Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage
Background Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool‐based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR...
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| Veröffentlicht in: | Helicobacter (Cambridge, Mass.) Mass.), 2016-08, Vol.21 (4), p.325-333 |
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| creator | Talarico, Sarah Safaeian, Mahboobeh Gonzalez, Paula Hildesheim, Allan Herrero, Rolando Porras, Carolina Cortes, Bernal Larson, Ann Fang, Ferric C. Salama, Nina R. |
| description | Background
Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool‐based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning.
Materials and Methods
Stool‐based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen‐tested stool samples collected at a US hospital.
Results
The stool‐based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool‐based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody‐positive individuals and four (16%) of 25 stools from CagA antibody‐negative individuals from Costa Rica. All 26 of these samples had a Western‐type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool.
Conclusions
The stool‐based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR‐based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool‐based ddPCR assays will facilitate future population‐based epidemiologic studies of this important human pathogen. |
| doi_str_mv | 10.1111/hel.12289 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4909588</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1808650834</sourcerecordid><originalsourceid>FETCH-LOGICAL-c5849-82fa06afca9ae2606acd653e1159357e2394fe86e4b9f38f930edb89fca377443</originalsourceid><addsrcrecordid>eNp1ks1uEzEURkcIREthwQsgS2xgMa1nPD_2BqkkJUFEhRQIEhvL8dyZuHXsYHtS8mY8Hk7SRoCEN76Szz2-lr8keZ7h0yyuswXo0yzPKXuQHGdlTtKS1PRhrDElaUEoO0qeeH-NMS5JwR4nR3lVVXVeZMfJr2kvTFBBBLUGNIQAMihrkDANGoGxYbNSpkO2RWPQStq5kAEcWm20dQq1zi7R52CtRr3fckNnVxoCGqouOjX6NLhCV7AGoT2aCafETq4MervzqIhMrGg8CgsR0MA6B1oE8OhWhQWSojtHM-V6DUbCdh5AA-FiWwdPk0dttMKzu_0k-fru4stgnE4-jt4PziepLGnBUpq3AleilYIJyKtYyqYqCWRZyUhZQ05Y0QKtoJizltCWEQzNnLLYQOq6KMhJ8mbvXfXzJTQSTHBC85VTS-E23ArF_z4xasE7u-YFw6ykNApe3Qmc_dGDD3ypvASthQHbe55RTKsy_tT2rpf_oNe2dyY-b0vlGcMVZZF6vaeks947aA_DZJhv88BjHvguD5F98ef0B_I-ABE42wO3SsPm_yY-vpjcK9N9h_IBfh46hLvhVU3qkn-7HPHpaHr54Tub8Bn5DSkw0qQ</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1802190689</pqid></control><display><type>article</type><title>Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage</title><source>Wiley-Blackwell Journals</source><source>MEDLINE</source><creator>Talarico, Sarah ; Safaeian, Mahboobeh ; Gonzalez, Paula ; Hildesheim, Allan ; Herrero, Rolando ; Porras, Carolina ; Cortes, Bernal ; Larson, Ann ; Fang, Ferric C. ; Salama, Nina R.</creator><creatorcontrib>Talarico, Sarah ; Safaeian, Mahboobeh ; Gonzalez, Paula ; Hildesheim, Allan ; Herrero, Rolando ; Porras, Carolina ; Cortes, Bernal ; Larson, Ann ; Fang, Ferric C. ; Salama, Nina R.</creatorcontrib><description>Background
Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool‐based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning.
Materials and Methods
Stool‐based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen‐tested stool samples collected at a US hospital.
Results
The stool‐based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool‐based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody‐positive individuals and four (16%) of 25 stools from CagA antibody‐negative individuals from Costa Rica. All 26 of these samples had a Western‐type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool.
