516
REFERENCES
Epidemiology of schizophrenia. In: Hafner H, Gattaz WF, Janzarik W, eds. In search for the causes of schizophrenia. Berlin: Springer-Verlag, 1987: 47-74. 2. Sartorius N, Jablensky A, Korten A, et al. Early manifestations and first contact incidence of schizophrenia in different cultures. Psychol Med 1986; 16: 909-28. 3. Odegaard O. Hospitalised psychoses in Norway: time trends, 1926-1965. Soc Psychiatry 1971; 6: 53-58. 4. Torrey EF. Schizophrenia: fixed incidence or fixed thinking? Psychol Med 1989; 19: 285-87. 5. Hare EH. Was insanity on the increase? Br J Psychiatry 1983; 142:
12.
Joyce PR. Changing trends in first admissions and readmissions for mania and schizophrenia in New Zealand. Aust NZ J Psychiatry 1987; 21:
13.
Odegaard O. The incidence of mental diseases as measured by census investigations versus admission statistics. Psychiatr Q 1952; 26:
1. Hafner H.
439-55. 6.
Eagles JM, Whalley LJ. Decline in the diagnosis of schizophrenia among first admissions to Scottish mental hospitals from 1969-1978. Br J Psychiatry 1985; 146: 151-54.
7. Dickson
WE, Kendell RE. Does maintenance lithium therapy prevent of mania under ordinary clinical conditions? Psychol Med
recurrences
1986; 16: 521-30. Eagles JM, Hunter D, McCance C. Decline in the diagnosis of schizophrenia among first contacts with psychiatric services in North-East Scotland, 1969-1984. Br J Psychiatry 1988; 152: 793-98. 9. Munk-Jorgensen P. Decreasing first admission rates of schizophrenia among males in Denmark from 1970 to 1984: changing diagnostic patterns? Acta Psychiatr Scand 1986; 73: 645-50. 10. Munk-Jorgensen P, Jorgensen P. Decreasing rates of first admission diagnoses of schizophrenia among females in Denmark 1970-1984. Acta Psychiatr Scand 1986; 74: 379-83. 11. Parker G, O’Donnell M, Walter S. Changes in the diagnosis of the functional psychoses associated with the introduction of lithium. Br J Psychiatry 1985; 146: 377-82. 8.
82-86.
212-18. 14. Hare EH. Aspects of the epidemiology of schizophrenia. Br J Psychiatry 1986; 149: 554-61. 15. Registrar General. Supplements on mental health. London: HM Stationery Office, 1952-60. 16. Ministry of Health. Hospitals and units in England and Wales: inpatient statistics from the Mental Health Enquiry. London: HM Stationery Office, 1964-69: statistical report series no 4, 5, 11, and 12. 17. Department of Health and Social Security. Psychiatric hospitals and units in England and Wales: inpatient statistics from the Mental Health Enquiry for the year 1970. London: HM Stationery Office, 1972: statistical and research report series no 4. 18. Symonds RL, Williams P. Lithium and the changing incidence of mania. Psychol Med 1981; 11: 193-96. 19. Cooper JE, Goodhead D, Craig T, Hams M, Howat J, Korer J. The incidence of schizphrenia in Nottingham. Br J Psychiatry 1987; 151: 619-26. 20. Hare EH. The changing content of psychiatric illness. J Psychosom Res 1974; 18: 283-89. 21. Bleuler M. The schizophrenic disorders: long-term patient and family studies (translated by S. M. Clemens). New Haven: Yale University Press, 1978. 22. Saugstad LF. Social class, marriage and fertility in schizophrenia. Schizophrenia Bull 1989; 15: 9-43. 23. Mahendra B. Where have all the catatonics gone? Psychol Med 1981; 11: 669-71.
Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies
This pilot study established that unlinked anonymous testing of dried blood spots routinely collected on Guthrie cards for neonatal screening is a feasible method for monitoring HIV prevalence in women at the time of delivery. The method was sensitive, specific, and less expensive than more conventional ELISAs. 114 515 dried blood spots taken from cards collected in three Thames regions were tested for antibody to HIV-1. 28 samples were confirmed to be antibody positive by western blot (seroprevalence 0·24 per 1000). Unlinked anonymous screening of newborn babies should be extended to monitor the spread of HIV infection in the heterosexual population and to target preventive strategies and provision of health
care.
Introduction In the US the prevalence of HIV infection in women at the time of delivery has been assessed anonymously by antiHIV testing of blood samples that have already been collected routinely from newborn babies.1,2 In the UK a
series of large multicentre surveys of unlinked anonymous that will include newborn babies will start soon.3 Anti-HIV in the serum of the newborn infant reflects transplacental antibody and is an indirect measure of maternal infection. It does not necessarily indicate fetal or neonatal infection.4,5 We report a preliminary study to establish the laboratory and logistic methods for anonymous testing of anti-HIV-1 in blood routinely collected from newborn babies.
testing
Subjects and
methods
Collection of samples. Blood samples, taken routinely by heel prick from newborn babies, were spotted onto filter paper (Guthrie
ADDRESSES: Department of Paediatric Epidemiology, Institute of Child Health, London WC1 1 EH (Prof C. S. Peckham, FFCM, A E. Ades, PhD, C. O’Connor); Section of Virology, Department of Medical Microbiology, School of Pathology, University College and Middlesex School of Medicine, London W1 (R. S Tedder, MRCPath, M. Briggs, BSc, N. Parra-Mejia, MSc); Department of Clinical Biochemistry, Hospitals for Sick Children, London (Prof M. Hjelm, MRCPath); and Department of Chemical Pathology, St Helier Hospital, Carshalton, Surrey, UK (A H Wilcox, MRCPath). Correspondence to Prof C. S. Peckham.
