Nuananong Jirakanjanakit

Insecticide Susceptible/Resistance Status in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand During 2003–2005

Abstract Susceptibility baselines and diagnostic doses of the technical grade insecticides deltamethrin, permethrin, fenitrothion, and propoxur were established based on Aedes aegypti (L.), Bora (French Polynesia), a reference susceptible strain. Field-collected Aedes mosquitoes from each part of Thailand were subjected to bioassay for their susceptibility to the diagnostic doses of each insecticide. Almost all Ae. aegypti collected were incipient resistant or resistant to deltamethrin and permethrin, except those from some areas of Songkhla (southern) and Phan district of Chiang Rai (northern) province. Susceptibility to fenitrothion was found in mosquitoes from Bangkok (central), Chonburi (eastern), Chiang Rai, Kanchanaburi (western), and Songkhla, whereas they were resistant in almost all areas of Nakhon Sawan (north central) and Nakhon Ratchasima (northeastern) provinces. Most of Ae. aegypti were susceptible to propoxur except those from Mae Wong, Nakhon Sawan province. Various levels of insecticide resistance and susceptibility in adjacent areas revealed a focal susceptible/resistance profile in the country. It could be noted that almost all of Ae. albopictus were susceptible to the insecticides tested at the same diagnostic doses. In conclusion, resistance to pyrethroids (permethrin and deltamethrin) has developed in Ae. aegypti in most of the collected areas, suggesting that an alternative choice of insecticide or other control measures should be applied.

Influence of larval density or food variation on the geometry of the wing of Aedes (Stegomyia) aegypti

Background and Method  Variation in wing length among natural populations of Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) is associated with different vectorial capacities. Geometric morphometrics allowed us to use a more powerful estimator of wing size (‘centroid size’), as well as to visualize the variation of wing shape, to describe the effects of density or food variation at larval stage on 20 anatomical landmarks of the wing of A. aegypti.

Frequency of Pyrethroid Resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

ABSTRACT: Thirty-two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda-cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda-cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda-cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.

Trend of Temephos Resistance in Aedes (Stegomyia) Mosquitoes in Thailand During 2003–2005

Abstract The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.

The micro-focus reduction neutralization test for determining dengue and Japanese encephalitis neutralizing antibodies in volunteers vaccinated against dengue

A micro-focus reduction neutralization test (mFRNT) was evaluated as an alternative test to the ordinary plaque reduction neutralization test (PRNT) for the determination of dengue virus and Japanese encephalitis virus neutralizing antibody responses in persons receiving dengue vaccine. The 2 tests were similar in terms of titres and ability to detect seroconversion. Although the neutralizing antibody titres obtained by mFRNT were slightly lower than those given by PRNT, the differences were less than two-fold, indicating than mFRNT was reliable. Reproducibility of mFRNT was confirmed by 10 replicate tests using the same control serum. Therefore, mFRNT may be useful in large-scale investigations of neutralizing antibody levels, for example, in young children forming part of an immunization programme; it can be performed quickly and is economical, requiring only a small volume of sera.

The use of Toxorhynchites splendens for identification and quantitation of serotypes contained in the tetravalent live attenuated dengue vaccine

Assurance of identity and quantity is an indispensable part of quality control in the manufacture of vaccines. Dengue-1 PDK13, dengue-2 PDK53, dengue-3 PGMK30F3 and dengue-4 PDK48 in the live attenuated tetravalent dengue vaccine were assayed by identification and quantitation in a mosquito system (Toxorhynchites splendens). Each serotype of dengue virus was identified by dengue specific monoclonal antibodies in the indirect fluorescent antibody test. Virus content was estimated by calculating the 50% mosquito infectious dose (MID50). Differences from 0 to +/-0.5 log10 were observed between the original monovalent titer and that from the blend which showed no significant difference at 95% confidence limit (P < 0.05). This result indicates that there is no interference between dengue serotypes in mosquitoes infected by intrathoracic inoculation with the virus mixture. It can be also concluded that this mosquito system can be used as an effective measure for infectivity titration of each component in the tetravalent dengue vaccine.

