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Editorial

Invited ReviewAllergy to House Dust Mites and AsthmaA Wan Omar, I Patimah & B Rusliza

Original PapersObesity and Associated Health Related Factors Among University Staffs in Serdang, MalaysiaL Rampal, P Saeedi, S Aminizadeh Bezenjani, MS Salmiah & O Norlijah

Toxicity Evaluation of Methanol Extract of Clitoria ternatea L. LeafL Kamilla, S Ramanathan, S Sasidharan & SM Mansor

Work-Related Hand Injuries: Type, Location, Cause, Mechanism and Severity in a Tertiary Hospital A Al-Husuny, L Rampal, A Manohar, MY Adon & AA Ahmad

The Effectiveness of Gentamicin against Acanthamoeba Cysts in VitroSA Noradilah, AG Mohamed Kamel, N Anisah, AR Noraina & S Yusof

Detection of Human Herpesvirus 6 (HHV-6) in Saliva of Healthy Adults in MalaysiaHL Choo, Y Shoji & CO Leong

Case ReportTrisomy X and Myelodysplastic Syndrome (MDS) with EosinophiliaRMT Eusni, CF Leong & S Salwati

Partial Epiglottic Edema Post Fish Bone IngestionM Irfan, AY Ali & Y Rohaizan

Lightning Strike Fatalities: Three Case Reports of Military Personnel in MalaysiaA Rozali, H Khairuddin, MS Sherina, LM Chia & K Yaakop

Acknowledgement

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Malaysian Journal of Medicine and Health Sciences Vol. 8 (2) June 2012


EDITORIAL

Obesity; Simple Diagnosis, Difficult Treatment

MZ AzharDept of Psychiatry, Faculty of Medicine & Health Sciences,

Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

In this issue of the journal there is an interesting and important topic of research on obesity. Obesity is fast becoming an important major health issue in Malaysia. Some factors that result in obesity among Malaysians especially children are suggested. Besides physical effects that are well know, the possible psychological effects are also suggested and a look at possible treatments for Malaysians is suggested.

Keywords: Obesity, Malaysian, depression, anxiety

*Corresponding Author: [email protected]

Malaysian Journal of Medicine and Health Sciences Vol. 8 (1) January 2012: 1-3

INTRODUCTIONObesity is often termed as having an overly high body mass index (BMI). When a person’s weight is extremely more than its ideal weight, we call it obesity. As Malaysia is rich in a variety of food, it has been reported that one in every ten children has the symptoms of obesity. There are many factors that result in obesity such as the role of the media, choices of food and parenting style. Fortunately, there are also several solutions to overcome these factors.

COMMON CAUSES AS SEEN BY LAY PERSONFirstly, children are supposedly active individuals. Television will catch their attention. So instead of playing outdoors, they would rather watch television programmes. The same happens to adults after work or on Sundays. Due to lack of exercise, the fat in the body builds up gradually to cause one to become obese.

Secondly, when it comes to the choice of food, “fast food” ranks top in children’s and Malaysian adults food choices. Fast food is high in sugar and carbohydrate. When children consume too much fast food, eventually the excess glucose will be converted into fat.

Thirdly, is the parenting style. Most parents are overworked and when they come home, the tired adults are too tired to prepare a meal. Most often they will just call for fast food delivery and it will be delivered to their door step. In addition, parents do not bother much about what their children consume. This gives freedom to the children to eat whatever they want which includes junk food, a contributor to obesity.

Fourthly, the actual causes of obesity are numerous. Time spent watching television, poor choices of food and parenting style are some of the causes of obesity among children. There are also genetic, environmental, biological and other social factors.

EFFECTS OF OBESITYThere are many well known physical effects of obesity that need not be elaborated. Most non-communicable diseases are associated with obesity. As such it is imperative to note that obesity does have mental and psychological effects.

Being overweight or underweight affects emotional health in several different ways. In fact, the mental health effects of obesity can be as damaging as the physical effects, according to recent obesity research.

The emotional and mental health issues caused by being overweight include social discrimination, low self-esteem, and even thoughts of ending life. However, research shows that being underweight can have even worse mental health effects. Here’s a summary of the emotional, mental, and social effects of being overweight and underweight;

Obese People Are More Prone to Depression and Anxiety

Obese women and men are less physically active because it’s not only more difficult to move around, it’s embarrassing to change in the locker rooms at the gym. Many obese people don’t feel comfortable in fitness classes because of their size and shape. This lack of physical activity can cause depression and anxiety (while physical activity reduces feelings of depression and anxiety).


MZ Azhar2

Overweight People Are Seen as Lacking in Willpower

Social discrimination includes the way society views you based on your appearance. The mental health effects of obesity include social discrimination – people often judge and mistreat individuals who are overweight. Obese people are seen as lazy and lacking in willpower, or incapable of looking after themselves properly. They’re socially undesirable, which increases their chances of anxiety and depression.

Overweight Students Have Poor College/University Attendance

Obesity research from the USA reveals that obese young women are half as likely to attend college as slim girls. The study which tracked nearly 11,000 American adolescents, found that young men did not differ from their non-obese peers in college attendance. Obese young women are also more likely to use drugs and alcohol than their slimmer peers.

The finding concludes: “those girls are far more vulnerable to the non-health risks of obesity reinforce the notion that body image is more important to girls’ self-concept and that social norms have greater effects on the education of girls than boys.”

The emotional health effects of obesity in girls are stronger than boys because girls are more tuned in to their appearance. Body image affects not only their self-esteem, but their levels of achievement as well.

Underweight People Are More Likely to be Depressed

A study from the Centre for Mental Health Research at the Australian National University studied the mental health effects of being overweight and underweight, and found that obese people struggle with depression and anxiety. Surprisingly, underweight people were more likely to deal with mental health problems.

The study suggests: “Underweight people also have the advantage in that they have less physical disability and physical ill-health than obese people, and that masks the underlying tendency to anxiety and depression, but when we extract out the physical ill-health component, we’re left with this picture that it’s the underweight that have the worst mental health.”

Whether you’re overweight or underweight, taking care of your emotional and mental health is one of the best things you can do for your body and life!

SUGGESTED TREATMENTFor the children mentioned above, parents need to limit the time spent on watching television. By limiting the time spent on television, children are more likely to be more active playing outside the home. Playing in the park enables them to move their limbs and fight the accumulation of fat. As children are easily tempted by eating fast food, parents need to be stricter with the choices of food their children consume. A balanced diet is needed for children’s growth. Instead of fast food, more vegetables and proteins are recommended. As vegetables have antioxidants which boost the body’s immune system and protein increases brain power, a healthier choice of food will produce healthier children. Busy parents should sacrifice some time preparing home cooked meals.

BEHAVIOUR MODIFICATION TECHNIQUESTreatment for obesity will be most successful if you create a long-term plan with your doctor. A reasonable goal might be to begin making lifestyle changes by increasing physical activity and eating healthy foods. Your initial goal should be to improve your health, not to achieve an ideal weight.

Guidelines suggest a goal of losing 10% of your body weight in 6 months. Doctors often recommend that people make lifestyle changes for at least 6 months before trying medicines or surgery. Counseling will also help if you use food to cope with depression, loneliness, anxiety, or boredom; you need to learn new skills to deal with those feelings.

Cut calories

Eating fewer calories while increasing activity is the best way to lose weight. For most adults, eating 1,200 to 1,500 calories a day for women and 1,500 to 1,800 calories a day for men is recommended for weight loss.

People often convince themselves that they don’t overeat. Keeping a food journal can help you find out how many calories you consume in a day. Then you can set a goal to cut out 500 to 1,000 calories a day. This will help you lose 1 to 2 pounds a week.

Limiting your calories to very low levels might seem like the way to quick weight loss, but it can have serious negative effects on your body and your ability to keep the weight off.

Research shows that limiting calories-not the types of foods you eat-causes more weight loss over the long term.


Obesity; Simple Diagnosis, Difficult Treatment 3

For example, cutting only carbohydrate or fat will not cause any more weight loss than a healthful and balanced low-calorie diet.

Eat healthier foods-don’t diet

Rather than focusing on a particular type of diet, try to eat healthier foods. Don’t try to restrict the foods you love. Eat less of them. Eat smaller portions.

Take a look at the dietary guidelines for good health.

Increase activity

Physical activity helps you burn more calories. Overall, experts recommend doing moderate or vigorous activity to get and stay healthy.

One of the best ways to increase your activity is by walking.Keep track of your steps with a step counter or pedometer. If you have a desk job, you may be surprised to see how

little you move in a typical day. Start with a goal of increasing your steps by 2,000 steps a day and work up to 10,000 to 12,000.

CONCLUSIONBeing obese is bad but is not the end of the world. There have been many research and techniques used to reduce weight. The key words in getting improvement from this unhealthy lifestyle illness are insight and motivation. The first is the understanding and awareness that one is having the problem and the second is the motivation to follow the strict behavior modification techniques to change to a healthier lifestyle. The change is not just with food but overall lifestyle changes. That seems to be the most difficult hurdle. It is not just the motivation of starting change but the also the motivation for maintaining that change.


Allergy to House Dust Mites and Asthma

1A Wan Omar*, 2I Patimah & 3B Rusliza1Department of Microbiology and Parasitology; Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia2Department of Biomedical Sciences; Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia3Department of Human Anatomy, Faculty of Medicine & Health Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

ABSTRACTHouse Dust mites are the most frequently encountered aeroallergens. Their role in sensitizing asthmatic patients was first reported in 1921, and the association of house dust mite allergy, asthma, and perennial rhinitis has been repeatedly corroborated. Asthma is a chronic respiratory disease that affects millions of people worldwide, and numerous scientific studies have shown that the prevalence of asthma is increasing. The most common dust mite species around the world include Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df), Euroglyphus maynei (Em) and Blomia tropicalis (Bt). Over the past three decades, many important allergens from these species have been identified and characterized at the molecular level. The biological function of several house dust mite allergens has been elucidated, with many of them showing enzymatic activity. Mite allergens remain one of the most studied, since house dust mites are very common in tropical and subtropical regions of the world. Therefore, it is very important to include mite antigens in routine diagnostic and therapeutic strategies for allergy and asthma, particularly in areas where mite exposure and sensitization are high. Recombinant DNA technology, as well as other molecular biology and immunological techniques, have played a fundamental role in advances towards a better understanding of the biology of house dust mites and their role in allergic diseases. This kind of study also contributes to the understanding of the complex immunologic mechanisms involved in allergic reactions. The development of effective diagnostic and therapeutic approaches depends on the continuity of research of house dust mite allergens. The objectives of this review are to describe the most important aspects of house dust mite allergy and to acquaint the scientific community with the latest findings pertaining to house dust mite allergens.

Keywords: Allergy, Asthma, House dust, Mite, Mite allergens

*Corresponding author: [email protected]

Malaysian Journal of Medicine and Health Sciences Vol. 8 (2) June 2012: 5-21

INTRODUCTIONHouse dust mites have been shown to be important sources of indoor allergens associated with asthma and other allergic conditions. Allergy covers a wide range of symptoms or reactions to substances in our environment, many of which have not been shown to have any connection with immunology. There is now good evidence to suggest that chronic exposure of allergic individuals to foreign proteins in houses is an important cause of two major chronic diseases: asthma and atopic dermatitis (AD). Atopic allergy describes those symptoms or diseases that are epidemiologically associated with, and thought to be caused by, immediate hypersensitivity to common inhaled allergens. Allergy is an exaggerated or hypersensitivity reaction of the immune system to specific external substances called allergens, which are usually harmless to most people. Common causes of allergy include animal dander, house dust mites, foods, pollen, insects, and chemical substances. Allergy to house dust mites is a condition that affects millions of people around the world.

Domestic mites, the main source of allergens in house dust, produce potent allergens that are capable of inducing sensitization and respiratory and cutaneous diseases. The most common species belong to the families Pyrogyphidae, Acaridae, Glycyphagidae, Echymopodidae, Chortoglyphidae, Cheyletidae, and Tarsonemidae. These arthropods have a worldwide distribution. The most important species are Dermatophagoides pteronyssinus, (Figure 1), D. farinae, D. siboney, D. microceras, Euroglyphus maynei, Acarus siro, Suidasia medanensis, Aleuroglyphus ovatus, Tyrophagus putrescentiae, Glycyphagus domesticus, Lepidoglyphus destructor, Blomia tropicalis, Chortoglyphus arcuatus, Cheyletus spp., and Tarsonemus spp. Storage mites belong to a wide range of families, genera, and species and are found in stored grain, barns, hay, and straw. Exposure to these mites and their allergens can also occur in


A Wan Omar, I Patimah & B Rusliza6

homes. Several species of storage mites have been identified in house dust worldwide. The term “domestic mites” applies to all mite species that can be found in the indoor environment and to which type I allergic sensitization has been demonstrated. In 1964, the role of a species of the genus Dermatophagoides in the etiology of bronchial asthma produced by the inhalation of house dust was proposed. Sensitization to domestic mites and asthma has since been recognized as a worldwide clinical problem. Cutaneous sensitivity to mite allergens has been demonstrated in 50% to 90% of asthmatic individuals. Several groups have identified nasal and bronchial reactivity in response to direct challenge with mite allergens in humans, supporting the idea that mite allergens play an important role in the pathogenesis of allergic respiratory diseases. Domestic mite extracts contain many allergens, which are grouped according to their homologies, or order of description. So far, approximately 19 groups of mite allergens have been characterized and/or sequenced.

Figure 1. House dust mite allergenicity. The various components of HDM, and their associated fecal pellets and dust, which activate the immune system to initiate an inflammatory response, are illustrated.

Source: Lisa G. Gregory and Clare M. Lloyd. Orchestrating house dust mite-associated allergy in the lung. Trends in Immunology September 2011, Vol. 32, No. 9 : 402 -411

Some common conditions caused by allergy to house dust mites include asthma, allergic rhinitis and conjunctivitis. Although allergy to house dust mites is not a direct threat to a person’s life, asthma could become a serious condition that, if not properly treated, could lead to death due to respiratory complications. Asthma is a chronic respiratory disease, often of allergic origin, that is characterized by continuous or paroxysmal labored breathing accompanied by wheezing, bronchial constriction and inflammation, and often by attacks of coughing or gasping. The prevalence of asthma in the United States and other countries has been increasing during the last ten years [1, 2, 3]. Diverse studies suggest that exposure to house dust mite allergens may be a primary cause or a risk factor in the development of asthma, and that it can act as a trigger for the exacerbation of the symptoms [4, 5, 6, 7, 8]. A 2002 study by Terreehorst and collaborators with 325 atopic patients showed that 92% of the asthmatic patients and 85% of the patients with atopic dermatitis had a history of allergic rhinitis, and that there was a high prevalence of nasal symptoms associated with sensitization to house dust mites in these patients. These investigators conclude in their study that asthma and atopic dermatitis are highly associated with allergic rhinitis, and that allergic rhinitis is a risk factor for asthma [9]. Other epidemiology surveys have reported similar results [10]. The American Academy of Allergy, Asthma and Immunology reports up to 78% of asthmatic patients with nasal symptom and up to 38% of allergic rhinitis patients with asthma [11]. Allergic rhinitis is the inflammation of the mucous membrane of the nose as a result of exposure to an allergen. It is a very common condition not as serious as asthma, but very debilitating and with a negative impact in the quality of life of the affected person. Allergic rhinitis caused by house dust mites is perennial, with symptoms appearing continuously or intermittently all throughout the year. People with allergic rhinitis or asthma often have a family or personal history of atopy. Atopy is the increased tendency, with a genetic origin, to produce immediate hypersensitivity reactions usually mediated by immunoglobulin E (IgE) antibodies against normally harmless substances. Diverse scientific


Allergy to House Dust Mites and Asthma 7

studies propose the atopy concept as the basis for a connection among allergic conditions such as atopic dermatitis, allergic rhinitis, asthma and conjunctivitis. Allergic conjunctivitis is the inflammation of the conjunctive membrane of the eye caused by airborne allergens that invade it, and atopic dermatitis is the inflammation of the skin as a result of a hypersensitive reaction. The onset of allergic rhinitis and asthma usually occur during childhood, adolescence or early adulthood. Symptoms may decrease in older people, but more often persist throughout their lifetime [9, 11]. Allergic and asthmatic people have to deal with their conditions in a daily basis, seeing their energy and productivity levels negatively affected and, therefore, their overall daily performance as well [12, 13]. Moreover, these conditions require scheduling frequent visits to their allergists and doctors, often having to take time from school and work. Allergic rhinitis and asthma may also influence adversely over other common activities such as sleeping, learning and social interactions [14]. Therefore, not only is the physical state of the person adversely affected but the emotional well-being as well.

BIOLOGY OF HOUSE DUST MITESHouse dust mites belong to the phylum Arthropoda (i.e., animals with external skeletons and jointed limbs), subphylum Chelicerata, class Arachnida, order Acari, and sub-order Astigmata (lacking specialized respiratory organs) [15, 16]. Contrary to what some people might believe, house dust mites are not closely related to insects, which belong to subphylum Uniramia. The morphology and physiology of house dust mites differ greatly from those of insects. For this reason, common insecticides used successfully to kill insects are ineffective for controlling house dust mite populations [17]. These microorganisms usually measure between 0.1 to 0.6 mm, so they are not visible to the unaided eye, for proper identification at least 10X magnification lens are necessary. Their bodies are oval-shaped and creamy to translucent white, and they have eight legs. The reproduction of house dust mites is sexual, with mating of a male and a female. The life cycle of house dust mites starts with the fertilized female laying a couple of eggs per day. Six-legged larvae hatch from the eggs and remain active for some time, then shed their integuments and become eight-legged resting protonymphs. The protonymphs also shed their integuments and become larger active tritonymphs.

Finally, the tritonymphs undergo another shedding of skin developing into active adult mites [18, 19, 20]. The period of time of this developmental cycle is uncertain, fluctuating from 2 to 6 weeks. The number of eggs a female may lay is also unclear, but it is estimated between 40 to 100 eggs over a six-week lifespan. Adult house dust mites may live between 2 to 5 months, depending on the environmental conditions. These microorganisms feed mainly from skin scales shed by humans and their pets, which are colonized by fungi, yeasts and bacteria, although they may also take advantage of other organic detritus that accumulates in homes. Competing or predating interactions between house dust mites and fungi are not clearly defined, but it was traditionally thought that fungi enhance rates of mite population increase [21]. Therefore, an approach considering fungi as a biotic factor for the control of house dust mites seems of little relevance yet. However, more research is necessary in this area and in the possibility of competitive interactions among different species of house dust mites. Proteins found in the metabolic waste products excreted in the feces by these microorganisms are the cause of the allergic reaction to them. A floating dust cloud seen in the light when dealing with bed clothing and similar materials may contain such waste products. House dust mites inhabit areas and items of the house, as well as the workplace, that comply with their survival requirements, such as carpets, curtains, mattresses, pillows, soft toys, books, and other pieces of upholstered furniture. Common house dust mites such as Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are inevitably found in every household, predominantly in areas of the world with high relative humidity (>45%) and warm temperatures, between 65 to 85°F. House dust mites satisfy their requirement of water by taking up vapor from the surrounding air and that is why many scientists suggest maintaining a low humidity indoors as a measure to reduce the allergen levels in houses. A study in India revealed that the population of mites during the summer was lower than the rainy season of September to October and the winter [22]. In this study, the investigators concluded that the extremely high temperatures of the summer season, the low relative humidity (around 25%) and the lack of rain were unfavorable environmental conditions for the growth and thriving of mites. They also found that the number of house dust mites present in the beds was higher than that of floor dust in most of the homes, concluding that it is probably due to the association of these mites with the human habitat. Moreover, they observed that old and humid houses with poor ventilation were more favorable for the survival of house dust mites in comparison with newer and well ventilated houses.

These findings of seasonal variation, house dust mite concentrations and type of dwelling were directly proportional to the severity of allergic attacks in different groups of allergic and asthmatic patients studied simultaneously. In another study of 1997 with asthmatic patients sensitized to house dust mites, the investigators found that changes in airway hyperresponsiveness and other immunological parameters are associated with allergen exposure and seasonal variations [23]. They found that there was a tendency to higher concentrations of house dust mite allergens during autumn than in spring.

In correlation with this finding was the interesting observation that in the sensitized patients the airway hyperresponsiveness and the concentrations of serum total IgE and house dust mite specific IgE were also higher in



autumn. Humidity was also higher during autumn compared with spring. Certain measures can be followed to control these microorganisms at home. For example, washing fabrics at least weekly in very hot water and using acaricides can kill dust mites. Frequent dry and wet vacuum cleaning helps, although most allergist will recommend getting rid of carpets as well as other similar home items. Putting special covers to mattresses and pillows and using mechanical ventilation systems or air conditioning in the house are also recommended measures.

Maintaining the relative humidity below 50% is a key factor for reducing dust mites levels [24, 25]. However, these and other meticulous control practices must be followed constantly and for indefinite time, an extremely difficult task for most people. Special mattress covers, acaricides, as well as other anti-allergen products to minimize contact with the dust mites can be expensive and not easily found in stores. These control methods should be used in combination and can have different effects in different homes. For example, the effectiveness of acaricides will depend on method of application, type of carpets and furniture, amount of dust in the house, and quality of post-treatment vacuum cleaning. Besides, control of these microscopic creatures has proven to be very difficult because they reproduce relatively rapidly. On the other hand, scientific studies have shown that asthmatic patients who are transferred to high altitudes or mite-free dwellings experienced reduced symptoms such as bronchial hyperactivity. Therefore, some scientists support dust mite avoidance measures as an effective alternative for controlling allergic diseases [26, 27]. Blomia tropicalis (Bt) belongs to the Glyciphagidae family. House dust mites in this family are characterized by numerous long dorsal setae (bristles), no dorsal shield, and no anal suckers. Their bodies are covered by minute papillae [28]. Members of the genus Blomia may be differentiated from other genera in this family because their legs lack a sub-tarsal scale present in the others, and also they have no claw. In addition, there are other subtle morphological differences among the species in this genus that allow differentiation of Bt from the other. These species are usually found in areas with very humid climate, such as the tropics and subtropics, including the island of Puerto Rico, although some of the members may be found in temperate regions [25, 26]. These mites have been traditionally referred to as “storage mites” or “stored products mites” because they have been found mainly in stored grain and flour, barns, hay and straw [27]. At the beginnings of scientific research with storage mites they were mostly associated with occupational allergic disease. Nevertheless, Bt and other Glyciphagid mites are also commonly found in homes nowadays, in rural and urban areas and therefore, are included in the “house dust mites” or “domestic mites” group [27]. The biology and ecology of Bt are the least studied among the major house dust mites. However, it has been found in several studies to be an important source of indoor allergens, mostly in the Southern hemisphere. Glycyphagid mites and most other common house dust mites have been also found in other habitats such as mammal and bird nests . This observation is not surprising considering that human habitats share certain characteristics such as heat, moisture and food availability with these other habitats that make them an ideal dwelling for mites. In a 1997 study in Puerto Rico, Bt was found as the second most common house dust mite with 31.6%, behind Dermatophagoides pteronyssinus with 45.6% [25, 26, 27].

