RESUMO
Per- and polyfluoroalkyl substances (PFASs) are a highly persistent, mobile, and bioaccumulative class of chemicals, of which emissions into the environment result in long-lasting contamination with high probability for causing adverse effects to human health and the environment. Within the European Biomonitoring Initiative HBM4EU, samples and data were collected in a harmonized way from human biomonitoring (HBM) studies in Europe to derive current exposure data across a geographic spread. We performed mixture risk assessments based on recent internal exposure data of PFASs in European teenagers generated in the HBM4EU Aligned Studies (dataset with N = 1957, sampling years 2014-2021). Mixture risk assessments were performed based on three hazard-based approaches: the Hazard Index (HI) approach, the sum value approach as used by the European Food Safety Authority (EFSA) and the Relative Potency Factor (RPF) approach. The HI approach resulted in the highest risk estimates, followed by the RPF approach and the sum value approach. The assessments indicate that PFAS exposure may result in a health risk in a considerable fraction of individuals in the HBM4EU teenager study sample, thereby confirming the conclusion drawn in the recent EFSA scientific opinion. This study underlines that HBM data are of added value in assessing the health risks of aggregate and cumulative exposure to PFASs, as such data are able to reflect exposure from different sources and via different routes.
Assuntos
Monitoramento Biológico , Fluorocarbonos , Adolescente , Humanos , Medição de Risco , Inocuidade dos Alimentos , BioacumulaçãoRESUMO
BACKGROUND: Perfluoroalkyl substances (PFAS) are man-made fluorinated chemicals, widely used in various types of consumer products, resulting in their omnipresence in human populations. The aim of this study was to describe current PFAS levels in European teenagers and to investigate the determinants of serum/plasma concentrations in this specific age group. METHODS: PFAS concentrations were determined in serum or plasma samples from 1957 teenagers (12-18 years) from 9 European countries as part of the HBM4EU aligned studies (2014-2021). Questionnaire data were post-harmonized by each study and quality checked centrally. Only PFAS with an overall quantification frequency of at least 60% (PFOS, PFOA, PFHxS and PFNA) were included in the analyses. Sociodemographic and lifestyle factors were analysed together with food consumption frequencies to identify determinants of PFAS exposure. The variables study, sex and the highest educational level of household were included as fixed factors in the multivariable linear regression models for all PFAS and each dietary variable was added to the fixed model one by one and for each PFAS separately. RESULTS: The European exposure values for PFAS were reported as geometric means with 95% confidence intervals (CI): PFOS [2.13 µg/L (1.63-2.78)], PFOA ([0.97 µg/L (0.75-1.26)]), PFNA [0.30 µg/L (0.19-0.45)] and PFHxS [0.41 µg/L (0.33-0.52)]. The estimated geometric mean exposure levels were significantly higher in the North and West versus the South and East of Europe. Boys had significantly higher concentrations of the four PFAS compared to girls and significantly higher PFASs concentrations were found in teenagers from households with a higher education level. Consumption of seafood and fish at least 2 times per week was significantly associated with 21% (95% CI: 12-31%) increase in PFOS concentrations and 20% (95% CI: 10-31%) increase in PFNA concentrations as compared to less frequent consumption of seafood and fish. The same trend was observed for PFOA and PFHxS but not statistically significant. Consumption of eggs at least 2 times per week was associated with 11% (95% CI: 2-22%) and 14% (95% CI: 2-27%) increase in PFOS and PFNA concentrations, respectively, as compared to less frequent consumption of eggs. Significantly higher PFOS concentrations were observed for participants consuming offal (14% (95% CI: 3-26%)), the same trend was observed for the other PFAS but not statistically significant. Local food consumption at least 2 times per week was associated with 40% (95% CI: 19-64%) increase in PFOS levels as compared to those consuming local food less frequently. CONCLUSION: This work provides information about current levels of PFAS in European teenagers and potential dietary sources of exposure to PFAS in European teenagers. These results can be of use for targeted monitoring of PFAS in food.
