Molecular Test Results Research Articles

The Impact of Diagnosis to Treatment Interval on the Outcome of Patients with Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) has historically been considered an oncologic emergency, requiring immediate intervention and initiation of therapy. Although urgency to initiate treatment has been debated, no guidelines exist to address the impact of diagnosis to treatment interval (DTI) in patients with AML. Recent FDA approvals have added treatment options using targeted therapy as monotherapy in older patients (e.g. - ivosidenib), or in combination with chemotherapy in younger patients (e.g.- midostaurin) in the newly diagnosed setting with improved outcomes. Unfortunately, results of molecular mutation testing often return several days after the initial diagnosis of AML has been made. While immediate induction is important to control disease and to minimize disease-related morbidity and mortality (Sekkers et al), molecular testing is becoming increasingly important for treatment decisions, and there may be benefit in waiting for these results. In addition, in 'real world settings', several barriers (e.g., lack of resources, correct diagnostic testing) often delay time to diagnosis, and initiation of therapy. We conducted a retrospective study in two academic centers in a major metropolitan area in the United States to evaluate the impact of DTI on the outcomes of patients with AML. Methods: We collected data from 196 patients who presented to George Washington University Hospital (120 patients) between June 2010 and July 2018 and Georgetown University hospital (76 patients) between January 2014 and December 2018. All patients were older than 18 years. Patients with acute promyelocytic leukemia and those who decided to pursue treatment elsewhere were excluded. Patients characteristics including age, gender, race, smoking past medical history and disease characteristics (Cytogenetics, FISH and NGS when available) were collected using retrospective chart review. Type of treatment received (low intensity vs high intensity), and the time from diagnosis to initiation of therapy were collected. We recorded mortality at day 30 and day 90. Multivariable logistic regression models were used to evaluate the association of DTI with mortality independently from patient age. Chi-square test was used to examine the association of 30- and 90-day mortality with DTI. Results: A total of 140 patients were used in the analysis, 71% of whom were treated within 5 days of diagnosis (DTI <1-5 days), 16% in 6-10 days, and 12% in >10 days. Incidence of 30-day mortality was 15%, 14%, and 0% (p=.24), while 90-day mortality was 28% 18%, and 13% for <1-5, 6-10, and >10 days respectively (p=.33). Neither association was statistically significant, and adjusting for age and type of therapy did not change the findings. Conclusion: Our data does not support that shorter DTI is associated with improved mortality regardless of age or type of therapy. Although there was no statistical significance, there was a numerical trend of improved mortality in patients with delayed DTI (6-10 days) compared to those with a DTI of <1-5 days. Larger prospective studies need to be conducted to investigate DTI in AML patients based on patient's age, disease characteristics, and treatment type. In particular, given the growing importance of knowing molecular mutations at diagnosis, used for both prognosis and incorporation of potential targeted agents, it will be important to understand how long we can safely wait to treat patients without affecting outcomes. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees.

Clinical Molecular Marker Testing Data Capture to Promote Precision Medicine Research Within the Cancer Research Network.

To evaluate health care systems for the availability of population-level data on the frequency of use and results of clinical molecular marker tests to inform precision cancer care. We assessed cancer-related molecular marker test data availability across 12 US health care systems in the Cancer Research Network. Overall, these systems provide care to a diverse population of more than 12 million people in the United States. We performed qualitative analyses of test data availability for five blood-based protein, nine germline, and 14 tissue-based tumor marker tests in each health care system's electronic health record and tumor registry using key informants, test code lists, and manual review of data types and output. We then performed quantitative analyses to estimate the proportion of patients with cancer with test utilization data and results for specific molecular marker tests. Health systems were able to systematically capture population-level data on all five blood protein markers, six of 14 tissue-based tumor markers, and none of the nine germline markers. Successful, systematic data capture was achievable for tests with electronic data feeds for test results (blood protein markers) or through prior manual abstraction by tumor registrars (select tumor-based markers). For test results stored in scanned image files (particularly germline and tumor marker tests), information on which test was performed and test results was not readily accessible in an electronic format. Even in health care systems with sophisticated electronic health records, there were few codified data elements available for evaluating precision cancer medicine test use and results at the population level. Health care organizations should establish standards for electronic reporting of precision medicine tests to expedite cancer research and facilitate the implementation of precision medicine approaches.

