Association between house dust mite (HDM) sensitisation and asthma control using skin prick test and HDM antigen specific IgE levels in saliva of Malaysian children

Background: Sensitisation to house dust mite (HDM) has been regarded as a major risk factor for development of asthma. This study was carried out to investigate the profiles of HDM sensitisation among Malaysian children with asthma. Material and Methods: The association between HDM sensitisation and control and severity of asthma was investigated. The salivary HDM specific IgE levels were quantified in different grades of control and severity of asthma in 125 unselected asthmatic children aged 5-12 years old attending the asthma follow-up clinic in Hospital Tuanku Ja’afar Seremban. An additional 29 non-asthmatic patients were selected as control. The skin prick test to assess sensitisation to Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) was performed on all the participants. A questionnaire regarding the control and severity of asthmatic symptoms of the subject was administered. Saliva was collected by voluntary spitting and ELISA was used to quantify the IgE specific to HDM antigen. Results: There was a significant association between sensitisation to DP and DF and the control of asthma. The association between DP sensitisation and severity of asthma just failed to reach a significant level although there is a clear trend for this. Significant association was found between DF sensitisation and severity. The HDM specific IgE in the saliva was significantly higher in asthmatic patients compared to non-asthmatic patients. There was no significant difference between the specific IgE levels in patients with different severity status of asthma. Conclusion: Salivary IgE levels may not be an appropriate indicator of the patients’ asthmatic condition in this study. However, it can be concluded that there is significant association between the sensitisation of HDM and the control and severity of asthma.

An intestinal parasitological survey among the Jehai Orang Aslis (aborigines) of the Temenggor forest, Perak state, Malaysia

Background: In Malaysia, the most common soiltransmitted helminth infections are A. lumbricoides, T. trichiura and hookworms. However, as there have been no extensive surveys on these infections, it is difficult to estimate with certainty the current overall incidence of infection with soil-transmitted helminths (STHs) among the Malaysian population including the Orang Aslis. Materials and Methods: A study was conducted to determine the infection rate of soil-transmitted helminths and intestinal protozoa among the Jehai Orang Aslis (Aborigines). The study was conducted between December 2005 and August 2006, in four Jehai villages of Perak State, Malaysia. A total of 175 stool samples was collected and personal identification such as name, age, household identification, and date of collection were recorded on the spot during collection. Faecal smears were stained with Trichrome for protozoa cysts and trophozoites and the modified Ziehl-Neelsen acid-fast method for the oocyst of Cryptosporidium and Isospora. Wet mounts with tincture of iodine of both stool samples (10% formalin and PVA) were also examined to detect cysts, ova and larva of intestinal helminths. Results: The prevalence rates of Trichuris trichiura, Ascaris lumbricoides and hookworm among the Jehai were 70.8%, 24.0%, and 10.9% respectively. The prevalence of Entamoeba coli, Entamoeba histolytica, Giardia intestinalis, Blastocystis hominis, and microsporidium was 40.6%, 33.7%, 25.7%, 91.4%, and 27.4% respectively. The difference in prevalence rates among the different age-groups and sex were found not significant. Children aged 0-9 years old had the highest prevalence rate of intestinal parasites and only 2 (1.1%) were free of any intestinal parasites. Conclusion: Intestinal parasitic infections were therefore still common among these people. Children aged 0-9 years old were found to have the highest infection rate of all the intestinal parasites examined. Further investigations are needed to determine more specific transmission of these infections, so that an attempt to control these infections can be made.

Phylogenetic analysis and identification of Sarcocystis spp. found in rodents in Peninsular Malaysia

Background: The tissue specimens used for extraction of DNA in this study were from rodents trapped in four states in Peninsular Malaysia, namely Kedah, Kelantan, Selangor and Johor. Methods: Histological sections of these rodent muscle tissues stained with hematoxylin and eosin showed infection with Sarcocystis spp. Based on these results, the current study was carried out to determine the phylogenetic relationship among the identified Sarcocystis spp. in these rodents. The formalin fixed paraffin embedded (FFPE) rodent muscle blocks were subjected to DNA extraction and followed with semi nested PCR targeting 5’ and 3’ regions of 18S rRNA of Sarcocystis spp. Results: Phylogenetic analysis showed two distinct groups of Sarcocystis spp. among the rodents in Peninsular Malaysia. Most of the identified Sarcocystis spp. were genetically closely related to Sarcocystis rodentifelis and Sarcocystis muris and were also observed to be genetically closely related to Sarcocystis sp. ex Columba livia and Sarcocystis sp. cyst type I ex Anser albifrons. Conclusion: Further classification to confirm these Sarcocystis spp. was not possible as only partial sequences of 18S rRNA was available and this was insufficient for optimal differentiation.

