Expect the unexpected: endocarditis caused by Legionella feeleii.

We report a fatal case of Legionella feeleii endocarditis in a post-lung transplant patient. The diagnosis was delayed, as routine microbiological testing of nonrespiratory specimens does not account for extrapulmonary Legionella, and urine antigen testing only reliably detects Legionella pneumophila serogroup 1. This case also illustrates the utility of molecular sequencing for blood culture-negative endocarditis.

Reflex Human Immunodeficiency Virus (HIV) Type 1 RNA Testing Enables Timely Differentiation of False-Positive Results From Acute HIV Infection.

Accurate, timely human immunodeficiency virus (HIV) diagnosis is critical. Routine HIV screening program data were examined before and after reflex HIV type 1 RNA testing. Reflex testing facilitated confirmation of reactive HIV screening assays (as true or false positives) (odds ratio, 23.7 [95% confidence interval, 6.7-83.4]; P < .0001), improving detection of acute HIV and reducing unconfirmed discordant results.

Bifidobacteria metabolize lactulose to optimize gut metabolites and prevent systemic infection in patients with liver disease.

Progression of chronic liver disease is precipitated by hepatocyte loss, inflammation and fibrosis. This process results in the loss of critical hepatic functions, increasing morbidity and the risk of infection. Medical interventions that treat complications of hepatic failure, including antibiotic administration for systemic infections and lactulose treatment for hepatic encephalopathy, can impact gut microbiome composition and metabolite production. Here, using shotgun metagenomic sequencing and targeted metabolomic analyses on 847 faecal samples from 262 patients with acute or chronic liver disease, we demonstrate that patients hospitalized for liver disease have reduced microbiome diversity and a paucity of bioactive metabolites, including short-chain fatty acids and bile acid derivatives, that impact immune defences and epithelial barrier integrity. We find that patients treated with the orally administered but non-absorbable disaccharide lactulose have increased densities of intestinal bifidobacteria and reduced incidence of systemic infections and mortality. Bifidobacteria metabolize lactulose, produce high concentrations of acetate and acidify the gut lumen in humans and mice, which, in combination, can reduce the growth of antibiotic-resistant bacteria such as vancomycin-resistant Enterococcus faecium in vitro. Our studies suggest that lactulose and bifidobacteria serve as a synbiotic to reduce rates of infection in patients with severe liver disease.

Utility of the Signal-to-Cutoff Ratio and Antigen Index from Fourth- and Fifth-Generation HIV-1/HIV-2 Antigen/Antibody Assays for the Diagnosis of Acute HIV Infection: Applicability for Real-Time Use for Immediate Initiation of Antiretroviral Therapy.

Identification of individuals with acute HIV infection (AHI) and rapid initiation of antiretroviral therapy (ART) are priorities for HIV elimination efforts. Fourth- and fifth-generation HIV-1/HIV-2 antigen (Ag)/antibody (Ab) combination assays can quickly identify patients with AHI, but false-positive results can occur. Confirmatory nucleic acid amplification testing (NAAT) may not be rapidly available. We reviewed the data for 127 patients with positive fourth-generation ARCHITECT and fifth-generation Bio-Plex immunoassay results who had negative or indeterminate confirmatory Ab testing results, which yielded 38 patients with confirmed AHI and 89 patients with false-positive results. The receiver operating characteristic (ROC) curves showed excellent discriminatory power, with an area under the curve (AUC) for the signal-to-cutoff (S/CO) ratio of 0.970 (95% confidence interval [CI], 0.935 to 1.00) and an AUC for the Ag index (AI) of 0.968 (95% CI, 0.904 to 1.00). A threshold of 3.78 for the S/CO ratio would maximize the sensitivity (96.3%) and specificity (93.4%). The threshold for AI was 2.83 (sensitivity of 100% and specificity of 96.4%). The S/CO ratio was significantly correlated with the viral load (Spearman correlation coefficient, 0.486 [P = 0.014]), but the AI was not. The viral loads were all high, with a median of >2.8 million copies/mL. Two false-positive results with AI and S/CO ratio values markedly higher than the medians were observed, indicating that biological false-positive results can occur. Review of the S/CO ratio or AI may be used to improve the accuracy of AHI diagnosis prior to confirmatory NAAT results being available.

