Prevalence of resistance to macrolides and aminoglycosides in Mycobacterium avium, M. abscessus, and M. chelonae identified in the Laboratorio Nacional de Referencia of Colombia from 2018 to 2022 / Prevalencia de la resistencia a macrólidos y aminoglucósidos en los complejos Mycobacterium avium y M. abscessus y en Mycobacterium chelonae identificados en el Laboratorio Nacional de Referencia de Colombia entre el 2018 y el 2022.

Introduction: The Mycobacterium chelonae species and the M. avium and M. abscessus complexes are emerging pathogens that cause mycobacteriosis. Treatment depends on the species and subspecies identified. The drugs of choice are macrolides and aminoglycosides. However, due to the resistance identified to these drugs, determining the microbe's sensitivity profile will allow clinicians to improve the understanding of the prognosis and evolution of these pathologies. Objective: To describe the macrolide and aminoglycoside susceptibility profile of cultures identified by Colombia's Laboratorio Nacional de Referencia de Mycobacteria from 2018 to 2022, as Mycobacterium avium complex, M. abscessus complex, and M. chelonae. Materials and methods. This descriptive study exposes the susceptibility profile to macrolides and aminoglycosides of cultures identified as M. avium complex, M. abscessus complex, and M. chelonae using the GenoType® NTM-DR method. Materials and methods: This descriptive study exposes the susceptibility profile to macrolides and aminoglycosides of cultures identified as M. avium complex, M. abscessus complex, and M. chelonae using the GenoType® NTM-DR method. Results: We identified 159 (47.3 %) cultures as M. avium complex, of which 154 (96.9 %) were sensitive to macrolides, and 5 (3.1 %) were resistant; all were sensitive to aminoglycosides. From the 125 (37.2 %) cultures identified as M. abscessus complex, 68 (54.4 %) were sensitive to macrolides, 57 (45.6 %) were resistant to aminoglycosides, and just one (0.8 %) showed resistance to aminoglycosides. The 52 cultures (15.5 %) identified as M. chelonae were sensitive to macrolides and aminoglycosides. Conclusions: The three studied species of mycobacteria have the least resistance to Amikacin. Subspecies identification and their susceptibility profiles allow the establishment of appropriate treatment schemes, especially against M. abscessus.

Introducción. Mycobacterium chelonae y los complejos Mycobacterium avium y M. abscessus, son agentes patógenos emergentes causantes de micobacteriosis. El tratamiento de esta infección depende de la especie y la subespecie identificadas. Los fármacos de elección son los macrólidos y aminoglucósidos, contra los cuales se ha reportado resistencia; por esta razón, el determinar el perfil de sensibilidad le permite al médico tratante comprender mejor el pronóstico y la evolución de estas infecciones. Objetivo. Describir los perfiles de sensibilidad ante macrólidos y aminoglucósidos, de los cultivos identificados como complejo Mycobacterium avium, complejo M. abscessus o especie M. chelonae, en el Laboratorio Nacional de Referencia de Micobacterias durante los años 2018 a 2022. Materiales y métodos. Se llevó a cabo un estudio descriptivo del perfil de sensibilidad a macrólidos y aminoglucósidos, de los cultivos identificados como complejo M. avium, complejo M. abscessus o M. chelonae, mediante la metodología GenoType® NTM-DR. Resultados. Los cultivos del complejo M. avium fueron 159 (47,3 %), de los cuales, 154 (96,9 %) fueron sensibles y 5 (3,1 %) resistentes a los macrólidos; todos fueron sensibles a los aminoglucósidos. Del complejo M. abscessus se estudiaron 125 (37,2 %) cultivos, 68 (54,4 %) resultaron sensibles y 57 (45,6 %) resistentes a los macrólidos; solo un cultivo (0,8 %) fue resistente a los aminoglucósidos. De M. chelonae se analizaron 52 cultivos (15,5 %), todos sensibles a los macrólidos y aminoglucósidos. Conclusiones. En las tres especies de micobacterias estudiadas, la resistencia contra la amikacina fue la menos frecuente. La identificación de las subespecies y los perfiles de sensibilidad permiten instaurar esquemas de tratamiento adecuados, especialmente en las micobacteriosis causadas por M. abscessus.