1993. Impact of Pharmacist Intervention in Response to Rapid Molecular Testing Results of Blood Cultures

BackgroundRapid diagnostic testing can identify bacterial pathogens and genetic markers of resistance. This information can aid in deescalating antimicrobial therapy based on local patterns prior to the return of final culture and susceptibility reports. In August of 2017 at our institution, pharmacists were incorporated in the review of these rapid diagnostic tests in real time with recommendations for changes to therapy paged to providers. The purpose of this study was to determine whether the inclusion of a pharmacist in this workflow reduces time to change in antimicrobial therapy.MethodsThis retrospective study over two periods in 2016 and 2018 analyzed the pre- and post-implementation of a workflow incorporating pharmacist review of positive blood cultures analyzed by rapid molecular assay. Patients were included if they were at least 18 years of age and were initiated on empiric antibiotics prior to test results. Patients were excluded if transitioned to hospice within 24 hours of test results, discharged prior to test results, if more than one infectious process or organism was identified, or if test results were positive for coagulase-negative staphylococci. The primary outcome of the study was the time to change to targeted antimicrobials. Secondary outcomes were rates of Clostridium difficile infection within 3 months and inpatient mortality. Categorical variables were evaluated using chi-squared or Fisher exact tests, and continuous variables were evaluated using Wilcoxon rank-sum tests.ResultsA total of 199 patients were included, with 98 and 101 patients in the pre- and post-implementation groups, respectively. The median time to change to targeted antimicrobials was significantly reduced with pharmacist intervention from 18.35 to 8.43 hours (P = 0.042). The groups had similar rates of C. difficile infection (1% vs. 0%, P = 0.492) and mortality (7.1% vs. 5%, P = 0.564). The post-group also had significant reductions in antibiotic days of therapy (10.5 vs. 9 days, P = 0.014) and intensive-care unit length of stay (3.04 vs. 1.44 days, P = 0.046).ConclusionIncorporating pharmacist review of rapid molecular results of blood cultures decreased time to change in antimicrobial regimen and reduced antibiotic days of therapy.Disclosures All authors: No reported disclosures.

Progressive Diagnosis of Chronic Myeloproliferative Neoplasms: A Case Study

Chronic myeloproliferative neoplasms are hematologic diseases characterized by the clonal proliferation of peripheral blood components. A series of laboratory tests are required for diagnosis. This patient is a 74-year-old male incidentally noted to have a leukocytosis, monocytosis, and rare blasts in his peripheral blood. He presented with a CBC showing 6.6 g/dL Hgb, 114fL MCV, 15.3x10(9)/L, WBC, 304.10(9)/L Platelet. WBC differential consisting of 21% neutrophils, 21% lymphocytes, 45% monocytes, 6% eosinophils, 4% basophils and 3% blasts. The peripheral blood smear showed hypochromic microcytic red cells with marked macrocytosis and slight polychromasia. In addition, atypical hypogranular neutrophils, and hypogranular platelets were revealed. A pathology consultation reported a bone marrow aspirate of 22% neutrophils, 6% metamyelocytes, 11% myelocytes, 1% promyelocytes, 12% eosinophils, 8% blasts, 18% normoblasts, 15% monocytes, and 7% lymphocytes. The M:E ratio was increased, 4:1, with 90% hypercellularity. There was a decreased quantity of erythroid precursors and an increased quantity of myeloid precursors. Blasts contained intermediate-sized nuclei, reticulated chromatin and variably granulated cytoplasm and megakaryocytes were markedly increased. Ancillary tests included both iron and reticulin staining of the bone marrow. Storage iron was decreased and reticulin fibers increased with a myelofibrosis grade 1 of 3. Flow cytometry immunophenotyping of the bone marrow revealed mildly increased myeloid blasts, increased mature monocytes, increased monoblasts/promonocytes with other abnormal findings consistent with a myeloid neoplasm with high grade features. In summary, the pertinent and unusual findings were the rare blasts, increased monocytes and basophils, and dysplastic neutrophils, and leukocytosis seen in the peripheral blood along with slightly increased blasts in the marrow. Along with additional cytogenetic and molecular testing results, the above laboratory finds were essential to the diagnosis of an unusual myelodysplastic/myeloproliferative neoplasm, Chronic Myelomonocytic Leukemia (CMML). The sequence of testing lead to the proper diagnosis and will aid in treatment options.

The Molecular Pathology of Eye Tumors: A 2019 Update Main Interests for Routine Clinical Practice.

Over the last few years, we have seen constant development of molecular pathology for the care of patients with cancer. The information obtained from molecular data has transformed our thinking about the biological diversity of cancers, particularly in the field of ophthalmic oncology. It has reoriented the way in which therapeutic decisions and decisions concerning patient surveillance are made, both in the area of pediatric cancers, including rhabdomyosarcoma and retinoblastoma, and adult cancers, such as uveal melanoma and lymphomas. A better definition of the molecular classification of these cancers and of the different biological pathways involved is essential to the understanding of both the pathologist and the onco-ophthalmologist. Molecular tests based on targeted or expanded analysis of gene panels are now available. These tests can be performed with tumor tissue or biofluids (especially blood) to predict the prognosis of tumors and, above all, the benefit of targeted therapies, immunotherapy or even chemotherapy. Looking for the BAP1 mutation in uveal melanoma is essential because of the associated metastatic risk. When treating retinoblastoma, it is mandatory to assess the heritable status of RB1. Conjunctival melanoma requires investigation into the BRAF mutation in the case of a locally advanced tumor. The understanding of genomic alterations, the results of molecular tests and/or other biological tests predictive of a therapeutic response, but also of the limits of these tests with respect to the available biological resources, represents a major challenge for optimal patient management in ophthalmic oncology. In this review, we present the current state of knowledge concerning the different molecular alterations and therapeutic targets of interest in ophthalmic oncology.

