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Genetically modified mosquito: the Malaysian public engagement experience

Subramaniam TS, Lee HL, Ahmad NW, Murad S

Biotechnol J, 2012 Nov;7(11):1323-7.
PMID: 23125042 DOI: 10.1002/biot.201200282

On December 21, 2010, 6000 genetically modified (GM) mosquitoes were released in an uninhabited forest in Malaysia. The purpose of the deliberate release was a limited “marked release and recapture” (MRR) experiment, a standard ecological method in entomology, to evaluate under field conditions, the flight distance and longevity of the sterile male Aedes aegypti strain OX513A(My1), a GM strain. As with any other GM technologies, the release was received with mixed responses. As the scientific community debate over the public engagement strategies for similar GM releases, dengue incidence continues to rise with a heavy toll on morbidity, mortality and healthcare budgets. Meanwhile the wild female Aedes aegypti continues to breed offspring, surviving and evading conventional interventions for vector control.
Interaction effects of temperature and food on the development of forensically important fly, Megaselia scalaris (Loew) (Diptera: Phoridae)

Zuha RM, Razak TA, Ahmad NW, Omar B

Parasitol Res, 2012 Nov;111(5):2179-87.
PMID: 22886544 DOI: 10.1007/s00436-012-3070-z

In forensic entomology, breeding of fly larvae in a controlled laboratory environment using animal tissue is a common technique to obtain insect developmental time for the estimation of postmortem interval. Previous studies on growth media are mostly on the effect of different diets on fly development. However, the interaction effects between temperature and food type used have not been explored. The objective of this study was to compare the use of cow's liver agar and raw liver on the development of a forensically important fly, Megaselia scalaris (Loew). This study also determined the interaction between different temperatures and different food types on the growth of this species. A total of 100 M. scalaris eggs were transferred into each of the two media mentioned above. Liver agar was prepared by adding dried ground liver into nutrient agar, whilst raw liver was naturally prepared from the same animal source. This experiment was conducted at 27, 30 and 33 °C in an incubator in a continuously dark condition. Length and weight of larvae, puparia and adult samples were determined. Total developmental times for larvae feeding on liver agar at each temperature were approximately 7-15 h slower than those feeding on raw liver. Survival rates were almost equal in both diets but were lower at 33 °C. Mean larva length in both diets did not differ significantly at all temperatures, but larvae feeding on liver agar had lower mean weight values than those in raw liver at 30 and 33 °C. The effect of temperature was significant in female puparia weight and male adult weight whereas the effect of diet types was significant in both male and female puparia size and weight. Interaction effects of temperature and food type on M. scalaris puparium size and adult weight were significant, indicating that puparium size and adult weight depended on both food type and temperature. This experiment highlighted the use of cow's liver agar as an alternative diet to breed M. scalaris in the laboratory and the importance of considering the interaction effect between temperatures and food types when deciding the most suitable medium in fly larva rearing.
Insecticidal Effects of Organotin(IV) Compounds on Plutella Xylostella (L.) Larvae. II. Inhibitory Potencies Against Acetylcholinesterase and Evidence for Synergism in Tests With Bacillus Thuringiensis(BER.) and Malathion

