RESUMEN
To meet the screening goal of WHO's 90-70-90 strategy aimed at eliminating cervical cancer (CC) by 2030, clinical validation of human papillomavirus (HPV) assays is essential to provide accurate and valid results through fulfilling three criteria of the international validation guidelines (IVGs). Previously, the clinical accuracy of the AmpFire® HPV Screening 16/18/HR assay (AmpFire assay) was reported but reproducibility data are lacking. Here, we aim to evaluate the intra- and inter-laboratory reproducibility of the AmpFire assay. The reproducibility of the isothermal AmpFire assay was assessed using 556 cervical cell samples collected from women attending CC screening and biobanked in a Belgian HPV national reference center. This assay detects HPV16, HPV18, and 12 other high-risk HPV (hrHPV) types (31/33/35/39/45/51/52/56/58/59/66/68) in aggregate. Lower 95% confidence interval bound around the assay's reproducibility should exceed 87%, with κ ≥ 0.50. Additionally, a literature review of the assay's clinical performance was performed. The AmpFire assay showed an excellent intralaboratory (96.4%, 95% CI:94.5-97.8%, κ = 0.920) and interlaboratory (95.3%, 95% CI:93.2-96.9%, κ = 0.897) reproducibility. One study demonstrated noninferior sensitivity of a prototype AmpFire assay targeting 15 hrHPV types (including HPV53) to detect CIN2+. However, clinical specificity became similar to the comparator after removing HPV53 from analyses. The low-cost and easy-to-use AmpFire assay presents excellent reproducibility and-after removing HPV53 from the targeted types-fulfills also clinical accuracy requirements. Inclusion of HPV53, which is not recognized as carcinogenic, comprises clinical specificity of screening assays.
Asunto(s)
Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Humanos , Reproducibilidad de los Resultados , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Femenino , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/virología , Papillomaviridae/aislamiento & purificación , Bélgica , Detección Precoz del Cáncer/métodos , Detección Precoz del Cáncer/normas , Adulto , Sensibilidad y Especificidad , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/normas , Técnicas de Diagnóstico Molecular/métodos , Cuello del Útero/virologíaRESUMEN
BACKGROUND: Detection of high-risk human papillomaviruses (hrHPV) is widely used at the first line of cervical cancer screening, requiring rigorous validation of the clinical performance of commercial kits designed for this indication. METHODS: Performance of the AmpFire HPV Screening 16/18/HR test (AF, Atila Biosystems) and the Hybrid Capture 2 test (HC2, Qiagen) for detecting hrHPV was cross-compared in 200 cervical samples in our institution. RESULTS: The global percentage of agreement between the 2 techniques was 95.0% (95%CI 92-98%) with a Cohen's kappa coefficient of 0.85 (95%CI 0.75-0.94). Ten samples showed discordant results between the 2 techniques in both directions (5 HC2+/AF- and 5 HC2-/AF+). Among possible explanations for these discrepancies was the detection of HPV66 and HPV53 genotypes in two samples, since these genotypes are targeted by the Ampfire test but not by the HC2 test, as well as intrinsic differences in analytical performance to target specific genotypes. CONCLUSIONS: A high level of agreement was observed between the two techniques, which encourages further testing in order to definitively validate the use of the Ampfire kit for primary cervical cancer screening.
Asunto(s)
Alphapapillomavirus , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Humanos , Femenino , Detección Precoz del Cáncer , Infecciones por Papillomavirus/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Alphapapillomavirus/genética , GenotipoRESUMEN
Men who have sex with men (MSM) are at high risk of sexually transmitted infections, among which HPV infections are particularly prominent. We took advantage of the MémoDépistages study to evaluate HPV distribution at anal and oropharyngeal sites in HIV-negative multipartner MSM. HPV DNA was detected in 82% (n = 344) of anal and 11% (n = 45) of oropharyngeal self-collected samples taken from 421 participants. Multiple HPV types were detected in 70% of anal samples, and single HPV types in 91% of oropharyngeal samples. HPV16 was the most frequent type detected in the anus, followed by HPV6, HPV51, and HPV52. HPV6, HPV16, and HPV11 were the most prevalent types in the oropharynx. HPV targeted by the nonavalent vaccine was detected in 71% and 50% of HPV-positive anal and oropharyngeal samples, respectively. The main risk factor associated with HPV detection was frequenting gay meeting places, living in large cities, and having an anal Chlamydia trachomatis/Neisseria gonorrhoeae infection. In this cohort of highly sexually active MSM, HPV detection was highly frequent and rendered them at high risk of precancerous and cancerous lesions. Universal vaccination against HPV before sexual debut is an important public health strategy to prevent HPV-associated cancers in this highly vulnerable population of HIV-negative MSM.
