Total Concordance Research Articles

Applicability of commercial multiplex kit for the diagnosis of: a multicenter investigation Clostridium difficile infections

Objective To evaluate the performance of Xpert C. difficile multiplex real-time PCR assay for diagnosis of Clostridium difficile infections in Chinese hospital settings. Methods This study was performed in Huashan Hospital, Ruijin Hospital, Beijing Hospital, Nanfang Hospital and Sir Run Run Shaw Hospital using a standard study protocol. Unique unformed stools from patients with acute hospital-acquired diarrhea were simultaneously analyzed by toxigenic anaerobic cultures and the Xpert C. difficile assay. All specimens displaying discordant results between the Xpert assay and toxigenic culture were sent for Sanger tcdB gene sequencing. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), total concordance rate, and 95% confidence interval (CI) were calculated before and after resolution of discordant results using SAS 9.3. Results A total of 745 stool specimens were collected and 46 were excluded due to failure of C. difficile recovery. The remaining 699 specimens were included. Compared to the results of toxigenic culture, the sensitivity, specificity, PPV, and NPV of Xpert C. difficile assay were 94.1% (144/153)(95%CI:89.1%-97.3%), 93.2% (509/546)(95%CI:96.7%-99.2%), 79.6% (144/181)(95%CI: 72.9%-85.2%)and 98.3% (509/518)(95%CI: 96.7%-99.2%), respectively. Both methods had a Kappa of 0.819. Xpert C. difficile assay showed sensitivity of 98.4%(62/63) (95% CI: 90.3%-99.9%) and specificity of 93.2%(509/546) (95% CI: 90.8%-95.2%) for toxin A-negative toxin B-positive strains. After the discordant results resolved by tcdB gene sequencing, PCR assay provided better performance with high sensitivity, specificity, positive predictive value, and negative predictive value [98.8% (171/173), 98.1% (516/526), 94.5% (171/181) and 99.6% (516/518), respectively]. Conclusions Compared to the results of toxigenic culture, the sensitivity, specificity and NPV of Xpert C. difficile assay were 94.1% (144/153) and 93.2% (509/546), respectively. With the results available within 1 h, Xpert C. difficile assay provides prompt and precise laboratory diagnosis in Chinese clinical settings. Key words: Clostridium difficile; Clostridium infections; Real-time polymerase chain reaction

Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications

To study whether embryonic cell-free DNA (cfDNA) in spent blastocyst media is representative of the chromosomal constitution of a blastocyst. Pilot prospective blinded study. Invitro fertilization center and genetics laboratory. A total of 115 trophectoderm (TE) biopsies and spent blastocyst media (SBM) from 46 patients with ages ranging from 32 to 46years, whose indications for preimplantation genetic testing of aneuploidy (PGT-A) were advanced maternal age, recurrent miscarriage, or recurrent implantation failure. Spent blastocyst media collection and TE biopsy. Concordance rates, sensitivity, and specificity between TE biopsies and SBM. Clinical outcomes in cases with euploid TE biopsies and euploid SBM compared with cases with euploid TE and aneuploid SBM. In general, the total concordance rate for ploidy and sex was 78.7%, and sensitivity and specificity were 94.5% and 71.7%, respectively. A significant increase for all parameters was observed for day 6/7 samples compared with day 5 samples, with day 6/7 samples showing total concordance for ploidy and sex of 84%, and sensitivity and specificity of 95.2% and 82.1%, respectively. Ongoing implantation rates in euploid TE/euploid SBM showed a threefold increase compared with euploid TE/aneuploid SBM (52.9% vs. 16.7%, respectively), without reaching significant differences. Interestingly, no miscarriages were observed when TE and SBM were euploidy concordant. These results offer a better understanding of the dynamics of cfDNA during embryo development and despite more basic research being needed, they are reassuring to consider in the future this noninvasive approach as an alternative to TE biopsy for PGT-A.