517
cards) and air-dried. The study included cards received in the North East and North West Thames regions since June 26, 1988, and those from the South West Thomas region since February, 1989. The sampling period ended on July 9, 1989. Approval was obtained from the ethical committees reponsible for the neonatal screening programmes. As maintenance of the screening programme was of primary concern, samples were only taken for anti-HIV-1testing after all tests and re-tests for metabolic disorders had been done. The time between heel prick and anti-HIV test was usually between 2 and 4 weeks (range 10 days-12 weeks). Cards were kept at room temperature without desiccation. A 2-5 mm diameter blood spot was punched out of the Guthrie card into a petrie dish marked with the name of the district health authority (DHA) where the mother was resident or, if the sample was taken in hospital, the DHA of the hospital. When there was no blood on the card a disc was still punched out. The spots were put into envelopes with the name of the DHA and the date, to the nearest week, when samples arrived in the neonatal screening laboratory. This was the only information retained with the blood spot. When the spots reached the testing laboratory they were kept at 4°C without desiccation until tested 4-5 days later. Elution of serum. Dried blood spots were eluted in a flatbottomed 96-well microtitre master plate in 100 µ1 elution buffer (phosphate-buffered saline, pH 7-2, containing 0-05% ’Tween 20’ and 0-05% sodium azide). The plates were then shaken slowly for 30 min at room temperature and left overnight at 4°C. Next morning they were put on a fast shaker for 3 min. Only 11 of the 12 rows in the plate were used for the blood samples; the remaining row was used for a titration across the endpoint of the positive control blood spot elution. Positive controls. A dilution of anti-HIV-1 positive serum in freshly drawn anti-HIV-1 negative whole blood was spotted onto Guthrie cards and allowed to dry at room temperature for 48 h. Control serum was also taken from this mixture after the clot had formed. A control dried blood spot was eluted in the first well of a row in each master plate and diluted in two-fold steps in 100 ltl volumes across the plate before transfer to the test plate. Anti-HIV-1assay. A modified gelatin particle assay (GPA, Fujirebio) was used.6 Briefly 30 pl of a 1 in 5 dilution was made of every eluate in V-welled microtitre plates with a manual loop diluting machine. The haemoglobin carried over from the eluates was checked by eye. Each plate contained dilutions of eluates from 88 dried blood spots and a series of 8 dilutions of the control eluate. 25 1 of a 1 in 10 dilution of antigen-coated gelatin particles was added to every well. The plates were left at room temperture for 15 min, centrifuged at 260 g for 2 min and placed on a light-box at an angle of 70°. The reactions were read after 10 min when the particles mixed with eluate containing anti-HIV remain as a discrete tight button. In comparison, particles in wells not containing anti-HIV streak out in an elongated "tear drop". Reactive eluates were re-tested. If again reactive they were titrated across the endpoint and aspirated from the master plate for storage at -20°C.
Fig 1-Effect of temperature and desiccation on anti-HIV-11 reactivity in dried blood spot samples taken from Guthrie cards.
Confirmatory western blot. 25 III of the repeatedly reactive eluates was diluted 1 in 3 in milk buffer and reacted with HIV-1 on a pre-blotted membrane in a ’Miniblotter’ (Immunetics). A western blot was considered positive if there was reactivity against proteins representing one or more of the gag or pol genes and one of the errv
genes.
Effects of storage. A batch of control dried blood spots was stored at room temperature or 4°C, with or without dessicant. At 0, 2, 4, 8, 14, and 19 weeks, four spots stored at each of the different conditions were eluted and the eluate frozen at - 20°C. Titrations of replicate eluates from each group were made in one test run at the end of the storage period.
Results Elution
efficiency
In studies (not reported here) of a series of dried blood spots and serum samples from 14 anti-HIV-1 seropositive individuals, the difference in titre between serum and dried blood spot anti-HIV-1 was found to be about 50-fold. During our study the mean difference between the control serum and control blood spots ranged from 30-fold to 133-fold (geometric mean reduction 51-2-fold).
Stability Dried blood spots lost least activity at room temperature when stored undesiccated (fig 1). By 10 weeks, the longest time between heel prick and anti-HIV-1testing, the loss of titre was 2-4-fold. Desiccation increased the rate of loss of activity. Storage at 4°C, desiccated or undesiccated, prevented significant loss of titre. Even after 19 weeks the reduction was only 2-fold.
