Detection Of anti-HIV Antibodies Research Articles

Magnitudes and Correlates of Human Immunodeficiency Virus, Hepatitis B Virus, and Syphilis among Pregnant Mothers Attending Antenatal Care in Addis Ababa, Ethiopia.

Background Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and syphilis are major sexually transmitted infections (STIs) among the general population in Ethiopia, which in turn result in a wide range of adverse pregnancy outcomes. Hence, it is important to determine the seroprevalence and risk factors of HIV, HBV, and syphilis infection among pregnant mothers attending antenatal care in Addis Ababa, Ethiopia. Method A cross-sectional study was conducted among 286 pregnant women from February 1, 2021, to March 30, 2021, in four selected public hospitals in Addis Ababa. Sociodemographic, risky sociocultural, behavioral, and hospital-related factors were collected using an interview-administered questionnaire. Detection of anti-HIV antibodies, hepatitis B surface antigen (HBsAg), and rapid plasma reagin (RPR) for syphilis was conducted. A binary logistic regression analysis was used to determine predictors of STIs using SPSS version 25. Result A total of 281 pregnant mothers with a mean age of 27.5 (SD 4.6) completed the study. Among the participants, the seroprevalence rates of HIV, HBV, and syphilis were 15 (5.3%), 9 (3.2%), and 5 (1.8%), respectively. Twenty six (9.3%) of the participants tested positive for any one of the STIs, and 3 (1.1%) of the women had HIV and syphilis coinfections. History of multiple sexual partners (AOR 3.42, 95% CI: 1.6-11.63) and STIs (AOR 3.7; 95% CI: 1.70-13.45) were significantly associated with HIV infection. Likewise, history of abortion (AOR 7.65, 95% CI: 1.17-49.74), tattooing (AOR 9.72, 95% CI: 1.41-66.73), and rape (AOR 9.72, 95% CI: 1.41-66.73) were significantly associated with hepatitis B virus infection. Husband history of multiple sexual partners (AOR 20.9, 95% CI: 1.8-241.8) was significantly associated with syphilis infection. The educational level of participants, history of tattooing (AOR 6.24, 95% CI: 1.79-21.7), and history of multiple sexual partners (AOR 5.15, 95% CI: 1.68-15.7) were independent predictors of infection with any one of the STIs. Conclusion There is still a high burden of HIV, HBV, and syphilis among pregnant mothers in Ethiopia. History of multiple sexual partners, abortion, rape, and tattooing was a significant predictor of STIs.

The relationship of antibodies detected in the Western Blot test with clinical and immunological stages in HIV-infected patients

Background/Aim: The laboratory diagnosis of human immunodeficiency virus (HIV) infection is based on detection of anti-HIV antibodies in an initial ELISA (enzyme-linked immunosorbent assay) test and its confirmation with the Western Blot (WB) procedure. Even though the specificity of WB is high for the detection of antibodies against various viral proteins, there are important differences in the timing of the appearance of antibody bands and intensities in different stages of HIV infection. The aim of this study was to evaluate antibodies detected in WB testing and the relationship to clinical and immunological stages in HIV-infected patients. Methods: Newly diagnosed 78 patients with HIV/AIDS (Acquired Immunodeficiency Syndrome) in our outpatient department between April 2009 and September 2012 were included in the study as a retrospective cohort. Age, gender, complaints, clinical signs, CD4+ T lymphocyte counts and HIV RNA level at diagnosis were collected retrospectively from medical records. WB band patterns obtained from the reference laboratory of the Istanbul Public Health Center were examined retrospectively. Results: Of the 78 HIV/AIDS cases, 68 (87.2%) were male. Mean age was 38.87(13.09) years (range, 17-83 years). Median CD4+ T lymphocyte count at diagnosis was 410.2 /mm³ (range, 3-1114). Mean HIV RNA level at diagnosis was 592.894 copies/ml. Rare band profiles were seen in 29.4% (23/78). According to World Health Organization (WHO) clinical staging, 59 (75.6%) patients were at stage I, 4 at stage II, 10 at stage III and 5 at stage IV. Gp120, gp160 and gp41, known as envelope glycoproteins in WB band antibodies, were seen in all patients. There was determined to be a decrease in the p17, p51, p55 and p39 bands in WB tests of the advanced grade (Grade IV) of HIV infection. Conclusions: Reduction of p17, p51, p55, p39 antibodies in advanced stages were related with the progression of HIV infection. This shows that WB test is an important parameter not only in the diagnosis of HIV infection, but also in the follow-up of clinical progression in the absence of these antibodies.

