Collaboration with Clinical Laboratory Positively Impacts Proper Test Utilization and Decreases Diagnostic Errors.

BACKGROUND: Diagnostic errors in clinical laboratory testing are extremely common and are major roadblocks in providing timely patient care. The purpose of this project was to investigate whether collaboration between the clinical laboratory, a diagnostic management team (DMT), and physicians who are ordering tests for a patient, resulted in improved test utilization by choosing wisely and better patient care in an academic medical center. METHODS: A retrospective study for a period of 24 months between 2017 and 2019 evaluated whether improvement of test ordering was achieved by timely interventions from the clinical laboratory and the coagulation DMT, resulting in fewer test selection errors. RESULTS: The results showed about 54% improvement in diagnostic errors for coagulation test selection in 634 patients evaluated for bleeding or thrombotic disorders by DMT when compared to previous studies. Furthermore, a total of approximately 2,400 coagulation test orders for patients that were done from July 2017 to July 2018 required intervention in 12% of the cases in the initial six months. When physician education was provided, intervention was needed in only approximately 4% of the cases, an improvement of 67% that was statistically significant at p-value < 0.05. Only 28% of the cases were associated with underutilization or failure to order required initial tests. The generated cost savings from prevention of over and underutilization of laboratory tests was in the order of ~ $16,000. CONCLUSIONS: The clinical laboratory and a DMT can function as an effective decision support system in decreasing errors in diagnostic test selection and facilitate knowledge among care providers regarding test results and interpretation, that may help in proper evidence-based guidelines and disease management.

Assessment of Platelet Function by Automated Light Transmission Aggregometry.

Light transmission aggregometry (LTA) has long been the historical "gold standard" of platelet function testing and is typically performed in specialized hemostasis laboratories due to its manual and labor intensive process. However, newer automated testing provides a means of standardization and ability to perform the testing in routine laboratories. Here we describe the measurement of platelet aggregation in the CS-Series™ (Sysmex Corporation, Kobe, Japan) and CN-Series™ (Sysmex Corporation, Kobe, Japan) routine blood coagulation analyzers. Differences in the methods for both analyzers are further described. For the CS-5100™ analyzer, the final diluted concentrations of the agonists are prepared by manual pipetting from reconstituted agonist solutions. These prepared dilutions are eight times concentrated with respect to the final working concentration of the agonists and appropriately diluted within the analyzer to achieve the desired concentration of agonists prior to testing. For the CN-6000™ analyzer, the dilutions of agonists and the final working concentrations are automatically prepared by the auto-dilution feature in the analyzer.

Is D-dimer Test Cost Effective and Time Sensitive for Diagnosis of Venous Thromboembolism in Emergency Department?

BACKGROUND: A D-dimer assay can be used to reduce unnecessary imaging when ruling out venous thromboembolism (VTE) in the Emergency Department (ED), thus potentially reducing patient visit times and costs. METHODS: This was a cross-sectional retrospective data analysis of an academic medical center ED visits between January 1 and June 30, 2019. ED visit length and VTE diagnostic cost were compared for visits with and without a D-dimer assay. The total sample size was 106 adult ED patients who were not at high risk of VTE and, of these, 27 encounters included D-dimer testing and 79 encounters did not. Outcomes were measured using independent samples t-tests to compare ED visit length and VTE diagnostic cost for ED visits with and without D-dimer tests. RESULTS: D-dimer testing had a moderate effect upon ED visit length, but it did not correspond to differences in ED visit length or VTE diagnostic cost. CONCLUSIONS: D-dimer testing was not statistically significant in improving the ED visit length or the VTE diagnostic cost compared to imaging studies for suspected VTE cases in the ED.

Overutilization and Underutilization of Thyroid Function Tests are Major Causes of Diagnostic Errors in Pregnant Women.

BACKGROUND: The failure to order the correct diagnostic test at the right time is one of the major contributing factors of diagnostic error. Excessive testing can lead to added economic burden and addressing underutilization is precarious as clinicians often fail to order the tests that would improve diagnosis, prognosis, and management. METHODS: A retrospective analysis of errors in test orders of thyroid function testing (TFT) in 321 pregnant women suspected of clinical and subclinical thyroid disorders was performed. Test selection was evaluated, and determinations were made about the extent of overutilization and underutilization of TFTs in reviewing each individual patient case by a Doctorate in Clinical Laboratory Science (DCLS) scholar. RESULTS: About 77% (247 cases) of the cases were found to have errors associated with test ordering for TFT. Of the cases reviewed, 18% cases were associated with overutilization, 53% of the cases were associated with underutilization, and 7% were associated with both (overutilization and underutilization). The annual cost burden because of ordering unnecessary tests was estimated to be approximately $13,000. The cost burden from errors resulting from not ordering a test would be of much greater magnitude but was difficult to estimate because underutilization has a ripple effect and may cause prolonged hospital stays, unnecessary medical bills, and delayed/ missed diagnosis leading to poor outcomes for patients. CONCLUSIONS: This study evaluated whether proper utilization of TFT were made at maternal health clinic locations of a large academic medical center in pregnant women to diagnose thyroid disorder and reported the issue of wastage of resources in the clinical laboratory. The study findings show significant errors in ordering of TFT for pregnant women in more than 75% of the cases that was based on evidence-based review of patient cases.

Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation.

Recent studies have shown that conjugation systems of Gram-negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we characterized the OMCC by focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMCpKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. The findings establish that OMCCs are highly structurally and functionally conserved - but also intrinsically conformationally flexible - scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function.

Mechanism of type-III protein secretion: Regulation of FlhA conformation by a functionally critical charged-residue cluster.

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well-conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.

An assessment of overutilization and underutilization of laboratory tests by expert physicians in the evaluation of patients for bleeding and thrombotic disorders in clinical context and in real time.

