1565
2. Centers for Disease Control. National HIV seroprevalence surveys. Atlanta: CDC, 1989. 3. Ippolito G, Stegagno M, Angeloni P, Guzzanti E. Anonymous HIV testing on newborns. JAMA 1990; 263: 36. 4. Hankins CA, Laberge C, Lapointe N, Lai Tung MT, Racine L, O’Shaughnessy M. HIV infection among Quebec women giving birth to live infants. Can Med Assoc J 1990; 143: 885-93. 5. McLaws M, Brown ARD, Cunningham PH, Imrie AA, Wilcken B, Cooper DA. Prevalence of maternal HIV infection based on anonymous testing of neonates, Sydney 1989. Med J Aust 1990; 153: 383-86. 6. European Collaborative Study. Children born to women with HIV-1 infection: natural history and risk of transmission. Lancet 1991; 337: 253-60. 7. Peckham CS, Tedder RS, Briggs M, et al. Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies. Lancet 1990; 335: 516-19. 8. Davison CF, Ades AE, Hudson CN, Peckham CS. Antenatal testing for human immunodeficiency virus. Lancet 1989; ii: 1442-44. 9. Hall SM, Glickman M. Report from the British Paediatric Surveillance Unit. Arch Dis Child 1990; 65: 807-09. 10. Berry TJ, Ades AE, Peckham CS. Too many ethical committees. Br Med J 1990; 301: 1274. 11. Wacholder S. Binomial regression in GLIM: estimating risk ratios and risk differences. Am J Epidemiol 1986; 123: 174-84. 12. Novick LF, Berns D, Stricof R, Stevens R, Pass K, Wethers J. HIV
seroprevalence in newborns in New York State. JAMA 1989;
261: 1745-50. 13. Centers for Disease Control. HIV prevalence estimates and AIDS case projections for the United States: report based upon a workshop. MMWR 1990; 39. 14. Downs AM, Ancelle-Park RA, Brunet J. Surveillance of AIDS in the European Community: recent trends and predictions to 1991. AIDS 1990; 4: 1117-24. 15. PHLS Communicable Disease Surveillance Centre. Acquired immune deficiency syndrome in England and Wales to end 1993. Projections using data to end September 1989. CDR January, 1990. 16. Krasinski K, Borrowsky W, Bebenroth D, Moore T. Failure of voluntary testing for human immunodeficiency virus to identify infected parturient women in a high-risk population. N Engl J Med 1988; 318: 185. 17. Barbacci M, Repke FT, Chaisson RE. Routine prenatal screening for HIV infection. Lancet 1991; 337: 709-11. 18. Dunn D, Hayes R. HIV screening in pregnancy. Lancet 1991; 337: 1218-19. 19. Meadows J, Jenkinson S, Catalan J, Gazzard B. Voluntary HIV testing in the antenatal clinic: differing uptake rates for individual counselling midwives. AIDS Care 1990; 2: 229-33. 20. Sherr L, Hedge B. The impact and use of written leaflets as a counselling alternative in mass antenatal HIV screening. AIDS Care 1990; 2: 235-45.
Prevalence of maternal HIV infection in Scotland based on unlinked anonymous testing of newborn babies
Dried blood spot samples from newborn babies have been successfully tested for HIV-1 antibody by the
particle agglutination method to assess the prevalence of infection in the mothers. In January, 1990, unlinked anonymous testing of Guthrie cards for HIV antibody was begun in Scotland. 99·6% of Scottish births were tested. 9 mothers spontaneously refused to allow testing of their baby’s blood. Samples were coded by district postcodes. Eluates of 65 773 dried blood spots were initially tested for HIV-1 antibody with the Fujirebio technique. Of the 31 positive samples 19 were confirmed to be positive by enzyme-linked radioimmunoassay and western blot (seroprevalence 0·29 per 1000). All these samples came from large metropolitan areas on the Prevalences were 2·5 per 1000 for Edinburgh city, 1·4 per 1000 for Dundee, and 0·7 per 1000 for Aberdeen. We identified as HIV-positive all babies known to be so in named testing programmes. HIV testing of Guthrie cards can be used to monitor HIV status in mothers who have just given birth. The use of district postcode data in sample identification will allow accurate targetting of prevention strategies and early detection of spread of infection by geographic area. east coast.
Introduction In the UK and USA the prevalence of HIV infection in the time of delivery has been assessed by
women at
antibody testing of blood samples that have routinely from newborn babies.1-3 The particle agglutination method of testing for HIV-1antibody on eluted dried blood spot samples has been successfully used in a large study.3 In the UK a series of large multicentre surveys of unlinked anonymous testing that include newborn babies have begun. HIV antibody in serum from these babies reflects maternal antibody status at delivery and does not record infant infection. We report the first year’s results of a definitive continuing seroprevalence survey of childbearing women in Scotland, using district postcode information to defme geographic location and spread of anonymous
been obtained
HIV-1 infection.