Assuntos
Ácidos Alcanossulfônicos , Poluentes Ambientais , Fluorocarbonos , Masculino , Feminino , Animais , Adolescente , Humanos , Peixes , Dieta , Modelos Lineares , Coleta de DadosRESUMO
The presence of Cryptosporidium and Giardia in 221 fecal samples from different species of Antarctic pinnipeds was investigated by immunofluorescence microscopy and PCR. Cryptosporidium, a skunk-like genotype, was detected only in a southern elephant seal. Giardia was not detected. This is the first report of a Cryptosporidium sp. in Antarctic marine mammals.
Assuntos
Cryptosporidium/isolamento & purificação , Giardia/isolamento & purificação , Focas Verdadeiras/microbiologia , Animais , Regiões Antárticas , Cryptosporidium/classificação , Cryptosporidium/genética , Fezes/microbiologia , Imunofluorescência , Microscopia de Fluorescência , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
The presence of Campylobacter spp. was investigated in 41 Antarctic fur seals (Arctocephalus gazella) and 9 Weddell seals (Leptonychotes weddellii) at Deception Island, Antarctica. Infections were encountered in six Antarctic fur seals. The isolates, the first reported from marine mammals in the Antarctic region, were identified as Campylobacter insulaenigrae and Campylobacter lari.
Assuntos
Infecções por Campylobacter/veterinária , Campylobacter/classificação , Campylobacter/isolamento & purificação , Otárias/microbiologia , Animais , Regiões Antárticas , Campylobacter/genética , Infecções por Campylobacter/microbiologia , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Focas Verdadeiras/microbiologia , Análise de Sequência de DNARESUMO
Besnoitia besnoiti was isolated from a skin biopsy of a chronically infected cow from central Spain. Zoites released from macroscopic cysts were adapted to its culture in vitro on a MARC-145 cell monolayer. Tachyzoites produced in vitro were either cryopreserved or used for genomic DNA isolation. A 2206 nt sequence containing 18S ribosomal RNA gene, internal transcribed spacer 1 (ITS 1), and a partial sequence of 5.8S ribosomal RNA gene was amplified by PCR and sequenced. This sequence showed a 99-100% identity to 18S, ITS1, and 5.8S sequences of B. besnoiti published in databases. After analysis by transmission and scanning electron microscopy of isolated bradyzoites and tachyzoites, it was observed that their ultrastructural morphology coincided with B. besnoiti. The isolate characterized in this study was identified as B. besnoiti on the basis of the disease produced, molecular characteristics, and morphology. The B. besnoiti isolate was denoted as BbSpain-1; it is the first isolate obtained and characterized in Spain and one of the first European isolates adapted to grow in vitro. The isolation and in vitro production of this B. besnoiti isolate offers a good opportunity to study general aspects of bovine besnoitiosis, including epidemiology, pathogenesis, and diagnosis of this re-emergent disease.
Assuntos
Coccidiose/veterinária , Sarcocystidae/isolamento & purificação , Dermatopatias Parasitárias/veterinária , Animais , Biópsia , Bovinos , Coccidiose/parasitologia , Sequência Consenso , DNA de Protozoário/química , DNA Espaçador Ribossômico/química , Feminino , Microscopia Eletrônica de Varredura/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 18S/genética , RNA Ribossômico 5,8S/genética , Sarcocystidae/genética , Sarcocystidae/ultraestrutura , Pele/parasitologia , Dermatopatias Parasitárias/parasitologia , EspanhaRESUMO
Mercury (Hg) is among the top 10 environmental chemicals of major public health concern (WHO). The Minamata Convention on Mercury (United Nations Environment Program, 2017), commits signing countries to control anthropogenic mercury emissions and reduce human exposure. Human biomonitoring (HBM) programs, are the most straight-forward approaches to get information on the actual exposure levels in the population and assess over time. We report here the results of a HBM study in a nationwide cross-section of Spanish adults (18-65y) as baseline values obtained before the Minamata Convention entered into force. Subsequent follow-ups will show if the Convention has been successful. The study includes 1880 blood samples, 1704 urine samples and 577 hair samples from all Spanish regions collected and analysed under a strictly quality controlled and quality assured protocol. The EU-DEMOCOPHES project demonstrated that fish and seafood are the major sources of mercury exposure and that the Spanish as well as the Portuguese populations have higher levels than other European countries. The data from the present study confirms this pattern at national level and that inhabitants in coastal regions have higher values than from inland regions. The geometric mean (GM) for blood is 6.35⯵g Hg/l, in urine is 1.11⯵g Hg/l and for hair is 1.91⯵g Hg/g. In an international comparison these values are not exceptional. Spanish concentrations fall into the group of Easter Mediterranean populations. Although information on gender, age, occupational sector, geographical area, sampling period and frequency of fish consumption is reported in the tables, the purpose of this paper has not been to analyse the determinants of exposure in detail but to provide baseline data for future assessments and for regional authorities.
