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2.
PLoS Negl Trop Dis ; 13(10): e0007750, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31661498

RESUMO

BACKGROUND: The World Health Organization WHO has estimated that in developed countries, up to 30% of the population may suffer from foodborne diseases each year, and that in developing countries up to 2 million deaths per annum can be attributed to cryptosporidiosis. Reports have already emphasized the role of immigrants in outbreaks of parasitic diseases especially those working in food processing industries. METHODOLOGY/PRINCIPAL FINDINGS: Herein we assessed Cryptosporidium spp. infections among immigrants in Qatar with a special focus on food handlers and housemaids. The overall prevalence of Cryptosporidium spp. by q-PCR among 839 asymptomatic subjects was 4.5%. Based on the Gp60 gene, the majority of isolates were identified as C. parvum subtype IIdA20G1b. The positive sample for C. hominis was subtyped as IeA12G3T3. Seven mixed infections were also identified (four C. parvum + C. hominis, and three C. parvum + C. meleagridis). The prevalence of Cryptosporidium spp. did not differ significantly between the sexes or age classes but varied significantly between subjects affiliated to different religions with the lowest prevalence among the Muslims. Multifactorial analysis retained also marked significance with education, income, and a house contents index. CONCLUSIONS/SIGNIFICANCE: Our results contribute to a better understanding of the epidemiology of cryptosporidiosis and the risk factors associated with the likelihood of carrying this infection among immigrant workers from developing countries.


Assuntos
Criptosporidiose/epidemiologia , Cryptosporidium/isolamento & purificação , Migrantes , Adolescente , Adulto , Criptosporidiose/diagnóstico , Cryptosporidium/genética , DNA de Protozoário/genética , Surtos de Doenças , Feminino , Genes de Protozoários , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Patologia Molecular , Prevalência , Catar/epidemiologia , Fatores de Risco , Adulto Jovem
3.
Ann Parasitol ; 65(3): 237-243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31599539

RESUMO

Rabbits are commonly reared by households and farmers in Nigeria as a source of meat, but there is no information available on Cryptosporidium genotypes occurring in rabbits in Nigeria. Fecal samples were collected from 107 rabbits and examined by modified Ziehl-Neelsen technique for the presence of Cryptosporidium oocysts. An infection rate of 3.7% (4/107) was obtained and all microscopy-positive samples were genotyped and subtyped to determine the circulating Cryptosporidium species using sequence analysis of the 18S rRNA gene and 60-kDa glycoprotein (gp60) gene, respectively. All the four microscopy-positive samples were identified as C. parvum by 18S rRNA gene. However, analysis of the gp60 gene revealed the presence of C. parvum subtype IIc, which is commonly found in humans in two isolates. These findings indicate natural infection of rabbits with C. parvum and underscore the need to investigate the probable role of animal hosts in the epidemiology of Cryptosporidium infection. This is the first report on genetic characterization of Cryptosporidium infecting rabbits in Nigeria.


Assuntos
Criptosporidiose , Cryptosporidium parvum , Doenças Parasitárias em Animais , Animais , Criptosporidiose/diagnóstico , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Fezes/parasitologia , Genótipo , Humanos , Nigéria/epidemiologia , Doenças Parasitárias em Animais/diagnóstico , Doenças Parasitárias em Animais/epidemiologia , Doenças Parasitárias em Animais/parasitologia , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Coelhos , Análise de Sequência de DNA , Sialoglicoproteínas/genética
4.
Comp Immunol Microbiol Infect Dis ; 66: 101333, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31437689

