Eco-virological survey of Aedes mosquito larvae in selected dengue outbreak areas in Malaysia.

UNLABELLED: BACKGROUND & OBJECTIVESI: Transovarial transmission of dengue virus in the Aedes vectors is now a well-documented phenomenon reported from many parts of the endemic areas in the world, which played an important role in initiating and maintaining the outbreak in human populations. This study investigated the factors affecting breeding habitats and the relationship with transovarial dengue virus in larvae of Aedes aegypti and Ae. albopictus. METHODS: Larval surveillance was conducted in dengue outbreak areas in Malaysia from 2008 until 2009. Sampling was carried out based on habitat type, water condition (substrate type), canopy coverage, temperature and pH at breeding habitats. RT-PCR was performed to detect presence of transovarial dengue virus in larvae collected in the study areas. RESULTS: A total of 789 breeding habitats were identified during this study and the majority of these breeding sites were plastic containers (57.46%). Aedes albopictus dominated most of the water condition surveyed, while Ae. aegypti indicated preference toward habitats with clear water. Aedes aegypti was selective in selecting ovipositional sites compared to Ae. albopictus where shaded areas were shown to be the most preferred. From a total of 363 mosquito larvae pools, 23 (6.3%) pools were positive for dengue virus where 18 of them were from Ae. albopictus and five were from Ae. aegypti mosquito larvae pools. INTERPRETATION & CONCLUSION: This study indicated the presence of transovarial transmission of dengue virus in immature Ae. aegypti and Ae. albopictus in the field. This study also showed that combination of water conditions, canopy coverage, temperature and pH of breeding habitats were the factors affecting the larval population. The study suggested that larval survey programme could serve as a tool not only to monitor the local dengue vector distribution but also to provide objective information for taking appropriate action by the community against dengue vectors.

Ochrobactrum anthropi catheter-related bloodstream infection: the first case report in malaysia.

A 60-year old Malay man presented with an intermittent lowgrade fever for 3 days and worsening of right ankle ulcers. He was known to have ischaemic heart disease, diabetes mellitus type 2, hypertension and end stage renal failure and was on regular haemodialysis. The organisms isolated from the peripheral and central blood specimens were identified as Ochrobactrum anthropi, a rarely encountered gramnegative bacillus with a unique antibiotic susceptibility profile. His condition improved after removal of the catheters and administration of antibiotic therapy. This case report highlights the importance of being wary of such rare, opportunistic pathogens and good communication between the microbiologists and the clinicians.

Comparative effects of thymol and vitamin E on nonalcoholic fatty liver disease in male Wistar rats.

Following the obesity epidemics, nonalcoholic fatty liver disease (NAFLD) has grown in prevalence and become a main cause of morbidity and death, intimately linked to cardiovascular disease, cancer, and cirrhosis. The key factor in the evolution of NAFLD is thought to be oxidative stress. Because most patients cannot change their lifestyle or dietary habits, a pharmaceutical strategy is now required to treat NAFLD. Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis, is treated with vitamin E. (NASH). Vitamin E is also a powerful antioxidant that has been demonstrated to lower oxidative stress in people with NAFLD. Thymol is a monoterpene phenol with a variety of pharmacological effects, however its anti-fatty liver properties have yet to be investigated. Despite the fact that oxidative stress is thought to have a role in the etiology of nonalcoholic steatohepatitis, antioxidant therapies have not been well studied in the treatment of nonalcoholic steatohepatitis. The goal was to learn more about vitamin E and thymol's biological activities, with a particular emphasis on their therapeutic effectiveness in NAFLD. Four groups of thirty-two adult male rats were formed (healthy control, thymol, Vit E, and fatty liver). For 28 days, rats were given either oral vitamin E (200 mg/kg) or thymol (50 mg/kg) randomly. The levels of ALT, AST, TNF- α, Ferritin, CK-MB enzymes, and MAPK gene expression were then determined in the serum. Based on a random effect model analysis, at the end of 28 days of therapy, ALT (41.43 U/L), AST (47.91 U/L), Ferritin (1.13 pg/dl), CK-MB (251.22 IU/L), TNF-α (95.39 pg/mL) (p≤0.001), and MAPK gene expression levels (p≤0.05) significantly reduced in both experimental groups compared with the fatty liver group. Vitamin E and thymol therapy is a safe, affordable, and effective therapeutic option in the fatty liver group. Patients with fatty liver disease should be encouraged to take vitamin E and Thymol supplements, which are both safe and affordable, because more effective new therapeutic options are lacking.

