Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.

S100A12 Is Part of the Antimicrobial Network against Mycobacterium leprae in Human Macrophages.

Triggering antimicrobial mechanisms in macrophages infected with intracellular pathogens, such as mycobacteria, is critical to host defense against the infection. To uncover the unique and shared antimicrobial networks induced by the innate and adaptive immune systems, gene expression profiles generated by RNA sequencing (RNAseq) from human monocyte-derived macrophages (MDMs) activated with TLR2/1 ligand (TLR2/1L) or IFN-γ were analyzed. Weighed gene correlation network analysis identified modules of genes strongly correlated with TLR2/1L or IFN-γ that were linked by the "defense response" gene ontology term. The common TLR2/1L and IFN-γ inducible human macrophage host defense network contained 16 antimicrobial response genes, including S100A12, which was one of the most highly induced genes by TLR2/1L. There is limited information on the role of S100A12 in infectious disease, leading us to test the hypothesis that S100A12 contributes to host defense against mycobacterial infection in humans. We show that S100A12 is sufficient to directly kill Mycobacterium tuberculosis and Mycobacterium leprae. We also demonstrate that S100A12 is required for TLR2/1L and IFN-γ induced antimicrobial activity against M. leprae in infected macrophages. At the site of disease in leprosy, we found that S100A12 was more strongly expressed in skin lesions from tuberculoid leprosy (T-lep), the self-limiting form of the disease, compared to lepromatous leprosy (L-lep), the progressive form of the disease. These data suggest that S100A12 is part of an innate and adaptive inducible antimicrobial network that contributes to host defense against mycobacteria in infected macrophages.

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

Validation of the PyloPlus Urea Breath Test System in pediatric patients.

IMPORTANCE: For the diagnosis and post-treatment monitoring of H. pylori infection, non-invasive testing methodologies improve patient comfort, particularly for children. Previously, only the BreathTek UBT had FDA approval for use in pediatric patients and required an adjustment calculation based on age, height, and weight of the patient. The purpose of this study was to evaluate the performance of the PyloPlus UBT assay in a pediatric population.

What the pox? Review of poxviruses affecting humans.

The re-emergence of human mpox with the multi-country outbreak and a recent report of borealpox (previously Alaskapox) resulting in one death has heightened awareness of the significance of the Poxviridae family and their zoonotic potential. This review examines various poxviruses affecting humans, with discussion of less commonly encountered Poxviridae members, including pathogenesis, epidemiology, and diagnostic methods. Poxvirus treatment is beyond the intended scope of this review and will not be discussed.

Characterization of SARS-CoV-2 Distribution and Microbial Succession in a Clinical Microbiology Testing Facility during the SARS-CoV-2 Pandemic.

The exchange of microbes between humans and the built environment is a dynamic process that has significant impact on health. Most studies exploring the microbiome of the built environment have been predicated on improving our understanding of pathogen emergence, persistence, and transmission. Previous studies have demonstrated that SARS-CoV-2 presence significantly correlates with the proportional abundance of specific bacteria on surfaces in the built environment. However, in these studies, SARS-CoV-2 originated from infected patients. Here, we perform a similar assessment for a clinical microbiology lab while staff were handling SARS-CoV-2 infected samples. The goal of this study was to understand the distribution and dynamics of microbial population on various surfaces within different sections of a clinical microbiology lab during a short period of 2020 Coronavirus disease (COVID-19) pandemic. We sampled floors, benches, and sinks in 3 sections (bacteriology, molecular microbiology, and COVID) of an active clinical microbiology lab over a 3-month period. Although floor samples harbored SARS-CoV-2, it was rarely identified on other surfaces, and bacterial diversity was significantly greater on floors than sinks and benches. The floors were primarily colonized by bacteria common to natural environments (e.g., soils), and benchtops harbored a greater proportion of human-associated microbes, including Staphylococcus and Streptococcus. Finally, we show that the microbial composition of these surfaces did not change over time and remained stable. Despite finding viruses on the floors, no lab-acquired infections were reported during the study period, which suggests that lab safety protocols and sanitation practices were sufficient to prevent pathogen exposures. IMPORTANCE For decades, diagnostic clinical laboratories have been an integral part of the health care systems that perform diagnostic tests on patient's specimens in bulk on a regular basis. Understanding their microbiota should assist in designing and implementing disinfection, and cleaning regime in more effective way. To our knowledge, there is a lack of information on the composition and dynamics of microbiota in the clinical laboratory environments, and, through this study, we have tried to fill that gap. This study has wider implications as understanding the makeup of microbes on various surfaces within clinical laboratories could help identify any pathogenic bacterial taxa that could have colonized these surfaces, and might act as a potential source of laboratory-acquired infections. Mapping the microbial community within these built environments may also be critical in assessing the reliability of laboratory safety and sanitation practices to lower any potential risk of exposures to health care workers.