Asunto(s)
Enfermedades del Ano , Infecciones por VIH , Infecciones por Papillomavirus , Minorías Sexuales y de Género , Masculino , Humanos , Canal Anal , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/epidemiología , Homosexualidad Masculina , Papillomavirus Humano 16 , Papillomaviridae/genética , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiologíaRESUMEN
High-risk (HR) human papillomavirus (HPV)-associated carcinogenesis is driven mainly by the overexpression of E7 and E6 oncoproteins following viral DNA integration and the concomitant loss of the E2 open reading frame (ORF). However, the integration of HR-HPV DNA is not systematically observed in cervical cancers. The E2 protein acts as a transcription factor that governs viral oncogene expression. The methylation of CpGs in the E2-binding sites (E2BSs) in the viral long control region abrogates E2 binding, thus impairing the E2-mediated regulation of E7/E6 transcription. Here, high-resolution melting (HRM)-PCR was developed to quantitatively analyze the methylation statuses of E2BS1, E2BS2, and the specificity protein 1 (Sp1)-binding site in 119 HPV16-positive cervical smears. This is a rapid assay that is suitable for the analysis of cervical samples. The proportion of cancer samples with methylated E2BS1, E2BS2, and Sp1-binding site CpGs was 47%, whereas the vast majority of samples diagnosed as being within normal limits, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions (HSIL) harbored unmethylated CpGs. Methylation levels varied widely, since some cancer samples harbored up to 60% of methylated HPV16 genomes. A pyrosequencing approach was used as a confirmation test and highlighted that quantitative measurement of methylation can be achieved by HRM-PCR. Its prognostic value deserves to be investigated alone or in association with other biomarkers. The reliability of this single-tube assay offers great opportunities for the investigation of HPV16 methylation in other HPV-related cancers, such as head and neck cancers, which are a major public health burden.
Asunto(s)
Metilación de ADN , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/metabolismo , Neoplasias del Cuello Uterino/virología , Sitios de Unión , Femenino , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa , Temperatura de TransiciónRESUMEN
Efficient primary (vaccination) and secondary (screening) prevention strategies have the potential to eliminate cervical cancer worldwide. In this context, surveillance of HPV infections remains mandatory to assess the efficacy and the impact of screening and vaccination policies. Therefore there is a need to safely store cervical samples to conduct long-term studies in vaccinated and non-vaccinated subjects. Up-dated data on cervical specimen preservation on FTA® cards indicate that HPV DNA can be safely retrieved after 54 months of storage. A concordance of 97 % was achieved between HPV genotypes detected in initial cervical samples and on FTA® cards 4.5 years later. Even if a drop in HPV viral loads was observed in some cases at 4.5 years, using FTA® cards for safe and long-term storage of cervical samples represents an interesting option.
Asunto(s)
Alphapapillomavirus , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , ADN Viral/genética , Femenino , Humanos , Infecciones por Papillomavirus/diagnóstico , Manejo de EspecímenesRESUMEN
Alveolar echinococcosis (AE) is a parasitic disease, due to Echinococcus multilocularis. Often compared to liver cancer, it develops by infiltration from its primary site to the surrounding tissue, and can then metastasize to other organs. Detection of circulating cell-free DNA (ccfDNA) is a useful analytical tool in oncology, for diagnosis, prognosis, and therapy monitoring. This study sought to investigate the presence of ccfDNA in patients with AE, and its potential usefulness for the evaluation of treatment efficiency. To achieve these aims, a quantitative PCR and a droplet digital PCR were developed to detect E. multilocularis ccfDNA. An AE animal model identified, for the first time, the presence of large quantities of ccfDNA. Samples from patients with AE (nâ¯=â¯31) were then analyzed twice, at diagnosis, and after three months of chemotherapy: about 25% were positive, almost always with very low concentrations of ccfDNA. These results confirmed that E. multilocularis produces ccfDNA, as solid tumors do, but detection may not yet be sufficient for AE diagnosis nor for the evaluation of treatment efficiency, due to the low levels of ccfDNA detected in patient serum.
