A Giardia varani Lavier, 1923-like flagellate was found in the feces of a captive water monitor, Varanus salvator, originally caught wild from an unknown location in Malaysia. The parasite is similar in size and shape to Giardia lamblia, except that median bodies are rare and cysts are binucleate. A description of both the trophozoite and cyst stage of this flagellate is provided.
An aquatic biomonitoring of Giardia cysts and Cryptosporidium oocysts in river water corresponding to five villages situated in three states in peninsular Malaysia was determined. There were 51.3% (20/39) and 23.1% (9/39) samples positive for Giardia and Cryptosporidium (oo)cysts, respectively. Overall mean concentration between villages for Giardia cysts ranged from 0.10 to 25.80 cysts/l whilst Cryptosporidium oocysts ranged from 0.10 to 0.90 oocysts/l. Detailed results of the river samples from five villages indicated that Kuala Pangsun 100% (9/9), Kemensah 77.8% (7/9), Pos Piah 33.3% (3/9) and Paya Lebar 33.3% (1/3) were contaminated with Giardia cysts whilst Cryptosporidium (oo)cysts were only detected in Kemensah (100 %; 9/9) and Kuala Pangsun (66.6%; 6/9). However, the water samples from Bentong were all negative for these waterborne parasites. Samples were collected from lower point, midpoint and upper point. Midpoint refers to the section of the river where the studied communities are highly populated. Meanwhile, the position of the lower point is at least 2 km southward of the midpoint and upper point is at least 2 km northward of the midpoint. The highest mean concentration for (oo)cysts was found at the lower points [3.15 ± 6.09 (oo)cysts/l], followed by midpoints [0.66 ± 1.10 (oo)cysts/l] and upper points [0.66 ± 0.92 (oo)cysts/l]. The mean concentration of Giardia cysts was highest at Kuala Pangsun (i.e. 5.97 ± 7.0 cysts/l), followed by Kemensah (0.83 ± 0.81 cysts/l), Pos Piah (0.20 ± 0.35 cysts/l) and Paya Lebar (0.10 ± 0.19 cysts/l). On the other hand, the mean concentration of Cryptosporidium oocysts was higher at Kemensah (0.31 ± 0.19 cysts/l) compared to Kuala Pangsun (0.03 ± 0.03cysts/l). All the physical and chemical parameters did not show significant correlation with both protozoa. In future, viability status and molecular characterisation of Giardia and Cryptosporidium should be applied to identify species and genotypes/subgenotypes for better understanding of the epidemiology of these waterborne parasites.
Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.
Giardia duodenalis is considered the most common intestinal parasite in humans worldwide. In Malaysia, many studies have been conducted on the epidemiology of giardiasis. However, there is a scarcity of information on the genetic diversity and the dynamics of transmission of G. duodenalis. The present study was conducted to identify G. duodenalis assemblages and sub-assemblages based on multilocus analysis of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes. Faecal specimens were collected from 484 Orang Asli children with a mean age of 7 years and examined using light microscopy. Specimens positive for Giardia were subjected to PCR analysis of the three genes and subsequent sequencing in both directions. Sequences were edited and analysed by phylogenetic analysis. G. duodenalis was detected in 17% (84 of 484) of the examined specimens. Among them, 71 were successfully sequenced using at least one locus. Genotyping results showed that 30 (42%) of the isolates belonged to assemblage A, 32 (45%) belonged to assemblage B, while discordant genotype results were observed in 9 specimens. Mixed infections were detected in 43 specimens using a tpi-based assemblage specific protocol. At the sub-assemblages level, isolates belonged to assemblage A were AII. High nucleotide variation found in isolates of assemblage B made subtyping difficult to achieve. The finding of assemblage B and the anthroponotic genotype AII implicates human-to-human transmission as the most possible mode of transmission among Malaysian aborigines. The high polymorphism found in isolates of assemblage B warrants a more defining tool to discriminate assemblage B at the sub-assemblage level.
Chemical inactivation of microorganisms is a common process widely employed in many fields such as in treatment of water, preservation in food industry and antimicrobial treatments in healthcare. For economy of applications and efficiency of treatment establishment the minimum dosage of breakpoint in the chemical application becomes essential. Even though experimental investigations have been extensive, theoretical understanding of such processes are demanding. Commonly employed theoretical analyses for the inactivation of microorganisms and depletion of chemicals include kinetics expressing the rates of depletion of chemical and microorganisms. The terms chemical demand (x) and specific disinfectant demand (alpha) are often used in theoretical modeling of inactivation. The value of specific disinfectant demand (alpha) has always been assumed to be a constant in these models. Intracellular concentration built up within the cells of the microorganisms during inactivation could lead to possible weakening effects of microorganisms thereby requiring lower doses as disinfection proceeds makes the assumption of constant alpha inaccurate. Model equations are formulated based on these observations co-relating the parameters alpha and x with a progressive inactivation (N/N(0)). The chemical concentration (C) is also presented in terms of the inactivation time (t) and the survival ratio (N/N(0)) for given pH and temperature conditions. The model is examined using experimentally verified Ct data of Giardia Cysts/chlorine system. The respective values of x for different survival ratios were evaluated from the data using MatLab software. Proposed model correlating for the disinfectant demand (x) with the survival ratio (N/N(0)) fits satisfactorily with those evaluated from data. The rate constants for different pH and temperature conditions are evaluated which showed compatibility with the Arrhenius model. The dependence of frequency factors with pH indicated compatibility with accepted models. The Ct values regenerated with the kinetic data shows a very accurate fit with published data.