New genetic biomarkers from transcriptome RNA-sequencing for Mycobacterium tuberculosis complex and Mycobacterium avium complex infections by bioinformatics analysis.

The study aims to accurately identify differentially expressed genes (DEGs) and biological pathways in mycobacterial infections through bioinformatics for deeper disease understanding. Differentially expressed genes (DEGs) was explored by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Unique DEGs were submitted on least absolute shrinkage and selection operator (LASSO) regression analysis. 1,057 DEGs from two GSE datasets were identified, which were closely connected with NTM/ latent TB infection (LTBI)/active TB disease (ATB). It was demonstrated that these DEGs are mainly associated with detoxification processes, and virus and bacterial infections. Moreover, the METTL7B gene was the most informative marker for distinguishing LTBI and ATB with an area under the curve (AUC) of 0.983 (95%CI: 0.964 to 1). The significantly upregulated HBA1/2 genes were the most informative marker for distinguishing between individuals of IGRA-HC/NTM and LTBI (P < 0.001). Moreover, the upregulated HBD gene was also differ between IGRA-HC/NTM and ATB (P < 0.001). We have identified gene signatures associated with Mycobacterium infection in whole blood, which could be significant for understanding the molecular mechanisms and diagnosis of NTM, LTBI, or ATB.

Single-cell transcriptomics by clinical course of Mycobacterium avium complex pulmonary disease.

Mycobacterium avium complex pulmonary disease (MAC-PD) has a heterogeneous clinical course. However, immune profiles associated with MAC-PD clinical course are limited. We performed single-cell RNA sequencing of peripheral blood mononuclear cells from 21 MAC-PD patients divided into three clinical courses: group A, spontaneous culture conversion; group B, stable disease without antibiotic treatment; and group C, progressive disease with antibiotic treatment. A lower proportion of NK cells and higher proportion of monocytes were noted in group C compared to combined groups A and B. The proportion of classical monocytes was higher in group C compared to groups A and B, while the proportion of non-classical monocytes decreased. EGR1, HSPA1A, HSPA1B, and CD83 were up-regulated in spontaneous culture conversion group A compared to progressive disease group C. Up-regulation of MYOM2 and LILRA4 and down-regulation of MT-ATP8, CD83, and CCL3L1 was found in progressive disease group C. PCBP1, FOS, RGCC, S100B, G0S2, AREG, and LYN were highly expressed in favorable treatment response compared to unfavorable response. Our findings may offer a comprehensive understanding of the host immune profiles that influence a particular MAC-PD clinical course and could suggest an immunological mechanism associated with the disease progression of MAC-PD.

Clinical and genomic features of Mycobacterium avium complex: a multi-national European study.

BACKGROUND: The Mycobacterium avium complex (MAC) comprises the most frequent non-tuberculous mycobacteria (NTM) in Central Europe and currently includes twelve species. M. avium (MAV), M. intracellulare subsp. intracellulare (MINT), and M. intracellulare subsp. chimaera (MCH) are clinically most relevant. However, the population structure and genomic landscape of MAC linked with potential pathobiological differences remain little investigated. METHODS: Whole genome sequencing (WGS) was performed on a multi-national set of MAC isolates from Germany, France, and Switzerland. Phylogenetic analysis was conducted, as well as plasmids, resistance, and virulence genes predicted from WGS data. Data was set into a global context with publicly available sequences. Finally, detailed clinical characteristics were associated with genomic data in a subset of the cohort. RESULTS: Overall, 610 isolates from 465 patients were included. The majority could be assigned to MAV (n = 386), MCH (n = 111), and MINT (n = 77). We demonstrate clustering with less than 12 SNPs distance of isolates obtained from different patients in all major MAC species and the identification of trans-European or even trans-continental clusters when set into relation with 1307 public sequences. However, none of our MCH isolates clustered closely with the heater-cooler unit outbreak strain Zuerich-1. Known plasmids were detected in MAV (325/1076, 30.2%), MINT (62/327, 19.0%), and almost all MCH-isolates (457/463, 98.7%). Predicted resistance to aminoglycosides or macrolides was rare. Overall, there was no direct link between phylogenomic grouping and clinical manifestations, but MCH and MINT were rarely found in patients with extra-pulmonary disease (OR 0.12 95% CI 0.04-0.28, p < 0.001 and OR 0.11 95% CI 0.02-0.4, p = 0.004, respectively) and MCH was negatively associated with fulfillment of the ATS criteria when isolated from respiratory samples (OR 0.28 95% CI 0.09-0.7, p = 0.011). With 14 out of 43 patients with available serial isolates, co-infections or co-colonizations with different strains or even species of the MAC were frequent (32.6%). CONCLUSIONS: This study demonstrates clustering and the presence of plasmids in a large proportion of MAC isolates in Europe and in a global context. Future studies need to urgently define potential ways of transmission of MAC isolates and the potential involvement of plasmids in virulence.