Ahmad NW, Huang TS, Balabaskaran S, Lo KM, Das VG

Met Based Drugs, 1994;1(1):1-17.
PMID: 18476213

Features of pesticide synergism and acetylcholinesterase (AChE) inhibition (in vitro) were studied using a selected range of organotin compounds against the early 4th instar larvae of a highly resistant strain of the diamondback moth (DBM), Plutella xylostella, a major universal pest of cruciferous vegetables.Fourteen triorganotin compounds were evaluated for their ability to enhance the toxicity of the microbial insecticide, Bacillus thuringiensis (BT) and of the commercial insecticide, Malathion to Plutella xylostella larvae. Supplemental synergism was observed with triphenyl- and tricyclopentyltin hydroxides in combinations with Bacillus thuringiensis. Increased synergism was observed with an increase in the number of cyclopentyl groups on tin in the mixed series, Cyp(n) Ph(3-n) SnX, where X = OH, and 1-(1,2,4-triazolyl). The combination of (p-chlorophenyl)diphenyltin N,N-dimethyldithiocarbamate at LD(10) and LD(25) concentrations with sublethal concentrations of Malathion as well as of tricyclohexyltin methanesulphonate at the 0.01% (w/v) concentration with Malathion exerted strong synergistic effects (supplemental synergism) with toxicity index (T.I) values of 7.2, 19.8 and 10.1, respectively.Studies on the in vitro inhibition of acetylcholinesterase prepared from the DBM larvae showed that while most of the triorganotin Compounds tested were without effect on the enzyme, compounds containing the thiocarbamylacetate or the dithiocarbamylacetate moieties demonstrated appreciable levels of inhibition, being comparable in efficacy to commercial grades of Malathion and Methomyl.
Fulltext A genomic approach to inferring kinship reveals limited intergenerational dispersal in the yellow fever mosquito

Jasper M, Schmidt TL, Ahmad NW, Sinkins SP, Hoffmann AA

Mol Ecol Resour, 2019 Sep;19(5):1254-1264.
PMID: 31125998 DOI: 10.1111/1755-0998.13043

Understanding past dispersal and breeding events can provide insight into ecology and evolution and can help inform strategies for conservation and the control of pest species. However, parent-offspring dispersal can be difficult to investigate in rare species and in small pest species such as mosquitoes. Here, we develop a methodology for estimating parent-offspring dispersal from the spatial distribution of close kin, using pairwise kinship estimates derived from genome-wide single nucleotide polymorphisms (SNPs). SNPs were scored in 162 Aedes aegypti (yellow fever mosquito) collected from eight close-set, high-rise apartment buildings in an area of Malaysia with high dengue incidence. We used the SNPs to reconstruct kinship groups across three orders of kinship. We transformed the geographical distances between all kin pairs within each kinship category into axial standard deviations of these distances, then decomposed these into components representing past dispersal events. From these components, we isolated the axial standard deviation of parent-offspring dispersal and estimated neighbourhood area (129 m), median parent-offspring dispersal distance (75 m) and oviposition dispersal radius within a gonotrophic cycle (36 m). We also analysed genetic structure using distance-based redundancy analysis and linear regression, finding isolation by distance both within and between buildings and estimating neighbourhood size at 268 individuals. These findings indicate the scale required to suppress local outbreaks of arboviral disease and to target releases of modified mosquitoes for mosquito and disease control. Our methodology is readily implementable for studies of other species, including pests and species of conservation significance.
Maggot debridement therapy with Lucilia cuprina: a comparison with conventional debridement in diabetic foot ulcers

Paul AG, Ahmad NW, Lee HL, Ariff AM, Saranum M, Naicker AS, et al.

Int Wound J, 2009 Feb;6(1):39-46.
PMID: 19291114 DOI: 10.1111/j.1742-481X.2008.00564.x

This is prospective case-control study of more than 18 months performed to assess the effectiveness of maggot debridement therapy (MDT) with the sterile larvae of Lucilia cuprina (a tropical blowfly maggot) for the treatment of diabetic foot ulcers. Literature thus far has only reported results with the temperate maggot, Lucilia sericata. This study documents outcome in diabetic foot wounds treated with maggot debridement versus those treated by conventional debridement alone. In this series of 29 patients treated with MDT, 14 wounds were healed, 11 were unhealed and 4 were classified under others. The control group treated by conventional debridement had 30 patients of which 18 wounds were healed, 11 unhealed and 1 classified under others. There was no significant difference in outcome between the two groups. The conclusion that can be made from this study is that MDT with L. cuprina is as effective as conventional debridement in the treatment of diabetic foot ulcers. It would be a feasible alternative to those at high risk for surgery or for those who refuse surgery.
Mating compatibility and competitiveness of transgenic and wild type Aedes aegypti (L.) under contained semi-field conditions

Lee HL, Vasan S, Ahmad NW, Idris I, Hanum N, Selvi S, et al.

