- Email: [email protected]
TRANSPLACENTAL TRANSMISSION OF HEPATITIS-B VIRUS BY SYMPTOM-FREE CHRONIC CARRIER MOTHERS
746 While
subjective manifestations are often open to interpretation, the lack of symptoms among bacteriuric patients taking tetracycline (including those who had previously reacted adversely to minocycline), and the absence of symptoms among contacts of those taking minocycline prophylactically, tend to support the validity of these observations. The data suggest that minocycline has a propensity for producing reversible vestibular side-effects, which requires more detailed and objective investigation. This work was supported by Research grant no. HD-03693 from the National Institute of Child Health and Human Development and training grant no. TO-1 AI-00068 from the National Institute of Allergy and Infectious Diseases. Requests for reprints should be addressed to L. W. L., Channing Laboratory, 774 Albany Street, Boston, Massachusetts 02118, U.S.A. REFERENCES 1. 2.
Guttler, R. B., Counts, G. W., Avent, C. K., Beaty, H. N.J. infect. Dis. 1971, 124, 199. Guttler, R. B., Beaty, H. N. Antimicrob. Agents Chemother. 1972, 1,
397. 3. Frisk, A. R., Tunevall, G. 1969. ibid. 1968, p. 335. 4. Holloway, W. J. Delaware med. J. 1970, 42, 333. 5. Devine, L. F., Johnson, D. P., Hagerman, C. R., Pierce, W. E., Rhode, S. L., Peckinpaugh, R. O. Am. J. Epidem. 1971, 93, 337. 6. Gould, W. J., Brookler, K. H. Archs Otolaryng. 1972, 96, 291. 7. Nelson, J. R. in Textbook of Medicine (edited by P. B. Beeson and W. McDermott); p. 174. Philadelphia, 1971.
TRANSPLACENTAL TRANSMISSION OF HEPATITIS-B VIRUS BY SYMPTOM-FREE CHRONIC CARRIER MOTHERS GEORGE PAPAEVANGELOU JAY HOOFNAGLE JENNY KREMASTINOU
Department of Hygiene and Epidemiology, University of Athens Medical School, Athens, Greece, and Bureau of Biologics, Food and Drug Administration, Bethesda,
Maryland, U.S.A.
Transplacental transmission of the hepatitis-B surface antigen (HBsAg) and of hepatitis-B virus was studied in 12 symptom-free chronic HBsAg carrier mothers and their 15 babies. HBsAg was present in the sera of the mothers throughout the study (at delivery and three and six months later). HBsAg was detected in the cord blood of 3 of the 15 children, but subsequently was not detected except in 1 child who first became HBsAgpositive at six months of age. Antibody to HBsAg Summary
was not
detected in the mothers,
nor
in their infants
during the first six months of life. Antibody to the core antigen (anti-HBc) was always present in carrier mothers and in the cord blood, but it disappeared from the sera of the newborns shortly after birth. These data indicate that children of chronic HBsAg carrier mothers generally do not become infected with hepatitis-B virus before or at birth despite the known intense exposure to HBsAg from the mother.
It is unclear whether the presence of HBAg in cord blood constitutes sufficient virus exposure to transmit type-B hepatitis to the child. Mothers with acute type-B hepatitis in late pregnancy or shortly after delivery often transmit overt or subclinical hepatitis B to their babies.3-’ However, there are conflicting of babies by mothers infections reports regarding who are symptom-free chronic HBsAg carriers.3,s-,: We have investigated the possibility of transplacental transmission of HBsAg and/or hepatitis B by taking serial blood-samples from symptom-free carrier mothers and their babies for six months after delivery and testing them for HBsAg, anti-HBs, and antibody to the hepatitis-B core antigen (anti-HB).
Materials and Methods
Serological Testing HBsAg was tested by counterelectrophoresis (C.E.P.)12 and by solid-phase radioimmunoassay (R.I.A.,’Ausria125 ’, Abbott Laboratories).13 Samples reactive for HBsAg by R.I.A. but negative by C.E.P. were tested for specificity by inhibition with human anti-HBs.14 Anti-HBs was assayed by passive hxmagglutination (Virgo Reagents, Electro-Nucleonics Inc.) 15 and by solid-phase radioimmunoassay (’ Ausab ’, Abbott Laboratories). Anti-HB was detected by a microtitre complement-fixation testJ6 Samples Tested We screened 428 pregnant women, seen in the obstetric clinics of the University of Athens, for HBsAg by C.E.P., and 14 (3%) were positive. 2 are not analysed here because cord bloods were not available. 3 of the positive mothers gave birth to another child after their first delivery. Thus, the study group consisted of 12 HBsAg-positive mothers and their 15 newborns. At parturition, maternal and umbilical cord bloods were drawn aseptically, and the sera were stored at -60°C until tested. The mothers and their babies were reexamined three and six months after delivery. All mothers remained HBsAg-positive. No liver biopsies were performed, but the absence of overt signs of liver disease and the lack of substantial biochemical abnormalities (raised serum-glutamic-pyruvic-transaminase values above 100 Karmen units) in the face of persistence of HBAg indicated that these mothers were chronic HBsAg carriers and did not have acute type-B hepatitis. Results
HBsAg HBsAg
was detected in the umbilical cord blood of 3 of the 15 children born to symptom-free carrier mothers. 1 of the cord bloods was positive by both C.E.P.’and R.I.A., while the other 2 were positive by R.I.A. only. At three months, HBAg was not detected in any of the newborns. At six months after birth, HBsAg was found in the serum of only 1 of the 15 children (case 1). This child was not HBsAg-positive at birth and at six months was only weakly positive
(titre 1/4, by
R.I.A.
only).
Arzti-HB, Anti-HB
Introduction
was not demonstrated in maternal or umbilical-cord blood of any case. It had not developed in any of the 15 newborns at three or six months after birth.
hepatitis-B surface antigen (HBsAg) has occasionally been reported in the cord blood of children born to mothers infected with the hepatitis-B virus.1-3
Anti-HB, Anti-HB was found in all carrier mothers in every blood-sample. Titres were high, ranging from 1 16
THE
747
HBsAg
AND ANTI-HBc TITRES IN CHRONIC HBsAg PRESENCE OF CARRIER MOTHERS AND IN THE CORD BLOOD OF THEIR BABIES
*
Detectable
by radioimmunoassay only.
1/1024 (see table). Furthermore, this antibody was present in the cord blood of each of the children born to these carrier mothers. The titre in the cord blood was similar to that of the mother and independent of the presence of HBgAg in the cord blood. At three months after birth, anti-HB was no longer detectable in any of the 15 children. All children remained negative for anti-HB when re-examined six months after delivery, except for case 1, where anti-HB reappeared at a very low titre (1/4) currently with the appearance of HB,Ag. to
Discussion These findings, together with other reports,1-3,17 indicate that HBsAg can be found in the cord blood of children born to chronic carrier mothers. HBsAg was found in 3 of the 15 cord bloods, although in only 1 was the titre of HBsAg similar to the titre in the mother’s serum (both reactive by C.E.P.). Thus, as might be expected, HBsAg (which is present in the serum in the form of 20 nm. spheres and tubules and as 42 nm. Dane particles) crosses the placental barrier poorly, if at all. The finding of this particulate antigen in cord bloods of only 3 of 15 cases suggests that HBgAg may cross into the fetal circulation only when the placental barrier breaks down, as must hap-
frequently during parturition. Although HBsAg was found in the sera of 3 children at birth, none of these 3 subsequently developed either biochemical or serological evidence of hepatitis B. In 12 other children whose sera were negative for HBsAg at birth, only 1 subsequently developed evidence of type-B hepatitis (in the form of low levels of HB,Ag and anti-HB six months after delivery). In fact, the appearance of HBsAg six months after delivery points to exposure to hepatitis-B virus after delivery rather than at birth. This infrequent occurrence of hepatitis-B infection among children born to chronic carrier mothers, despite known exposure to HBsAg in 3 and probable exposure to HBsAg in the rest, accords with published reports.8-11 In contrast to this are the reports of frequent hepatitis-B infections in children born to mothers acutely ill with type-B hepatitis at or around the time of delivery. There are several possible explanations for this
pen
