Transplacental transmission of hepatitis C virus in HIV-negative mother

Res. Virol. 1998, 149, 229-234

0 INSTITUT PASTEURIELSEVIER Paris 1998

Transplacental transmission of hepatitis C virus in HIV-negative mothers S. Agha (*)(I), L.S. Sherif (*I, M.A.

Mansoura

Allam

(*) and M. Fawzy (*I

(I) Clinical Pathology Department and (21 Obstetrics and Gynecology Department, Faculty of Medicine, Mansoura University, Mansoura

(Egypt)

SUMMARY

The aim of this work was to evaluate transplacental transmission of hepatitis C virus (HCV) in HIV-negative pregnant women who were HCV-PCR-positive, and also to determine the serotypes of the virus in these cases. Therefore, 767 pregnant women were screened for anti-HCV antibodies, hepatitis B surface antigen (HBsAg) and anti-HIV antibodies. HCV PCR was performed for HCV-positive women. Those who were PCR-positive were tested for anti-HCV IgM. Neonates of PCR-positive mothers were tested for virus transmission by the PCR test. Virus serotyping was done for mothers and neonates. Anti-HCV antibodies were detected in 105 out of 767 (13.7%) pregnant women. PCR was positive in 16 out of 67 HCV-positive women (26.9 %). Transplacental transmission occurred in 11.l % of HIV-negative pregnant women. HCV type 4 predominates in Egypt (63.3%). Mothers who are PCR-positive and have high aspartate aminotransferase and positive anti-HCV IgM are most likely to transmit HCV to their babies. Key-words: HCV, Pregnancy, HIV; Transmission.

INTRODUCTION

Blood transfusion and intravenous drug abuse are considered to be the main pathways of transmission of hepatitis C virus (HCV) (Bell et al., 1990 ; Meyer and Gordon, 1991). Perinatal transmission of HCV is known to occur, but whether the predominant route is transplacental, during birth or through breast milk necessitates further investigation (Nagata et al., 1992). Coinfection

of HCV

with human

immuno-

SubmittedMarch 5, 1998, acceptedMay 26, 1998. (*) Corresponding author.

deficiency virus (HIV) is thought to be one of the risk factors in vertical transmission of HCV. This is because immunosuppression secondary to maternal HIV infection could result in increased HCV replication and might lead to more efficient transmission to the infant (Giovannini et al., 1990). The aim of this study was to evaluate the transplacental transfer of HCV in HIV-negative/HCV-PCR-positive pregnant women and to determine the virus serotype in these cases.

S. AGHA

230 MATERIALS

AND METHODS

This study was conducted on 767 pregnant women who presented at the antenatal care unit of the Obstetrics Department of Mansoura University Hospital, Mansoura, Egypt. The study began in May, 1996 and ended in January, 1997. Written consent was obtained from women participating in the study. All the pregnant women enrolled in the study were screened for antibodies to hepatitis C virus (anti-HCV antibodies) by the “ELISA Second Generation kit” (Abbott HCV EIA 2nd generation, Abbott Diagnostic Division, Germany). The women were also screened for hepatitis B surface antigen (HBsAg) by “ET1-Mak-3” (Sorin Biomedica Diagnostic S.P.A., Italy) and antibodies to human immunodeficiency virus (anti-HIV) using “Cobas CoreAnti-HIV-l,HIV-2” (EIA DAGS, Switzerland). Pregnant women positive for anti-HCV antibodies were subjected to complete history taking, general and abdominal examinations for manifestations of liver disease.These women were further investigated for detection of HCV RNA in the serum by the reverse transcription polymerase chain reaction (RT-PCR) using the “Access RT-PCR system” (Promega, USA). Briefly, HCV RNA was extracted by the addition of 200 l.~lof serum to 200 ~1 of guanidine thiocyanate and 40 l.tl sodium acetate. After mixing, 400 ~1 of phenol chloroform isoamyl was added and tubes were centrifuged. Isopropanol in a volume of 500 ~.tl was added to the aqueous phase and tubes were incubated at -20°C overnight. Pellets were then washed by ethyl alcohol and dried at 56°C for 45 min. RNA was resuspendedin 20 pl of RNase-free water. The tube containing PCR reaction reagents only was used as a negative control. Two rounds of amplification were performed. In round 1, 5 ~1 of extracted RNA was added to the master mix containing all reagents required for RT amplification, and exposed to a “Hybaid” thermal cycler (HB-TR 1 ; Hybaid Ltd., Middlesex, UK) according to the following protocol. One cycle of 48°C for 45 min and 95°C for 5 min, 5 cycles of 95°C for 1 min, 60°C for 45 s and 72°C for 2 min and one cycle of 72°C for 5 min. In round 2, 2 ~1 of RT-PCR product was added to the master mix containing all reagents required for nested PCR and exposed to the Hybaid thermal cycler using the above protocol except for the first cycle, which was of 95°C for 5 min. Detection was carried out by