Neglected Mycobacterium Avium Complex Infection in a Patient with Prolonged Pneumonia.

BACKGROUND: Non-tuberculous mycobacterial pulmonary infections (NTM-PD) are becoming increasingly common in clinical practice, and early detection and accurate determination of the infecting pathogen is crucial for subsequent treatment. We report a case of NTM-PD in a healthy middle-aged female with Mycobacterium tuberculosis complex group (MAC) infection confirmed by mNGS examination. METHODS: Appropriate laboratory tests, chest CT scan, bronchoscopic alveolar lavage fluid (BALF) examination, and macrogenomic next-generation sequencing (mNGS) were performed to establish the diagnosis. RESULTS: Chest CT showed multiple inflammatory lesions in the right middle lobe, and BALF sent for mNGS finally confirmed the diagnosis of MAC infection. After symptomatic treatment with azithromycin combined with ethambutol and rifampicin, the patient improved and was discharged from the hospital. CONCLUSIONS: In patients with pulmonary infections, pathogens should be clarified early to determine the diagnosis. mNGS of BALF samples have high specificity in detecting pathogens of infectious diseases, especially complex mixed infectious disease pathogens.

Transcriptome analysis of long non-coding RNAs in Mycobacterium avium complex-infected macrophages.

Mycobacterium avium complex (MAC) is a non-tuberculous mycobacterium widely distributed in the environment. Even though MAC infection is increasing in older women and immunocompromised patients, to our knowledge there has been no comprehensive analysis of the MAC-infected host-cell transcriptome-and particularly of long non-coding RNAs (lncRNAs). By using in vitro-cultured primary mouse bone-marrow-derived macrophages (BMDMs) and Cap analysis of gene expression, we analyzed the transcriptional and kinetic landscape of macrophage genes, with a focus on lncRNAs, during MAC infection. MAC infection of macrophages induced the expression of immune/inflammatory response genes and other genes similar to those involved in M1 macrophage activation, consistent with previous reports, although Nos2 (M1 activation) and Arg1 (M2 activation) had distinct expression profiles. We identified 31 upregulated and 30 downregulated lncRNA promoters corresponding respectively to 18 and 26 lncRNAs. Upregulated lncRNAs were clustered into two groups-early and late upregulated-predicted to be associated with immune activation and the immune response to infection, respectively. Furthermore, an Ingenuity Pathway Analysis revealed canonical pathways and upstream transcription regulators associated with differentially expressed lncRNAs. Several differentially expressed lncRNAs reported elsewhere underwent expressional changes upon M1 or M2 preactivation and subsequent MAC infection. Finally, we showed that expressional change of lncRNAs in MAC-infected BMDMs was mediated by toll-like receptor 2, although there may be other mechanisms that sense MAC infection. We identified differentially expressed lncRNAs in MAC-infected BMDMs, revealing diverse features that imply the distinct roles of these lncRNAs in MAC infection and macrophage polarization.

Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

Employing Multicolor Melting Curve Analysis to Rapidly Identify Non-Tuberculous Mycobacteria in Patients with Bronchiectasis: A Study from a Pulmonary Hospital in the Fuzhou District of China, 2018-2022.

Non-tuberculous mycobacteria (NTM) infection is common in bronchiectasis, with rising incidence globally. However, investigation into NTM in bronchiectasis patients in China remains relatively limited. This work aimed to identify and understand the features of NTM in bronchiectasis patient in Fuzhou district of China. The pulmonary samples were collected from 281 bronchiectasis patients with suspected NTM infection in Fuzhou, 2018-2022. MPB64 antigen detection was employed for the preliminary evaluation of NTM. Further NTM identification was realized using gene chip and gene sequencing. Among 281 patients, 172 (61.21%) patients were NTM-positive (58.72%) according to MPB64 antigen detection, with females (58.72%) outnumbering males (41.28%) and the highest prevalence in the age group of 46-65 years. In total, 47 NTM single infections and 3 mixed infections (1 Mycobacterium tuberculosis complex-M. intracellulare, 1 M. avium-M. intracellulare, and 1 M. abscessus-M. intracellulare) were identified through multicolor melting curve analysis (MMCA), which was compared with gene sequencing results. Both methods suggested Mycobacterium (M.) intracellulare, M. abscessus, and M. avium as the primary NTM species affecting bronchiectasis patients. M. intracellulare and M. abscessus were more frequent in females than males with the highest prevalence in the age group of 46-65 years according to MMCA. This research provides novel insights into the epidemiological and clinical features of NTM in bronchiectasis patients in Southeastern China. Significantly, M. intracellulare, M. abscessus, and M. avium were identified as the major NTM species, contributing to a better understanding and management of bronchiectasis accompanied by NTM infection.

Bigger problems from smaller colonies: emergence of antibiotic-tolerant small colony variants of Mycobacterium avium complex in MAC-pulmonary disease patients.

BACKGROUND: Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria that includes Mycobacterium avium and Mycobacterium intracellulare. MAC pulmonary disease (MAC-PD) poses a threat to immunocompromised individuals and those with structural pulmonary diseases worldwide. The standard treatment regimen for MAC-PD includes a macrolide in combination with rifampicin and ethambutol. However, the treatment failure and disease recurrence rates after successful treatment remain high. RESULTS: In the present study, we investigated the unique characteristics of small colony variants (SCVs) isolated from patients with MAC-PD. Furthermore, revertant (RVT) phenotype, emerged from the SCVs after prolonged incubation on 7H10 agar. We observed that SCVs exhibited slower growth rates than wild-type (WT) strains but had higher minimum inhibitory concentrations (MICs) against multiple antibiotics. However, some antibiotics showed low MICs for the WT, SCVs, and RVT phenotypes. Additionally, the genotypes were identical among SCVs, WT, and RVT. Based on the MIC data, we conducted time-kill kinetic experiments using various antibiotic combinations. The response to antibiotics varied among the phenotypes, with RVT being the most susceptible, WT showing intermediate susceptibility, and SCVs displaying the lowest susceptibility. CONCLUSIONS: In conclusion, the emergence of the SCVs phenotype represents a survival strategy adopted by MAC to adapt to hostile environments and persist during infection within the host. Additionally, combining the current drugs in the treatment regimen with additional drugs that promote the conversion of SCVs to RVT may offer a promising strategy to improve the clinical outcomes of patients with refractory MAC-PD.

Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection.

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

First isolation of Mycobacterium saskatchewanense from medical devices.