difference between mothers acutely and chronically infected. First, it is possible that these symptom-free chronic carrier mothers are not infectious for type-B hepatitis-i.e., that their HBsAg circulates in noninfectious forms. However, all experimental or epidemiological evidence indicates that all HBgAg-positive patients are infectious. The infectivity of chronic HBgAg carriers has been well documented in studies of post-transfusion hepatitis 18 The hypothesis that some HBgAg-positive patients (especially those with acute viral hepatitis, type B) are more infectious than others (such as those with the chronic carrier state) is appealing but unsupported by experimental data. Second, the possibility exists that only non-infectious forms of HBsAg (20 nm. spheres and tubules) ordinarily cross the placental barrier and that the Dane particle or the intact form of the infectious virus does not enter the fetal circulation. This hypothesis would not, however, explain why women with acute type-B hepatitis frequently transmit the disease to their newborns, whereas women with the chronic HBsAg carrier state do not. Finally, it is possible that these babies resist infection with this virus because they possess some antiviral, protective factor perhaps present at birth and disappearing shortly thereafter. This factor responsible for the lack of hepatitis-B infections that these children exhibit subsequent to exposure at or around the time of delivery might be synthesised by the infant in utero in response to the exposure’ to HBsAg from the mother or by the symptom-free chronic HBsAg carrier mother and transferred through the placenta. This is an attractive hypothesis, but the suggested protective factor has yet to be identified. Anti-HBs is thought to be the antibody responsible for recovery and resistance to reinfection in type-B hepatitis.16,19 This antibody, however, was not found in cord blood of children born to these chronic carrier mothers, nor was it present at three and six months. Our findings show that anti-HB readily crosses the placental barrier. Anti-HB was found in all chronic carrier mothers as well as in the cord blood of their children at birth. The -rapid disappearance of this antibody from the sera of the newborns with time suggests that this antibody was maternal in origin and that its presence did not indicate infection or replication of the virus in the fetus. This was further documented by the lack of appearance of anti-HBs in these children. Whether anti-HB, present at the time of birth and thus at the time of intense exposure to HBsAg, was the unknown factor responsible for their subsequent avoidance of infection is not known. Certainly chronic HBsAg carriers generally have high titres of anti-HB, whereas patients with acute type-B hepatitis do not develop anti-HB until one to three months after the first appearance of HBsAg in the serum." These differences in presence and titre of anti-HB among acutely and chronically infected mothers may account for the differences in frequency of transmission of hepatitis-B infection. On the other hand, the presence of long-standing and high titres of anti-HBe in chronic HBsAg carriers suggests that this antibody is ineffective in removing the virus. This makes the presence of anti-HB an insufficient explanation for the absence of transmission of hepati-
748 tis-B virus from chronic carrier mothers to their newborns. Follow-up of these and other infants may provide more information on their subsequent susceptibility Such studies are now in to hepatitis-B infection. progress. This study
was supported in part by a grant from the Office of Scientific Research and Development, Greek Ministry of We thank all those who made sera Culture and Sciences. available or who helped in the collection of samples. Requests for reprints should be addressed to G. P., 52 Skoufa Street, Athens 135, Greece.
REFERENCES 1. 2. 3.
4.
5. 6. 7.
Keys, F., Hobel, C., Oh, W., Gitnick, G., Hewitt, L. Clin. Res. 1971, 19, 184. Krech, U., Sonnabend, W., Kistler, G., Mäder, A. Vox sang. 1973, 24, 55. Schweitzer, I. L., Mosley, J. W., Ashcaval, M., Edwards, V., Overby, L. B. Gastroenterology, 1973, 65, 277. Turner, G., Field, A. M., Lasheen, R., Todd, M., Bruce-White, G. J. clin. Path. 1970, 23, 826. Wright, R., Perkins, J., Bower, B., Jerrome, D. Br. med. J. 1970, iv, 719. Cossart, Y. E., Hargroves, F. D., March, S. P. Am. J. Dis. Child. 1972, 123, 376. Merril, D., Dubois, R., Kohler, P. New Engl. J. Med. 1972, 287, 1280.
Skinhøj, P., Olesen, H., Cohn, J., Mikkelsen, M. Acta path. microbiol. scand. 1972, 80, 362. 9. Gillespie, A., Dorman, D., Walker-Smith, J. A., Yu, J. S. Lancet, 1970, ii, 1081. 10. Charalambidis, B., Hadjiyannis, S. Paper presented at 2nd Panhellenic Congress of Gastroenterology, held in Athens, Greece, 8.
in 1971. 11. 12. 13. 14.
