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    Flowchart of the studied pregnant women population and their offspring (November 2001 to October 2004).

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    HCV subtype distribution among 64 HCV-RNA positive pregnant women in Yaoundé, Cameroon.

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LOW RISK OF MOTHER-TO-CHILD TRANSMISSION OF HEPATITIS C VIRUS IN YAOUNDÉ, CAMEROON: THE ANRS 1262 STUDY

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  • 1 Centre Pasteur du Cameroun, Yaoundé, Cameroun; Laboratoire de Virologie, CHU Toulouse Purpan, Toulouse, France; Centre Mère Enfant, Fondation Chantal Biya, Yaoundé, Cameroun; Maternité Principale, Hôpital Central, Yaoundé, Cameroun

To assess mother-to-child transmission (MTCT) of hepatitis C virus (HCV) in Cameroon, 5,008 pregnant women were screened for HCV antibodies. Eighty-nine (1.8%) were HCV-antibody (HCV-Ab) positive. Among these, 7 (7.9%) were HBsAg positive, 6 (6.7%) HIV-positive, and one (1.1%) was co-infected by both hepatitis B virus (HBV) and HIV. Sixty-eight (76%) out of 89 HCV-Ab positive pregnant women were HCV-RNA positive. The HCV genotype determination indicated the predominance of genotype 4 (45.3%), followed by the genotypes 1 (28.1%) and 2 (26.6%). The mean HCV-RNA levels of 41 women at the time of delivery was 4.8 (range 0.06–34.7) × 106 RNA copies/mL. Finally, 35 women delivered 36 live children. None of those screened at 6 weeks and 6 months of age were HCV-RNA positive. The failure to detect HCV vertical transmission suggests that the mother-to-child transmission (MTCT) is not a major route of HCV transmission in Cameroon.

INTRODUCTION

Hepatitis C virus (HCV) infection is a major worldwide public health problem. The World Health Organization (WHO) estimates that 3% (nearly 170 million people) of the world’s population are chronically infected with HCV and that it accounts for around 20% of cases of acute hepatitis and 70% of cases of chronic hepatitis.1,2 Africa is reported to have the highest HCV prevalence rate.3

Although direct percutaneous inoculation is the most efficient mode of transmission of HCV, several studies have demonstrated that sexual, household, occupational, and vertical transmission of HCV may also be of importance.4 The overall risk of infection with respect to perinatal transmission has not been fully elucidated, but studies suggest that in utero, peri-and post-natal transmission via close contact, saliva, and breast milk/breastfeeding are all possible.58 Most studies report a rate of vertical transmission of about 5% among HCV-RNA positive pregnant women.9,10 Concomitant HIV infection, high load of HCV-RNA (up to 1 million copies/mL) may both significantly increase the risk of HCV vertical transmission.1113

Most of these studies have been conducted in the Northern Hemisphere. In Africa, the vertical transmission of HCV was mainly investigated in Egypt and Morocco6,1417 with rates of transmission from HCV-RNA positive mothers ranging from 0% to 36%. In west-central African countries, where HIV, hepatitis B virus (HBV), and HCV infections are endemic, one study conducted in Tanzania among 980 pregnant women has reported a seroprevalence of 5%. In this study, only one child was found to be HCV-RNA positive at 18 months of age.18

To estimate the rate and to appreciate risk factors associated with the mother-to-child transmission (MTCT) of HCV in Cameroon, 5,008 pregnant women living in Yaoundé, the capital city of Cameroon, have been screened for HCV infection. Prevalence of HIV and HBV co-infections, qualitative and quantitative HCV viremia and genotypes were further investigated among HCV-antibody positive pregnant women. Children born to HCV-RNA positive women have been prospectively screened for HCV infection at 6 weeks and 6 and 15 months of age.

MATERIALS AND METHODS

Study design.

