HBV superinfection in HCV chronic carriers: A disease that is frequently severe but associated with the eradication of HCV

Abstract

The impact of hepatitis B virus (HBV) superinfection in hepatitis C virus (HCV) chronic carriers was evaluated in a long-term follow-up study on 29 chronic anti-HCV carriers with acute hepatitis B (AVH-B) (Case group BC) and 29 anti-HCV negative patients with AVH-B (Control group B), pair-matched for age (±5 years), sex, and risk factors for the acquisition of HBV infection. Patients in Case group BC and those in Control group B showed similar initial HBV viral load and a similar trend of becoming negative for HBV-DNA. AVH-B showed a severe course more frequently in Case group BC than in Control group B (34.5% versus 6.9%, P < 0.05). Of the 28 patients in Case group BC alive at the end of the acute illness (one death from liver failure), 24 were followed up for 2-6 years, median 5 years: 22 patients became HBsAg-negative and two progressed to HBsAg-positive chronic hepatitis. HCV-RNA was undetectable in all patients during AVH-B; in the 24 patients with a long-term follow-up, HCV-RNA was detected in seven (29.2%) after 1 year, in 14 (58.3%) after 2 years, and in 18 (75%) after 3-6 years. The six patients who eradicated chronic HCV infection, compared with 18 showing reactivation of HCV replication, had higher values of aspartate aminotransferase and alanine aminotransferase and a higher prevalence of cases with severe AVH-B (83.3% versus 22.2%, P < 0.05). Conclusions: Although it can be life-threatening, HBV superinfection in HCV chronic carriers may lead to clearance of chronic HCV infection, especially in patients with severe AVH-B. (HEPATOLOGY 2009.)

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection is characterized by a reciprocal inhibition of the viral genomes, a severe clinical presentation, and an infrequent response to antiviral treatment.1–9 This reciprocal inhibition was observed also in acute HBV/HCV coinfection.10, 11

Models of HBV and HCV superinfection have also been investigated. A marked inhibition of the preexisting viral replication has been observed both in HBV chronic carriers with superimposed acute hepatitis C12–15 and in HCV chronic carriers with HBV superinfection,16–18 both models being frequently characterized by a severe clinical course.15, 18 In a study on HBV superinfection in HCV chronic carriers, the inhibition exerted by HBV on the HCV genome persisted during a follow-up of 1 year in half of the patients,18 but a comprehensive evaluation of the virus interaction and clinical outcome was hampered by the lack of information on HCV replication before HBV superinfection by the low sensitivity of the HBV-DNA assay used and the short follow-up period.

The present article describes a case-control study performed on 29 HCV chronic carriers with HBV superinfection and 29 anti-HCV negative pair-matched controls who developed acute hepatitis B in the same period. Most of the patients with HBV superinfection had been observed for at least 1 year before the onset of acute hepatitis B and then followed up for 2-6 years (median 5 years) in a clinical and virological study performed applying the improved methodology.

ALT, alanine aminotransferase; anti-HBc IgM, hepatitis B core antibody IgM; AST, aspartate aminotransferase; AVH-B, acute hepatitis B; HAV, Hepatitis A Virus; HBeAb, hepatitis B e antibody; HBeAg, hepatitis B e antigen; HBsAb, hepatitis B surface antibody; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HIV, human immunodeficiency virus; IgM, immunoglobulin M; PCR, polymerase chain reaction.

Patients and Methods

Patients.

A total of 115 patients with acute viral hepatitis B (AVH-B), first observed within the 3rd week from the onset of symptoms, were enrolled in the study at one of the two participating Units of Infectious Diseases from January 2000 to July 2005. These two units, one in Naples and one in Caserta, had been using the same clinical and laboratory approach for years and had cooperated in several clinical investigations.

All 115 patients had detectable serum hepatitis B surface antigen (HBsAg) and immunoglobulin M (IgM) to hepatitis B core antigen (anti-HBc IgM) and lacked antibodies to hepatitis D virus (anti-HDV) and to human immunodeficiency virus (anti-HIV); they showed serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum values at least 12 and 20 times higher than the upper level of normal, respectively. Forty-seven of the 115 patients (40.8%) were drug abusers. No patient had been vaccinated against HBV.