Double-Antigen Lateral Flow Immunoassay for the Detection of Anti-HIV-1 and -2 Antibodies Using Upconverting Nanoparticle Reporters.

Rapid diagnostic tests (RDTs) are often used for the detection of anti-human immunodeficiency virus (HIV) antibodies in remote locations in low- and middle-income countries (LMIC) with low or limited access to central laboratories. The typical format of an RDT is a lateral flow assay (LFA) with visual interpretation prone to subjectivity. This risk of misinterpretation can be overcome with luminescent upconverting nanoparticle reporters (UCNPs) measured with a miniaturized easy-to-use reader instrument. An LFA with UCNPs for anti-HIV-1/2 antibodies was developed and the assay performance was evaluated extensively with challenging patient sample panels. Sensitivity (n = 145) of the UCNP-LFA was 96.6% (95% CI: 92.1–98.8%) and specificity (n = 309) was 98.7% (95% CI: 96.7–99.7%). Another set of samples (n = 200) was used for a comparison between the UCNP-LFA and a conventional visual RDT. In this comparison, the sensitivities for HIV-1 were 96.4% (95% CI: 89.8–99.3%) and 97.6% (95% CI: 91.6–99.7%), for the UCNP-LFA and conventional RDT, respectively. The specificity was 100% (95% CI: 96.4–100%) for both assays. The developed UCNP-LFA demonstrates the applicability of UCNPs for the detection of anti-HIV antibodies. The signal measurement is done by a reader instrument, which may facilitate automated result interpretation, archiving and transfer of data from de-centralized locations.

Evaluation of dried blood spot samples for screening of hepatitis C and human immunodeficiency virus in a real-world setting

Both hepatitis C virus (HCV) infection and human immunodeficiency virus (HIV) infection are underdiagnosed, particularly in low-income countries and in difficult-to-access populations. Our aim was to develop and evaluate a methodology for the detection of HCV and HIV infection based on capillary dry blood spot (DBS) samples taken under real-world conditions. We carried out a cross-sectional study of 139 individuals (31 healthy controls, 68 HCV-monoinfected patients, and 40 HCV/HIV-coinfected patients). ELISA was used for anti-HCV and anti-HIV antibody detection; and SYBR Green RT-PCR was used for HCV-RNA detection. The HIV serological analysis revealed 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The HCV serological analysis revealed a sensitivity of 92.6%, specificity of 100%, PPV of 100%, and NPV of 79.5%. Finally, the HCV-RNA detection test revealed a detection limit of 5 copies/µl with an efficiency of 100% and sensitivity of 99.1%, specificity of 100%, PPV of 100%, and NPV of 96.9%. In conclusion, our methodology was able to detect both HCV infection and HIV infection from the same DBS sample with good diagnostic performance. Screening for HCV and HIV using DBS might be a key strategy in the implementation of national programs for the control of both infections.

Performance validation of western blot for anti-HIV antibody detection in blood samples collected on filter paper (DBS)

ABSTRACT Introduction: For conducting field studies on human immunodeficiency virus (HIV) serodiagnosis, the use of samples collected on filter paper [dried blood spot (DBS)] is profitable owing to the simplicity in handling and delivering them. Because of these characteristics, the use of DBS is recommended for studies in which the goal is to increase the population access to diagnostic testing, including HIV infection screening. For HIV diagnosis, the conventional strategy firstly uses an enzyme immunoassay as screening test, and the positive sample is confirmed on a complementary assay, as western blotting (WB). Objective: This study aimed at evaluating the analytical performance of WB assay for analyzing DSB samples. Method: One hundred eighteen blood samples collected in filter paper were analyzed by a modified WB. These samples derived from the SampaCentro Study, and they were HIV-antibody-positive in a screening test. In order to assess the reliability of these results, the assay performance was previously certified by employing the sample panel (reference panel) of Instituto Adolfo Lutz (IAL), which consisted of whole blood and plasma paired samples. Results: All of the DBS samples [100% (118/118)] showed the band pattern compatible with the criterion for HIV positivity established by the Ministry of Health. To validate the WB testing, the reciprocal relationship between antibody reactivity of DBS samples and of the respective plasma samples was investigated. Conclusion: WB optimization allowed the achievement of accurate results, and the easy DBS sample collection, transportation and storage will support the use of this immunodiagnostic technique for conducting HIV/acquired immunodeficiency syndrome (Aids) epidemiological surveys.

