Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

Granulomas in Pediatric Liver Biopsies: Single Center Experience.

BACKGROUND: Granulomas in pediatric liver biopsies (GPLB) are rare with the largest pediatric cohort reported over 25 years ago. METHODS: Single-center retrospective study of GPLB. RESULTS: Seventeen liver biopsies from 16 patients with granulomas were identified (9 boys, 56%) with a median age of 13 years (range: 1-18) for which the most common indication was the presence of a nodule/mass (47%). Significant comorbidities were seen in 13 patients (81%) and included: liver transplant (25%), history of a neoplasm (25%), autoimmune hepatitis (6%), Crohn disease (6%), bipolar disorder (6%), severe combined immunodeficiency (6%), and sickle cell disease (6%). Eleven patients were taking multiple medications at the time of biopsy. Granulomas were more commonly pan-acinar (11 cases) followed by subcapsular (4 cases), portal (1 case), and periportal (1 case). Necrosis was seen in 10 cases (59%). GMS stain was positive in 2 cases for Histoplasma-like yeast; microbiological cultures were negative in all cases (no: 4). A 18S and 16S rRNA gene sequencing performed in 15 cases revealed only 1 with a pathogenic microorganism, Mycobacterium angelicum. CONCLUSION: In our experience, GPLB are heterogenous with only 3 cases having an identifiable infectious etiology and many of the remaining cases being associated to multiple medications, suggesting drug-induced liver injury as possible etiology.

Taxonomic Changes for Human Viruses, 2020 to 2022.

The classification of viruses remains relevant to several disciplines, including clinical virology. Since the original publication of this review in 2019, many known viruses have undergone taxonomic revisions, and several novel human and animal viruses have been described. Here, we provide an update to our previous reviews of taxonomic changes for disease-causing viruses of humans, covering changes that occurred between 2020 and 2022. As with previous editions, this update was informed by recent advances in virus taxonomy made by the International Committee on Taxonomy of Viruses; the changes and additions noted herein are not all-inclusive.

Microbiology of bile aspirates obtained at ERCP in patients with suspected acute cholangitis.

BACKGROUND: The cornerstone of treatment for acute cholangitis is source control with biliary drainage and early antibiotics. The primary aim of this study was to describe the microbiology of bile aspirate pathogens obtained at the time of endoscopic retrograde cholangiopancreatography (ERCP) in patients suspected of having acute cholangitis. METHODS: In this single-center retrospective study, patients were included if a bile aspirate was collected at ERCP for suspicion of acute cholangitis, from 1 January 2010 to 31 December 2016. RESULTS: There were 721 ERCP procedures for suspected acute cholangitis with bile culture results, with 662 positive bile cultures (91.8 %). Pathogens included: Enterococcus species (spp.) 448 (67.7 %); Klebsiella spp. 295 (44.6 %); Escherichia coli 269 (40.6 %); Pseudomonas spp. 52 (7.9 %); and anaerobes 64 (9.7 %). Susceptibility of Klebsiella pneumoniae and E.coli isolates to ciprofloxacin was 88 % and 64 %, respectively. Extended-spectrum beta-lactamases and carbapenem resistance were found in 7.9 % and 3.6 % of Enterobacteriaceae, respectively. There were 437 concurrent blood cultures, of which 174 were positive (39.8 % of cultures drawn). Prior biliary endoscopic sphincterotomy (ES) was evident in 459 ERCP cases (63.7 %), and was associated with increased frequency of Klebsiella spp., Pseudomonas aeruginosa, Enterobacter spp., and Enterococcus spp. Prior biliary ES significantly increased the probability of vancomycin-resistant Enterococcus (VRE). CONCLUSIONS: The vast majority of bile cultures (91.8 %) were positive. The susceptibilities of E.coli and K.pneumoniae to ciprofloxacin are lower than historically noted. A notable portion of cultures contained pathogenic drug-resistant organisms. Prior biliary ES is associated with a higher frequency of certain organisms and higher frequency of VRE.

Excessive Matrix Metalloproteinase-1 and Hyperactivation of Endothelial Cells Occurred in COVID-19 Patients and Were Associated With the Severity of COVID-19.

