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Original Articles

Vertical Transmission of Hepatitis C Virus in a Cohort of 2,447 HIV-Seronegative Pregnant Women: A 24-Month Prospective Study

Ceci, Oronzo*; Margiotta, Marcella; Marello, Fiorino*; Francavilla, Ruggiero; Loizzi, Pasquale*; Francavilla, Antonio; Mautone, Angela; Impedovo, Luigi*; Ierardi, Enzo; Mastroianni, Maria*; Bettocchi, Stefano*; Selvaggi, Luigi*

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Journal of Pediatric Gastroenterology and Nutrition: November 2001 - Volume 33 - Issue 5 - p 570-575
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Abstract

Hepatitis C virus (HCV) is a RNA virus that accounts for most cases of viral hepatitis in adults, and the recognition that it can be transmitted perinatally deserves particular attention by pediatricians. Vertical transmission is the most frequent mode of HCV acquisition in the pediatric setting. Mother-to-infant transmission has been reported to occur in 0% to 44% of children born to HCV-infected mothers (1–6). Maternal risk factors for vertical transmission of HCV infection are human immunodeficiency virus (HIV) coinfection, high HCV viral load, and vaginal delivery (7–11). Vertical transmission of the virus may take place either in utero (transplacental transfer), perinatally, or, rarely, through breast-feeding (9,12,13). Maternal anti-HCV antibodies (immunoglobulin G) are transferred passively to the fetus and may be present in the infants until 12 to 15 but not beyond 18 months of age (14,15).

Early studies were fraught with in-house methods that resulted in false-positive results for HCV-RNA (2); however, nowadays the availability of reliable commercial assays for HCV-RNA determination and the improved in-house methods allow us to better define the rate of HCV vertical transmission, and the polymerase chain reaction (PCR) method is being increasingly used to document suspected vertical HCV transmission in young infants.

The aim of this prospective study was to investigate the incidence of HCV vertical transmission and spontaneous viral clearance in a cohort of infants born to HCV-positive/HIV-negative women from Southern Italy.

MATERIAL AND METHODS

Study Population and Sample Collection

Between January 1995 and June 1997, HCV antibodies were sought with a second-generation enzyme-linked immunosorbent assay (AXSYM HCV 3.0 Abbott Laboratories, Abbott Park, IL) in 2447 consecutive pregnant women attending our Institute. All HCV-positive and HIV-negative women were enrolled to the study after a signed informed consent. The mothers were assessed for presence of the following HCV-related risk factors: blood product exposure before 1992, intravenous drug abuse, acupuncture, tattoo, incarceration, body piercing, and sexual partner with a history of drug abuse or of HCV infection. They were all tested for serum HCV-RNA and for HCV genotype and viral load measurement if they tested HCV-RNA positive. Indication for cesarean section was based on obstetric reasons, independently of maternal HCV status. Maternal ages, parity, complications during pregnancy, gestational age, mode of delivery, and birth weight were recorded. All HCV-positive women decided not to breast-feed.

Follow-up of the infants included clinical review, measurement of alanine aminotransferase (ALT; normal value <40 IU/L) and HCV RNA at birth, 4, 8, 12, 18, and 24 months of age. Blood samples were stored immediately after venesection at −80°C and tested in batches. HCV genotype was determined in HCV RNA-positive infants. At the end of the follow-up anti-HCV antibodies were also tested by a recombinant immunoblot assay (Chiron RIBA HCV 3.0, Chiron Corporation, Emeryville, CA).

Predictors of Transmission

Vertical transmission was defined as the presence of anti-HCV antibodies beyond 18 months of age or HCV RNA positivity on at least two separate tests (13). The predictive value of eight variables previously reported to be associated with vertical HCV transmission has been evaluated (6–9). They included presence of HCV maternal risk factors (exposure to blood products and intravenous drug addiction), HCV viral load, HCV genotype, gestational age, mode of delivery, and birth weight.