DERMATOPHAGOIDES AND OTHER SPECIESDermatophagoides species of house dust mites belong to the family Pyroglyphidae and are the predominant ones in temperate and tropical regions of the world, including North America and Latin America. This genus is the most studied as it is evident in the scientific literature. Members of the Pyroglyphidae family are characterized by the presence of anal suckers, an anterior dorsal shield, and body with “fingerprint” pattern of striations and setae of variable length. The term “house dust mites” has been traditionally used to include mainly members of the Pyroglyphidae family that live permanently and almost exclusively in house dust. However, in the Second International Workshop on Mite Allergens and Asthma (1990) it was recommended to use the term “domestic mites” to include this family and also the Glycyphagidae family, to which Bt belongs [25, 26, 27]. Among the members of the Pyroglyphidae family, the most common are Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), both with worldwide distribution. From the 49 species of Pyroglyphidae dust mites, 13 have been found in house dust while the others live mainly in bird nests or feathers. In Europe, Japan, North America, New Zealand and Australia the predominant Pyroglyphidae genera are Dermatophagoides and Euroglyphus, which are closely related. In fact, Dp is the most commonly dust mite found in homes in Europe, followed by Df and Euroglyphus maynei (Em) [27, 29]. Em has also been found in Korea, China, India, New Guinea, and Southern United States. In Puerto Rico, Dp, Df and Em are the most common species of house dust mites, along with Bt from the Glyciphagidae family. Another species in this family that is being studied is Dermatophagoides siboney, which has been found also in tropical environments, including Cuba and Puerto Rico [25, 27, 28].

THE ROLE OF DUST MITES IN ALLERGIC DISEASESDust mites are the most frequently encountered aeroallergens in temperate climates. Their role in sensitizing asthmatic patients was first reported in 1921, and the association of house dust mite allergy, asthma, and perennial rhinitis has been repeatedly corroborated [30, 31]. Accordingly, the treatment of asthma and perennial rhinitis frequently includes immunotherapy with dust mite extract [32, 33]. In a 1967 study, dust mites of the Dermatophagoides genus were



identified as the single most important allergen in house dust. Mites are Arachnids (members of the spider family) with Dermatophagoides farinae the species encountered in North America and Dermatophagoides pternoyssinuscommon in Europe [34]. Presently, both species are found worldwide. The house dust mite life cycle includes three larval stages, and the total life span is about 3 months. Their principal habitat is fomites—carpet, fabric, upholstery, pillows and mattresses, and there are approximately 100 living mites in each gram of house dust. Their diet consists of human epidermal scale, animal dander, and trace nutrients. The more bare skin present in a home, the more dust mites are likely to be present in that environment. Because mites cannot take in fluids by mouth, however, their primary source of water is ambient water vapor. The upper humidity limit is constrained by the occurrence of mold growth, which can inhibit mite development, particularly at relative humidities above 88%. The survival of active adult mites is limited to 4 to 11 days at a relative humidity below 50% at 25oC. The most allergenic material is, in fact, mite fecal matter. The amount of fecal matter produced increases with increasing relative humidity, and the highest levels of allergens found in the environment usually correspond to optimal humidity conditions [35]. A 1992 study reported that home air conditioning, by reducing relative humidity, reduces the dust mite population as compared to homes without air conditioning or dehumidification. Der pI and Der pII are the major dust mite respiratory antigens, and importantly, they can persist in the environment long after the death of the mites [36]. Dust mite antigens also include other allergenic components such as acaridials, dialdehydes, cysteine proteases, trypsin, eicosapentanoic acid, and citral [37, 38].

House dust proteases exert profound effects on epithelial cells and promote allergic sensitization. These effects include disruption of intercellular adhesion, increased paracellular permeability, and initiation of cell death [39]. Allergen avoidance is recognized as an integral part of the management of patients with asthma along with antiasthma drugs and immunotherapy [40]. The National Institute of Environmental Health Sciences recently reported that over 45% of American homes have bedding with dust mite allergen concentrations that exceed the level necessary for allergic sensitization. They found that certain simple steps—allergen-proof mattresses and pillow covers, weekly laundering of other bedding, careful vacuuming and dry steam cleaning of bedroom carpets and upholstery—can significantly reduce the levels of dust mite allergens in bedrooms [41]. In addition, most studies have noted that when indoor humidity is kept below 50%, mite populations do not grow to significant levels.

DUST MITES AND ATOPIC DERMATITISThe ubiquity and allergenicity of the dust mite should make it suspect as another possible trigger for exacerbations of the eczematous itchy skin of some atopics. In addition, dust mites have the capability of acting as irritants contact haptens, and IgE antigens on the atopic- impaired, epidermal barrier layer. A 1932 paper reported that patients with atopic eczema improved when they were placed in a dust-free environment, a finding confirmed subsequently by many investigators [42, 43, 44, 46]. A 1982 study reported positive patch test results when patients with AD were patch tested to purified allergen [47]. A PubMed (Medline) search listed almost 60 articles addressing the role of patch testing patients with AD. All but one of these papers support a positive correlation (16–92% positivity) between dust mites and their putative role as a trigger for exacerbations of AD [48]. Despite this overwhelmingly supportive literature there still remains significant skepticism, primarily among dermatologists, regarding the relevance of these findings to the overall management of patients with AD—perhaps as an antithetic reaction to the zealous support voiced by so many allergists toward the role of aeroallergens in AD.

More recently, other investigators have tested patients with AD for dust mite contact sensitivity, and all have similarly reported positive results in a subset of patients [49, 50, 51, 52]. The variability in the number of positive patch test results in patients with AD in different studies probably results from different testing techniques used by each investigator. The major flaws in these studies have been the lack of standardized dust mite patch-test material, patient selection bias, and a lack of uniformity in testing. While the lyophilized Der p1 and Der p2 aqueous material used for skin prick testing was used by all the early investigators, many of the later investigators (this author included) have been using a protocol utilizing mite whole body material [53, 54, 55, 56].

HOUSE DUST MITE ALLERGENSThe Allergen Nomenclature Sub-Committee, International Union of Immunological Societies (World Health Organization) establishes guidelines for the identification of a molecule as an allergen [57]. Only allergens with a frequency of IgE reactivity above 5% will be included in the nomenclature. Also, an allergen may be classified as a “major” or a “minor” allergen depending on whether more or less than 50% of the patients tested reacted with the corresponding allergen specific IgE in the given system. Of course, there are inherent factors that impose some uncertainty to the determination of IgE prevalence such as choice of test system, criteria for selection of patients, geographic region, environmental conditions and others. Therefore, researchers are encouraged to perform their analysis with a substantial number of patients whenever possible. The Sub-Committee recommends analyzing the defined component for allergenic activity with at least 20-30 human sera from highly allergic individuals [58].

During the past three decades it has been well demonstrated by different scientific investigations that allergens



from house dust mites constitute a major etiologic factor of allergies and asthma in many countries around the world [59]. Extensive studies have been conducted in search of a better understanding of the biological, chemical and structural properties of dust mite allergens as well as other factors that might be influencing or determining their allergenicity. Thanks to advances in molecular biology technology, the biological function and structural properties of many allergens have been elucidated, although many investigations with allergens are still in progress or are yet to be undertaken. Although the biological function of allergens has not been shown to be the only or the main responsible factor for their allergenicity, it may facilitate the immunological milieu required for specific sensitization toward an allergen or enhance the ability of the protein to trigger an IgE antibody response. For example, scientists have observed that allergenic molecules with enzymatic activity, such as cysteine proteases, irritate the mucosal surface facilitating their own processing The best characterized allergens of house dust mites are those in group 1, which have been identified as cysteine proteases. One of the most studied allergens from this group, Der p 1, seems to enhance allergenicity by several mechanisms such as increasing the permeability of the respiratory mucosa, enhancing antigen processing, promoting IgE synthesis, and augmenting TH2 cell responses [60]. Der p 1 was shown to cleave the low affinity receptor for IgE, CD23, and this action seems to disrupt the negative feedback regulation of IgE synthesis mediated by this receptor [61]. However, enzyme function is not essential to trigger IgE responses, as other types of biological functions have been found for several allergens from house dust mites and other sources. Other proteins from house dust mites that have shown to be allergenic include group 3 allergens which are serine proteases, group 4 (Der p 4) with amylase activity, group 6 allergens (Der p 6) identified as chymotrypsin-like proteases, group 10 as a tropomyosin (a structural protein), group 13 as a fatty acid binding protein, and allergens from the groups 2, 5, 7, and 12, all with unknown biologic function [62, 63]. The sensitizing dose of an allergen has been debated in several studies, but in the Indoor Allergens and Asthma: Report of the Third International Workshop it was established as 2 mg allergen/g of dust (100 mites/g). The structural stability of allergens may also play an important role in the allergic response toward them, as has been shown in some studies where IgE epitopes have been altered or the three-dimensional structure of the protein has been split. Allergens have different structures and are classified in different protein families according to their biological function, suggesting that there may be few or no common structural features or intrinsic properties between allergens making them allergenic. However, it seems that there is an important connection between biological function, structural integrity and IgE binding capacity for an individual allergen to keep its allergenicity [64]. Other factors cannot be ruled out as participants of the sensitization and the allergic reaction toward an allergen, such as genetic predisposition or defects in the regulation of IgE responses of the individual, other possible adjuvants such as hormones, bacterial and viral infections, and the route and degree of exposure. In view of these implications, recombinant allergens altered by site-directed mutagenesis to remove IgE epitopes may represent a valuable tool for further research and more effective allergen immunotherapy.

IMMUNOLOGICAL MECHANISM OF HOUSE DUST MITE ALLERGYThe development of allergy starts when the individual is in contact with the allergen for the first time and becomes hypersensitive to it. When the allergen invades the mucous membrane of the nasal passages an immediate allergic response occurs (Figure 2). The most accepted mechanism indicates that the allergen is presented to T helper-2 (TH2) lymphocytes by antigen presenting cells (APC), and the TH2 cells respond by releasing cytokines such as interleukin-4 (IL-4) and interleukin-5 (IL-5) which attract inflammatory cells to the airways, such as the mast cells, basophils, and eosinophils. TH2 cells activate B cells of the immune system, which produce a specific antibody, IgE, which is overproduced in subsequent allergic reactions. IgE antibodies attach to membrane receptors in mucosal mast cells and activate them. The allergen binds those antibody molecules causing the release of mast cell granules to the outside of the cell. These granules contain chemical inflammatory substances such as histamine and leukotrienes, which trigger the array of symptoms that comprise the allergic reaction. At the same time, mast cells secrete IL-4, which stimulates B cells to produce more IgE. In chronic allergic asthma the allergen induces activation of submucosal mast cells in the lower air passages, leading to bronchial constriction and an increase in fluid and mucus secretions which make breathing more difficult. These phenomena are a result of a late-phase reaction, characterized by the continuous synthesis and release of chemical mediators such as cytokines and leukotrienes from the activated mast cells and eosinophils. Mast cells also secrete IL-5, which further stimulates eosinophils for their release of more inflammatory mediators. A similar late-phase reaction occurs in chronic allergic rhinitis [65, 66, 67, 68].

It is not well known what structural features or what functional properties of allergens confer them the capacity to modulate the immune response toward the TH2 cell activation, which stimulates an IgE response [69, 70]. However, although much research is still necessary, it has been observed that many common allergens share typical characteristics such as enzymatic activity, relatively low molecular weight (less than 60-70 kDa) and high solubility. Moreover, it has also been shown that factors extrinsic to the allergen are involved, such as genetic predisposition to overproduce IgE (atopy) and the particular immune response of the individual, the nature or degree of exposure to the allergen and other environmental factors [70, 71].



Figure 2. Proposed model for the innate initiation of the allergic response induced by HDM.Der p 1 and the Der p 2-LPS complex, together with adjuvant-like contaminating molecules (including LPS, B-glucan or chitin), can activate unknown protease-sensitive receptors as well as PRR expressed by epithelial cells. Altogether, these stimulations lead to the production of chemokines and cytokines that not only attract and activate DCs but promote also an influx of inflammatory leukocytes to trigger eosinophilia, neutrophilia, airway remodeling and AHR. Thanks to the increasing permeability of the epithelial barrier following cleavages of tight junction proteins by Der p 1, submucosal DCs can be also directly activated by components of HDM. There is an immediate release of Cys-LT, potent mediators of bronchial smooth muscle constriction and IL-6. Activated DC will mature and migrate to mediastinal lymph nodes to present the allergen to naïve T cells. The cytokine milieu (notably TSLP, low IL-12 concentration) will drive the differentiation of naïve T cells into Th2 cells producing the cytokines IL-4, IL-5 and IL-13, the critical effectors of the salient features of asthma as the allergen- specific IgE antibody and the recruitment and activation of eosinophils. The presence of IL-6 will induce Th polarization skewing to Th17 cells which accentuate the airway pathology through notably the action of Th17 cytokines as IL-17 on the airway smooth muscles. TLR, Toll-like receptor; CCL, CC-chemokine ligand; GMCSF, granulocyte/macrophage colony stimulating factor; TSLP, thymic stromal lymphopoietin; Cys-LT, cysteinyl leukotriene; SCF, stem cell factor; DC-SIGN, dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin; MCP-1, monocyte chemotactic protein-1.

Source: Alain Jacquet. New Insights into the Molecular Basis of the House Dust Mite-Induced Allergic Response The Open Allergy Journal 2009; 2: 38-44



The most common symptoms of allergic rhinitis and allergic asthma caused by inhaled house dust mite allergens include sneezing, itchy eyes, ears and nose, local edema, nasal secretions, nasal congestion, itchy palate and throat, cough, shortness of breathing, and wheezing.

ROLE OF IgE IN ANTIGEN PRESENTATIONThe IgE/mast cell pathway, when first acknowledged, elucidated the mechanisms of anaphylaxis and the so called atopic diathesis—especially for respiratory symptoms [72]. However, dermatologic manifestations, namely eczematous skin lesions, could not be adequately explained by the IgE/mast cell flow of events.

Yet, few would deny the existence of a small subset of patients, who associated exacerbations of their eczema with recognized IgE triggers such as foods, animal dander, mold, and even pollens [73]. Was elevated IgE in eczema patients merely an epiphenomenon, an indicator of other concurrent atopic disease or did it in fact play a role in inducing AD lesions? It is only recently that IgE receptors have been found not only on mast cells, but also on Langerhans cells and other dendritic antigen- presenting cells of atopic individuals, and it is this finding that has led to studies ultimately showing a direct role for IgE in AD pathogenesis [74, 75].

Dendritic antigen-presenting cells have been shown to be necessary for initiating and controlling the immunologic response to pathogens present at the interface with the environment [76]. They have a significant capacity to take up and process antigens in this location, but their ability to prime T cells is limited until they undergo phenotypic changes and become effective immunostimulatory cells. Maturation of dendritic cells occurs as they migrate to regional lymph nodes. They begin to express coreceptors and elaborate chemokines that enhance their ability to present antigen to T cells. The high-affinity IgE receptor (Fc_RI) that on mast cells facilitates the release of inflammatory mediators plays a different role on Langerhans cells. In AD lesional skin, Langerhans cells expressing Fc_RI are present in higher numbers than in the skin of nonatopics; these cells have been shown to be essential in the sensitization phase of AD.5 Moreover, the presence of Fc_RI_ Langerhans cells bearing IgE molecules is a prerequisite for provoking eczematous lesions in aeroallergen patch tests in atopic patients [77]. We now understand that activated Th2 cells are the effector cells for the early stages of AD, and similarly activated Th1 cells account for the later phases [78]. Thus, we now have a rational mechanism for the chronicity of AD (in a subset of patients) triggered, and then maintained by IgE antigens which include aeroallergens such as dust, animal danders, etc. The second deterrent to appreciating the putative role of contactants as triggers of AD is that an additional diagnosis, such as allergic contact dermatitis, is not sought once atopy is recognized. Moreover, atopic patients are considered by many clinicians to be less prone to sensitization by contact allergens. On the contrary, atopic patients are indeed sensitized just as frequently to contactants as their nonatopic counterparts under many circumstances [79]. In fact, numerous reports document that they react equally to most common allergens, eg, fragrances, rubber accelerators, lanolin, formaldehyde, and others [80, 81]. Nickel is the most common contact allergen with equal frequency in atopic and nonatopic subjects [82]. Most recently, allergy to topical corticosteroids has been increasingly reported in individuals with AD because of the frequent use of such agents on the impaired epidermal layer of these patients. Nonfluorinated corticosteroids are especially likely to cause this type of reaction [83]. The possibility of corticosteroid allergy should be suspected clinically when there is worsening of eczema at corticosteroid-treated sites.

CLINICAL AND LABORATORY TESTS FOR DETECTIONOF ALLERGEN SPECIFIC IgE ANTIBODIES

Allergy Skin Tests

Immediate hypersensitivity skin tests are used to identify specific IgE sensitization [84]. The skin is marked for testing with a panel of appropriate allergens for the patient, selected on the basis of the clinical history and knowledge of the allergens commonly found in the locality. Positive and negative comparator tests using histamine and saline can be performed to prove that the skin is capable of demonstrating a positive reaction and to prevent the interpretation of false-positive results occurring as a result of dermatographism. A drop of allergen solution is placed onto the skin at each mark, and a fresh fine sterile needle or lancet is used to gently prick the skin through each drop, introducing a minute volume of allergen solution into the dermis. After 10-15 minutes the results are interpreted by reference to the control tests. Provided that there is no wheal response to the negative control, the presence of a raised wheal at the site of the allergen skin prick test of 3 mm or greater in diameter indicates the presence of IgE antibodies specific to that allergen. Taken in conjunction with the clinical history, the results of skin prick testing can confirm a diagnosis of IgE-mediated disease and identify causal allergens. Skin prick tests are particularly reliable for inhalant allergens. However, the variations in reaction between tests and testers limits its use with experienced personnel. Intracutaneous tests are used in some geographical areas and for some suspected allergens, e.g., drugs. The method is more sensitive than the skin prick test but carries more risk of systemic reactions and often gives false positive reactions. The method may be indicated when allergen extracts are not strong enough to give positive skin prick test reactions.



Radioallergosorbent tests (RAST)

The discovery of IgE allowed the development of immuno-assays for IgE and IgE-antibodies, enabling direct and objective measurement of the extent and specificity of the immune response [85, 86]. In RAST, allergens are linked to paper discs or polyurethane caps (CAP - RAST) and are reacted with the individual’s serum. Binding of IgE specific to that allergen is detected by the use of an enzyme linked anti-human IgE antibody in a colorimetric reaction. Results of RAST testing show a very good corelation between the presence of IgE antibody in serum and positive skin and provocation tests, as well as symptoms of allergy. Positive RAST results to a specific allergen demonstrate specific IgE sensitization but are not proof that the allergen is the cause of clinical symptoms.

The measurement of allergen specific IgE antibodies in serum is of similar diagnostic value to that of skin tests but has a much higher reproducibility and is not influenced by ongoing symptoms or treatment, eg, antihistamines or anti-inflammatory therapy. In some instances, especially in food allergic individuals where, in rare cases, even skin prick testing with minute amounts of allergen might cause an anaphylactic reaction, RAST using blood samples is a safe method to determine levels of specific IgE antibodies. RAST is also the test of choice for individuals who have widespread eczema, which precludes skin prick testing.

Approximately 500 different allergens are now available for RAST-based allergy diagnosis [58, 59, 60]. In addition to classical pollen, dander and food allergens, drugs, occupational chemicals and recombinant allergens are available. The general availability of well standardized in-vitro allergy tests has greatly improved the quality of allergy diagnosis.

The use of extracts allows the identification of the source of the allergen but not the specific molecule to which the patient is allergic to, nor the determination of IgE levels against a particular allergen. Current diagnosis and treatment of dust mite allergy is mainly based on the use of crude mite extracts. A more effective approach would be to make a diagnosis and design a therapy according to the patient’s allergen reactivity profile. This approach would allow the identification of the specific mite allergenic components causing the disease and the measurement of IgE levels against them. It is in this scenario that the design and availability of recombinant allergens play a fundamental role.

Measurement of total IgE, not IgE antibodies, in serum, secretion or on cell surfaces is of little diagnostic value. The reason is that mitogenic factors in viruses (e.g., Cytomegalovirus - CMV), bacteria (e.g., Staphylococcus), helminths (e.g., Ascaris, Schistosoma) and adjuvant factors in air pollution (e.g., cigarette smoke, and diesel exhaust) stimulate the production of IgE molecules without initiating any allergen specific IgE-sensitization. However, production of IgE-antibodies will increase the total IgE level slightly and thus an increased total-IgE in cord blood is a high sensitivity but low specificity predictor of allergy.

TREATMENT OF HOUSE DUST MITE ALLERGYDuring the past ten to fifteen years, scientists have been investigating new approaches to the treatment of house dust mite allergy. A variety of medications for minimizing allergy symptoms are currently available. However, these medications have to be taken indefinitely and many of them can cause side effects such as drowsiness, which affects the patient’s daily performance. Other more effective and non-drowsy medications have to be prescribed and can also be very expensive. Control and avoidance measures at home have to be followed aggressively, and implementation of these practices can be very difficult.

This treatment literally reduces your sensitivity to the house dust mite. Desensitising injections (extracts) contain a small quantity of house dust mite. The body defends itself by producing protective antibodies. If enough are produced in the body, the next time you come in contact with house dust mites they will protect you. Not unlike immunisation.

Immunotherapy

Immunotherapy, or hyposensitization, with dust mite allergens is another common preventive therapeutic approach [87]. The patient is injected over a period of 1-4 years with increasing doses of the mite extract until reaching a maintenance dose, starting once a week and reducing the frequency to one or two injections a month depending on the patient’s response [88, 89]. The aim of this procedure is to gradually induce tolerance to the mite allergens in the patient’s immune system. Scientific studies have shown that immunotherapy is an effective treatment, but also indicate that the precise mechanisms involved in immunotherapy are still unclear. Some studies have shown that after the immunotherapy there was a down-regulation in the overall allergen-specific production of inflammatory cytokines such as IL-4, IL-5 and interferon-gamma (IFN-g) [90]. Proposed mechanisms include the switching of the allergic immune reactivity from a TH2-type response to a TH1-type (i.e. immune deviation), peripheral T cell unresponsiveness (anergy), or possibly deletion of allergen-reactive lymphocytes (Figure 3). Immunotherapy can be expensive, but it has the advantage of being aimed at the cause of the allergy and not only the symptoms, and thereby can eliminate or reduce dramatically the allergic reaction. It has been documented through extensive studies that immunotherapy may be one of the best therapeutic strategies for dust mite allergy. However, it has been observed that the use of dust mite extracts may involve some disadvantages. One of them is the possibility of anaphylaxis (severe life-threatening allergic reaction),



although this is apparently minimized with the application of very small doses over a long period. Additionally, in mite extracts it is difficult to standardize mixtures of allergenic and nonallergenic components, including proteins, carbohydrates and nucleic acids [91, 92]. The quality of the extract is influenced by several factors such as the extraction procedure and storage conditions. Certain allergens may not be well represented or may even be degraded during the preparation of the mite extract.

Figure 3. Mechanisms of allergen-specific immunotherapy and the role of regulatory T cells in allergic diseases. An allergen is taken up by regional dendritic cells leading to the induction of regulatory T cells. These cells suppress allergic responses directly and indirectly by the following mechanisms. 1. Suppression of mast cells, basophils and eosinophils. 2. Suppression of effector T cells. 3. Suppression of inflammatory cell migration to tissues and tissue inflame-mation. 4. Suppression of mucus production. 5. Suppression of inflammatory dendritic cells and induction of tolerogenic dendritic cells. 6. Suppression of allergen-specific IgE and induction of IgG4 from B cells.

Source: Fujita et al. Clinical and Translational Allergy 2012, 2:2 http://www.ctajournal.com/content/2/1/2

Allergy medications

The first treatment for controlling dust mite allergy is avoiding dust mites as much as possible. When you minimize your exposure to dust mites, you should expect to have allergic reactions that are less often or less severe. However, it’s impossible to completely eliminate dust mites from the environment, hence the need of medications to control symptoms [93]. One of the following medications is needed to improve nasal allergy symptoms [94, 95]. • Antihistamines reduce the production of an immune system chemical that is active in an allergic reaction. These

drugs relieve itching, sneezing and runny nose. Prescription antihistamine tablets include fexofenadine (Allegra) and desloratadine (Clarinex). Azelastine (Astelin, Astepro) and olopatadine (Patanase) are prescription antihistamines taken as a nasal spray. Over-the-counter antihistamine tablets (Claritin, Zyrtec, others), as well as antihistamine syrups for children, also are available.