Anti-HIV-1prevalence 115 876 dried blood spots were punched from Guthrie cards. 408 were excluded since their origin was either outside the three regions in the study or was unknown, none were positive for anti-HIV-1. Of the 115 468 blood spots eligible for analysis, 953 contained insufficient blood (less than 50% coverage of the punched disc); the remaining 114 515 (99-2%) were tested. The eluates from 30 dried blood spots were repeatedly reactive in the screening test. 26 of these titrated to endpoints greater than 1 in 100 (fig 2). 28 of the 30 eluates showed antibody to env glycoproteins and to at least one of
Fig 2-Distribution of anti-HIV-1 titre in 30 repeatedly reactive samples. All 26 sera with titres over 100 were of those with titres below 100.
screen-
positive on western blot, as were 2
518
SEROPREVALENCE BY LOCATION OF HEALTH DISTRICT
the two groups of other structural proteins on western blot. These included 2 of the 4 eluates with GPA titres below 100. The remaining 2 with low GPA titres showed no antibody when western blotted and were considered not to be anti-HIV-1 positive. The prevalence of anti-HIV was 0-24 per 1000 (28/114 515). The seroprevalence was highest in inner London and lowest outside the metropolitan area (table). There was no evidence that seroprevalence had changed during the study, either overall or within any region.
Discussion
prevalence per 1000 of anti-HIV-1 was 0,24, considerably lower than the 1-3 reported in a study of 92 Italian hospitalsthe 2-1in Massachusetts,1 and the 6-6 in New York stated The gradient we found, of increasing prevalence from home counties through outer city to inner city, has also been observed in North American studies. Anti-HIV in the newborn infant does not imply that the infant is infected. About 1 in 4 seropositive newborn babies
The overall
will be infected.4°5 On this basis the neonatal HIV-1 infection rate in London over the study period would be 6 per 100 000 livebirths, an incidence comparable with that of
phenylketonuria. We have demonstrated the feasibility of screening all newborn infants for anti-HIV-1. The modification of a commercially available agglutination assay had a similar sensitivity to the ELISAs used by the Centres for Disease Control in their national HIV screen of dried blood spots (unpublished data). All but 2 of our 30 repeatedly screentest reactive sera were confirmed by western blot. Repeat reactivity is therefore highly predictive and only a low proportion of repeatedly reactive sera will fail to be identified by western blot as being anti-HIV-1 positive. More significantly a high specificity (at least 99-996%) is implied by the lowest seroprevalence, which was observed outside London. The reagent costs, including western
blotting were 0 12 per sample. Surveillance of anti-HIV-1prevalence in newborn babies or pregnant women can effectively monitor the prevalence of HIV infection in the heterosexual population.9 The advantage of neonatal testing is that Guthrie cards are collected in central laboratories on an almost universal basis. They are likely to be representative of women delivering babies and not biased by self selection. In a review of congenital hyperthyroid screening in 1983, only one out of 493 consecutive cases was missed.lO However, babies born to mothers known to be HIV-infected might be more likely to be missed. Instead of using Guthrie cards, we could have tested samples taken for routine antenatal rubella testing. This would have entailed much more work since specimens are kept at different sites and stored as serum. In addition, some women at higher risk of HIV, such as intravenous drug users, are more likely to be excluded from routine rubella screening because they may present for the first time either late in pregnancy or already in labour. However, antenatal
testing would identify those HIV-infected women whose pregnancies are terminated or who abort spontaneously. Seroprevalence estimates from anonymous testing are likely to be more reliable than those from voluntary testing. In New York only 40% of those who were offered antenatal testing agreed, and those found to be positive only disclosed additional risk factors afterwards." In another study 1000 women were anonymously screened, but only half of the 50 who admitted risk activity agreed to be tested; voluntary screening detected only 14% of those who were infected.12 Similar findings have been reported in studies of patients attending sexually transmitted disease clinics.13-15 A major concern is that anonymous testing precludes any of infected women. However, anonymous studies have revealed unexpectedly high local prevalence of HIV infection, which increases awareness among health professionals and underlines the need for voluntary testing and additional support for those found to be infected. We emphasise that unlinked anonymous testing does not replace voluntary testing with consent in antenatal clinics. 16 Neonatal HIV testing has been widely accepted in the USA where it is being extended to more than 40 statesand the Canadian Royal Society of Medicine has also recommended anonymous sampling. In the UK the Department of Health supports unlinked anonymous testing for HIV and sees no ethical or legal objections: "The legal advice given to the Department of Health is that provided the patient’s blood is used for the original test for which consent was obtained it is unnecessary to tell patients (either individually or through a public notice in a clinic) that any blood residue might be tested unlinked and anonymously".9 However, it is felt that individuals have the right to opt out of anonymous studies.3 It has also been suggested that informed consent should be sought from the health care workers involved. Either of these procedures are likely to lead to patient selection bias. For an infection with a prevalence of less than 1 in 1000, even a 99-9% compliance could lead to serious underestimates of seroprevalence. The policy of allowing patients to opt out of unlinked anonymous testing is to be regretted and it will be essential to record the proportion that do so. Further studies are being done to compare the seroprevalence obtained by neonatal screening with the results of other HIV reporting schemes, including anonymous antenatal testing and reports of HIV infection in pregnant women and children. care
This study was supported by the Medical Research Council. We thank the staff of the neonatal screening laboratories for their assistance.