Fourth generation assays for HIV testing

ABSTRACTIntroduction: Laboratory diagnosis of HIV infection is essential for the prevention of infection and the identification of infected individuals who could benefit from highly active antiretroviral therapy. Since the release of the first assays for the detection of anti-HIV antibodies, the technology of immunoassays has improved.Areas covered: Fourth generation assays – simultaneously detecting HIV p24 antigen and antibodies – have been developed and have been a major improvement in the detection of HIV infection, with a reduction of the diagnostic window. Studies have provided definite evidence for their clinical utility. Combination assays with separate results for anti-HIV antibodies and p24 antigen have been developed.Expert Commentary: In conclusion, fourth generation assays are an effective tool for the laboratory diagnosis of HIV infection. The ADVIA Centaur HIV Ag/Ab Combo assay is in line with most recent fourth generation assays and its clinical utility has been assessed.

A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti- HIV Antibodies and Viral RNA

ObjectiveWe developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest.MethodsWe first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention.ResultsThe finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample.ConclusionThe ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for the "Test and Treat" and "Treatment as Prevention" approaches for controlling the HIV epidemic.

Gingival crevicular fluid: As a diagnostic marker in HIV positive patients.

Objective:To determine the utility of gingival crevicular fluid (GCF) for the detection of anti-HIV antibodies in human immunodeficiency virus (HIV)-infected individuals using enzyme linked immunosorbent assay (ELISA) with respect to their CD4 counts.Materials and Methods:The study was carried out between 37 seropositive (37GCF, 10 saliva) and 37 seronegative (GCF) individuals for a period of 7-8 months in GDC Nagpur. Thirty-seven GCF and 10 whole saliva samples were collected from the same patient. GCF samples were collected from gingival crevice with the help of Kimble disposable microcapillary. Saliva was collected by asking the patient to bend forward. The drooling saliva was collected in a sterile bottle and stored at Minus 20°C (-20°C). After the clinical observations the data were collected and tabulated for statistical analysis.Results:When compared with serum, the sensitivity, specificity, and positive and negative predictive values of GCF were 100% respectively.Conclusion:All the above findings are suggestive of GCF being a better diagnostic medium than saliva.

Long-Term Persistence of Vaccine-Induced HIV Seropositivity in Healthy Volunteers

Objectives: To assess the long-term serological impact of HIV preventive vaccine trial participation, vaccineinduced HIV seropositivity (VISP) was evaluated and related factors were investigated. The anti-HIV antibody reactivity ratio distribution was estimated. Methods: ANRS COHVAC is an open national prospective multicentre cohort study including healthy volunteers who received at least one dose of vaccine candidate of ANRS HIV preventive vaccine trials since 1992. VISP was studied in a cross-sectional study at the time of the cohort’s initial visit, starting in 2008. Anti-HIV antibody detection was performed using the ABBOTT ARCHITECT® HIV Ag/Ac Combo Enzyme Immunoassay (EIA) in a centralized laboratory. A ratio greater than or equal to 1 was considered to define HIV seropositivity. Results: 293 participants were evaluated for a median period of 6 years (range: 2-18 years) after their inclusion in vaccine preventive trials. The frequency of VISP was estimated at 7.2% (21 out of 293) for all volunteers, and 69.0% (20 out of 29) for volunteers who received recombinant HIV-1 envelope protein, after a median period of 16.6 years after immunization (range: 16.3-18.4). The ARCHITECT test ratio among positive volunteers was low, with a median of 3.02 (range: 1.02 -14.04). Conclusion: Healthy volunteers should be informed of possible VISP persistence for nearly 17 years, following HIV envelope vaccination inducing antibody responses. A single, routine serology test is unable to differentiate between VISP and a recent HIV infection. The combination of different technologies, applicable to resource-limited settings, is needed to distinguish vaccine-induced seropositivity from an HIV infection.

A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2

This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1env, incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu 3+ chelate or with nanoparticles doped with the Eu 3+ chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu 3+ chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios.

Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity

A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.

