Begoña Bailo

Molecular Genotyping of Giardia duodenalis Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid, Spain

Background 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. Methods 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 β-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed. Principal findings 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. Conclusions 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.

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR-RFLP and nested-PCR of ribosomal DNA ITS1 region.

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1-PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1-PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.

Detection and molecular characterisation of Giardia duodenalis, Cryptosporidium spp. and Entamoeba spp. among patients with gastrointestinal symptoms in Gambo Hospital, Oromia Region, southern Ethiopia

To assess the prevalence and genetic diversity of the enteric protozoa species G. duodenalis, Cryptosporidium spp. and Entamoeba histolytica in individuals with gastrointestinal symptoms compatible with infections by these pathogens seeking medical attention in a rural area in southern Ethiopia.

Prevalence and Genetic Diversity of Giardia duodenalis and Cryptosporidium spp. among School Children in a Rural Area of the Amhara Region, North-West Ethiopia.

Backgroud 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. Methods 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 β-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. Principal Findings 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. Conclusions 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.

Detection and molecular characterization of Giardia duodenalis in children attending day care centers in Majadahonda, Madrid, Central Spain.

AbstractInfections by the protozoan enteroparasites Giardia duodenalis and Cryptosporidium spp are a major cause of morbidity in children attending day care facilities in developed countries. In this cross-sectional study, we aimed to estimate the occurrence and genotype frequencies of these pathogens in children attending day care centers in Majadahonda, Central Spain. To do so, single stool samples were obtained from 90 children and tested for the presence of G duodenalis and Cryptosporidium spp by conventional microscopy and immunochromatography. Positive results by these techniques were subsequently confirmed by immunofluorescence microscopy. G duodenalis-positive samples were subjected to molecular characterization studies by multilocus sequence-based genotyping of the glutamate dehydrogenase and &bgr;-giardin genes of the parasite. G duodenalis assemblages were confirmed by restriction fragment length polymorphism analyses and sequencing. A socioepidemiological questionnaire was used to identify variables potentially associated with giardiasis/cryptosporidiosis in the population of children under investigation. Overall, G duodenalis and Cryptosporidium spp were detected in 15.5% and 3.3% of stool samples, respectively. Giardiasis and cryptosporidiosis were found in 3/3 and 2/3 day care centers, respectively, affecting mainly infants aged 13 to 24 months. A total of 8 G duodenalis isolates were confirmed as subassemblage BIV, all of them belonging to asymptomatic children. Attempts to genotype Cryptosporidium isolates failed. None of the variables considered could be associated with higher risk of infection with giardiasis or cryptosporidiosis. These results clearly indicate that asymptomatic infections with G duodenalis and Cryptosporidium spp are frequent in <3-year-old children in Central Spain.

Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in Northern Spain

Domestic dogs and cats may act as natural reservoirs of a large number of zoonotic pathogens, including the enteric parasites Giardia duodenalis and Cryptosporidium spp., the most relevant protozoan species causing gastrointestinal disease worldwide. A cross-sectional epidemiological study aiming to assess the prevalence and molecular diversity of G. duodenalis and Cryptosporidium spp. was conducted in an animal rescue centre in the province of Álava (Northern Spain). A total of 194 and 65 faecal dropping samples from individual dogs and cats, respectively, were collected between November 2013 and June 2016. G. duodenalis cysts and Cryptosporidium spp. oocysts were detected by direct fluorescence microscopy and PCR-based methods targeting the small subunit ribosomal RNA gene of these parasites. Overall, G. duodenalis and Cryptosporidium spp. were detected in 33% (63/194) and 4.1% (8/194) of dogs, and 9.2% (6/65) and 4.6% (3/65) of cats, respectively. G. duodenalis and Cryptosporidium co-infections were observed in 1.5% (3/194) of dogs, but not in cats. No significant differences in infection rates could be demonstrated among dogs or cats according to their sex, age group, status, or geographical origin. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of G. duodenalis allowed the characterization of 19 canine isolates that were unambiguously assigned to sub-assemblages AII (n=7), BIII (n=1), and BIV (n=7), and assemblages C (n=3) and D (n=1). Two feline isolates were genotyped as assemblages A and F, respectively. No mixed assemblage or sub-assemblage infections were identified. C. canis (n=5) and C. hominis (n=1) were the Cryptosporidium species found in dogs, whereas C. felis (n=1) was identified in cats. The finding of G. duodenalis sub-assemblages AII, BIII, and BIV circulating in dogs (but not cats) may have zoonotic potential, although most of the AII and BIV isolates sub-genotyped corresponded to genetic variants not previously found in Spanish human populations. Dogs may also act as novel suitable hosts for C. hominis. We recommend to considerer companion animals as sentinel surveillance system for zoonotic giardiasis and cryptosporidiosis in order to minimize the risk of spreading of these parasitic diseases among the human population.

