Diagnostic Accuracy of the Abbott SD Bioline MPT64 antigen test for identification of MTB Complex in a U.S. Clinical Mycobacteriology Laboratory.

Identification of the Mycobacterium tuberculosis complex (MTBC) from culture and differentiation from other non-tuberculous mycobacterial species is required for rapid diagnosis and accurate treatment. However, gaps exist in culture-based identification of acid-fast bacilli (AFB) positive cultures for rapid rule-out of MTBC in the United States. The SD Bioline™ MPT64 (Abbott Inc, South Korea) lateral flow assay (LFA) has high sensitivity and specificity for the detection of MTBC in liquid culture but has not been evaluated in a clinical mycobacteriology laboratory in the United States. We conducted a diagnostic accuracy study of this LFA for detection of MTBC versus NTMs on AFB positive cultures. A total of 362 tests were performed, with a sensitivity and specificity of 100 % (362/362) across all tests. The SD Bioline MPT64 assay provides accurate test results with AFB-positive liquid cultures and could fill the current gap for rapid rule-out of MTBC in U.S.-based clinical laboratories.

A Brief Update on Mycobacterial Taxonomy, 2020 to 2022.

This brief update provides details regarding two newly described species in the genus Mycobacterium that were identified from humans or associated with human disease and have been validly published for the period January 2020 through October 2022.

An analytic feasibility study of the BD MAX™ MDR-TB assay for testing of non-sputum specimens for detection of the Mycobacterium tuberculosis complex (MTBC) and isoniazid (INH) and rifampin (RIF) resistance.

Rapid diagnosis of tuberculosis and drug resistance in extrapulmonary specimens can be challenging. The BD MAX™ multidrug resistant (MDR)-TB assay (BD MAX™) has demonstrated high sensitivity and specificity for the detection of the Mycobacterium tuberculosis complex (MTBC) as well as resistance to INH and Rifampin (RIF) in pulmonary specimens but has not been rigorously assessed in extrapulmonary samples. We evaluated the diagnostic accuracy of the BD MAX™ assay for the detection of MTBC and drug resistance in extrapulmonary specimens spiked with MTBC from the Johns Hopkins strain collection. A total of 1083 tests were performed across multiple sample types, with an overall percent agreement of 94.8% (795/839) for detection of MTBC and 99% (379/383) and 96.4% (323/335) for determination of INH and RIF resistance-conferring mutations, respectively. The BD MAX™ assay provides same day detection of MTBC and drug-resistance results and could be a beneficial diagnostic test in extrapulmonary sample types.

Clinical Validation of the Xpert MTB/RIF Test for Identification of the Mycobacterium tuberculosis Complex in Acid-Fast Bacillus Smear-Positive MGIT Broth Cultures.

The identification of the Mycobacterium tuberculosis complex (MTBC) from smear-positive broth cultures can be achieved using several methods, including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from acid-fast bacilli (AFB) smear-positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampin resistance. The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear-positive MGIT 960 broth cultures (Becton, Dickinson, Sparks, MD). We sought to validate the Xpert MTB/RIF test for use with AFB smear-positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false-positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampin resistance from AFB smear-positive MGIT 960 broth cultures.

SARS-CoV-2 Supply Shortages and Tuberculosis Diagnostics: Current Issues Requiring Immediate Solutions.

The SARS-CoV-2 pandemic has strained manufacturing capacity worldwide, resulting in significant shortages of laboratory supplies both directly and indirectly. Such shortages include probe-based kits for detection of the Mycobacterium tuberculosis complex from positive liquid broth cultures. These shortages and possible loss of this particular assay have consequences for laboratory testing algorithms and public health in the United States. As there are no FDA-approved, commercially available options that currently exist which could immediately fill this gap, laboratories must identify alternatives and plan for modifying current testing algorithms to accommodate this change.

Global phylogenomic analyses of Mycobacterium abscessus provide context for non cystic fibrosis infections and the evolution of antibiotic resistance.

Mycobacterium abscessus (MAB) is an emerging pathogen that leads to chronic lung infections. To date, the global population structure of non-cystic fibrosis (CF) MAB and evolutionary patterns of drug resistance emergence have not been investigated. Here we construct a global dataset of 1,279 MAB whole genomes from CF or non-CF patients. We utilize whole genome analysis to assess relatedness, phylogeography, and drug resistance evolution. MAB isolates from CF and non-CF hosts are interspersed throughout the phylogeny, such that the majority of dominant circulating clones include isolates from both populations, indicating that global spread of MAB clones is not sequestered to CF contexts. We identify a large clade of M. abscessus harboring the erm(41) T28C mutation, predicted to confer macrolide susceptibility in this otherwise macrolide-resistant species. Identification of multiple evolutionary events within this clade, consistent with regain of wild type, intrinsic macrolide resistance, underscores the critical importance of macrolides in MAB.