Serologic Investigation

HCV antibodies were assessed by a recombinant immunoblot assay (Chiron RIBA HCV 3.0, Chiron Corporation, Emeryville, CA) at birth and at 12, 18, and 24 months. The samples were considered “positive” or “indeterminate” when they displayed an optical density reading greater than the cut-off of at least two or one of the four antigens, respectively. ALT values were assessed using a Hitachi 747 system (Boehringer Mannheim, Germany).

Virologic Investigation

RNA was extracted from 0.2 mL of serum by a single-step acid guanidinium thiocyanate-phenol-chloroform method (16). HCV-RNA detection was performed with reverse transcriptase “nested” PCR with primers deduced from the 5´ non-coding region (17). Complementary DNA was synthesized with 50 pmol of the antisense outer primer and Moloney Murine Leukaemia virus reverse transcriptase (Superscript, BRL). The first PCR was performed for 38 cycles (94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute.) in 0.1 mL of solution containing 2.5 units of Taq polymerase (Taq-Gold Perkin Elmer-Cetus, Norwalk, CT) and 50 pmol of sense outer primer. For “nested “PCR target sequences belonging to the highly conserved 5´ non-coding region of the HCV genome were chosen: outer primers were 5´-TCCAGAGCATCTGGCACGT-3´ (antisense) and 5´-GCCATGGCGTTAGTATGAGT-3´ (sense). Inner primers were 5´-GTGCAGCCTCCAGGACCCCC-3´ (sense) and 5´-TATCCCACGAACGCTCACGGC-3´ (antisense). The probe was 5´-CCATAGTGGTCTGCGGAACCGGTGAGTACA-3´. Product size was 260 bp for the outer product and 210 bp for the inner target. Sequentially, 1 μL of the first PCR product was amplified for 25 cycles using the same protocol with the “inner primers” (50 pmol). Samples (10 μL) from second-round amplifications were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining, followed by the Southern blot hybridization assay with the γ- 32 P–labeled probe and autoradiography.

To detect contamination at each step of the procedure, HCV PCR was performed on negative control sera coextracted with test sera, serum-free lysis buffer used for RNA extraction, and the PCR mixture alone. Adherence to anticontamination protocols was strict, with particular attention to separation of pre-PCR and post-PCR steps (18). Samples from mothers and their infants were always tested twice, and in case of discordance a reliable commercial assay for HCV-RNA was used according to the manufacturer's instructions (Amplicor HCV, Roche Diagnostics SpA Milano, Italy).

Quantification of HCV-RNA in sera collected from viremic mothers was performed using the branched DNA signal amplification assay (bDNA, Quantiplex HCV RNA 2.0 Assay; Chiron Corporation, Emeryville, CA) according to the manufacturer's instructions. HCV RNA genotyping, according to classification by Simmonds, was evaluated by line-probe assay (Innolipa, Immunogenetics, Ghent, Belgium).

Statistical Analysis

The analysis of variance was used for multivariate analysis, and the Kruskal-Wallis Multiple Comparison Z-Value Test was used for comparison between the groups. Fixer exact test was used as appropriate. A P value <0.05 was considered significant.

RESULTS

Of the 2,447 pregnant women tested, 78 (3.2%) were anti-HCV positive and 60 (2.5%; 77% of all anti-HCV positive women) were HCV-RNA positive. Four HIV-positive/HCV-positive women (0.2%) were excluded. Median maternal age was 30 years (range, 21–42 years). Risk factors for HCV infection, maternal viral load, genotypes, gestational age, mode of delivery, and birth weights are shown in Table 1.

TABLE 1
TABLE 1:
Characteristics of the 60 HCV-RNA positive mothers

The infants born to 18 HCV RNA-negative mothers were also HCV RNA-negative at birth. Of these 18 newborns, 9 remained HCV-RNA negative and had no detectable anti-HCV antibodies at 24 months. The remaining nine were HCV-RNA negative at 12 months but then became lost to a further follow-up.

HCV-RNA data of the infants born to HCV-RNA–positive mothers are presented in Table 2. Thirty of 60 (50%) remained HCV RNA-negative during follow-up (group A), 22 (36.7%) were HCV RNA-positive on one occasion (group B), and 8 (13.3%) were HCV RNA-positive on at least two consecutive tests (group C). Two children (3.3%) from group C remained persistently HCV RNA-positive from the eighth to twenty-fourth month.