Between November 2001 and April 2003, 5,008 pregnant women (mean age, 25.6 years; range, 13–46 years), attending antenatal care at the mother and child center of the Chantal Biya Foundation (CBF) in Yaoundé and the Etoudi maternity ward, were screened for HCV infection. This study, approved by the Cameroon National Ethical Committee and local health authorities, was conducted in the framework of the Public Health Pilot Program (PHPP) for the reduction of the mother-to-child transmission of HIV-1 in Yaoundé, Cameroon.19

After counseling and written informed consent, HCV-positive pregnant women were enrolled in a specific post-test. During the post-test session, information regarding HCV infection was given and another blood sample was collected for serological confirmation of HCV and screening of HIV and HBV infection as well as for liver function test (ALAT). HCV plasma samples were then stored at −80°C for further analysis. HCV-positive pregnant women were then followed up till delivery. A last blood sample was taken from mothers just before or immediately after delivery and stored at −80°C for HCV-RNA quantification. After delivery, blood samples of the newborns were collected at Week 6 and Month 6 and subjected to qualitative HCV-RNA detection. At 15 months of age, sera from children were screened for the presence of HCV antibodies. Children were considered HCV-infected if they were found HCV-RNA positive at Week 6 and/or at Month 6.

As described elsewhere,19 HIV-infected pregnant women received nevirapine (NVP: 200 mg orally) at the onset of labor and 2 mg/kg NVP syrup was given to their babies within the first 72 hours of life on the maternity ward.

To prevent perinatal HBV transmission, hepatitis B vaccine was administered to newborns of HBV-infected mothers within 24 hours after birth, at 1 month, and at 6 months of age.20

Serological and biochemical tests.

The presence of anti-HCV antibodies was checked using a commercial third-generation ELISA (MONOLISA anti-HCV plus version 2, Biorad, Marnes-La-Coquette, France). A ratio (R) of optical densities (OD) for each sample was calculated by dividing its OD with the cutoff value. As previously described,21 samples were scored as positive if the ratio (R) was equal to or above 6.

HIV serological status was assessed by means of two ELISA tests (Genscreen Plus Version 2; Bio-Rad, Marnes la Coquette, France, and Wellcozyme HIV-1 Recombinant; Abbott, Rungis, France) and confirmed by immunoblot analysis (New Lav Blot I and II, Bio-Rad).

The presence of the surface antigen of hepatitis B virus (HBsAg) was determined by a commercial ELISA kit (MONOLISA Ag HBs plus, Bio-Rad). Positive HBsAg samples were then tested for hepatitis B e-antigen (HBeAg) and antibody to HBeAg (anti-HBe) by using the MONO-LISA HBe kit (Bio-Rad). The alanine aminotransferase (ALAT) level was determined semiautomatically by using the commercial kits Enzyline ALAT/GPT (BioMérieux, Marcy-l’Etoile, France).

HCV-RNA detection.

HCV-RNA detection was performed on conserved 5′ noncoding region by real-time RT-PCR using TaqMan technology on Light Cycler as recently described22 with little modifications. Briefly, the HCV-RNA was extracted from 200 μL of plasma by High Pure Viral Nucleic Acid (Roche Diagnostics Systems, Meylan, France). RT-PCR was performed on 6.2 μL of extract RNA using Light Cycler RNA Master Hybridation Probe kit (Roche Diagnostics Systems) with 0.5 μM of the primers R130 (5′-CGG GAG AGC CAT AGT GG-3′) and R290 (5′-AGT ACC ACA AGG CCT TTC G-3′), 0.2 ′M of the probe R6-Taq-148 (5′-6-FAM-CTG CGG AAC CGG TGA AGT ACA C-TAMRA-3′), and 3.25 mM of MnOAC2. PCR conditions were 61°C for 20 minutes, 95°C for 2 minutes, followed by 45 cycles with 3 seconds at 95°C and 20 seconds at 62°C, and finally 30 seconds at 40°C. The limit of detection using this technique was 1,000 copies/mL of plasma.

