444. Better Efficiency, Same Accuracy: Point-of-Care PCR for the Detection of Group A streptococcus in Noninvasive Skin Infections

Background Group A streptococcus (GAS) is a common cause of skin and soft-tissue infections (SSTIs). Current diagnostic techniques are culture-based and time intensive, requiring the prescription of empiric antibiotics before results are available. New detection tools are needed to hasten the diagnosis and appropriate treatment of SSTIs. The Cobas® Liat® System is a point of care (POC), real-time PCR system developed by Roche Molecular Diagnostics and is used in the United States and Europe to detect GAS from throat swabs within 15 minutes. We evaluated the feasibility and performance characteristics of POC for the detection of GAS in non-severe SSTIs. Methods Wound swabs collected from patients presenting to the Whiteriver Indian Health Service Hospital with non-severe SSTIs requiring only outpatient treatment were eligible for inclusion. Two swabs were collected: one swab was cultured on sheep’s blood agar, and the other swab was tested using POC. Compared with culture, we determined the sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) for POC to detect GAS in wound samples. We performed chart reviews 30-days from eligibility to assess the potential impact of POC systems on antibiotic use and healthcare utilization for SSTIs. Results To date, we have tested 100 (25%) of our target 400 samples (enrollment will be complete in August 2019). Of the 100 samples, 50 (50%) tested positive for GAS by POC, all of which were culture positive for GAS, 49 tested negative by POC (2 after a first invalid result), all of which tested culture negative for GAS (table), and 1 had an invalid POC result even after repeat testing (culture positive for MRSA only) and was excluded from further analysis. Among samples with a valid POC result, POC SN was 100%, SP was 100%, PPV was 100%, and NPV was 100%. The most common mono-infections were MRSA (22%), GAS (18%), and CoNS (6%). Among GAS cases, MSSA (32%) and MRSA (18%) co-infection was common. Conclusion POC PCR is highly sensitive and specific for the detection of GAS in non-severe SSTIs. To our knowledge, this is the first prospective study to use this technology for wound samples. POC PCR methods have the potential to accelerate identification of SSTI pathogens and improve antibiotic prescribing. Disclosures All authors: No reported disclosures.

LB21. The Seattle Flu Study: A Community-Based Study of Influenza

Background Influenza epidemics and pandemics cause significant morbidity and mortality. An effective response to a potential pandemic requires the infrastructure to rapidly detect and contain new and emerging flu strains at a population level. The objective of this study was to use data gathered simultaneously from community and hospital sites to develop a model of how flu enters and spreads in a population. Methods In the 2018–2019 season, we enrolled individuals with respiratory illness from community sites throughout the Seattle area, including homeless shelters, childcare facilities, Seattle-Tacoma International Airport, workplaces, college campuses, clinics, and at home (Figure 1). We collected data and nasal swabs from individuals with at least two respiratory symptoms. Additionally, we collected residual nasal swabs and data from individuals who sought care at four regional hospitals. Home-based self-testing for influenza and prediction models for influenza were piloted. Swabs were tested with a multiplex molecular assay, and influenza whole-genome sequencing was performed. Geospatial mapping and computational modeling platforms were developed to characterize regional spread of respiratory pathogens. Results A total of 18,847 samples were collected in the 2018–2019 season. Of those tested to date, 291/3,653 (8%) community and 2,393/11,273 (21%) hospital samples have influenza detected. Of the community enrollments, 39% had influenza-like illness. Community enrollees were in age groups not well-represented from hospitals. Influenza A/H3N2 activity peaked on college campuses and homeless shelters 2 weeks before the peak in hospitals. We observed multiple independent introductions of influenza strains into the city and evidence of sustained transmission chains within the city (Figures 2 and 3). Conclusion Utilizing the city-wide infrastructure we developed, we observed the introduction of influenza A/H3N2 into the community before the hospital and evidence of transmissions of unique strains into and within the Seattle area. These data provide the blueprint for implementing city-wide, community-based surveillance systems for rapid detection, real-time assessment of transmission patterns, and interruption of spread of seasonal or pandemic strains. Disclosures Helen Y. Chu, MD MPH, Merck (Advisor or Review Panel member), Michael Boeckh, MD PhD, Ablynx (Consultant, Grant/Research Support), Ansun Biopharma (Consultant, Grant/Research Support), Bavarian Nordic (Consultant), Gilead (Consultant, Grant/Research Support), GlaxoSmithKline (Consultant), Vir Bio (Consultant, Grant/Research Support), Janet A. Englund, MD, Chimerix (Grant/Research Support), GlaxoSmithKline (Grant/Research Support), MedImmune/Astrazeneca (Grant/Research Support), Meissa Vaccines (Consultant), Merck (Grant/Research Support),Novavax (Grant/Research Support), Sanofi Pastuer (Consultant), Matthew Thompson, MD, Alere Inc. (Research Grant or Support), Roche Molecular Diagnostics (Consultant, Research Grant or Support, Speaker’s Bureau), . Other Authors: No reported disclosures.