Potent antibody-mediated neutralization limits bacteriophage treatment of a pulmonary Mycobacterium abscessus infection.

An 81-year-old immunocompetent patient with bronchiectasis and refractory Mycobacterium abscessus lung disease was treated for 6 months with a three-phage cocktail active against the strain. In this case study of phage to lower infectious burden, intravenous administration was safe and reduced the M. abscessus sputum load tenfold within one month. However, after two months, M. abscessus counts increased as the patient mounted a robust IgM- and IgG-mediated neutralizing antibody response to the phages, which was associated with limited therapeutic efficacy.

Current Updates on Mycobacterial Taxonomy, 2018 to 2019.

This minireview provides an updated overview of taxonomic changes for the genus Mycobacterium, with a focus on new species identified from humans or those associated with human disease for the period of 2018 to 2019.

Activity of Various Essential Oils Against Clinical Dermatophytes of Microsporum and Trichophyton.

Dermatophytoses account for nearly a quarter of all fungal infections worldwide. These difficult to treat infections of the skin, hair, and nails, are growing more resistant to conventional antifungal treatments, and when treatable, often require prolonged therapeutic regimens. For centuries, essential oils have been used to treat a variety of ailments. In this study, we evaluated the clinical effects in vitro of 65 essential oils and 21 essential oil blends against various clinical species/strains of dermatophytes from two primary genera, Microsporum and Trichophyton. Our aim: To determine the overall activity of a wide range of essential oils against a number of clinical strains of dermatophytes. For all assays, 16 clinically derived species/strains of dermatophytes were used. The activity of each essential oil was assessed using a modified disk-diffusion assay over a period of 21 days of incubation vs. standard antifungal drugs. Subsequently, we determined the minimum inhibitory dilution possible for the most potent essential oils and performed combination testing to determine if synergy could be demonstrated with sub-inhibitory concentrations. We also assessed the effect of repeated vs. single applications. Of all the essential oils tested, cassia, cilantro, cinnamon, thyme, and oregano were the most potent along with one blend, DDR Prime; all genera/species tested were completely inhibited for 21 days following a single application. Many of the other oils tested exhibited temporal differences in activity where significant inhibition was observed ≤10 days of incubation which declined by day 21. Synergistic combinations were achieved with oregano and cilantro, cassia, or cinnamon bark; rose and cassia were also synergistic. Repeat application maintained complete inhibition for citronella, lemon myrtle, and litsea out to 21 days, but not lemon grass or On Guard. More study is necessary to understand the ways essential oils inhibit the growth of dermatophytes. Comprehensive research aimed at understanding the mechanism of action of essential oils and their components may provide the basis for a natural alternative to topical antifungal drugs. Such research could be envisioned to target optimal combinations and determine the timing between applications to provide for maximum inhibition of recurrence or growth.

A Benchtop Automated Sputum-to-Genotype System Using a Lab-on-a-Film Assembly for Detection of Multidrug-Resistant Mycobacterium tuberculosis.

Automated genotyping of drug-resistant Mycobacterium tuberculosis (MTB) directly from sputum is challenging for three primary reasons. First, the sample matrix, sputum, is highly viscous and heterogeneous, posing a challenge for sample processing. Second, acid-fast MTB bacilli are difficult to lyse. And third, there are hundreds of MTB mutations that confer drug resistance. An additional constraint is that MTB is most prevalent where test affordability is paramount. We address the challenge of sample homogenization and cell lysis using magnetic rotation of an external magnet, at high (5000) rpm, to induce the rotation of a disposable stir disc that causes chaotic mixing of glass beads ("MagVor"). Nucleic acid is purified using a pipet tip with an embedded matrix that isolates nucleic acid ("TruTip"). We address the challenge of cost and genotyping multiple mutations using 203 porous three-dimensional gel elements printed on a film substrate and enclosed in a microfluidic laminate assembly ("Lab-on-a-Film"). This Lab-on-a-Film assembly (LFA) serves as a platform for amplification, hybridization, washing, and fluorescent imaging, while maintaining a closed format to prevent amplicon contamination of the workspace. We integrated and automated MagVor homogenization, TruTip purification, and LFA amplification in a multisample, sputum-to-genotype system. Using this system, we report detection down to 43 cfu/mL of MTB bacilli from raw sputum.

