Hepatitis C virus (HCV) is a hepatotropic RNA virus that causes progressive liver damage, which in turn can lead to cirrhosis and hepatocellular carcinoma. According to the World Health Organization (WHO), as of 2019, about 58 million people worldwide have chronic hepatitis C, and only 21% of those with chronic hepatitis C are diagnosed. In order to eliminate viral hepatitis as a public health threat, the mainland issued the Action Plan for Eliminating the Public Health Hazards of Hepatitis C (2021-2030) in 2021, which requires increasing the detection rate of hepatitis C, and achieving full coverage of nucleic acid testing for HCV antibody positive people. In the Global Health Sector Viral Hepatitis Strategy 2016-2021 released by the World Health Organization (WHO) in 2016, the goal of "eliminating viral hepatitis as a major public health hazard by 2030" aims to diagnose more than 90% of people infected with HCV by 2030. In order to achieve this goal as soon as possible, new technologies and strategies for HCV infection detection have emerged and gradually been applied in recent years. In addition, with the continuous development of direct-acting antiviral drugs (DAAs), the majority of hepatitis C patients can be cured, and the simplified HCV infection detection and diagnosis process can shorten the turnaround time from visit to treatment, so that hepatitis C patients can be treated as quickly as possible. This article refers to the latest guidance documents and research progress on HCV infection detection at home and abroad, and reviews the laboratory detection methods and strategies for HCV infection.
1Test markers for HCV infection
HCV can cause both acute and chronic infections. Acute HCV infection refers to the detection marker of HCV infection within 6 months after exposure and infection with HCV, according to the natural process of HCV infection, HCV RNA can be detected in peripheral blood 1~2 weeks after exposure to HCV, followed by HCV core antigen (HCV cAg) p22 and HCV antibody. In the absence of treatment, some people with HCV infection can spontaneously clear HCV infection within 6 months of infection, while others become chronic. The dynamics of HCV infection detection markers are shown in Figure 1.
Note: a, self-limited HCV infection, b, chronic HCV infection.
Fig.1 Dynamic changes of HCV infection detection markers in peripheral blood
2Laboratory testing methods for HCV infection
According to the Diagnosis of Hepatitis C (WS213-2018), the laboratory diagnosis of HCV infection mainly relies on HCV antibody and HCV RNA detection. Among them, HCV antibody detection includes enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), rapid detection test (RDT), western blot test (WB) and recombinant western blot assay (RIBA), and nucleic acid detection includes real-time PCR (qPCR) and transcription-mediated amplification (TMA). If nucleic acid testing is not available, HCV cAg testing is used as an alternative to HCV RNA for diagnosing viremia after acute HCV infection, chronic infection, or reinfection, and has comparable clinical sensitivity as HCV RNA testing. Table 1 shows the number of HCV test methods and test products approved by the National Medical Products Administration and the U.S. Food and Drug Administration (FDA) in mainland China.
2.1 HCV screening test methods
The first-generation HCV screening reagent is based on the principle of indirect method, using the recombinant c100-3 epitope in the NS4 region to identify HCV infected patients. On this basis, the second-generation screening reagent added nucleocapsid C and NS3 targets, which improved the sensitivity and specificity of the detection, and the detection window was 7~8 weeks. The third-generation antibody screening reagent based on the double-antigen sandwich method is currently the most commonly used reagent for HCV screening, with a clinical sensitivity and specificity of more than 95%. The fourth-generation screening test can detect HCV antibodies and core antigens at the same time, further shortening the detection window of HCV to about 4 weeks, and is more suitable for early screening detection of HCV. The progress of the main detection performance of HCV screening reagents is shown in Table 2.
ELISA and CLIA are currently the most commonly used screening tests in laboratories. ELISA has high clinical sensitivity and specificity, and is a routine method for screening HCV in disease control systems, hospitals, blood banks and other institutions. CLIA is an epitope-specific antibody assay that typically has similar sensitivity and specificity to third-generation ELISAs. At present, CLIA has become the mainstream technology for HCV screening due to its wide dynamic range, semi-quantitative, highly automated, accurate test results, and short turnaround time.