Alexiou, D., Papaevangelou, G., Papadatos, C., Georgiopoulos, F., Kremastinou, J. Pediatrie, 1973, 28, 733. Pesendorfer, F., Krassnitzky, O., Wewalka, F. Klin. Wschr. 1970, 48, 58. Ling, C. M., Overby, L. R. J. Immun. 1972, 109, 834. Prince, A. M., Brotman, B., Jass, D., Ikram, H. Lancet, 1973,
i, 1346. 15. Vyas, G. N., Shulman, N. R. Science, 1970, 170, 332. 16. Hoofnagle, J. H., Gerety, R. J., Barker, L. F. Lancet, 1973, ii, 869. 17.
Papaevangelou, G., Kremastinou, J., Prevedourakis, C., Kaskarelis,
D. Archs Dis. Childh. (in the press). 18. Gocke, D. J. J. Am. med. Ass. 1972, 219, 1165. 19. Krugman, S., Giles, J. P., Hammond, J. ibid. 1971,
218, 1665.
PITFALLS IN THE STUDY OF HEPATITIS A
Langley Court,
Beckenham, Kent BR3 3BS F. W. GAY*
Department of Pathology, Queens University of Belfast,
Belfast, Northern Ireland T. G. WREGHITT
Langley Court, Beckenham,
Using
Kent
the methods of double diffusion
Sin um ary agar gel and immune electron microscopy, patients with infectious hepatitis showed seroconversion to several unidentified fæcal antigens. This is in agreement with previous findings that antibody to bacterial, viral, and dietary antigens is raised in patients with liver disease. Because of the multiplicity of the immune reaction associated with infectious hepatitis, simple serological techniques will always have pitfalls associated with them. *
Present address: London W1.
Materials and Methods Patients Patients notified to us by general practitioners in the Belfast area (September to November, 1973) were seen A first sample of blood was taken, and fxces at home. The families were then followed up for 5-6 obtained. weeks to obtain specimens from fresh contact cases as early as possible in the illness and even in some instances before jaundice had appeared. A second blood sample was taken between 2 and 6 weeks after the first specimen had been obtained. Cases for this study (see table) were selected as being of classical presentation and incubation period for infectious hepatitis; all developed jaundice, showed typical liver-enzyme rises, and were Australiaantigen-negative by standard gel-diffusion tests. For control purposes faecal specimens were obtained from a variety of other liver diseases, including obstructive jaundice caused by carcinoma of the pancreas, chronic active (lupoid) hepatitis, and drug-induced hepatitis. Faeces and sera were stored at -20°C.
Preparation of Fcecal Extracts 20% suspensions of foeces in phosphate-buffered saline were prepared and used as starting material in all experiments. Faecal concentrates were obtained by centrifuging the 20% suspensions for 30 minutes at 10,000 r.p.m. in an M.S.E. high-speed 18 centrifuge and the supernatant from this step concentrated 25 times in a’minicon’
(Amicon, Massachusetts, U.S.A.).
J. D. ALMEIDA Wellcome Research Laboratories,
THE discovery of Australia antigen, now known as B antigen, as a laboratory marker for B has advanced considerably the knowledge of this diseased Unfortunately, no such marker has been discovered for hepatitis A (infectious hepatitis), even although many different techniques, including epidemiology,22 serology,3 tissue culture,4 animal models, and, most recently, immune electron microscopy,6 have been applied to its study. Further, even worse than yielding no results, many of these techniques have produced conflicting findings. We have undertaken a study of paired sera and fxcal specimens from an outbreak of infectious hepatitis in Belfast. Specimens were examined both by immune electron microscopy and double diffusion in agar gel.
hepatitis hepatitis
Department of Virology, Middlesex Hospital,
Immune Electron Microscopy 0-5 ml. amounts of faecal concentrate were mixed with 0-2 ml. of either the first (acute) or second (convalescent) serum and these were then left to react overnight at 4°C. The mixtures were then centrifuged for one hour at 15,000 r.p.m. and the pellet used for negative staining, which was carried out in the usual manner using 3 % phosphotungstic acid adjusted to pH 6 with N NaOH. Grids were examined immediately after preparation in a Philips 300
electron 57,000 times.
