Multilocus genotyping of Giardia duodenalis in Southwestern Iran. A community survey.
ABSTRACT: Giardia duodenalis is one of the main enteric pathogens associated with diarrheal disease. In developing countries, giardiasis is a major public health concern, particularly in children under five years of age. This study aimed to evaluate the occurrence and genetic diversity of G. duodenalis causing human infections in Shushtar County, Southwestern Iran. Individual faecal specimens were collected from 1,163 individuals (male/female ratio: 0.9; age range 2-75 years) with (n = 258) and without (n = 905) gastrointestinal symptoms living in rural and urban settings during the period 2017-2018. Conventional (sucrose flotation and microscopy) methods were used for the initial detection of G. duodenalis cysts in faecal specimens. Microscopy-positive samples were confirmed by PCR amplification and sequencing of the small subunit rRNA (ssu rRNA) gene of the parasite. A multilocus genotyping (MLG) scheme targeting the triose phosphate isomerase (tpi), the glutamate dehydrogenase (gdh), and the beta-giardin (bg) genes was used for genotyping purposes. Giardia duodenalis cysts were detected in 7.7% (90/1,163) of samples by microscopy, of which 82 were confirmed by ssu-PCR. Successful amplification and sequencing results were obtained for 23.2% (19/82), 9.8% (8/82), and 8.5% (7/82) of the confirmed samples at the tpi, gdh, and bg loci, respectively. MLG data for the three loci were available for two samples only. Out of the 24 samples genotyped at any loci, 50% (12/24) were identified as assemblage A and the remaining half as assemblage B. Overall, AII was the most prevalent sub-assemblage detected (41.7%, 10/24), followed by BIII (25.0%, 6/24), discordant BIII/BIV (5/24) or AII/AIII (2/24) sequences, and BIV (1/24). No significant correlation was demonstrated between a given assemblage/sub-assemblage and the occurrence of clinical symptoms. No genotypes adapted to animal hosts other than humans (e.g. assemblages C-F) were found circulating in the investigated human population, suggesting that transmission of human giardiasis in this Iranian region is primarily of anthroponotic nature. Further molecular-based studies are needed to confirm and expand these results, and to ascertain the presence and public health relevance of the parasite in environmental (e.g. drinking water) samples.
Project description:Giardia duodenalis is an important intestinal protozoan in humans worldwide with high infection rates occurring in densely populated and low resource settings. The parasite has been recorded to cause diarrhea in children. This study was carried out to identify G. duodenalis assemblages and sub-assemblages in children presenting with diarrhea in Kenya.A total of 2112 faecal samples were collected from children aged ? 5 years and screened for the presence of Giardia cysts using microscopy. A total of 96 (4.5%) samples were identified as Giardia positive samples and were genotyped using glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and ?-giardin loci.The three markers successfully genotyped 72 isolates and grouped 2 (1.4) isolates as Assemblage A, 64 (88.9) as Assemblage B and 7 (9.7%) consisted of mixed infections with assemblage A and B. A further analysis of 50 isolates using GDH Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) categorized 2 assemblage A isolates as sub-assemblage AII while 6 and 14 assemblage B isolates were categorized into sub-assemblage BIII and BIV respectively. A mixed infection with sub-assemblage BIII and BIV was recorded in 28 isolates. Over half (55.6%) of Giardia infections were recorded among the children between 13 to 48 months old.This paper reports the first data on the assemblages and sub-assemblages of Giardia duodenalis in children representing with diarrhea in Kenya.
Project description:Intestinal parasites are common in the Moroccan population. Enteroparasites in children from four schools in urban and rural areas of Tetouan (Morocco) were studied to treat these children and to design prevention and control programs. A total of 673 children were examined. The prevalence of parasitized children was 51%. The average number of enteroparasites was half in urban areas than in rural areas. Multiple parasitism appeared in 30% of the samples presenting two, three, or four parasites. The most prevalent parasite was Blastocystis hominis (64%). Giardia duodenalis was the most frequent pathogen, with an overall prevalence of 20% (24% in rural areas and 16% in urban areas). Other pathogenic enteroparasites were Cyclospora cayetanensis (5% in rural and urban areas), Iodamoeba butschlii, Hymenolepis spp., Trichuris trichiura and Enterobius vermicularis, with prevalence lower than 2%. In this work, G. duodenalis genotypes were molecularly characterized by a study of the glutamate dehydrogenase (gdh) and 18S rRNA genes. This is the first study of molecular characterization of G. duodenalis in Moroccan children, and the sequence analysis revealed both Assemblage A (AII) and Assemblage B (BIII, BIV), with the predominance of Assemblage BIV (73%).
