Diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance by fluorescence in situ hybridization from formalin-fixed, paraffin-embedded gastric biopsy specimens

A reliable diagnostic test for Helicobacter pylori is important in clinical practice and research. The ideal diagnostic test for H. pylori should be sensitive, specific, and cost-effective. Helicobacter pylori resistance to clarithromycin is a common reason for failure of eradication therapy. The aim of this study was to evaluate the fluorescent in situ hybridization (FISH) method to detect H. pylori and determine clarithromycin resistance in formalin-fixed, paraffin-embedded gastric biopsy specimens. One hundred seventeen gastric biopsy specimens from patients with dyspepsia were examined for the presence of H. pylori by conventional culture, FISH, and histopathological methods. A set of fluorescent-labeled oligonucleotide probes binding to either H. pylori 16S rRNA or 23S rRNA sequences were used for FISH analysis. Phenotypic antibiotic susceptibilities of the isolates were tested using the Epsilometer test method (E test). Helicobacter pylori was detected in 70 of 117 biopsy specimens by histopathological examination and FISH, whereas it was detected in 47 specimens by culturing. Histopathology and FISH techniques failed to identify H. pylori in 1 biopsy sample isolated by culture. Clarithromycin resistance was found in 11 of 46 H. pylori isolates using the E test method. All of the phenotypic resistance measurements of isolates were correlated with genotypic clarithromycin resistance. Eleven clarithromycin-resistant strains were identified by FISH. The diagnosis of H. pylori infection and the determination of clarithromycin resistance in formalin-fixed, paraffin-embedded specimens using FISH is promising because it provides a rapid, reliable, and culture-independent diagnosis.Key words: Helicobacter pylori, clarithromycin resistance, FISH.

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Detection of Helicobacter pylori and Determination of Clarithromycin Susceptibility Using Formalin-Fixed, Paraffin-Embedded Gastric Biopsy Specimens by Fluorescence In Situ Hybridization

Helicobacter ◽

10.1111/j.1523-5378.2007.00483.x ◽

2007 ◽

Vol 12 (2) ◽

pp. 136-141 ◽

Cited By ~ 20

Author(s):

Özlem Yilmaz ◽

Ebru Demiray ◽

Sait Tümer ◽

O?uz Altungöz ◽

Kutsal Yörüko?lu ◽

...

Keyword(s):

Helicobacter Pylori ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Gastric Biopsy ◽

Biopsy Specimens ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Helicobacter pylori Mutations Detected by Next-Generation Sequencing in Formalin-Fixed, Paraffin-Embedded Gastric Biopsy Specimens Are Associated with Treatment Failure

Journal of Clinical Microbiology ◽

10.1128/jcm.01834-18 ◽

2019 ◽

Vol 57 (7) ◽

Cited By ~ 5

Author(s):

Behtash G. Nezami ◽

Mehul Jani ◽

David Alouani ◽

Daniel D. Rhoads ◽

Navid Sadri

Keyword(s):

Treatment Failure ◽

Gastric Biopsy ◽

23S Rrna ◽

Content Type ◽

Biopsy Specimens ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

H Pylori ◽

Risk Of Treatment ◽

Formalin Fixed

ABSTRACT Helicobacter pylori antibiotic resistance is widespread and increasing worldwide. Routine detection of H. pylori mutations that invoke antimicrobial resistance may be a useful approach to guide antimicrobial therapy and possibly avert treatment failure. In this study, formalin-fixed, paraffin-embedded (FFPE) gastric biopsy specimens from a cohort of individuals from northern Ohio in the United States were examined using a next-generation sequencing (NGS) assay to detect H. pylori mutations that are known to confer resistance to clarithromycin, levofloxacin, and tetracycline. From January 2016 to January 2017, 133 H. pylori-infected gastric biopsy specimens were identified histologically and subsequently analyzed by NGS to detect mutations in gyrA, 23S rRNA, and 16S rRNA genes. The method successfully detected H. pylori in 126 of 133 cases (95% sensitivity). Mutations conferring resistance were present in 92 cases (73%), including 63 cases with one mutation (50%) and 29 cases with mutations in multiple genes (23%). Treatment outcomes were available in 58 cases. Sixteen of the 58 cases failed therapy (28%). Therapy failure correlated with the number of mutated genes: no failure in cases with no mutations (0/15), 19% (5/27) failure in cases with one gene mutation, and 69% (11/16) failure in cases with more than one mutated gene. Common 23S rRNA mutations (A2142G or A2413G) were present in 88% (14/16) of failed cases as opposed to in only 10% (4/42) of eradicated cases (P < 0.001). This NGS assay can be used on remnant specimens collected during standard-of-care testing to detect mutations that correlate with increased risk of treatment failure. A prospective study is needed to determine if the risk of treatment failure can be decreased by using this assay to guide antibiotic therapy.