Conducting research that is both ethical and responsive to the health needs of a developing country

There is no substantial difference in conducting research that is both ethical and responsive to the health needs in developing and developed nations. Differences are in financial constraints, technological expertise in identification and addressing needs, and in the perception of equal partnership of all stakeholders. There will be differences in emphasis of research but this is slowly blurred due to globalisation. Public health emergencies in developing countries need timely and effective global collaborative research to implement control strategies. Research needs should be based on predictive models with learning from past emergencies, technological advances, strategic critical appraisal of local and global health information, and dialogue with all stakeholders. Adequate funding will be challenging and resources from national, international and aid foundations will be needed. Issues associated with such funding include deployment of international rapid response teams, collaborating researchers, transfer of technology, and intellectual property ownership. While all types of research ranging from basic, applied, clinical studies, meta-analysis, and translational research are relevant, the relative importance and specific allocation of resources to these may differ. Is the choice related to responsiveness or based on researchers’ perception of their contributions to evidence-based practice and research? Ethical issues relating to vulnerable groups, risk distribution, quality issues, research integrity and oversight are just as important. Internationally funded research including clinical trials must be sensitive to such issues to avoid allegations of exploitation. Thus the potential of utilisation and buy-in of research findings and recommendations must be considered.

Effect of Traditional Chinese Medicine Complex for diabetes (TCM-D™) on experimentally induced diabetic mice

Background: We previously evaluated the biochemical changes induced by the local product TCM for diabetes (TCM-D™) on blood glucose levels and other biochemical changes in normal mice fed orally with the recommended human dose (30 ml/kg daily) and ten times this dose for eight weeks. TCM-D™ is an aqueous extract of the roots of Trichosanthes kirilowii Maxim, Paeonia lactiflora Pall, Glycyrrhiza uranlensis Fisch. and Panax ginseng Meyer (red) combined at the dry weight proportions of 36%, 28%, 18% and 18% respectively. The study showed that at these dosages the blood glucose levels as well as the body weights in treated mice were significantly reduced when compared with pretreatment values and control animals. The present study evaluated the effect of the extract in a mouse model of Type 1 diabetes mellitus. Methods: TCM-D™ extract was prepared as a 10x concentrate and given orally at 0.3 ml/100 g and 1.5 ml/100 g to mice which were experimentally induced diabetic with intraperitoneal injections of streptozotocin (5 mg/100g) in sodium citrate (pH 4.5). Control diabetic mice were dosed with extract diluent (distilled water). Results: At the doses studied the compound did not show any significant lowering of the glucose levels in a mouse model of Type 1 diabetes. There were significant increases in the alanine aminotransferase (ALT) and creatinine levels which were most likely due to the treatment with the compound. There were no significant changes in the aspartate aminotransferase (AST) and blood urea levels due to the treatment. Neither was there any significant effect on the weight of the treated animals due to the treatment. Conclusions: It is concluded that TCM-D™ did not have any significant blood glucose lowering effect on streptozotocin induced diabetic mice when fed orally at 1-5 times the recommended human dose. Further work is needed to determine if the extract has any significant effect in a mouse model with Type 2 diabetes mellitus.

Blood glucose level and other biochemical changes induced in normal mice by oral Traditional Chinese Medicine complex for diabetes (TCM-D™)