Determination of HCV genotypes and subtypes.

Genotypes and subtypes were determined by phylogenetic analysis of the NS5b region sequences (382 bp) as previously described.21,23 Sequences in FASTA format were aligned using Clustal X version 1.83 software.24 Genetic distances were measured with the Kimura-2 parameter model implemented in MEGA version 2.1.25 Neighbor-joining phylogenetic trees and bootstrap analysis were performed using MEGA program. One thousand replicates were used in the bootstrap analysis of neighbor-joining trees, and values above 700 (70%) were considered to support the grouping.26 Sixty-four NS5b nucleotide sequences (390 bp) have been submitted to GenBank Database under accession numbers AY265431 to AY26545127 and AY685012 to AY685052.

HCV-RNA quantification.

Quantification of HCV-RNA in plasma collected just before or after delivery from viremic mothers was performed using the recent and more sensitive version of the branched DNA signal amplification assay (bDNA 3.0, VERSANT HCV RNA 3.0 Assay; Bayer Diagnostics, Puteaux, France) according to the manufacturer’s instructions.28

Statistical analysis.

The characteristics of patients were compared by parametric tests as appropriated, using the statistical program Epi-info, version 6 (Centers for Disease Control and Prevention, Atlanta, GA). P values < 0.05 were considered statistically significant.

RESULTS

HCV seroprevalence and HIV/HBV co-infections among HCV-Ab positive pregnant women.

Eighty-nine out of 5,008 (1.8%) pregnant women (95% CI1.4–2.2%) were found HCV antibody positive (Figure 1). Seventy-five out of 89 (84.3%) were seropositive for HCV alone, whereas 14 (15.7%) were co-infected. Among these, 6 of 89 (6.7%; 95% CI 2.8–14.6%) were also seropositive for HIV, 7 of 89 (7.9%; 95% CI 3.5–16.1%) were HBsAg positive, and 1 of 89 (1.1%) (95% CI 0.1–7.0%) was both HBsAg and HIV-positive. None of the 8 HBsAg-positive pregnant women was HBeAg positive.

Viremia and genotype distribution among HCV-Ab positive pregnant women.

Eighty-nine HCV-Ab positive women were screened for the presence of HCV-RNA. As indicated on Figure 1, HCV-RNA was detected in 68 of 89 (76%) HCV-Ab positive pregnant women (95% CI 66.0–84.5%). The determination of the HCV genotypes by sequence analysis within the NS5b region of HCV was then investigated. Sixty-four PCR products have been directly sequenced and compared with corresponding HCV-NS5b sequences from Los Alamos database. As indicated in Table 1, 29 of 64 (45.3%) were of genotype 4, 18 (28.1%) of genotype 1, and 17 (26.5%) of genotype 2.

To further evaluate the genetic diversity within genotypes, nucleotide sequences were compared with those previously reported and submitted to Los Alamos database.

Among HCV genotype 4 (Figure 2A), 22 of 29 (76%) were of subtype 4f, 2 of 29 sequences were of subtype 4p, 2 of 29 of subtype 4t, and one sequence of 4e. The two remaining sequences were unclassified.

Among genotype 1 (Figure 2B), 9 of 18 NS5b sequences clustered within the subtype 1e, 3 of 18 sequences were of subtype 1h, and 4 of 18 sequences branch with non-significant value of bootstrap with the subtype 1l. The two last sequences did not branch with any currently classified sequences.

The genotype 2 sequences (Figure 2C) formed a monophyletic group together with subtypes 2h, 2l, and unclassified sequences already described in West African countries.2931

Pregnant women’s follow-up.

