Anti‐ENA antibody profiles in patients with hepatitis C virus infection

Abstract

Background

The presence of antinuclear antibodies (ANA) has been described following hepatitis C virus (HCV). Very few studies have investigated the presence of anti‐extractable nuclear antigens (ENA) in HCV infection.

Methods

The aim of this study was to assess the prevalence of ENA antibodies in 100 patients with HCV infection compared to the prevalence of ENA antibodies in 100 healthy control patients. Sera from patients were tested for ENA using a multiplex microbead immunoassay. Sera positive for ENA were subsequently tested for ANA using an indirect immunofluorescence assay and titered if positive.

Results

Fourteen (14%) of the 100 patients with HCV infection had anti‐ENA antibodies: four each showed anti‐SSA antibodies and anti‐dsDNA antibodies, three each had RNP antibodies and Scl‐70 antibodies, and one each had anti‐SSB, centromere B, Sm, and Sm/RNP antibodies. Ten of the 14 patients positive for anti‐ENA were positive by indirect immunofluorescence staining (IFA) with titers ranging from 1:40 to 1:160. Five had antinuclear patterns, one had combined antinuclear and cytoplasmic patterns, and four only had a cytoplasmic pattern. Three of the 100 healthy control patients had ANA positive titers (1:80 and 1:320) and anti‐ENA antibodies: one anti‐Scl‐70 and two anti‐RNP.

Conclusion

The prevalence of anti‐ENA antibodies was significantly higher in the patients with HCV infections than in the healthy controls. Other studies of anti‐ENA profiles in patients with HCV infection have identified similar patterns of positivity for anti‐SSA, anti‐SSB, anti‐dsDNA, anti‐RNP, anti‐ Sm/RNP, Scl‐70, centromere B, and anti‐Sm.

1. INTRODUCTION

Autoantibody testing is performed to help diagnose patients with clinical symptoms indicative of possible autoimmune diseases. Antinuclear antibodies (ANA) are present in many systemic autoimmune conditions such as systemic lupus erythematosis (SLE).1 However, a positive ANA test may also be seen with nonautoimmune inflammatory diseases, including infections.

Hepatitis C virus (HCV) infection is often associated with extrahepatic symptoms such as arthralgias, arthritis, vasculitis, and sicca syndrome that could signify a rheumatic disease.2 Autoantibodies are commonly found in patients with HCV. Several investigators have studied whether patients with HCV and with a positive ANA result have a different disease profile,3, 4 while others have concluded that the ANA positivity is an immunological epiphenomenon that has no influence on the response to therapy or histology.5, 6 However, reports of ANA prevalence range vary widely from 3% to 41%. The most useful studies of hepatitis C and ANA positivity, however, are those that directly compare chronic hepatitis C patients to control groups using the same methodology.7, 8, 9 In these studies, the ANA positivity rate in the HCV patient group varied from 12%‐17.6% vs 3%‐4% in the control group. All of these studies, however, used the standard method indirect immunofluorescence assay (IFA) as the methodology to screen for the detection of ANA.

A positive ANA screening by IFA often leads to further laboratory testing to detect the presence of antibodies, such as anti‐dsDNA antibodies or anti‐extractable nuclear antigens (anti‐ENA) antibodies, useful for the diagnosis of autoimmune diseases. Recently, many clinical diagnostic laboratories have introduced multiplex immunoassays for simultaneous multiple analysis to determine a profile of multiple antibodies for detecting ANA including the dsDNA and the anti‐ENA antibodies. There are only a few studies that have investigated the anti‐ENA profiles of patients with HCV infection. These studies used standard enzyme‐linked immunoassay (ELISA) and recombinant immunoblot assays to measure the anti‐ENA antibodies. Lobreglio et al., Garcia‐Carrasco et al., and D'Amico et al. detected anti‐SSA and anti‐SSB antibodies in patients with HCV infection.10, 11, 12 Batchoun additionally reported a high prevalence of anti‐ENA antibodies in patients infected with HCV who were undergoing hemodialysis. The prevalence of anti‐ENA antibodies was significantly higher in patients with HCV infection undergoing hemodialysis, compared to hepatitis C‐positive blood bank donors.13

In this study, we aim to further analyze the profile of autoantibodies in hepatitis C‐positive patients compared to a healthy control population using a new multiplex immunoassay that detects multiple autoantibodies simultaneously.

