ABSTRACT
BACKGROUND: Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex. RESULTS: The cross-sectional study carried out with > 200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations. CONCLUSIONS: Our study shows the association of R284K and TAM1 mutations in individuals failing therapy with tenofovir/emtricitabine, and unveils a novel mechanism by which secondary mutations are selected in the context of drug-resistance mutations.
Subject(s)
Adenine/analogs & derivatives , Deoxycytidine/analogs & derivatives , Drug Resistance, Viral , HIV Infections/virology , HIV Reverse Transcriptase/genetics , HIV-1/genetics , Mutation, Missense , Organophosphonates/administration & dosage , Adenine/administration & dosage , Adenine/pharmacology , Anti-HIV Agents/administration & dosage , Anti-HIV Agents/pharmacology , Deoxycytidine/administration & dosage , Deoxycytidine/pharmacology , Emtricitabine , HIV Infections/drug therapy , HIV Reverse Transcriptase/metabolism , HIV-1/drug effects , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Organophosphonates/pharmacology , Selection, Genetic , Sequence Analysis, DNA , Tenofovir , Treatment FailureABSTRACT
INTRODUCTION: To evaluate the new automated system cobas 4800 CT/NG test for detection of Chlamydia trachomatis in urogenital specimens. MATERIAL AND METHODS: We analyzed 696 specimens (488 swabs from urethral or cervical specimens, and 208 urines) to detect C. trachomatis. The results of the cobas 4800 CT/NG test (c4800) were compared to those obtained with Cobas AMPLICOR CT/NG test (CAM). Discordant results were analyzed with a conventional PCR assay and microchip electrophoresis system in agarose gel, MultiNA. RESULTS: We made two simultaneous analyses. In the first one, we compared the results obtained with swab specimens using the c4800 system and CAM. In this case, the sensitivity, the specificity, the positive and negative predictive values (PPV and NPV) were: 77.9%, 100%, 100% and 96% respectively. In the second one, we compared the results obtained for urine and its corresponding swab specimens on the c4800. The values obtained were: 100%, 98.9%, 92.9% and 100% respectively. The kappa values of these comparisons were: 0.857 for swab specimens on the c4800 and CAM, and 0.957 for urine versus swab specimens on the c4800. CONCLUSIONS: The results obtained with c4800 system were completely comparable with those obtained with CAM. We also noted an excellent correlation with these results when we compared swab specimens with their urine samples in the c4800 system. Therefore this sample type could be used routinely to diagnose infections in men and women.
Subject(s)
Chlamydia trachomatis/isolation & purification , Cervix Uteri/microbiology , Female , Humans , Male , Mycology/methods , Urethra/microbiology , Urine/microbiologyABSTRACT
The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10(1)-10(5) conidia ml(-1)), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5-20 conidia using conidial suspensions. The linear range was from 60 to 6 x 10(7) fg. The Tm ranged from 67.34 to 70.7 degrees C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.
Subject(s)
Aspergillosis , Aspergillus/classification , Aspergillus/isolation & purification , Fungemia , Polymerase Chain Reaction/methods , Aspergillosis/diagnosis , Aspergillosis/microbiology , Aspergillus/genetics , DNA, Fungal/analysis , DNA, Fungal/isolation & purification , Fungemia/diagnosis , Fungemia/microbiology , Humans , Mycological Typing Techniques , Sensitivity and Specificity , Species SpecificityABSTRACT
BACKGROUND: HPV testing in cervical cancer screening has been proposed as an alternative or complementary to cytology in women older than 30 years. However, adequate clinical sensitivity and specificity are crucial for a new test to be implemented. Hybrid Capture 2 (HC2) has proved good clinical performance in selecting women at risk for high-grade intraepithelial lesions with a high sensitivity and specificity. cobas HPV Test has been recently launched and its performance in different clinical settings needs to be determined. OBJECTIVES: The aim of this study was to evaluate the cobas HPV Test for the detection of cervical HPV infection in a population of women in Catalonia (Spain) using HC2 as a reference. MATERIALS AND METHODS: Cervical liquid cytology samples from 958 women have been studied. Sensitivity was analyzed in 60 samples from patients with a high-grade intraepithelial lesion (≥ CIN2) on histology and specificity was determined in 898 samples from women with no ≥ CIN2. All cases had HC2 and cobas HPV Test performed. Statistical analyses of sensitivity, specificity and comparison between HC2 and cobas HPV Test by a non-inferiority test were applied. RESULTS: Sensitivity of HC2 and cobas HPV Test for detecting ≥ CIN2 proved identical (98.3%) while specificity was 85.3% and 86.2% respectively. The non-inferiority test demonstrated that cobas HPV Test surpassed 90% sensitivity and 98% specificity of HC2. CONCLUSION: The cobas HPV Test results fulfilled sensitivity and specificity requirements for HPV based cervical cancer screening and for the triage of minor cytological abnormalities, allowing its introduction in clinical settings.
Subject(s)
Early Detection of Cancer/methods , Human Papillomavirus DNA Tests/methods , Papillomavirus Infections/diagnosis , Papillomavirus Infections/epidemiology , Uterine Cervical Neoplasms/epidemiology , Uterine Cervical Neoplasms/virology , Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Sensitivity and Specificity , Spain/epidemiologyABSTRACT
The greater prevalence of human papillomavirus (HPV) types 16 and 18 compared to the other high-risk HPV types of cervical cancer led to the development of clinical tests that detect both types separately from other genotypes. One method is the Roche Cobas 4800 HPV test, which is based on a real-time PCR. The aim of this study was to evaluate the performance of the Cobas 4800 HPV test for detecting genotypes 16 and 18 by comparing the results with those obtained in a combination of the Roche Amplicor HPV assay and the Roche Linear Array (LA) HPV genotyping assay. Excellent concordance was found between both methods (92.7%, kappa value=0.872). The Cobas 4800 HPV test could be used as a single test for identifying HPV types 16 and 18 directly from clinical specimens.
