Role of Line Immunoassay in the Diagnosis of Early HIV Infection: A Diagnostic Case

WOS: 000324607700019PubMed ID: 23971936Combined p24 antigen-HIV antibody fourth-generation assays that identify most of the early HIV infections have been used extensively worldwide for several years. This poses challenges for the traditional algorithm of line immunoassay (LIA) confirmation. LIA tests are useful methods with their high specificity and their ability to differentiate HIV-1 from HIV-2, but they are reactive days after the fourth generation enzyme immunoassays. With acute HIV infection, high levels of infectious virus are detectable in serum and genital secretions. The rate of transmission during acute HIV infection is higher than the established HIV infection, for this reason, new HIV testing strategies need to focus on sensitivity, especially for this highly contagious phase immediately after infection. Serum sample of a patient sent to Ege University Hospital Clinical Virology Laboratory was repeatedly reactive with low signal/cutoff ratios with two different commercial fourth generation enzyme immunoassays (Architect HIV Ag/Ab Combo Reagent Kit, Abbott, Germany and Vidas HIV Duo Quick, Biomerieux, France). The sample was non-reactive with the LIA (INNO-LIA HIV I/II Score, Innogenetics, Belgium) and HIV RNA (RealTime HIV-1 Amplification Reagent Kit, Abbott, USA) result was positive (4.1 x 10(5) copies/ml). With the presentation of this case, the role of LIA in the diagnosis of early HIV infection and its place in test algorithms were questioned

Comparison of immunofluorescence assay and multiplexed microparticle-based immunoassay for detecting

WOS: 000254720500042PubMed ID: 18215427A new multiplexed microparticle-based immunoassay was compared with the immunofluorescence assay that is used widely for detecting EBV-specific antibodies in immunocompetent patients. Serum samples of 162 patients submitted for routine EBV diagnosis were tested for viral capsid antigen IgM, viral capsid antigen IgG and serological profile interpretations with both systems. The result concordances were 94.2%, 93.6%, and 92.1%, respectively. Multiplexed microparticle-based immunoassay can be an alternative to immunofluorescence assay especially in laboratories receiving large numbers of samples. (C) 2007 Elsevier B.V. All rights reserved

Hepatitis delta virus (HDV) genotypes in patients with chronic hepatitis: molecular epidemiology of HDV in Turkey

WOS: 000243560400015PubMed ID: 16678465Objective: Analysis of hepatitis delta virus (HDV) isolates from around the world has indicated that there are at least three phylogenetically distinct genotypes with different geographic distributions. The aim of this study was to determine the distribution of HDV genotypes by direct sequencing in patients with chronic delta hepatitis in Izmir, Turkey. Design and methods: Serum samples from 32 chronic hepatitis patients (21 mates, 11 females; mean age 44.2 years, range 23-70 years) with anti-delta positivity were analyzed for hepatitis B and C serologies. After reverse transcription, cDNA of partial delta antigen was amplified by in-house nested PCR. The products of the HDV PCR were bidirectionally sequenced with internal primers using Big Dye Terminator DNA Sequencing Kit (Applied Biosystems, CA, USA) and ABI Prism 310 Genetic Analyzer (Perkin Elmer, USA). Nucteotide sequences of HDV were compared with previously reported sequences and aligned by using ClustalW (1.82). Results: HDV-RNA was positive in 26 (81.3%) of 32 anti-delta positive samples. Comparison of the HDVsequences with published sequences of HDV genotypes 1, 11, and III indicated that all were closely related to HDV genotype I isolates. Similarity among isolated sequences ranged from 84% to 96%. Conclusion: HDV genotyping was successfully performed by direct sequencing of the amplicons obtained from routine HDV-RNA screening PCR tests. All of the HDV isolates from the chronic delta hepatitis patients included in this study were found to be genotype I. (c) 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved

Analysis of Hepatitis B Virus (HBV) preS1, preS2 and S Gene Regions from Patient Groups Infected with HBV Genotype D

