Antibiotics susceptibility is the most important single factor for HP eradication, and therefore, the detection of resistance is essential. The gold standard for detecting antibiotic resistance is culture based. However, these methods require at least 20 ~ 72 hours, low yield and limited accessibility due to their laboratory requirements. The culture methods are gradually being replaced by molecular biological methods such as PCR due to these limitations.[14] Recent PCR methods are faster, simpler, and more economical [15, 16]. PCR methods can find evidence of HP infection from the conventional stain or culture test-negative samples. The recent European guideline recommends clarithromycin susceptibility testing, if available through molecular techniques or culture, before prescribing any clarithromycin-containing therapy.[5] Guidelines recommend quadruple treatment when there is no information regarding clarithromycin resistance. The increased number of antibiotics could lead to disturbance of gut microbiota and resistance of other bacteria.[17] The PCR methods allow the use of the optimized triple therapy for 60–90% of patients. [5]
In South Korea, DPO-PCR is one of the available methods for testing clarithromycin resistance. Seeplex H. pylori-ClaR ACE Detection is a commercially available DPO-PCR kit that detect H. pylori, A2143G, and A2142G mutations. DPO-PCR showed its concordance with E-test regarding clarithromycin susceptibility in 95.3% of cases. [18] The eradication rates in clarithromycin-susceptible subjects determined by DPO-PCR were between 89.2 and 97.4%. However, for subjects with clarithromycin-resistant strains determined by DPO-PCR, Bismuth quadruple therapy is more effective with eradication rates of 91.7–93.5% for subjects with clarithromycin-resistance determined by DPO-PCR. [11, 19, 20]
Allplex™ is a recent multiplex quantitative real-time reverse transcription PCR (qRT-PCR) assay that uses multiple detection temperature techniques (MuDT).[21] Compared to DPO-PCR, Allplex ™ is faster and likely to be more accurate because it does not need the western blot process. Allplex™ is commercially available in European Union countries, Brazil, Ukraine, and Kenya.
Among the 125 specimens with confirmed HP infection in gene sequencing, 99.2% and 96.0% were confirmed with Allplex™ and DPO-PCR, respectively. Allplex™ showed 100% sensitivity, 97.6% specificity, 98% PPV, and 100% NPV for detecting HP infection in a French study.[12], similar to this study.
The point mutations by whole genome sequencing were well correlated with the clarithromycin resistance phenotype in a European study (congruence 99%)[22] In Korea, A2143G mutation revealed 85.7% of concordance with the phenotype (6 out of 7 strains)[10], and A2142G mutation showed 100% of concordance with the phenotype (20 out of 20 strains).[23] These point mutations resulted in eradication failure with the clarithromycin-based regimen.[23–25]
In this study, we compared the diagnostic performance of Allplex™ and DPO-PCR using gene sequencing as a reference test. Allplex™ showed better diagnostic performance for HP detection with a higher accuracy rate and kappa agreement levels. Allplex™ also showed better diagnostic performance for the detection of HP mutations. Our results suggest that Allplex™ can be considered for the diagnosis of HP and mutations.
This study has several limitations. First, the sample size was relatively small.
Second, this study did not investigate the phenotype of clarithromycin resistance with HP culture. However, many studies reported an almost 100% of match rate between genotype and phenotype of the point mutations A2142G, A2142C, and A2143G[22, 26] Third, A2142C mutation was not found in this study, confirming its low prevalence in Korea from a previous study, (1 out of 431 strains)[25] In the United states, the positivity rates of A2142G and A2143G mutations range from 5.3 to 14% and from 23.8 to 80%, respectively, and those of the A2142C mutation ranges from 0 to 4%.[27–30] In Europe, the A2142G and A2143G mutations range from 9.6 to 32.6% and from 25.0 to 44.1%, respectively, and A2142C mutation was reported to range from 1.6 to 1.9%.[31, 32] In Japan, the A2142G and A2143G mutations range from 2.0 to 2.3% and from 53.6 to 67.4%, respectively.[33, 34] Notably, most clarithromycin resistance cases in South Korea were from the A2143G mutation.[35, 36]
Lastly, we did not study the other mutations which account for the clarithromycin resistance. Other than the three mutations in this study, A2115G, A2142T, G2141A, and T2182C were reported to influence clarithromycin resistance.[37, 38] However, in the previous studies, the most common mechanism of point mutations in clarithromycin resistance is preventing macrolides from binding sites were A2143G (69.8%), A2142G (11.7%), and A2142C (2.6%).[37]
In conclusion, Allplex™ showed comparable diagnostic performance with direct gene sequencing and non-inferior diagnostic performance to DPO-PCR. Further clinical research is required to confirm whether the increase in the diagnostic performance of Allplex™ leads to effective eradication therapy is effective.