Subject(s)
Cervix Uteri/virology , Human papillomavirus 16/genetics , Human papillomavirus 18/genetics , Papillomavirus Infections/virology , Polymerase Chain Reaction/methods , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction/methods , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , DNA, Viral/genetics , DNA, Viral/isolation & purification , Female , Genotype , Human papillomavirus 16/isolation & purification , Human papillomavirus 18/isolation & purification , Humans , Nucleic Acid Amplification Techniques/methods , Reproducibility of ResultsABSTRACT
We have evaluated 696 samples (488 swabs and 208 urine specimens) with the cobas 4800 (c4800) CT/NG Test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in swab and urine specimens. c4800 results were compared with those obtained from COBAS AMPLICOR (CAM) CT/NG Test. Discordant results were reanalyzed with the MultiNA system and compared with clinical data. For C. trachomatis detection by both methods, we obtained 93.8%, 100%, 100%, and 99.1% for sensitivity, specificity, and positive and negative predictive values, respectively. For urine specimens analyzed in c4800, our results were 96.6%, 100%, 100%, and 99.4%, respectively. For N. gonorrhoeae detection, swab results were:88.0%, 100%, 100%, and 99.4%. For urine specimen, results obtained were 100%, 100%, 100%, and 100%. Reanalyses were all concordant between both methods. c4800 results were comparable with those obtained with the CAM system. We had an excellent correlation between swab and urine specimens analyzed by c4800.
Subject(s)
Bacteriological Techniques/methods , Chlamydia trachomatis/isolation & purification , Genitalia/microbiology , Molecular Diagnostic Techniques/methods , Neisseria gonorrhoeae/isolation & purification , Sexually Transmitted Diseases, Bacterial/diagnosis , Urine/microbiology , Chlamydia trachomatis/genetics , Female , Humans , Male , Neisseria gonorrhoeae/genetics , Sensitivity and Specificity , Sexually Transmitted Diseases, Bacterial/microbiologyABSTRACT
Introducción: Se ha evaluado el nuevo sistema automatizado cobas 4800 CT/NG test para la detección de Chlamydia trachomatis (C. trachomatis) en muestras urogenitales. Material y métodos: Se analizaron 696 muestras (488 exudados uretrales y cervicales y 208 orinas) para la detección de ADN de C. trachomatis. Los resultados del cobas 4800 CT/NG test (c4800) se compararon con los obtenidos por el Cobas AMPLICOR CT/NG test (CAM). Las discordancias se analizaron mediante PCR convencional y electroforesis en gel de agarosa en microchips, MultiNA. Resultados: Se realizaron dos análisis simultáneos, el primero de ellos fue comparar los resultados obtenidos con los exudados en el c4800 y en CAM. En este caso, la sensibilidad, la especificidad y los valores predictivos positivo (VPP) y negativo (VPN) fueron del 77,9, el 100, el 100 y 96%, respectivamente. El segundo análisis fue comparar los resultados obtenidos para las muestras de orina con los obtenidos con sus correspondientes exudados en el c4800. Los valores obtenidos para sensibilidad, especificidad, VPP yVPN fueron: 100, 98,9, 92,9 y 100%, respectivamente. Los valores kappa de estas comparaciones fueron:0,857 exudados en c4800 y CAM y 0,957 para orina frente a exudados en c4800. Conclusiones: Los resultados obtenidos con c4800 demuestran que son equiparables con los obtenidos con CAM. Asimismo, observamos una correlación excelente al comparar las muestras de exudados con su correspondiente muestra de orina en c4800, por lo que se puede emplear dicha muestra de forma sistemática en el diagnóstico de estas infecciones tanto en varón como en mujeres (AU)
Introduction: To evaluate the new automated system cobas 4800 CT/NG test for detection of Chlamydiatrachomatis in urogenital specimens. Material and methods: We analyzed 696 specimens (488 swabs from urethral or cervical specimens, and208 urines) to detect C. trachomatis. The results of the cobas 4800 CT/NG test (c4800) were compared to those obtained with Cobas AMPLICOR CT/NG test (CAM). Discordant results were analyzed with a conventional PCR assay and microchip electrophoresis system in agarose gel, MultiNA. Results: We made two simultaneous analyses. In the first one, we compared the results obtained withs wab specimens using the c4800 system and CAM. In this case, the sensitivity, the specifity, the positive and negative predictive values (PPV and NPV) were: 77.9%, 100%, 100% and 96% respectively. In the second one, we compared the results obtained for urine and its corresponding swab specimens on the c4800. The values obtained were: 100%, 98.9%, 92.9% and 100% respectively. The kappa values of these comparisons were: 0.857 for swab specimens on the c4800 and CAM, and 0.957 for urine versus swab specimens on the c4800. Conclusions: The results obtained with c4800 system were completely comparable with those obtained with CAM .We also noted an excellent correlation with these results when we compared swab specimens with their urine samples in the c4800 system. Therefore this sample type could be used routinely to diagnose infections in men and women (AU)