Comparison of circulating DNA in malignant neoplasia from diverse locations: Investigating a diagnostic role

Context: Circulating free DNA (cfDNA) analysis has emerged as novel noninvasive diagnostic biomarker in several solid tumors. Raised levels have been reported in several malignancies and may correlate with clinicopathological and treatment response. The current study was designed to assess the diagnostics of cfDNA in different tumor types of malignancies correlating with tumor (T), nodes (N), and metastases (M) stage. Design: Serum samples were collected from treatment naïve cases with histologically diagnosed tumors including 23 brain tumors, 48 breasts, 50 gallbladder carcinoma (GBC), 13 lungs, 68 oral squamous cell carcinoma (OSCC), and 25 normal controls. CfDNA was quantified with real-time polymerase chain reaction (PCR), Invasive ductal carcinoma (IDC) using beta-globin gene amplification. Cut off values for diagnostics were calculated using receiver operating curve analysis. Results: Contrary to other cfDNA studies where it was postulated that cfDNA would not cross the blood–brain barrier and reach the systemic circulation, we found detectable cfDNA in glioma with median (Q1–Q3) of 349.22 ng/ml (19.87–1276.58). Median cfDNA concentration in breast, gallbladder, lung, oral and normal controls was 328.72 (128.38–624.44), 778.50 (589.88–1864.35), 348.73 (194.67–483.61), 386.27 (47.88–959.67), and 74.12 (49.66–120.00), respectively. Grades I and II glioma had significantly lower levels compared to Grades III and IV (P = 0.0001). Significant difference in median cfDNA values in IDC and GBC was observed with increasing tumor grades, stage, T stage, nodal stage and metastasis and with stage of OSCC cases. Conclusion: CfDNA levels showed good diagnostic discrimination in glioma, GBC, breast, lung carcinoma, and OSCC. Significant increase in titers was evident with increase in cancer stage from I to IV in breast, GBC and OSCC.

Evaluación de una reacción en cadena de la polimerasa en tiempo real simple y rápida para la cuantificación del ADN del virus de la hepatitis B / Evaluation of a simple and fast real time polymerase chain reaction assay for quantification of hepatitis B virus DNA

Introducción: Los ensayos para cuantificar el ADN del virus de la hepatitis B (VHB) o carga viral son imprescindibles en el diagnóstico y en el seguimiento de los pacientes con hepatitis B crónica; de ahí que estén disponibles estuches diagnósticos para esta función. En el presente estudio se muestra la validación de SUMASIGNAL VHB (un paso), el cual es un sistema de reacción en cadena de la polimerasa en tiempo real (RCP-TR) para la cuantificación del genoma del VHB, propuesto por el Centro de Inmunoensayo. Objetivo: Evaluar el desempeño analítico de SUMASIGNAL VHB (un paso). Métodos: Se utilizó un panel de 80 muestras de suero bien caracterizadas y el Tercer Estándar Internacional de la OMS para las técnicas de amplificación de ácidos nucleicos del virus de la hepatitis B. Se determinaron las características del ensayo como especificidad clínica, especificidad analítica (reactividad cruzada), rango lineal o linealidad y exactitud, precisión intraensayo y comparación con un ensayo de referencia. Resultados: La especificidad analítica y clínica fue del 100 por ciento. Al evaluar la linealidad y exactitud con un estándar de referencia de la OMS, se obtuvo que la totalidad de las diferencias entre los Log10 del valor obtenido y el de referencia resultaron inferiores a 0,5 Log10 (r= 0,9977 y r2= 0,9954). Además, se obtuvieron bajos coeficientes de variación intraensayo. La evaluación comparativa con el estuche comercial Artus HBV RG PCR kit mostró una correlación fuerte (r= 0,8882). Conclusiones: SUMASIGNAL VHB (un paso) es un ensayo fácil de realizar manualmente, es rápido e incluye reactivos de extracción de ácidos nucleicos. Teniendo en cuenta la validez del método para el uso previsto, puede ser recomendado para su introducción en el diagnóstico, la vigilancia y la indicación de tratamiento en los pacientes con hepatitis B crónica(AU)