Mycobacterium saskatchewanense is a species of pigmented slow-growing Non-Tuberculous Mycobacteria (NTM), positive for Mycobacterium avium complex (MAC) by AccuProbe system. MAC organisms have frequently been isolated from different medical devices. This is the first study reporting isolation of M. saskatchewanense from medical devices and highlights the importance of correctly identifying the NTMs that often colonize sanitary water. GenoType Mycobacterium CM CE-IVD kit (CM) was used as the first step of NTM strain identification, and all positive cultures were found to be components of MAC. Then, GenoType NTM-DR CE-IVD kit (NTM-DR) was used to differentiate the different species. Sub-culture on solid media were used for: (i) phenotypical confirmation by colony morphology and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry; (ii) molecular confirmation by Next Generation Sequencing. All positive cultures were identified as M. intracellulare by CM and NTM-DR assays, whereas colony morphology showed bright yellow scotochromogenic growth. MALDI-TOF analyses identified the strains as M. saskatchewanense with a high score, and identification was confirmed by NGS analysis based on the hsp-65 region. This paper suggests that it is important to actively monitor NTM contamination in medical devices that use sanitary water, to prevent the possibility of patients becoming infected.

Evaluating amikacin minimum inhibitory concentration in trailing growth for Mycobacterium avium complex.

BACKGROUND: Amikacin is a first-line drug that must be evaluated when performing an antimycobacterial susceptibility test (AST) for Mycobacterium avium complex (MAC). However, the presence of sporadic trailing growth in MAC makes determining the precise point for reading its minimal inhibitory concentration (MIC) challenging. METHODS: Susceptibility was re-tested for 134 MAC clinical isolates using the Sensititre SLOMYCOI panel, the rrs gene was sequenced, and amikacin exposure history was investigated. The MIC50, MIC90, and the epidemiological cut-off value (ECOFF) were calculated using the EUCAST method. RESULTS: After re-testing and ignoring trailing growth, of the 22 M. intracellulare isolates originally classified as resistant to amikacin according to the CLSI guideline, 10 strains were reclassified as intermediate and four as susceptible. Similarly, from the seven resistant M. avium strains, one was reclassified as intermediate and four as susceptible. No rrs gene mutations were detected in any isolates, including resistant strains. When ignoring trailing growth, the calculated MIC50, MIC90, and ECOFF values closely aligned with the EUCAST MIC distribution. CONCLUSION: To maintain the current CLSI breakpoint, trailing growth should be ignored when reading the amikacin MIC of MAC. To read the MIC at complete bacterial inhibition, the CLSI breakpoint needs to be raised.

Correlation of drug exposure and bacterial susceptibility with treatment response for Mycobacterium avium complex lung disease: protocol for a prospective observational cohort study.

INTRODUCTION: The burden of Mycobacterium avium complex (MAC) lung disease is increasing globally and treatment outcome is in general poor. Therapeutic drug monitoring has the potential to improve treatment outcome by ensuring adequate drug exposure. However, very limited population-based studies exist for MAC lung disease. This study aims to describe the distribution of drug exposure for key antimycobacterial drugs at population level, and to analyse them in relationship to treatment outcome in patients with MAC lung disease. METHODS AND ANALYSIS: A prospective cohort aiming to include 100 adult patients diagnosed with and treated for MAC lung disease will be conducted in Shanghai Pulmonary Hospital, China. Blood samples will be collected after 1 month MAC treatment for measurement of macrolides, rifamycin, ethambutol, amikacin and/or fluoroquinolones, using a validated liquid-chromatography tandem mass spectrometry method. Respiratory samples will be collected at inclusion and once every 3 months for mycobacterial culture until treatment completion. Minimum inhibitory concentration (MIC) determination will be performed using a commercial broth microdilution plate. In addition to mycobacterial culture, disease severity and clinical improvement will be assessed from the perspective of lung function, radiological presentation and self-reported quality of life. Whole genome sequencing will be performed for any longitudinal isolates with significant change of MIC to explore the emergence of drug resistance-conferring mutations. The relationship between drug exposure and treatment outcome will be analysed and potential confounders will be considered for adjustment in multivariable models. Meanwhile, the associations between drug exposure in relation to MIC and markers of treatment response will be explored using Cox proportional hazards or binary logistic regression models, as appropriate. ETHICS AND DISSEMINATION: This study has been approved by the ethics committee of Shanghai Pulmonary Hospital (No. K22-149Z). Written and oral informed consent will be obtained from all participants. The study results will be submitted to a peer-reviewed journal. TRIAL REGISTERATION NUMBER: NCT05824988.