2. MATERIALS AND METHODS

2.1. Human sera

Between April 14, 2016 and July 5, 2016, a total of 100 serum samples positive for HCV IgG and 100 serum samples negative for HCV IgG were tested for anti‐ENA. ANA testing was performed on samples positive for anti‐ENA. The electronic medical record was reviewed on the HCV IgG samples to exclude samples from patients with a history of infection with other hepatitis viruses, other recent infections, and a prior diagnosis of autoimmune disease. For the 100 consecutive serum samples negative for HCV IgG, the electronic medical record was reviewed to exclude samples from patients with medical illnesses including HIV infection, recent acute viral or bacterial infection, history of hepatitis or liver disease, autoimmune, rheumatologic, inflammatory, or chronic kidney diseases.

Procedures were followed in accordance with ethical standards established by the Medical University of South Carolina (MUSC) in accordance with the Helsinki Declaration of 1975. The protocol used was approved by the MUSC Institutional Review Board (no. 54963) to meet the Health Information Portability and Accountability Act guidelines. Specimens were stored at 2‐8°C and run within a few days of receipt for the BioPlex anti‐ENA testing or ANA testing.

2.2. Screening for hepatitis C virus infection by serology

Hepatitis C virus antibodies IgG and IgM were measured using the Abbott EIA, a chemiluminescent microparticle immunoassay (CMIA) performed on the Abbott ARCHITECT i2000 SR Immunassay System (Abbott Laboratories, Abbott Park, IL, USA). Specimens with signal to cut‐off ratio (S/CO) values ≥1.00 are considered reactive by the ARCHITECT anti‐HCV assay.

2.3. HCV quantitation

Hepatitis C virus quantitation of RNA was performed on the Abbott m2000 Real Time System (Abbott Park, IL, USA) using real‐time polymerase chain reaction (PCR).

2.4. ANA testing

Antinuclear antibodies were performed according to the manufacturer's instructions with a 1:40 screening dilution using the Kallestad HEp‐2 Cell Line Substrate (Bio‐Rad Laboratories, Hercules, CA, USA).

All specimens displaying greater than 1+ fluorescence at the 1:40 screening dilution were titered to endpoint. Both antinuclear and cytoplasmic observations of fluorescence were reported.

2.5. ENA profile

The BioPlex 2200 (BioPlex) system (Bio‐Rad Laboratories) was used to measure a panel of autoantibodies (“ANA screen”) as according to the manufacturer's instructions. The panel includes autoantibodies to dsDNA, chromatin, ribosomal P, SSA/Ro (52 kDa and 60 kDa), SSB/La, Sm, Sm/RNP, RNP (A and 68), Scl‐70, Jo‐1, and centromere B. Autoantibody specificities were reported as positive using a cut‐off level of antibody that corresponded to the 99th percentile of values obtained from normal controls provided by the manufacturer.

The cut‐off level for negative results for autoantibodies to dsDNA was ≤4 Index Units (IU)/mL, indeterminate 5‐9 IU/mL, and positive ≥10 IU/mL. The cut‐off level for negative results for the ENA autoantibodies was <1.0 Antibody Index (AI)/mL with positive results ≥1.0 AI/mL.

2.6. Statistical analysis

Comparisons of groups were performed using a Yates' corrected chi‐square test.

Additional statistical analyses were performed using an Excel spreadsheet (Microsoft Corp., Redmond, WA, USA) and SPSS software (SPSS Inc. Chicago, IL, USA).

