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Long-Term Preservation and Storage of Faecal Samples in Whatman^® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.

ABSTRACT: Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman^® Filter Cards were comparatively assessed: the FTA^® Classic Card, the FTA^® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (-20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman^® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman^® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.

SUBMITTER: Koster PC

PROVIDER: S-EPMC8151430 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Few data are available on the transmission dynamics of intestinal protozoa in children in welfare institutes. In this study, fecal specimens were collected from 396 children in a welfare institute in Shanghai, China during December 2011 (207 specimens), June 2012 (78 specimens), and September 2013 (111 specimens), and examined for Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by PCR analysis of the small subunit rRNA, triosephosphate isomerase, and internal transcribed spacer genes, respectively. The Cryptosporidium hominis and G. duodenalis assemblage A identified were further subtyped by multilocus sequence typing. Altogether, Cryptosporidium was detected in 39 (9.8%) children, with infection rates of 11.6% (24/207), 9.0% (7/78), and 7.2% (8/111) in December 2011, June 2012, and September 2013, respectively. Infection rates were higher in children of 0-12 months (20.4% compared to 0-7.3% in other age groups, P = 0.0001) and those with diarrhea (17.9% compared to 7.7% in those with no diarrhea, P = 0.006). In contrast, G. duodenalis was detected in 161/396 (40.7%), with infection rates of 48.3% (100/207), 35.9% (28/78), and 29.7% (33/111) in December 2011, June 2012, and September 2013, respectively. There were no significant gender- or diarrhea-associated differences, but the G. duodenalis infection rate in children of 13-24 months (50%) was significantly higher than in the age groups of 0-12 months and > 48 months (29.8-36.5%, P = 0.021). Co-infection of Cryptosporidium and G. duodenalis was seen in 19 (4.8%) children, but no E. bieneusi infection was detected in this study. All Cryptosporidium-positive specimens belonged to the subtype IaA14R4 of C. hominis, while all G. duodenalis-positive specimens belonged to sub-assemblage AII. Both were the same subtypes in a previous outbreak of cryptosporidiosis and giardiasis in a hospital ward hosting children from the welfare institute. Results of the study indicate that there was a persistent occurrence of limited C. hominis and G. duodenalis subtypes in the small enclosed community, with differences in age distribution and association with diarrhea occurrence between cryptosporidiosis and giardiasis.

Project description:BACKGROUND: Cryptosporidium and Giardia are important causes of diarrhea diseases in humans and animals worldwide, and both of them are transmitted by the fecal-oral route, either by direct contact or by the ingestion of contaminated food or water. The role of flies in the mechanical transmission of Cryptosporidium and Giardia has been receiving increasing attention. To date, no information is available in China about the occurrence of Cryptosporidium and Giardia in flies. We here investigated Cryptosporidium and Giardia in flies on dairy farms in Henan Province, China, at the genotype and subtype levels. METHODS: Eight hundred flies were randomly collected from two dairy farms from July 2010 to September 2010 and were divided evenly into 40 batches. The fly samples were screened for the presence of Cryptosporidium and Giardia with nested PCR. Cryptosporidium was genotyped and subtyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The identity of Giardia was determined by sequence analyzing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and ?-giardin (bg) genes. RESULTS: Forty batches of flies had 10% of contamination with Cryptosporidium or Giardia, with a mixed infection of the two parasites in one batch of flies. The Cryptosporidium isolates were identified as C. parvum at the SSU rRNA locus, and all belonged to subtype IIdA19G1 at the gp60 locus. The Giardia isolates were all identified as assemblage E of G. duodenalis at the tpi, gdh, and bg loci. One novel subtype of assemblage E was identified based on the gdh and bg loci. CONCLUSIONS: This is the first molecular study of Cryptosporidium and Giardia in flies identified at both genotype and subtype levels. SSU rRNA and gp60 sequences of C. parvum in flies was 100% homologous with those derived from humans, suggesting flies act as an epidemiological vector of zoonotic cryptosporidiosis. The variable PCR efficiencies observed in the analysis of Giardia at different loci suggest that we should use the multilocus genotyping tool in future studies to increase the detection rate, and importantly, to obtain more complete genetic information on Giardia isolates.

