Multiplex PCR-based Assay Research Articles

An Efficient Multiplex PCR-Based Assay as a Novel Tool for Accurate Inter-Serovar Discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin.

Salmonella enterica serovars Enteritidis, Pullorum/Gallinarum, and Dublin are infectious pathogens causing serious problems for pig, chicken, and cattle production, respectively. Traditional serotyping for Salmonella is costly and labor-intensive. Here, we established a rapid multiplex PCR method to simultaneously identify three prevalent Salmonella serovars Enteritidis, Pullorum/Gallinarum, and Dublin individually for the first time. The multiplex PCR-based assay focuses on three genes tcpS, lygD, and flhB. Gene tcpS exists only in the three Salmonella serovars, and lygD exists only in S. Enteritidis, while a truncated region of flhB gene is only found in S. Pullorum/Gallinarum. The sensitivity and specificity of the multiplex PCR assay using three pairs of specific primers for these genes were evaluated. The results showed that this multiplex PCR method could accurately identify Salmonella Enteritidis, Pullorum/Gallinarum, and Dublin from eight non-Salmonella species and 27 Salmonella serovars. The least concentration of genomic DNA that could be detected was 58.5 pg/μL and the least number of cells was 100 CFU. Subsequently, this developed method was used to analyze clinical Salmonella isolates from one pig farm, one chicken farm, and one cattle farm. The results showed that blinded PCR testing of Salmonella isolates from the three farms were in concordance with the traditional serotyping tests, indicating the newly developed multiplex PCR system could be used as a novel tool to accurately distinguish the three specific Salmonella serovars individually, which is useful, especially in high-throughput screening.

Rapid and Specific Detection of the Escherichia coli Sequence Type 648 Complex within Phylogroup F.

The Escherichia coli sequence type 648 complex (STc648) is an emerging lineage within phylogroup F-formerly included within phylogroup D-that is associated with multidrug resistance. Here, we designed and validated a novel multiplex PCR-based assay for STc648 that took advantage of (i) four distinctive single-nucleotide polymorphisms in icd allele 96 and gyrB allele 87, two of the multilocus sequence typing alleles that define ST648; and (ii) the typical absence within STc648 of uidA, an E. coli-specific gene encoding β-glucuronidase. Within a diverse 212-strain validation set that included 109 STs other than STc648, from phylogroups A, B1, B2, C, D, E, and F, the assay exhibited 100% sensitivity (95% confidence interval [CI], 82% to 100%) and specificity (95% CI, 98% to 100%). It functioned similarly well in two distant laboratories that used boiled lysates or DNAzol-purified DNA as the template DNA. Thus, this novel multiplex PCR-based assay should enable any laboratory equipped for diagnostic PCR to rapidly, accurately, and economically screen E. coli isolates for membership in STc648.

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Clinical validation of a novel real-time human papillomavirus assay for simultaneous detection of 14 high-risk HPV type and genotyping HPV type 16 and 18 in China.

In the present study, we describe the laboratory workflow and the clinical validation of a novel multiplex real-time PCR-based HPV assay in China. The cross-sectional validation analysis showed that this assay worked well for detection of 14 HR-HPV types and identification of HPV 16 and 18 in a single sensitive assay that is suitable for both clinical usage and high-throughput cervical screening purposes. We predict that this accurate, high-throughput and low-cost HPV assay can greatly reduce the heavy economic burden of HPV detection in China.

