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Saturday 16 November 1985

IMPORTANCE OF WESTERN BLOT ANALYSISIN PREDICTING INFECTIVITY OF

ANTI-HTLV-III/LAV POSITIVE BLOOD

JUAN I. ESTEBANCHANG-CHIH TAIJOHN W. D. KAY

J. WAI-KUO SHIHANNE J. BODNERHARVEY J. ALTER

Department of Transfusion Medicine, Clinical Center, NationalInstitutes of Health, Bethesda, Maryland; Biotech ResearchLaboratories Inc, Rockville, Maryland; and Litton Bionetics,

Laboratory Products Division, Charleston, South Carolina, USA

Summary Stored donor and recipient sera from pros-pective studies of post-transfusion hepatitis

were analysed for the presence of human T-cell lymphotropicvirus type-III/lymphadenopathy associated virus (HTLV-III/LAV) antibodies as determined by enzyme-linkedimmunosorbent assays (ELISA). Of 3961 donor samplesgiven to 461 patients, only 2 (0·05%) contained specificHTLV-III/LAV antibodies as determined by an avidin-biotin-enhanced western blot tech nique. Anti-HTLV-

III/LAV was measured before and 3 and 6 months aftertransfusion in 295 recipients of anti-HTLV-III-negativeblood, 7 recipients of ELISA-positive blood which waswestern blot negative, and 2 recipients of ELISA-positiveblood confirmed as specific by western blot. Only the last 2recipients became infected with HTLV-III/LAV, as assessedby antibody seroconversion (p<0&middot;0001). Serocon versionoccurred early (6 and 8 weeks after transfusion) and wascharacterised first by antibody to p24 and later by anti body top41. AIDS has not developed in either patient, but one has aT4/T8 ratio of 0&middot;4 and impaired mitogen responses; thesecond patient has no evidence of immune dysfunction 4years after exposure. This study confirms that HTLV-III/LAV infection can be transmitted by blood transfusionand supports the advisability of anti-HTLV-III/LAV testingof all blood donors. It also confirms the validity of westernblot testing for HTLV-III/LAV specificity and suggests thatELISA-positive, western-blot-negative blood may not be infectious.

Introduction

THERE is strong epidemiological evidence that the

acquired immunodeficiency syndrome (AIDS) can betransmitted by transfusion of blood and blood products. 1- 6However, no prospective study of recipients of anti-HTLV-III/LAV (human T-cell lymphotropic virus typeIII/lymphadenopathy associated virus) positive blood hasbeen reported, and the extent to which the presence of anti-HTLV-III/LAV in a symptomless donor correlates withtransmission of HTLV-III/LAV infection to the recipient isstill unresolved.We report here our HTLV-III/LAV serological findings in

donor and recipient serum samples obtained during prospective studies of post-transfusion hepatitis.

Subjects and Methods

Patients and Donors

461 heart surgery patients who had previously been entered inprospective studies of post-transfusion hepatitis conducted at theNational Institutes of Health (NIH) between 1977 and 1984 wereselected for the present study. A presurgery sample was availablefrom all patients and additional samples were obtained once or twicea week in the first 3 months after transfusion, monthly for the next 3months, and then again at 9 months. These patients received bloodproducts from 4251 volunteer donors (range 1 to 38, mean 9 - 2 perpatient), who had donated blood either to the Washington RedCross or directly to the NIH Blood Bank. Stored frozen samplesfrom 3961 of these donors (93% of the total) were available for antibody testing. The annual distribution of the donor-recipientpopulation studies is shown in table i.

