In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.
A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 10² and 10³ leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine.
A study of chikungunya virus was carried out to establish Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) as a rapid detection technique of the virus. The susceptibility of lab-colonized Aedes aegypti to chikungunya virus was also determined. Artificial membrane feeding technique was used to orally feed the mosquitoes with a human isolate of chikungunya virus. A total of 100 fully engorged female Ae. aegypti were obtained and maintained for 7 days. Seventy of them survived and then pooled at 10 individuals per pool. Total RNA was extracted from the samples and RT-PCR amplifications were carried out. Five out of 7 pools showed positive PCR band at 350-bp, indicating Ae. aegypti is a potential vector of chikungunya virus. The minimum infection rate (MIR) was 71% within these laboratory colonies. RT-PCR is a sensitive technique that is useful in detecting infected mosquitoes in epidemic areas. This technique can de used as a rapid detection method and provide an early virologic surveillance systems of chikungunya virus infected mosquitoes.
Peritonitis still remains a serious complication with high rate of morbidity and mortality in patients on CAPD. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early and optimal treatment. The aim of this study was to use 16S rRNA and ITS gene sequencing to identify common bacterial and fungal pathogens directly from the peritoneal fluid without culturing. Ninety one peritoneal fluids obtained from 91 different patients on CAPD suspected for peritonitis were investigated for etiological agents by 16S rRNA and ITS gene sequencing. Data obtained by molecular method was compared with the results obtained by culture method. Among the 45 patients confirmed for peritonitis based on international society of peritoneal dialysis (ISPD) guidelines, the etiological agents were identified in 37(82.2%) samples by culture method, while molecular method identified the etiological agents in 40(88.9%) samples. Despite the high potential application of the 16S rRNA and ITS gene sequencing in comparison to culture method to detect the vast majority of etiological agents directly from peritoneal fluids; it could not be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in Gene Bank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.
Colorectal carcinoma ranks third among ten leading causes of cancer in Malaysia. The colorectal carcinoma tumourigenesis involves the inactivation of tumour suppressor genes, and activation of proto-oncogenes. The p53 is one of the tumour suppressor genes that is involved in the colorectal carcinogenesis. The p53 gene is located on human chromosome 17p13.1 and comprises of 11 exons. Deficiencies in the p53 gene can cause the cancerous cells to spread to distant organs such as liver, lungs, lymph nodes, spine and bone. The most common p53 abnormalities that can lead to the metastasis of colorectal tumours are mutation and deregulation of the gene. In this study, nine colorectal carcinoma samples were used to establish a simple and sensitive strategy in the study on in vivo p53 expression by using realtime LightCycler SYBR Green I technology.
The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.
The United Nations High Commissioner for Refugees (UNHCR) reports over 80 million people are displaced worldwide with approximately 26.3 million categorized as refugees and over a million residing temporarily in South East Asia. Despite the lack of national legislative framework in place for refugees and asylum seekers (RAS), Malaysia hosts approximately 178,140 as registered with UNHCR and the majority originate from Myanmar. In this review, we examine refugees from South East Asia, particularly from Myanmar that have contributed to the largest influx of refugees to this region with a focus on their health status. The present study traces barriers to the health care of refugees in the country of asylum and also the challenges faced by these communities in accessing health services.