Transgenic Res, 2013 Feb;22(1):47-57.
PMID: 22700207 DOI: 10.1007/s11248-012-9625-z

We conducted the world's first experiments under semi-field conditions (ACL-2 field house) to assess the mating competitiveness of genetically sterile RIDL male mosquitoes (513A strain). The field house is a state-of-the-art, fully-contained trial facility, simulating the living space for a household of 2-4 people in Peninsular Malaysia. Ten genetically sterile RIDL male A. aegypti mosquitoes competed with ten wild type males inside this field house to mate with ten wild type females. Hatched larvae from mated females were screened under a fluorescent microscope for genetic markers to determine if they were fathered by RIDL male or wild type male, and all results were cross-checked by PCR. Two such experiments were conducted, each repeated sufficient number of times. All strains were on a Malaysian lab strain background for the first experiment, while the RIDL males alone were on a recently-colonised Mexican strain background for the second experiment. A total of 52 % of the matings were with RIDL males in the first experiment, while 45 % of the matings were with RIDL (Mexican) males in the second experiment. Statistically, this is not significantly different from 50 % of the matings expected to take place with RIDL males if the latter were as competitive as that of the wild type males. This shows that A. aegypti RIDL-513A has excellent mating competitiveness under semi-field conditions, verifying earlier trends obtained in small lab cages. We also observed high mating compatibility between recently-colonised Mexican RIDL males and lab-reared Malaysian wild type females.
Insecticide susceptibility status of field-collected Aedes (Stegomyia) aegypti (L.) at a dengue endemic site in Shah Alam, Selangor, Malaysia

Rong LS, Ann AT, Ahmad NW, Lim LH, Azirun MS

Southeast Asian J Trop Med Public Health, 2012 Jan;43(1):34-47.
PMID: 23082552

Biweekly ovitrap surveillance (OS) was conducted for a year (August 2007 - September 2008) at two different dengue endemic sites in Shah Alam, Selangor, Malaysia, 50 km from Kuala Lumpur. Aedes aegypti collected from these 2 locations were raised to the F3 stage and subjected to a WHO standard bioassay method to determine lethal time (LT) against pyrethroids (permethrin 0.75%, cyfluthrin 0.15%), organophosphates (malathion 5.0%, fenitrothion 1.0%), carbamates (propoxur 0.1%, bendiocarb 0.1%) and organochlorine (DDT 4.0%). Insecticide susceptibilities were analyzed for one year. Aedes aegypti were resistant to DDT with a mortality range of 0 - 13.3% throughout the year at both sites. Susceptibilities to pyrethroids and carbamates varied throughout the year. In contrast, susceptibilities to pyrethroids and carbamates varied throughout the year: resistant to propoxur, bendiocarb and permethrin with mortality of < 80% in most months; but, showed incipient resistant to cyfluthrin in most months. Mosquitoes were consistently susceptible to malathion and fenitrothion, with complete mortality during most months. They were especially susceptible to malathion with LT50 values of 21.32 - 36.37 minutes, suggesting effectiveness of malathion for control of dengue.
Infestation with the cat flea, Ctenocephalides felis felis (Siphonaptera: Pulicidae) among students in Kuala Lumpur, Malaysia

Chin HC, Ahmad NW, Lim LH, Jeffery J, Hadi AA, Othman H, et al.

Southeast Asian J Trop Med Public Health, 2010 Nov;41(6):1331-4.
PMID: 21329307

This paper reports six male undergraduate students living at a local university hostel who were infested with cat fleas (Siphonaptera: Pulicidae: Ctenocephalides felis felis) in February 2009. All of them suffered from maculopapular rashes and severe pruritus after the bites. Investigation revealed the presence of a stray cat in the hostel building; five of the students had a history of contact with the cat. Six cat fleas were collected at the hostel and identified as C. felis felis. Most of the students were not aware of this infestation and did not seek medical treatment.
Fulltext Aural myiasis in a neonate in peninsular Malaysia

Ahmad NW, Ismail A, Jeffery J, Ibrahim S, Hadi AA, Ibrahim MN, et al.