RESUMO

To obtain information about the occurrence and genotype distribution of G. intestinalis and C. parvum in Austrian cattle, faecal samples from diarrhoeic calves younger than 180 days of age originating from 70 farms were examined. Of the 177 faecal samples, 27.1% were positive for Giardia cysts (immunofluorescence microscopy) and 55.4% for Cryptosporidium oocysts (phase-contrast microscopy). Positive samples were characterized by nested PCR for Giardia, 83.3% (triosephosphate isomerase; tpi) and 89.6% (ß-giardin; bg) were positive, while the Cryptosporidium nested PCR returned 92.5% (60-kDa glycoprotein) positive results. Sequence analysis revealed one assemblage A-positive sample and 30 (bg) respectively 29 (tpi) assemblage E-positive samples for G. intestinalis. For C. parvum four subtypes within the IIa family (IIaA15G2R1, n = 29; IIaA19G2R2, n = 3; IIaA21G2R1, n = 2; IIaA14G1R1, n = 1) could be differentiated. Validation of two immunochromatographic point-of-care tests resulted in a sensitivity of 29.2% and 77.6%; a specificity of 98.4% and 91.1% for the detection of Giardia intestinalis and Cryptosporidium parvum, respectively. Results confirm the widespread occurrence of both protozoa in diarrhoeic calves in Austria.


Assuntos
Doenças dos Bovinos/parasitologia , Cryptosporidium parvum/genética , Diarreia/veterinária , Giardia lamblia/genética , Sistemas Automatizados de Assistência Junto ao Leito/normas , Fatores Etários , Animais , Áustria , Bovinos , Criptosporidiose/diagnóstico , Cryptosporidium parvum/isolamento & purificação , Diarreia/parasitologia , Fezes/parasitologia , Genótipo , Giardia lamblia/isolamento & purificação , Giardíase/diagnóstico , Giardíase/veterinária , Imunoensaio/normas
5.
Eur J Clin Microbiol Infect Dis ; 38(9): 1709-1717, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31302785

RESUMO

To investigate long-term health sequelae of cryptosporidiosis, with especial reference to post-infectious irritable bowel syndrome (PI-IBS). A prospective cohort study was carried out. All patients with laboratory-confirmed, genotyped cryptosporidiosis in Wales, UK, aged between 6 months and 45 years of age, over a 2-year period were contacted. Five hundred and five patients agreed to participate and were asked to complete questionnaires (paper or online) at baseline, 3 and 12 months after diagnosis. The presence/absence of IBS was established using the Rome III criteria for different age groups. Two hundred and five of 505 cases completed questionnaires (40% response rate). At 12 months, over a third of cases reported persistent abdominal pain and diarrhoea, 28% reported joint pain and 26% reported fatigue. At both 3 and 12 months, the proportion reporting fatigue and abdominal pain after Cryptosporidium hominis infection was statistically significantly greater than after C. parvum. Overall, 10% of cases had sufficient symptoms to meet IBS diagnostic criteria. A further 27% met all criteria except 6 months' duration and another 23% had several features of IBS but did not fulfil strict Rome III criteria. There was no significant difference between C. parvum and C. hominis infection with regard to PI-IBS. Post-infectious gastrointestinal dysfunction and fatigue were commonly reported after cryptosporidiosis. Fatigue and abdominal pain were significantly more common after C. hominis compared to C. parvum infection. Around 10% of people had symptoms meriting a formal diagnosis of IBS following cryptosporidiosis. Using age-specific Rome III criteria, children as well as adults were shown to be affected.


Assuntos
Criptosporidiose/complicações , Criptosporidiose/diagnóstico , Síndrome do Intestino Irritável/parasitologia , Dor Abdominal/etiologia , Adolescente , Adulto , Artralgia/etiologia , Criança , Pré-Escolar , Cryptosporidium/genética , Diarreia/parasitologia , Fadiga/etiologia , Feminino , Seguimentos , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Inquéritos e Questionários , Adulto Jovem
6.
Rev Inst Med Trop Sao Paulo ; 61: e28, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31241657

