Improved detection of circulating antigen in cattle infected with Taenia saginata metacestodes.

In order to improve the sensitivity and the specificity of an existing IgM monoclonal antibody-based ELISA (Brandt et al., 1992) for the detection of circulating antigen in the sera of cattle infected with T. saginata metacestodes, a modified sandwich ELISA was developed. Monoclonal antibodies (MAbs) of the IgG isotype were produced against the excretory-secretory (ES)-products of T. saginata metacestodes. Since it was shown that the affinity of these IgG MAbs for ES antigen was higher than that of the IgM MAbs, the latter were replaced by two IgG1 MAbs (158C11 and 60H8). Furthermore, heat treatment of the sera significantly increased the OD-values of ES-spiked serum samples as compared to nontreated samples. It also decreased the number of false positive reactions. When the original IgM MAb-based ELISA was compared with the IgG MAb-based ELISA using heat treated sera from animals harbouring more than 50 living metacestodes of T. saginata, the sensitivity increased from 56% with the former to 92% with the latter assay. Only a small percentage of animals carrying less than 50 cysts were detected both with the ELISA using IgG or IgM MAbs. The specificity of the IgM- and IgG MAb-based ELISAs was 93.4% and 98.7% respectively.

Prevalence of cysticercosis in epileptics and members of their families in Burundi.

In the province of Bururi in Burundi, 103 epileptics and 72 control subjects from the same households were examined for cysticercosis. Antigen was detected by enzyme-linked immunosorbent assay in 4.9% of epileptic persons and in 4.2% of controls. Antibody was detected by enzyme-linked electroimmunotransfer blot assay (EITB) in 11.7% of epileptics and in 2.8% of controls. Neither difference was statistically significant, nor was a history of taeniasis significantly more frequent in epileptics than in controls. However, cysticercosis was significantly more frequently diagnosed by EITB in people with a history of taeniasis than in those without such a history. The prevalence of taeniasis in schoolchildren ranged between 0 and 1.0%. Meat inspection detected cysticercosis in 2% and 39% of pigs in 2 localities, respectively.

Evaluation and comparison of the Amplicor HIV-1 PCR kit with an 'in-house' nested PCR.

BACKGROUND: The polymerase chain reaction (PCR), a powerful gene amplification technique, is moving rapidly from the research laboratory into routine clinical use. OBJECTIVES: To evaluate the specificity and sensitivity of a commercially available PCR kit, the Amplicor HIV-1 PCR kit (AMP) and to compare it with an in-house nested PCR, which amplified part of the POL gene (POL(n)). STUDY DESIGN: A total of 517 samples were tested by AMP, including 159 fresh whole blood specimens from HIV-1 antibody positive Europeans and 358 archival samples (338 seropositive and 20 seronegative individuals) originating from 35 different countries in Africa and Europe. We compared the performance of AMP on the archival samples with POL(n). RESULTS: The overall sensitivity and specificity as compared to HIV-1 serology were 93% and 100%, and 96% and 100%, for AMP and the in-house PCR, respectively. Repeat testing on co-cultured lymphocytes increased the sensitivity of AMP to 95%. CONCLUSIONS: AMP is a rapid, and easy to use commercially available PCR kit, since only one amplification of the test sample is required. Moreover, the use of radioactivity is omitted, and reading of the test can be done with a spectrophotometer. The use of at least one additional primer pair may increase confidence in distinguishing a positive and negative sample by both PCR techniques.

Quantification of human immunodeficiency virus in plasma by RNA PCR, viral culture, and p24 antigen detection.

A semiquantitative PCR technique for detecting human immunodeficiency virus type 1 (HIV-1) RNA in plasma was compared with quantitative viral culture and p24 antigen detection in plasma. Ninety-three samples from 20 symptomatic, 10 asymptomatic, and 10 seronegative individuals were tested. For most of the seropositive patients, consecutives samples were examined. Viral RNA was extracted from plasma by the method described by Boom et al. (R. Boom, C.J. A. Sol, M. M. M. Salimans, C.L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990). The RNA PCR was the most sensitive method (100 and 74% sensitivity for symptomatic and asymptomatic patients, respectively) and produced less divergent results with the consecutive samples from individual patients compared with the other techniques. All samples positive by viral culture or p24 antigen assay were also positive in the RNA PCR. For each of the three assays, the number of positive results obtained correlated with the disease stage. The estimated mean number of HIV-1 RNA copies was significantly higher in symptomatic patients (22,750 copies per ml) than in asymptomatic patients (1,820 copies per ml). It was also higher in samples positive for viral culture than in culture-negative samples. No close correlation was found between the amount of HIV-1 RNA and the amount of p24 antigen or the titer of infectious virus in plasma or between this titer and the level of p24 antigen. The plasma RNA PCR may be a useful additional marker of disease progression and may be valuable for monitoring the effects of antiviral therapy.