1730. Outcomes of Patients With Detectable Cytomegalovirus (CMV) DNA at Randomization in the Double-blind, Placebo-Controlled Phase 3 Trial of Letermovir (LET) Prophylaxis for CMV-Seropositive Allogeneic Hematopoietic-Cell Transplantation (HCT) Recipients

Background LET prophylaxis through HCT Week 14 was highly effective in preventing clinically significant CMV infection (CS-CMVi), had a good safety profile, and was associated with lower all-cause mortality by HCT Week 24 compared with placebo (PBO). Patients with detectable CMV DNA at randomization were excluded from the trial’s efficacy analyses (NCT02137772). Here we report the outcomes of these patients. Methods We compared patients randomized 2:1 and treated with LET or PBO who had detectable CMV DNA at randomization (n = 70) to those with undetectable CMV DNA (n = 495; primary efficacy population, PEP). CS-CMVi was defined as CMV viremia requiring antiviral preemptive therapy (PET) or CMV disease; patients with missing data were imputed as events. PET was prescribed blinded to study drug. We analyzed CS-CMVi incidence, CMV viral load (VL) kinetics, and mortality using post study vital status. Detectable, nonquantifiable CMV VL (<151 c/mL) was imputed as 150 c/mL. Results Of 70 patients with detectable CMV DNA at randomization (48 LET, 22 PBO), CMV VL was 150 c/mL in 63 patients (range, 150–716). All patients had undetectable CMV VL ≤5 days before randomization. Baseline characteristics were similar to the PEP, except for more patients with myeloablative conditioning (62.9% vs. 48.3%) and longer median days post-HCT to start of study drug (15 days vs. 8 days). Median study drug exposure was 70 days (range, 1–113) in LET group and 14 days (range, 7–99) in PBO group. By HCT Week 14, CS-CMVi occurred in 15 (31.3%) LET-treated patients and 17 (77.3%) PBO patients; CS-CMVi with imputed events were 22 (45.8%) in LET group and 20 (90.9%) in PBO group (difference –44.8%; 95% CI, –64.7% to –24.8%; P < 0.0001). Median CMV VL at time of PET was 413 c/mL (range, 150–31,847) and was similar between groups. Eight patients had quantifiable CMV VL (range, 171–1,728 c/mL) 1 week after starting study drug: 6 did not receive PET (5 LET [10.4%], 1 PBO [4.5%]). CMV VL was undetectable subsequently; other 2 withdrew from study. One (2.1%) LET-treated patient developed breakthrough CMV viremia with a UL56 C325W mutation. HCT Week 48 all-cause mortality was 26.5% in LET and 40.9% in PBO (figure). Conclusion LET prevented CS-CMVi compared with PBO among patients with detectable CMV DNA at randomization. Disclosures F. M. Marty, Merck: Consultant and Investigator, Consulting fee, Research support and Speaker honorarium. Astellas: Consultant and Investigator, Consulting fee and Research support. Chimerix: Consultant and Investigator, Consulting fee and Research support. Fate Therapeutics: Consultant, Consulting fee. GlaxoSmithKline: Consultant, Consulting fee. LFB: Consultant, Consulting fee. Roche Molecular Diagnostics: Consultant, Consulting fee. Shire: Consultant and Investigator, Consulting fee and Research support. Cidara: Investigator, Research support. Ansun: Investigator, Research support. Gilead: Investigator, Research support. WHISCON: Investigator, Research support. P. Ljungman, Merck: Investigator, Research support. AiCuris: Consultant, Consulting fee. Astellas: Investigator, Research support. Oxford Immunotec: Consultant and Investigator, Consulting fee and Research support. R. F. Chemaly, Merck: Consultant and Investigator, Consulting fee and Research support. Chimerix: Consultant and Investigator, Consulting fee and Research support. Astellas: Consultant, Consulting fee. Novartis: Investigator, Research support. Oxford Immunotec: Consultant, Consulting fee. H. Wan, Merck: Employee and Shareholder, Salary. V. L. Teal, Merck: Employee and Shareholder, Salary. J. Butterton, Merck: Employee and Shareholder, Salary. W. W. Yeh, Merck: Employee and Shareholder, Salary. R. Y. Leavitt, Merck: Employee and Shareholder, Salary. C. Badshah, Merck: Employee and Shareholder, Salary.