Subjects and methods Collection of samples and data-Blood samples, taken routinely by heel prick from babies around the 7th day of life, were spotted onto filter paper (Guthrie cards) and air dried. The nurse who took the sample entered information on the card, which included the baby’s date of birth and the mother’s address. All cards for Scotland were then sent to the Scottish Inborn Errors Screening Laboratory at Stobhill Hospital, Glasgow. Ethical approval-We approached twenty ethics committees in the fifteen health board areas in Scotland. Our study included cards ADDRESSES: Department of Child Health, Royal Hospital for Sick Children, Glasgow, UK (Prof F. Cockburn, FRCP, D. M. Tappin, MRCP); Scottish Inborn Errors Screening Laboratory, Stobhill General Hospital, Glasgow (R. W. A. Girdwood, FRCPath, R Kennedy, FIMLS, A J Brown, BA Biol); Regional Virus Laboratory, Ruchill Hospital, Glasgow (E A. C. Follett, MRCPath). Correspondence to Dr D. M Tappin, Department of Child Health, Royal Hospital for Sick
Children, Yorkhill, Glasgow G3 8SJ, UK
1566
PLACE OF BIRTH OF HIV-ANTIBODY-POSITIVE BABIES DURING 1990
Glasgow=all mothers living in Glasgow postcode districts G1-84, Edinburgh = Edinburgh city, EH1-17, Dundee=DD1-5, Aberdeen = AB1, 2
from 99-6% of babies born in Scotland during 1990. Births from Orkney island (0-4%) were not included because the island’s ethics committee felt that women whose babies tested positive should be told, which was not our study design. Posters informing patients that residual blood samples may be anonymously tested for HIV antibody had been widely distributed in Scotland.
Postcoding of cards and recording of data-Since our main maintenance of the metabolic screening programme, were only tat;en for HIV-1antibody testing after all tests samples and re-tests for metabolic disorders had been done. During this process all duplicate cards were so marked. Cards were kept at room temperature without desiccation. With the use of postcode directories, the first three digits of the mother’s district postcode were recorded on the card. Duplicate cards were not included. The calendar quarter of birth and the district postcode were then recorded on computer, apart from records for Orkney and for babies whose mothers spontaneously opted out. The computer then produced a random number for each batch. A 3 mm diameter disc was punched out of one of the blood spots on the Guthrie card into a 96-well microtitre (Sterilin, Feltham, UK) plate which was marked with the random number. Computer information was stored at the Royal Hospital for Sick Children, Glasgow, and the microtitre plates were tested at Stobhill Hospital.
concern was
Elution of serum-Dried blood discs were eluted in flatbottomed 96-well microtitre master plates in 100 µl elution buffer (phosphate-buffered saline, pH 7-2, containing 005% "Tween 20" and 0-005 % sodium azide). The plates were shaken at 200 cycles per min for 30 min at room temperature and left overnight at 4°C, following which they were shaken for 3 min at 500 cycles per min. Controls were dried blood spots-high positive, low positive, and negative-from the Centers for Disease Control (CDC), Atlanta; a serum sample supplied with the Fujirebio HIV-antibody assay which was used at dilutions of 1 in 4 and 1 in 32; and in-house positive-control blood spots from the regional virus laboratory, Ruchill Hospital. HIV-antibody assay-A modified gelatin particle assay (GPA, Fujirebio) was used.3,4 30 µl of a 1 in 5 dilution was made of every eluate in v-welled microtitre plates with a multichannel pipette. 25 µl ofa1 in 5 dilution of antigen-coated gelatin particles was added to every well. The plates were left at room temperature for 15 min, centrifuged at 260g for 2 min, and placed at an angle of 70° on a light box. The reactions were read after 10 min: the particles mixed with the eluate containing HIV antibody remained as a discrete tight button, whereas those not containing HIV antibody became an elongated "tear drop". Reactive eluates were titrated across the endpoint and aspirated from the master plate for storage at - 20°C.
Confirmatory tests-At the regional virus laboratory enzymelinked immunosorbent assay (ELISA) and western blot tests were used to confirm the presence of HIV-1and HIV-2 antibody in all samples reactive in the screening test. We used an IgG antibody capture ELISA for HIV-1 and HIV-2 antibody (GACELISA), developed at the virus reference laboratory, Central Public Health Laboratory, London.5,6 10 ul samples of the repeatedly reactive eluates were diluted in 90 µl of diluent and incubated for 30 min at 37°C in microtitre wells coated with rabbit antibody to human IgG. After incubation and washing, an enzyme-conjugated recombinant
antigen was added and incubation continued for 60 min. Further washing was followed by incubation with substrate (20 min at 37°C) and amplifier (10 min at 37°C). The reaction was stopped by addition of 50 µl of 2 mol/1 sulphuric acid, and absorbance at 492 nm was measured. The cut-off was set at three times the negative mean. Complete western blots for HIV-1 were done in all repeatedly reactive eluates that were also reactive in the GACELISA system or within 10% of the cutoff. 25 ul of eluate was diluted in 50 µl of milk buffer and run on a preblotted membrane (Organon Teknika, Cambridge) in a ’Miniblotter’ aparatus’ (Biometra, Manchester). The blots were developed with conjugates supplied by Dupont in their HIV-1western blot kit. The conditions for positivity were the same as those used by Peckam et al3-ie, samples were judged positive if bands were seen representing reactivity against one or more of the GAG or POL genes and one of the ENV genes.