Entamoeba histolytica, the causative agent of human amoebiasis remains a significant cause of morbidity and mortality in developing countries and is responsible for up to 100,000 deaths worldwide each year. Entamoeba dispar, morphologically indistinguishable from E. histolytica is more common in humans in many parts of the world. Similarly Entamoeba moshkovskii, which was long considered to be a free-living amoeba is also morphologically identical to E. histolytica and E. dispar, and is highly prevalent in some E. histolytica endemic countries. Humans are the host of infection and there would not appear to be other meaningful animal reservoirs of E. histolytica. Entamoeba. histolytica can be present in sewage and contaminated water. The infection is mainly transmitted via ingestion of water or food contaminated by faeces containing E. histolytica cysts. Clinical features of amoebiasis range from asymptomatic colonization to amoebic dysentery and invasive extraintestinal amoebiasis, which is manifested most commonly in the form of abscesses in liver and lungs. The epidemiology of amoebiasis has dramatically changed since the separation of E. histolytica and E. dispar species and the worldwide prevalence of these species has not been estimated until recently. Morever, E. moshkovskii, another morphologically indistinguishable human parasitic Entamoeba was not mentioned or considered as a contributor to the prevalence figures in endemic areas. Amoebiasis is still a major health problem especially in aboriginal settlements and amongst people living in remote area in Malaysia. However, until now there is only one data currently available to indicate the true prevalence and incidence of E. histolytica and E. dispar. Further studies are needed to determine the burden of E. histolytica, E. dispar and E. moshkovskii infections in Malaysia. In the present review, we briefly summarize all methods use in diagnosing Entamoeba species, ranging from microscopic identification to molecular detection such as culture and isoenzyme analysis, antibody detection tests, antigen detection tests, immunochromatographic assays, conventional PCR, real-time PCR and loop-mediated isothermal amplification (LAMP).
The parasitic protozoan fauna in sixty-six anurans comprising of Duttaphrynus melanostictus, Phrynoidis juxtaspera, Hylarana erythraea and Polypedates leucomystax collected from Zoo Negara Malaysia was investigated. The distribution and prevalence rate of parasitic species in the digestive tract and blood were examined. Seven species of intestinal protozoa (Opalina ranarum, Cepedea dimidiata, Nycthetorus cordiformis, Entamoeba ranarum, Iodamoeba butschlii, Endamoeba blattae, and Tritrichomonas sp.) and two species of blood protozoa (Lankesterella sp. and Trypanosoma sp.) were recorded. Opalina ranarum was the most common protozoan found in the rectum and intestine (prevalence rate: 34.8%) infecting all host species, with P. juxtaspera heavily infected with the parasite, whereas Tritrichomonas sp. was the least prevalent intestinal species infecting only D. melanostictus. Both Lankesterella sp. and Trypanosoma sp. were found in the blood of H. erythraea.
Naegleria fowleri is a free-living amoeba, known as a causative agent for a fatal disease of the central nervous system (CNS) in man such as Primary amoebic meningoencephalitis (PAM). Factors contributing to its pathogenicity and its distribution in the environment have been investigated by previous researchers. In case of its pathogenicity, several enzymes such as phospolipase A and sphingomyelinase, have been proposed to probably act as aggressors in promoting PAM but no study so far have been conducted to investigate the presence of proteinase enzyme in this amoeba although a 56kDa cystein proteinase enzyme has been identified in Entamoeba histolytica as an important contributing factor in the amoeba's virulence. In this preliminary study, a pathogenic amoeba, Naegleria fowleri (strain NF3) was examined for the presence of proteinases. Samples of enzymes in this amoeba were analysed by electrophoresis using SDS-PAGE-gelatin gels. The results showed that this amoeba possesses at least two high molecular weight proteinases on gelatin gels; their apparent molecular weights are approximately 128 kDa and approximately 170 kDa. Band of approximately 128 kDa enzyme is membrane-associated and its activity is higher at alkaline pH compared with lower pH; at lower pH, its activity is greatly stimulated by DTT. The approximately 170 kDa band enzyme appears to be inactivated at pH 8.0, at lower ph its activity is higher and DTT-dependance. The activity of this enzyme is partially inhibited by inhibitor E-64 but markedly inhibited to antipain suggesting it belongs to the cysteine proteinase group.
