[Strengthening the study of chronic hepatitis E].

This paper summarizes the incidence, modes of transmission, diagnosis, treatment and prevention of chronic hepatitis E.

Rapid tests for HIV, syphilis, and chronic hepatitis in a prison population in a prison complex in Salvador (BA), Brazil. / Testes rápidos de HIV, sífilis e hepatites crônicas na população carcerária em um complexo penitenciário de Salvador (BA), Brasil.

This study aimed to quantitatively analyze the results of rapid tests for Human Immunodeficiency Virus (HIV), Syphilis, and Chronic Hepatitis in the prison population in a prison complex in Salvador (BA), Brazil. This cross-sectional study consisted of a sample of men incarcerated from August 2018 to August 2020 submitted to rapid tests. Descriptive statistics and prevalence ratios with respective 95% confidence intervals were employed to analyze data. A total of 6,160 men were studied. Most were black and brown (93.1%) and resided in Salvador (BA), Brazil (65.8%), with predominantly elementary schooling level (65.3%). Five hundred eighty-one (9.4%) people deprived of their liberty were positive for one or more STIs, and Syphilis was the most prevalent (80%). The variables age greater than 25 years [PR = 1.37 95%CI (1.17-1.61)] and schooling level without Higher Education [PR = 2.16 95%CI (1.04-4.49)] were associated with a higher positivity rate in tests, while not sharing drugs was a protective factor for test positivity [PR = 1.28 95%CI (1.07-1.53)]. We concluded that there was a low prevalence of STIs in the sample studied, and Syphilis was the most prevalent.

O objetivo do estudo foi analisar quantitativamente resultados de testes rápidos de vírus da imunodeficiência humana (HIV), sífilis e hepatites crônicas na população carcerária em complexo penitenciário de Salvador (BA). Trata-se de um estudo transversal. A amostra foi composta por homens privados de liberdade no período de agosto de 2018 a agosto de 2020, com testes rápidos sendo realizados. Para análise dos dados foi utilizada estatística descritiva e razão de prevalência com os respectivos intervalos de confiança de 95%. Foram estudados 6.160 homens, com maioria (93,1%) de pretos e pardos, residentes de Salvador (65,8%), com escolaridade predominante de ensino fundamental (65,3%). Das pessoas privadas de liberdade, 581 (9,4%) obtiveram resultado reagente para uma ou mais IST, sendo sífilis a mais prevalente (80%). As variáveis idade maior de 25 anos [RP = 1,37 IC95% (1,17-1,61)] e nível de escolaridade sem presença de ensino superior [RP = 2,16 IC95% (1,04-4,49)] se mostraram associadas à maior taxa de positividade nos testes, enquanto o não compartilhamento de drogas em algum momento da vida mostrou ser fator protetor à positividade nos testes [RP = 1,28 IC95% (1,07-1,53)]. Conclui-se que existiu uma baixa prevalência das IST na amostra estudada, sendo sífilis a mais prevalente.

Serum protein electrophoresis in 26 dogs with chronic hepatitis.

Serum protein electrophoresis (SPE) shows the distribution of protein fractions, helping clinicians to characterize some pathologic processes. Information is lacking in the literature about SPE alterations in dogs with chronic hepatitis (CH). Our aim was to describe SPE alterations in canine CH, to compare SPE results to histologic scores, and to study SPE trends during follow-up. We reviewed retrospectively case data from dogs with a histologic diagnosis of CH. Only cases with SPE, CBC, and serum chemistry results available were included. Dogs were divided into subgroups based on histologic necroinflammatory activity (A) and fibrosis (F) scores (groups A0-1 and A>1; groups F<2 and F≥2). We included 26 dogs; 15 had follow-up SPE. The most common SPE alterations at admission were hypoalbuminemia (n = 16), increases in α1-globulins (n = 11), γ-globulins (n = 11), α2-globulins (n = 8), ß2-globulins (n = 7), and ß1-globulins (n = 6), and decreased albumin:globulin (A:G) ratios (n = 20). Four of 11 dogs had ß-γ bridging. Groups with higher A and F scores had higher ß2-globulins. Eleven of 15 dogs with a post-treatment SPE had a decrease in γ-globulins and increase in A:G ratio compared to their T0, although there was no statistically significant difference. Although further studies are warranted, SPE may be useful for monitoring canine CH.

Chronic hepatitis E: Advancing research and patient care.