Asunto(s)
Ácidos Nucleicos Libres de Células/sangre , ADN de Helmintos/sangre , Equinococosis/diagnóstico , Echinococcus multilocularis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Ácidos Nucleicos Libres de Células/genética , ADN de Helmintos/genética , Equinococosis/sangre , Equinococosis/parasitología , Echinococcus multilocularis/aislamiento & purificación , Gerbillinae , HumanosRESUMEN
Cystic echinococcosis (CE) is a neglected zoonotic disease, diagnosed through clinical findings, imaging techniques, and serology, for which many serological tests are available. Here we report a rapid unit assay, the immunochromatographic VIRapid® HYDATIDOSIS test (Vircell, Granada, Spain), potentially suitable for laboratories in low-prevalence or poorly equipped areas. This test was evaluated with a large retrospective cohort (224 sera), including patients suffering from CE or from other parasitic or liver diseases. The test was also assessed in routine conditions with a prospective cohort (115 sera) in areas where both cystic and alveolar echinococcoses have been diagnosed. Its performance (in terms of sensitivity, specificity, and both positive and negative likelihood ratios) was similar to an ELISA based on a crude antigen. Our study shows that this test performs adequately in the diagnostic process, when used with caution, especially regarding cross-reactivity with other parasitic diseases.
Asunto(s)
Cromatografía de Afinidad/métodos , Pruebas Diagnósticas de Rutina/métodos , Equinococosis/diagnóstico , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
PURPOSE: The only drugs available to treat alveolar echinococcosis (AE) are mostly parasitostatic and in many cases prescribed for life. Decision criteria for discontinuation rely on the absence of parasitic viability. The aim of the present study is to search for candidate proteins that may exhibit good potential as biomarkers for viability. EXPERIMENTAL DESIGN: Sixteen serum samples (five healthy controls, 11 patients with AE), are used. AE-patients are classified into three groups "Cured" (n = 2), "ABZ-responders" (n = 4) and "ABZ-nonresponders" (n = 5). Immunoreactive proteins from vesicular fluid (VF) are identified and quantified by LC-MS/MS analysis after immunoprecipitation (IP) using all 16 serum samples. RESULTS: Shotgun analysis of VF lead to the identification of 107 E. multilocularis proteins. Comparative proteomics reveal nine proteins more abundant in IP eluates from ABZ-nonresponder patients (cathepsin b, prosaposin a preprotein, actin modulator protein, fucosidase alpha L1 tissue, gluthatione-S-tranferase, beta galactosidase, elongation factor 2, H17g protein tegumental antigen, and NiemannPick C2 protein). CONCLUSIONS AND CLINICAL RELEVANCE: Detection of antibodies against these proteins by ELISA could be helpful to monitor the course of alveolar echinococcosis under albendazole (ABZ) treatment.
Asunto(s)
Biomarcadores/metabolismo , Equinococosis/tratamiento farmacológico , Echinococcus multilocularis/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Albendazol/uso terapéutico , Animales , Western Blotting , Echinococcus multilocularis/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The analysis of KRAS mutations has become a prerequisite for anti-epidermal growth factor receptor therapy in patients with metastatic colorectal cancers. KRAS mutations are associated with resistance to treatment by monoclonal antibodies such as cetuximab and panitumumab and thus are correlated with a shorter progression-free survival. BRAF mutations also may play a role in treatment decisions. The widespread use of these targeted therapies has generated the need to develop cost-effective methods for routine KRAS and BRAF analysis. The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations. Thus 110 routinely formalin-fixed and paraffin-embedded tissue blocks were tested by each method. The SNaPshot analysis detected KRAS and BRAF codon 600 mutations in, respectively, 34.5% (n = 38) and 10% (n = 11) of these tissue blocks. These results were confirmed by direct DNA sequencing and by high-resolution melting analysis. The costs and time constraints of each detection method were compared at the same time. In conclusion, our newly designed multiplex SNaPshot assay is a fast, inexpensive, sensitive, and robust technique for molecular diagnostic practices and patient selection.