Mother-to-infant transmission of hepatitis C virus

L-selectin is frequently expressed by leukaemic blast cells. Our data suggest that measurement of CSF sL-selectin may help to detect meningeal involvement in patients with L-selectin+ acute leukaemia and in deciding when and for how long to treat. We thank Dr V von Fliedner, the Swiss Group for Clinical and Experimental Cancer Research, and Prof F Regli for providing patient samples, and Dr B Burnand for statistical advice. This work was supported by grant 31-33792.92 from the Swiss National Foundation for Scientific Research and the Mushamp Foundation.

References 1

Pinkel D, Woo S. Prevention of meningeal leukemia in children. Blood

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Bleyer W. Central nervous system leukemia. Pediatr Clin North Am 1988; 35: 789-14. Mahmoud H, Rivera G, Hancock M, et al. Low leukocyte counts with blast cells in cerebrospinal fluid of children with newly diagnosed acute lymphoblastic leukemia. N Engl J Med 1993; 329: 314-19. Ortega J, Nesbit M, Sather H, Robison L, D’Angio, Hammond G. Long-term evaluation of CNS prophylaxis trial—treatment comparisons and outcome after CNS relapse in childhood ALL: a report from the Childrens Cancer Study Group. J Clin Oncol 1987; 5:

1994; 84: 355-66.

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1646-54. Ochs J, Rivera G, Aur R, Hustu H, Berg R, Simone J. Central nervous system morbidity following an initial isolated central nervous system relapse and its subsequent therapy in childhood acute lymphoblastic leukemia. J Clin Oncol 1985; 3: 622-26. Saikia T, Dhabhar B, Iyer R, et al. High incidence of meningeal

leukemia in lymphoid blast crisis of myelogenous leukemia. Am J Hematol 1993; 43: 10-13. 7 Bigner SH, Johnston NW. The cytopathology of cerebrospinal fluid I: nonneoplastic conditions, lymphoma and leukemia. Acta Cytol 1981; 25:335. 8 Nies B, Thomas L, Friereich E. Meningeal leukemia: a follow up study. Cancer 1965; 18: 546. 9 Lasky LA. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science 1992; 258: 962-68. 10 Adams DH, Shaw S. Leucocyte-endothelial interactions and regulation of leucocyte migration. Lancet 1994; 343: 831-36. 11 Springer T. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994; 76: 301-14. 12 Spertini O, Schleiffenbaum B, White-Owen C, Ruiz P Jr, Tedder TF. ELISA for quantitation of L-selectin shed from leucocytes in vivo. J Immunol Methods 1992; 156: 115-23. 13 Donelly SC, Haslett C, Dransfield I, et al. Role of selectins in development of adult respiratory distress syndrome. Lancet 1994; 344: 215-19. 14 Spertini O, Callegari P, Cordey A-S, et al. High levels of the shed form of L-selectin (sL-selectin) are present in patients with acute leukemia and inhibit blast cell adhesion to activated endothelium. Blood 1994; 84:1249-56. 15 Benett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia: a report of the FrenchAmerican-British Cooperative Group. Ann Intern Med 1985; 103: 620-24. 16 Dunn O. Multiple contrasts using rank sums. Technometrics 1964; 6: 241-52. 17 Komada Y, Ochiai H, Shimizu K, Azuma E, Kamiya H, Sakurai M. Shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells. Blood 1990; 76: 112-16.

risk of mother-toinfant transmission of HCV is unclear.1-5 We report our prospective study on mother-to-infant transmission of HCV Between January, 1990, and June, 1993, 21 516 consecutive

vertical/perinatal modes. However, the

Mother-to-infant transmission of C virus

hepatitis

the risk of mother-to-infant transmission of C virus (HCV), we followed up 116 babies of antihepatitis HCV positive mothers, of whom 22 were coinfected with HIV and 94 had HCV alone. None of the babies whose mothers had HCV alone acquired HCV, while 8 babies (36%; p<0·001) of mothers co-infected with HIV acquired HCV (5 babies) or HCV and HIV (3). There was no association between any specific maternal HCV genotype and enhanced risk of neonatal infection. HCV-RNA levels were in mothers with HIV significantly higher (p<0·05) coinfection than in those with HCV alone. These data indicate that maternal HIV status correlates with enhanced level of viraemia which favours neonatal infection.

