In-house Quantitative Polymerase Chain Reaction Research Articles

Standardisation and validation of an in-house quantitative real-time polymerase chain reaction (qPCR) assay for the diagnosis of Clostridioides difficile infection

ObjectivesClostridioides difficile is an emerging enteric pathogen that causes nosocomial diarrhoea in adults. The excessive cost of commercial molecular tests restricts the access of developing countries to its diagnosis. This study aimed to develop and validate in-house quantitative polymerase chain reaction (qPCR) targeting the C. difficile toxin B gene (tcdB) using two detection methodologies—SYBR Green and hydrolysis probes—for the diagnosis of C. difficile infection (CDI). MethodsGlutamate dehydrogenase (GDH) plus toxigenic culture was the standard reference diagnostic method. The SYBR Green method and hydrolysis probes were used to study 392 samples simultaneously to assess the diagnostic value of these real-time PCR assays in detecting CDI from clinical samples. ResultsThe SYBR Green and hydrolysis probe assays showed 97.9% and 87.5% sensitivity; 99.1% and 100.0% specificity; 94.0% and 100.0% positive predictive value; 99.7% and 98.3% negative predictive value; and 99.0% and 98.5% accuracy, respectively. ConclusionsThe two qPCR methodologies evaluated could offer an adequate tool as part of an algorithm in the laboratory diagnosis of CDI.

Cost minimization analysis of an in-house molecular test for cytomegalovirus in relation to a commercial molecular system

IntroductionCytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. ObjectiveThis study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. Study designThe methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n=30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. ResultsThe in-house assay had a limit of detection and quantification of 60.3IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. ConclusionsThese results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Comparative evaluation of a laboratory-developed real-time PCR assay and RealStar\xae Adenovirus PCR Kit for quantitative detection of human adenovirus

BackgroundHuman adenoviruses are common causes of community-acquired respiratory tract and enteric infections. Severe disseminated infections with high mortality rates may be seen in immunocompromised individuals. An accurate and cost-effective quantitative assay is essential not only for laboratory diagnosis of adenoviral infections, but also for monitoring of response to antiviral treatment. The diagnostic performance of an in-house quantitative polymerase chain reaction assay was compared to a commercial system.MethodsThe analytical sensitivity, specificity, linearity, precision and accuracy of an in-house adenovirus quantitative polymerase chain reaction assay were evaluated against the RealStar® Adenovirus PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany), using 122 clinical specimens and 18 proficiency testing samples.ResultsLinear regression analysis of the quantitative results by the in-house assay showed the dynamic range from 2.60 to 9 log10 (plasma) and 2.94 to 9 log10 (viral transport medium) copies/mL, with the coefficient of determination (R2) of 0.996 and 0.998, respectively. A dilution series demonstrated the limits of detection and lower limits of quantification for plasma were 2.06 log10 and 2.60 log10 copies/mL and those for viral transport medium were 2.31 log10 and 2.94 log10 copies/mL respectively. The precision of the in-house assay was highly reproducible among runs with coefficients of variance ranging from 0.07 to 3.21% for plasma and 0.17% to 2.11% for viral transport medium. A comparison of 52 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house assay and the RealStar® Adenovirus PCR Kit (R2 = 0.984), with an average bias of − 0.16 log10 copies/mL.ConclusionsThe in-house adenovirus assay is a sensitive and reliable assay with lower cost for the detection and quantification of adenoviral DNA when compared to the RealStar® Adenovirus PCR Kit.

MicroRNA-671-3p inhibits the development of breast cancer: A study based on in vitro experiments, in-house quantitative polymerase chain reaction and bioinformatics analysis.

MicroRNAs (miRNAs or miRs) are highly conserved small noncoding RNA molecules involved in gene regulation. An increasing number of studies have demonstrated that miRNAs act as oncogenes or antioncogenes in various types of cancer, including breast cancer (BC). However, the exact role of miR-671-3p in BC has not yet been reported. In the present study, in vitro experiments were implemented to explore the effects of miR-671-3p on the proliferation and apoptosis of BC cells, and reverse transcription-quantitative polymerase chain reaction was conducted using in-house clinical BC samples to address the expression level and clinical value of miR-671-3p in BC. Simultaneously, miR-671-3p target genes were collected, and subsequent bioinformatics analyses were executed to probe the potential signaling pathway through which miR-671-3p influenced the occurrence and progression of BC. According to the results, the expression level of miR-671-3p was lower in BC tissues compared with that in adjacent non-tumorous tissues (P=0.048), and the area under the curve was 0.697 (95% confidence interval=0.538-0.856), with a sensitivity and specificity of 0.818 and 0.579, respectively. Forced miR-671-3p expression in the BC cell line MDA-MB-231 evidently arrested cell proliferation and induced cell apoptosis. Furthermore, in silico enrichment analyses suggested that miR-671-3p may be involved in the initiation and progression of BC through the targeting of genes associated with the Wnt signaling pathway. In conclusion, the present study findings suggested that miR-671-3p may function as a tumor suppressor in BC by influencing the Wnt signaling cascade, which provides a prospective molecular target for the therapy of BC.

