Luigina Terlenghi

Hepatitis C virus core antigen: Analytical performances, correlation with viremia and potential applications of a quantitative, automated immunoassay

BACKGROUND Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. OBJECTIVE To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. STUDY DESIGN Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. RESULTS The HCV Ag assay showed a specificity of 100%, a good precision (CV<10%) and excellent dilution linearity (r>0.999). The sensitivity (3 fmol/L) corresponds to 700-1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r=0.713 vs. Siemens bDNA (523 specimens), r=0.736 vs. Roche Cobas TaqMan (356 specimens) and r=0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. CONCLUSIONS HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.

Avidity Index for Anti-HIV Antibodies: Comparison between Third- and Fourth-Generation Automated Immunoassays

ABSTRACT The development of assays for detecting recent HIV infections has become crucial for analyzing trends in infection in different populations, both for surveillance and prevention activities. The anti-HIV avidity index (AI), measured with third-generation immunoassays (which detect anti-HIV antibody), has been shown to be an accurate tool for discriminating recent HIV infections (<6 months) from established infections (≥6 months). We compared a third-generation immunoassay (AxSYM HIV 1/2 gO; Abbott Diagnostics) to a fourth-generation immunoassay (Architect HIV Ag/Ab Combo; Abbott Diagnostics; which detects anti-HIV antibody and p24 antigen) in terms of AI performance in distinguishing between recent and established HIV infections. A total of 142 samples from 75 HIV-infected individuals with an estimated date of seroconversion were assayed. The two assays showed the same accuracy in identifying a recent infection (91.5%), using an AI cutoff of 0.80, although Architect HIV Ag/Ab Combo was slightly more sensitive (89.4% versus 84.8%; P > 0.05) and yet less specific (93.4% versus 97.4%; P > 0.05). The correlation between assays was high (r = 0.87). When 20 specimens falling in the gray zone around the cutoff point (0.75 ≤ AI ≤ 0.84) were excluded, the accuracy of AI with Architect HIV Ag/Ab Combo was 94.7%, and the concordance between the two assays was 99.2%. The anti-HIV AI is a serological marker that accurately discriminates recent from established HIV infections. It can be successfully applied on fully automated fourth-generation HIV Ab/Ag immunoassays, which have several advantages, including increased throughput, high reproducibility, no need for specific technical skills, and easy comparability of results obtained in different settings.

Liquid competition radioimmunoassay for the detection and quantitation of the HIV p24

Productive infection of permissive cell cultures by HIV has been detected by different assays of which the measurement of reverse transcriptase (RT) activity has been considered highly specific and sensitive. Here we describe the production and characterization of a mouse hybridoma cell line, MB12, secreting monoclonal antibodies to HIV p24, the major core protein, and the use of this monoclonal antibody to develop a type specific indirect liquid competitive radioimmunoassay (RIA) capable of providing earlier detection of the replicating virus than the RT assay. This assay also provides a quantitative analysis of HIV p24, which can be used to study the viral replication in permissive cell cultures. The ease of methodology and the adaptability of the competitive RIA to various assay conditions make this immunoassay suitable for the study of HIV expression in infected cell cultures.

Evaluation of the expression of IFN-γ in lymphocytes using a monoclonal antibody and flow cytometry