BACKGROUND: Systemic vascular injury occurs in coronavirus disease 2019 (COVID-19) patients; however, the underlying mechanisms remain unknown. METHODS: To clarify the role of inflammatory factors in COVID-19 vascular injury, we used a multiplex immunoassay to profile 65 inflammatory cytokines/chemokines/growth factors in plasma samples from 24 hospitalized (severe/critical) COVID-19 patients, 14 mild/moderate cases, and 13 healthy controls (HCs). RESULTS: COVID-19 patients had significantly higher plasma levels of 20 analytes than HCs. Surprisingly, only 1 cytokine, macrophage migration inhibitory factor (MIF), was among these altered analytes, while the rest were chemokines/growth factors. Additionally, only matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor A (VEGF-A) were significantly elevated in hospitalized COVID-19 patients when compared to mild/moderate cases. We further studied MMP-1 enzymatic activity and multiple endothelial cell (EC) activation markers (soluble forms of CD146, intercellular adhesion molecule 1 [ICAM-1], and vascular cell adhesion molecule 1 [VCAM-1]) and found that they were highly dysregulated in COVID-19 patients. CONCLUSIONS: COVID-19 patients have a unique inflammatory profile, and excessive MMP-1 and hyperactivation of ECs are associated with the severity of COVID-19.

Evaluation of the WASPLab Segregation Software To Automatically Analyze Urine Cultures Using Routine Blood and MacConkey Agars.

Automation of the clinical microbiology laboratory has become more prominent as laboratories face higher specimen volumes and understaffing and are becoming more consolidated. One recent advancement is the use of digital image analysis to rapidly distinguish between chromogenic growth for screening bacterial cultures. In this study, colony segregation software developed by Copan (Brescia, Italy) was evaluated to distinguish between significant growth and no growth of urine cultures plated onto standard blood and MacConkey agars. Specimens from 3 sites were processed on a WASP instrument (Copan) and incubated on the WASPLab platform (Copan), and plates were imaged at 0 and 24 hours postinoculation. Images were read by technologists following validated laboratory protocols (VLPs), and results were recorded in the laboratory information systems (LIS). Image analysis performed colony counts on the 24-hour images, and results were compared with the VLP. A total of 12,931 urine cultures were tested and analyzed with an overall sensitivity and specificity of 99.8% and 72.0%, respectively. After secondary review, 91.1% of manual-positive/automation-negative specimens were due to expert rules that reported the plate as contaminated or growing only normal flora and not due to threshold counts. Nine specimens were found to be manual-positive/automation-negative; a secondary review demonstrated that the results of 8 of these specimens were due to growth of microcolonies that were programmed to be ignored by the software and 1 were due to a colony count near the limit of significance. Overall, the image analysis software proved to be highly sensitive and can be utilized by laboratories to batch-review negative cultures to improve laboratory workflow.

Multicenter Evaluation of the QIAstat-Dx Respiratory Panel for Detection of Viruses and Bacteria in Nasopharyngeal Swab Specimens.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

Multicenter Evaluation of the BioFire FilmArray Pneumonia/Pneumonia Plus Panel for Detection and Quantification of Agents of Lower Respiratory Tract Infection.

The ability to provide timely identification of the causative agents of lower respiratory tract infections can promote better patient outcomes and support antimicrobial stewardship efforts. Current diagnostic testing options include culture, molecular testing, and antigen detection. These methods may require collection of various specimens, involve extensive sample treatment, and can suffer from low sensitivity and long turnaround times. This study assessed the performance of the BioFire FilmArray Pneumonia Panel (PN panel) and Pneumonia Plus Panel (PNplus panel), an FDA-cleared sample-to-answer assay that enables the detection of viruses, atypical bacteria, bacteria, and antimicrobial resistance marker genes from lower respiratory tract specimens (sputum and bronchoalveolar lavage [BAL] fluid). Semiquantitative results are also provided for the bacterial targets. This paper describes selected analytical and clinical studies that were conducted to evaluate performance of the panel for regulatory clearance. Prospectively collected respiratory specimens (846 BAL and 836 sputum specimens) evaluated with the PN panel were also tested by quantitative reference culture and molecular methods for comparison. The PN panel showed a sensitivity of 100% for 15/22 etiologic targets using BAL specimens and for 10/24 using sputum specimens. All other targets had sensitivities of ≥75% or were unable to be calculated due to low prevalence in the study population. Specificity for all targets was ≥87.2%, with many false-positive results compared to culture that were confirmed by alternative molecular methods. Appropriate adoption of this test could provide actionable diagnostic information that is anticipated to impact patient care and antimicrobial stewardship decisions.