Quality
control-From
May, 1990, controls
were
supplied by
CDC every month. Each batch consisted of 12 unknown dried blood spot samples. We found no false-positive or false-negative results with our system in 5 of these batches. This is part of the worldwide HIV dried blood spot quality control and provides a check on the sensitivity of the Fujirebio kit screening test. Effects of storage on stability of dried blood spot sample-A batch of CDC quality control dried blood spots was stored at room temperature without desiccant and at 4°C and - 20°C with dessicant. At 1,2,3,4, and 5 weeks after receipt, the batch of spots was repunched, eluted, and retested, and no change in antibody titre was seen.
Collaboration
Study-The
with
Edinburgh
Edinburgh
Perinatal
Transmission
collaborators
provided us with HIV-positive births during 1990 in
information for all known Edinburgh, and with risk category data for these births.
Results excluded since their origin was either outside Scotland (64), and 9 cards were not Orkney (259) tested because of parental refusal. 65 781 dried blood discs were punched from Guthrie cards, 5 of which contained insufficient blood and 3 did not elute from the blood spots. The remaining 65 773 (99-6%) were tested. A total of 31 eluates was reactive in the screening test. Of the 31 eluates, 19 had titres greater than 100 and 20 were positive in the GACELISA test, including the 19 with high titre. These 19 eluates showed antibody to env glycoproteins and to at least one of the two groups of structural proteins on western blot, and were therefore regarded as true positives. 1 sample that was positive in the GACELISA and another within 10% of the cutoff value could not be confirmed as positive by western blot. The prevalence of HIV antibody was thus 0-29 per 1000 childbearing women for Scotland as a whole. All affected mothers lived in metropolitan areas on the east coast of Scotland. 18 of the 19 mothers lived in three large cities, Edinburgh, Dundee, and Aberdeen. Period prevalence for mothers of infants who survived the first week of life, from these cities, was 2-5 per 1000,11.4 per 1000, and 0-7 per 1000, respectively. We emphasise that no antibody-positive babies were born from 16 308 deliveries in greater Glasgow (west Scotland) (table). We identified 13 HIV-antibody-positive babies in Edinburgh, and 13 such babies born in 1990 were known to the Edinburgh Perinatal Transmission Study (N. Croft, personal communication); Guthrie cards had been received at Stobhill hospital for all 13 babies. 7 mothers were intravenous drug users (IVDUs), 5 were partners of IVDUs, and 1 had no high risk associations. In Dundee a study of antenatal HIV antibody testing was ended in July, 1990,8 and 2 HIV-antibody-positive babies were expected. Both mothers were at low risk for HIV infection. In 323 cards
were or
1567
The prevalence of HIV-1antibody was more than eight times the average for Scotland in Edinburgh, nearly five times the average in Dundee, and more than twice the average in Aberdeen. The use of district postcodes allowed accurate localisation of high prevalence areas for targeting of prevention strategy. All but 1 of the HIV-positive mothers resided in these three large cities, and the other lived in a substantial town, all on the east coast of Scotland. Accurate geographic location of present prevalence has several advantages. Spread of infection to a new area can be identified at an early stage, and this has happened in Aberdeen during our study. Two further HIV-antibodypositive births were found in Aberdeen during the first quarter of 1991. The progress of the epidemic in specific areas can be monitored yearly, and comparison between population groups would be much easier if geographic location is well defined, but each study, we emphasise, should use the same system-ie, postcode district. We feel that the gradient of prevalence referred to in earlier reports 1,3,7 merely reflects poor discrimination of small high
termination of pregnancy is done at about 12 weeks’ gestation. Therefore, births from January, 1990, to January, 1991, are comparable with terminations from July, 1989, to July, 1990.) If we extrapolated our results to all heterosexual women we would have a large error from terminations, without consideration of other factors. Future unlinked anonymous testing to cover therapeutic abortion, when coupled with information from continued Guthrie-testing will provide a more complete picture. Since our data relating to Edinburgh and Dundee showed only 1 case of HIV that had not been identified by the named testing programmes we believe that antenatal patients opting out of named testing and proceeding to term do not form a high-risk group. Furthermore, information from the Edinburgh paediatric services shows that almost all the HIV-antibody-positive babies were born to high-risk mothers. 80% of HIV-positive pregnancies in Dundee were in high-risk women. This information is encouraging and shows that little spread of HIV infection has occurred to the presumed low-risk general population in Edinburgh or Dundee and that none has happened in Glasgow. This emphasises the importance of prevention of spread of infection in drug abusers. In Glasgow, HIV prevalence in anonymous and voluntary testing of drug addicts remains below 1-5% (J. Emslie, S. Haw, personal communication) whereas in Edinburgh and Dundee it is 38%"* and 40%,11
prevalence areas.
respectively.
