Navigational Bronchoscopy versus Computed Tomography-guided Transthoracic Needle Biopsy for the Diagnosis of Indeterminate Lung Nodules: protocol and rationale for the VERITAS multicenter randomized trial.

Background: Lung nodule incidence is increasing. Many nodules require biopsy to discriminate between benign and malignant etiologies. The gold-standard for minimally invasive biopsy, computed tomography-guided transthoracic needle biopsy (CT-TTNB), has never been directly compared to navigational bronchoscopy, a modality which has recently seen rapid technological innovation and is associated with improving diagnostic yield and lower complication rate. Current estimates of the diagnostic utility of both modalities are based largely on non-comparative data with significant risk for selection, referral, and publication biases. Methods: The VERITAS trial (na V igation E ndoscopy to R each Indeterminate lung nodules versus T ransthoracic needle A spiration, a randomized controlled S tudy) is a multicenter, 1:1 randomized, parallel-group trial designed to ascertain whether electromagnetic navigational bronchoscopy with integrated digital tomosynthesis is noninferior to CT-TTNB for the diagnosis of peripheral lung nodules 10-30 mm in diameter with pre-test probability of malignancy of at least 10%. The primary endpoint is diagnostic accuracy through 12 months follow-up. Secondary endpoints include diagnostic yield, complication rate, procedure duration, need for additional invasive diagnostic procedures, and radiation exposure. Discussion: The results of this rigorously designed trial will provide high-quality data regarding the management of lung nodules, a common clinical entity which often represents the earliest and most treatable stage of lung cancer. Several design challenges are described. Notably, all nodules are centrally reviewed by an independent interventional pulmonology and radiology adjudication panel relying on pre-specified exclusions to ensure enrolled nodules are amenable to sampling by both modalities while simultaneously protecting against selection bias favoring either modality. Conservative diagnostic yield and accuracy definitions with pre-specified criteria for what non-malignant findings may be considered diagnostic were chosen to avoid inflation of estimates of diagnostic utility. Trial registration: ClinicalTrials.gov NCT04250194.

Use of the chinchilla model for nasopharyngeal colonization to study gene expression by Moraxella catarrhalis.

Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames (ORFs) encoding proteins that are involved in a truncated denitrification pathway or in the oxidative stress response, as well as several putative transcriptional regulators. Additionally, 200 M. catarrhalis genes were found to be downregulated when this bacterium was introduced into the nasopharynx. These downregulated genes included ORFs encoding several well-characterized M. catarrhalis surface proteins including Hag, McaP, and MchA1. Real-time reverse transcriptase PCR (RT-PCR) was utilized as a stringent control to validate the results of in vivo gene expression patterns as measured by DNA microarray analysis. Inactivation of one of the genes (MC ORF 1550) that was upregulated in vivo resulted in a decrease in the ability of M. catarrhalis to survive in the chinchilla nasopharynx over a 3-day period. This is the first evaluation of global transcriptome expression by M. catarrhalis cells in vivo.

Identification of gene products involved in the oxidative stress response of Moraxella catarrhalis.

Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990-3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by exogenous hydrogen peroxide (H2O2). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated in M. catarrhalis strain O35E, resulting in an increase in sensitivity to killing by H2O2 in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H2O2. DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identified M. catarrhalis genes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing of M. catarrhalis O35E katA and ahpC mutants for their abilities to scavenge exogenous H2O2 showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations into M. catarrhalis strain ETSU-4 showed that the growth of a ETSU-4 katA mutant was markedly inhibited by the addition of 50 mM H2O2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain.

Identification of a bacteriocin and its cognate immunity factor expressed by Moraxella catarrhalis.

BACKGROUND: Bacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing. RESULTS: Moraxella catarrhalis strain E22 containing plasmid pLQ510 was shown to inhibit the growth of M. catarrhalis strain O35E. Two genes (mcbA and mcbB) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (mcbC) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (mcbI) immediately downstream from mcbC encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the mcbI gene in M. catarrhalis O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the mcbA, mcbB, mcbC, and mcbI ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several M. catarrhalis strains including O12E. Inactivation of the mcbA, mcbB, or mcbC ORFs in M. catarrhalis O12E eliminated the ability of this strain to inhibit the growth of M. catarrhalis O35E. In co-culture experiments involving a M. catarrhalis strain containing the mcbABCI locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth. CONCLUSION: This is the first description of a bacteriocin and its cognate immunity factor produced by M. catarrhalis. The killing activity of the McbC protein raises the possibility that it might serve to lyse other M. catarrhalis strains that lack the mcbABCI locus, thereby making their DNA available for lateral gene transfer.

Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase.

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.