WOS: 000428835600003PubMed ID: 29642827Mutations in preS and S gene regions of hepatitis B virus genome may cause immune escape and diagnostic escape HBV mutants. The aim of this study was to determine preS1, preS2 and S gene regions of HBV from HBV infected patient groups by sequence analysis and contribute to the relevant literature. Nucleic acid sequence analysis of preS and S genes of HBV PCR products from 56 archived plasma samples sent to Ege University Faculty of Medicine Medical Microbiology Department Molecular Virology laboratory, for HBV tests were determined by chain termination reaction. Amino acid (aa) sequences were compared with the reference sequences obtained from GenBank. Plasma samples belonged to four groups of patients: A- Chronic HBV infected patients with typical HBV serological profiles (22 samples), B-HBV infected patients with atypical HBV serological profiles (26 samples), C- HBV re-infected patients after liver transplantation (5 samples), D- Seroconversion phase following acute HBV infection (3 samples). One of two vaccine escape mutant samples was also diagnostic escape mutant; the other diagnostic escape mutant was isolated from anti-HBc positive sample. All of the sequences were determined as genotype D. HBsAg subtypes were determined as; two ayw1, six ayw3, two mix, 46 ayw2. Among the 304 codons analysed between preS 33rd and S 162nd amino acids; aa variants were determinedin 105 codons (34.5%). Sequences can be found in GenBank with accession numbers FJ001941-FJ001996. At least one aa variation was detected in 48 of 56 samples (85.7%). The amino acid variants were as follows; PreS1: A33T, A39T, P41 K, D44del, D5ON, T51P, D54N, L65P/M, F67L, W77T, A81 S, Q82E, I84T, L851/M, Q86H/T, L88S, A90T/V, N91K/del, A95P, S96A, T97I/A, N98K, Q100K, S101T, S109T, P110S, N114D/E, PreS2: M1V, Q2R, S5H, F8S, H9Q, Q13L, D14N, R16K, R18K, G19S/D, F22L/S, S28T, G30E, N33T, V39A, P41H/L, I42T/L, I45T, F46Y, S47L, R48K, I49T, D51V/G, P52L, A53V, L54R/G, N55K; S gene: E2D, I4F, F8L, G10A, V14A, F20S, L22del, R24K, P29L, Q30K, N40S, F41del, G44E, T45L, T46P, V47A, L49R, Q54R, P56L, S64F, P70A, M75I, C76Y, R79H, I81T, F83C, L88P, L94S, Y100F, Q101H/R, M103L, L104F, L109I/M, I110L, G112S/R, S113N/P, S114A/del, T115I, T116N, T118A/K, P120A/T, T123A, in "a" determinant; T126I, Q129H/R, T131N, M133T, Y134N, S136Y, S143L/M/T, D144E, G145A/R. Deletions were also found in all three preS/S gene regions. The highest number of aa variations were detectedin the isolated anti-HBc positive sample (in 24 codons), followed by liver transplant group (8-13 codons). Point mutation was detected in the preS2/S promoter CCAAT box. Major hydrophilic region (MHR) variants were determined in 41.1% of 56 samples. The highest number of MHR variants belonged to atypical HBV serological profile group (group B; 61.5%) and liver transplantation group with HBV re-infection (all C group). Among the diagnostic escape and immune escape mutant (anti-HBs positive) samples, reported MHR and "a" determinant mutations were detected. In conclusion, the study population carries HBV preS/S variants; MHR and "a" determinant variant rates are high among diagnostic or immune escape mutants. It is important to evaluate the mutant detection performance of HBsAg tests

Comparison of Two Anti-HIV Confirmatory Assays: Recombinant HIV 1/2 Line Immunoassay and Geenius HIV 1/2 Confirmatory Test

WOS: 000582397200009PubMed: 33107290The main purpose in the diagnosis of human immunodeficiency virus (HIV) infection is to rapidly and accurately identify people with HIV infection. It is recommended that samples that are repeatedly reactive should be verified/supported according to the classical algorithms of international and national guidelines. the recombinant "line immunoassay test (LIA)", which has been used for many years, is studied with the accumulated samples to be cost and labor-effective. in this study, the supplemental recombinant HIV 1/2 LIA (INNO-LIA (R), Fujirebio, Ghent, Belgium) used to confirm and differentiate the diagnosis of HIV-1 and HIV-2 infections, and an immunochromatographic supplemental test (Geenius (TM) (Bio-Rad Laboratories, Marnes-la-Coquette, France) which can provide faster results were compared. One hundred fifty serum samples sent to Ege University Faculty of Medicine Hospital Medical Virology Laboratory with anti-HIV 1/2 and p24 antigen positive and indeterminant results and three HIV-1 positive external quality control samples were included in the study. Samples were tested both with the Geenius (TM) HIV 1/2 (Bio-Rad Laboratories, Marnes-la-Coquette, France) and recombinant HIV 1/2 LIA (INNO-LIA (R), Fujirebio, Ghent, Belgium). HIV 1 viral load was evaluated by using Abbott real-time HIV-1 test in Abbott m200sp system (Abbott Molecular, Wiesbaden, Germany) in plasma samples. in both assays, the results were consistent in 147 samples (96.08%). Six samples that have discordant results were as follows: one sample was LIA HIV-1 positive and Geenius indeterminate, two samples were LIA indeterminant and Geenius HIV-1 positive, and in three samples, LIA was indeterminate and Geenius negative. in two EIA reactive samples (2/97, 2.06%) and three EIA negative samples (3/53, 5.66%) LIA results were indeterminant. Geenius test, on the other hand, correctly identified HIV positive and negative samples. the immunochromatographic test could be used in the diagnostic algorithm of HIV infection, due to its short application time, not being labor intensive, its ability to distinguish HIV-1/2, its high sensitivity/specificity compared to LIA, and the compliance with LIA. However, it should be noted that in acute HIV infection, all analytical antibody tests, become reactive later than the fourth generation enzyme immunoassays

