Application Of Serum HBV RNA In Disease Assessment And Drug Resistance Monitor In Chronic HBV-infected Patients

Background and AimChronic HBV infection is a global public health problem.Intrahepatic covalently closed circular DNA(cccDNA)is the root cause of HBV being difficult to completely eliminate and causing different clinical outcomes.However,detection of cccDNA relies on liver biopsy and is difficult to be clinically popularized.Although ccc DNA cannot be directly detected in peripheral blood,other serum indicators can be used to reflect the levels of ccc DNA.At present,nucleos(t)ide analogs(NAs)are the main anti-HBV treatment,but they cannot eliminate ccc DNA directly.The vast majority of chronic HBV-infected patients require long-term medication.Although the probability of drug resistance is greatly reduced after the use of NAs with high barrier to resistance,due to the inevitability and complexity of HBV mutations,the emergence of drug-resistant strains is actually an unavoidable clinical problem in the long-term treatment of NAs.Different from hepatitis B surface antigen(HBs Ag)and HBV DNA,HBV RNA is only derived from ccc DNA transcription and not affected by NAs.It can more accurately reflect the intrahepatic ccc DNA status.However,there is no universally standardized method for HBV RNA detection,and the research focusing on disease assessment and NAs resistance monitor in chronic HBV-infected patients is still lacking.We first established a method for quantitative detection of serum HBV RNA by droplet digital PCR(dd PCR),and used a commercial real-time fluorescent quantitative PCR(q PCR)method for comparative evaluation to ensure the accuracy of the detection results.Then we observed the untreated chronic HBV-infected patients'serum HBV RNA levels in different clinical phenotypes,and analyzed the dynamic characteristics of serum HBV RNA and drug resistance mutations in treatment-na?ve patients who initiated entecavir therapy but developed genotypic resistance(GR)or partial virological response(PVR)in subsequent treatment.This study may provide HBV RNA as a new tool for disease assessment and monitoring drug resistance mutations during NAs therapy.Methods1.Serum HBV RNA levels were detected by dd PCR and commercial q PCR in chronic HBV-infected patients,and the correlation and consistency of the two methods were analyzed by linear regression analysis and Bland-Altman analysis.A case of high concentration HBV RNA sample was used for gradient dilution,the two methods were parallelly tested and repeated 5 times in each gradient to determine the repeatability and sensitivity.2.A retrospective cohort including 149 untreated HBV-infected patients was divided into four clinical phenotypes:hepatitis B envelop antigen(HBe Ag)positive with normal alanine transaminase(ALT;EPNA)or with elevated ALT(EPEA),HBe Ag-negative with normal ALT(ENNA)or with elevated ALT(ENEA).Serum HBV RNA levels were quantified by qPCR method and liver biopsy was performed in those with undetectable serum HBV DNA or RNA.Univariate logistic regression analysis to explore the risk factors associated with elevated ALT in HBe Ag-negative patients.3.Serum HBV RNA levels were detected in 31 cases of untreated chronic HBV-infected patients with HBe Ag-negative,HBV DNA-positive,normal or minimally elevated ALT(<2×upper limit of the normal,ULN).Scheuer scoring system was used to determine liver inflammation degree.Inflammation grade(G)?2 was judged as significant hepatitis,and the receiver operating curve(ROC)was used to explore the diagnostic value of HBV RNA levels in identifying HBe Ag-negative active hepatitis status.4.In the hepatitis database of our hospital,we screened 5 CHB patients who were initially treated with ETV.GR mutation was detected in serum HBV DNA by first generation sequencing.At the end of observation,3 patients developed genotyp ic resistance(GR)during the antiviral time and 2 patients did not develop GR but PVR were included.Serum samples collected from the biological sample bank across the initial time point to the follow-up end point were detected by dd PCR method for quantitating HBV RNA levels.Next we observed t dynamic characters of HBs Ag,HBV DNA,and HBV RNA.The third generation sequencing(TGS)was used for determining serum HBV RNA reverse transcription region mutation in several follow-up points for drug resistance monitor.Result1.The linear regression coefficients(R²)of the two serum HBV RNA detection methods in the untreated and treated group were 0.912 and 0.874,respectively,and the 95%limits of agreement were(-1.430,1.506)and(-1.533,1.648),respectively.The gradient dilution experiment