A stable hybridoma cell line secreting specific antibodies against human interferon-gamma (IFN-gamma) and designated IGMB-14 has been established. It belongs to the IgG1, kappa subclass and it reacts in Western blot with the 28 kDa and 56 kDa polypeptides present in two different affinity purified IFN-gamma preparations. Peripheral blood mononuclear cells (PBMC) from a healthy individual, stimulated in vitro by PHA, were analysed for IFN-gamma production both when viable and following fixation. The presence of cytoplasmic or surface IFN-gamma was visualized by an indirect immunofluorescence assay using monoclonal antibody (MAb) IGMB-14 and a single laser FACS-III fluorescence-activated cell sorter. The staining permitted the detection of newly synthesized cytoplasmic IFN-gamma molecules in lymphocytes at day 1 after PHA stimulation and surface IFN-gamma at day 2. IFN-gamma was expressed on almost all the CD4+ lymphocytes as shown by a double staining technique. The specificity of the reaction was confirmed by Western blots and abolishing IFN-gamma staining by pretreatment of MAb IGMB-14 with IFN-gamma. The presence of surface IFN-gamma was also visualized on freshly isolated PBMC from two patients suffering from measles and AIDS but not on PBMC from a healthy individual. The experiments showed that this immunofluorescent method is useful for the detection, enumeration, and phenotypic characterization of IFN-gamma-producing cells in vitro and, in addition, for evaluating the presence of PBMC expressing IFN-gamma on their surface during a viral disease.

Comparative Evaluation of BDProbeTec ET, LCx and PACE 2 Assays for the Detection of Chlamydia trachomatis in Urogenital Specimens

Chlamydia trachomatis is probably the most widespread sexually transmitted disease in Western industrialised countries, with a trail of dramatic consequences. The organism is notoriously difficult to detect, which accounts for its spread and makes it so unpredictable, but once detected, the infection can be treated easily. Chlamydial infections are usually asymptomatic, or they cause mild or nonspecific symptoms and signs that are not likely to be noticed. Approximately 70% of women with endocervical infections and up to 50% of men with urethral infections are asymptomatic and therefore unlikely to seek medical attention [1]. Thus, chlamydia has become known as the silent epidemic. It is the most frequently identified single cause of pelvic inflammatory disease, which affects an estimated 15–40% of women [2]. Yet, effective screening for this agent can lead to prompt treatment and prevent complications. Non-culture, non-amplification methods such as enzyme immunoassay and probe hybridization (PACE 2 and PACE 2 PCA assays; Gen-Probe, USA) are now the most widely used tests for Chlamydia trachomatis screening [3, 4, 5, 6]. New amplification assays are gaining wider use because they have proven to be more sensitive than nonamplification tests and cultures [5, 6, 7, 8]. Additionally, they offer the advantage that urine can be used as an alternative specimen for testing [6, 7]. The purpose of the study presented here was to evaluate two commercially available amplification methods, the ligase chain reaction (LCx assay; Abbott Laboratories, USA) and the strand displacement amplification method (BDProbeTec Et; [BDPT] Becton Dickinson, USA), and compare them with two standard probe hybridization assays (PACE 2 and PACE 2PCA) using female endocervical and male urethral swabs from a population at low risk of chlamydia infection. Also reported here are the results of a preliminary study based on our analysis of the diagnostic performance of the two commercial amplification assays using first-void urine samples from male and female patients. The study population included 300 patients (239 women and 61 men), aged 18–50 years (mean age, 24 years) treated at Spedali Civili Hospital in Brescia, Italy, between March and June 2001. Fifty subjects (37 women and 13 men) aged 28–50 years, suffered from probable infertility (defined as the inability to achieve pregnancy after normal unprotected intercourse over a 1-year period); these patients were asymptomatic and had received no antimicrobial therapy in the previous month. The remaining 202 women, aged 18–45 years, were seen for the following reasons: routine examination during pregnancy (113 patients), routine gynecologic care (56 patients), and pregnancy ending in premature delivery and abortion (33 patients). The remaining 48 men, aged 28–50 years, were seen for clarification of a variety of symptoms. Preliminary information including patient age, reason(s) for examination, patient complaints and clinical assessment, and antibiotic use during the previous 21 days was obtained from each patient by the hospital’s nursing staff. Three endocervical or urethral samples were collected from each of the 300 patients according to the gender of the patient (239 women and 61 men). The first swab was used for Gram staining and then for BDPT. The second swab was used for a DNA probe (PACE 2), and the third swab was used for the LCx assay. A subset of 50 patients (37 women and 13 men) also provided urine samples, which were assayed by both amplification methods (BPDT and LCx). All samples were transferred to the laboratory in the respective manufacturer’s transport medium and stored according to the respective manufacturer’s instructions prior to testing. All tests were performed according to each manufacturer’s directions. C. Pollara ()) · L. Terlenghi · M. A. De Francesco · F. Gargiulo · F. Perandin · N. Manca Institute of Microbiology and Virology, Spedali Civili Hospital, Piazza Spedali Civili 1, 25123 Brescia, Italy e-mail: pollara@bshosp.osp.unibs.it Tel.: +39-030-3995860 Fax: +39-030-3996071