Aberdeen the 2 HIV-antibody-positive babies we identified had not been detected by virological, obstetric, or paediatric services (Regional Virus Laboratory, unpublished data).
Discussion
We estimated that we tested 99-6% of Scottish births. This figure has been confirmed by provisional figures from the Registrar General, Scotland (Annual report, 1990). An important advantage of anonymous testing over named testing is that patients who refuse testing are much less likely to bias the data. Even if all 9 refusers in our study were HIV positive, prevalence rates would be little altered and only two vertical transmissions would be expected.9 Refusals were sporadic throughout the year-one was in Edinburgh and the others in low prevalence areas. Our results have shown that concern that babies born to mothers at high risk of HIV infection would be missed by the Guthrie card system is ill-founded. All babies known to be HIV-positive were identified by such testing. We are confident that we can monitor fully HIV status in childbearing women in
Scotland. The modified Fujirebio kit we used was highly sensitive and
satisfies the CDC quality control criteria for monitoring. Specificity was also high, with only 12 of 65 773 samples showing reactivity that could not be confirmed by western blot. The cost of the complete system including all reagents, staff, and equipment was only 0-70 per sample. At present this test only screens for HIV-1 antibody. A combined test incorporating HIV-2 antibody is being investigated in Japan. This should be available for use at the end of 1991. So far no HIV-2 antibody-positive individuals
have been identified in Scotland. All Guthrie cards for Scotland
are
received in
one
laboratory. However, we could not include women who had spontaneous or therapeutic abortions, stillbirths, or perinatal deaths. When our results were compared with those of the antenatal
study in Dundee,8 which included therapeutic abortions, we identified 3 HIV-antibodypositive births-2 were expected from HIV-antibodypositive pregnancies in the Dundee study. However, our investigation could take no account of the number of HIV-positive women who decided not to continue their pregnancy. 8 HIV-positive women had terminations in Dundee during a comparable period. (Therapeutic
We thank the many midwives, health visitors, and others involved in sample collection, and obstetricians, paediatricians, general practitioners, and administrative and clerical staff for their help; Miss C. Benison for her help in postcoding and entering all the data at Stobhill Hospital; Mr J. Taylor who designed and modified the computer programme; Mrs S. Martin and Miss J. Coote for the confirmation tests; Dr N. Croft, Dr J. Y. Q. Mok, Dr F. D. Johnstone, and Dr R. Smith for important data; Dr D. Reid, Dr J. Emslie, and Dr D. Goldberg and the Communicable Diseases (Scotland) Unit for their help in liaison with the Department of Health, the Scottish Home and Health Department, and the Medical Research Council; and the Ethics Committees or their help and constructive comments. Dr J. V. Parry and Dr P. P. Mortimer kindly provided the IgG antibody capture ELISA. This study was supported by the Medical Research Council
(grant no 8908795C). REFERENCES 1. Hoff
R, Berardi VP, Weiblen BJ, Mahoney-Trout L, Mitchell ML, Grady GF. Seroprevalence of human immunodeficiency virus among childbearing women. N Engl J Med 1988; 318: 525-30. 2. Centres for Disease Control. CDC surveillance supplements. MMWR 1989; 38: (no S-4). 3. Peckham CS, Tedder RS, Briggs M, et al. Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies. Lancet 1990; 335: 516-19. JAJ, Salker R, Challis P, Contreras M. Gelatin particle agglutination assay for HIV antibodies: a rapid economical modification with increased sensitivity. Med Lab Sci 1989; 46: 135-40. 5. Parry JV, Perry KR, Mortimer PP. Sensitive assays for viral antibodies in saliva: an alternative to tests on serum. Lancet 1987; ii: 72-75. 6. Connell JA, Parry JV, Mortimer PP, et al. Accurate assays for anti-HIV in urine. Lancet 1990; 335: 1366-69. 7. Norvick LF, Berns D, Stricof R, Stevens R, Pass K, Wethers J. HIV seroprevalence in New York State. JAMA 1989; 261: 1745-50. 8. Scrimgeour JB, Patel NB, Reid D, Smith R. HIV antibody seroprevalence in an antenatal population and its relationship to self reported behavioural risk. (Proceedings of the VI International Conference on AIDS, 1990, San Fransisco.) Abstract 3145. 9. Ades AE, Newell ML, Peckham CS. European Collaborative study. Children born to women with HIV-1 infection: natural history and risk transmission. Lancet 1991; 337: 253-60. 10. Peutherer JF, Edmonds E, Simmonds P, Dickson JD, Bath GE. HTLV-III antibody in Edinburgh drug addicts. Lancet 1985; ii: 4. Barbara
1129-30. 11.