Sensitivity of 8 CE (European Community)-approved rapid disposable tests for anti-HIV antibody detection during and after seroconversion

We evaluated the sensitivity for the anti-HIV antibody detection of the 8 rapid disposable tests (RDTs) EC approved in 2008. Methods A panel of 100 native serum samples collected from HIV-1 subtype B or non-B, HIV-1 group O or HIV-2-infected patient and 3 commercial seroconversion panels were tested. 50 sera from HIV-negative patients were included to blind reading and RDT specificity. Results All native samples HIV-1 subtype B were reactive; an HIV-1 non-B subtype gave false-negative results with 3 of the 8 tests. False-negative results on HIV-1 group O samples were observed with one RDT. All the HIV-2 samples were detected. Seroconversion sample reactivity ranged from 60 to 86.7% according to the tests. Conclusion Despite their lower performances relative to ELISA tests during the HIV seroconversion period, RDT may be of interest in case of chronic infection.

Fast electrochemical detection of anti-HIV antibodies: Coupling allosteric enzymes and disk microelectrode arrays

Here a novel electrochemical method for the rapid detection of anti-HIV antibodies in serum is presented. The novelty lies in the combination of allosteric enzymes and coulometry to yield a fast, simple and reliable HIV diagnostic method. We have used a previously developed β-galactosidase enzyme that is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind the enzyme, the 3D conformation changes positively affecting the performance of the active site and, consequently, the enzyme activity is stimulated. Using 4-aminophenyl β- d-galactopyranoside (PAPG) as substrate yields p-aminophenol (PAP), which is reversibly oxidised at a very mild potential, ca. 0.37 V vs. Ag/AgCl over a range of electrode materials within the working pH range of β-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 μM for 4-aminophenol (PAP). The presence of anti-HIV antibodies results in enzyme activity increases above 50% which, combined with the sensitivity and response time afforded by the microelectrode arrays, allowed for the diagnosis of HIV in sera samples within an hour.

Transactional sex is the driving force in the dynamics of HIV in Accra, Ghana.

To delineate the population attributable fraction (PAF) of transactional sex in prevalent cases of HIV infection in the male adult population of Accra, Ghana. Cross-sectional study of clients who visited a sex worker (SW), of boyfriends of SW and of male personnel in prostitution venues. A questionnaire was administered and urine obtained for detection of anti-HIV antibodies. The PAF of prevalent HIV acquired from SW was calculated using a combination of data from this survey of clients, from on-going SW surveys, the national HIV surveillance system and the national census. HIV prevalence was 4.9% (8/162) among clients of mobile SW, 15.8% (53/335) among clients of home-based SW, 17.5% (10/57) among personnel and 32.1% (9/28) among boyfriends. A condom was used in 90% of intercourses, according to clients. Non-use of a condom was clustered in selected locations and independently associated with older age of client, frequency of intercourse with SW in the last year and current urethritis. Among the male population of Accra aged 15-59 years, 84% of prevalent cases of HIV were attributable to transactional sex. A sensitivity analysis showed that under various assumptions PAF varied between 47% and 100%. In Accra, approximately four-fifths of prevalent cases of HIV in adult males were acquired from SW. Comprehensive interventions providing education, condoms and treatment for sexually transmitted diseases for SW and their clients should be approached as other public health priorities and provided in all cities, large and small, of West Africa.

Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale

Many recombinant proteins partially aggregate into inclusion bodies during production in Escherichia coli in batch culture on defined medium. Production on complex medium, however, effectively prevented inclusion body formation of a β-galactosidase-HIVgp41 fusion protein for detection of anti-HIV antibodies which is produced at 42 °C under control of a temperature-inducible expression system. Cells pre-conditioned by cultivation on complex medium before induction showed faster growth, higher product concentration and reduced inclusion body formation even when producing on defined medium. In contrast, for human basic fibroblast growth factor (hFGF-2) produced under control of the phage T7-promoter, medium composition could not reduce inclusion body formation even at 30 °C. Here, slow production in high-cell density fed-batch mode using a defined medium with limited glucose feeding enabled the accumulation of 50 mg product per gram cell dry mass exclusively in the soluble cell fraction, resulting in a volumetric concentration of more than 4 g per litre hFGF-2. With the β-galactosidase fusion protein produced in fed-batch, over 100 mg of product per gram cell dry mass accumulated in the soluble cell fraction. With a cell density of 100 g cell dry mass per litre, this resulted in a volumetric concentration of 10 g per litre of soluble β-galactosidase-HIVgp41 fusion protein. Thus, two approaches to balance heterologous protein production and host physiology are presented, which fit the needs of lab bench or pilot plant, respectively.