• Corticosteroids delivered as a nasal spray can reduce inflammation and control symptoms of hay fever. These drugs include fluticasone (Flonase), mometasone furoate (Nasonex), triamcinolone (Nasacort) and ciclesonide (Omnaris). Nasal corticosteroids provide a low dose of the drug and have a much lower risk of side effects compared with oral corticosteroids.

• Decongestants can help shrink swollen tissues in your nasal passages and make it easier to breathe through your nose. Some over-the-counter allergy tablets combine an antihistamine with a decongestant. Oral decongestants can



increase blood pressure and shouldn’t be taken if you have severe high blood pressure or cardiovascular disease. In men with an enlarged prostate, the drug can worsen the condition. Talk to your doctor about whether you can safely take a decongestant. Over-the-counter decongestants taken as a nasal spray may briefly reduce allergy symptoms. The use of a decongestant spray for more than three days in a row can contribute to congestion.

• Cromolyn sodium prevents the release of an immune system chemical and may reduce symptoms. You need to use this over-the-counter nasal spray several times a day, and it’s most effective when used before signs and symptoms develop. Cromolyn sodium doesn’t have serious side effects.

• Leukotriene modifiers block the action of certain immune system chemicals. Your doctor may prescribe this prescription tablet, montelukast (Singulair). Possible side effects include headache. Less common side effects include abdominal pain, cough, dental pain and dizziness.

Other therapies

• Nasal lavage is the use of a saltwater (saline) rinse for your nasal passages. Your doctor may suggest a saline rinse to help lessen congestion, sneezing and postnasal drip. You can purchase over-the-counter saline sprays or nasal lavage kits with devices, such as squeeze bottles, to administer a rinse. You can make your own solution with 1/8 teaspoon (5 milliliters) of table salt in 8 ounces (237 milliliters) of distilled or purified water. Mix the ingredients together and store the solution at room temperature, and remix another batch after a week. Lavage your nose daily.

Lifestyle and home remedies

Avoiding exposure to dust mites is the best strategy for controlling dust mite allergy. While house dust mite cannot completely be eliminated from homes, their numbers can be reduced significantly by adopting the following instructions and measures [96, 97, 98]. • Use allergen-proof bed covers. Cover your mattress and pillows in dust-proof or allergen-blocking covers. These

covers, made of tightly woven fabric, prevent dust mites from colonizing or escaping from the mattress or pillows. Encase box springs in allergen-proof covers.

• Wash bedding weekly. Wash all sheets, blankets, pillowcases and bedcovers in hot water that is at least 130 F (54.4 C) to kill dust mites and remove allergens. If bedding can’t be washed hot, put the items in the drier for at least 20 minutes at a temperature above 130 F (54.4 C) to kill the mites. Then wash and dry the bedding to remove allergens. Freezing non washable items for 24 hours also can kill dust mites, but this won’t remove the allergens.

• Keep humidity low. Maintain a relative humidity between 30 and 50 percent in your home. A dehumidifier or air conditioner can help keep humidity low, and a hygrometer (available at hardware stores) can measure humidity levels.

• Choose bedding wisely. Avoid bedcovers that trap dust easily and are difficult to clean frequently.

• Buy washable stuffed toys. Wash them often in hot water and dry thoroughly. Also, keep stuffed toys off beds.

• Remove dust. Use a damp or oiled mop or rag rather than dry materials to clean up dust. This prevents dust from becoming airborne and resettling.

• Vacuum regularly. Vacuuming carpeting and upholstered furniture removes surface dust — but vacuuming isn’t effective at removing most dust mites and dust mite allergens. Use a vacuum cleaner with a double-layered microfilter bag or a high-efficiency particulate air (HEPA) filter to help decrease house-dust emissions from the cleaner. If your allergies are severe, leave the area being vacuumed while someone else does the dirty work. Stay out of the vacuumed room for 20 minutes after vacuuming.

• Cut clutter. If it collects dust, it also collects dust mites. Remove knickknacks, tabletop ornaments, books, magazines and newspapers from your bedroom.

• Remove carpeting and other dust mite habitats. Carpeting provides a comfortable habitat for dust mites. This is especially true if carpeting is over concrete, which holds moisture easily and provides a humid environment for mites. If possible, replace wall-to-wall bedroom carpeting with tile, wood, linoleum or vinyl flooring. Consider replacing other dust-collecting furnishings in bedrooms, such upholstered furniture, nonwashable curtains and horizontal blinds.

• Air purifiers. Air purifiers collect airborne dust in your home and can help with controlling dust if you also maintain vigorous cleaning practices. But purifiers won’t remove dust mites because the mites are too heavy to remain airborne long enough to be filtered through an air purifier. Some dust mites may be airborne right after cleaning, but they quickly settle again onto surfaces.



• Acaricides. Various chemicals have been used to control mite populations. Products containing benzyl benzoate, benzoic acid, pyrethroids, and pirimiphos methyl, among others, are effective acaricides. Denaturating agents, such as tannic acid, reduce allergen levels in carpets but do not kill mites

CONCLUSIONIt is clear that properties of the allergen dictate features of the immune response and therefore ensuing pathology. The complexity of HDM induces a multifaceted immune response involving both the innate and adaptive arms of the immune system, activated by enzymatic protease activity and ligand binding to C-type lectin, protease-activated and Toll-like receptors at mucosal surfaces in the lung. Until the immunologic aberrations of atopy can be interdicted, treatment of the clinical symptoms resulting from the activation of relevant effector cells remains essentially symptomatic. Namely, the sneezing of allergic rhinitis is quelled by antihistamines, which antagonize the effect of the histamine released by activated, atopic mast cells and basophils. Unfortunately, many afflicted atopics are only half-heartedly instructed to avoid the recognized triggers that initiate the inflammatory cascade that sets off their symptoms. Progress will probably come in a variety of directions, e.g. both in the design of regimes for reducing exposure and in new approaches to immunotherapy. The recognition and understanding of the contribution of innate and adaptive pathways might well lead to the development of new strategies for therapeutic intervention that will play a role in the future treatment of asthma and other allergic diseases.

ACKNOWLEDGEMENTSincere thanks to Professor Dr. Norlijah Othman, Dean, Faculty of Medicine and Health Sciences, University Putra Malaysia for giving permission to publish this paper. The authors wish to thank all who have directly and indirectly contributed to our understanding of house dust mite allergy and asthma.

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Obesity and Associated Health Related Factors Among University Staffin Serdang, Malaysia

1L Rampal*, 2P Saeedi, 1S Aminizadeh Bezenjani, 1MS Salmiah & 3O Norlijah1Department of Community Health, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia2Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia3Department of Pediatrics, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

INTRODUCTIONOverweight and obesity are defined as increased body weight for height, and body mass index (BMI) is the most common indicator of overweight and obesity [1]. Obesity is a well-established risk factor for cardiovascular disease in the general population[2, 3]. The risk of coronary heart disease, ischemic stroke, type 2 diabetes, cancers of the breast, colon, prostate and other organs grows steadily with increasing body mass. Relationship between overweight or obesity and cardiovascular morbidity, CVD mortality and total mortality has been reported by several prospective epidemiological studies [4, 5, 6, 7]. Being chronically overweight contributes to osteoarthritis - a major cause of disability[8]. A moderate decrease in weight (10% to 15%) has been shown to significantly improve the health of 90% of the obese patients[9]. It has reached epidemic proportions globally, and evidence suggests that the situation is likely to get worse. It has been estimated that 1.46 billion adults aged 20 and older, (1.41-1.51 billion) worldwide had BMI of 25 kg/m2 or greater, of these 205 million males (193-217 million) and 297 million females (280-315 million) were obese in 2008. The mean population BMI varied substantially between nations. The age-standardized prevalence of obesity was 9.8% in males and 13.8% in females in 2008, which were nearly twice the 1980 prevalence of 4.8% for males and 7.9% for females [10]. Recent evidence shows that the prevalence of obesity has increased worldwide over the past 30 years in both rich and poor countries, and in all segments of society[10]. Malaysia is a middle income country with a multi-ethnic population. In Malaysia, the prevalence of obesity amongst Malaysians 18 years and above has increased from 4.4% in 1996 [11] to 12.3% in 2004 [12] and 14.0% in 2006[13]. As no or very little data is available on the extent of the problem of obesity in the university, this study has been conducted with the aim to determine the prevalence of obesity and factors associated with it among university staff in Serdang.



ABSTRACTIntroduction: Obesity is a well-established risk factor for coronary heart disease, ischemic stroke, type 2 diabetes, cancers of the breast, colon, prostate and other organs. Objectives: To determine the prevalence of obesity and associated factors among university staffs. Methods: A cross sectional study was carried out among university staffs of University Putra Malaysia using a self-administered validated pre-tested questionnaire. Weight was measured using a digital bathroom scale (TANITA Model HD 319) and height was measured using a SECA Body Meter Model 206. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m2). A p value of <0.05 was considered to be statistically significant. Overweight was defined as a body mass index (BMI) of 25 to 29.9 kg/m2 and obesity as a BMI of equal or more than 30 kg/m2. Results: The mean age of the 454 university staffs was 42.86 years. The overall mean BMI was 24.52 ± 4.43 kg/m2, ranged 16.12 to 36.57 (25.69 ± 3.69 kg/m2 for males and 23.31 ± 4.81 kg/m2 for females). The prevalence of overweight and obesity was 31.1% (40.3% males and 21.5% females) and 11.8% (12.1% males and 11.7% females) respectively. After adjusting for all the variables in the logistic regression model, gender, age, occupation, smoking, alcohol intake and physical inactivity were the main predictors of obesity. Conclusions: The prevalence of overweight and obesity is very high among the university staffs. There is a need for a comprehensive integrated non-communicable disease prevention program. There is also a need to establish proactive networks for building up capacity in research and training, mobilizing contributions from within the country and overseas.

Keywords: Obesity, prevalence, associated factors, university staff, Malaysia


L Rampal, P Saeedi, S Aminizadeh Bezenjani, MS Salmiah & O Norlijah24

MATERIAL & METHODSStudy Design/Study Population

A cross-sectional study was carried out among the university staffs of University Putra Malaysia. Data was collected from February 2010 to May 2010. A list of all Malaysian staff aged 30 years and above employed in UPM served as a sampling frame. The sample was selected using probability sampling technique using table of random numbers to ensure that every one had an equal chance of being chosen. The study was confined to respondents aged 30 years and above so that the results could be compared with the previous National surveys. A validated self-administered, pre-tested questionnaire on respondent’s age, gender, ethnicity, educational level, hypertension status, smoking status, alcohol consumption, and physical activity was used to collect the data. Weight, height and blood pressure measurements were also recorded.

Body Mass Index (BMI)

Weight was measured using a digital bathroom scale (TANITA Model HD 319) and height was measured using a SECA Body Meter Model 206. The respondents were requested to stand barefoot on the middle of the weighing machine, with the head looking straight in front, arms by the side. When the reading of the weighing machine was stable, the weight was recorded. After each respondent, the weighing machine was reset to zero. It was checked frequently by the use of a known weight. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m2). It was classified into different categories using the National Institutes of Health- National Heart, Lung, and Blood Institute clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults. In this study overweight is defined as a body mass index (BMI) of 25 to 29.9 kg/m2 and obesity as a BMI of equal or more than 30 kg/m2 [14].

Blood Pressure

Blood pressure was measured after the respondents had rested for at least 5 minutes using a standard mercury sphygmomanometer. The respondents were examined in a seated position with the arm placed at the heart level. Two blood pressure measurements were taken for each respondent by one of the investigators who is a qualified nurse, and the average used in the analysis. Hypertension status of the subjects was assessed based on the US Seventh Joint National Committee report on the prevention, detection, evaluation, and treatment of high BP[15]. Hypertension was defined as an average of two blood pressure readings at single occasion of systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg or a self–reported diagnosis and current treatment for hypertension with antihypertensive medication and/or by self-reports of a medical diagnosis of hypertension and current treatment for hypertension with antihypertensive medication.

Smoking status

An ever smoker (includes both current and ex- smokers) was defined as a person who reported that they have smoked more than 100 cigarettes in their entire life. A current smoker was defined as someone who presently smokes daily and has smoked within the last 30 days. An ex-smoker was defined as someone who has stopped smoking.

Ethics

Before conducting the study ethical approval was obtained from the Faculty of Medicine and Health Sciences, University Putra Malaysia. Informed consent was also obtained from each respondent before data collection.

Statistical analysis

Statistical analysis was carried out using SPSS version 20.0. Independent sample t-test was used to compare the means between two groups, whereas, one way ANOVA was used to compare the means between more than two groups. Logistic regression was used to determine the main predictors of obesity among respondents. Result of logistic regression was expressed as odds ratio and 95% CI. A p value of <0.05 was considered to be statistically significant.

RESULTSThe socio- demographic characteristics of respondents by gender are shown in Table 1. The mean age of 454 respondents was 42.86 ± 9.62, and ranged from 30 to 68 years. The majority of both male and female respondents were Malays. Most of the respondents were married (84.8%), with academic position (53.7%) and had high level of income (≥ RM 6000). Among the males 19.9% were current smokers. None of the females were current smokers. The majority of the respondents (80.8%) were never smokers. The prevalence of hypertension was 34.4% (45.5% in males and 22.9% in


Obesity and Associated Health Related Factors Among University Staff in Serdang, Malaysia 25

Table 1. Characteristics of respondents by gender

Characteristics Male (n= 231) n (%) Female (n= 223) n (%) Total (n= 454) n (%)

Age (years)30-39 79 (34.2) 126 (56.5) 205 (45.2)40-49 69 (29.9) 58 (26.0) 127 (28.0)50-59 63 (27.3) 37 (16.6) 100 (22.0)≥ 60 20 (8.6) 2 (0.9) 22 (4.8)

EthnicityMalay 186 (80.5) 206 (92.4) 392 (86.3)Chinese 30 (13.0) 10 (4.5) 40 (8.8)Indian 15 (6.5) 7 (3.1) 22 (4.9)

Marital statusSingle 24 (10.4) 35 (15.7) 59 (13.0)Married 206 (89.2) 179 (80.3) 385 (84.8)Divorced 1 (0.4) 3 (1.3) 4 (0.9)Widow 0 (0.0) 6 (2.7) 6 (1.3)

Educational levelPrimary 4 (1.7) 3 (1.4) 7 (1.5)Secondary 44 (19.1) 69 (30.9) 113 (24.9)Tertiary 183 (79.2) 151 (67.7) 334 (73.6)

Occupation Academic 152 (65.8) 92 (41.3) 244 (53.7)Administrative 18 (7.8) 52 (23.3) 70 (15.4)Clerical 4 (1.7) 34 (15.2) 38 (8.4)Technician 7 (3.1) 0 (0.0) 7 (1.6)Supportive 50 (21.6) 45 (20.2) 95 (20.9)

Income (RM)Low (< 3000) 46 (19.9) 53 (23.8) 99 (21.8)Medium (3000-5999) 71 (30.7) 96 (43.0) 167 (36.8)High (≥ 6000) 114 (49.4) 74 (33.2) 188 (41.4)

Smoking statusCurrent smokers 46 (19.9) 0 (0.0) 46 (10.1)Former smokers 39 (16.9) 2 (0.9) 41 (9.1)Never smokers 146 (63.2) 221 (99.1) 367 (80.8)

Hypertension statusNormotensive 126 (54.5) 172 (77.1) 298 (65.6)Hypertensive 105 (45.5) 51 (22.9) 156 (34.4)

females). The results show that the overall mean BMI of respondents was 24.52 ± 4.43 kg/m2, ranged 16.12 to 36.57 kg/m2. Table 2a and 2b show the distribution of BMI by socio-demographic factors. Bivariate analysis using one way ANOVA and independent t test showed that overall mean BMI was significantly associated with age, gender, marital status, educational level and income. Males had significantly higher mean BMI (25.69 ± 3.69 kg/m2) as compared to females (23.31 ± 4.81 kg/m2). The mean BMI significantly increased with age among both males (F= 14.72, p< 0.001) and females (F= 43.23, p< 0.001). The results show that in males the mean BMI increased steadily till the age of 59 and then showed a decline. However, in females the mean BMI increased steadily with age. In males, the mean BMI was higher up to age 49 years compared to females. Interestingly, for respondents aged 50 and above the BMI for females was higher than the males. Using Post Hoc-Tukey test for comparing all the age groups for males showed that there was a significant difference in the mean BMI between age groups 30-39 and 40-49 (p= 0.025), 30-39 and 50-59 (p<



Table 2a. BMI distribution by socio-demographic characteristics (both gender)

Mean Median 95% CI SD F, t value P value

Gender Male 25.69 25.23 25.21 - 26.17 3.69 t= 5.914 <0.001*Female 23.31 21.85 22.67 - 23.94 4.81

Age (years)30-39 22.28 21.85 21.73 - 22.83 4 F= 58.33 <0.001*40-49 24.83 24.28 24.21 - 25.45 3.5250-59 28.15 27.78 27.40 - 28.89 3.75≥ 60 27.1 27.49 25.73 - 28.47 3.09

EthnicityMalay 24.47 23.91 24.02 - 24.92 4.51 F= 0.915 0.401Chinese 25.34 25.24 23.99 - 26.69 4.22Indian 23.91 24.25 22.47 - 25.35 3.24

Marital statusMarried 24.77 24.44 24.33 - 25.21 4.37 t= -2.768 0.007*Not married 23.12 22.23 22.02 - 24.22 4.58

Educational levelPrimary 21.18 20.86 17.56 - 24.80 3.91 F= 8.240 <0.001*Secondary 25.82 25.43 24.93 - 26.71 4.77Tertiary 24.15 23.79 23.70 - 24.61 4.23

OccupationAcademic 24.71 24.45 24.21 - 25.21 3.93 t= -0.965 0.335Non academic 24.3 23.57 23.63 - 24.98 4.96

IncomeLow 23.75 22.78 22.83 - 24.67 4.62 F= 5.893 0.003*Medium 24.04 23.62 23.33 - 24.75 4.65High 25.35 25.16 24.78 - 25.93 4.01

* p< 0.05

Figure 1. Distribution of Body Mass Index by Age and Gender



Table 2b. BMI distribution by socio-demographic characteristics among males and females

Characteristics Mean Median 95% CI SD F, t value P value

Male Age (years)

30-39 23.97 23.57 23.13 - 24.82 3.75 F= 14.725 <0.001*40-49 25.57 24.64 24.83 - 26.31 3.0950-59 27.75 27.35 26.86 - 28.63 3.52≥ 60 26.45 27.16 25.34 - 27.56 2.36

EthnicityMalay 25.65 25.22 25.09 - 26.21 3.88 F= 0.731 0.489Chinese 26.2 25.81 25.08 - 27.33 3Indian 25.21 24.61 23.88 - 26.54 2.4

Marital statusMarried 25.99 25.64 25.51 - 26.47 3.5 t= 3.608 <0.001*Not married 23.24 22.43 21.44 - 25.03 4.34

Educational levelPrimary 22.21 23.29 14.45 - 29.97 4.87 F= 2.192 0.114Secondary 26.18 25.9 25.00 - 27.36 3.87Tertiary 25.65 25.14 25.13 - 26.18 3.6

OccupationAcademic 25.75 25.29 25.22 - 26.27 3.24 t= 0.297 0.788Non academic 25.59 24.64 24.60 - 26.59 4.45

Income Low 24.02 23.1 22.73 - 25.32 4.37 F= 43.238 <0.001*Medium 25.77 25.14 24.98 - 26.55 3.32High 26.32 26.09 25.68 - 26.96 3.43

Female Age (years)

30-39 21.22 20.48 20.55 - 21.89 3.79 F= 43.238 <0.001*40-49 23.96 22.9 22.95 - 24.96 3.8150-59 28.83 29.21 27.47 - 30.18 4.07≥ 60 33.64 33.64 25.95 - 41.33 0.85

EthnicityMalay 23.41 22.06 22.75 - 24.06 4.78 F= 0.839 0.434Chinese 22.75 20.32 18.31 - 27.19 6.2Indian 21.11 21 18.19 - 24.03 3.15

Marital statusMarried 23.37 21.82 22.65 - 24.08 4.83 t= 0.379 0.705Not married 23.06 22.04 21.61 - 24.51 4.75

Educational levelPrimary 19.81 20.86 14.01 - 25.61 2.33 F= 11.595 0.010*Secondary 25.59 25.4 24.32 - 26.86 5.28Tertiary 22.33 21.39 21.65 - 23.01 4.23

OccupationAcademic 23 21.8 22.09 - 23.91 4.37 t= 0.816 0.415Non academic 23.52 22 22.64 - 24.40 5.1

IncomeLow 23.51 22.17 22.17 - 24.85 4.85 F= 1.139 0.322Medium 22.77 21.37 21.74 - 23.80 5.08High 23.86 23.02 22.84 - 24.88 4.38

* p< 0.05



Table 3. Respondent’s BMI by health-related characteristics

Characteristics Mean Median 95% CI SD F, t value P value

Both gender Smoking status

Current smoker 25.86 24.96 24.75 - 26.98 3.74 F= 7.857 <0.001*Former smoker 26.51 26.12 25.35 - 27.66 3.66Never smoked 24.13 23.59 23.67 - 24.59 4.51

Alcohol consumptionCurrent drinker 24.64 24.28 22.67 - 26.61 3.55 F= 1.035 0.356Former drinker 26.03 26.72 23.75 - 28.32 4.44Never drank 24.46 24.08 24.03 - 24.88 4.46

Activity LevelInactive 24.88 25.32 24.08 - 25.67 4.51 F= 4.160 0.016*Insufficiently active 23.21 22.89 22.19 - 24.22 4.48Sufficiently active 24.74 24.12 24.21 - 25.28 4.33

Hypertension status Normotensive 22.83 22.63 22.39 - 23.26 3.8 t= 13.207 <0.001* Hypertensive 27.76 27.75 27.17 - 28.34 3.72

Male Smoking status

Current smoker 25.86 24.96 24.75 - 26.98 3.74 F= 0.908 0.405Former smoker 26.34 26.11 25.17 - 27.50 3.6Never smoked 25.47 25.14 24.86 - 26.07 3.7

Alcohol consumptionCurrent drinker 25.41 24.82 23.56 - 27.26 3.05 F= 0.315 0.73Former drinker 26.45 26.72 24.26 - 28.64 3.62Never drank 25.66 25.23 25.15 - 26.18 3.74

Activity LevelInactive 26.5 26.26 25.49 - 27.51 3.63 F= 1.636 0.197Insufficiently active 25.32 26.31 24.15 - 26.48 3.06Sufficiently active 25.49 24.54 24.87 - 26.10 3.8

Hypertension status Normotensive 24.05 23.83 23.50 - 24.61 3.14 t= 8.436 <0.001* Hypertensive 27.66 27.49 27.01 - 28.31 3.34

Female Smoking status

Current smoker (Nil) N.A. N.A. N.A. N.A. t= -1.941 0.054Former smoker 29.84 29.84 0 - 70.41 4.51Never smoked 23.25 21.82 22.61 - 23.88 4.78

Alcohol consumptionCurrent drinker 19.62 19.62 0 - 42.19 2.51 F= 0.747 0.475Former drinker 24.67 25.33 13.47 - 35.87 7.03Never drank 23.31 21.92 22.67 - 23.96 4.78

Activity LevelInactive 23.74 22.59 22.64 - 24.84 4.74 F= 2.540 0.081Insufficiently active 21.93 19.96 20.55 - 23.31 4.75Sufficiently active 23.64 22.11 22.69 - 24.59 4.82

