A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.
A survey was undertaken to investigate the prevalence of intestinal parasites from different groups of mammals housed in a zoological garden in Malaysia. A total of 197 faecal samples were collected randomly from various primates (99), hoofed mammals (70) and feline (28). It was discovered that 89.3% of feline, 54.5% of primates and 45.7% of hoofed mammals were infected with intestinal parasites. Intestinal parasites found in primates were Balantidium coli (19.2%), Cryptosporidium spp. (14.1%), hookworm (10.1%), Trichuris spp. (5.1%), Ascaris (4.0%) and Blastocystis spp. (2.0%). For hoofed mammals, hookworm had the highest prevalence (34.3%) followed by Trichuris spp. and Cryptosporidium spp. (5.7%). Meanwhile, for feline, Toxocara cati was the most prevalent (64.3%), followed by Cryptosporidium spp. (14.3%), Spirometra spp. (7.1%), and hookworm (3.6%). Animals that were infected were all asymptomatic with low parasite load. Routine monitoring of the presence of parasites in animals kept in the zoo is imperative in assisting zoo management in the formulation and implementation of preventive and control measures against the spread of infectious parasitic diseases among animals within the zoo or to humans.
Q fever is a disease caused by Coxiella burnetii. It is a disease of public health concern in many parts of the world. In this study, we described the seroprevalence of Q fever among selected populations of Orang Asli (OA), indigenous people, many of whom live within the forest fringe areas of Peninsular Malaysia. Serum samples were obtained from 887 OA participants from selected villages. Samples were analyzed for the presence of IgG antibodies reactive against C. burnetii by enzyme-linked immunosorbent assay. Statistical methods were used to identify possible associations between seropositivity for C. burnetii and a number of demographic variables obtained from the questionnaires. In total, 9.6% (n = 85/887) of the serum samples were reactive to C. burnetii. Statistical results suggest that elderly male OA residing in OA village, Bukit Payung, were most likely to be tested seropositive for C. burnetii. This study suggests that OA are at a significant risk of contracting C. burnetii infection, and both demographic and geographic factors are important contributors to this risk. Further prospective studies are needed to establish the true burden of C. burnetii infection within the indigenous population as well as within Peninsular Malaysia as a whole.
Mosquitoes are principal vectors of major vector-borne diseases. They are widely found throughout urban and rural areas in Malaysia. They are responsible for various vector-borne diseases such as dengue, malaria, filariasis and encephalitis. A total of 158 mosquito larvae specimens were collected from the National Zoo, Malaysia, from 11 types of breeding habitats during the study period from end of May 2007 to July 2007. Aedes albopictus was the predominant species (35.4%), followed by Tripteroides aranoides (26.6%), Lutzia halifaxii (11.4%), Aedes alboscutellatus (10.1%), Aedes caecus (8.9%), Armigeres spp. (4.4%), Malaya genurostris (2.5%) and Culex vishnui (0.6%). It is important to have a mosquito free environment in a public place like the zoo. Routine larval surveillance should be implemented for an effective mosquito control program in order to reduce mosquito population.
Limited data is available on the prevalence of parasitic infections among the island communities in Malaysia with most studies performed between 1960s-1980s. This study was conducted to determine the current prevalence status of parasitic infections among communities living in Pangkor Island Peninsular Malaysia. A total of 131 stool and 298 serum samples were collected and subjected to microscopic examination for intestinal protozoa and helminths and detection of Toxoplasma gondii antibodies using commercial ELISA kits respectively. In addition, thin and thick peripheral blood films were microscopically screened for the presence of Plasmodium spp. and microfilariae respectively. The overall prevalence of intestinal parasitic infections among Pangkor Island community was 9.9% (13/131) with T. trichiura (5.3%) being the most common intestinal parasite detected. Toxoplasmosis was reported in almost 60% of the community with the seroprevalence being significantly high among females (64.7%) compared to males (52.8%) (p=0.038). None of those examined samples were infected with intestinal sarcocystosis, malaria and filariasis. This study revealed that the prevalence of intestinal parasitic infections among Pangkor Island community has been greatly reduced compared to that reported 35 years ago. Massive improvements in the socioeconomic status, personal hygiene, water facilities and sanitation may have contributed to the low prevalence of parasitic infections in this community. Nevertheless, further studies still need to be performed to determine the possible risk factors for the high prevalence of toxoplasmosis in this community.
