Improvement in the performance of HIV screening kits.

SUMMARY: The aim of this study was to assess the performance of HIV screening kits introduced over a 12-year period. HIV kits used by the National Blood Service (NBS) were assessed in the context of other HIV kits employed by diagnostic and reference laboratories. Thirty-three HIV screening kits were assessed and 13 had the potential to be used by the NBS. Specimens applied to NBS evaluations included 2000 HIV-negative specimens collected from blood donors, 200 HIV-positive specimens and 21 seroconversion panels, with larger numbers applied to the latter two categories prior to implementation of Communauté Européennes (CE) marking. The 33 HIV kits gave repeat reactive rates, based on HIV-negative specimens, of between 0% and 0.8% (and between 0% and 0.2% for kits relevant to the NBS). When examined for diagnostic sensitivity, the 33 kits gave sensitivities between 99.78% and 100%. Kits relevant to NBS gave sensitivities of 100% except one kit, which failed to detect one anti-HIV-2-positive specimen. Twenty-six kits were compared for detection of primary HIV infection. Of these, the 10 combined HIV antigen/antibody kits examined were more sensitive than other formats and have been exclusively adopted by NBS where operational considerations allow. Their added seroconversion sensitivity makes them the screening method of choice for populations at increased risk, e.g. in sexually transmitted infection (STI) clinics. The regular review of evaluation results has demonstrated a continuing improvement over time in the performance of HIV screening kits and contributed to advances in blood safety.

Comparative evaluation of 15 serological assays for the detection of syphilis infection.

Fifteen commercial syphilis kits were assessed against the same moderately sized specimen panel that included 114 serum and plasma specimens from syphilis cases and 249 specimens from unselected blood donors. The 114 specimens from syphilis cases comprised 40 from cases of primary syphilis, 43 from cases of secondary syphilis, 19 from cases of early latent syphilis, and 12 from cases of late latent syphilis. Of the 15 kits, ten were enzyme immunoassays, four were Treponema pallidum haemagglutination assays, and one was a T. pallidum particle agglutination assay. Thirteen of the 15 kits gave final specificities of 100%; the other two kits were repeatedly reactive with one to two specimens. Initial sensitivities ranged from 93.9 to 99.1%. Most variation between kits was observed in results for the groups with untreated primary and treated late latent disease, although the differences were not statistically significant. The comparative data on kit performance derived from this study is useful for examining syphilis testing guidelines and for making informed purchasing decisions.

Towards error-free HIV diagnosis: guidelines on laboratory practice.

Error can have multiple causes. These guidelines set out the most common reasons for inaccuracies in HIV testing and indicate how they can be avoided. Emphasis is placed on laboratory procedures as during over 15 years experience they, rather than the kits and reagents, have proved to be the most frequent source of error.

Simple/rapid test devices for anti-HIV screening: do they come up to the mark?

Thirteen simple/rapid test devices (S/RTDs) for the detection of antibodies to HIV 1 and HIV 2 were assessed. Ninety-two specimens in four categories were used and results with the thirteen S/RTDs were compared with those obtained with six currently available commercial laboratory-based anti-HIV 1/2 EIAs. Seven of the 13 S/RTDs scored all 26 blood donors' specimens as unreactive, and 11 correctly identified all the 25 "straightforward" anti-HIV positive specimens. False negative results arose when testing by Uni-Gold HIV and SeroCard HIV, which gave 72 and 68 correct positive observations, respectively, out of 75. No S/RTD detected seroconversion earlier than the most sensitive EIAs, but four S/RTDs performed similarly to most of the EIAs. On the low-titre panel specimens, six S/RTDs were less sensitive than the least sensitive EIA and, in contrast to four of the six EIAs, only one S/RTD was able correctly to identify all the positive specimens. A manufacturing problem was identified that allowed the HIV antigen-sensitised area on the membrane of two SeroCard HIV devices to be misaligned with the device's reading window so that the reaction was almost entirely obscured. As long as small numbers of specimens were involved, most S/RTDs required considerably less time and less equipment than EIAs, but overall they were slightly less sensitive. Their use in various health settings and for confirmatory procedures is discussed.

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 degrees 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).

Variation in the sensitivity of HBsAg screening kits.