HCV rapid tests are simple to operate, do not require specialized testing equipment, and can usually obtain simple qualitative results by visual interpretation within 30 minutes. Most rapid tests use fingerstick blood and can be performed by trained primary health workers. Therefore, the use of rapid tests in resource-limited settings where laboratory testing is not available or in outreach testing can help promote access to HCV testing, enhance referrals, and reduce attrition during follow-up of infected patients.
2.2 HCV antibody confirmatory testing methods
The confirmatory detection methods for HCV antibodies mainly include WB and RIBA. Due to the high specificity of confirmatory tests, WB and RIBA are often used as complementary tests to serological tests to confirm the presence of HCV-specific antibodies in samples that respond to screening tests. However, in the actual detection work, the antibody confirmatory detection test process is complex, the detection cycle is long, the results are difficult to interpret and subjective, and the antibody confirmatory test results are often uncertain. On the other hand, positive results for WB and RIBA can only indicate HCV infection or spontaneous recovery after infection, and cannot be used to distinguish between current HCV infection. With the advent of molecular assays and the increased sensitivity of assays, the U.S. Centers for Disease Control and Prevention updated its clinical and laboratory guidelines for HCV infection testing in 2013 to not recommend the continued use of HCV WB and RIBA in testing, but rather the use of other registered pathogen detection reagents, such as HCV RNA or antigen tests, for supplemental testing for HCV infection.
2.3 HCV nucleic acid detection methods
HCV nucleic acid detection includes qualitative and quantitative detection of HCV RNA. HCV nucleic acid qualitative testing can quickly and sensitively detect viral nucleic acid, which can be used for blood donor screening and clinical diagnosis. HCV nucleic acid quantitative detection is mainly used for antiviral therapy monitoring and efficacy evaluation. At present, HCV nucleic acid detection reagents are mainly based on qPCR and TMA.
2.3.1 Qualitative detection of HCV RNA
Qualitative detection of HCV RNA plays an important role in the diagnosis and monitoring of HCV infection. Qualitative testing provides rapid, sensitive detection of HCV RNA, providing evidence that viral RNA is below a cut-off point. The detection limit of HCV RNA qualitative detection reagent based on qPCR technology can be as low as less than 10 IU/mL, which is the most commonly used detection method for HCV RNA qualitative detection. TMA uses the combined action of reverse transcriptase and RNA polymerase to amplify the gene of interest at a constant temperature. The method offers high amplification efficiency, short analysis times, and flexible throughput, eliminating the need for specialized thermal cyclers, and good agreement between TMA and qPCR results, but TMA has a higher analytical sensitivity and can detect HCV RNA in samples that cannot be detected by qPCR.
2.3.2 Quantitative detection of HCV RNA
HCV RNA quantitative detection technology is mainly based on qPCR technology, which quantifies HCV RNA by real-time monitoring of the fluorescent signal generated by the degradation of fluorescent probes. With the development of HCV nucleic acid detection methods, the lower limit of quantification of quantitative detection reagents is equivalent to that of qualitative reagents, which is the main detection method of HCV nucleic acid detection. At present, most of the registered HCV RNA quantitative detection reagents have a minimum detection limit of less than 50 IU/mL, which can accurately detect most HCV infections. In addition, quantitative testing can quantify the viral load of HCV in infected patients, which can be used to determine HCV RNA levels at baseline and during treatment, and is more suitable for monitoring and efficacy evaluation of antiviral therapy.
The WHO estimates that most people with HCV infection have a viral load greater than 10 000 IU/mL, and for areas where laboratory testing is not possible or where sample transport is difficult, HCV RNA testing using dried blood spot (DBS) samples may be considered to diagnose HCV viremia. However, there are currently no registered reagents for the detection of HCV RNA in DBS samples. The nucleic acid detection reagents registered for plasma and serum have been used for the detection of DBS, which have good sensitivity and specificity, and have good correlation and consistency with the test results of serum and plasma samples in clinical practice.
2.4 HCV antigen detection methods
HCV cAg p22 is a nucleocapsid peptide of HCV that is released into plasma during viral assembly and can be detected early in HCV infection and throughout HCV infection. HCV cAg testing can be used as an alternative method to diagnose current HCV infection when nucleic acid testing is not available. Compared with HCV nucleic acid detection, core antigen testing is based on serology, which has a shorter turnaround time and lower testing cost, and is more suitable for use in primary testing laboratories.