DETECTION OF PORCINE CIRCOVIRUS TYPE 3 IN RUSSIAN PIG HOLDINGS

Diseases associated with porcine circoviruses (mainly with porcine circovirus type 2) have various manifestations, are common in pigs in countries having well-developed pig industry and responsible for significant economic losses. Porcine circovirus type 3 (PCV-3) causing systemic inflammation of unknown etiology in animals was detected the USA in 2015. Later, data on PCV-3 detection in Asia, Europe and South America were published. Analysis of literature data on current epidemic situation on PCV-3 infection in foreign countries as well as the disease clinical manifestations and postmortem lesions are described. Results of molecular and genetic tests of biomaterials collected from pigs in 51 holdings located in 28 regions of the Russian Federation are presented. A total of 280 samples of biological materials of different types (organs, tissues, stillborn piglets) collected from domestic pigs with respiratory, reproductive and neurological disorders, dermatitis and from emaciated pigs were tested and PCV-3 genome was detected in 11 samples from 9 holdings located in 5 regions of the Russian Federation. Porcine circovirus type 3 was detected in lung, bronchial and mediastinal lymph node, spleen tissues from grower and fattening piglets, adult pigs and aborted fetuses. Samples that were positive for PCV-3 DNA when tested with molecular methods (PCR, real-time PCR) were tested for other pathogens. The following pathogens were also detected in 6 out of 11 samples (55%): Actinobacillus pleuropneumoniae, Mycoplasma hyorhinis, Streptococcus suis, Haemophilus parasuis, Mycoplasma hyopneumoniae and Pasteurella multocida. Porcine circovirus type 2 was detected in one sample. Presented test results are indicative of probable combined etiology of respiratory and reproductive disorders in tested pigs that results in various clinical manifestations. Grower and fattening piglets were found to be the most susceptible to PCV-3-associated disease. Further studies are required for identification of actual PCV-3 pathogenicity and its prevalence in the territory of the Russian Federation.

1558P - Biomarker status as a mediator of age-related overall survival (OS) in advanced non-small cell lung cancer (aNSCLC)

BackgroundRisk models for NSCLC have identified age as a prognostic factor, but the relevance of molecular biomarkers to age-related outcomes is undefined. We explored the prevalence of biomarkers by age group and the impact of biomarker status on age-related differences in OS among aNSCLC patients (pts) in a real-world population. MethodsPts with stage IIIB, IV or recurrent metastatic NSCLC (diagnosed 1/1/11 to 11/30/18) were identified in a US-based, EHR-derived, deidentified database (Flatiron Health). Cox proportional hazard regressions evaluated OS by age at aNSCLC diagnosis (dx) (< vs≥50 years), controlling for gender, histology, race, stage, dx year, smoking status, visit site, region and PDL1 status. Sequential and subgroup regressions modeled ALK+, EGFR+ and ROS1+ biomarker status as mediators of the age effect on OS. ResultsOur sample included 41,024 pts (median age 69 years); median OS was 11 months. Pts <50 years lived longer than pts ≥50 years (HR: 0.83; p<0.001). Molecular testing and positive results for ALK, EGFR and ROS1 were higher in age <50 (Table); this finding was maintained for non-squamous pts (p≤0.001). Within ALK+, EGFR+ or ROS1+ subgroups, age was not associated with OS. Sequential variable analysis found 35% of the association between age and OS may be explained by ALK, EGFR and ROS1 biomarker status. ConclusionsYoung aNSCLC pts have longer OS than older pts; this benefit is significantly mediated through biomarker status. Pts with mutations had similar OS regardless of age group. As older pts are tested less often and have a lower prevalence of mutations, future work should explore whether increased testing in older pts may mitigate the age-related OS disparity.Table1558P Baseline characteristics by age at aNSCLC Dx*TableAll, n (%) N=41,024< 50 y, n (%) n=1381≥50 y, n (%) n=39,643Female19,301 (47)716 (52)18,585 (47)Non-squamous histology28,203 (69)1092 (79)27,111 (68)Non-smoker4723 (12)323 (23)4400 (11)ALK tested23,618 (58)948 (69)22,670 (57)ALK+†625 (2.6)106 (11)519 (2.3)EGFR tested25,276 (62)1000 (72)24,276 (61)EGFR+†3009 (12)162 (16)2847 (12)ROS1 tested12,310 (30)514 (37)11,796 (30)ROS1+†113 (0.92)12 (2.3)101 (0.86)*All age comparisons p<0.001;†among tested Legal entity responsible for the studyFlatiron Health. FundingFlatiron Health. DisclosureA.B. Cohen: Shareholder / Stockholder / Stock options, Full / Part-time employment: Flatiron Health, an independent subsidiary of Roche; Shareholder / Stockholder / Stock options: Roche. B.D.A.P. Neri: Shareholder / Stockholder / Stock options, Full / Part-time employment: Flatiron Health, an independent subsidiary of the Roche group. B.J.S. Adamson: Full / Part-time employment, Provisional patent filed: Flatiron Health, an independent subsidiary of Roche; Non-remunerated activity/ies, Provisional patent filed: University of Washington; Research grant / Funding (self): American Foundation for Pharmaceutical Education; Research grant / Funding (institution): PhRMA Foundation; Leadership role: International Society for Pharmacoeconomics and Outcomes Research; Advisory / Consultancy: Salutis Consulting; Advisory / Consultancy: Fred Hutchinson Cancer Research Center; Advisory / Consultancy: Institute for Disease Modeling, Intellectual Ventures; Advisory / Consultancy: Boston Scientific; Shareholder / Stockholder / Stock options: Roche. C.M. Scanlon: Shareholder / Stockholder / Stock options, Full / Part-time employment: Flatiron Health, an independent subsidiary of Roche. C. Gross: Honoraria (self), Travel / Accommodation / Expenses: Flatiron Health, Inc., which is an independent subsidiary of the Roche Group; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Johnson & Johnson. N.J. Meropol: Shareholder / Stockholder / Stock options, Full / Part-time employment: Flatiron Health, Inc., which is an independent subsidiary of the Roche Group; Shareholder / Stockholder / Stock options: Roche. R.A. Miksad: Shareholder / Stockholder / Stock options, Full / Part-time employment: Flatiron Health, Inc., which is an independent subsidiary of the Roche Group; Shareholder / Stockholder / Stock options: Roche; Advisory / Consultancy: De Luca Foundation.

