Normalized real-time PCR for diagnosis of H. pylori infection

Al-Moayad, Ebtisam E.; Alghalibi, Saeed M.; Al-Shamahy, Hassan Abdulwahab; Nasher, Akram Thabet; Al-Hebshi, Nezar N.

doi:10.5339/qmj.2014.19

Cited by 4 publications

(8 citation statements)

References 15 publications

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“…It was found that the sensitivity and/or specificity obtained using ureA-qPCR in this study was higher than in previous reports. 21,31,33,44 A higher +LR (8.72 versus 4.3) and good -LR (0.04 versuss 0.63) was obtained in this study compared with that of Ramirez-Lazaro et al 45 The qPCR method shows a statistically significant higher percentage of H. pylori positivity compared to conventional methods (23.8% versus 14.7%, p<0.0001). Agreement between the tests according to Kappa analysis was at substantial degree (0.67) for qPCR and conventional methods.…”

supporting

confidence: 43%

“…32 H. pylori DNA load in gastric biopsy was expressed as the quotient between the copy number of microorganisms and copy number of human cells in each gastric sample 31 multiplied by 100 to provide the number of copies of H. pylori DNA per 100 human cells. 33 The limit of detection of the ureA qPCR assay was determined using 10 serially diluted DNA samples of H. pylori strain C55. DNA concentration ranging from 100 ng/µL to 0.01 fg/µL were used as a template for qPCR.…”

Section: Detection Of H Pylori In Biopsy Samples By Qpcrmentioning

confidence: 99%

“…The cut-off point was determined and used to distinguish positive from negative results. The optimal cut-off point was defined as positive by the presence of any number of microorganisms (real-time PCR>0 microorganism/human cell) proposed by Saez et al, 31 while Al-Moayad et al 33 suggested a cutoff value of one bacterial per 100 human cells to distinguish cases from controls. Due to differences in laboratory practices, it is not feasible to directly compare the suggested cut-off point.…”

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confidence: 99%

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Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy

Binmaeil¹,

Hanafiah²,

Rose³

et al. 2021

IDR

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Aims and Objectives: More than half of the world's population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycinresistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin-and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies. Materials and Methods: Specific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay's sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies. Results: In this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and −LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively. Conclusion:The effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin-and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.

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supporting

confidence: 43%

Section: Detection Of H Pylori In Biopsy Samples By Qpcrmentioning

confidence: 99%

mentioning

confidence: 99%

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Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy

Binmaeil¹,

Hanafiah²,

Rose³

et al. 2021

IDR

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“…giving results within 24–72 hours, but its sensitivity was considerably lower (32%). As said before, RUT sensitivity is greatly influenced by bacterial density, requiring a minimum number of the organisms per biopsy, and varies significantly from one study to another [ 24 , 26 ].…”

Section: Discussionmentioning

confidence: 99%

Utility of normalized genome quantification of Helicobacter pylori in gastric mucosa using an in-house real-time polymerase chain reaction

et al. 2017

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Traditional diagnostic assays for Helicobacter pylori detection have their limitations. Molecular methods can improve both diagnosis and understanding of gastric diseases. Here we describe an in-house quantitative real-time polymerase chain reaction (q-rt-PCR) for the detection of H. pylori in gastric biopsies which has been developed and has a detection limit of 10 copies, the specificity of which was tested against other gastric colonizer bacteria. In this study, 199 gastric biopsies from adults with different clinical gastric symptoms were examined. Biopsies were obtained during endoscopy and the following tests performed: rapid urease testing (RUT), culture and q-rt-PCR. H. pylori bacterial load expressed as bacterial load per 105 cells was calculated using a standard curve. H. pylori was isolated in 41% of patients, RUT was positive in 32% and bacterial genome was detected in 45% (p = 0.010). Concordance between traditional invasive microbiological methods used together and q-rt-PCR was almost 100%. Bacterial load in patients with positive RUT was significantly higher than those where it was negative (p<0.0001). There were also significant differences between bacterial load in patients with more than one positive assay versus those where only one method was positive (p = 0.006). The in-house q-PCR developed here is quick and inexpensive, and allows accurate diagnosis of H. pylori infection. It also permits normalized bacterial load quantification, which is important to differentiate between asymptomatic colonisation and infection.

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“…Polymerase chain reaction (PCR) has also been applied for H. pylori detection. Al-Moayad et al[83] concluded that standardized PCR allows an accuracy superior to that observed in RUT, with improved detection in specimens with lower bacterial charge[84]. However, the necessity of endoscopy is an important limitation of the three methods mentioned above, and the advances in non-invasive diagnostic techniques have strengthened the idea of prioritizing the use of diagnostic alternatives for which endoscopy is dispensable.…”

Section: Clinical Managementmentioning

confidence: 99%

Pathogenesis and clinical management of Helicobacter pylori gastric infection

Brito

Silva

Soares

et al. 2019

WJG

184

146

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Helicobacter pylori (H. pylori) is a gram-negative bacterium that infects approximately 4.4 billion individuals worldwide. However, its prevalence varies among different geographic areas, and is influenced by several factors. The infection can be acquired by means of oral-oral or fecal-oral transmission, and the pathogen possesses various mechanisms that improve its capacity of mobility, adherence and manipulation of the gastric microenvironment, making possible the colonization of an organ with a highly acidic lumen. In addition, H. pylori presents a large variety of virulence factors that improve its pathogenicity, of which we highlight cytotoxin associated antigen A, vacuolating cytotoxin, duodenal ulcer promoting gene A protein, outer inflammatory protein and gamma-glutamyl transpeptidase. The host immune system, mainly by means of a Th1-polarized response, also plays a crucial role in the infection course. Although most H. pylori-positive individuals remain asymptomatic, the infection predisposes the development of various clinical conditions as peptic ulcers, gastric adenocarcinomas and mucosa-associated lymphoid tissue lymphomas. Invasive and non-invasive diagnostic methods, each of them with their related advantages and limitations, have been applied in H. pylori detection. Moreover, bacterial resistance to antimicrobial therapy is a major challenge in the treatment of this infection, and new therapy alternatives are being tested to improve H. pylori eradication. Last but not least, the development of effective vaccines against H. pylori infection have been the aim of several research studies.

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Normalized real-time PCR for diagnosis of H. pylori infection

Cited by 4 publications

References 15 publications

Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy

Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy

Utility of normalized genome quantification of Helicobacter pylori in gastric mucosa using an in-house real-time polymerase chain reaction

Pathogenesis and clinical management of Helicobacter pylori gastric infection

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