Introduction: Assays to quantify hepatitis B virus (HBV) DNA or viral load are indispensable for the diagnosis and follow-up of patients with chronic hepatitis B, hence the availability of diagnostic kits for this purpose. The present study deals with the validation of HBV SUMASIGNAL (one step), a real time polymerase chain reaction (RT-PCR) system for quantification of the HBV genome proposed by the Immunoassay Center. Objective: Evaluate the analytical performance of HBV SUMASIGNAL (one step). Methods: Use was made of a panel of 80 well characterized serum samples and the Third WHO International Standard for hepatitis B virus nucleic acid amplification techniques. Determination was performed of assay characteristics such as clinical specificity, analytical specificity (cross-reactivity), linear range or linearity and accuracy, intra-assay precision and comparison with a reference assay. Results: Analytical and clinical specificity was 100 percent. Evaluation of linearity and accuracy with a WHO reference standard revealed that all the differences between the log10 of the value obtained and the reference value were lower than 0.5 log10 (r= 0.9977 and r2= 0.9954). The intra-assay variation coefficients obtained were low. Comparative evaluation with the commercial Artus HBV RG PCR kit showed a strong correlation (r= 0.8882). Conclusions: The assay HBV SUMASIGNAL (one step) is easy to conduct manually, fast and includes reagents for nucleic acid extraction. Based on the validity of the method for the use in mind, it may be recommended for incorporation into the diagnosis, surveillance and treatment of patients with chronic hepatitis B(AU)

Quantification of microbial DNA in laboratory environment during DNA extraction / 生物工程学报

Metagenomic sequencing provides a powerful tool for microbial research. However, traditional experimental DNA extraction process will inevitably mix with environmental microorganisms which float in the air. It is still unclear whether the mixed environmental microbial DNA will heavily affect the metagenomic results of samples with extremely low microbial content. In this study, we first collected environmental bacteria in the laboratory and quantified the mixed environmental microbial DNA content during DNA extraction based on a qPCR-based quantification assay. We then extracted DNA from pure water in order to determine the mixed microbial taxons during extraction under open environment. At last, we extracted total DNA from a skin sample in a Biosafety cabinet or under open laboratory environment, to assess the impact of the mixed environmental microorganisms on the metagenomic results. Our results showed that DNA extraction under open laboratory environment in Beijing region resulted in 28.9 pg contaminant, which may accout for 30% of total DNA amount from skin samples. Metagenomic analysis revealed that the main incorporated environmental taxons were Cutibacterium acnes and Escherichia coli. Tens of environmental bacteria were foisted in the skin DNA samples, which largely decreased the relative abundance of dominant species and thus deteriorated the result accuracy. Therefore, analyzing microbial composition of samples with extremely low DNA content should better performed under aseptic environment.

Bubaline Diaphragm Matrix: Development and Clinical Assessment into Cattle Abdominal Hernia Repair

Abstract The purpose of the study was to develop a xenogenic bubaline diaphragm matrix (BDM) for abdominal hernia repair. A fresh diaphragm was decellularized using aqueous sodium dodecyl sulfate (SDS) solutions (0.5-4% w/v) over a period. Acellularity was confirmed histologically and characterized by Masson's trichrome staining, scanning electron microscopy (SEM), DNA quantification, agarose gel electrophoresis, and Fourier-transform infrared spectroscopy. The BDM was used for clinical abdominal hernia repair in six cattle. Clinical, hemato-biochemical and antioxidant parameters were evaluated to assess biocompatibility of xenogenic BDM. Histologically, the diaphragm treated with 2% SDS for 48 h showed complete acellularity and orderly arranged collagen fibers. The SEM confirmed preservation of collagen structure and integrity. The DNA content was significantly (P < 0.05) reduced in BDM (33.12 ± 5.40 ng/mg) as compared to the native diaphragm (443.96 ± 162.60 ng/mg). DNA extracts from BDM show considerable removal of DNA material, with absence of DNA band in agarose gel. The FTIR spectrum of BDM has shown all characteristic transmittance peaks of bovine skin collagen indicating preserved collagen structure. Six cattle with BDM implant recovered uneventfully and remained sound at least upto 6 months. Hemato-biochemical and antioxidant findings were unremarkable. Bubaline diaphragm matrix shows excellent repair efficiency and biocompatibility for abdominal hernia repair in cattle without complications.