Of the 115 patients with acute HBV infection, 29 had been anti-HCV positive with abnormal AST/ALT serum levels for at least 1 year. They formed Case group BC; of these 29, 24 (82.8%) patients had also been tested and found positive for plasma HCV-RNA. Of the remaining 86 anti-HCV negative patients, 29, pair-matched for age (±5 years), sex, and risk factors for the acquisition of HBV infection were chosen to form a control group for Case group BC in a long-term case-control study; this control group was named Control group B. Fourteen of the 29 patients in Case group BC and 6 of the 29 in Control group B came from our preliminary study18 and were included in the present investigation on the basis of the availability of plasma samples stored frozen at −40°C for reevaluation of HBV and HCV viral load with the improved methodology.

At the first observation a sample of plasma was obtained from all patients and stored at −40°C. Serial samples of plasma were obtained at 5-day intervals for 23 patients in Case group BC and 27 in Control group B; a total of 85 and 108 samples of plasma, respectively, were obtained, stored in small fractions at −40°C, and never thawed until used for this investigation.

Patients were considered to have developed severe hepatitis if they showed porto-systemic encephalopathy and/or ascites and/or a progressive reduction in prothrombin activity below 35%.19–21

A long-term follow-up of at least 2 years for Case group BC and 6 months for Control group B was planned; plasma samples were obtained at 6-month intervals also during this follow-up, stored in small fractions at −40°C, and never thawed until used for this investigation.

Patients in Control group B were considered to have recovered from acute hepatitis when they showed a steady normalization of AST/ALT serum levels and a clearance of serum HBsAg within the sixth month from the onset of symptoms; the persistence of HBsAg in serum for more than 6 months was considered a sign of progression to chronicity. HBsAg serum clearance within 6 months indicated recovery from acute hepatitis B also in patients in Case group BC, regardless of a normalization of serum aminotransferases.

The criteria to define the eradication of HCV infection in patients in Case group BC were not preestablished because a clearance of circulating anti-HCV in patients who have recovered can take an unpredictably long time, and, also, a steady HCV-RNA plasma clearance with normalization of serum aminotransferases does not exclude a further reactivation.

Methods.

HAV, HBV, HCV, HDV, and HIV serum markers were detected using commercial immunoenzymatic assays (Abbott Laboratories, North Chicago, IL, for HBsAg, anti-HBs, anti-HBc [total and IgM], anti-HAV [total and IgM], and anti-HIV 1 and 2; DiaSorin, Saluggia, VC, Italy, for anti-HDV IgG and anti-HDV IgM; Ortho Diagnostic Systems, Neckargemund, Germany, for anti-HCV). Liver function tests were performed applying routine methods.

Plasma of all patients in the study was tested for HBV-DNA. Viral DNA was extracted from 200 μL of plasma using microspin columns (QIAamp DNA Blood kit, Qiagen, Hilden, Germany). The DNA extracted was analyzed for the presence of the HBV genome by performing a real-time polymerase chain reaction (PCR) with a wide range of linearity22 in a Light-cycler 1.5 (Roche Diagnostics, Branchburg, NJ) using forward (5′-TTTTTCACCTCTGCCTAATCATC-3′ nucleotide [nt] 8-30) and reverse primers (5′-ACCCACCCAGGTAGCTAGAGTCAT-3′ nt 306-289) located in the core region of HBV, and a 3′-fluorescein (FL)-labeled probe (5′-CCAAAGCCACCCAAGGCAC-FL-3′) and a 5′-Red640 (R₆₄₀)-labeled probe (5′-R₆₄₀-TTGGAGGCTTGAACAGTAGGACATGA-p-3′). An external standard curve was used to quantify HBV genomes present in the samples; the standard is a PCR product cloned with the TA cloning system (k2000-01, Invitrogen, Carlsbad, CA); 8 μL of the appropriately diluted plasmid was used to generate the standard curve. Using a commercial HBV-DNA positive control of known amount (Accurun 325 HBV DNA, series 300, BBI Diagnostics, West Bridgewater, MA) according to previous investigations,23, 24 the detection limit of this method is estimated at around 40 copies/mL.