Assuntos
Exposição Ambiental/estatística & dados numéricos , Poluentes Ambientais/metabolismo , Mercúrio/metabolismo , Adulto , Monitoramento Ambiental , Poluentes Ambientais/sangue , Poluentes Ambientais/urina , Feminino , Cabelo/química , Humanos , Masculino , Mercúrio/sangue , Mercúrio/urina , EspanhaRESUMO
Neospora caninum is a cyst-forming parasite that causes abortion in cattle. Despite this parasite's ubiquitous distribution and wide host range, the number of N. caninum isolates obtained to date is limited. In vitro isolation of the parasite is arduous and often unsuccessful. In addition, most isolates have been obtained from clinically affected hosts and therefore could be biased towards more virulent isolates. In this report, an improved isolation approach from transplacentally infected newborn calves was undertaken and 9 new isolates were obtained. Moreover, a microsatellite technique was applied to investigate the genetic diversity of these isolates. Most isolates showed specific genetic profiles. However, the Nc-Spain10 isolate was identical to the previously described Nc-Spain1H isolate and Nc-Spain3H was identical to Nc-Spain4H. These isolates were likely to have identical genotypes because they were isolated from distinct calves of the same herd. Future pathogenic characterization of these isolates will contribute to the investigation of the relationship between isolate virulence and the outcome of infection, as well as other epidemiological features, such as transmission.
Assuntos
Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , Variação Genética , Neospora/genética , Alelos , Animais , Anticorpos Antiprotozoários/sangue , Bioensaio , Bovinos , Células Cultivadas , Coccidiose/parasitologia , Feminino , Genótipo , Masculino , Camundongos , Camundongos Nus , Repetições de Microssatélites/genética , Neospora/classificação , Neospora/isolamento & purificação , Espanha , Especificidade da EspécieRESUMO
The characterization of Cryptosporidium using DNA extracted from whole faecal samples collected from 2414 humans with diarrhoea in England between 1985 and 2000 where cryptosporidial oocysts were detected using conventional methods is described. Characterization was achieved by PCR/RFLP and DNA sequencing of fragments of the Cryptosporidium oocyst wall protein and the 18S rDNA genes. Cryptosporidium parvum was detected in 56.1 % of cases, Cryptosporidium hominis in 41.7 % and a mixture of C. parvum and C. hominis in 0.9 %. In the remainder of cases, Cryptosporidium meleagridis (0.9 %), Cryptosporidium felis (0.2 %), Cryptosporidium andersoni (0.1 %), Cryptosporidium canis (0.04 %), Cryptosporidium suis (0.04 %) and the Cryptosporidium cervine type (0.04 %) were detected.
Assuntos
Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/genética , Diarreia/microbiologia , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Cryptosporidium/classificação , Cryptosporidium/isolamento & purificação , Cryptosporidium/patogenicidade , DNA de Protozoário/genética , Inglaterra/epidemiologia , Fezes/parasitologia , Genes de Protozoários , Humanos , Lactente , Dados de Sequência Molecular , Proteínas de Protozoários/genética , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Especificidade da EspécieRESUMO
The complete amino acid sequence of human placental oxytocinase (placental leucine aminopeptidase) has been determined by cDNA cloning and sequencing. Oxytocinase is a type II integral membrane protein of 1025 amino acid residues, consisting of an acidic intracellular region of 110 amino acids followed by a hydrophobic transmembrane segment of 22 residues and 893 extracellular residues containing the characteristic Zn2+ coordination sequence element His-Glu-Xaa-Xaa-His-(18 residues)-Glu found in gluzincins. Two sets of cDNA clones with different 5'-ends were isolated and suggested to represent different spliced products of 3.6 kb (mature mRNA) and 12 kb, respectively. Oxytocinase mRNA is present in large amounts in placenta, heart and skeletal muscle and in small amounts in brain, kidney, liver and pancreas. A conserved sequence element, the GAMEN motif, which distinguishes the aminopeptidase family among gluzincins from other gluzincins, has been identified.