Prevalence of human parvovirus B19 in Chinese plasma pools for manufacturing plasma derivatives.

BackgroundHuman parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. To ensure the quality and safety of plasma-derived products, European regulations, Plasma Protein Therapeutics Association (PPTA) standard and FDA guidelines require testing of manufacturing plasma for parvovirus B19 DNA to limit the load of this virus. In China, however, there have been no related documentation and technical guiding principles for monitoring B19V, moreover, an adequate level of information on the prevalence of B19V in Chinese plasma donations is not available.FindingsBy using an in-house quantitative polymerase chain reaction (qPCR) assay adapted for all three genotypes of B19V, 235 source plasma pools from three regional different Chinese manufacturers of blood products were screened and quantified. Results showed that 71.91 % (169/235) of plasma pools were contaminated by B19V, with the concentrations of 5.18 × 102–1.05 × 109 IU/mL. Approximately 31.95 % of the DNA-positive plasma pools were only moderately contaminated (<104 IU/mL), while 68.05 % contained >104 IU/mL.ConclusionsThe high level of B19V in plasma pools could present a great risk in plasma derivatives. Therefore, the implementation of B19V NAT (Nucleic Acid Testing) assays capable of detecting all B19V genotypes and discard donations with high titer B19V DNA for Chinese blood products manufacturers seems to be necessary.

How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India

Development of a reliable, cost-effective cytomegalovirus quantitative polymerase chain reaction (QPCR) is a priority for developing countries. Manufactured kits are expensive, and availability can be inconsistent. Development of an in-house QPCR kit that is reliable and quality assured requires significant effort and initial investment. However, the rewards of such an enterprise are manifold and include an in-depth understanding of molecular reactions, and expertise in the development of further low-cost molecular kits. The experience of an oncology centre in Eastern India has been shared. Hopefully, this would provide a brief roadmap for such an initiative. Staff with adequate understanding of molecular processes are essential along with vital infrastructure for molecular research and development.

The genomic and seroprevalence of human bocavirus in healthy Chinese plasma donors and plasma derivatives.

Human bocavirus (HBoV) is a novel parvovirus identified in 2005. It has mostly been detected in respiratory and enteric infections and has not been studied large scale in blood products in relation to transfusion. An in-house quantitative polymerase chain reaction (Q-PCR) was developed to test HBoV DNA in plasma and plasma derivatives. Plasma samples (n = 6096) collected from healthy donors, 241 plasma pools, and 326 plasma derivatives were screened for HBoV DNA by Q-PCR. Positive samples were confirmed by nested PCR and further amplified for sequence analysis and phylogenetic studies. The prevalence of immunoglobulin (Ig)G and IgM specific to HBoV structural proteins was measured by enzyme-linked immunosorbent assay in 209 samples grouped according to virus load (Group 1, HBoV DNA >10(4) copies/mL; Group 2, HBoV DNA >5 × 10(2) copies/mL but below 10(4) copies/mL; Group 3,HBoV DNA negative). The genomic prevalence of HBoV in the plasma donors was 9.06%, ranging from 5.01 × 10(2) to 3.02 × 10(6) copies/mL. HBoV-specific IgG and IgM were detected at 20.00 and 7.50% in Group 1, at 20.29 and 2.90% in Group 2, and at 13.00 and 4.0% in Group 3, respectively. Phylogenetic analyses proved that HBoV Genotype 1 was the prevalent genotype in Chinese plasma donors. Low levels of HBoV DNA were detectable at high prevalence in Chinese plasma donors and plasma derivatives. Further study is needed to determine whether HBoV screening is necessary.