OR19-1 Heritability and Etiological Overlap in Seven Autoimmune Diseases: A Population-Based Swedish Twin Study

With few exceptions, autoimmune diseases (AIDs) are genetically complex disorders. Familial aggregation of AIDs is common, and it is therefore not surprising that many genetic risk markers in autoimmunity are pleiotropic 1. Despite this, etiologic overlap across AIDs has not yet been explored in an unbiased fashion. We therefore used the Swedish Twin Registry to study heritability and overlap between seven AIDs known to cluster. Using data on 116,320 twins of known zygosity, we identified 1,957 subjects with a diagnosis of Hashimoto’s thyroiditis, 762 with atrophic gastritis, 723 with celiac disease, 645 with Grave’s disease, 420 with type-1 diabetes, 192 with vitiligo and 31 with autoimmune Addison’s disease. Heritability for individual disorders ranged from 0.97 for Addison´s disease to 0.47 for pernicious anemia using AE-models (additive genetic and unique environmental effects). Probandwise concordance rates ranged from 0.71 in monozygotic twins (MZ) with Addison’s disease to 0 in dizygotic twins (DZ) with Addison’s disease and vitiligo. Rates were higher in MZ than in DZ pairs for all seven diseases. Probandwise pseudo-concordance was defined as the proportion of co-twins with a different AID among twins with index AID. Total probandwise concordance was defined as the proportion of co-twins with (any of the studied) AIDs among twins with index AID. The ratio of probandwise pseudo-concordance to total probandwise concordance was used as a measure of autoimmune clustering. This ratio was highest for Addison’s disease (MZ 0.54, DZ 1.0) and vitiligo (MZ 0.46, DZ 1.0), lower for Hashimoto’s thyroiditis (MZ 0.29, DZ 0.29) and lowest for Celiac disease (MZ 0.12, DZ 0.31). In the context of the seven AIDs explored here, probandwise concordance rates for individual diseases underestimate the burden of autoimmunity. This effect is considerable for disorders with significant etiological overlap, such as Addison’s disease and vitiligo, but marginal in celiac disease, which has little etiological overlap. The genetic influence on disease occurrence for individual disorders is high, with heritability estimates ranging from 0.97 to 0.47. 1. Cotsapas C, Voight BF, Rossin E, et al. Pervasive sharing of genetic effects in autoimmune disease. PLoS Genet 2011;7(8):e1002254. doi: 10.1371/journal.pgen.1002254

Root proximity of miniscrews at a variety of maxillary and mandibular buccal sites: Reliability of panoramic radiography.

To assess the root proximity and the insertion angles of miniscrews after miniscrew placement at a variety of maxillary and mandibular buccal sites using cone-beam computed tomography (CBCT) and to determine the differences in root proximity between CBCT and panoramic radiography (PR). This retrospective study included 50 patients (mean age, 22.0 ± 4.5 years) who underwent postoperative CBCT and PR after miniscrew placements for intermaxillary fixation in orthognathic surgery. Twelve miniscrews were placed in the buccal bone of each patient: at sites between the central incisor and lateral incisor (SII), sites between the canine and first premolar (SCP), and sites between the second premolar and first molar (SPM) on the right and left sides of the mandible and maxilla. The insertion angles were measured on CBCT, and the root proximity was assessed on CBCT and PR. The mean vertical placement angles ranged from 84.27° to 95.12°, and the mean horizontal placement angles ranged from 90.93° to 101.1°. The rates of no contact between the root and the miniscrew were 68.0% in the SII, 50.5% in the SCP, and 57.8% in the SPM, which were significantly different (P = .000). The total concordance rate between PR and CBCT was 41.3%. Clinicians should use extreme caution during placement of miniscrews in the SCP. There are limitations on the use of PR for evaluating the root proximity of miniscrews.

