Anti-HCV seroprevalence and risk factors of hepatitis C virus infection in Moroccan population groups

Res. Virol. 1996, 147,247-255

0 INSTITUT PASTEURIELSEVIER

Paris 1996

Anti-HCV seroprevalence and risk factors of hepatitis C virus infection in Moroccan population groups S. Benjelloun,

B. Bahbouhi,

S. Sekkat,

A. Bennani,

N. Hda and A. Benslimane

(*)

Institut Pasteur du Maroc, Casablanca (Maroc)

SUMMARY

Hepatitis C virus (HCV) seroprevalence and transmission routes were investigated in several groups of the Moroccan population. This study showed a low HCV seroprevalence in the Moroccan general population. However, haemodialysis patients and haemophiliacs were at higher risk of having HCV infection, since the prevalences were, respectively, 35.1 and 42.4% in comparison with the blood donors’ prevalence (1.1 %I. These results indicated that parenteral exposure is the transmission pathway of HCV. To investigate the possibility of vertical HCV transmission, a cohort of healthy, unselected pregnant women were included in the study. A prevalence of 1% was found among them. Seven newborns were anti-HCV-positive, although, when RT-PCR was used to search for HCV RNA in their sera, none of them was viraemic. These data indicated that anti-HCV antibodies were passively acquired in these cases. We concluded that vertical transmission is absent when mothers are at low risk of contracting other parenterally or sexually transmitted diseases. Three percent of a group of patients of a centre for sexually transmitted diseases were repeatedly anti-HCV-positive, suggesting the possible sexual transmission of HCV. When screening 116 sera of anti-HIV-positive subjects, 19.8% were anti-HCV-positive. Furthermore, 17.9% of the sixty-seven patients who were proven to have sexually contracted HIV were also anti-HCV-positive. These data might reflect a likely cotransmission of these two viruses, hence suggesting HIV is a cofacter for HCV sexual transmission, as previously reported. Key-words: Hepatitis C, AIDS; HCV antibodies, RT-PCR, Seroprevalence, STD.

INTRODUCTION

A hepatitis C virus (HCV) cDNA clone was isolated from a chimpanzee chronically infected with a contaminated human factor VIII concentmte in 1989 (Choo et af., 1989). The molecular

Submitted

November

6, 1995, accepted February 9, 1996.

(*) Corresponding author.

Morocco,

characterization of the HCV genome led to the development of a serological assay for the diagnosis of HCV infection (Choo et al., 1989; Kuo et al., 1989). Results indicated HCV to be the major causative agent of bloodbome non-A,nonB hepatitis (Choo et al., 1989; 1990). Subsequent

248

S. BENJELLOUN

inprovements in diagnosing hepatitis C and the rapidly evolving description of the HCV genome have contributed to an understanding of the epidemiologic characteristics, natural history and response to treatment of chronic hepatitis C infection. While approximately 90% of cases of post-transfusional hepatitis are now known to be caused by HCV, transfusions account for only 5 to 10 % of new cases of hepatitis C. Other sources of percutaneous exposure, mostly from intravenous drug use (Kiyosawa et al, 1990) but also related to haemodialysis (Zeldis et al., 1990), organ transplantation (Konig et al., 1992) and occupational exposure (Kiyosawa et al., 1990) account for a significant number of cases. Vertical and sexual transmission have also been implicated as risk factors for hepatitis C infection. In many epidemiologic studies, however, known risk factors have still not been identified in up to 40% of hepatitis C cases (Alter et al., 1993). We report here the investigation of anti-HCV antibody prevalence in several groups in the Moroccan population, and we have determined the population(s) at high risk of acquiring HCV infection. PATIENTS

AND METHODS

Patients

One thousand healthy volunteer blood donors (818 males, 182 females) with a mean age of 29.8 years (range, 18-50) were included. They were recruited from the Blood Transfusion Centre in Casablanca. Stored plasma samples from 67 adults (age range, 18-32) and 51 children (age range, 3-17) with haemophilia, who are regularly examined at the haemophilia treatment centre in Casablanca, were available. Most of these patients received blood products made from pooled plasma. In addition, samples collected from a cohort of 67 males and 47 females treated in a haemodialysis unit were selected for this study. The mean age was

