Multilocus genotyping of Giardia duodenalis in Malaysia

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.     

Giardia duodenalis infection and wastewater irrigation in Pakistan

The risk of Giardia duodenalis (Giardia) infection in farmers using untreated wastewater in agriculture was investigated in the city of Faisalabad, Pakistan, through a cross-sectional study. The study found a significantly increased risk of (asymptomatic) Giardia infection in wastewater farming households when compared with farming households using regular (non-wastewater) irrigation water (OR 3.3, 95% CI 2.5-4.4). Textile labourers who were employed in the city of Faisalabad but who lived in the same village as the wastewater farmers showed a risk of Giardia infection in between that of wastewater and non-wastewater farming households (OR 2.4, 95% CI 1.9-3.1). This study suggests that exposure to wastewater with high Giardia concentrations carries an increased risk for (asymptomatic) Giardia infection.     

Giardia duodenalis: enhanced growth in cell culture

Growth of Giardia duodenalis in broth and in animals has been studied in considerable detail. In contrast, the kinetics of growth in cell culture have been little evaluated. In this study, in vitro growth of G. duodenalis was evaluated in cell culture, primarily using mouse McCoy cells in vials. The media used were Giardia broth (TYI-G), Trichomonas vaginalis broth (TYI-T), and standard cell culture media (CMGA) alone and in combination (2 parts by volume CMGA to one part of TYI broth). Addition of cell culture enhanced the sensitivity of the systems in detecting low numbers of G. duodenalis. Growth was identified consistently with inocula less than or equal to 10/ml, and often with a calculated 10-1/ml inoculum with CMGA/TYI-T and CMGA/TYI-G with cells, and with TYI-G with and without cells. The 2 preferred systems for sensitivity and growth were CMGA/TYI-G with cells and TYI-G with cells. The pH fell minimally in the growth systems and, if CMGA was in the media, cell monolayers remained intact and viable throughout the experiment. In preliminary experiments, cell cultures did not allow growth of one strain of G. muris. These cell culture systems may be useful for detection of low numbers of non-laboratory adapted trophozoites, and should be useful in evaluating the interaction of G. dudodenalis with cells in culture.     

Mixed Giardia duodenalis assemblage infections in children and adults in South India

SUMMARY The assemblages of Giardia duodenalis in 25 children with and 25 children without diarrhoea and 24 adults with gastrointestinal symptoms in South India were determined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting the glutamate dehydrogenase (gdh), β-giardin and triosephosphate isomerase (tpi) genes was used. The tpi PCR was the most sensitive and detected G. duodenalis in all 74 microscopy-positive samples, while gdh and β-giardin PCR were positive in 62·2% and 56·8% of the samples. Assemblage B was predominant in both children and adults (82·4%) followed by assemblage AII (9·4%); assemblage AI was not detected. Infections with both assemblages A and B (detected by tpi PCR-RFLP) were seen exclusively in children and the mixed assemblage BIII and BIV (detected by gdh PCR-RFLP) was more common in children than adults (P=0·058).     

Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis

To address the source of infection in humans and public health importance of Giardia duodenalis parasites from animals, nucleotide sequences of the triosephosphate isomerase (TPI) gene were generated for 37 human isolates, 15 dog isolates, 8 muskrat isolates, 7 isolates each from cattle and beavers, and 1 isolate each from a rat and a rabbit. Distinct genotypes were found in humans, cattle, beavers, dogs, muskrats, and rats. TPI and small subunit ribosomal RNA (SSU rRNA) gene sequences of G. microti from muskrats were also generated and analyzed. Phylogenetic analysis on the TPI sequences confirmed the formation of distinct groups. Nevertheless, a major group (assemblage B) contained most of the human and muskrat isolates, all beaver isolates, and the rabbit isolate. These data confirm that G. duodenalis from certain animals can potentially infect humans and should be useful in the detection, differentiation, and taxonomy of Giardia spp.     