HIV avidity index performance using a modified fourth-generation immunoassay to detect recent HIV infections

Abstract Background: Detecting recent HIV infections is important to evaluate incidence and monitor epidemic trends. We aimed to evaluate the diagnostic performance and accuracy of the avidity index (AI) for discriminating for recent HIV infections. Methods: We collected serum samples from HIV-1 positive individuals: A) with known date of infection (midpoint in time between last HIV-negative and first HIV-positive test); B) infected for >1 year. Samples were divided into two aliquots: one diluted with phosphate buffered saline (PBS) and the other with 1 M guanidine. Both aliquots were assayed by the Architect HIV Ag/Ab Combo 4th generation assay (Abbott). We compared AI found in recent (RI=<6 months from seroconversion) and established (EI) infections. The diagnostic accuracy was evaluated by receiver operating characteristic (ROC) curve analysis. The proportion of samples misclassified as recent (FRR) was calculated. Results: In total, 647 samples were collected: 455 in group A (51.6% RI and 48.4% EI) and 192 in group B. Among these, sixteen samples were from elite controllers, 294 from treated patients, 328 from patients infected with non-B subtypes. Samples before antiretroviral initiation showed a mean AI significantly lower among RI compared to EI (0.66+0.28 vs. 1.00±0.12; p<0.000). The FRR was 0% using a cut-off of ≤0.70. An extremely low FRR was observed among elite controllers, samples with low VL or CD4. HIV subtype had no impact on AI misclassifications. All individuals in group A reached the AI threshold of 0.80 within 24 months after seroconversion. Conclusions: The AI is an accurate serological marker for discriminating recent from established HIV infections and meets WHO requirements for HIV incidence assays.

Trigger-oriented HIV testing at Internal Medicine hospital Departments in Northern Italy: an observational study (Fo.C.S. Study)

Abstract Background: Early detection of undiagnosed HIV infected patients is of paramount importance. The attitude of Italian hospital-based Internal Medicine physicians to prescribe HIV testing following the detection of HIV-associated signs, symptoms and behaviours (triggers) has been reported to be poor. The aim of the study is to quantify the extent of the missed opportunities for early HIV diagnosis in Internal Medicine Departments (IMD). Methods: Patients admitted to IMD of a General University Hospital in Italy in March–June 2013 were interviewed using a structured questionnaire investigating the presence of triggers for HIV testing, including patient’s characteristics, symptoms and conditions associated with HIV infection. HIV tests performed during hospitalisation were recorded. Results: HIV testing was performed in 73 (6.6%) out of 1113 hospitalisations (1072 patients), providing positive results in three cases (4.1%). All of them presented ≥1 triggers. Conversely, 853 triggers were identified in 528 hospitalisations with at least one trigger (47.4%). The proportion of hospitalisations where an HIV testing was prescribed was 3.1%, 9.5% and 16.0% in the presence of zero, one-to-two or more triggers, respectively. Age <70 years, female gender, length of hospital stay, haematological disease, HBV infection, multiple sexual partners and lymphadenopathy were predictors of HIV testing by logistic regression analysis. Conclusions: Although chances of an HIV test being performed in patients hospitalised in IMD increases along with the number of triggers, the number of tests being performed in people presenting with triggers is unacceptably low and requires educational interventions in order to obtain individual and public health advantages.