ALT AST HBsAg HCV

= = = =

alanine aminotransferase. aspartate aminotransferase. hepatitis B surface antigen. hepatitis C virus.

ET AL. electrophoresis on 2% agarose gel in TAE buffer and gel was viewed on transilluminator. Neonates of PCR-positive mothers were tested for HCV RNA using RT-PCR on blood samplescollected immediately from peripheral veins of babies to avoid both contamination with maternal blood and to exclude lactation as a route of transmission. Liver function tests including serum alanine amino-transferase (ALT), asparatate aminotransferase (AST), bilirubin and albumin were performed for PCR-positive women and their neonates (bioMerieux kits, France). An anti-HCV IgM assay was done for PCR-positive mothers (Abbott HCV IgM EIA). In this test, the HCV structural (core) antigen was used. Serotyping of HCV was done for PCR-positive mothers and their neonates(Murex HCV Serotyping l-6 Assay, Murex Diagnostics limited, UK). In this test, synthetic peptides representing the variable antigenic region from NS4 of HCV types 1, 2, 3, 4, 5 and 6 were used. One strip of eight wells was used for each sample or control. An appropriate competing solution (l-6 or “all”) was added to the relevant wells. Readings were obtained at 450 nm and results were calculated. Results were statistically analysed on IBM compatible computers using SPSS/PC+Package (SPSS Incorp, Chicago, IL).

RESULTS In this work, 767 pregnant women were screened for HBsAg, anti-HCV and anti-HIV antibodies. All cases were negative for HIV. HBsAg

and anti-HCV

were positive

in 18

(2.35 %) and in 105 (13.69 %) cases, respectively (table I). Six cases were positive for both HBsAg

and anti-HCV;

two of them did not

complete the follow-up and the remaining four cases were negative for PCR. Among the 67 anti-HCV-positive

pleted

PCR-positive

cant difference

HIV RT-PCR TAE

pregnant women who com-

the follow-up

= = =

study, 18 (26.87 %) were

(table II). A statistically

signifi-

was observed between the num-

human immunodeficiency virus. reverse transcription/polymerase Tris-acetate-EDTA.

chain reaction.

TRANSPUCENTAL

Table I. Prevalence of anti-HCV,

HBsAg and anti-HIV

Number Anti-HCV HBsAg Anti-HIV CI = confidence

95%

13.69%

CI

0%

CI

99%

11.26-16.12 1.28-3.42 0

2.35%

10.49-16.89 0.94-3.74

0

interval.

Table II. PCR versus ELISA

in HCV-positive

pregnant women.

Negative PCR

Positive ELISA No.

%

No.

67

100%

49

CI=confidence

Positive PCR %

73.13%

No. 18

%

95%

CI

16.25-37.48

26.87%

99%

CI

12.9-40.8

interval.

bers of cases having high AST (6) among the 18 PCR-positive mothers and the numbers of cases having high AST (4) among the 49 PCRnegative mothers. Other clinical and laboratory data showed no statistically significant differences (table III). Two out of eighteen PCR-positive mothers transmitted the virus to their babies (2/18, 11.11 %) (table IV). The two PCR-positive neonates had statistically highly

Table III. Clinical

(No.=49)

Jaundice Liver cirrhosis Ascites Bilirubin Albumin Elevated ALT (>45 U/ml) Elevated AST (>4OU/ml) were 49, 51 and 48; (**)

significant levels of ALT and AST compared with the sixteen PCR-negative neonates. For ALT, the mean level was 90 + 14.14 and 35.56k12.81, and for AST, it was 93.5k38.98 and 39.912 18.63, respectively (table V). Serotype 4 was the most prevalent in the eighteen PCR-positive mothers (15/18, 83.3 %) and antiHCV IgM was positive in 6/18 subjects (33.3 %) (table VI).

data and liver function tests in 67 PCR-tested pregnant women. PCR-negative

(*) Levels

antibodies among 767 pregnant women.