Prevalence and molecular characterization of Giardia duodenalis in dogs in Aydin,Turkey

The purpose of the present study was to investigate the prevalence and molecular characterization of Giardia duodenalis among dogs in Aydin, Turkey. A total of 473 faecal samples from dogs were collected. The overall prevalence of G. duodenalis was 18.8%. Higher infection rates were observed in dogs younger than three months old, from shelters, and with diarrhoea. Faecal samples of 89 dogs, diagnosed Giardia-positive by microscopy, were found positive by nested PCR. The β-giardin nested PCR assay revealed assemblage B in all samples (100%), whereas 38 of the samples were mixed with assemblage A (42%). Sequence analysis of isolates indicated sub-genotypes A3 and B4 which have been previously detected in human isolates from Turkey. The results of the present study indicated the relatively high prevalence of giardiasis and the presence of the zoonotic sub-genotypes suggesting the important role of dogs as potential reservoirs of human infections.     

Occurrence and molecular characterization of Giardia duodenalis in child population from Colombia

Giardia duodenalis is one of the most prevalent human intestinal parasite, with children living in developing countries being particularly at risk of infection. The occurrence and molecular diversity of G. duodenalis was investigated in stools specimens from 307 individuals aged one to nineteen years in Colombia. Samples were collected in three educational establishments (n: 163) and two hospital laboratories (n: 144) from urban and rural areas. Feces were concentrated using a biphasic sedimentation method and wet mounts of the sediment were examined by light microscopy. G. duodenalis assemblages and sub-assemblages were determined on positive samples by PCR of the triose phosphate isomerase (tpi), β-giardin (bg) and small-subunit (ssu) rRNA genes. G. duodenalis infection was detected by microscopy in 23 individuals (7.5%). The protozoan was more prevalent among specimens collected in educational establishments (11.6%) than in those obtained from hospital laboratories (2.8%). Infection was most common in individuals from urban areas and children aged 1–5 years. No significant association between diarrhea and infection could be demonstrated. Twenty Giardia-positive samples were successfully allocated to assemblage B (n: 11), sub-assemblage AII (n: 7), and assemblage A (n: 2). Results indicate the potential for transmission of G. duodenalis infection in children attending educational establishments and individuals from urban areas, where transmission seems to be primarily anthroponotic.     

Correlation of Giardia duodenalis assemblages with clinical and epidemiological data in Cuban children

Giardia duodenalis is one of the most frequent intestinal parasitic infections in children worldwide. To date, eight main assemblages of G. duodenalis have been described, but only A and B genetic groups are known to infect humans. In Cuba, this parasite has most clinical impact on children. The aim of this investigation was genetic characterization of G. duodenalis isolated from children with giardiasis diagnosed at the Paediatric Hospital “William Soler” between 2010 and 2011, and to compare the genetic results with clinical and epidemiological data. A total of 103 stool samples from 452 children were positive for G. duodenalis and co-infections with other parasites were noted in 5 cases. Assemblage identification was carried out by the amplification of a fragment of the triosephosphate isomerase (tpi) gene. Sub-assemblages of assemblage A (AI and AII) were identified by a nested PCR using the intergenic spacer (IGS) region of ribosomal deoxyribonucleic acid gene as a target. DNA from 90 of 103 (87.4%) samples was successfully amplified by PCR–tpi. The prevalence of assemblages A and B was 40% and 42%, respectively. Infections with both assemblages were reported in 16 cases. No associations between epidemiological information and assemblage was detected, but assemblage B was significantly (P < 0.01) more frequently found in children with diarrhea, flatulence or abdominal pain than assemblage A. Sub-assemblage AII accounted for the majority of cases (86.5%).     

High genetic diversity of Giardia duodenalis assemblage E in Chinese dairy cattle

Giardia duodenalis is a common protozoan parasite that can infect humans and animals. Although previous studies demonstrated that the assemblage E of G. duodenalis is prevalent in cattle, studies on its genetic diversity were mostly based on single loci and very few involved multilocus analysis. To better understand the genetic variability and structure of G. duodenalis assemblage E in Chinese dairy cattle, 651 multilocus sequences derived from nine provinces (Gansu, Guangdong, Henan, Jiangsu, Ningxia, Shaanxi, Shanghai, Sichuan and Xinjiang) of China were analyzed in this study. Results showed that a total of 220 haplotypes were identified in the G. duodenalis assemblage E, with a high haplotype diversity (Hd = 0.97225) and low nucleotide diversity (π = 0.00259). The genetic differentiation index (FST) and gene flow (Nm) results indicated low degree of genetic differentiation, implying frequent genetic communication. Combined with the analysis of molecular variance (AMOVA), genetic variation within populations (81.7%) was higher than that among populations (18.3%), indicating low degree of genetic differentiation between populations. Such low rates of gene differentiation supported no significant correlations with geographical divisions. Moreover, both negative Tajima's D and Fu's FS values of neutrality tests and unimodal curve of mismatch distribution analyses indicated that G. duodenalis assemblage E population in Chinese dairy cattle had experienced demographic expansion. Overall, these findings contribute to an improved understanding of the population genetics and evolutionary biology of G. duodenalis assemblage E and assist in its control in cattle.     