Botanical quality control of digestive tisanes commercialized in an urban area (Bahía Blanca, Argentina)

Digestive herbal teas in the form of monoherbal infusions or in mixtures are the most commonly used form of phytotherapy in Bahía Blanca (Argentina). This practice is based on family tradition reinforced by current trends reverting to the ingestion of natural products. The objective of this work was to analyze the botanical quality of the digestive mixtures sold in Bahía Blanca as fine cut mixtures (in tea bags) and thick cut mixtures (fragmented herbs). Samples of seven mixtures sold in the form of tea bags and four as bags of fragmented herbs were studied under stereoscopic and optical microscopes and micrographic studies were carried out following conventional protocols. Observed characteristics were compared with those in the reference bibliography for verified genuine monoherbs. In the studied products a total of 21 different species were found. There was total concordance between the species declared on the label and those identified in the samples in six of the mixtures in the form of tea bags and in only one of the bags of fragmented herbs. Adulterations were found in four of the samples analyzed (one in a tea bag and three in the fragmented mixtures). The labels on only two of the tea bag samples presented complete and correct information; in the remaining samples irregularities were found related to: the taxonomic identification of the species, the absence of a lot number, date of expiry, the plant parts used and proportions of the herbs etc. The findings underline the great importance of quality controls as a tool to protect the rights of phytotherapy consumers.

Inaccuracy of Thyroid to Background Uptake Ratio in Evaluating Technetium-99m-pertechnetate Thyroid Uptake and Establishing an Improved Algorithm.

Objective(s):The aim of this study was to explore the accuracy of thyroid to background uptake ratio (UR) in the evaluation of 99mTc-pertechnetate thyroid uptake (TcTU) and establishment of an improved algorithm. Methods:This study was conducted on the thyroid images of 322 patients with thyroid diseases and 67 controls. For the purpose of the study, URs of the images were calculated, and then corrected by standardized thyroid area size to establish a corrected uptake ratio (CUR). Subsequently, the accuracy between UR and CUR was compared.Results:The results of linear regression using weighted least squares (using TcTU as a dependent variable and CUR, UR, or thyroid area size as independent variables) showed that CUR (t=105.5, P=0.000), UR (t=31.9, P=0.000), and thyroid area size (t=15.9, P=0.000) are influential factors of TcTU. Furthermore, the standardized coefficient of CUR (β=0.983) was obviously higher than those of UR (β=0.851) and thyroid area size (β=0.629). The linear goodness-of-fit between CUR and TcTU (R=0.983) was better than that between UR and TcTU (R=0.851). In addition, the total concordance rate between CUR and TcTU (96.7%) was significantly higher than that between UR and TcTU (83.0%; χ2=42.9, P=0.000). Discordance rates of CUR in large thyroid area (1.4% vs. 13.4%, χ2=17.0, P=0.000) and small thyroid area (3.3% vs. 42.2 %, χ2=44.3, P=0.000), were significantly lower than that of UR. In the abnormal thyroid areas, the discordance rates of UR obviously increased as compared to those of CUR. The UR overestimated the thyroid uptake in small thyroid areas and underestimated it in large thyroid areas.Conclusion:Based on the findings, CUR is more accurate than UR in measuring 99mTcO4ˉ thyroid uptake; accordingly, it is more significant in the diagnosis of thyroid disease.

PRM273 - METHODOLOGICAL CONSIDERATIONS ON THE RECOMMENDATIONS TO CONDUCT AND REPORT SYSTEMATIC REVIEWS IN MEDICAL LITERATURE: SYSTEMATIC REVIEW

Several recommendations are available to help conducting and/or reporting a systematic review. This systematic review aims to identify and characterize the published recommendations to conduct and/or to report a systematic review of medical interventions. This systematic review followed the Cochrane Collaboration and the Preferred Reporting Items for Systematic Reviews and Meta-analyses guide. A search was carried out in MEDLINE and EMBASE databases, using “systematic review” indexed term. No language restrictions or publication dates were applied. The search comprised all recommendations to conduct and/or report a systematic review assessing a medical intervention. Data on methodology of each recommendation were extracted. Descriptive analysis was performed. Fifty recommendations were identified. Almost 60% of retrieved references were published in the last 4 years. Recommendations to both conduct and report a systematic review were issued in 42% studies. A total concordance on the methods to conduct and/or report a systematic review was not observed among the recommendations. Literature search and data assessment were recommended in more than 90% of recommendations. An a priori protocol was only recommended in 54% of recommendations. Forty-two percent of recommendations endorsed their methods to any subject related with medical interventions. Half of the studies did not specify the study designs to be included on systematic reviews. Despite the recommendations to conduct and/or report systematic reviews offer guidance on the systematic search and selection and analysis of data, they can differ on other aspects. Such heterogeneity can have methodological implications on the results and their interpretation.