Hypertension status Normotensive 21.93 21.11 21.33 - 22.53 3.99 t= 9.212 <0.001* Hypertensive 27.95 29 26.70 - 29.20 4.43

* p< 0.05



Table 4. Prevalence of obesity by age, gender and ethnicity

CharacteristicUnderweight

< 18.49 Normal weight

18.50-24.99Pre-obese

25.00-29.99Obese class I

30.34.99Obese class II

35-39.99

Age

30-39 41 (20.0) 118 (57.6) 33 (16.1) 12 (5.8) 1 (0.5)

40-49 3 (2.3) 73 (57.5) 41 (32.3) 10 (7.9) 0 (0.0)

50-50 0 (0.0) 20 (20.0) 51 (51.0) 23 (23.0) 6 (6.0)

≥ 60 0 (0.0) 4 (18.2) 16 (72.7) 2 (9.1) 0 (0.0)

Total (n= 454) 44 (9.7) 215 (47.4) 141 (31.1) 47 (10.3) 7 (1.5)

Gender

Male (n= 231) 3 (1.3) 107 (46.3) 93 (40.3) 25 (10.8) 3 (1.3)

Female (n= 223) 41 (18.4) 108 (48.4) 48 (21.5) 22 (9.9) 4 (1.8)

Ethnicity

Malay (n= 392) 39 (9.9) 188 (48.0) 118 (30.1) 40 (10.2) 7 (1.8)

Chinese (n= 40) 4 (10.0) 14 (35.0) 15 (37.5) 7 (17.5) 0 (0.0)

Indian (n= 22) 1 (4.5) 13 (59.1) 8 (36.4) 0 (0.0) 0 (0.0)

0.001), 30-39 and 60 years and above (p= 0.021) and 40-49 and 50-59 (p= 0.002). However, there was no significant difference in the mean BMI between age groups 40-49 and 60 years and above and 50-59 and 60 years and above. For females a significant difference was noted in mean BMI between all age groups except between respondents who were 50-59 and 60 years and above. It was also noted that the male respondents who were married had significantly higher mean BMI than unmarried males (t= 3.60, p< 0.001), whereas, no significant association was found among females. Males mean BMI increased significantly by their income level (F= 43.23, p< 0.001). Using Post Hoc-Tukey test for males showed that there was a significant difference in the mean BMI between low and medium income (p= 0.03) and low and high income (p= 0.001), but no significant difference was found between medium and high income groups. This association was not significant among females. In addition, significant association was noted between females mean BMI and education (F= 11.59, p= 0.01). Comparing all the education groups showed that there was a significant difference in the mean BMI between secondary and tertiary groups (p< 0.001). No significant association was found between other education groups (Post Hoc-Tukey test). Table 3 shows the distribution of BMI by health-related factors. Overall, significant difference was found between mean BMI with smoking status, activity level and hypertension status. The mean BMI was significantly higher among both males (t= 8.43, p< 0.001) and females (t= 9.21, p< 0.001) who had hypertension. Table 4 shows that 31.1% (40.3% males and 21.5% females) of the respondents were overweight and 11.8% (12.1% males and 11.7% females) were obese. The majority of the respondents who were between 30-39 and 40-49 had normal weight, whereas, the majority of the respondents between 50 to 59 years old and 60 years and above were overweight or obese. Although the results showed a clear gender difference in the prevalence of overweight with more males than females, there was no significant gender difference in the prevalence of obesity. Findings also showed that 30.1% of Malays, 37.5% of Chinese and 36.4% of Indian staffs were overweight. About 12.0% of Malays, 17.5% of Chinese and none of the Indians were obese (BMI ≥ 30 kg/m2). Table 5 shows the results of the logistic regression analysis. After adjusting for all the variables in the model, gender, age, occupation, smoking, alcohol intake and physical inactivity were the main predictors of obesity.

The overall accuracy of this model to predict the subjects having obesity was 89.0% and the area under ROC curve was 77.7%. Females were 2.34 times more likely to be obese, those who were 50 years and above were 8.55 times more likely to be obese as compared to those aged below 50 years (OR= 8.552, 95% CI= 3.93 – 18. 64), non-academic staff were 2.51 times more likely to be obese as compared to academic staff (OR= 2.51, 95% CI= 1.24 – 5.10), current smokers were 3.21 times more likely to be obese as compared to never smokers (OR= 8.552, 95% CI= 1.09 – 9.48), those ever had consumed alcohol were 5.09 times more likely to be obese as compared to never consumed alcohol (OR= 5.09, 95% CI= 1.65 – 15.67) and those who were physically inactive were 2.35 times more likely to be obese as compared to those physically active (OR= 2.35, 95% CI= 1.09 – 5.05).



DISCUSSION In Malaysia, cardiovascular diseases have been the leading cause of death for the past 40 years [16]. The most important CVD risk factors are high blood pressure, obesity, high blood cholesterol, cigarette smoking, diabetes, physical inactivity and having an unhealthy reaction to stress. The prevalence of high blood pressure, cigarette smoking, diabetes, physical inactivity amongst Malaysians is high. The prevalence of hypertension amongst adults aged 30 years and above has increased from 32.9% in 1996[11] to 40.5% in 2004[16] and to 42.6% in 2006[13]. In a study carried out by Rampal et al. in 2010 among 1,778 school children aged 13-17 years in the Putrajaya, showed that the overall prevalence of pre-hypertension and hypertension was 11.1% and 11.6% respectively [17]. The prevalence of hypertension was significantly higher in the adolescents who were overweight or obese. The results of their study also showed that there was a significant correlation between BMI and hypertension (r = 0.52, r2 = 0.27, p= 0.001) [17]. In this study overall percentage of overweight and obesity was 31.1% and 11.8% respectively as compared to the national prevalence of 29.1% and 14% [13]. In this study those who had non-academic position were more likely than staffs with academic position to be obese. It is possible that those with academic position may have more knowledge about obesity and its complications and they try to follow a healthier lifestyle as compared to the non-academic staff. Also, obese respondents were around 3 times more likely than non-obese to be smoker. Being a smoker is due to following an unhealthy lifestyle. Therefore smokers may also not pay attention to their weight status as an important factor related to their health status. This could suggest that they may not have an appropriate diet. Therefore, their energy intake and also their knowledge about a normal and balanced diet should be considered in future studies. Staffs with hypertension were around nine times more likely than normal staffs to be obese. BMI increased significantly with age, and this was consistent with previously published studies. [18, 19, 20] It is also interesting to note that among the males staff only 19.9% were current smokers and none of the females were current smokers. This is very low as compared

Table 5. Logistic regression analysis of the factors associated with obesity

Variables β Adjusted OR 95% CI P value

Gender

Male 1

Female 0.85 2.34 1.036 - 5.289 0.041

Age Group

30 to less than 40 1

40 to less than 50 0.193 1.213 0.500 – 2.945 0.669

50 and above 2.146 8.552 3.924 – 18. 636 < 0.001

Occupation

Academic Staff 1

Non-academic Staff 0.921 2.511 1.237 – 5.096 0.011

Smoking

Never 1

Former 0.995 2.706 0.901 – 8.123 0.076

Current 1.166 3.209 1.086 – 9.477 0.035

Alcohol Intake

Never 1

Ever 1.627 5.089 1.652 – 15.673 0.005

Physical Activity

Active 1

Insufficiently Active -0.136 0.872 0.252 – 3.017 0.829

Inactive 0.853 2.347 1.091 – 5.047 0.029

Nagelkerke R Square = 0.218; Hosmer and Lemeshow Test, p = 0.197; the overall accuracy of this model to predict the subjects having obesity was 89.0%; area under ROC curve = 77.7%.



to the national prevalence of current smokers of 46.4% in males and 1.6% in females [13]. This could either be due to the fact that tobacco control measures taken by the university and declaring the whole university as a ‘Smoke Free University’ have shown results or due to under reporting as smoking is regarded as a socially unacceptable behavior in the university. The prevalence of hypertension (another risk factor for NCD) in this study was 34.4% (45.5% in males and 22.9% in females) among university staff which is lower than the national prevalence of 42.6% (41.7% in males and 43.4% in females) [13].

As the prevalence of overweight and obesity is high among the university staff, a comprehensive integrated NCD prevention program should be implemented. Meta-analyses of Randomized Controlled Trials (RCTs) have shown that a weight-reducing diet, combined with exercise, produces significant weight loss, reduces total cholesterol and LDL-cholesterol, increases HDL-cholesterol, and improves control of blood pressure and diabetes [21, 22]. A meta-analysis of RCTs found that a net weight reduction of 5.1 kg (95% CI 4.25 to 6.03 kg), resulting from restricted energy intake, increased physical activity or both, reduced systolic blood pressure by 4.44 mmHg (95% CI 2.95 to 5.93 mmHg) and diastolic blood pressure by 3.57 mmHg (95% CI 2.25 to 4.88 mmHg) [23]. There should be more intensive inter-sectoral and intra sectoral collaboration as well as community partnership and ownership. There is also a need to establish proactive networks for building up capacity in research and training, mobilizing contributions from within the country and overseas.

In conclusion, the prevalence of overweight and obesity was 31.1% (40.3% males and 21.5% females) and 11.8% (12.1% males and 11.7% females) respectively. Logistic regression analysis showed that obesity was associated with gender, age, occupation, smoking, alcohol intake and physical inactivity.

ACKNOWLEDGMENTWe would like to thank the Dean of the Faculty of Medicine and Health Sciences, Registrar University Putra Malaysia and all the staff involved in this study for their assistance, support and cooperation.

REFERENCES[1] The National Health Service Information Centre, Lifestyles Statistics. Statistics on obesity, physical activity and

diet: England 2010.

[2] Rimm EB, Stampfer MJ, Giovannucci E, et al. Body size and fat distribution as predictors of coronary heart disease among middle-aged and older US men. Am J Epidemiol 1995; 141: 1117-1127.

[3] World Health Organization. Preventing Chronic Disease - A Vital Investment. WHO and the Public Health Agency of Canada. World Health Organization: Geneva, Switzerland 2005.

[4] McGee DL, Diverse Populations Collaboration. Body mass index and mortality: A meta-analysis based on person-level data from twenty-six observational studies. Ann Epidemiol 2005; 15(2): 87-97.

[5] Zhou BF. Effect of body mass index on all-cause mortality and incidence of cardiovascular diseases - report for meta-analysis of prospective studies open optimal cut-off points of body mass index in Chinese adults. Biomed Environ Sci 2002; 15(3): 245-252.

[6] Wilson PW et al. Overweight and obesity as determinants of cardiovascular risk: The Framingham experience. Arch Intern Med 2002; 162(16): 1867-1872.

[7] Calle EE et al. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med 2003; 348(17): 1625-1638.

[8] World Health Organization. Global health risks: Mortality and burden of disease attributable to selected major risks. Geneva 2009.

[9] Willett WC, Manson JE, Stampfer MJ, Danaei G, Lin JK, Paciorek CJ. Weight, weight change, and coronary heart disease in women: Risk within the ‘normal’ weight range. JAMA 1995; 273: 461-465.

[10] Finucane MM, Steven GA, Cowan MJ, et al. National, regional, and global trends in body-mass index since 1980: systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants. Lancet 2011; 377(9765): 557- 67.



[11] Ministry of Health Malaysia. The Second National Health and Morbidity Survey 1996. Ministry of Health Malaysia NHMS II Report 1997.

[12] Rampal L, Rampal S, Khor GL, et al. A national study on the prevalence of obesity among 16,127 Malaysians. Asia Pac J Clin Nutr 2007; 16(3): 561-566.

[13] Ministry of Health Malaysia. The Third National Health and Morbidity Survey 2006. Ministry of Health Malaysia NHMS III Report 2007.

[14] NIH. Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults NIH Publication No. 98-4083, 1998.

[15] U.S. Department of Health and Human Services National Institutes of Health, National Heart, Lung, and Blood Institute. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure 2004

[16] Rampal L, Rampal S, Azhar MZ, Rahman AR. Prevalence, awareness, treatment and control of hypertension in Malaysia: A national study of 16,440 subjects. Public Health 2008; 122: 11-18.

[17] Rampal L, Ng KC, Nur I I, Farah I Z, Mohammad N I et al., Prevalence of Hypertension among Malay Adolescents, Putrajaya, Malaysia 2010. Malaysian J of Med and Health Sciences 2011; 7 (2): 53-60

[18] Aekplakorn W, Chaiyapong Y, Neal B, Chariyalertsak S, et al. Prevalence and determinants of overweight and obesity in Thai adults: Results of the Second National Health Examination Survey. J Med Assoc Thai 2004; 87(6): 685-93.

[19] Kantachuvessiri A, Sirivichayakul C, KaewKungwal J, Tungtrongchitr R, Lotrakul M. Factors associated with obesity among workers in a metropolitan waterworks authority. Southeast Asian J Trop Med Public Health 2005; 36(4): 1057-65.

[20] Martin AR, Nieto JMM, Ruiz JPN, Jimenez LE. Overweight and obesity: The role of education, employment and income in Spanish adults. Appetite 2008; 51: 266-272.

[21] Avenell A et al. What are the long-term benefits of weight reducing diets in adults? A systematic review of randomized controlled trials. J Hum Nutr Diet 2004 Aug;17(4): 317-35.

[22] Avenell A et al. What interventions should we add to weight reducing diets in adults with obesity? A systematic review of randomized controlled trials of adding drug therapy, exercise, behavior therapy or combinations of these interventions. J Hum Nutr Diet 2004; 17(4): 293-316

[23] Neter JE et al. Influence of weight reduction on blood pressure: A meta-analysis of randomized controlled trials. Hypertension 2003; 42(5): 878-884.


Toxicity Evaluation of Methanol Extract of Clitoria ternatea L. Leaf

1L Kamilla, 1S Ramanathan, 2S Sasidharan* & 1SM Mansor1Center for Drug Research, Universiti Sains Malaysia,

11800 Minden, Penang, Malaysia2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,

11800 Minden, Penang, Malaysia.

ABSTRACTThe Clitoria ternatea (Fabaceae) root, seed, and leaf are commonly used in Ayurvedic medicine in Malaysia and Indonesia. The methanol leaf extracts of C. ternatea was tested for toxicity by means of brine shrimp lethality test and acute oral toxicity assay. In the brine shrimp lethality test, the leaf extract were non-toxic or showed weak lethality (LC

50 > 1 mg/ml) at the 6 h, 12 h and 24 h incubation period.

Nevertheless, at the 48 h incubation time, the leaf extract exhibited moderate toxicity activity with LC50

values of 0.49 mg/ml. In the acute toxicity study using mice, the median lethal dose (LD50

) of the extract was found greater than 2000 mg/kg, and we found no pathological changes by means of macroscopic examination of tissues of mice treated with the extract. We conclude that the C. ternatea leaf extract is not toxic in mice and brine shrimp.

Keywords: Clitoria ternatea, toxicity study, brine shrimp assay, acute oral toxicity

*Corresponding author: [email protected], [email protected]


INTRODUCTIONClitoria ternatea L. (Fabaceae), commonly called Shankapushpi in Ayurvedic medicine, is used traditionally to treat various ailments [1]. The roots, seeds and leaves of C. ternatea are commonly used in Ayurvedic medicine. The herb had been used for centuries as a memory enhancer, nootropic, antistress, anxiolytic, antidepressant, anticonvulsant, tranquilizing and sedative agent. C. ternatea young shoots, leaves, flowers and tender pods are eaten as a vegetable in Kerala (India) and in Philippines [2]. In Malaysia, the flowers used used to impart a bright blue color to rice cakes and the leaves to impart a green color to food. In addition, C. ternatea has a relatively well documented neuropharmacological action which justifies its use in Central Nervous System (CNS) diseases in Ayurvedic medicine. C. ternatea has been used in ‘Medhya Rasayana’ a rejuvenating recipe used for the treatment of neurological disorders and thought to strengthen a person’s intellectual capacities [2]. C. ternatea is a potential candidate for enhancing learning and memory, Clitoria ternatea Linn. root extract treatment used during a growth spurt period enhances learning and memory in rats. In traditional medicine transmitted orally or in writing (esp. Ayurveda) various therapeutic effects have been attributed to roots, leaves and seeds of C. ternatea [2]. Its extracts possess a wide range of pharmacological activities including antimicrobial, antipyretic, anti-inflammatory, analgesic, diuretic, local anaesthetic, antidiabetic, insecticidal, blood platelet aggregation inhibiting and vascular smooth muscle relaxant properties [2].

Plants are known to generate several chemical constituents, which are naturally toxic to bacteria and fungi. Despite significant progress made in molecular biology and medicinal chemistry that leads to the development of new drugs, plants have been a potential source in the discovery of bioactive compounds for development into clinical agents [4]. In a recent study, leaf extract of C. ternatea showed to be effective against Aspergillus niger, a pathogenic microorganism which cause significant infection in immunocompromised hosts and severely ill-patients [5].

There is a high degree of concern regarding the safe use of plant extracts and for this reason, preclinical and clinical toxicological evaluation of these extracts are needed [6]. With the aim to assure the safety of the extract, and also due to the scarcity of literature information about C. ternatea extract toxicity, an initial toxicological evaluation using brine shrimp assay and an acute oral toxicity of the methanol extract were carried out in this study.

MATERIALS AND METHODSPlant material

Raw and mature C. ternatea leaves were collected in Seberang Jaya, Penang, Malaysia in January 2008. The leaves were then washed with water to remove dirt prior to the drying process (40oC for 3 days). The authencity work was carried out by Dr S. Sreeramanan, a botanist from School of Biological Sciences, Universiti Sains Malaysia where the herbarium was deposited with a voucher number of 11006.


L Kamilla, S Ramanathan, S Sasidharan & SM Mansor34

Preparation of plant extracts

The leaf extract was prepared by maceration of dried powdered plant material in methanol solvent for 3 days [7]. Two hundred g of powdered leaf was macerated in 400 mL methanol under stirring conditions for 72 h. The macerated extract was then filtered through No. 1 Whatman filter paper. The methanol crude extract was vaporized to dryness using the rotary evaporator (BUCHI Rotary Evaporator R-110) and freeze dried.

Experimental animals

Swiss albino mice (20-25g) of both male and female (nulliparous and non pregnant) were used for the assessment of the acute oral toxicity. The animals were kept in a plastic cage with ventilated stainless steel cover and allowed to adjust to the laboratory environment over a period of one week before the commencement of the experiment. The animals were supplied with food and water ad libitum. The experimentals protocols were approved by Universiti Sains Malaysia Animals Ethics Committee (USM/PPSF/50(063)JLD 3).

Brine shrimp, Artemia salina assay

The Brine shrimp lethality test was carried out as described by Meyer [8]. Brine shrimp eggs, Artemia salina was hatched in artificial seawater (prepared by dissolving 38g of sea salt in 1L distilled water). After 24 h incubation at room temperature (22-29oC), the larvae (nauplii) were attracted to one side of the vessel with a light source and collected with pipette. Extract was dissolved in 2% aqueous Tween 80 (v/v). Seawater was placed in all the test tubes. A two-fold dilution was carried out to obtain a concentration of 10 mg/mL - 0.0195 mg/mL of the extract. A suspension of nauplii containing 15 larvae was counted and added into each test tube and incubated for 24 h. At the 6 h and 12 h, the test tubes were examined and the number of dead nauplii in each tube was counted and recorded. The mortality rate (%) was calculated by dividing the number of dead shrimps with total number shrimps and multiplied with 100 %. Lethality concentration fifty (LC

50 values) for the extract was calculated using the equation given for the

graph. The general toxicity activity was considered weak when the LC50

was between 0.5 and 1 mg/mL, moderate when the LC

50 was between 0.1 and 0.5 mg/mL, and designated as strong when the LC

50 ranged from 0 to 0.1 mg/ml

[9]. Potassium dichromate and 2 % aqueous Tween 80 was use as positive and negative control respectively.

ACUTE ORAL TOXICITY ASSAYSighting study

The sighting study was conducted as per OECD-420 guidelines [10]. The animals fasted for 4 hour although still they were allowed free access to water before the commencement of the experiments [11]. Doses of 50, 300 and 2000 mg/kg of leaf extract were given orally to the mice. The control animals received 2% aqueous Tween 80.

Acute oral main study

The acute oral study was performed as per OECD [11] guidelines [12]. The animals will fast for 4 hour although they will be allowed free access to water before the commencement of the experiment. The animals used for the main study will be divided into 4 groups with each group containing six mice. The male control group (A) and female control group (B) will receive 2 % aqueous Tween 80 orally in constant volume of 10 mL/kg. While the male test group (C) and female test group (D) will be treated with 2000 mg/kg of leaf extract.

Observation of animals

All animal were observed individually after dosing at least once during the first 30 minutes periodically during the first 24 hours, with special attention given to first 4 hours and daily thereafter for a total of 14 days. The visual observations included changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system as well as somatomotor activity and behavioral pattern. Individual weight of animals was determined shortly before the test substance was administered and at least weekly thereafter. Animal found to show sign of toxicity, severe pain or enduring signs of severe distress were humanely killed. All test animals (including those that died during the test or were removed from the study for animal welfare reasons) were subjected for gross necropsy. The appropriate dose was then selected and used for the main study.

Histopathological studies

Slices of liver, kidney, lung, spleen and heart were fixed in 4% formaldehyde, embedded in paraffin wax, sectioned at 8 µm and stained with haematoxylin and eosin. Detailed microscopic examination was carried out in those organs of both control and treatment groups of both sexes.


Toxicity of Clitoria ternatea 35

Data analysis

The mean results of brine shrimp mortality against the logarithms of concentrations were plotted using the Microsoft Excel computer program, which also gives the regression equations. The regression equations were used to calculate LC

50 value. The data for body weight and organ weight were expressed as the mean ± standard error of the mean

(SEM). Body weight evolution and weight of the organs from the control and the test groups were compared using the t–test and two way ANOVA run on the software SPSS for Windows.Values of p < 0.05 were considered significant. t–test and two way ANOVA run on the software SPSS for Windows.Values of p < 0.05 were considered significant. t

RESULTS AND DISCUSSIONMost of the herbal products used in traditional medicine have solid scientific support with regard to their effectiveness. There is little information available on the possible risks of C, ternatea to human health Based on their long-term exploit by humans one might expect C, ternatea used in traditional medicine to have low toxicity. However, recent investigations have revealed that many plants used as food or in traditional medicine showed toxic effects in in vitroassays. This raises concern about the potential toxicity resulting from the application of such plants by the consumers. Therefore, this research work aimed to assess the potential hazards of C. ternatea methanol extract by using brine shrimp lethality assay and Acute oral toxicity assay.

Brine shrimp lethality assay

Brine shrimp assay has been an excellent method for preliminary investigations of toxicity and to screen medicinal plants popularly used in traditional medicine. This method was used in this study because it is rapid, inexpensive and only utilizes small amount of extract [4]. Moreover, a positive correlation between the lethality to brine shrimp and the corresponding oral lethal dose in mice of medicinal plants has been demonstrated by Parra et al. [13].

The brine shrimp assay has proven to be a convenient system for evaluating the toxicity of plant extract since the larvae sensitive to various compounds in the crude extract. Hence, brine shrimp toxicity assay was carried out using the leaf extract at 6th h, 12th h, 24th h and 48th h incubation time. The lethal concentration (LC

50) of the leaf extract for 6

h, 12 h, 24 h and 48 h exposure were > 10 mg/mL, > 10mg/ml, 1.46 mg/ml and 0.49 mg/mL respectively.