Conclusions
The stool‐based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR‐based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool‐based ddPCR assays will facilitate future population‐based epidemiologic studies of this important human pathogen.</description><identifier>ISSN: 1083-4389</identifier><identifier>EISSN: 1523-5378</identifier><identifier>DOI: 10.1111/hel.12289</identifier><identifier>PMID: 26667241</identifier><language>eng</language><publisher>England: Blackwell Publishing Ltd</publisher><subject>Adolescent ; Adult ; Aged ; Antigens ; Antigens, Bacterial - analysis ; Antigens, Bacterial - genetics ; Bacteria ; bacterial load ; Bacterial Load - methods ; Bacterial Proteins - analysis ; Bacterial Proteins - genetics ; cagA gene ; Child ; Child, Preschool ; Costa Rica ; Droplet digital PCR ; Feces - microbiology ; Female ; Genotyping Techniques - methods ; H. pylori ; Helicobacter Infections - microbiology ; Helicobacter pylori ; Helicobacter pylori - genetics ; Helicobacter pylori - isolation & purification ; Humans ; Infant ; Male ; Middle Aged ; Polymerase Chain Reaction - methods ; RNA, Ribosomal, 16S - genetics ; Sensitivity and Specificity ; stool-based assay ; Ulcers ; United States ; Virulence Factors - analysis ; Virulence Factors - genetics ; Young Adult</subject><ispartof>Helicobacter (Cambridge, Mass.), 2016-08, Vol.21 (4), p.325-333</ispartof><rights>2015 John Wiley & Sons Ltd</rights><rights>2015 John Wiley & Sons Ltd.</rights><rights>Copyright © 2016 John Wiley & Sons Ltd</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5849-82fa06afca9ae2606acd653e1159357e2394fe86e4b9f38f930edb89fca377443</citedby><cites>FETCH-LOGICAL-c5849-82fa06afca9ae2606acd653e1159357e2394fe86e4b9f38f930edb89fca377443</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fhel.12289$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fhel.12289$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>230,314,780,784,885,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26667241$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Talarico, Sarah</creatorcontrib><creatorcontrib>Safaeian, Mahboobeh</creatorcontrib><creatorcontrib>Gonzalez, Paula</creatorcontrib><creatorcontrib>Hildesheim, Allan</creatorcontrib><creatorcontrib>Herrero, Rolando</creatorcontrib><creatorcontrib>Porras, Carolina</creatorcontrib><creatorcontrib>Cortes, Bernal</creatorcontrib><creatorcontrib>Larson, Ann</creatorcontrib><creatorcontrib>Fang, Ferric C.</creatorcontrib><creatorcontrib>Salama, Nina R.</creatorcontrib><title>Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage</title><title>Helicobacter (Cambridge, Mass.)</title><addtitle>Helicobacter</addtitle><description>Background
Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool‐based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning.
Materials and Methods
Stool‐based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen‐tested stool samples collected at a US hospital.
Results
The stool‐based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool‐based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody‐positive individuals and four (16%) of 25 stools from CagA antibody‐negative individuals from Costa Rica. All 26 of these samples had a Western‐type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool.
Conclusions
The stool‐based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR‐based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool‐based ddPCR assays will facilitate future population‐based epidemiologic studies of this important human pathogen.</description><subject>Adolescent</subject><subject>Adult</subject><subject>Aged</subject><subject>Antigens</subject><subject>Antigens, Bacterial - analysis</subject><subject>Antigens, Bacterial - genetics</subject><subject>Bacteria</subject><subject>bacterial load</subject><subject>Bacterial Load - methods</subject><subject>Bacterial Proteins - analysis</subject><subject>Bacterial Proteins - genetics</subject><subject>cagA gene</subject><subject>Child</subject><subject>Child, Preschool</subject><subject>Costa Rica</subject><subject>Droplet digital PCR</subject><subject>Feces - microbiology</subject><subject>Female</subject><subject>Genotyping Techniques - methods</subject><subject>H. pylori</subject><subject>Helicobacter Infections - microbiology</subject><subject>Helicobacter pylori</subject><subject>Helicobacter pylori - genetics</subject><subject>Helicobacter pylori - isolation & purification</subject><subject>Humans</subject><subject>Infant</subject><subject>Male</subject><subject>Middle Aged</subject><subject>Polymerase Chain Reaction - methods</subject><subject>RNA, Ribosomal, 16S - genetics</subject><subject>Sensitivity and Specificity</subject><subject>stool-based assay</subject><subject>Ulcers</subject><subject>United States</subject><subject>Virulence Factors - analysis</subject><subject>Virulence Factors - genetics</subject><subject>Young Adult</subject><issn>1083-4389</issn><issn>1523-5378</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1ks1uEzEURkcIREthwQsgS2xgMa1nPD_2BqkkJUFEhRQIEhvL8dyZuHXsYHtS8mY8Hk7SRoCEN76Szz2-lr8keZ7h0yyuswXo0yzPKXuQHGdlTtKS1PRhrDElaUEoO0qeeH-NMS5JwR4nR3lVVXVeZMfJr2kvTFBBBLUGNIQAMihrkDANGoGxYbNSpkO2RWPQStq5kAEcWm20dQq1zi7R52CtRr3fckNnVxoCGqouOjX6NLhCV7AGoT2aCafETq4MervzqIhMrGg8CgsR0MA6B1oE8OhWhQWSojtHM-V6DUbCdh5AA-FiWwdPk0dttMKzu_0k-fru4stgnE4-jt4PziepLGnBUpq3AleilYIJyKtYyqYqCWRZyUhZQ05Y0QKtoJizltCWEQzNnLLYQOq6KMhJ8mbvXfXzJTQSTHBC85VTS-E23ArF_z4xasE7u-YFw6ykNApe3Qmc_dGDD3ypvASthQHbe55RTKsy_tT2rpf_oNe2dyY-b0vlGcMVZZF6vaeks947aA_DZJhv88BjHvguD5F98ef0B_I-ABE42wO3SsPm_yY-vpjcK9N9h_IBfh46hLvhVU3qkn-7HPHpaHr54Tub8Bn5DSkw0qQ</recordid><startdate>201608</startdate><enddate>201608</enddate><creator>Talarico, Sarah</creator><creator>Safaeian, Mahboobeh</creator><creator>Gonzalez, Paula</creator><creator>Hildesheim, Allan</creator><creator>Herrero, Rolando</creator><creator>Porras, Carolina</creator><creator>Cortes, Bernal</creator><creator>Larson, Ann</creator><creator>Fang, Ferric C.