Molecular Diagnostic Results Research Articles

Intensivists\u2019 beliefs about rapid multiplex molecular diagnostic testing and its potential role in improving prescribing decisions and antimicrobial stewardship: a qualitative study

BackgroundRapid molecular diagnostic tests to investigate the microbial aetiology of pneumonias may improve treatment and antimicrobial stewardship in intensive care units (ICUs). Clinicians’ endorsement and uptake of these tests is crucial to maximise engagement; however, adoption may be impeded if users harbour unaddressed concerns or if device usage is incompatible with local practice. Accordingly, we strove to identify ICU clinicians’ beliefs about molecular diagnostic tests for pneumonias before implementation at the point-of-care.MethodsWe conducted semi-structured interviews with 35 critical care doctors working in four ICUs in the United Kingdom. A clinical vignette depicting a fictitious patient with signs of pneumonia was used to explore clinicians’ beliefs about the importance of molecular diagnostics and their concerns. Data were analysed thematically.ResultsClinicians’ beliefs about molecular tests could be grouped into two categories: perceived potential of molecular diagnostics to improve antibiotic prescribing (Molecular Diagnostic Necessity) and concerns about how the test results could be implemented into practice (Molecular Diagnostic Concerns). Molecular Diagnostic Necessity stemmed from beliefs that positive results would facilitate targeted antimicrobial therapy; that negative results would signal the absence of a pathogen, and consequently that having the molecular diagnostic results would bolster clinicians’ prescribing confidence. Molecular Diagnostic Concerns included unfamiliarity with the device’s capabilities, worry that it would detect non-pathogenic bacteria, uncertainty whether it would fail to detect pathogens, and discomfort with withholding antibiotics until receiving molecular test results.ConclusionsClinicians believed rapid molecular diagnostics for pneumonias were potentially important and were open to using them; however, they harboured concerns about the tests’ capabilities and integration into clinical practice. Implementation strategies should bolster users’ necessity beliefs while reducing their concerns; this can be accomplished by publicising the tests’ purpose and benefits, identifying and addressing clinicians’ misconceptions, establishing a trial period for first-hand familiarisation, and emphasising that, with a swift (e.g., 60–90 min) test, antibiotics can be started and refined after molecular diagnostic results become available.

Isolated- and Beckwith-Wiedemann syndrome related- lateralised overgrowth (hemihypertrophy): Clinical and molecular correlations in 94 individuals.

The congenital imprinting disorder, Beckwith-Wiedemann syndrome (BWS) is associated with variable clinical features including hemihypertrophy/lateralised overgrowth (LO) and embryonal tumour predisposition. BWS-associated (epi)genetic alterations occur in a subset of patients with isolated LO (ILO), leading to the concept of BWS spectrum disorder (BWSp). We investigated the relationship between clinical features and molecular diagnostic results in a cohort with LO using the BWSp international consensus group (BWSICG) clinical scoring system. Clinical/molecular findings in 94 previously-unreported patients with LO referred for BWSp molecular studies were reviewed retrospectively. The BWSICG score was assigned and diagnostic rate calculated. BWSp-associated (epi)genetic alteration was identified in 15/94 (16%). The molecular diagnostic rate by MS-MLPA (blood DNA) for BWS-related molecular findings in patients with LO was positively correlated with the BWSICG score. 3/48 with ILO had a molecular alteration. No individuals with ILO had developed an embryonal tumour at last follow up. Among a cohort of individuals with LO referred for BWSp molecular testing, the BWSICG score correlated with diagnostic yield. The embryonal tumour risk in children with ILO and negative molecular testing appeared very low, however longer- and more complete follow up is required to better define tumour risks in this group.