HBV genotyping was performed by Line-Probe-Assay (INNO-LIPA HBV Genotyping; Innogenetics, Zwigndrecht, Belgium).

Plasma HCV-RNA was tested in all anti-HCV-positive sera. Viral RNA was extracted from 140 μL of plasma samples using a microspin column (QIAamp RNA viral kit for plasma, Qiagen). The RNA extracted was analyzed for the presence of the HCV genomes by performing a real-time PCR with a wide range of linearity25 in a Light-cycler 1.5 (Roche Diagnostics) using forward (5′-AGCGTCTAGCCATGGCGT-3′ nt 74-91) and reverse primers (5′-CAAGCACCCTATCAGGCAGT-3′ nt 308-288) located in the 5-UTR region of HCV, and a 3′-FL-labeled probe (5′-GCAGCCTCCAGGACCCCCC-FL 3′) and a 5′-R₆₄₀-labeled probe (5′-R₆₄₀-CCCGGGAGAGCCATAGTGGTCTC-p-3′). An external standard curve was used to quantify HCV genomes present in the samples; the standard is a PCR product cloned with TA cloning system (Invitrogen k2000-01); 8 μL of the properly diluted plasmid was used to generate the standard curve. Using an HCV-RNA positive control of known amount (Accurun 305 HCV RNA, Series 150, BBI Diagnostics) according to a previous investigation,24 the detection limit of this method is estimated at around 40 IU/mL.

HCV genotyping was performed by Line-Probe-Assay (INNO-LIPA HCV II, Innogenetics), which allows the identification of the six major genotypes and their subtypes (1a, 1b, 2a/2c, 2b, 3a, 3b, 3c, 4a, 4b, 4c/4d, 4f, 4h, 5a, and 6a), according to the classification based on the amplification of the NS5 region of the HCV genome.

Results

Acute Hepatitis B.

Patients in Case group BC were first observed during acute hepatitis B 4-16 days (median 7) after the onset of symptoms and those in Control group B 3-20 days (median 6) after (Table 1). Both groups had the same prevalence of males (96.5%) and drug abusers (89.7%). The median age was 29 years (range 18-55) in Case group BC and 27 years (range 19-54) in Control group B (Table 1).

Table 1. Comparative Analysis of Demographics and Initial Laboratory and Clinical Characteristics of Acute Hepatitis B Between Patients with Preexisting Chronic HCV Infection (Case Group BC) and Those Without (Control Group B)

Characteristic	Case Group BC	Control Group B
A versus B: P < 0.05; C versus D: P < 0.01. AST, aspartate aminotransferase; ALT, alanine aminotransferase; n.v., normal value. * Severe hepatitis: a prothrombin activity lower than 35% and/or the development of porto-systemic encephalopathy and/or ascites.
Number of cases	29	29
Age, years, median (range)	29 (18–55)	27 (19–54)
Males, n (%)	28 (96.5)	28 (96.5)
With history of
i.v. drug abuse, n (%)	26 (89.7)	26 (89.7)
unsafe heterosexual contact, n (%)	3 (10.3)	3 (10.3)
Day of first observation after the onset of symptoms, median (range)	7 (4–16)	6 (3–20)
AST, IU/L, (mean ± SD), n.v. 15-35	1998 ± 1118	1863 ± 1312
ALT, IU/L, (mean ± SD), n.v. 15-35	2783 ± 1396	3116 ± 2184
Prothrombin activity, %, median (range)	62 (11–105)	64 (11–110)
Total bilirubin, mg/dL (mean ± SD)	13.8 ± 6.0	15.2 ± 6.9
Patients with:
jaundice, n (%)	28 (96.6)	27 (93.1)
prothrombin activity<35%, n (%)	8 (27.6)	2 (6.9)
ascites, n (%)	2 (6.9)	0
porto-systemic encephalopathy, n (%)	2 (6.9)	1 (3.5)
severe hepatitis,* n (%)	10 (34.5)^A	2 (6.9)^B
HBeAg/anti-HBe:
+/−, n (%)	2 (6.9)	12 (41.4)
+/+, n (%)	2 (6.9)	4 (13.8)
−/−, n (%)	3 (10.3)	2 (6.9)
−/+, n (%)	22 (75.9)^C	11 (37.9)^D
HBV viral load, copies/mL (mean ± SD)	7.1 × 10^8 ± 2.5 × 10^9	1.6x10^8 ± 4.5 × 10^9
HBV genotype,
D, n (%)	28 (96.6)	29 (100)
F, n (%)	1 (3.4)	0