Twenty-five out of 68 HCV-RNA positive women were lost to follow-up before delivery either because they were not referred to the study pediatrician or because of moving. There were 2 spontaneous miscarriages (4.65%; 95% CI 0.8–17.1%) at 5 months of pregnancy. Among the remaining 41 women, 5 were HIV/HCV infected, 3 HBV/HCV infected, and one (M5230N) was HIV/ HBV/HCV infected (Table 2). Seventeen out of 41 harbored HCV genotype 4, 14 HCV genotype 2, and 9 HCV genotype 1, and the genotype was not determined for one woman (M6440N) (Table 2). At delivery, the mean gestational age was 38 weeks (range 32–42 weeks). Thirty-nine (95%) mothers delivered vaginally, whereas two (5%) were subjected to emergency caesarean section because of nonprogression of labor or post-term pregnancy (gestational age more than 42 weeks).

The mean ALAT level among the 41 HCV-positive women during pregnancy was 32 IU/L (range 13–96 IU/L). The ALAT level was in the normal range for 36 of the 41 HCV-positive mothers. Five women who presented abnormal ALAT values were HCV-Ab positive only (Table 2).

HCV RNA plasma viral load was determined for the 41 HCV-infected women during labor or immediately after delivery (Table 2). The mean plasma HCV viral load of all the 41 mothers was 4.8 (range 0.06 to 34.7) × 106 RNA copies/mL. Twenty-two of the 41 women (54%) presented a HCV viral load greater than 1 million copies per mL (Table 2) and were considered at high risk for HCV MTCT.11,13 Viral load was neither statistically different between HIV-positive nor negative women (P = 0.43), nor between HBV positive and negative women (P = 0.70). In the same way, the difference was not statistically significant between mothers delivering live birth or stillbirth children (P = 0.80).

Children’s follow-up.

Forty-one consenting women delivered 36 children (21 boys and 15 girls, including one pair of twins) that were screened for HCV infection at 6 weeks of age. There were five (12.2%, 95% CI 4.6–27.0%) stillbirths and one baby died at 4 weeks of age (Figure 1). The mean birth weight was 3,200 g (range 2,100–5,050 g).

To early diagnose HCV infection among children born to HCV-infected mothers, the presence of HCV-RNA was assessed among the 36 babies at 6 weeks of age. As indicated on Figure 1, none of the 36 babies (0%; 95% one-sided CI 0–8%) was HCV-RNA positive.

Three of 36 babies died between 4 and 6 months. These three children, born to HIV/HCV infected mothers, were HIV infected despite nevirapine prophylaxis to prevent HIV-1 MTCT.19 None of the three babies born to HBsAg positive mothers was HBsAg positive.

The 33 children still alive at 6 months remained HCV-RNA negative (Figure 1). Twenty-two of them, followed up to and tested at Month 15, remained negative (data not shown).

DISCUSSION

We report here the largest HCV MTCT study conducted to date in a west-central African country. A total of 5,008 pregnant women living in Yaoundé, the capital city of Cameroon, were screened for HCV infection. None of the 36 live children born from 35 HCV-RNA positive mothers tested positive for HCV-RNA at Week 6, and Month 6 remained negative.

The 1.8% of HCV seroprevalence reported among pregnant women in this study is comparable to the 1% to 2.6% prevalence rates previously reported among those in West African countries such as in Guinea and Côte d’Ivoire.29,32 By contrast, This 1.8% seroprevalence rate appears lower than the rates, ranging between 3.9% and 13%, reported in other African countries such as in southern Tanzania, Egypt, Congo, and Malawi6,18,33,34 and elsewhere in Cameroon (6.8%).35 Whether these disparities may be due to different epidemiologic methods or different mode of HCV transmission within these countries is unclear. Nevertheless, it should be pointed out that the choice of the serological algorithm to determine the HCV seroprevalence is of great importance in developing countries where intercurrent infections contribute to false-positive EIA results.36 Indeed, we, and others, have proposed a cost-efficient HCV serodiagnosis based on a single third-generation enzyme immunoassay (G3 EIA) allowing to reduce false-positive results.21,32