Background: A number of Traditional Chinese Medicine (TCM) preparations are being used for the treatment of diabetes mellitus. Some components of these preparations have biochemical effects other than those of lowering blood glucose and indeed have been used for other medical indications in traditional practice. The primary objective of the study was to determine the effect of the oral mixture of Traditional Chinese Medicine for diabetes (TCM-D™ complex) on blood glucose level and the biochemical changes if any, on the liver (ALT, AST, gamma-GT, albumin, globulin) and renal (blood creatinine, urea) functions in normal mice. The oral mixture is an aqueous extract of four wellknown traditional Chinese medicinal herbs and consists of Trichosanthes kirilowii Maxim., Paeonia lactiflora Pall.,Glycyrrhiza uranlensis Fisch., and Panax ginseng (red) CA Meyer in the proportion of 36%, 28%, 18%, and 18% respectively of the dry weight. These herbs have been shown to have blood glucose lowering activity and have been used for other traditional medicinal purposes. The safety of the combination was evaluated in the present study. Methods: Experimental Balb/c mice were treated orally via gastric tube with the extract at daily doses equivalent to 1 and 10 times the recommended human dose for 8 weeks. Blood glucose and other biochemical profiles were monitored at pre-treatment and monthly posttreatment until killed. Results: When compared to pre-treatment levels, the blood glucose levels were significantly lower in treated animals compared to those in the control group. At the recommended TCM-D™ dose the levels in treated animals were significantly lower than that of control animals and at pre-treatment. When compared with pre-treatment, the glucose levels were lowest at Week 8 of treatment, the mean levels being 111.23%, 83.32% and 70.33% in control, and in animals given 1 x and 10 x the recommended TCM-D™ dosage respectively. The blood glucose lowering effect was also associated with a significant weight loss in treated animals. There were transient increases in AST and ALT levels but these reverted to normal at Week 8 of treatment. The levels of bilirubin, g-GT, albumin, creatinine and blood urea were also not significantly different at Week 8 from pre-treatment levels in all groups. Conclusion: Even at 10 times the dosage recommended for humans, TCM-D™ did not affect the liver and renal functions of treated animals. Treated and control animals remained healthy and normal throughout the period of observation.

Pathology of lymphatic filariasis

Developing and adult worms of the human lymphatic filarial parasites (Wuchereria bancrofti, Brugia malayi, and Brugia timori) are located mainly in the lymphatic system and occasionally in aberrant sites like subcutaneous and conjunctival cysts. Lymphatic pathology ranging from dilatation of lymphatic channels and lymphangiectasia are detected on ultrasonography in apparently healthy, amicrofilaraemic, but filarial antigen positive individuals in endemic areas. Microfilariae are distributed in various organs and may be associated with immune mediated pathology at these sites; tropical pulmonary eosinophilia is characterized by intense immune mediated destruction of microfilariae in the lung parenchyma. In the spleen and other sites, nodular granulomatous lesions can occur where microfilariae are trapped and destroyed. The finding of Wolbachia endosymbionts in all stages of lymphatic filarial parasites has provided new insight on the adverse reactions associated with anti-filarial chemotherapy. Inflammatory molecules mainly lipopolysaccharide (LPS)-like molecules released from endosymbionts on death of the parasites are largely responsible for the adverse reactions encountered during anti-filarial chemotherapy. Prenatal tolerance or sensitization to parasite derived molecules can immune-modulate and contribute to both pathology and susceptibility/resistance to infection. Pathological responses thus depend not only on exposure to filarial antigens/infection, but also on host-parasiteendosymbiont factors and to intervention with antifilarial treatment. Treatment induced or host mediated death of parasites are associated with various grades of inflammatory response, in which eosinophils and LPS from endosymbionts play prominent roles, leading to death of the parasite, granulomatous formation, organization and fibrosis. The non-human primate (Presbytis spp.) model of Brugia malayi developed for the tertiary screening of anti-filarial compounds has provided unique opportunities for the longitudinal study of the pathology associated with lymphatic filariasis. The pathology in this non-human primate model closely follows that seen in human lymphatic filarial infections and correlates with clinical evidence of lymphatic pathology as detected with ultrasonography. These studies also show that successful treatment as detected by loss of motility and calcification of worms on ultrasonography is associated with reversal of early dilatations of lymphatic channels.

Mentorship in the research setting

Abstract: The research mentorship programme is unique in that it is a planned journey undertaken by the mentor and mentee, preferably with well-defined milestones along the journey. During the journey, familiar landmarks will be pointed out by the mentor. In path-finding situations the experience and wisdom of the mentor and the critical appraisal of both mentor and mentee will contribute to learning from the encounter. In most mentor-mentee partnerships, a formal acceptance to the relationship, well-defined landmarks measuring progress in the journey, regular appraisal of the skills developed and acquired, and phased, judicious modification in the individual roles of that relationship will be required. Although there is no consensus on the elements of mentorship, there are some strategies which can contribute to the success of the relationship. Critical success factors include convergence of the research area within the broad expertise of the research mentor. The research mentor should have a proven research track record and is committed to serve in that official capacity. The research mentoring process is dynamic and characteristics of both mentor and mentee contribute to the robustness of that relationship. The mentee would have identified some attributes of the mentor that are desirable and is willing to work hard to achieve, build on, and improve upon. In the research setting endpoint measurements of success will be based on recognition of the research standing of the mentee, measurable outcomes such as number of papers in top tier journals, citation indices, etc. consultancies attracted as well as invitations to deliver plenaries in scientific conferences, patents filed and research findings translated and applied, and other measures of research productivity. In the pursuit of research excellence the mentee would have imbibed values of professionalism and ethics in research and would have constantly kept in mind that to be successful, the mentee would be able to excel beyond his mentor and that the next generation of researchers will seek mentorship from him.