At the first observation, no difference was found between patients in Case group BC and those in Control group B regarding the prevalence of HBV-DNA positive patients or HBV-DNA titers (mean ± SD: 7.1 × 10⁸ ± 2.5 × 10⁹ versus 1.6 × 10⁸ ± 4.5 × 10⁹ copies/mL) (Table 1). The same comparison was made also at the subsequent timepoints during the acute stage of the illness and no differences were observed with regard the reduction in the titers and the clearance of HBV-DNA (Fig. 1). However, at the time of the first observation, patients in Case group BC more frequently than those in Control group B had seroconverted to anti-HBe (75.9% versus 37.9%, P < 0.01) (Table 1). HBV genotype D was identified in all patients but one in Case group BC and in all in Control group B (Table 1).

Figure 1. HBV viral load during acute hepatitis B, according to the presence or absence of chronic HCV infection.

Focusing on Case group BC, all 24 patients who had been HCV-RNA-positive for at least 1 year before the onset of HBV superinfection became HCV-RNA negative during AVH-B. More precisely, HCV-RNA was found to be negative in nine (81.8%) of the 11 tested within the 10th day from the onset of symptoms, in 18 (85.7%) of the 21 tested from days 11-20, in 100% of the 21 tested from days 21-45, and in 100% of the 21 patients tested after day 45. Also, the remaining five anti-HCV-positive patients in Case group BC, for whom the HCV-RNA status before the onset of HBV superinfection was unknown, were HCV-RNA negative during AVH-B.

A severe clinical presentation was observed in 10 (34.5%) of the 29 patients in Case group BC and in two (6.9%) of the 29 in Control group B (P < 0.05) (Table 1); orthotopic liver transplantation became essential for two of these 10 patients in Case group BC and for one of the two in Control group B. Of the three patients who developed massive liver failure, two were successfully transplanted, but the third (Case group BC) died because no liver was available for transplantation.

Long-term Follow-up Study.

Of the 29 patients in Case group BC, one died of liver failure during the acute illness, 24 (82.8%) were followed for 2-6 years, median 5 years, and four dropped out due to lack of compliance. Of these 24, 22 patients became HBsAg-negative and recovered from AVH-B; 14 (63.6%) of these 22 seroconverted to anti-HBs. The remaining two patients progressed to HBsAg chronic carriage: one seroconverted to anti-HBe-positive with HBV-DNA undetectable and HCV-RNA titer = 7.1 × 10⁵ IU/mL at the end of a 4-year follow-up and the other remained HBeAg-positive with HBV-DNA titer = 7.4 × 10⁵ copies/mL and HCV-RNA negative after a 5-year follow-up.

All 19 patients who had been HCV-RNA-positive for at least 1 year before AVH-B and who were followed for 2 years or more became HCV-RNA-negative during HBV superinfection; of these 19, HCV-RNA again became detectable at month 6 of follow-up in one, at month 12 in four, at month 18 in seven, at month 24 in 10, and at month 30 in 13. Also, the remaining five patients with a follow-up, for whom HCV-RNA was never determined before HBV superinfection, were HCV-RNA-negative during AVH-B, but HCV-RNA was detectable at month 6 of follow-up in one, at month 12 in three, and at month 18 in all five patients, strongly indicating that these five had been HCV-RNA-positive before HBV superinfection. Therefore, altogether, of the 24 patients in Case group BC with a follow-up, two (8.3%) had turned positive by month 6 of follow-up, seven (29.2%) by month 12, 12 (50%) by month 18, 14 (58.3%) by month 24, and 18 (75%) by month 30 (Table 2). The six (25%) patients who were still negative at month 30 remained so at months 36, 42, and 48 (Table 2); of these six, four were tested and found negative also at month 60 and two at month 72. Thus, the six patients who were still HCV-RNA-negative after 3 years remained so even after 5 or 6 years of follow-up, strongly suggesting that HCV eradication can be achieved in about a quarter of cases. Worthy of mention is the observation that of the six patients who eradicated HCV chronic infection, one cleared both circulating HCV-RNA and anti-HCV (Fig. 2).