HIV screening and confirmation: a simplified and less expensive testing algorithm.

In this study we investigated the performance of fourteen different assays capable of simultaneously detecting antibodies to HIV-1 and HIV-2, referred to as combined screening assays (CSAs), on a panel of 371 sera, with a prevalence of 51.5% and 1.3% for HIV-1 and HIV-2 antibodies respectively. The geographic distribution of the sera was as follows; Europe (121), Africa (203) and Latin America (47). These sera were collected from different clinical groups of patients; Asymptomatic (36), AIDS-Related Complex/AIDS patients (18), infected individuals with generalised lymphadenopathy (12), blood donors (149), and subjects with unknown clinical status (156). The Dupont Western blot (WB) kit for detection of HTLV-III antibodies and the Pasteur new Lav-Blot II kit were used for the confirmation of HIV-1 and HIV-2 infection respectively. Of the 14 tests studied, 9 were enzyme linked immunosorbent assays (ELISAs), and 5 were non-Elisa tests requiring visual reading. An alternative approach for HIV antibody testing was studied restrospectively, whereby sera positive in an initial CSA (A) were retested on a second CSA (B), that was different from the first. The use of WB was limited to sera that gave discrepant (A+B-) results in the two CSAs. A positive result in both CSAs was reported as anti-HIV positive. A negative result in the first CSA was reported anti-HIV negative. Sensitivity, specificity, cost, and the delta (delta) values (delta values of the ELISA assays) were taken into consideration when selecting suitable pairs of assays. All the ELISAs scored 100% sensitivity, but for the non-ELISAs, the sensitivity ranged from 96.0% to 100%. The specificity for the ELISAs and non-ELISAs varied from 87.4% to 100% and from 51.4% to 100% respectively. Delta (delta) values for the ELISAs ranged from 3.82 to 136.68 and from -1.15 to -3.08 for the anti-HIV positive and anti-HIV negative populations respectively. Of the 121 test combinations studied, 9 (7.4%) pairs yielded 100% sensitivity and specificity and 61 (50.4%) pairs of CSAs required further testing on WB. This implies 100% positive predictive value, at a cost that was on average 6 times less, and a testing time that was 5 times faster than the conventional algorithm. We conclude that there are several combinations of pairs of CSAs that can be used in the alternative algorithm that can provide accurate results at a much lower cost than the conventional algorithm requiring confirmation by WB of all initially reactive CSA results.(ABSTRACT TRUNCATED AT 400 WORDS)

Alternative confirmatory strategy for anti-HIV antibody detection.

Enzyme immuno assays for simultaneous screening of antibodies to HIV-1 and HIV-2 (EIA HIV-1 + HIV-2) have recently been developed. Confirming all reactive EIA HIV-1/2 screening results by Western blot (WB) for HIV-1 and HIV-2 antibodies is expensive. Six different EIA HIV-1/2 screening assays and one supplemental Line immuno assay (INNO-LIA HIV-1/HIV-2 Ab (LIA)) for confirmation of reactive EIA HIV-1/2 screening assay results were carried out on a panel of 400 sera of which 13% were HIV-1- and 2.5% were HIV-2-positive. The LIA was used as the 'gold standard'. Retrospectively, the results of the six EIA HIV-1/2 were evaluated in pairs (A and B), applying B to those sera reactive in A. A+B+ results were reported as positive. A+B- were either interpreted as negative or the LIA result of A+B- was accepted as the final result. At least seven of the EIA HIV-1/2 pairs gave rise to no false-positive or -negative results. This strategy was 5 times faster and resulted in a budget on average 50% lower than that of the conventional strategy. Further investigation of these alternative confirmatory strategies, in which the proposed algorithms are applied in sequential use of the different screening assays, are needed under field conditions in developing countries.

Detection of HIV-1 antibodies in blood specimens spotted on filter-paper.