Targeted next-generation sequencing of the ATP7B gene for molecular diagnosis of Wilson disease

ObjectivesIn recent years, next-generation sequencing (NGS) technologies, which enable high throughput sample processing at relatively lower costs, are adopted in both research and clinical settings. A multiplex PCR-based NGS assay to identify mutations in the ATP7B gene for routine molecular diagnosis of Wilson disease was evaluated in comparison with the gold standard direct Sanger sequencing. Design and methodsFive multiplex PCRs to amplify the partial promoter, 5′ untranslated and the entire coding regions of the ATP7B gene were designed. Indexed paired-end libraries were generated from the pooled amplicons using Nextera XT DNA Sample Preparation Kit and subjected to NGS on the MiSeq platform. DNA from the peripheral blood of 12 patients with Wilson disease, 2 B-lymphocyte cell lines and 3 external quality assurance samples were sequenced by the MiSeq and Sanger sequencing. ResultsComplete coverage was achieved across the targeted bases without any drop-out sequences. The observed read depth in a single run with 20 samples was >100X. Comparison of the NGS results against Sanger sequencing data on a panel of clinical specimens, cell lines and European Molecular Genetics Quality Networks (EMQN) quality assurance samples showed 100% concordance in identifying pathogenic mutations. ConclusionWith the capability of generating relatively higher throughput in a short time period, the NGS assay is a viable alternative to Sanger sequencing for detecting ATP7B mutations causally linked to Wilson disease in the clinical diagnostic laboratory.

Development of multiplex PCR assay for authentication of Cornu Cervi Pantotrichum in traditional Chinese medicine based on cytochrome b and C oxidase subunit 1 genes

This study describes a method for discriminating the true Cervus antlers from its counterfeits using multiplex PCR. Bioinformatics were carried out to design the specific alleles primers for mitochondrial (mt) cytochrome b (Cyt b) and cytochrome C oxidase subunit 1 (Cox 1) genes. The mt DNA and genomic DNA were extracted from Cervi Cornu Pantotrichum through the modified alkaline and the salt-extracting method in addition to its counterfeits, respectively. Sufficient DNA templates were extracted from all samples used in two methods, and joint fragments of 354 bp and 543 bp that were specifically amplified from both of true Cervus antlers served as a standard control. The data revealed that the multiplex PCR-based assays using two primer sets can be used for forensic and quantitative identification of original Cervus deer products from counterfeit antlers in a single step.

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Beetles Protaetia brevitarsis seulensis Kolbe (Coleoptera: Cetoniidae) and Allomyrina dichotoma Linn. (Coleoptera: Scarabaeidae) are widely used in traditional medicine, and the number of insect-rearing farms is increasing in South Korea. The purpose of this study was to establish a multiplex PCR-based assay for rapid simultaneous detection of multiple pathogens causing insect diseases. Six insect parasites such as fungi Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae), bacteria Bacillus thuringiensis Berliner (Bacillales: Bacillaceae), Pseudomonas aeruginosa Migula (Pseudomonadales: Pseudomonadaceae), and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae), and Oryctes rhinoceros nudivirus were chosen based on the severity and incidence rate of insect diseases in South Korea. Pathogen-specific primers were designed and successfully applied for simultaneous detection of multiple infectious agents in farm-bred insects P. b. seulensis and A. dichotoma using multiplex PCR and high resolution capillary electrophoresis. Our results indicate that multiplex PCR is an effective and time-saving method for simultaneous detection of multiple infections in insects, and the QIAxcel capillary electrophoresis system is useful for quantitative evaluation of the individual impact of each infectious agent on the severity of insect disease. The approach designed in this study can be utilized for rapid and accurate diagnostics of infection in insect farms.

Colorectal carcinomas with KRAS codon 12 mutation are associated with more advanced tumor stages.