To

assess

pregnant women (mean age 28 years, range 16-44) attending several antenatal clinics in Lombardy, Italy, were screened at delivery for HCV antibody. For babies of anti-HCV-positive mothers, blood tests and laboratory and clinical evaluations were scheduled at birth, about every 3 months during the first year of follow-up, and then every 6 months. All those infants of antiHCV-positive mothers without HIV infection and with normal alanine aminotransferase were delivered vaginally and nursed. Babies of mothers with abnormal enzymes or co-infection with HIV were not breastfed. Presence of viral RNA in maternal serum could not be used as a criterion for exclusion of nursing, since testing was retrospective on delivery samples stored frozen. HCV antibodies were looked for by EIA (Ortho Diagnostic Systems), and repeatedly reactive samples were confirmed by RIBA (Chiron). Viral RNA was assayed in mothers at delivery and in their infants at least twice (mean 3-4, range 2-9) by reverse-transcriptase PCR with nested primers derived from the 5’ non-coding region of the HCV genome. Only results reproducible in at least two independent experiments were considered. HCV-RNA was measured by a nucleic-acid hybridisation assay based on signal amplification of branched DNA (Quantiplex HCV-RNA, Chiron). HCV genotypes were determined with a line-probe assay (INNO-LiPA-HCV,

Innogenetics). Lancet 1995; 345: 289-91

Hepatitis C virus (HCV) is the major cause of both parenterally transmitted and cryptogenic or community acquired non-A, non-B hepatitis. Inability to detect parenteral risk factors in many patients with acute and chronic hepatitis C suggests that HCV can be transmitted by other than overt percutaneous exposure, such as *Participants listed

at end of paper

Babies with high alanine aminotransferase (>45 IU/L) were examined for HBsAg and for IgM antibodies to hepatitis A virus, cytomegalovirus, and Epstein-Barr virus (Abbott). HIV-antibody was detected by EIA and confirmed by western blot (Sorin Biomedica). The clinical stage of HIV was assigned according to the CDC classification.’

250 (1-2%) mothers were anti-HCV positive by EIA and RIBA, and 195 (78%) gave informed consent to study entry. We considered only 116 babies who were followed up for at least a year (mean 18-8 months, range 12-46), including 22 babies of mothers co-infected with 289

aminotransferase was abnormal (158 IU/L) between 5 and 6 months of age and then returned to normal, while HCV-RNA was detected only in the serum collected at 6 months. For baby 5, only two samples were available for PCR and both were positive, with alanine aminotransferase also being abnormal (106 IU/L). In baby 6, enzyme peaked (82 IU/L) at 3 months and HCVRNA was detected on two occasions (at 8 and 24 hepatitis, ND=not done, ALT=alanine aminotransferase. Table: HCV and HIV status at delivery of mothers who transmitted HCV infection CAH=chromc active

HIV (14 class II, 2 class III, and 6 class IV C2) and 94 born to mothers with HCV alone. Follow-up rates of the two cohorts of babies were similar. All mothers of the first group were intravenous drug users and 6 (27%) had been diagnosed with chronic active hepatitis. Of the mothers of the second group, 8 (9%) had chronic persistent hepatitis, 15 (16%) had abnormal alanine aminotransferase (mean 168 IU/L, 75-445) at delivery, and 5 (5%) reported clinical histories of selflimiting hepatitis. The remaining 66 (70%) showed no signs or symptoms of acute or chronic liver disease. 16 of the 94 (17%) were drug addicts, 12 (13%) had been given transfusions, and 5 (5%) were sexual partners of drug addicts; 61 (65%) had had no apparent HCV exposure.

HCV-RNA was detected in 46 of the 94 (49%) mothers with anti-HCV alone and in 18 of the 22 (82%) with antiHCV and anti-HIV. None of the 94 babies of mothers with anti-HCV alone became infected with HCV. During follow-up, all these children lost maternal anti-HCV by 1 year of age, were normal by clinical and laboratory examinations, and were persistently HCV-RNA negative in the subsequent measurements. 71 (76%) of these babies, including 23 born to HCV-RNA positive mothers, remained uninfected although they were breastfed. 8 babies of the 22 (36%) mothers who were co-infected with HIV acquired HCV infection, becoming HCV-RNA positive with abnormal alanine aminotransferase (mean peak 137-3 IU/L, range 59-259). All 8 were born to the 18 (44%) mothers who were HCV-RNA positive at delivery. The HCV and HIV statuses of the 8 transmitting mothers are shown in the table. 3 babies (3, 7, and 8) acquired HIV and HCV infections, whereas the other 5 babies were infected with HCV alone. Antibody to HCV was persistently present from birth onward in 4 babies (figure). 2 babies lost maternal antibody at 6 months of age and remained negative thereafter, whilst 2 became anti-HCV-negative and then again positive (ex-novo

months). During follow-up none of these babies became icteric or developed HBsAg or IgM antibodies to hepatitis A virus, cytomegalovirus, or Epstein-Barr virus. The HCV genotypes of infected babies matched those of their mothers. There were no differences in the doubly infected women in the distribution of genotypes between mothers whose babies became infected (table) and those who did not transmit infection (4 women had genotype 1 a, while 2 had lb, 2a, and 3a). Titres of HCV-RNA in the 18 mothers co-infected with HIV and of 10 mothers with HCV alone (all intravenous drug users and 4 also had chronic persistent hepatitis) were significantly higher in the doubly infected women than in those with HCV alone (median 77-55X105 equivalents per mL [range