Parasit Vectors, 2009;2(1):63.
PMID: 20003466 DOI: 10.1186/1756-3305-2-63

Myiasis is a pathological condition in humans and animals caused by various species of dipterous larvae. Myiasis which occurs in a newborn baby is referred as neonatal myiasis. It is a rare condition and there are only a few reports to date. A case of neonatal aural myiasis in a two day old infant is reported in this paper.
Fulltext Predation on pupa of Chrysomya rufifacies (Marquart) (Diptera: Calliphoridae) by parasitoid, Exoristobia philippinensis Ashmead (Hymenoptera: Encyrtidae) and Ophyra spinigera larva (Diptera: Muscidae)

Chin HC, Ahmad NW, Lim LH, Jeffery J, Omar B, Dhang CC, et al.

Trop Biomed, 2009 Dec;26(3):369-72.
PMID: 20237454

A forensic entomological study was conducted using monkey carcasses (Macaca fascicularis Raffles) that were placed in either an outdoor or indoor environment at a coastal area in Tanjung Sepat, Selangor, Malaysia during May until August 2008. We collected pupae of Chrysomya rufifacies (Marquart) from the carcasses and kept them individually. The emergence of 13 parasitic microhymenopteran, from one of the pupae occurring within a week were identified as Exoristobia philippinensis Ashmead (Hymenoptera: Encyrtidae). Another observation was made whereby a pupa of C. rufifacies was predated by a muscid larva, Ophyra spinigera (Stein). The larva squeezed into the pupa and consumed the contents. This paper report C. rufifacies as a new host record for E. philippinensis in Malaysia and highlighted the predatory behavior of O. spinigera larva in natural environment.
Fulltext First report of the signal fly, Scholastes sp. (Diptera:Platystomatidae) visiting animal carcasses in Malaysia

Dhang CC, Heo CC, McAlpine D, Kurahashi H, Ahmad NW, Mohamad AM, et al.

Trop Biomed, 2008 Dec;25(3):264-6.
PMID: 19287370

Signal fly, Scholastes sp. (Diptera: Platystomatidae) was observed associated with animal carcasses in Malaysia. The first observation was on a monkey carcass, which was killed by using a handgun and immediately placed in a forested area in Gombak, Selangor while the second observation was on a pig that died of natural causes and whose carcass was placed in an oil palm plantation in Tanjung Sepat, Selangor. Both animal carcasses were visited by Scholastes sp. flies during the fresh decomposition period. However, the role Scholastes flies in the decomposition process remains unknown. In this paper, we report the occurrence of Scholastes sp. on animal carcasses in Malaysia for the first time.
Hirudotherapy in Wound Healing

Nair HKR, Ahmad NW, Lee HL, Ahmad N, Othamn S, Mokhtar NSHM, et al.

Int J Low Extrem Wounds, 2022 Dec;21(4):425-431.
PMID: 32815407 DOI: 10.1177/1534734620948299

Leeches are hermaphrodite, bloodsucking parasitic worms usually found in places with fresh water. Leech therapy existed 3000 years, and it is being used at a different scope. Several species of leeches have been used in medicine, and the most common species used is Hirudo medicinalis. Leeches suck the excess blood, reduce the swelling in the tissues, and promote healing by allowing fresh oxygenated blood to reach the area until normal circulation can be restored. Pain relief from leech therapy is rapid, effective, and long-lasting in many conditions. The aim of this study was to evaluate the efficacy and duration of healing utilizing sterile medicinal leeches, Hirudinaria manillensis, in the management of pain and wound healing. Leech was taken out from its sterile tube by using a pair of non-tooth sterile plastic forceps and gloved hands. Each leech was left in place for as long as it was feeding. Leeches were removed only after they became detached from the patient. The specimen jars containing the used leeches were sealed in either a biohazard bag or in a small yellow clinical waste bin liner securely fastened with a cable tie. The leech was killed by using 70% alcohol prior to disposal into a yellow hazard bin, which undergoes incineration. All 3 patients had improvements in their condition, especially in terms of reduction in the pain and improvement in their sense of balance. All the wounds healed well. Therefore, leech therapy is effective in reducing pain and increasing perfusion to allow the wounds to heal quickly. However, a more robust trial is needed to show significance as the sample size is small.
Maggot debridement therapy to treat hard-to-heal diabetic foot ulcers: a single-centre study

Nair HK, Ahmad NW, Ismail AA, Alabed AAA, Zheming BO, Kaur G, et al.