BackgroundKRAS mutation occurs in 35%-40% of colorectal cancer (CRC). The aim of our study was to evaluate the pathological and molecular features of specific KRAS mutated colorectal carcinomas. KRAS and BRAFV600E mutation tests were performed in 762 primary tumors from a consecutive cohort study of Chinese CRC patients.MethodsDNA mismatch repair (MMR) status was determined by immunohistochemistry (IHC) staining. Assessment of KRAS and BRAF V600E mutational status was performed using a multiplex allele-specific PCR-based assay.ResultsMutations of KRAS (34.8%) and BRAFV600E (3.1%) were nearly mutually exclusive. Both KRAS- and BRAF- mutated tumors were more likely to be located at proximal colon than wild-type (WT) carcinomas. KRAS-mutated carcinomas were more frequently observed in female patients (47.5% vs 37.1%, p = 0.005) and mucinous differentiation (34.7% vs 24.8%, p = 0.004), but have no difference between lymph node (LN) metastases and among pTNM stages. Whereas, BRAF-mutated carcinomas more frequently demonstrated histologic features such as proximal location (60.9% vs 20.9%, p = 0.001), low-grade histology (43.5% vs 18.0%, p = 0.005), mucinous differentiation (69.6% vs 25.9%, p = 0.001) and deficient MMR (dMMR) (21.7% vs 7.6%, p = 0.03). In particular, KRAS codon 12 mutated carcinomas had increased lymph node metastasis (odds ratio [OR] = 1.31; 95% confidence interval [CI] = 1.04 to 1.65; P = 0.02) and were more likely in higher disease stage (III-IV) than that of WT carcinomas (OR = 1.30; 95% CI = 1.03 to 1.64; P = 0.03). However, there were no significant differences in lymph node metastasis and disease stage between KRAS codon 13 mutated carcinoma and WT carcinoma patients.ConclusionsIn summary, KRAS codon 12 mutation, but not codon 13 mutation, is associated with lymph node metastasis and higher tumor stages.

Two novel frameshift mutations in BRCA2 gene detected by next generation sequencing in a survey of Spanish patients of breast cancer.

To analyze BRCA1 and BRCA2 genes using a cost-effective and rapid approach based on next generation sequencing (NGS) technology. A population of Spanish cancer patients with a personal or familial history of breast and/or ovarian cancer was analyzed for germline mutations in BRCA1 and BRCA2 genes. The methodology relies on a 5 multiplex PCR assay coupled to NGS. Ten pathogenic mutations (four in BRCA1 and six in BRCA2 gene) were identified in a Spanish population. The deletion c.1792delA, in exon 10, and the duplication c.5869dupA, in exon 11 of BRCA2 gene were not previously reported and should be considered as pathogenic due to its frameshift nature. Two novel frameshift mutations in BRCA2 gene were detected using the multiplex PCR-based assay following by NGS.

A multiplex PCR assay for the simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis

IntroductionFor developing countries, sexually transmitted infections (STIs) and their complications are ranked in the top 5 disease categories for which adults seek medical treatment. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) are the three most common STIs worldwide, with TV accounting for over half of the cases. In developing countries, traditional methods for diagnosing STIs are laborious, often not very sensitive, and have a long turnaround time with most recent commercially available diagnostic tests targeting one or, at most, two of these STIs at a time. Here, we describe the development of a highly sensitive, rapid and affordable sample-to-answer multiplex PCR-based assay for the simultaneous detection of Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis. Materials and MethodsWe designed a multiplex PCR assay for the detection of 4 targets (CT, TV, NG, and process/PCR control) using melt curve analysis. To establish the limit of detection (LOD) for each pathogen, we used previously extracted and quantified TV, NG, and CT genomic DNA (Vircell, Spain). For each target, the LOD was determined by lowering its copy number while increasing the other two STI loads in a stepwise fashion. The process/PCR control remained constant in the optimized assay and was spiked into each sample before extraction. For a concordance study, we tested urine, vaginal and rectal swab specimens from 26 patients positive for one or more of the tested STIs. In addition, 56 liquid cytology specimens (Thinprep) were used to assess specificity. ResultsThis assay has a turnaround time of less than 2h and has a limit of detection as low as 7–31 copies for each STI in the presence of the other 2 targets. Our assay also demonstrated 100% concordance with 26 known clinical samples from urine, vaginal and rectal swab specimens. TV, NG, CT, and our process/PCR control were consistently identified at 78°C, 82.3°C, 85.7°C, and ~92°C, respectively. When applied to DNA extracted from residual Thinprep specimens, the assay was negative in 54/56 samples. Two samples were found to be co-infected with CT. ConclusionsOur multiplex assay combines a rapid and cost-effective approach to molecular diagnostics with the versatility required for use within a variety of laboratory settings. These performance characteristics make this multiplex STI assay highly suitable for use in a clinical laboratory.