DEPC HBV HCV HIV

= = = =

diethylpyrocarbonate. hepatitis B virus. hepatitis C virus. human immunodeficiency

ET AL. 45 years (range 28-65). The haemodialysis patients were not infected by the human immunodeficiency virus (HIV). Nine of them were carriers of hepatitis

B virus (HBV) surface antigen (HBsAg), against HBV.

To evaluate the risk of vertical transmission of HCV, 676 healthy pregnant women attending a public hospital were systematically screened for HCV serum antibodies. The seven offspring of the seven anti-HCV antibody-positive and viraemic mothers who gave their consent were tested at birth and at approximately three months for anti-HCV antibodies and HCV RNA by RT-PCR (reverse transcription/polymerase chain reaction). To estimate the possibility of sexual transmission of HCV, 2,088 patients with a history of at least one episode of sexually transmitted diseases (STD) were also included. These patients were recruited from the Centre for STD in the Moroccan Pasteur Institute in Casablanca, and a questionnaire was completed for each subject. Finally, to evaluate the prevalence of anti-HCV antibodies in HIV-seropositive patients, and further, to determine the extent to which sexual HCV transmission is correlated to HIV infection, 116 HIVinfected patients were enrolled in this study. Specimens were collected during an epidemiological

investigation,

the purpose of which was to determine

HIVseroprevalence and associated Morocco. A detailed questionnaire

risk factors in that provided

clinical

data was com-

as well as epidemiological

pleted through a personal interview. Sixty-seven out of 116 patients were sex partners of HIV-infected subjects. As the epidemiological investigation revealed no parenteral exposure such as transfusions, tatooing or intravenous drug use, it was assumed that these patients acquired HIV infection through sexual contact. Characteristics of the patients study are summarized in table I.

enrolled

in this

ELBA The presence of anti-HCV antibodies was determined by a second generation enzyme-linked immu-

nosorbent assay which used the recombinant

fusion

peptide antigens (Wellcozyme Murex, UK). A repeat assay was performed on all positive sera to ensure reproducibility. Repeatedly positive sera were further tested by either “Ribatest” (Ortho

PCR RT-PCR STD virus.

while all

others were immunized

= = =

polymerase chain reaction. reverse transcription/PCR. sexually transmited disease

HEPATITIS

C VIRUS INFECTION

IN MOROCCAN

system, USA) or Western blot (Wellcozyme Murex, UK). According to the manufacturer’s instructions, the sample was regarded as positive by the latter tests if an appreciable reactivity to any two antigen bands was recorded. DNA synthesis

POPULATION

GROUPS

249

0.5 mM/l of dNTP, 10 mM/l Hepes, 0.2 mM/l EDTA, 200 units of Moloney murine leukaemia virus reverse transcriptase (Gibco BRL) and 20 units of RNase inhibitor (Boehringer). cDNA synthesis was performed at 37°C for 1 h. The reaction was stopped for 5 min at 95°C (Sellner et al., 1992), and 5 yl of cDNA was added to a mixture containing 50 pmol of each of the outer primers (sense, antisense) and 1 unit “Ampli-Taq DNA polymerase” (Perkin Elmer Cetus). The first reaction was run with 25 cycles of 1 min at 92°C for denaturation, 1 min at 55°C for annealing and 1 min at 72°C for extension, with a final elongation step of 5 min at 72°C. The second PCR was performed in a 50-pl mixture containing 5 pl of the first reaction product and the corresponding inner primer pair.

and nested PCR

PCR was first performed on repeatedly antiHCV-positive mothers. Newborns of viraemic mothers were tested by PCR. Some indeterminate anti-HCV sera were also tested for HCV RNA by RT-PCR. Recommended precautions were taken to avoid DNA contamination (Kwok and Higuchi, 1989). RNA was isolated from 2OOpl serum using the following modification of the thiocyanate-phenolchloroform method (Chomczynski and Sacchi, 1987). After the first extraction with lysis buffer, the aqueous phase was reextracted with I volume of chloroform : isoamylalcohol (24: l), and the nucleic acids were precipitated with isopropanol. The pellet was washed with cold 70% ethanol, dried at room temperature and dissolved in 20~1 of DEPC-treated water.

