| 1. |
- Ankarklev, Johan, et al.
(författare)
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A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination
- 2018
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Ingår i: Infection, Genetics and Evolution. - : Elsevier BV. - 1567-1348 .- 1567-7257. ; 60, s. 7-16
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Tidskriftsartikel (refereegranskat)abstract
- Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.
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| 2. |
- Ankarklev, Johan, 1980-, et al.
(författare)
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Allelic sequence heterozygosity in single Giardia parasites
- 2012
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.
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| 3. |
- Ankarklev, Johan, et al.
(författare)
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Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children
- 2012
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Ingår i: PLOS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2735. ; 6:8, s. e1780-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.Methodology/Principal Findings: Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection.Conclusions/Significance: This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection.
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| 4. |
- Ankarklev, Johan, et al.
(författare)
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Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates
- 2015
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.
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| 5. |
- Bjelkmar, Par, et al.
(författare)
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Early outbreak detection by linking health advice line calls to water distribution areas retrospectively demonstrated in a large waterborne outbreak of cryptosporidiosis in Sweden
- 2017
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Ingår i: BMC Public Health. - : BIOMED CENTRAL LTD. - 1471-2458. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skelleftea municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skelleftea in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially.
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| 6. |
- Bujila, Ioana, et al.
(författare)
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Cryptosporidium chipmunk genotype I : An emerging cause of human cryptosporidiosis in Sweden
- 2021
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Ingår i: Infection, Genetics and Evolution. - : Elsevier. - 1567-1348 .- 1567-7257. ; 92
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Tidskriftsartikel (refereegranskat)abstract
- Most cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or Cryptosporidium hominis. However, more uncommon species are increasingly being recognised to cause infection in humans. Here we report that Cryptosporidium chipmunk genotype I, which has various rodents as its natural host, is the third most common source of human cryptosporidiosis in Sweden. We also describe the first small outbreak of cryptosporidiosis caused by Cryptosporidium chipmunk genotype I and report the first case of zoonotic transmission of Cryptosporidium chipmunk genotype I from a red squirrel to a human. Cryptosporidium chipmunk genotype I was identified in 20 human cases, including 16 sporadic cases, three outbreak-related cases, and one zoonotic case, as well as in two squirrel samples. Gp60 subtyping which was successful for 19 human cases and two squirrel samples showed that all samples harboured the same subtype, XIVaA20G2T1. The work presented here suggests that red squirrel is a natural host of Cryptosporidium chipmunk genotype I and that infection with Cryptosporidium chipmunk genotype I is an emerging cause of domestic cryptosporidiosis in Sweden and a potential source of outbreaks.
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| 7. |
- Bujila, Ioana, et al.
(författare)
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Cryptosporidium species and subtypes identified in human domestic cases through the national microbiological surveillance programme in Sweden from 2018 to 2022
- 2024
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Ingår i: BMC Infectious Diseases. - : BMC. - 1471-2334. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Background The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. A national microbiological surveillance programme was implemented in Sweden in 2018 in order to increase knowledge of the molecular epidemiology of human cryptosporidiosis to better understand transmission patterns and potential zoonotic sources. This article summarises the results of the first five years of the surveillance programme. Methods Cryptosporidium-positive faecal and DNA samples from domestically acquired infections were collected from clinical microbiological laboratories in Sweden. Species and subtype determination was performed using 60 kDa glycoprotein and/or small subunit ribosomal RNA gene analysis. Results Between 2018 and 2022, 1654 samples were analysed and 11 different species were identified: C. parvum (n = 1412), C. mortiferum (n = 59), C. hominis (n = 56), C. erinacei (n = 11), C. cuniculus (n = 5), C. meleagridis (n = 3), C. equi (n = 2), C. ubiquitum (n = 2), and one each of C. canis, C. ditrichi and C. felis. Subtyping revealed seven subtype families of C. parvum (new subtype families IIy and IIz) and 69 different subtypes (11 new subtypes). The most common C. parvum subtypes were IIdA22G1c, IIdA24G1, IIdA15G2R1 and IIaA16G1R1b. For C. hominis, four different subtype families and nine different subtypes (two new subtypes) were identified. For additional species, two new subtype families (IIIk and VId) and nine new subtypes were identified. All successfully subtyped C. mortiferum cases were subtype XIVaA20G2T1, confirming previous findings in Sweden. Several outbreaks were identified of which the majority were foodborne and a few were due to direct contact with infected animals. Conclusion Infection with C. parvum is the leading cause of human cryptosporidiosis acquired in Sweden, where more than 90% of domestic cases are caused by this zoonotic species and only a small proportion of cases are due to infection with other species. The rodent-associated C. mortiferum is considered an emerging zoonotic species in Sweden and the number of domestically acquired human cases has surpassed that of infection with C. hominis. A high diversity of species and subtypes, as well as diversity within the same subtype, was detected. Also, cryptosporidiosis appears to affect adults to a great extent in Sweden.