Giardia intestinalis merupakan parasit kosmopolitan dan infeksinya tersebar luas di seluruh dunia terutamanya di negara membangun yang tahap sanitasinya rendah dan kekurangan bekalan air yang bersih. Seramai 71 orang kanak-kanak Orang Asli dari Pos Lenjang, Pahang telah terlibat di dalam kajian ini. Sampel feses dikumpul dan diperiksa bagi mengesan infeksi G. intestinalis dengan menggunakan tiga teknik diagnosis iaitu teknik apusan langsung, konsentrasi formalin-eter dan perwarnaan trikrom. Prevalens infeksi Giardia intestinalis di kalangan kanak-kanak Orang Asli di Pos Lenjang, Pahang adalah tinggi iaitu 43.7%. Dari segi jantina, prevalens infeksi hampir sama di kalangan kanak-kanak perempuan (45.0%) berbanding kanak-kanak lelaki (41.9%). Infeksi juga didapati lebih banyak berlaku di kalangan kanak-kanak bersekolah (48.6%) berbanding kanakkanak pra-sekolah (38.2%).
Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays.
The aim of this study was to explore the utilization of polymeric membrane for bio-sensing application in most efficient and rapid way. Customization of membrane formulation via phase separation study to modify its morphologies and properties enable the detection of different pathogens in a specific manner. Experimental findings (FESEM, through-pore distribution, porosity, capillary flow test and protein binding test) verified the predictions of faster capillary flow time and higher membrane's protein binding by the addition of cellulose acetate and nitrocellulose to the membrane casting dope, respectively. Throughout the phase separation study, the potential phase behavior was investigated, which was correlating various membrane structures to its performances for potential pathogens detection in water.
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.
In the present study, 310 faecal samples from goats from eight different farms in Malaysia were tested for the presence of Giardia using a PCR-coupled approach. The nested PCR for SSU amplified products of the expected size (∼200 bp) from 21 of 310 (6.8%) samples. Sixteen of these 21 products could be sequenced successfully and represented six distinct sequence types. Phylogenetic analysis of the SSU sequence data using Bayesian Inference (BI) identified Giardia assemblages A, B and E. The identification of the 'zoonotic' assemblages A and B suggests that Giardia-infected goats represent a possible reservoir for human giardiasis in Malaysia.
We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n514) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/93/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n510) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV) and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies.
This study was conducted to determine the genotypes of Giardia duodenalis isolated from human faecal samples at Pos Betau, Pahang, Malaysia. Faecal specimens were collected and examined for G. duodenalis cysts using Trichrome staining techniques. Molecular identification was carried out by the amplification of a region of the small subunit of the nuclear ribosomal RNA (SSU rRNA) gene using nested PCR and subsequent sequencing. The sequences from 15 isolates from G. duodenalis were subjected to phylogenetic analysis (including appropriate outgroups) using the neighbor-joining and maximum parsimony methods. The trees identified G. duodenalis assemblages A and B, with a predominance of assemblage B. The predominance of anthroponotic genotypes indicates the possibility of anthroponotic transmission of these protozoa in this Semai Pahang Orang Asli community.
We used a combined microscopy-molecular approach to determine the occurrence and identities of waterborne Giardia sp. cysts isolated from 18 separate, 10l grab samples collected from a Malaysian zoo. Microscopy revealed that 17 of 18 samples were Giardia cyst positive with concentrations ranging from 1 to 120 cysts/l. Nine (52.9%) of the 17 cyst positive samples produced amplicons of which 7 (77.8%) could be sequenced. Giardia duodenalis assemblage A (6 of 7) and assemblage B (1 of 7), both infectious to humans, were identified at all sampling sites at the zoo. The presence of human infectious cysts raises public health issues, and their occurrence, abundance and sources should be investigated further. In this zoo setting, our data highlight the importance of incorporating environmental sampling (monitoring) in addition to routine faecal examinations to determine veterinary and public health risks, and water monitoring should be considered for inclusion as a separate element in hazard analysis, as it often has a historical (accumulative) connotation.