168O - Quantification of Cell Free DNA as a Prognostic Factor in Advanced NSCLC

ABSTRACT Purpose To investigate the prognostic importance of baseline circulating cell free DNA (cfDNA) in plasma from patients with advanced Non Small Cell Lung Cancer (NSCLC) prior to first-line chemotherapy. Material and methods The inclusion criteria were; histopathologically verified advanced NSCLC, indication for first-line chemotherapy and performance status (PS) 0-2. Treatment comprised carboplatin (area under the curve (AUC)5) iv day 1, and vinorelbine 30 mg/m2 iv day 1 and 60mg/m2 po day 8, q3w. A maximum of six courses was given. Plasma was obtained from a pre-treatment EDTA blood-sample. The number of cfDNA alleles at baseline was assessed by an in-house quantitative polymerase chain reaction (qPCR). Results A total of 246 patients were included, all receiving at least 1 course (median: 4). The overall response rate was 26%. The number of cfDNA alleles ranged from 480 to 1048500 per ml with a median value of 3200 and a 75th percentile of 6040 alleles per ml. The progression free survival (PFS) by intention to treat (ITT) was 5.4 months and the overall survival (OS) 8.9 months. The baseline level of cfDNA was strongly correlated to outcome. The median PFS was 2.9 months in patients with high levels (> the 75th percentile) compared to 5.7 months in patients with low levels ( Conclusion High levels of cfDNA seem to have a strong prognostic influence as estimated by both PFS and OS in patients with newly diagnosed advanced NSCLC. Combined with PS it outlines a group of patients with minimal or no benefit of chemotherapy, though further investigations are warranted. Disclosure All authors have declared no conflicts of interest.

1654P - Quantitative Measurement of KRAS Mutated Alleles in Plasma as Prognostic Marker in Metastatic KRAS Mutant Colorectal Cancer

ABSTRACT Purpose We investigated the prognostic value of quantitative measures of KRAS mutated alleles (pKRAS) in plasma from patients with metastatic colorectal cancer (mCRC) prior to treatment with pemetrexed and gemcitabine in a phase II clinical trial. Methods Inclusion criteria comprised; histopathologically verified mCRC with KRAS mutant chemotherapy resistant disease, indication for third-line chemotherapy, adequate organ function and performance status. Patients received pemetrexed (initially 500 mg/m2 q3w) combined with Gemcitabine (1250 mg/m2 day 1 and 8) until progression or unacceptable toxicity. RECIST version 1.1, NCI-CTCAE version 4.0 and Kaplan-Meier statistics were used for evaluation of endpoint measures. Plasma was obtained from a pre-treatment EDTA blood-sample, and the number of pKRAS alleles was assessed by an in-house quantitative polymerase chain reaction. Results Forty patients were included during April 2010-Aug 2011. The median number of cycles was 3 (range 0-12). Thirty-six percent of patients obtained disease stabilisation, but no objective responses were observed. The median PFS and OS were 2.8 (range 2.1-4.0) and 5.4 (range 4.3-7.0) months (mo), respectively. Pre-treatment blood samples and archival tumor tissue were available for analysis in all patients. Thirty-three mutations were detectable in the peripheral blood, whereas 7 patients had primary tumor mutations and wildtype status according to the blood sample. The median level of pKRAS alleles was 900 alleles/ml. When divided into quartiles of pKRAS, significantly shorter PFS and OS were revealed with increasing levels of alleles. The median PFS was 2.1 mo (95%CI 1.9-2.1) in patients with high levels (> median) compared to 4.2 mo (95%CI 2.5-5.1) in those with lower levels ( Conclusion A high pre-treatment level of pKRAS alleles had a detrimental effect on both PFS and OS, whereas low levels were correlated to a better chance of disease stabilisation. This confirms our data from two previous study cohorts and pKRAS could therefore serve as a new prognostic marker in KRAS mutant mCRC. Disclosure All authors have declared no conflicts of interest.

Systematic Evaluation of DNA-Based Quantitative-Polymerase Chain Reaction (Q-PCR) Assays to Track Treatment Response in Patients with JAK2-V617F Associated Myeloproliferative Neoplasms: A Joint European LeukemiaNet/ MPN&MPNr-EuroNet Study