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. Centers for Disease Control. CDC surveillance supplements. MMWR 1989; 38: (no S-4). 3. Heptonstall J, Gill ON. The legal and ethical basis for unlinked anonymous HIV testing. Comm Dis Rep 1989; 48: 3-6. 4. European Collaborative Study. Mother-to-child transmission of HIV infection. Lancet 1988; ii: 1039-43. 5. Blanche S, Rouzioux C, Guihard Moscato M-L, et al. A prospective study of infants bom to women seropositive for human immunodeficiency virus type 1. N Engl J Med 1989; 320: 1643-48. 6. Barbara 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. 7. Ippolito G, Stegagno M, Costa F, Angeloni P, Angeloni U, Guzanti E. Detection of anti-HIV antibodies in newborns: a blind serosurvey in 92
519
l’enfant.
parturient women in a high-risk population. N Engl J Med 1988; 318:
Paris, Nov 27-30, 1989: 141 (abstr). 8. Novick LF, Bems D, Stricof R, Stevens R, Pass K, Wethers J. HIV seroprevalence in newborns in New York State. JAMA 1989; 261:
185. 13. Hull HF, Bettinger CJ, Gallaher MM, Keller NM, Wilson J, Mertz GJ. Comparison of HIV-antibody prevalence in patients consenting to and declining HIV-antibody testing in an STD clinic. JAMA 1988; 260:
Italian hospitals. Les
implications
du SIDA pour la
mere et
1745-50. 9. Gill ON, Adler MW, Day NE. Br Med J 1989; 299: 1295-98.
Monitoring the prevalence of HIV.
10. Grant DB, Smith I. Survey of neonatal screening for primary hypothyroidism in England, Wales and Northern Ireland 1982-4. Br Med J 1988; 296: 1355-58. 11. Minkoff HL, Holman S, Beller E, Delke I, Fishbone A, Landesman S. SUNY Health Science Center routinely offered prenatal HIV testing.
N Engl J Med 1988; 319: 1018. 12. Krasinski K, Borrowsky W, Bebenroth D, Moore T. Failure of voluntary testing for human immunodeficiency virus to identify infected
935-38. 14. Landesman S, Minkoff H, Helman S, McCalla S, Syn O. Serosurvey of human immunodeficiency virus infection in parturients. JAMA 1987; 258: 2701-03. 15. Evans BA, McCormack SM, Bond RA, MacRae KD, Thorp RW. Human immunodeficiency virus infection, hepatitis B virus infection and sexual behaviour of women attending a genitourinary clinic. Br Med J 1988; 296: 473-75. 16. Royal College of Obstetricians and Gynaecologists. Report of the RCOG sub-committee on problems associated with AIDS in relation to obstetrics and gynaecology. London: RCOG, 1987.
CLINICAL PRACTICE Extent, distribution, and mammographic/
histological
To
correlations of breast ductal carcinoma in situ
potential of breast-conserving (DCIS), 82 mastectomy specimens were studied by Egan’s serial subgross method. 42 (51%) of the tumours were larger than 50 mm and only 12 (15%) were assess
the
treatment for ductal carcinoma in situ
smaller than 20 mm; the size distribution was not affected by the mode of detection (mammography 52 cases, clinical examination 30). All but 1 case showed only 1 region of tumour. 66% of tumours involved one breast quadrant, 23% extended over more than one quadrant, and 11% were centrally located. Mammographic estimates, based on the extent of microcalcifications, frequently underestimated the histological size of tumours, the extent of the discrepancy being related to the histological type—8/50 predominantly comedo DCIS showed a discrepancy greater than 20 mm with 15/32 compared predominantly micropapillary/cribriform. In view of the frequently large size, adequate excision of many DCIS will require a wide excision involving up to a whole quadrant.
Introduction Until now the standard treatment for ductal carcinoma in situ of the breast (DCIS, intraductal carcinoma) has been ablative surgery, with a cure rate of nearly 100%. However,
the results of breast-conserving treatment rather than mastectomy for the management of early invasive breast cancer are promising. This development has created controversy, in that mastectomy is still recommended for patients with non-invasive intraductal cancers (ie, at an earlier stage than any invasive cancer), whereas many patients with invasive cancers are offered breast-conserving treatment. The increasing use of mammography in routine diagnosis of breast disorders and the introduction of mammographic mass screening programmes1 have expanded the scale of the problem by raising the rate of detection of DCIS three or four fold to as much as 15-20% of all mammographically detected cancers .1,3 The main reservation about conservative treatment of DCIS is that its distribution in the breast is assumed to be multicentric. In patients with DCIS, treated by lumpectomy and subsequent mastectomy, residual tumour was found in 30-60% of mastectomy specimens 4 The effect of radiotherapy on this residual DCIS is not yet clear.6,7 Therefore, the surgeon should ideally make every attempt to resect all disease, with histologically adequate margins, ADDRESSES: Departments of Pathology Schuurmans Stekhoven, PhD), Radiology (J and Epidemiology (A. L. M. Verbeek, MD), Hospital, and Department of Pathology,
(R. Holland, MD, J. H. H. C. L Hendriks, MD), Radboud University Canisius Wilhelmina Hospital (M. Mravunac, MD), Nijmegen, the Netherlands. Correspondence to Dr R. Holland, Department of Pathology, Radboud University Hospital, 6500 HB Nijmegen, the Netherlands.
