Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

766 Abstracts 41117-41121

12th World AIDS Conference

Objective: To evaluate interlaboratory variation with a universal SPT template
on T-cell subset percentages among the Clinical Trial Network laboratories.
Method: A new analytical template was developed to fit clinical flow cytometers in use in Canada both for 3 and 4-color applications. This novel template
is based on fluorescence triggering and double anchor gating of CD45/CD3.
The recommended monoclonal antibody combinations were CD45/CD3/CD4 and
CD45/CD3/CD8 or CD45/CD3/CD4/CD8 for 3 and 4-color applications respectively. Two multi site surveys were launched to evaluate the interlaboratory
variation. The new template was compared to the traditional light scatter or
CD45 gating templates. On each occasion three fresh blood specimens, HIV
positive and negative, were analyzed by all laboratories utilizing both the new and
traditional templates.
Results: The average standard deviation (AvegSD) of the first survey was 2.2
and 2.5 for the conventional and new approach respectively. The AvegSD of the
second survey was 2.5 and 2.8 respectively.
Conclusion: The universal SPT template is effective. It provides both absolute
and percentage of T lymphocyte subsets without compromising the excellent
reproducibility the CTN has already obtained with the conventional template.
41117 1 Sensivity evaluation of Vironostika Uni-Form II plus 0
with samples from Thailand
Thippawan Chuenchitra. 'AFRIMS 315/6 Rajvith Rd., Bangkok 10401, Thailand
Objective: To determine the sensitivity of Vironostika HIV Uni-Form II plus O with
subtyped samples from Thailand.
Materials & Methods: A panel of 178 samples known to be positive for HIV
antibody (Abbott screening test, Cambridge Biotech Western Blot) was tested.
Samples were collected from subjects who referred to the Joint Clinical Research
Center, Bangkok, Thailand. Based on differential PCR, 170 samples were subtype E and 8 were subtype B. To assess its sensitivity, samples were tested with
Vironostika HIV Uni-Form II plus 0 (Organon Teknika, Boxtel, The Netherlands).
Results: All 8 subtype B and all 170 subtype E serum samples tested positive
with Vironostika HIV Uni-Form II plus 0.
Conclusion: Vironostika HIV Uni-Form II plus 0 correctly identified anti-HIV
positive samples from Thai individuals infected with HIV-1 subtypes B and E.
These subtypes are commonly found in Thailand. The results support its use as
a HIV antibody screening test in this country.
S41118 HIV-screening with the Elecsys~ automated analyser:
Testing for HIVAg within 18 minutes with a sensitivity
of 3 pg/ml
Urban Schmitt, D. Schlieper, B. Upmeier, W. Melchior, E. Faatz. Boehringer
Mannheim GMBH, Nonnenwald, Penzberg, Germany
Background: Testing for HIVAg normally takes more than 2 hours and consumes
at least 100 jl of sample. Also most of the HIVAg assays have to be performed
manually using microtiter plates, introducing the risk of mixing up samples. We
therefore developed a HIVAg assay for the Elecsys~ automated analyser with the
very short reaction time of 18 minutes and positive sample identification.
Methods and Results: In order to achieve broad HIV-subtype reactivity we
selected 4 monoclonal antibodies, reactive with recombinant p24 and virus culture
material of the subtypes A to I and 0. The four antibodies are not competing
with each other since they each recognise different epitopes of p24. By using
two of the anti-p24 MAbs in Biotin-labelled form together with Streptavidin coated
magnetic particles and two of the anti-p24 MAbs in Ruthenium-labelled form (for
the Electrochemiluminescence detection), we could establish a HIVp24Ag assay
with a total assay time of 18 minutes, a sample volume of 50 pl and a sensitivity
of 3 pg/ml.
First results with the Elecsys" HIVAg (Boehringer Mannheim GmbH) show
that the assay is earlier in the detection of HIV infection compared to the best
HIVAg assays presently available. The Elecsys~ HIVAg also has a better HIV 1
subtype reactivity (tested with viral culture supernatants) and some HIV 2 p26
crossreactivity. To enhance sensitivity with samples that might contain immune
complexes, an acid pretreatment to dissolve the immune complexes is possible.
Specificity tested with blood donors and samples of hospitalised patients was
found to be >99.7% (initially reactive) and 100% (confirmed by neutralisation
assay).
Conclusion: The Elecsys~ HIVAg is a fully automated and very rapid assay
with the high sensitivity of 3 pg/ml and a very good specificity.
41119 Anti HIV1/2 mass testing, what should be done?
Alexandre M. Oliveira1, C.C. Hofling2, M.C. Ramos2, S.A.A. Chaves3. 1 R.
Uruguaiana, 552 Apto. 41, 13026-001-Campinas-S. R, 2Universidade Estadual
de Campinas, Campinas; 3Hospital Das Clinicas-Unicamp, Campinas, S.P,
Brazil
Objectives: In order to evaluate the need for subsequent tests to establish HIV

infection diagnosis, a large population was tested against HIV1/2 antibodies using
both ELISA (DAGS) and WESTERN BLOT (WB) analysis.
Methods: Two sources of individuals was used, a large general outpatient/inpatient care hospital, and an anonymous testing service. A total of 15526
patients was screened. A qualitative result of the ELISA was defined as a function
of the cut off (>1-positive; <1 negative; >0.95 and <1 intermediate). The same
quantitative data was also used to calculate the crude probability of a given result.