The lower detection limit of HCV antigen detection reagent can reach as low as 3 fmol/L (0.06 pg/mL), which is equivalent to the detection limit of HCV nucleic acid detection of 500~3 000 IU/mL, and can detect more than 95% of chronic HCV infection. A meta-study has shown that HCV antigen detection reagents have high sensitivity and specificity, and there is a high correlation between HCV cAg and HCV RNA when the viral load exceeds 3 000 IU/mL.
At present, there is still controversy as to whether core antigen testing can be used as a follow-up tool for patients treated with DAA, especially for the detection of recurrence or reinfection. Studies have evaluated the clinical utility of HCV cAg testing before and after treatment, and some of these studies have shown that HCV antigen test results are extremely accurate. However, other studies have found that antigen test results are inconsistent with nucleic acid test results 12 weeks after the end of treatment, resulting in misclassification of infected people: 3 infected patients were detectable for HCV RNA but had a negative antigen test, and 1 infected person had a positive antigen test but could not detect HCV RNA. Twenty-four weeks after the end of treatment, HCV RNA and HCV antigen test results were consistent.
Although there are limitations in detecting patients with low HCV viral load, HCV antigen testing may be a better option for screening for HCV infection in high-risk patients who require regular testing, and in those with HCV infection who are immunocompromised or congenitally immunocompromised (eg, HIV-infected, chronically on dialysis, organ transplant, or congenital immunodeficiency).
2.5 New methods for the detection of HCV infection
With the continuous development of detection technology, rapid nucleic acid detection methods such as loop-mediated isothermal amplification technology and recombinase polymerase amplification technology have been applied to the rapid detection of HCV RNA, which have a short detection cycle, high sensitivity and specificity for HCV of different genotypes, and will not cross-react with other pathogens, so that HCV nucleic acid detection results can be obtained quickly and accurately.
In recent years, occult HCV infection has been proposed, characterized by the presence of HCV RNA in infected individuals with negative HCV RNA tests in serum samples, hepatocytes, or peripheral blood mononuclear cells (PBMCs). Digital PCR can be used to monitor changes in HCV RNA in PBMCs after treatment in infected patients who have achieved a sustained virologic response to DAA treatment and have shown that the viral load in PBMCs is very low.
3Development of HCV testing strategies
3.1 Mainland HCV infection testing process
In recent years, the World Health Organization (WHO) has recommended simplifying the testing and diagnosis process for HCV infection, recommending HCV nucleic acid (quantitative or qualitative) testing directly after a positive HCV serology test to identify chronic HCV infection. The U.S. Centers for Disease Control and Prevention has issued guidelines that state that testing for HCV infection should include testing for HCV antibodies using an FDA-approved test for HCV antibodies, followed by a nucleic acid test method for testing HCV RNA in antibody-positive individuals.
In 2023, the Chinese Center for Disease Control and Prevention (CDC) revised the Technical Specifications for Laboratory Testing of Hepatitis C Virus, which simplifies the testing process for HCV infection in mainland China, including HCV antibody screening tests and further nucleic acid tests after a positive screening result (Figure 2). Laboratory-based antibody testing is preferred for HCV antibody screening testing, and rapid testing is recommended in populations where testing is limited or where rapid availability of test results can facilitate subsequent treatment. HCV RNA testing is recommended for samples with a negative HCV antibody test in the past 6 months or at follow-up visits if the subject has a history of HCV exposure within the past 6 months, and HCV RNA testing may be considered if the participant is immunocompromised. Samples with a positive antibody test should be tested for HCV and retested with another antibody test if a false-positive antibody test result is suspected.
Note: *For individuals who may have been exposed to HCV in the past 6 months, HCV RNA testing or follow-up testing for HCV antibodies is recommended. For immunocompromised individuals, HCV RNA testing may be considered. **If a false-positive antibody test is suspected, another HCV antibody test may be used. HCV RNA testing can be done using a laboratory-based nucleic acid test or a point-of-care nucleic acid test. HCV antigen testing may also be used when HCV RNA testing is not available. Individuals who may have been exposed to HCV in the past 6 months, or who have clinical evidence of HCV infection, or who are concerned about sample quality, can repeat HCV RNA testing or follow-up observation.