We report a case of Hymenolepis diminuta infection in a 2-year-old Malaysian child. This case was initially reported as 'normal' after the examination of proglottids shed from the anus of the child at a private laboratory on two occasions. The putative proglottids shed was then referred to the Parasite Southeast Asia Diagnostic (Para:SEAD) Laboratory, Department of Parasitology, Faculty of Medicine, University of Malaya for further examination. Microscopic examination confirmed that the child was infected with H. diminuta based on the characteristic eggs found in the proglottids. She was treated with a single dose praziquantel (20 mg/kg of body weight) and recovered well.
Human immunodeficiency virus (HIV)-infected individuals have greater susceptibility to infections by a myriad of microorganisms which can cause significant morbidity and mortality compared to immunocompetent individuals. Of these microbial infections, intestinal parasitic infections (IPIs) however are receiving less attention than bacterial and viral infections, hence, the lack of information of parasitic infections in HIV individuals. Prevalence of IPIs among 346 HIV-infected individuals in Malaysia was determined in this study. The overall prevalence of intestinal parasitic infections (IPIs) was 37.9% (131 of 346) with protozoa infections (18.8%) being more common compared to helminth infections (7.5%). Observed protozoa include Entamoeba histolytica/dispar (16.8%), Cryptosporidium parvum (12.4%), Isospora belli (10.1%), Cyclospora cayetanensis (4.9%) and Giardia duodenalis (intestinalis) (3.2%) whilst helminthes which were detected comprised of Ascaris lumbricoides (13.9%), Trichuris trichiura (6.4%) and hookworms (0.6%). Among those 131 infected, 50.4% had multiple infections and 48.9% had single parasitic infection. The CD4 counts were significantly lower (i.e., 200 cells/mm³) in patients harbouring IPIs. Of those individuals infected with intestinal parasites, 49% were intravenous drug users and 58% were not on any antiretroviral therapy. Most were asymptomatic and had concurrent opportunistic infections (OIs) mainly with Mycobacterium tuberculosis infection. These results confirmed that IPIs are ubiquitous among HIV-infected individuals, especially those presenting with low CD4 T cells counts, and provide useful insights into the epidemiology of these infections among HIV-infected patients in Malaysia. It is therefore recommended, that diagnosis of these intestinal parasitic pathogens should be conducted on a routine basis for better management of gastrointestinal illnesses among HIV individuals.
In the last few years in Malaysia, dengue fever has increased dramatically and has caused huge public health concerns. The present study aimed to establish a spatial distribution of dengue cases in the city of Kuala Lumpur using a combination of Geographic Information System (GIS) and spatial statistical tools. Collation of data from 1,618 dengue cases in 2009 was obtained from Kuala Lumpur City Hall (DBKL). These data were processed and then converted into GIS format. Information on the average monthly rainfall was also used to correlate with the distribution pattern of dengue cases. To asses the spatial distribution of dengue cases, Average Nearest Neighbor (ANN) Analysis was applied together with spatial analysis with the ESRI ArcGIS V9.3 programme. Results indicated that the distribution of dengue cases in Kuala Lumpur for the year 2009 was spatially clustered with R value less than 1 (R = 0.42; z-scores = - 4.47; p < 0.001). Nevertheless, when this pattern was further analyzed according to month by each zone within Kuala Lumpur, two distinct patterns were observed which include a clustered pattern (R value < 1) between April to June and a dispersed pattern (R value > 1) between August and November. In addition, the mean monthly rainfall has not influenced the distribution pattern of the dengue cases. Implementation of control measures is more difficult for dispersed pattern compared to clustered pattern. From this study, it was found that distribution pattern of dengue cases in Kuala Lumpur in 2009 was spatially distributed (dispersed or clustered) rather than cases occurring randomly. It was proven that by using GIS and spatial statistic tools, we can determine the spatial distribution between dengue and population. Utilization of GIS tools is vital in assisting health agencies, epidemiologist, public health officer, town planner and relevant authorities in developing efficient control measures and contingency programmes to effectively combat dengue fever.
