Marianne Lebbad

From mouse to moose: multilocus genotyping of Giardia isolates from various animal species.

Giardia intestinalis is a protozoan parasite that consists of seven genetically distinct assemblages (A to G). Assemblage A and B parasites have been detected in a wide range of animals including humans, while the other assemblages (C to G) appear to have a narrower host range. However, the knowledge about zoonotic transmission of G. intestinalis is limited. To address this question, 114 Giardia isolates from various animals in Sweden including pets, livestock, wildlife and captive non-human primates were investigated by a sequence-based analysis of three genes (beta-giardin, glutamate dehydrogenase and triose phosphate isomerase). Assemblage A infections were detected in nine ruminants, five cats and one dog, while three sheep were infected with both assemblages A and E. Multilocus genotypes (MLGs) were defined for assemblage A, and three of these MLGs have previously been detected in Giardia isolates from humans. The newly described sub-assemblage AIII, until now reported mainly in wild hoofed animals, was found in one cat isolate. Assemblage B occurred in three monkeys, one guinea pig and one rabbit. The rabbit isolate exhibited sequences at all three loci previously detected in human isolates. The non-zoonotic assemblages C, D, E, F or G were found in the remaining 83 G. intestinalis isolates, which were successfully amplified and genotyped, generating a wide variety of both novel and known sub-genotypes. Double peaks in chromatograms were seen in assemblage B, C, D and E isolates but were never observed in assemblage A, F and G isolates, which can reflect differences in allelic sequence divergence. No evidence of genetic exchange between assemblages was detected. The study shows that multilocus genotyping of G. intestinalis is a highly discriminatory and useful tool in the determination of zoonotic sub-groups within assemblage A, but less valuable for subtyping assemblages B, C, D and E due to the high frequency of double peaks in the chromatograms. The obtained data also suggest that zoonotic transmission of assemblages A and B might occur to a limited extent in Sweden.

Dominance of Giardia assemblage B in León, Nicaragua

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.

Multilocus genotyping of human Giardia isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children.

Background Giardia intestinalis is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. G. intestinalis consists of eight genetically distinct genotypes or assemblages, designated A–H, and assemblages A and B can infect humans. Giardiasis has been classified as a possible zoonotic disease but the role of animals in human disease transmission still needs to be proven. We tried to link different assemblages and sub-assemblages of G. intestinalis isolates from Swedish human patients to clinical symptoms and zoonotic transmission. Methodology/Principal Findings Multilocus sequence-based genotyping of 207 human Giardia isolates using three gene loci: ß-giardin, glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was combined with assemblage-specific tpi PCRs. This analysis identified 73 patients infected with assemblage A, 128 with assemblage B, and six with mixed assemblages A+B. Multilocus genotypes (MLGs) were easily determined for the assemblage A isolates, and most patients with this genotype had apparently been infected through anthroponotic transmission. However, we also found evidence of limited zoonotic transmission of Giardia in Sweden, since a few domestic human infections involved the same assemblage A MLGs previously reported in Swedish cats and ruminants. Assemblage B was detected more frequently than assemblage A and it was also more common in patients with suspected treatment failure. However, a large genetic variability made determination of assemblage B MLGs problematic. Correlation between symptoms and assemblages was found only for flatulence, which was significantly more common in children less than six years of age infected with assemblage B. Conclusions/Significance This study shows that certain assemblage A subtypes are potentially zoonotic and that flatulence is connected to assemblage B infections in young children. Determination of MLGs from assemblages A and B can be a valuable tool in outbreak situations and to help identify possible zoonotic transmission.

Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden

In November 2010, ≈27,000 (≈45%) inhabitants of Östersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens.

Pivmecillinam and adverse birth and neonatal outcomes: a population-based cohort study.