Effects of ribavirin and hydroxyurea on oral infection of Aedes aegypti (L.) with dengue virus.

This study was conducted to determine the inhibitory effects of ribavirin and hydroxyurea on dengue virus replication in Aedes aegypti mosquitoes. Female Ae. aegypti mosquitoes were infected with dengue-2 virus and fed ribavirin at a dose of 0.3 mg/ml and/or hydroxyurea at a dose of 6 mg/ml via artificial membrane feeding technique. The virus in infected mosquitoes was isolated using C6/36 cell culture. Peroxidase-antiperoxidase (PAP) staining was used to detect dengue-infected C6/36 cells and to quantify the level of infection by determining the presence of infected cells. In mosquitoes treated with ribavirin alone, hydroxyurea alone or both drugs in combination had reductions in dengue infection rates of 87.72, 89.47 and 95.61%, respectively. The mortalities of female Ae. aegypti mosquitoes fed with these drugs were significantly higher than the control. Ribavirin also had an inhibitory effect on the fecundity of female Ae. aegypti mosquitoes.

Late onset group b beta-hemolytic streptococcus infection in a neonate manifesting as a urinary tract infection: a rare clinical presentation.

Group B beta-hemolytic streptococcus (GBS) sepsis is a serious bacterial infection in neonates, with significant morbidity and mortality. We report here a neonate with late onset GBS infection manifesting as a urinary tract infection (UTI) in an infant presenting with prolonged neonatal jaundice. The pathogenesis of this late onset is postulated.

Habitat characterization and mapping of Anopheles maculatus (Theobald) mosquito larvae in malaria endemic areas in Kuala Lipis, Pahang, Malaysia.

In Peninsular Malaysia, a large proportion of malaria cases occur in the central mountainous and forested parts of the country. As part of a study to assess remote sensing data as a tool for vector mapping, we conducted entomological surveys to determine the type of mosquitoes, their characteristics and the abundance of habitats of the vector Anopheles maculatus in malaria endemic areas in Pos Senderot. An. maculatus mosquitoes were collected from 49 breeding sites in Pos Senderot. An. maculatus preferred to breed in water pockets formed on the bank of rivers and waterfalls. The most common larval habitats were shallow pools 5.0-15.0 cm deep with clear water, mud substrate and plants or floatage. The mosquito also preferred open or partially shaded habitats. Breeding habitats were generally located at 100-400 m from the nearest human settlement. Changes in breeding characteristics were also observed. Instead of breeding in slow flowing streams, most larvae bred in small water pockets along the river margin.

Indoor and outdoor residual spraying of a novel formulation of deltamethrin K-Othrine® (Polyzone) for the control of simian malaria in Sabah, Malaysia.

Since 2000, human malaria cases in Malaysia were rapidly reduced with the use of insecticides in Indoor Residual Spray (IRS) and Long-Lasting Insecticide Net (LLIN). Unfortunately, monkey malaria in humans has shown an increase especially in Sabah and Sarawak. The insecticide currently used in IRS is deltamethrin K-Othrine® WG 250 wettable granule, targeting mosquitoes that rest and feed indoor. In Sabah, the primary vector for knowlesi malaria is An. balabacensis a species known to bite outdoor. This study evaluates an alternative method, the Outdoor Residual Spray (ORS) using a novel formulation of deltamethrin K-Othrine® (PolyZone) to examine it suitability to control knowlesi malaria vector in Sabah, compared to the current method. The study was performed at seven villages in Sabah having similar type of houses (wood, bamboo and concrete). Houses were sprayed with deltamethrin K-Othrine® (PolyZone) at two different dosages, 25 mg/m2 and 30 mg/m2 and deltamethrin K-Othrine® WG 250 wettable granule at 25 mg/m2, sprayed indoor and outdoor. Residual activity on different walls was assessed using standard cone bioassay techniques. For larval surveillances, potential breeding sites were surveyed. Larvae were collected and identified, pre and post spraying. Adult survey was done using Human Landing Catch (HLC) performed outdoor and indoor. Detection of malaria parasite in adults was conducted via microscopy and molecular methods. Deltamethrin K-Othrine® (PolyZone) showed higher efficacy when sprayed outdoor. The efficacy was found varied when sprayed on different types of wall surfaces. Deltamethrin K-Othrine® (PolyZone) at 25 mg/m2 was the most effective with regards to ability to high mortality and effective knock down (KD). The vector population was reduced significantly post-spraying and reduction in breeding sites as well. The number of simian malaria infected vector, human and simian malaria transmission were also greatly reduced.