TABLE 2
TABLE 2:
HCV-RNA and RIBA data of babies born to HCV-RNA–positive mothers

The percentage of anti-HCV antibodies in infants born to anti-HCV and HCV-RNA–positive mothers at 0, 12, and 24 months is reported in Table 3. If we focus on group C, it seems clear that all infants at birth are anti-HCV positive as a result of maternal antibody persistence. At 12 months, six of eight (75%) infants in group C were still positive for anti-HCV, whereas at month 24, recombinant immunoblot assay (RIBA) was positive in two children (3.33%; both HCV-RNA positive), indeterminate in two (3.33%; both HCV-RNA negative), and negative in four (6.66%; all HCV-RNA negative).

TABLE 3
TABLE 3:
Percentage of anti-HCV antibody positivity in infants born to anti-HCV–positive mothers

Multivariate analysis has shown that three of eight variables studied have a significant value in predicting vertical HCV transmission. All children in group C were born to mothers with HCV risk factors in contrast to 13 of 30 in group A (100% vs. 43.3%;P < 0.004). In group C patients, maternal viremia was significantly greater than in group A (6.90 ± 5.87 mEq HCV-RNA/mL × 10 6 vs. 3.93 ± 2.94 mEq HCV-RNA/mL × 10 6 ;P < 0.05) (Table 4). Three of 8 infants of group C were born to blood-transfused mothers compared with 2 of 30 in group A (37.5% vs. 6.7% respectively;P < 0.05). No association between HCV vertical transmission and the other variables studied was found. No children in groups A and B had clinical signs of liver disease or abnormal ALT levels during the study, whereas ALT was occasionally elevated in children in group C.

TABLE 4
TABLE 4:
Maternal viral load in the three groups of infants studied

The follow-up data at 24 months show that all children in group A and B were HCV RNA and anti-HCV negative. In group C two children (3.33%) were RIBA and HCV RNA-positive, two (3.33%) were HCV RNA-negative and RIBA “indeterminate,” and four (6.66%) became HCV RNA and RIBA negative (Table 5).

TABLE 5
TABLE 5:
Characteristics of mother/infant couples in group C

DISCUSSION

This long-term prospective study shows an overall rate of HCV vertical transmission of 13.3% followed by a spontaneous viral clearance in more than two thirds of the cases within the second year. It also supports the hypothesis of transient HCV viremia without infection and vertical transmission without clinical evident disease. In concordance with the previous studies a high maternal viral load (4,5,7,10,19) and possession of HCV risk factors (11,20) were the variables predictive of HCV vertical transmission in our patients.

HCV vertical transmission has been extensively investigated in mothers coinfected with HIV and hepatitis B virus (5,8,9), whereas less is known about HIV-negative mothers (11,14). Roudot-Thorval et al. (3), found no evidence of vertical transmission in infants born to HCV-positive/HIV-negative mothers. However, in their series, 9 of 17 (53%) mothers were HCV RNA-negative, and it is possible that the number of HCV-RNA–positive mothers was inadequate to ascertain a low-risk phenomenon such as HCV vertical transmission. The high rate of vertical transmission (13.3%) in our study contrasts with the findings of Resti (3.2%) (11) and Conte (5.1%) (6); however, if we consider our sustained HCV infection rate (3.3%), the results are similar to theirs.

To minimize the impact of false-positive HCV testing when counseling on HCV vertical transmission, it has been recently suggested to consider only HCV-infected infants with at least two separate HCV-RNA positive tests or with anti-HCV antibodies detectable beyond the age of 18 months (13). This algorithm seems reasonable; however, we often found infants who became HCV-RNA negative after having been persistently positive. That children may spontaneously clear the HCV seems evident from the Vogt study (21), and the rate of viral clearance could be even higher in neonates (22). In our series HCV disappeared in six of the eight (75%) patients who acquired the infection as confirmed by two HCV-RNA negative results after a 6-month interval. Our data show that the rate of vertical transmission (and therefore of spontaneous viral clearance) may depend on the time of HCV-RNA testing, because if we had not tested infants at months 8 or 12 or both, transient infections would have not been apparent. Clinically speaking, these transient infections may not be important, but when determining the frequency of viral transmission, they should not be excluded. The major concern with our results is to explain why six infants in group C did not have persistent anti-HCV positivity in their 24-month sample, whereas the two infants with persistent viremia did.

