Performance of microbiological tests for tuberculosis diagnostic according to the type of respiratory specimen: A 10-year retrospective study.

Background: The microbial diagnosis of tuberculosis (TB) remains challenging and relies on multiple microbiological tests performed on different clinical specimens. Polymerase chain reactions (PCRs), introduced in the last decades has had a significant impact on the diagnosis of TB. However, questions remain about the use of PCRs in combination with conventional tests for TB, namely microscopy and culture. We aimed to determine the performance of microscopy, culture and PCR for the diagnosis of pulmonary tuberculosis according to the type of clinical specimen in order to improve the diagnostic yield and to avoid unnecessary, time and labor-intensive tests. Methods: We conducted a retrospective study (2008-2018) on analysis (34'429 specimens, 14'358 patients) performed in our diagnostic laboratory located in the Lausanne University Hospital to compare the performance of microbiological tests on sputum, induced sputum, bronchial aspirate and bronchoalveolar lavage (BAL). We analysed the performance using a classical "per specimen" approach and a "per patient" approach for paired specimens collected from the same patient. Results: The overall sensitivities of microscopy, PCR and culture were 0.523 (0.489, 0.557), 0.798 (0.755, 0.836) and 0.988 (0.978, 0.994) and the specificity were 0.994 (0.993, 0.995), 1 (0.999, 1) and 1 (1, 1). Microscopy displayed no significant differences in sensitivity according to the type of sample. The sensitivities of PCR for sputum, induced sputum, bronchial aspirate and BAL were, 0.821 (0.762, 0.871), 0.643 (0.480, 0.784), 0.837 (0.748, 0.904) and 0.759 (0.624, 0.865) respectively and the sensitivity of culture were, 0.993 (0.981, 0.998), 0.980 (0.931, 0.998), 0.965 (0.919, 0.988), and 1 (0.961, 1) respectively. Pairwise comparison of specimens collected from the same patient reported a significantly higher sensitivity of PCR on bronchial aspirate over BAL (p < 0.001) and sputum (p < 0.05) and a significantly higher sensitivity of culture on bronchial aspirate over BAL (p < 0.0001). Conclusions: PCR displayed a higher sensitivity and specificity than microscopy for all respiratory specimens, a rational for a smear-independent PCR-based approach to initiate tuberculosis microbial diagnostic. The diagnosis yield of bronchial aspirate was higher than BAL. Therefore, PCR should be systematically performed also on bronchial aspirates when available.

An Automated Dashboard to Improve Laboratory COVID-19 Diagnostics Management.

Background: In response to the COVID-19 pandemic, our microbial diagnostic laboratory located in a university hospital has implemented several distinct SARS-CoV-2 RT-PCR systems in a very short time. More than 148,000 tests have been performed over 12 months, which represents about 405 tests per day, with peaks to more than 1,500 tests per days during the second wave. This was only possible thanks to automation and digitalization, to allow high throughput, acceptable time to results and to maintain test reliability. An automated dashboard was developed to give access to Key Performance Indicators (KPIs) to improve laboratory operational management. Methods: RT-PCR data extraction of four respiratory viruses-SARS-CoV-2, influenza A and B and RSV-from our laboratory information system (LIS), was automated. This included age, gender, test result, RT-PCR instrument, sample type, reception time, requester, and hospitalization status etc. Important KPIs were identified and the visualization was achieved using an in-house dashboard based on the open-source language R (Shiny). Results: The dashboard is organized into three main parts. The "Filter" page presents all the KPIs, divided into five sections: (i) general and gender-related indicators, (ii) number of tests and positivity rate, (iii) cycle threshold and viral load, (iv) test durations, and (v) not valid results. Filtering allows to select a given period, a dedicated instrument, a given specimen, an age range or a requester. The "Comparison" page allows a custom charting of all the available variables, which represents more than 182 combination. The "Data" page, gives the user an access to the raw data in tables format, with possibility of filtering, allowing for a deeper analysis and data download. Informations are updated every 4 h. Conclusions: By giving a rapid access to a huge number of up-to-date information, represented using the most relevant visualization types, without the burden of timely data extraction and analysis, the dashboard represents a reliable and user-friendly tool for operational laboratory management. The dashboard represents a reliable and user-friendly tool improving the decision-making process, resource planning and quality management.

TL1A selectively enhances IL-12/IL-18-induced NK cell cytotoxicity against NK-resistant tumor targets.