WB results were based upon what bands were present and defined as positive,
probable positive, probable negative, negative, and indeterminate. The overall
prevalence of infection as given by the ELISA was 0.82. The ELISA predicted
0.957 of the cases as defined by the WB, and ruled out 0.984 of the non-infected
persons.
ELISA Western Blot
Positive Prob positive Prob negat Negative Indeterm Total
Positive 245 69 1 6 7 328
Negative 1 1 52 9 63
Indeterm. 5 2 7
ELISA-DAGS distinguished very well the infected from the non-infected population. The vast
majority of the cases (99.72%) had low (<0.9) or high (>10) absorbance/cut off ratios. Only a
small number (0.28%) had low antibody titers as predicted by the result of the absorbance, and
were probably responsible for the false positive results
Conclusions: The automated ELISA-DAGS proved to be a reliable, very
predictive test (both positive and negative), and suitable for mass screening test.
41120 Rapid, third-generation one-step immunoassay for HIV
antibodies, useful in developing countries
Jesus Benitez1, O. Ganzo2, J. Rivero3, J. Gavilondo2. 1Cir. Gen. Engineening &
Biotechnology, PO Box, Havana; CIGB, Havana; Santigo de Las Vegas
Sanatorium, Havana, Cuba
Objective: To develop a one-step immunochromatographic assay (ICA) for the
detection of HIV-1 and HIV-2 antibodies in biological fluids based on the antigen
sandwich principle.
Methods: The recombinant antigens p24-h, gp41-O were expressed in E.coli
as IL-2 fusion proteins using a vector bearing the tryptophan promoter and T4 terminator. While the recombinant p24 comprises the whole sequence of the natural
HIV-1 protein, our gp41 contains the N-terminal segment of the transmembranic
HIV-1 glycoprotein and the immunodominant epitope of the same protein in the
subtype 0. The detection of antibodies to HIV-2 was enhanced with the inclusion
of a synthetic peptide that comprises aminoacids 595-612 of the HIV-2ROD gp36.
Recombinant antigens were purified up to 85-90% using selective precipitation
procedures and ion metal affinity chromatography. Recombinant p24 and gp41
and the synthetic peptide were conjugated to colloidal gold. Plastic backed nitrocellulose sheets were line-coated sequentially with a mixture of antigens and
a mixture of Mabs to these antigens using an automated printing apparatus.
Conjugated antigens were dried into a porous wick and placed at the sample end
of the sheet, close to the antigen's line. An absorbent paper pad was placed at
the other end of the sheet. The sheet was finally cut in strips using an automated
cutting machine. The reactive strip is dipped in 200 ftL of undiluted human serum,
and the presence of antibodies to HIV is indicated through the formation of two
red lines on the test region.
Results: We studied a panel of 87 blood donor sera, 107 HIV-1 and 9 HIV-2
positive samples. One hundred percent of sensitivity and specificity were achieved
in this preliminary evaluation. At present we are involved in a wider evaluation of
the test with serum, blood and saliva samples and in a real time stability study at
different temperatures.
Conclusion: The high values obtained for the sensitivity and specificity in
addition to the autonomy of the rapid assay with respect to the presence of sophisticated equipment and the supply of electricity makes this assay an alternative
to the ELISAs in the conditions of both rural and urban areas of many developing
countries.
411211 Seroprevalence of HIV infection among street children in
Maiduguri
Edemenang D. Okpudo-ltata1, T.O. Harry2, M. Bulterys3, A. Abimiku4. 1Dept of
2University of Maiduguri Teach Hospital, Maiduguri, Nigeria; 3University of New
Mexico, Albuquerque; 4University of Maryland, Baltimore, USA
Objectives: To determine the prevalence of HIV infection among, street children in Maiduguri and to further determine the risk factors associated with HIV
transmission among them.
Design: Cross-sectional study.
Methods: Participants were street children aged between 6 and 20. A simple standardized, questionnaire was designed and administered to collect basic
demographic data and to also identify the prevailing risk factors associated with
HIV transmission among the children. Blood samples were taken from each of
them and processed for HIV screening. All samples were initially screened for
HIV antibodies using commercial enzyme-linked immunosorbent assay (ELISA;
Genelavia-mixt, Diagnostics Pasteur, Psteur, Paris, France). All ELISA-reactive specimens were repeatedly tested and confirmed by a rapid HIV-1/2 assay based on
latex agglutination (Capillus HIV-1/2, Cambridge Diagnostics, Galway, Ireland).
Results: A total of 203 children were recruited for the study. 124 (61.1%) were

boys and 28 (22.6%) of them had had heterosexual contacts, while 6 (4.8%) of
the boys confessed to having homosexual contacts. Similarly 79 (38.9%) were
girls and 44 (55.7%) of them had had heterosexual contacts. Minimum age for
sexual contact was 8 for girls and 11 for boys. Overall, 157 (77.3%) of the children
had "local surgery" namely scarification, circumcision, tonsillectomy etc. (usually
performed with unsterilized locally made instruments). 2 (2.5%) girls and 1 (0.8%)
boy were positive for HIV-1.