Expression, purification, and characterization of an anti-RBCFab-p24 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood

A fusion protein A41Fabp246 consisting of Fab of an anti-RBC MAb A41 and amino acids 8-153 of HIV-1 p24 (p246) fused at the C-terminus of A41Fd was purified following assembly of A41Fdp246 with A41 LC in vitro, using a denaturation– renaturation protocol and a 4-step column chromatography procedure. The highly purified, monomeric A41Fabp246 was then evaluated for hemagglutination-based detection of anti-p24 antibodies using sera from HIV-infected individuals. This derivative of p24 is devoid of maximum homology region and the C-terminal domain of p24, which is responsible for oligomerization of p24, but retains full complement of immunodominant epitopes. This new fusion protein in combination with fusion proteins consisting of monovalent fragment of another anti-human RBC antibody fused to immunodominant regions of envelope glycoproteins of HIV-1 and HIV-2 should be useful in preparing a cocktail of reagents for highly sensitive detection of anti-HIV antibodies in whole blood.

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli beta-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active beta-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIV-infected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.

Cutaneous Dendritic Cells Are Main Targets in Acute HIV-1-Infection

Acute human immunodeficiency virus (HIV) infection is a transient illness that typically presents with mucocutaneous and constitutional symptoms. It is soon followed by seroconversion with the detection of anti-HIV antibodies in the peripheral blood. To better understand the pathogenetic events leading to this clinical picture, we sought to investigate the (immuno)histologic features of the skin rash occurring in an acutely infected person. A skin biopsy of an acutely infected person was investigated histologically and immunohistologically using paraffin-embedded tissue sections. Interface dermatitis with pronounced vacuolization of the basal keratinocytes was a prominent histological finding. The inflammatory infiltrate was composed of CD3+/CD8+ T cells with coexpression of Granzyme B7 and TIA-1, and CD68+ histiocytes/dendritic cells. CD1a+ intraepidermal Langerhans cells (LC) were significantly decreased and individual LC co-expressed HIV-p24 antigens as evidenced in double labeling experiments. HIV-infected LC were demonstrated in close apposition to cytotoxic T cells.This study provides the first definitive evidence for infection of LC at extramucosal sites in this very early stage of disease. Our findings emphasize the critical role of dendritic cells as a virus reservoir and the skin as a major site of HIV replication during the course of the disease.

Isoelectricfocusing and reverse blotting as a diagnostic tool in pediatric HIV infection.

Early diagnosis of perinatally acquired HIV-infection is based on either direct HIV detection--by means of viral culture and/or PCR--or anti-HIV antibody detection. However, due to the passive, transplacental passage of maternal immunoglobulin G, antibody detection is nor reliable until 15-18 months of age. In this regard, clonotypic analysis of specific antibodies performed by isoelectricfocusing and reverse blotting (IEF-RB) can be very helpful, as it recognizes possibly different patterns between mother and infant. We used IEF-RB in order to analyze the kinetics of development of anti-HIV antibodies in infants born to seropositive mothers. Sera from ten mother/infant pairs (all mothers were HIV-infected) were retrospectively analyzed in order to detect different patterns, between mother and infant, in anti-gp120 V3-loop clonotype. We diagnosed the real HIV status of the examined infants no later than month 6 and in one case as early as month 2. Considering the small size of sample number, these data are preliminary and should be confirmed by larger scale studies. However, they show IEF-RB, when applied to infants born to seropositive mothers, may be useful in evaluating the infants' dynamics of anti-HIV humoral immune response.

HIV-Antibody Detection in Vitreous Humor and Serum by Enzyme Immunosorbent Assay and Particle Agglutination Test

To compare enzyme immunosorbent assay (EIA) and the particle agglutination (PA) test for the detection of antibodies to the human immunodeficiency virus (HIV) in cadaveric vitreous humor and serum. Seventeen cadavers presenting pathologic signs of acquired immunodeficiency syndrome (AIDS) at autopsy and 33 without AIDS signs were evaluated. Consistency (k) and significance (p) between the diagnostic tests were established. The right and left eyes of each of 48 bilaterally tested cadavers presented identical diagnostic results in all laboratory tests. All tests were positive for antibodies in the AIDS cadavers except for two EIA-negative vitreous results. Three autopsy-negative cadavers showed by both EIA and PA testing positive results in serum and negative in vitreous. Two of them showed a serum-positive Western blot. Serum EIA anti-HIV antibody detection test is the current gold standard for evaluating corneal donor material. Neither the PA test nor EIA should be used for vitreous testing because of their low sensitivity.