RESUMO

Cryptosporidium spp. is a pathogenic protozoan present in the gastrointestinal tract of several hosts. This protozoan was originally classified as within the Coccidia Class and has recently been reclassified to gregarine based on studies that observed the evolutionary phases from the process of excision and sequencing of the 18S rRNA gene. Molecular biology techniques have become diagnostic tools and have also been used to understand the epidemiology of Cryptosporidium spp., since several species of this genus are very similar morphologically and morphometrically. Molecular techniques have been used in the identification of parasites, at the species and subtypes levels and to study disease transmission. The laboratory diagnosis of human cryptosporidiosis can be made by parasite detection methods, such as optical microscopy, antigens or genetic material detection, as well as serum antibodies raised to Cryptosporidium spp. Molecular methods were developed and allowed, not only an extensive revision of the taxonomy, but also an improvement in the laboratory diagnosis. In Brazil, there are few reports of Cryptosporidium spp. outbreaks in humans and all of them took place in nurseries. A few epidemiological studies developed in Brazil have used molecular methods for the detection of Cryptosporidium spp., as well as genotyping studies of their species and subtypes. The use of real-time PCR, together with microscopy and immunochromatography techniques, would result in a more precise diagnosis of cryptosporidiosis. The analysis of genotypes, subtypes and clonality of Cryptosporidium could be useful to understand and define the prognosis and severity of infections.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , DNA de Protozoário/genética , Fezes/parasitologia , Animais , Brasil/epidemiologia , Criptosporidiose/diagnóstico , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , DNA de Protozoário/análise , Genótipo , Humanos , RNA Ribossômico/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
8.
Am J Trop Med Hyg ; 101(1): 160-163, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31115293

RESUMO

Cryptosporidium is a parasite that causes watery diarrhea among both children and adults. However, because many physicians do not routinely ask for Cryptosporidium diagnostic test, cryptosporidiosis prevalence is likely underestimated. The current study investigated the prevalence of cryptosporidiosis among children admitted to the hospital with gastrointestinal symptoms. Stool sample was collected from each child and subjected to routine microbiological culture. Cryptosporidium presence was tested by three different methods: real-time PCR (RT-PCR), quick antigen, and microscopic examination with acid-fast staining. Each positive specimen was further tested with BioFire FilmArray Multiplex PCR (bioMérieux SA, Marcy-l'Etoile, France) to determine that Cryptosporidium is the only pathogen in the sample. Demographic and epidemiological data were collected from the patients' medical records. Of 291 patient stool samples, nine were positive for Cryptosporidium hominis or Cryptosporidium parvum. The average age of the nine Cryptosporidium-positive cases was 2.3 years, lower than the average age of the study population (4.2 years). Of the positive cases, 66.7% were Arabs and 33.3% were Jews. The main complaint of children with Cryptosporidium was diarrhea. Regarding the laboratory methods for Cryptosporidium identification, of the positive samples, 100% (9/9) were identified by RT-PCR, 88% (8/9) were positive by antigen test, and only 67% (6/9) were positive by microscopic examination with acid-fast staining. Because of the low incidence of Cryptosporidium among patients in our facility, the prevalence of cryptosporidiosis still cannot be established. Nevertheless, among the other pathogens found in stool samples, Cryptosporidium is the second leading cause of hospitalization due to gastrointestinal disease in children in our area.


Assuntos
Criptosporidiose/diagnóstico , Criptosporidiose/epidemiologia , Cryptosporidium/isolamento & purificação , Pré-Escolar , Criptosporidiose/parasitologia , Diarreia/epidemiologia , Diarreia/parasitologia , Fezes/parasitologia , Humanos , Israel/epidemiologia , Prevalência
9.
PLoS One ; 14(4): e0215068, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958837