Severe COVID-19 infection is associated with aberrant cytokine production by infected lung epithelial cells rather than by systemic immune dysfunction.

The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Implementation of a Sample Pooling Strategy for the Direct Detection of SARS-CoV-2 by Real-Time Polymerase Chain Reaction During the COVID-19 Pandemic.

OBJECTIVES: To report our institutional experience in devising and implementing a pooling protocol and process for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) testing over a 3-month period in the fall of 2020. METHODS: The widespread testing implemented in the United States for detecting SARS-CoV-2 infection in response to the coronavirus disease 2019 pandemic has led to a significant shortage of testing supplies and therefore has become a major impediment to the public health response. To date, several institutions have implemented sample pooling, but publications documenting these experiences are sparse. Nasal and nasopharyngeal samples collected from low-positivity (<5%) areas were tested in pools of five on the Roche cobas 6800 analyzer system. Routine SARS-CoV-2 RT-PCR turnaround times between sample collection to result reporting were monitored and compared before and after sample pooling implementation. RESULTS: A total of 4,131 sample pools were tested over a 3-month period (during which 39,770 RT-PCR results were reported from the Roche system), allowing our laboratory to save 13,824 tests, equivalent to a conservation rate of 35%. A 48-hour or less turnaround time was generally maintained throughout the pooling period. CONCLUSIONS: Sample pooling offers a viable means to mitigate shortfalls of PCR testing supplies in the ongoing pandemic without significantly compromising overall turnaround times.

Universal screening for Clostridioides difficile at an urban academic medical center.

We implemented universal inpatient Clostridioides difficile screening at an 800-bed hospital. Over 3 years, 2,010 of 47,048 screening tests (4.2%) were positive, with significantly higher rates of C. difficile colonization on transplant units than medical-surgical units: 5.4% (152 of 2,801) versus 4.3% (880 of 20,564), respectively (P = .005). Compliance with screening ranged from 79% to 96%.

Cording in Disseminated Mycobacterium chelonae Infection in an Immunocompromised Patient.

Cording is a phenomenon in which acid fast bacilli grow in parallel and was previously used as a means of presumptive microscopic identification of Mycobacterium tuberculosis (TB). However, this process has been shown in multiple other nontuberculous mycobacterial (NTM) species. Here we present the case of an immunocompromised adult who presented with wrist pain, weight loss, and cough. A positron emission tomography scan showed uptake in the right ulna, multiple soft tissue sites, and the left lung. Biopsies and cultures were obtained from multiple sites, and the patient was ultimately diagnosed with disseminated Mycobacterium chelonae infection. The organism showed cording in culture. As seen in this patient, cording may occur in multiple NTM species and is not reliable as the sole indicator of the presence of TB.

Clinical Performance of the Point-of-Care cobas Liat for Detection of SARS-CoV-2 in 20 Minutes: a Multicenter Study.

Highly accurate testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the point of care (POC) is an unmet diagnostic need in emergency care and time-sensitive outpatient care settings. Reverse transcription-PCR (RT-PCR) technology is the gold standard for SARS-CoV-2 diagnostics. We performed a multisite U.S. study comparing the clinical performance of the first U.S. Food and Drug Administration (FDA)-authorized POC RT-PCR for detection of SARS-CoV-2 in 20 min, the cobas Liat SARS-CoV-2 and influenza A/B nucleic acid test, to the most widely used RT-PCR laboratory test, the cobas 68/8800 SARS-CoV-2 test. Clinical nasopharyngeal swab specimens from 444 patients with 357 evaluable specimens at five U.S. clinical laboratories were enrolled from 21 September 2020 to 23 October 2020. The overall agreement between the Liat and 68/8800 systems for SARS-CoV-2 diagnostics was 98.6% (352/357). Using Liat, positive percent agreement for SARS-CoV-2 was 100% (162/162) and the negative percent agreement was 97.4% (190/195). The Liat is an RT-PCR POC test that provides highly accurate SARS-CoV-2 results in 20 min with performance equivalent to that of high-throughput laboratory molecular testing. Rapid RT-PCR testing at the POC can enable more timely infection control and individual care decisions for coronavirus disease 2019.

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Clinical Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 Nucleic Acid Amplification Tests for Diagnosing Coronavirus Disease 2019.