REFERENCES
Epidemiology of schizophrenia. In: Hafner H, Gattaz WF, Janzarik W, eds. In search for the causes of schizophrenia. Berlin: Springer-Verlag, 1987: 47-74. 2. Sartorius N, Jablensky A, Korten A, et al. Early manifestations and first contact incidence of schizophrenia in different cultures. Psychol Med 1986; 16: 909-28. 3. Odegaard O. Hospitalised psychoses in Norway: time trends, 1926-1965. Soc Psychiatry 1971; 6: 53-58. 4. Torrey EF. Schizophrenia: fixed incidence or fixed thinking? Psychol Med 1989; 19: 285-87. 5. Hare EH. Was insanity on the increase? Br J Psychiatry 1983; 142:
12.
Joyce PR. Changing trends in first admissions and readmissions for mania and schizophrenia in New Zealand. Aust NZ J Psychiatry 1987; 21:
13.
Odegaard O. The incidence of mental diseases as measured by census investigations versus admission statistics. Psychiatr Q 1952; 26:
1. Hafner H.
439-55. 6.
Eagles JM, Whalley LJ. Decline in the diagnosis of schizophrenia among first admissions to Scottish mental hospitals from 1969-1978. Br J Psychiatry 1985; 146: 151-54.
7. Dickson
WE, Kendell RE. Does maintenance lithium therapy prevent of mania under ordinary clinical conditions? Psychol Med
recurrences
1986; 16: 521-30. Eagles JM, Hunter D, McCance C. Decline in the diagnosis of schizophrenia among first contacts with psychiatric services in North-East Scotland, 1969-1984. Br J Psychiatry 1988; 152: 793-98. 9. Munk-Jorgensen P. Decreasing first admission rates of schizophrenia among males in Denmark from 1970 to 1984: changing diagnostic patterns? Acta Psychiatr Scand 1986; 73: 645-50. 10. Munk-Jorgensen P, Jorgensen P. Decreasing rates of first admission diagnoses of schizophrenia among females in Denmark 1970-1984. Acta Psychiatr Scand 1986; 74: 379-83. 11. Parker G, O’Donnell M, Walter S. Changes in the diagnosis of the functional psychoses associated with the introduction of lithium. Br J Psychiatry 1985; 146: 377-82. 8.
82-86.
212-18. 14. Hare EH. Aspects of the epidemiology of schizophrenia. Br J Psychiatry 1986; 149: 554-61. 15. Registrar General. Supplements on mental health. London: HM Stationery Office, 1952-60. 16. Ministry of Health. Hospitals and units in England and Wales: inpatient statistics from the Mental Health Enquiry. London: HM Stationery Office, 1964-69: statistical report series no 4, 5, 11, and 12. 17. Department of Health and Social Security. Psychiatric hospitals and units in England and Wales: inpatient statistics from the Mental Health Enquiry for the year 1970. London: HM Stationery Office, 1972: statistical and research report series no 4. 18. Symonds RL, Williams P. Lithium and the changing incidence of mania. Psychol Med 1981; 11: 193-96. 19. Cooper JE, Goodhead D, Craig T, Hams M, Howat J, Korer J. The incidence of schizphrenia in Nottingham. Br J Psychiatry 1987; 151: 619-26. 20. Hare EH. The changing content of psychiatric illness. J Psychosom Res 1974; 18: 283-89. 21. Bleuler M. The schizophrenic disorders: long-term patient and family studies (translated by S. M. Clemens). New Haven: Yale University Press, 1978. 22. Saugstad LF. Social class, marriage and fertility in schizophrenia. Schizophrenia Bull 1989; 15: 9-43. 23. Mahendra B. Where have all the catatonics gone? Psychol Med 1981; 11: 669-71.
Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies
This pilot study established that unlinked anonymous testing of dried blood spots routinely collected on Guthrie cards for neonatal screening is a feasible method for monitoring HIV prevalence in women at the time of delivery. The method was sensitive, specific, and less expensive than more conventional ELISAs. 114 515 dried blood spots taken from cards collected in three Thames regions were tested for antibody to HIV-1. 28 samples were confirmed to be antibody positive by western blot (seroprevalence 0·24 per 1000). Unlinked anonymous screening of newborn babies should be extended to monitor the spread of HIV infection in the heterosexual population and to target preventive strategies and provision of health
care.
Introduction In the US the prevalence of HIV infection in women at the time of delivery has been assessed anonymously by antiHIV testing of blood samples that have already been collected routinely from newborn babies.1,2 In the UK a
series of large multicentre surveys of unlinked anonymous that will include newborn babies will start soon.3 Anti-HIV in the serum of the newborn infant reflects transplacental antibody and is an indirect measure of maternal infection. It does not necessarily indicate fetal or neonatal infection.4,5 We report a preliminary study to establish the laboratory and logistic methods for anonymous testing of anti-HIV-1 in blood routinely collected from newborn babies.
testing
Subjects and
methods
Collection of samples. Blood samples, taken routinely by heel prick from newborn babies, were spotted onto filter paper (Guthrie
ADDRESSES: Department of Paediatric Epidemiology, Institute of Child Health, London WC1 1 EH (Prof C. S. Peckham, FFCM, A E. Ades, PhD, C. O’Connor); Section of Virology, Department of Medical Microbiology, School of Pathology, University College and Middlesex School of Medicine, London W1 (R. S Tedder, MRCPath, M. Briggs, BSc, N. Parra-Mejia, MSc); Department of Clinical Biochemistry, Hospitals for Sick Children, London (Prof M. Hjelm, MRCPath); and Department of Chemical Pathology, St Helier Hospital, Carshalton, Surrey, UK (A H Wilcox, MRCPath). Correspondence to Prof C. S. Peckham.