microscope
at
a
plate magnification
of
Immunodiffusion Simple Ouchterlony double diffusion in agarose was used for these tests. Agarose A 37 supplied by L’Industrie Biologique Francaise was used. This was made up as a 0-9% solution in ’Tris’ E.D.T.A. buffer. The template employed was six wells surrounding one, the wells having a diameter of 3 mm. and the spaces between them 4 mm. Gel templates were arranged so that each fxcal extract was able to react with both the acute and convalescent serum. Occasionally, in order to obtain greater density of precipitin lines, antigen and antibody wells were double filled. Gels were left overnight at room temperature and the precipitin pattern photographed next
subjective manifestations are often open to interpretation, the lack of symptoms among bacteriuric patients taking tetracycline (including those who had previously reacted adversely to minocycline), and the absence of symptoms among contacts of those taking minocycline prophylactically, tend to support the validity of these observations. The data suggest that minocycline has a propensity for producing reversible vestibular side-effects, which requires more detailed and objective investigation. This work was supported by Research grant no. HD-03693 from the National Institute of Child Health and Human Development and training grant no. TO-1 AI-00068 from the National Institute of Allergy and Infectious Diseases. Requests for reprints should be addressed to L. W. L., Channing Laboratory, 774 Albany Street, Boston, Massachusetts 02118, U.S.A. REFERENCES 1. 2.
Guttler, R. B., Counts, G. W., Avent, C. K., Beaty, H. N.J. infect. Dis. 1971, 124, 199. Guttler, R. B., Beaty, H. N. Antimicrob. Agents Chemother. 1972, 1,
397. 3. Frisk, A. R., Tunevall, G. 1969. ibid. 1968, p. 335. 4. Holloway, W. J. Delaware med. J. 1970, 42, 333. 5. Devine, L. F., Johnson, D. P., Hagerman, C. R., Pierce, W. E., Rhode, S. L., Peckinpaugh, R. O. Am. J. Epidem. 1971, 93, 337. 6. Gould, W. J., Brookler, K. H. Archs Otolaryng. 1972, 96, 291. 7. Nelson, J. R. in Textbook of Medicine (edited by P. B. Beeson and W. McDermott); p. 174. Philadelphia, 1971.
TRANSPLACENTAL TRANSMISSION OF HEPATITIS-B VIRUS BY SYMPTOM-FREE CHRONIC CARRIER MOTHERS GEORGE PAPAEVANGELOU JAY HOOFNAGLE JENNY KREMASTINOU
Department of Hygiene and Epidemiology, University of Athens Medical School, Athens, Greece, and Bureau of Biologics, Food and Drug Administration, Bethesda,
Maryland, U.S.A.
Transplacental transmission of the hepatitis-B surface antigen (HBsAg) and of hepatitis-B virus was studied in 12 symptom-free chronic HBsAg carrier mothers and their 15 babies. HBsAg was present in the sera of the mothers throughout the study (at delivery and three and six months later). HBsAg was detected in the cord blood of 3 of the 15 children, but subsequently was not detected except in 1 child who first became HBsAgpositive at six months of age. Antibody to HBsAg Summary
was not
detected in the mothers,
nor
in their infants
during the first six months of life. Antibody to the core antigen (anti-HBc) was always present in carrier mothers and in the cord blood, but it disappeared from the sera of the newborns shortly after birth. These data indicate that children of chronic HBsAg carrier mothers generally do not become infected with hepatitis-B virus before or at birth despite the known intense exposure to HBsAg from the mother.
It is unclear whether the presence of HBAg in cord blood constitutes sufficient virus exposure to transmit type-B hepatitis to the child. Mothers with acute type-B hepatitis in late pregnancy or shortly after delivery often transmit overt or subclinical hepatitis B to their babies.3-’ However, there are conflicting of babies by mothers infections reports regarding who are symptom-free chronic HBsAg carriers.3,s-,: We have investigated the possibility of transplacental transmission of HBsAg and/or hepatitis B by taking serial blood-samples from symptom-free carrier mothers and their babies for six months after delivery and testing them for HBsAg, anti-HBs, and antibody to the hepatitis-B core antigen (anti-HB).