P1.16-30 Impact of Patient TKI Copayments on Insurance Expenditure in Advanced EGFR or ALK Positive Non-Small Cell Lung Cancer (NSCLC)

To influence treatment choice and control expenditures, insurance plans impose cost sharing policies for expensive oral tyrosine kinase inhibitors (TKIs). We evaluated the effect of patient TKI copayments on insurance expenditures among patients with EGFR and ALK positive advanced NSCLC receiving TKIs. We identified EGFR+ and ALK+ NSCLC patients in the Washington State SEER registry using natural language processing on registry pathology reports, followed by manual confirmation of molecular test results. We linked registry records with commercial and Medicare claims. Eligible patients had stage IV NSCLC diagnosed between 01/01/2010 and 12/31/2016, EGFR exon 19 deletions or L858R mutations, or ALK + by FISH, ≥ 12 months of insurance enrollment, survival of ≥6 months, and ≥ 1 pharmacy claims for EGFR or ALK TKIs with FDA approval in the study period. We estimated monthly TKI copayments by subtracting the drug amount paid from the amount allowed by insurance in pharmacy claims. We used claims to calculate lifetime total and drug costs from the insurance perspective. Covariates included sociodemographic characteristics, insurance type, comorbidity, mutation type, and receipt of chemotherapy and immunotherapy. We used generalized linear models with gamma family and log link to estimate the adjusted effect of TKI copayment of $0 vs. above $0 on insurer expenditures in 2016 US dollars. Of 103 eligible patients, median age was 69; 66% were female; 72% were White, median household income was $69,951; 54% had Medicare, 85% were EGFR+, and 51% received chemotherapy or immunotherapy. Mean monthly TKI copayment was $312 (range= $0 to $5,913). Mean total and drug reimbursements were $234,294 and $114,253. Table 1 shows the adjusted effect of TKI copayments on insurance expenditures. Higher TKI copayments were not associated with lower insurance expenditures. Eliminating TKI copayments would reduce patient financial burden and not adversely impact insurer spending.Table 1Effect of Patient TKI Copayments on Insurance Expenditures, Adjusted for Age, Race, Comorbidity, Insurance Type, Mutation Type, Receipt of Chemotherapy and Immunotherapy (EGFR TKIs: erlotinib, gefitinib, afatinib, osimertinib; ALK TKIs: crizotinib, alectinib, ceritinib, brigatinib).TKI Copayment (n=103)Adjusted Mean Total Expenditure (95% CI)P valueAdjusted Mean Drug Expenditure (95% CI)P value$0 (n=82)$230,989 ($188,958; $273,019)Ref.$110,171 ($88,150; $132,194)Ref.Above $0 (n=21)$264,963 ($182,424; $347,502)0.44$132,707 ($83,206; $182,208)0.38 Open table in a new tab

Generating timely molecular diagnostic test results: workflow comparison of the cobas® 6800/8800 to Panther