517
cards) and air-dried. The study included cards received in the North East and North West Thames regions since June 26, 1988, and those from the South West Thomas region since February, 1989. The sampling period ended on July 9, 1989. Approval was obtained from the ethical committees reponsible for the neonatal screening programmes. As maintenance of the screening programme was of primary concern, samples were only taken for anti-HIV-1testing after all tests and re-tests for metabolic disorders had been done. The time between heel prick and anti-HIV test was usually between 2 and 4 weeks (range 10 days-12 weeks). Cards were kept at room temperature without desiccation. A 2-5 mm diameter blood spot was punched out of the Guthrie card into a petrie dish marked with the name of the district health authority (DHA) where the mother was resident or, if the sample was taken in hospital, the DHA of the hospital. When there was no blood on the card a disc was still punched out. The spots were put into envelopes with the name of the DHA and the date, to the nearest week, when samples arrived in the neonatal screening laboratory. This was the only information retained with the blood spot. When the spots reached the testing laboratory they were kept at 4°C without desiccation until tested 4-5 days later. Elution of serum. Dried blood spots were eluted in a flatbottomed 96-well microtitre master plate in 100 µ1 elution buffer (phosphate-buffered saline, pH 7-2, containing 0-05% ’Tween 20’ and 0-05% sodium azide). The plates were then shaken slowly for 30 min at room temperature and left overnight at 4°C. Next morning they were put on a fast shaker for 3 min. Only 11 of the 12 rows in the plate were used for the blood samples; the remaining row was used for a titration across the endpoint of the positive control blood spot elution. Positive controls. A dilution of anti-HIV-1 positive serum in freshly drawn anti-HIV-1 negative whole blood was spotted onto Guthrie cards and allowed to dry at room temperature for 48 h. Control serum was also taken from this mixture after the clot had formed. A control dried blood spot was eluted in the first well of a row in each master plate and diluted in two-fold steps in 100 ltl volumes across the plate before transfer to the test plate. Anti-HIV-1assay. A modified gelatin particle assay (GPA, Fujirebio) was used.6 Briefly 30 pl of a 1 in 5 dilution was made of every eluate in V-welled microtitre plates with a manual loop diluting machine. The haemoglobin carried over from the eluates was checked by eye. Each plate contained dilutions of eluates from 88 dried blood spots and a series of 8 dilutions of the control eluate. 25 1 of a 1 in 10 dilution of antigen-coated gelatin particles was added to every well. The plates were left at room temperture for 15 min, centrifuged at 260 g for 2 min and placed on a light-box at an angle of 70°. The reactions were read after 10 min when the particles mixed with eluate containing anti-HIV remain as a discrete tight button. In comparison, particles in wells not containing anti-HIV streak out in an elongated "tear drop". Reactive eluates were re-tested. If again reactive they were titrated across the endpoint and aspirated from the master plate for storage at -20°C.
Fig 1-Effect of temperature and desiccation on anti-HIV-11 reactivity in dried blood spot samples taken from Guthrie cards.
Confirmatory western blot. 25 III of the repeatedly reactive eluates was diluted 1 in 3 in milk buffer and reacted with HIV-1 on a pre-blotted membrane in a ’Miniblotter’ (Immunetics). A western blot was considered positive if there was reactivity against proteins representing one or more of the gag or pol genes and one of the errv
genes.
Effects of storage. A batch of control dried blood spots was stored at room temperature or 4°C, with or without dessicant. At 0, 2, 4, 8, 14, and 19 weeks, four spots stored at each of the different conditions were eluted and the eluate frozen at - 20°C. Titrations of replicate eluates from each group were made in one test run at the end of the storage period.
Results Elution
efficiency
In studies (not reported here) of a series of dried blood spots and serum samples from 14 anti-HIV-1 seropositive individuals, the difference in titre between serum and dried blood spot anti-HIV-1 was found to be about 50-fold. During our study the mean difference between the control serum and control blood spots ranged from 30-fold to 133-fold (geometric mean reduction 51-2-fold).
Stability Dried blood spots lost least activity at room temperature when stored undesiccated (fig 1). By 10 weeks, the longest time between heel prick and anti-HIV-1testing, the loss of titre was 2-4-fold. Desiccation increased the rate of loss of activity. Storage at 4°C, desiccated or undesiccated, prevented significant loss of titre. Even after 19 weeks the reduction was only 2-fold.