Clinical reassessment of human embryo ploidy status between cleavage and blastocyst stage by Next Generation Sequencing.

One of the most important limitations of genetic testing in preimplantation embryos is embryonic mosaicism, especially when performed on D3 with only a single blastomere evaluated. Previous publications, using Array-Comparative Genomic Hybridization (a-CGH) to compare day 3 (D3) biopsies versus trophectoderm biopsies for the analysis of aneuploid embryos, showed similar high concordance rates per embryo diagnosis for D3 biopsies and trophectoderm biopsies. Next generation sequencing (NGS) was introduced lately as a new technique for preimplantation genetic testing for aneuploidies (PGT-A). Using this technique, this retrospective descriptive study evaluated the degree of the concordance of the diagnosis between preimplantation human cleavage stage (D3) and blastocyst stage (D5) embryos. Double biopsies on D3 and D5 were performed on 118 embryos, reaching blastocyst stage on D5 and had not been selected for transfer. As the fertilization law of the United Arab Emirates does not allow embryo freezing, also surplus euploid embryos after D 3 biopsy were included.Analysis of the NGS results from D3 and D5 embryo biopsies showed a total concordance rate per embryo diagnosis of 85.6% for euploid and aneuploid embryos. The concordance rates per embryo chromosomal pattern for embryo diagnosed as aneuploid at both biopsy stages was 82.2%. However, the status regarding the affected chromosomes was not identical on D3 and D5. Hence, the total concordance rate between D3 biopsy and D5 biopsy was limited to 67.8%.This current study clearly demonstrated that the concordance rates between D3 and D5 biopsies in aneuploid and euploid embryos are lower than previously reported.

The Discordance of Gene Mutations between Circulating Tumor Cells and Primary/Metastatic Tumor Tissue

Non-invasive methods are widely recommended with precision medicine. Circulating tumor cells (CTCs) are suggested to be able to solve the many limitations present in tumor tissue. As sequencing methods are being improved, studies about mutations in CTCs are burgeoning. However, major questions remain to be answered about whether the mutations in the CTCs represent the mutations in primary tumor tissue and metastatic tumor tissue and how to apply the clinical utility of the mutations in CTCs. In the searched articles, we compared the genes mutations between CTCs and tumor tissue, and extracted data about the heterogeneity of the mutational status in CTCs and the change in mutations of CTCs before and during treatment. For mutations detected in single genes, we calculated the concordance of the mutations between the CTCs and primary tumor tissue. For mutations detected in multiple genes, we calculated the concordance of the mutations between the CTCs and primary/metastatic tumor tissue. The heterogeneity of the mutational status is clear in CTCs. For mutations detected in a single gene, the total concordance of mutations is 53.05%. For mutations detected in multiple genes, the concordance of mutations is extremely high and different. Before mutations in CTCs can find widespread clinical utility, we should clarify the following subjects: amplification and sequencing errors, the definition of mutations in sequenced single CTCs, the heterogeneity of the mutational status in single CTCs, and the discordance of mutations between CTCs and tumor tissue.Therefore, the clinical application of CTCs mutations might be realized after considerably more research. Funding: This work was supported by National Institutes of Health (R21HG006173, R43HG007621, HL117929). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study was also funded by grants from the National Natural Science Foundation of China (81472823), the Fundamental Research Funds for Xi’an Jiaotong University (xjj2015093), the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University, China (XJTU1AHCR2014-007), and Major Basic Research Project of Natural Science of Shaanxi Provincial Science and Technology Department (2017ZDJC-11). Competing interests: The authors have declared no conflicts of interest.