Figure 2 Routine HCV testing process
HCV RNA testing can be done using a qualitative or quantitative laboratory-based test, or point-of-care testing. If the test result is negative, indicating that HCV is not present, if the subject has a history of HCV exposure in the past 6 months, clinical evidence suggests HCV infection, or if there is concern about the quality of the sample, the HCV RNA test or follow-up observation can be repeated. When HCV RNA testing is not available, HCV antigen testing can be used, and highly sensitive HCV antigen testing can be used as an alternative to HCV RNA for diagnosing acute or chronic HCV infection.
Due to the presence of mother-transmitted antibodies, HCV infection in infants and young children under 18 months of age can only be confirmed by HCV RNA testing. It is recommended to perform 2 HCV RNA tests during the 2~6 months of birth and 6 months of age in the infant, if HCV RNA is detected in both times, it indicates chronic HCV infection, if the first test is negative, it indicates that it is not infected with HCV, if HCV RNA is detected for the first time, it is not detected in the retest, and it needs to be followed up to 18 months of age, and the HCV routine testing process should be tested.
3.2 HCV核酸即时检测（POCT）
POCT refers to a type of testing method that can be performed at the bedside, in the field or at home, and can quickly obtain test results. In 2022, the World Health Organization (WHO) officially added POCT nucleic acid testing to the HCV testing process, considering it to be comparable to the detection limit of laboratory testing and can be used as an alternative to HCV RNA laboratory testing and cure testing. As of the end of November 2023, 1 HCV POCT quantitative detection product has passed WHO pre-qualification, FDA registration, CE certification and registration with the National Medical Products Administration of China.
The clinical sensitivity and specificity of POCT detection methods can reach more than 99%, and the coincidence rate between POCT and conventional nucleic acid detection methods is more than 97%, indicating that the performance of POCT and conventional nucleic acid detection methods is comparable, and it can be effectively replaced in areas where laboratory nucleic acid testing cannot be carried out, and the accessibility of HCV infection testing services can be expanded. POCT, on the other hand, can quickly return test results, and can be used in combination with HCV antibody rapid tests to diagnose current HCV infection on the same day, reducing the turnaround time from HCV antibody screening to initiation of treatment.
HCV testing and diagnostics are the entry point for HCV elimination, and studies have shown that decentralizing HCV diagnostics and other services can significantly improve the link between HCV testing and care and treatment services. Compared with laboratory nucleic acid detection methods, POCT equipment is small and portable, which can avoid the quality degradation caused by long-distance transportation of samples, help disperse HCV diagnosis, improve the detection rate of HCV, and promote the detection and treatment of HCV infected patients.
3.3 触发检测（reflex test）
In order to speed up the diagnosis of HCV infection and start treatment quickly, the latest WHO recommendation is to perform HCV RNA testing immediately after a positive HCV antibody test, that is, the test is triggered. Triggered testing is characterized by the fact that individuals can complete the diagnosis of HCV infection through one clinical visit, which is divided into laboratory-based triggered testing process and primary clinic-based triggered testing process.
Laboratory-based trigger testing process: Individuals only need to complete 1 blood sampling in 1 clinical visit. If the laboratory antibody test is positive, use the remaining sample (or aliquot sample) for follow-up HCV RNA or antigen testing. If the HCV antibody test result is positive and the HCV RNA or antigen test result is returned at the same time, no further follow-up or blood sampling is required.
Trigger testing process based on primary clinics: Individuals are required to complete 2 blood collections in 1 clinical visit. Primary clinics first use the HCV Antibody Rapid Test to test the peripheral whole blood sample, and if the test result is positive, the subject will be collected for the second time (venous blood or peripheral whole blood). The 2-time blood samples can be used for laboratory-based HCV RNA or antigen testing, and can also be used for HCV RNA point-of-care nucleic acid testing in primary clinics.
Triggering testing is necessary to achieve the goal of hepatitis C elimination. Triggered testing based on laboratories and primary clinics can increase the chances of HCV viral load testing in HCV-infected patients, minimize the possibility of detection delay and follow-up loss, improve diagnostic efficiency, and improve the clinical care and treatment of HCV RNA-positive patients. A study in the United States showed that after HCV-triggered testing was implemented in the emergency department, the proportion of HCV antibody-positive patients receiving nucleic acid testing increased from 55% to 84%, the acceptance of nucleic acid testing increased significantly, and the proportion of patients with HCV infection was diagnosed, and the triggered testing reduced the turnaround time of post-diagnosis treatment of patients, and clinical care could be initiated within 30 minutes. Another study evaluated the impact of trigger testing on patient clinical care and found a 28% increase in the proportion of patients receiving treatment after HCV-triggered testing, and a reduction in median treatment time from 71 to 52 days, significantly improving referral for patients with HCV infection.