Anti-HIV-1prevalence 115 876 dried blood spots were punched from Guthrie cards. 408 were excluded since their origin was either outside the three regions in the study or was unknown, none were positive for anti-HIV-1. Of the 115 468 blood spots eligible for analysis, 953 contained insufficient blood (less than 50% coverage of the punched disc); the remaining 114 515 (99-2%) were tested. The eluates from 30 dried blood spots were repeatedly reactive in the screening test. 26 of these titrated to endpoints greater than 1 in 100 (fig 2). 28 of the 30 eluates showed antibody to env glycoproteins and to at least one of
Fig 2-Distribution of anti-HIV-1 titre in 30 repeatedly reactive samples. All 26 sera with titres over 100 were of those with titres below 100.
screen-
positive on western blot, as were 2
518
SEROPREVALENCE BY LOCATION OF HEALTH DISTRICT
the two groups of other structural proteins on western blot. These included 2 of the 4 eluates with GPA titres below 100. The remaining 2 with low GPA titres showed no antibody when western blotted and were considered not to be anti-HIV-1 positive. The prevalence of anti-HIV was 0-24 per 1000 (28/114 515). The seroprevalence was highest in inner London and lowest outside the metropolitan area (table). There was no evidence that seroprevalence had changed during the study, either overall or within any region.
Discussion
prevalence per 1000 of anti-HIV-1 was 0,24, considerably lower than the 1-3 reported in a study of 92 Italian hospitalsthe 2-1in Massachusetts,1 and the 6-6 in New York stated The gradient we found, of increasing prevalence from home counties through outer city to inner city, has also been observed in North American studies. Anti-HIV in the newborn infant does not imply that the infant is infected. About 1 in 4 seropositive newborn babies
The overall
will be infected.4°5 On this basis the neonatal HIV-1 infection rate in London over the study period would be 6 per 100 000 livebirths, an incidence comparable with that of
phenylketonuria. We have demonstrated the feasibility of screening all newborn infants for anti-HIV-1. The modification of a commercially available agglutination assay had a similar sensitivity to the ELISAs used by the Centres for Disease Control in their national HIV screen of dried blood spots (unpublished data). All but 2 of our 30 repeatedly screentest reactive sera were confirmed by western blot. Repeat reactivity is therefore highly predictive and only a low proportion of repeatedly reactive sera will fail to be identified by western blot as being anti-HIV-1 positive. More significantly a high specificity (at least 99-996%) is implied by the lowest seroprevalence, which was observed outside London. The reagent costs, including western
blotting were 0 12 per sample. Surveillance of anti-HIV-1prevalence in newborn babies or pregnant women can effectively monitor the prevalence of HIV infection in the heterosexual population.9 The advantage of neonatal testing is that Guthrie cards are collected in central laboratories on an almost universal basis. They are likely to be representative of women delivering babies and not biased by self selection. In a review of congenital hyperthyroid screening in 1983, only one out of 493 consecutive cases was missed.lO However, babies born to mothers known to be HIV-infected might be more likely to be missed. Instead of using Guthrie cards, we could have tested samples taken for routine antenatal rubella testing. This would have entailed much more work since specimens are kept at different sites and stored as serum. In addition, some women at higher risk of HIV, such as intravenous drug users, are more likely to be excluded from routine rubella screening because they may present for the first time either late in pregnancy or already in labour. However, antenatal
testing would identify those HIV-infected women whose pregnancies are terminated or who abort spontaneously. Seroprevalence estimates from anonymous testing are likely to be more reliable than those from voluntary testing. In New York only 40% of those who were offered antenatal testing agreed, and those found to be positive only disclosed additional risk factors afterwards." In another study 1000 women were anonymously screened, but only half of the 50 who admitted risk activity agreed to be tested; voluntary screening detected only 14% of those who were infected.12 Similar findings have been reported in studies of patients attending sexually transmitted disease clinics.13-15 A major concern is that anonymous testing precludes any of infected women. However, anonymous studies have revealed unexpectedly high local prevalence of HIV infection, which increases awareness among health professionals and underlines the need for voluntary testing and additional support for those found to be infected. We emphasise that unlinked anonymous testing does not replace voluntary testing with consent in antenatal clinics. 16 Neonatal HIV testing has been widely accepted in the USA where it is being extended to more than 40 statesand the Canadian Royal Society of Medicine has also recommended anonymous sampling. In the UK the Department of Health supports unlinked anonymous testing for HIV and sees no ethical or legal objections: "The legal advice given to the Department of Health is that provided the patient’s blood is used for the original test for which consent was obtained it is unnecessary to tell patients (either individually or through a public notice in a clinic) that any blood residue might be tested unlinked and anonymously".9 However, it is felt that individuals have the right to opt out of anonymous studies.3 It has also been suggested that informed consent should be sought from the health care workers involved. Either of these procedures are likely to lead to patient selection bias. For an infection with a prevalence of less than 1 in 1000, even a 99-9% compliance could lead to serious underestimates of seroprevalence. The policy of allowing patients to opt out of unlinked anonymous testing is to be regretted and it will be essential to record the proportion that do so. Further studies are being done to compare the seroprevalence obtained by neonatal screening with the results of other HIV reporting schemes, including anonymous antenatal testing and reports of HIV infection in pregnant women and children. care
This study was supported by the Medical Research Council. We thank the staff of the neonatal screening laboratories for their assistance.