Transcription analysis of differentially expressed genes in insecticide-resistant Aedes aegypti mosquitoes after deltamethrin exposure

ABSTRACT: Aedes aegypti mosquitoes are resistant to various insecticides, including pyrethroids, throughout Thailand. We previously reported that Ae. aegypti from Mae Wong district, Nakhon Sawan Province in north-central Thailand, were resistant to insecticides, including pyrethroids (deltamethrin and permethrin), organophosphates and carbamates, and that high levels of detoxification enzymes were present. In the present study we used the method of suppression by subtractive hybridization to determine differential expression of genes in Mae Wong Ae. aegypti that survived the exposure to increasing doses (∼ 1.5 - 2 × 10-5M) of deltamethrin beyond the diagnostic dose compared to unexposed mosquitoes. Screening of 350 cDNA clones from the suppression subtractive library by cDNA array hybridization revealed that 58 clones were over-expressed in the mosquito that survived high dose deltamethrin. The over-expressed cDNA insert sequences corresponded to 11 functional genes, five hypothetical protein genes, and five sequences of unknown function that could be located on the supercontig of the Ae. aegypti genome. The functional genes are those coding for cuticular proteins, muscle proteins, proteins related to controlling the release of synaptic vesicles, and other genes such as heat shock protein and small subunit ribosomal RNA. Over-expression of tomosyn and myosin light chain kinase genes was verified using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), confirming their increased expression in response to deltamethrin exposure in insecticide-resistant Ae. aegypti.

Laboratory assays and field dengue vaccine evaluation at Ratchaburi province, Thailand: a preliminary result.

Laboratory-based epidemiological studies have been initiated in a cohort of school-aged children in Ratchaburi province, Thailand in preparation for imminent dengue vaccine field trials. In these studies, levels of neutralising antibodies were determined by the plaque reduction neutralisation test (PRNT) while virological confirmation was performed using RT-PCR or mosquito inoculation. Serological confirmation was performed using IgM and IgG ELISA. The incidence rate of dengue in the Ratchaburi cohort was 1635 per 100,000 during 2005-2006. Among 3547 patients in this cohort, 331 were classified with febrile disease and 58 with dengue. All four dengue serotypes were observed in Ratchaburi province. Efforts to identify the infecting serotype in symptomatic patients, based solely upon the neutralising antibody response, were complicated by cross- neutralising antibodies. In addition, 20 symptomatic and 49 asymptomatic cases were identified among 897 subjects investigated during 2005-2006, a ratio of 1:2.5. Serological analysis of asymptomatic dengue infections demonstrated boosting of immune responses due to subclinical infections with dengue or Japanese encephalitis. These results demonstrate that laboratory-confirmed dengue disease in areas of high transmission can be established. Epidemiological data of this kind are critical to dengue vaccine efficacy trials.

Genetic structure and gene flow among Aedes aegypti (Diptera: Culicidae) populations from central Thailand.

Abstract Isozymes from five wild-caught populations of Aedes aegypti (L.) were compared using starch gel electrophoresis to estimate rates of gene flow between and among geographically close mosquito populations. Ae. aegypti were collected from five different locations in Bangkok, Thailand. One collection was obtained from central Bangkok (Huai-Khwang); the other four samples were obtained from surrounding areas (Districts Latkrabang, Ratburana, Laksi, and Bangkok Noi). Based on 24 loci (17 enzyme systems), only minor genetic differentiation was observed between all five populations. The highest percentage of polymorphic loci (34.3) was observed from the central Bangkok population; the least percentage of polymorphism (20.0) was seen from Laksi (north Bangkok). This study indicates that a large effective migration rate exists among all five populations. No fixed genetic differences were detected.

Carrier Design for Astaxanthin Delivery

Astaxanthin is a natural carotenoid pigment with powerful antioxidant capacity which has been reported for beneficial effects on human health to prevent cancers and anti-degenerative diseases. Bioavailability of astaxanthin is however limited due to insoluble and instable properties of its lipophilic nature. This study describes the cytotoxicity and astaxanthin delivery using a surfactant (tween 80) and penetration enhancers (methyl -cyclodextrin and liposome) to enhance astaxanthin bioavailability in human hepatocellular carcinoma cell line. Toxicity of tween 80 and methyl -cyclodextrin increased with concentration. Liposome did not alter cell viability relative to untreated controls, regardless of concentration tested. Cellular uptake among delivery vehicles was compared at 24 hours and was found to be higher than that of astaxanthin without a vehicle. The cellular uptake rate was highest when liposome was used as the delivery vehicle. This finding aids in the development of design criteria for vehicles for delivery of antioxidants to cells.