Abstract▪2812▪This icon denotes a clinically relevant abstractSince the discovery of the JAK2-V617F mutation, the relative level of the mutant allele has been widely studied to gain further insights into the biology of myeloproliferative neoplasms and to establish utility in predicting clinical outcome. Mutant allele burden has been correlated with risk of thrombotic events in essential thrombocythemia, severity of the disease phenotype in polycythemia vera (PV) and survival in primary myelofibrosis (PMF). JAK2-V617F has also been quantified to assess disease response, with significant reductions in allele burden reported in PV patients following interferon (IFN) α-2b and pegylated IFN α-2a treatment. Moreover, serial DNA-based quantitative polymerase chain reaction (Q-PCR) assays have been used after allogeneic transplantation for myelofibrosis to predict outcome and guide donor lymphocyte infusion. With quantification of JAK2-V617F being considered an endpoint in a number of trials with novel agents including JAK2 inhibitors, it is important that assays are robust, mutant-specific and afford a suitable level of sensitivity. However, numerous JAK2-V617F Q-PCR assays have now been published, which based on experience of molecular monitoring in other hematological malignancies, are likely to vary markedly in their performance. Indeed, this concern was confirmed by the results of a quality control (QC) exercise involving 5 European LeukemiaNet (ELN) laboratories, each with their own established JAK2-V617F Q-PCR assay, who reported markedly differing levels of mutant allele percentage (ranging from 23–80%) in a QC sample distributed by UK NEQAS that comprised a 1 in 30 dilution of HEL cells (which harbor multiple copies of JAK2-V617F and lack the wild type [WT] JAK2 allele) in K562 cells (WT JAK2). To address this issue, a joint initiative was established within European LeukemiaNet between the Chronic Myeloproliferative Diseases and Minimal Residual Disease working groups (WP9 & WP12) to systematically evaluate a range of published and “in-house” JAK2-V617F assays, with the aim of identifying sensitive mutant-specific assays that perform similarly on different Q-PCR platforms – factors of fundamental importance for reliable tracking of disease response. The study involved 12 expert centers - in 6 European countries and included 1 US laboratory, which between them used Q-PCR platforms from 3 different manufacturers. The study involved central distribution of DNA from serial dilutions of JAK2-V617F mutant cell lines (HEL &/or UKE-1) in K562 and plasmid standards for JAK2 WT, JAK2-V617F and independent control genes (albumin, cyclophilin). Reagents for each assay were coded and also centrally distributed; reaction conditions for each coded assay were provided. Laboratories with established published or in-house Q-PCR assays were encouraged to perform these in parallel with the coded test assays on the distributed QC materials, to control for local variations in test conditions. Data from each QC round were submitted by participating laboratories for independent central analysis (Dept. of Genetics, King’s College London); conclusions were drawn concerning the performance of the various assays before breaking the code to reveal their identities. Overall, 7 successive QC rounds were conducted, evaluating 9 assays (6 published, 3 unpublished “in-house”). Six assays were eliminated, due to insufficient/inferior sensitivity (n=5) or inconsistent performance (n=1). Of the 3 assays with better performance profile, the assay published by Lippert et al (Blood 2006;108 :1865) reliably quantified JAK2-V617F across the diagnostic range, with consistent results obtained between laboratories irrespective of Q-PCR platform. The 2 remaining assays (Larsen et al, Br J Haematol. 2007;136 :745 & in-house assay from the Oppliger Leibundgut lab [Bern]), afforded better sensitivity, capable of detecting a 0.08–0.008 % level of JAK2-V617F in DNA preparations from HEL and K562 cells. The latter assays were tested on serial post-transplant samples from 2 patients allografted for JAK2-V617F mutant PMF, successfully identifying the presence of residual disease, complementing chimerism analysis to predict clinical outcome. In conclusion, this ELN study has identified robust assays suitable for quantifying JAK2-V617F in clinical trials and which merit further investigation as a tool to guide management of patients undergoing allogeneic transplant. Disclosures:No relevant conflicts of interest to declare.

JC virus load in progressive multifocal leukoencephalopathy: analysis of the correlation between the viral burden in cerebrospinal fluid, patient survival, and the volume of neurological lesions.

JC virus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating central nervous system infection that mainly affects patients with acquired immunodeficiency syndrome. The diagnostic value of the detection of JCV DNA in cerebrospinal fluid (CSF) has been proved. A correlation between the JCV burden in CSF and the PML prognosis has been proposed. To our knowledge, the present study is the first to examine JCV burden in CSF in relation to the magnitude of neurological damage. An in-house quantitative polymerase chain reaction assay was used for measurement of the JCV burden in CSF samples from 12 patients with PML. A wide variation in JCV load (6.4 log) was found among the patient CSF samples, a finding that makes JCV load measurements worthwhile. Virus load values of >4.68 log were associated with shorter patient survival time. No correlation was found between the virus load values and the global volume of brain tissue damaged. Our data suggest that factors other than the volume of neurological lesions influence the shedding of JCV in the CSF.