Urquart GED, Scott SS, Wooldridge E, et al. Human immunodeficiency virus (HIV) in intravenous drug abusers in Tayside. Comm Dis Soc 1987; 9: 5-10.
2. Centers for Disease Control. National HIV seroprevalence surveys. Atlanta: CDC, 1989. 3. Ippolito G, Stegagno M, Angeloni P, Guzzanti E. Anonymous HIV testing on newborns. JAMA 1990; 263: 36. 4. Hankins CA, Laberge C, Lapointe N, Lai Tung MT, Racine L, O’Shaughnessy M. HIV infection among Quebec women giving birth to live infants. Can Med Assoc J 1990; 143: 885-93. 5. McLaws M, Brown ARD, Cunningham PH, Imrie AA, Wilcken B, Cooper DA. Prevalence of maternal HIV infection based on anonymous testing of neonates, Sydney 1989. Med J Aust 1990; 153: 383-86. 6. European Collaborative Study. Children born to women with HIV-1 infection: natural history and risk of transmission. Lancet 1991; 337: 253-60. 7. Peckham CS, Tedder RS, Briggs M, et al. Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies. Lancet 1990; 335: 516-19. 8. Davison CF, Ades AE, Hudson CN, Peckham CS. Antenatal testing for human immunodeficiency virus. Lancet 1989; ii: 1442-44. 9. Hall SM, Glickman M. Report from the British Paediatric Surveillance Unit. Arch Dis Child 1990; 65: 807-09. 10. Berry TJ, Ades AE, Peckham CS. Too many ethical committees. Br Med J 1990; 301: 1274. 11. Wacholder S. Binomial regression in GLIM: estimating risk ratios and risk differences. Am J Epidemiol 1986; 123: 174-84. 12. Novick LF, Berns D, Stricof R, Stevens R, Pass K, Wethers J. HIV
seroprevalence in newborns in New York State. JAMA 1989;
261: 1745-50. 13. Centers for Disease Control. HIV prevalence estimates and AIDS case projections for the United States: report based upon a workshop. MMWR 1990; 39. 14. Downs AM, Ancelle-Park RA, Brunet J. Surveillance of AIDS in the European Community: recent trends and predictions to 1991. AIDS 1990; 4: 1117-24. 15. PHLS Communicable Disease Surveillance Centre. Acquired immune deficiency syndrome in England and Wales to end 1993. Projections using data to end September 1989. CDR January, 1990. 16. Krasinski K, Borrowsky W, Bebenroth D, Moore T. Failure of voluntary testing for human immunodeficiency virus to identify infected parturient women in a high-risk population. N Engl J Med 1988; 318: 185. 17. Barbacci M, Repke FT, Chaisson RE. Routine prenatal screening for HIV infection. Lancet 1991; 337: 709-11. 18. Dunn D, Hayes R. HIV screening in pregnancy. Lancet 1991; 337: 1218-19. 19. Meadows J, Jenkinson S, Catalan J, Gazzard B. Voluntary HIV testing in the antenatal clinic: differing uptake rates for individual counselling midwives. AIDS Care 1990; 2: 229-33. 20. Sherr L, Hedge B. The impact and use of written leaflets as a counselling alternative in mass antenatal HIV screening. AIDS Care 1990; 2: 235-45.
Prevalence of maternal HIV infection in Scotland based on unlinked anonymous testing of newborn babies
Dried blood spot samples from newborn babies have been successfully tested for HIV-1 antibody by the
particle agglutination method to assess the prevalence of infection in the mothers. In January, 1990, unlinked anonymous testing of Guthrie cards for HIV antibody was begun in Scotland. 99·6% of Scottish births were tested. 9 mothers spontaneously refused to allow testing of their baby’s blood. Samples were coded by district postcodes. Eluates of 65 773 dried blood spots were initially tested for HIV-1 antibody with the Fujirebio technique. Of the 31 positive samples 19 were confirmed to be positive by enzyme-linked radioimmunoassay and western blot (seroprevalence 0·29 per 1000). All these samples came from large metropolitan areas on the Prevalences were 2·5 per 1000 for Edinburgh city, 1·4 per 1000 for Dundee, and 0·7 per 1000 for Aberdeen. We identified as HIV-positive all babies known to be so in named testing programmes. HIV testing of Guthrie cards can be used to monitor HIV status in mothers who have just given birth. The use of district postcode data in sample identification will allow accurate targetting of prevention strategies and early detection of spread of infection by geographic area. east coast.
Introduction In the UK and USA the prevalence of HIV infection in the time of delivery has been assessed by
women at
antibody testing of blood samples that have routinely from newborn babies.1-3 The particle agglutination method of testing for HIV-1antibody on eluted dried blood spot samples has been successfully used in a large study.3 In the UK a series of large multicentre surveys of unlinked anonymous testing that include newborn babies have begun. HIV antibody in serum from these babies reflects maternal antibody status at delivery and does not record infant infection. We report the first year’s results of a definitive continuing seroprevalence survey of childbearing women in Scotland, using district postcode information to defme geographic location and spread of anonymous
been obtained
HIV-1 infection.