First, we believe it is extremely important to distinguish between a “negative” and an “indeterminate” RIBA test. Indeed, there is evidence to suggest the patients with an indeterminate RIBA 3.0 pattern without HCV-RNA detection (HCV-RNA positive in a previous occasion) might have just recovered from a self-limiting HCV infection and lost part of their circulating antibodies (23). This scenario may apply for patients 1 and 2 but not for patients 3, 6, 7, and 8 (Table 5), who were negative for the RIBA at month 24 even though they were HCV-RNA positive on two separate occasions.

Seronegative HCV infection (absence of a serologic response to HCV polypeptides) is a well-known entity; indeed, as much as 10% of viremic immunocompetent subjects do not have HCV antibodies (24). This phenomenon has been reported also in vertically infected immunocompetent infants (25) and in children (26). Thaler et al. (25) found that all eight of the children they studied who were born to anti-HCV-positive and HCV-RNA–positive mothers had HCV-RNA on several occasions during their first year of life, but none of these children actively produced anti-HCV antibodies. Similar experience comes from Japan (27) and Europe (28).

There are several possible explanations for a seronegative HCV infection. First, a delayed antibody response in the infant may be secondary to the presence of maternal blocking antibodies that may prevent an antibody response (2). Second, if persistent viral infection occurred early enough in pregnancy, there may have been partial immunologic tolerance to the expressed viral antigens; alternatively, the nonstructural antigens used in the antibody test may not be sufficiently antigenic to elicit a detectable antibody response from a relatively immature immune system (29). Third, a seronegative HCV infection may reflect a very transient low-grade viremia by milder and fewer quasi species than their respective mothers (30) without the ensuing antibody response to HCV (31). A similar experience comes from vertically HIV infected children in whom short periods of HIV viremia have been described in absence of HIV antibodies, suggesting that viral clearance in perinatally infected infants may be possible without de novo humoral antibodies response (32,33). We consider the hypothesis of false-positive results on testing the infants at any scheduled visit unlikely, because PCR testing was run on separate occasions, it was always duplicated, and in case of discordance it was confirmed by the Amplicore HCV assay.

None of our children showed persistent ALT abnormalities or clinical signs of liver disease during the first 2 years of life, suggesting that a vertically acquired infection may not be followed by a clinically evident disease. However, this observation, in the absence of histologic information does not have any prognostic value, because biochemical markers of liver inflammation generally do not correlate with HCV RNA levels and histologic changes (34).

Identifying predictors of vertical HCV transmission is of major importance for family counseling. Previous studies have reported a higher incidence of infection in mothers who were HIV-positive (5,8,9,19), had higher viral load (4,5,7,10,19), and belonged to HCV risk categories (11,20). The role of mode of delivery (6,9,19), breast-feeding (9), gestational age, and birth weight (9,14) is still controversial. Our study suggests that the mothers with HCV risk factors have a greater chance of transmitting the infection (P < 0.004), especially if they have had a blood transfusion (P < 0.05), and that maternal viral load is positively correlated with the vertical transmission (P < 0.05). Of interest, although statistically not significant, the two children who were HCV RNA-positive at 24 months had been born to the mothers with the highest viral load in the whole series (Table 4). We found no evidence that HCV genotype, gestational age, mode of delivery, or birth weight influence mother-to-infant HCV transmission.

The more we learn about HCV in long-term studies, the more we realize that there is a higher spontaneous HCV clearance than previously believed, especially in vertically infected infants.

Acknowledgments:

The authors thank Nedim Hazdic, M.D. (Department of Child Health, King's College Hospital, London), for his precious comments and illuminate criticism.

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Keywords:

Hepatitis C virus; Vertical transmission; HCV RNA titer; Spontaneous viral clearance; HCV/HIV co-infection

© 2001 Lippincott Williams & Wilkins, Inc.