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. Centers for Disease Control. CDC surveillance supplements. MMWR 1989; 38: (no S-4). 3. Heptonstall J, Gill ON. The legal and ethical basis for unlinked anonymous HIV testing. Comm Dis Rep 1989; 48: 3-6. 4. European Collaborative Study. Mother-to-child transmission of HIV infection. Lancet 1988; ii: 1039-43. 5. Blanche S, Rouzioux C, Guihard Moscato M-L, et al. A prospective study of infants bom to women seropositive for human immunodeficiency virus type 1. N Engl J Med 1989; 320: 1643-48. 6. Barbara 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. 7. Ippolito G, Stegagno M, Costa F, Angeloni P, Angeloni U, Guzanti E. Detection of anti-HIV antibodies in newborns: a blind serosurvey in 92
519
l’enfant.
parturient women in a high-risk population. N Engl J Med 1988; 318:
Paris, Nov 27-30, 1989: 141 (abstr). 8. Novick LF, Bems D, Stricof R, Stevens R, Pass K, Wethers J. HIV seroprevalence in newborns in New York State. JAMA 1989; 261:
185. 13. Hull HF, Bettinger CJ, Gallaher MM, Keller NM, Wilson J, Mertz GJ. Comparison of HIV-antibody prevalence in patients consenting to and declining HIV-antibody testing in an STD clinic. JAMA 1988; 260:
Italian hospitals. Les
implications
du SIDA pour la
mere et
1745-50. 9. Gill ON, Adler MW, Day NE. Br Med J 1989; 299: 1295-98.
Monitoring the prevalence of HIV.
10. Grant DB, Smith I. Survey of neonatal screening for primary hypothyroidism in England, Wales and Northern Ireland 1982-4. Br Med J 1988; 296: 1355-58. 11. Minkoff HL, Holman S, Beller E, Delke I, Fishbone A, Landesman S. SUNY Health Science Center routinely offered prenatal HIV testing.
N Engl J Med 1988; 319: 1018. 12. Krasinski K, Borrowsky W, Bebenroth D, Moore T. Failure of voluntary testing for human immunodeficiency virus to identify infected
935-38. 14. Landesman S, Minkoff H, Helman S, McCalla S, Syn O. Serosurvey of human immunodeficiency virus infection in parturients. JAMA 1987; 258: 2701-03. 15. Evans BA, McCormack SM, Bond RA, MacRae KD, Thorp RW. Human immunodeficiency virus infection, hepatitis B virus infection and sexual behaviour of women attending a genitourinary clinic. Br Med J 1988; 296: 473-75. 16. Royal College of Obstetricians and Gynaecologists. Report of the RCOG sub-committee on problems associated with AIDS in relation to obstetrics and gynaecology. London: RCOG, 1987.
CLINICAL PRACTICE Extent, distribution, and mammographic/
histological
To
correlations of breast ductal carcinoma in situ
potential of breast-conserving (DCIS), 82 mastectomy specimens were studied by Egan’s serial subgross method. 42 (51%) of the tumours were larger than 50 mm and only 12 (15%) were assess
the
treatment for ductal carcinoma in situ
smaller than 20 mm; the size distribution was not affected by the mode of detection (mammography 52 cases, clinical examination 30). All but 1 case showed only 1 region of tumour. 66% of tumours involved one breast quadrant, 23% extended over more than one quadrant, and 11% were centrally located. Mammographic estimates, based on the extent of microcalcifications, frequently underestimated the histological size of tumours, the extent of the discrepancy being related to the histological type—8/50 predominantly comedo DCIS showed a discrepancy greater than 20 mm with 15/32 compared predominantly micropapillary/cribriform. In view of the frequently large size, adequate excision of many DCIS will require a wide excision involving up to a whole quadrant.
Introduction Until now the standard treatment for ductal carcinoma in situ of the breast (DCIS, intraductal carcinoma) has been ablative surgery, with a cure rate of nearly 100%. However,
the results of breast-conserving treatment rather than mastectomy for the management of early invasive breast cancer are promising. This development has created controversy, in that mastectomy is still recommended for patients with non-invasive intraductal cancers (ie, at an earlier stage than any invasive cancer), whereas many patients with invasive cancers are offered breast-conserving treatment. The increasing use of mammography in routine diagnosis of breast disorders and the introduction of mammographic mass screening programmes1 have expanded the scale of the problem by raising the rate of detection of DCIS three or four fold to as much as 15-20% of all mammographically detected cancers .1,3 The main reservation about conservative treatment of DCIS is that its distribution in the breast is assumed to be multicentric. In patients with DCIS, treated by lumpectomy and subsequent mastectomy, residual tumour was found in 30-60% of mastectomy specimens 4 The effect of radiotherapy on this residual DCIS is not yet clear.6,7 Therefore, the surgeon should ideally make every attempt to resect all disease, with histologically adequate margins, ADDRESSES: Departments of Pathology Schuurmans Stekhoven, PhD), Radiology (J and Epidemiology (A. L. M. Verbeek, MD), Hospital, and Department of Pathology,
(R. Holland, MD, J. H. H. C. L Hendriks, MD), Radboud University Canisius Wilhelmina Hospital (M. Mravunac, MD), Nijmegen, the Netherlands. Correspondence to Dr R. Holland, Department of Pathology, Radboud University Hospital, 6500 HB Nijmegen, the Netherlands.