Prevalence of Sarcocystis spp. in rodents in Peninsular Malaysia

The aim of the study was to assess the prevalence of Sarcocystis infection in wild and peri-urban rodents in some states in Peninsular Malaysia. The thigh muscle from these rodents were formalin preserved, sectioned and stained with hematoxylin and eosin and examined under light microscopy. Of the 146 muscle tissue examined only 73 were positive for Sarcocystis infection. Morphological identification showed the presence of some new morphological types to be present. Different species of Sarcocystis were seen in the sections but more extensive studies are needed to identify them to species level.

Detection of glycoproteins from human erythrocytes of different ABO blood groups infected with Plasmodium falciparum

Background: Many proteins released by cells to the blood and other fluids are glycoproteins. One set of glycoproteins carry the ABO blood group determinants and glycoproteins have been shown to be vital in determining the structure and organization of plasma membranes. There is evidence suggesting their important role in cell-to-cell contact, adhesion, hormone interaction and vital transformation. Differences in proteins and glycoproteins in the different human blood groups may influence the invasion process of Plasmodium falciparum. The objectives of the study were to determine whether there are any changes in proteins and glycoproteins of red blood cells upon infection by P. falciparum and whether these protein and glycoprotein changes differ in the various ABO blood groups. Methods: A Malaysian strain of P. falciparum was cultured in vitro in red blood cells from A, B, O and AB blood groups. Protein and glycoprotein profiles of uninfected and P. falciparum- infected red blood cells from the different human ABO blood groups were analyzed by SDS-PAGE. For protein bands, the gels were stained with Coomassie blue while glycoproteins were visualized following staining of gels using GelCode® Glycoprotein Staining Kit. Results: Cell membranes of P. falciparum infected erythrocytes from different ABO blood groups have different glycoprotein profiles compared to uninfected cells. All the infected samples showed a prominent protein band of molecular weight 99 kDa which was not present in any of the uninfected samples while a 48 kDa band was seen in four out of the seven infected samples. The erythrocyte cell membranes of A and AB blood groups showed different glycoprotein profiles upon infection with P. falciparum when compared to those from blood groups B and O. Conclusion: The two glycoproteins of molecular weights 99 kDa and 48 kDa should be further studied to determine their roles in the pathogenesis of malaria and as potential targets for drug and vaccine development

Research Ethics, Governance, Oversight And Public Interest

A better educated public has started to challenge the way decisions are made in medical research activities. Although Institutional and National Guidelines on Research are in place, there are fears that Institutional Review Boards (IRBs) and funding agencies are only fairly active in scientific and ethical reviews of research proposals but not on oversight of projects after their initiation. These issues are integral to good research governance and researchers and custodians of research ethics must ensure that public interest is not compromised. Medical progress is based on research including human experimentation carried out according to guiding principles as enunciated in the Declaration of Helsinki (2000), but the quality of compliance with the Declaration is an important issue. Better choice and appropriate training of members of IRBs to improve the quality of decision making and governance processes are urgently needed. Competency in evaluation of proposals requires not only the appropriate scientific knowledge but also access to relevant preclinical and other data. Unfortunately, the completeness and quality of such data may not be adequate. Public interest demands that injury to trial subjects in clinical trials is minimized if not avoided completely. Unfortunately this is not always possible with trials where novel biological modes of action are tested. A more robust evaluation mechanism for project approval may minimize but not completely avoid injury to subjects; thus insurance cover to provide care and compensation to subjects must be compulsory. The decision to approve or reject a project must be based on the balance of potential risks and benefits, taking into consideration justifiable distributive risks to target communities and populations. Economic considerations should never be the primary focus, especially when there are real concerns that the migration of early phase clinical trials including vaccine trials to developing countries is based on the perceived less stringent ethical requirements and oversight there.