Utilizing 34 348 severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) nucleic acid amplification test (NAAT) results from 2 health systems, we estimated the clinical sensitivity of a single SARS-CoV-2 NAAT. We found that SARS-CoV-2 NAAT has 82%-97% sensitivity for diagnosing coronavirus disease 2019 among symptomatic patients.

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies.

As the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 86 samples from SARS-CoV-2 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 76 tested negative and 10 tested positive for IgA (88.4% agreement, 95% CI: 79.9-93.6) while 84 tested negative and 2 tested positive for IgG (97.7% agreement, 95% CI: 91.9-99.6). Of 82 samples from SARS-CoV-2 PCR-positive patients, 14 tested negative and 68 tested positive for IgA (82.9% agreement, 95% CI: 73.4-89.5) while 27 tested negative and 55 tested positive for IgG (67.1% agreement, 95% CI: 56.3-76.3). Of samples collected ≥4 days after positive PCR, 38 of 42 (90.5% agreement, 95% CI: 77.9-96.2) were positive for IgA, and 42 of 42 (100% agreement, 95% CI: 91.6-100) were positive for IgG, respectively. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrated good sensitivity for detection of IgA and excellent sensitivity for detection of IgG antibodies from samples collected ≥4 days, after COVID-19 diagnosis by PCR. This assay demonstrated good specificity for IgA and excellent specificity for IgG and demonstrated only borderline cross reaction in 2 of the 28 samples from patients with common human coronaviruses infection, types NL63 and OC43.

Rapid pathogen identification and antimicrobial susceptibility testing in in vitro endophthalmitis with matrix assisted laser desorption-ionization Time-of-Flight Mass Spectrometry and VITEK 2 without prior culture.

PURPOSE: Prompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture. Similarly, automated antimicrobial susceptibility test systems (AST, VITEK 2) provide accurate antimicrobial susceptibility profiles. In this proof-of-concept study, we apply these technologies for the direct identification and characterization of pathogens in vitreous samples, without culture, as an in vitro model of infectious endophthalmitis. METHODS: Vitreous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus) and incubated at 37°C. Vitreous endophthalmitis samples were centrifuged and pellets were directly analyzed with MALDI-TOF MS and VITEK 2 without prior culture. S. aureus colonies that were conventionally grown on culture medium were used as control samples. Time-to-identification, minimum concentration of bacteria required for identification, and accuracy of results compared to standard methods were determined. RESULTS: MALDI-TOF MS achieved accurate pathogen identification from direct analysis of intraocular samples with confidence values of up to 99.9%. Time from sample processing to pathogen identification was <30 minutes. The minimum number of bacteria needed for positive identification was 7.889x106 colony forming units (cfu/µl). Direct analysis of intraocular samples with VITEK 2 gave AST profiles that were up to 94.4% identical to the positive control S. aureus analyzed per standard protocol. CONCLUSION: Our findings demonstrate that the direct analysis of vitreous samples with MALDI-TOF MS and VITEK 2 without prior culture could serve as new, improved methods for rapid, accurate pathogen identification and targeted treatment design in infectious endophthalmitis. In vivo models and standardized comparisons against other microbiological methods are needed to determine the value of direct analysis of intraocular samples from infectious endophthalmitis with MALDI-TOF MS and VITEK 2.

Comparison of 3 Nucleic Acid Amplification Tests and a Rapid Antigen Test with Culture for the Detection of Group A Streptococci from Throat Swabs.

BACKGROUND: Recently, the US Food and Drug Administration cleared 3 nucleic acid amplification test (NAAT) assays for detection of Streptococcus pyogenes [group A Streptococcus (GAS)] in pharyngeal specimens. However, there are limited studies evaluating the performance of these NAAT assays. METHODS: We compared the results of 3 NAATs (cobas Liat, Luminex Aries, and Cepheid Xpert Xpress) and a rapid antigen assay (Quidel QuickVue in-line strep A) with the accepted gold standard method, bacterial culture. RESULTS: Sixty-eight throat swab specimens collected between August and October 2017 were tested. Compared to bacterial culture, the sensitivities, specificities, positive predictive value, and negative predictive value for detecting GAS were as follows: cobas Liat: 100%, 97.4%, 96.7%, and 100%; Cepheid Xpert: 100%, 97.4%, 96.7%, and 100%; Luminex Aries: 95.2%, 100%, 100%, and 95.5%. The Quidel QuickVue in-line strep A assay showed poor sensitivity, detecting only 5.2% of culture-positive specimens. CONCLUSION: The 3 NAATs have high sensitivity when compared with bacterial culture for detection of GAS. With rapid turnaround time and ease of use, these tests can be considered as reliable point-of-care tests for the diagnosis of GAS, replacing the need for back-up culture.