Serological AssaysDonor samples were tested for anti-HTLV-III antibodies by the

use of commercial enzyme linked immunosorbent assay (ELISA)test kit (’HTLV-Ill Bio EnzaBead’, Litton Bionetics, Charleston,South Carolina). Samples that were positive on the first test were retested in duplicate. For confirmation of sensitivity, a subset of 1956donor samples collected between 1980 and 1984 were tested by

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TABLE 1-ANNUAL DISTRIBUTION OF THE DONOR-RECIPIENTPOPULATION STUDIED

*Numbers in parentheses refer to mean number of donors per recipient.

another pre-licensure ELISA microtitre method (Biotech ResearchLaboratories Inc, Rockville, Maryland) as previously described.8Recipient samples obtained before surgery and 3 and 6 months

after surgery from 295 recipients of ELISA-negative blood transfused between 1980 and 1984 were tested by ELISA. All availablesamples from patients who received ELISA-positive blood weretested in duplicate by the use of two ELISA assays (HTLV-111 BioEnzaBead and ’Virgo HTLV-III ELISA’, Electronucleonics

Columbia, Maryland). Samples found to be positive were titrated toend-point dilution and retested by the same methods. All serareactive by ELISA were tested by an avidin-biotin immunoblottingassay (western blot) as previously described.9

Immunological Assessment

Assays for T-cell subsets and immune responses to phytohaemag-glutinin (PHA), concanavalin A (con A), pokeweed mitogen(PWM), and tetanus toxoid were performed by standard techniquesas previously described. 10 0

Results

9 of 3961 donors (0 - 2%) were positive on initial ELISA test(table II). Of these, only 4 (0-1%) remained positive when retested in duplicate and only 2 (0 . OS&deg;Io) when tested bywestern blot. The other ELISA assay used to test the subset of1956 donor sera obtained between 1980 and 1984 detectedthe same two western-blot-confirmed, ELISA-positivesamples but no additional true positive samples. The firstconfirmed positive donor was detected in 1981 and the otherin 1984. Both were positive on western blot for antibodiesagainst p24, p41, and p64.No antibody seroconversion occurred in 295 recipients of

ELISA-negative blood, in 5 recipients of blood that wasELISA positive only on initial testing, and in 2 recipients ofblood that was consistently ELISA positive but negative onwestern blot (table ill). In contrast, the only 2 patients who received ELISA-positive blood that was confirmed as specificby western blot, had anti-HTLV-III/LAV antibodies 6 and 8weeks after transfusion. The difference in antibody seroconversion rates was statistically significant when recipients ofELISA-positive/western-blot-negative blood were comparedwith recipients of western-blot-positive blood (0/7 versus 2/2;p = 0 . 02 by one-tailed Fisher’s exact test) and when all 302recipients of western-blot-negative blood were comparedwith recipients of western-blot-positive blood (0/302 vs 2/2;p<0-0001).

’

The first recipient (R-1) of western-blot-positive blood wasa 56-year-old white male who underwent a coronary arterybypass graft in May, 1981, and received three units of packedred cells, one of which was anti-HBc, anti-HBs, and anti-

TABLE II-ANNUALIZED PREVALENCE OF HTLV-III/LAV ANTIBODY I,

VOLUNTEER BLOOD DONORS

*HTLV-111 Bio EnzaBead, Litton Bionetics.

TABLE III-DONOR ANTI-HTLV-11! STATUS VERSUS RECIPIENT

INFECTION

WB = Western blot; nt = not tested.*Includes 5 non-repeatable ELISA+ +a+b vs c: p<O,OOOl.b vs c: p=O’02.

HTLV-IIIILAV positive. Antibodies to p24 first appeared atpost-transfusion week 6 and those against p4l and p64 atweek 14; all antibodies persisted for the entire follow-upperiod (fig 1). HTLV-III/LAV antibodies were first detectedby ELISA assay 1 at week 6, when only antibody to p24 waspresent, whereas ELISA assay 2 did not detect antibodiesuntil week 10, which was around the time of first appearanceof antibody to the envelope peptide p41. In both assays, anti.body titres increased after the appearance of antibody to p41,reaching a peak of 1/9600 in the last available sample 9months after transfusion.The second patient (R-2) was a 72-year-old white female

who underwent aortic valve replacement in January, 1984,and received 15 units of blood, 3 of which were anti-HBc and

WEEKS AFTER TRANSFUSION

Fig 1-Comparison of anti-HTLV-1IIILAV serological profile in

patient R-1 as detected by two ELISA assays and western blot.