Orang Asli are the indigenous minority peoples of peninsular Malaysia. Despite proactive socioeconomic development initiated by the Malaysian Government in upgrading the quality of life of the Orang Asli communities since 1978, they still remained poor with a current poverty rate of 76.9%. Poverty exacerbates the health problems faced by these communities which include malnourishment, high incidences of infectious diseases (eg. tuberculosis, leprosy, malaria) and the perpetual problem with intestinal parasitic infections. Studies reported that the mean infection rate of intestinal parasitic infections in Orang Asli communities has reduced from 91.1% in 1978, to 64.1% in the subsequent years. Although the results was encouraging, it has to be interpreted with caution because nearly 80% of studies carried out after 1978 still reported high prevalence (i.e. >50%) of soil-transmitted helminthiases (STH) among Orang Asli communities. Prior to 1978, hookworm infection is the most predominant STH but today, trichuriasis is the most common STH infections. The risk factors for intestinal parasitic infections remained unchanged and studies conducted in recent years suggested that severe STH infections contributed to malnutrition, iron deficiency anaemia and low serum retinol in Orang Asli communities. In addition, STH may also contribute to poor cognitive functions and learning ability. Improvements in socioeconomic status in Malaysia have shown positive impact on the reduction of intestinal parasitic infections in other communities however, this positive impact is less significant in the Orang Asli communities. In view of this, a national parasitic infections baseline data on morbidity and mortality in the 18 subgroups of Orang Asli, will assist in identifying intervention programmes required by these communities. It is hope that the adoption of strategies highlighted in the World Health Organisation- Healthy Village Initiatives (WHO-HVI) into Orang Asli communities will ensure the whole mechanism of delivery and empowerment by the government agencies become more efficient and productive in alleviating intestinal parasitic infections in these communities.
Cryptosporidium and Giardia are two important pathogenic parasites that have caused many waterborne outbreaks which affected hundreds of thousands of people. Contamination from effluent discharged by sewage treatment plants have been implicated in previous waterborne outbreaks of Cryptosporidium and Giardia. This study evaluated the reduction of Cryptosporidium and Giardia (oo)cysts in two sewage treatment plants (STPA and STPB) in Malaysia which employed different treatment processes for a period of a year. Raw sewage influents and treated sewage effluents were concentrated by repeated centrifugation, subjected to sucrose density flotation and concentrated to a minimal volume depending upon the levels of contaminating debris. Cryptosporidium oocysts and Giardia cysts were enumerated using epifluorescence microscopy. The parasite concentrations in raw sewage were 18-8480 of Giardia cysts/litre and 1-80 of Cryptosporidium oocysts/litre. In treated sewage, the concentration of parasites ranged from 1-1462 cysts/litre and 20-80 oocysts/ litre for Giardia and Cryptosporidium respectively. Statistical analysis showed that sewage treatment process which employed extended aeration could reduce the concentration of Cryptosporidium and Giardia (oo)cysts significantly but treatment process which encompasses aerated lagoon could only reduce the concentration of Giardia cysts but not Cryptosporidium oocysts significantly. This phenomenon is of great concern in areas whereby effluent of sewage treatment plants is discharged into the upstream of rivers that are eventually used for abstraction of drinking water. Therefore, it is important that wastewater treatment authorities rethink the relevance of Cryptosporidium and Giardia contamination levels in wastewater and watersheds and to develop countermeasures in wastewater treatment plants. Further epidemiological studies on the occurrence and removal of pathogenic organisms from excreta and sewage are also recommended, in order that the public health risks can be defined and the most cost effective sewage treatment options developed.