Percentage

105 18 0

231

HCV TRANSMISSION

PCR-positive (No.=18)

1 1 1.1521.58

0 0 0 1.08+1.01

3.64kO.54 3'*'

3.85kO.59 4Y)

2

4'***'

72, 47, 51 and 56; (***)

(j(****,

43, 51, 49 and 59; (****)

56, 63, 46, 85, 65 and 61

P 0.384 0.541

0.541 0.337 0.184 0.144 0.029

S. AGHA ET AL.

232

Table IV. Incidence of mother-to-infant

PCR-positive mothers

Newborns PCR-negative No. %

No.

%

18

100% CI=confidence

16

PCR-positive

88.89%

No.

%

2

11.11%

95%

CI

O-25.63

99%

CI

O-30.19

interval.

Table V. Comparison

Maternal Maternal Maternal Maternal Neonatal Neonatal Neonatal Neonatal

transmission of HCV RNA.

of liver function tests (ALT, AST, bilirubin and albumin) relation to neonatal PCR.

ALT AST bilirnbin albumin ALT AST bilirubin albumin

PCR-negative neonates (16)

PCR-positive neonates (2)

35.56+13.X9 44.63k33.74 1.04~1.07 3.8kO.6 35.56k12.81 39.91k18.63 3.25k3.84 4.21kO.27

38.5Ok12.02 65.50227.58 1.35kO.21 4.2kO.8 90.00~14.14 93.5Ok38.98 4.75-1-4.6 4.45kO.64

Table VI. Anti-HCV

IgM and HCV serotypes in PCR-positive mothers.

Serotypes : 1 2 4

Anti-HCV IgM : positive negative

No.

%

2/18

11.1

l/18 15/18

5.6 83.3

6118

33.3 66.7

12/18

in mothers and neonates in

DISCUSSION

Perinatal transmission of hepatitis C virus (HCV) is known to occur, but the route of transmission remains obscure. Therefore, 767 pregnant women were screened for anti-HCV antibodies, HBsAg and anti-HIV antibodies. Out of

P 0.438

0.119 0.053 0.479 0.023 0.032 0.479 0.561

767 pregnant women, 105 (13.69 %) were positive for anti-HCV antibodies by second generation ELISA (table I), a prevalence which is comparable to that reported by other authors in Egypt (El-Sahly et al., 1991; El-Zayadi et al., 1991). They showed that the prevalence of antiHCV antibodies varies from 5 to 20% in a healthy population. The prevalence of anti-HCV in pregnant women in this study was higher than that recorded in developed and developing countries: 6.8 % in Cameroon (Ndumbe et al., 1994), 1.3 % in Taiwan (Lin et al., 1995), 1.9 % in Puerto Rico (Deseda et al., 1995), 4.3 in the USA (Silverman et al., 1993), 3.9 % in France (Marcellin et al., 1993) and 0.7% in northern Italy (Marranconi et al., 1994). The high prevalence reported in this study may be due to the fact that the sample represented high-risk cases presenting at the antenatal care unit, where women are more likely to have been exposed to risk factors for HCV infection. Also, within this