Polymorphisms of genes involved in lipid metabolism and risk of chronic kidney disease in Japanese - cross-sectional data from the J-MICC study.

BackgroundChronic kidney disease (CKD) is known to be one of the causes of cardiovascular disease and end-stage renal disease. Among the several treatable risk factors of CKD, that of dyslipidemia is relatively controversial. To clarify the association of polymorphisms in genes involved in lipid metabolism with the risk of CKD in the Japanese population, we used cross-sectional data from the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study.MethodsA total of 3,268 men and women, aged 35–69 years, were selected from J-MICC Study participants for inclusion in this study. Twenty-eight candidate single nucleotide polymorphisms (SNPs) were selected in 17 genes associated with the risk of lipid metabolism disorders, and genotyping of the subjects was conducted using the multiplex PCR-based invader assay. The prevalence of CKD was determined for stages 3–5 (defined as estimated glomerular filtration rate <60 ml/min/1.73 m2).ResultsLogistic regression analysis revealed that SNPs APOA5 T − 1131C (rs662799), APOA5 T1259C (rs2266788), TOMM40 A/G (rs157580), and CETP TaqIB (rs708272) were significantly associated with CKD risk in those individuals genotyped, with age- and sex-adjusted odds ratios (ORs) per minor allele (and 95% confidence intervals (CIs)) of OR 1.22 (95% CI: 1.06–1.39), 1.19 (1.03–1.37), 1.27 (1.12–1.45), and 0.81 (0.71–0.92), respectively. Analysis of the gene–environment interaction revealed that body mass index (BMI) was a significant effect modifier for APOA5 T − 1131C (rs662799) and a marginally significant effect modifier for APOA5 T/C (rs2266788), with the interaction between BMI ≥30 and individuals with at least one minor allele of each genotype of OR 10.43 (95% CI: 1.29–84.19) and 3.36 (0.87–13.01), respectively.ConclusionsFour polymorphisms in APOA5, TOMM40, and CETP were shown to be significantly associated with CKD risk, and a significant interaction between the two APOA5 SNPs and BMI on CKD risk was also demonstrated. This suggests the future possibility of personalized risk estimation for this life-limiting disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-511X-13-162) contains supplementary material, which is available to authorized users.

Abstract 4696: Identification of driver mutations and gene amplifications in resected asophageal adenocarcinoma: Impact on clinical care