The hepatitis E virus (HEV) was initially thought to exclusively cause acute hepatitis. However, the first diagnosis of chronic hepatitis E in transplant recipients in 2008 profoundly changed our understanding of this pathogen. We have now begun to understand that specific HEV genotypes can cause chronic infection in certain immunocompromised populations. Over the past decade, dedicated clinical and experimental research has substantiated knowledge on the epidemiology, transmission routes, pathophysiological mechanisms, diagnosis, clinical features and treatment of chronic HEV infection. Nevertheless, many gaps and major challenges remain, particularly regarding the translation of knowledge into disease prevention and improvement of clinical outcomes. This article aims to highlight the latest developments in the understanding and management of chronic hepatitis E. More importantly, we attempt to identify major knowledge gaps and discuss strategies for further advancing both research and patient care.

Advanced sequencing approaches detected insertions of viral and human origin in the viral genome of chronic hepatitis E virus patients.

The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. There, infections with HEV-genotype 3 (HEV-3) are predominant which can progress to chronicity in immunocompromised individuals. Persistent infection and antiviral therapy can select HEV-3 variants; however, the spectrum and occurrence of HEV-3 variants is underreported. To gain in-depth insights into the viral population and to perform detailed characterization of viral genomes, we used a new approach combining long-range PCR with next-generation and third-generation sequencing which allowed near full-length sequencing of HEV-3 genomes. Furthermore, we developed a targeted ultra-deep sequencing approach to assess the dynamics of clinically relevant mutations in the RdRp-region and to detect insertions in the HVR-domain in the HEV genomes. Using this new approach, we not only identified several insertions of human (AHNAK, RPL18) and viral origin (RdRp-derived) in the HVR-region isolated from an exemplary sample but detected a variant containing two different insertions simultaneously (AHNAK- and RdRp-derived). This finding is the first HEV-variant recognized as such showing various insertions in the HVR-domain. Thus, this molecular approach will add incrementally to our current knowledge of the HEV-genome organization and pathogenesis in chronic hepatitis E.

Diagnostic value of gamma-glutamyl transpeptidase to alkaline phosphatase ratio combined with gamma-glutamyl transpeptidase to aspartate aminotransferase ratio and alanine aminotransferase to aspartate aminotransferase ratio in alpha-fetoprotein-negative hepatocellular carcinoma.

BACKGROUND: The purpose of the study was to evaluate the diagnostic value of gamma-glutamyl transpeptidase to alkaline phosphatase ratio (GAPR) combined with gamma-glutamyl transpeptidase to aspartate aminotransferase ratio (GAR) and alanine aminotransferase to aspartate aminotransferase ratio (AAR) in alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). METHODS: A total of 925 AFP-negative patients, including 235 HCC patients, 213 chronic hepatitis (CH) patients, and 218 liver cirrhosis (LC) patients, as well as 259 healthy controls were enrolled in this study. The differences of laboratory parameters and clinical characteristics were analyzed by Mann-Whitney U or Kruskal-Wallis H-test. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of GAPR, GAR, and AAR in AFP-negative HCC (AFP-NHCC) patients. RESULTS: GAPR, GAR, and AAR were important parameters closely related to AFP-NHCC. The combination of GAPR, GAR, and AAR was most effective in differentiating AFP-NHCC group from control group (AUC = 0.875), AFP-negative CH group (AUC = 0.733), and AFP-negative LC group (AUC = 0.713). GAPR combined with GAR and AAR exhibited a larger AUC than single ratio or pairwise combination for distinguishing AFP-NHCC group with TNMⅠstage, BCLC stage A, and tumor size less than 3 cm. The diagnostic value of GAPR combined with GAR and AAR was higher in AFP-NHCC and was also reflected in the TNM stage, Barcelona Clinic Liver Cancer (BCLC) stage and tumor size. CONCLUSIONS: GAPR combined with GAR and AAR were effective diagnostic markers of AFP-NHCC, especially in patients with good liver function, early stage or small size.

Differences in autoimmune hepatitis based on inflammation localization.