Extract and Controls Lethality Concentration fifty (LC50) (mg/ml)

6 hr 12 hr 24 hr 48 hr

LeafPotassium dichromate

> 10 + 0.211.11 + 0.04

> 10 + 0.190.38 + 0.01

1.46 + 0.020.024 + 0.001

0.49+ 0.010.013 + 0.005

The LC50

was obtained by linear regression equations. LC50 value between 0 and 0.1 mg/ml was considered toxic. Data are mean ± SEM

Table 1. Toxic effects of the Clitoria ternatea leaf methanol extract after 6 h and 12 h using brine shrimp lethality assay.

According to Padmaja [9], the general toxicity activity was considered weak when the LC50

was between 500 and 1000 µg/mL, moderate when the LC

50 was between 100 and 500 µg/mL, and designated as strong when the LC

50

ranged from 0 to 100 µg/mL. Hence, the toxicity activity of leaf extracts at 6 h, 12 h and 24 h against the Brine shrimps were considered weak. Meanwhile, at 24 h exposure, the leaf extracts exhibited a moderate level of toxicity activity against the Brine shrimps. This preliminary toxicity results show that leaf extract is not toxic to brine shrimps after a short period of exposure. The results of the current study further confirmed the traditional claims of this plant in folk medicine.

Acute oral toxicity assay

Further evidence of the brine shrimp assay results was obtained from acute oral toxicity study in mice. Toxicological evaluation is carried out in various experimental animals to predict toxicity and to provide guidelines for selecting a ‘safe’ dose in humans. Some of the factors are capable to interfere with the toxicity of medicinal plants extract. The factors include the intrinsic property of the botanical drug or it happens during the process of extract preparation [14].

In this study, the acute oral toxicity of leaf extract of C. ternatea was evaluated by employing the OECD [11]

guideline that allows the evaluation of lethality potential and possible toxicant effects developed in a short period of time. Extract with 50, 300, and 2000 mg/kg body weight doses were administered as recommended by OECD guidelines [11]. Administration of further higher doses was considered physiologically unsound and is not generally



recommended. The doses tested in this study did not cause any mortality or any signs of acute toxicity in the tested animals in short

term (i.e. 48 h) and long term (i.e. 14 days) observation period. The dose did not cause any appreciable alterations in water and food intake (data not shown) during 2 weeks in both sexes. There were no significant differences (P>0.05) between the weekly mean body weight between control and the animals treated with extract except for day 1 the control group shows heavier mean weight body compared to group treated with 2000 mg/kg body weight of extract for both sex. Furthermore, body weight gain throughout the observation period among the treated animals was comparable to their respective controls. No sex-related differences were evident in either species. In addition, there were no significant differences in the mean organ weight between control and animals treated with 50, 300, and 2000 mg/kg body weight.

Treatment group

Dose(mg/kg)

Weekly mean body weight (g) Mean weight gain (g)Day 1 Day 7 Day 14

Male Control *23.54 ± 0.24 25.73 ±0.72 27.20 ±0.86 3.66 + 0.21

50 21.33 ± 0.32 24.86 ±0.42 26.83 ±0.66 5.50 + 0.41

300 24.90 ± 0.22 25.62 ±0.42 26.54 ±0.52 1.64 + 0.10

2000 *21.55 ± 0.45 24.15 ±0.57 25.18 ± 0.67 3.63 + 0.20

Female Control *20.27 ± 0.14 21.79 ± 0.30 23.67 ± 0.42 3.40 + 0.22

50 22.41 ± 0.55 23.36 ± 0.42 25.45 ± 0.37 3.04 + 0.19

300 24.50 ± 0.35 25.76 ± 0.51 26.98 ± 0.32 2.48 + 0.17

2000 *21.47 ± 0.45 22.61 ± 0.55 23.85 ± 0.38 2.38 + 0.20

Data are mean ± SEM; n=6 for control and 2000 mg/kg; * p < 0.05.

Table 2. Mean body weight of mice after 14 days treatment with leaf extract of C. ternatea

Treatment group

Dose(mg/kg)

Mean organ weight (g)

Heart Lung Liver Kidney Spleen

Male Control 0.13 ± 0.01 0.21 ± 0.01 1.79 ± 0.12 0.39 ± 0.02 *0.20 ± 0.02

50 0.12 ± 0.02 0.20 ± 0.01 1.64 ± 0.01 0.44 ± 0.01 0.09 ± 0.02

300 0.13 ± 0.01 0.32 ± 0.02 2.07 ± 0.02 0.43 ± 0.01 0.15 ± 0.02

2000 0.13 ± 0.00 0.25 ± 0.01 1.98 ± 0.11 0.44 ± 0.03 *0.14 ± 0.2

Female Control 0.10 ± 0.00 0.18 ± 0.01 1.32 ± 0.06 0.28 ± 0.01 0.08 ± 0.00

50 0.11 ± 0.01 0.16 ± 0.02 1.14 ± 0.05 0.24 ± 0.01 0.08 ± 0.00

300 0.11 ± 0.02 0.23 ± 0.02 1.65 ± 0.06 0.33 ± 0.02 0.07 ± 0.00

2000 0.10 ± 0.01 0.19 ± 0.02 1.29 ± 0.11 0.25 ± 0.15 0.09 ± 0.01

Data are mean ± SEM; n=6 for control and 2000 mg/kg; * p < 0.05.

Table 3. Mean organ weight of mice after 14 days treatment with leaf extract of C. ternatea

The acute toxicity study indicated that 2 % Tween 80 suspension of C. ternatea extract is not toxic when administered orally to experimental animals. Therefore, the acute minimum fatal dose of C. ternatea leaf extract for mice is higher than 2000 mg/kg body weight which indicates some high level of safety margin in oral formulation. Gross examination at autopsy and histopathological evaluations of the various organs stained with hematoxylin and



eosin revealed no significant differences (Figure 1; Figure 2) for both sexes. In comparison, the study conducted by Jothy et al. [15] revealed similar results using Cassia fistula seed extract at dose 5000 mg/kg. No toxic symptoms or mortality were observed in any animals, which lived up to 14 days after the administration of methanol seeds extract at single dose level of 5000 mg/kg body weight in their study. They also reported that the behavioural patterns of animals were observed first 6 hours and then 14 hours after the administration and the animals in both vehicle treated and extract-treated groups were normal and did not display significant changes in behavior, skin effects, breathing, impairment in food intake and water consumption, postural abnormalities and hair loss [15].

Figure 1.Figure 1. Histological examination of heart (a), kidney (b) liver (c) and lung (d) of female Histological examination of heart (a), kidney (b) liver (c) and lung (d) of female mice. A = alveoli, M = myocardium, B = bronchiole, CV = central vein, RC = renal capsule, 1: treated, and 2: control



Based on the results of the brine shrimp lethality test (LC50

> 1 mg/ml) and acute oral toxicity studies, it was concluded that a dose of 2000 mg/kg body weight of the extracts of C. ternateaconcluded that a dose of 2000 mg/kg body weight of the extracts of C. ternateaconcluded that a dose of 2000 mg/kg body weight of the extracts of leaf extract given orally appeared to be non-toxic. However, there must be a point at which it can be concluded that a test substance is practically non-toxic or non-lethal after an acute exposure. The dose of 2000 mg/kg body weight for acute oral toxicity is generally considered to be very high. Thus C. ternatea leaf extracts have a high margin of safety. However, further toxicity studies are needed to determine the effects of this extract on chronic oral toxicity, on animal fetus, on pregnant animals, and their reproductive capacity to complete the safety profile of this extract.

Figure 2.Figure 2. Histological examination of heart (a), kidney (b) liver (c) and lung (d) of male Histological examination of heart (a), kidney (b) liver (c) and lung (d) of male mice. A = alveoli, M = myocardium, B = bronchiole, CV = central vein, RC = renal capsule, 1: treated, 2: control



ACKNOWLEDGEMENTThis research project was supported by the Research University Grant and Short Term Grant Scheme (304 / CDADAH / 638128). The author (L. Kamilla) would also like to acknowledge the financial assistance provided by USM Fellowship, Universiti Sains Malaysia.

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289-290.

[2] Mukherjee PK, Kumar V, Kumar NS, Heinrich M. The Ayurvedic medicine Clitoria ternatea - From traditional use to scientific assessment. J Ethnopharmacol 2008; 120: 291-301.

[3] SharmaRK, Bhagwan D. Agnivesa’s Caraka Samhita, 3. Varanasi, Chaukhambha Orientalia, 1988: p. 46.

[4] Bastos MLA, Lima MRF, Conserva LM, Andrade VS, Rocha EMM, Lemos RPL. Studies on the antimicrobial activity and brine shrimp toxicity of Zeyheria tuberculosa (Vell.) Bur. (Bignoniaceae) extracts and main constituents. Annals of Clinical Microbiology and Antimicrobials 2009; 8: 1-6.

[5] Kamilla L, Mansor SM, Ramanthan S, Sasidharan S. Effects of Clitoria ternatea leaf extract on growth and morphogenesis of Aspergillus niger. Microsc Microanal 2009; 15: 1-7.

[6] Sasidharan S, Darah I, Jain K. In Vivo and In Vitro toxicity study of Gracilaria changii. Pharm Biol 2008, 46: 413-417.

[7] Cos P, Vlietinck AJ, Berge DV, Maes L. Anti-infective potential of natural products: How to develop a stronger in vitro ‘proof-of-concept’. J Ethnopharmacol 2006; 106: 290-302.

[8] Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med 1982; 45: 31-34.

[9] Padmaja R, Arun PC, Prashanth D, Deepak M, Amit A, Anjana M. Brine shrimp lethality bioassay of selected Indian medicinal plants. Fitoterapia 2002; 73: 508-510.

[10] OECD (Organization of Economic Co-operation Development). Guideline for Testing of Chemical: 420 Acute Oral Toxicity-Fixed Dose Procedure: Acute Oral Toxicity. France, OECD, 2001: 1-14.

[11] OECD (Organization of Economic Co-operation Development). Guideline for Testing of Chemical: 423 Acute Oral Toxicity-Fixed Dose Procedure: Acute Oral Toxicity. France, OECD, 2001; pp. 1-14.

[12] Pandurangan A, Khosa RT, Hemalatha S. Evaluation of anti-inflammatory and analgesic activity of root extract of Solanum trilobatum Linn. Iran J Pharm Res 2008; 7: 217-221.

[13] Parra AL, Yhebra RS, Sardinas IG, Buela LI. Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD

50 value) in mice, to determine oral acute toxicity of plant extracts.

Phytomedicine 2001 9: 395-400.

[14] Barros S, Ropke CD, Sawada TCH, Silva VVD, Pereira SMM, Baros SBDM. Assessment of acute and subchronic oral toxicity of ethanolic extract of Pothomorphe umbellate L. Miq (Pariproba). Brazilian J Pharm Sci 2005; 41: 53-61.

[15] Jothy SL, Zakaria Z, Chen Y, Lau YL, Latha LY, Sasidharan S. Acute Oral Toxicity of Methanolic Seed Extract of Cassia fistula in Mice. Molecules 2011; 16(6): 5268-5282.


Work-Related Hand Injuries: Type, Location, Cause, Mechanism and Severity in a Tertiary Hospital

1A Al-Husuny, 1L Rampal*, 2A Manohar, 3MY Adon & 1AA Ahmad1Department of Community Health, Faculty of Medicine and Health Science, Universiti Putra Malaysia,

43400 Serdang, Selangor, Malaysia2Department of Orthopaedics, Faculty of Medicine and Health Science, Universiti Putra Malaysia,

43400 Serdang, Selangor, Malaysia3Environmental Health Research Center, Institute of Medical Research,

Kuala Lumpur

ABSTRACT Introduction: Work-Related Hand Injuries (WRHIs) may result in disability and diminished productivity and cause economic impacts not only to the individual, worker’s families and industries, but to the local community as well. Objectives: To determine the prevalence of severe Work-Related Hand Injuries (WRHIs) and factors associated at a tertiary hospital. Methods: A pre-tested validated questionnaire was used to obtain data. All patients 18 years and above with WRHIs seen at a tertiary hospital between January 2010 and June 2010 were included in the study. Data was analysed using SPSS version 18. Results: Out of the 297 industrial accidents, 74 (24.9%) were WRHIs. Among those with WRHIs, (47.3%) of them had severe hand injuries. The overall mean age of the respondents was 30.36 (± 9.54 SD) years. Majority (82.5%) of the injuries occurred between Mondays to Friday. Majority (70.1%) of hand injuries were caused by machine and 48.6% of the hand injuries occurred when the hand was caught in the operating part of the machine. Majority (62.1%) of the respondents had fingers’ injuries and 32.4% had open fracture. Bivariate analysis showed that there was significant association between severity of WRHIs and locations of injury, mechanisms of injury, sources of injury and sectors of industry (p < 0.05). Logistic regression analysis showed that WRHIs was significantly associated with source of injury and sector of industry. Respondents with hand injury resulted while operating on mechanical machine was 26 times more likely to report severe WRHIs than those with other sources of their hand injury like (sharp tool, heavy door, and wet floor). Respondents working in metal-machinery industries were eight times more likely to report severe WRHIs than those who working in other sectors of industry like (wood-furniture, constriction, food preparing, service and automotive). Conclusions: WRHIs contributed to 24.9% of all industrial accidents seen at the emergency department and orthopaedic clinic and 47.3% of the respondents with WRHIs had severe hand injuries. Severity of WRHIs was significantly associated with sources of injury and sectors of industry.

Keywords: Prevalence, Severe, Work-Related Hand Injuries



INTRODUCTIONHands play an important role in performing everyday activities. The hand is the most common anatomical site to be injured at work and constitutes 30% of all occupational accidents received at emergency department [1-3]. Work-Related Hand Injuries (WRHIs) occur most commonly in jobs that involve intensive hand activity like manufacturing, construction and food-preparing factories [2]. WRHIs may result in disability and diminished productivity and cause economic impacts not only to the individual, worker’s families and industries, but to the local community as well [4]. Risk factors associated with occurrence of WRHIs can be categorized into injury-related factors, work-related factors, workplace-related factors, medical history, social habits and those related to socio-demographic characteristics [5]. Most severe hand injuries commonly occur among the machine operators and machine maintenance workers [6]. Sorock (2002) reported that machines accounted for 37% of WRHIs [7]. Despite the fact that WRHIs contribute to a significant workload at the emergency rooms and orthopaedic clinics, information about WRHIs is lacking in many countries. The aim of the study was to determine the prevalence of severe WRHIs and its factors associated at a tertiary hospital.


A Al-Husuny, L Rampal, A Manohar, MY Adon & AA Ahmad42

MATERIALS AND METHODSStudy design/Study Location

This hospital based cross sectional study was carried out in a tertiary Hospital.

Study population

All accidents (i.e. industrial and non-industrial) cases aged 18 years and above, seen at the emergency room, the orthopaedic ward, the general surgery ward and the orthopaedic outpatient clinic in tertiary Hospital between January 2010 and June 2010 were included in this study.

Instruments and procedures

A pre-tested validated questionnaire was used to obtain data. The questionnaire was pretested on 30 patients not included in the sample. The outcome variable was WRHIs and the independent variables were age, gender, ethnicity, marital status, types of injury, location of injury, mechanisms of injury, hand activities at the time of injury, causes of injury and time of injury, source of injury, types of injury, location of injury and mechanism of injury.

Modified Hand Injury Severity Score (MHISS) [8]

MHISS was used to determine the severity of hand injury through evaluating the four hand components i.e. Integument, Skeletal, Motor and Neurovascular bundles (ISMN) and quantify injuries to hand, wrist and forearm. MHISS was chosen for use in this study because MHISS is a standardized tool that provides quantifiable and comparable measures of hand injury severity [9]. It has been devised based on the previously-described HISS scoring system [10], with the advantages that MHISS permits quantification of both hand and forearm injuries by assuming that the hand, wrist and forearm are one functional unit. In addition, MHISS quantifies injuries to the neurovascular bundles rather than only the nerve in the HISS scoring system. Each ISMN component contains both absolute scores and scores which are weighted according to the functional importance of the affected ray. Table 1 shows the individual digit weighting factors [10]. For instance, thumb injuries are given a greater weighting than little finger injuries. The total score for each component is doubled by the presence of additional factors such as wound contamination, a compound fracture, crush or avulsion. In amputations, all missing structures are scored as damaged. The overall MHISS is the total of the scores for each ISMN component. The MHISS score is grouped into four categories: Minor, Moderate, Severe or Major Injury, as described for the HISS by Campbell and Kay [10] (MHISS <20 = minor, MHISS 21–50= moderate, MHISS 51–100= severe and MHISS 101and more = major). In the present study, the respondents were categorised into two levels of injury severity using MHISS. Those with MHISS of more than 50 were classified as ‘with severe hand injuries’. Those respondents with a score of 50 or less than 50 were classified as ‘with not severe hand injuries’.

Digit Weighting factors

Thumb x 6lndex x 2Middle x 3Ring x 3Little x 2

*(Campbell & Kay, 1996) [10]

Table 1. The individual digit weighting factors

Statistical analysis

Statistical analysis was carried out using SPSS version 18. Categorical variables were presented as frequencies and percentages. Continuous variables were presented as means with their 95% confidence interval (CI). The Pearson’s chi-square test (x2) test was used to determine the associations between categorical variables. Independent sample t-test was used to compare means between two groups. Binary Logistic regression was used for multivariate analysis. A p-value of < 0.05 was considered as statistically significant.


Work-Related Hand Injuries: Type, Location, Cause, Mechanism and Severity in a Tertiary Hospital 43

Ethical Approval

Approval from the Faculty of Medicine and Health Science, University Putra Malaysia human research committee and approval from the Ministry of Health National Institutes of Health were received before commencement of the study. Informed consent was also obtained from the each respondent before data was collected.

RESULTSA total of 297 industrial-related accidents were seen at the tertiary hospital (emergency room, orthopaedic ward, general surgery ward, and orthopaedic outpatient clinic) from January 2010 to June 2010. Of these 297 industrial-related accidents, 74 (24.9%) were WRHIs. Table 2 shows the prevalence of severe WRHIs by age. Out of the total 74 respondents with WRHIs, 35 respondents had severe WRHIs giving a prevalence of 47.3%. The overall mean age of those respondents with severe WRHIs was 30.20 ± 10.66 years and of those respondents with not severe WRHIs was 30.50 ± 8.54 years. This difference in the mean age was not statistically significant (t= 0.14, df =72 and p = 0.89).

Table 2. Prevalence of severe WRHIs by age

Age group (years) Severe (%) Not severe (%) Total (%)

18-25 yrs 15 (57.7) 11 (42.3) 2626-35 yrs 10 (34.5) 19 (65.5) 2936-45 yrs 5 (50.0) 5 (50.0) 1046 yrs and above 5 (55.6) 4 (44.4) 9Total 35 (47.3) 39 (52.7) 74

Table 3. Types and locations of WRHIs by severity

Variable Severe (%) Not severe (%) Total (%)

Types of injuryOpen fracture 10 (41.7) 14 (58.3) 24 (32.4)Closed fracture 2 (46.7) 10 (83.3) 12 (16.2)Crush hand 11 (68.6) 5 (31.4) 16 (21.5)Amputation 10 (100.0) 0 (0) 10 (13.5)Laceration 1 (20.0) 4 (80.0) 5 (6.8)Finger tips injury 0 (0) 3 (100.0) 3 (4.1)Degloving injury 1 (33.3) 2 (66.7) 3 (4.1)Avulsion 0 (0) 1 (100.0) 1 (1.4)Total 35 (47.3) 39 (52.7) 74 (100.0)

Locations of injuryMultiple fingers 14 (87.5) 2 (12.5) 16 (21.6)Forearm 4 (25.0) 12 (75.0) 16 (21.6)Index finger 7 (46.7) 8 (53.3) 15 (20.2)Thumb 4 (57.3) 3 (42.7) 7 (9.5)Little finger 2 (33.4) 4 (66.6) 6 (8.1)Ring finger 2 (40.0) 3 (60.0) 5 (6.8)Middle finger 1 (25.0) 3 (75.0) 4 (5.4)Palm of the hand 1 (33.4) 2 (66.6) 3 (4.1)Back of the hand 0 (0) 2 (100.0) 2 (2.7)Total 35 (47.3) 39 (52.7) 74 (100.0)



Table 4. Sources of injury, occupational categories and sectors of industry, mechanisms of injury and hand activities at the time of injury by severity of WRHIs

Variable Severe (%) Not severe (%) Total (%)

Sources of injury Machine 31 (59.6) 21(40.4) 52 (70.1)Wet floor 1(7.7) 12 (92.3) 13 (17.6)Heavy door 2 (40.0) 3 (60.0) 5 (6.8)Sharp tool 1 (25.0) 3 (75.0) 4 (5.5)Total 35 (47.3) 39 (52.7) 74 (100.0)

Occupational categoriesMachine operators 15(51.7) 14(48.3) 29 (39.1)Manual materials handlers 2(20.0) 8(80.0) 10 (13.5)Carpenters 6 (66.6) 3 (33.4) 9 (12.2)Construction workers 3 (33.3) 6 (66.7) 9 (12.2)Machinery maintenance workers 4 (66.7) 2 (33.3) 6 (8.1)Food preparers 1 (25.0) 3 (75.0) 4 (5.4)Sheet metal workers 3 (75.0) 1 (25.0) 4 (5.4)Welders 1 (25.0) 2 (75.0) 3 (4.1)

Sectors of industry Metal-machinery 22(55.0) 18(45.0) 40 (54.1)Wood-furniture 7(70.0) 3 (30.0) 10 (13.5)Construction 3(33.4) 6 (66.6) 9 (12.1)Food preparing 3 (42.9) 4 (57.1) 7 (9.5)Services 0 (0) 6 (100.0) 6 (8.1)Automotive 0 (0) 2 (100.0) 2 (2.7)

Mechanisms of injuryHand caught inside machine 22 (61.4) 14 (38.6) 36 (48.6)Cutting 8 (50.0) 8 (50.0) 16 (21.7)Hand hit by hard objects 4 (28.9) 10 (71.1) 14 (18.9)Fall 0 (0) 6 (100.0) 6 (8.1)Piercing 1 (50.0) 1 (50.0) 2 (2.7)

Hand activities at the time of injuryWorking on machine 22 (53.8) 19 (46.2) 41(55.4)Handling objects 5 (31.4) 11 (68.6) 16 (21.5)Lifting objects 1 (14.5) 6 (85.5) 7 (9.5)Working on powered hand tool (circular saw) 5 (100.0) 0 (0) 5 (6.8)Carrying objects 2 (50.0) 2 (50.0) 4 (5.4)Working on non-powered hand tool (knife) 0 (0) 1 (100.0) 1(1.4)

Injury characteristics

Types and locations of the injuries

Table 3 shows the types and locations of the injuries. Open fracture injuries constituted 32.4% of all injuries. This was followed by crush hand injuries (21.5%), closed fracture (16.2%), amputation (13.5%), laceration (6.8%), finger tips injuries (4.1%), skin flap (4.1%) and avulsion (1.4%). Only 21.6% of the injuries was multiple fingers injury. Other site of injury were forearm (21.6%), thumb (9.5%), and palm and back of the hand (6.8%).