To further characterize HCV strains circulation among HCV-Ab positive pregnant women, the presence of HCV-RNA, as well as HCV genotypes were assessed. Seventy-six percent of HCV-Ab positive pregnant women tested positive for HCV-RNA and were considered at risk for HCV MTCT.9 A similar rate has been reported among pregnant women in Italy and Cote d’Ivoire5,7,32 and correlates with the viremia generally reported among HCV-Ab positive individuals becoming chronic carriers.37 Attempts to amplify the NS5b encoding gene by PCR were successful for 64 out of 68 HCV-RNA positive women. As previously shown,21,27,38,39 we confirm the presence of HCV genotypes 1, 2, and 4 in Cameroon. Genotypes 1 and 4 were highly heterogeneous, containing many previously described subtypes as 1e, 1l, 1h 4f, 4p, 4t, 4e, and numerous unclassified sequences, whereas genotype 2 sequences were more homogeneous. Whether the high genetic diversity within genotypes 1 and 4 is linked to ancient circulation remains to be confirmed. In the same way, whether the low genetic diversity within HCV genotype 2 reflects a more recent introduction in western Africa needs to be clarified. Most importantly, the impact of a high HCV genetic diversity in Cameroon on diagnosis, response to therapy, and severity of related liver diseases urgently needs further investigation.

Failure to detect mother-to-child transmission of HCV in this study is consistent with that reported in some studies in France,40,41 the United States,42 Sweden,43 Italy,44 Japan,8 Morocco,16 and recently in Egypt.15 As suggested by Jabeen and colleagues,45 the favorable outcome of pregnancy and the low transmission rate of HCV may be explained partially by the endogenous production of interferon (alpha, beta, and gamma), as already described in the placental environment.4648

Mother-to-child transmission of HCV has been linked to the presence of high HCV-RNA in the mother’s blood.10 Recent studies indicate that quantification of plasma HCV viral load may help assess the risk of HCV MTCT. HCV MTCT risk has been shown to increase with HCV-RNA load greater than 1 million copies per milliliter.11,13 In the current study, no transmission occurred among 22 pregnant women presenting HCV-RNA load greater than 1 million copies per milliliter. However, the number of mother-child pairs was limited and warrants additional work to further appreciate the impact of the viral load on HCV MTCT. Although previous studies have reported higher HCV-RNA level and faster progression of HCV-related liver disease49 among individuals co-infected with HIV, we did not observe significant difference in HCV-RNA load among pregnant women whether HIV co-infected or not. As well, we did not find any significant difference in HCV-RNA load among pregnant women whether co-infected with HBV or not. The inhibition exerted by HBV on HCV replication has been shown in chronic HBV/HCV co-infections.50 However, the small number of HIV/HCV and HBV/HCV co-infected pairs in this study precludes any conclusion. Gervais and colleagues51 reported a decreased (or a normalization) of serum ALAT level during pregnancy in HCV-infected women due to dilution effect. We found that 88% (95% CI 73.0–95.4%) of HCV-RNA positive pregnant women had ALAT level within normal limit. The question of the usefulness of liver function determination to monitor HCV MTCT can still be raised. We found no significant difference in gestational age, birth weight, and mode of delivery between HCV-RNA positive and negative mothers. Although high, the neonatal mortality rate we report here (12.2%; 95% CI 4.6–27.0%) does not significantly differ (P = 0.11) from the rate observed in 2003 at the maternity ward of Yaoundé Central Hospital (5.3%; 95% CI 4.6–6.2%) (unpublished data).