Efficiency of the novel quenching-probe PCR method to detect 23S rRNA mutations in children with Mycoplasma pneumoniae infection

An automated rapid molecular diagnostic kit (Smart Gene Myco) was recently developed for individual detection of Mycoplasma pneumoniae (MP) genes. This new testing approach requires no special equipment and skills and can be completed within 50 min. We prospectively evaluated this diagnostic kit, along with other conventional tests, for pneumonia diagnosis in children. Samples from 98 children (50 boys and 48 girls; aged 1–14 years; mean: 4.7 ± 2.1 years; median: 4 years) clinically diagnosed with pneumonia were tested for MP using real-time polymerase chain reaction (RT-PCR) as a reference method. Results from three molecular diagnostic tests, serum anti-MP antibodies, and MP culture were compared to RT-PCR data. Among the 98 children, 38 were positive for MP. All molecular diagnostic results showed complete concordance with the RT-PCR data. The sensitivity of the culture was 64%, whereas the sensitivities of the ImmunoCard Mycoplasma and SERODIA Myco II kits were lower (39% and 29%, respectively). Furthermore, a significant positive correlation was found between MP copy numbers and the culture test sensitivity (r = 0.95, p = 0.048). Macrolide-resistance mutations in the 23S ribosomal RNA gene were detected in 24 of 38 children using Smart Gene Myco based on quenching-probe PCR, which was confirmed by direct sequencing, revealing all mutations as A2063G. This is the first study to evaluate the clinical utility of the Smart Gene Myco kit, demonstrating that it is a fast and reliable method to support timely therapeutic decisions in children with MP pneumonia.

National Proficiency Testing of Molecular Diagnostics for Tuberculosis and Drug Resistance Detection - China, 2014-2019.

What is already known about this topic? Proficiency testing (PT) is a key component of quality assurance and is essential in ensuring accurate laboratory diagnosis of tuberculosis (TB) and drug-resistant TB. The National Tuberculosis Reference Laboratory (NTRL) developed a novel PT panel to test laboratories’ proficiency for molecular TB diagnostic assays throughout the TB laboratory network. A total of 6 PT rounds for molecular diagnostics were conducted by NTRL from 2014 to 2019. What is added by this report? PT conducted using artificial sputum specimens increased from 120 in the first round to 1,835 in the sixth round. Overall, laboratories demonstrated good proficiency for MTB and drug-resistance detection by molecular diagnostics, which is evident from the qualification rates over the six rounds: 95%, 97%, 96%, 93%, 93%, and 97%, respectively. What are the implications for public health practice? The use of artificial sputum specimens for PT panel production to test TB molecular diagnostics in China is feasible. Most of the participating laboratories provided reliable molecular diagnostic results for MTB and drug-resistance detection. The TB laboratory network can be instrumental in implementing PT expansion and improving the quality of TB molecular diagnosis in China.

Validity of whole genomes sequencing results in neoplasms in precision medicine

ObjectiveTo compare the whole genomes sequencing (WGS) results in the 100K Genomes project with the results of routine molecular diagnostics in precision medicine.Materials and methodsWe analysed 374 cancers including a...

Molecular detection of Leishmania infantum, Babesia vogeli, and Rangelia vitalii in dogs from the Embu-Guaçú municipality around the Guarapiranga Reservoir, São Paulo

Vector-borne diseases are of great importance worldwide in human and veterinary medicine, as several parasites can cause disease in multiple different hosts. In this work, we investigated the prevalence of some vector-borne parasitic diseases in dogs in Embu-Guacu, Sao Paulo, Brazil. Molecular diagnosis was performed by PCR using oligonucleotide markers specific for protozoans with significance in both animal and human health, such as Leishmania infantum , Rangelia vitalii , and Babesia vogeli . Molecular diagnostic results showed a high occurrence (16.25%, 13/80) of Babesia vogeli , but negative results for Leishmania infantum and Rangelia vitalii . Further studies should be conducted with a larger number of dogs, and vector control and educational measures should be implemented within the population to minimize the impact of vector-borne diseases on animal health.

Survey of protozoan vector-borne diseases in dogs from Atlantic Rainforest fragment around Billings Dam, São Paulo, Brazil

ABSTRACT: Vector-borne diseases are currently one of the biggest public health concerns worldwide. Dogs, being the closest companion animals to humans, are considered the main reservoir of some of these diseases in the urban environment. Therefore, the study of the disease behavior in dogs can help to understand the disease affecting human health. Serological and molecular diagnoses of Babesia vogeli, Rangelia vitalli, Leishmania infantum, and other trypanosomatids, were performed by immunochromatographic and PCR assays, respectively, on dogs in a dog shelter located in an Atlantic Forest fragment near the Billings Dam, São Bernardo do Campo, São Paulo-Brazil. Our molecular diagnostic results showed a high prevalence of Babesia vogeli, at 20.9% (17/81). No other protozoan was detected in any of the tests. Determining the prevalence of major vector-borne diseases is essential to establish preventive and control measures for zoonotic diseases in animals kept in shelters, in order to minimize the impact of vector-borne diseases on animal health.