Abstract BACKGROUND: Adenocarcinoma of the distal esophagus and esophagogastric junction (EGJ) have received much attention because of increasing frequency and poor outcome despite multimodality therapy. Driver mutations and gene amplifications in resected esophageal adenocarcinoma are not widely investigated. Opportunity exists to discover actionable mutations in resected esophageal adenocarcinoma. METHODS: We analyzed driver mutations harbored in coding regions of 25 selected genes (∼95 point mutations) using a multiplex PCR-based assay. EGFR, HER2, MET, and PI3K amplifications were tested using fluorescent in situ hybridization in patients who underwent esophagectomy for distal esophagus and EGJ adenocarcinoma from 2009 to 2013. Disease free survival (DFS) and overall survival (OS) were compared between patients with TP53, KRAS, PI3K, BRAF, APC mutant and wild-type tumors using Kaplan-Meier methods and Cox regression models. The impact of HER2 expression and outcomes were also analyzed. RESULTS: Of 54 distal esophagus and EGJ adenocarcinoma patients, 5 different driver mutations were observed in 39 patients, including TP53 (11/39, 28.2%), KRAS (6/39,15.4%), PI3K (2/11,5.1%), BRAF(1/39, 2.6%) and APC (2/39,5.1%). Double driver mutations (3/39, 7.9%) were shared in 3 patients with TP53 and KRAS (1/39, 2.6%), TP53 and PI3K (1/39, 2.6%), KRAS and PI3K (1/39, 2.6%), respectively. Amplifications in HER2 (10/45, 22.2%), MET (1/34, 2.9%) and EGFR (2/17, 11.8%) were observed where directed therapy was altered in 5 patients (1 patient received induction herceptin and 4 patients received adjuvant herceptin). In the targeted patients with HER2 amplification who received herceptin, mean progression-free survival was 18.8 (range 7-41 months) vs. 8.9 (1-28 months) [p=0.089] and OS was 35.7 (range 26-46 months) vs. 24.2 (range 20-29 months) [p=0.053], compared to herceptin naive patients. We did not identify any significant difference in DFS and OS (inclusive of all stages) between TP53, KRAS or any mutation compared to wild type patients. DISCUSSION: Oncogenic driver mutations are under-investigated in resected distal esophageal and EGJ adenocarcinoma. Different therapeutic opportunities may become available if routine tumor genotyping is employed in the treatment of esophageal adenocarcinoma. We recommend the routine use of driver mutation and gene amplification analysis for esophageal cancer. Citation Format: Haiyu Zhou, Luis Tapias Vargas, Justin Elliot, Douglas J. Mathisen, Michael Lanuti. Identification of driver mutations and gene amplifications in resected asophageal adenocarcinoma: Impact on clinical care. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4696. doi:10.1158/1538-7445.AM2014-4696

Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002.

Tumor somatic mutation analysis is part of the standard management of metastatic lung cancer. However, physicians often have to deal with small biopsies and consequently with challenging mutation testing. Circulating free DNA (cfDNA) is a promising tool for accessing the tumor genome as a liquid biopsy. Here, we evaluated next-generation sequencing (NGS) on cfDNA samples obtained from a consecutive series of patients for the screening of a range of clinically relevant mutations. A total of 107 plasma samples were collected from the BioCAST/IFCT-1002 lung cancer study (never-smokers cohort). Matched tumor DNA (tDNA) was obtained for 68 cases. Multiplex PCR-based assays were designed to target specific coding regions in EGFR, KRAS, BRAF, ERBB2, and PI3KCA genes, and amplicon sequencing was performed at deep coverage on the cfDNA/tDNA pairs using the NGS IonTorrent Personal Genome Machine Platform. CfDNA concentration in plasma was significantly associated with both stage and number of metastatic sites. In tDNA, 50 mutations (36 EGFR, 5 ERBB2, 4 KRAS, 3 BRAF, and 2 PIK3CA) were identified, of which 26 were detected in cfDNA. Sensitivity of the test was 58% (95% confidence interval, 43%-71%) and the estimated specificity was 87% (62%-96%). These data demonstrate the feasibility and potential utility of mutation screening in cfDNA using IonTorrent NGS for the detection of a range of tumor biomarkers in patients with metastatic lung cancer.

The feasibility to use minimal tumor samples for mutation analysis by multiplex PCR-based assay.

e22060 Background: Several targeted agents and gene analysis are important for treatment of non-small cell lung cancer (NSCLC). The single gene examination is not sufficient to make the decision of...

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population.

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48×10(-21), higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA. In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.

Developing an SCAR and ITS reliable multiplex PCR-based assay for safflower adulterant detection in saffron samples