ABSTRACTBackground: Molecular diagnostic tests for HBV, HCV and HIV-1 and other pathogens are widely used for clinical management. Practical issues related to workflow and labor requirements need to be characterized to inform selection of the most appropriate system.Research design and methods: We compared the workflow of two high-throughput systems: cobas 6800 (Roche) and Panther (Hologic), using average mid-size laboratory test volumes for five different assays (HIV-1, HBV, HCV, HPV or TV, and CT/NG).Results: Set-up time, time to first results, time to last results, and total hands-on time for cobas 6800 was 0.40, 2.47, 7.12, and 0.98 hours, respectively; on the Panther system, these times were 0.75, 2.7, 9.1, and 1.48 hours. Fifty-seven samples had results available at the first time point on cobas 6800 compared to 5 samples on the Panther system. The Panther system required more manual steps including several with potential risks of contamination or error. The number of reagents items required was 5 for cobas 6800 and 40 for the Panther system.Conclusions: Both systems provided a high level of automation. The cobas 6800 platform had shorter start up, time to first result, time to last result and hands-on times than the Panther system.

Quaternized chitosan-Matrigel-polyacrylamide hydrogels as wound dressing for wound repair and regeneration

Hydrogels could be promising wound healing dressings that maintain a moist environment in the wound site and accelerate wound healing. However, the lack of antibacterial effect, suitable mechanical property and adhesiveness limits their applications. Here, we designed a quaternized chitosan-Matrigel-polyacrylamide (QCS-M-PAM) hydrogel with multi-functions. The morphology, swelling ratio, mechanical test, antimicrobial property, hemostatic performance and biocompatibility of the hybrid hydrogel were investigated in vitro and vivo. The hybrid hydrogel showed a three-dimensional (3D) microporous structure, high swelling ratio, excellent stretchable and compressive property, similar modulus to human skin, good adhesiveness, and low cytotoxicity. The results of histology and molecular testing in vivo demonstrated that the hybrid hydrogel could significantly enhance wound healing, collagen deposition, and induce skin adnexal regeneration by upregulating anti-inflammatory factors, and downregulating proinflammatory factors. Together, the present antibacterial hydrogels with hemostatic and adhesive properties are considered to have promising potential used as wound dressings for full-thickness skin defect.

Subcortical heterotopic gray matter brain malformations: Classification study of 107 individuals.

ObjectiveTo better evaluate the imaging spectrum of subcortical heterotopic gray matter brain malformations (subcortical heterotopia [SUBH]), we systematically reviewed neuroimaging and clinical data of 107 affected individuals.MethodsSUBH is defined as heterotopic gray matter, located within the white matter between the cortex and lateral ventricles. Four large brain malformation databases were searched for individuals with these malformations; data on imaging, clinical outcomes, and results of molecular testing were systematically reviewed and integrated with all previously published subtypes to create a single classification system.ResultsReview of the databases revealed 107 patients with SUBH, the large majority scanned during childhood (84%), including more than half before 4 years (59%). Although most individuals had cognitive or motor disability, 19% had normal development. Epilepsy was documented in 69%. Additional brain malformations were common and included abnormalities of the corpus callosum (65/102 [64%]), and, often, brainstem or cerebellum (47/106 [44%]). Extent of the heterotopic gray matter brain malformations (unilateral or bilateral) did not influence the presence or age at onset of seizures. Although genetic testing was not systematically performed in this group, the sporadic occurrence and frequent asymmetry suggests either postzygotic mutations or prenatal disruptive events. Several rare, bilateral forms are caused by mutations in genes associated with cell proliferation and polarity (EML1, TUBB, KATNB1, CENPJ, GPSM2).ConclusionThis study reveals a broad clinical and imaging spectrum of heterotopic malformations and provides a framework for their classification.

High diagnostic yield of direct Sanger sequencing in the diagnosis of neuronal ceroid lipofuscinoses.

BackgroundNeuronal ceroid lipofuscinoses are neurodegenerative disorders. To investigate the diagnostic yield of direct Sanger sequencing of the CLN genes, we reviewed Molecular Genetics Laboratory Database for molecular genetic test results of the CLN genes from a single clinical molecular diagnostic laboratory.MethodsWe reviewed electronic patient charts. We used consent forms and Research Electronic Data Capture questionnaires for the patients from outside of our Institution. We reclassified all variants in the CLN genes.ResultsSix hundred and ninety three individuals underwent the direct Sanger sequencing of the CLN genes for the diagnosis of neuronal ceroid lipofuscinoses. There were 343 symptomatic patients and 350 family members. Ninety‐one symptomatic patients had molecular genetic diagnosis of neuronal ceroid lipofuscinoses including CLN1 (PPT1) (n = 10), CLN2 (TPP1) (n = 33), CLN3 (n = 17), CLN5 (n = 7), CLN6 (n = 10), CLN7 (MFSD8) (n = 10), and CLN8 (n = 4) diseases. The diagnostic yield of direct Sanger sequencing of CLN genes was 27% in symptomatic patients. We report detailed clinical and investigation results of 33 NCL patients. Juvenile onset CLN1 (PPT1) and adult onset CLN6 diseases were nonclassical phenotypes.ConclusionIn our study, the diagnostic yield of direct Sanger sequencing was close to diagnostic yield of whole exome sequencing. Developmental regression, cognitive decline, visual impairment and cerebral and/or cerebellar atrophy in brain MRI are significant clinical and neuroimaging denominators to include NCL in the differential diagnosis.