LXRα is uniquely required for maximal reverse cholesterol transport and atheroprotection in ApoE-deficient mice.

The liver X receptor (LXR) signaling pathway is an important modulator of atherosclerosis, but the relative importance of the two LXRs in atheroprotection is incompletely understood. We show here that LXRα, the dominant LXR isotype expressed in liver, plays a particularly important role in whole-body sterol homeostasis. In the context of the ApoE(-/-) background, deletion of LXRα, but not LXRß, led to prominent increases in atherosclerosis and peripheral cholesterol accumulation. However, combined loss of LXRα and LXRß on the ApoE(-/-) background led to an even more severe cholesterol accumulation phenotype compared to LXRα(-/-)ApoE(-/-) mice, indicating that LXRß does contribute to reverse cholesterol transport (RCT) but that this contribution is quantitatively less important than that of LXRα. Unexpectedly, macrophages did not appear to underlie the differential phenotype of LXRα(-/-)ApoE(-/-) and LXRß(-/-)ApoE(-/-) mice, as in vitro assays revealed no difference in the efficiency of cholesterol efflux from isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled macrophages revealed a marked defect in fecal sterol efflux in LXRα(-/-)ApoE(-/-) mice. Mechanistically, this defect was linked to a specific requirement for LXRα(-/-) in the expression of hepatic LXR target genes involved in sterol transport and metabolism. These studies reveal a previously unrecognized requirement for hepatic LXRα for optimal reverse cholesterol transport in mice.

Analysis of the FAST™ system for expedited identification and antimicrobial susceptibility testing of bloodborne pathogens.

The high morbidity and mortality of sepsis can be impacted by expediting identification (ID) and antibiotic susceptibility testing (AST) of causative bacteria. We evaluated the Qvella FAST™ System which creates a Liquid Colony™ (LC) from blood cultures that can be used to expedite results by 24 to 48 hours. We analyzed 289 LC samples and found that there were 17 (5.9%) that resulted in no ID. One hundred percent of the LC samples that produced an ID were concordant with SOC identification. Gram-positive bacteria showed a categorical agreement (CA) of 99.5%, with 3 minor errors (minE), and no major errors (majE) or very major errors (VME), and essential agreement (EA) of 98.9%. For Gram-negatives, the CA was 97.8% and the EA was 98.5% with 31 minE, 0 majE, and 2 VME. The FAST-System™ can accelerate ID and AST by 24 to 48 hours with potential positive impacts on time to effective therapy for sepsis.

Transience of interference in an immunoassay measuring serum levels of beta-D-glucan.

Some sera tested for 1-3-beta-D-glucan to identify invasive fungal infections exhibit interference. To assess interference transience, we evaluated results for 426 patients with an interference sample followed by a later sample. Interference was transient for 73% of patients (later sample negative or positive); median time between samples was 8 days.

Comparison of two nucleic acid amplification tests (NAATs) and two antigen tests for detection of SARS-CoV-2 from upper respiratory specimens.