3. RESULTS

3.1. Prevalence of anti‐ENA antibodies in patients positive for HCV antibodies

Fourteen of the 100 HCV antibody‐positive patients (14.0%) were also positive for anti‐ENA antibodies (Table 1), while only three of the 100 HCV antibody‐negative patients were positive for anti‐ENA antibodies (P=.012). The mean age of all the HCV antibody‐positive patients was 49. 4 (±16.0 SD) and was not statistically different than the mean age 55.1 (±14.8 SD) for patients positive for both HCV antibodies and anti‐ENA antibodies (P>.05) (Table 2). The mean age of the healthy controls was 35.2 (±19.2 SD), while the mean age of the three healthy controls that were positive for anti‐ENA antibodies was 26.0 (±11.8 SD).

Table 1.

Demographic and laboratory features of HCV‐positive patients positive for anti‐ENA and or ANA

	Age	Gender	RNA	Genotype	HCV	IFA	Pattern	Sm	Sm/RNP	RNP	SSA	SSB	Scl‐70	dsDNA	CentB	Major diagnosis
1	61	M	Pos	NP†	16.12	1:40	Cytoplasmic fiber pattern	1.1 Lowa			1.0 Low					Chronic HCV Hepatocellular Carcinoma
2	67	M	Pos	3	12.59	1:40	Homogenous		1.0 Low	3.4 Mod.				22 Low		Chronic kidney disease
3	65	M	Pos	NP	11.58	1:160	Speckled			3.8 Mod.						Chronic HCV
4	62	M	Pos	1a	13.02	Neg	N/A			1.0 Low						Chronic HCV
5	60	M	Pos	1a	14.51	Neg	N/A				1.0 Low					Chronic HCV
6	53	M	NP	NP	10.01	Neg	N/A				3.2 Mod.					Trauma/Vehicular
7	62	F	Neg	NP	2.22	1:80	Speckled cytoplasm				7.1 High					Hypertension
8	62	M	Pos	3	6.56	1:40	Cytoplasmic fiber pattern					3.8 Mod.				Chronic HCV Hepatocellular Carcinoma
9	65	M	NP	NP	2.32	Neg	N/A						1.6 Low	1.7 Low		Gall stones
10	17	F	NP	NP	1.1	1:80	Speckled						1.2 Low			Arthritis
11	51	F	Pos	1a	12.81	1:80	Cytoplasmic fiber pattern						5.3 High			Heart failure
12	53	M	Pos	3	12.1	1:40	Speckled							28 Low		Chronic HCV
13	28	F	Pos	1a	16.08	1:40	Homogenous							27 Low		Trauma/Vehicular
14	65	M	Neg	NP	13.61	1:160	Centromere with cytoplasmic fiber pattern								1.0 Low	Chronic kidney disease
15	58	M	Pos	1b	5.92	1:160	Speckled									Chronic HCV

NP, Not performed; N/A, Not applicable.

^aLow=Low Positive, Mod.=Moderate Positive, High=High Positive.

Table 2.

Clinical characteristics, HCV antibody levels and specific anti‐ENA antibodies in HCV‐positive and healthy control patients

Patient groups	N	Age (mean±SD)	M/F	HCV antibody ratio (mean±SD)	Antibody [N (%)]
					Anti‐SSA	Anti‐RNP	Scl‐70
HCV antibody positive	100	49.4±16	1.5	11.2±3.8	4 (4%)	3 (3%)	3 (3%)
HCV and anti‐ENA positive	14	55.1±14.8	2.5	10.3±5.2	4 (28.6%)	3 (21.4%)	3 (21.4%)
Healthy controls	100	35.2±19.2	0.47	Negative	0	2 (2%)	1 (1%)
Anti‐ENA positive healthy controls	3	26.0±11.8	0.50	Negative	0	2 (66%)	1 (33%)