Project description:The flagellate protozoan Giardia duodenalis is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating G. duodenalis isolates in the country.In this longitudinal molecular epidemiological study we report the diversity and frequency of the G. duodenalis assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013-January 2015. G. duodenalis cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. G. duodenalis assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (GDH) and ?-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed.Of 188 confirmed positive samples from 178 giardiasis cases a total of 124 G. duodenalis isolates were successfully typed at the GDH and/or the BG loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in G. duodenalis assemblage B, but not assemblage A, isolates at both the GDH and BG loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of G. duodenalis found in individuals with symptomatic giardiasis in the population under study.Human giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent G. duodenalis assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of G. duodenalis.
Project description:Giardia duodenalis is an enteric parasite commonly detected in children. Exposure to this organism may lead to asymptomatic or symptomatic infection. Additionally, early-life infections by this protozoan have been associated with impaired growth and cognitive function in poor resource settings. The Global Enteric Multicenter Study (GEMS) in Mozambique demonstrated that G. duodenalis was more frequent among controls than in diarrhoeal cases (?3 loosing stools in the previous 24 hours). However, no molecular investigation was conducted to ascertain the molecular variability of the parasite. Therefore, we describe here the frequency and genetic diversity of G. duodenalis infections in children younger than five years of age with and without diarrhoea from the Manhiça district in southern Mozambique enrolled in the context of GEMS. Genomic DNA from 757 G. duodenalis-positive stool samples by immunoassay collected between 2007-2012, were reanalysed by multiplex PCR targeting the E1-HP and C1-P21 genes for the differentiation of assemblages A and B. Overall, 47% (353) of the samples were successfully amplified in at least one locus. Assemblage B accounted for 90% (319/353) of all positives, followed by assemblage A (8%, 29/353) and mixed A+B infections (1%, 5/353). No association between the presence of a given assemblage and the occurrence of diarrhoea could be demonstrated. A total of 351 samples were further analysed by a multi-locus sequence genotyping (MLSG) approach at the glutamate dehydrogenase (gdh), ß-giardin (bg) and triose phosphate isomerase (tpi) genes. Overall, 63% (222/351) of samples were genotyped and/or sub-genotyped in at least one of the three markers. Sequence analysis revealed the presence of assemblages A (10%; 23/222) and B (90%; 199/222) with high molecular diversity at the nucleotide level within the latter; no mixed infections were identified under the MLSG scheme. Assemblage A sequences were assigned to sub-assemblages AI (0.5%, 1/222), AII (7%, 15/222) or ambiguous AII/AIII (3%, 7/222). Within assemblage B, sequences were assigned to sub-assemblages BIII (13%, 28/222), BIV (14%, 31/222) and ambiguous BIII/BIV (59%, 132/222). BIII/BIV sequences accumulated the majority of the single nucleotide polymorphisms detected, particularly in the form of double peaks at chromatogram inspection. This study demonstrated that the occurrence of gastrointestinal illness (diarrhoea) was not associated to a given genotype of G. duodenalis in Mozambican children younger than five years of age. The assemblage B of the parasite was responsible for nine out of ten infections detected in this paediatric population. The extremely high genetic diversity observed within assemblage B isolates was compatible with an hyperendemic epidemiological scenario where infections and reinfections were common. The obtained molecular data may be indicative of high coinfection rates by different G. duodenalis assemblages/sub-assemblages and/or genetic recombination events, although the exact contribution of both mechanisms to the genetic diversity of the parasite remains unknown.
Project description:Giardia duodenalis is one of the most important and widespread gastrointestinal parasites in the world. Despite its relevance as a causative agent of diarrhea, asymptomatic giardiasis occurs frequently, especially in low resources settings in which children are exposed to many risk factors. Based on microscopic examination and the polymerase chain reaction (PCR) amplification and sequencing of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes, we assessed G. duodenalis occurrence and genetic diversity in isolates of children attending a daycare center and living in low income families, in an economically successful region. Considering both, microscopic examination and PCR/sequencing methods, the overall prevalence of Giardia infection was 51.4%, with the highest frequency in children aged 1-4 years old (p<0.05). Genotyping of 50 isolates revealed that the assemblage A was found in 60% of the samples (30/50), followed by the assemblage B in 38% (19/50) and 2% of mixed-assemblage infections (1/50). At the sub-assemblage level, isolates genotyped as A were AII and among isolates B, BIII and BIV were identified. Both assemblages A and B were detected in children of all age groups, however assemblage A was more prevalent. The detection of anthroponotic assemblages and sub-assemblages (AII, BIII and BIV) reinforces human-to-human transmission, mainly in children of all age groups when they have not yet received toilet training, making them more vulnerable to infection.