Subjects and methods Collection of samples and data-Blood samples, taken routinely by heel prick from babies around the 7th day of life, were spotted onto filter paper (Guthrie cards) and air dried. The nurse who took the sample entered information on the card, which included the baby’s date of birth and the mother’s address. All cards for Scotland were then sent to the Scottish Inborn Errors Screening Laboratory at Stobhill Hospital, Glasgow. Ethical approval-We approached twenty ethics committees in the fifteen health board areas in Scotland. Our study included cards ADDRESSES: Department of Child Health, Royal Hospital for Sick Children, Glasgow, UK (Prof F. Cockburn, FRCP, D. M. Tappin, MRCP); Scottish Inborn Errors Screening Laboratory, Stobhill General Hospital, Glasgow (R. W. A. Girdwood, FRCPath, R Kennedy, FIMLS, A J Brown, BA Biol); Regional Virus Laboratory, Ruchill Hospital, Glasgow (E A. C. Follett, MRCPath). Correspondence to Dr D. M Tappin, Department of Child Health, Royal Hospital for Sick
Children, Yorkhill, Glasgow G3 8SJ, UK
1566
PLACE OF BIRTH OF HIV-ANTIBODY-POSITIVE BABIES DURING 1990
Glasgow=all mothers living in Glasgow postcode districts G1-84, Edinburgh = Edinburgh city, EH1-17, Dundee=DD1-5, Aberdeen = AB1, 2
from 99-6% of babies born in Scotland during 1990. Births from Orkney island (0-4%) were not included because the island’s ethics committee felt that women whose babies tested positive should be told, which was not our study design. Posters informing patients that residual blood samples may be anonymously tested for HIV antibody had been widely distributed in Scotland.
Postcoding of cards and recording of data-Since our main maintenance of the metabolic screening programme, were only tat;en for HIV-1antibody testing after all tests samples and re-tests for metabolic disorders had been done. During this process all duplicate cards were so marked. Cards were kept at room temperature without desiccation. With the use of postcode directories, the first three digits of the mother’s district postcode were recorded on the card. Duplicate cards were not included. The calendar quarter of birth and the district postcode were then recorded on computer, apart from records for Orkney and for babies whose mothers spontaneously opted out. The computer then produced a random number for each batch. A 3 mm diameter disc was punched out of one of the blood spots on the Guthrie card into a 96-well microtitre (Sterilin, Feltham, UK) plate which was marked with the random number. Computer information was stored at the Royal Hospital for Sick Children, Glasgow, and the microtitre plates were tested at Stobhill Hospital.
concern was
Elution of serum-Dried blood discs were eluted in flatbottomed 96-well microtitre master plates in 100 µl elution buffer (phosphate-buffered saline, pH 7-2, containing 005% "Tween 20" and 0-005 % sodium azide). The plates were shaken at 200 cycles per min for 30 min at room temperature and left overnight at 4°C, following which they were shaken for 3 min at 500 cycles per min. Controls were dried blood spots-high positive, low positive, and negative-from the Centers for Disease Control (CDC), Atlanta; a serum sample supplied with the Fujirebio HIV-antibody assay which was used at dilutions of 1 in 4 and 1 in 32; and in-house positive-control blood spots from the regional virus laboratory, Ruchill Hospital. HIV-antibody assay-A modified gelatin particle assay (GPA, Fujirebio) was used.3,4 30 µl of a 1 in 5 dilution was made of every eluate in v-welled microtitre plates with a multichannel pipette. 25 µl ofa1 in 5 dilution of antigen-coated gelatin particles was added to every well. The plates were left at room temperature for 15 min, centrifuged at 260g for 2 min, and placed at an angle of 70° on a light box. The reactions were read after 10 min: the particles mixed with the eluate containing HIV antibody remained as a discrete tight button, whereas those not containing HIV antibody became an elongated "tear drop". Reactive eluates were titrated across the endpoint and aspirated from the master plate for storage at - 20°C.
Confirmatory tests-At the regional virus laboratory enzymelinked immunosorbent assay (ELISA) and western blot tests were used to confirm the presence of HIV-1and HIV-2 antibody in all samples reactive in the screening test. We used an IgG antibody capture ELISA for HIV-1 and HIV-2 antibody (GACELISA), developed at the virus reference laboratory, Central Public Health Laboratory, London.5,6 10 ul samples of the repeatedly reactive eluates were diluted in 90 µl of diluent and incubated for 30 min at 37°C in microtitre wells coated with rabbit antibody to human IgG. After incubation and washing, an enzyme-conjugated recombinant
antigen was added and incubation continued for 60 min. Further washing was followed by incubation with substrate (20 min at 37°C) and amplifier (10 min at 37°C). The reaction was stopped by addition of 50 µl of 2 mol/1 sulphuric acid, and absorbance at 492 nm was measured. The cut-off was set at three times the negative mean. Complete western blots for HIV-1 were done in all repeatedly reactive eluates that were also reactive in the GACELISA system or within 10% of the cutoff. 25 ul of eluate was diluted in 50 µl of milk buffer and run on a preblotted membrane (Organon Teknika, Cambridge) in a ’Miniblotter’ aparatus’ (Biometra, Manchester). The blots were developed with conjugates supplied by Dupont in their HIV-1western blot kit. The conditions for positivity were the same as those used by Peckam et al3-ie, samples were judged positive if bands were seen representing reactivity against one or more of the GAG or POL genes and one of the ENV genes.