Described are the results of an international collaborative study to evaluate the use of whole blood samples spotted on filter-paper (BSP) for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). BSP samples were collected from 40 patients at risk for HIV-1 infection and tested blindly using commercially available HIV antibody test kits, either specifically manufactured or modified for this purpose. Parallel serum samples were also collected, and the antibody reactivity was defined and confirmed by Western blot. The results demonstrate that recovery of antibodies from BSP samples after elution can be comparable to that from serum. Some kits can be easily adapted to test BSP samples, while others cannot. At present, detection of HIV antibodies in BSP samples should therefore be carried out using kits specifically manufactured for this purpose or by the development of a modified protocol using a panel of BSP and their corresponding serum specimens.

A simplified and less expensive strategy for confirming anti HIV-1 screening results in a diagnostic laboratory in Lubumbashi, Zaire.

The conventional algorithm for HIV testing based on the confirmation of all positive anti-HIV screening reactions by Western blot (WB) is too expensive for developing countries. We investigated the validity of confirming positive screening assay reactions by a second screening test, limiting the use of the supplemental assay to the discrepant test results (algorithm 3), or screening all sera with 2 different assays and retesting all discrepant results by a supplemental assay (algorithm 4) on a panel of 519 sera in a regional reference laboratory in Lubumbashi, Zaire. Combining the Vironostika anti-HTLV-III ELISA with HIV Chek 1 + 2 or Clonatec Rapid HIV 1/2 Ab on all samples and retesting the discrepant results in WB or a line immunoassay (INNO-LIA) (algorithm 4), yielded a sensitivity of 100% and specificities of 98.4% and 99.0% respectively, at costs of 7.3 US $ and 9.3 US $ per test, respectively, for a 40% prevalence of HIV antibody positive samples. The conventional algorithm scored a sensitivity of 97.1% and a specificity of 100% for 11.3 US $ per test. The testing strategy of combining HIV Chek 1 + 2 and Clonatec Rapid HIV 1/2 Ab, an interesting option for small isolated centra, had a 96.6% sensitivity, but yielded only a slightly better specificity of 99.0%, as compared to 97.8% for HIV Chek alone. The price of combining the two simple assays using algorithm 3 was 6.8 US $ per test, using algorithm 4 was 10.6 US $. HIV testing strategies based on ELISA and a simple HIV test are a valuable alternative for reference laboratories faced with a high prevalence of HIV positive samples.

Evaluation of a line immunoassay for simultaneous confirmation of antibodies to HIV-1 and HIV-2.

An anti-HIV-1/HIV-2 line immunoassay (LIA), using peptides and recombinant antigens was evaluated against commercially available Western blot tests for HIV-1 and HIV-2 antibodies. Two thousand one hundred and ten sera of European, African, and South American origin were used in the evaluation. The panel included 1066 sera with antibodies to HIV-1, 192 sera with antibodies to HIV-2, and 64 sera with antibodies to both. Using Western blot results interpreted according to the WHO criteria as a reference standard, the overall specificity obtained by this LIA was 100% and the sensitivity was 99.77% (97.51-100% for 95% confidence limits) when sera dually reactive in Western blot were included. Of the three sera negative in the LIA but positive in HIV-1 WB, two could be retested in a radioimmunoprecipitation assay and were negative. When dually reactive sera in the Western blot (WHO) were included, the LIA yielded 9.9% indeterminate results as compared with 15.5% for both assays (chi 2 = 29.30; p less than 0.001). Although only one HIV-2 specific peptide antigen (gp36) was used, the LIA yielded a specificity of 100% and a sensitivity of 100% as compared with the HIV-2 Western blot assay. When indeterminate results were included, the overall agreement between the LIA and the HIV-1 and HIV-2 Western blot (WHO criteria) was 89.9% and 90.1% respectively. These results indicate that the LIA provides reliable simultaneous detection of antibodies to HIV-1 and HIV-2, and at a cost which is substantially lower than the cost of Western blot tests.

Simplified and less expensive confirmatory HIV testing.

The conventional approach to human immunodeficiency virus (HIV) antibody testing, which relies on confirmation of all initially positive screening results using a Western blot assay, is expensive. In an alternative approach, we retested sera that were positive in an initial screening assay using a second screening assay, which differed from the first, and limited the use of Western blot to those sera that gave discrepant results in the two screening assays. This resulted in 100% sensitivity and specificity at a cost that was, on average, 6.1 times less than that of the conventional approach. This level of sensitivity and specificity was also achieved at a cost that was 9.0 times less than the conventional approach if the Western blot was replaced by a third screening assay that differed from the previous two. Retesting positive sera using the same assay did not increase the accuracy of the results obtained by testing the sera only once.