Mycobacterium avium Complex Infections: Detailed Phenotypic and Functional Immunological Work-Up Is Required despite Genetic Analyses.

INTRODUCTION: Cervical scrofulous lymphadenitis due to Mycobacterium avium complex (MAC) in immunocompetent adults is a rare disease. The presence of MAC infections demands meticulous clinical evaluation of patients along with detailed phenotypic and functional evaluation of their immune system including next-generation sequencing (NGS) analyses of target genes. METHODS: Exact clinical histories of the index patients both suffering from retromandibular/cervical scrofulous lymphadenitis were obtained along with phenotypic and functional immunological evaluations of leukocyte populations followed by targeted NGS-based sequencing of candidate genes. RESULTS: Immunological investigations showed normal serum immunoglobulin and complement levels, but lymphopenia, which was caused by significantly reduced CD3+CD4+CD45RO+ memory T-cell and CD19+ B-cell numbers. Despite normal T-cell proliferation to a number of accessory cell-dependent and -independent stimuli, the PBMC of both patients elaborated clearly reduced levels of a number of cytokines, including IFN-γ, IL-10, IL-12p70, IL-1α, IL-1ß, and TNF-α upon TCR-dependent T-cell stimulation with CD3-coated beads but also superantigens. The IFN-γ production deficiency was confirmed for CD3+CD4+ helper and CD4+CD8+ cytotoxic T cells on the single-cell level by multiparametric flow cytometry irrespective of whether PMA/ionomycin-stimulated whole blood cells or gradient-purified PBMC was analyzed. In the female patient L1, targeted NGS-based sequencing revealed a homozygous c.110T>C mutation in the interferon-γ receptor type 1 (IFNGR1) leading to significantly reduced receptor expression on both CD14+ monocytes and CD3+ T cells. Patient S2 presented with normal IFNGR1 expression on CD14+ monocytes but significantly reduced IFNGR1 expression on CD3+ T cells, despite the absence of detectable homozygous mutations in the IFNGR1 itself or disease-related target genes. Exogenous addition of increasing doses of IFN-γ resulted in proper upregulation of high-affinity FcγRI (CD64) on monocytes from patient S2, whereas monocytes from patient L1 showed only partial induction of CD64 expression after incubation with high doses of IFN-γ. CONCLUSION: A detailed phenotypic and functional immunological examination is urgently required to determine the cause of a clinically relevant immunodeficiency, despite detailed genetic analyses.

Virulence of Mycobacterium intracellulare clinical strains in a mouse model of lung infection - role of neutrophilic inflammation in disease severity.

BACKGROUND: Mycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice. RESULTS: We classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs. CONCLUSIONS: The virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments.

Accurate subspecies-level identification of clinically significant Mycobacterium avium and Mycobacterium intracellulare by whole-genome sequencing.

Whole genome sequencing (WGS) of Mycobacterium avium complex (MAC) isolates in the clinical laboratory setting allows for rapid and reliable subspecies identification of a closely related complex of human pathogens. We developed a bioinformatics pipeline for accurate subspecies identification and tested 74 clinical MAC isolates from various anatomical sites. We demonstrate that reliable subspecies level identification of these common and clinically significant MAC isolates, including M. avium subsp. hominissuis (most dominant in causing lower respiratory tract infections in our cohort), M. avium subsp. avium, M. intracellulare subsp. intracellulare, and M. intracellulare subsp. chimaera, can be achieved by analysis of only two marker genes (rpoB and groEL/hsp65). We then explored the relationship between these subspecies and anatomical site of infection. Further, we conducted an in silico analysis and showed our algorithm also performed well for M. avium subsp. paratuberculosis but failed to consistently identify M. avium subsp. silvaticum and M. intracellulare subsp. yongonense, likely due to a lack of available reference genome sequences; all the 3 subspecies were not found in our clinical isolates and rarely reported to cause human infections. Accurate MAC subspecies identification may provide the tool and opportunity for better understanding of the disease-subspecies dynamics in MAC infections.