The protein profile of serum samples from patients with amoebic liver abscess (ALA) was compared to those of normal individuals to determine their expression levels and to identify potential surrogate disease markers. Serum samples were resolved by two dimensional electrophoresis (2-DE) followed by image analysis. The up and down-regulated protein spots were excised from the gels and analysed by MS/MS. The concentration of three clusters of proteins i.e. haptoglobin (HP), α1-antitrypsin (AAT) and transferrin in serum samples of ALA patients and healthy controls were compared using competitive ELISA. In addition, serum concentrations of HP and transferrin in samples of patients with ALA and pyogenic liver abscess (PLA) were also compared. The results of the protein 2-DE expression analysis showed that HP cluster, AAT cluster, one spot each from unknown spots no. 1 and 2 were significantly up-regulated and transferrin cluster was significantly down-regulated in ALA patients' sera (p<0.05). The MS/MS analysis identified the unknown protein spot no.1 as human transcript and haptoglobin and spot no. 2 as albumin. Competitive ELISA which compared concentrations of selected proteins in sera of ALA and healthy controls verified the up-regulated expression (p<0.05) of HP and the down-regulated expression (p<0.01) of transferrin in the former, while there was no significant difference in AAT expression (p> 0.05). However, when ALA and PLA samples were compared, competitive ELISA showed significant increased concentration of HP (p<0.05) while transferrin levels were not different. In conclusion, this study showed that HP is a potential surrogate disease marker for ALA.
The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.
Nosocomial blood stream infection (or nosocomial bacteremia) is a common problem in hospitals worldwide, including Malaysia. A three-year prospective cohort study (October 2003-March 2007) of the incidences, risk factors, and patterns of the microorganisms causing bacteremia was conducted using a validated surveillance form in three intensive care units (ICUs) in Malaysia. Center for Disease Control criteria were used to diagnose bacteremia. Patients were monitored from admission until the end point of study, which was the first detection of bacteremia in the blood in each patient. The frequency of occurrence of bacteremia with clinical symptoms was 10.7% (n=23). Bacteremia was observed to occur within a mean length of stay of 10 days in ICU. The rate of device-related infection was 10.4% per device utilization days with a device utilization rate of 95.9%/1000 patient days. The total number of patient days was 2309 and the period of device utilization was 2211 days. The common bacteria detected were extended-spectrum beta-lactamases (ESBLs) Klebsiella pneumoniae (n=6); Pseudomonas aeruginosa (n=6); Acinetobacter species (n=5); Methicillin-resistant Staphylococcus aureus (MRSA)(n=3); and (non- ESBL) Klebsiella pneumoniae (n=2). Multivariable analysis using Cox Proportional Hazard Model showed that the predictors for developing bacteremia were cancer, MRSA carriage, duration of central venous catheter (CVC) infusion, frequency change of CVC, and the administration of hydrocortisone drugs. These results indicate that a combination of nursing and medical interventions as well as patients' severity of illness could lead to bacteremia in ICU. Strategic implementation of quality assurance measures in ICUs could help to control this problem.
Treatment Failure with chloroquine is one of the challenges that faced the dedicated efforts to eradicate malaria This study aims at investigating the impact of treatment failure with chloroquine on the progression of the disease-induced histo-pathogenic and immunogenic outcomes. To achieve this, Rane's protocol with modifications was applied on a model of Plasmodium berghei ANKA infected ICR mice to determine the dose response curve of chloroquine and to screen the treatment impact on the disease progression. Chloroquine was given at 1, 5, 10, 15 and 20 mg/kg once the parasitemia reached to 20-30% (the experimental initiation point). During the subsequent days, the mice were monitored for changes in the clinical signs, hematology parameters and the progress of the parasitemia until the parasitemia reached to 60-70% (the experimental termination point) or up to 10 days after chloroquine administration in case of achieving a complete eradication of the parasite. At the end, the mice were exsanguinated and their blood and organs were collected for the biochemistry and the histology study. A complete eradication of the parasite was achieved at 20 mg/kg while recrudescence was observed at the lower doses. At 1 mg/kg, the parasite growth was comparable to that of the positive control. The histo-pathogenic and immunogenic changes were stronger in the groups that experienced recrudescence (at 5 and 10 mg/kg). All in all, the study highlights the possibility of having a worsened clinical condition when chloroquine is given at its sub-therapeutic doses during malaria treatment.