There are numerous tests available for acute diagnosis of SARS-CoV-2, the virus that causes the disease COVID-19. These tests fall into two main groups: nucleic acid amplification tests (NAATs) and antigen-based assays. We evaluated the clinical performance of two rapid antigen assays (BD Veritor System for Rapid Detection of SARS CoV-2 and Abbott BinaxNOW COVID-19 Ag Card) and one NAAT (Hologic Aptima SARS CoV-2 Assay) by comparing them with the initial test of record, the Roche cobas SARS-CoV-2 assay; the antigen tests were also compared to Aptima. We tested remnant frozen specimens from patients suspected of SARS-CoV-2 infections (either due to symptoms or exposure) on the comparator platforms to evaluate assay performance across a wide range of positive results, including cobas cycle threshold (Ct) values ranging between 12 and 35. We tested 250 previous positive and 50 previous negative specimens and found 95.6% positive percent agreement (PPA) with the Aptima assay. The few discrepancies between the NAATs occurred only when Ct values were >32. Agreement was much lower for the rapid antigen tests, with 45.2%/47.3% PPA for the Veritor and 47.0%/47.0% PPA for the Binax compared to cobas/Aptima. Discrepancies occurred when cobas Ct values were >20 for Veritor and >25 for Binax. The negative percent agreement (NPA) was 100% for all assay comparisons. These data indicate similar performance between the cobas and Aptima NAATs but demonstrate that antigen-based assays may be insufficient to diagnose SARS-CoV-2 infection when lower levels of the virus are shed.

Clinical Whole Genome Sequencing for Clarithromycin and Amikacin Resistance Prediction and Subspecies Identification of Mycobacterium abscessus.

Mycobacterium abscessus infections are an emerging health care concern in patients with chronic pulmonary diseases, leading to high morbidity and mortality. One major challenge is resistance to clarithromycin, a cornerstone antibiotic with high efficacy. Therefore, treatment is primarily guided by phenotypic susceptibility results of clarithromycin, which requires extended incubation to assess for inducible resistance. Resistance mechanisms for clarithromycin include induction of erm(41) and mutations in the 23S rRNA gene (rrl). In addition, mutations in the 16S rRNA encoding gene (rrs) can confer high-level amikacin resistance, another essential drug in the treatment of M. abscessus infections. Herein, we developed a clinical whole genome sequencing (WGS) assay for clarithromycin resistance based on rrl and erm(41) gene sequences and amikacin resistance based on the rrs sequence in M. abscessus, as well as subspecies identification. Genotypic-based predictions were determined for 104 isolates from 68 patients. The overall accuracy of genotypic prediction for clarithromycin compared with phenotypic susceptibility results was 100% (95% CI, 96.45%-100%). For amikacin, we also obtained 100% accuracy (95% CI, 96.52%-100%). The high concordance between the genotypic and phenotypic results demonstrates that a WGS-based assay can be used in a clinical laboratory for determining resistance to clarithromycin and amikacin in M. abscessus isolates. WGS can also provide subspecies identification and high-definition phylogenetic information for more accurate M. abscessus strain typing.

Deceiving Phenotypic Susceptibility Results on a Klebsiella pneumoniae Blood Isolate Carrying Plasmid-Mediated AmpC Gene blaDHA-1.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) frequently causes hospital-acquired infections and is associated with high morbidity and mortality. CRKP can have multiple resistance mechanisms and only a few can be routinely detected by commercial molecular or phenotypic assays making surveillance for CRKP particularly challenging. In this report, we identified and characterized an unusual non-carbapenemase-producing CRKP carrying a rare plasmid-borne inducible AmpC gene, blaDHA-1 . The isolate was recovered from blood culture of a 67-year-old female presenting with sepsis post bladder surgery and ureteral stent removal. The primary isolate displayed an indeterminate susceptibility pattern for ceftriaxone by broth microdilution, but was susceptible by disk diffusion with one colony growing within the zone of inhibition. The ceftriaxone resistant colony was sub-cultured and had a minimum inhibitory concentration (MIC) of 2 ug/ml for imipenem (intermediate) and a zone size of 18 mm for ertapenem (resistant), but remained susceptible to cefepime and meropenem. Further phenotypic characterization of this sub-cultured isolate showed carbapenemase activity. Whole genome sequencing (WGS) revealed the presence of two subpopulations of a K. pneumoniae (MLST sequence type 11) from the primary blood culture isolate: one pan-susceptible to beta-lactams tested and the other resistant to the 3rd generation cephalosporins and ertapenem. WGS analysis identified the resistant K. pneumoniae harboring IncFIB(K) and IncR plasmids and the presence of plasmid-borne beta-lactam resistance genes blaOXA-1 and blaDHA-1, an inducible AmpC gene. Additional resistance genes against quinolones (aac(6')-Ib-cr, oqxA, oqB), aminoglycoside (aph(3')-Ia), sulfonamide (sul1), and tetracycline (tet(A)) were also identified. DHA-1 positive K. pneumoniae have been previously identified outside the US, particularly in Asia and Europe, but limited cases have been reported in the United States and may be underrecognized. Our study highlights the importance of using both extended phenotypic testing and WGS to identify emerging resistance mechanisms in clinical Enterobacterales isolates with unusual antimicrobial resistance patterns.