Three children, born from HIV/HCV co-infected mothers, died during follow-up. They were HIV infected despite nevi-rapine (NVP) prophylaxis. Despite the effectiveness of NVP to reduce peripartum HIV MTCT,52 the rate of HIV MTCT observed in this study among HIV/HCV co-infected mothers (60%, 95% CI 17.0–92.7%) was significantly higher (P < 0.05) than the rate recently reported in the pilot public health program for the reduction of HIV-1 MTCT in Yaoundé (10.6%; 95% CI 5.1–16.0%).19 Similar observations have been made in the United States where the rate of HIV MTCT was significantly higher among HCV co-infected HIV-infected pregnant women despite Zidovudine prophylaxis.53 Whether NVP prophylaxis is less effective in cases of HIV/HCV co-infections needs to be further investigated. Indeed, we cannot exclude that HCV infection might increase the risk of in utero HIV MTCT during the first months of pregnancy. Elsewhere, pregnant women infected with both HIV and Plasmodium falciparum have been reported to be at an increased risk of high HIV-RNA concentration,54 which could be related to higher MTCT of HIV.55,56 Whether such a phenomenon also occurs with HCV/HIV co-infection needs further investigation.

Despite a prevalence rate of 1.8% of HCV-Ab positive among pregnant women in Yaoundé, Cameroon, and the high prevalence of co-infections with HIV and/or HBV, we did not observe any MTCT of HCV. The failure to detect HCV vertical transmission in our study suggests that the contribution of MTCT in the spread of HCV may be negligible in Cameroon, and other routes of transmission should be considered.

Table 1

Genotype distribution and serological status of 64 HCV-RNA positive pregnant women in Yaoundé, Cameroon

Genotype distribution
Serological status124Total
HCV+15142352
HCV+/HIV+2046
HCV+/HBsAg+1225
HCV+/HIV+/HBsAg+0101
Total18172964
Table 2

Delivery parameters of 41 HCV-RNA positive pregnant women during pregnancy

MothersGenotypeHBVHIVViral load (106 RNA copies/ml)Alat (UI/ml) N < 50
NA: non amplified.
M4753N1e1,0N
M7747N1e0,2N
M4684N1ePos0,7N
M5405N1e0,1N
M6086N1h0,3N
M7269N1h0,81.5N
M5186N1l1,5N
M5051N1uPos2,1N
M7777N1u0,1N
M4959N2uPos11,0N
M4548N2u13,3N
M6787N2u0,21.5N
M7360N2u0,2N
M4831N2uPos0,2N
M7930N2u26,4N
M5401N2u3,30N
M7649N2u0,41.1N
M7841N2u4,0N
M7905N2u5,7N
M4488N2u6,2N
M5230N2uPosPos0,8N
M7881N2u7,6N
M5480N2u0,8N
M6560N4f1,3N
M5177N4f1,4N
M376E4f13,92N
M7480N4fPos15,3N
M7863N4f22,61.1N
M6872N4f2,4N
M7858N4f3,5N
M7920N4f0,5N
M6310N4f4,9N
M7335N4f0,7N
M3645N4fPos0,8N
M5574N4f7,8N
M4962N4fPos0,8N
M7516N4f0,1N
M5225N4f0,98N
M6977N4u3,1N
M6732N4pPos34,7N
M6440NNA0,4N
Figure 1.
Figure 1.

Flowchart of the studied pregnant women population and their offspring (November 2001 to October 2004).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 2; 10.4269/ajtmh.2005.73.460

Figure 2.
Figure 2.

HCV subtype distribution among 64 HCV-RNA positive pregnant women in Yaoundé, Cameroon.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 2; 10.4269/ajtmh.2005.73.460

*

Address correspondence to Eric Nerrienet, Laboratoire de virologie, Centre Pasteur du Cameroun, BP 1274 Yaoundé, Cameroon. E-mail: nerrienet@pasteur.yaounde.org. Present address: HIV and Hepatitis Unity, Institut Pasteur du Cambodge, BP983 Phnom Penh, Kingdom of Cambodia. E-mail: enerrienet@pasteur-kh.org