A previous study unexpectedly showed an increased, statistically imprecise, risk of low Apgar score in children of women redeeming prescriptions for pivmecillinam in late pregnancy. To improve statistical precision we extended the previous dataset with data for 5 more y, and in addition added more neonatal outcomes. We thus examined the risk of adverse birth and neonatal outcomes among pregnant users of pivmecillinam based on population-based registries in North Jutland County, Denmark. We included 63,659 women with a live birth, or stillbirth after the 28th week of gestation. 2031 had redeemed prescriptions for pivmecillinam any time during pregnancy, 559 in the first trimester and 371 within 28 d before delivery. Adjusted odds ratios were: birth defects 0.83 (95% confidence interval (95% CI) 0.53-1.32) for exposure during first trimester, preterm delivery 0.96 (95% CI 0.79-1.18) and low birth weight 0.79 (95% CI 0.52-1.20) for exposure any time during pregnancy, and stillbirth 1.19 (95% CI 0.30-4.80), low Apgar score 1.17 (95% CI 0.37-3.66), hypoglycaemia 1.03 (95% CI 0.53-2.00), and respiratory distress syndrome 0.79 (95% CI 0.38-1.68) for exposure within 28 d before delivery. Use of pivmecillinam during pregnancy did not appear to increase the risk of adverse birth and neonatal outcomes; however, statistical precision is still low.An outbreak of cryptosporidiosis among visitors to a public swimming pool occurred in the late summer of 2002. We performed a retrospective cohort study, including 3 cohorts, A) 178 school-children who visited the pool on a single occasion, B) 263 arbitrarily chosen school children, aged 6–12 y, and their household members, living within the municipality where the outbreak occurred, and C) an additional 28 individuals with laboratory confirmed cryptosporidiosis. The outbreak lasted 4 weeks and affected an estimated 800–1000 individuals. The primary attack rate was 40–50%. The median incubation period was 5 d (range 2–13 d). The secondary attack rate was 8–10%. Diarrhoea was reported by 93% of the patients, abdominal pain 89%, nausea 73%, and fever 40%. Fifty-four percent had<5 loose stools and 20% had>10 loose stools per d. The duration of symptoms was 4–10 d for 52% and>10 d for 34% of the cases. This is the first reported outbreak of pool associated cryptosporidiosis in Sweden and emphasizes the importance of proper control routines of swimming pools with continuous assessment of the quality of the water sources and filtration processes.

Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children

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.

Intestinal parasites in HIV-2 associated AIDS cases with chronic diarrhoea in Guinea-Bissau.

Previous studies from African countries where HIV-1 infection is prevalent have shown that infections with Cryptosporidium parvum, Isospora belli and microsporidia are frequently associated with chronic diarrhoea in AIDS patients. The information about the occurrence of these parasites in HIV-2 associated AIDS cases with chronic diarrhoea is limited. We have performed a study of stool parasites in patients from Guinea-Bissau, the country with the highest prevalence of HIV-2 in the world. Stool specimens from 52 adult patients with chronic diarrhoea of which 37 were HIV-positive and fulfilling the clinical criteria of AIDS (five HIV-1, 28 HIV-2 and four dually infected with HIV-1 and HIV-2) were screened for parasitic infections. Twenty five percent of the HIV-2 positive patients were infected with C. parvum, 11% with I. belli and 11% with microsporidia, all three parasites were seen only in HIV-positive patients. The three patients with microsporidiosis, all HIV-2 infected, are to our knowledge the first cases reported from Guinea-Bissau. Other stool parasites such as Blastocystis hominis, hookworm and Strongyloides stercoralis were observed both among HIV-positive and HIV-negative patients.

Zoonotic transmission of Cryptosporidium meleagridis on an organic Swedish farm.

We believe that we present the first evidence of zoonotic transmission of the bird parasite, Cryptosporidium meleagridis. Despite being the third most common cause of human cryptosporidiosis, an identified zoonotic source has not been reported to date. We found Cryptosporidium oocysts in pigs, sheep/goats, hens and broiler chickens on a farm with suspected zoonotic transmission. By DNA analysis we identified C. meleagridis in samples from one human, three chickens and one hen. Sequencing of the ssrRNA and 70kDa Heat Shock Protein (HSP) genes showed identical C. meleagridis sequences in the human and chicken samples, which is evidence of zoonotic transmission. The HSP70 sequence was unique.

PCR differentiation of Entamoeba histolytica and Entamoeba dispar from patients with amoeba infection initially diagnosed by microscopy

Amoebiasis is a notifiable disease in Sweden and 400–500 cases are reported annually to the Swedish Institute for Infectious Disease Control (SMI). The true number of patients with Entamoeba histolytica infection is unknown as diagnosis mainly relies on cyst detection by microscopy. The main purpose of this study was to estimate the proportions between E. histolytica and E. dispar in patients with amoebic infection, using established PCR technologies. Secondly, we aimed to evaluate the usefulness of ethanol as a transport medium for samples forwarded for Entamoeba-PCR. Faecal samples from 207 patients with initial diagnosis of E. histolytica/E. dispar were referred to SMI for species differentiation. The PCR analysis showed that 165 patients were positive for E. dispar, whereas only 10 patients were positive for E. histolytica. No mixed infections were observed. The remaining 32 patients were negative both by microscopy and by PCR. Ethanol fixation was evaluated on 168 paired samples (transported unfixed or fixed in ethanol). Ethanol was found to be a useful transport medium as in 8 cases only the fixed sample was PCR-positive. This study shows that few patients in Sweden are infected with E. histolytica. The ability to differentiate E. dispar from E. histolytica should reduce the number of unnecessarily treated patients.

A Foodborne Outbreak of Cyclospora Infection in Stockholm, Sweden

During May and June 2009 an outbreak of Cyclospora cayetanensis infection involving 12 laboratory-confirmed and 6 probable cases was detected in Stockholm County, Sweden. Imported sugar snap peas from Guatemala were the suspected vehicle, based on information obtained from patient questionnaires. This is the first reported outbreak of cyclosporiasis in Sweden and the second in Europe.