J Wound Care, 2021 12 01;30(Sup12):S30-S36.
PMID: 34882006 DOI: 10.12968/jowc.2021.30.Sup12.S30

OBJECTIVE: Maggot debridement therapy (MDT) has seen a resurgence in recent years in the treatment of hard-to-heal wounds, as a result of rising antibiotic resistance. The sterilised larvae of Lucilia cuprina have been used in MDT in Malaysia since 2003, with encouraging results for the treatment of hard-to-heal diabetic wounds. We report a case series of 30 patients selected from our clinic by convenient sampling with diabetic lower limb ulcers treated with MDT. The average age of patients receiving MDT was >50 years. Of the 30 patients in the study, nine were female and 21 were male. All patients had underlying diabetes, two patients had leg ulcers and 28 patients had diabetic foot ulcers. Sterilised Lucilia cuprina larvae were applied via a standard method of 10 maggots per square centimetre and dressed with sterile gauze. The study endpoint was defined as ≤5% coverage with slough or necrotic tissue following three successive applications of MDT. In this study, maximum debridement of wounds was achieved in 96.6% (29 patients) of our patients, with ≤5% coverage with slough or necrotic tissue, in addition to a reduction in wound-related pain, as assessed by a visual analogue scale. No adverse events were reported. The findings of this study support the use of MDT as a safe, efficacious, and cost-effective method of managing diabetic wounds.
Fulltext Identification of potential vectors of Dirofilaria immitis and Brugia pahangi (Spirurida: Filariidae): First observation of infective third-stage larva of B. pahangi in Culex quinquefasciatus (Diptera: Culicidae)

Vinnie-Siow WY, Low VL, Tan TK, Wong ML, Leong CS, Ahmad NW, et al.

Pathog Glob Health, 2022 Sep;116(6):356-364.
PMID: 35287548 DOI: 10.1080/20477724.2022.2035624

Information on the mosquito species that transmit canine filariosis is scanty. Hence, an experimental study was conducted to identify the potential vectors responsible for the transmission of D. immitis Leidy and B. pahangi Buckley & Edeson. A total of 367 mosquitoes belonging to six species containing both laboratory and field strains (i.e. Aedes togoi Theobald, Aedes aegypti Linnaeus, Aedes albopictus Skuse, Culex quinquefasciatus Say, Culex vishnui Theobald and Anopheles dirus Peyton & Harrison) were used in this study. All mosquitoes were artificially fed on either D. immitis or B. pahangi microfilariae (mfs) infected blood by using the Hemotek™ membrane feeding system. Out of 367 mosquitoes, 228 (64.9%) were fully engorged. After feeding on D. immitis (20%) and B. pahangi (33%) mfs positive blood, the mortality rates for Cx. quinquefasciatus were found to be slightly lower than that of other species of mosquitoes. On the other hand, majority of An. dirus were found to be incapable to withstand the infection of mfs as the mortality rates were relatively high (D. immitis = 71.4%; B. pahangi = 100.0%). Brugia pahangi was detected in Ae. togoi and Cx. quinquefasciatus with infection rates of 50% and 25%, respectively. Aedes togoi was the only species infected with D. immitis with an infection rate of 69%. Our results showed that Ae. togoi was an excellent experimental vector for both D. immitis and B. pahangi. This study also documented the observation of B. pahangi, for the first time in the head region of Cx. quinquefasciatus under a laboratory setting.
Fulltext Measuring the effectiveness of integrated vector management with targeted outdoor residual spraying and autodissemination devices on the incidence of dengue in urban Malaysia in the iDEM trial (intervention for Dengue Epidemiology in Malaysia): study protocol for a cluster randomized controlled trial

Saadatian-Elahi M, Alexander N, Möhlmann T, Langlois-Jacques C, Suer R, Ahmad NW, et al.