Assuntos
Cistinil Aminopeptidase/química , Placenta/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cistinil Aminopeptidase/genética , DNA Complementar/química , Feminino , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.
Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Criptosporidiose/veterinária , Cryptosporidium/classificação , Fezes/parasitologia , Proteínas de Protozoários/genética , Animais , Bovinos , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , Genes de RNAr , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 18S/genéticaRESUMO
An unusual genotype of Cryptosporidium was identified in the faeces of six human patients by PCR/RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene. Conventional microscopy showed oocysts indistinguishable in size from those of Cryptosporidium parvum, which reacted with two different commercially available anti-oocyst monoclonal antibodies. The isolates were further characterised by PCR/RFLP analysis of the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) genes as well as by DNA sequencing of the COWP and the TRAP-C1 gene fragments and of two regions of the 18S rRNA gene. Sequence analysis of the COWP, TRAP-C1, and 18S rRNA gene fragments confirmed that this genotype is genetically distinct from C. parvum. 18S rRNA gene sequences were found to be identical to those published for Cryptosporidium meleagridis.
Assuntos
Cryptosporidium/genética , Fezes/parasitologia , Genes de Protozoários , Sequência de Aminoácidos , Animais , Cryptosporidium/isolamento & purificação , Genótipo , Humanos , Dados de Sequência MolecularRESUMO
DNA was extracted from faecal samples collected from 1680 patients in which Cryptosporidium oocysts were recognised by light microscopy. DNA from faeces from five of these patients failed to amplify by PCR three gene fragments--the Cryptosporidium oocyst wall protein (COWP) gene, the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) gene and the thrombospondin-related adhesive protein of Cryptosporidium-2 (TRAP-C2) gene--with primers designed from C. parvum sequences. However, DNA from these five patients did amplify cryptosporidial 18S rDNA gene fragments and a heat-shock protein (HSP70) gene fragment was also amplified from four of them. The purpose of this study was to characterise further the Cryptosporidium associated with infection in these patients. DNA sequence analysis of 18S rDNA genes showed that four of these patients were infected by C. felis, and the remaining one by an as yet un-named Cryptosporidium species designated the 'dog type' (C. dt). Infection by C. felis was further confirmed in all four patients by DNA sequence analysis of the HSP70 gene. Oocysts present in all five samples reacted strongly with two anti-cryptosporidial oocyst monoclonal antibodies, except for the C. dt, which was tested with only one of the antibodies. Two of the patients infected by C. felis had underlying illness; one 8-year-old male had an undefined severe inherited underlying condition, and the second patient, a 32-year-old male, was HIV positive. Two of the remaining three patients (two females aged 1 and 2 years, respectively) were apparently immunocompetent (one infected with C. felis and one with the C. dt). No information was obtained for the fifth patient. The patient infected by C. dt had a recent history of travel to Africa. This is the first report of infection with these two Cryptosporidium species in immunocompetent patients, and in any patient in the UK.