Detection of BRAF mutations in melanoma: Rate of mutation detection at codon 600 using Sanger sequencing as compared to the cobas 4800 method.

8596 Background: Detection of BRAF V600 mutations is currently a prerequisite for approved use of vemurafenib in patients with metastatic melanoma. The cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics), a PCR-based assay approved to aid in selecting patients for vemurafenib therapy, primarily detects V600E. It is also reported to detect V600K, which has been associated with vemurafenib response as well. We compared the mutation detection rate of the cobas assay with that of Sanger sequencing. Methods: 125 de-identified FFPE tissues submitted for BRAF mutation analysis that all showed histologically-confirmed melanoma were tested. BRAF mutations were detected using both the cobas kit and bidirectional Sanger sequencing using BigDye kits (Applied Biosystems). DNA was extracted from 5-um sections without macrodissection using the cobas DNA extraction kit (for the cobas test) or from 5-10-um sections using Agencourt extraction kits (Beckman Coulter) following macrodissection. Results: The two methods showed agreement in 104/125 (83.2%) of cases (Table). Sanger sequencing detected V600 dinucleotide mutations in 9 samples that were negative by the cobas assay. Sanger sequencing produced no results in 10 cases owing to suboptimal PCR, including 2 that were positive by the cobas assay. The cobas assay produced 2 invalid results, including 1 that was positive for V600E by Sanger.The cobas assay detected 7/11 V600K mutations. Conclusions: Overall agreement between cobas and Sanger sequencing was 83.2%. The Sanger method had higher analytic sensitivity, resulting in nine additional V600 mutations not called by cobas compared to the two seen by cobas but not Sanger sequencing. Thus, 16% (9/57) more patients would be identified as candidates for vemurafenib therapy using the Sanger method. [Table: see text]

High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR

The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies.The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and β-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67).An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed.The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.

Association between Chlamydia psittaci infection and extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT)-lymphomas