Histopathology is essential for the diagnosis and evaluation of disease activity of autoimmune hepatitis (AIH). We aimed to elucidate the characteristics of AIH from the localization of inflammation. We re-evaluated a nationwide survey that was performed in Japan in 2018 of AIH patients diagnosed between 2014 and 2017. A total of 303 patients were enrolled, and the clinical and treatment characteristics were compared between the patients with predominantly portal inflammation (230 patients) or lobular inflammation (73 patients). AIH patients with lobular inflammation had a higher probability of being diagnosed with acute hepatitis than those with portal inflammation. Liver enzyme levels were higher in patients with lobular inflammation, whereas immunoglobulin G levels were higher in patients with portal inflammation. The prevalence of an alanine aminotransferase level < 30 U/L after 6 months of treatment was significantly higher in patients with lobular inflammation than in those with portal inflammation (81.7% vs. 67.3%, P = 0.046). The localization of inflammation may be useful for evaluating the onset of AIH.

Canine copper-associated hepatitis: A retrospective study of 17 clinical cases.

Background: Copper-associated hepatitis (CAH) is a well-documented chronic hepatic disease in dogs. In some breeds, the disease results from an inherited defect in copper metabolism. In others, it is unclear whether its acummulation is a primary or secondary condition. Reports of copper accumulation in dog breeds that are not genetically predisposed are increasing. Aim: To describe the epidemiology, clinical and laboratory findings, liver biopsy techniques, and treatment response in dogs with CAH. Methods: A retrospective study was performed, drawing upon medical records from CAH dogs at a Veterinary Referral Hospital in Paris, France. The diagnosis of CAH had been confirmed in these patients by positive rhodanine staining of hepatic tissue obtained through biopsy. Medical records were mined for the following data: age at presentation, sex, breed, chief presenting complaints, abdominal ultrasound (US) findings, and rhodanine staining pattern. Results: A total of 17 dogs were included in the study. Median age at presentation was 8-year old (4-11). No sex predisposition was found. Terriers (4/17) and German Shepherd Dogs (GSD, 3/17) were overrepresented. American Staffordshire Terriers and Beauceron had not previously appeared in case reports on CAH; two of each breed were identified in this study. Clinical signs of affected dogs were non-specific. An incidental identification of increased liver-enzymes was observed in 5/17 dogs. A heterogeneous, mottled liver was frequently described (5/17) on abdominal US. Liver biopsies were performed by US-guided percutaneous approach in 10/17 dogs, laparoscopy and laparotomy in 6/17 and 1/17, respectively. The rhodanine staining pattern was centrilobular (zone 3) in 8/17 dogs and periportal (zone 1) in 3/17 dogs. The pattern was considered multifocal in 6/17 dogs. Conclusion: Increased liver enzymes may be the only clinical finding in dogs with copper-associated hepatitis, reflecting the silent progression of this disease. Centrilobular pattern of rhodanine staining was observed in the majority of cases suggesting the primary condition of the disease. Results of this study are consistent with the current literature, which reports that terriers and GSD are predisposed to CAH. This is the first description of CAH in Beauceron and American Staffordshire Terrier dogs.

aMAP risk score predicts hepatocellular carcinoma development in patients with chronic hepatitis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with chronic hepatitis. In this international collaboration, we sought to develop a global universal HCC risk score to predict the HCC development for patients with chronic hepatitis. METHODS: A total of 17,374 patients, comprising 10,578 treated Asian patients with chronic hepatitis B (CHB), 2,510 treated Caucasian patients with CHB, 3,566 treated patients with hepatitis C virus (including 2,489 patients with cirrhosis achieving a sustained virological response) and 720 patients with non-viral hepatitis (NVH) from 11 international prospective observational cohorts or randomised controlled trials, were divided into a training cohort (3,688 Asian patients with CHB) and 9 validation cohorts with different aetiologies and ethnicities (n = 13,686). RESULTS: We developed an HCC risk score, called the aMAP score (ranging from 0 to 100), that involves only age, male, albumin-bilirubin and platelets. This metric performed excellently in assessing HCC risk not only in patients with hepatitis of different aetiologies, but also in those with different ethnicities (C-index: 0.82-0.87). Cut-off values of 50 and 60 were best for discriminating HCC risk. The 3- or 5-year cumulative incidences of HCC were 0-0.8%, 1.5-4.8%, and 8.1-19.9% in the low- (n = 7,413, 43.6%), medium- (n = 6,529, 38.4%), and high-risk (n = 3,044, 17.9%) groups, respectively. The cut-off value of 50 was associated with a sensitivity of 85.7-100% and a negative predictive value of 99.3-100%. The cut-off value of 60 resulted in a specificity of 56.6-95.8% and a positive predictive value of 6.6-15.7%. CONCLUSIONS: This objective, simple, reliable risk score based on 5 common parameters accurately predicted HCC development, regardless of aetiology and ethnicity, which could help to establish a risk score-guided HCC surveillance strategy worldwide. LAY SUMMARY: In this international collaboration, we developed and externally validated a simple, objective and accurate prognostic tool (called the aMAP score), that involves only age, male, albumin-bilirubin and platelets. The aMAP score (ranged from 0 to 100) satisfactorily predicted the risk of hepatocellular carcinoma (HCC) development among over 17,000 patients with viral and non-viral hepatitis from 11 global prospective studies. Our findings show that the aMAP score had excellent discrimination and calibration in assessing the 5-year HCC risk among all the cohorts irrespective of aetiology and ethnicity.