Table 2. Plasma HCV-RNA During a 2-Year to 6-Year Follow-Up in the 24 Patients* in Case Group BC Who Became HCV-RNA-Negative by the End of AVH-B

Time of Follow-up	HCV-RNA
	Turned Positive, n (%)	Remained Negative, n (%)
* Of the 29 patients in the Case group BC, one died of liver failure, four dropped out, and 24 were followed for 2-6 years. ↑ None of these six patients turned positive after five (four patients tested) and six (two patients tested) years.
By the 6^th month	2 (8.3)	22 (91.7)
By the 12^th month	7 (29.2)	17 (70.8)
By the 18^th month	12 (50)	12 (50)
By the 24^th month	14 (58.3)	10 (41.7)
By the 30^th month	18 (75)	6 (25)
By the 36^th month	18 (75)	6 (25)
By the 48^th month	18 (75)	6 (25)↑

Figure 2. Clinical outcome for 24 patients in Case group BC after a 2-6 year (median 5) follow-up. Of the 29 patients in Case group BC, one died of liver failure, four dropped out, and 24 were followed for 2-6 years. Grading and staging are expressed according to Ishak's scoring system.

To identify the initial characteristics predictive of the virological outcome, the six patients who showed eradication of chronic HCV infection were compared with the 18 with reactivation of this infection during the follow-up (Table 3) and the only differences observed were higher values of aminotransferases (AST: 3,417 ± 2,090 versus 1,619 ± 1,214 IU/L, P < 0.05; ALT: 5,291 ± 3,809 versus 2,364 ± 1,374 IU/L, P < 0.01) and a higher prevalence of severe AVH-B (83.3% versus 22.2%, P < 0.05) in the first subgroup; thus, of the 24 patients followed for at least 2 years, five (55.6%) of the nine patients with severe AVH-B eradicated HCV chronic infection, whereas this outcome was observed only in one (6.7%) of the 15 patients with “normal” AVH-B; this difference is statistically significant and of clinical relevance.

Table 3. Analysis of Demographic, Biochemical, Virological, and Clinical Characteristics Recorded at the First Timepoint During HBV Superinfection as Predictive Factors of the Outcome of HCV Infection in the 24 Patients in Case Group BC with a Long-Term Follow-Up

Characteristic	With HCV Eradication	With Temporary Clearance of Plasma HCV-RNA
Statistical analysis: A versus B: P < 0.05; C versus D: P < 0.01; E versus F: P < 0.05. AST, aspartate aminotransferase; ALT, alanine aminotransferase, n.v., normal value. * Severe hepatitis: a prothrombin activity lower than 35% and/or the development of porto-systemic encephalopathy and/or ascites.
Number of patients	6	18
Age, years, median (range)	29 (24–33)	28 (21–37)
Males, n (%)	6 (100)	18 (100)
AST, IU/L (mean ± SD), n.v. 15-35	3417 ± 2090 ^A	1619 ± 1214^B
ALT, IU/L, (mean ± SD), n.v. 15-35	5291 ± 3809^C	2364 ± 1374^D
Total bilirubin, mg/dL, (mean ± SD)	12.2 ± 5.7	12.9 ± 6,5
With severe hepatitis,* n (%)	5 (83.3)^E	4 (22.2) ^F
With seroconversion to anti-HBe, n (%)	1 (16.7)	4 (22.2)
HBV-DNA load, copies/mL (mean ± SD)	5.4 × 10^8 ± 4.1 × 10^8	1.2 × 10^9 ± 3.6 × 10^9
HBV genotype D, n (%)	6 (100)	18 (100)