7568 Background: MALT-lymphomas are often associated with infectious organisms. Microbial species associated with MALT lymphomas are: Helicobacter pylori (H.pylori), Campylobacter jejuni, Borrelia burgdorferi, and Hepatitis C Virus. Recent studies showed evidence of Chlamydia psittaci (C. psittaci) infection in 80% of ocular adnexal lymphoma and tumor regression following eradication therapy. We studied the presence of C. trachomatis, C. pneumoniae, and C. psittaci DNAs in MALT lymphomas of various sites and in non-malignant controls. Methods: MALT lymphomas from the following sites were selected for analysis: salivary gland (n=13), thyroid gland (n=4), skin (n=2), ocular adnexa (n=2), stomach (n=6), intestinum (n=4). Samples of Sjoegren syndrome (n=10) and samples of H. pylori positive gastritis (n=7) were included as non-malignant controls. Macrodissected tissue containing >80% lymphoma cells from paraffin embedded tissue was processed for DNA isolation. For the presence of C. psittaci and C. pneumoniae DNA real time PCR assays were used. For C. trachomatis a commercially available PCR assay (Roche Molecular Diagnostics) was employed. All samples were tested twice. A sample was regarded as positive if at least one positive result was obtained. Results: C. psittaci was found at variable frequencies in MALT lymphomas of different sites: 4/13 (31%) salivary gland, 2/4 (50%) thyroid gland, 2/2 (100%) skin, 2/2 (100%) ocular adnexa, 1/6 (17%) gastric, and 0/4 intestinal MALT lymphomas. Among the non-malignant specimens, C. psittaci DNA was detected in 4/10 (40%) samples of Sjoegren syndrome and in 0/7 samples of H. pylori positive gastritis. All nongastric MALT lymphomas tested were negative for C. trachomatis and C. pneumoniae. Conclusion: Our data reveal a clear association of C. psittaci infection with extragastric MALT lymphoma. However, no evidence could be established for C. trachomatis and C. pneumoniae infection. Moreover, the high prevalence of Sjoegren syndromes - the most frequent precursor lesion for MALT lymphomas of salivary gland and ocular adnexa - may suggest an involvement of C. psittaci induced antigenic driven lymphomagenesis in a significant proportion of extragastric MALT lymphomas. No significant financial relationships to disclose.

Analytical Performance Evaluation of a Linear Array-Based β-Hemoglobinopathy Mutation Assay.

Routine clinical evaluation of β-globin phenotypes usually includes HPLC screening and alkaline/acid electrophoresis and/or isoelectric focusing. Although common structural variants are easily identified in the routine environment, specific β-thalassemia mutations are not and these can be the basis for prognosis and also for subsequent prenatal diagnosis for at-risk family members. Herein, we report evaluation of the performance of a linear array-based β- hemoglobinopathy mutation assay provided by Roche Molecular Diagnostics (Pleasanton, CA) that detects 6 β-globin variants (S, C, E, Knossos, D-Los Angeles and O-Arab) and 42 β-thalassemia mutations (90% predicted detection in any at-risk population group). The assay consists of one multiplex PCR with biotinylated primers, amplicon hybridization to a reverse line blot array, and colorimetric detection of a streptavidin-HRP conjugate. We tested 50 archived, abnormal and 92 sequential clinical samples, all previously tested in our routine assays. The results of the DNA and HPLC/electrophoresis were 100% consistent with the linear array, although Hb-S, C, D-Los Angeles and E were the only structural variants present in our collection. We verified the presence of Hb-S and excluded Hb-O-Arab in an ambiguous compound heterozygote for which HPLC and electrophoresis could not distinguish possible Hb-C-Harlem from Hb-O-Arab. Of 15 putative β-thalassemia minor samples, 10 had 6 different β-thalassemia mutations. Based on our experience, the daily throughput for a single technologist performing this mutation analysis is approximately 200 samples; it is, thus, an appropriate second-tier assay for newborn screening programs. The assay is robust, reliable and is also an excellent screen prior to sequencing for rare mutations.

Fully Automated Detection of Hepatitis C Virus RNA in Serum and Whole-Blood Samples

ABSTRACT In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.

Assessment of Use of the COBAS AMPLICOR System with BACTEC 12B Cultures for Rapid Detection of Frequently Identified Mycobacteria

The use of the COBAS AMPLICOR System (Roche Molecular Diagnostics, Basel, Switzerland), the only automated system for PCR testing, was evaluated for a rapid identification of mycobacteria with positive BACTEC 12B cultures. Two hundred ninety-six specimens with a growth index of ≥30 were analyzed for the presence of Mycobacterium tuberculosis complex, Mycobacterium avium, andMycobacterium intracellulare. Compared to traditional methods and provided that samples with PCR inhibition are retested at a 1:10 dilution, the sensitivity and specificity of the COBAS AMPLICOR System with BACTEC 12B cultures were 100 and 98%, respectively. The COBAS AMPLICOR method is rapid and reliable for identifying the most common mycobacteria in cultures.