RESUMO

BACKGROUND: Multiplex molecular panels are relentlessly replacing conventional methods for the detection of enteric pathogens from stool samples in clinical and research laboratories. Here we evaluated four commercial multiplex real-time PCR assays for the detection of Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica. METHODS: The diagnostic performance of the Gastroenteritis/Parasite Panel I (Diagenode), the RIDAGENE Parasitic Stool Panel (R-Biopharm), the Allplex Gastrointestinal Parasite Panel 4 (Seegene) and the FTD Stool Parasites (Fast Track) real-time PCR methods was assessed against a reference panel of 126 well-characterized DNA samples including Cryptosporidium hominis (n = 29), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 47), Entamoeba histolytica (n = 3), other parasite species (n = 20), and apparently healthy subjects (n = 24). PRINCIPAL FINDINGS: Obtained diagnostic sensitivities ranged from 53-88% for Cryptosporidium hominis/parvum, and from 68-100% for G. duodenalis. The R-Biopharm method achieved the best performance for the detection of Cryptosporidium hominis/parvum both in terms of diagnostic sensitivity (87.5%) and detection limit (a 100-fold increase compared to other tests). The Fast Track method was particularly suited for the detection of G. duodenalis, achieving a 100% sensitivity and a detection limit at least 10-fold superior. Detection of E. histolytica was similarly achieved by all compared methods except Diagenode. CONCLUSIONS: Diagnostic performance varied largely depending on the method used and the targeted pathogen species. Factors including test sensitivity/specificity, cost, patient population surveyed, laboratory workflow, and diagnostic algorithm should be carefully considered when choosing the most appropriate multiplex PCR platform.


Assuntos
Criptosporidiose/diagnóstico , Testes Diagnósticos de Rotina/métodos , Diarreia/diagnóstico , Entamebíase/diagnóstico , Fezes/parasitologia , Giardíase/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Bioensaio , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , Diarreia/epidemiologia , Diarreia/parasitologia , Entamoeba histolytica/genética , Entamoeba histolytica/isolamento & purificação , Entamebíase/parasitologia , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Giardíase/parasitologia , Humanos
10.
Parasitol Res ; 118(6): 1937-1942, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30976968

RESUMO

Protozoa of the genus Cryptosporidium are common parasites of domestic and wild animals-mammals, birds, reptiles, and fishes. The invasive forms are thick-walled oocysts, which can be present in water supplies, on fruits, vegetables, or in the soil contaminated with feces. In this work, we describe three cases of middle-aged persons with massive Cryptosporidium hominis infection and chronic diarrhea with no immunological abnormalities and no history of previous travels to tropical countries. The lesions discovered during colonoscopy within the large intestine-cryptitis and the histopathological changes were related to massive cryptosporidiosis. All these statements indicate necessity of parasitological stool examination in cases with chronic diarrhea in which no etiological agents are detected, but not only in HIV positive individuals. Parasite's eradication leads to symptom disappearance as well as improvement of histopathological mucosa alterations.


Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium/isolamento & purificação , Diarreia/diagnóstico , Diarreia/parasitologia , Oocistos/isolamento & purificação , Animais , Criptosporidiose/parasitologia , Cryptosporidium/genética , DNA de Protozoário/genética , Água Potável/parasitologia , Fezes/parasitologia , Feminino , HIV , Infecções por HIV/complicações , Humanos , Intestino Grosso/parasitologia , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Viagem , Abastecimento de Água
11.
Biosens Bioelectron ; 135: 137-144, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31005765

RESUMO

Foodborne illnesses are a major contributor to misery and health challenges in both rich and poor nations. Illnesses from pathogens such as Escherichia coli and Cryptosporidium parvum oocysts account for most of the cases of diarrhea in the world. Many standard methods exist for detecting these pathogens in water. However, these standard methods do not readily translate to the detection of the same pathogens in food. Detection techniques for pathogens in food are often inadequate, due to their inability to completely separate pathogens from food matrices. In this paper, we present a technique to separate and detect both Escherichia coli cells and Cryptosporidium parvum oocysts that have been embedded in ground meat. We achieve this objective by combining enzymatic digestion of the meat, hydrodynamic cavitation to disassemble pathogens from the meat, immunomagnetic separation to purify meat samples and indirect electrochemical detection of the target pathogens. Our use of hydrodynamic cavitation to separate pathogens is compared against an industry standard separation technique. Results indicate that the use of hydrodynamic cavitation amplifies the detection capabilities of our sensing technique and is overall comparable to or better than conventional stomacher sample preparation.


Assuntos
Cryptosporidium parvum/isolamento & purificação , Escherichia coli O157/isolamento & purificação , Análise de Alimentos/métodos , Carne Vermelha/microbiologia , Animais , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/métodos , Bovinos , Criptosporidiose/diagnóstico , Criptosporidiose/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Análise de Alimentos/economia , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/microbiologia , Hidrodinâmica , Separação Imunomagnética/economia , Separação Imunomagnética/métodos , Fatores de Tempo
12.
Artigo em Inglês | MEDLINE | ID: mdl-30886833

RESUMO

Objectives: The potential linkage between Cryptosporidium spp. infection and colorectal human cancer was suggested by limited reports showing higher prevalence of C. parvum and C. hominis in patients with colon cancer. Here we conducted research concerning presence of Cryptosporidium spp. in malignant tissue collected from patients with colorectal cancer. Methods: Cancerous colon tissue samples collected from 145 non-HIV infected patients with colorectal cancer were screened for Cryptosporidium spp. by immunofluorescence antibody test and genus-specific nested polymerase chain reaction followed by sequencing. Results: Screened pathogen was found in cancerous tissue originating from immunocompetent man with colon adenocarcinoma. Genotyping revealed presence of Cryptosporidium meleagridis. The presence of Cryptosporidium life cycle stages (oocysts and endogenous stages) in colon carcinoma tissue was confirmed by genus-specific FITC-labeling. Conclusions: Herein, we report on a C. meleagridis infection of a colon adenocarcinoma in an immunocompetent patient. This is the first report of C. meleagridis infection in the human colon and first evidence of active development of this species in cancer tissue.


Assuntos
Adenocarcinoma/complicações , Neoplasias do Colo/complicações , Criptosporidiose/diagnóstico , Cryptosporidium/isolamento & purificação , Cryptosporidium/classificação , Cryptosporidium/genética , Genótipo , Humanos
14.
Am J Trop Med Hyg ; 100(1): 78-80, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30457100

RESUMO

In high prevalence settings, cryptosporidiosis is commonly implicated as a cause of a gastroenteritis syndrome in the organ transplant population. Stool microscopy is predominant diagnostic modality. Therapeutic options in this group of patients are limited, making their management exceptionally challenging. We describe a case of a renal transplant recipient with cryptosporidiosis confirmed by the stool FilmArray gastrointestinal panel (GIP) nucleic acid-based assay and stool microscopy, describe our institutional experience in diagnosing cryptosporidiosis in a low-prevalence setting, and review the available literature on management of this condition in the organ transplant population. In a low-prevalence setting, the GIP can serve as a rapid screening tool in the diagnosis of cryptosporidiosis.


Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium/isolamento & purificação , Fezes/parasitologia , Transplante de Rim , Adulto , Antiprotozoários/uso terapêutico , Criptosporidiose/tratamento farmacológico , Cryptosporidium/genética , Gerenciamento Clínico , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Prevalência , Singapura
15.
Parasitol Int ; 69: 8-12, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30391553

RESUMO

The infection of neonatal calves with Cryptosporidium parvum can have a huge economic impact because diarrhea caused by the parasite sometimes results in death. A serodiagnostic system will be helpful in the diagnosis of C. parvum infection. CpP23 is commonly used as an antigen for enzyme-linked immunosorbent assay (ELISA); however, some positive sera show low reactivities, as shown in this study. Herein, we focused on three other antigens, CpGP15, CpP2 and CpGP60, in addition to CpP23, to detect C. parvum-specific antibodies in cattle sera. CpP23 and CpGP15 showed substantial ability to discriminate between positive (n = 10) and negative (n = 10) control cattle sera. Unlike our previous report, both the sensitivity and the specificity were 100% when the two antigens were employed for the ELISA. The newly developed ELISA was applied to a total of 344 sera obtained from 9 cattle farms. Two farms among them had suffered from C. parvum infections before, and were regarded as the C. parvum-positive farms. The positive rates of antibodies against CpP23 and CpGP15 in the C. parvum-positive farms were 42.7% and 49.8%, respectively, whereas the positive rate for either of the antigens was 63.0% in the farms. In contrast, 14.3% and 9.8% were positive for CpP23 and CpGP15 in the C. parvum-negative farms, respectively, whereas 18.8% was positive for either of the antigens. This study revealed that the ELISAs employing both of CpP23 and CpGP15 can avoid false-negative results and are useful for monitoring of the C. parvum infection in cattle farms.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doenças dos Bovinos/diagnóstico , Criptosporidiose/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/sangue , Criptosporidiose/sangue , Cryptosporidium parvum/imunologia , Diarreia/parasitologia , Ensaio de Imunoadsorção Enzimática/veterinária , Fazendas , Fezes/parasitologia , Japão , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/veterinária
16.
Diagn Microbiol Infect Dis ; 93(1): 33-36, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30122511

RESUMO

Microscopy is the gold standard for the diagnosis of gastrointestinal parasites but is time-consuming and dependent on operator skills. Rapid diagnostic tests represent alternative methods but most evaluations have been conducted on a limited number of samples preventing their implementation in the clinical setting. We evaluated a new CE-IVD marked immunochromatographic assay (Crypto/Giardia K-SeT®, Coris Bioconcept) for the detection of G. intestinalis and Cryptosporidium spp. in 2 phases (retrospective and prospective) on a set of 482 stool samples including rare Cryptosporidium species. Besides G. intestinalis, this test could represent a rapid and reliable alternative to the modified Ziehl-Neelsen staining for the diagnosis of cryptosporidiosis (sensitivity/specificity were 89.2%/99.3% and 86.7%/100% for G. intestinalis and Cryptosporidium resp.), reducing diagnostic delays. Such strategy would also be time-saving by avoiding wet mount microscopy and concentrations steps, being particularly appropriate for laboratories having little expertise in microscopy or not able to implement molecular diagnostic methods.


Assuntos
Criptosporidiose/diagnóstico , Fezes/parasitologia , Giardíase/diagnóstico , Imunoensaio , Testes Imediatos , Animais , Antígenos de Protozoários/análise , Cryptosporidium/imunologia , Cryptosporidium/isolamento & purificação , Testes Diagnósticos de Rotina , Giardia/imunologia , Giardia/isolamento & purificação , Humanos , Enteropatias Parasitárias/diagnóstico , Estudos Prospectivos , Estudos Retrospectivos , Sensibilidade e Especificidade
17.
Acta Biochim Biophys Sin (Shanghai) ; 51(1): 104-111, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30544221

RESUMO

Cryptosporidium parvum is an important zoonotic parasite that causes significant economic loss in the animal husbandry industry, especially the cattle industry. As there is no specific vaccine or drug against Cryptosporidium, a rapid and accurate method for the detection of C. parvum is of great significance. In this study, colloidal gold strips were developed based on Cryptosporidium parvum virus 1 (CSpV1) for the detection of C. parvum infection in cattle fecal samples. The colloidal gold solution was prepared by reducing trisodium citrate and the CSpV1 #5 monoclonal antibody was labeled with colloidal gold. A polyclonal antibody against the CSpV1 capsid protein and an anti-mouse IgG antibody were coated on the colloidal gold strips for use in the test and control lines, respectively. Our results showed that the detection sensitivity in fecal samples was up to a 1:64 dilution. There was no cross-reaction with Cryptosporidium andersoni or Giardia in the fecal samples. The different preservation conditions (room temperature, 4°C, and 37°C) and preservation time (7, 30, 60, and 90 days) were analyzed. The data showed that the strips could be preserved for 90 days at 4°C and for 60 days at room temperature or 37°C. The colloidal gold strips were used to detect the samples of 120 clinical fecal in Changchun, China. The results indicated that the rate of a positive test was 5% (6/120). This study provides a rapid and accurate method for detecting C. parvum infection in cattle and humans.


Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Fezes/parasitologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/virologia , Criptosporidiose/diagnóstico , Criptosporidiose/virologia , Cryptosporidium parvum/virologia , Fezes/virologia , Coloide de Ouro/química , Humanos , Concentração de Íons de Hidrogênio , Vírus de RNA/imunologia , Vírus de RNA/fisiologia , Sensibilidade e Especificidade , Zoonoses/diagnóstico , Zoonoses/parasitologia , Zoonoses/virologia
18.
Am J Trop Med Hyg ; 99(6): 1613-1618, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30398140

RESUMO

Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards. DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction. For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.


Assuntos
Cryptosporidium/isolamento & purificação , DNA de Protozoário/genética , Entamoeba histolytica/isolamento & purificação , Giardia lamblia/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/normas , Fitas Reagentes/análise , Bangladesh/epidemiologia , Criança , Pré-Escolar , Criptosporidiose/diagnóstico , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/classificação , Cryptosporidium/genética , DNA de Protozoário/isolamento & purificação , Entamoeba histolytica/classificação , Entamoeba histolytica/genética , Entamebíase/diagnóstico , Entamebíase/epidemiologia , Entamebíase/parasitologia , Fezes/parasitologia , Giardia lamblia/classificação , Giardia lamblia/genética , Giardíase/diagnóstico , Giardíase/epidemiologia , Giardíase/parasitologia , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Papel , Carga Parasitária , Sensibilidade e Especificidade , Extração em Fase Sólida/métodos
19.
Korean J Parasitol ; 56(5): 419-427, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30419727

RESUMO

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAMTM, HEXTM, Cy5TM, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×103 copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.


Assuntos
Bacteriófago T4 , Criptosporidiose/diagnóstico , Criptosporidiose/parasitologia , Cryptosporidium parvum/isolamento & purificação , Cyclospora/isolamento & purificação , Ciclosporíase/diagnóstico , Ciclosporíase/parasitologia , Diarreia/parasitologia , Fezes/parasitologia , Giardia lamblia/isolamento & purificação , Giardíase/diagnóstico , Giardíase/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real , Cryptosporidium parvum/genética , Cyclospora/genética , DNA de Protozoário , Giardia lamblia/genética , Humanos , Viagem
20.
Parasite ; 25: 48, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30230444

RESUMO

Although microscopic examination of stool samples remains the reference method for the diagnosis of intestinal protozoal infections, these techniques are time-consuming and require operators who are experienced and well trained. Molecular biology seems to offer performances at least equivalent in terms of sensitivity and specificity for certain parasites. This study aimed to compare three multiplex PCR assays on 93 prospectively collected positive stools (prospective cohort) and a panel of 12 more Cryptosporidium-positive samples (Cryptosporidium panel). On the prospective cohort, the sensitivity was 89%, 64% and 41% for Giardia sp. detection for BD MaxTM, G-DiaParaTM and RIDA®GENE, respectively and 75%, 100% and 100% for C. parvum/hominis detection. The sensitivity of the RIDA®GENE assay for all Cryptosporidium species was 100%, and for D. fragilis 71%. All the techniques obtained the same results for E. histolytica detection, with one positive sample. All species in the Cryptosporidium panel were identified by the RIDA®GENE PCR. The BD MaxTM and G-DiaParaTM assays detected only C. parvum/hominis with the exception of one positive sample for C. meleagridis. No assay showed satisfactory results for all parasites simultaneously, and the DNA extraction seems to be the critical step. More studies are needed to standardize this procedure.


Assuntos
Enteropatias Parasitárias/diagnóstico , Intestinos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Protozoários/diagnóstico , Bioensaio , Criptosporidiose/diagnóstico , Criptosporidiose/parasitologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , Entamoeba histolytica/genética , Entamoeba histolytica/isolamento & purificação , Entamebíase/diagnóstico , Entamebíase/parasitologia , Fezes/parasitologia , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Giardíase/diagnóstico , Giardíase/parasitologia , Humanos , Enteropatias Parasitárias/parasitologia , Técnicas de Diagnóstico Molecular/instrumentação , Estudos Prospectivos , Infecções por Protozoários/parasitologia , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
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