BACKGROUND: Diagnostic error is extremely common in the USA and likely around the world. A major reason for the diagnostic error is both the overutilization and the underutilization of laboratory tests. Using a panel of two to four experts in coagulation, test selection was reviewed in clinical context and in real time, and consensus determinations were made to derive conclusions about the extent of overutilization and underutilization. METHODS: Two hundred cases of patients being evaluated for bleeding or thrombotic issues were presented at each daily meeting of the diagnostic management team, and a review of each case for appropriate utilization of tests was completed. RESULTS: Two hundred randomly selected cases revealed 77.5% diagnostic errors (155 cases). Sixteen percent were associated with overutilization of laboratory tests, 44% were associated with underutilization, and 17.5% were associated with both. The annual cost burden estimated for overutilization alone in one institution of 450 beds was on the order of $20,000. The cost burden for the delay in diagnosis or the misdiagnosis in cases with underutilization is orders of magnitude greater ($200,000 or more), but it is impossible to determine the cost of a misdiagnosis in an individual case because it can produce many different clinical outcomes. CONCLUSIONS: This was a rare opportunity for experts in a given field to review cases in real time and in clinical context and provide immediately a consensus answer about test utilization. The results of this study show errors in test selection in nearly 75% of the cases evaluated.

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates.

UNLABELLED: Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. IMPORTANCE: For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here, we supply genetic and biochemical evidence that a helical bundle, designated the all-alpha domain (AAD), of T4SS receptors functions as a substrate specificity determinant. We show that AADs from two substrate receptors, Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC, bind DNA without sequence or strand preference but specifically bind the cognate relaxases responsible for nicking and piloting the transferred strand through the T4SS. We propose that interactions of receptor AADs with DNA-processing factors constitute a basis for selective coupling of mobile DNA elements with type IV secretion channels.

Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus infection.

UNLABELLED: The Middle East respiratory syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hypersensitize cells to infection when an S-cleaving protease(s) was present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans. IMPORTANCE: MERS is a frequently fatal disease that is caused by a zoonotic CoV. The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals and found that human, camel, and horse receptors sensitized cells to MERS-CoV infection more robustly than goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell surface lung protease greatly increases susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.

DNA substrate-induced activation of the Agrobacterium VirB/VirD4 type IV secretion system.

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.

Isolation of bacterial type IV machine subassemblies.

The bacterial type IV secretion systems (T4SSs) deliver DNA and protein substrates to bacterial and eukaryotic target cells generally by a mechanism requiring direct contact between donor and target cells. Recent advances in defining the architectures of T4SSs have been made through isolation of machine subassemblies for further biochemical and ultrastructural analysis. Here, we describe a protocol for isolation and characterization of VirB protein complexes from the paradigmatic VirB/VirD4 T4SS of Agrobacterium tumefaciens. This protocol can be adapted for isolation of T4SS subassemblies from other gram-negative bacteria as well as gram-positive bacteria. The biological importance of isolated T4SS subcomplexes can be assessed by assaying for copurification of trapped or cross-linked substrates. This can be achieved with a modified form of the chromatin immunoprecipitation (ChIP) assay termed transfer DNA immunoprecipitation (TrIP). Here, a TrIP protocol is described for recovery of formaldehyde-cross-linked DNA substrate-channel subunit complexes from cells employing T4SSs for conjugative DNA transfer.

Chemotaxis signaling protein CheY binds to the rotor protein FliN to control the direction of flagellar rotation in Escherichia coli.

The direction of rotation of the Escherichia coli flagellum is controlled by an assembly called the switch complex formed from multiple subunits of the proteins FliG, FliM, and FliN. Structurally, the switch complex corresponds to a drum-shaped feature at the bottom of the basal body, termed the C-ring. Stimulus-regulated reversals in flagellar motor rotation are the basis for directed movement such as chemotaxis. In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. In response to the chemotaxis signaling molecule phospho-CheY (CheY(P)), the motors can switch to clockwise (CW) rotation, causing dissociation of the filament bundle and reorientation of the cell. CheY(P) has previously been shown to bind to a conserved segment near the N terminus of FliM. Here, we show that this interaction serves to capture CheY(P) and that the switch to CW rotation involves the subsequent interaction of CheY(P) with FliN. FliN is located at the bottom of the C-ring, in close association with the C-terminal domain of FliM (FliM(C)), and the switch to CW rotation has been shown to involve relative movement of FliN and FliM(C). Using a recently developed structural model for the FliN/FliM(C) array, and the CheY(P)-binding site here identified on FliN, we propose a mechanism by which CheY(P) binding could induce the conformational switch to CW rotation.

Subunit organization and reversal-associated movements in the flagellar switch of Escherichia coli.

Bacterial flagella contain a rotor-mounted protein complex termed the switch complex that functions in flagellar assembly, rotation, and clockwise/counterclockwise direction control. In Escherichia coli and Salmonella, the switch complex contains the proteins FliG, FliM, and FliN and corresponds structurally with the C-ring in the flagellar basal body. Certain features of subunit organization in the switch complex have been deduced previously, but details of subunit organization in the lower part of the C-ring and the molecular movements responsible for motor switching remain unclear. In this study, we use cross-linking, binding, and mutational experiments to examine subunit organization in the bottom of the C-ring and to probe movements that occur upon switching. The results show that FliN tetramers alternate with FliM C-terminal domains to form the bottom of the C-ring in an arrangement that closely reproduces the major features observed in electron microscopic reconstructions. When motors were switched to clockwise rotation by a repellent stimulus, cross-link yields were altered in a pattern indicating relative movement of FliN and FliM(C). These results are discussed in the framework of a structurally grounded hypothesis for the switching mechanism.