Correlation of Immunohistochemistry and Fluorescence in Situ Hybridization for HER-2 Assessment in Breast Cancer Patients: Single Centre Experience

BACKGROUND:Accurate assessment of HER-2 is imperative in selecting patients for targeted therapy. Most commonly used test methods for HER-2 are immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). We evaluated the concordance between FISH and IHC for HER-2 in breast cancer samples using Food and Drug Administration approved tests.MATERIAL AND METHODS:Archived paraffin tissue blocks from 73 breast cancer patients were used. HER-2 immunostaining was performed using Ventana anti–HER-2 monoclonal antibody. The FISH assay was performed using PathVysion™ HER-2 DNA Probe Kit.RESULTS:Of the 73 cases 68.5% were IHC 0/1+, 15.07% were IHC 2+ and 16.44% were IHC 3+. Successful hybridisation was achieved in 72 cases. HER-2 FISH amplification was determined in 16.67% cases. Ten IHC 3+ and two IHC 2+ cases were FISH positive. Two of the IHC 3+ cases were FISH negative. Concordance rate was 100%, 18.18% and 83.33% for IHC 0/1+, 2+ and 3+ group, respectively. Total concordance was 84.72%, kappa 0.598 (p < 0.0001). The sensitivity of IHC in detecting IHC 2+ and IHC 3+ cases was 16.7% and 83.3%, and the specificity was 85% and 96.67%, respectively.CONCLUSION:The consistency between the methods was highest for IHC negative and lowest for IHC equivocal cases. The immunohistochemistry showed high sensitivity for IHC 2+/3+ cases and high specificity for IHC 3+ cases. Our results support the view that false-positive rather than false-negative IHC results are a problem with HER-2/IHC testing, and that IHC should be used as an initial screening test, but IHC 2+/ 3+ results should be confirmed by FISH.

Comparative Study of Fine Needle Aspiration Cytology and Histopathology in Grading Breast Carcinoma

Grading of tumors in Fine Needle Aspiration Cytology (FNAC) is a useful tool for selecting therapy and prognosis in breast carcinoma patients. A total of 38 patients of Infiltrating ductal carcinoma of breast who underwent FNAC and mastectomy were cytologically and histologically graded (employing Taniguchi's cytological grading system and Scarff Bloom Richardson grading method respectively).Statistical analysis was done using 'Z' test and 'χ 2 ' test. The lymph nodes were also studied for detection of metastasis. Total concordance between Cytological grade(CG) and Histological grade(HG) was seen in 78.95% of cases and Positive correlation between the two grading systems was also noted. Lymph node metastasis was seen in 52.63% of cases which was maximum in grade III cases. Cytological grading of breast carcinoma correlates well with histological grading and it may be useful in the prognostic evaluation of the cases without additional morbidity or expense to the patient.

Same-Day Identification and Antimicrobial Susceptibility Testing of Bacteria in Positive Blood Culture Broths Using Short-Term Incubation on Solid Medium with the MicroFlex LT, Vitek-MS, and Vitek2 Systems.

BackgroundEarly and appropriate antibiotic treatment improves the clinical outcome of patients with septicemia; therefore, reducing the turn-around time for identification (ID) and antimicrobial susceptibility test (AST) results is essential. We established a method for rapid ID and AST using short-term incubation of positive blood culture broth samples on solid media, and evaluated its performance relative to that of the conventional method using two rapid ID systems and a rapid AST method.MethodsA total of 254 mono-microbial samples were included. Positive blood culture samples were incubated on blood agar plates for six hours and identified by the MicroFlex LT (Bruker Daltonics) and Vitek-MS (bioMeriéux) systems, followed by AST using the Vitek2 System (bioMeriéux).ResultsThe correct species-level ID rates were 82.3% (209/254) and 78.3% (199/254) for the MicroFlex LT and Vitek-MS platforms, respectively. For the 1,174 microorganism/antimicrobial agent combinations tested, the rapid AST method showed total concordance of 97.8% (1,148/1,174) with the conventional method, with a very major error rate of 0.5%, major error rate of 0.7%, and minor error rate of 1.0%.ConclusionsRoutine implementation of this short-term incubation method could provide ID results on the day of blood culture-positivity detection and one day earlier than the conventional AST method. This simple method will be very useful for rapid ID and AST of bacteria from positive blood culture bottles in routine clinical practice.

