Evaluation of the intercept oral specimen collection device with HIV assays versus paired serum/plasma specimens.

Oral fluid has many advantages over blood-based techniques: it is less invasive, eliminates the occupational risk associated with needle stick accidents and collection can be self-administrated. Each individual test is packaged with a corresponding collection device. This study tested the suitability of the Intercept Oral Specimen Collection Device for different HIV diagnostic tests: three different rapid HIV tests and two adapted ELISAs, which were evaluated and compared with a gold standard on blood. In addition a total IgG quantification was performed to demonstrate the quality of the specimen. HIV antibodies were detected with a sensitivity of 100%, 99.3%, 98.6%, 100% and 95.7% for, DPP, OraQuick, Aware, Genscreen and Vironostika respectively using the Intercept Collection Device. Respective specificities were 100%, 100%, 99.3%, 97.3% and 100%.

Using conventional HIV tests on oral fluid.

There is need for more evaluations of non-invasive tests in order to broaden the reach of testing programs and to perform large scale epidemiological studies. In this study, three different human immunodeficiency virus (HIV) enzyme linked immunosorbent assays (ELISAs) and one line immunoassay were evaluated to detect HIV antibodies in oral fluid samples. Specimens were collected, after informed consent was obtained, with the Oracol (MMD, Worcester, England) device. A total IgG quantitation test was performed to demonstrate the quality of the sample. Assessment of a modified protocol of the Vironostika HIV Ag/Ab, Enzygnost Anti-HIV 1/2 Plus Genscreen HIV-1/2 Version 2 and a line immune confirmatory assay the INNO-LIA HIV I/II score was done, using oral fluid specimens of 325 HIV positive and negative individuals. For the ELISAs, the addition of an extra internal oral fluid control was evaluated as well as different cut-offs, time between sampling and testing and the effect of drinking water just before sampling. Finally, the confirmatory test and some testing algorithms and combination of tests were discussed. The results obtained from the oral fluid specimens were compared with the gold standard on paired serum specimens. Firstly, there was no significant difference observed between the use of the kit controls and the oral fluid controls. New protocols and calculation of cut-offs were defined for two of the three ELISAs. High sensitivities and specificities were obtained with all three ELISAs without any statistical difference between the three tests. Secondly, no statistically significant difference was observed when samples were stored for different time periods between sampling and testing, meaning that a period of seven days at room temperature before testing is still acceptable. Thirdly, drinking water before sample collection did not interfere with the testing, although lower optical densities were observed. None of the positive samples were missed. In addition, the line immunoassay INNO-LIA HIV I/II score test is a promising test for confirmation of reactive oral fluid specimen, but more samples need to be validated in order to adapt the interpretation rules specifically for oral fluid specimens. Different choices/algorithms adapted for the purpose of testing can be proposed. In conclusion, it can be said that the commercial ELISAs with adapted protocol and cut-off values are suitable tools for making HIV test performance accessible to people. With this non-invasive sampling method, more eligible individuals can and will be selected for further HIV test on blood.

A venue-based HIV prevalence and behavioural study among men who have sex with men in Antwerp and Ghent, Flanders, Belgium, October 2009 to March 2010.

This venue-based, cross-sectional study reports on human immunodeficiency virus (HIV) prevalence and behaviour of 649 men who have sex with men (MSM) in Antwerp and Ghent, Flanders, Belgium, from October 2009 to March 2010. Using time-location sampling, we found that HIV prevalence in MSM who attended different types of venue ranged from a high of 14.5% (95% CI: 8.9­20.1; n=22 in cruising venues to 4.9% (95% CI: 1.9­7.9; n=10) in more general gay venues to 1.4% (95% CI: 0.0­3.6; n=3) at younger MSM venues. Of those who tested HIV positive (n=35, five were unaware of their HIV status or self-reported as being HIV negative. One in five respondents were of non-Belgian nationality. The results showed relatively high rates of testing for HIV (52.2%; 95 % CI: 47.8­56.2; n=288) and other sexually transmitted infections (STIs) (57.4%; 95% CI: 52.6­62.0; n=248) in the last 12 months. A majority of the men (n=233) used condoms consistently during their last anal sexual contact with a casual partner; however, HIV-positive men who were aware of their serostatus (n=30) reported less condom use with casual partners. This is the first such study in Belgium and the results constitute the evidence base for local, targeted interventions. Furthermore, our findings underscore the need for European cross-border cooperation to prevent HIV infection and other STIs among MSM.