Materials and Methods
Serological Testing HBsAg was tested by counterelectrophoresis (C.E.P.)12 and by solid-phase radioimmunoassay (R.I.A.,’Ausria125 ’, Abbott Laboratories).13 Samples reactive for HBsAg by R.I.A. but negative by C.E.P. were tested for specificity by inhibition with human anti-HBs.14 Anti-HBs was assayed by passive hxmagglutination (Virgo Reagents, Electro-Nucleonics Inc.) 15 and by solid-phase radioimmunoassay (’ Ausab ’, Abbott Laboratories). Anti-HB was detected by a microtitre complement-fixation testJ6 Samples Tested We screened 428 pregnant women, seen in the obstetric clinics of the University of Athens, for HBsAg by C.E.P., and 14 (3%) were positive. 2 are not analysed here because cord bloods were not available. 3 of the positive mothers gave birth to another child after their first delivery. Thus, the study group consisted of 12 HBsAg-positive mothers and their 15 newborns. At parturition, maternal and umbilical cord bloods were drawn aseptically, and the sera were stored at -60°C until tested. The mothers and their babies were reexamined three and six months after delivery. All mothers remained HBsAg-positive. No liver biopsies were performed, but the absence of overt signs of liver disease and the lack of substantial biochemical abnormalities (raised serum-glutamic-pyruvic-transaminase values above 100 Karmen units) in the face of persistence of HBAg indicated that these mothers were chronic HBsAg carriers and did not have acute type-B hepatitis. Results
HBsAg HBsAg
was detected in the umbilical cord blood of 3 of the 15 children born to symptom-free carrier mothers. 1 of the cord bloods was positive by both C.E.P.’and R.I.A., while the other 2 were positive by R.I.A. only. At three months, HBAg was not detected in any of the newborns. At six months after birth, HBsAg was found in the serum of only 1 of the 15 children (case 1). This child was not HBsAg-positive at birth and at six months was only weakly positive
(titre 1/4, by
R.I.A.
only).
Arzti-HB, Anti-HB
Introduction
was not demonstrated in maternal or umbilical-cord blood of any case. It had not developed in any of the 15 newborns at three or six months after birth.
hepatitis-B surface antigen (HBsAg) has occasionally been reported in the cord blood of children born to mothers infected with the hepatitis-B virus.1-3
Anti-HB, Anti-HB was found in all carrier mothers in every blood-sample. Titres were high, ranging from 1 16
THE
747
HBsAg
AND ANTI-HBc TITRES IN CHRONIC HBsAg PRESENCE OF CARRIER MOTHERS AND IN THE CORD BLOOD OF THEIR BABIES
*
Detectable
by radioimmunoassay only.
1/1024 (see table). Furthermore, this antibody was present in the cord blood of each of the children born to these carrier mothers. The titre in the cord blood was similar to that of the mother and independent of the presence of HBgAg in the cord blood. At three months after birth, anti-HB was no longer detectable in any of the 15 children. All children remained negative for anti-HB when re-examined six months after delivery, except for case 1, where anti-HB reappeared at a very low titre (1/4) currently with the appearance of HB,Ag. to
Discussion These findings, together with other reports,1-3,17 indicate that HBsAg can be found in the cord blood of children born to chronic carrier mothers. HBsAg was found in 3 of the 15 cord bloods, although in only 1 was the titre of HBsAg similar to the titre in the mother’s serum (both reactive by C.E.P.). Thus, as might be expected, HBsAg (which is present in the serum in the form of 20 nm. spheres and tubules and as 42 nm. Dane particles) crosses the placental barrier poorly, if at all. The finding of this particulate antigen in cord bloods of only 3 of 15 cases suggests that HBgAg may cross into the fetal circulation only when the placental barrier breaks down, as must hap-
frequently during parturition. Although HBsAg was found in the sera of 3 children at birth, none of these 3 subsequently developed either biochemical or serological evidence of hepatitis B. In 12 other children whose sera were negative for HBsAg at birth, only 1 subsequently developed evidence of type-B hepatitis (in the form of low levels of HB,Ag and anti-HB six months after delivery). In fact, the appearance of HBsAg six months after delivery points to exposure to hepatitis-B virus after delivery rather than at birth. This infrequent occurrence of hepatitis-B infection among children born to chronic carrier mothers, despite known exposure to HBsAg in 3 and probable exposure to HBsAg in the rest, accords with published reports.8-11 In contrast to this are the reports of frequent hepatitis-B infections in children born to mothers acutely ill with type-B hepatitis at or around the time of delivery. There are several possible explanations for this
pen