An Overview of Tuberculosis-Designated Hospitals in China, 2009-2015: A Longitudinal Analysis of National Survey Data.

Design A national tuberculosis- (TB-) designated hospital survey was conducted in 2015 to identify significant changes since 2009 in implementation of TB-testing services within hospitals of various types and administrative levels in various regions in China. Methods In 2015, all TB-designated hospitals were required to complete questionnaires designed by the National Clinical Center for TB. Community hospitals also completed simplified questionnaires as part of the study. Results Overall, in 2015 there were 1685 TB-designated hospitals in China, consisting of 1335 (79.2%) county-level hospitals and 350 (20.8%) hospitals at the prefecture level and above. The percentage of counties with TB-designated hospitals in the western region (57.4%) was significantly lower than corresponding percentages for eastern and middle regions (70.3% and 96.5, respectively). Based on data recorded on hospital surveys in both 2009 and 2015, significant differences were noted between years in proportions of general hospitals with TB wards and of specialized infectious disease hospitals (P < 0.01). Of 1256 county-level laboratories conducting smear microscopy, only 979 (79%) performed external quality control evaluations of test results in 2015. For prefecture-level hospitals, 70% (234/334), 76% (155/203), and 67% (66/98) of hospitals obtained external quality control validations of smear microscopy, phenotypic DST, and molecular test results, respectively. Conclusions Although China's health reform efforts have resulted in improved TB patient access to quality health care, more attention should be paid to balancing the distribution of medical facilities across different regions. In addition, laboratory capabilities and quality control systems should be strengthened to ensure delivery of high-quality laboratory services by TB-designated hospitals throughout China.

A multi-institutional experience in vascular Ehlers-Danlos syndrome diagnosis

ObjectiveVascular Ehlers-Danlos syndrome (vEDS) is a rare disorder and 1 of 13 types of EDS. The syndrome results in aortic and arterial aneurysms and dissections at a young age. Diagnosis is confirmed with molecular testing via skin biopsy or genetic testing for COL3A1 pathogenic variants. We describe a multi-institutional experience in the diagnosis of vEDS from 2000 to 2015. MethodsThis is a multi-institutional cross-sectional retrospective study of individuals with vEDS. The institutions were recruited through the Vascular Low Frequency Disease Consortium. Individuals were identified using the International Classification of Diseases-9 and 10-CM codes for EDS (756.83 and Q79.6). A review of records was then performed to select individuals with vEDS. Data abstraction included demographics, family history, clinical features, major and minor diagnostic criteria, and molecular testing results. Individuals were classified into two cohorts and then compared: those with pathogenic COL3A1 variants and those diagnosed by clinical criteria alone without molecular confirmation. ResultsEleven institutions identified 173 individuals (35.3% male, 56.6% Caucasian) with vEDS. Of those, 11 (9.8%) had nonpathogenic alterations in COL3A1 and were excluded from the analysis. Among the remaining individuals, 86 (47.7% male, 68% Caucasian, 48.8% positive family history) had pathogenic COL3A1 variants and 76 (19.7% male, 19.7% Caucasian, 43.4% positive family history) were diagnosed by clinical criteria alone without molecular confirmation. Compared with the cohort with pathogenic COL3A1 variants, the clinical diagnosis only cohort had a higher number of females (80.3% vs 52.3%; P < .001), mitral valve prolapse (10.5% vs 1.2%; P = .009), and joint hypermobility (68.4% vs 40.7%; P < .001). Additionally, they had a lower frequency of easy bruising (23.7% vs 64%; P < .001), thin translucent skin (17.1% vs 48.8%; P < .001), intestinal perforation (3.9% vs 16.3%; P = .01), spontaneous pneumothorax/hemothorax (3.9% vs 14%, P.03), and arterial rupture (9.2% vs 17.4%; P = .13). There were no differences in mortality or age of mortality between the two cohorts. ConclusionsThis study highlights the importance of confirming vEDS diagnosis by testing for pathogenic COL3A1 variants rather than relying on clinical diagnostic criteria alone given the high degree of overlap with other forms genetically triggered arteriopathies. Because not all COL3A1 variants are pathogenic, the interpretation of the genetic testing results by an individual trained in variant assessment is essential to confirm the diagnosis. An accurate diagnosis is critical and has serious implications for lifelong screening and treatment strategies for the affected individual and family members.