Current Concepts in the Epidemiology, Diagnosis, and Management of Histoplasmosis Syndromes.

Histoplasmosis is a global disease endemic to regions of all six inhabited continents. The areas of highest endemicity lie within the Mississippi and Ohio River Valleys of North America and parts of Central and South America. As a result of climate change and anthropogenic land utilization, the conditions suitable for Histoplasma capsulatum are changing, leading to a corresponding change in epidemiology. The clinical manifestations of histoplasmosis are protean, variably resembling other common conditions such as community-acquired pneumonia, tuberculosis, sarcoidosis, Crohn's disease, or malignancy. Making a successful diagnosis is contingent on a thorough understanding of epidemiology, common clinical presentations, and best testing practices for histoplasmosis. While most subclinical or self-limited diseases do not require treatment in immunocompetent patients, all immunocompromised patients and those with progressive disseminated disease or chronic pulmonary disease should be treated. Liposomal amphotericin B is the preferred agent for severe or disseminated disease, while itraconazole is adequate for milder cases and "step-down" therapy following response to amphotericin B. In this review, we discuss the current evidence-based approaches to the epidemiology, diagnosis, and management of histoplasmosis.

Multicenter Clinical Evaluation of the Automated Aries Bordetella Assay.

Molecular methods offer superior sensitivity and specificity and reduce testing turnaround time from days to hours for detection of Bordetella pertussis and Bordetella parapertussis In this study, we evaluated the performance of the automated PCR-based Aries Bordetella Assay, which detects both B. pertussis and B. parapertussis directly from nasopharyngeal swab specimens. The limits of detection (LoDs) were 1,800 CFU·ml-1 for B. pertussis and 213 CFU·ml-1 for B. parapertussis The assay detected 16/18 unique B. pertussis/B. parapertussis strains. Of 71 potentially cross-reacting organisms, 5 generated false positives in 1/6 replicates; none of 6 additional Bordetella spp. were erroneously detected. Specimens were stable at 20 to 25°C for at least 10 h, at 4 to 8°C for 10 days, and at temperatures not exceeding -70°C for 6 months. Of 1,052 nasopharyngeal specimens from patients with suspected pertussis, 3.0% (n = 32) were B. pertussis positive and 0.2% (n = 2) were B. parapertussis positive. Combining these data with Aries Bordetella Assay data from 57 nasopharyngeal samples with previously confirmed B. pertussis or B. parapertussis data and with data from 50 contrived B. parapertussis samples, the proportions of positive and negative agreement of the respective Aries assays with the reference assays were 97.1% and 99.0% for B. pertussis and 100% and 99.7% for B. parapertussis The Aries Bordetella Assay provides accurate detection and distinction of B. pertussis and B. parapertussis infections within 2 h. (This study has been registered at ClinicalTrials.gov under registration no. NCT02862262.).

Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes.

A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 µg/ml. This concentration significantly reduced bacterial viability, while 40 µg/ml killed all cells of the A. baumannii ATCC 19606(T) and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606(T) and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-µg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606(T) or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media.

Francisella philomiragia Bacteremia in a Patient with Acute Respiratory Insufficiency and Acute-on-Chronic Kidney Disease.

Francisella philomiragia is a very uncommon pathogen of humans. Diseases caused by it are protean and have been reported largely in near-drowning victims and those with chronic granulomatous disease. We present a case of F. philomiragia pneumonia with peripheral edema and bacteremia in a renal transplant patient and review the diverse reports of F. philomiragia infections.

Comparison of the next-generation Xpert MRSA/SA BC assay and the GeneOhm StaphSR assay to routine culture for identification of Staphylococcus aureus and methicillin-resistant S. aureus in positive-blood-culture broths.