The second PCR product was run on 2 % agarose gel electrophoresis. The DNA was stained with ethidium bromide and visualized in UV light. A band of 211 basepairs indicated a positive reaction. Statistical analysis Comparisons of proportion were made using the x2 test. Significance was set at a 0.05 level.

The primers for the cDNA synthesis and the nested PCR were from the conserved S-non-coding region of the HCV genome (Okamoto et al, 199Oa, b) : outersense primer : SGCCATGGCGTTAGTATGAGT-3’; outerantisense primer: 5’-TGCACGGTCTACGAGACCBZ-3’; innersense primer : S-GTGCAGCCTCCAGGACCCCC-3’; innerantisease primer: SGGGCACTCGCAAGCACCCTAT-3’. For cDNA synthesis, extracted RNA was reverse-transcribed in 20 pl cDNA reaction mixture containing 50 pmol of the outer antisense primer,

RESULTS Sera from 1,000 blood donors who served as controls were tested, and 1l/ 1,000 (1.1%) were anti-HCV-positive (table I). As was expected, a higher prevalence of anti-HCV antibodies was

Table I. Prevalence of HCV antibodies.

Group

No. tested

Blood donors@) Haemodialysis patients Haemophiliacs Pregnant women STD patients Anti-HIV + patients

1,000 114 118 676 2,088 116

Total

4,112

Age (years) median range 29.8 45 18.5 28 29.6 31 -

ND = non-determined; Ab = antibody. Sex ratio (ME): males infected/females infected. ~1 Control group ; cb) statistically significant difference

1S-50 28-65 3-32 20-45 15-51 22-4 1 -

Sex

HCV Ab+

M

F

818 67 118 1,178 ND

182 47 676 910 ND

11 40 50 7 62 23

2,181

1,815

193

; (h*’ p < 0.0 I ; (h**) p < 0.00 I.

(%I (1.1) (35.1)@**) (42.4)cb**) (1.0) (3.0)(t)“’ ( 19.8)cb**’ -

Sex ratio WV 4.5 1.4 OS -

250

S. BENJELLOUN

found in haemodialysis and haemophiliac patients’ samples : respectively, 40/l 14 (35.1%) and 50/l 18 (42.4 %) were anti-HCV-positive (table I). In order to study the vertical transmission of HCV, 676 pregnant women were tested for antiHCV antibodies, and 7 (1.0 %) were positive (table I). The latter were proven to be viraemic by RT-PCR. At birth, 7/7 infants had anti-HCV antibodies. The titres were similar to those of the mothers, and the Riba- patterns were also similar in the mothers and their offspring. All the newborns had normal transaminase levels. The anti-HCV antibodies decreased at three months. The seven babies were repeatedly tested for HCV RNA from birth to three months of age. None of them was positive for HCV RNA in the PCR assay at any time during follow-up (table II).

Table

II. Follow-up of HCV status in infants of seven anti-HCV-antibodyand viral RNA-seropositive mothers. HCV status

Confirmatory Infant Infant 1 at birth 3 months Infant 2 at birth 3 months Infant 3 at birth 3 months Infant 4 at birth 3 months Infant 5 at birth 3 months Infant 6 at birth 3 months Infant 7 at birth 3 months All

alanine

ELISA-2

test

PCR

+++ 4.