New ß-Lactamase Inhibitors Nacubactam and Zidebactam Improve the In Vitro Activity of ß-Lactam Antibiotics against Mycobacterium abscessus Complex Clinical Isolates.

The new diazabicyclooctane-based ß-lactamase inhibitors avibactam and relebactam improve the in vitro activity of ß-lactam antibiotics against bacteria of the Mycobacterium abscessus complex (MABC). Here, we evaluated the in vitro activities of two newer diazabicyclooctane-based ß-lactamase inhibitors in clinical development, nacubactam and zidebactam, with ß-lactams against clinical isolates of MABC. Both inhibitors lowered the MICs of their partner ß-lactams, meropenem (8-fold) and cefepime (2-fold), respectively, and those of other ß-lactams, similar to prior results with avibactam and relebactam.

In Vitro Activity of New Tetracycline Analogs Omadacycline and Eravacycline against Drug-Resistant Clinical Isolates of Mycobacterium abscessus.

Tigecycline is used in multidrug regimens for salvage therapy of Mycobacterium abscessus infections but is often poorly tolerated and has no oral formulation. Here, we report similar in vitro activity of two newly approved tetracycline analogs, omadacycline and eravacycline, against 28 drug-resistant clinical isolates of M. abscessus complex. Since omadacycline and eravacycline appear to be better tolerated than tigecycline and since omadacycline is also formulated for oral dosing, these tetracycline analogs may represent new treatment options for M. abscessus infections.

A survey of antimicrobial resistance in Enterobacteriaceae isolated from the Chesapeake Bay and adjacent upper tributaries.

In recent years, the rise in antimicrobial resistance (AR) in the healthcare setting as well as the environment has been recognized as a growing public health problem. The Chesapeake Bay (CB) and its upper tributaries (UT) is a large and biologically diverse estuary. This pilot study evaluated the presence of AR of gram-negative bacteria isolated from water samples collected at various sites of the Chesapeake Bay. Bacterial organisms were identified and antimicrobial susceptibility testing was performed by phenotypic and genotypic methods. Ninety-two distinctly different gram-negative bacteria were identified; Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, and Escherichia coli were most often isolated. Serratia marcescens was more frequently isolated in samples from the UT compared to the CB. Antimicrobial resistance was more frequently detected in organisms from the CB by phenotypic and genotypic methods. Antimicrobial resistance to ampicillin, imipenem, tetracycline, and chloramphenicol were the most frequently observed resistance patterns. ACT-1, CMY, and SHV genes were the most frequently detected resistance genes, with predominance in organism isolated from the CB. The results from this study emphasize the importance for further developing comprehensive surveillance programs of AR in bacterial isolates in the various environments, such as recreational and other water systems.

In Vitro Activity of the New ß-Lactamase Inhibitors Relebactam and Vaborbactam in Combination with ß-Lactams against Mycobacterium abscessus Complex Clinical Isolates.

Pulmonary disease due to infection with Mycobacterium abscessus complex (MABC) is notoriously difficult to treat, in large part due to the intrinsic resistance of MABC strains to most antibiotics, including ß-lactams. MABC organisms express a broad-spectrum ß-lactamase that is resistant to traditional ß-lactam-based ß-lactamase inhibitors but inhibited by a newer non-ß-lactam-based ß-lactamase inhibitor, avibactam. Consequently, the susceptibility of MABC members to some ß-lactams is increased in the presence of avibactam. Therefore, we hypothesized that two new non-ß-lactam-based ß-lactamase inhibitors, relebactam and vaborbactam, would also increase the susceptibility of MABC organisms to ß-lactams. The objective of the present study was to evaluate the in vitro activity of various marketed ß-lactams alone and in combination with either relebactam or vaborbactam against multidrug-resistant MABC clinical isolates. Our data demonstrate that both ß-lactamase inhibitors significantly improved the anti-MABC activity of many carbapenems (including imipenem and meropenem) and cephalosporins (including cefepime, ceftaroline, and cefuroxime). As a meropenem-vaborbactam combination is now marketed and an imipenem-relebactam combination is currently in phase III trials, these fixed combinations may become the ß-lactams of choice for the treatment of MABC infections. Furthermore, given the evolving interest in dual ß-lactam regimens, our results identify select cephalosporins, such as cefuroxime, with superior activity in the presence of a ß-lactamase inhibitor that are deserving of further evaluation in combination with these carbapenem-ß-lactamase inhibitor products.

