HTLV-III ANTIBODIES

1152

suspected by others, who have also observed reversion to seronegativity in the presence of high titres ofpirus.3 Virus isolation from antibody-negative persons has also been reported.4,5

been

JAN C. ULSTRUP Microbiology Laboratory, Oslo City Hospital, Ulleval, 0407 Oslo 4, Norway

KJELL SKAUG KARL J. FIGENSCHAU IVAR

ØRSTAVIK

Section for Infectious Diseases,

Department of Medicine, Oslo City Hospital Ullevål against AIDS Central Health Council, Oslo

JOHAN N. BRUUN

Relief Clinic

1.

Anti-HTLV-Ill tests (A, B, and C). *Molecular

weight

on

serial

serum

samples from three individuals

in thousands.

tEIA=ELISA test. A = Abbott, 0 = Organon, P = Pasteur, W = Wellcome, D=Du

Pont.

B=borderline

result.

IF=immunofluorescence

test.

NS = non-specific result. LAS= lymphadenopathy syndrome. No western blot ofNov 8, 1985, serum from patient B.

ELISA

Cooper DA, Gold J, Maclean P, et al. Acute AIDS retrovirus infection: Definition ofa

clinical illness associated with seroconversion. Lancet 1985, i 537-40. 2 Barin F, MacLane MF, Allan JS, Lee TH, Groopman JE, Essex M Virus envelope proteins of HTLV-III represents major target antigen for antibodies in AIDS patients. Science 1985; 228: 1094-96. 3. Levy JA, Kaminsky LS, Morrow WJW, et al. Infection by the retrovirus associated with the acquired immunedificiency syndrome: clinical, biological, and molecular features. Ann Intern Med 1985; 103: 694-99. 4. Salahuddm SZ, Groopman JE, Markham PD, et al HTLV-III in symptom-free seronegative persons. Lancet 1984; ii: 1418-20. 5. Groopman JE, Hartzband PI, Shulman L, et al Antibody seronegative human T-lymphotropic virus type III (HTLV III)-infected patients with acquired immunedeficiency syndrome or related disorders. Blood 1985; 66: 742-44.

July, 1983, were all negative; the November EVALUATION OF EIGHT ELISA KITS FOR THE positive in the Du Pont ELISA only, but the DETECTION OF ANTI-LAV/HTLV-III ANTIBODIES December specimen was also positive in the Organon and Wellcome ELISAs (see figure). In February, 1984, all ELISA tests were SIR,-Eight ELISA kits for detecting antibody to LAV/HTLV-III have been evaluated by a study group of the French National Society positive, as was the immunofluorescence test. However, in the western blot, faint gp 160 and gp 120 bands and a band in the 55 and of Blood Transfusion. The results presented here are from an the 41 regions could be discerned in the first specimen (July, 1983). evaluation, from July, 1985, of the Litton Bionetics, Behring (ENI), The high sensitivity of this western blot test was confirmed in two Wellcome, and Dupont de Nemours kits and a new kit from other patients. Patient B, a homosexual man born in 1949, was Diagnostics Pasteur (only one viral antigen well) and from a second evaluation done earlier this year with the Abbott, Diagnostics admitted to hospital for an acute febrile illness with Pasteur (two wells, viral and cellular antigen), and Organon kits. An lymphadenopathy 2-3 weeks after alleged exposure. His first specimen (Nov 8, 1985) was negative in all tests, but 6 days later . earlier study of these last three kits, done between March and May, 1985, before they were marketed in France, has already been there was distinct antibody activity against gpl60, p24, and p17in the western blot, which gives a seroconversion time of 20-27 days. published.t The evaluation was in two parts-one for samples from blood ELISA tests (Organon, Wellcome, Du Pont) were negative. On Nov donors screened individually by five or six blood transfusion centres 18 the Du Pont test was the only ELISA test to be positive. On Nov and the other for a panel of 55 specimens tested simultaneously by 21 gp120 and several other bands were distinct in the western blot, seven to ten laboratories, except for the Wellcome kit which, and the gpl60, p24, and p17bands were much stronger. The because of the shortage of materials, was used in only one centre for ELISA The latest taken showed borderline results. serum, Organon blood donors and by two laboratories for the panel. All the ELISA on Jan 8, 1986, one month after discharge, showed in addition p31, the and Three ELISA tests and techniques were used strictly in accordance with the 53, 55, and 64 bands. gp41, recommendations of the manufacturer, and the results were immunofluorescence test were all positive. Patient C, a woman born in 1961, was found during a routine expressed by optical density (OD) ratios, as OD sample/OD cut-off value (OD cut-off/OD sample for the Wellcome competitive assay). investigation of intravenous drug users to have discordant ELISA For the Organon kit, the cut-off value was the latest one proposed by results (Organon and Du Pont positive with low extinction values, this firm-ie, 0-22 (4 N + P). Wellcome negative). 19 days later (Dec 4, 1985) extinction values Blood donor samples reactive in an ELISA test were retested in were increased in the Organon and Du Pont ELISA tests and seroconversion was observed with the Wellcome test. The duplicate in the same laboratory. All the repeatedly reactive samples immunofluorescence test was non-specific in both specimens but (ie, those which three times gave a ratio above 1) were sent to the western blotting revealed well-developed bands in both specimens. coordinating laboratory for western blot analysis. Where the western blot was uninterpretable (antibody to core proteins without Our results indicate that these western blot strips are very sensitive for demonstrating antibodies against HTLV-III. Gpl60 antibody to envelope proteins), radioimmunoprecipitation assay was used. When a confirmatory test was negative, the sample was and gp120 bands are not usually seen when a western blot is used. labelled as a false positive (table). Most of the sera from blood donors However, these antibodies are usually well developed in the were fresh so different samples were tested in the different kits; the radioimmunoprecipitation test, and they have been claimed to be exception was the 3031 identical samples tested with the Behring, present most consistently in the sera of infected people2 and to be the last to disappear if AIDS develops. These bands seem to be very Dupont, and Litton kits which had been tested earlierl and kept frozen. No significant difference was observed in the frequency of specific too. false positive between fresh and frozen samples except for the To increase the specificity of the ELISA test manufacturers often Behring test (0 -33% fresh, 2 -97% frozen). put the cut-off values many standard deviations from the mean, and The panel of 55 specimens consisted of 17 negative samples, 25 this may be the reason why some sera register as negative. With low levels of antibody immunofluorescence staining is very faint, which positive samples (3 in duplicate), and 13 dilutions (3 in duplicate) of 4 positive samples. All the positive samples came from symptomless makes it difficult to decide on a positive result. There was a clear difference between the time to seroconversion carriers, in contrast to Mortimer and colleagues’ comparative between patients B and C, who converted within a few days, and study.2Other details about this panel and the results of confirmatory tests have been published. This panel was tested in patient A, who had asthenia and other symptoms for 4-5 months before he seroconverted in ELISA. We wonder if this might be due duplicate and the results of the seven to ten laboratories taking part were collated in the coordinating laboratory. A mean ratio was to specific immune complexes in the blood. Such complexes have tests

