Keith R. Perry

SENSITIVE ASSAYS FOR VIRAL ANTIBODIES IN SALIVA: AN ALTERNATIVE TO TESTS ON SERUM

Paired serum and saliva specimens were tested by conventional assays and by IgG-capture radioimmunoassays (GACRIA) and enzyme-linked immunosorbent assays (GACELISA) for antibody to hepatitis A virus (HAV, 100 pairs), human immunodeficiency virus (HIV, 53), hepatitis B virus core (HBc, 62), and rubella virus (30). Conventional assays failed to detect viral antibodies in the saliva of 93 of 119 seropositive subjects. However, GACRIA detected the antibodies in both serum and saliva of all subjects seropositive by conventional tests, except 2 saliva specimens false-negative for anti-HBc and 1 false-negative for anti-rubella-virus. For anti-HIV and anti-HBc serum and saliva GACRIA reactivities did not differ significantly, but anti-HAV and anti-rubella-virus GACRIA reactivities were stronger in serum than saliva. GACRIA and GACELISA results on saliva for the four antibodies correlated closely; for anti-HAV and anti-HIV GACELISA and GACRIA were equally accurate. For both saliva and serum, GACRIA was superior to an IgA-capture assay in detecting anti-HAV and anti-HIV.

Clinical trial with inactivated hepatitis A vaccine and recommendations for its use.

OBJECTIVE--To compare the reactogenicity and immunogenicity of an inactivated hepatitis A vaccine in two different immunisation schedules. DESIGN--Randomised trial. SETTING--One London teaching hospital. SUBJECTS--104 healthy adult volunteers (71 men, 33 women aged 19-60). INTERVENTIONS--Hepatitis A vaccine to group 1 (54 volunteers) at 0, 1, and 2 months and to group 2 (50) at 0, 1, and 6 months. MAIN OUTCOME MEASURES--Symptoms at and after each dose; liver function, hepatitis A virus specific serum immune response; and responses in saliva and parotid fluid in immunised volunteers and subjects with natural immunity. RESULTS--The vaccine was well tolerated; 97% (96/99) and 100% of those immunised developed serum antibody after one and two doses of vaccine respectively. Geometric mean titres increased progressively after each dose and were significantly higher in men but not women in group 2 after the third dose (ratio between geometric mean titres 0.265, 95% confidence interval 0.18 to 0.39; p less than 0.001). At one year this group-sex interaction was absent; geometric mean titres for both sexes were significantly higher in group 2 (ratio 0.330, 0.227 to 0.478; p less than 0.0001). Antibody responses were not significantly different between the groups at two years. Compared with naturally infected subjects immunised volunteers developed poor or undetectable virus specific IgG and IgA responses in saliva and parotid fluid. CONCLUSIONS--The vaccine was safe and highly immunogenic, and the differences in the immune responses in saliva and parotid fluid are unlikely to affect its efficacy.

Risk of hepatitis A infection in sewage workers

OBJECTIVE: To evaluate the risk of hepatitis A virus (HAV) infection among sewage workers from occupational exposure to raw sewage. METHODS: An analytical cross sectional study of 241 company employees with possible occupational exposure to sewage in a large water and sewerage company was carried out. Previous exposure to hepatitis A virus infection was assessed, as were its associations with possible risk factors. RESULTS: Frequent occupational exposure to raw sewage was a significant risk factor for HAV infection, independently of other known risk factors (odds ratio 3.73, 95% confidence interval 1.48 to 9.37). Of 50 employees who reported occupational exposure to raw sewage most of the time, 30 (60%) had had HAV infection. CONCLUSION: Employees who are likely to be at risk of frequent exposure should have their immunity ensured. The salivary assay for IgG anti-HAV used in the study was highly specific and would be suitable for prevaccination testing of older employees, who are more likely to be immune.

Effect of hepatitis A vaccination schedules on immune response

An inactivated hepatitis A vaccine was given to 104 seronegative volunteers aged between 19 and 60 years according to two schedules: 0, 1 and 2 months or 0, 1 and 6 months. The vaccine was well tolerated and 97 and 100% of vaccinees developed a serum antibody response following a single and two doses of vaccine respectively. Geometric mean titres increased progressively after each dose; responses following the 0, 1, 6 month schedule were significantly higher at one year but, among those tested at two years, these differences were less marked. Vaccinees, when compared with naturally infected persons, developed poor or undetectable hepatitis-A-virus-specific immunoglobulin G and A antibody responses in saliva and parotid fluid. Such differences are, however, unlikely to affect the protective efficacy of the vaccine.