Sources of injury, occupational categories and sectors of industry, mechanisms of injury and hand activities at the time of injury by severity of WRHIs



Table 5. Association between severity of WRHIs and risk factors

VariableWRHIs

χ2χ2χ dfP

valuesSevere (%) Not Severe (%) Total

Locations of injury

Finger injuries 26(56.5) 20(43.5) 46 4.15 1 0.042*

Other sites of injury 9(32.1) 19(67.9) 28

Sources of injury

Machine 31(59.6) 21(40.4) 52 9.05 1 0.003*

Other sources 4(18.2) 18(81.8) 22

Mechanisms of injury

Hand caught inside machine 22(61.1) 14(38.9) 36 4.342 1 0.037*

Other mechanisms 13(34.2) 25(65.8) 38

Sectors of industry

Metal-machinery 22(55.0) 18(45.0) 40 7.807 2 0.020*

Wood-furniture 7(70.0) 3(30.0) 10

Other industry sectors 6(25.0) 18(75.0) 24

Types of injury

Open fracture 10 (41.7) 14 (58.3) 24 3.77 2 0.152

Crush hand 11(68.8) 5(31.2) 16

Other types of injury 14(41.2) 20(58.8) 34

Hand activities at the time of injury

Working on machine 22(53.7) 19(46.3) 41 2.319 2 0.314

Handling objects 5(31.2) 11(68.8) 16

Other hand activities 8(47.1) 9(52.9) 17

Day of the week

Working days 28(45.9) 33(54.1) 61 0.046 1 0.83

Weekend 7(53.8) 6(46.2) 13

Time of injury

8.00 am- 12.00 pm 13(43.3) 17(56.7) 30 0.44 2 0.803

12.00 pm- 2.00 pm 5(45.5) 6(54.5) 11

After 2.00 pm 17(51.5) 16(48.5) 33

Occupation categories

Machine operators 15(51.7) 14(48.3) 29 3.457 2 0.178

Manual materials handlers 2(20.0) 8(80.0) 10

Other occupations 18(51.4) 17(48.6) 35

*p*p* is significant when p<0.05



Table 6. Logistic regression analysis

Predictors β Adjusted OR 95% CI p value

Sources of injury

Other causes 1

Machine 3.25 25.84 1.69 - 393.37 0.019*

Sectors of industry

Other sectors 1

Metal-machinery 2.12 8.36 1.22 – 57.23 0.031*

Locations of injury

Other site of injury 1

Fingers 0.91 2.47 0.58 – 10.60 0.22

Types of injury

Other types of injury 1

Open fracture -1.06 0.34 0.09 – 1.38 0.13

Mechanisms of injury

Other mechanisms

Hand caught inside machine 0.53 1.7 0.38 - 7.62 0.48

Time of injury

12.00 pm-2.00 pm (Lunch break) 1

8.00 am- 12.00 pm -0.95 0.39 0.04 - 3.60 0.4

After 2.00 pm -0.77 0.46 0.05 - 4.51 0.51

Day of the week

Weekend 1

Working days -0.63 0.53 0.09 - 3.25 0.49

Hand activities at the time of injury

Other activities 1

Working on machine -2.1 0.12 0.01 - 1.88 0.13

Handling objects -1.75 0.17 0.01 - 2.54 0.2

Occupational categories

Other occupations 1

Machine operators -2.44 0.12 0.01 – 1.87 0.13

Manual materials handlers -2.19 0.17 0.01 – 2.54 0.2

Nationality

Others 1

Malaysian -1.54 0.21 0.04 - 1.09 0.06

Marital status

Married 1

Single 0.09 1.09 0.29 - 4.11 0.89

significant level p< 0.05



Table 4 shows the sources of injury, occupational categories and sectors of industry, mechanisms of injury and hand activities at the time of injury by severity of WRHIs. Majority (70.1%) of the respondents had injuries resulting during operation on mechanical machine. Only 39.1% of respondents were machine operators. Other occupational categories affected were manual material handlers (13.5%), carpenter (12.2%), construction workers (12.2%), machinery maintenance workers (8.1%), food preparers (5.4%), sheet metal workers (5.4%), and welders (4.1%). Majority (54.1%) of the respondents were working in metal-machinery industry. Other sectors of industry were represented by wood-furniture (13.5%), construction (12.1%), food preparing (9.5%), services (8.1%) and automotive (2.7%).

Only (48.6%) of the hand injuries occurred when the hand was caught inside the machine. Other mechanisms of injury occurred by cutting mechanism (21.7%), when (the hand hit by hard objects) mechanism (18.9%), and fall and piercing mechanisms of injury constituted (10.8%). The results also show that the majority (55.4%) of the hand injuries occurred while workers were working on the machine, and as hand activity at the time of injury. Other activities represented by handling objects (21.5%), lifting objects (9.5%), working on powered hand tool (6.8%), carrying object (4.1%), and working on non-powered hand tool (1.4%).

Day of the week and time of accident of WRHIs by severity

The day of the week and time of accident of WRHIs by severity was also examined. The majority (82.5%) of the injuries occurred during working days. Only (44.6%) of the WRHIs occurred between 2.00 pm and 8.00 am, (40.5%) of the WRHIs occurred after 8.00 am and before 12.00 noon, while (14.9%) of the WRHIs occurred between 12.00 noon and 2.00 pm.

Factors associated with severe WRHIs

Bivariate analysis showed that there was a significant association between severity of WRHIs and locations of injury, mechanisms of injury and sources of injury.

Logistic regression analysis

Table 6 shows the logistic regression. Only two independent variables showed significant contribution to the model (sources of injury and sectors of industry). The strongest predictor of reporting severe WRHIs was sources of injury. Respondents with hand injury resulted while operating on mechanical machine was 26 times more likely to report severe WRHIs than those with other sources of their hand injury like (sharp tool, heavy door, and wet floor). The results also showed that respondents working in metal-machinery industries were eight times more likely to report severe WRHIs than those who working in other sectors of industry like (wood-furniture, constriction, food preparing, service and automotive).

DISCUSSION Work-Related injuries represent significant rates of acute hand injuries seen in emergency services [2,11,12]. Therefore, the concept of hand injury severity assessment is focused on Work-Related injuries. The influence of multiple risk factors has been associated with severity of hand injuries. This study shows that severity of the hand injury is significantly related to the presence of a risk or a protective factor.

In our study, only 74 patients had WRHIs (24.9%) from a total of 297 industrial-related accidents seen at the tertiary hospital. However, these finding were different from Serinken study at tertiary hospital in Western Turkey who found that (32.7%) of occupational injuries were WRHIs [3]. The finding of this study shows that, the proportion of severe WRHIs is (47.3%) from all WRHIs. These findings are consistent with that of severe WRHIs among workers reported by Urso - Baiardia in United Kingdom [8]. In the present study, the majority (62.1%) of injuries that occurred to the fingers either single or multiple were associated with severe WRHIs. Meanwhile, the majority (67.9%) of other sites of injuries, like injury to forearm, thumb and back and palm of the hand were found to be associated with non-severe WRHIs. However, these findings are not in agreement with Sorock (2002) who reported that, majority of injuries that occurred to the fingers have been associated with non severe WRHIs [7]. Using of MHISS allows classification of injuries according to the hand components involved into lacerations, open fracture (comminuted fracture), closed fracture, crush hand injuries and amputations (complete or partial) [8]. In the present study, 32.1% of the respondents presented with open fracture and 17.0% presented with lacerations, finger tips injuries and avulsions. However, the findings of this study did not support the previous research by Serinken et al., (2008) who reported that lacerations, finger tips, cuts and abrasions were found to be the most frequent types of hand injuries (40%) of all WRHIs. Hand and fingers caught inside operating machine was the main mechanism (48.6%) responsible for fingers injuries. In contrast, injuries to the forearm and palm or back of the hand tend to be caused by mechanisms like (when hand hit by heavy object, fall, cutting and piercing), which have been found to be associated in the majority (65.8%) with non severe WRHIs. Working on mechanical machine was the most common source of WRHIs. The respondents with injuries



resulted while operating mechanical machine were 26 time more liable to have severe WRHIs than the respondents that had injuries resulted by (sharp tool, wet floor and heavy door). The findings of this current study were consistent with previous studies [6, 7 & 13], who found that all the WRHIs cases resulted while operating machines, were of severe cases. In this study, the highest frequency (43.4%) of WRHIs occurred after 2.00 pm inclusive of respondents who worked at night shift. However, 41.5% of the WRHIs occurred between 8.00 am and 12.00 noon, and 15.1% occurred between 12.00 noon and 2.00 pm. However, these findings were not in agreement with the findings of the previous studies, which reported that the highest frequency of hand injuries occurred in the first four hours of working from 08.00 am to 12.00 pm [3, 14 & 15]. However, these studies reported the time of patient’s admission to the emergency room, while this current study depended on the actual time of accident. Workers belonging to the metal-machinery industries sector were eight times more likely to be associated with severe WRHIs than other workers belonging to sectors of industry like; wood-furniture, construction, services, food preparing and automotive. However, these finding do not support the previous research by Serinken et al. (2008) who reported that, only 41.1% of the injuries happened in metal-machinery industries [3]. This difference in sectors of industry can be attributed to the types of industries found in the area of study.

CONCLUSIONWRHIs contributed to 24.9% of all industrial accidents seen at the emergency department and orthopaedic clinic and 47.3% of the respondents with WRHIs had severe hand injuries. Severity of WRHIs was significantly associated with sources of injury and sectors of industry. Respondents with hand injury resulted while operating on mechanical machine was 26 times more likely to report severe WRHIs than those with other sources of their hand injury like (sharp tool, heavy door, and wet floor). Respondents working in metal-machinery industries were eight times more likely to report severe WRHIs than those working in other sectors of industry like; wood-furniture, constriction, food preparing, service and automotive.

ACKNOWLEDGEMENTWe would like to thank Professor Dr Norlijah Othman, Dean, Faculty of Medicine and Health Sciences, University Putra Malaysia for giving us permission to publish this paper. We also would like to thank the Ministry of Health National Institutes of Health for approval to conduct this study. We would also like to acknowledge and to thank all staff involved in this study for their support and cooperation.

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[6] Trybus M, Lorkowski J, Brongel L, Hladki W. Causes and consequences of hand injuries. The American Journal of Surgery 2006; 192(1): 52-57.

[7] Sorock GS, Lombardi DA, Hauser RB, Eisen EA, Herrick RF, Mittleman MA Acute traumatic occupational hand injuries: Type, location, and severity. J Occup Environ Med (2002); 44(4): 345-351.

[8] Urso-Baiarda F, Lyons RA, Laing JH, Brophy S, Wareham K, Camp D Prospective evaluations of the Modified Hand Injury Severity Score in predicting return to work. International Journal of Surgery 2008; 6(1): 45-50.

[9] Chen YH, Lin HT, Lin YT, Chao YH, Lin CH et al. Self-perceived health and return to work following work-



related hand injury. Occupational Medicine Lond 2012; doi: 10.1093/ occmed/ kqr215.

[10] Campbell DA & Kay SPJ. The hand injury severity scoring system. The Journal of Hand Surgery: Journal of the British Society for Surgery of the Hand 1996; 21(3), 295-298.

[11] Centers for Disease Control. Surveillance for non fatal occupational injuries treated in hospital emergency departments - United States, 1996. MMWR 1998; 47:302-6.

[12] Larsen CF, Mulder S, Johansen AM, Stam C. The epidemiology of hand injuries in The Netherlands and Denmark. Eur J Epidemiol 2004; 19: 323-7.

[13] Oleske DM, Hahn JJ. Work-related injuries of the hand: data from an occupational injury/illness surveillance system. Journal of Community Health 1992; 17(4): 205-219.

[14] Lombardi DA, Sorock GS, Hauser R, Nasca PC, Eisen EA, Herrick RF, et al. Temporal factors and the prevalence of transient exposures at the time of an occupational traumatic hand injury. Journal of Occupational and Environmental Medicine 2003; 45(8): 832-840.

[15] MacDonald II, Smith L, Lowe SL, & Folkard S. Effects on Accidents of Time into Shift and of Short Breaks between Shifts. International Journal of Occupational and Environmental Health 1997; 3 (Supplement 2), S40-S45.


The Effectiveness of Gentamicin against Acanthamoeba Cysts in Vitro

1SA Noradilah*, 2AG Mohamed Kamel, 3N Anisah, 3AR Noraina & 3S Yusof 1Universiti Sains Islam Malaysia, Level 13, Menara B, Pesiaran MPAJ,

55100 Pandan Indah, Kuala Lumpur2Universiti Kebangsaan Malaysia, Department of Biomedical Science,

Faculty of Health Sciences, Kuala Lumpur.3Universiti Kebangsaan Malaysia, Department of Parasitology,

Faculty of Medicine, Kuala Lumpur.

ABSTRACTAcanthamoeba is a free-living protozoa which causes serious ocular problem. Acanthamoeba keratitis is becoming more prevalent amongst contact lens wearers and it can cause loss of vision and blindness if not treated properly. The objective of this research is to determine the effectiveness of gentamicin against six Acanthamoeba spp. isolates, of which three were clinical isolates (HS 6, HKL 95, HTH 73) and three environmental isolates (SMAL 7, SMAL 8, TTT 9). Cyst suspension from the chosen isolates were exposed to gentamicin. After 48 hours of incubation at temperature of 30°C and 37ºC, each mixture was filtered and filtration membrane was put onto non-nutrient agar laid with Escherichia coli. The agar plates were incubated for three days at 30oC and 37oC and the plates were examined daily until day 14 to look for the presence of Acanthamoeba trophozoites under inverted microscope. The presence of trophozoites indicated the ineffectiveness of gentamicin. Gentamicin was found to be effective against Acanthamoeba cysts from all the test strains at both incubation temperatures. The minimum cysticidal concentration (MCC) mean value of gentamicin was 0.193 mg/mL at 30oC and 0.229 mg/mL at 37oC. So, we concluded that gentamicin has cysticidal potential towards Acanthamoeba.

Keywords : Acanthamoeba, sensitivity, gentamicin

*Corresponding author: [email protected], [email protected]


INTRODUCTIONSmall free-living amoebae belonging to the genera Acanthamoeba occurs world-wide. It has been isolated from seawater, ocean sediment and chlorinated swimming pools which can contain up to 104 amoebae per litre as Acanthamoeba are resistant to chlorine, other types of water and sewage outfalls.[1] In addition, a relationship between the presence of Acanthamoeba and faecal indicator bacteria has been found in samples of ocean sediment.[2] Because of the wide spread distribution of Acanthamoeba, human contact with the organism is inevitable and frequent.[3]

Acanthamoeba may cause a serious eye infection known as Acanthamoeba keratitis in healthy individuals which occurs in two separate forms. In the first form, the pathogen is restricted to the epithelium and there is a good chance of recovery. In the second form, the parasite enters the stroma, where it causes necrosis and intense inflammation which leads to blindness if not properly treated. [4]

The first-line treatment for Acanthamoeba keratitis is topical therapy with biguanides such as polyhexamethylene biguanide or in combination with diamidenes such as propamidine, neomycin (aminoglycosides) and imidazoles. Evaluation of the safety and efficacy of neomycin–polymyxin B–gramicidin ophthalmic solution (Neotracin; Cilag) when it was administered with 0.1% propamidine isethionate has also been carried out.[5] However, these drops are not always available and chronic use is associated with significant ocular surface toxicity.[6] Furthermore, a combination of drug therapy has been shown to have high success rate only if the disease is diagnosed early. In addition, the treatment for Acanthamoeba keratitis using antimicrobial and antiviral therapy is troublesome.[7]

Neomycin in combination with other antimicrobial agents is the first line treatment for Acanthamoeba keratitis, therefore, this study was performed to investigate the potential efficacies of gentamicin, also an aminoglycosides against Acanthamoeba cysts and to determine the effectiveness of gentamicin towards Acanthamoeba cysts at different incubation temperature. In this study, the focus is on the cyst of Acanthamoeba as it is resistant towards disinfectant, temperature variation and dessication.[8] Hopefully this study will help everyone especially contact lens wearers, optometrists and ophtalmologists, so that Acanthamoeba keratitis can be avoided or properly treated. Misdiagnosis and mistreatment of Acanthamoeba keratitis may lead to resistance development in Acanthamoeba cysts and severe eye condition.


SA Noradilah, AG Mohamed Kamel, N Anisah, AR Noraina & S Yusof 52

MATERIALS AND METHODSAcanthamoeba source

Acanthamoeba isolates were obtained from the Acanthamoeba Culture Laboratory, Universiti Kebangsaan Malaysia by subculture from clinical (HS 6, HKL 95, HTH 73) and environmental isolates (SMAL 7, SMAL 8, TTT 9).

Gentamicin

Gentamicin was obtained from The Tun Hussein Onn National Eye Hospital and used before the expiratory dates.

Sensitivity test

The method was modified from the standard method proposed by Narasimhan et al. [9]. Acanthamoeba cysts suspension was vortexed for one minute to ensure the cysts were thoroughly mixed in the Page Saline solution. Essentially, double dilutions of gentamicin were performed in microtitre plates with five uL cysts at a concentration of 1 × 105 cysts per 100 µl of medium per well. The gentamicin concentration ranges from 0.031 mg/mL to 1 mg/mL. Microtitre plates were then incubated at 30oC and 37oC for 48 hours.

Two positive controls were prepared; one with Page Saline solution mixed with cyst and the other one with hydrogen peroxide mixed with cyst. Two negative controls were also prepared; one with Page Saline solution only and the other one with gentamicin only to ensure they are free from any contaminations. The tests were processed in duplicate.

The mixture of gentamicin and Acanthamoeba cyst, positive and negative controls were filtered respectively using the filtration unit which consists of millipore, vacuum pump and nitrate cellulose membrane as the main component. The nitrate cellulose membrane measured 0.45µm. Microtiter well was rinsed again using Page Saline solution to detach any remaining cyst. Rinsing was done twice. After filtration, the nitrate cellulose membrane was put onto the non-nutrient agar plates seeded with heat-killed Escherichia coli as source of food for Acanthamoeba. The agar plates were then incubated at 30oC and 37oC for three days.

The membrane was taken out from the agar. Each agar plate was examined daily under the inverted microscope for the presence of trophozoite. Observation was done daily until day 14 to confirm the result to be negative for the presence of trophozoite. Any trophozoite observation indicates the ineffectiveness of gentamicin.

In this study, the focus was given to Acanthamoeba cysts as they are more resistant against any treatment or antimicrobial agents exposed to them. The lowest antimicrobial concentration preventing trophozoites formation after 14 days incubation was taken as the minimum cysticidal concentration (MCC).

RESULTSThe minimum cysticidal concentration (MCC) value for gentamicin against Acanthamoeba cysts are shown in Table 1. The mean MCC value for all clinical isolates when incubated at 30oC was 0.250 mg/mL. At 37oC, the mean MCC value for isolates HS 6 and HKL 95 was 0.250 mg/mL while for HTH 73 strain, the MCC was 0.375 mg/mL.

Table 1. Minimum cysticidal concentration (MCC) value for gentamicin tested against Acanthamoeba isolates.

Strain

MCC value (mg/mL)

Incubation temperature 30°C Incubation temperature 37°C

First test Second test Mean First test Second test Mean

HS 6 0.250 0.25 0.250 0.250 0.250 0.250

HKL 95 0.250 0.250 0.25 0.250 0.250 0.25

HTH 73 0.250 0.250 0.250 0.250 0.5 0.375

SMAL 7 0.125 0.125 0.125 0.250 0.125 0.188

SMAL 8 0.250 0.125 0.188 0.125 0.125 0.125

TTT 9 0.125 0.063 0.094 0.125 0.25 0.188

Mean MCC (mg/mL) 0.193 0.229

The mean MCC for environmental isolates were a bit different from the MCC obtained when gentamicin were tested against the clinical isolates. At incubation temperature of 30oC, the MCC obtained for SMAL 7 strain was 0.125


The Effectiveness of Gentamicin against Acanthamoeba Cysts in Vitro 53

mg/mL while SMAL 8 was 0.188 mg/mL. TTT 9 strain showed the lowest MCC, which was 0.094 mg/mL. When exposed to gentamicin at 37oC, MCC value obtained for isolate SMAL 7 and TTT 9 was 0.188 mg/mL whilst SMAL 8 was 0.125 mg/mL. Generally, by reading the MCC value, the environmental isolates were found to be more sensitive towards gentamicin compared to the clinical isolates.

Using unpaired t-test, it was found that there was no significant difference in the effectiveness of gentamicin against Acanthamoeba cysts at 30°C and 37°C, where t(10) =-0.810, p>0.05.

Positive and negative controls

No trophozoite was observed until day 14 when the cysts were exposed to hydrogen peroxide. On the other hand, trophozoites were seen in the plate containing cysts added with Page amoebic saline.

Negative control was prepared with Page amoebic saline alone, to ensure that the saline was free from any contamination. There were no cysts or trophozoites observed until day 14 for the negative control. Gentamicin was also tested to ensure they are free from any contamination and in good condition before sensitivity testing was carried out. No cysts or trophozoites were observed for these plates.

DISCUSSIONGentamicin was isolated in 1963 by Weinstein and his colleague from a fungus found in soil, Micromonospora purpura (Actinomycetes). It was then introduced in United States in 1969.[10]

Gentamicin 40mg/mL contains 40mg gentamicin sulphate and 4.4µg/mL benzalkonium choride as preservative. The mode of action is similar to neomycin as both are aminoglycosides. The aminoglycosides causes codon misread by binding to the ribosomal subunit 30S, stops the peptidyl-tRNA translocation from the recipient to donor area and interrupts protein synthesis.[11]

Gentamicin is widely used for the treatment of severe infection caused by bacteria. It is active against gram negative bacteria and Streptococcus aureus. It is inactive against anaeorobic bacteria and less active against Streptococcus haemolyticus and pneumococcus.[12]

In this study, gentamicin has been serially diluted and tested against Acanthamoeba cysts to determine the minimum cysticidal concentration (MCC). Double dilution was performed and sensitivity test of gentamicin against Acanthamoeba cysts at 30°C gave mean MCC value of 0.193 ± 0.07 mg/mL for all tested strains. Meanwhile, the mean MCC value obtained from the sensitivity test at 37oC was 0.229 ± 0.09 mg/mL. Ghani [13] evaluated the effectiveness of gentamicin on Acanthamoeba clinical isolates and discovered the mean MCC as 13.33 mg/ml (range 10-20 mg/ml). The result obtained in our study differed from the mean MCC value obtained by Ghani et al. This may be due to different isolates used which may show different levels of sensitivity towards gentamicin.

As there are no other in vitro studies done by other researchers to evaluate the effectiveness of gentamicin against Acanthamoeba, so, no detail comparisons can be made with this study. Gentamicin as compared to neomycin has never been used for the treatment of Acanthamoeba keratitis. However, in this study, gentamicin was found to have cysticidal potential.

CONCLUSIONFrom this study, it can be concluded that gentamicin has potential cysticidal efficacy with minimum cysticidal concentration (MCC) of 0.193 ± 0.07 mg/mL at 30°C and 0.229 ± 0.09 mg/mL at 37oC. Further investigations need to be carried out to evaluate the effectiveness of gentamicin againts many other isolates from different sources.

REFERENCES[1] Seal DV. Acanthamoeba keratitis update—incidence, molecular epidemiology and new drugs for treatment. Eye

2003; 17: 893-905.

[2] Schroeder JM, Booton GC, Hay J, Niszl I, Seal DV, Markus MB Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoebae from humans with keratitis and sewage sludge. J Clin Microbiol 2001; 39: 1903-1911.

[3] Illingworth CD, Cook SD Acanthamoeba keratitis. Surv Ophthalmol 1998; 42: 493-508.

[4] Kumar R, Lloyd D. Recent Advances in the Treatment of Acanthamoeba Keratitis. Clin Infect Dis. 2002; 35 (4): 434-441.

[5] Hargrave SL, McCully JP, Husseini Z. Results of a trial of combined propamidine isethionate and neomycin


SA Noradilah, AG Mohamed Kamel, N Anisah, AR Noraina & S Yusof 54

therapy for Acanthamoeba keratitis: Brolene study. Ophthalmol 1999; 106: 952-7.

[6] Larkin DF, Kilvington S, Dart JK. Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide. Ophthalmol 1992; 99(2): 185-91.