PIP: After HIV antibodies had been detected by various assays in the sera of 164 people (Europeans, Africans, and South Americans), microbiologists from the Institute of Tropical Medicine in Antwerp, Belgium used simple and inexpensive assays to confirm HIV infection. They retested sera that were positive in the 1st screening with a different assay. They used the Western blot assay only if contradictory results occurred in the 2 assays. The alternative approach developed 100% sensitivity and specificity. Further the cost of this approach averaged 6.1 times less than if they confirmed positive results with the Western blot and 9 times less than if they used this conventional approach if a 3rd assay, different from the 2 previous assays, replaced the Western blot. In addition, when the researchers retested positive sera with the same assay as the 1st, the accuracy of the results did not improve. In fact, the researchers highlighted that the 1st assay should be more sensitive than the following assays, since the 1st assay is the factor that limits the sensitivity of the combination. Researchers should follow this study with similar research in resource limited settings with different epidemiologic patterns of HIV seroprevalence. In conclusion, this alternative approach may result in accurate, less expensive, more rapid and/or less equipment reliant testing for HIV infection.

Comparative evaluation of 36 commercial assays for detecting antibodies to HIV.

Summarized are the results of an assessment of the major operational characteristics of 36 commercially available assays for detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and/or type 2 (HIV-2). For this purpose, 20 enzyme-linked immunosorbent assays (ELISAs), 11 simple immunoassays with visual reading, four supplemental assays, and one discriminatory assay were assessed using a panel of 537 sera (65% of which were of African, 26% of European, and 9% of South American origin); the prevalence of HIV-1 was 39.1% and of HIV-2, 15.7%. The following operational parameters of the assays were investigated: ease of performance; suitability for use in small blood collection centres; sensitivity and specificity; positive predictive values at different prevalences; inter-reader variability for simple assays whose results were read visually; the proportion of indeterminate results; and, for some of the ELISA assays, delta-values, as quantitative measures of sensitivity and specificity. The results will be of use to health policy decision-makers, managers of national AIDS prevention and control programmes, directors of blood banks, and laboratory specialists in the selection of appropriate HIV antibody assays.

PIP: Microbiologists at the Institute of Tropical Medicine in Antwerp, Belgium used sera from 537 people (65% Africans, 26% Europeans, and 9% South Americans) to compare commercial assays for detecting HIV antibodies. These assays included 20 ELISAs, 11 simple assays, 4 supplemental assays, and 1 discriminatory assay. HIV-1 seroprevalence was 39.1% and 15.7% for HIV-2. Basically the sensitivity of the assays were very good and equal, except the sensitivity of the Peptide HIV ELISA assay was considerably lower. In fact, on the most part, the sensitivities were higher than the specificities. But only 4 assays had significantly lower specificities than sensitivities. A high number of false positive reactions occurred in the African sera with these 4 assays which emphasizes the need to use African sera to evaluate HIV antibody kits. The higher the positive and negative Delta values the more likely the assay can accurately identify antibody positive and antibody negative sera respectively. The Elavia Mixt (HIV-1+2) and the Wellcozyme HIV-1+2 had the highest positive Delta value while Abbott recombinant HIV-1/HIV-2 EIA had the lowest positive value. Du Pont HIV-1/HIV-2 had the lowest negative value. No significant difference existed in determining sensitivity and specificity by visually reading the results between the simple assays. Interreader variability ranged from 0.8-31.7% with Recodot and Genie HIV-1 and HIV-2 having the highest variability. Further no significant difference existed in sensitivity and specificity between the ELISAs. The Ancoscreen supplemental assay did not have high sensitivity and specificity when used on African sera. Further INNO-LIA HIV-1/HIV-2 Ab test detected both HIV-1 and HIV-2 antibodies at the same time and ranked lower in indeterminant results than the Western blot.

Macrofilaricidal activity of oral flubendazole on Brugia pahangi.

The macrofilaricidal effect of orally administered flubendazole on Brugia pahangi was studied in the multimammate rat, Mastomys natalensis. A micronized formulation was incorporated in the routine feed for 2 weeks, starting 16 weeks after infection. In the treated rats the microfilarial density dropped to zero 20 weeks after infection, whereas it remained high in untreated animals. When killed between 40 and 41 weeks after infection, the treated group yielded a mean of 2 male and 0.16 female worms per rat, while the untreated group had means of 12 and 15.75 respectively. Flubendazole had no direct microfilaricidal effect.