2802. Occupational Exposure to the Ugandan Strain of Zika Virus in a Laboratory Worker in the United States: Clinical Presentation, Viral Persistence, and Antibody Response

BackgroundA laboratory worker suffered an accidental needle stick resulting in infection with the Ugandan strain (MR766) of Zika virus (ZIKV), a strain that has rarely been studied in humans. We report the clinical presentation and outcomes, molecular and serological diagnostic results, and immunological response. A 34-year-old Brazilian-born female laboratory researcher, presented with malaise, skin rash, myalgia and joint pain 10 days after an accidental needle stick while inoculating a mouse with ZIKV-MR766. On physical examination she had bilateral maculopapular rash on the cheeks, and tender effusions at the metacarpal and proximal interphalangeal joints and ankles. Symptoms and signs resolved within 3 weeks. ZIKV infection was confirmed by Nucleic Acid Amplification Test (Lab Corp®) in urine. Serological testing using the ZIKV IgM ELISA test from Lab Corp®, and a confirmatory plaque reduction neutralization test (PRNT) in accordance with the Centers for Disease Control and Prevention (CDC), results were negative.MethodsWhole blood, plasma, urine, saliva, and a vaginal swab were collected from day (D) 14 post exposure (PE) to D104 PE. A novel, antibody competition-based ZIKV diagnostic test (highly specific for ZIKV antibodies) was performed in serum, and detection of ZIKV-MR766 genomic RNA was performed in all body fluids longitudinally.ResultsAntibody response revealed broad IgM response to both ZIKV-Paraiba (strain from the 2015 outbreak) and ZIKV-MR766 during the acute phase of the infection, suggesting cross-reactivity. There was no cross-reactivity against dengue or yellow fever viruses. An IgG response was detected against both ZIKV strains and increased until D104 PE. ZIKV RNA was detected in whole blood, saliva, urine, and the vaginal swab at D14 PE. At D20 PE, virus was only detectable in whole blood at a value of less than 37 copies per mL. At D23 PE, there was no detectable virus. (figure).ConclusionThis case highlights the potential for ZIKV occupational exposure. Findings may be useful for the development of diagnostic tests against ZIKV as we were able to accurately determine time of exposure, presence of virus in body fluids, development of symptoms, and antibody responses after a well-documented infection. Disclosures All authors: No reported disclosures.

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.

Abstract 1676: A hybrid ontology and graph system enabling precision oncology applications through real-time incorporation of omics biomarkers into a scalable real-world data platform

Abstract Background: The ability to produce timely, clinically actionable recommendations is a cornerstone of a robust precision oncology program. This requires the assimilation of rapidly changing precision oncology data that flows asynchronously from various EMR and lab sources. A key challenge is harmonization of omics biomarkers from hospital-based and commercial testing labs, rendering these omics data interpretable and comparable between patients, labs and reports. To address this challenge, we describe an automated system leveraging a hybrid ontology and graph structure to permit the rapid harmonization and interpretation of molecular diagnostic results across multiple labs. Methods: A two-part, fully automated system to ingest and harmonize omics biomarker data from hospital-based and commercial testing labs was developed. First, an ontology-based omics lexicon provides annotation of discrete laboratory values provided electronically. The ontology model is Manchester language-based with data stored in a relational document store. Second, a graph structure serves as the knowledge model and relates the annotated lab values to 12 types of clinically informative biomarkers, including sequence and copy number variants, tumor mutational burden, MSI, gene rearrangements, wild type, and expression biomarkers. The graph structure leverages the ontology to fully describe hierarchical and categorical data associated with each biomarker. Access to the omics knowledge model uses a RESTful API and relational database architecture. Acquisition of molecular data uses custom JSON schemas and a RESTful API. Results: Feeds from 7 commercial and hospital-based labs resulted in the receipt of >12,000 molecular diagnostic reports containing >2 million individual biomarker observations, with integration of each report achieved within 2 minutes of electronic data receipt. As of Nov. 2018, 81.2% of reports had at least one biomarker observation, while the remaining reports lacked results. Within these biomarker observations, 95.8% were represented in the ontology/graph structure, resulting in successful linkage to ~22,000 distinct harmonized biomarkers out of more than 12 million in the knowledge base. Of the linked, harmonized variant biomarkers, 4% are class-level (e.g. BRAF mutation) with the remainder reflecting more specificity (e.g. BRAF V600E). Within the set of more specific biomarkers, 54% represent a protein altering variant biomarker. Conclusions: We have built an automated, scalable system using a novel ontology/graph structure to reliably and automatically harmonize and integrate omics data into a real-world oncology precision medicine platform. Proof of concept validation has been demonstrated in over 12,000 patient reports, with the results being used to enable clinical trials matching and outcomes research. Citation Format: James E. Shima, James L. Chen, Matthew J. Glick, Ryan A. Warrier, Eric C. Abruzzese, Walter C. Mankowski, Jonathan Hirsch. A hybrid ontology and graph system enabling precision oncology applications through real-time incorporation of omics biomarkers into a scalable real-world data platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1676.