Project description:BackgroundDespite their wide occurrence, cryptosporidiosis and giardiasis are considered neglected diseases by the World Health Organization. The epidemiology of these diseases and microsporidiosis in humans in developing countries is poorly understood. The high concentration of pathogens in raw sewage makes the characterization of the transmission of these pathogens simple through the genotype and subtype analysis of a small number of samples.Methodology/principal findingsThe distribution of genotypes and subtypes of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in 386 samples of combined sewer systems from Shanghai, Nanjing and Wuhan and the sewer system in Qingdao in China was determined using PCR-sequencing tools. Eimeria spp. were also genotyped to assess the contribution of domestic animals to Cryptosporidium spp., G. duodenalis, and E. bieneusi in wastewater. The high occurrence of Cryptosporidium spp. (56.2%), G. duodenalis (82.6%), E. bieneusi (87.6%), and Eimeria/Cyclospora (80.3%) made the source attribution possible. As expected, several human-pathogenic species/genotypes, including Cryptosporidium hominis, Cryptosporidium meleagridis, G. duodenalis sub-assemblage A-II, and E. bieneusi genotype D, were the dominant parasites in wastewater. In addition to humans, the common presence of Cryptosporidium spp. and Eimeria spp. from rodents indicated that rodents might have contributed to the occurrence of E. bieneusi genotype D in samples. Likewise, the finding of Eimeria spp. and Cryptosporidium baileyi from birds indicated that C. meleagridis might be of both human and bird origins.Conclusions/significanceThe distribution of Cryptosporidium species, G. duodenalis genotypes and subtypes, and E. bieneusi genotypes in urban wastewater indicates that anthroponotic transmission appeared to be important in epidemiology of cryptosporidiosis, giardiasis, and microsporidiosis in the study areas. The finding of different distributions of subtypes between Shanghai and Wuhan was indicative of possible differences in the source of C. hominis among different areas in China.

Project description:Molecular diagnostic tools have played an important role in improving our understanding of the transmission of Cryptosporidium spp. and Giardia duodenalis, which are two of the most important waterborne parasites in industrialized nations. Genotyping tools are frequently used in the identification of host-adapted Cryptosporidium species and G. duodenalis assemblages, allowing the assessment of infection sources in humans and public health potential of parasites found in animals and the environment. In contrast, subtyping tools are more often used in case linkages, advanced tracking of infections sources, and assessment of disease burdens attributable to anthroponotic and zoonotic transmission. More recently, multilocus typing tools have been developed for population genetic characterizations of transmission dynamics and delineation of mechanisms for the emergence of virulent subtypes. With the recent development in next generation sequencing techniques, whole genome sequencing and comparative genomic analysis are increasingly used in characterizing Cryptosporidium spp. and G. duodenalis. The use of these tools in epidemiologic studies has identified significant differences in the transmission of Cryptosporidium spp. in humans between developing countries and industrialized nations, especially the role of zoonotic transmission in human infection. Geographic differences are also present in the distribution of G. duodenalis assemblages A and B in humans. In contrast, there is little evidence for widespread zoonotic transmission of giardiasis in both developing and industrialized countries. Differences in virulence have been identified among Cryptosporidium species and subtypes, and possibly between G. duodenalis assemblages A and B, and genetic recombination has been identified as one mechanism for the emergence of virulent C. hominis subtypes. These recent advances are providing insight into the epidemiology of waterborne protozoan parasites in both developing and developed countries.

Project description:BackgroundIn a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays.Methodology/principal findingsCryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1-10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67-55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79-11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16-7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58-0.95).Conclusions/significanceThis is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites.

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Long-Term Preservation and Storage of Faecal Samples in Whatman^® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.

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Long-Term Preservation and Storage of Faecal Samples in Whatman® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.

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Long-Term Preservation and Storage of Faecal Samples in Whatman^® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.