Second-generation RIBA to confirm diagnosis of HCV infection

912

drinkers. The excess mortality in non-drinkers compared with light drinkers (1-2 per day), after adjustment for age and smoking, is about 10% for all causes and about 20% for coronary heart disease. This result contrasts with the 40% excess in total mortality in non-drinkers reported by Klatsky et aP and Marmot et al.4 We suspect that the burden of ill health is higher in non-drinkers and occasional/irregular drinkers in both the "healthy" and "sick" men in this study. If our hypothesis is to be refuted, then the burden of disease by alcohol-intake category must be given.5 Cullen also takes me to task for my review of the Nurses Health Study.6 While the trends of relative risk for coronary heart disease with increasing alcohol intake were statistically significant, these trends were strongly dependent on higher rates in the non-drinkers and heavy drinkers, with little trend inbetween. Since non-drinking women had the highest prevalence rates for diabetes, obesity, high blood cholesterol, and hypertension and the lowest rates of current smoking (which suggests a high proportion of ex-smokers), it is evident that in this study, as in many others, non-drinkers have the heaviest burden of ill-health, especially for cardiovascular-related disease. Clearly, non-drinkers are not a satisfactory baseline either for women or men. In these alcohol-mortality studies, data must be presented on the burden of ill health in the various drinking

categories. Department of Public Health and Primary Care, Royal Free Hospital School of Medicine, London NW3 2PF, UK

A. G. SHAPER

review of prospective studies. Br J Addict 1990; 85: 837-47. 2. Boffetta P, Garfunkel L. Alcohol drinking and mortality among men enrolled in an American Cancer Society Prospective study. Epidemiology 1990, 1: 342-48. 3. Klatsky A, Armstrong M, Friedman GD. Relations of alcoholic beverage use to subsequent coronary artery disease hospitalization Am J Cardiol 1986; 58: 710-14. 4. Marmot MG, Rose G, Shipley M, Thomas BJ. Alcohol-mortality, a U-shaped curve. Lancet 1981; i: 580-83. 5. Wannamethee G, Shaper AG. Men who do not drink. Int J Epidemiol 1988; 17: 201-10 6. Stampfer HJ, Colditz GA, Willett WC, Speitzer FE, Hennekens OH. A prospective study of moderate alcohol consumption and the risk of coronary disease in women. N Engl J Med 1988; 319: 267-73.

1.

Shaper AG. Alcohol and mortality a

Second-generation RIBA to confirm diagnosis of HCV infection SIR,-Dr Marcellin and colleagues (March 2, p 551) report the of hepatitis-C-virus (HCV) antibody in patients with chronic non-A, non-B hepatitis. They detected a large number of false-negative results with the ELISA in comparison with the second-generation recombinant immunoblot assay (RIBA). There have been concerns about the specificity1-3 of the ELISA, and the first RIBA could not be considered a real confirmatory test because of the large number of indeterminate results.4 The secondgeneration RIBA has two additional antigens on the strips, C33c (non-structural) and C22-3 (HCV-core associated). We have

prevalence

evaluated both recombinant assays on two groups of sera-75 samples submitted to our laboratory from patients with liver disorders and with repeatedly positive ELISAs (optical densities [OD] 0’589-3’387; mean OD/cut-off ratio 37) and 16 ELISApositive sera from 77 patients on haemodialysis. In the first group, 48 (64%) sera were reactive, 12 (16%) indeterminate (10 reactive to the 5-1-1 band, 2 to C100-3), and 15 (20%) negative when tested with the first-generation RIBA. For low values of OD (ELISA ratio below 5), no statistical correlation was observed between OD and likelihood of a positive result with the RIBA. This correlation was significant when the ELISA ratio was above 5 (95% vs 52%, p < 001). All the indeterminate results and 3 of the negative samples with the first-geneation RIBA were reactive with RIBA-2. Samples reactive to only one antigen in the RIBA should be taken as potentially infective.s No indeterminate results were obtained with RIBA-2. The ELISA ratio of the 12 sera that were negative with both tests ranged from 15 to 7(mean 3-4). There was no direct correlation between this ratio and the confirmatory results, suggesting that all ELISA-positive results should be confirmed, irrespective of the OD. In the second group of sera, 7 reactive, 4 indeterminate, and 5

negative results were obtained with the first-generation assay. With RIBA-2, all 4 indeterminate samples were reactive and of the 5 initially negative sera, 1 was reactive, 2 indeterminate, and 2 negative. The prevalence of anti-HCV in this group of patients was 21% by ELISA and 16% after confirmation with RIBA-2. These results accord with previous reports6 and suggest the presence of HCV antibody even with normal transaminase activities. The false-positive rate with the ELISA was 15%, with RIBA-2 as the true confirmatory test. The second-generation RIBA seems to have resolved difficulties with indeterminate patterns and false-negative results in the first generation assay, and could be a reliable confirmatory test for the detection of HCV antibodies. ALBERTO LEÓN RAFAEL CANTON MATILDE ELÍA MARISA MATEOS