The challenge of chronic hepatitis E in liver transplant recipients: Failure of sofosbuvir plus ribavirin therapy / Problema de la hepatitis E crónica en los receptores de trasplante de hígado: fracaso del tratamiento con sofosbuvir más ribavirina

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RNA Profiling Analysis of the Serum Exosomes Derived from Patients with Chronic Hepatitis and Acute-on-chronic Liver Failure Caused By HBV.

Hepatitis B virus (HBV) is the main causative viral agent for liver diseases in China. In liver injury, exosomes may impede the interaction with chromatin in the target cell and transmit inflammatory, apoptosis, or regeneration signals through RNAs. Therefore, we attempted to determine the potential functions of exosomal RNAs using bioinformatics technology. We performed RNA sequencing analysis in exosomes derived from clinical specimens of healthy control (HC) individuals and patients with chronic hepatitis B (CHB) and acute-on-chronic liver failure caused by HBV (HBV-ACLF). This analysis resulted in the identification of different types and proportions of RNAs in exosomes from the HC individuals and patients. Exosomes from the CHB and HBV-ACLF patients showed distinct upregulation and downregulation patterns of differentially expressed genes compared with those from the HC subjects. Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway analysis further confirmed different patterns of biological functions and signalling pathways in CHB and HBV-ACLF. Then we chose two upregulated RNAs both in CHB and HBV-ACLF for further qPCR validation. It confirmed the significantly different expression levels in CHB and HBV-ACLF compared with HC. Our findings indicate selective packaging of the RNA cargo into exosomes under different HBV attacks; these may represent potential targets for the diagnosis and treatment of HBV-caused liver injury.

Four-year long (2014-2017) clinical and laboratory surveillance of hepatitis E virus infections using combined antibody, molecular, antigen and avidity detection methods: Increasing incidence and chronic HEV case in Hungary.

BACKGROUND: Hepatitis E virus (HEV) is a pathogen of viral hepatitis. Since 2006, the number of reported HEV cases has ten-fold increase in Hungary. OBJECTIVES: The aim of this clinical and laboratory surveillance study was to analyse and confirm HEV IgM-positive sera with different methods in four consecutive years (2014-2017) in Hungary. STUDY DESIGN: Between 2014 and 2017, a total of 1439 sera samples were tested for HEV from in/out-patients with unknown hepatitis from university and county hospitals and general practitioners from three counties in Southwest Hungary (covered population: Σ894.000 persons) using combined antibody (serology), various molecular (RT-PCR and RT-qPCR), novel antigen (Ag) and avidity detection methods. RESULTS: Total of 162 (11.3%) of the 1439 sera were HEV IgM-positive including 13 (8%) HEV RT-PCR-positive (confirmed as HEV genotype 3 sub-genotypes 3a/c/e/f/i in genus Orthohepevirus A) with up to 1.1383 × 108 RNA copy/ml, 30 (18.5%) HEV Ag-positive and 16 with low avidity index for HEV, respectively. Total of 6 samples were positive simultaneously with the combined four methods and 31 with three methods. If the quotient of serum sample's OD/cut-off of anti-HEV ELISA IgM and IgG scores is higher than ≥1 it predisposes for acute HEV infection. No rat or ferret HEV RNA (genus Orthohepevirus C) were identified from these specimens by RT-PCR. During our surveillance period a 68-year-old professional (meat-packing) hunter with kidney transplantation and immunosuppressive therapy was confirmed and treated as the first documented case of chronic HEV infection in Hungary. CONCLUSION: This four-year-long clinical and laboratory surveillance highlights the increasing importance of acute and chronic HEV infections in Hungary and supports the use of confirmatory assays for laboratory diagnosis of HEV in human.