A study to determine the contribution of Giardia cysts and Cryptosporidium oocysts from cattle farms was carried out at the Langat Basin. This study investigated the contribution of cattle farms, located near Sungai Langat and Sungai Semenyih, towards river contamination with these cysts and oocysts. The findings showed that out of 24 samples of water taken from Sungai Semenyih, 4.2% was positive for Giardia cysts with a concentration of 1.3 cysts/L and 20.8% were positive with Cryptosporidium oocysts with a range of 0.7 - 2.7 oocysts/L. At Sungai Langat, from the 43 samples taken, 23.3% were positive for Giardia cysts with a range of 1.5 - 9 cysts/L whereas 11.6% were positive with Cryptosporidium oocysts with a range of 2.5 - 240 oocysts/L. Isolation of cysts and oocysts in bovine faecal materials revealed that 14.6% of faecal samples were positive for Giardia cysts which had a range of 75 - 1.3x104 cysts/g and 25% were positive for Cryptosporidium oocysts with a range of 50 - 3.9x105 oocysts/g. From the cattle wastewater, 98% were positive with oocysts and 6.7% with cysts. The concentrations were between 20 - 3.1x103 oocysts/mL for Cryptosporidium and 4 - 75 cysts/mL for Giardia. Given that the prevalence of Cryptosporidium and Giardia are high amongst the cattle and the positive findings of the (oo)cysts in the river samples, it could be deduced that there is a very high possibility of the cattle farms contaminating the river with Giardia cysts and Cryptosporidium oocysts. Viability study of Cryptosporidium oocysts in the surrounding soil and pond within the cattle farm showed that the viability of Cryptosporidium oocysts decreased with time. It was estimated that it will take 52 days for all the oocysts from both environment to be non-viable. With a viability rate of approximately 2 months in a cattle farm setup, river water contaminated with Cryptosporidium oocysts has a high chance of acting as an agent of transmission. As cattle farms are also inhabited by the owners and their families, this problem may pose a threat to humans (e.g. children) especially if they are dependent on the river water as their source of water for their daily activities.
This study was conducted to reassess the risk factors for giardiasis in communities of the Orang Asli (indigenous people) in Pahang, Malaysia. Stool samples were collected from 321 individuals (2-76 years old; 160 males, 161 females). Data were collected via laboratory analysis of faecal samples and a pre-tested standard questionnaire. River water samples were tested for Giardia cysts and Cryptosporidium oocysts. The overall prevalence of G. intestinalis infection was 23.7%. Children < or =12 years old had the highest infection rate and have been identified as a high risk group (odds ratio (OR)=6.2, 95% CI 1.5-27.0, P<0.005). The risk of getting giardiasis also appeared to be significantly associated with drinking piped water (OR=5.1, 95% CI 0.06-0.7, P<0.005) and eating raw vegetables (OR=2.4, 95% CI 0.2-0.6, P<0.005). In conclusion, sociodemographic factors have always been associated with the high prevalence of Giardia infections in Malaysia. However, the present study also highlights the need to look into the possibility of other risks such as water and food transmission routes. In future, it is necessary that these two aspects be considered in control strategies.
We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 10(2) CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 10(3) CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.
Despite great development in socioeconomic status throughout 50 years of independence, Malaysia is still plagued with soil-transmitted helminthiases (STH). STH continue to have a significant impact on public health particularly in rural communities. In order to determine the prevalence of STH among rural Orang Asli children and to investigate the possible risk factors affecting the pattern of this prevalence, fecal samples were collected from 292 Orang Asli primary schoolchildren (145 males and 147 females) age 7-12 years, from Pos Betau, Kuala Lipis, Pahang. The samples were examined by Kato-Katz and Harada Mori techniques. Socioeconomic data were collected using pre-tested questionnaires. The overall prevalence of ascariasis, trichuriasis, and hookworm infections were 67.8, 95.5 and 13.4%, respectively. Twenty-nine point eight percent of the children had heavy trichuriasis, while 22.3% had heavy ascariasis. Sixty-seven point seven percent of the children had mixed infections. Age > 10 years (p = 0.016), no toilet in the house (p = 0.012), working mother (p = 0.040), low household income (p = 0.033), and large family size (p = 0.028) were identified as risk factors for ascariasis. Logistic regression confirmed low income, no toilet in the house and working mother as significant risk factors for ascariasis. The prevalence of STH is still very high in rural Malaysian communities. STH may also contribute to other health problems such as micronutrient deficiencies, protein-energy malnutrition and poor educational achievement. Public health personnel need to reassess current control measures and identify innovative and integrated ways in order to reduce STH significantly in rural communities.