</creator><creator>Salama, Nina R.</creator><general>Blackwell Publishing Ltd</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>C1K</scope><scope>K9.</scope><scope>5PM</scope></search><sort><creationdate>201608</creationdate><title>Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage</title><author>Talarico, Sarah ; Safaeian, Mahboobeh ; Gonzalez, Paula ; Hildesheim, Allan ; Herrero, Rolando ; Porras, Carolina ; Cortes, Bernal ; Larson, Ann ; Fang, Ferric C. ; Salama, Nina R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5849-82fa06afca9ae2606acd653e1159357e2394fe86e4b9f38f930edb89fca377443</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Adolescent</topic><topic>Adult</topic><topic>Aged</topic><topic>Antigens</topic><topic>Antigens, Bacterial - analysis</topic><topic>Antigens, Bacterial - genetics</topic><topic>Bacteria</topic><topic>bacterial load</topic><topic>Bacterial Load - methods</topic><topic>Bacterial Proteins - analysis</topic><topic>Bacterial Proteins - genetics</topic><topic>cagA gene</topic><topic>Child</topic><topic>Child, Preschool</topic><topic>Costa Rica</topic><topic>Droplet digital PCR</topic><topic>Feces - microbiology</topic><topic>Female</topic><topic>Genotyping Techniques - methods</topic><topic>H. pylori</topic><topic>Helicobacter Infections - microbiology</topic><topic>Helicobacter pylori</topic><topic>Helicobacter pylori - genetics</topic><topic>Helicobacter pylori - isolation & purification</topic><topic>Humans</topic><topic>Infant</topic><topic>Male</topic><topic>Middle Aged</topic><topic>Polymerase Chain Reaction - methods</topic><topic>RNA, Ribosomal, 16S - genetics</topic><topic>Sensitivity and Specificity</topic><topic>stool-based assay</topic><topic>Ulcers</topic><topic>United States</topic><topic>Virulence Factors - analysis</topic><topic>Virulence Factors - genetics</topic><topic>Young Adult</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Talarico, Sarah</creatorcontrib><creatorcontrib>Safaeian, Mahboobeh</creatorcontrib><creatorcontrib>Gonzalez, Paula</creatorcontrib><creatorcontrib>Hildesheim, Allan</creatorcontrib><creatorcontrib>Herrero, Rolando</creatorcontrib><creatorcontrib>Porras, Carolina</creatorcontrib><creatorcontrib>Cortes, Bernal</creatorcontrib><creatorcontrib>Larson, Ann</creatorcontrib><creatorcontrib>Fang, Ferric C.</creatorcontrib><creatorcontrib>Salama, Nina R.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Helicobacter (Cambridge, Mass.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Talarico, Sarah</au><au>Safaeian, Mahboobeh</au><au>Gonzalez, Paula</au><au>Hildesheim, Allan</au><au>Herrero, Rolando</au><au>Porras, Carolina</au><au>Cortes, Bernal</au><au>Larson, Ann</au><au>Fang, Ferric C.</au><au>Salama, Nina R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage</atitle><jtitle>Helicobacter (Cambridge, Mass.)</jtitle><addtitle>Helicobacter</addtitle><date>2016-08</date><risdate>2016</risdate><volume>21</volume><issue>4</issue><spage>325</spage><epage>333</epage><pages>325-333</pages><issn>1083-4389</issn><eissn>1523-5378</eissn><abstract>Background
Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool‐based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning.
Materials and Methods
Stool‐based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen‐tested stool samples collected at a US hospital.
Results
The stool‐based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool‐based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody‐positive individuals and four (16%) of 25 stools from CagA antibody‐negative individuals from Costa Rica. All 26 of these samples had a Western‐type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool.
Conclusions
The stool‐based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR‐based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool‐based ddPCR assays will facilitate future population‐based epidemiologic studies of this important human pathogen.</abstract><cop>England</cop><pub>Blackwell Publishing Ltd</pub><pmid>26667241</pmid><doi>10.1111/hel.12289</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Helicobacter (Cambridge, Mass.), 2016-08, Vol.21 (4), p.325-333 |
| issn | 1083-4389 1523-5378 |
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| source | Wiley-Blackwell Journals; MEDLINE |
| subjects | Adolescent Adult Aged Antigens Antigens, Bacterial - analysis Antigens, Bacterial - genetics Bacteria bacterial load Bacterial Load - methods Bacterial Proteins - analysis Bacterial Proteins - genetics cagA gene Child Child, Preschool Costa Rica Droplet digital PCR Feces - microbiology Female Genotyping Techniques - methods H. pylori Helicobacter Infections - microbiology Helicobacter pylori Helicobacter pylori - genetics Helicobacter pylori - isolation & purification Humans Infant Male Middle Aged Polymerase Chain Reaction - methods RNA, Ribosomal, 16S - genetics Sensitivity and Specificity stool-based assay Ulcers United States Virulence Factors - analysis Virulence Factors - genetics Young Adult |
| title | Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage |
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