specimen

-

GEORG PETERSEN

was

in

1153 FREQUENCY OF FALSE POSITIVES

IN BLOOD DONORS AND

NONOXYNOL-9 AND HTLV-III

FREQUENCY

OF CORRECT RESULTS IN PANEL SERA

SIR,-Our letteron a collaborative project between the Mariposa Foundation and the Centers for Disease Control should have indicated that nonoxynol-9 concentrations in spermicides range from 1% to 12 - 507o (not 5%). We neglected to note that HTLVIII/LAV inactivation is very rapid (less than 60 s), an important consideration in evaluating the potential of nonoxynol-9 spermicides in reducing sexual transmission of HTLV-III. As evidence mounts that the virus may be sexually transmitted within host lymphocytes, and not just in free form, the importance of destruction of lymphocytes by 5% concentrations of nonoxynol-9 (present in some brands of spermicides) should not be overlooked. Mariposa Education and Research Foundation,

BRUCE VOELLER

Topanga, California 90290, USA

al. Inactivation of HTLV-III/LAV-infected cultures of normal human lymphocytes by nonoxynol-9 in vitro. Lancet 1985; ii: 1422.

1. Hicks

*Figures in parentheses are rates per 1000. t Doubtful results in parentheses.

The members of the SNTS study group who took part in this study were: F. Barin, Hopital Bretonneau, Tours; J. Baudelot, CTS Bobigny; M. Gueguen, CTS Rennes; C. Janot, CTS Nancy; J. M. Lemaire, CTS Montpellier; M. Maniez, CTS Lille; F. Mesnier, CTS Bordeaux; M. L. North, CTS Strasbourg; C. Rouzioux, Hopital Claude Bernard, Paris; W. Smilovici, CTS Toulouse.