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Fast and Accurate Quantitative Detection of Helicobacter pylori and Identification of Clarithromycin Resistance Mutations in H. pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.41.10.4573-4577.2003 ◽

2003 ◽

Vol 41 (10) ◽

pp. 4573-4577 ◽

Cited By ~ 50

Author(s):

C. Lascols ◽

D. Lamarque ◽

J.-M. Costa ◽

C. Copie-Bergman ◽

J.-M. Le Glaunec ◽

...

Keyword(s):

Helicobacter Pylori ◽

Real Time ◽

Real Time Pcr ◽

Gastric Biopsy ◽

Quantitative Detection ◽

Resistance Mutations ◽

Biopsy Specimens ◽

Clarithromycin Resistance ◽

H Pylori

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Determination of Helicobacter pylori Virulence Genes in Gastric Biopsies by PCR

ISRN Gastroenterology ◽

10.1155/2013/606258 ◽

2013 ◽

Vol 2013 ◽

pp. 1-4 ◽

Cited By ~ 8

Author(s):

Tamer Essawi ◽

Wail Hammoudeh ◽

Israr Sabri ◽

Walid Sweidan ◽

Mohammad A. Farraj

Keyword(s):

Helicobacter Pylori ◽

Virulence Genes ◽

Gastric Biopsy ◽

Strong Indication ◽

Specific Primers ◽

Biopsy Specimens ◽

Pcr Identification ◽

Gastric Biopsies ◽

H Pylori

Aim. The aim of this study was to identify the presence of H. pylori in biopsy specimens from symptomatic patients by PCR. In addition, the rate of cagA, vacA, iceA1, and iceA2 virulence genes was determined. Materials and Methods. One hundred antral gastric biopsy specimens were collected during endoscopy from patients suffering from gastroduodenal symptoms. The samples were collected by the gastroenterologists in their own clinics in Ramallah, Palestine. DNA was extracted from the biopsies and subsequently used for PCR identification of H. pylori and the virulence genes using specific primers. Results. The rate of positive H. pylori in the collected biopsies was 44%. The rates of the virulence genes in this sample: cagA, vacA, iceA1, and iceA2 were 65.9%, 40.9%, 63.6%, and 84.1%, respectively. Conclusion. The iceA2 gene was the most frequent in this study. Much research is necessary to determine the presence of an association of this gene with gastric pathology. Variation in the rates of the iceA gene in different countries is a strong indication of its geographical distribution. This study would provide important information regarding the prevalence of virulence genes (vacA, cagA, iceA1, and iceA2) in H. pylori strains in the sample tested in this country.

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Quantitative radioimmunohistochemical measurements of p185(erbB-2) in frozen tissue sections.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.11.8918900 ◽

1996 ◽

Vol 44 (11) ◽

pp. 1251-1259 ◽

Cited By ~ 12

Author(s):

J R Reeves ◽

J J Going ◽

G Smith ◽

T G Cooke ◽

B W Ozanne ◽

...

Keyword(s):

Tumor Biology ◽

Breast Carcinomas ◽

Tissue Sections ◽

Biopsy Specimens ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Interstudy Variability ◽

High Degree ◽

The Relationship ◽

Formalin Fixed

The relationship between expression of the c-erbB-2 proto-oncogene and the biology of breast cancer has been investigated widely, most studies using immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This technique is at best semiquantitative and there is a high degree of interstudy variability because of its subjective nature and poor methodological standardization. The relationship between the levels of expression and biology can be examined thoroughly only with an accurately quantitative technique. We have developed a radioimmunohistochemical assay to measure p185(erbB-2) in tissue biopsy specimens. The method involves incubating frozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and grain counting with image analysis. Sections of cell pellets with known c-erbB-2 levels are processed with each batch of samples as internal calibration standards. We have quantified c-erbB-2 expression in 60 breast carcinomas and compared the results with conventional immunohistochemistry. Radioimmunohistochemistry measured receptor levels throughout the range of expression in breast carcinomas, whereas conventional immunohistochemistry detected the protein only in the highest expressing tumors. The quantitative, objective data produced by radioimmunohistochemistry allow a more thorough evaluation of the relationship between c-erbB-2 expression and tumor biology. This technique may have applications in other fields where quantitative data is required and relevant monoclonal antibodies are available.

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