Table 1 lists the major diagnosis for the HCV‐positive/anti‐ENA‐positive patients in this study. Six of the 14 HCV‐positive, anti‐ENA‐positive patients had a diagnosis of chronic HCV hepatitis. Of all the 100 HCV‐positive patients, 14 had a diagnosis of chronic HCV hepatitis, 13 with chronic kidney disease, eight with hepatic failure or cirrhosis, four with acute HCV hepatitis, eight with liver disease/abnormal liver function, nine with trauma, and eight with alcohol or drug abuse. One patient was screened for HCV because of complaints of arthritis in the knees and elbows (17‐year‐old female; Table 1). Inflammatory arthritis and connective tissue disease was ruled out in this patient by a rheumatologist. The remaining patients were screened for HCV because of various health reasons including pregnancy, sexually transmitted disease screening, or routine health checks. No connective tissue diseases associated with the presence of these ENA antibodies were diagnosed in these patients.

There was no significant difference between the prevalence of anti‐ENA antibodies among the HCV‐positive male patients compared with the female patients (P=.517). The male:female ratio was 1.5, with 16.7% of the males also positive for anti‐ENA antibodies and 10% of the females also positive for anti‐ENA antibodies. Three of the 100 HCV (3.0%) antibody‐negative healthy control patients were positive for anti‐ENA antibodies. The male:female ratio for the entire healthy control group was 0.47.

The mean S/CO ratio HCV antibody value for all of the 100 HCV antibody‐positive patients was 11.2 (±3.8 SD) (Table 2). The mean S/CO ratio for the HCV antibody‐positive, anti‐ENA‐positive patients was 10.3 (±5.2 SD).

3.2. HCV RNA detection

Detection of HCV RNA by PCR was performed in 80 of the 100 HCV antibody‐positive patients and was positive in 55 patients (68.8%) with a mean copy number of 1.27×10⁶. In the HCV antibody‐positive patients that were positive for anti‐ENA, HCV RNA detection was performed in 11 of the 14 patients and was positive in 9 (75.0%) (Table 1) with a mean copy number of 1.07×10⁶. HCV genotyping was performed in 25 of the 55 RNA‐positive, HCV antibody‐positive patients with 14 (56.0%) as genotype 1a, four (16.0%) as genotype 1b, six (24.0%) as genotype 3, and one (4.0%) as genotype 1. Four (57.1%) of the seven genotyped RNA‐positive, anti‐ENA were genotype 1a and three (42.9%) were genotype 3 (Table 1).

3.3. Anti‐ENA patterns in patients positive for HCV antibodies

The most predominant anti‐ENA pattern in the HCV‐positive patients was anti‐SSA or anti‐dsDNA (Table 1 and Figure 1A), with four patients each (28.6%) being positive for these antibodies. This was followed by anti‐RNP and Scl‐70, with three patients (21.4%) positive for these antibodies. Two of the healthy controls (one male and one female) were also positive for anti‐RNP, while one healthy control (female) was positive for anti‐Scl‐70 (Figure 1B).

Figure 1.

(A) Extractable nuclear antigens (ENA) profile results of hepatitis C‐positive patients positive for anti‐ENA. (B) ENA profile results of healthy hepatitis C‐negative patients positive for anti‐ENA

Ten of the 14 HCV antibody‐positive patients were positive by IFA: five had antinuclear patterns, one had combined antinuclear and cytoplasmic patterns, and four had a cytoplasmic pattern (Table 1). Titers ranged from 1:40 to 1:160, with various nuclear and cytoplasmic patterns present, including: speckled, homogenous, and centromere ANA patterns along with cytoplasmic fiber or speckled patterns. There were three speckled ANA patterns and two homogenous ANA patterns. The predominant cytoplasmic pattern was homogenous. One HCV antibody‐positive patient was negative for anti‐ENA, but positive by IFA for ANA with a speckled antinuclear pattern and a titer of 1:160 (Table 1).