Use of the ELISA (Em2-Em18) and Western Blotting Methods on Diagnosis of Alveolar Echinococcosis

Objective: Alveolar echinococcosis (AE) is one of the most lethal parasitic zoonoses in the Northern Hemisphere, and early serological detection is important to start treatment and to improve survival. A total of 50 sera samples of patients diagnosed as having various diseases were examined for by two different serological diagnostic methods. Methods: Em2-Em18 ELISA (Bordier Affinity Products, Crissier, Switzerland) and Echinococcus Western Blot immünoglobulin G (IgG) (LDBIO Diagnostics, Lyon, France) were used for analyisis. Results: A high titer of antibodies was found in 9 of 10 patients diagnosed as having AE with Em2-Em18 ELISA, in 2 of 21 patients with cystic echinococcosis, in 1 of 2 patients with fascioliasis and in 1 patient with chronic hepatitis. The Echinococcus Western Blot IgG test, used as a confirmatory test, showed IgG antibody in 85.7% (18/21) of patients with CE, while all serum samples of 10 patients with AE were evaluated as positive. This method yielded an incorrect diagnosis in the patient with chronic hepatitis and in the patient with granulomatous inflammation with caseification. Samples taken from patients with liver-related diseases and other parasitic-related diseases were found to be negative. Conclusion: The serological methods used in the study were found to be important in the early diagnosis of alveolar echinococcosis in the endemic areas, since it could be used in sero-epidemiological studies.

Chronic hepatitis E after kidney transplantation with an antibody response suggestive of reinfection: a case report.

BACKGROUND: Hepatitis E virus (HEV) infection is now recognized as a major cause of acute hepatitis worldwide. HEV specific antibodies develop shortly after infection and are thought to confer protection. CASE PRESENTATION: We report an immunocompromised patient who developed chronic HEV infection despite the presence of high level antibodies. HEV infection was detected using RT-PCR upon diagnostic evaluation due to increased liver enzymes. Upon retrospective analysis of stored serum samples we found that the patient was HEV RNA positive since 7 months. Chronic HEV infection was successfully treated with ribavirin. CONCLUSIONS: In conclusion, the patient suffered from a chronic course of HEV infection, which was successfully treated with ribavirin. Our case underlines the importance of RT-PCR for HEV diagnostics in immunosuppressed patients and supports the notion that HEV antibodies do not confer universal protection. Counseling patients at risk for chronic HEV infection seems advisable. The role of the humoral and T-cell mediated immune response in cases of HEV reinfection deserves further study.

El tratamiento antiviral no mejora la ateromatosis subclínica en pacientes con hepatitis crónica por virus de la hepatitis C / Antiviral treatment does not improve subclinical atheromatosis in patients with chronic hepatitis caused by hepatitis C virus

Introducción: La infección crónica por el virus de la hepatitis C (VHC) es un factor de riesgo para desarrollar placas de ateroma, aunque se desconoce el posible efecto al eliminar el virus. Nuestro objetivo fue analizar si tras 12 meses de la erradicación del VHC por antivirales de acción directa (AAD) mejoraba la ateromatosis subclínica y existía modificación en la composición de las placas. Materiales y métodos: Estudio prospectivo que incluyó 85 pacientes con infección crónica por VHC en diferentes estadios de fibrosis, sometidos a AAD. Se excluyeron pacientes con antecedentes cardiovasculares, diabetes y enfermedad renal. Se realizó ecografía arterial (carótidas y femorales) para diagnosticar placa de ateroma (definida como grosor íntima-media≥1,5mm) y se analizó su composición (porcentaje de lípidos, fibrosis y calcio con software HEMODYN4) al inicio del estudio y tras 12 meses de finalizar la terapia. Resultados: Tras el seguimiento no se detectaron cambios en el grosor íntima-media (0,65mm vs. 0,63mm, p=0,240) ni en la presencia de placas (65,9%vs. 71,8%, p=0,063). Tampoco hubo modificación significativa en la composición de las mismas ni del territorio vascular afecto, observándose un aumento del perfil lipídico en sangre (p<0,001) tras 12 meses del tratamiento. Estos resultados se confirmaron en subgrupos por gravedad de enfermedad hepática. Discusión: La erradicación del VHC por AAD no mejora las placas de ateroma ni varía su composición, independientemente de la fibrosis hepática. Se precisan más estudios prospectivos que evalúen el riesgo residual cardiovascular tras la erradicación viral