A putative serine protease of T. spiralis (TsSP) was expressed in Escherichia coli and its potential as a diagnostic antigen was primarily assessed in this study. Anti-Trichinella IgG in serum samples from T. spiralis different animal hosts (mice, rats, pigs and rabbits) were detected on Western blot analysis with rTsSP. Anti-Trichinella antibodies were detected in 100% (30/30) of experimentally infected mice by rTsSP-ELISA. Cross-reactions of rTsSPELISA were not found with sera from mice infected with other parasites (S. erinaceieuropaei, S. japonicum, C. sinensis, A. cantonensis and T. gondii) and sera from normal mice. There was no statistical difference in antibody detection rate among mice infected with the encapsulated Trichinella species (T. spiralis, T. nativa, T. britovi, and T. nelsoni) (P>0.05). The results of rTsSP-ELISA showed that serum specific antibody IgG in mice infected with 100 or 500 T. spiralis muscle larvae (ML) were detectable early at 7-8 dpi, but not detected by ML ES antigen-ELISA prior to 10-12 dpi. Specific anti-Trichinella IgG was detected in 100% (18/18) of infected pigs by rTsSP-ELISA and ES-ELISA, but no specific antibodies was not detected in 20 conventionally raised normal pigs by two antigens. The results showed the rTsSP had the potential for early serodiagnosis of animal Trichinella infection, however it requires to be assayed with early infection sera of swine infected with Trichinella and other parasites.
This is a survey of 120 children for scabies and head lice infestations in a welfare home in Pulau Pinang. Children from this welfare home (Rumah Kanak-Kanak Taman Bakti, Kepala Batas, Pulau Pinang) were randomly selected. Majority of them were Malays (72.5%) and the rest were Indians. The infestation rates were highest in the 10-12 years age group with 46% and 70% for scabies and head lice respectively. Head lice was more commonly seen in girls (65%) than boys (29%). Scabies was more commonly seen in boys (50%) than girls (16%). Overall prevalence rate for scabies was 31% and for head lice infestation was 49%.
The objectives of this study were to determine the occurrence of Campylobacter spp. in live chickens sold at wet markets in Selangor, Malaysia and the multidrug resistance (MDR) profiles of the isolates. Cloacal swabs were taken from the chickens before slaughter and their caecal mucosae were swabbed after slaughter. Of the 90 chickens examined, 68 (75.6%) were positive for Campylobacter. Campylobacter were recovered from caecal swabs (53/90) and cloacal swabs (34/90) and Campylobacter coli (46 isolates) were identified slightly more than Campylobacter jejuni (41 isolates), but these differences were not significant (p<0.05). The most frequently observed resistance was to cephalothin (95.5%), followed by tetracycline (80.8%), erythromycin (51.4%), enrofloxacin (42.4%) and gentamicin (24.4%). Multidrug resistance (resistant to four or more antibiotics) was detected in 35.3% isolates. Campylobacter jejuni showed nine resistance profiles and the most common was to gentamicin-eryhtromycin-enrofloxacin-cephalothin-tetracycline (32.4%) combination while C. coli showed six profiles, with cephalothin-tetracycline (32.2%) combination being most common.