Utilization of whole genome sequencing for resolution of discrepant Mycobacterium tuberculosis drug susceptibility results: A case report.

A 44-year-old woman undergoing therapy for acute promyelocytic leukemia (APL) developed disseminated tuberculosis. Mycobacterium tuberculosis (TB) was isolated from the blood and sputum. Initial drug susceptibility testing (DST) of the blood isolate revealed resistance to isoniazid and ethambutol but the sputum isolate showed no resistance. Due to drug resistance concerns, the patient was treated with multiple second and third-line drugs, and suffered from drug side effects. To further investigate the DST discrepancies, whole genome sequencing (WGS) was performed on both isolates. No known resistance mutations to first line or second line drugs were identified in either isolate, which was confirmed by additional susceptibility testing performed by a different reference laboratory and the California Department of Public Health (CDPH) laboratory. Treatment was reduced to a simpler and less toxic regimen due to these investigations. WGS is shown to be a valuable tool for resolving discordant phenotypic DST results of TB isolates and has the potential to provide accurate and timely results guiding appropriate therapy in the clinical setting.

Validation, Implementation, and Clinical Utility of Whole Genome Sequence-Based Bacterial Identification in the Clinical Microbiology Laboratory.

The application of next-generation sequencing extends from microbial identification to epidemiologic insight and antimicrobial resistance prediction. Despite this potential, the roadblock for clinical laboratories lies in implementation and validation of such complex technology and data analysis. Herein, a validation study used whole-genome sequencing (WGS) for pan-bacterial identification (ID) in a clinical laboratory, and assessed its clinical relevance. A diverse set of 125 bacterial isolates, including a subset without genus (25) and/or species (10) ID, were analyzed by de novo assembly and reference genome mapping. The 16S rRNA, rpoB, and groEL genes were used for ID. Using WGS, 100% (89 of 89) and 89% (79 of 89) of isolates were identified at the genus and species-levels, respectively. WGS also provided improved results for 71% (25/35) isolates originally reported with genus-only or descriptive IDs. Chart review identified cases in which improved genus and/or species-level ID by WGS may have had a positive impact on patient care. Reasons included the use of an ineffective antibiotic owing to unclear ID, use of antibiotics when not clinically indicated, and help with an outbreak investigation. The implementation of next-generation sequencing in a clinical microbiology setting is a challenging but necessary task. This study provides a model for the validation and implementation of bacterial ID by WGS in such a setting.

Molecular Mirror Technology Facilitates High-Throughput, Accurate SARS-CoV-2 Testing.