Evaluation of a rapid and simple fourth-generation HIV screening assay for qualitative detection of HIV p24 antigen and/or antibodies to HIV-1 and HIV-2.

The performance was assessed of a new, rapid, visual and qualitative immunoassay for the detection of HIV p24 antigen (Ag) and antibodies (Ab) to HIV-1 and HIV-2. Characterised serum or plasma specimens from patients diagnosed with HIV infection were tested: 179 samples of known Ab-positive patients harbouring different subtypes of HIV-1 (n=154) and HIV-2 (n=25) and 200 samples from individuals not infected with HIV. The assay's Ag sensitivity was assessed by testing HIV seroconversion panels (n=10) and primary HIV infection specimens (n=57). In addition, the influence of the genetic variability of HIV-1 on Ag detection was evaluated using dilutions of culture supernatants infected with different subtypes (n=50). The performance of the rapid test was compared to a "gold standard" testing algorithm with the use of a single Ag ELISA and with the Vironostika((R)) HIV Uni-Form II Ag/Ab test, a fourth-generation ELISA. The new assay, the Determine HIV-1/2 Combo demonstrated 100% (98.2-100.0) Ab specificity (200/200) and 100% (98.0-100.0) Ab sensitivity (179/179). In these samples, the observed Ag sensitivity was 86.6% (58/67) with the Determine HIV-1/2 Combo test and 92.5% (62/67) with the Vironostika compared to the reference single Ag ELISA. The assay could not detect Ag in one group O, one subtype F and two subtype H cell supernatant isolates. None of the HIV-2 Ag could be detected.

Low specificities of HIV diagnostic tests caused by Trypanosoma brucei gambiense sleeping sickness.

The accuracy of diagnostic tests for HIV in patients with tropical infections is poorly documented. Human African trypanosomiasis (HAT) is characterized by a polyclonal B-cell activation, constituting a risk for false-positive reactions to diagnostic tests, including HIV tests. A retrospective study of the accuracy of HIV diagnostic tests was performed with 360 human African HAT patients infected with Trypanosoma brucei gambiense before treatment and 163 T. b. gambiense-infected patients 2 years after successful treatment in Mbuji Mayi, East Kasai, Democratic Republic of the Congo. The sensitivities, specificities, and positive predictive values (PPVs) of individual tests and algorithms consisting of 3 rapid tests were determined. The sensitivity of all tests was 100% (11/11). The low specificity (96.3%, 335/348) and PPV (45.8%, 11/24) of a classical seroconfirmation strategy (Vironostika enzyme-linked immunosorbent assay [ELISA] followed by line immunoassay) complicated the determination of HIV status, which had to be determined by PCR. The specificities of the rapid diagnostic tests were 39.1% for Determine (136/348); 85.3 to 92.8% (297/348 to 323/348) for Vikia, ImmunoFlow, DoubleCheck, and Bioline; and 96.6 to 98.3% (336/348 to 342/348) for Uni-Gold, OraQuick, and Stat-Pak. The specificity of Vironostika was 67.5% (235/348). PPVs ranged between 4.9 and 64.7%. Combining 3 different rapid tests resulted in specificities of 98.3 to 100% (342/348 to 348/348) and PPVs of 64.7 to 100% (11/17 to 11/11). For cured HAT patients, specificities were significantly higher for Vironostika, Determine, Uni-Gold, and ImmunoFlow. T. b. gambiense infection decreases the specificities of antibody detection tests for HIV diagnosis. Unless tests have been validated for interference with HAT, HIV diagnosis using classical algorithms in untreated HAT patients should be avoided. Specific, validated combinations of 3 HIV rapid tests can increase specificity.

Pediatric human immunodeficiency virus screening in an African district hospital.

In order to evaluate alternative tests and strategies to simplify pediatric human immunodeficiency virus (HIV) screening at the district hospital level, a cross-sectional exploratory study was organized in the Democratic Republic of the Congo. Venous and capillary phlebotomies were performed on 941 Congolese children, aged 1 month to 12 years (153 children under 18 months and 788 children more than 18 months old). The HIV prevalence rate was 4.7%. An algorithm for children more than 18 months old, using serial rapid tests (Determine, InstantScreen, and Uni-Gold) performed on capillary blood stored in EDTA tubes, had a sensitivity of 100.0% (95% confidence interval [CI], 88.9 to 100.0%) and a specificity of 100.0% (95% CI, 99.5 to 100.0%). The results of this study suggest that the ultrasensitive p24 antigen assay may be performed on capillary plasma stored on filter paper (sensitivity and specificity, 100.0%; n=87) instead of venous plasma (sensitivity, 92.3%; specificity, 100.0%; n=150). The use of glucolets (instruments used to perform capillary phlebotomies), instead of syringes and needles, may reduce procedural pain and the risk of needle stick injuries at a comparable cost. Compared to the reference, HIV could have been correctly excluded based on one rapid test for at least 90% of these children. The results of this study point towards underutilized opportunities to simplify phlebotomy and pediatric HIV screening.

