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Although commensal bacteria are crucial in maintaining immune homeostasis of the intestine, the role of commensal bacteria in immune responses at other mucosal surfaces remains less clear. Here, we show that commensal microbiota composition critically regulates the generation of virus-specific CD4 and CD8 T cells and antibody responses following respiratory influenza virus infection. By using various antibiotic treatments, we found that neomycin-sensitive bacteria are associated with the induction of productive immune responses in the lung. Local or distal injection of Toll-like receptor (TLR) ligands could rescue the immune impairment in the antibiotic-treated mice. Intact microbiota provided signals leading to the expression of mRNA for pro-IL-1β and pro-IL-18 at steady state. Following influenza virus infection, inflammasome activation led to migration of dendritic cells (DCs) from the lung to the draining lymph node and T-cell priming. Our results reveal the importance of commensal microbiota in regulating immunity in the respiratory mucosa through the proper activation of inflammasomes.

The global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown. We compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay. Of the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%). We found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.

Abstract Objectives Phone calls to the microbiology laboratory can be to clarify culture results and provide education, but those calls also interrupt laboratory workflow. We characterized calls that the laboratory received and developed targeted comments to educate providers. Methods Calls were logged and characterized, and we developed comments to address common call subjects. We applied the new comments to cultures and logged calls over the same interval the subsequent year. Data before and after implementation were analyzed. Results Call volume decreased from 496 calls to 419 calls after implementation. There was a significant difference in level of training among callers (P < .005), but the nature of the calls did not change. Laboratory response showed an increase in release of previously generated data (eg, suppressed susceptibility results). Comments specifically developed to address intrinsic antibiotic resistance and common susceptibility patterns did not decrease call volume. Conclusions Implementation of comments in the laboratory information system decreased call volume, but targeted comments were less effective than anticipated.

Abstract Objectives Despite extensive research on procalcitonin (PCT)-guided therapy in lower respiratory tract infections, the association between PCT and bacterial pneumonia remains unclear. Methods We evaluated retrospectively the performance of PCT in patients presenting with lower respiratory tract infection symptoms and grouped by seven diagnoses. All patients had microbial testing, chest imaging, and CBC counts within 1 day of PCT testing. Results Median PCT level in patients diagnosed with bacterial pneumonia was significantly higher than in patients diagnosed with other sources of infections or those not diagnosed with infections. Median PCT levels were not different among patients grouped by type or quantity of pathogen detected. They were significantly higher in patients with higher pathogenicity scores for isolated bacteria, those with abnormal WBC count, and those with chest imaging consistent with bacterial pneumonia. A diagnostic workup that included imaging, WBC count, and Gram stain had an area under the receiver operating characteristic curve of 0.748, and the addition of PCT increased it to 0.778. Conclusions PCT was higher in patients diagnosed with bacterial pneumonia. Less clear is its diagnostic ability to detect bacterial pneumonia over and above imaging and laboratory data routinely available to clinicians.

In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the peptide-loading complex (PLC). We provide evidence for an MHC class I/CRT intermediate before PLC engagement and examine the nature of that chaperone interaction in detail. To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC subcomplex, excluding the Transporter Associated with Antigen Processing, from purified, recombinant proteins. ERp57 disulfide linked to the class I-specific chaperone tapasin and CRT were the minimal PLC components required for MHC class I association and peptide loading. Mutations disrupting the interaction of CRT with ERp57 or the class I glycan completely eliminated PLC activity in vitro. By using the purified system, we also provide direct evidence for a role for UDP-glucose:glycoprotein glucosyltransferase 1 in MHC class I assembly. The recombinant Drosophila enzyme reglucosylated MHC class I molecules associated with suboptimal ligands and allowed PLC reengagement and high-affinity peptide exchange. Collectively, the data indicate that CRT in the PLC enhances weak tapasin/class I interactions in a manner that is glycan-dependent and regulated by UDP-glucose:glycoprotein glucosyltransferase 1.

[...]it will be important to ascertain the performance of the Karius test in different patient populations and through on-going validation studies. Depending on patient population and local testing practices, Pneumocystis jirovecii, Mycobacterium tuberculosis, Mycoplasma pneumoniae, human herpes viruses, Legionella longbeachae, and Borrelia hermsii may not be included in first- or second-line testing. Among nonculture based assays available, Karius is the first (and currently only) vendor offering plasma cfDNA mNGS for infectious disease diagnosis; however, mNGS analyses of cerebrospinal fluid and respiratory specimens and tissue-based targeted PCR and sequencing are also available. [...]we cannot ignore the cost of these assays.