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 (0.5 IU mL-1) and the NIBSC/UKBTS HBsAg Monitor Sample (0.125 IU mL-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.

A collaborative case control study of sporadic hepatitis A in England.

A case control study of sporadic hepatitis A was carried out in 201 districts in England from July 1990 to June 1991. The aims were to determine the risk factors associated with the infection and to identify individuals or groups who might benefit from prophylactic measures, such as human normal immunoglobulin or hepatitis A vaccine. Factors associated with risk of hepatitis A included travel abroad (odds ratio (OR) 19.8; 95% confidence interval (CI) 4.87-80.6), a household contact with hepatitis A (OR 13.5; 95% CI 6.49-28.0) and sharing a household with a child aged 3 to 10 years (OR 1.57; 95% CI 1.1-2.22). This study provided no clear evidence of increased risk in health care workers, teachers, or other occupational groups. A non-significant trend towards an increased risk in nursery nurses and child minders aged 20 to 29 years was observed. Pre-exposure prophylaxis with hepatitis A vaccine may be considered for people who travel frequently to areas where hepatitis A is highly or moderately endemic. Post-exposure prophylaxis with human normal immunoglobulin should be given to contacts of known cases in accordance with national guidelines. Immunoglobulin alone has been recommended in outbreaks, but the use of vaccine alone or combined with immunoglobulin should be evaluated.

Investigation of a hepatitis A outbreak in a primary school by sequential saliva sampling.

BACKGROUND: An outbreak of hepatitis A occurred in a primary school (children aged 4-11 years), starting in the Autumn of 1990 and terminating some 5 months later after some spread into the local community. OBJECTIVES: The objectives were to monitor the spread of the virus within the primary school over time, to document infection in asymptomatic individuals and the efficacy of using saliva for HAV antibody detection in young children as an acceptable screening method by using the Salivette method and ordinary cotton tipped swabs. STUDY DESIGN: Serial saliva samples were taken over a period of months and anti-HAV IgM and IgG antibodies measured by radioimmunoassay. RESULTS: Twenty-seven children in the school and nine individuals from the surrounding community acquired hepatitis A. Twenty-one (78%) of the 27 children were symptomatic, as were all the affected adults. The cotton-tipped swabs were found to be as effective a method as Salivette at diagnosing infection in those in whom the methods were compared. CONCLUSIONS: Despite many reports stating that children are more likely to be asymptomatic with HAV infection we found the majority to report significant symptoms. Young children do not easily accept serum sampling as a method for diagnosis or epidemiological studies, and we show that saliva sampling is an effective and acceptable diagnostic method.

The risk of hepatitis A from sewage contamination of a water supply.

The drinking water supply of a town became contaminated with sewage. The town's population was surveyed to determine the baseline prevalence of hepatitis A antibodies and to establish whether an associated outbreak of hepatitis A had occurred. Samples of saliva were obtained from 540 people in 200 randomly selected households, and tested for IgG and IgM antibodies to hepatitis A. Fifty-six per cent (279/495) were susceptible to hepatitis A and 43% (213/495) immune as a result of previous infection. Immunity was directly related to age; those who were immune were significantly older than those who were susceptible (mean ages: 43.5 years and 19.0 years; p < 0.0001). Six people were found to have had a recent infection with hepatitis A, but exposure to hepatitis A during the water pollution incident was possible in only one case. The results offer no evidence that this incident caused an outbreak of hepatitis A, but the study has provided useful epidemiological data on hepatitis A.

HIV infection in injecting drug users attending centres in England and Wales, 1990-1991.