TRANSPLACENTAL

group, bilharziasis, which is thought to be a risk factor for HCV infection (El-Sahly et al., 1991), is endemic. Only 18 pregnant women out of 767 (2.35 %) were HBsAg-positive, indicating that the prevalence of HBV infection is much lower than that of HCV infection. There were no HIVpositive cases. PCR was performed in order to determine the presence of HCV-RNA in the 67 HCV-positive pregnant women who completed the follow-up study. Only eighteen mothers (26.87%) were PCR-positive (table II). It is interesting to note that ELISA readings in the 49 PCR-negative mothers were quite low. On the other hand, primers used in this experiment gave positive results in 81 % of HCV-positive patients with chronic C hepatitis (data not shown). Correlating the two findings, we confirm that the primers are conserved in the sequences of HCV serotype 4. The presence of HCV RNA among pregnant women with anti-HCV antibodies was variable in different studies: 38.5 % in France (Marcellin et al., 1993) and 86% in Taiwan (Lin et al., 1995). No significant differences were detected among PCR-positive and PCR-negative pregnant women in terms of the presence of jaundice, liver cirrhosis, ascites, serum bilirubin, serum albumin and serum ALT (table III). Taking 40 U/ml as a cut-off level for AST, a significant difference between PCR-negative and -positive mothers was observed. In this study, among the 18 neonates of PCRpositive mothers, 2 infants were PCR-positive (table IV), indicating that HCV was transmitted vertically at a rate of 11.11 %. The route of transmission of HCV may be transplacental, intrapartum or following delivery, through breast milk. In these two PCR-positive infants, lactation was excluded as a route of transmission, as the neonatal blood sample was taken immediately after delivery. Also, the intraparturn route was excluded because these two neonates had elevated serum ALT and AST levels (table V), indicating that they were infected long before delivery. This might indicate that maternal-neonatal transmission of HCV in the positive cases could only be transplacental. A simi-

HCV TRANSMISSION

lar observation was reported by Cilla (1992), Lam et al. (1993) and Ohto (1994).

233

et al. et al.

Coinfection of HCV with HIV was considered to be an important risk factor for the vertical transmission of HCV (Giovannini et al., 1990). Novati et al. (1992) found HCV RNA in 50% of infants born to HCV- and HIV-positive mothers, and they suggested that HCV is transmitted to infants only when the mother has both HCV and HIV. In this study, infection with HIV was not observed to be a necessary factor for HCV transmission. Similarly, Lam et al. (1993) and Ohto et al. (1994) have shown that perinatal transmission of HCV could occur even in the absence of HIV coinfection. The elevated levels of serum AST and ALT in the two neonates who were PCR-positive and the elevated levels of AST in their mothers (table V) are predictors of maternal-foetal transmission of HCV. Therefore, in circumstances in which PCR is not available, elevated AST in women with anti-HCV antibodies could suggest the presence of HCV RNA in their blood. Also, elevated ALT and AST in neonates of PCR-positive mothers could predict virus transmission from mother to infant. Anti-HCV IgM was positive in 6/18 PCR-positive mothers (33.3 %) (table VI). The two mothers who transmitted the virus to their infants were anti-HCV-IgMpositive. Serotyping of HCV in the 18 PCRpositive mothers revealed that HCV type 4 was present in 15 cases (83.3 %), type 1 in 2 (11.1%) and type 2 in case 1 (5.6 %). The two neonates were infected with HCV type 4. The high prevalence of HCV serotype 4 (83.3 %) confirmed the observation of Simmonds et al. (1993) that HCV genotype 4 predominates in Egypt* We conclude that maternal-foetal transmission of HCV may be predicted if pregnant women are positive for both PCR and anti-HCV IgM and if the AST is high.

Acknowledgements This study was supported by a grant from Mansoura University, Mansoura, Egypt.

S. AGHA

Transmission transplacentaire du virus de I’hbpatite C chez des m&-es VIH nkgatives Sur 767 femmes enceintes VIH n&gatives et HBsAg nkgatives, 105 (13,69%) ont des IgM antiVHC. Sur 67 femmes anti-VHC positives, 18 (26.9 %) sont positives en PCR pour I’ARN viral. La transmission transplacentaire a Ctk observCe chez 2 des 18 mbres PCR positives (1 1,l %). Le sCrotype 4 est prkdominant en Egypte (83,3 %). La transmission du VHC apparait plus importante chez les m&es PCR positives et qui ont des IgM anti-VHC ainsi qu’un taux ClevC d’aspartate-aminotransferase. Mats-cl&s:

VHC, Grossesse,VIH; Transmission.

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