Department of Microbiology, Hospital Ramón y Cajal, 28034 Madrid, Spain

1 Theilmann L, Blazek M, Goeser T, Gmelin K, Kommerell B, Fiehn W False-positive anti-HCV tests in rheumatoid arthritis. Lancet 1990, 335: 1346 2 Boudart D, Lucas JC, Muller JY, LeCauer D, Planchon B, Harousseau JL. False-positive hepatitis C virus antibody tests m paraproteinaemia Lancet 1990; 336: 63. 3. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R HepatitisC virus antibodies in chronic active hepatitis: pathogenic factor or false-positive result? Lancet 1990; 335: 754-57. 4. Menitove J, Richard WA, Destree M Early US experience with anti-HCV kit m blood donors. Lancet 1990; 336: 244-45. 5. Bellobuono A, Mozzi F, Petrini G, Zanella A, Sirchia G Infectivity of blood that is immunoblot intermediate reactive on hepatitis C virus antibody testing. Lancet 1990; 336: 309 6. Mondelli MU, Cristina G, Filice G, Rondanelli EG, Piazza V, Barbien C Anti-HCV positive patients m dialysis units? Lancet 1990; 336: 244.

SiR,—We disagree with Dr Van der Poel and colleagues’ (Feb 9, 317) conclusion that "the new 4-RIBA [four-antigen recombinant immunoblot assay] represents a candidate

p

confirmation test to discriminate between infective and noninfective HCV [hepatitis C virus] C-100 ELISA-positive blood donors". The HCV C-100 ELISA (Ortho) assay was the first test for the detection of antibodies to HCV, and was based on one HCV antigen. A new assay is being introduced, HCV C-200 C-22 EIA UBI (Nuclear Laser Medicine, Milan), which reacts with antibodies to three HCV antigens. We tested 420 blood samples from 350 healthy volunteers, 50 intravenous drug users, and 20 patients with high alanine transferase values with the two ELISA systems and with 4-RIBA (Chiron). 61 samples were ELISApositive-47 with HCV C-100 ELISA and 57 with HCV C-200 C-22 EIA; 43 were positive with both assays. With the 4-RIBA assay, of the 61 reactive samples, 50 were positive, 5 indeterminate, and 6 negative. There were 18 discrepancies:

No positive with : HCV C-100 HCV C-200 C-22

4 14

No

4-RIBA. No mdeter-

No

positive

mmate

negatme

-

-

8

4 42

Our preliminary data show that the new HCV C-200 C-22 EIA UBI assay is both more specific and more sensitive than HCV C-100 ELISA. We conclude that 4-RIBA could also represent a candidate confirmation test to discriminate between infective and non-infective HCV C-200 C-22 EIA-positive blood samples. Provincial Laboratory of and Immunisation,

Hygiene

38100 Trento, Italy, and Ospedale Civile, Arco

DANILA BASSETTI VINCENZO CUTRUPI BIANCA DALLAGO PIETRO ALFONSI

SIR,-Dr van der Poel and colleagues suggest the new fourantigen recombinant immunoblot assay (4-RIBA) for antibodies to hepatitis C virus (HCV) as a candidate confirmation test to discriminate between infective and non-infective HCV C-100 ELISA-positive blood donors. Our results support this suggestion. In the 4-RIBA (Ortho Diagnostic) two recombinant HCV antigens, C33c from the non-structural and C22 from the core

913

ANTI-HCV RESULTS IN IMPLICATED AND NON-IMPLICATED DONORS

conventional smallpox vaccine were a new product it would be unlikely to get a product licence for general use. The results reported by Cooney et al may aid our understanding of the factors that determine the human virulence of vaccinia virus. Animal studies have shown that vaccinia recombinants which have the foreign gene inserted into the vaccinia thymidine kinase gene, and which are therefore TK-, tend to be attenuated.3.4However, the recombinant used by Cooney et al was also TK - ,sand produced results in man similar to that of the conventional TK+ vaccine. In fact in the two subjects tested who had not previously had smallpox vaccine the effect if anything was more severe than the conventional vaccine. Thus, whatever its attenuating effect in animals, it seems that inactivation of the thymidine kinase gene of vaccinia does not necessarily attenuate the virulence of the virus for man. University of Liverpool, Department of Medical Microbiology, Liverpool L69 3BX, UK

