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Background: Serological markers such as HBsAg and anti-HIV may be present in serum at very high concentrations and this may give rise to erroneous diagnoses due to cross-contamination., Objectives: To investigate poor equipment maintenance and use, including contamination with human serum, as a potential source of erroneous assay results., Study Design: The potential of microtitre plate washers and micropipettors to transfer material between microplate wells and between specimens was examined. For the study of micropipettors we recruited 19 UK diagnostic laboratories., Results: Four out of seven plate washers in use, until adjusted, had the potential to cause false positive HBsAg reactions. The centering of the probes that delivered the wash fluid, delivery pressure, wash volume and the use of a pre-programmed card to direct the washing procedure were important variables. We investigated soiling of tip cones of micropipettors. In every laboratory human IgG could be detected in at least a third of eluates from micropipettor tip cones; only 31 (14%) of 222 showed no evidence of contamination with human serum. Only one laboratory submitted eluates devoid of specific antibodies. Anti-HAV was the marker most commonly found (n = 68), followed by HBsAg (n = 27) and anti-HIV (n = 20). Seven micropipettor eluates from two laboratories were radioactively contaminated., Conclusions: Recommended precautions are regular checking, cleaning and servicing of equipment, care in interpreting weak reactions, reference back to serum left on the clot of the original specimen and testing of a follow up specimen. Poorly maintained immunoassay equipment can readily generate false positive results due to low-level cross-contamination, particularly with the current highly sensitive HBsAg and anti-HIV assays.

Background: Babies born to HIV-infected mothers retain anti-HIV of maternal origin until 15-18 months of age. Because of this, HIV proviral DNA and p24 antigen measurements have become the methods of choice for timely diagnosis of HIV infection in infancy. They are, however, too expensive for widespread use in the developing world., Objective: To evaluate a simple, inexpensive serological method for diagnosing mother-child transmission of HIV, in an African population, which takes account of the effects of placental transfer of maternal antibody and continued exposure to HIV through breast-feeding., Study Design: Plasma specimens for a prospective study of mother-to-infant transmission of HIV in rural Zaire were collected at birth, 3, 6, 9, 12, 18 and 24 months from 21 infected infants (PP group), 21 uninfected infants (PN group) born to seropositive mothers and 21 control infants (NN group) born to uninfected mothers. The specimens were retrospectively tested for IgG, IgM and IgA anti-HIV by immunoglobulin class-specific capture EIAs, and by a commercial anti-HIV EIA., Results: In neonatal specimens, IgA and IgM anti-HIV were present, respectively, in 13 of 14 (97%) and 8 of 14 (57%) of the PP group and in 6 of 11 (55%) and 2 of 11 (18%) of the PN group. Later, at 3 months and older, IgA and IgM anti-HIV were only detected in the PP group. They peaked at 18 months (93%) and 24 months (67%) respectively. Of the 21 PP group children, 8 (38%) were transiently IgG anti-HIV-negative in the first year, indicating that infection had probably taken place after birth; four of the 8 had no detectable IgA anti-HIV during the first year. None of the specimens collected from the NN group babies were reactive for IgA, IgM or IgG anti-HIV., Conclusions: IgA and IgM anti-HIV may be passively transferred across the placenta. Where breast-feeding is prevalent, about half of the transmissions may occur after birth, thus delaying the diagnosis of mother-child transmission. Nevertheless, this simple, cheap IgA anti-HIV, EIA identified 65% of transmissions by 9 months of age, and 93% at 18 months of age. It is a more useful marker than IgM anti-HIV, and gave a much more rapid answer than did tests for IgG anti-HIV seroreversion.

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.

Background: The possibility that saliva could be used for HIV screening and diagnosis has been known since 1986. Despite the obvious advantages over venepuncture of ease of collection, safety, compliance and cost, interest in salivary testing has grown relatively slowly. Several studies have demonstrated that salivary anti-HIV testing can be highly accurate, particularly if specimen collection procedure are optimal., Objective: To review current knowledge about the detection of anti-HIV in oral fluids, with an emphasis on the identification of optimal procedures., Study Design: In the light of existing published data, the factors leading to accurate salivary diagnosis of HIV infection were identified and reviewed., Results: To achieve the best results it is essential to collect oral fluid specimens that are rich in IgG. Most IgG in the oral cavity derives from the crevicular space between the gums and the teeth, and not from salivary glands. Available methods for collecting salivary specimens are discussed. Until these collection methods are fully validated, individual specimens or at least clinical ones found anti-HIV negative should be tested for total IgG before being reported on. There is a lack of proven confirmatory methods for salivary anti-HIV and this problem is reviewed. Salivary anti-HIV testing has been employed mostly for surveillance, but life insurance applicants are increasingly screened in this way and clinical applications are under active consideration. With appropriate safeguards, diagnostic and pre-blood donation salivary testing could be introduced shortly. The necessary technology is also available to develop rapid single-use salivary tests. This would bring anti-HIV testing closer to the patient., Conclusions: Salivary tests for anti-HIV offer advantages of convenience, economy and safety, and are more acceptable to subjects than blood tests. Further evaluation of the collection devices and assays, the introduction of safeguards against inadequate sampling and the development of suitable confirmatory assays are required. When these deficiencies have been met, salivary tests may supersede tests on serum for HIV and also other infections.