difference between mothers acutely and chronically infected. First, it is possible that these symptom-free chronic carrier mothers are not infectious for type-B hepatitis-i.e., that their HBsAg circulates in noninfectious forms. However, all experimental or epidemiological evidence indicates that all HBgAg-positive patients are infectious. The infectivity of chronic HBgAg carriers has been well documented in studies of post-transfusion hepatitis 18 The hypothesis that some HBgAg-positive patients (especially those with acute viral hepatitis, type B) are more infectious than others (such as those with the chronic carrier state) is appealing but unsupported by experimental data. Second, the possibility exists that only non-infectious forms of HBsAg (20 nm. spheres and tubules) ordinarily cross the placental barrier and that the Dane particle or the intact form of the infectious virus does not enter the fetal circulation. This hypothesis would not, however, explain why women with acute type-B hepatitis frequently transmit the disease to their newborns, whereas women with the chronic HBsAg carrier state do not. Finally, it is possible that these babies resist infection with this virus because they possess some antiviral, protective factor perhaps present at birth and disappearing shortly thereafter. This factor responsible for the lack of hepatitis-B infections that these children exhibit subsequent to exposure at or around the time of delivery might be synthesised by the infant in utero in response to the exposure’ to HBsAg from the mother or by the symptom-free chronic HBsAg carrier mother and transferred through the placenta. This is an attractive hypothesis, but the suggested protective factor has yet to be identified. Anti-HBs is thought to be the antibody responsible for recovery and resistance to reinfection in type-B hepatitis.16,19 This antibody, however, was not found in cord blood of children born to these chronic carrier mothers, nor was it present at three and six months. Our findings show that anti-HB readily crosses the placental barrier. Anti-HB was found in all chronic carrier mothers as well as in the cord blood of their children at birth. The -rapid disappearance of this antibody from the sera of the newborns with time suggests that this antibody was maternal in origin and that its presence did not indicate infection or replication of the virus in the fetus. This was further documented by the lack of appearance of anti-HBs in these children. Whether anti-HB, present at the time of birth and thus at the time of intense exposure to HBsAg, was the unknown factor responsible for their subsequent avoidance of infection is not known. Certainly chronic HBsAg carriers generally have high titres of anti-HB, whereas patients with acute type-B hepatitis do not develop anti-HB until one to three months after the first appearance of HBsAg in the serum." These differences in presence and titre of anti-HB among acutely and chronically infected mothers may account for the differences in frequency of transmission of hepatitis-B infection. On the other hand, the presence of long-standing and high titres of anti-HBe in chronic HBsAg carriers suggests that this antibody is ineffective in removing the virus. This makes the presence of anti-HB an insufficient explanation for the absence of transmission of hepati-
748 tis-B virus from chronic carrier mothers to their newborns. Follow-up of these and other infants may provide more information on their subsequent susceptibility Such studies are now in to hepatitis-B infection. progress. This study
was supported in part by a grant from the Office of Scientific Research and Development, Greek Ministry of We thank all those who made sera Culture and Sciences. available or who helped in the collection of samples. Requests for reprints should be addressed to G. P., 52 Skoufa Street, Athens 135, Greece.
REFERENCES 1. 2. 3.
4.
5. 6. 7.
Keys, F., Hobel, C., Oh, W., Gitnick, G., Hewitt, L. Clin. Res. 1971, 19, 184. Krech, U., Sonnabend, W., Kistler, G., Mäder, A. Vox sang. 1973, 24, 55. Schweitzer, I. L., Mosley, J. W., Ashcaval, M., Edwards, V., Overby, L. B. Gastroenterology, 1973, 65, 277. Turner, G., Field, A. M., Lasheen, R., Todd, M., Bruce-White, G. J. clin. Path. 1970, 23, 826. Wright, R., Perkins, J., Bower, B., Jerrome, D. Br. med. J. 1970, iv, 719. Cossart, Y. E., Hargroves, F. D., March, S. P. Am. J. Dis. Child. 1972, 123, 376. Merril, D., Dubois, R., Kohler, P. New Engl. J. Med. 1972, 287, 1280.
Skinhøj, P., Olesen, H., Cohn, J., Mikkelsen, M. Acta path. microbiol. scand. 1972, 80, 362. 9. Gillespie, A., Dorman, D., Walker-Smith, J. A., Yu, J. S. Lancet, 1970, ii, 1081. 10. Charalambidis, B., Hadjiyannis, S. Paper presented at 2nd Panhellenic Congress of Gastroenterology, held in Athens, Greece, 8.
in 1971. 11. 12. 13. 14.
Alexiou, D., Papaevangelou, G., Papadatos, C., Georgiopoulos, F., Kremastinou, J. Pediatrie, 1973, 28, 733. Pesendorfer, F., Krassnitzky, O., Wewalka, F. Klin. Wschr. 1970, 48, 58. Ling, C. M., Overby, L. R. J. Immun. 1972, 109, 834. Prince, A. M., Brotman, B., Jass, D., Ikram, H. Lancet, 1973,
i, 1346. 15. Vyas, G. N., Shulman, N. R. Science, 1970, 170, 332. 16. Hoofnagle, J. H., Gerety, R. J., Barker, L. F. Lancet, 1973, ii, 869. 17.
