Diagnostic accuracy of a novel lateral flow assay for histoplasmosis.

Antigen testing is an important diagnostic tool for histoplasmosis but has limited availability globally. We evaluated the OIDx urine lateral flow antigen assay among 204 persons suspected to have histoplasmosis. Among patients with proven histoplasmosis, sensitivity was 33.3% (3/9, 95% CI 7.5%-70.1%) and specificity 80.5% (157/195, 95% CI 74.3%-85.8%). The MiraVista urine antigen test had better specificity (96.9%) and equal sensitivity. The OIDx test demonstrated 33.3% (3/9) positive agreement and 84.0% (163/194) negative agreement with the MiraVista test. These results should be considered in the context of our low HIV prevalence population with a mixture of pulmonary and disseminated disease.

We evaluated a new lateral flow antigen test for the diagnosis of histoplasmosis. Proven/probable cases were mostly pulmonary disease making antigen tests likely to be less sensitive in this population. The test had similar sensitivity to the established antigen test but was less specific.

Evaluation of Alinity m CMV assay performance for detecting CMV in plasma, cerebrospinal fluid, and bronchoalveolar lavage specimens.

Accurate detection and quantification of cytomegalovirus (CMV) is crucial to preventing adverse outcomes in immunocompromised individuals. Current assays were developed for use with plasma specimens, but CMV may be present in bronchoalveolar lavage (BAL) fluid and cerebrospinal fluid (CSF). We evaluated the performance of the Abbott Alinity m CMV assay compared to the Abbott RealTime CMV assay for quantification of CMV in plasma, BAL, and CSF specimens. To evaluate clinical performance, 190 plasma, 78 BAL, and 20 CSF specimens were tested with the Alinity m assay and compared to the RealTime assay. The Alinity m CMV assay showed high precision (SD <0.01 to 0.13) for all 3 specimen types. Clincal plasma and BAL specimens with quantifiable CMV DNA demonstrated strong correlation to RealTime CMV assay results (r2 = 0.9779 for plasma, r2 = 0.9373 for BAL). The Alinity m CMV assay may be useful for quantification of CMV in plasma, BAL, and CSF specimens.

Cefazolin as a predictor of urinary cephalosporin activity in indicated Enterobacterales.

Traditionally, cephalothin susceptibility results were used to predict the susceptibility of additional cephalosporins; however, in 2013-2014, the Clinical and Laboratory Standards Institute (CLSI) revisited this practice and determined that cefazolin is a more accurate proxy than cephalothin for uncomplicated urinary tract infections (uUTIs). Therefore, a cefazolin surrogacy breakpoint was established to predict the susceptibility of seven oral cephalosporins for Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in the context of uUTIs. Clinical microbiology laboratories face several operational challenges when implementing the cefazolin surrogacy breakpoint, which may lead to confusion for the best path forward. Here, we review the historical context and data behind the surrogacy breakpoints, review PK/PD profiles for oral cephalosporins, discuss challenges in deploying the breakpoint, and highlight the limited clinical outcome data in this space.

Working with the Electronic Health Record and Laboratory Information System to Maximize Ordering and Reporting of Molecular Microbiology Results.

Molecular microbiology assays have a higher cost of testing compared to traditional methods and need to be utilized appropriately. Results from these assays may also require interpretation and appropriate follow-up. Electronic tools available in the electronic health record and laboratory information system can be deployed both preanalytically and postanalytically to influence ordering behaviors and positively impact diagnostic stewardship. Next generation technologies, such as machine learning and artificial intelligence, have the potential to expand upon the capabilities currently available and warrant additional study and development but also require regulation around their use in health care.

Evaluation of the Diagnostic Accuracy and Clinical Utility of Fungal Profile Plus Polymerase Chain Reaction Assay in Pulmonary Infections.

Background: Pulmonary infections due to Aspergillus, Mucorales, and Nocardia have high morbidity and mortality, in part due to diagnostic challenges. Commercially available molecular assays on bronchoalveolar lavage fluid (BALF) may have increased sensitivity over currently available diagnostic options. Our aim was to characterize the diagnostic performance of assays for each of these pathogens in our patient population. Methods: The medical records of patients whose BALF was tested by polymerase chain reaction (PCR) for Aspergillus, Mucorales, and Nocardia between 2019 and 2021 were reviewed in a cross-sectional manner. European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) definitions of "proven," "probable," and "possible" infection were used, including histopathology, serology, and culture. We used (1) "proven" or "probable" infection by EORTC criteria, (2) improvement or stabilization on targeted antimicrobial therapy, and (3) absence of a more likely diagnosis as the reference standard. Results: The Aspergillus PCR assay demonstrated the highest agreement with the diagnostic reference standard, with 31.25% (10/32) sensitivity and 97.17% (206/212) specificity. Positive and negative predictive values were 62.50% (10/16) and 90.35% (206/228), respectively. No Mucorales or Nocardia infections were identified by the diagnostic reference standard, so the sensitivity could not be calculated. The specificity of Mucorales and Nocardia targets was 98.35% and 96.69%, respectively. Conclusions: Our data demonstrated relatively poor clinical sensitivity for all 3 constituent PCR assays in our patient population, suggesting a limited role for this test in the diagnosis of Aspergillus, Mucorales, or Nocardia.