OBJECTIVE: To monitor trends in HIV infection and associated risk behaviours in injecting drug users (IDU) in England and Wales. DESIGN: Ongoing voluntary unlinked anonymous cross-sectional survey. METHOD: IDU attending centres in 1990 and 1991 were invited to complete a brief questionnaire requesting demographic and behavioural information, and to provide a saliva sample to be tested for antibodies to HIV and to the core antigen of hepatitis B virus (HBV). RESULTS: In 1990, 1.2% (19 out of 1543) of samples from 33 centres, and in 1991 1.8% (25 out of 1417) of samples from 37 centres contained antibody to HIV. Antibody t9 HBV core-antigen was found in 33 and 31% of IDU in 1990 and 1991, respectively. The prevalence of HIV infection in IDU attending centres in London (4.2%) was higher than in those attending centres elsewhere (0.8%). The prevalence of HIV infection in 1991 varied between individual centres from 0 to 10.6%, and at many centres outside London no IDU were infected with HIV. In the same year the prevalence of past infection with HBV varied from 14 to 54%, and IDU who had evidence of HBV infection were found among attenders in nearly all centres. The prevalences of sharing injecting equipment and risky sexual behaviour were high at many centres. The prevalence of HIV infection was higher in IDU who had started to inject in 1985 or earlier, than in those who started injecting later. In each year, approximately half the IDU surveyed reported having had a voluntary confidential HIV-antibody test, and the prevalence of HIV infection was five times higher in those tested than in those who had not been tested. CONCLUSIONS: HIV prevalence in IDU attending centres in England and Wales was low in 1990-1991. There is some indication that IDU have modified their injecting or sexual behaviour, but even at existing reduced levels of risk behaviour, transmission can occur in HIV is introduced into previously unexposed groups.

Detection of measles, mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay.

Antibody capture radioimmunoassays were developed for detecting virus specific IgM (MACRIA) and IgG (GACRIA) to measles, mumps, and rubella and used to investigate saliva as an alternative specimen to serum for diagnosis. Saliva was collected from 63 patients with measles, 19 with mumps, and 150 with rubella, which were all clinically diagnosed and serologically confirmed. Virus specific IgM was detected in 92% of measles, 75% of mumps, and 100% of rubella saliva samples collected during the first week of illness. Between 1 and 5 weeks after onset virus specific IgM was detected in 100% of saliva specimens. After the 5th week the proportion of reactive specimens declined. The specificity of the MACRIA tests was established by testing saliva samples collected from blood donors for measles (88), mumps (88), and rubella IgM (91). All of the saliva specimens tested for measles and rubella specific IgM were unreactive, 1/88 specimens tested for mumps specific IgM contained significant reactivity. Saliva specimens collected from acute cases of MMR were tested in all 3 MACRIAs. A small proportion of saliva samples contained detectable IgM of more than one virus infection. Rubella and measles specific IgG was detected in the saliva of all cases from the 4th or 5th day of illness, respectively. Detection of mumps specific IgG was less successful. We have demonstrated that virus specific IgM can be reliably detected in saliva samples collected from acute cases of measles, mumps, and rubella and identified 1-5 weeks after onset of illness as the optimum time for collection of samples.

Simple haemadherence test for the detection of class-specific immunoglobulins to hepatitis A virus.

The ability of hepatitis A virus (HAV) to agglutinate human erythrocytes was used to develop IgM and IgG antibody capture haemadherence tests (MACHAT and GACHAT). Haemadherence was dependent on the pH of the red cell suspension and was best in the pH range 5.4 to 5.8. The tests were applied to serum, urine, and saliva specimens from individuals susceptible to, or with recent or past infection with HAV. Haemadherence test reactivities were compared with results obtained with IgM and IgG antibody capture radioimmunoassay (MACRIA and GACRIA) and competitive radioimmunoassay (COMPRIA). For 339 serum specimens examined, the sensitivity and specificity of MACHAT were 98.2% and 99.6%, respectively, and of GACHAT 99.1% and 100.0%. For 303 urine specimens, the sensitivity and specificity of MACHAT were 99.1% and 100.0%, and of GACHAT 100% for both. On initial testing, accuracy on saliva specimens was considerably less. For 2,819 saliva specimens, the sensitivity and specificity of MACHAT were 85.7% and 97.2% and of GACHAT 90.4% and 94.7%. The haemadherence test is a simple, inexpensive method which is satisfactory for use on serum and urine specimens. MACHAT and GACHAT can be used for epidemiological investigations, e.g., hepatitis A outbreaks and, in conjunction with a confirmatory test, for clinical diagnostic testing.

The detection in urine specimens of IgG and IgM antibodies to hepatitis A and hepatitis B core antigens.