Clinical diagnosis versus autopsy diagnosis in head trauma

Abstract The correct and complete diagnosis is essential for the adequate care and the favourable clinical evolution of the patients with head trauma. Purpose: To identify the error rate in the clinical diagnosis of head injuries as shown in comparison with the autopsy diagnosis and to identify the most common sources of error. Material and method: We performed a retrospective study based on data from the medical files and the autopsy reports of patients with head trauma who died in the hospital and underwent forensic autopsy. We collected: demographic data, clinical and laboratory data and autopsy findings. To quantify the concordance rate between the clinical diagnosis of death and the autopsy diagnosis we used a 4 classes classification, which ranged from 100% concordance (C1) to total discordance (C4) and two classes of partial discordance: C2 (partial discordance in favour of the clinical diagnosis- missing injuries in the autopsy reports) and C3 (partial discordance in favor of the necroptic diagnosis- missing injuries in the medical files). Data were analyzed with SPSS version 20.0. Results: We analyzed 194 cases of death due to head injuries. We found a total concordance between the clinical death diagnosis and autopsy diagnosis in 30.4% of cases and at least one discrepancy in 69.6% of cases. Increasing the duration of hospitalization directly correlates with the amount of the imaging investigations and these in turn correlates with an increased rate of diagnosis concordance. Among the patients with stage 3 coma who associated a spinal cord injury, we found a partial diagnosis discordance in 50% of cases and a total discordance in 50% of cases, possibly due to the need for conducting emergency imaging investigation and the need for surgical treatment. In cases with partial and total discordant diagnosis, at least one lesion was omitted in 45.1% of the cases. The most commonly omitted injuries in C2 cases were subdural hematoma, intracerebral hematoma and ventricular hemorrhage (21.6%). In C3 cases the most frequently omitted injuries were subarachnoidian hemorrhage and skull base fractures (17.9%). Conclusions: The clinical cause of death is not always concordant with the autopsy diagnosis. Autopsy may identify the inconsistencies in diagnosis, the injuries frequently skipped and the factors favoring the discordance rate between the clinical death diagnosis and the autopsy diagnosis, making it a valuable tool for improving the clinical care of the patients with head trauma.

La nouvelle classification 2015 des adénocarcinomes pulmonaires : reproductibilité au sein d’un service Tunisien spécialisé en pathologie thoracique

The new classification of lung cancer contains modifications of terminology and a new subdivision of the tumors with the most relevant modifications concerning the group of adenocarcinomas. The latter has been increasing and represents nowadays the most frequent type. Our aim was to assess the reproducibility of the new classification through the experience of a Department of Pathology specialized in thoracic pathology. Our study included initially 106 cases diagnosed as adenocarcinomas and reviewed by 2 pathologists and 1 referee. Five cases were ruled out because they corresponded to squamous carcinoma according to the immunohistochemical findings. The same number of slides was reviewed without a limit of time. Statistical analysis was performed using the SPSS software. The Kappa index was estimated and a second coefficient: rho was analyzed. A total concordance was noticed in 82 cases (81.2%) and a discordance was noticed in 19 cases (18.8%). The agreement degree was good with an index Kappa estimated to 0.743 and a rho index reaching 0.763. Our study highlights the good reproducibility of the 2015 WHO classification of lung cancer among a trained team. Whereas, in order to improve the reproducibility of such a classification, even in non specialised departments, a training of the pathologists is necessary in order to highlight the prognostic impact of this classification.