A total of 1,885 blood and stool samples of four main protozoan parasitic infections were retrospectively reviewed from January, 2000 to April, 2004. Eleven of the 1,350 stool samples were shown positive for Cryptosporidium and Giardia infections; one of the 5 cases was clinically diagnosed as gastrointestinal cryptosporidiosis, while 6 cases were giardiasis. In patients with giardiasis, children were among the high-risk groups, making up 66.7% of these patients. The common presenting signs and symptoms were: diarrhea (83.3%), loss of appetite (83.3%), lethargy (83.3%), fever (66.7%), nausea/vomiting (50.0%), abdominal pain (16.7%), dehydration (16.7%) and rigor and chills (16.7%). Metronidazole was the drug of choice and was given to all symptomatic patients (83.3%). For the blood samples, 28 of the 92 peripheral smears for Plasmodium spp infection were diagnosed as malaria. The age range was from 4 to 57, with a median of 32.5 years. The sex ratio (M:F) was 3.6:1, while the age group of 30-44 years was the most commonly affected in both sexes. The majority of patients were foreigners (60.7%) and non-professional (39%). Plasmodium vivax (71%) infection was the most common pathogen found in these patients, along with a history of traveling to an endemic area of malaria (31%). The predominant presenting signs and symptoms were: fever (27%), rigor and chills (24%), nausea/vomiting (15%) and headache (8%). Chloroquine and primaquine was the most common anti-malarial regimen used (78.6%) in these patients. The seroprevalence of toxoplasmosis in different groups was 258/443 (58%): seropositive for IgG 143 (32.3%); IgM 67 (15%); and IgG + IgM 48 (10.8%). The age range was from 1 to 85, with a mean of 34 (+/- SD 16.6) years. The predominant age group was 21 to 40 years (126; 28.4%). The sex ratio (M:F) was 1.2:1. Subjects were predominantly male (142; 32%) and the Malay (117; 26.4%). Of these, 32 cases were clinically diagnosed with ocular toxoplasmosis. The range of age was from 10 to 56 years with a mean of 30.5 (+/- SD 12.05) years. The sex ratio (M:F) was 1:1.7. The majority were in the age group of 21 to 40 years, female (20; 62.5%), and Malay (17; 53%). They were also single (16; 50%), unemployed (12; 37%), and resided outside Kuala Lumpur (21; 65.6%). The more common clinical presentations were blurring of vision (25; 78%), floaters (10; 31%) and pain in the eye (7; 22%). We found that funduscopic examination (100%) and seropositivity for anti-Toxoplasma antibodies (93.7%) were the main reasons for investigation. Choroidoretinitis was the most common clinical diagnosis (69%), while clindamycin was the most frequently used antimicrobial in all cases. Among HIV-infected patients, 10 cases were diagnosed as AIDS-related toxoplasmic encephalitis (TE) (9 were active and 1 had relapse TE). In addition, 1 case was confirmed as congenital toxoplasmosis.