Molecular Identification of Giardia duodenalis Isolates from Fars Province,Iran

Background

Giardia duodenalis is one of the most common human intestinal protozoan parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. The present study aimed to identify the prevalence of G. duodenalis isolates and determine the most common of its assemblages in the patients referring to health centers and hospitals in Fars province, Iran that will be subjected to further molecular investigation.

Methods

We collected 1000 human fecal samples from health centers and hospitals in Shiraz, Iran in a one year period from September 2009 to August 2010. Microscopic examination for the presence of G. duodenalis cysts and trophozoites was performed by direct wet mount before and after the concentration techniques. Extraction of DNA was performed by Phenol-Chloroform-Isoamylalcohol (PCI). G. duodenalis-positive specimens were analyzed by PCR. A fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the forward primer RH11 and the reverse primer RH4. Genotyping was performed using sequence analysis of G. duodenalis glutamate dehydrogenase gene using primers GDHeF, GDHiF, and GDHiR.

Results

The prevalence of Giardia infection was 10.7% (107/1000) examined based on microscopic examination. PCR identified 80% (40/50) of the samples as positive for G. duodenalis based on SSU-rDNA amplification on sucrose gradient samples. Besides, genotyping results indicated 32 isolates (80%) as assemblage AII and 8 isolates (20%) as assemblage BIII and BIV based on the DNA sequence analysis of the glutamate dehydrogenase locus of G. duodenalis.

Conclusion

The findings of this study emphasize that Iran (Fars Province) is a favorable area for giardiasis with an anthroponotic infection route.     

Giardiasis in NSW: Identification of Giardia duodenalis assemblages contributing to human and cattle cases,and an epidemiological assessment of sporadic human giardiasis

Two genetic assemblages (A and B) of the protozoan parasite species, Giardia duodenalis, infect humans, domestic animals and wildlife. In New South Wales, Australia, over 2000 sporadic human giardiasis cases are reported annually, but parasite sources and links between sporadic cases are unknown. This study describes G. duodenalis assemblages contributing to human and cattle cases in NSW, and examines demographic, spatial, and temporal distributions of NSW human infections and G. duodenalis assemblages. Genotyping by PCR-restriction fragment length polymorphism of the glutamate dehydrogenase (gdh) gene identified G. duodenalis assemblage B as the most common (86%) cause of infection among human cases (n = 165). Approximately 37% of cattle DNA samples were PCR positive (18S rRNA, gdh), and G. duodenalis assemblages E (69%) or B (31%) were identified from these samples. Human assemblage A was more common among older age groups, and seasonality in the geographic dispersal of human assemblage A was observed. The results of this study indicate G. duodenalis assemblage B is highly prevalent among humans in NSW, and the potential for cross-species transmission exists between humans and cattle in this region. Spatio-temporal and demographic distributions of human assemblage A and B are highlighted, and risk factors associated with these dispersal patterns warrants further research.     

Prevalence and multilocus genotypes of Giardia duodenalis infecting pigs in Ogun state,Nigeria

Giardia duodenalis is an intestinal flagellated protozoan parasite that is infectious to humans and a wide range of animals worldwide. While varying prevalence rates have been reported in pigs worldwide, there are currently no published reports on the genotypes of Giardia infecting pigs in any African country. The present study is on the prevalence and genotypes of G. duodenalis in 209 pigs raised on four farms in Ogun State Nigeria. Using an enzyme-linked immunosorbent assay (ELISA) kit, Giardia duodenalis coproantigens were detected on all farms and in 25.4% (53/209) of pigs sampled. However, there was no significant influence (p > 0.05) of age, sex and stool consistencies of the pigs on the distribution of the infection. Genotyping of Giardia duodenalis in all ELISA-positive samples, achieved by the amplification of the small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi) and beta giardin (bg) genes, identified 14 and 37 assemblage B and E isolates respectively while mixed infection by both assemblages was recorded in two isolates. Novel nucleotide substitutions were identified in four assemblage B isolates at the ssu rRNA locus. Genetic diversity was observed among the assemblage B isolates after multiple alignment analyses of the gdh, tpi and bg sequences whereby sub-assemblages BII (n = 2), BIII (n = 9) and BIV (n = 3) were identified. The assemblage B isolates from pigs in this study were phylogenetically related to isolates from humans, marmoset and cattle while the assemblage E isolates were related to isolates from sheep, goats and cattle. These findings suggest that pigs in southwest Nigeria predominantly harbour G. duodenalis isolates that could be infectious to other animal species and to a lesser extent, isolates that may be of zoonotic importance.     

Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children

Data on the relationship between the two genotypes of Giardia duodenalis that infect humans, assemblages A and B, their clinical presentation and intestinal inflammation are limited. We analyzed 108 stool samples previously collected for a diarrhoeal study among Brazilian children, representing 71 infections in 47 children. Assemblage B was most prevalent, accounting for 43/58 (74.1%) infections, while assemblage A accounted for 9/58 (15.5%) infections and 6/58 (10.3%) infections were mixed (contained both assemblage A and B). There was no significant difference in diarrhoeal symptoms experienced during assemblage A, B or mixed infections. Children with assemblage B demonstrated greater variability in G. duodenalis cyst shedding but at an overall greater level (n=43, mean 3.6 x 10(5), range 5.3 x 10(2)-2.5 x 10(6)cysts/ml) than children infected with assemblage A (n=9, mean 1.4 x 10(5), range 1.5 x 10(4)-4.6 x 10(5)cysts/ml; P=0.009). Children with mixed infections shed more cysts (mean 8.3 x 10(5), range 3.1 x 10(4)-2.8 x 10(6)cysts/ml) than children with assemblage A or B alone (P=0.069 and P=0.046 respectively). This higher rate of cyst shedding in children with assemblage B may promote its spread, accounting for its increased incidence. Additionally, second and third infections had decreasing faecal lactoferrin, suggesting some protection against severity, albeit not against infection, by prior infection.     

Giardia duodenalis infection and anthropometric status in preschoolers in Salvador, Bahia State, Brazil

The aim of this study was to estimate the association between Giardia duodenalis infection and anthropometric deficits, as measured by weight-for-age and height-for-age. This cross-sectional study included 629 children from 12 to 48 months of age, selected from 30 geographic areas in the city of Salvador, Bahia State, Brazil. Poisson regression and linear regression were used for the multivariate statistical analyses. G. duodenalis was diagnosed in 13.5% of the children. The children's breastfeeding duration and living conditions (garbage collection and paved streets or sidewalks) modified the effect of G. duodenalis infection on anthropometric status. Among infected children, there were statistically significant associations between weight deficit and shorter breastfeeding (PR=2.22; 95%CI: 1.56-3.14) and inadequate paving of streets and sidewalks (PR=2.00; 95%CI: 1.37-2.92), while height deficit was associated with deficient public garbage collection (PR=2.21; 95%CI: 1.31-2.51). In the linear regression, the association with the anthropometric indicators remained positive and statistically significant. The child's unhealthy living environment aggravated the negative effect of G. duodenalis infection on anthropometric status, and breastfeeding was a protective factor in the outcome.     

Investigation of Possible Correlation between Giardia duodenalis Genotypes and Clinical Symptoms in Southwest of Iran

Background

Giardia duodenalis is one of the most important human enteric parasites throughout the world. Clinical symptoms of this parasite vary from asymptomatic infection to chronic diarrhea. Still it is not clear, whether different types of pathogenesis are due to different strains of organism or to variable host factors. The purpose of this study was to investigate possible correlation of clinical symptoms with assemblages among symptomatic and asymptomatic cases collected from southwest of Iran.

Methods

Fecal samples were collected from 100 symptomatic and asymptomatic cases, which were positive for G. duodenalis. The samples were subjected to semi-nested PCR and RFLP for gdh gene.

Results

Among symptomatic patients, 54% had mixed genotypes AII and BIII, 28% and 18% of samples indicated assemblages BIII and AII, respectively. In contrast, among asymptomatic cases, 64%, 26% and 10%samples had mixed genotypes, BIII and AII assemblages, respectively. Statistical analysis using Chi- Square test showed that there was no significant correlation between assemblage and clinical symptoms in current study.