Comparative evaluation of eight commercial enzyme linked immunosorbent assays and 14 simple assays for detection of antibodies to HIV.

To evaluate the performance of 22 assays for the detection of antibodies to HIV. Twenty-two assays for the combined detection of antibodies to HIV-1 and HIV-2, were evaluated on the same panel of serum specimens of diverse origin. Eight of the assays were ELISAs and the remaining 14 were simple, assays read visually. The specimen panel consisted of anti-HIV positive and negative samples from Africa (n=192), Europe (n=206), Asia (n=99) and Latin America (n=98). In addition to estimations of sensitivity and specificity, the assays were assessed, using a novel scoring system, for their ease of performance and for their suitability for use in small laboratories and clinics. The sensitivities of the assays in terms of seroconversion were assessed using series of specimens collected from nine individuals undergoing seroconversion. Eight ELISAs and eight of 14 simple assays had sensitivities and specificities of >99 and 95%, respectively. The results of these evaluations will be of assistance to those responsible for the selection of appropriate anti-HIV assays according to laboratory circumstances, the purpose of the testing and the population being tested.

Evaluation of four HIV antigen tests.

A direct comparison of different HIV antigen assays is very helpful in making an informed choice, not only for the testing laboratories but also for healthcare workers in the developing world who are looking for reliable and inexpensive tests/methods in the follow-up of their treated patients. As a follow-up to the study published previously [Fransen K., Martens G., Stynen D., Goris A., Nys P., Nkengasong J., Heyndrickx L., Janssens W., van der Groen G., 1997. J. Med. Virol. 53, 31--35] where only two tests have been compared, four different commercial methods for HIV antigen determination in plasma and supernatant of cell cultures have now been evaluated on a limited sample size (88): COULTER HIV-1 p24 Antigen Assay (Coulter), (Test 1) INNOTEST HIV Antigen mAb (Innogenetics) (Test 2), Genetic Systems HIV-1 Ag EIA (Sanofi-Pasteur(1)) (Test 3) and VIDAS HIV P24 II (bioMérieux) (Test 4). Of the four tests used in this study, Test 2 was by far the most sensitive test. In a population of 88 follow-up samples from 35 different patients representing all stages of infection, the test detected confirmed p24 antigen at least once in 85.7% (30/35) of these patients, versus Test 3 in 74.3% (26/35), Test 4 in 71.4% (25/35), and Test 1 in 48.6% (17/35) of the patients. Test 2 detected confirmed p24 antigen in 84.9% of the follow-up samples, followed by Test 4 (65.9%), Test 3 (64.8%) and Test 1 (39.8%). Finally, Test 2 also proved best for detecting genetically diverse isolates.

Evaluation of an agglutination HIV-1 + 2 antibody assay.

Studies have shown that an HIV (HIV-PA) agglutination assay (Serodia) for the detection of antibody to human immunodeficiency virus (HIV) can be as sensitive and as specific as enzyme-linked immunosorbent assay (ELISA). However, since this HIV assay was designed to detect antibody to the HIV-1 virus, a substantial number of HIV-2 positive sera are missed by this assay. Since the HIV-2 has now been found throughout the world this test is becoming less suitable. The new HIV-1 + 2 assay version (HIV-1 + 2 PA) was evaluated in 300 sera, which contained 50 HIV-1, 40 HIV-2 and 10 HIV-1/HIV-2 antibody positive samples, and a sensitivity and specificity of 100% and 99%, respectively, was obtained. Whereas all HIV-2 positive sera were detected by the new HIV-1 + 2 version, 26% (13/50) were missed by the old version of the agglutination test. It is concluded that the HIV-1 + 2 PA assay is a promising instrument free assay which can be used for screening purposes in areas where both HIV-1 and HIV-2 are present.

Eradication of hantavirus infection among laboratory rats by application of caesarian section and a foster mother technique.

