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Development of a high throughput human stool specimen processing method for a molecular Helicobacter pylori clarithromycin resistance assay.

ABSTRACT: It has become critical to detect Helicobacter pylori (H. pylori) infection due to the link to gastric cancer with some strains. These strains are also increasing in resistance to antibiotics with clarithromycin leading the way as the first line treatment. Resistance to clarithromycin has been shown to correlate with the A2142G, A2142C, and A2143G mutations on the rrl gene. In the last few decades, non-invasive specimens, such as stool, have been a reliable alternate to gastric biopsy for immunoassay tests. More recently, it has been proven feasible for stool to be used in molecular based tests. Many of the core laboratories in the United States need a high throughput sample preparation to run this test. Here, a high throughput assay is compared to a previously published manual sample prep H. pylori molecular based assay. Using the Magna Pure 96 (Roche), at least 96 stool species and 96 biopsy specimens can be tested in an 8-hour shift of a clinical lab. The high throughput sample prep had a positive percent agreement (PPA) of 87% compared to the manual sample prep using the same testing configuration. The genotype predictions from the high throughput assay matched genotype predictions from the manual sample prep with the same stool sample 92% of the time. A concordance rate of 89% was observed with genotype predictions from the high throughput assay of the same patient stool and biopsy. In stool samples from the high throughput assay, there was 100% concordance between the quantitative polymerase chain reaction (qPCR)-derived genomic prediction and DNA sequencing data. The high throughput workflow can get more patients tested faster in addition to detection of mutations associated with clarithromycin resistance.

SUBMITTER: Clines N

PROVIDER: S-EPMC6913962 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR).The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion.The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin.The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

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Development of a high throughput human stool specimen processing method for a molecular Helicobacter pylori clarithromycin resistance assay.

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