[7] Moore MB, McCulley JP, Netwon C, Cobo LM, Foulks GN, O’day DM, Johns KJ, Driebe WT, Epstein RJ, Doughmas DJ Acanthamoeba keratitis : A growing problem in soft and hard contact lens wearer. Ophtalmol 1987; 94: 1654-1661

[8] Kamel AGM, Faridah HA, Norazah A, Noor Rain A, John H, David S. A case of waterborne contact lens associated Acanthamoeba keratitis from Malaysia: Successful treatment with chlorehexidine and propamidine. Inter Med J 2000; 7(1): 63-65.

[9] Narasimhan, Sandhya MS, Madhavan, Hajib N, Lily T. Development and application of an in vitro susceptibility test for Acanthamoeba species isolated from keratitis to polyhexamethylene biguanide and chlorhexidine. The J of Cornea and Ext Dis 2001; 21(2): 203-205

[10] Ravindra F, Jayakody RL. Gentamicin sulfate. Inchem 1993; 211: 123

[11] Kadurugamuwa J. Surface action of gentamicin on Pseudomonas aeruginosa J. Bacteriol 1993; 175: 5798-5805

[12] Ravindra F, Jayakody RL. Gentamicin sulfate. Inchem 1994; 211: 123

[13] Ghani MKA, Shirley GHT, Anisah N, Putri N, Yusof S, Noraina R, Norazah A. Invitro susceptibility test for Acanthamoeba sp. isolated from clinical specimens against chlorhexidine, propamidine isethionate, gentamicin and chloramphenicol. Euro Cong of Clin Microbiol and Infect Dis. 2010


Detection of Human Herpesvirus 6 (HHV-6) in Saliva of Healthy Adults in Malaysia

1HL Choo, 2Y Shoji* & 3CO Leong1School of Postgraduate Studies and Research, International Medical University,

School of Pharmacy and Health Sciences, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.2School of Dentistry, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

3School of Pharmacy and Health Sciences, International Medical University, Bukit Jalil,57000 Kuala Lumpur, Malaysia.

INTRODUCTIONHuman herpesvirus 6 (HHV-6) is an emerging pathogen that was first isolated from patients with lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS).[1] HHV-6 is classified in the Betaherpesvirinae subfamily, along with human cytomegalovirus (HCMV) and HHV-7. There are 2 distinct HHV-6 variants, HHV-6A and HHV-6B.[2] Both variants displayed >90% identical DNA sequence with the exception of a few genes or regions.[3, 4] Despite the high degree of similarity in their DNA content, HHV-6A and HHV-6B have distinct biological properties and association with specific pathological conditions.[5]

HHV-6B has been identified as the cause of exanthem subitum (roseola), a typically mild eruptive childhood disease that is occasionally complicated by development of meningitis, meningoencephalitis, chronic hepatitis or idiophatic thrombocytopenic purpura.[6] When primary infection occurs in teenagers or in adults, the presence of the virus has been associated with hepatitis, encephalitis, mononucleosis syndrome, chronic fatigue syndrome, fatal disseminated infection and, more recently, multiple sclerosis.[6] In contrast, infection with HHV-6A is generally asymptomatic and appears to be unrelated to any specific pathology.[6]

Following primary infection, HHV-6A and HHV-6B are thought to persist for life as a latent form in peripheral blood mononuclear cells (PBMCs), macrophages, and vascular endothelial cells and as a low-level chronic replicating form located in oropharyngeal epithelial cells.[7] A large body of evidence suggests that HHV-6 may act as an opportunistic agent in patients with immunodeficiencies, particularly those who have undergone bone marrow or organ transplantation and human immunodeficiency virus (HIV) infected individuals.[6] A role of HHV-6 as a cofactor in the progression of HIV infection toward full-blown AIDS has also been proposed.[8]

Despite the interest in HHV-6 as an important pathogen, only limited number of population-based studies has evaluated the prevalence of HHV-6 infection in healthy population of developing countries in Asia. This pilot study aimed to investigate the prevalence of HHV-6 and its variant infection in young healthy adults in our population.

MATERIALS AND METHODSPatients and samples

A total of 36 healthy adults (15 males, 21 females) with a median age of 21 years (range, 19 to 23 years) were recruited as volunteers in the present pilot study. Saliva samples (5 ml) were collected through mouth rinses with water. All samples were maintained on ice, divided into 1 ml aliquots and stored at -80oC until use. All subjects were in good



ABSTRACT Background: Human herpesvirus-6 (HHV-6) levels have been considered as markers for various diseases. The aim of this study was to evaluate the prevalence of HHV-6 infection in healthy adults in Malaysia. Methods: The level of HHV-6 in saliva was investigated in 36 healthy adults, age 19 to 23 years, at Kuala Lumpur, Malaysia using variant-specific TaqmanTM quantitative real-time PCR (qPCR). Results: The amount of HHV-6 DNA in the saliva of healthy adults ranged from negative to 10,000 HHV-6 genomes/ml of saliva (median, 360 genomes/ml of saliva). Of the 36 samples tested, 30 (83%) contained HHV-6 DNA. HHV-6B was the only variant detected in the saliva of all the positive cases. Conclusions: The detection of HHV-6 DNA in saliva by real-time PCR assay provides a sensitive and specific quantitation of HHV-6. Our pilot study suggests the wide prevalence of HHV-6 in saliva from healthy adults.

Keywords: Herpesvirus 6, HHV-6, prevalence, real-time PCR, saliva


HL Choo, Y Shoji & CO Leong56

general health and did not have histories of liver or kidney dysfunction, symptoms of acute illness (i.e., fever, sore throat, body aches, and diarrhea), or visible oral lesions at the time of enrollment. The study was approved by the International Medical University (Malaysia) Institutional Review Board, and all subjects provided written informed consent as part of the study protocol.

Real-time Quantitative PCR.

DNA in saliva was extracted from 0.5 ml samples using the QIAamp UltraSens Virus kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Real time-PCR was used to detect and quantify HHV-6 in saliva as described previously.[9, 10] Briefly, the 25 µl reaction mixture contained 2 µl of purified DNA from saliva, 12.5 µl TaqMan PCR master mix, 200 nM of each primer, and 100 nM probe specific for HHV-6 (Applied-Biosystem, Carlsbad, CA, USA). The sequence of the forward primer was: 5’-GACAATCACATGCCTGGATAATG-3’. The sequences of the reverse primers specific for HHV-6A, HHV-6B and both variants were: 5’-TGGTAATGGACTAATTGTGTGTTGTTTTA-3’; 5’-TGGTAATGGACTAAGTGT GCGTTATTTTC-3’ and 5’-TGTAAGCGTGTGGTAATGGACTAA-3’, respectively. The TaqMan® probe was 5’-FAM-AGCAGCTGGCGAAAAGTGCTGTGC-TAMRA-3’. All PCR reactions were performed using a Biorad iQ5 real-time PCR detector system (Bio-Rad, Richmond, CA, USA) and data analyzed using Biorad iQ5 Optical System Software V1.0 as described previously.[11-13] The conditions for all PCR reactions were as follows: 50oC for 2 min, 95°C for 10 min, and 45 cycles of 95oC for 15 s and 60oC for 1 min. Purified DNAs from HHV-6A (GS strain) and HHV-6B (Z29 strain) (Source Bioscience, Nottingham, UK) were used as standard controls. Determination of standard curves for both DNAs was based on triplicate tests over a range of 1 to 104 copies for each reaction. HHV-6 DNA quantitation in saliva was normalized per ml of saliva. The detection limit of the assay were 10 copies of viral genomes per ml of saliva for detection of HHV-6 and 40 copies of genomes per ml of saliva for detection of HHV-6A or HHV-6B without any cross reactivity.

SupplementSupplementFigure 1. Standard curve obtained for HHV-6 quantification. Purified

HHV-6A and HHV-6B viral DNA were used to construct the standard curve for (A) overall HHV-6 and (B) variant-specific HHV-6 quantification. Five-fold serial dilutions ranging from 1 copy to 104 copies of DNA were tested in triplicate, and the mean Ct values were plotted against copy number


Detection of Human Herpesvirus 6 (HHV-6) in Saliva of Healthy Adults in Malaysia 57

STATISTICAL ANALYSIS.The proportions of positive cases obtained for male and female subjects were compared using Fisher’s exact test and Mann-Whitney U test. Statistical significance was determined at the P < 0.05 level (two-tailed). All data were analyzed with use of the SPSS statistical analysis software V18.

RESULTSUsing the highly sensitive qPCR, we demonstrated that 30/36 (83%) healthy adults were positive for HHV-6 in their saliva. Out of the 36 subjects, 14/15 (93%) male and 16/21 (76%) female were tested positive for HHV-6 DNA. The amounts of HHV-6 DNA per milliliter of saliva vary from 100 to 10,000. Male adults appeared to have higher level of HHV-6 DNA in their saliva (more than 2-fold) compared to female adults. The difference however was not statistically significant.

Table 1. HHV-6 DNA levels in 36 healthy individuals

Subject no. Age Gender HHV-6/ml saliva HHV-6A HHV-6B

1 22 Female - n.d. n.d.2 20 Female - n.d. n.d.3 21 Female - n.d. n.d.4 21 Female - n.d. n.d.5 21 Female - n.d. n.d.6 21 Female 95 Negative Positive7 21 Female 166 Negative Positive8 20 Female 173 Negative Positive9 21 Female 185 Negative Positive10 21 Female 275 Negative Positive11 20 Female 311 Negative Positive12 20 Female 312 Negative Positive13 21 Female 325 Negative Positive14 20 Female 396 Negative Positive15 20 Female 544 Negative Positive16 22 Female 604 Negative Positive17 20 Female 749 Negative Positive18 20 Female 1,158 Negative Positive19 21 Female 1,290 Negative Positive20 23 Female 2,769 Negative Positive21 21 Female 4,896 Negative Positive22 21 Male - n.d. n.d.23 19 Male 89 Negative Positive24 21 Male 96 Negative Positive25 22 Male 164 Negative Positive26 22 Male 276 Negative Positive27 20 Male 490 Negative Positive28 22 Male 599 Negative Positive29 20 Male 751 Negative Positive30 22 Male 984 Negative Positive31 23 Male 1,024 Negative Positive32 20 Male 1,284 Negative Positive33 20 Male 1,781 Negative Positive34 20 Male 3,598 Negative Positive35 21 Male 4,758 Negative Positive36 20 Male 8,208 Negative Positive

n.d. Not determined



Figure 1. Amount of HHV-6 DNA in saliva of male and female adults. HHV-6 DNA in saliva from healthy adults was determined using real-time PCR. Detection limit was determined to be 10 copies genome/ml of saliva. , amount of HHV-6 DNA of each subject; median value median value

To further evaluate the prevalence of different HHV-6 variants in healthy adults, saliva samples that were positive for HHV-6 were further analyzed for HHV-6A and HHV-6B using variant specific qPCR. We found that 30/30 (100%) of the HHV-6 positive healthy adults were positive for HHV-6B in their saliva. The level of HHV-6B detected was consistent with the overall level of HHV-6, suggesting that HHV-6B is the major variant that is presence in the saliva of the healthy adults. This result was further confirmed by the absence of HHV-6A DNA in all the samples being tested. Our results therefore suggest a high prevalence of HHV-6B in saliva of healthy adults in the Malaysian population.

DISCUSSIONThe HHV-6 infection is widespread. Its prevalence in healthy adult populations has been reported to range from 0% to 100% depends on the method of detection, sample types, age and geographical locale. A meta-analysis of 4,137 cases from 43 studies reported between 1988 to 2007 reveals that the prevalence of HHV-6 in the sera of healthy individuals was 67.9% (2122/3125) using indirect immunofluorescence assays (IFA) versus 8.8% (16/182) using PCR. Similarly, detection of HHV-6 DNA by PCR in various tissues also yielded a highly variable result (41.1% in PBMCs vs 76.2% in saliva) in healthy populations. It is well accepted that HHV-6 infection usually occurs before 2 years of age and the HHV-6-specific IgG can be detected in almost all newborns, but the prevalence declines to less than 10% by 4 to 5 months of age, increases to 65% by 1 year of age and to greater than 90% by 13 to 36 months of age.[14, 15] Seroprevalence studies in Japan, England, and the United States also demonstrated that infection with HHV-6 is common in the Western world but may differ in population in other geographic/ethnic groups.[16]

Table 2. Summary of HHV-6 DNA levels in 36 healthy individuals

Male Female Overall P Value

HHV-6 positive 14/15 (93%) 16/21 (76%) 30/36 (83%) 0.185a

HHV-6 genome/ml saliva

Mean 1,607 ± 2,273 679 ± 1,158 1,065 ± 1,746 0.163b

Median 751 311 360 0.109c

a Fisher’s exact test (2-tailed)b Student’s t-test (2-tailed)c Mann-Whitney U test (2-tailed)



Table 3. Meta-analysis of HHV-6 prevalence in healthy individuals

Source Methods HHV-6 prevalence, n (%) Population Ref

Serum Immunoassay1 37/460 8% Austria [32]15-Aug 53% - [33]16-Oct 63% - [34]29/45 64% Slovakia [35]33/49 67% Melanesia [35]

297/430 69% China [36]243/333 73% Thailand [37]332/434 76% Brazil [38]502/600 84% Malaysia [30]153/180 85% Sweden [39]185/210 88% Thailand [40]41/43 95% - [41]59/60 98% Hungary [42]

187/234 HHV-6A 58-80% HHV-6A Malaysia [31]178/234 HHV-6B 49-76% HHV-6B

Serum PCR2 0/49 0% - [43]Jan-46 2% - [44]Jan-38 3% - [45]20-Jan 5% Kuwait [46]29-Dec 41% - [47]

PBMC3 PCR 29-Jan 3% - [48]29-Jan 3% - [49]May-67 7% - [50]12/150 8% Latvia [51]Apr-44 9% - [52]17-Apr 24% - [53]Dec-42 29% - [54]14/46 30% - [44]

25-Sep 36% - [55]16-Jun 38% Turkey [56]23-Oct 43% - [57]

176/238 74% Thailand [58]18/20 90% - [59]18/20 90% - [21]43/44 98% - [22]

Saliva PCR 19/28 68% - [60]19/28 68% - [61]28/38 74% - [62]26/34 76% - [63]18/20 90% - [21]18/20 90% - [29]

Tear PCR 20-Jan 5% - [64]Skin PCR 13-Mar 23% - [65]Tonsils PCR 69/69 100% - [66]

1 IFA, indirect immunofluorescence assays (IFA) 2 PCR includes qPCR, nested-PCR and competitive PCR3 PBMC, peripheral blood mononuclear cells



Although HHV-6 antibody titers have been widely used in early clinical studies to identify subgroups of patients with active HHV-6 infection on the assumption that anti-HHV-6 immunoglobulin G (IgG) or IgM titers correlated with viral activity [17-21], a number of recent reports suggests the contrary.[14, 22, 23]. While IgM antibodies have been used to confirm a case of HHV-6 associated roseola or febrile seizures during primary infection, which typically occurs before the age of two, the HHV-6 IgM test is not very useful for adults because it is not produced during viral reactivation.[14]

Similarly, elevated IgG antibodies to HHV-6 as an indicator for active, chronic infection also raises controversy as individuals vary in the way they respond to the virus.[14]

Since the majority of healthy individuals have detectable levels of latent virus in their white blood cells, PCR DNA tests of whole blood are not useful unless the test is quantitative, and the absolute level of virus can be compared to a healthy population. When the virus is found in the serum or plasma it is considered a sign of active infection. However, unlike most viral infections where large number of virions spill into the plasma when the virus is replicating, HHV-6 is spread largely from cell-to-cell or directly through the cells walls.[24, 25] Hence, very little free virus spilled in the serum, even in an active infection.

Accumulating evidence suggests that HHV-6 may establish a life-long latent and/or persistent infection in the salivary glands and shedding in saliva of normal healthy individuals.[26-29] Indeed, HHV-6 DNA has been detected in saliva of healthy and HIV positive individuals by PCR with a much higher sensitivity and specificity than IFA or ELISA.[21]

To date, only 2 studies have reported the seroprevalence of HHV-6 in Malaysia, ranging from 49-84% in healthy population.[30, 31] Both studies analyzed sera obtained from a broad-range of age group using IFA. Since HHV-6 has been reported to persist in salivary secretions,[15] we are interested to evaluate the level of HHV-6 DNA in saliva of healthy adult using a highly sensitive and variant-specific qPCR. Our results showed that 83% of the healthy adults were infected with HHV-6, consistent with the previous study reported by Chua et al.[30] The levels of HHV-6 DNA range from 100 to 10,000 genome copies per milliliter of saliva.

Further analysis using variant specific qPCR revealed that HHV-6B was the only variant detected in the saliva of the healthy individuals while HHV-6A was not found in the saliva. This result is in contrast to a report by Yadav et al. which showed at least 58% of Malaysia population is positive for HHV-6A.[31] The main reason for the discrepancy could be due to the differences in method of analysis and sample types. While we analyzed HHV-6 prevalence using saliva and qPCR, Yadav et al. were using serum and IFA. [31] Therefore, we do not rule out the possibility that HHV-6A might be present in other tissues other than oral mucosa or saliva as it has been reported to have a greater tropism in PBMCs or neurons.[6] Regardless, our study suggests the wide prevalence of HHV-6, particularly HHV-6B, among healthy population and suggests that saliva could be the main route of HHV-6 transmission.

CONCLUSIONOur pilot study shows that 83% (30/36) of the Malaysian healthy adults recruited in this small cohort study is infected with HHV-6. Although asymptomatic in healthy adults, the reactivation of the virus has been reported to cause life threatening diseases in immunocompromised individuals such as organ transplant or stem cell recipients and HIV patients. Although there is no drug approved specifically for treatment of HHV-6 infection, drugs used to treat closely related cytomegalovirus (CMV), such as ganciclovir (Cytovene), foscarnet (Foscavir) or cidofovir (Vistide), are widely used in patients with severe HHV-6 reactivation.

In conclusion, we demonstrated that the detection of HHV-6 DNA in saliva by qPCR provides a noninvasive, rapid, sensitive and specific quantitation of HHV-6 in biological samples. This technique will aid to the monitoring of HHV-6 transmission and reactivation in high risk individuals (i.e. immunocompromised patients) which can lead to serious complications. Our study also suggests the wide prevalence of HHV-6 in healthy individuals in Malaysia. Further work is being planned for the detection and quantitation of HHV-6 in diseased individuals using the baseline levels established in this pilot study.

ACKNOWLEDGEMENTThe authors would like to acknowledge Vincent Han Hing Lee, Zeng Zhou Tan and Noor Azura Hani Abdul Razak (School of Dentistry, International Medical Univiersity, Malaysia) for samples collection. The study was supported by the grant IMU 209/2010, Malaysia.

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[36] Wu Z, Mu G, Wang L. Seroprevalence of human herpesvirus-6 in healthy population in two provinces of north China. Chin Med Sci J 1997; 12: 111-114.

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[39] Linde A, Dahl H, Wahren B, Fridell E, Salahuddin Z, Biberfeld P. IgG antibodies to human herpesvirus-6 in children and adults and in primary Epstein-Barr virus infections and cytomegalovirus infections. J Virol Methods 1988; 21: 117-123.

[40] Bhattarakosol P, Pancharoen C, Mekmullica J. Seroprevalence of anti-human herpes virus-6 IgG antibody in children of Bangkok, Thailand. Southeast Asian J Trop Med Public Health 2001; 32: 143-147.

[41] Carricart SE, Bustos D, Biganzoli P, Nates SE, Pavan JV. Isotype immune response of IgG antibodies at the persistence and reactivation stages of human herpes virus 6 infection. J Clin Virol 2004; 31: 266-269.

[42] Stiller I, Pusztai R, Sombor E, Orosz L, Pal A, Tarodi B. Prevalence and avidity of human herpesvirus-6 specific IgG antibodies in pregnant women in Hungary. Acta Microbiol Immunol Hung 2006; 53: 25-34.

[43] Alvarez-Lafuente R, De las Heras V, Bartolome M, Picazo JJ, Arroyo R. Relapsing-remitting multiple sclerosis and human herpesvirus 6 active infection. Arch Neurol 2004; 61: 1523-1527.

[44] Alvarez-Lafuente R, Martin-Estefania C, de Las Heras V, Castrillo C, Picazo JJ, Varela de Seijas E, Gonzalez RA. Active human herpesvirus 6 infection in patients with multiple sclerosis. Arch Neurol 2002; 59: 929-933.

[45] Broccolo F, Drago F, Paolino S, Cassina G, Gatto F, Fusetti L, Matteoli B, Zaccaria E, Parodi A, Lusso P et al.Reactivation of human herpesvirus 6 (HHV-6) infection in patients with connective tissue diseases. J Clin Virol 2009; 46: 43-46.

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[52] Sandhoff T, Kleim JP, Schneweis KE. Latent human herpesvirus-6 DNA is sparsely distributed in peripheral blood lymphocytes of healthy adults and patients with lymphocytic disorders. Med Microbiol Immunol 1991;



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Trisomy X and Myelodysplastic Syndrome (MDS) with Eosinophilia

1RMT Eusni*, 2CF Leong & 2S Salwati1Department of Pathology, Faculty of Medicine & Health Sciences, University Putra Malaysia,

2Department of Pathology, Faculty of Medicine, National University of Malaysia.

INTRODUCTIONMyelodysplastic syndromes (MDS) are a group of clonal haematopoietic stem cell diseases characterized by dysplasia and ineffective haematopoiesis in one or more of the major myeloid cell lines [1]. It occurs predominantly in elderly individuals. MDS with eosinophilia is a rare entity under the syndromes. Most of the patients with these entities has chromosomal abnormalities, in particular chromosome 7. These patients usually have poor survival due to rapid evolution to acute leukaemia. We report here a case of a young patient who had myelodysplastic syndrome and with underlying abnormal cytogenetic findings involving trisomy X and presence of a marker chromosome at chromosome 11.

CASE REPORTIn our institution we were presented with a 21-year-old Malaysian student with worsening symptoms of anaemia and bleeding tendencies (spontaneous bruises, gum bleeding, menorrhagia) of 2-month duration. There were no other sources of bleeding tendencies or any evidence of blood loss. Physical examination revealed she had pallor and hepatosplenomegaly. Other systems examination was unremarkable.

Initial investigations showed severe anaemia with haemoglobin level of 52 G/L,her total white cell count was 10.2 X 109/L and her platelet count was 67 X 109/L. Differential count showed moderate eosinophilia of 1.8 X 109/L. Peripheral blood picture showed hypochromic microcytic anaemia, thrombocytopenia and eosinophilia with dysplastic features (multilobulated and hypogranularity). Biochemical investigations and bone marrow aspirate Perl’s stain revealed iron deficiency state. A bone marrow aspirate revealed hypercellular marrow with dysplastic features seen in erythroid, granulocytic & megakaryocytic lineage (Figure 1). Eosinophil precursors were increased up to 5% and morphologically dysplastic. The trephine biopsy showed dysplastic and megaloblastic features in the erythroid and megakaryocytic lineage. Abnormal localisations of immature precursors (ALIPs) were not seen. Eosinophil precursors were increased. The chromosomal analysis using conventional cytogenetic banding (G banding) initially showed presence of an extra chromosome X. For further confirmation, the investigation then proceeded further to multicoloured fluorescent in situ hybridization (M-FISH) which confirmed a trisomy X with an additional presence of a marker chromosome in chromosome 11. Microbiological & serological tests did not provide any evidence of bacterial or parasitic infection, nor autoimmune disease, to suggest secondary causes of her eosinophilia. Thus, a final diagnosis of iron deficiency anaemia with underlying myelodysplastic syndrome with eosinophilia, associated with trisomy X was proposed. Her eosinophilia was persistently elevated up to 4.6 X 109/L after 1 month of presentation. She was treated with prednisolone for six months and eosinophils count normalized following the treatment. Her



ABSTRACTWe reported a young patient with myelodysplastic syndrome (MDS) with eosinophilia, in which her chromosomal analysis revealed the presence of trisomy X and a marker chromosome at chromosome 11. The technique used to detect the chromosomal abnormalities is a multicoloured –fluorescent in situ hybridization technique (M-FISH). Our observation suggested that these underlying chromosomal abnormalities were probably responsible for her development of MDS with eosinophilia.