12-56-351-7X10’’] vs 9.337 X 105 [<3-5-104-8XlO’]; U test). However, in the 18 HIV-co-infected p<0’05, mothers, there was no significant difference in HCV-RNA between the 8 who transmitted infection (100.9X105 equivalents per mL, 12.56-327.7X105) and the 10 mothers who did not (66.77 X 105, 19’04-351.7X 105). Our results showed that mother-to-infant transmission of HCV was uncommon when the mother was not co-infected with HIV. The finding that anti-HCV antibody is very rare among Italian childrenindirectly supports the hypothesis that the vertical/perinatal route is of little epidemiological importance in our country. That 71 babies remained uninfected despite breastfeeding indicates that this route is not an efficient mechanism of perinatal transfer of HCV, at least when the mother has no signs of liver disease. However, when the mother was simultaneously infected with HCV and HIV, the potential risk of viral transmission became higher (p<0.001, Fisher’s exact test). In our setting, 36% (8/22) of babies of intravenous-drug-abusing mothers who were positive for both anti-HIV and anti-HCV acquired infection and the percentage increased to 44% (8/18) for babies whose mothers were also HCV-RNA positive at delivery. These data indicate that maternal HIV status predicts HCV infection in the baby. Also, intravenous drug use itself as

production). For the 3 babies who acquired HCV and HIV, HCVRNA was first detected between 3 and 6 months of age and remained persistently positive with fluctuating levels of alanine aminotransferase in 2 cases (7 and 8). In baby number 3, viral RNA and elevated alanine aminotransferase (200 IU/L) were found up to 8 months of age, and had disappeared at 18 months, when enzyme was normal. For the 5 who acquired HCV alone, HCVRNA was positive at 6 months of age with intermittently elevated alanine aminotransferase in baby 1. In baby 2, HCV-RNA was first detected at 6 months of age, was negative up to 24 months, and positive again between 29 and 42 months, with enzyme peaks at 6 (259 IU/L) and 24 (147 IU/L) months of age. In baby 4, alanine 290

Figure: HCV

markers

Shaded bars=periods of positivity for anti-HCV signs=positive and negative PCR.

by

EIA. Plus and minus

well as HIV may be a major predisposing factor for perinatal transmission of HCV. 29 However, in our setting, of the 16 drug-using women without HIV none transmitted infection to their babies. HCV-RNA levels of drug-using mothers with HIV coinfection were significantly higher than in those with HCV alone, although the ranges overlapped. Maternal viraemia and rate of HCV infection in the infant has been reported to be correlated.1O Active maternal liver disease may also enhance the risk of viral HCV transmission.2,10 In our study, 4 of the 8 mothers of infected babies had been diagnosed with chronic active hepatitis, but 4 had normal alanine aminotransferase at delivery. However, since histological diagnoses were not available for the 4 transmitting mothers with normal enzyme at delivery, they may have been highly infectious healthy carriers or had chronic liver disease in remission phase. Although our study was small, we did not find any association between any specific maternal HCV genotype and an increased rate of neonatal infection. The finding that vertical/perinatal transmission is enhanced when the mother is co-infected with HCV and HIV has important public health implications, especially in urban areas in which most drug abusers are infected with both viruses. Participants in the study group: S Bresciani, M G Marin, D Padula, A Rodella (Brescia); I Bulgarelli, F Chiodo, E Magliano, G Miotto, M L Muggiasca, E Pilloton, R Pozzoli, F Pregliasco, L Romano, C Stringhi (Milan); F D’Agostino, F Paolillo (Lodi); and B Zapparoli (Monza). This work was supported in part by a grant from the Italian Ministry of Health, Centro Studi, Rome. We thank Dr L Stuyver for supplying part of the reagents for genotyping.