Hantavirus antibodies were demonstrated by the indirect immunofluorescent antibody assay, in the serum of inbred strains of laboratory rats, during the period 1973-1982, at the Unit of Experimental Immunology in the Catholic University of Louvain, Brussels, Belgium. LOU rats, as well as immunocytomas, which were requested by laboratories in the U.K. and The Netherlands, were supplied at a time when the infection was unknown and unsuspected in Europe. Hantavirus-infected laboratory rats were rendered free of virus through re-derivation by caesarian section and suckling by virus-free foster mothers. Immunocytomas were tested for the presence of hantaviruses by implantation into seronegative laboratory rats. The strain of hantavirus causing the laboratory infection was clearly different from the one circulating in free-living bankvoles in Belgium. The exchange of laboratory rats and rat tumours in relation to the potential risk of laboratory-acquired hantavirus infection, is discussed.

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.

Confirmation and differentiation of antibodies to human immunodeficiency virus 1 and 2 with a strip-based assay including recombinant antigens and synthetic peptides.

We evaluated the use of the INNO-LIA HIV-1/HIV-2 Ab test (LIA HIV; Innogenetics) for the confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The test includes three recombinant HIV-1 proteins: p24 (gag), p17 (gag), and endonuclease (p31; pol), in combination with two synthetic peptides derived from the env gene of HIV-1 and one synthetic peptide selected from the env gene of HIV-2. Analysis of 450 sera from blood donors, 220 sera from patients with non-HIV pathology, and 28 Western blot (WB) p24-only reactive sera revealed no false-positive results, and the rate of indeterminate results was substantially lower than that with WB. Testing of 334 WB-confirmed HIV antibody-positive sera (309 HIV-1; 25 HIV-2) revealed no false-negative results. In two of seven seroconversion panels tested, LIA HIV detected the presence of HIV antibodies before WB did. In the other five panels, LIA HIV and WB confirmed the presence of HIV antibodies in the same sample. The LIA HIV assay therefore appears well suited for routine confirmation of the presence of HIV-1 and HIV-2 antibodies.

Line immunoassay and enzyme-linked line immunofiltration assay for simultaneous detection of antibody to two treponemal antigens.

Two enzyme immunoassays, the line immunoassay (LIA) and the enzyme-linked line immunofiltration assay (ELLIFA), were studied for suitability in the serodiagnosis of syphilis. In both assays, antibody to treponemes was detected using the recombinant DNA derived treponemal protein TmpA and the purified axial filament derived from the Reiter treponeme. The antigens were applied in parallel lines onto nitrocellulose membranes. The sensitivity and specificity of both assays were compared with that of the Treponema pallidum hemagglutination assay (TPHA), the fluorescent treponemal antibody absorption test, and the axial filament and TmpA enzyme-linked immunosorbent assays. The sensitivity and specificity of the LIA and the ELLIFA were found to be comparable to that of the TPHA using serum samples from 65 untreated syphilitic patients, 95 patients treated for syphilis and 60 blood donors, except in the case of the LIA using axial filament. This latter test was slightly less sensitive in primary and early latent syphilis than the TPHA. In the LIA procedure, serum antibodies to two antigens could be detected simultaneously within two hours. This assay may be useful for fieldwork. In the ELLIFA procedure, antibodies to the two antigens could be detected simultaneously within 15 minutes. The ELLIFA procedure may provide a multiple antigen test with a very short assay operation time.

Partial characterization of a Hantavirus isolated from a Clethrionomys glareolus captured in Belgium.

A Hantavirus was isolated in Vero-E6 cells from lungs of a free living bank vole (Clethrionomys glareolus) captured in Turnhout, Province of Antwerp--Northern part of Belgium. With help of monoclonal antibodies the Belgian Hantavirus isolate could be clearly differentiated from Hantaan virus strain 76-118, Prospect Hill virus strain PH1 and SR11, a Hantavirus isolated from laboratory Wistar rat in Japan, but not from the nephropathia epidemica virus strain Hällnäs.

Immunoperoxidase assay for the detection of specific IgG antibodies to Hantaan virus.

A technique, using indirect immunoperoxidase antibody (IPA), was developed for the detection of IgG antibody to Hantaan virus. The same protein A-peroxidase conjugate was used with mouse, rat and human sera. The IPA technique employs glass slides with air-dried gamma-ray-inactivated and acetone-fixed Hantaan-infected Vero-E6 cells. Antibody titers detected by IPA was comparable to those detected by the indirect fluorescent antibody technique.