Myelodysplastic syndrome (MDS) is a condition whereby there is ineffective production of haematopoietic stem cells and poor quality of cells produced. The cause can either be a primary bone marrow problem, de novo or therapy related. Most MDS cases are secondary rather than primary. Many chromosomal abnormalities have been found in cases of myelodysplastic syndrome. We described a case of MDS with eosinophilia in association with presence of trisomy X and a marker chromosome in chromosome 11.

Keywords: myelodysplastic syndrome, eosinophilia, trisomy X


RMT Eusni, CF Leong & S Salwati66

anaemia responded partially to haematinics therapy and blood product support. Her thrombocytopenia was worsening. Due to her young age and the presence of clonal chromosomal abnormalities, she was planned for allogeneic peripheral blood stem cell transplant (alloPBSCT) with an HLA matched siblings. However, she defaulted follow up when the option was made known to her.

Figure 1. Bone marrow aspirate reveal hypercellular with dysplasia and megaloblastic changes seen in the erythropoietic and granulopoietic cell lines

Figure 2. Cytogenetic G banding showing an extra chromosome X (arrow) ,47,XXX

Figure 3. Further analysis using multicoloured fluorescent in situ hybridization (M-FISH) revealed trisomy X and a presence of marker chromosome at chromosome 11 (arrow)

Figure 4. The presence of trisomy X using CEPX, a specific probe targeted towards the centromere region of chromosome X

DISCUSSIONMyelodysplastic syndrome (MDS) is a condition whereby genetically injured haemopoietic stem cells populate the marrow and lead to ineffective production of haemopoietic cells and reduction in quality of cells produced. MDS with eosinophilia is not a well recognized entity. Cytogenetic abnormalities common in MDS with eosinophilia are trisomy 8 [1] and monosomy 7 [2]. However, the presence of primary MDS with eosinophilia & trisomy X was not reported before except for a report by Wan TS et al (2002) whereby he noted the presence of eosinophilia and trisomy X in et al (2002) whereby he noted the presence of eosinophilia and trisomy X in et alwhich the patient developed acute leukaemia and she succumbed during induction treatment probably due to release of granules in eosinophils leading to diffuse alveolar damage and pulmonary hemorrhage leading to her death [3]. The occurrence of these cytogenetic findings ie trisomy 8,monosomy 7 and trisomy X generally confers poor prognosis in term of shorten survival and rapid evolution toward acute leukaemia except for del5q which generally confers good prognosis but in del5q, eosinophilia was not a common association [4].

The M-FISH technique used in this case had added value whereby an additional presence of a marker chromosome was identified in chromosome 11 which was not detected using GTG- banding technique. Patients presented with MDS with eosinophilia should be thoroughly investigated to rule out the secondary causes of eosinophilia, as shown


Trisomy X and Myelodysplastic Syndrome (MDS) with Eosinophilia 67

in this case and a complete cytogenetic analysis, which may or may not include M-FISH as the presence or absence of cytogenetic abnormality is helpful in the prognosis and management of these cases. However, the decision to embark on such specialized tools will depend on the technical skills available in the laboratory, the costs involved and the intensity of labour and it has to be weighed whether it will provide useful extra diagnostic information, which help to prognosticate and with the management of these cases.

Prognosis in MDS is related to the morphological classification, cytogenetic findings and percentage of blasts in the bone marrow aspirates.[4] Using these criteria, the International Prognostic Scoring System (IPSS) has developed a scoring system in predicting survival and evolution to acute leukemia i.e: low 0, intermediate (INT) 1: 0.5-1, INT 2: 1.5-2.0 and high >2.5[4]. As for this patient she would fall into the INT 1 risk group, with a score of 1, based on the bilineage cytopenia (anaemia and thrombocytopenia- a score of 0.5, bone marrow blast of < 5%, - a score of 0, and cytogenetic findings of intermediate risk: all other abnormalities, - a score of 0.5). Some investigators have put morphology of MDS with refractory cytopenias and multilineage dysplasia, as a high-risk morphology. The frequency of evolution into acute leukemia is 11% [4]. However, considering her age is less than 60 her prognosis may be better. The overall median survival is 33 - 41 months [4]. In British Journal of Hematology (BJH) guidelines for adult MDS, median survival for INT 1 less than 60 years old is 5.2 years [4]. A recent study by Friedrich et al (2010) has reported et al (2010) has reported et althat presence of eosinophilia and/or basophilia as part of presentation in MDS has an impact whereby the survival is shorter in this group of patient as compared to those without eosinophilia and /or basophilia [5].

As for this case the presence of more than 1 cytogenetic abnormality add to the poor prognosis in myelodysplastic syndrome[4]. Hence, the option for this patient was allogeneic stem cell transplantation, as stated above.

The association of MDS with eosinophilia and trisomy X is yet to be known. Further studies need to be done to precisely understand the association of trisomy X and development of MDS with eosinophilia.

In conclusion, the association of trisomy X and marker chromosome in chromosome 11 with MDS with eosinophilia has not been reported before and this adds to the list of clonal cytogenetic abnormalities present in primary myelodysplastic syndrome. Hence, further study is needed to look into the mechanisms behind the cytogenetic abnormalities leading to MDS with eosinophilia.

REFERENCES[1] Doorduijn JK, Van Lom K, Lowenberg B. Eosinophilia and granulocytic dysplasia terminating in acute myeloid

leukaemia after 24 years. British Journal of Haematology 1996; 95: 531-4.

[2] Kwong YL, Chan LC. Involvement of eosinophils in acute myeloid leukaemia with monosomy 7 demonstrated by fluorescence in situ hybridization. British Journal of Haematology 1994; 88: 389-91.

[3] Wan TS, Yip SF, Yeung YM ,Chan LC, Ma SK. Fatal diffuse alveolar damage complicating acute myeloid leukemia with abnormal eosinophils and trisomy X, Ann Hematol 2002; 81: 167-9.

[4] Guidelines for the diagnosis and therapy of adult Myelodysplastic Syndrome, Br J Hematol 2003, 120, 187-200.

[5] Friedrich W, Ulrich G, Michael K, Thomas N, Sabine B, Philip G et al, Evaluation of the prognostic significance of eosinophilia and basophilia in a larger cohort of patients with myelodysplastic syndromes. Cancer 2010; 116: 2372-81.


Partial Epiglottic Edema Post Fish Bone Ingestion

1M Irfan*, 1AY Ali & 2Y Rohaizan1Department of Otorhinolaryngology-Head & Neck Surgery, School of Medical Sciences,

Universiti Sains Malaysia Health Campus, 16150 Kota Bharu, Kelantan, Malaysia2Department of Radiology, School of Medical Sciences,

Universiti Sains Malaysia Health Campus, 16150 Kota Bharu, Kelantan, Malaysia

INTRODUCTIONEpiglottitis is an inflammation of the epiglottis which is usually acute in nature and almost always secondary to infection. It is rarely due to trauma caused by a foreign body. Foreign body in the throat especially fish bone tend to be impacted in the soft tissues such as tongue, tonsils and cricopharyngeus muscle, rather than on the cartilaginous structure such as the epiglottis. Direct trauma to the epiglottis, though have been reported are usually due to the iatrogenic intubation or airway surgical procedures. We report a case of partial epiglottic edema post foreign body ingestion

CASE SUMMARYA 52-year-old Malay gentleman with no known medical illness presented with history of pain during swallowing. He admitted to having a foreign body sensation in the throat, and he had a history of fish bone accidental ingestion 3 hours prior to admission. There was no associated dysphagia and breathing difficulty.

Oral examination revealed normal findings. Flexible nasopharyngolaryngoscopy performed exhibited left sided swollen epiglottis. The vallecula space on the left medial to the midline was obliterated, with the swollen epiglottis touching the base of tongue. There was no foreign body seen. A lateral neck radiograph obtained, showed the appearance of thumb-sign shadow of epiglottis, with suspicious small fragment of opacity at the superior part to the cricoid cartilage.



ABSTRACTA fish bone (foreign body) in the throat is a common presentation in an otolaryngology practice. Commonly the fish bone can be visualized and removed in a clinic setting. More distal foreign body impaction such as at the cricopharyngeus level will need direct laryngoscopy and removal under general anesthesia. It is not uncommon to have patient presented with residual symptom of post foreign body ingestion. Findings such as traumatized mucosa and embedded bone with slough on the mucosal surface are commonly encountered. We report a case of post foreign body ingestion presented with odynophagia and laryngoscopy showed a partially swollen epiglottis. The symptom recovered with conservative management.

Keywords: Epiglottis, hematoma, foreign body, pharynx, fish bone

Figure 1. Endoscopic view of the epiglottis showed the lingual surface was half-swollen on the left side


M Irfan, AY Ali & Y Rohaizan70

He was admitted for close observation of the airway status and started on intravenous antibiotics, dexamethasone and was prepared for direct laryngoscopy and esophagoscopy on the next day under general anesthesia. However during review on the following day, the patient claimed that the symptom had already subsided. There was no pain on swallowing saliva. There was no foreign body sensation either. He was very comfortable. A repeat laryngoscopy revealed only minimal bruises on lingual surface of epiglottis and complete resolution of the edema.

The patient was subsequently discharged and asked to continue with the oral antibiotics and then for the follow-up visit with clinic for review. However, the patient defaulted the follow-up.

DISCUSSIONEpiglottis is an uncommon site for fish bone impaction among the oral cavity or oropharyngeal structures, owing to its anatomy built up. The epiglottis is made up of cartilage and only covered by a layer of mucosa on both its lingual and pharyngeal surfaces. However, due to the loose and vascular mucosa around the epiglottic area, any inflammation, irritation or allergic reaction may rapidly cause oedema and vascular engorgement.[1]

The epiglotis is one of the main structure in the oropharynx and its abnormality especially increment in size will accommodate a significant portion of the oropharyngeal space. Thus, any patient with suspected enlargement of epiglottis, whether due to hematoma or edema such as in acute trauma or infection should be admitted and treated as life-threatening condition as it may result in complete upper airway obstruction and sudden death.[2]

Epiglottic swelling which is related to acute epiglottitis can be appreciated in a plain lateral neck radiograph, showing a classical ‘thumb sign’ opacity of the epiglottis. It is commonly employed as a screening imaging when epiglottitis is suspected. Besides that, bedside ultrasonography at an Emergency Department could be a valuable tool to detect pathological enlargement of the epiglottis. Ultrasound may be used in unstable patients for diagnosing epiglottitis because it is cheap, rapid, non-invasive and does not aggravate the patient’s symptoms.[3] The more reliable diagnostic strategy is to perform direct laryngoscopy or flexible nasopharyngolaryngoscopy as it can clearly show the suspected pathologic epiglottis.[1]

It is not uncommon for a patient to have symptoms of acute epiglottitis with suspected ingestion of a foreign body or the sensation of a ‘lump’ in the throat although later no foreign body is found.[1] This can be due to the fact that most patients were worried, as past history of foreign body ingestion had contributed directly to the symptom. Without knowing the more sinister condition and complication of epiglottitis, the presentation sometimes may be delayed. It can be more dangerous if the patient attempts to remove the foreign body on their own as there is a reported case of finger sweep to remove pharyngeal foreign body which ended up with epiglottic edema.[4]

In view of the impending airway obstruction, the patient should be admitted and closely observed. However if observation facilities and personnel skilled in airway management are available, routine prophylactic intubation of adult patients with acute epiglottitis is unnecessary. It is because such procedure, performed as an elective procedure,

Figure 2. Lateral neck radiograph showed swollen epiglottis with obliteration of normal valleculla space with a possible small piece of curved fish bone embedded in the soft tissue at the C5 level


Partial Epiglottic Edema Post Fish Bone Ingestion 71

will end up with longer hospital stay and greater morbidity due to other causes.[5] If the signs of impending airway obstruction is pronounced, the patient can be either intubated by endotracheal route, or tracheostomized.

In conclusion, presentation of odynophagia with positive history of foreign body should alert the treating physician to the possibility of epiglottic injury. Prompt diagnosis can be achieved from adequate history, lateral neck radiograph and laryngoscopic examination. Throughout the course of the investigation, the patient should be closely observed in view of sudden airway obstruction.

REFERENCES[1] Chung CH. Acute epiglottitis presenting as the sensation of a foreign body in the throat. Hong Kong Med J 2000;

6(3): 322-4.

[2] Asrar L, Oyetunji N, Raza SS, Zeyaulhaque I. Foreign body in the vallecula presenting as acute epiglottitis with unilateral supraglottitis. Saudi Med J 2005; 26(9): 1449-52.

[3] Bektas F, Soyuncu S, Yigit O, Turhan M. Sonographic diagnosis of epiglottal enlargement. Emerg Med J 2010; 27(3): 224-5.

[4] Kabbani M, Goodwin SR. Traumatic epiglottis following blind finger sweep to remove a pharyngeal foreign body. Clin Pediatr (Phila) 1995; 34(9): 495-7.

[5] Park KW, Darvish A, Lowenstein E. Airway management for adult patients with acute epiglottitis: A 12-year experience at an academic medical center (1984-1995). Anesthesiology 1998; 88: 254-61.


Lightning Strike Fatalities:Three Case Reports of Military Personnel in Malaysia

1A Rozali*, 2H Khairuddin, 3MS Sherina, 4LM Chia & 1K Yaakop1Health Services Division of Malaysian Armed Forces, Kuala Lumpur

23rd Infantry Division Headquarters, Terendak Camp, Melaka3Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor

494 Armed Forces Hospital, Terendak Camp, Melaka.

INTRODUCTIONLightning hazards are known causes of weather related injuries and death among civilians, [1] as well as among military personnel in developed and developing countries.[2, 3] Lightning is a natural atmospheric discharge that occurs between regions of net positive and net negative electric charges, and may cause injury or sudden death through direct strike, flash discharge (splash), contact current (step voltage) or blunt trauma. The most dramatic effects on the human body involve the cardiovascular and central nervous systems which may lead to significant morbidity and mortality.[4]

A retrospective study in Malaysia from 1996-2005 by Murty et al revealed that there were 27 fatal cases of et al revealed that there were 27 fatal cases of et allightning strikes which involved mostly foreign workers (59.3%) who worked as construction workers (40.7%). Most of the victims were brought in dead (37.0%) to the hospital.[1] A report from the Morbidity and Mortality Weekly Report (MMWR) of the Center for Disease Control and Prevention in 2002 revealed that approximately 350 active servicemen had been injured, with one death, due to lightning strikes between 1998 to 2001 in all United States military installations.[2] According to a report in the Malaysian Armed Forces, there have been about 9 deaths due to lightning strikes with single or multiple medical complications from 1985 to 2010.[3]

Malaysia is among the tropical countries in the world which has recorded the highest rates of lightning activity. The Meteorological Department of Malaysia recorded a few areas in Peninsular Malaysia that have had extremely high rates of 180 to 200 thunderstorm days per year as compared to other countries.[5] This significant number of lightning activities may increase the incidence of injuries and deaths related to lightning strikes. In view of the scarcity of reports, we would like to present three cases of sudden deaths due to lightning strikes among three military personnel. These incidents occurred while the personnel were carrying out their duties in operational areas in Malaysia. The aim of this paper is to highlight the hazards of lightning strikes and to prevent morbidity and mortality related to lightning strikes among military personnel in Malaysia.

CASE REPORTCases 1 & 2: These cases involved two soldiers, aged 21 years old and 33 years old respectively, who were struck by lightning (direct strikes) at the same time. The incident occurred while both of them, together with two other military personnel were patrolling in the frontline ‘forward location’ of Pos Batu Layar, Sabah. Both victims were patrolling by the sea side, under the Rhu trees with surrounding bushes. There was light rain with thunderclaps. After the lightning strike, the victims were found to be unconscious and brought to the nearest hospital, but were declared dead on arrival. The post mortem report confirmed that they died due to cardiac arrest secondary to lightning strike.



ABSTRACTLightning strikes have been known to cause fatal injuries. However, these cases have not been adequately highlighted in Malaysia. This paper reports on three cases of military personnel who lost their lives after being struck by lightning while on operational duties. It is extremely important to implement adequate safety measures and ensure that all military personnel are adequately trained on emergency procedures, such as Cardio Pulmonary Resuscitation (CPR) to prevent casualties of lightning strikes in future. This paper addresses several issues to avoid similar occurrences; the importance of taking extra safety precautions and recognizing signs of lightning strikes, as well as the immediate administration of CPR on victims.

Keywords: Lightning-strikes, Military-personnel, Operational-duty, Safety-measures


A Rozali, H Khairuddin, MS Sherina, LM Chia & K Yaakop74

Case 3: A 22 year old military personnel was struck by lightning while he and a few other military personnel from the same group (section size troopment) were having lunch under a tent. There was light rain with lightning and thunder. The deceased and the other military personnel were thrown to the ground when lightning struck their tent. The deceased was found to be unconscious and was immediately brought to the nearby hospital. He was pronounced dead on arrival. The post mortem report confirmed that he had cardiac arrest secondary to lightning strike.

DISCUSSION All victims were military personnel deployed in operational areas in Sabah, East Malaysia. Military personnel are more prone to being struck by lightning due to their operational duties outdoors and in all types of weather conditions. Military attires worn during operations include a complete set of gear, support accessories, and equipments that include personal weapons. Most of the gear, accessories and equipments are made from metal; such as buckles, knives, radio equipments and cooking utensils. It has been reported that lightning easily strikes if certain types of metals are attached to the body. [4] It is worse if the ground in that area also contains certain metals and minerals which attract lightning. Since military duties require personnel to move in groups of at least two, cases of lightning strikes among military will usually involve multiple casualties in each incident.

The findings of this report are consistent with the study by Murthy et al, where the lightning strikes occurred in the evenings at the end of the year when the victims had taken shelter under trees.[1] In this paper, both victims (Cases 1 & 2) were struck by lightning when they sought shelter under the trees. In case 3 however, the victim was struck while he was in a tent.

All victims had sudden loss of consciousness after being struck by lightning. Edlich et al. described that the most dramatic effects of lightning strikes involve the cardiovascular as well as central nervous systems, in which cardiopulmonary arrest is the most common cause of death. This probable mechanism is due to primary injuries or burns of the myocardium without coronary artery occlusion. This then induces vasomotor spasm from direct sympathetic stimulation resulting in severe loss of pulse in the extremities. The vasoconstriction may also be associated with transient paralysis. Immediate resuscitation of people struck by lightning greatly affects the prognosis.[4]

In view of this, immediate action should be taken to attend to victims who are struck by lightning. Military paramedic and personnel must be able to recognize the signs and symptoms of lightning strikes, as well as be aware that immediate resuscitation of these cases as essential to save lives. In the above cases, all three victims were brought to the nearest government hospitals for continued resuscitation. As there was no record of any resuscitation done at the scene of the incidents, we were unable to ascertain if adequate resuscitative measures were performed prior to their arrival at the hospitals. Therefore, we strongly recommend that military paramedic and personnel should be competent in administering Cardio Pulmonary Resuscitation (CPR), such as securing the airway, as well as in reviving breathing and maintaining the circulation (ABC). Medical emergency guidelines, including CPR techniques should also be reviewed and implemented at operational field sites as these incidents often occur in remote areas. These areas are very far from the nearest hospitals and emergency facilities, with the possibility of poor roads and trails. Therefore, it is very difficult to ascertain the duration of the journey to transport the victims to the hospitals.

In order to prevent further mishaps, we would like to propose that precautions be taken when there is rain and thunder while carrying out duties at operational sites. All levels of command must be informed about not having foot patrols when it rains, especially in thunderstorms (unless during emergencies or conflict situations). Personnel should be advised to stay indoors or under an enclosed shed or guard house, instead of under open tents. The current advice on avoiding being in open spaces and under trees during a thunderstorm should be reinforced. The best place to be is in an enclosed building. All military personnel should be aware of the hazards of raining and lightning, and should take reasonable precautions to protect themselves and those under their command from becoming victims of lightning strikes.

We hope that by publishing this report, all cases of lightning strikes can be avoided in the future. More importantly preventive measures should be implemented and reinforced as soon as possible to avoid such occurrences among Malaysian military personnel. Monitoring of such cases, should they unfortunately occur again should be properly recorded with adequate compensation to the victims’ families who have lost their breadwinners.

ACKNOWLEDGEMENTWe are grateful to the Director General of the Health Services Division, Malaysian Armed Forces for his support and permission to publish this paper.

REFERENCES[1] Murty OP, Chong KK, Mohammed Husrul AH, Ranjeev Kumar NK, Wan Yuhana MY. Fatal lightning strikes in

Malaysia: A review of 27 fatalities. Am J Forensic Med Pathol 2009; 30(3): 246-251.


Lightning Strike Fatalities: Three Case Reports of Military Personnel in Malaysia 75

[2] Center of Disease Control and Prevention (CDC). Lightning associated injuries and deaths among military personnel –United States, 1998 - 2001. MMWR Weekly, September 27, 2001/51(38); 859-862.

[3] Rozali A. Report on lightning fatalities among military personnel in Malaysian Armed Forces, 1985 - 2010. Kuala Lumpur: Malaysian Armed Forces (Unpublished).

[4] Edlich RF, Farinholt HM, Winters KL, Britt LD & Long WB. Modern concepts of treatment and prevention of lightning injuries. J Long Term Eff Med Implants 2005; 15(2): 185-96.

[5] Andrew S, Elizaberth T. Land of lightning. The Star Sunday 17, 2009.


77

Acknowledgement Vol. 8, No. 2 June 2012

The Editorial Board of the Medicine and Health Sciences gratefully acknowledge the following individuals for reviewing the papers submitted for publication consideration:

Prof. Dr. Yasmin Abdul Malik International Medical University

Prof. Dr. Azhar Md. Zain Universiti Putra Malaysia

Prof. Dr. Hj. Hamidon Hj. Basri Universiti Putra Malaysia

Prof. Dr. Wan Omar Abdullah Universiti Putra Malaysia

Prof. Dr. Elizabeth George Universiti Putra Malaysia

Prof. Dr. Zamberi Sekawi Universiti Putra Malaysia

Prof. Dr. Sherina Sidik Universiti Putra Malaysia

Prof. Dr. Muhammad Nazrul Hakim Abdullah Universiti Putra Malaysia

Assoc. Prof. Dr. Johnson Stanslas Universiti Putra Malaysia

Assoc. Prof. Dr. Rosline Hassan Universiti Sains Malaysia

Dr. Anita Abdul Rahman Universiti Putra Malaysia

Dr. Intan Nureslyna Samsudin Universiti Putra Malaysia

Dr. Sethu Thakachy Subha Universiti Putra Malaysia

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Corporate Author: Center for Disease Control and Prevention. Pertussis surveillance-United States. 1986-1988. Morb Mort Wkly Rep 1990; 39: 57-66.

Books and Other MonographsPersonal Author(s): Hoftbrand AV, Pettit JE, Moss PAH. Essential Haematology (4th ed). Cambridge: Blackwell Science, 1995.

Corporate Author: World Health Organization, International Agency for Research on Cancer. World Cancer Report. Lyon: IARC Press, 2003.

Editor, Compiler, Chairman as Author: Gibney MJ, Margetts BM, Kearney JM et al. (eds). Public health nutrition. Oxford: Blackwell Science, 2004.

Chapter in Book: White R. Stigmatization of mentally ill medical students – some strategies to tackle stigmatization and discrimination. In: Crisp AH (eds). Every family in the land. Understanding prejudice and discrimination against people with mental illness. London: Society of Medicine Press, 2003: Chap 9 part 2.

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