Magnetic model MMTC-2.2 of ITER tokamak complex

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC) is a progressive myocardial disease that primarily affects the right ventricle. It develops predominantly at young age, and the first symptom is often sudden cardiac death (SCD) associated with malignant ventricular arrhythmia. Diagnosis using standard cardiac assessment may be hampered due to slight and nonspecific clinical signs at early stage of the disease, particularly in relatives of the patient. Molecular genetic testing can provide more information for clinical decision making. Here we report a patient with a clinical diagnosis of ARVC who was found to have a new frame shift mutation in the PKP2 gene through molecular genetic testing using Next Generation Sequencing methods. Subsequent family assessment showed that all three of the proband’s children also carried this mutation. The results of molecular diagnostics allowed us to assess the risk of developing ARVC and SCD in relatives of the proband, as well as to set up individual cardiac assessment protocols. Results obtained emphasize the importance of family screening when a pathogenic mutation is detected in the primary patient, and demonstrate the efficiency of genetic testing in cardiological practice.

OA 10.07 Genomic Profile of Cell-Free DNA from Sputum, Plasma, Urine and Tumor Tissue and Correlation with Clinical Effect in Advanced NSCLC

The detection of driver gene mutation based on tumor tissue can instruct target therapy and conduct molecular monitoring after drug-resistance in advanced NSCLC, but many patients have no access to this kind of test because of inadequate tumor tissue or inability to tolerate the invasive test. Some studies have explored the value of EGFR mutation test in body fluids such as plasma, urine and sputum from NSCLC patients. But the sensitivity based on individual liquid specimen is poor compared with gold standard---tissue. We detect multi-genes in multi-liquid samples in parallel to investigate the Consistency and complementarity of genetic profile in different liquid samples and it’s correlation with efficacy of the real world therapy in advanced NSCLC. The patients newly diagnosed with NSCLC and first-generation EGFR-TKI acquired drug-resistance were enrolled into our research (NCT:02778854) prospectively, the pre-treatment samples including tumor tissue, plasma, urine and sputum were collected. We conducted capture-based NGS (next generation sequencing) on all of these samples from 50 patients with a ctDNA panel covering significant exons and introns from 400 human genes including EGFR, KRAS, ALK, ROS1, c-MET and other important genes in the tumor related singling pathways such as PI3K-AKT-mTOR, JAK-STAT, Notch, Wnt and so on. Patients recruited in our experiment have been given unique treatment such as targeted treatment or chemotherapy according to the clinical examination. The final molecular diagnostic results of all clinical liquid or tissue specimen are supposed to be correlated with clinical response data. (Applied for Late-Breaking Abstract) This section is not applicable now because the sequencing and complex data analysis is in progress. Therefore, we will submit the final results as late-breaking abstract. Section not applicable. We expect to figure out the molecular diagnostic value of different body fluid compared with tumor tissue. we are able to analyze for correlation of the genomic profile derived from liquid samples and respective tissue results and clinical response of each patient.