The occurrence of a coccidian parasite, Cryptosporidium, among birds in the Kuala Lumpur National Zoo was investigated in this study. A hundred bird fecal samples were taken from various locations of the zoo. Fecal smears prepared using direct smear and formalin ethyl acetate concentration technique were stained with modified Ziehl-Neelsen stain. Samples positive for Cryptosporidium with Ziehl-Neelsen stain were later confirmed using the immunofluorescence technique and viewed under the epifluorescence microscope. Six species of bird feces were confirmed positive with Cryptosporidium oocysts. They included Wrinkled Hornbill (Aceros corrugatus), Great Argus Pheasant (Argusianus argus), Black Swan (Cygnus atratus), Swan Goose (Anser cygnoides), Marabou Stork (Leptoptilos crumeniferus), and Moluccan Cockatoo (Cacatua moluccencis). These birds were located in the aviary and lake, with the Moluccan Cockatoo routinely used as a show bird. Results obtained in this study indicated that animal sanctuaries like zoos and bird parks are important sources of Cryptosporidium infection to humans, especially children and other animals.
A total of 66 fecal specimens obtained from patients infected with human immunodeficiency virus (HIV) from Kajang Hospital were screened for Cryptosporidium oocysts. The fecal specimens were concentrated using the formalin ethyl acetate concentration technique, stained with modified Ziehl-Neelsen and confirmed with immunofluorescence stain. It was established that 2 (3.0%) were positive for Cryptosporidium. The two cases involved a Chinese local man (with diarrhea) and an Indonesian foreigner (without diarrhea). A higher index of suspicion for clinical cryptosporidiosis in HIV patients, including those with chronic weight loss with or without diarrhea, is recommended. In addition, laboratory testing for Cryptosporidium in HIV-infected patients is highly recommended in order to have a better understanding of the epidemiology and management of the disease in Malaysia.
A survey of the river water frequently used by the Temuan Orang Asli (aborigine) indicated that 66.7% of the river water samples were Giardia cyst positive and 5.6% were Cryptosporidium oocyst positive. Although Giardia cysts were detected in samples from all the sites (e.g. upstream, midstream, and downstream), Cryptosporidium was only present in one river water sample taken from downstream from a village. The only sample of upstream water which contained Giardia cysts had a concentration of 0.7 cysts/l. All samples taken from midstream contained cysts with a mean concentration of 9.8 +/- 6.6 cysts/l (range = 1-20 cysts/l). Eighty-three point three percent of the samples collected from downstream contained cysts and 16.7% had oocysts. The average concentration of cysts was 12.9 +/- 16.4 cysts/l (range = 0-44 cysts/l), whereas the oocyst concentration was 0.4 oocysts/l. All river samples tested positive for the presence of E. coli, indicating fecal contamination. The results of this study imply that the river system is contaminated with fecal-oral transmitted parasites. The river water, used by the Orang Asli, is a probable route for Giardia and Cryptosporidium transmission in this community. Long term strategies, incorporating health education regarding personal hygiene, and provision of toilets and the importance of their proper usage, need to be embraced by this community in order to control the spread of these parasites.
A survey was carried out to determine the prevalence of bronchial asthma and their contributing risk factors among Orang Asli subgroups living in Malaysia using IUATLD questionnaire and spirometry without being discriminatory towards age or gender. Of the 1171 distributed questionnaires, 716 (61.1%) comprising of 62.7% Semai Pahang, 51.3% Temiar, 74.2% Mah Meri, 65.6% Semai Perak, 53.6% Temuan, 53.8% Semelai, 61.1% Jakun and 67.4% Orang Kuala subgroups completed their questionnaire and were included in the data analysis. Participants comprised 549 (76.7%) children and 167 (23.3%) adults, age between 1 to 83 years old, 304 (42.5%) males and 412 (57.5%) females. The overall prevalence of bronchial asthma was 1.4% of which 1.5% was children, 1.3% adults, 1.0% male and 1.7% female, respectively. Of the 8 subgroups surveyed, 5 out of 10 confirmed asthma cases were Semai Pahang, followed by 3 cases among Mah Meri, and one case each among Temuan and Semai Perak subgroups, respectively. This study also demonstrated that the prevalence of self-reported and confirmed bronchial asthma tend to be higher among those who had close contact with pets, smoking individuals and among those who had a family history of asthma.