Refractoriness of Aedes aegypti (Linnaeus) to dual infection with dengue and chikungunya virus.

In this study, artificial membrane feeding technique was used to orally feed Aedes aegypti with dengue and chikungunya viruses. Virus detection was carried out by reverse transcriptase polymerase chain reaction. The study did not detect dual infection of Ae. aegypti with dengue and chikungunya virus from the same pool or from individual mosquitoes. Oral receptivity of Ae. aegypti to chikungunya virus was higher than that of dengue virus.

The effect of extrinsic incubation temperature on development of dengue serotype 2 and 4 viruses in Aedes aegypti (L.).

Dengue 2 and 4 viruses obtained from dengue-infected patients were maintained in a C6/36 Aedes albopictus Skuse cell line and used to infect adult female Aedes aegypti mosquitoes. Each serotype was mixed separately with fresh human erythrocytes and fed to adult female mosquitoes using an artificial membrane feeding technique. Fully engorged mosquitoes were selected and retained at 26 degrees C, 28 degrees C and 30 degrees C to observe dengue virus development in Aedes vectors. Virus detection was carried out by reverse-transcriptase polymerase chain reaction (RT-PCR). The virus was first detected on Day 9 at 26 degrees C and 28 degrees C and on Day 5 at 30 degrees C for both dengue 2 and 4. The study shows the incubation period of the viruses decreased when the extrinsic incubation temperature increases.

Stress- and non-stress-mediated mechanisms are involved in pain-induced apoptosis in hippocampus and dorsal lumbar spinal cord in rats.

Chronic pain has been reported to induce apoptosis. Both chronic excitation of neural pathways involved in pain transmission and control and the stress of pain may be potentially involved in apoptosis induced by pain. Here, we have investigated their possible role in pain-induced apoptosis. Inflammatory pain was induced by injection of formalin in intact and adrenalectomized (ADX) rats. Following exposure to repeated injections of 5% formalin, we detected Bax, Bcl-2, pro-caspase and activated caspase-3 proteins using immunoblotting. The results were compared with those obtained from animals suffered from chronic immobilization stress (IMO). These results showed an increased ratio of Bax/Bcl-2 and activated caspase-3 in hippocampus and dorsal lumbar spinal cord of animals treated with pain and IMO stress; these effects were reduced in ADX animals. On the other hand, the remaining apoptotic effect of pain in adrenalectomized rats was also significant. We surmise that both chronic neural activation and the stress induced by pain are involved in pain-induced apoptosis.

Effects of single administration of morphine on G-protein mRNA level in the presence and absence of inflammation in the rat spinal cord.

Antinociceptive potency of opioids is greater against various noxious stimuli in animals with peripheral inflammation. Opioid agonists stimulate activation of G-protein-coupled receptor. Changes in the resting levels of G-protein subtypes could have an effect on intracellular signalling pathways. The present study was designed to investigate the effects of analgesic morphine treatment on the level G-protein subunits mRNA in the presence and absence of inflammation. Our results showed that the carrageenan administration increased G-protein subunits. Administration of analgesic dose of morphine alone and in the presence of inflammation induced different alterations in the levels of G-protein mRNA. Taken together, the results obtained using real time RT-PCR suggested that G-protein genes expression levels following the acute administration of morphine between animals with and without inflammation could influence, at least in part, analgesic responsiveness.

Persistency of transovarial dengue virus in Aedes aegypti (Linn.).

A study was conducted to examine the persistency of transovarial dengue virus type 2 (DEN-2) in a Selangor strain of Aedes aegypti mosquitoes. Two hundred 4-5 day old female mosquitoes were fed with blood containing dengue virus. The infected mosquitoes were reared to the 7th generation; each generation was screened for the virus using immunological staining methods. The virus was detectable until the 5th generation but absent in the 6th and the 7th generations. Therefore, dengue virus type 2 can be transmitted transovarially in Aedes aegypti mosquitoes until the fifth generation under laboratory conditions.

Calcium phosphate supplementation increases faecal Lactobacillus spp. in a randomised trial of young adults.