Assuntos
Cryptosporidium/isolamento & purificação , Fezes/parasitologia , Adulto , Animais , Criança , Pré-Escolar , Cães , Inglaterra , Feminino , Proteínas de Choque Térmico HSP70/genética , Humanos , Masculino , RNA Ribossômico 18S/genéticaRESUMO
OBJECTIVES: To genetically characterize an unusual genotype of Cryptosporidium from the stools of humans with diarrhoea and to identify risk factors in the affected patients. METHODS: DNA was extracted from human faeces where Cryptosporidium oocysts were detected by light microscopy. Cryptosporidial gene fragments from six different loci were analysed by PCR alone, PCR/RFLP and by DNA sequencing. Oocysts were characterized by light and immunofluorescence microscopy and epidemiological data was collected from the affected patients. RESULTS: Analysis of the Cryptosporidium oocyst wall protein (COWP) gene amplified from > 2000 human faecal samples identified 19 patients all of which produced an unusual RFLP profile. Subsequent DNA sequence analysis of this and an additional four genetic loci (including 18S rRNA sequences) confirmed these as a homogeneous group which was genetically distinct from Cryptosporidium parvum. The isolates were identified as Cryptosporidium meleagridis since the gene sequences were identical to those from this species recovered from birds. Conventional microscopy showed oocysts indistinguishable from C. parvum and reacted strongly with two different commercially available anti-oocyst monoclonal antibodies. None of the patients showed risk factors unusual for cryptosporidiosis; however, ten of the cases occurred during the summer/autumn, six had a history of foreign travel, four were co-infected with Giardia, two were HIV positive, and six were without identifiable immunocompromising factors. CONCLUSIONS: This study further confirms that C. meleagridis, in addition to C. parvum, is involved in human disease. The study also highlights the lack of basic information on the host range of this genus of parasites, the complexity of the transmission routes involved in human cryptosporidiosis, and the value of molecular techniques in identify hitherto unrecognised differences in Cryptosporidium from human faeces.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium/genética , Fezes/parasitologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Criptosporidiose/parasitologia , Cryptosporidium/patogenicidade , Primers do DNA , Feminino , Genótipo , Humanos , Masculino , Microscopia de Fluorescência , Microscopia de Polarização , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Fatores de Risco , Análise de Sequência de DNARESUMO
In a previous study, farm and stray dogs were considered potential high risk populations of Neospora caninum infection in Spain. Consequently, we decided to investigate the significance of N. caninum infection in these populations. Specific antibodies were detected in 120 out of 275 dog sera (43.6%), with titres ranging from 1:50 to 1:800. Differences in seroprevalence between farm (47.5%, 67/141) and stray (39.5%, 53/134) dogs were not significant (P>0.05; χ(2) test), but farm dogs showed significantly higher titres (P<0.01; Student's t-test). N. caninum seroprevalence in farm dogs was associated with increasing age (P<0.01; χ(2) test) and dogs with free access to the farm were more likely to be seropositive than controlled-dogs (P<0.05; χ(2) test). The presence of anti-N. caninum antibodies was more often detected in dogs from farms with 5-20% N. caninum within-herd seroprevalence (56.9%, 37/65) than those from farms with 0-5% seroprevalence (38%, 23/60) (P<0.05; χ(2) test). We microscopically observed N. caninum-like oocysts in the faeces from one farm dog, but the number of oocysts was very low, and the aetiology could not be confirmed. Also, parasite isolation was attempted from fresh neural tissue from stray dogs but was unsuccessful.
Assuntos
Coccidiose/veterinária , Neospora/isolamento & purificação , Animais , Anticorpos Antiprotozoários/sangue , Coccidiose/epidemiologia , Coccidiose/parasitologia , Cães , Neospora/imunologia , Oocistos , Estudos Soroepidemiológicos , Espanha/epidemiologiaRESUMO
A method was developed for extracting cryptosporidial DNA from stained fecal smears on glass microscope slides. The correct genotype of Cryptosporidium parvum was amplified by PCR from 89 (85%) of 105 smears following conventional staining but not from negative controls. This technique may have applications for analysis of other infectious agents.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium parvum/classificação , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Animais , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , DNA de Protozoário/análise , Genótipo , Humanos , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , Coloração e Rotulagem/métodosRESUMO
We developed a sensitive nested polymerase chain reaction procedure for the Cryptosporidium oocyst wall protein (COWP) gene. Amplification and genotyping were successful in 95.2% of 1,680 fecal samples, 77.6% by the unnested and 17.6% by the nested COWP procedure. The COWP gene was amplified from 2,128 fecal samples: 71 from livestock animals and 2,057 from humans. This series included 706 cases from seven drinking water-associated outbreaks and 51 cases from five swimming pool-associated outbreaks, as well as 1,300 sporadic cases.
