Molecular Epidemiology Study Of The Genotypic Mutations Conferring Resistance To Nucleos(t)ide Analogues From Clinical Specimens Of Patients With Hepatitis B

ObjectiveNucleos(t)ide analogues(NAs) are main anti-HBV drugs, but long-term application may cause resistance and affect curative effect. We detect the hepatitis B virus(HBV) drug resistance mutations against NAs to get a comprehensive understanding of the status of NAs resistance mutations, provide basis for detection and monitoring of NAs resistance mutations, design more effective therapeutic strategies, improve antiviral curative effect and reduce or delay resistance, MethodsSerum HBV DNA of patients with HBV infection was extracted, HBV P gene was amplified by nested PCR, and the results were analyzed by sequence analysis software Bioedit. 19 resistance mutation sites were detected, including primary drug resistance mutation sites(rtA181, rtT184, rtS202, rtM204, rtN236, rtM250), secondary/compensatory mutation sites(rtL80, rtV173, rtL180) and putative antiviral resistance mutation sites(rtV84, rtS85, rtV207, rtS213, rtV214, rtQ215, rtL229, rtI233, rtP237, rtN/H238), and covering main HBV drug resistance mutations against NAs. ResultsAmong 2223 serum samples from the patients with HBV infection, the genetic mutations conferring resistance to NAs were detected in 1202(54.07%)specimens; out of those, the mutations of primary drug resistance sites were detected in 980(44.08%) patients. The mutation detection rates were shown age- and region-dependent, i.e., the positive rates increased with ages, and the rates were higher in northern than those in southern China. The constituent ratios of primary drug resistance mutation sites rtM204, rtA181, rtN236, rtT184, rtS202, rtM250 were 51.00%, 27.27%, 13.87%, 4.25%, 13.87% and 0.88%. Multiple-site mutations(66.20%) were detected more than single-site(33.80%) among the clinical HBV isolates. 100% rtM204 V accompanied with rtL180 M, while 35.55 % rtM204 I accompanied with rtL180M(χ2=260.54, P<0.05); 8.7%rtM204V accompanied with rtL80I/V, while 50.58%rtM204I accompanied with rtL80I/V(χ2=116.70, P<0.05); 14.17% rtM204 V accompanied with rtV173 L, while 2.6%rtM204I accompanied with rtV173L(χ2=28.27, P<0.05). A complex composition of mutations was revealed. A total of 32 mutation modes were identified and the rtM204I/V+rtL180M was the highest proportion, followed by rtA181T/V/S. The rates of drug resistance mutation were in the ranking of LAM>ADV>ETV. Multidrug resistance mutations were detected in 48(2.2%) patients. Conclusions1 The mutation detection rate is high and shown age- and region-dependent.2 Many kinds of drug resistance mutation sites are detected and rtM204V/I is the most common, followed by rtA181T/V. Multiple-site mutations are detected more than single-site. rtM204V/I often accompany with other mutations in different probability.3 The composition of mutation modes is complex and a total of 32 mutation modes are identified. Cross resistance even multidrug resistance exists between these drugs.4 Resistance mutations detection has important guiding significance on clinical medication. If conditions admitted, resistance mutations should be detected for identifying resistance mutation types. Complex mutation mode and higher multidrug resistance ratio prompt us to detect mutation sites as comprehensive as possible.