The aim of the studies was to determine the effects of calcium carbonate and calcium phosphate supplementation on faecal Lactobacillus spp., with and without a probiotic supplement, in healthy adults. Study 1 comprised of a randomised, double-blind, crossover design; participants (n=15) received 2 capsules/d of 250 mg elemental calcium as calcium carbonate (Ca1) and calcium phosphate (Ca2) each for 2-week periods, with 2-week baseline and washout periods. Study 2 was a randomised, double-blind, crossover design; participants (n=17) received 2 capsules/d of Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011 (probiotic) alone, the probiotic with 2 capsules/d of Ca1, and probiotic with 2 capsules/d of Ca2 each for 2-week periods with 2-week baseline and washout periods. In both studies, stools were collected during the baseline, intervention and washout periods for Lactobacillus spp. quantification and qPCR analyses. Participants completed daily questionnaires of stool frequency and compliance. In Study 1, neither calcium supplement influenced viable counts of resident Lactobacillus spp., genome equivalents of lactic acid bacteria or stool frequency. In Study 2, faecal Lactobacillus spp. counts were significantly enhanced from baseline when the probiotic was administered with Ca2 (4.83±0.30, 5.79±0.31) (P=0.02), but not with Ca1 (4.98±0.31) or with the probiotic alone (5.36±0.31, 5.55±0.29) (not significant). Detection of L. helveticus R0052 and L. rhamnosus R0011 was significantly increased with all treatments, but did not differ among treatments. There were no changes in weekly stool frequency. Calcium phosphate co-administration may increase gastrointestinal survival of orally-administered Lactobacillus spp.

Detection of giardine gene in local isolates of Giardia duodenalis by polymerase chain reaction (PCR).

Giardia duodenalis is an intestinal parasite that causes diarrhoea and malabsorption in children. The parasite also infects AIDS patients with a weak immune system. A study was carried out on six local isolates of Giardia duodenalis (110, 7304, 6304, M007, 2002 and 6307) from faeces of Orang Asli patients admitted to the Gombak Hospital. WB, a reference pathogenic strain from human and G. muris from a wild mouse, were commercially obtained from the American Type Culture Collection (ATCC). All the isolates were cultured axenically in TYI-S-33 medium. Two sets of primers were used for the techniques: primers LP1 and RP1 and primers LP2 and RP2. The sets of primers amplified giardine gene of 171 bp and 218 bp in sizes respectively. The study showed that the two sets of primers could detect G. duodenalis to the genus and species level specifically.

Microdroplet application of mosquitocidal Bacillus thuringiensis using ultra-low-volume generator for the control of mosquitos.

In an effort to develop a more effective technique in dispersing a microbial control agent, Bacillus thuringiensis (Bt), a truck-mounted ultra low volume (ULV) generator (Scorpion) was used to disperse B. thuringiensis israelensis (Bti) and Bti with malathion. Complete larval and adult mortalities for all tested mosquito species within the first 70-80 feet from the ULV generator were achieved. Beyond that distance less than 50% mortality was achieved as insufficient sprayed particles reached the area. A minimum of 10(3) Bti colony forming units per ml is required to cause 100% larval mortality. The sprayed Bti larvicidal toxins were persistent in the test water 7 days post ULV. The effectiveness of B. thuringiensis jegathesan (Btj), a new mosquitocidal Bt serotype was also evaluated. Similar mortality results as Bti were achieved except that the Btj toxins underwent degradation in the test water, since less than 50% less in larval mortality was observed in 7 days post ULV samples. This ULV method has the potential to disperse Bt and malathion effectively for a simultaneous control of mosquito adults and larvae.

Health status of Orang Asli (aborigine) community in Pos Piah, Sungai Siput, Perak, Malaysia.

A study of health status of Orang Asli population (based on physical examination findings) was conducted in 4 villages in Pos Piah, Sungai Siput Perak, Malaysia. In all 356 individuals between 4 months-72 years old (178 males and 178 females) participated in this study. Poor general health status, physical and mental handicaps were seen in 7.8%, 0.3% and 0.3% of the population, respectively. About one-fifth of the population had dental caries. Splenomegaly, hepatomegaly and hepatosplenomegaly were among the commonest abnormalities with the occurrence rates of 19.8%, 13.7% and 6.7%, respectively, being detected in the population. About one-fifth of the population showed signs suggestive of protein-energy deficiency; whilst less than 5% showed signs indicative of riboflavin, iodine and iron deficiencies. Vitamin A deficiency was the commonest nutritional deficiency identified in this community with almost 38.4% of them showing signs of the deficiency. The commonest skin infection was scabies.