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| 8. |
- Casmo, Veronica, et al.
(författare)
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Occurrence of Cryptosporidium spp. and Cystoisospora belli among adult patients with diarrhoea in Maputo, Mozambique
- 2018
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Ingår i: Heliyon. - : ELSEVIER SCI LTD. - 2405-8440. ; 4:9
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Tidskriftsartikel (refereegranskat)abstract
- Infections with Cryptosporidium spp. and Cystoisospora belli are important causes of diarrhoea in HIV patients. Nevertheless, information concerning these two parasites is scarce in many African countries, including Mozambique. In this study occurrence of Cryptosporidium spp. and C. belli was investigated by microscopy of stool specimens from 108 adult diarrhoeal patients, most with a confirmed HIV diagnosis. The Cryptosporidium isolates were further characterized by molecular methods. Cryptosporidium and C. belli oocysts were found in 8.3% (9/108), and 25.0% (27/108) of the study participants, respectively. Species identification was possible for all Cryptosporidium isolates with available DNA. The following Cryptosporidium species were detected (number of cases within parentheses): C. parvum (3), C. hominis (3), C. felis (1), and C. hominis/C. parvum (1). Subtyping targeting the gp60 gene revealed two C. hominis isolates with subtype IaA23R3, one C. parvum isolate with IIcA5G3d, and one with IIeAl2G1. In summary the occurrence of C. hominis and anthroponotic subtypes of C. parvum indicates that the main route of Cryptosporidium transmission in the present study population was human to human (direct or via food and water). The high prevalence of C. belli highlights the need for early diagnosis of this parasite, for which a treatment exists.
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| 9. |
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| 10. |
- Lebbad, Marianne, et al.
(författare)
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Dominance of Giardia assemblage B in León, Nicaragua.
- 2008
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Ingår i: Acta Tropica. - : Elsevier BV. - 0001-706X .- 1873-6254. ; 106:1, s. 44-53
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Tidskriftsartikel (refereegranskat)abstract
- Giardiasis is a major problem in León, Nicaragua, yet despite this no data are available regarding the prevalence of different Giardia genotypes in this area. To address this question, a molecular analysis of Giardia isolates from humans and dogs living in the same area in León, Nicaragua was performed. Giardia isolates from 119 Nicaraguan patients and 8 dogs were successfully genotyped using single and/or nested beta-giardin PCR with subsequent restriction length fragment polymorphism (RFLP) analysis. The analyses of human samples yielded 94 (79%) assemblage B isolates and 25 (21%) assemblage A isolates. Only the non-human-associated assemblages C and D were found in the dog samples. Sixteen isolates with assemblage A pattern, 26 isolates with assemblage B pattern and all dog isolates were further characterized by sequencing the nested beta-giardin PCR product and by molecular analyses of the glutamate dehydrogenase (gdh) gene. Within the study area the assemblage A isolates were highly genetically homogenous, showing only sub-genotypes A2 (n=3) or A3 (n=13) at the beta-giardin locus and AII only at the gdh locus while assemblage B showed a high genetic polymorphism at both loci. Seven different sub-genotypes were identified within 13 of the sequenced assemblage B beta-giardin isolates. The remaining 13 sequenced assemblage B-isolates appeared to contain several different variants of the beta-giardin gene since the chromatograms displayed one to seven double peaks. The gdh sequences showed an even higher polymorphism since only 2 of 26 assemblage B isolates were without double peaks. Two mixed infections between assemblage A and B were found when the gdh gene was analyzed. Polymorphisms were also observed in the dog-associated assemblages C and D, but to a lesser extent than in assemblage B.
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