Figure 2 shows a comprehensive analysis of virological, clinical, and histological data of the 24 patients with a long-term follow-up after HBV superinfection. Of the six patients who eradicated HCV chronic infection, five cleared both HBV and HCV infections and recovered; of these five, one also cleared circulating anti-HCV (Fig. 2). The remaining patient of these six is the one mentioned above who eradicated HCV infection but progressed to HBsAg-positive chronic hepatitis (Fig. 2). Of the 18 patients who became HCV-RNA-positive, 17 recovered from AVH-B but remained anti-HCV/HCV-RNA-positive (Fig. 2). Of these 17, four showed steadily normal and 13 steadily abnormal ALT serum values. Of these 13 patients with HCV-related chronic hepatitis, nine agreed to undergo liver biopsy, which showed different degrees of grading and staging (Fig. 2), and four refused. The remaining patient of the 18 is the one mentioned above who developed chronic hepatitis by HBV and HCV (Fig. 2).

Thus, at the end of the follow-up period a percutaneous liver biopsy was indicated in 15 patients and performed in 11. To evaluate the impact of HBV superinfection in these 11 patients, we formed a control group of 22 patients with anti-HCV/HCV-RNA-positive chronic hepatitis with no previous or ongoing HBV infection. For each of these 11 patients in Case group BC, two HBsAg/anti-HBs/anti-HBc-negative patients with chronic HCV infection were selected from those who underwent liver biopsy at the same centers in the same period, pair-matched for age (±5 years), sex, HCV genotype, risk factor for parenteral acquisition of HCV infection, and duration of intravenous drug addiction (±5 years); this new group of patients was named Control group C. The data from this comparison show only a few biochemical and histological differences, not significant for the statistical analysis (Table 4).

Table 4. Comparative Analysis of Demographic, Biochemical, Virological, and Histological Characteristics Recorded at the Time of Liver Biopsy in 11 Patients in Case Group BC and in 22 in the Control Group C

Characteristic	Case Group BC	Control Group C
A versus B: P = 0.1; C versus D: P = 0.07. AST, aspartate aminotransferase; ALT, alanine aminotransferase; n.v.: normal value. Histological Activity Index (HAI) and fibrosis score are expressed according to Ishak's scoring system.
Number of patients	11	22
Age, years, median (range)	30 (21–37)	29.5 (20–42)
Males, n (%)	11 (100)	22 (100)
With history of intravenous drug addiction, n (%)	11 (100)	22 (100)
Duration of intravenous drug addiction, years, median (range)	2 (1–6)	2 (1–7)
With HCV genotype, n (%)
1	5 (45.6)	10 (45.6)
2	1 (9)	2 (9)
3	4 (36.4)	8 (36.4)
Not determined	1 (9)	2 (9)
AST, IU/l (mean ± SD), n.v. 15-35	58 ± 49	51 ± 34
ALT IU/l (mean ± SD), n.v. 15-35	71 ± 37	66 ± 34
Liver histology:
HAI (mean ± SD)	6.3 ± 2.3^A	5.1 ± 2.4^B
With HAI ≥7, n (%)	5 (45.5)	6 (20.7)
Fibrosis score (mean ± SD)	2.3 ± 1.1^C	1.6 ± 1.0^D
With fibrosis score ≥3, n (%)	4 (36)	2 (22.7)

Of the 29 patients with AVH-B in Control group B, 21 (72.4%) were followed for at least 6 months and 8 were considered dropouts due to lack of compliance. Of these 21 patients, one progressed to HBV chronic carriage (HBV-DNA 8.3 × 10³ copies/mL) with normal AST and ALT serum values and 20 recovered and cleared the HBV infection; 14 (70%) of these 20 seroconverted to anti-HBs-positive.

Discussion

The data from the present study show that HBV superinfection in patients with chronic HCV infection was responsible for a strong inhibition of HCV replication, which led to the eradication of HCV in a quarter of the patients. In fact, six out of 24 patients who were still HCV-RNA-negative 3 years after HBV superinfection remained so even at the 4th year, and at the 5th and 6th year for those tested. At the time of HBV superinfection, the differences distinguishing patients who subsequently eradicated HCV infection from those showing only a temporary inhibition of HCV were a higher prevalence of cases with severe acute hepatitis B and a higher level of serum aminotransferases in the first subgroup, differences that were statistically significant and of clinical relevance. Thus, extensive acute hepatocellular necrosis, although life-threatening, may lead to a clearance of chronic HCV infection. The inhibition exerted by HBV superinfection on HCV chronic replication was really impressive in one patient who became HCV-RNA and anti-HCV-negative soon after acute hepatitis B, an early sign of HCV eradication.