The objective was to estimate the prevalence of intestinal protozoa among the aborigines and to determine the problems regarding the infection. The study was carried out in January 2006 in Pos Senderut, Pahang, Malaysia. Samples of faeces were collected from children and adults and these were fixed in PVA and trichrome staining was carried out. From the 130 individuals studied, 94 (72.3%) were positive with at least one intestinal protozoa. Nine intestinal protozoa namely Blastocystis hominis, Giardia lamblia, Entamoeba histolytica, Entamoeba coli, Endolimax nana, Entamoeba hartmani, Entamoeba polecki, Iodamoeba butschlii and Chilomastix mesnili were detected. The prevalent species were B. hominis (52.3%), followed by G. lamblia (29.2%), E. coli (26.2%) and E. histolytica (18.5%). The other species ranged from 1.5 to 10.8%. Among the positive samples, mixed infection with E. histolytica and G. lamblia was 3.8%, E. histolytica and B. hominis was 15.4%, G. lamblia and B. hominis was 17.7%. Triple infection of E. histolytica, G. lamblia and B. hominis was 3.1%. The infection was more prevalent in children below 10 years age group (45.4%) and lowest in the age above 60 years (3.8%). The high prevalence was attributable to poor environmental management, poor personal hygiene and lack of health education.
Intestinal parasitic infections are among the most common diseases affecting mankind causing major public health problems to billions of people living in developing countries. The aim of this study is to determine the prevalence of intestinal parasites in various communities residing in different habitats in Malaysia and compare the findings with 101 studies conducted over the past 42 years (1970-2013). A cross-sectional study design was conducted with the aid of a questionnaire to collect relevant information about the study population. Faecal samples were examined using the direct smear and formal ether sedimentation techniques. A total of 342 children were examined amongst whom 24.6% were positive for intestinal parasitic infections. Results showed that 32.3% of rural children, 20.6% of urban squatters and 5.4% of children from flats were positive for one or more parasites. The most common parasite encountered was Trichuris trichiura (20.2%) followed by Ascaris lumbricoides (10.5%) and hookworm (6.7%). No case of hookworm was reported in urban children whereas 12.2% of rural children were positive. The most common protozoan parasite detected was Entamoeba coli (3.2%) followed by Giardia intestinalis (1.8%), Entamoeba histolytica (1.8%) and Blastocystis hominis (1.2%). Nearly one-fifth (18.4%) of the children had single infection followed by double (12.0%) and triple infections (1.2%). Orang Asli (indigenous) children (44.3%) had the highest infection rate followed by Indians (20.2%), Malays (14.0%) and Chinese (11.9%). Twenty-eight studies carried out on plantation communities with regards to intestinal parasitic infections in Malaysia from 1970 to 2013 showed a steady decline in the prevalence rate ranging from 95.0% in the seventies to 37.0 % in 2012. Intestinal parasitic infections were more common in Orang Asli communities with prevalence ranging from over 90% in the seventies and fluctuating below 70% in most studies between 2000 to 2013 except for two studies that showed a prevalence of 98.2% and 100%. The prevalence rate among urban squatters, urban residents and those living in flats showed dramatic decrease in prevalence rate.
A survey was undertaken to investigate the prevalence of intestinal and blood parasites among wild rats in urban area of Kuala Lumpur, Malaysia. A total of 137 stool and blood samples were collected from wild rats from Sentul and Chow Kit areas. Five species of rats were captured and supplied by Kuala Lumpur City Hall. The most common was Rattus rattus diardii (Malayan Black rat), 67%, followed by Rattus norvegicus (Norway rat), 10%, Rattus argentiventer (rice-field rat), 10%, Rattus tiomanicus (Malaysian field rat), 9% and Rattus exulans (Polynesian rat), 4%. Rattus rattus diardii is commonly known to live in human environment and they are normally identified as pests to human community. More male rats were captured (61%) compared to female (39%). Out of 137 samples, 81.8% samples were positive with intestinal parasites, with 86.2% from Sentul area and 78.5% from Chow Kit area. Six different parasites were detected. The most common intestinal helminth parasite detected was Nippostrongylus brasiliensis (80.3%), followed by Hymenolepis nana (23.4%), Capillaria hepatica (13.9%) and Hymenolepis diminuta (2.9%). Intestinal protozoan detected was Entamoeba histolytica/E. dispar (8.8%). Trypanosoma lewisi (1.5%) was the only blood parasite detected.