A bloodstream infection with Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a serious condition that carries a high mortality rate and is also associated with significant hospital costs. The rapid and accurate identification and differentiation of methicillin-susceptible S. aureus (MSSA) and MRSA directly from positive blood cultures has demonstrated benefits in both patient outcome and cost-of-care metrics. We compare the next-generation Xpert MRSA/SA BC (Xpert) assay to the GeneOhm StaphSR (GeneOhm) assay for the identification and detection of S. aureus and methicillin resistance in prospectively collected blood culture broths containing Gram-positive cocci. All results were compared to routine bacterial culture as the gold standard. Across 8 collection and test sites, the Xpert assay demonstrated a sensitivity of 99.6% (range, 96.4% to 100%) and a specificity of 99.5% (range, 98.0% to 100%) for identifying S. aureus, as well as a sensitivity of 98.1% (range, 87.5% to 100%) and a specificity of 99.6% (range, 98.3% to 100%) for identifying MRSA. In comparison, the GeneOhm assay demonstrated a sensitivity of 99.2% (range, 95.2% to 100%) and a specificity of 96.5% (range, 89.2% to 100%) for identifying S. aureus, as well as a sensitivity of 94.3% (range, 87.5% to 100%) and a specificity of 97.8% (range, 96.1% to 100%) for identifying MRSA. Five of six cultures falsely reported as negative for MRSA by the GeneOhm assay were correctly identified as positive by the Xpert assay, while one culture falsely reported as negative for MRSA by the Xpert assay was correctly reported as positive by the GeneOhm assay.

Cutaneous blastomycosis masquerading as pyoderma gangrenosum.

Cutaneous blastomycosis (CB) is associated with a variety of skin manifestations. Among other entities, CB may be mistaken for pyoderma gangrenosum due to overlap of findings on histopathologic examination. We report a case of CB, initially diagnosed as pyoderma gangrenosum and treated with steroids, leading to disseminated blastomycosis and acute respiratory distress syndrome (ARDS).

Utilizing confocal microscopy and topical natamycin in metarhizium keratitis of Caribbean origin.

A 32-year-old contact lens-wearing man with recent travel history to the Caribbean was referred for a corneal infiltrate in the left eye that worsened following 1-week of steroid-antibiotic therapy. Corneal cultures were obtained and sent to our facility's clinical microbiology laboratory for analysis. Same-day in vivo confocal microscopy revealed fungal elements. Nucleic acid sequencing performed on the isolated determined it to be a member of the entomopathogenic genus Metarhizium. Over the course of 3 months, the patient's corneal infiltrate ultimately resolved following topical natamycin 5 % therapy. This is the first reported case to have originated in the Caribbean and to utilize in vivo confocal microscopy to aid diagnosis. Our case also supports previous reports of success with natamycin therapy in treatment of Metarhizium sp. keratitis.

Candida auris outbreak at a tertiary care hospital during the COVID-19 pandemic.

BACKGROUND: Candida auris (C auris) is a fungal pathogen that has the potential for environmental persistence leading to outbreaks in health care settings. There has been a worldwide surge in C auris outbreaks during the COVID-19 pandemic. In this report, we describe an outbreak of C auris, its control, patient outcomes, and lessons learned. METHODS: The outbreak occurred in a 600-bed adult academic tertiary care hospital. Contact tracing was initiated immediately after identification of the index case and surveillance testing for C auris was obtained from patients who were exposed to the index case. Infection prevention measures were closely followed. RESULTS: A total of 560 cultures were performed on 453 unique patients between August 2021 and December 2021. Of those, 31 cultures (5.5%) were positive for C auris; 27 (87.1%) were colonized with C auris, while 4 patients developed a clinical infection (12.9%). The secondary attack rate was 6.8% (31/453). The 30-day all-cause mortality rate for all patients who tested positive for C auris was 9.7%. DISCUSSION: C auris can cause protracted outbreaks that result in colonization and invasive infections. Multidisciplinary work to improve adherence to infection prevention measures as well as targeted admission screening are essential to limit outbreaks.

Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.