Hurthle cell predominance impacts results of Afirma gene expression classifier and ThyroSeq molecular panel performance in indeterminate thyroid nodules

Molecular tests such as the Afirma gene expression classifier (GEC) and mutational panels (such as ThyroSeq) have been introduced to help risk stratify cytologically indeterminate thyroid nodules with the aim to reduce the number of unnecessary thyroidectomies. Some reports have suggested that samples with Hurthle cell predominance have higher false-positive rates on GEC testing, but data are limited. We reviewed thyroid nodules with indeterminate (Bethesda III/IV) cytology at our institution. Patient demographics, cytologic and histologic diagnoses (where available), and molecular test results were collected. GEC was performed on 202 nodules, and ThyroSeq was performed on 81 nodules. In the GEC cohort, 66% of nodules with Hurthle cell predominance yielded "suspicious" result vs 46% of nodules without Hurthle cell predominance, with risk of malignancy (ROM) for surgically resected nodules of 16% and 33%, respectively. In ThyroSeq cohort, 8% of nodules with Hurthle cell predominance yielded a high-risk mutation vs 19% of nodules without Hurthle cell predominance, with ROM of 50% and 33%, respectively. For ThyroSeq molecular panel, while it did not appear that there was an increase in rate of high-risk mutations detected in the samples with Hurthle cell predominance, small numbers limit the generalizability of these results. For the GEC cohort, indeterminate thyroid nodules with predominance of Hurthle cells showed an increased rate of "suspicious" results compared to samples without Hurthle cell predominance. The ROM for GEC "suspicious" nodules with Hurthle cell predominance on surgical resection was lower in our study. Repeat FNA may be of use in patients with these types of nodules. In the context of a Hurthle cell predominant lesion, positive results on molecular testing may not carry a high rate of malignancy.

Hürthle cell lesions on thyroid fine needle aspiration cytology: Molecular and histologic correlation.

Hürthle cell lesions often pose diagnostic challenges, despite their common occurrence on thyroid fine-needle aspiration cytology (FNAC). The associated molecular alterations are also not well understood. Therefore, our study aimed to delineate the molecular profile of Hürthle cell lesions classified as Bethesda Categories III or IV (atypia of undetermined significance (AUS) or suspicious for follicular neoplasm (SFN)) on FNAC and to correlate this molecular profile with surgical resection findings. This study consisted of 188 Hürthle cell lesions with indeterminate cytology and ThyroSeq® v2/v3 molecular testing results. Surgical follow-up was available for 33 cases. The majority of indeterminate Hürthle cell lesions had negative ThyroSeq® results (61%) and were benign on available surgical follow-up. The most prevalent mutations involved the RAS gene (21%), which were associated with benign lesions, non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), and malignancy. The remaining mutations involved less than 18% of the cases, including PAX8/PPARG (3.7%), TSHR (3.7%), EIF1AX (2.7%), MET (2.1%), PTEN (1.6%), clonal copy number alteration (1.6%), TERT (1.1%), and 0.5% each of GNAS, PIK3CA, and TP53 mutations. On follow-up, 45% were benign, 24% were NIFTP, and 30% were malignant. The malignant cases had different molecular alterations. No single molecular alteration defines cytologically indeterminate Hürthle cell lesions; the majority of cases have low-risk or no molecular alterations and are benign on follow-up. These findings suggest that molecular testing may be useful, but is not definitive, in determining which cases may be managed conservatively; additional studies are needed to fully determine the negative predictive value in ruling out malignancy.

Primary hyperoxaluria diagnosed after kidney transplantation failure: lesson from 3 case reports and literature review

BackgroundPrimary hyperoxaluria (PH) is a rare inborn disorder of the metabolism of glyoxylate, which causes the hallmark production oxalate and forms insoluble calcium oxalate crystals that accumulate in the kidney and other organs. Since the manifestation of PH varies from recurrent nephrolithiasis, nephrocalcinosis, and end-stage renal disease with age at onset of symptoms ranging from infancy to the sixth decade, the disease remains undiagnosed until after kidney transplantation in some cases.Case presentationHerein, we report 3 cases of PH diagnosed after kidney transplantation failure, providing the comprehensive clinical course, the ultrasonic image of renal graft and pathologic image of the biopsy, highlighting the relevance of biopsy findings and the results of molecular genetic testing. We also focus on the treatment and the unfavorable outcome of the patients. Meanwhile, we review the literature and show the additional 10 reported cases of PH diagnosed after kidney transplantation. Additionally, we discuss the progressive molecular understanding of the mechanisms involved in PH and molecular therapy.ConclusionsOverall, the necessity of preoperative screening of PH in all patients even with a minor history of nephrolithiasis and the importance of proper treatment are the lessons we learn from the 3 cases, which prompt us to avoid tragedies.

Novel pan-serotype control RNA for dengue virus typing through real-time reverse transcription-polymerase chain reaction

Dengue virus (DENV) is the causative agent of one of the most important febrile illnesses worldwide. Four DENV serotypes are responsible for a broad clinical spectrum of the disease. Positive controls are costly and required for the validation of molecular test results of DENV serotyping. In this study, we describe the in silico design of the qDENV-Control plasmid with the target sequences to oligonucleotides and probes widely used for DENV serotyping, and the subsequent production of qDENV Control RNA by T7-driven run-off in vitro transcription. The qDENV Control RNA was successfully used to validate the positive and negative DENV serotyping results, allowing its incorporation in routine in-house protocols for virologic surveillance. This Control RNA allowed the absolute quantification of viral RNA copies from unknown samples as required in several fundamental studies.