Matched MeSH terms: Giardia/genetics*; Giardia/growth & development
Giardia duodenalis causes diarrhoea and malabsorption. The objectives of the study were to detect local isolates of G. doudenalis by polymerase chain reaction (PCR) and to determine their restriction fragment length polymorphisms (RFLP). G. doudenalis isolated from stools of patients from Hospital Orang Asli Gombak were cultured axenically using TYI-S-33 medium with 10% foetal calf serum. The commercially designed primer-pair 432/433 was used to amplify a 0.52 kb segment known to encode the homologous cysteine-rich trophozoite surface antigen (tsp11 and tsa417). Results showed that the primer-pair 432/433 could amplify the target region of the local isolates. RFLP study on the identical isolates showed that all the restriction enzymes tested ( HindIII, ClaI, PstI and Kpn) gave a banding pattern similar to that of the WB strain a reference pathogenic strain from human. The reference pathogenic strain were commercially obtained from the American Type Culture Collection (ATCC).
Giardia duodenalis is a protozoan parasite that can cause significant diarrhoeal diseases. Knowledge of population genetics is a prerequisite for ascertaining the invasion patterns of this parasite. In order to infer evolutionary patterns that could not be uncovered based on the morphological features, a population genetic study with the incorporation of molecular marker was carried out to access the genetic structure of G. duodenalis isolated from the Malaysian population and the global populations.
The influx of low skilled workers from socioeconomically deprived neighbouring countries to Malaysia has raised concerns about the transmission of communicable gastrointestinal diseases such as giardiasis and cryptosporidiosis to the local population. Therefore, a cross sectional study was conducted to investigate the prevalence of both diseases and the genetic diversity of these pathogens in the migrant population. Microscopic examination of faecal samples from 388 migrant workers involved in five working sectors were screened and 10.8% (n = 42) were found to be positive with Giardia spp. and 3.1% (n = 12) with Cryptosporidium spp. infections. PCR amplicons at the triosephosphate isomerase (tpi) gene were successfully obtained for Giardia duodenalis from 30 (30/388; 7.73%) samples with assemblages AII and B in 13 (13/30; 43.3%) and 17 (17/30; 56.7%) positive samples, respectively. Nine samples (9/388; 2.3%) were identified as Cryptosporidium parvum using PCR-RFLP analysis. Country of origin, duration of residence in Malaysia and working sectors significantly influenced G. duodenalis assemblage AII infections amongst the targeted population. Meanwhile, C. parvum infection was significantly associated with those working in the food service sector. Despite the low presence of pathogenic G. duodenalis and C. parvum in the study population, the results highlight the risk of anthroponotic foodborne and waterborne transmission and therefore call for implementation of control strategies through improvements in personal hygiene and sanitation standards.
Giardia duodenalis is an intestinal parasite that causes diarrhoea and malabsorption in children. The parasite also infects AIDS patients with a weak immune system. A study was carried out on six local isolates of Giardia duodenalis (110, 7304, 6304, M007, 2002 and 6307) from faeces of Orang Asli patients admitted to the Gombak Hospital. WB, a reference pathogenic strain from human and G. muris from a wild mouse, were commercially obtained from the American Type Culture Collection (ATCC). All the isolates were cultured axenically in TYI-S-33 medium. Two sets of primers were used for the techniques: primers LP1 and RP1 and primers LP2 and RP2. The sets of primers amplified giardine gene of 171 bp and 218 bp in sizes respectively. The study showed that the two sets of primers could detect G. duodenalis to the genus and species level specifically.
This study is the first report on the occurrence of Giardia and Cryptosporidium (oo)cysts in recreational rivers water from Malaysia. It was carried out in water samples at two rivers, 'Sungai Congkak' and 'Sungai Batu', located in Selangor State. The occurrence of both Giardia lamblia and Cryptosporidium parvum (oo)cysts was higher in Sungai Congkak (50% or 15/30 and 10% or 3/30 respectively) than Sungai Batu (16% or 5/30 and 3.3% or 1/30 respectively). The mean density of cysts/L was 0.72 in Sungai Congkak and 0.023 in Sungai Batu, and that of oocysts/L was 0.023 in Sungai Congkak and 0.0033 in Sungai Batu, showing that the occurrence of Giardia was higher and more frequent than Cryptosporidium in both rivers. Sungai Congkak also showed higher faecal coliforms count (ranging from 0.48x10³ to 73x10³ CFU/100 mL) than Sungai Batu (0.41x10³ to 16x10³ CFU/100 mL). On the other hand, the Giardia and Cryptosporidium (oo)cysts and faecal coliforms were more concentrated at the downstream station, followed by midstream and upstream stations which might be due to human factors where settlements and recreation areas were located around and between midstream and downstream stations. The (oo)cysts and faecal coliforms also increased during public holidays due to the significantly higher number of visitors (bathers) compared with the week days. All the parameters (physical, faecal coliforms and rainfall) did not show consistent significant correlation (based on r values of Pearson correlation analysis) with both protozoa, therefore these parameters are not suitable as indicator for the presence of Giardia and Cryptosporidium (oo)cysts in both rivers.
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.