Introduction: Chronic infection with hepatitis C virus is a risk factor for developing atheromatous plaques, although the possible effect of virus clearance is unknown. Our aim was to determine whether or not subclinical atheromatosis improved and there was any modification in the composition of the plaques 12 months after eradication of hepatitis C virus by direct-acting antiviral agents. Materials and methods: Prospective study that included 85 patients with chronic hepatitis C virus infection in different stages of fibrosis who were on direct-acting antiviral agents. Patients with a cardiovascular history, diabetes and kidney disease were excluded. An arterial ultrasound (carotid and femoral) was performed to diagnose atheromatous plaques (defined as intima-media thickness ≥1.5mm) and the composition (percentage of lipids, fibrosis and calcium with HEMODYN4 software) was analysed at the beginning of the study and 12 months after stopping the therapy. Results: After follow-up no changes were detected in the intima-media thickness (0.65mm vs. 0.63mm, P=.240) or in the presence of plaques (65.9% vs 71.8%, P=.063). There was also no significant change in their composition or affected vascular territory, with an increase in blood lipid profile (P<.001) after 12 months of treatment. These results were confirmed in subgroups by severity of liver disease. Discussion: The eradication of hepatitis C virus by direct-acting antiviral agents does not improve the atheroma plaques and nor does it vary their composition, regardless of liver fibrosis. More prospective studies are needed to evaluate residual cardiovascular risk after virus eradication

Development and potential application of a simultaneous multiplex assay of Golgi protein 73 and alpha-fetoprotein for hepatocellular carcinoma diagnosis.

OBJECTIVE: Detecting a single serum marker, such as Golgi protein 73 (GP73) or alpha-fetoprotein (AFP), may not meet the requirements for the early diagnosis of hepatocellular carcinoma (HCC) due to low sensitivity and specificity. Therefore, this study aimed to develop a simultaneous multiplex assay of GP73 and AFP. PATIENTS AND METHODS: Anti-human GP73- and AFP-coupled microsphere beads and biotin-labeled detectable antibodies were prepared to develop a multiplex assay of GP73 and AFP using the Luminex xMAP technology. The assay was evaluated for cross-reactivity, standard curve, sensitivity, range of detection, and precision. Additionally, the assay was used to determine the levels of serum GP73 and AFP in healthy controls and patients with chronic hepatitis, liver cirrhosis, and HCC. RESULTS: The multiplex assay was successfully developed to simultaneously detect GP73 and AFP without cross-reactivity. The sensitivity for GP73 detection was 0.215 ng/mL and that for AFP detection was 0.666 ng/mL. The ranges of GP73 and AFP detection were 0.98-861.08 ng/mL and 2.01-1848.73 ng/mL, respectively. The intra- and inter-assay coefficients of variation (CVs) were <10%, indicating good precision, with recovery rates of 75-125%. The levels of serum GP73 in healthy controls, chronic hepatitis patients, liver cirrhosis patients, and HCC patients were 61.64 ± 30.60 ng/mL, 208.4 ± 99.42 ng/mL, 183.7 ± 82.78 ng/mL, and 214.1 ± 160.5 ng/mL, respectively. The levels of serum AFP in healthy controls, chronic hepatitis patients, liver cirrhosis patients, and HCC patients were 24.87 ± 14.52 ng/mL, 134.4 ± 216.5 ng/mL, 66.45 ± 133.4 ng/mL, and 891.4 ± 1278 ng/mL, respectively. The receiver operating characteristic (ROC) results showed that the area under the curves (AUC) for the combination of GP73 and AFP was 0.972, which was larger than the AUC for each marker. The sensitivity and specificity of the combined detection of GP73 and AFP for the diagnosis of HCC were 90.91% and 98.86%, respectively. The multiplex assay demonstrated a good correlation with enzyme-linked immunosorbent assay (ELISA), with correlation coefficients of 0.818 and 0.982 for GP73 (p<0.001) and AFP (p<0.001), respectively. CONCLUSIONS: A multiplex assay for the simultaneous detection of GP73 and AFP with high sensitivity and accuracy was developed for the diagnosis of HCC. This assay may provide a reliable reference for the early diagnosis of HCC.