Investigation of drug resistance against protease, reverse transcriptase, and integrase inhibitors by next-generation sequencing in HIV-positive patients

WOS: 000585023500001PubMed: 33026651Our aim was to investigate the mutations in protease (PR), reverse transcriptase (RT), and integrase (IN) gene regions in human immunodeficiency virus (HIV) using a single amplicon via next-generation sequencing (NGS). the study included plasma samples from 49 HIV-1-positive patients, which were referred for HIV-1 drug resistance testing during 2017. A nested polymerase chain reaction (PCR) was performed after the RNA extraction and one-step reverse transcription stages. the sequencing of the HIV genome in the PR, RT, and IN gene regions was carried out using MiSeq NGS technology. Sanger sequencing (SS) was used to analyze resistance mutations in the PR and RT gene regions using a ViroSeq HIV-1 Genotyping System. PCR products were analyzed with an ABI3500 GeneticAnalyzer (Applied Biosystems). Resistance mutations detected with NGS at frequencies above 20% were identical to the SS results. Resistance to at least one antiretroviral (ARV) drug was 22.4% (11 of 49) with NGS and 10.2% (5 of 49) with SS. At least one low-frequency resistance mutation was detected in 18.3% (9 of 49) of the samples. Low-frequency resistance mutations resulted in virological failure in only one patient. the cost of the analyses was reduced by sample pooling and multiplex analysis using the MiSeq system. This is the first study in Turkey to use NGS technologies for the detection of resistance mutations in all three gene (PR, RT, IN) regions using a single amplicon. Our findings suggest that NGS is more sensitive and cost-effective than the SS method

Hopelessness in Turkish cancer inpatients: The relation of hopelessness with psychological and disease-related outcomes

WOS: 000266230900002PubMed ID: 19246240The purpose of this Study was to investigate the hopelessness level and the relationship of depression, anxiety and disease-related factors to the presence of hopelessness among Turkish patients with cancer. Ninety-five patients hospitalized for cancer treatments were recruited for current study. Data were collected by using a demographic questionnaire, the Pain Numeric Rating Scale, the Beck Hopelessness Scale, and the Hospital Anxiety Depression Scale. The mean hopelessness score was 5.20 +/- 4.39. There were significant differences in terms of hopelessness between the patients who had metastasis and pain as compared with those without metastasis and pain (p < 0.05). There were also found that significant correlation between hopelessness and depression and between hopelessness and anxiety (r = 0.721; r = 0.645, respectively, p < 0.001). Foreword stepwise multiple regression analysis revealed that the independent predictors of hopelessness were depression score and thr presence of metastasis (F = 55.133: p < 0.001). The findings suggest that levels of hopelessness among cancer patients with pain and metastasis are higher than among those without pain and metastasis, and that the severity of pain, anxiety, and depression is positively correlated with hopelessness level. The assessment of hopelessness, pain, anxiety and depression levels of the patients with cancer should be an essential part of health care practice. Therefore, when arranging care assessment, to evaluate hopelessness could help professionals to appropriately refer patients to further psychological care resources. (C) 2009 Elsevier Ltd. All rights reserved

Restriction fragment length polymorphism analysis and direct sequencing for determination of HBV genotypes in a Turkish population

WOS: 000256866500005PubMed ID: 18623983The aim of this study was to analyze the restriction fragment length polymorphism and direct sequencing results in genotyping of hepatitis B virus from a Turkish population in a clinical virology laboratory. Serum samples of 54 chronic hepatitis B patients attending the Ege University Hospital were studied. Sequences of partial S gene PCR products were analysed and RFLP was performed. Fifty-three isolates could be identified by direct sequencing as genotype D. One sample needed to be cloned and determined as genotype D. Forty-two isolates were genotyped as D with RFLP according to published determinative patterns. Twelve isolates had undefined patterns. Eight of them suggested a mixture of isolates with different patterns and cloning of these samples confirmed the presence of heterogeneous isolates. Four isolates with undefined pattern were determined as genotype D by direct sequencing. All the studied isolates were genotype D. The results of this studied population suggest that RFLP is suitable for HBV genotyping in a routine clinical virology laboratory setting. However sequence analysis and even cloning may be needed to clarify indeterminate results