THE INFLUENCE OF DENTAL STATUS ON THE DETECTION OF IgG CLASS ANTI-VIRAL ANTIBODIES IN HUMAN SALIVA

An antibody-capture radioimmunoassay was used to measure levels of IgG class antibodies to rubella and hepatitis A viruses in serum and saliva of 30 edentulous, 30 partially dentate and 31 dentate individuals. The prevalence of seropositivity for rubella was 98.9 per cent and for hepatitis A 73.6 per cent. The serum reactivities were generally greater than those for saliva. There were 8 false-negative results for saliva out of the 182 tests performed, of which 4 were in the edentulous group, 3 in the partially dentate and 1 in the dentate group. For both rubella and hepatitis A virus antibodies the (geometric) mean ratios between the saliva and serum reactivities were similar across the three dental groups. The values for sensitivity, specificity and positive predictive value suggest that assay of saliva for antiviral IgG antibody is a satisfactory technique regardless of dental status.

Variation in the sensitivity of HBsAg screening kits

Summary. Fifteen HBsAg kits from 14 manufacturers were assessed. Their sensitivity was evaluated by testing 150 HBsAg‐positive sera, sera from four donors who were low‐level HBsAg carriers, and sequential specimens from 22 seroconverting individuals together with dilutions of six of these specimens. The British HBsAg Working Standard (0.5IUmL‐1) and the NIBSC/UKBTS HBsAg Monitor Sample (0.125IUmL‐1) were also tested. Five assays failed to detect one of the 150 routine HBsAg‐positive sera. Four assays (Auszyme Monoclonal; Monolisa Ag HBs 2nd generation; Murex HBsAg; Ortho HBsAg Test Systems 3) were able to detect HBsAg in all but one of the six sera from low‐level carriers, whereas one assay (MicroTrak II HBsAg) detected only one of the six. The most sensitive kit (Monolisa Ag HBs 2nd generation) detected HBsAg in 79 specimens from the seroconversion panels; four other kits detected HBsAg in at least 70 specimens, seven in 60–69, two in 50–59 and the least sensitive in 31. Further analysis of the findings on seroconverters indicated a median reduction in the duration of HBsAg detection of 5 days or more for four assays when compared with the most sensitive assay. One kit (Auszyme Monoclonal) detected HBsAg in 15 of the 18 dilutions prepared from the seroconversion specimens, whereas three kits detected HBsAg in fewer than 10 dilutions. Two kits gave negative reactions with the British HBsAg Working Standard on all of five occasions and six were consistently unreactive with the NIBSC/UKBTS HBsAg Monitor Sample; only three kits (Bioelisa, Enzygnost, Murex) were always reactive. There is therefore substantial variation in sensitivity among the HBsAg kits currently available.

False negativity by an anti-HIV assay kit (IMx 8B32) and evaluation of its replacement (IMx 8C98)

False negativity in a commercial anti‐HIV kit (IMx HIV‐1/HIV‐2 3rd Generation Plus (code 8B32) was investigated, and the kit that superseded it (IMx HIV‐1/HIV‐2 III Plus, code 8C98) was evaluated. In a comparison on 574 freshly collected anti‐HIV‐1‐positive specimens, 97.2% were more reactive in 8C98 than in 8B32; 35.5% were more than twice as reactive and 8.5% were more than four times as reactive. In 8B32, the signal from 55 specimens selected because of weak reactivity was enhanced 1.5 to 8.8 times by preliminary heating at 56°C for 30 min. The reactivity of the 55 heated sera was then similar to that of the same specimens tested without heat treatment in the 8C98 assay. Reactivity in 8B32 was also increased in 66 of 76 (at least twofold in 20) randomly chosen anti‐HIV‐positive serum specimens by the addition of EDTA (10 mM final concentration). One of these specimens was false negative (signal:cutoff (S:CO) ratio 0.76) in 8B32, though its reactivity was restored by addition of EDTA (S:CO ratio 9.54). These findings indicate that the inhibitory effect that originally led to false negative findings in 8B32 was probably due to complement activity, and that the same activity was present in the freshly collected specimens used here to evaluate the replacement IMx anti‐HIV assay (8C98). The specimen panel employed to evaluate 8C98 included 1,892 anti‐HIV‐positive and 779 anti‐HIV‐negative specimens. There were no false negative reactions. The lowest S:CO ratio observed was 6.2 and only 17 (0.2%) anti‐HIV‐positive specimens gave ratios less than 10. Nine unreproducible false positive reactions arose, all possibly attributable to specimen carryover by the IMx instrument. The performance of 8C98 was also compared with that of 10 other current anti‐HIV kits using 21 sets of seroconversion specimens (127 specimens in total), and five performance assessment panels (92 specimens in total) comprised mostly of single bleeds from recent seroconverters. IMx 8C98 was the second most sensitive assay. We found no evidence that the 8C98 kit was prone to the effect that had given rise to false negative results in its predecessor (8B32). J. Med. Virol. 56:138–144, 1998.