Project description:Giardia duodenalis and Cryptosporidium spp. are enteric protozoan causing gastrointestinal illness in humans and animals. Giardiasis and cryptosporidiosis are not formally considered as neglected tropical diseases, but belong to the group of poverty-related infectious diseases that impair the development and socio-economic potential of infected individuals in developing countries.We report here the prevalence and genetic diversity of G. duodenalis and Cryptosporidium spp. in children attending rural primary schools in the Bahir Dar district of the Amhara Region, Ethiopia. Stool samples were collected from 393 children and analysed by molecular methods. G. duodenalis was detected by real-time PCR, and the assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and ?-giardin genes of the parasite. Detection and identification of Cryptosporidium species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene.The PCR-based prevalences of G. duodenalis and Cryptosporidium spp. were 55.0% (216/393) and 4.6% (18/393), respectively. A total of 78 G. duodenalis isolates were successfully characterized, revealing the presence of sub-assemblages AII (10.3%), BIII (28.2%), and BIV (32.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.7% and 15.4% of the isolates, respectively. An additional five (6.4%) isolates were assigned to assemblage B. No mixed infections of assemblages A+B were found. Extensive genetic variation at the nucleotide level was observed within assemblage B (but no within assemblage A), resulting in the identification of a large number of sub-types. Cryptosporidium diversity was demonstrated by the occurrence of C. hominis, C. parvum, and C. viatorum in the population under study.Our data suggest an epidemiological scenario with an elevated transmission intensity of a wide range of G. duodenalis genetic variants. Importantly, the elevated degree of genetic diversity observed within assemblage B is consistent with the occurrence of intra-assemblage recombination in G. duodenalis.
Project description:Giardia duodenalis is a common enteric protozoan that infects a range of hosts including humans and other mammals. Multilocus genotyping of G. duodenalis in captive non-human primates (NHPs) from zoos in China is limited. In this study, we evaluated 302 NHP fecal samples collected from 32 different NHP species. The primates were from 12 zoos distributed across eight provinces and two municipalities (Chongqing and Beijing) of China. The overall infection rate was 8.3% (25/302). The six G. duodenalis-positive zoos and their infection rates were: Suzhou Zoo (40.0%, 4/10), Yangzhou Zoo (22.2%, 2/9), Dalian Zoo (16.7%, 4/24), Chengdu Zoo (12.8%, 6/47), Guiyang Forest Wildlife Zoo (12.1%, 7/58), and Changsha Zoo (4.7%, 2/43). Molecular analysis of three loci, beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh), showed high genetic heterogeneity, and seven novel subtypes (BIII-1, MB10-1, WB8-1, B14-1, MB9-1, DN7-1, and BIV-1) were detected within assemblage B. Additional analysis revealed 12 different assemblage B multilocus genotypes (MLGs), one known MLG and 11 novel MLGs. Based on phylogenetic analysis, 12 assemblage B MLGs formed two main clades, MLG-SW (10-12, 18) and MLG-SW (13, 14, 16, 17), the other four MLG-SW (15, 19, 20, 21) were scattered throughout the phylogenetic tree in this study. Using multilocus genotyping, this study expands our understanding of the occurrence of Giardia infection and genetic variation in Giardia in captive non-human primates from zoos in China.
Project description:Giardia duodenalis is one of the most common causes of diarrhea in humans with about 250-300 million cases per year. It is considered to be a species complex comprising of eight genetic assemblages (A to H), with assemblages A and B being the major causes of human infections. In this study we carried out genotypic characterization of G. duodenalis isolates detected in asymptomatic school-going children aged 3-16?years. Between May and September 2017, a total of 329 fecal samples were collected from school-going children from Chawama compound of Lusaka City and were screened for Giardia by microscopic examination. All microscopically positive fecal samples were analyzed by semi-nested polymerase chain reaction (PCR) targeting the glutamate dehydrogenase (gdh) gene. Genotyping of amplified PCR products was conducted by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Microscopically, Giardia was found in 10% (33/329) of fecal samples. The PCR-RFLP analysis of the gdh gene revealed assemblages A and B in 27.3% (9/33) and 72.7% (24/33), respectively. Furthermore, analysis with restriction enzymes identified sub-assemblages AII (27.3%, 9/33), BIII (12.1%, 4/33), BIV (51.5%, 17/33) and mixed infections of BIII and BIV (9.1%, 3/33). Phylogenetic analysis showed the clustering of 27.6% (8/29) and 72.4% (21/29) of Zambian Giardia gdh gene sequences into assemblages A and B, respectively. This study has revealed the presence of both assemblage A and B and that spread of G. duodenalis in school-going children appears to be mostly through anthroponotic transmission. To our knowledge, this is the first report of genotypic characterization of G. duodenalis identified in Zambia.