An Update on Mycobacterial Taxonomy, 2016-2017.

This minireview provides an update on recent taxonomic changes for the genus Mycobacterium with an emphasis on newly identified species isolated from humans or associated with human disease.

Automated broth-based systems versus the MYCOTB plate for antimicrobial susceptibility testing of the Mycobacterium tuberculosis complex: challenges in interpretation.

We examined categorical agreement between automated mycobacterial susceptibility testing methods (Mycobacterial Growth Indicator Tube [MGIT] 960 System and the VersaTREK Mycobacteria Detection and Susceptibility System) which are based on single critical concentration (CC) "breakpoints" and a commercial microbroth dilution method (Sensititre Mycobacterium tuberculosis MIC Plate [MYCOTB]) which provides an MIC value. Mycobacterium tuberculosis isolates (n=355) were tested against three first-line antimycobacterial agents (ethambutol [EMB], isoniazid [INH], rifampin [RIF]) using the MYCOTB plate and either the MGIT 960 (site 1, n=142) or VersaTREK (site 2, n=213) systems. Overall categorical agreement was 96.8%. When stratified by drug and CC-defined susceptible and resistant isolates, concordance ranged from 75% to 100%. Interpretation of MIC-based results versus established CC-based results was challenging for drugs whose CC was not represented by an exactly equivalent concentration in the manufacturer-defined dilutions on the MYCOTB plate (EMB, INH). We propose interpretations of MYCOTB plate MICs using the currently available plate configuration.

Activities of Dual Combinations of Antibiotics Against Multidrug-Resistant Nontuberculous Mycobacteria Recovered from Patients with Cystic Fibrosis.

Patients with cystic fibrosis (CF) are at risk for recurrent pulmonary infections due to increased viscosity of airway secretions, leading to persistent colonization with pathogenic bacteria, including nontuberculous mycobacteria (NTM). Extensive antibiotic use for treatment of infections has led to increasing antimicrobial resistance, which is a significant barrier to the treatment of NTMs. We examined the in vitro activity of several antibiotics against a selection of the most drug-resistant clinical isolates of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium avium complex recovered from CF patients at our institution, as well as paired combinations of antibiotics against a subset of M. abscessus strains, to determine whether they exhibit synergy in inhibiting bacterial growth. Most isolates displayed resistance to at least six of the nine antibiotics tested for which phenotypic interpretation is available, and elevated minimum inhibitory concentrations (MICs) were observed for many of the other drugs. The major exception was clofazimine, which had relatively low MICs for most isolates across all species. When synergy testing was performed by using paired combinations of drugs, clofazamine and clarithromycin exhibited 100% synergy for all combinations tested, as did amikacin, with the exception of one isolate. These results suggest that synergistic antibiotic combinations are capable of overcoming drug resistance in vitro, and laboratories might consider implementation of synergy testing in multidrug-resistant (MDR)-NTM organisms to guide treatment decisions in the setting of extensive antimicrobial resistance.

Using Reduced Inoculum Densities of Mycobacterium tuberculosis in MGIT Pyrazinamide Susceptibility Testing to Prevent False-Resistant Results and Improve Accuracy: A Multicenter Evaluation.

The primary platform used for pyrazinamide (PZA) susceptibility testing of Mycobacterium tuberculosis is the MGIT culture system (Becton Dickinson). Since false-resistant results have been associated with the use of this system, we conducted a multicenter evaluation to determine the effect of using a reduced cell density inoculum on the rate of false resistance. Two reduced inoculum densities were compared with that prescribed by the manufacturer (designated as "BD" method). The reduced inoculum methods (designated as "A" and "C") were identical to the manufacturer's protocol in all aspects with the exception of the cell density of the inoculum. Twenty genetically and phenotypically characterized M. tuberculosis isolates were tested in duplicate by ten independent laboratories using the three inoculum methods. False-resistant results declined from 21.1% using the standard "BD" method to 5.7% using the intermediate ("A") inoculum and further declined to 2.8% using the most dilute ("C") inoculum method. The percentages of the resistant results that were false-resistant declined from 55.2% for the "BD" test to 28.8% and 16.0% for the "A" and "C" tests, respectively. These results represent compelling evidence that the occurrence of false-resistant MGIT PZA susceptibility test results can be mitigated through the use of reduced inoculum densities.