The intensity of the specific antigen-antibody reactions in western Momsassessed visually and asigned a score from (+) to + + +, (+) indicating bareudetectable reaction; + clearly detectable; + + strong, and + + + very strong

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anti-HBs positive; 1 of these 3 was also positive for anti-HTLV-III/LAV.

Immediately after surgery there was an early and transientpeak of antibodies (ELISA titres 1/300 to 1/1200) directedagainst p41 and p64 proteins, consistent with passive transferof antibodies from donor to recipient (fig 2). De-novo produc-tion of antibodies to p24 and p41 first appeared at week 8 and10, respectively, and were associated with a sharp rise in anti-body titre, which continued to increase to 1/9600 (1 :1200 inassay 2) by week 24, reaching a plateau which persisted tilllast tested, more than 1 year after transfusion. Although noneof her donors was positive for HBsAg, 28 weeks after transfu-sion she had serological and biochemical evidence of hepatitisB (fig 2, lower panel). Despite apparent serological resolutionof her hepatitis B, serum transaminase began to rise again atweek 46 and has been elevated since that time.

Neither recipient belonged to a high-risk group for AIDS.Recipient R-l, though still anti-HTLV-III/LAV positive, re-mains clinically well 4 years after the implicated transfusion,with no physical evidence of AIDS or AIDS related complex(ARC) and with a normal T4cell number, T4T/8 ratio, andproliferative responses to PHA, con-A, PWM, and tetanustoxoid. Patient R-2 is clinically well without physical mani-festations of disease 1-year after transfusion. However, she re-mains anti-HTLV-III positive and has reduced T4 cells

(4131p1), a reversed T4/T8 ratio (0’ 42), depressed T-cell mito-gen response, and no recall response to tetanus toxoid.

Discussion

We have prospectively assessed the risk ofHTLV-III/LAVinfection in cardiac surgery patients receiving an average of9.2 units of volunteer donor blood. Of 3961 donors tested byELISA, only 0-1% were reproducibly positive and only 2donors (0’ 05%) had specific antibodies as assessed by a sensi-tive avidin-biotin western blot. This low antibody prevalenceis probably a true reflection of the low AIDS risk of the volun-teer, primarily suburban, donor population tested.The key element of this study is not the prevalence of anti-

body, but the fact that only the recipients of ELISA-positiveblood that was confirmed as specific by western blot becameinfected with the HTLV-III/LAV agent. This finding indi-cates that blood that is specifically positive for anti-HTLV-III/LAV antibody may transmit the AIDS virus but unitswhich are not repeatedly ELISA positive or which are non-specific by western blot are unlikely to do so. It also suggeststhat blood donors should not be notified of an anti-HTLV-

III-positive result until the specificity of that result is con-firmed by western blot or an equivalent method.Antibody seroconversion occurred early (6 and 8 weeks)

after parenteral exposure, similar to that observed in experi-mentally infected chimpanzees.6 These data suggest that al-though AIDS may not develop for a long time after transfu-sion, the rapid appearance of antibodies allows early post-transfusion identification of individuals at risk of transfusion-associated AIDS. The first antibody response was to the p24

Fig 2-Anti-HTLV-III/LAV status as detected by ELISA and western blot (upper panel) and ALTlevels and HBV serological profile (lower panel) in patient R-2.

Western blot intensity as in figure 1.

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core protein. In patient R-l, antibodies to the surface glyco-protein p41 did not appear until 9 weeks later, while inpatient R-2, the interval between the appearance of p24 andp4l was only 2 weeks. It is of interest that three consecutivesamples from patient R-1 remained negative in one of the twoELISA assays used for recipient testing, when only antibodyto p24 was present. An ELISA assay which does not detectp24 specificity might not detect antibody early during thecourse of infection and an infectious donor that carried onlythis antibody might thus not be identified. Such a possibilityhas been reported" but the frequency with which it occurs isnot yet known.