P166 Validation of a next generation sequencing based HLA typing system

Aim Very high resolution typing of HLA genes by Next Generation Sequencing (NGS) methods is becoming mainstream in histocompatibility laboratories for the workup of haematopoietic stem cell and solid organ transplant patients and donors. Determining the full sequence for class I and extended exon coverage for class II genes produces HLA types with far fewer ambiguities than traditional Sanger sequencing based typing (SBT) and other lower resolution typing methods. In addition NGS brings benefits of higher throughput and lower cost. Methods We have validated the Illumina TruSight HLAv2 typing kit for HLA-A, B, C, DRB1/3/4/5, DQA1, DQB1, DPA1 and DPB1. A total of 151 samples were tested following the method described by the vendor. All method steps were carried out manually. Testing comprised 6 batches of 24 samples and 1 batch of 12 samples, which were made up of 73 local DNA samples typed previously by a combination of SBT, SSP and SSO, 54 DNA samples from the UCLA reference panel and 24 samples from the 17th International HLA Workshop Study blinded proficiency panel. Reference types for all eleven loci were not available for all local samples, four digit types were available for all eleven loci for the UCLA samples. Sequence analysis and genotype calling was performed using Illumina Assign for Trusight HLA v2.1 and GenDX NGSEngine. HLA type concordance rate, ambiguity rate and accuracy were examined. Results Total percent concordance between the reference type (n = 130) and genotype determined by Assign was as follows: A: 100; B: 99.2; C: 99.2; DRB1: 97.6.5; DRB345: 98.4; DQA1: 99.2; DQB1: 100; DPA1: 100; and DPB1: 97.6. No allele (1st, 2nd and 3rd field) or genotype ambiguity was found for HLA-A, B, C, DRB345 DQB1 or DPA1. An ambiguous type was determined in 4 cases for DRB1, 3 cases for DQA1 and 29 cases for DPB1. In the case of DRB1 and DPB1 this was due to lack of part of the gene sequence and for DQA1 it was due to poor read depth in those samples. Average read depth across all loci was 223. Repeat rates varied between 1.3% (A & B) and 10.9% (DPA1). Conclusions The Illumina TruSight HLAv2 typing kit provides a suitable first pass high resolution typing system for stem cell and solid organ transplant patients and donors. Accuracy is excellent and the ambiguity levels are very low. We intend to introduce this method for routine HLA typing.

Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma.

BackgroundCurrently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis.MethodsA droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations.ResultsThe established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%-79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases.ConclusionsThe TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers.

Nasopharyngeal pertussis toxin IgA antibodies in the diagnosis of pertussis in Australian community patients

Nasopharyngeal aspirate (NPA) Bordetella pertussis-specific IgA antibody assay using whole-cell (WC) antigen has previously been shown to have promise in the diagnosis of patients with suspected pertussis. Recently, the use of WC assays in serum have been replaced by pertussis toxin (PT) because of specificity concerns. In this study, PT and WC B. pertussis-specific IgA antibody was assayed in 491 NPAs. Specimens also had molecular testing for the presence of B. pertussis and B. parapertussis as per the usual laboratory protocol. Positive concordance of the two serological assays was 51.2%, negative concordance was 67.5% and total concordance was 75.8%. 99 of 119 discordant specimens were resolved by utilising the B. pertussis polymerase chain reaction (PCR) result and clinical status, and yielded a sensitivity of 57.6% and a specificity 97.7% for WC, with 90.2% and 93.1%, respectively, for the PT assay (p<0.00025 and 0.025-0.01). In contrast, the sensitivity of PCR was only 19.1% in this cohort. We conclude that specificity is not a significant issue for mucosal pertussis-specific IgA assays using WC, but the superior sensitivity of the PT assay favours the latter method. This assay, combined with PCR assays, should significantly improve the diagnosis of pertussis cases.

MP22-08 ESTIMATED GLOMERULAR FILTRATION RATE: DO WE MEASURE THE REAL RENAL FUNCTION OR ARE WE STILL GROPING IN THE DARK?

MP22-08 ESTIMATED GLOMERULAR FILTRATION RATE: DO WE MEASURE THE REAL RENAL FUNCTION OR ARE WE STILL GROPING IN THE DARK?

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients.

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) extracted from the plasma of advanced non-small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) -sensitive (19DEL, L858R) and TKI-resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR-TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra-sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR-TKI decision-making for advanced NSCLC patients.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.