Antimicrobial Stewardship Review of Automated Candidemia Alerts Using the Epic Stewardship Module Improves Bundle-of-Care Adherence.

BACKGROUND: Antimicrobial stewardship interventions utilizing real-time alerting through the electronic medical record enable timely implementation of the bundle of care (BOC) for patients with severe infections, such as candidemia. Automated alerting for candidemia using the Epic stewardship module has been in place since July 2015 at our medical center. We sought to assess the impact of these alerts. METHODS: All adult inpatients with candidemia between April 1, 2011, and March 31, 2012 (pre-intervention), and June 30, 2016, and July 1, 2017 (post-intervention), were evaluated for BOC adherence. We also evaluated the impact on timeliness to initiate targeted therapy, length of stay (LOS), and 30-day mortality. RESULTS: Eighty-four patients were included, 42 in the pre- and 42 in the post-intervention group. Adherence to BOC was significantly improved, from 48% (pre-intervention) to 83% (post-intervention; P = .001). The median time to initiation of therapy was 4.8 hours vs 3.3 hours (P = .58), the median LOS was 24 and 18 days (P = .28), and 30-day mortality was 19% and 26% (P = .60) in the pre- and post-intervention groups, respectively. CONCLUSIONS: Antimicrobial stewardship program review of automated alerts identifying patients with candidemia resulted in significantly improved BOC adherence and was associated with a 1.5-hour reduction in time to initiation of antifungal therapy. No significant change was observed with 30-day mortality or LOS.

Improved rates of antimicrobial stewardship interventions following implementation of the Epic antimicrobial stewardship module.

We evaluated the impact of the Epic antimicrobial stewardship module (EAM) on the number of interventions, antimicrobial usage, and clinical outcomes. Use of the EAM allowed us to significantly increase the number of ASP antimicrobial reviews and interventions while maintaining a sustained impact on antimicrobial utilization.

Misdiagnosis of Bordetella bronchiseptica Respiratory Infection as Bordetella pertussis by Multiplex Molecular Assay.

Multiplex polymerase chain reaction (PCR) tests are useful for the rapid detection of pathogens, though diagnostic challenges may arise. We report 2 immunocompromised patients with Bordetella bronchiseptica respiratory infection misdiagnosed as Bordetella pertussis using PCR, including discussion of transmission, diagnostic testing, clinical implications, and infection control considerations.

Use of the Accelerate Pheno System for Identification and Antimicrobial Susceptibility Testing of Pathogens in Positive Blood Cultures and Impact on Time to Results and Workflow.

The Accelerate Pheno system uses automated fluorescence in situ hybridization technology with morphokinetic cellular analysis to provide rapid species identification (ID) and antimicrobial susceptibility testing (AST) results for the most commonly identified organisms in bloodstream infections. The objective was to evaluate the accuracy and workflow of bacterial and yeast ID and bacterial AST using the Accelerate Pheno system in the clinical microbiology laboratory. The consecutive fresh blood cultures received in the laboratory were analyzed by the Accelerate Pheno system within 0 to 8 h of growth detection. ID/AST performance, the average times to results, and workflow were compared to those of the routine standard of care. Of the 232 blood cultures evaluated (223 monomicrobial and 9 polymicrobial) comprising 241 organisms, the overall sensitivity and specificity for the identification of organisms were 95.6% and 99.5%, respectively. For antimicrobial susceptibility, the overall essential agreement was 95.1% and categorical agreement was 95.5% compared to routine methods. There was one very major error and 3 major errors. The time to identification and the time to susceptibility using the Accelerate Pheno system were decreased by 23.47 and 41.86 h, respectively, compared to those for the standard of care. The reduction in hands on time was 25.5 min per culture. The Accelerate Pheno system provides rapid and accurate ID/AST results for most of the organisms found routinely in blood cultures. It is easy to use, reduces hands on time for ID/AST of common blood pathogens, and enables clinically actionable results to be released much earlier than with the current standard of care.