Recipient R-2 may have been simultaneously infected withthree distinct viral agents. She had unequivocal serologicalevidence of infection with the hepatitis B virus (HBV) andwith HTLV-III/LAV. Infection with a non-A, non-B hepati-tis agent is suggested by a low-level but discrete alanineaminotransferase (ALT) peak at week 12 and by the reappear-ance and persistence of ALT elevation after apparent re-covery from hepatitis B.

Significantly, neither HTLV-III/LAV-infected patient hasshown any clinical evidence of AIDS or the AIDS-related

complex (ARC). This may reflect the long incubation periodof transfusion-related AIDS, now estimated to have a medianof approximately 4 years.12 Another interpretation for theabsence of disease in these patients is that AIDS or ARC de-velops in only a minority of HTLV-III/LAV infected indivi-duals. Thus far, AIDS or ARC has developed in less than20% of prospectively followed, symptomless, anti-HTLV-III/LAV positive hemophiliacs and homosexuals. 13, 14 It is notpossible at present to predict the final outcome in our infectedrecipients. It is, however, of considerable concern that reci-pient R-2 has reduced T4 cell number and impaired immuno-logical responses, findings consistent with those observedearly in the course of AIDS.This study adds support to the advisability of widescale

testing programs for anti-HTLV-III/LAV antibody detectionin all blood and plasma donors. In combination with intensi-fied surveillance and education of blood donors and self-exclusion of those at risk, antibody screening should

markedly reduce the frequency of transfusion-associated-AIDS. The impact of such testing may not, however, be evi-dent for several years because many blood recipients have al-ready been infected and AIDS may subsequently develop in a.proportion of these.

’

We thank Dr David Alling (National Institute of Allergy and InfectiousDiseases) for statistical assistance; Mr Pearce Youngbar (Litton Bionetics,Laboratory Products Division), Ms Jacqueline Melpolder, and Ms LenitaHudson (Department of Transfusion Medicine, NIH) for technical assistance;and Ms Sandy Higgins and Ms Sue Allyn for doing the immunological assays.

Correspondence should be addressed to H. J. A., Department of Transfu-sion Medicine, Clinical Center, Building 10A, Room 1E33, National Instituteof Health, Bethesda, Maryland 20892, USA.

REFERENCES

1. Ammann AJ, Cowan MJ, Wara DW, et al. Acquired immunodeficiency in an infant:possible transmission by means of blood products Lancet 1983; i: 956-58.

2. Curran JW, Lawrence DN, Jaffe H, et al. Acquired immunodeficiency syndrome(AIDS) associated with transfusions. N Engl J Med 1984, 310: 69-75.

3 Feorino PM, Kalyanaraman VS, Haverkos HW, et al Lymphadenopathy associatedvirus infection of a blood donor-recipient pair with acquired immunodeficiency syn-drome. Science 1984, 225: 69-72.

4. Feorino PM, Jaffe HW, Palmer E, et al. Transfusion-associated acquired immunodefi-ciency syndrome. Evidence for persistent infection in blood donors. N Engl J Med1985; 312: 1293-96.

5. Groopman JE, Salahuddin SZ, Sarngadharan MG, et al. Virologic studies in a case oftransfusion-associated AIDS. N Engl J Med 1984, 311: 1419-22.

6 Alter HJ, Eichberg JW, Masur H, et al. Transmission of HTLV-III infection fromhuman plasma to chimpanzees An animal model for AIDS Science 1984; 226:549-52.

7. Alter HJ, Purcell RH, Feinstone SM, Holland PV, Morrow AG. Non-A, non-B hepa-titis: A review and interim report of an ongoing prospective study. In: Vyas GN.Cohen SN, Schmid R, eds. Viral hepatitis. Philadelphia: Franklin Institute Press1978: 359-69.