References 1 2

3 4 5

Thaler MM, Park C-K, Landers DV, et al. Vertical transmission of hepatitis C virus. Lancet 1991; 338: 17-18. Kuroki T, Nishiguchi S, Fukuda K, et al. Mother-to-child transmission of hepatitis C virus. J Infect Dis 1991; 164: 427-28. Reinus JF, Leikin EL, Alter HJ, et al. Failure to detect vertical transmission of hepatitis C virus. Ann Intern Med 1992; 117: 881-86. Wejstal R, Widell A, Mansson AS, et al. Mother-to-infant transmission of hepatitis C virus. Ann Intern Med 1992; 117: 887-90. Lam JPH, McOmish F, Burns SM, et al. Infrequent vertical transmission of hepatitis C virus. J Infect Dis 1993; 167: 572-76. Puoti M, Zonaro A, Ravaggi A, et al. Hepatitis C virus RNA and antibody response in the clinical course of acute hepatitis C virus infection. Hepatology 1992; 4: 877-81. Centers for Disease Control and Prevention. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR 1992; 41 (RR-17): 1-19. Romanò L, Azara A, Chiaramonte M, et al. Low prevalence of antiHCV antibody among Italian children. Infection 1994; 22: 350-51. Weintrub PS, Veereman-Wauters G, Cowan MJ, Thaler MM. Hepatitis C virus infection in infants whose mothers took street drugs intravenously. J Pediatr 1991; 119: 869-74. Ohto H, Terazawa S, Sasaki N, et al. Transmission of hepatitis C virus from mothers to infants. N Engl J Med 1994; 330: 744-50.

Effect of neonatal circumcision on pain responses during vaccination in boys

Using data from one of our randomised trials, we investigated post-hoc whether male neonatal circumcision is associated with a greater pain response to routine vaccination at 4 or 6 months. Pain response during routine vaccination with diphtheria-pertussis-tetanus (DPT) alone or DPT followed by Haemophilus influenzae type b conjugate (HIB) was scored blind. 42 boys received DPT and 18 also received HIB. After DPT, median visual analogue scores by an observer were higher in the circumcised group (40 vs 26 mm, p=0·03) After HIB, circumcised infants had higher behavioural pain scores (8 vs 6, p=0·01) and cried longer (53 vs 19 s, p=0·02). Thus neonatal circumcision may affect pain response several months after the event. Lancet 1994; 344: 291-92

randomised controlled trial of a topical anaesthetic cream (EMLA, eutectic mixture of local anaesthetics; Astra) on infant pain in routine diphtheria-pertussistetanus (DPT) vaccination, boys had higher pain scores than girls.’ If this sex difference is a real effect, it may be partly related to previous experience with acute pain, such as circumcision. Circumcised babies have short-term alterations in behaviour, sleep patterns, frequency of feeding, crying, fussiness, and heart rate.2-7 Effects beyond the first few hours after the painful event have not been investigated. We have done a post-hoc analysis from our trial’ to investigate whether circumcision was associated with pain scores during vaccination. In

our

Healthy boys aged 4-6 months (mean 5) who were vaccinated alone, or DPT and Haemophilus influenzae type b conjugate (HIB), were included. DPT was administered intramuscularly on the upper thigh 60 min after treatment with EMLA or placebo. Infants who also received HIB were given an with DPT

Institute of Virology (Prof A R Zanetti PhD, E Tanzi PhD) and Paediatrics Department IV, University of Milan (S Paccagnini MD), Prof N Principi MD); Institute of Chemistry, Medical School, University of Brescia (G Pizzocolo BSc); Department of Obstetrics and Gynaecology, Hospital "L Mangiagalli", University of Milan (M L Caccamo MD); Department of Paediatrics, Hospital "NiguardaCà Granda", Milan (Prof E D’Amico MD); Blood Transfusion Centre, Hospital "Maggiore", Lodi (G Cambiè MD); and Laboratory of Clinical Pathology, Hospital "S Gerardo", Monza, Italy (L Vecchi MD)

intramuscular dose several minutes after the DPT and on the opposite leg without EMLA or placebo. All injections were made by one of two paediatricians. During the DPT injection, an observer and the paediatrician who administered the vaccine (both unaware of treatment allocations) rated the infant’s pain response on a 100 mm ungraded visual analogue scale (VAS: 0=no pain, 100=worst possible pain). We videotaped the infants during their one or two injections, until they settled. A coder who was unaware of the treatments rated the pain responses for all vaccinations on a modified behavioural pain scale (face, cry, and body movements). The total pain score was obtained by adding the scores of the components and varied between 0 and 10.1 Baseline pain scores, post-procedural scores, net pain (ie, difference between baseline and post-vaccination score), and duration of cry were assessed for all injections. Infants who had undergone painful procedures that were not routine (eg, lumbar puncture, surgery) were excluded from analyses. Infants who were in discomfort before the vaccine (ie, baseline behavioural pain scores greater than 2) were also excluded from analysis of pain for that vaccination. Statistical differences were calculated with Mann-Whitney, X2, or Fisher’s exact tests. Correlations were done with Spearman’s method. Of the 42 boys who received DPT, 30 (71%) had been

Correspondence to: Prof A R Zanetti, Institute of Virology, University of Milan, Via C Pascal 38, 20133 Milan, Italy

circumcised. No significant differences were found between circumcised and uncircumcised infants in

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