Comparison of the QIAGEN artus HBV QS-RGQ Assay With the Roche COBAS AmpliPrep/COBAS TaqMan HBV Assay for Quantifying Viral DNA in Sera of Chronic Hepatitis B Patients

BACKGROUND: Hepatitis B virus DNA quantification is essential for managing chronic hepatitis B (CHB). We compared the performance of artus HBV QS-RGQ (QIAGEN GmbH, Germany) and CAP/CTM v2.0 HBV assays (Roche Molecular Diagnostics, USA) in CHB patients. METHODS: A comparative evaluation between two assays was performed with 508 clinical serum samples. Precision, linearity, and the limit of detection (LOD) of QS-RGQ assay was evaluated by using the WHO standard 97/750 and clinical samples. RESULTS: Detection rates and viral loads as determined QS-RGQ assay were significantly lower than those from the CAP/CTM v2.0 assay (52.8% vs 60.6%; 3.55±1.77 IU/mL vs 4.18±1.89 IU/mL, P<0.0001). The kappa coefficient between qualitative results was 0.79 (95% confidence interval, 0.74 to 0.85). Bland-Altman plot found a mean difference of (QS-RGQ − CAP/CTM v2.0)=−0.63 log₁₀ IU/mL (95% limit of agreement, −1.48 to 0.22). Repeatability and total imprecision (% CV) of the QS-RGQ assay were 1.0% and 1.1% at 2,000 IU/mL, and 0.7% and 1.4% at 20,000 IU/mL, respectively. Linearity of this assay ranged from 31.6 to 1.0±10⁷ IU/mL, and the LOD was 2.95 IU/mL. CONCLUSIONS: The artus HBV QS-RGQ assay showed good performance but significantly decreased detection rate and viral load compared with CAP/CTM v2.0 assays. This assay recommends using plasma; however, we used stored serum because of the retrospective study design. Usually HBV DNA quantification is performed in plasma or serum, but sample type and clinical relevance of quantitative values should be considered when determining the clinical application of this reagent.

Reacción en cadena de la polimerasa en tiempo real para la cuantificación del ADN del virus de la hepatitis B / Real-time polymerase chain reaction assay for Hepatitis B virus DNA quantification

Introducción: los niveles de ADN viral en muestras de suero son un marcador útil para monitorear la progresión de la enfermedad y la respuesta al tratamiento en pacientes con hepatitis B crónica; de ahí que se comercialicen estuches diagnósticos para esta función, con la desventaja de ser costosos. Objetivos: desarrollar y evaluar el desempeño analítico de un sistema de reacción en cadena de la polimerasa en tiempo real para la cuantificación del ADN del virus de la hepatitis B. Métodos: se utilizaron cebadores que amplifican un fragmento del gen C y sonda de hidrólisis en el equipo LightCycler 1.5. Se construyó una curva estándar y se evaluó su eficiencia. Se utilizaron 272 muestras de suero para ensayos de especificidad analítica y clínica, especificidad y exactitud genotípica, coeficientes de variación intraensayo e interensayo, comparación con un estuche comercial y con la reacción en cadena de la polimerasa cualitativa para el virus de la hepatitis B. Resultados: la curva estándar mostró excelente correlación lineal (r= -1) y valores muy bajos de error a lo largo de varias magnitudes de concentración de ADN diana. La especificidad analítica y clínica fue de 100 %, en tanto que al evaluar la especificidad y exactitud genotípica, se obtuvo que las diferencias entre los Log10 del valor obtenido y el de referencia eran inferiores a 0,5 Log10. El límite de detección por análisis de Probit se estimó en 16,41 UI/µL con un rango dinámico de cuantificación de hasta 10(8) UI/mL. El sistema mostró bajos coeficientes de variación intraensayo (0,16 a 1,45 %) e interensayo (0,9 a 2,62 %). La comparación con el estuche comercial artus HBV LC PCR kit mostró una correlación de r= 0,964 y r²= 0,929; con la reacción en cadena de la polimerasa cualitativa se confirmó la mayor sensibilidad y ventajas de la reacción en cadena de la polimerasa en tiempo real. Conclusiones: el ensayo cumple con los requisitos para la cuantificación del ADN del virus de la hepatitis B, que demuestra ser específico, sensible y reproducible. Su aplicación permitirá un mejor diagnóstico y seguimiento de los pacientes con hepatitis B crónica en Cuba.