All three HCV antibody‐negative healthy control patients that were positive for anti‐ENA antibodies were positive for ANA by IFA; a 13‐year‐old male and a 36‐year‐old female were positive at a titer of 1:80 speckled pattern with an anti‐ENA against RNP. Both had low positive values for the anti‐RNP, 1.1 AI, and 1.7 AI, respectively. However, a 29‐year‐old female had an ANA titer of 1:320 homogenous pattern with a low positive (1.1 AI) anti‐ENA against Scl‐70 alone.

4. DISCUSSION

Hepatitis C virus infection is often associated with extrahepatic symptoms such as arthritis, vasculitis and sicca syndrome that might suggest an immune mediated or rheumatic disease.2 The occurrence of ANA positivity in HCV‐positive patients has been extensively studied while only a few studies have studied anti‐ENA antibodies in HCV‐positive patients. D'Amico et al.12 found 46.3% of 69 patients with HCV‐positive hepatitis had anti‐ENA antibodies using an ELISA. They specifically found anti‐SSA in 23.2% and anti‐SSB in 20.3%. However, in a study of 62 patients with sicca syndrome by Jorgensen et al.,14 of the 12 patients with both HCV infection and sicca syndrome (19%), only one patient had anti‐SSA antibodies.

We found 14% of all of our HCV‐positive subjects were positive for anti‐ENA. The highest prevalence autoantibody was anti‐SSA with four subjects positive for anti‐SSA (4%). D'Amico also found that the highest prevalence anti‐ENA antibody in their study was anti‐SSA. However, our overall percentage of anti‐ENA positivity is much lower than found in the study by D'Amico et al., which was 46.3%. The D'Amico study focused on a patient population of chronic hepatitis C‐infected patients with more severe disease including patients with persistently elevated liver enzymes and liver needle biopsies consistently showing chronic active hepatitis. If the analysis of patients in our study is limited to the chronic hepatitis C patients (13 patients out of the 100 HCV infected patients), our results more closely match the results in the D'Amico et al. study with five of the 13 (38.5%) patients being positive for anti‐ENA, 30.8% positive for anti‐SSA, and 7.7% positive for anti‐SSB.

The anti‐ENA test in our study was the BioPlex “ANA screen” test, a multiplex assay designed to compare in sensitivity and specificity to the ANA screen at a titer of 1:80. Comparison to a 1:80 was used since ANA titers less than or equal to 1:80 may have variable clinical significance.15 In this respect, the 14% positivity rate in our study compares relatively well to other studies in which the ANA positivity rate for HCV serologically positive patients was 12%‐17.9%.7, 8, 9

Additionally, we found anti‐ENA positivity to SSB, Scl‐70, RNP, anti‐dsDNA, Sm, Sm/RNP, and centromere B in our HCV‐positive patients. Batchoun et al.13 similarly found anti‐SSA, SSB, Sm, Sm/RNP, Scl‐70, dsDNA, and Scl‐70 in HCV‐positive patients undergoing hemodialysis, and only anti‐SSA, SSB, Sm, and Scl‐70 in their HCV‐positive patients not undergoing hemodialysis.

Studies comparing the BioPlex multiplex assay with IFA generally show good sensitivity, specificity, and agreement. A recent study showed 82.2% positive agreement, 83.7% negative agreement, and 83.3% overall agreement of the BioPlex multiplex system with IFA.16 In our study, the BioPlex multiplex system appeared to be more sensitive than the IFA, with only 10 of the 14 anti‐ENA‐positive samples positive for IFA. Four of the positive IFA samples showed a cytoplasmic IFA pattern. Increased rates of ANA positivity with cytoplasmic pattern on IFA have been previously reported for hepatitis infections, including hepatitis A and hepatitis B.17, 18 In summary, our study emphasizes the relatively high prevalence of anti‐ENA antibodies in HCV antibody‐positive patients when compared to healthy controls. This correlates well with the high prevalence of positive ANA that has been observed in the past in this population.