Abstract 1784: Early feasibility and development of multiplexed, single-reaction assays for ALK, ROS1 and RET novel ddPCR RNA fusions

Abstract We have previously described a targeted genomic Laboratory Developed Test (LDT) that includes variant specific Droplet Digital™ PCR (ddPCR) testing for EGFR, KRAS and BRAF in plasma. This test supports the rapid delivery of molecular diagnostic test results, with >95% of tests results delivered in 72 hours of receipt in our Laboratory. This test then meets the key clinical need for the delivery of results that can result in faster treatment decisions. Additionally, the test may be of utility for those patients who need mutation results quickly or for whom tissue may be unavailable or insufficient for molecular testing. This is especially true for patients diagnosed with non-small cell lung cancer (NSCLC). In this report we will update on new test concepts created using the recently available design software engine for ddPCR assays. Specifically, we will describe studies on the development of single-reaction, multiplexed assays for the respective detection of ROS1 (up to 11 variants), RET (up to 8 variants) and EML4-ALK (v1 - v3). Design considerations, specificity and sensitivity, as well as reproducibility and robustness studies for these complex assays will be reviewed. Similar studies were conducted for the development of the commercially available test for the EML4-ALK fusion variants. EML4-ALK concordance studies compared the fusions found in blood with known positives and negatives found using FISH and PCR based methods (n=24 evaluable matched pair samples). Clinical sensitivity, specificity and concordance were 85%, 100% and 92% respectively. In this study we also report on test performance of the ALK RNA fusion test over 3 consecutive months of testing. Of note, we have delivered greater than 95% of tests (n = 272 samples) with an observed positive sample frequency of 2%. The robust detection of rare variant, RNA fusions from plasma within 72 hours represents a molecular testing option of value to patients with NSCLC and their physicians. Citation Format: Hestia Mellert, Kristin Alexander, Leisa Jackson, Galen Roda, Samantha Cooper, Dianna Marr, Stephen J. Jones, Nia Charrington, Gary Pestano. Early feasibility and development of multiplexed, single-reaction assays for ALK, ROS1 and RET novel ddPCR RNA fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2017-1784

Abstract 5681: Utility of a targeted NSCLC genomic test for cfDNA variants in the community setting

Abstract Time to treatment can have significant impact on progression of cancer, and treatment decisions rely heavily upon genomic and proteomic testing. However, multiple factors can delay the availability of molecular diagnostic test results, including the use of tissue-based testing. Almost one third of patients with non-small cell lung cancer (NSCLC) are either not candidates for biopsies, or have insufficient tissue samples from their initial biopsy. Additionally, most physicians in the community setting want to initiate treatment quickly but do not have access to on-site molecular testing at their practice. As a result, physicians may delay their decision to commence treatment with specific targeted therapies or treat before the mutation status is known. The goal of this study was to assess the utility of a blood-based cell-free (cf) nucleic acid Laboratory Developed Test (LDT) in the community setting. The tests utilize Droplet Digital™ PCR (ddPCR) technology to detect the EGFR sensitizing mutations L858R and exon 19 deletion (E746 - A750), the EGFR resistance mutation (T790M), the KRAS mutations G12C, G12V and G12D, and the BRAF V600E mutation. Patient samples for testing were collected and shipped at ambient room temperature using a Biodesix® whole blood sample collection kit and processed at the Biodesix laboratory. Metrics were reviewed from the real-time measurement of established molecular diagnostic markers in the plasma of patients with NSCLC. For this study we analyzed results from greater than 4,000 patient cases (~24,000 individual variants). Greater than 70% of test orders were received from physician practices that self-identified as community based. Test mutation status results were reported within 72 hours of sample receipt from the physician’s office (95%). The percentage of tests requested that were positive for each variant category were 9% for EGFR sensitizing, 9% for EGFR resistance, 11% for KRAS, and 1% for BRAF. Notably, we generated test results successfully for 98% of tests submitted. We have developed fast, highly robust and sensitive blood-based assays to expedite time to treatment and expand the laboratory testing options for patients with NSCLC. Citation Format: Westen Hahn, Scott Thurston, Leisa Jackson, Amanda Weaver, Cherie Tschida, Brent Sage, Kristin Alexander, Tunee Pelletier, Nia Charrington, Samantha Cooper, Dianna Marr, Paul M. Bowling, Hestia Mellert, Gary Pestano. Utility of a targeted NSCLC genomic test for cfDNA variants in the community setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5681. doi:10.1158/1538-7445.AM2017-5681