Introduction: viral DNA levels in serum samples are a useful marker to monitor the disease progression and the treatment response in patients with chronic hepatitis B. Commercial kits for this purpose are available, but they are considerably expensive. Objectives: to evaluate the analytical performance of a real-time polymerase chain reaction (RT-PCR) assay for Hepatitis B virus DNA quantification. Methods: specific primers to the gene C and TaqMan chemistry in a LightCycler 1.5 equipment was used. A standard curve was made and evaluated. Two hundred and seventy-two serum samples were used to assess the clinical and analytical specificity, the genotypic accuracy and specificity, the intra-assay and interassay coefficients of variation and the comparison with a commercial assay and with the qualitative PCR. Results: the standard curve showed a strong linear correlation (r= -1) and low error values in the tested target DNA concentration. Analytical and clinical specificities were 100 %. Genotype accuracy and specificity showed that the differences between the results obtained by RT-PCR assay and those of the reference assay were less than 0.5 Log10. The 95% HBV DNA detection end-point assessed by Probit analysis was 16.41 IU/µL with a dynamic range of quantification of 10(8) IU/mL. Intra-assay and interassay coefficients of variation ranged from 0.16 to 1.45 % and 0.9 to 2.62 % respectively. The RT-PCR assay correlated well with those from a commercial assay (r= 0.964 and r²= 0.929) and with the HBV qualitative PCR, thus confirming its better sensitivity and advantages. Conclusions: the RT-PCR assay is well suited to monitoring HBV DNA levels showing to be sensitive, specific and reproducible. Its application in the clinical practice ensures a better diagnosis and management of patients with chronic hepatitis B in Cuba.

A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes / Ensaio quantitativo em tempo real para o DNA do vírus da hepatite B (HBV) desenvolvido para detectar todos os genótipos do HBV

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.

O vírus da Hepatite B (HBV) é uma das principais causas de doença crônica do fígado no mundo. Além do genótipo, a análise quantitativa do HBV é amplamente utilizada para monitorar a progressão da doença e o tratamento. Em locais com recursos escassos, métodos baratos para o monitoramento da carga viral são desejáveis e, em países em desenvolvimento, sua utilidade já foi demonstrada para outros vírus, como o da Hepatite C e HIV. Neste trabalho, descrevemos a validação de um teste de PCR em Tempo Real para a quantificação do DNA do HBV utilizando sondas Taqman/MGB. Os oligos e sondas foram escolhidos usando um alinhamento contendo seqüências de todos os genótipos do HBV para garantir uma amplificação igual de todos eles. O teste possui um controle interno e foi padronizado com um painel internacional de HBV. Sua eficácia foi testada comparando-se os resultados com outros dois métodos: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) e outro PCR em tempo real realizado em um laboratório de referência. As reprodutibilidades intra e inter-ensaio foram determinadas e a média dos valores de CV obtidos foram de 0,12 e 0,09, respectivamente. O teste foi validado com uma ampla faixa dinâmica e amplificou com eficiência os diferentes genótipos de HBV, fornecendo uma boa opção para a quantificação de rotina do DNA do HBV, com um protocolo barato e confiável.

Serum Hepatitis B Virus DNA Quantitative Analysis Using Polymerase Chain Reaction in Patients with Chronic Hepatits B Virus Infection / 대한진단검사의학회지

BACKGROUND: The treatment using more potent antiviral agents for the hepatitis B virus (HBV) infection has been performed widely and a highly sensitive quantification method using the polymerase chain reaction (PCR) that measures the HBV viral genome in sera is available. In this study, the HBV DNA level in each disease status including the inactive HBsAg carrier is evaluated. METHODS: Samples were obtained from 227 patients with chronic HBV infection that were grouped into chronic hepatitis (111), liver cirrhosis (71), and inactive HBsAg carrier (45). Quantification of HBV DNA was performed using the automated Cobas Amplicor HBV monitor test(TM). RESULTS: Among the chronic hepatitis B group (9.76 x 10(7) copies/mL), the liver cirrhosis group (4.88x10(5) copies/mL), and the inactive carrier group (3.18 x10(3)copies/mL), the medians of serum HBV DNA levels were significantly different from one group to another (P=0.000). Also, the median of HBV DNA levels in the patients with positive HBeAg (1.77 x10(8) copies/mL) was significantly higher than that of negative HBeAg (2.71 x 10(4) copies/mL) (P=0.000). In the patients with negative HBeAg, HBV DNA level in the inactive carrier group (Median 3.18 x 10(3) copies/mL) was significantly lower than that of the chronic hepatitis group (Median 2.2 x 10(5) copies/mL (P=0.000). CONCLUSIONS: The serum HBV DNA level varied among different disease groups, particularly according to HBeAg positivity. 40% of the chronic hepatitis group with negative HBeAg had HBV DNA levels below 10(5) copies/mL. Therefore, the quantitative analysis of HBV DNA using this sensitive and automated PCR method would be useful in detecting viral proliferation.