Clinical features of malaria in Orang Asli population in Pos Piah, Malaysia.

A malaria survey was conducted to examine the presence of common clinical features of malaria in individuals living in an endemic area of malaria. The overall infection rate was 11.0% with 7.5% and 3.5% infected with Plasmodium vivax and Plasmodium falciparum respectively. The mean parasitaemia level of both species was 2905.9 parasites/microliter blood, with the mean parasitaemia level of P. vivax and P. falciparum at 682.7 parasites/microliter blood and 6981.7 parasites/microliter blood respectively. The infection rates were higher in the younger age group. Hepatomegaly, hepatosplenomegaly and clinical anaemia were significantly associated with malaria. None of the patients were febrile. In conclusion, in low endemic areas, the presence of clinical anaemia, hepatomegaly and hepatosplenomegaly in afebrile individuals could be considered as useful criteria for the presence of asymptomatic parasitaemia. It is important to carry out laboratory diagnostic investigations, to ensure all the asymptomatic parasitaemia which act as reservoirs are detected and treated.

Current insecticide susceptibility status of Malaysian Anopheles maculatus Theobald to malathion, permethrin, DDT and deltamethrin.

Chemical insecticides are still considered as important control agents for malaria vector control. However, prolonged use of these chemicals may select mosquito vectors for resistance. In this study, susceptibility status of adult Anopheles maculatus collected from 9 localities in peninsular Malaysia, viz., Jeli, Temerloh, Pos Banun, Senderut, Jeram Kedah, Segamat, Kota Tinggi, Kluang and Pos Lenjang were determined using the standard WHO bioassay method in which the adult mosquitoes were exposed to standard insecticide impregnated papers malathion, permethrin, DDT and deltamethrin--at pre-determined diagnostic dosage. Deltamethrin was most effective insecticide among the four insecticides tested, with the LT50 of 29.53 min, compared to malathion (31.67 min), DDT (47.76 min) and permethrin (48.01 min). The effect of all insecticides on the laboratory strain was greater (with all insecticides demonstrated LT50 < 1 hour) than the field strains (deltamethrin 32.7, malathion 53.0, permethrin 62.0, DDT 67.4 min). An. maculatus exhibited low degree of resistance to all test insecticides, indicating that these chemical insecticides are still effective in the control of malaria vector.

Aedes larval population dynamics and risk for dengue epidemics in Malaysia.

Early detection of a dengue outbreak is an important first step towards implementing effective dengue interventions resulting in reduced mortality and morbidity. A dengue mathematical model would be useful for the prediction of an outbreak and evaluation of control measures. However, such a model must be carefully parameterized and validated with epidemiological, ecological and entomological data. A field study was conducted to collect and analyse various parameters to model dengue transmission and outbreak. Dengue prone areas in Kuala Lumpur, Pahang, Kedah and Johor were chosen for this study. Ovitraps were placed outdoor and used to determine the effects of meteorological parameters on vector breeding. Vector population in each area was monitored weekly for 87 weeks. Weather stations, consisting of a temperature and relative humidity data logger and an automated rain gauge, were installed at key locations in each study site. Correlation and Autoregressive Distributed Lag (ADL) model were used to study the relationship among the variables. Previous week rainfall plays a significant role in increasing the mosquito population, followed by maximum humidity and temperature. The secondary data of rainfall, temperature and humidity provided by the meteorological department showed an insignificant relationship with the mosquito population compared to the primary data recorded by the researchers. A well fit model was obtained for each locality to be used as a predictive model to foretell possible outbreak.

Effectiveness of aminophylline prophylaxis of renal impairment after coronary angiography in patients with chronic renal insufficiency.

This study was done to investigate whether aminophylline reduces the incidence of contrast induced nephropathy (CIN) after coronary angioplasty. Sixty patients who had serum creatinine concentration of > 1.3 mg/dl randomly received 250 mg IV aminophylline or placebo 30 minutes before coronary angiography. Serum creatinine and blood urea nitrogen were determined immediately before (base line) and at 24 and 48 hours after administration of contrast medium. The primary end point was the incidence of CIN. The incidence of CIN was 20% in placebo group and 13.3% in aminophylline group; older age was significantly associated with CIN: In this study, we could not demonstrate the prophylactic effect of a single infusion of 250 mg aminophylline, 30 minutes before administration of contrast media. A larger trial that incorporates the evaluation of clinically relevant outcomes is required to more adequately assess the role for aminophylline in CIN prevention.