Saffron (Crocus sativus L.), one of the most important and expensive medicinal spice products traded internationally, is subject to adulteration by design or default with safflower stamens and corn stigmas, leading to poor quality of saffron samples. The present study aims at the development of specific, sensitive and reproducible PCR-based markers to detect these adulterants in traded saffron. Six putative RAPD markers generated by random primers, OPA-14, MG-11, MG-12 and AJ-05, were identified as saffron specific by comparative RAPD analysis of genuine saffron, safflower and corn. These specific RAPD markers were cloned, sequenced and six pairs of SCAR primers were designed. Specific designed primers were able to amplify reproducible saffron DNA with expected sizes and no amplification in corn and safflower DNA. In this study, a primer pair was also designed based on ITS sequences for specific amplification of safflower DNA. PCR reactions were also specifically amplified 613 bp of ITS region in safflower genome. The multiplex PCR assays were further established for the joint use of some SCAR and ITS markers efficiently. The special feature of this new molecular method was technically rapid and convenient practically and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method could be used as a helpful assistant tool for the identification of adulterant saffron samples. This study described the development of a new SCAR and ITS maker-based multiplex PCR assay for the rapid molecular detection of substitutes in saffron.

Rapid detection of blaVIM-1–37 and blaKPC1/2–12 alleles from clinical samples by multiplex PCR-based assays

VIM and KPC are two major families of carbapenemases involved in nosocomial outbreaks of multidrug-resistant Gram-negative bacilli. To rapidly detect blaVIM- and blaKPC-encoding strains, three multiplex PCR-based methods were designed and validated: (i) a real-time PCR to detect all reported VIM alleles, namely blaVIM-1–19, 23–37; (ii) a real-time PCR to identify blaVIM-type and blaKPC carbapenemases in an ultrarapid single reaction; and (iii) a standard PCR to amplify and sequence all VIM alleles. All three methods detected 33 VIM-positive samples among 107 Gram-negative isolates with imipenem and meropenem minimum inhibitory concentrations ≥1mg/L. The three methods displayed 100% sensitivity, specificity and concordance. Sequencing of the blaVIM amplicons revealed that 30 samples encoded blaVIM-1 and 3 samples encoded blaVIM-2. The real-time assay, optimised for the simultaneous detection of blaVIM and blaKPC, identified 3 and 12 isolates positive for both blaVIM/blaKPC and for blaKPC, respectively. The analytical sensitivity of the real-time assays was linear over 6 log dilutions, with a reproducible detection limit of 1 CFU. No cross-reactivity was detected. The developed assays provide powerful tools for rapid identification of VIM and KPC carbapenemase producers, therefore contributing to the prevention and containment of resistance dissemination.

Association Between ABCB1 (MDR1) Gene Polymorphism and Unresponsiveness Combined Therapy in Chronic Hepatitis C virus

BackgroundTo treat viral infection of chronic hepatitis C (CHC) is a main strategy to prevent progression of liver disease, and cancer. Some patients with CHC have failed to respond to the common antiviral therapy in different populations.ObjectivesIn the current study it was aimed to find out the possible role of multiple drug resistance gene1 (MDR1) in non-responder patients with CHC infection in Turkish population.Patients and MethodsPeripheral blood-EDTA samples were used for total genomic DNA isolation. In total of 55 patients with chronic hepatitis C and positive results for genotype 1 [31 male (56.4%), 24 female (43.6%) and mean age-min-max; 56.9 ± 9.66 (39-71)]; 19 responder (34.5%), 21 non responder (38.2%), and 15 recurrence (27.3%) were included in the presented results. Functional MDR1 gene was genotyped by multiplex PCR-based reverse-hybridization Strip Assay method, and some samples were confirmed by direct sequencing.ResultsOur results indicate that MDR1 gene polymorphism is strongly associated with non-responder patients and those with recurrent chronic hepatitis C during conventional drug therapy when compared to the responder patients. Homozygous of the TT genotype for MDR1 exon 26 polymorphism was at 2.0-fold higher risk of non-responder than patients with CC and CT.ConclusionsThe homozygous MDR1 3435TT genotype which encodes the xenobiotic transporter P-glycoprotein may be associated with a poor antiviral response in HCV chronicity during conventional therapy, and large-scale studies are needed to validate this association.

Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay

The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR) assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 °C for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised.

Early Mixed T-Lymphocyte and Myeloid Chimerism Is Predictive of Disease Recurrence Following Allogeneic Stem Cell Transplantation for AML/MDS.