Quality
control-From
May, 1990, controls
were
supplied by
CDC every month. Each batch consisted of 12 unknown dried blood spot samples. We found no false-positive or false-negative results with our system in 5 of these batches. This is part of the worldwide HIV dried blood spot quality control and provides a check on the sensitivity of the Fujirebio kit screening test. Effects of storage on stability of dried blood spot sample-A batch of CDC quality control dried blood spots was stored at room temperature without desiccant and at 4°C and - 20°C with dessicant. At 1,2,3,4, and 5 weeks after receipt, the batch of spots was repunched, eluted, and retested, and no change in antibody titre was seen.
Collaboration
Study-The
with
Edinburgh
Edinburgh
Perinatal
Transmission
collaborators
provided us with HIV-positive births during 1990 in
information for all known Edinburgh, and with risk category data for these births.
Results excluded since their origin was either outside Scotland (64), and 9 cards were not Orkney (259) tested because of parental refusal. 65 781 dried blood discs were punched from Guthrie cards, 5 of which contained insufficient blood and 3 did not elute from the blood spots. The remaining 65 773 (99-6%) were tested. A total of 31 eluates was reactive in the screening test. Of the 31 eluates, 19 had titres greater than 100 and 20 were positive in the GACELISA test, including the 19 with high titre. These 19 eluates showed antibody to env glycoproteins and to at least one of the two groups of structural proteins on western blot, and were therefore regarded as true positives. 1 sample that was positive in the GACELISA and another within 10% of the cutoff value could not be confirmed as positive by western blot. The prevalence of HIV antibody was thus 0-29 per 1000 childbearing women for Scotland as a whole. All affected mothers lived in metropolitan areas on the east coast of Scotland. 18 of the 19 mothers lived in three large cities, Edinburgh, Dundee, and Aberdeen. Period prevalence for mothers of infants who survived the first week of life, from these cities, was 2-5 per 1000,11.4 per 1000, and 0-7 per 1000, respectively. We emphasise that no antibody-positive babies were born from 16 308 deliveries in greater Glasgow (west Scotland) (table). We identified 13 HIV-antibody-positive babies in Edinburgh, and 13 such babies born in 1990 were known to the Edinburgh Perinatal Transmission Study (N. Croft, personal communication); Guthrie cards had been received at Stobhill hospital for all 13 babies. 7 mothers were intravenous drug users (IVDUs), 5 were partners of IVDUs, and 1 had no high risk associations. In Dundee a study of antenatal HIV antibody testing was ended in July, 1990,8 and 2 HIV-antibody-positive babies were expected. Both mothers were at low risk for HIV infection. In 323 cards
were or
1567
The prevalence of HIV-1antibody was more than eight times the average for Scotland in Edinburgh, nearly five times the average in Dundee, and more than twice the average in Aberdeen. The use of district postcodes allowed accurate localisation of high prevalence areas for targeting of prevention strategy. All but 1 of the HIV-positive mothers resided in these three large cities, and the other lived in a substantial town, all on the east coast of Scotland. Accurate geographic location of present prevalence has several advantages. Spread of infection to a new area can be identified at an early stage, and this has happened in Aberdeen during our study. Two further HIV-antibodypositive births were found in Aberdeen during the first quarter of 1991. The progress of the epidemic in specific areas can be monitored yearly, and comparison between population groups would be much easier if geographic location is well defined, but each study, we emphasise, should use the same system-ie, postcode district. We feel that the gradient of prevalence referred to in earlier reports 1,3,7 merely reflects poor discrimination of small high
termination of pregnancy is done at about 12 weeks’ gestation. Therefore, births from January, 1990, to January, 1991, are comparable with terminations from July, 1989, to July, 1990.) If we extrapolated our results to all heterosexual women we would have a large error from terminations, without consideration of other factors. Future unlinked anonymous testing to cover therapeutic abortion, when coupled with information from continued Guthrie-testing will provide a more complete picture. Since our data relating to Edinburgh and Dundee showed only 1 case of HIV that had not been identified by the named testing programmes we believe that antenatal patients opting out of named testing and proceeding to term do not form a high-risk group. Furthermore, information from the Edinburgh paediatric services shows that almost all the HIV-antibody-positive babies were born to high-risk mothers. 80% of HIV-positive pregnancies in Dundee were in high-risk women. This information is encouraging and shows that little spread of HIV infection has occurred to the presumed low-risk general population in Edinburgh or Dundee and that none has happened in Glasgow. This emphasises the importance of prevention of spread of infection in drug abusers. In Glasgow, HIV prevalence in anonymous and voluntary testing of drug addicts remains below 1-5% (J. Emslie, S. Haw, personal communication) whereas in Edinburgh and Dundee it is 38%"* and 40%,11
prevalence areas.
respectively.