Sanguine,

et

SLOW-RELEASE ASPIRIN AND PROSTAGLANDIN INHIBITION

calculated for each sample and the percentage of negative (ratio below 0 -90), doubtful (0 -90 or more but below I -00), and positive results (1 -00 or more) was established for the three categories in the panel (negative, positive, positive dilutions). The ’percentage of correct results is given in the table. The incorrect results found in the 17 negative samples of the panel related to single specimens only: no 46 for the Abbott (8 positive results/8, mean ratio 130) and Litton (1/8; 0 -60) tests; no 55for the Behring (8/8; 1 -32) and Organon (5/7; 1 -04) kits; and no 30 for the Dupont test (3/10; 0 ° 78). The negative and doubtful results found in the 25 positive samples in the panel were observed with the same specimen (no 23).* This study shows that the Wellcome kit followed by the two Pasteur kits are the most specific of the eight kits tested. Sensitivity is much more diifficult to compare. As judged by the recognition of undiluted and diluted positive samples of the panel, the most sensitive tests would seem to be the two Pasteur kits, then Wellcome, then Dupont, and then Abbott. But this evaluation, on only a few positive samples, is not rigorous since there are great qualitative and quantitative variations in anti-LAV/HTLV-III in different specimens.3,4 Western blot analysis showed, for example, that sample 25 recognised very clearly p25 and very poorly envelope proteins; the recognition pattern of sample 47 was the other way round. Since the first situation may arise more often than the second in blood donations-because antibodies to the major core protein appear first after exposure3-5-we are doing further comparisions of ELISA tests in individuals who have lately seroconverted.

Centre National de Transfusion 6 rue Alexandre Cabanel, 75739 Paris, France

DR, Martin LS, Getchell JP,

ANNE-MARIE COUROUCE, for the Retrovirus Study Group of the French National Society of Blood Transfusion (SNTS)

SIR,-The use of low-dose, slow-release aspirin for the ideal antithrombotic effect has been widely proposed. 1-4 It has been suggested that the slow administration of low doses of aspirin may permit cumulative presystemic acetylation of platelet cyclooxygenase with resultant inhibition of the formation of the vasoconstrictor and platelet aggregating agent, thromboxane A2 - 2,3 6 Because of gut, liver,5 and extrahepatic metabolism,6 concentrations of aspirin reaching the peripheral blood after dosing with these formulations may be too low to inhibit vascular cyclo’oxygenase and reduce the formation of the vasodilator and platelet antiaggregating agent, prostacyclin.2,3 52 volunteers were used to examine the hypothesis of a selectivity from low-dose, slow-release aspirin formulations. They took one of three aspirin formulations for 10 days. The formulations were soluble aspirin (’Aspro Clear’; Nicholas), a 100 mg aspirin plus glycine tablet (’Cardiprin’; Reckitt & Colman), and a slow release formulation (Smith, Kline and French). Over 24 h the slow release formulation yielded relatively constant plasma aspirin and salicylate concentrations. Blood and urine were collected on days 0, 6, and 10. The blood (1ml) was incubated in a plain glass tube at 37°C for 1 h and the resultant serum was analysed by 125Iradioimmunoassay (New England Nuclear) for TXB2, the stable hydrolysis product of TXAz. The urine was analysed for the stable hydrolysis product of prostacyclin, 6-keto-PGFIa (relative to creatinine excretion), by column chromatography I radioimmunoassay (New England Nuclear). Validation for the analytical procedures was based on the consistency of control values (mean±SEM) for TXB2 (354 - 5-t27 -1 ng/ml) and 6-keto-PGFIa (146±7 ng/mg creatinine) with normal values reported using gas chromatography/mass spectrometry

and 25

(TXB2 300±40 ng/MI;7 6-keto-PGFIa 140±25 ng/mg creatinine8). Currently we are validating our assays directly by gas chromatography/mass spectrometry. The table summarises the results. The serum TXB2 generated is highly dependent on the aspirin dose (p<0 ’01, analysis of variance), TXBzbeing expressed as a % of control values. Urinary 6-ketoCOMPARISON OF SERUM TXB, AND URINARY

6-KETO-PGF,a

PRODUCTION AFTER DAILY DOSES OF ASPIRIN FOR TEN DAYS

*A table giving mean ratios for sample no 23 and for the dilutions of 4 positive samples may be obtained from The Lancet-ED. L. 1 Courouce

AM, et le groupe d’étude de la SNTS. Evaluation de 3 trusses immunoenzymatiques de dépistage des anticorps anti-LAV. Comparaison avec des tests de confirmation. Revue Fr Transf Immuno-hématol 1985, 28: 325-44. 2 Mortimer PP, Parry JV, Mortimer JY. Which anti-HTLV III-LAV assays for screening and confirmatory testing? Lancet 1985; ii: 873-77. 3 Lange JMA, Coutinho RA, Krone WJA, et al. Distinct IgG recognition patterns during progression of subclinical and clinical infection with lymphadenopathy associated virus/human T lymphotropic virus. Br Med J 1986; 292: 228-30. 4. Schupbach J, Haller O, Vogt M, et al. Antibodies to HTLV III in Swiss patients with AIDS and pre-AIDS and-in groups at risk for AIDS. N Engl J Med 1985; 312: 265-70. 5. Esteban JI, Shih JW-K, Tai C-C, et al. Importance of western-blot analysis in predicting infectivity of anti-HTLV-III/LAV positive blood. Lancet 1985; ii: 1083-86.

Values,

as mean

(and SEM), expressed as percentage of control values (day 0) (n=5-7). (SK&F); tsoluble aspirin (Nicholas); 4:Cardiprin (Reckitt &

*Slow-release aspirin

Colman)