To determine whether there is a delay between the appearance of anti-HIV in serum/plasma and its detection in saliva and urine, salivary and urine specimens were collected from nine individuals who, on the basis of increasing IgG anti-HIV reactivity, Western blot band patterns and presence of strong IgM anti-HIV reactivity in their serum specimens, were believed to have recently become anti-HIV-positive. Serum from 8 of these patients and 3 commercial panels of plasma specimens collected during seroconversion were diluted to mimic the low immunoglobulin concentrations present in saliva and urine and tested in Wellcozyme HIV 1 + 2 GACELISA and four commercial EIAs intended for testing serum specimens. The 9 pairs of saliva and urine specimens were collected between 4 and 43 days (median 24 days) after the first evidence of seroconversion. All were reactive by Wellcozyme HIV 1 + 2 GACELISA and gave optical density/cut off (OD/CO) ratios in the range 3.8 to 9.8 (median 5.2) for dribbled saliva and 2.4 to 10.1 (median 6.3) for urine. Salivary specimens taken with commercial collection devices gave OD/CO ratios in the range 1.6 to 10.6 (median 5.9). In the serum/plasma specimens Wellcozyme HIV 1 + 2 GACELISA detected anti-HIV at higher dilutions than the other assays, often with a 100-fold or more difference. Saliva and urine specimens were all strongly reactive by Wellcozyme HIV 1 + 2 GACELISA. We therefore predict that it would first detect anti-HIV in salivary and urine specimens at about the same time as it becomes detectable in serum/plasma but that other commercial EIAs would not.

The serological reaction to HIV infection is almost invariably a dynamic progression towards strong reactivity to a wide range of viral antigens. Serological diagnosis should therefore be based either on the presence of this wide range in a single specimen or on the demonstration of increasing activity between two specimens collected at an interval of two or more weeks and tested in parallel. When a specimen is reactive a second should in any case be collected to check the identity of the first. These precepts are sometimes being ignored, especially in non-clinical testing, e.g., for purpose of life assurance. Two recent cases in which there were false but unchanging enzyme immunoassay and Western blot reactions that might have been interpreted as positive illustrate the potential for error.

The accuracy and ease of use of three automated test systems (Behring Opus, Boehringer Mannheim ES300 and bioMerieux Vidas) and their associated assays (Opus HIV 1 + 2, Enzymun-Test Anti-HIV 1 + 2 and Vidas HIV 1 + 2) for the detection of anti HIV-1 and anti HIV-2 in human serum and plasma were investigated by testing them against a panel of 364 well-characterised specimens. The panel included specimens confirmed to contain anti HIV-1 (n = 65) and anti HIV-2 (n = 25), specimens collected during HIV-1 seroconversion (n = 40), specimens (n = 116) which had given spurious reactions in a variety of anti-HIV screening assays, specimens from patients infected with anti HTLV-I (n = 18) and specimens from unselected blood donors (n = 100). Excluding the seroconversion specimens, all three assays were 100% sensitive. Observed specificities ranged from 98.3% to 99.6%. For the seroconversion specimens the Vidas HIV 1 + 2 was the most sensitive overall. All three assays were as sensitive and specific as most of the conventional anti-HIV enzyme immunoassays currently available. The Opus and Vidas systems would be convenient for clinical laboratories handling small to medium numbers of specimens per day. The Boehringer Mannheim ES300 is designed for larger scale use and required more familiarisation.

Serum specimens (n = 17668) from UK antenatal patients in the Thames Regions were tested by Wellcozyme HIV 1/2 EIA singly and in pools of 6, 12 and 24: 35 (0.2%, 1 in 505) were confirmed as anti-HIV positive. The pools of 12 were also tested for anti-HIV 1/2 by IAF Biochem, Behring and Diagnostics Pasteur EIAs. All 35 positive specimens were easily detectable after pooling in groups of 12. The false positive rate for Wellcozyme was nearly halved compared with individual testing (1 in 309 false positive compared with 1 in 174). For the other assays false positive rates on pools of 12 were: IAF Biochem 1 in 193, Behring 1 in 140, Diagnostics Pasteur 1 in 1547. Twenty-two known anti-HIV 2-positive sera were detected by all four EIAs when diluted as in pools of 6 and 12, but by only three EIAs in pools of 24 and 48. Pooling in groups of 6 did not seem to delay detection of HIV 1 seroconversion, but pooling in groups of 12, 24 and 48 might delay it by 1, 2 and 3 weeks respectively. For this study the effect of pooling in groups of 12 would have been a reagent saving of 87-91% and a labour saving of about 50%. Because of the low HIV incidence and rarity of specimens collected around seroconversion in UK, little, if any, loss of sensitivity would result from it. Pooling in groups of 12 has therefore been chosen for the screening of anonymous antenatal specimens in the UK.