Comparison of diagnostic performance of five molecular assays for detection of SARS-CoV-2.

We compared the performance of the Abbott Real Time SARS-CoV-2 assay (Abbott assay), Aptima™ SARS-CoV-2 assay (Aptima assay), BGI Real-Time SARS-CoV-2 assay (BGI assay), Lyra® SARS-CoV-2 assay (Lyra assay), and DiaSorin Simplexa™ COVID assay for SARS-CoV-2 detection. Residual nasopharyngeal samples (n = 201) submitted for routine SARS-CoV-2 testing by Simplexa assay during June-July 2020 and January 2021 were salvaged. Aliquots were tested on other assays and compared against the CDC 2019-nCoV Real-Time RT-PCR assay. Viral load in positive samples was determined by droplet digital PCR. Among 201 samples, 99 were positive and 102 were negative by the CDC assay. The Aptima and Abbott assays exhibited the highest positive percent agreement (PPA) at 98.9% while the BGI assay demonstrated the lowest PPA of 89.9% with 10 missed detections. Negative percent agreement for all 5 platforms was comparable, ranging from 96.1% to 100%. The performance of all five assays was comparable.

Activity of pulmonary vancomycin exposures versus planktonic and biofilm isolates of methicillin-resistant Staphylococcus aureus from cystic fibrosis sputum.

Vancomycin is commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections in patients with cystic fibrosis (CF) lung disease. However, there are limited data to support the in vitro activity of this agent against MRSA isolated from CF sputum. The primary objective of this study was to evaluate the activity of vancomycin at pulmonary concentrations (intravenous and inhaled) against four clinical MRSA CF sputum isolates in planktonic and biofilm time-kill (TK) experiments. Vancomycin minimum inhibitory concentrations (MICs) were determined for these isolates at standard inoculum (SI) (~106 CFU/mL) and high inoculum (HI) (~108 CFU/mL) as well as in biofilms cultivated using physiological medium representing the microenvironment of the CF lung. Vancomycin concentrations of 10, 25, 100 and 275 µg/mL were evaluated in TK experiments against planktonic MRSA at varying inocula and versus biofilm MRSA. Vancomycin MICs increased from 0.5 µg/mL when tested at SI to 8-16 µg/mL at HI. Vancomycin MICs were further increased to 16-32 µg/mL in biofilm studies. In TK experiments, vancomycin displayed bactericidal activity (≥3 log10 killing at 24 h) against 1/4 and 0/4 planktonic MRSA isolates at SI and HI, respectively, whereas vancomycin was bactericidal against 0/4 isolates against MRSA biofilms. Based on these findings, vancomycin monotherapy appears unlikely to eradicate MRSA from the respiratory tract of patients with CF, even at high concentrations similar to those observed with inhaled therapy. Novel vancomycin formulations with enhanced biofilm penetration or combination therapy with other potentially synergistic agents should be explored.

Comparison of two FDA-cleared EIA assays for the detection of Coccidioides antibodies against a composite clinical standard.

Coccidioidomycosis is a disease endemic to the southwestern United States, parts of Mexico, and Central and South America. Diagnosis of the disease is commonly delayed because of the lack of prompt testing and the dearth of reliable diagnostic tests. Culture and nucleic acid testing require a specimen, yet the typical patient presents with a dry cough and no sputum. Serologic methods depend on an effective antibody response by the patient, but antibody production may be unreliable or delayed until several weeks after initial symptom onset. Most published reports of serologic assays compare them to traditional serologic tests such as complement fixation and immunodiffusion. We sought to characterize the performance of two commercially available serologic tests, Meridian Premier and IMMY Omega, against a composite clinical reference standard to determine the sensitivity and specificity of these tests in detecting whether antibody is likely present in clinical specimens. The composite reference standard included symptoms, radiologic findings, and serologic results from complement fixation and immunodiffusion. For the Meridian test, sensitivity and specificity respectively were 69.4% and 95.4% for immunoglobulin G (Ig G) and 57.1% and 70.4% for immunoglobulin M (IgM). For the IMMY assay, sensitivity and specificity respectively were 53.1% and 96.7% for IgG and 34.7% and 85.5% for IgM.

Unilateral Phrenic Nerve Palsy in Infants with Congenital Zika Syndrome.

Since the first identification of neonatal microcephaly cases associated with congenital Zika virus infection in Brazil in 2015, a distinctive constellation of clinical features of congenital Zika syndrome has been described. Fetal brain disruption sequence is hypothesized to underlie the devastating effects of the virus on the central nervous system. However, little is known about the effects of congenital Zika virus infection on the peripheral nervous system. We describe a series of 4 cases of right unilateral diaphragmatic paralysis in infants with congenital Zika syndrome suggesting peripheral nervous system involvement and Zika virus as a unique congenital infectious cause of this finding. All the patients described also had arthrogryposis (including talipes equinovarus) and died from complications related to progressive respiratory failure.