Project description:Giardia and Cryptosporidium are important causes of diarrhoea in Bangladesh. The high prevalence of both parasites in humans and cattle in rural Bangladesh and the common use of water ponds by village inhabitants and their animals suggest a potential for zoonotic transmission. Direct transmission of Giardia and Cryptosporidium between cattle and their handlers and indirect transmission through water ponds was investigated. Faecal/stool samples were collected from 623 calves and 125 calf handlers in a cross-sectional survey. In two villages, water samples were collected monthly from water ponds and faecal/stool samples were collected monthly from inhabitants and their cattle. Giardia cysts and Cryptosporidium oocysts were detected in water samples and in faecal/stool samples and positive samples were genotyped, to determine their human or animal origin. The prevalence of Giardia and Cryptosporidium in calves was 22% and 5% respectively. In calf handlers, the prevalence of Giardia and Cryptosporidium was 11.2% and 3.2% respectively. Both in the cross-sectional survey and in the longitudinal study in the villages, G. duodenalis assemblage E was most prevalent in calves, while in humans assemblage AII, BIII and BIV were found. In cattle, Cryptosporidium parvum, C. bovis and C. andersoni were identified, but no Cryptosporidium sequences were obtained from humans. Giardia and Cryptosporidium were detected in 14/24 and 12/24 water samples respectively. G. duodenalis assemblage E and BIV (-like), as well as C. andersoni and C. hominis were identified. Although the presence of Giardia and Cryptosporidium in both water ponds suggests that water-borne transmission of Giardia and Cryptosporidium is possible, the genotyping results indicate that there is no significant direct or indirect (water-borne) transmission of Giardia between cattle and people in this area of rural Bangladesh. No conclusions could be drawn for Cryptosporidium, because of the low number of sequences that were obtained from human and water samples.
Project description:BACKGROUND:Intestinal protozoan parasites are major contributors to the global burden of gastrointestinal disease causing significant socioeconomic consequences. Children living in resource-poor settings with restricted access to water and sanitary services are particularly at risk of these infections. METHODS:A prospective, community-based, cross-sectional survey was conducted in Paraná (southern Brazil) between May 2015 and May 2016. A total of 766 stool samples were individually collected from volunteers (male/female ratio: 0.99; age range: 0-76 years) and used for investigating the presence of intestinal helminth and protozoan species by routine microscopic procedures including the Kato-Katz and modified Ritchie concentration methods and the Ziehl-Neelsen stain technique. Quantitative real-time PCR confirmed microscopy-positive samples for Giardia duodenalis and the assemblages and sub-assemblages determined by multilocus sequence-based genotyping of the glutamate dehydrogenase (gdh) and ?-giardin (bg) genes of the parasite. Identification of Blastocystis subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene (SSU rDNA) of this heterokont microorganism. RESULTS:Overall, 46.1% (353/766) of the participants were infected/colonised by at least one intestinal parasite/commensal species. Protozoan and helminth species were detected in 42.7% and 10.1% of the surveyed population, respectively. Blastocystis sp. (28.2%), Endolimax nana (14.9%), and Giardia duodenalis (11.0%) were the most prevalent species found among protozoans and Ascaris lumbricoides (5.0%), Trichuris trichiura (4.6%) and hookworms (1.0%) among helminths. A total of 38 G. duodenalis-positive samples were genotyped at gdh and bg markers, revealing the presence of the sub-assemblages AII (47.4%), AII/AIII (2.6%), BIII (5.3%), BIV (26.3%) and BIII/BIV (13.1%). Two samples (5.3%) were only identified as assemblage B. AII was predominantly found in females aged 5-9 years and was associated with a higher likelihood of reporting gastrointestinal symptoms. A total of 102 Blastocystis-positive samples were successfully subtyped at the SSU rRNA gene revealing the presence of ST1 (36.3%), ST2 (15.7%), ST3 (41.2%), ST4 (2.9%), ST6 (1.0%) and ST8 (2.9%). CONCLUSIONS:Data presented here indicate that enteric parasites still represent a pressing health concern in Paraná, Brazil, probably due to sub-optimal water, sanitation and hygiene conditions. A mostly anthroponotic origin is suspected for G. duodenalis and Blastocystis sp. infections.