In the present investigation chronic HCV replication did not affect the HBV viral load but seroconversion to anti-HBe occurred earlier in Case group BC, suggesting a weak inhibitory effect of chronic HCV replication on HBV acute synthesis. Similarly, HCV superinfection inhibits HBV chronic replication, which may in turn induce a weak reduction of acute HCV synthesis.14, 15

The mechanisms by which HBV and HCV interact remain substantially unknown. There are data suggesting that HCV clearance in patients with HBV or HBV/HDV superinfection may be due to the newcomer virus-specific T-cell response targeting HBV and/or HDV-infected liver cells,26, 27 supporting the hypothesis that a virus-specific T-cell response may inhibit viral replication by nonspecific cytolytic and noncytolytic mechanisms.28 Interesting contributions have come from experimental models. In HuH-7 cell cultures the HCV core protein has an inhibitory effect on HBV expression and replication.2 Moreover, HCV replication is inhibited in vivo29 and in vitro30, 31 by interferon-γ, interferon-α, and tumor necrosis factor-α, suggesting that HBV superinfection may inhibit chronic HCV replication by producing inhibitory cytokines. In favor of a nonspecific mechanism as the key factor in inhibiting viral replication are the data from studies demonstrating that HAV superinfection may be responsible for a marked inhibition of a preexisting HCV or HBV chronic replication, leading in some cases to the clearance of one of these two viruses and to the remission of the associated liver disease.32, 33 Similarly, the patients with self-limiting HCV infection, compared with those with persistent chronic HCV infection, more frequently showed previous HAV or HBV infection.34 Thus, besides subtle regulatory mechanisms between the viruses, an additional contribution to the inhibition of chronic HCV or HBV replication may be given by acute hepatocellular necrosis itself due to the superinfecting virus (HBV or HCV or HAV), most probably regarding not only cells infected by the new virus but also those infected by the preexisting virus. In support of this hypothesis is the observation that in the present study AST and ALT serum values were significantly higher in patients who subsequently eradicated HCV than in those with a reactivation of HCV chronic infection.

In general, the sequence and strength (whether acute or chronic) of HBV and HCV infections should be considered among factors influencing the virological expression of a superinfection.4, 14, 15, 17, 18, 35 However, the virological and clinical outcome is dominated by the tendency of HCV to become chronic and by the ability of HCV chronic infection to persist in a form of latency during HBV superinfection and to later reactivate in three-quarters of the cases. In fact, in the model of HBV superinfection in HCV chronic carriers in this study, 8.3% of patients with a 2-year to 6-year follow up progressed to chronic carriage of both viruses, 54.2% developed HCV-related chronic hepatitis, 16.7% became HCV chronic carriers with steadily normal aminotransferases, and 20.8% recovered and cleared both infections. For the model of HCV superinfection in HBV chronic carriers, conclusive data come from the largest study on this topic: in the 93 HBsAg chronic carriers investigated, HCV superinfection was frequently severe (45% of cases), and was associated with the progression of HCV infection to chronicity in 67% of cases and to HBsAg serum clearance in 10.9%.14 The data from that study and our present study support the long-term virological and clinical dominance of HCV in both models of superinfection, due to the intrinsic characteristics of both viruses.

The data emerging from the present investigation indicate that HBV superinfection is frequently life-threatening in HCV chronic carriers, thus raising the concern that this clinical event could become an emerging healthcare problem in countries with a wide spread of both HBV and HCV infection.

Further efforts should be made to extend the use of HBV vaccination in patients with chronic HCV infection. This practice is recommended by several international and national healthcare institutions36–38 but remains poorly applied worldwide, in particular in developing countries where the risk of HBV superinfection in HCV chronic carries is presumed to be high.

Acknowledgements

We thank the registered nurses Angela Di Fonzo, Michela Picozzi, Lorenza Scialla, and all the nurses for their invaluable technical assistance.