Evaluation of a Commercial Multiplex Molecular Panel for Diagnosis of Infectious Meningitis and Encephalitis.

Rapid and accurate laboratory tests are important for the timely diagnosis and treatment of central nervous system infections. The FilmArray meningitis/encephalitis (ME) panel (BioFire Diagnostics, Salt Lake City, UT) is an FDA-cleared, multiplex molecular panel that allows the detection of 14 pathogens (bacterial [n = 6], viral [n = 7], and fungal [n = 1] pathogens) from cerebrospinal fluid (CSF). In this study, we evaluated the performance characteristics of the FilmArray ME panel using clinical, residual CSF samples (n = 291) that tested positive by a routine method(s) (e.g., bacterial culture, individual real-time PCR assay) for a pathogen represented on the ME panel. Of note, a subset (n = 76) of the CSF specimens was collected during the prevaccine era and had been characterized as positive for a bacterial pathogen. The FilmArray ME panel demonstrated an overall percent positive agreement (PPA) of 97.5% (78/80) for bacterial pathogens, 90.1% (145/161) for viruses, and 52% (26/50) for Cryptococcusneoformans/C. gattii Despite the low overall agreement (52%) between the ME panel and antigen testing for detection of C. neoformans/C. gattii, the percent positive agreement of the FilmArray assay for C. neoformans/C. gattii was 92.3% (12/13) when the results were compared directly to the results of routine fungal smear or culture. The FilmArray ME panel offers a rapid (∼60-min), syndrome-based approach for the detection of select meningitis and encephalitis pathogens.

Isolated cerebral mucormycosis caused by Rhizomucor pusillus.

A 61-year-old man with relapsing chronic lymphocytic leukaemia, status post allogeneic stem cell transplant and multiple chemotherapy regimens presented to the emergency room after suffering a grand mal seizure. His evaluation revealed a 1.5-2 cm ring-enhancing left temporal lobe brain lesion on the CT scan. This brain lesion was resected and the histopathology revealed an invasive fungal organism resembling mucormycosis. Amplification and sequencing of the 28S ribosomal RNA gene identified the organism as Rhizomucor pusillus The patient was treated with liposomal amphotericin B 5 mg/kg every 24 hours for 4 weeks, and then was transitioned to oral posaconazole. Serial brain imaging at 1 and 3 months, while on therapy, showed significant improvement.

Laboratory Diagnosis of Infective Endocarditis.

Infective endocarditis is life-threatening; identification of the underlying etiology informs optimized individual patient management. Changing epidemiology, advances in blood culture techniques, and new diagnostics guide the application of laboratory testing for diagnosis of endocarditis. Blood cultures remain the standard test for microbial diagnosis, with directed serological testing (i.e., Q fever serology, Bartonella serology) in culture-negative cases. Histopathology and molecular diagnostics (e.g., 16S rRNA gene PCR/sequencing, Tropheryma whipplei PCR) may be applied to resected valves to aid in diagnosis. Herein, we summarize recent knowledge in this area and propose a microbiologic and pathological algorithm for endocarditis diagnosis.

The role of multiplex molecular panels for the diagnosis of gastrointestinal infections in immunocompromised patients.

PURPOSE OF REVIEW: An increasing number of laboratories have implemented multiplex molecular panels for the diagnosis of gastrointestinal infections. This review focuses on recent data addressing the performance of US Food and Drug Administration-cleared multiplex gastrointestinal panels and discusses the advantages and limitations of these tests in the immunocompromised population. RECENT FINDINGS: Testing for gastrointestinal pathogens using multiplex molecular panels increases sensitivity and detection of coinfections compared with routine testing methods. Furthermore, multiplex panels reduce turnaround time and may allow for more informed decisions regarding treatment and infection control measures. However, the routine use of multiplex gastrointestinal panels has led to an increase in the detection of certain organisms, such as enteroaggregative Escherichia coli and sapovirus, which many clinical laboratories did not specifically test for in the past. This has created a degree of confusion on how to best interpret the results of multiplex panels, especially in the immunocompromised host. SUMMARY: Multiplex molecular panels provide a rapid and sensitive tool for the diagnosis of infectious diarrhea, and may allow for more timely decisions regarding the management of immunosuppressed patients. However, there are limitations associated with multiplex panels, including the interpretation of results and the cost associated with testing. Clinical microbiologists should work closely with clinicians to develop evidence-based algorithms to guide test utilization in this area.

RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction.

Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease.

Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs.

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ~20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an "expanded" rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.

Human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium.

Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.

Respiratory syncytial virus engineered to express the cystic fibrosis transmembrane conductance regulator corrects the bioelectric phenotype of human cystic fibrosis airway epithelium in vitro.

Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.