BackgroundInvasive fungal infections (IFI) cause severe symptoms that affect immunocompromised and transplant patient populations. Antifungal therapies vary depending on the pathogenic species, and delays in diagnosis can lead to graft loss and an increase in morbidity and mortality. Therefore, rapid identification of fungi causing IFI is critical for informing antifungal therapy. Such actionable genus/species information can be obtained quickly via Next-generation Sequencing (NGS). In this study, an NGS assay was developed to identify fungal species responsible for IFI, allowing for selection of effective antifungal therapies.MethodsInternal transcribed spacer (ITS) regions 1 and 2 were used for fungal identification. Primers were taken from published research and/or designed/modified by assessment in fungal sequence alignments. A DNA sequence database was compiled and a reference-assisted assembly approach utilizing % sequence ID and % coverage was developed for species identification. End-point PCR was conducted on DNA extracted from 19 pathogenic fungal species, and mixed communities (MC) for preliminary sensitivity and inclusivity. Sensitivity was assessed using dilutions of template DNA into the PCR reaction.ResultsNGS data of 14 individual species and 4 MC passed quality control checks. Using only ITS1 and ITS2, species identification was expected for 10 of 14 individuals. We observed species identification in 9 individual samples, and 13 were identified within the top 5 results. All individuals were identified to genus. In MC analyses, combinations of 3, 4, 6, and 10 fungal species resolved 100% of the genera present, but failed to resolve species adequately with only 2 loci evaluated. Unexpectedly, 3 tested Aspergillus spp. were correctly identified with this limited data in both single and MC samples. The lower limit of detection was assessed at 5,000 genomic equivalents/mL of eluate.ConclusionThe inclusivity and sensitivity demonstrated here of an NGS approach for identification of etiological agents of IFI support this assay’s potential utility as an aid in the treatment of IFI in at-risk patient groups. This assay allows for rapid identification (<4 days) of fungal species to aid clinicians in improving case outcomes.Disclosures All authors: No reported disclosures.
Background Influenza (flu) infections affect a large subset of the population every year and have significant impacts on the health of patients, especially those with weak or compromised immune systems such as the elderly, children, cancer patients, and transplant recipients. Baloxavir marboxil was approved in October 2018 as a novel antiviral therapeutic for treating flu. During clinical trials, mutations were identified at the I28 codon of the polymerase acidic (PA) protein that greatly increased the resistance of a flu strain to this novel drug. In this study, a qPCR was developed and validated to identify these resistance mutations, allowing for guided therapy based on the resistance profile of the strain. Methods Flu A sequences (6,175) of the PA gene from the NCBI Influenza Virus Database collected over the last 5 years were compiled and aligned. Primers and probes were designed to target the I38 codon of the PA gene, and specific probes for each codon yielding a resistant amino acid mutation (I38T, -M, and -F) were designed. Locked nucleic acid (LNA) bases were used to increase the specificity of the probes. A combination of clinical flu specimens, laboratory strains, and synthetic constructs of each potential resistance mutation were used to validate the precision, sensitivity, and accuracy of the assay in nasopharyngeal swabs. Results Precision of the cycle threshold (Ct) values for each detector was determined to have a standard deviation of less than 3 for inter-assay and less than 2 for intra-assay replicates. Sensitivity was determined to be 800 copies/mL in nasopharyngeal swabs. Accuracy was found to be 92.3%. A single laboratory strain from the H1N1 2009 epidemic showed cross-reactivity with both wild-type and resistant probes, but no circulating clinical H1N1 samples tested showed this response. Conclusion The precision, sensitivity, and accuracy of a qPCR for resistance mutations to baloxavir marboxil support this assay’s utility as an aid in the treatment of flu in at-risk patient groups. This assay allows for rapid detection (<24 hours) of resistance markers to aid clinicians in improving flu case outcomes. Disclosures All authors: No reported disclosures.
BackgroundAntiviral resistance to human cytomegalovirus (CMV) is a growing concern for immunocompromised patients on prolonged antiviral regimens, and CMV remains the most clinically significant infection following allogeneic hematopoietic-cell transplantation. Letermovir targets subunit 2 of the viral terminase complex (UL56) and is approved for CMV prophylaxis in adult stem cell transplant recipients. Resistance to letermovir is conferred by point mutations in the UL56 gene, and with the potential clinical need for antiviral resistance testing, we have developed a UL56 sequencing assay covering 23 identified resistance mutations. Here we summarize the performance characteristics of the UL56 antiviral resistance assay.MethodsThis assay uses automated nucleic acid extraction followed by CMV UL56-specific polymerase chain reaction (PCR). PCR products are subjected to cycle sequencing and capillary electrophoresis, and the resulting sequences are analyzed for the The presence of known resistance mutations between codons 229 and 369 of the UL56 gene. The assay’s limit of detection (LOD), precision and accuracy were validated in accordance with accepted regulatory standards using multiple laboratory and clinical CMV strains.ResultsThe LOD was determined to be 99 IU/mL. Precision was demonstrated with multiple sample replicates over three days of testing, with 100% amino acid concordance within the region of interest (ROI). The assay also accurately identified 100% of amino acids within the ROI of 30 unique CMV-positive de-identified clinical samples. While some polymorphisms were detected, no mutations conferring resistance were identified in the clinical samples tested, which is in agreement with the literature indicating that naturally occurring polymorphisms in the UL56 gene have not been shown to confer resistance to letermovir.ConclusionThe CMV UL56 antiviral resistance assay was shown to be a rapid and sensitive means of detecting mutations conferring letermovir resistance. This expands current CMV antiviral resistance testing, which includes UL54 and UL97 sequencing, and provides physicians with the ability to monitor for the emergence of antiviral resistance mutations to all current FDA-approved anti-CMV drugs.Disclosures J. Grantham, Viracor Eurofins Clinical Diagnostics: Employee, Salary. J. Nutt, Viracor Eurofins Clinical Diagnostics: Employee, Salary. A. Tyler, Viracor Eurofins Clinical Diagnostics: Employee, Salary. E. Bixler, Viracor Eurofins Clinical Diagnostics: Employee, Salary. M. Altrich, Viracor Eurofins Clinical Diagnostics: Employee, Salary. S. Kleiboeker, Viracor Eurofins Clinical Diagnostics: Employee, Salary.
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