Tests to diagnose acute SARS-CoV-2 infection are at the center of controlling the COVID-19 pandemic. Rapid tests benefit from providing quick results but suffer from lower sensitivity, while PCR tests usually take longer to provide more reliable results and can be difficult to scale to meet population needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA) using nucleic acid extraction and a nucleic acid extraction-free method to determine its ability to identify SARS-CoV-2 in nasal specimens from individuals suspected of having SARS-CoV-2. We compared the MMA using nucleic acid extraction to the emergency use authorization (EUA)-approved TaqPath reverse transcriptase PCR (RT-PCR) assay to determine its performance characteristics. From 412 total specimens (including 115 previous positives and 297 previous negatives), we found that the positive percent agreement (PPA) was 99.1% (confidence interval [CI], 97.4% to 100.0%) and the negative percent agreement (NPA) was 99.3% (95% CI, 98.4% to 100.0%) for SARS-CoV-2 detection. Using the extraction-free method, we analyzed 109 specimens (51 previous positives and 58 previous negatives) and found that the PPA for the more rapid version of the assay was 87.8% (95% CI, 78.5% to 96.9%) and the NPA was 100.0% (95% CI, 100.0%) for virus detection. The extraction method has performance comparable to what is observed in many PCR-based assays. The extraction-free method has lower PPA but has the advantage of being more rapid and having a higher throughput. Our data offer a proof of concept that nuclear magnetic resonance (NMR) detection can be used in SARS-CoV-2 diagnostic testing and may allow for alternative supply chains to increase testing options. IMPORTANCE Accurate diagnostics for SARS-CoV-2 infections have been critical for responding to the COVID-19 pandemic. Both high-sensitivity/specificity PCR-based tests and lower-sensitivity/specificity rapid antigen assays have been the subject of worldwide supply chain limitations as individual facilities and countries have struggled to meet their population testing needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA), which uses nuclear magnetic resonance to detect the presence of SARS-CoV-2 nucleic acids both with and without full nucleic acid extractions. We found that compared to a U.S. emergency use authorization (EUA) approved assay (TaqPath) that uses reverse transcriptase PCR (RT-PCR), the MMA had high PPA and NPA with full nucleic acid extractions, and acceptable positive percent agreement (PPA) and negative percent agreement (NPA) with an extraction-free protocol. In a landscape marred by supply chain shortages across the world, altered SARS-CoV-2 detection methods such as the MMA can add to testing supplies while providing quality SARS-CoV-2 testing results.

An Unusual Carbapenem Resistant Escherichia coli Carrying Plasmid-mediated AmpC and Mutated ompC in A Patient with Recurrent Urinary Tract Infections.

We describe a case of carbapenem resistant E. coli isolated from urine in an 87-year-old woman with recurrent urinary tract infections. Using whole genome sequencing (WGS), we identified the carbapenem resistance mechanism to be a combination of ompC porin loss and plasmid-mediated AmpC gene blaCMY-2 , which was not detected by routine molecular and phenotypic carbapenemase assays. Our case raises a concern for the limitation of current CRE screening tools for emerging resistance mechanisms and demonstrates the utility of WGS as a better tool for characterization of CRE in the clinical setting.

Conserved Oligomeric Golgi (COG) Complex Proteins Facilitate Orthopoxvirus Entry, Fusion and Spread.

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1-COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.

Amplicon-Based Next-Generation Sequencing for Detection of Fungi in Formalin-Fixed, Paraffin-Embedded Tissues: Correlation with Histopathology and Clinical Applications.

Invasive fungal infections are increasing in prevalence because of an expanding population of immunocompromised individuals. To reduce morbidity and mortality, it is critical to accurately identify fungal pathogens to guide treatment. Current methods rely on histopathology, fungal culture, and serology, which are often insufficient for diagnosis. Herein, we describe the use of a laboratory-developed internal transcribed spacer-targeted amplicon-based next-generation sequencing (NGS) assay for the identification of fungal etiology in fungal stain-positive formalin-fixed, paraffin-embedded tissues by using Illumina MiSeq. A total of 44 specimens from 35 patients were included in this study, with varying degrees of fungal burden from multiple anatomic sites. NGS identified 20 unique species across the 54 total organisms detected, including 40 molds, 10 yeasts, and 4 dimorphic fungi. The histopathologic morphology and the organisms suspected by surgical pathologist were compared with the organisms identified by NGS, with 100% (44/44) and 93.2% (41/44) concordance, respectively. In contrast, fungal culture only provided an identification in 27.3% (12/44) of specimens. We demonstrated that NGS is a powerful method for accurate and unbiased fungal identification in formalin-fixed, paraffin-embedded tissues. A retrospective evaluation of the clinical utility of the NGS results also suggests this technology can potentially improve both the speed and the accuracy of diagnosis for invasive fungal infections.