+ +

-

+ -

+ -

-

++ +

-I-

++ +

+ +

+

+ +

+ +

+ + levels

were

To evaluate the possibility of sexual transmission of HCV, 2,088 patients with STD, whose risk factors were mainly previous history of STDs, multiple sexual partners or prostitution, were tested for anti-HCV. An overall anti-HCV antibody prevalence of 3.0% (62/2,088) was found (table I). In HIV-positive individuals, the overall prevalence of anti-HCV was high; 23 out of 116 (19.8%) HIV-infected patients tested were antiHCV-positive (table I). As mentioned above, 67/l 16 were HIV-infected through sexual contact and among them, 12 (17.9%) were anti-HCVpositive (table III).

DISCUSSION The analysis of the genomic sequence of HCV has facilitated the development of a number of diagnostic assays for testing circulating antibodies in samples from patients with HCV infection. This has enabled study of the epidemiological distribution of HCV infection in many areas of the world, as well as of the routes of HCV transmission. HCV is the major cause of parenterahy transmitted non-A,non-B hepatitis. However, approximately half of the patients with type C hepatitis do not have a history of parenteral exposure (Alter et al., 1989; Kiyosawa et al., 1990). Our results indicate

Table III. according

that in Morocco,

the epi-

Seroprevalence of anti-HCV antibodies to sexual transmission in HIV-seropositive subjects. No. tested

No. HCV Ab+

Percent (So)

33 14

3 5

9.1 35.7

Heterosexual couples STDs

11

3

27.3

9

1

11.1

Total

67

12

17.9

Risk groups

+

+++ ++

aminotransferase

-

+

ET AL.

Bisexual Multiple

partners

normal.

HEPATITIS

C VIRUS INFECTION

IN MOROCCAN

demiological status of HCV infection is similar to that observed in other areas of the world, with a prevalence of 1.1% among adult volunteer blood donors and higher prevalences among the risk groups. Previous studies of blood donors reported various prevalence rates such as 0.98% in Japan (Yamaguchi et aE., 1994), 0.46% in Portugal (Santos et al., 1993) and 1.7% in Saudi Arabia (Abdelaal et al., 1994). Parenteral HCV transmission is now well documented. Haemodialysis and haemophiliac patients are often multiply transfused and are thus frequently infected by hepatotropic viruses, mainly hepatitis C. This is also reflected by our findings, since the prevalence of anti-HCV antibodies in haemodialysed patients was as high as 35.1%. Similarly, a high prevalence of anti-HCV antibodies compared with that of blood donors has been reported in haemodialysis patients in various geographic areas (5 to 85% worldwide) (Pol et al., 1992, 1993, Nordenfelt et al., 1993 ; Yamaguchi et al., 1994; Hayashi et d., 1994a, b). This high frequency of HCV infection is related to the number of blood transfusions and the duration of dialysis (Kallinowski et aZ., 1991; Muller et ul., 1992). Therefore, the diffusion of nonA,non-B hepatitis in dialysis units appears to be mainly transfusion-related, but there is also a significant risk of environmental contamination related to violation of infection control measures. The strict observation of the general recommendations for the prevention of hepatitis diffusion is crucial and seems to be a reasonable and effective approach. Our results also show a high prevalence of anti-HCV antibodies in haemophiliac patients, and thus are consistent with previous studies suggesting that HCV-associated chronic liver disease is a major complication of chronic blood product treatment in individuals with haemophilia (Esteban et al, 1989; Makris et al., 1990). At least 85-90% of cases of post-transfusion hepatitis are due to this virus (Esteban et al., 1990). This virus also causes most cases of sporadic or community-acquired non-A,non-B hepatitis not associated with transfusion (Alter et al., 1990). The transmission of HCV by direct percutaneous exposure to blood is well documented.