The U.S. Centers for Disease Control and Prevention and the American Association of Public Health Laboratories have identified HCV-triggered testing as a high-priority goal in the field of HCV testing. However, due to internal constraints and the high cost of nucleic acid testing, laboratories may not be able to directly trigger testing on infected person samples. At this stage, trigger testing is still a gap in laboratory testing.
3.4 Self-testing
In 2021, the WHO recommended HCV self-testing as an additional method for HCV testing services. Self-testing is the process by which individuals collect samples for testing and read the results in a private environment to understand their infection status.
HCV self-testing process: Individuals are self-tested with HCV antibody rapid test kits, and if the test result is positive, further testing by a professionally trained tester to identify chronic HCV infection and treatment evaluation; if the test result is negative, a comprehensive determination should be made in combination with the recent exposure risk, and for people with recent potential exposure risk, re-testing should be considered, and regular re-testing should be considered if there is a persistent risk.
At present, the application experience of HCV infection self-testing is very limited, but it is a potentially important method to expand testing in the future: (1) less than 1/4 of people with chronic HCV infection know their infection status, (2) HCV self-testing can cover HCV infected patients who are not accessible by existing testing services and want privacy protection, and (3) most people at high risk of HCV infection can correctly use HCV self-testing reagents, which can help diagnose more HCV infection.
4. Outlook
Chronic HCV infection is a growing global public health problem. Early diagnosis and timely treatment of HCV infection is an important means to stop the progression of chronic HCV infection to cirrhosis and HCV-associated hepatocellular carcinoma. With the continuous development of detection technology and the promotion and implementation of new detection strategies, laboratory testing for HCV infection has been steadily developing in the direction of more sensitive, faster and more accurate. However, the current laboratory testing for HCV infection still faces challenges. First of all, the proportion of patients with chronic HCV infection who are diagnosed and treated is very low, and there are still nearly 10,000 new HCV infections in the world every year. On the other hand, how to integrate existing testing technologies and testing strategies in the routine testing of HCV infection, and simplify the process of HCV laboratory testing, so as to shorten the turnaround time for determining current HCV infection and reduce the proportion of infected people lost to follow-up.
Antiviral treatment for patients with chronic HCV infection has entered the pan-genotype era of DAA, with more than 95% of hepatitis C patients cured, and there will be more new laboratory testing technologies and strategies to help achieve the strategic goal of eliminating viral hepatitis by 2030. At present, point-of-care nucleic acid testing technology can greatly shorten the turnaround time of testing, diagnose HCV infection on the same day, understand the effect of antiviral treatment in time, detect drug resistance, and improve treatment compliance. Triggering testing can reduce the number of clinical visits of infected patients, increase the chances of HCV infected patients to obtain nucleic acid testing, timely detect current HCV infection, increase the proportion and efficiency of diagnosis of HCV infected patients, improve the clinical care and treatment of patients, and reduce loss to follow-up. Self-testing can help improve the willingness of the tested to take the initiative to test, expand the coverage of testing, and promote early diagnosis and early treatment of individuals. The detection of multiple pathogens through one test can expand the proportion of testing and detect possible HCV infection as soon as possible.
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引证本文 Citation
WANG Yu, XING Wenge, LIU Zhongfu, et al . Laboratory testing methods and strategies for hepatitis C virus infection[J]. Journal of Clinical Hepatobiliary Diseases, 2024, 40(4): 672-678
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Journal of Clinical Hepatobiliary Diseases, Issue 4, 2024 "Elimination of Hepatitis C Virus Infection" (Executive Editor-in-Chief: Chen Hongsong/Rao Huiying)
Rao Huiying/Chen Hongsong: Current status and elimination process of hepatitis C virus infection in China
Expert Forum: Management of hepatitis C in pregnant women and children
Zuo Li: Elimination of hepatitis C virus infection in the hemodialysis population
Hu Peng: Hepatitis C virus infection and organ transplantation
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