Conclusion

High prevalence of mixed infection in both groups may affect this conclusion, therefore further study in more details are necessary to clarify these finding. Additionally, it is important to carry out investigations regarding human host factors as well.     

Prevalence of Giardia duodenalis assemblages and sub-assemblages in symptomatic patients from Damascus city and its suburbs

Giardia duodenalis is one of the most important human enteric parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. However, there is limited information on the prevalent genetic variability of G. duodenalis in Syria. This study aimed to evaluate the predominance of G. duodenalis assemblages/sub-assemblages causing humans infection in the city of Damascus and its suburbs. 40 symptomatic giardiasis patients were recruited in this study. Fecal samples were genotyped using PCR/RFLP assay targeting the β-giardin and glutamate dehydrogenase (gdh) genes. HaeIII, BspL1 and RsaI restriction enzymes were used to differentiate between G. duodenalis assemblages/sub-assemblages. Our data showed that 65% of isolates were of assemblage A; 45% belonged to sub-assemblage AII and 20% to sub-assemblage AI. Assemblage B was detected in 27.5% of isolates; 12.5% fit in sub-assemblage BIV, 5% fit in sub-assemblage BIII and 10.5% fit in Discordant genotype BIII/BIV. Mixed genotypes (AII+BIII and AI+BIV) were identified in 3 isolates (7.5%). Significant correlation was found between Giardia AII sub-assemblage and weight loss symptom (P-value = 0.05) as well as between contact with domestic animals (cats, P-value = 0.027). Moreover, a significant correlation was found between sub-assemblage AI and livestock breeding (P-value = 0.000). In conclusion genotyping of human Giardia duodenalis isolates suggests anthroponotic transmission for the route of infection in Damascus and its suburbs. Further studies are needed to screen a wide geographic areas in Syria and to estimate the prevalence of G. duodenalis infection in our population.     

Integronlike structures in Campylobacter spp. of human and animal origin

Resistance to antimicrobial agents used to treat severe Campylobacter spp. gastroenteritis is increasing worldwide. We assessed the antimicrobial resistance patterns of Campylobacter spp. isolates of human and animal origin. More than half (n = 32) were resistant to sulphonamide, a feature known to be associated with the presence of integrons. Analysis of these integrons will further our understanding of Campylobacter spp. epidemiology.     

Giardia duodenalis infection: risk factors for children living in sub-standard settlements in Brazil

The objective of the present study was to characterize Giardia duodenalis infection among children living in sub-standard settlement areas in the municipality of Juiz de Fora, Minas Gerais State, Brazil. The cross-sectional epidemiological study included 590 children from 1 to 5 years of age. Data were collected from one child per selected family through home interviews with the parent or guardian and parasitological examination of stool samples. Thirty-one putative risk factors concerning family structure, socioeconomic status, and environmental factors were analyzed by multivariate logistic regression. Prevalence of G. duodenalis infection was 18% (106 children). Four potential risk factors were heavily associated with G. duodenalis infection: number of under-five children in the same household, index child's birth order, existence of a bathroom in the home, and drinking water source.     

Ability of a small wild rodent (Peromyscus maniculatus) to host strains of human‐type Giardia duodenalis

Wild deermice (Peromyscus maniculatus) were trapped in a forested area of the Fraser Valley of British Columbia, Canada. Animals were tested for parasites and treated with metronidazole to ensure absence of pre‐existing Giardia infection. Sera were collected from all animals and tested by enzyme‐linked immunosorbent assay (ELISA) to rule out previous infections with Giardia. Cysts from one of two G. duodenalis strains associated with human infections were inoculated into different groups of deermice. Cysts were tested for viability at the time of inoculation. Five different cyst concentrations and appropriate controls were included in each group of thirteen deermice used for each strain. After a suitable incubation period, feces from test and control animals were examined for cysts or trophozoites using both the formol‐ether concentration method and microscopic examination of iron‐hematoxylin stained preparations. Three of the four deermice inoculated with 1 × 10⁵ cysts of an isolate associated with an outbreak of human giardiasis were found to be infected. No infection was detected in any of the deermice inoculated with the Giardia strain isolated from a symptomatic child. The isolate that infected small rodents in these experiments only did so at a higher inoculum and in juvenile deermice. We confirm that wild deermice may act as hosts for some human source strains of the G. duodenalis morphological group.