Environmental Health And Building Related Illnesses

Malaysia has good environmental laws to protect the outdoor environment and public health. However there are no laws governing indoor air quality (IAQ) and the knowledge among the public about its importance is also lacking. Environmental professionals think it is not a priority and this influences the policy decisions in the country. Therefore there is a need to create awareness by way of research, education and other promotional activities. What is much needed at this time is the establishment of standards for the conduct of risk assessment studies. To establish standards we need reliable data which can be used to develop appropriate guidelines for the purpose of mitigation and adaptation programmes. IAQ can have significant influence on health resulting in drop in productivity and economy of a country. It has been estimated that in the US, building related illnesses (BRI) symptoms have a relationship with decrease (3 to 5%) in work performance in an affected population resulting in an annual loss of US$60 billion in revenue. However, based on efficient management programmes they have also projected that the potential annual savings can be in the region of US$10 to 30 billion. This establishes that fact that good management programmes based on efficient guidelines is of economic value to a country and wellbeing of the population. The IMU has embarked on a research programme to collect the much-needed data for the framing of a good IAQ guideline for Malaysia.

Pharmacogenomics In Drug Therapy And Interaction: The Role Of Cytochrome P450

Pharmacogenomics (or pharmacogenetics), the study of the effects of genetic differences on a person’s response to drugs, can help in optimizing drug efficacy and minimizing adverse drug reactions. Interperson difference in drug metabolism is one of the important consequences of such genetic variation. This variation is determined in part by mutations in cytochrome P450 enzymes (CYPs). IMU is part of a major collaborative research project in the area of phamacogenetics and drug metabolism. Working together with USM and UiTM, our group has, since 2000, generated useful population database on genetic polymorphism of various CYP isoforms. We have successfully genotyped three major ethnic groups, Malay, Indian and Chinese for their allelic frequency of important isoforms. These include CYP2D6, CYP2C9, CYP2C8 and CYP2A6. Data generated so far collectively have contributed to our effort in mapping and constructing genomic database for Malaysian population. Since early 2002, our research has been focusing on developing in vitro methods in studying the functional consequences of genetic polymorphism of CYP enzymes. Using site-directed mutagenesis, CYP mutants, carrying nucleotide changes as reported in known alleles in human populations, were generated and expressed in E. coli system, and the expressed recombinant proteins were characterized using enzyme assays to determine the functional consequences of mutations. We have established a series of HPLC (high performance liquid chromatography)-based and fluorescence-based assays to investigate CYP activities. Assays that have been developed include tolbutamide methylhydroxylase, paclitaxel 6a-hydroxylase, dextromethorphan O-demethylation, testosterone 6b-hydroxylation and coumarin 7-hydroxylase assays. These assays serve as activity markers allowing comparison of catalytic activities of mutant proteins generated. Another focus of our work is to use the developed assays as a screening tool to investigate drug-herb interactions. This was achieved by co-incubation of herbal extracts and active constituents with the probe substrates in the assays followed by characterization of the kinetic behaviors of the enzymes involved using various pharmacokinetic parameters such as Km, Vmax, IC50 and Ki. This work is currently carried out with collaboration from the Institute for Medical Research (IMR) and is supported by MOSTI’s eScienceFund under RM9. It is envisaged that this screening work will give us insights on the potential of the commonly used herbs to cause pharmacokinetic interactions with other drug substrates, and allow us to elucidate the mechanisms involved in the interactions.

Seroprevalence of Malassezia furfur in an urban population in Malaysia

An in-house prepared M. furfur antigen was used to carry out a seroprevalence study in an urban population in Malaysia by indirect immunofluorescence assay. Of the 800 serum samples from all ages screened, 738 samples were positive for M. furfur specific IgG, giving an overall seropositive rate of 92.3%. There was no significant difference in the seropositive rates among the different gender group and races. However, there was a statistical significant difference in the seropositive rate among different age groups with a lower rate (73%) for the age group 5 years old and below, which increased rapidly to 99% for the 16 to 20 years old age group but declined slightly for the oldest age group. The degree of seropositivity, which semi-quantitatively reflect the anti-M. furfur specific IgG titre, did not show any significant difference among the gender and racial groups. On the other hand, there was a significant difference in the degree of seropositivity among the various age groups, with the 16 to 20 years old age group having the highest antibody titre and the extreme of age groups having the lower antibody titre.