Quantitative Detection And Size Determination Of Hepatitis C Virus RNA In Urine Of Patients With Chronic Hepatitis C

Hepatitis C is a global public health problem.It has been estimated that more than 170 million people worldwide have chronic hepatitis C infections,and approximately 10-20%of them will develop severe liver diseases such as cirrhosis and hepatocellular cancer.Its causative agent,hepatitis C virus(HCV),is a small,positive-sense,single-stranded RNA virus.The detection and quantification of HCV RNA is indispensable for hepatitis C management.Quantitative HCV nucleic acid testing has become a key parameter in treatment response surveillance and in clinical decision-making during HCV antiviral therapy.Blood is the dominant sample type used in the detection of HCV RNA because of the parenteral mode of HCV transmission.However,reports have shown that approximately 15-40%of patients with hepatitis C have no identifiable parenteral source of infection.HCV RNA has been detected in body fluids other than blood to evaluate possible non-parenteral routes of HCV infection,and has been reported to test positive in body fluids such as saliva and urine,but the detection rate varies greatly.Urine is an ultra-noninvasive sample source that is relatively time and cost efficient.Particularly for newborns,children,and patients requiring repeated sampling to monitor antiviral therapy efficacy,urine has remarkable advantages over blood,including noninvasive sampling,convenient collection,and low biological risk to the staff.Several studies have reported HCV RNA detection in the urine of patients with hepatitis C,although with varying success,reporting detection rates of 0-56.5%.However,the underlying mechanism of HCV RNA shedding in urine and in what form it is present in urine remain unclear.Extrahepatic replication of HCV has not been found to occur in the urinary system,indicating that local replication and excretion of HCV are impossible.Short-fragmented nucleic acids from circulation have been reported to exist in urine.It is reasonable to speculate that urinary HCV RNA may exist as short fragments,since they may transport through the glomerular filtration system,which allows complexes<6.4 nm in diameter and with a molecular weight<70 kDa.However,previously reported isolation and PCR-based detection methods of HCV RNA detection in urine were not designed for relatively short RNA fragments.Therefore,the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear.To overcome the drawbacks of previously developed methods for HCV RNA detection in urine and to more sensitively and accurately measure urine HCV RNA levels,a novel real-time PCR assay with a modified isolation method and short amplicon(62 bp)designed for short HCV RNA fragments was developed in this study.The limit of detection,precision,linearity,and specificity of the assay were evaluated and demonstrated high-quality performance.The study included 95 patients with chronic hepatitis C,of whom 60 were positive for plasma HCV RNA and the other 35 were negative.HCV RNA in the urine of 95 patients with chronic hepatitis C was quantified using the newly established detection system.The results showed that 36 out of the 60 samples(60%)with positive plasma HCV RNA were able to detect urine HCV RNA,and urine HCV RNA was not detected in 35 samples with negative plasma HCV RNA.To investigate the effect of amplicon size on detection efficiency and the size distribution of urinary HCV RNA fragments,four different-sized amplicons were used in the quantification of HCV RNA in urine.Four different amplicons were tested,and only the 62-bp and 157-bp amplicons could detect urine HCV RNA,with the 62-bp amplicon significantly more efficient than 157-bp amplicon(P<0.001).HCV RNA was undetectable in all urine samples with the 222-and 304-bp amplicons.The size determination results revealed that the relative concentration of detectable HCV RNA dropped by a median of nearly 90%when the amplicon size increased from 62 to 157 bp,and was not detected at higher amplicon sizes.These results reflected the fragmented nature of urinary HCV RNA,indicating that HCV RNA molecules in the urine of patients infected with HCV were short RNA fragments,with most shorter than 157 bp.With the 62-bp assay,patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels(P<0.001).and plasma and urine concentrations were significantly positively correlated(R2=0.708,P<0.001).In conclusion,this study established a new reverse transcription real-time quantitative PCR assay for the quantitative detection of HCV RNA in the urine of patients with chronic hepatitis C through the improvement of nucleic acid extraction methods and the application of short amplicon.The fragment size distribution of HCV RNA in urine and its clinical application value were also explored.The results showed that the novel RT-qPCR assay developed in this study greatly increases the sensitivity of urine HCV RNA detection,and reveals the presence of short HCV RNA fragments in urine.The positive correlation between urine and plasma HCV RNA concentrations indicates a potential role for urinary HCV RNA as an ultra-noninvasive marker for diagnosis and monitoring treatment response in viremic patients with hepatitis C.