INTRODUCTION: TL1A (TNFSF15) augments IFN-gamma production by IL-12/IL-18 responsive human T cells. Its ligand, death domain receptor 3 (DR3), is induced by activation on T and NK cells. Although IL-12/IL-18 induces DR3 expression on most NK cells, addition of TL1A minimally increases IFN-gamma production. METHODS: (51)Chromium release and flow cytometric analysis were used to determine whether the TL1A-DR3 pathway is implicated in tumor cell lysis. Our aim was to determine whether the TL1A-DR3 pathway is implicated in tumor cell lysis. RESULTS: TL1A had no additional effect on IL-12/IL-18-induced cytotoxicity against an NK-susceptible tumor (K562); however, it promoted cytotoxicity against NK-resistant targets susceptible to lysis only by activated NK cells. DISCUSSION: With IL-12/IL-18 activation, TL1A increased CD107a expression on NK cells which led to enhanced lysis of Daudi by PBMC and purified NK cells. To a lesser degree, TL1A increased lysis of colorectal adenocarcinoma epithelial derived lines (WiDr and SW837) by IL-12/IL-18-activated cells. CONCLUSION: TL1A increased cytotoxicity of IL-12/IL-18-activated NK cells against target cells dependent on NK activation for lysis and could function in vivo as a key co-activator of NK cytotoxicity.

Microbial induction of inflammatory bowel disease associated gene TL1A (TNFSF15) in antigen presenting cells.

TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease patients. Neutralizing anti-mouse TL1A Ab attenuates chronic colitis in two T-cell driven murine models, suggesting that TL1A is a central modulator of gut mucosal inflammation in inflammatory bowel disease. We showed previously that TL1A is induced by immune complexes via the Fc gamma R signaling pathway. In this study, we report that multiple bacteria, including gram negative organisms (E. coli, E. coli Nissle 1917, Salmonella typhimurium), gram positive organisms (Listeria monocytogenes, Staphylococcus epidermidis), partial anaerobes (Campylobacter jejuni), and obligate anaerobes (Bacteroides thetaiotaomicron, Bifidobacterium breve, Clostridium A4) activate TL1A expression in human APC, including monocytes and monocyte-derived DC. Bacterially induced TL1A mRNA expression correlates with the detection of TL1A protein levels. TL1A induced by bacteria is mediated in part by the TLR signaling pathway and inhibited by downstream blockade of p38 MAPK and NF-kappaB activation. Microbial induction of TL1A production by human APC potentiated CD4(+) T-cell effector function by augmenting IFN-gamma production. Our findings suggest a role for TL1A in pro-inflammatory APC-T cell interactions and implicate TL1A in host responses to enteric microorganisms.

The 3'-flap pocket of human flap endonuclease 1 is critical for substrate binding and catalysis.

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3'-extrahelical nucleotide (3'-flap) binding pocket. When presented with 5'-flap substrates having a 3'-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3'-flap on substrates reduced Km and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3'-flap, and the absence of a 3'-flap from a 5'-flap substrate was more detrimental to hFEN1 activity than removal of the 5'-flap or introduction of a hairpin into the 5'-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3'-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5'-phosphorylated product. Together the results indicate that the presence of a 3'-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.

Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways.

Flap endonuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, of which some are seemingly contradictory, is of considerable interest. To date, at least 20 proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a C-terminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the C-terminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.

Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments.

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

Coactivator-associated arginine methyltransferase-1 enhances nuclear factor-kappaB-mediated gene transcription through methylation of histone H3 at arginine 17.

Coactivator-associated arginine methyltransferase-1 (CARM1) is known to enhance transcriptional activation by nuclear receptors through interactions with the coactivators p160 and cAMP response element binding protein-binding protein (CBP) and methylation of histone H3 at arginine 17 (H3-R17). Here, we show that CARM1 can act as a coactivator for the transcription factor nuclear factor-kappaB (NF-kappaB) and enhance NF-kappaB activity in a CBP (p300)-dependent manner. This enhancement in 293T cells was abolished by cotransfection with a specific short hairpin RNA targeted to knockdown CARM1. Chromatin immunoprecipitation demonstrated CARM1 recruitment in vivo to the promoters of NF-kappaB p65-regulated genes along with CBP and steroid receptor coactivator-1. This was accompanied by an increase in histone H3-R17 methylation as well as H3-K9 and H3-K14 acetylation, and a decrease in H3-citrulline. Immunoprecipitation with anti-p65 antibody revealed that CARM1 physically interacts with NF-kappaB p65. Furthermore, we demonstrated the physiological significance by observing that similar events occurred when THP-1 monocytic cells were stimulated with TNF-alpha or with S100b, a ligand for the receptor of advanced glycation end products, both of which are associated with diabetic complications and also known inducers of NF-kappaB and inflammatory genes in monocytes. These results demonstrate that CARM1 participates in NF-kappaB-mediated transcription through H3-R17 methylation and support a nonnuclear receptor-associated function for CARM1. They also demonstrate for the first time that CARM1 occupancy, histone H3-R17 methylation, and citrullination are regulated at the promoters of inflammatory genes in monocytes, thereby suggesting a novel role for histone arginine modifications in inflammatory diseases.