P2.03b-036 Analysis of Potentially Targetable Mutations in 821 Patients with Squamous cell Lung Cancer Undergoing Routine NGS-Based Molecular Diagnostics: Topic: Biomarkers

Molecular multiplex diagnostics is increasingly integrated now in routine diagnostics of lung adenocarcinoma (LAD). Although targetable aberrations are predominantly found in LAD, they have also been reported in squamous cell lung carcinoma (SQLC). We here present results of routine molecular multiplex diagnostics of advanced stage SQLC obtained within the German Network Genomic Medicine (NGM) and compare them with results reported previously in early stage SQLC in The Cancer Genome Atlas (TCGA) LUSC cohort. Tumor biopsies of 821 patients consecutively diagnosed within NGM were analyzed with next-generation parallel sequencing (NGS). The panel consisted of 102 amplicons and 14 genes: KRAS, PIK3CA, BRAF, EGFR, ERBB2, NRAS, DDR2, TP53, ALK, CTNNB1, MET, AKT1, PTEN and MAP2K1. In subsets of patients, fluorescence in-situ hybridization (FISH) was performed for amplification detection of FGFR1 and MET. We queried the TCGA dataset with respect to the panel used and compared the findings. For NGM patients, therapy and outcome are also reported. In addition to the expected frequencies of TP53, DDR2, PTEN and PIK3CA mutations, we detected EGFR mutations in 3.2% and BRAF mutations in 1.8%. Unlike the TCGA dataset, where the frequencies were 2.8% and 3.9%, respectively, the detected mutations in the NGM cohort included also activating targetable mutations (i.e., EGFR del19 and L858R, and BRAF V600E). FISH data revealed presence of MET amplification in 14.2% and of FGFR1 amplification in 20.0%. The association and correlation of these aberrations with clinical findings and prognosis as well as with PD-L1 expression status and mutational load will be presented. Our data give an overview on the presence and clinical characteristics of targetable mutations in advanced SQLC and show, that such mutations occur in a substantial amount of patients. Thus, molecular multiplex diagnostics might be indicated also in SQLC in order to use all therapeutic options available in these patients.

Occurrence of Pseudocowpox virus associated to Bovine viral diarrhea virus-1, Brazilian Amazon.

In 2011, an outbreak of severe vesicular disease occurred in the state of Pará, Amazon region. Besides proliferative or verrucous lesions, cattle showed atypical clinical signs such as diarrhea and leading to death. The animals were submitted to clinical, pathological and molecular diagnosis, and laboratory tests have confirmed the presence of Pseudocowpox virus (PCPV), a Parapoxvirus genus member, and have also found Bovine viral diarrhea virus-1 (BVDV-1), probably causing persistent infection. The results of molecular diagnostics, followed by sequencing data demonstrated the circulation of both viruses (PCPV and BVDV-1) in an area previously affected by another poxvirus, as Vaccinia virus.The cocirculation between PCPV and BVDV-1 indicates a major concern for animal health because the clinical presentation can be a severe disease. This is the first detection of PCPV in the Brazilian Amazon.

Rapid Detection of FKS-Associated Echinocandin Resistance in Candida glabrata.

A novel and highly accurate diagnostic assay platform was established for rapid identification of FKS mutations associated with echinocandin resistance in Candida glabrata The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay for FKS1 and FKS2 was developed to identify within 3 h the most common and clinically relevant resistance-associated mutations, including 8 FKS1 HS1 (wild type [WT], S629P, F625S, D632Y, D632E [T1896G], D632E [T1896A], I634V, and F625F) and 7 FKS2 HS1 (WT, F659del, F659S, F659V, F659L, S663P, and S663F) genotypes. A blinded panel of 188 C. glabrata clinical isolates was tested by both assays. The molecular diagnostic results from the dual assay were 100% concordant with data obtained from DNA sequencing. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify echinocandin-resistant C. glabrata and holds promise as a surrogate diagnostic method to better direct echinocandin therapy.