8. Weiss SH, Goedert JJ, Sarngadharan MG, et al. Screening tests for HTLV-III (AIDSagent) antibodies: specificity, sensitivity and applications. JAMA 1985, 253:221-25.

9. Alexander SS, Tai CC, Ting RL, et al. Western blot analysis of human T-celllympho-tropic virus III proteins by using an avidin-biotin system. Proceedings of the 85thAnnual Meeting of the American Society of Microbiology. Las Vegas, March 3-7,1985; 297 (abstract).

10. Lane HC, Whalen G, Fauci AS. In-vitro evaluation of human lymphocyte function InWeir DM, Herzenberg LA, eds. Handbook of experimental immunology. Edinburgh: Blackwell (in press).

11. Petricciani JC, Seto B, Wells M, et al. An analysis of serum samples positive for HTLVIII antibodies. N Engl J Med 1985; 313: 47-48.

12. Peterman TA, Jaffe HW, Feorino PM, et al. Transfusion-associated AIDS in theUnited States. International Conference on Acquired Immunodeficiency Syndrome(AIDS). Atlanta, Georgia. April 14-17, 1985 65 (abstract).

13. Eyester ME, Goedert JJ, Sarngadharan MG, et al Development and early natural his-tory of HTLV-III antibodies in persons with hemophilia. JAMA 1985, 253.2219-23.

14. Goedert JJ, Weiss SH, Biggar RJ, et al. Natural history of HTLV-III seropositivepersons from AIDS risk groups. International Conference on Acquired Immunode-ficiency Syndrome (AIDS). Atlanta, Georgia April 14-17, 1985: 65 (abstract)

DIET AND RISK FACTORS FOR CORONARYHEART DISEASE IN ASIANS IN NORTHWEST

LONDON

P. M. MCKEIGUEA. M. ADELSTEINM. J. SHIPLEY

R. A. RIEMERSMA

M. G. MARMOTS. P. HUNT

S. M. BUTLERP. R. TURNER

Department of Medical Statistics and Epidemiology,London School of Hygiene and Tropical Medicine; Department ofCommunity Medicine, University College London and Middlesex

Hospital Medical School; University of Edinburgh CardiovascularResearch Unit; and Department of Chemical Pathology, St Thomas’

Hospital Medical School, London

Summary Asian immigrants to England and Waleshave high mortality from coronary heart

disease but low mortality from colon cancer. A survey ofAsians in the London boroughs of Brent and Harrow wasundertaken with the object of investigating this. Comparedwith the British population, the Asians consumed lesssaturated (S) fat and cholesterol and more polyunsaturated(P)fat and vegetable fibre. The P/S ratio of the Asians’ diet was0 &middot;85 compared with 0&middot;28 in the British population: this wasreflected in the very high linoleic acid content of their plasmalipids. The plasma total cholesterol and high-density-lipoprotein cholesterol of Asian men was similar to that of aBritish comparison group; the concentrations in Asianwomen were much lower than in British women. Smokingrates were low in both Asian men and Asian women. The highrates of coronary heart disease in Asian immigrants are notexplained by the levels of these risk factors.

Introduction

IMMIGRANTS to England and Wales from the Indiansubcontinent have higher morbidity and mortality fromcoronary heart disease than the England and Wales

average.I-3 In the years 1970-72 the standardised mortalityratio (SMR) was 119 for men and 128 for women. 2,3 Incontrast, they have a low SMR for colon cancer-54 for menand 59 for women. High mortality ratios for coronary heartdisease and low mortality from colon cancer over the years1970-78 were found in all four main ethnic groups of Asian

immigrants-Gujaratis, Punjabis, Southerners, andMuslims.4 An excess of coronary heart disease in Indianscompared with other ethnic groups has also been reported