Assuntos
Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Animais , Cryptosporidium/crescimento & desenvolvimento , Genótipo , RNA Ribossômico 18S/genética , Água/parasitologia , Abastecimento de ÁguaRESUMO
Cryptosporidium present in 1,705 fecal samples from humans and 105 from livestock animals were analyzed by PCR-restriction fragment length polymorphism of the Cryptosporidium oocyst wall protein. Overall, genotype 1 (human exclusive type) was detected in 37.8% of the samples from humans, genotype 2 (broad host range) was detected in 61.5%, a third genotype designated genotype 3 (Cryptosporidium meleagridis) was detected in 0.3%, and both genotypes 1 and 2 were recovered from 0.4%. All samples from livestock yielded genotype 2. Among 469 patients infected during eight drinking water-related outbreaks, five outbreaks were predominantly due to genotype 1, and three were due to genotype 2. Fifty-four samples were collected from patients involved with five swimming pool-associated outbreaks: two outbreaks were due to genotype 1, one was due to genotype 2, and the remaining two involved both genotypes 1 and 2. Among 26 family outbreaks and 1 children's nursery outbreak (2 to 3 members per group), the same genotype was recovered from the different members of each outbreak: 13 were due to genotype 1, and 14 were due to genotype 2. In eighteen patients reporting contact with animals and/or farms, genotype 1 was recovered from one patient and genotype 2 was recovered from the remaining 17. Among the sporadic cases, there were distinct geographical and temporal variations in the distribution of the genotypes. The spring peak in cases was due to genotype 2. Genotype 1 was significantly more common in patients infected during the late-summer-autumn peak and in those with a history of foreign travel.
Assuntos
Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , Fezes/parasitologia , Animais , Animais Domésticos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Genes de Protozoários , Genótipo , Humanos , Epidemiologia Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Reino Unido/epidemiologiaRESUMO
Samples of whole feces in which Cryptosporidium oocysts were recognized by hospital laboratories were collected from 218 patients with diarrhea. All samples were reexamined by light microscopy, and oocysts were detected in 211 samples. A simple and rapid procedure for the extraction of DNA from whole feces was developed, and this was used to amplify fragments of the Cryptosporidium outer wall protein (COWP), the thrombospondin-related adhesive protein C1 (TRAP-C1), and the 18S rRNA genes by PCR. For seven samples oocysts were not detected by microscopy and DNA failed to be amplified by the three PCR procedures. Among the 211 samples "positive" by microscopy, the sensitivities of PCRs for the 18S rRNA, COWP, and TRAP-C1 gene fragments were 97, 91, and 66%, respectively. The sensitivities of all three PCR procedures increased with increasing numbers of oocysts as observed by microscopy. Two genotypes of the COWP and TRAP-C1 genes can be detected by PCR-restriction fragment length polymorphism analysis. With this series of samples, the same genotypes of the COWP and TRAP-C1 genes always segregated together. A combined genotyping data set was produced for isolates from 194 samples: 74 (38%) were genotype 1 and 120 (62%) were genotype 2. Genotype 2 was detected in a significantly greater proportion of the samples with small numbers of oocysts, and genotype 1 was detected in a significantly greater proportion of the samples with larger numbers of oocysts. There were no significant differences in the distribution of the genotypes by patient sex and age. The distribution of the genotypes was significantly different both in patients with a history of foreign travel and in those from different regions in England.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/genética , Diarreia/parasitologia , Adolescente , Adulto , Idoso , Animais , Criança , Cryptosporidium/classificação , Feminino , Genes de Protozoários , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , RNA Ribossômico 18S/genéticaRESUMO
Polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the Cryptosporidium parvum outer wall protein (COWP) gene was applied to specimens collected from 95 patients with cryptosporidiosis associated with two suspected waterborne outbreaks, 46 sporadic human cases and 62 infected livestock from other areas, and 12 patients infected with other gastrointestinal parasites. Ninety-six per cent of C. parvum isolates from patients linked to the two suspected waterborne outbreaks were of genotype 1; all the isolates from livestock were of genotype 2. Isolates from 59% of the sporadic human infections were of genotype 1 and 35% were of genotype 2. Specimens from two patients yielded both genotypes. Specimens from patients infected with other parasites yielded no amplicons.
Assuntos
Criptosporidiose/epidemiologia , Cryptosporidium parvum/genética , Surtos de Doenças , Abastecimento de Água , Animais , Criptosporidiose/parasitologia , Cryptosporidium parvum/classificação , DNA de Protozoário/genética , Inglaterra/epidemiologia , Fezes/parasitologia , Genes de Protozoários , Genótipo , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Ruminantes/parasitologiaRESUMO
An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalis assemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpi gene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpi gene of G. duodenalis assemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalis genotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.