Abstract 3075 Background:Allogeneic stem cell transplantation (allo-SCT) is a potential curative therapy for patients with relapsed or refractory acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the risk for disease recurrence following transplant remains high. The ability to identify patients likely to relapse may allow for preemptive interventions in high-risk patients. The goal of hematopoietic transplantation is to eradicate the recipient myeloid leukemia cells and restore hematopoiesis and immunity with donor cells. Donor lymphoid cells mediate an important graft-vs.-leukemia effect. Post transplant peripheral blood (PB) chimerism analysis represents one potential tool for predicting disease recurrence, although the relationship between mixed chimerism and disease relapse is not well defined. Methods:We conducted a retrospective review of patients with AML/MDS who underwent allo-SCT with fludarabine/busulfan based conditioning regimens at The University of Texas MD Anderson Cancer Center between 2001 and 2011. PB chimerism was assessed between post-transplant days +90–120 using a multiplex PCR-based microsatellite polymorphism assay. Cox’s proportional hazards regression was used on univariate and multivariate (MV) analysis to evaluate the impact of chimerism of T-lymphocytes and myeloid cells in PB, as well as patient-, disease-, and transplant-related variables on the rate of disease progression and progression-free survival (PFS). Survival and PFS were assessed in landmark analysis starting on day +120. The cut off levels for assessment of chimerism were based on the respective quartiles of distribution of T-lymphocytes and myeloid cells in the study population. Results:483 patients who underwent allo-SCT for AML/MDS with fludarabine/busulfan based conditioning regimens between 2001–2011 were analyzed. Within this cohort, 378 patients were alive and without evidence of disease progression on day +120 and were eligible for study evaluation. Patients with disease progression or death within 3 weeks of chimerism assessment were excluded from analyses assessing the impact of chimerism on outcomes. 158 patients were in CR1, 66 in CR2, and 154 had active disease at the time of transplantation. PB T-lymphocyte and myeloid donor cell chimerism data between days +90–120 were available for 265 (70%) and 286 (76%) patients, respectively. The median follow-up time among surviving patients was 54 months (range, 5–126). Progression-free survival from day +120 was 56% (95% CI 39–52) at 3 years, and 46% (95% CI 39–52) overall. On univariate analysis, mixed T-lymphocyte chimerism of ≤87% (HR=1.8, P 0.03, Figure 1) and myeloid chimerism ≤98% (HR=2.4, P 0.005, Figure 2) were significantly associated with a higher rate of 3-year disease progression. These cut-off points were based on the 25th percentile of the respective distributions of T-lymphocyte and myeloid chimerism in the study population. Additional adverse factors included poor-risk cytogenetics (HR=1.5, P 0.04) and disease status other than first or second remission at the time of transplant (HR=2, P 0.002). All factors remained significant on MV analysis, with the exception of myeloid chimerism, which became only marginally significant (HR=1.96, P 0.06). Mixed T-lymphocyte (HR=1.5, P 0.05) and myeloid (HR=1.9, P 0.02) chimerism were also associated with significantly lower 3-year PFS on univariate analysis, but were no longer significant (HR=1.5, 95% CI 0.95–2.3 and HR=1.6, 95%, CI 0.9–2.8, respectively) after adjustment for disease status at transplant. Disease status other than first or second remission at transplant was the only significant predictor of 3-year PFS on MV analysis (HR=1.6, P=0.03). Stem cell source (PB vs. BM), donor type (match-related donor vs. other), age (>50 vs. ≤50 years), and diagnosis (AML vs. MDS) did not impact the rate of disease progression or disease free survival. Conclusion:Mixed T-lymphocyte and myeloid chimerism assessed on day +120 post SCT are associated with the rate of disease progression independently of disease status at transplant. The use of chimerism assessments may be useful in selecting patients at high-risk for relapse for preemptive therapeutic approaches. [Display omitted] [Display omitted] Disclosures:Champlin:Otsuka: Research Funding.