POPULATION

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251

In general, persons with well recognized risk factors, including blood or blood product recipients, haemodialysis patients and health care workers with occupational exposure to blood, do not account for all the cases of acute hepatitis. The transmission route for sporadic cases remains controversial, although person-to-person transmission by sexual behaviour or by non-sexual household contact, including vertical transmission, has been implicated (Alter et al., 1990; Akahane et al., 1992). A cohort of 676 healthy, unselected pregnant women were enrolled in this study. We found a seroprevalence of 1 %, which is similar to that reported in Japan (1.1%) (Kojima and Yamanaka, 1994). Evaluating the risk of transmission of HCV from infected women to their newborn infants on the basis of published studies is particular-y difficult, since the studies were limited in the number of cases and duration of follow-up. In the present, prospective, study of infants born to unselected anti-HCV-seropositive mothers, we did not show any vertical transmission of HCV. At birth, antiHCV antibodies were present in the sera of the newborns, and their ELISA titres and confnmatory patterns were similar to those of their mothers. The anti-HCV antibodies decreased at 3 months, suggesting the passive transmission of anti-HCV antibodies during pregnancy (Wejstal et al., 1990; Thaler et al., 1991). We used nested RT-PCR to detect HCV genomic RNA in these seven newborn infants of anti-HCV-positive mothers, and no samples yielded a positive result. Thus, our findings support data in previous studies concerning unselected pregnant women, suggesting that vertical transmission is rare or absent. However, our findings may be limited by the small number of mother-infant pairs. The estimated risk of vertical HCV transmission varies widely in the literature (0 to 100%) according to the study population and the test used, as summarized in table IV (Rouzioux et al., 1990; Novati et al., 1992; Giovanini et al., Lancet, 1990; 1:1216-1217, correspondence; Reesink, H.W. et al., Luncet, 1990; 1:1216-1217, correspondence; Kurochi, T. et al., J. Infect. Dis., 199 1; 164:427-428, correspondence; Chen, D.S.

252

S. BENJELLOUN Table IV. Comparison

Authors Rouzioux et al. Wejstal et al. Giovannini(*) Reesink(*) Thaler et al. Klmcy

Novati et al. Our study Proportion of HIV/high tion ; NM = not mentioned. (*I Letters to the editor;

Year of publication

of studies of mother-to-infant

No. of cases (mother/infant)

1990 1990 1990 1990 1991 1991 1991 1992 exp. = proportion

ET AL.

42142 S/l 1 25125 1707 8f8 9113 8/8 8/8 717 of anti-HIV-seropositive

transmission

of HCV.

Proportion of HIV/high exp.

HCV RNA testing

loo%/NM 100 %INM 37.5 %/lOO% NM 0% lOO%/0%

No No No No Yes Yes Yes Yes Yes

mothers/proportion

of mothers

highly

% of transmission 24 9.1 44.0 5.9 100.0 69.2 0 50.0 0 exposed

to HCV

infec-

see text for references.

et al., J. Infect. Dis., 199 1; 164:428-429, correspondence). High rates of vertical transmission (40 to 100%) were observed when mothers had detectable serum anti-HIV antibodies or were highly exposed to parenterally acquired disease (Thaler et al., 1991; Giounnini, M. et al, Luncet, 1990; 1: 1166, correspondence). Indeed, it has been suggested that maternal HIV infection may favour HCV vertical transmission. One plausible explanation for the difference in rates of transmission in highly or poorly exposed women is a difference in level of C viraemia. It is conceivable that anti-HIV-positive women have a high rate of HCV replication because of immunodeficiency or viral interaction between HCV and HIV (Novati et al., 1992 ; Alter et al., 1990). This would imply that women who are anti-HIV-negative and whose behaviour does not put them at risk for repeated HCV contamination would have lower levels of C viraemia. If the risk of vertical transmission of HCV were indeed dose-related, this could be a satisfactory explanation for the divergent results in the literature. The quantification of viral HCV RNA will enable the testing of this hypothesis (Roudot-Thoraval et al., 1993). While sexual activity has been implicated in the transmission of hepatitis B infection (Alter et al., 1989) its role as a mechanism for the transmission of HCV is less clear (Alter et al., 1989;