Aberdeen the 2 HIV-antibody-positive babies we identified had not been detected by virological, obstetric, or paediatric services (Regional Virus Laboratory, unpublished data).
Discussion
We estimated that we tested 99-6% of Scottish births. This figure has been confirmed by provisional figures from the Registrar General, Scotland (Annual report, 1990). An important advantage of anonymous testing over named testing is that patients who refuse testing are much less likely to bias the data. Even if all 9 refusers in our study were HIV positive, prevalence rates would be little altered and only two vertical transmissions would be expected.9 Refusals were sporadic throughout the year-one was in Edinburgh and the others in low prevalence areas. Our results have shown that concern that babies born to mothers at high risk of HIV infection would be missed by the Guthrie card system is ill-founded. All babies known to be HIV-positive were identified by such testing. We are confident that we can monitor fully HIV status in childbearing women in
Scotland. The modified Fujirebio kit we used was highly sensitive and
satisfies the CDC quality control criteria for monitoring. Specificity was also high, with only 12 of 65 773 samples showing reactivity that could not be confirmed by western blot. The cost of the complete system including all reagents, staff, and equipment was only 0-70 per sample. At present this test only screens for HIV-1 antibody. A combined test incorporating HIV-2 antibody is being investigated in Japan. This should be available for use at the end of 1991. So far no HIV-2 antibody-positive individuals
have been identified in Scotland. All Guthrie cards for Scotland
are
received in
one
laboratory. However, we could not include women who had spontaneous or therapeutic abortions, stillbirths, or perinatal deaths. When our results were compared with those of the antenatal
study in Dundee,8 which included therapeutic abortions, we identified 3 HIV-antibodypositive births-2 were expected from HIV-antibodypositive pregnancies in the Dundee study. However, our investigation could take no account of the number of HIV-positive women who decided not to continue their pregnancy. 8 HIV-positive women had terminations in Dundee during a comparable period. (Therapeutic
We thank the many midwives, health visitors, and others involved in sample collection, and obstetricians, paediatricians, general practitioners, and administrative and clerical staff for their help; Miss C. Benison for her help in postcoding and entering all the data at Stobhill Hospital; Mr J. Taylor who designed and modified the computer programme; Mrs S. Martin and Miss J. Coote for the confirmation tests; Dr N. Croft, Dr J. Y. Q. Mok, Dr F. D. Johnstone, and Dr R. Smith for important data; Dr D. Reid, Dr J. Emslie, and Dr D. Goldberg and the Communicable Diseases (Scotland) Unit for their help in liaison with the Department of Health, the Scottish Home and Health Department, and the Medical Research Council; and the Ethics Committees or their help and constructive comments. Dr J. V. Parry and Dr P. P. Mortimer kindly provided the IgG antibody capture ELISA. This study was supported by the Medical Research Council
(grant no 8908795C). REFERENCES 1. Hoff
R, Berardi VP, Weiblen BJ, Mahoney-Trout L, Mitchell ML, Grady GF. Seroprevalence of human immunodeficiency virus among childbearing women. N Engl J Med 1988; 318: 525-30. 2. Centres for Disease Control. CDC surveillance supplements. MMWR 1989; 38: (no S-4). 3. Peckham CS, Tedder RS, Briggs M, et al. Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies. Lancet 1990; 335: 516-19. JAJ, Salker R, Challis P, Contreras M. Gelatin particle agglutination assay for HIV antibodies: a rapid economical modification with increased sensitivity. Med Lab Sci 1989; 46: 135-40. 5. Parry JV, Perry KR, Mortimer PP. Sensitive assays for viral antibodies in saliva: an alternative to tests on serum. Lancet 1987; ii: 72-75. 6. Connell JA, Parry JV, Mortimer PP, et al. Accurate assays for anti-HIV in urine. Lancet 1990; 335: 1366-69. 7. Norvick LF, Berns D, Stricof R, Stevens R, Pass K, Wethers J. HIV seroprevalence in New York State. JAMA 1989; 261: 1745-50. 8. Scrimgeour JB, Patel NB, Reid D, Smith R. HIV antibody seroprevalence in an antenatal population and its relationship to self reported behavioural risk. (Proceedings of the VI International Conference on AIDS, 1990, San Fransisco.) Abstract 3145. 9. Ades AE, Newell ML, Peckham CS. European Collaborative study. Children born to women with HIV-1 infection: natural history and risk transmission. Lancet 1991; 337: 253-60. 10. Peutherer JF, Edmonds E, Simmonds P, Dickson JD, Bath GE. HTLV-III antibody in Edinburgh drug addicts. Lancet 1985; ii: 4. Barbara
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