showed that the detection rates of dd PCR and q PCR methods were 100%when the serum HBV RNA was?2.0 log₁₀ copies/m L,and the CV values were between2.29%-10.8%and 3.11%-7.33%,respectively.However,when the serum HBV RNA level is lower than 2.0 log₁₀ copies/m L,the detectable rate gradually decreases with the increase of the serial dilution,the lower limit of detection(LLoD)was 61 copies/m L and 15 copies/m L,respectively.On the whole,dd PCR and q PCR methods have a significant linear correlation between the detectable results and dilution gradient,and the R² are 0.895 and 0.938,respectively,P<0.0001.2.The detectable serum HBV RNA levels(log₁₀ copies/m L)in EPNA,EPEA,ENNA and ENEA were 6.02±1.48,6.54±1.27,2.51±0.78 and 3.54±1.25,respectively.The low level(<2.0 log₁₀ copies/m L)comprised mainly of ENNA phenotype(76.9%),while the high level(>6.0 log₁₀ copies/m L)was HBe Ag-positive patients(98.1%).In EPNA,EPEA,and ENEA,serum HBV RNA and serum HBV DNA were significantly positively correlated,and the correlation coefficients(r)were 0.868(P=9.480×10^-9),0.687(P=2.621×10^-8),0.769(P=6.686×10^-9),respectively.In the ENNA phenotype,there is no significant correlation between the two biomarkers(r=0.324,P=0.075).In the EPNA and EPEA phenotypes,serum HBV RNA and HBs Ag showed a significant positive correlation,and the correlation coefficients were 0.777(P=2.999×10^-6)and 0.637(P=4.993×10^-7),respectively,but in the HBe Ag-negative phenotype,there is no such significant correlation.Serum HBV DNA and RNA were both independent risk factors associated with elevated ALT in HBe Ag-negative patients,with odds ratios(OR)of 2.650(1.640-4.282),P=6.823×10^-5 and 2.570(1.541-4.286),P=2.971×10^-4,respectively.Seven serum HBV DNA-undetectable but RNA-detectable patients underwent liver biopsy,showing moderate or severe liver inflammation(G?2).3.Serum HBV RNA level can distinguish significant liver inflammation,the area under the receiver operating curve(AUC)is 0.737(0.549,0.925),P=0.029,but HBV DNA can not(P=0.248).According to the maximum Youden index,the best cut-off value of HBV RNA for identifying significant liver inflammation is 2.81 log₁₀ copies/m L,the sensitivity is 73.7%,the specificity is 66.7%,and the accuracy is 71.0%.4.GR1,GR2,and GR3 were determined resistant mutations at the 208 weeks,186weeks,and 142 weeks after antiviral initiation,respectively.Before this time point,the serum HBV RNA levels in 3 GR patients were sustained>4.0 log₁₀ copies/m L.Two PVR patients'serum HBV RNA>6.0 log₁₀ copies/m L during the observation.After switching to or adding TDF,the serum HBV RNA levels in patient GR1 and GR2 were decreased 1.63log₁₀ and 2.58 log₁₀in the next 26 and 22 weeks,respectively,and the HBV DNA levels decreased from 4.40 log₁₀ and 3.44 log₁₀ to LLo D,respectively.But HBs Ag was barely decreased(+0.03 log₁₀ and-0.02 log₁₀,respectively).The drug-resistant mutations detected in the HBV RNA were consistent with the results of detection in HBV DNA.Using TGS for long-term monitor of HBV RNA RT region shows that no mutations were found before the resistance time point in patient GR1 and GR2,while patient GR2 had rt L180M+M204I mutations at the start of treatment.Conclusion1.The ddPCR and qPCR are both reliable methods for serum HBV RNA quantitation.The detection of low-concentration serum samples is difficult,but it can be improved by repetition.2.Serum HBV RNA levels in different clinical phenotypes are varying,indicating that this biomarker can be used for disease assessment.Both serum HBV RNA and HBV DNA are independent risk factors associated with elevated ALT,so it is necessary to detect HBV RNA in patients with undetectable serum HBV DNA.Serum HBV RNA can complement HBV DNA for reflecting liver inflammation and provide a reference for clinical intervention.3.In untreated HBe Ag-negative patients with normal or minimally elevated ALT(<2×ULN),serum HBV RNA can be used to distinguish the status of liver inflammation but HBV DNA cannot.Therefore,the serum HBV RNA may provide a proof for initiating antiviral therapy.4.Serum HBV RNA levels and the sequencing analysis of HBV RNA are sui table for long-term monitoring antiviral efficacy and the occurrence of genotypic resistance mutations in chronic hepatitis B patients.In conclusion,serum HBV RNA,as a virology indicator,can be used for liver inflammation assessment in HBe Ag-negative patients whose serum HBV DNA undetectable or ALT<2×ULN;Serum HBV RNA is also suitable for the antiviral efficacy assessment and drug resistance monitor in NAs therapy,thus helping clinicians as a proof to generate individualized management and therapeutic strategies for patients.