The use of urine as a noninvasive specimen for the diagnosis of hepatitis A (HAV) and hepatitis B (HBV) virus infections was investigated. Specimens of urine were collected at the same time as blood or saliva specimens, or singly in cases of previously serologically confirmed recent infection. The specimens were tested for IgG and IgM anti-HAV and anti-HBc by immunoglobulin class-specific capture radioimmunoassays (GACRIA and MACRIA). On the basis of assays on urine specimens it was possible to distinguish between individuals who were susceptible or immune to HAV or who had recently been infected with HAV. Using assays on 327 corresponding saliva specimens as reference tests, the observed sensitivity and specificity of tests on urine specimens by anti-HAV GACRIA were 98.9% and 99.1%, respectively, and by anti-HAV MACRIA were 95.8% and 99.6%, respectively. IgM and IgG anti-HBc were detected readily in the urine of 35 acute or recent cases of hepatitis B but were not found in the urine of seronegative individuals. Of the urine specimens from 52 individuals who were HBsAg carriers or who had had long past HBV infections, 49 contained detectable IgG anti-HBc. Of urine specimens from 42 HBsAg carriers, 11 contained raised IgM anti-HBc levels. Urine, which is a convenient specimen to collect, can be used to study outbreaks of hepatitis A, to ascertain the HAV immune status of individuals, to differentiate hepatitis A from hepatitis B, and to identify individuals who have been naturally exposed to HBV.

Salivary antibody testing in a school outbreak of hepatitis A.

During a community-wide outbreak of hepatitis A in Gloucester, UK there was a high attack rate in children attending two city primary schools and a pre-school centre sharing the same site. In September 1990, saliva specimens were collected from 478 (85%) of the 562 children. The prevalence of antibody to hepatitis A virus (anti-HAV), as determined by saliva testing, was 29.6%; highest prevalences were seen in 5-6-year-olds and in children from that area of the city at the centre of the community-wide outbreak. The proportion of immune children with a history of clinical hepatitis varied with age from 1 in 42.7 of under-5-year-olds to 1 in 4.7 of 8-10-year-olds. Six children who received prophylaxis with human normal immune globulin (HNIG) because they were household contacts of cases subsequently became infected. Since there was evidence of transmission outside the school environment it is unlikely that a policy of universal prophylaxis within the schools would have stopped the outbreak. Mass prophylaxis in school outbreaks is only likely to be effective if most transmission is occurring at school and if the target population can be clearly defined. Salivary antibody testing is a simple, practical and acceptable procedure in young children. Salivary antibody surveys in conjunction with vaccination against hepatitis A should provide a cost-effective method for control of future outbreaks.

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.

An outbreak of hepatitis A: school toilets as a source of transmission.

An outbreak of hepatitis A associated with a Middle school involved 23 cases; 17 were pupils attending the Middle school, one was a teacher, one was a relative of a case, and four were from the associated First school, of whom three had siblings in the Middle school. The probable source case was a male pupil infected by a sibling who had contracted hepatitis A while abroad on holiday. A questionnaire survey and salivary IgG and IgM anti-HAV testing of the pupils demonstrated a statistically significant association between infection and the use of a changing room toilet for defecation. An inspection of the school showed that toilets lacked toilet paper, soap and hand towels. Advice was given to pupils, parents and staff on hygiene. Human normal immunoglobulin was administered to susceptible family contacts, pupils and staff at the school. The school outbreak might have been prevented if the source case for the school had been given immunoglobulin when his sibling developed hepatitis A.

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.

Hepatitis A outbreak involving bread.

An outbreak of hepatitis A involved more than 50 residents of a group of villages in the late spring and summer of 1989. The only food that was common to all the laboratory-confirmed cases was bread, purchased either unwrapped or as rolls, sandwiches or filled rolls, and supplied either directly from one shop or indirectly through its subsidiary outlets. It was concluded that this bread was the most likely vehicle of transmission of the hepatitis A virus and that the bread was contaminated by soiled hands which were inadequately washed because of painful skin lesions. Comprehensive control measures were successful in limiting further spread of the infection. This outbreak highlights the transmissibility of hepatitis A virus on food. The use of disposable gloves when handling food which is to be consumed without further cooking would prevent transmission of this or other infectious agents by this route.

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.