Concerns persist regarding possible false-negative results that may compromise COVID-19 containment. Although obtaining a true false-negative rate is infeasible, using real-life observations, the data suggest a possible false-negative rate of ˜2.3%. Use of a sensitive, amplified RNA platform should reassure healthcare systems.

A novel electronic consult (e-consult) system for a pathology and laboratory medicine service (PLMS) was implemented in 2015 at a high-complexity Veterans Administration health care facility. Consults were previously made through direct provider communication without documentation in the medical record. To evaluate the utilization trends of the laboratory e-consult system at the Department of Veterans Affairs Connecticut facility during the first 2 years since inception. E-consultation involves pathology and laboratory medicine resident review followed by attending pathologist review and cosignature. E-consults to the pathology and laboratory medicine service from 2015 to 2017 were reviewed to record type of consult, requesting department, patient location, and turnaround time. The pathology and laboratory medicine service received 351 e-consults from 2015 to 2017. The volume varied by subsection: hematology and coagulation (215 of 351; 61%), chemistry (109 of 351; 31%), blood bank (19 of 351; 6%), and microbiology/virology (8 of 351; 2%). Hematology and coagulation consults were entirely for peripheral blood smear review (215 of 215; 100%). Chemistry consults were placed for toxicology/drugs of abuse (81 of 109; 74%), test utilization (17 of 109; 16%), or nontoxicology (11 of 109; 10%). Three services placed the majority of consults: primary care (279 of 351; 80%), hematology/oncology (39 of 351; 11%), and psychiatry (27 of 351; 8%). The median turnaround time for completion of e-consults was 1.2 days. Since e-consult implementation, the mean number of consults increased from 8.6/mo in 2015 to 18.1/mo in 2017, peaking in the last quarter of analysis in 2017 with a mean of 25.3 consults/mo. This novel e-consult system improved accessibility to and documentation of answers to laboratory questions and increased the visibility of the pathology and laboratory medicine service. Future goals include development of outcomes-based measures to better assess the clinical impact of e-consults.

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol‐oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide‐linked to ERp57 within the cell. Tapasin upregulation by interferon‐γ induces sequestration of the vast majority of ERp57 into the MHC class I peptide‐loading complex. The rate of tapasin–ERp57 conjugate formation is unaffected by the absence of β 2 ‐microglubulin (β 2 m), and is independent of calnexin or calreticulin interactions with monoglucosylated N‐linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N‐terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide‐bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide‐loading complex.

Background: Data in adults and older children demonstrate repeat blood cultures (BlCx) are not always necessary. Indications for repeat BlCx include Staphylococcus aureus or yeast in the initial blood culture, or the presence of a central venous catheter (CVC). Blood collection in premature babies can be challenging and there are little data regarding when repeat BlCxs are necessary after an initial positive. The goal of this study is to determine risk factors for persistent bloodstream infection (BSI) to determine when unnecessary blood cultures can be avoided. Methods: The Yale New Haven Children’s Hospital NNICU is a 68-bed level 4 unit. Babies in the NNICU with a positive blood culture from 8/1/16 to 12/31/21 were included. Persistent BSI was defined as a repeat positive BlCx with the same organism >48 hrs. after the original culture. A BlCx > 7 days after the original BlCx was considered a new event. Babies who died within 48 hrs. of the initial culture were excluded. In preliminary analysis we did not distinguish between true BSI and contamination. Data were extracted from the medical record by the Yale Data Analytics Team and by manual chart review. Data were stored in excel for descriptive statistics. Additional statistical analysis in SPSS is on-going to account for multiple variables. Results: 142 babies had a positive BlCx with 122 babies alive at 48 hrs. and included in the study. These 124 babies had 139 positive BlCx growing 145 organisms. Persistent BSI occurred in 17.3% (24/139) of BlCxs. Factors associated with persistence in univariate analyses included the presence of a CVC and recovery of S. aureus. (Table 1) No babies with either streptococcal infection or early onset sepsis had persistent BSI. (Table 1) Additional variables under evaluation in a multiple regression model to determine the probability of persistent BSI include other sources of infection, white blood cell count at the time of BlCx, congenital heart disease, immunosuppressive agents such as steroids and whether empiric antibiotic therapy was appropriate. We will also define probable contaminants and repeat the analyses with and without these BSI episodes. Conclusions: Preliminary analysis shows that neonates have similar risk factors for persistent BSI as adults including the presence of a CVC and the recovery of S. aureus that require repeat BlCx to confirm clearance. For babies with streptococcal infection, repeat BlCx may not be routinely required. Current work is examining additional potential risk factors in multi-variable models.