Papaevangelou, G., Kremastinou, J., Prevedourakis, C., Kaskarelis,
D. Archs Dis. Childh. (in the press). 18. Gocke, D. J. J. Am. med. Ass. 1972, 219, 1165. 19. Krugman, S., Giles, J. P., Hammond, J. ibid. 1971,
218, 1665.
PITFALLS IN THE STUDY OF HEPATITIS A
Langley Court,
Beckenham, Kent BR3 3BS F. W. GAY*
Department of Pathology, Queens University of Belfast,
Belfast, Northern Ireland T. G. WREGHITT
Langley Court, Beckenham,
Using
Kent
the methods of double diffusion
Sin um ary agar gel and immune electron microscopy, patients with infectious hepatitis showed seroconversion to several unidentified fæcal antigens. This is in agreement with previous findings that antibody to bacterial, viral, and dietary antigens is raised in patients with liver disease. Because of the multiplicity of the immune reaction associated with infectious hepatitis, simple serological techniques will always have pitfalls associated with them. *
Present address: London W1.
Materials and Methods Patients Patients notified to us by general practitioners in the Belfast area (September to November, 1973) were seen A first sample of blood was taken, and fxces at home. The families were then followed up for 5-6 obtained. weeks to obtain specimens from fresh contact cases as early as possible in the illness and even in some instances before jaundice had appeared. A second blood sample was taken between 2 and 6 weeks after the first specimen had been obtained. Cases for this study (see table) were selected as being of classical presentation and incubation period for infectious hepatitis; all developed jaundice, showed typical liver-enzyme rises, and were Australiaantigen-negative by standard gel-diffusion tests. For control purposes faecal specimens were obtained from a variety of other liver diseases, including obstructive jaundice caused by carcinoma of the pancreas, chronic active (lupoid) hepatitis, and drug-induced hepatitis. Faeces and sera were stored at -20°C.
Preparation of Fcecal Extracts 20% suspensions of foeces in phosphate-buffered saline were prepared and used as starting material in all experiments. Faecal concentrates were obtained by centrifuging the 20% suspensions for 30 minutes at 10,000 r.p.m. in an M.S.E. high-speed 18 centrifuge and the supernatant from this step concentrated 25 times in a’minicon’
(Amicon, Massachusetts, U.S.A.).
J. D. ALMEIDA Wellcome Research Laboratories,
THE discovery of Australia antigen, now known as B antigen, as a laboratory marker for B has advanced considerably the knowledge of this diseased Unfortunately, no such marker has been discovered for hepatitis A (infectious hepatitis), even although many different techniques, including epidemiology,22 serology,3 tissue culture,4 animal models, and, most recently, immune electron microscopy,6 have been applied to its study. Further, even worse than yielding no results, many of these techniques have produced conflicting findings. We have undertaken a study of paired sera and fxcal specimens from an outbreak of infectious hepatitis in Belfast. Specimens were examined both by immune electron microscopy and double diffusion in agar gel.
hepatitis hepatitis
Department of Virology, Middlesex Hospital,
Immune Electron Microscopy 0-5 ml. amounts of faecal concentrate were mixed with 0-2 ml. of either the first (acute) or second (convalescent) serum and these were then left to react overnight at 4°C. The mixtures were then centrifuged for one hour at 15,000 r.p.m. and the pellet used for negative staining, which was carried out in the usual manner using 3 % phosphotungstic acid adjusted to pH 6 with N NaOH. Grids were examined immediately after preparation in a Philips 300
electron 57,000 times.
microscope
at
a
plate magnification
of
Immunodiffusion Simple Ouchterlony double diffusion in agarose was used for these tests. Agarose A 37 supplied by L’Industrie Biologique Francaise was used. This was made up as a 0-9% solution in ’Tris’ E.D.T.A. buffer. The template employed was six wells surrounding one, the wells having a diameter of 3 mm. and the spaces between them 4 mm. Gel templates were arranged so that each fxcal extract was able to react with both the acute and convalescent serum. Occasionally, in order to obtain greater density of precipitin lines, antigen and antibody wells were double filled. Gels were left overnight at room temperature and the precipitin pattern photographed next