Tor et al., 1990; Eyster et aZ., 1991; Everhart et al., 1990). To better understand the role of sexual behaviour in the transmission of HCV, we screened 2,088 patients attending the Pasteur Institute of Morocco’s STD Centre, for evidence of HCV infection: 62 were anti-HCV-antibodypositive (3.0%). This prevalence is 3-fold higher than that of controls (1.1%). No pat-enter-al exposure was found in the seropositive patients with STDs in this study. In contrast, the number of sex partners was strongly associated with anti-HCV test results. This is consistant with preliminary reports from other countries. Anti-HCV antibodies were detected in 7.7% (USA, Weinstock et al., 1993) and 4.7 % (West Germany, Hess et al., 1989) of patients with sexually transmitted diseases. These data support the possibility of sexual transmission of HCV. Therefore, sexual behaviour may explain some cases of HCV infection not related to transfusion. Eyster et al. (1991), in a study of haemophiliacs and their spouses, suggested that concurrent HIV infection may be a cofactor for HCV sexual transmission. To assess this hypothesis, we tested 116 HIV-infected patients for anti-HCV antibodies. An overall significantly higher anti-HCV prevalence of 19.8% resulted. On the other hand, 67/l 16 were proven to have sexually acquired HIV infection, and among them, twelve were anti-HCV-positive

HEPATITIS

C VIRUS INFECTION

IN MOROCCAN

(17.9%). This fact is probably a reflection of the same mode of transmission of these two viruses. Therefore, it suggested that HIV may be a cofactor for sexual transmission of HCV. As mentioned above, one possible explanation is that immune response suppression due to HIV infection would result in an increase in HCV viraemia. Thus, sexual transmission of HCV would very likely occur (Oyster et al, 1991). On the other hand, preliminary data reported that anti-HCV prevalence in HIV-infected patients might be underestimated because of loss of the humoral immune reponse, called “seroreversion” (Ragni ef aZ., 1993). In conclusion, our results, in conjunction with others, suggest that besides being a parenterally transmitted infection, HCV can be sexually transmitted. Thus, the introduction of systematic antiHCV screening tests as well as efforts towards the prevention of AIDS and other sexually transmitted diseases is important in reducing the spread of HCV.

Acknowledgements We would like to thank R. Guinet for rich and useful comments.

SCroprCvalence anti-VHC et facteurs de risque de I’infection par le virus de 1’hCpatite C dans une population marocaine Divers groupes de la population marocaine ont fait l’objet d’une Ctude de sCropr&alence des anti-

corps anti-VHC.

Chez les donneurs de sang, elle

ttait de 1,l %. Des sbropr&vaIences plus ClevCes ont CtC retrouvCes chez les htmodialysts et les hemophiles, respectivement de 35,l et 42,4%, soulignant ainsi l’importance de la transmission par voie sanguine de ce virus. Pour l’ttude de la transmission verticale du VHC, des femmes enceintes saines 2 terme, choisies au hasard, ont CtC incluses dans ce travail. La s&opr&aIence des anticorps anti-VHC au sein de ce groupe Ctait de 1%. Parrni les sept nouveau-& de m&es infectCes, aucun n’Ctait virCmique (ARN VHC nkgatif par RT-PCR). Ce r&ultat serait en faveur d’une absence de transmission verticale chez les femmes non expostes aux facteurs de risques habituels (MST, toxicomanie par voie veineuse).

POPULATION

GROUPS

253

Par contre, 3 % des patients consultant pour MST prksentaient des anticorps anti-VHC. Cette prCvalence est 3 fois plus ClevCe que celle retrouvte chez les donneurs de sang et Ies femmes enceintes. La prtvalence des anticorps anti-VHC chez 116 sCropositifs pour le VIH &ait de 19.8%. Parmi 67 patients ayant contract6 le VIH par voie sexuelle, 17.9 % prksentaient egalement des anticorps antiVHC. Cette pr6valence elevke chez ce dernier groupe de patients renforcerait le r81e du VIH comme cofacteur dans la transmission sexuelle du VHC.

Mats-cl&s: VHC, RT-PCR,

HCpatite C, SIDA ; Anticorps antiSCropr&valence, Maroc, MST.

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