isopoda); Daphnia pulex (Cladocera); Cloeon dipterum (Ephemeroptera); Plea leachi (Heteroptera); and Eristalis sp., Chaoborus crystallinus, Chironomus thummi, and Psychoda alternata (Diptera). In addition, bioassays were performed with mosquito larvae (Aedes aegypti, Anopheles stephensi, and Culex pipiens). Psychoda alternata larvae were very susceptible, with LC50/LC90 values comparable to those of mosquito larvae (about 10(3)-10(5) spores/ml). The tests with Chaoborus crystallinus larvae showed significant mortality rates at high concentrations, but generally not before 4 or 5 days after treatment. The remaining nontarget organisms did not show any susceptibility. The investigation confirms the specificity of C.b.m. to nematocerous Diptera.
Two soap formulations, both containing 20% deet and one each containing permethrin at 0.5 and 1.0%, respectively, were applied to exposed arms and legs of volunteers as personal protection against outdoor human biting mosquitoes in six locations on Penang Island, Malaysia. The predominant mosquito species collected from these locations were Aedes albopictus, Mansonia uniformis, Culex gelidus, Anopheles lesteri and Armigeres subalbatus. Efficacy and residual effects up to 4 hours indicated good protection against these species. Reduction in mosquito landing-biting rates in treated groups ranged from 83.8 to 100.0%. At high densities, small percentages of Ma. uniformis and An. lesteri landed or bit on treated skin. Use of the soap formulations in terms of cost-effectiveness, safety and overall vector control strategy for some tropical diseases is discussed.
Mosquito-borne diseases have been increasing at an alarming rate over the past decades. In Malaysia, one finds several important mosquito-borne diseases such as Japanese encephalitis, dengue, malaria, and chikungunya. Mosquito surveillance and control programs are the most effective way of detecting and controlling mosquito-borne diseases, but these programs are less effective without an aware and well-informed general public. In 2014 we used a questionnaire to evaluate the extent of awareness of basic mosquito biology and mosquito-borne diseases in 6 villages, Kampung Masjid, Kampung Teluk Gedung, Kampung Teluk Dalam, Kampung Ujung Kelawai, Kampung Sungai Pinang Besar, and Kampung Sungai Pinang Kechil on Pangkor Island, Malaysia. A total of 1,012 individuals responded to the questionnaire, consisting of 790 Malay (78.1%), 164 Chinese (16.2%), and 58 Indian (5.7%). More than 60% (Malay = 73.7%, Chinese = 64.0%, Indian = 79.3%) of the respondents were familiar with basic mosquito biology and practiced personal protection against mosquito bites, and the association was statistically significant (P = 0.02). However, the majority of the respondents had limited knowledge on mosquito-borne diseases, and this varied significantly among the 3 ethnic groups (P = 0.0001). Our recommendations are to improve and intensify public health education outreach programs to the island residents and to encourage community participation in vector control programs.
Toxorhynchites splendens larvae are a natural predator of dengue vector mosquito larvae, Aedes albopictus. This study was carried out to evaluate the predation rate of Tx. splendens third instar larvae on Ae. albopictus larvae in 24 h. Each predator was offered prey at a density between 10 to 50 individuals. Predation rate of Tx. splendens were also tested with two manipulated factors; various types of container and different water volumes. The experiment was evaluated in man-made containers (tin cans, plastic drinking glasses and rubber tires) and natural container (bamboo stumps) which were filled with different water volumes (full, half full, 1/4 full, and 1/8 full). The prey density and the characteristics of the container were found as significant factors which influence the predation rate of Tx. splendens. The predator consumed significantly more prey at higher prey densities (40 and 50 preys) compared to the lowest density (10 preys) (F=3.935, df=4, p=0.008). The results showed significantly higher consumption in horizontal shaped container of rubber tire than in vertical shape of bamboo stumps (F=3.100, df=3, p=0.029). However, the water volume had no significant effect on predation rate of Tx. splendens (F=1.736, df=3, p=0.162). We generally suggest that Tx. splendens is best to be released in discarded tires or any other containers with horizontal shape design with wide opening since Tx. splendens can become more effective in searching prey in this type of container design. This predator is also a suitable biocontrol candidates to be introduced either in wet and dry seasons in Malaysia.
Thirty isolations of Tembusu virus and four of Sindbis virus were obtained from approximately 280 000 mosquitoes collected between October 1968 and February 1970 in Sarawak, particularly from K. Tijirak, a Land Dyak village 19 miles South of Kuching. Twenty-two isolations of Tembusu virus and two of Sindbis virus were from Culex tritaeniorhynchus; two of Tembusu virus and two of Sindbis virus came from Culex gelidus. Tembusu virus was active throughout the year at K. Tijirak, the highest infection rates in C. tritaeniorhynchus being in January-March and May-August, when the C. tritaeniorhynchus population was declining and ageing. These results confirm that C. tritaeniorhynchus is the principal arthopod host of Tembusu virus in Sarawak. Antibody studies suggest that birds, particularly domestic fowl, are probably vertebrate maintenance hosts of Tembusu and Sindbis viruses in Sarawak.