Trials, 2021 May 30;22(1):374.
PMID: 34053466 DOI: 10.1186/s13063-021-05298-2

BACKGROUND: In common with many South East Asian countries, Malaysia is endemic for dengue. Dengue control in Malaysia is currently based on reactive vector management within 24 h of a dengue case being reported. Preventive rather than reactive vector control approaches, with combined interventions, are expected to improve the cost-effectiveness of dengue control programs. The principal objective of this cluster randomized controlled trial is to quantify the effectiveness of a preventive integrated vector management (IVM) strategy on the incidence of dengue as compared to routine vector control efforts.
METHODS: The trial is conducted in randomly allocated clusters of low- and medium-cost housing located in the Federal Territory of Kuala Lumpur and Putrajaya. The IVM approach combines: targeted outdoor residual spraying with K-Othrine Polyzone, deployment of mosquito traps as auto-dissemination devices, and community engagement activities. The trial includes 300 clusters randomly allocated in a 1:1 ratio. The clusters receive either the preventive IVM in addition to the routine vector control activities or the routine vector control activities only. Epidemiological data from monthly confirmed dengue cases during the study period will be obtained from the Vector Borne Disease Sector, Malaysian Ministry of Health e-Dengue surveillance system. Entomological surveillance data will be collected in 12 clusters randomly selected from each arm. To measure the effectiveness of the IVM approach on dengue incidence, a negative binomial regression model will be used to compare the incidence between control and intervention clusters. To quantify the effect of the interventions on the main entomological outcome, ovitrap index, a modified ordinary least squares regression model using a robust standard error estimator will be used.
DISCUSSION: Considering the ongoing expansion of dengue burden in Malaysia, setting up proactive control strategies is critical. Despite some limitations of the trial such as the use of passive surveillance to identify cases, the results will be informative for a better understanding of effectiveness of proactive IVM approach in the control of dengue. Evidence from this trial may help justify investment in preventive IVM approaches as preferred to reactive case management strategies.
TRIAL REGISTRATION: ISRCTN ISRCTN81915073 . Retrospectively registered on 17 April 2020.
Relationship between rainfall and Aedes larval population at two insular sites in Pulau Ketam, Selangor, Malaysia

Wee LK, Weng SN, Raduan N, Wah SK, Ming WH, Shi CH, et al.

Southeast Asian J Trop Med Public Health, 2013 Mar;44(2):157-66.
PMID: 23691624

Two insular settlements (Kampung Pulau Ketam and Kampung Sungai Lima) were selected to study the population dynamics of Aedes aegypti and Aedes albopictus mosquitoes, vectors of dengue and chikungunya infections. Ovitrap surveillance was conducted between October 2007 and October 2008. There was an inverse negative association between ovitrap index and rainfall at the time of collection, probably because rainfall increased the number of available oviposition sites. Rainfall and ovitrap index were positively associates the 25th day after rainfall occurred. A minor, second peak was observed from the 38th to the 42nd day. The first peak was consistent with the minimum 18-day period between the hatching of eggs to the first oviposition. The second minor peak could be due to the second gonotrophic cycle of the female mosquitoes. Rainfall is an important environmental factor associated with Aedes breeding at the study sites.
Fulltext Introduction of Aedes aegypti mosquitoes carrying wAlbB Wolbachia sharply decreases dengue incidence in disease hotspots

Hoffmann AA, Ahmad NW, Keong WM, Ling CY, Ahmad NA, Golding N, et al.

iScience, 2024 Feb 16;27(2):108942.
PMID: 38327789 DOI: 10.1016/j.isci.2024.108942

Partial replacement of resident Aedes aegypti mosquitoes with introduced mosquitoes carrying certain strains of inherited Wolbachia symbionts can result in transmission blocking of dengue and other viruses of public health importance. Wolbachia strain wAlbB is an effective transmission blocker and stable at high temperatures, making it particularly suitable for hot tropical climates. Following trial field releases in Malaysia, releases using wAlbB Ae. aegypti have become operationalized by the Malaysian health authorities. We report here on an average reduction in dengue fever of 62.4% (confidence intervals 50-71%) in 20 releases sites when compared to 76 control sites in high-rise residential areas. Importantly the level of dengue reduction increased with Wolbachia frequency, with 75.8% reduction (61-87%) estimated at 100% Wolbachia frequency. These findings indicate large impacts of wAlbB Wolbachia invasions on dengue fever incidence in an operational setting, with incidence expected to further decrease as wider areas are invaded.

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