DERRICK BAXBY

1. Fenner F, Henderson DA, Arita I, et al. Smallpox and its eradication. Geneva: WHO, 1988 2. Buller RML, Smith GL, Cremer K, et al. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative

phenotype. Nature 1985; 317:

i

I

I

I

i

*Upper limrt of normal 40 U/I, ALT= alanine transferase t4-RIBA testing done on follow-up samples 20-35 mo postdonation +=pos!tMe,-=negat!ve

(structural) region of the HCV genome, have been coated in bands on nitrocellulose strips in addition to the 5-1-1 and C-100 antigens present in the first generation RIBA. The antigen band intensities are compared with weak positive and moderate positive control bands, and a response of 1 + or greater (up to 4 + ) to any two or more HCV antigens is interpreted as a positive result. We have tested samples from 9 implicated donors in our prospective post-transfusion hepatitis study,* each of whom was associated with 1 of the 9 post-transfusion HCV cases. In addition, 26 non-implicated C-100 ELISA positive donations, not associated with recipient hepatitis, were analysed. All 26 non-implicated ELISA-positive donor samples were non-reactive for the antigens C33c and C22 in 4-RIBA (table). All the implicated donors, however, showed reactivity for both antigens, including donors 2, 5, and 8, who were not detected by ELISA and were non-reactive for antigens 5-1-1 and C-100 in RIBA. In view of our experience, antibodies to the new antigens C33c and C22 seem to predict infectivity with high sensitivity, and their addition in the primary HCV screening assay is important. FREJA EBELING Finnish Red Cross Blood Transfusion Service, SF-00310 Helsinki, Finland

RUTH NAUKKARINEN GUNNAR MYLLYLÄ JUHANI LEIKOLA

1. Ebeling F, Naukkarinen R, Leikola J. Recombinant immunoblot assay for hepatitis C virus antibody as predictor of infectivity. Lancet 1990; 335: 982-83.

813-14.

3. Buller RML, Moss B Genetic basis for vaccinia virus virulence. In: Quinnan GV Jr, ed. Vaccinia viruses as vectors for vaccine antigens. New York: Elsevier, 1985: 37-46. 4. Rodriguez D, Rodriguez J-R, Rodriguez JF, et al. Highly attenuated vaccinia virus mutants for the generation of safe recombinant viruses. Proc Natl Acad Sci USA 5. Hu

1989; 86: 1287-91. S-L, Kosowski SG, Dallrymple JM. Expression of AIDS virus envelope gene in recombinant vaccinia viruses. Nature 1986; 320: 537-40.

Exacerbations of asthma in salmeterol

patients on

SIR,-Sears and colleagues’ paper on the effects of regular fenoterol’ has prompted concern about the safety of long-term use of regular p-agonists. On the basis of a small crossover trial Sears et al proposed that long-term treatment with regular fenoterol led to a deterioration in asthma and its control. They also suggested that such an effect might apply to other p-agonists. On the basis of data on over 2000 patients during the clinical development of salmeterol, a long acting inhaled &bgr;z-agonist, we drew different conclusions (Jan 5, p 43). Nevertheless it is pertinent that these concerns are raised now, since guidelines are being drawn up worldwide for the management of asthma, coinciding with the advent of new longer acting inhaled &bgr;z-agonists. Because salmeterol controls symptoms so effectively there is a possibility that it might mask any underlying deterioration. If so, the number of exacerbations would be a useful indicator of disease control. For the purpose of this study, an exacerbation was defined as any worsening of an asthma symptom recorded as an adverse event. Serious exacerbations were defined as those that caused death, were life-threatening, or resulted in hospital admission. In several large controlled clinical trials during the development of salmeterol the number of serious exacerbations was less in patients treated with salmeterol (12%,n 4658) than in those on control treatments such as other 0-agonists or theophyllines (2%; =

n = 3466).

Safety of recombinant vaccinia vaccines SIR,-Dr Cooney and colleagues’ report (March 9, p 567) of a trial of a recombinant vaccinia vaccine is of great interest for at least two reasons other than the fact that the vaccine expresses an HIV

antigen. Their conclusion that the recombinant vaccine is safe is based on direct comparison of the effects of the vaccine and conventional smallpox vaccine; both vaccines gave similar results. Thus for the recombinant to be considered safe it follows that Cooney et al regard conventional smallpox vaccine as safe-a conclusion with which many will disagree. The side-effects of smallpox vaccination are well known,’ and most workers interested in the development of recombinant vaccinia vaccines have emphasised the need for the vector to be attenuated further if such vaccines are to gain acceptance 2’ It is not altogether fanciful to suggest that if

a

A more detailed analysis of exacerbation rates in patients treated with salmeterol has been done for one of the pivotal development studies in which inhaled salmeterol 50 Ilg twice daily (n 361) was compared with salbutamol (n 358) over one year in patients with =

=

NUMBER OF PATIENTS WITH ASTHMA EXACERBATIONS