Impact of Rapid mecA Polymerase Chain Reaction Rapid Diagnostic Testing for Staphylococcus aureus in a Pediatric Setting

Background Rapid mecA testing, in combination with antimicrobial stewardship intervention, has improved outcomes in adults with Staphylococcus aureus (SA) bacteremia. In pediatric patients, data comparing rapid molecular testing to conventional susceptibility testing for methicillin resistance in SA are limited, and benefits have been observed only in combination with a coordinated real-time antimicrobial stewardship response. The objective of this study was to determine the impact of rapid mecA detection on time to optimal therapy in patients at a pediatric institution without a specific antimicrobial stewardship intervention. Methods This was a retrospective cohort study at a freestanding children’s hospital including patients with a blood culture positive for SA. Outcomes before and after the implementation of rapid mecA detection via use of GeneXpert MRSA/SA BC (Cepheid, Sunnyvale, CA) were compared. The primary outcome was time to optimal therapy, which was defined as vancomycin alone for methicillin-resistant SA or nafcillin, oxacillin, or cefazolin alone for methicillin-susceptible SA. Results One hundred one patients were included, with 61 patients in the preimplementation period receiving conventional susceptibility testing only and 40 patients in the postimplementation period receiving rapid molecular mecA testing. The median (interquartile range) time (hours) to optimal therapy from culture collection was decreased from 61.5 (47.8–68.1) preimplementation to 42.5 (21.9–56.6; P = 0.003) postimplementation. Conclusions Despite lack of a protocol for response to molecular mecA test results, time to optimal therapy and vancomycin use were significantly decreased. Future studies are needed to assess the impact on infection-related outcomes and antibiotic adverse effects.

Comparison of Serological and Molecular Tests for Diagnosis of Caprine Arthritis Encephalitis and Clinical Evaluation of Mammary Glands of Infected Dairy Goats

Background: Caprine Arthritis Encephalitis (CAE) is a disease that causes productive losses in dairy goat flocks due to the reduction in milk production, followed by lesions in joints and mammary glands. An early diagnosis is essential, considering that there is frequent occurrence of asymptomatic animals. Hence, this study aimed to perform a comparison of immunological and molecular based diagnostic tests, represented by Agar Gel Immunodiffusion (AGID), Western Blot (WB) and nested Polymerase Chain Reaction (nPCR). In addition, the mammary glands (MG) of dairy goats were clinically evaluated. Material, Methods &amp; Results: Blood collection and clinical examination were performed in 1191 dairy goats of 12 farms located in Northeastern and Southeastern regions of Brazil. Serological (AGID, WB) and molecular (nPCR) test results were compared and the data, along with MG alterations, were analyzed using Epi-info 7 and WinEpiscope 2.0. Seroprevalence in AGID test was 41.14% (490/1191). In WB, 51.47% (613/1191) of animals were seropositive and nPCR detected 69.44% (827/1191) positive animals. Hence, WB was more sensitive (P &lt; 0.001) than AGID. However, nPCR detected more positive animals than AGID (P &lt; 0.001) and WB (P &lt; 0.001). The analysis of mammary glands revealed that 105 out of 1096 nanny goats presented alterations, of which 101 presented altered consistency, 16 presented elevated temperatures and 60 had enlarged retromammary lymph nodes. There was significant statistic difference (P &lt; 0.05) only when comparing the results of serological tests with MG alterations.Discussion: In general, AGID technique is most frequently used when screening flocks for the disease due to the practicality and low cost this test presents. However, the results demonstrated that AGID detected the lowest number of positive animals. This low sensitivity that the test presented may be attributed to its antigen-antibody interaction mechanism, considering that agar gel precipitation requires multiple interactions. In addition, WB was more effective than AGID in detecting antibodies. On the other hand, nPCR was important for the detection of infected animals that serological tests failed to detect. The intermittence of immunological response observed in the serological tests may be explained by the variation of antibodies levels that may occur during life. Likewise, viral compartmentalization would justify the intermittent detection of proviral DNA. Hence, the results can be influenced by the viral intermittence, test sensitivity, late seroconversion and statistic values that can be calculated (sensitivity, specificity, positive predictive value, negative predictive level and kappa). Crossing the results of the diagnostic tests with the different mammary gland alterations, it was shown that there was a statistically significant difference (P &lt;0.05) only in the comparison of the results of the serological tests with GM alterations. Everything indicates that the humoral or cellular immune system being on stimulus is more propitious to find these changes. In conclusion, WB was more sensitive than AGID and, considering that nPCR can detect a larger number of animals infected with the goat lentivirus, it must be associated with a sensible serological test, such as Western Blot. In addition, infected animals have alterations in MG, which is more frequent in cases with positive serological results.