Unexpected finding of feline-specific Giardia duodenalis assemblage F and Cryptosporidium felis in asymptomatic adult cattle in Northern Spain

Giardia duodenalis and Cryptosporidium spp. are common enteric protozoan parasites in production animals, including cattle. Typically, both the clinical outcome of these infections and the distribution of G. duodenalis assemblages and Cryptosporidium species are age-dependent, with the occurrence of diarrhoeal disease being mainly associated with young animals and sub-clinical, low intensity infections being predominantly found in adult animals. To investigate the prevalence and genetic diversity of G. duodenalis and Cryptosporidium spp. in asymptomatic adult cattle, a total of 362 faecal samples were collected in four farms in the province of Álava, Northern Spain, between November 2011 and December 2012. The presence of G. duodenalis was estimated by real-time PCR, and the assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-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. Overall, G. duodenalis and Cryptosporidium spp. were detected in 68 (18.8%) and 45 (12.4%), respectively, of the 362 animals tested. Strikingly, four isolates representing two novel sub-types of G. duodenalis assemblage F were identified at the gdh, but not the bg, locus. This is the first report describing the presence of the feline-specific G. duodenalis assemblage F in bovine isolates. Additionally, five (three novel and two known) sub-types of G. duodenalis assemblage E were also identified at the gdh locus and a single one (assigned to sub-assemblage EII) at the bg locus. Of nine Cryptosporidium isolates, four (including a novel sub-type) were assigned to the cat-specific C. felis, two were typed as C. bovis, and the remaining three were only characterized at the genus level. Data presented here provide epidemiological and molecular evidence demonstrating that the host range specificity of G. duodenalis assemblages and Cryptosporidium species may be wider than previously anticipated.

No molecular epidemiological evidence supporting household transmission of zoonotic Giardia duodenalis and Cryptosporidium spp. from pet dogs and cats in the province of Álava, Northern Spain

The role of pet dogs and cats as suitable source of human infections by the diarrheagenic protozoan parasites Giardia duodenalis and Cryptosporidium spp. has been a topic of intense debate for long time and still remains a largely unsolved problem. In this cross-sectional molecular epidemiological survey we attempted to investigate whether zoonotic (or zooanthroponotic) disease transmission was occurring among humans and domestic dogs and cats sharing the same spatial and temporal setting in both rural and urban areas of the province of Álava, Northern Spain. A total of 268 (including 179 human, 55 canine, and 34 feline) individual faecal specimens were obtained from 63 family households during February-March and November-December 2014. Detection of G. duodenalis cysts and Cryptosporidium spp. oocysts was achieved by direct fluorescence microscopy (DFAT) and PCR-based methods targeting the small subunit (SSU) ribosomal RNA gene of the parasites. Giardia-positive isolates were subsequently sub-genotyped at the glutamate dehydrogenase (GDH) and β-giardin (BG) genes. Overall, G. duodenalis infections were identified in 3.4% (6/179) of humans, 29% (16/55) of dogs, and 5.9% (2/34) of cats, respectively. Cryptosporidium spp. infections were detected in 1.1% (2/179) of humans, 5.5% (3/55) of dogs, and 8.8% (3/34) of cats, respectively. Simultaneous infections in human and canine/feline hosts by G. duodenalis or Cryptosporidium spp. were only demonstrated in a single household in which a cat and its owner tested positive for Cryptosporidium by DFAT, but this result could not be confirmed by SSU-PCR. Infections were homogeneously distributed among the studied human or animal populations irrespectively of their sex, age group, or geographical region of origin. Inadequate washing of raw vegetables and fruits was the only risk factor significantly associated to a higher likelihood of having human giardiosis/cryptosporidiosis. Molecular characterization of G. duodenalis isolates revealed the presence of sub-assemblage BIV in a single human isolate. All dog (n=3) and cat (n=2) isolates successfully genotyped were assigned to canine- and feline-specific assemblages C and F, respectively. No mixed assemblage or sub-assemblage infections could be demonstrated. Regarding Cryptosporidium, C. canis was found infecting dogs (n=2), and C. felis a single cat. Attempts to amplify and characterize Cryptosporidium human isolates failed repeatedly. Our results suggest that pet dogs and cats do not seem to play a significant role as suitable reservoirs of human giardiosis or cryptosporidiosis in the province of Álava. We conclude, therefore, that zoonotic transmission of giardiosis or cryptosporidiosis among pet dogs and cats and their owners in this geographical region is very likely a rare event.

Characterization of the Taenia spp HDP2 sequence and development of a novel PCR-based assay for discrimination of Taenia saginata from Taenia asiatica

A previously described Taenia saginata HDP2 DNA sequence, a 4-kb polymorphic fragment, was previously used as the basis for developing PCR diagnostic protocols for the species-specific discrimination of T. saginata from T. solium and for the differentiation of T. saginata from T. asiatica. The latter was shown subsequently to lack the required specificity, so we undertook genetic studies of the HDP2 sequence from T. saginata and T. asiatica to determine why, and to develop a novel HDP2-PCR protocol for the simultaneous unambiguous identification of human taeniids. Sequencing and further analysis of the HDP2 DNA fragments of 19 Asiatic isolates of T. saginata and T. asiatica indicated that the HDP2 sequences of both species exhibited clear genomic variability, due to polymorphic variable fragments, that could correspond to the non-transcribed region of ribosomal DNA. This newly observed polymorphism allowed us to develop a novel, reproducible and reliable HDP2-PCR protocol which permitted the simultaneous discrimination of all T. saginata and T. asiatica isolates examined. This species-specific identification was based on, and facilitated by, the clear size difference in amplicon profiles generated: fragments of 1300 bp, 600 bp and 300 bp were produced for T. asiatica, amplicons of 1300 bp and 300 bp being obtained for T. saginata. Control T. solium samples produced one amplicon of 600 bp with the HDP2-PCR protocol. The assay has the potential to prove useful as a diagnostic tool in areas such as South East Asia where T. saginata, T. asiatica and T. solium coexist.

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts.