AIM： To evaluate the usefulness of stooI-PCR test for diagnosis of Helicobacter pylori （H pylon） infection in pediatric populations. METHODS： Based on endoscopic features （including nodular gastritis, erosive duo...AIM： To evaluate the usefulness of stooI-PCR test for diagnosis of Helicobacter pylori （H pylon） infection in pediatric populations. METHODS： Based on endoscopic features （including nodular gastritis, erosive duodenitis and ulcer） and/or a positive rapid urease test （RUT） obtained during endoscopy, 28 children from a group of children admitted to the Children＇s Medical Center of Tehran for persistent upper gastrointestinal problems were selected to compare biopsy-based tests with stool- PCR. Their gastric activity and bacterial density were graded by the updated Sydney system, and their first stool after endoscopy was stored at -70℃. Biopsies were cultured on modified campy-blood agar plates and identified by gram-staining, biochemical tests, and PCR. Two methods of phenol-chloroform and boiling were used for DNA extraction from H pylori isolates. Isolation of DNA from stool was performed using a stool DNA extraction kit （Bioneer Inc, Korea）. PCR was performed using primers for detection of vacA, cagA, and 16srRNA genes in both isolates and stool. RESULTS： Sixteen out of 28 child patients （57%） were classified as H pylori positive by biopsy-based tests, of which 11 （39%） were also positive by stool- PCR. Sensitivity and specificity of stool-PCR was 62.5% and 92.3% respectively. H pylori was observed in histological sections for 10 out of 11 stool-positive patients. Association was observed between higher score of H pylori in histology and positivity of stool- PCR. Also association was observed between the more severe form of gastritis and a positive stool-PCR. CONCLUSION： Association between higher score of H pylori in histology and a positive stool-PCR make it a very useful test for detection of H pylori active infection in children. We also suggest that a simple stool-PCR method can be a useful test for detection of Hpylori virulence genes in stool.展开更多
Colorectal carcinoma(CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves APC/β-catenin characterized by c...Colorectal carcinoma(CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves APC/β-catenin characterized by chromosomal instability resulting in the accumulation of mutations. The second pathway is characterized by lesions in DNA mismatch repair genes.Aberrant DNA methylation in selected gene promoters has emerged as a new epigenetic pathway in CRC development. CRC screening is the most efficient strategy to reduce death. Specific DNA methylation events occur in multistep carcinogenesis.Epigenetic gene silencing is a causative factor of CRC development. DNA methylations have been extensively examined in stool from CRC and precursor lesions. Many methylated genes have been described in CRC and adenoma, although no definite DNA methylation biomarkers panel has been established. Multiple DNA methylation biomarkers, including secreted frizzled-related protein 2, secreted frizzled-related protein 1, tissue factor pathway inhibitor 2, vimentin, and methylguanine DNA methyltransferase, have been further investigated, and observations have revealed that DNA methylation biomarkers exhibit with high sensitivity and specificity. These markers may also be used to diagnose CRC and adenoma in early stages. Real time polymerase chain reaction(q PCR) is sensitive, scalable, specific, reliable, time saving, and cost effective. Stool exfoliated markers provide advantages, including sensitivity and specificity. A stool q PCR methylation test may also be an enhanced tool for CRC and adenoma screening.展开更多
Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected...Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.展开更多
Introduction and Objective: The genus Bifidobacterium can generally be found in quantity in the habitats such as human and animal gastrointestinal tract, dental caries, vagina and oral cavity. The aim of this study wa...Introduction and Objective: The genus Bifidobacterium can generally be found in quantity in the habitats such as human and animal gastrointestinal tract, dental caries, vagina and oral cavity. The aim of this study was to isolate Bifidobacterium from stool and determine their inhibitory effect against some pathogens. Materials and Methods: 130 samples were collected by wet swabs and kept in sterile tubes containing MRS broth media. And Bifidobacterium isolated from stool was enriched in Man-Rogosa-Sharpe medium (MRS) broth and isolated by growing on MRS agar medium and characterized by phenotypic characteristics and PCR technique at genus and species levels. The antimicrobial substance was extracted from ethyl acetate solvent and the antimicrobial activity against some pathogenic bacteria, such as Salmonella typhi and Shigella sonnei, were investigated. Results: Eleven Bifidobacterium bifidum and four Bifidobacterium adolescentis, which were isolated from fresh stool, were identified by PCR. Antimicrobial substance from MRS broth medium was extracted. This antimicrobial compound showed a potent inhibitory activity against four tested bacteria. These bacteria produced acetic acid and lactic acid as inhibitory substances that were different from bacteriocins. Conclusion: Fresh stool may be used as a source of antimicrobial lactic acids bacteria, Bifidobacterium bifidum and adolescentis as two probiotics can establish themselves in gut and urogenital tract to prevent the human body from adverse effects of pathogens.展开更多
Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of...Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.展开更多
文摘AIM： To evaluate the usefulness of stooI-PCR test for diagnosis of Helicobacter pylori （H pylon） infection in pediatric populations. METHODS： Based on endoscopic features （including nodular gastritis, erosive duodenitis and ulcer） and/or a positive rapid urease test （RUT） obtained during endoscopy, 28 children from a group of children admitted to the Children＇s Medical Center of Tehran for persistent upper gastrointestinal problems were selected to compare biopsy-based tests with stool- PCR. Their gastric activity and bacterial density were graded by the updated Sydney system, and their first stool after endoscopy was stored at -70℃. Biopsies were cultured on modified campy-blood agar plates and identified by gram-staining, biochemical tests, and PCR. Two methods of phenol-chloroform and boiling were used for DNA extraction from H pylori isolates. Isolation of DNA from stool was performed using a stool DNA extraction kit （Bioneer Inc, Korea）. PCR was performed using primers for detection of vacA, cagA, and 16srRNA genes in both isolates and stool. RESULTS： Sixteen out of 28 child patients （57%） were classified as H pylori positive by biopsy-based tests, of which 11 （39%） were also positive by stool- PCR. Sensitivity and specificity of stool-PCR was 62.5% and 92.3% respectively. H pylori was observed in histological sections for 10 out of 11 stool-positive patients. Association was observed between higher score of H pylori in histology and positivity of stool- PCR. Also association was observed between the more severe form of gastritis and a positive stool-PCR. CONCLUSION： Association between higher score of H pylori in histology and a positive stool-PCR make it a very useful test for detection of H pylori active infection in children. We also suggest that a simple stool-PCR method can be a useful test for detection of Hpylori virulence genes in stool.
文摘Colorectal carcinoma(CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves APC/β-catenin characterized by chromosomal instability resulting in the accumulation of mutations. The second pathway is characterized by lesions in DNA mismatch repair genes.Aberrant DNA methylation in selected gene promoters has emerged as a new epigenetic pathway in CRC development. CRC screening is the most efficient strategy to reduce death. Specific DNA methylation events occur in multistep carcinogenesis.Epigenetic gene silencing is a causative factor of CRC development. DNA methylations have been extensively examined in stool from CRC and precursor lesions. Many methylated genes have been described in CRC and adenoma, although no definite DNA methylation biomarkers panel has been established. Multiple DNA methylation biomarkers, including secreted frizzled-related protein 2, secreted frizzled-related protein 1, tissue factor pathway inhibitor 2, vimentin, and methylguanine DNA methyltransferase, have been further investigated, and observations have revealed that DNA methylation biomarkers exhibit with high sensitivity and specificity. These markers may also be used to diagnose CRC and adenoma in early stages. Real time polymerase chain reaction(q PCR) is sensitive, scalable, specific, reliable, time saving, and cost effective. Stool exfoliated markers provide advantages, including sensitivity and specificity. A stool q PCR methylation test may also be an enhanced tool for CRC and adenoma screening.
基金supported by a research grant from the Office of the Vice-Chancellor for Research and Development,University of the Philippines-Diliman(Grant No.101007 PNSE)to W.L.R.and H.J.S
文摘Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.
文摘Introduction and Objective: The genus Bifidobacterium can generally be found in quantity in the habitats such as human and animal gastrointestinal tract, dental caries, vagina and oral cavity. The aim of this study was to isolate Bifidobacterium from stool and determine their inhibitory effect against some pathogens. Materials and Methods: 130 samples were collected by wet swabs and kept in sterile tubes containing MRS broth media. And Bifidobacterium isolated from stool was enriched in Man-Rogosa-Sharpe medium (MRS) broth and isolated by growing on MRS agar medium and characterized by phenotypic characteristics and PCR technique at genus and species levels. The antimicrobial substance was extracted from ethyl acetate solvent and the antimicrobial activity against some pathogenic bacteria, such as Salmonella typhi and Shigella sonnei, were investigated. Results: Eleven Bifidobacterium bifidum and four Bifidobacterium adolescentis, which were isolated from fresh stool, were identified by PCR. Antimicrobial substance from MRS broth medium was extracted. This antimicrobial compound showed a potent inhibitory activity against four tested bacteria. These bacteria produced acetic acid and lactic acid as inhibitory substances that were different from bacteriocins. Conclusion: Fresh stool may be used as a source of antimicrobial lactic acids bacteria, Bifidobacterium bifidum and adolescentis as two probiotics can establish themselves in gut and urogenital tract to prevent the human body from adverse effects of pathogens.
文摘Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.