Pre-examination factors affecting molecular diagnostic test results and interpretation: A case-based approach

BackgroundMultiple organizations produce guidance documents that provide opportunities to harmonize quality practices for diagnostic testing. The International Organization for Standardization ISO 15189 standard addresses requirements for quality in management and technical aspects of the clinical laboratory. One technical aspect addresses the complexities of the pre-examination phase prior to diagnostic testing. MethodsThe Committee for Molecular Diagnostics of the International Federation for Clinical Chemistry and Laboratory Medicine (also known as, IFCC C-MD) conducted a survey of international molecular laboratories and determined ISO 15189 to be the most referenced guidance document. In this review, the IFCC C-MD provides case-based examples illustrating the value of select pre-examination processes as these processes relate to molecular diagnostic testing. Case-based examples in infectious disease, oncology, inherited disease and pharmacogenomics address the utility of: 1) providing information to patients and users, 2) designing requisition forms, 3) obtaining informed consent and 4) maintaining sample integrity prior to testing. ConclusionsThe pre-examination phase requires extensive and consistent communication between the laboratory, the healthcare provider and the end user. The clinical vignettes presented in this paper illustrate the value of applying select ISO 15189 recommendations for general laboratory to the more specialized area of Molecular Diagnostics.

Clinical presentation and outcome of riboflavin transporter deficiency: mini review after five years of experience.

IntroductionRiboflavin (vitamin B2) is absorbed in the small intestine by the human riboflavin transporters RFVT1 and RFVT3. A third riboflavin transporter (RFVT2) is expressed in the brain. In 2010 it was demonstrated that mutations in the riboflavin transporter genes SLC52A2 (coding for RFVT2) and SLC52A3 (coding for RFVT3) cause a neurodegenerative disorder formerly known as Brown-Vialetto-Van Laere (BVVL) syndrome, now renamed to riboflavin transporter deficiency. Five years after the diagnosis of the first patient we performed a review of the literature to study the presentation, treatment and outcome of patients with a molecularly confirmed diagnosis of a riboflavin transporter deficiency.MethodA search was performed in Medline, Pubmed using the search terms ‘Brown-Vialetto-Van Laere syndrome’ and ‘riboflavin transporter’ and articles were screened for case reports of patients with a molecular diagnosis of a riboflavin transporter deficiency.ResultsReports on a total of 70 patients with a molecular diagnosis of a RFVT2 or RTVT3 deficiency were retrieved. The riboflavin transporter deficiencies present with weakness, cranial nerve deficits including hearing loss, sensory symptoms including sensory ataxia, feeding difficulties and respiratory difficulties which are caused by a sensorimotor axonal neuropathy and cranial neuropathy. Biochemical abnormalities may be absent and the diagnosis can only be made or rejected by molecular analysis of all genes. Treatment with oral supplementation of riboflavin is lifesaving. Therefore, if a riboflavin transporter deficiency is suspected, treatment must be started immediately without first awaiting the results of molecular diagnostics.

NCCN Oncology Research Program's Investigator Steering Committee and NCCN Best Practices Committee Molecular Profiling Surveys.

With advances such as next-generation sequencing (NGS) increasing understanding of the basis of cancer and its response to treatment, NCCN believes it is important to understand how molecular profiling/diagnostic testing is being performed and used at NCCN Member Institutions and their community affiliates. The NCCN Oncology Research Program's Investigator Steering Committee and the NCCN Best Practices Committee gathered baseline information on the use of cancer-related molecular testing at NCCN Member Institutions and community members of the NCCN Affiliate Research Consortium through 2 separate surveys distributed in December 2013 and September 2014, respectively. A total of 24 NCCN Member Institutions and 8 affiliate sites provided quantitative and qualitative data. In the context of these surveys, "molecular profiling/diagnostics" was defined as a panel of at least 10 genes examined as a diagnostic DNA test in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Results indicated that molecular profiling/diagnostics are used at 100% of survey respondents' institutions to make patient care decisions. However, challenges relating to reimbursement, lack of data regarding actionable targets and targeted therapies, and access to drugs on or off clinical trials were cited as barriers to integration of molecular profiling into patient care. Frameworks for using molecular diagnostic results based on levels of evidence, alongside continued research into the predictive value of biomarkers and targeted therapies, are recommended to advance understanding of the role of genomic biomarkers. Greater evidence and consensus regarding the clinical and cost-effectiveness of molecular profiling may lead to broader insurance coverage and increased integration into patient care.