To confirm the clinical usefulness of Nested PCR-QP using stool specimens, we designed two clinical studies. The first was a method using the residual solution of the H. pylori stool antigen test for adolescents, as it was difficult to perform esophagogastroduodenoscopy (EGD) in adolescents, and the second was a method using culture testing for adults.
1. Nested PCR-QP detection sensitivity and correlation with eradication therapy
The subjects were middle school students in Saga Prefecture who underwent H. pylori testing between August and December 2017 under our program to eliminate gastric cancer. Consent to participate in the study was obtained from 71 middle school students. These 71 students who tested positive in the urine H. pylori antibody test (RAPIRAN®; Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) as the primary test underwent stool H. pylori antigen testing (Testmate Rapid Pylori Antigen®; Wakamoto Pharmaceutical Co., Ltd. Tokyo, Japan) as the secondary test. Among these students, the secondary test results were positive for 57 and negative for 14. Thus, the 57 patients who tested positive in the secondary test were judged to be infected with H. pylori. We then performed Nested PCR-QP measurements using the leftover specimens from the stool antigen tests that the 57 students testing positive had undergone. DNA sequencing analysis was performed for all 57 samples by carrying out Nested PCR-QP using PCR-direct sequencing. Students who tested positive in the stool antigen test underwent a 7-day treatment comprising 20 mg vonoprazen (VPZ, Takeda Pharmaceutical Co., Ltd. Tokyo, Japan), 750 mg amoxicillin (AMPC), and 200 mg clarithromycin twice a day, regardless of the results regarding CAM resistance mutations. Confirmation of the eradication therapy was conducted using the urea breath test (UBIT® tablet, 100 mg and POC One®; Otsuka Electronics Co., Ltd. Hirakata, Japan).
2. Nested PCR-QP in relation to culture test and drug susceptibility test
Considering that EGD was not eagerly performed in asymptomatic children, we planned another study on adults at the Imamura Hospital (Tosu, Japan). The subjects were 23 adults who were diagnosed with H. pylori infection on the basis of the results of rapid urease test (RUT; Helicocheck®; Institute of Immunology, Co., Ltd., Tochigi, Japan) and culture testing performed at the hospital, where they had undergone EGD between February 2018 and September 2018. The culture testing and drug susceptibility testing (H. pylori drug sensitivity test: H. pylori MIC measurement) was performed by BML, Inc. We utilized the breakpoints recommended by the Japan Society of Chemotherapy for CAM and amoxicillin  and the breakpoints listed in the EUCAST Trial breakpoint table v.8.1 for metronidazole (MNZ) . Stool specimens were used in the stool H. pylori antigen test, and the remnant solution of those who tested positive was used to perform Nested PCR-QP measurements.
3. Nested Pcr-qp
Nested PCR-QP is a novel genetic analysis method that analyses 23S rRNA genetic mutations (A2142C, A2142G, and A2143G) that are associated with CAM resistance in H. pylori. The basic components are the Nested PCR and QProbe . The sample used in Nested PCR-QP is stool H. pylori antigen test remnant solution. DNA is extracted from the Nested PCR-QP samples measuring 100 µL using QIAamp® DNA Mini kit(QIAGEN GmbH, Hilden, Germany)to obtain 150 µL of DNA solution. This DNA solution is the 1st PCR template for Nested PCR-QP.
The 1st PCR primers are Hp23S 1835F and Hp23S 2327R in accordance with the method described by Noguchi et al . The 1st PCR utilizes a 1 µL template created using a T100 Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) and is performed using Ampdirect plus BIOTAQ HS DNA Polymerase (Shimadzu corp., Kyoto, Japan). The PCR reaction protocol was as follows: After primer annealing for 10 min at 95℃, 36 cycles were performed at 94℃ for 30 s, 50℃ for 60 s, and 72℃ for 60 s.
The 1st PCR reaction mixture diluted 20-fold in sterilized water was used in the 2nd PCR template; the 2nd PCR primer was the newly designed HP F1(5′-CCAGAGATTCAGTGAAATTGTAGTGGAGGTG-3′)、HP R3(5′-GGCTCCATAAGAGCCAAAGCCCTTAC-3′), and the probe utilized was the newly designed HP QP1(5′-CCGCGGCAAGACGGAAAGAC-BODIPY-3′). The 2nd PCR and melting curve analysis were performed using a 5 µL template from LightCycler Nano (Roche Diagnostics K.K., Tokyo, Japan) and KOD DNA Polymerase (Toyobo Co., Ltd, Osaka, Japan). The PCR reaction protocol was as follows: After primer annealing for 2 min at 98℃, 65 cycles were performed at 95℃ for 5 s and 55℃ for 15 s. Melting curve analysis was performed as follows: After the 2nd PCR, warming was performed from 40℃ to 80℃ at a rate of 0.09℃/s, and measurements were obtained using fluorescent light with a wavelength of 510–528 nm. Melting curve analysis using fluorescence quenching allowed us to determine that there were no mutations when the fluorescence quenching inflection point was between 67℃ and 71℃ (wild type) but there were mutations between 59℃ and 63℃ (A2142G, A2143G) and between 63℃ and 67℃ (A2142C).
4. Statistical Analysis
The effectiveness of eradication therapy was compared between the Nested PCR-QP mutation group and the non-mutation group using chi-square test. Statistical significance was set at a P-value of < 0.05. Statistical analyses of the data were performed using JMP Pro13 (SAS Institute Inc., Cary, NC, USA).