Authors’ addresses: Richard Njouom, Centre Pasteur du Cameroun, BP1274 Yaoundé, Cameroun, Telephone: 237 223 10 15, Fax: 237 223 15 64, E-mail: njouom@pasteur-Yaounde.org. Christophe Pasquier, Laboratoire de Virologie, CHU Purpan, Place du Dr Baylac, TSA40031, 31059 Toulouse Cedex 9, France, Telephone: (33) 5 61 77 24 63, Fax: (33) 5 61 77 25 42, E-mail: pasquier.c@chu-toulouse.fr. Ahidjo Ayouba, Centre Pasteur du Cameroun, BP1274 Yaoundé, Cameroun, Telephone: 237 223 10 15, Fax: 237 223 15 64, E-mail: ayouba@pasteur-Yaounde.org. (Present address: Institut Pasteur de Paris, Unité de Biologie des Rétrovirus, 25 rue du Dr Roux, 75724 Paris Cedex 15, France, E-mail: ayouba@pasteur.fr). Mathurin Cyrille Tejiokem, Centre Pasteur du Cameroun, BP1274 Yaoundé, Cameroun, Telephone: 237 223 10 15, Fax: 237 223 15 64, E-mail: tejiokem@pasteur-yaounde.org. Aurelia Vessiere, Centre Pasteur du Cameroun, BP1274 Yaoundé, Cameroun, Telephone: 237 223 10 15, Fax: 237 223 15 64, E-mail: vessiere@pasteur-yaounde.org. Jermie Mfoupouendoun, Centre Pasteur du Cameroun, BP1274 Yaoundé, Cameroun, Telephone: 237 223 10 15, Fax: 237 223 15 64. Gilbert Tene, Centre Mère Enfant, Fondation Chantal Biya, BP 1936 Yaoundé, Cameroun. Telephone: 237 999 02 23, E-mail: giltene@yahoo.fr. Nicole Eteki, Maternité Principale, Hôpital Central de Yaoundé, BP 87 Yaoundé, Cameroun, Telephone: 237 777 13 98, E-mail: etekinicole@yahoo.fr. Marcel Monny Lobe, Centre Mère Enfant, Fondation Chantal Biya. BP1936, Yaoundé, Cameroun, Telephone: 237 222 50 84. Jacques Izopet, Laboratoire de Virologie, CHU Purpan, Place du Dr Baylac, TSA40031, 31059 Toulouse Cedex 9, France, Telephone: (33) 5 61 77 24 63, Fax: (33) 5 61 77 25 42, E-mail: izopet.j@chu-toulouse.fr. Eric Nerrienet, Centre Pasteur du Camer-oun, BP1274 Yaoundé, Cameroun, Telephone: (237) 223 10 15, Fax: (237) 223 15 64, E-mail: nerrienet@pasteur-yaounde.org. (Present address: Institut Pasteur du Cambodge, 5, boulevard Monivong, Phnom Penh, BP983, Kingdom of Cambodia, Telephone: (855) 23 368 036, E-mail: enerrienet@pasteur-kh.org).

Acknowledgments: Richard Njouom is a recipient of a fellowship from the French Ministry for Foreign Affairs. We are very grateful to Viviane Mogoun Tchuente for her technical assistance and to the personnel of the Chantal Biya Foundation and the maternity wards of Etoudi and of Central Hospital of Yaoundé, Cameroon.

Financial support This work was funded by the ANRS agency of France (project no. 1262).

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Author Notes

Reprint requests: E. Nerrienet, Laboratoire de virologie, Centre Pasteur du Cameroun, BP 1274 Yaoundé, Cameroon, Telephone: (237) 223 18 03, Fax: (237) 223 15 64, E-mail: nerrienet@pasteur.yaounde.org. (Present address: HIV and Hepatitis Unity, Institut Pasteur du Cambodge, BP983 Phnom Penh, Kingdom of Cambodia, Fax: (855) 12 725 606, E-mail: enerrienet@pasteur-kh.org.)
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