A Rapid Screening Assay for Clarithromycin-Resistant Mycobacterium avium Complex Using Melting Curve Analysis with Nonfluorescent Labeled Probes.

Mycobacterium avium complex (MAC) thrives in various environments and mainly causes lung disease in humans. Because macrolide antibiotics such as clarithromycin or azithromycin are key drugs for MAC lung disease, the emergence of macrolide-resistant strains prevents the treatment of MAC. More than 95% of macrolide-resistant MAC strains are reported to have a point mutation in 23S rRNA domain V. This study successfully developed a melting curve assay using nonfluorescent labeled probes to detect the MAC mutation at positions 2058 to 2059 of the 23S rRNA gene (AA genotype, clarithromycin susceptible; TA, GA, AG, CA, AC, and AT genotypes, clarithromycin resistant). In the AA-specific probe assay, the melting peak of the DNA fragment of the AA genotype was higher than that of DNA fragments of other genotypes. Melting temperature (Tm) values of the AA genotype and the other genotypes were about 80°C and 77°C, respectively. DNA fragments of each genotype were identified correctly in six other genotype-specific probes (TA, GA, AG, CA, AC, and AT) assays. Using genomic DNA from six genotype strains of M. avium and four genotype strains of M. intracellulare, we confirmed that all genomic DNAs could be correctly identified as individual genotypes according to the highest Tm values among the same probe assays. These results indicate that this melting curve-based assay is able to determine MAC genotypes at positions 2058 to 2059 of the 23S rRNA gene. This simple method could contribute to the rapid detection of clarithromycin-resistant MAC strains and help to provide accurate drug therapy for MAC lung disease. IMPORTANCE Since macrolide antibiotics such as clarithromycin or azithromycin are key drugs in multidrug therapy for Mycobacterium avium complex (MAC) lung diseases, the rapid detection of macrolide-resistant MAC strains has important implications for the treatment of MAC. Previous studies have reported a correlation between drug susceptibility testing and the mutation of macrolide resistance genes. In this study, we developed a novel melting curve-based assay using nonfluorescent labeled probes to identify both clarithromycin-resistant M. avium and M. intracellulare with mutations in the 23S rRNA gene, which is the clarithromycin or azithromycin resistance gene. This assay contributed to not only the detection of MAC mutations but also the determination of all genotypes at positions 2058 to 2059 of the 23S rRNA gene. Furthermore, because nonfluorescent labeled probes are used, this assay is more easily and more immediately available than other methods.

Sex-Specific Associations Between Susceptibility to Mycobacterium avium Complex Lung Disease and Programmed Cell Death 1 Gene Polymorphisms.

BACKGROUND: Mycobacterium avium complex lung disease (MAC-LD) preferentially occurs in postmenopausal women and may have immune exhaustion involving the programmed cell death 1 (PD-1) pathway. It is still unknown whether sex-specific associations between susceptibility to MAC-LD and programmed cell death 1 gene (PDCD1) polymorphisms exist. METHODS: Adult patients with MAC-LD (n = 152) and controls (n = 167) were included at 2 medical centers in Taiwan. Five single-nucleotide polymorphisms in PDCD1 genes were genotyped, and their associations with MAC-LD and soluble PD-1 protein were analyzed, especially in sex subgroups. RESULTS: PDCD1 rs2227982 polymorphism was associated with increased risk of MAC-LD in women (adjusted odds ratio for AA vs AG vs GG, 2.205 [95% confidence interval, 1.108-4.389]; P = .02), and the rs10204525 TT genotype was associated with low risk in men (TT vs TC and CC, 0.396 [.176-.890]; P = .02). Compared with men with rs10204525 TT, women with rs2227982 AG and with AA had 2.7- and 5.0-fold increased risks, respectively. Soluble PD-1 levels were lower in the female subgroup with rs2227982 AG and AA than in the remainder (median level [interquartile range], 46.7 [33.7-71.5] pg/mL vs 66.2 [48.6-101.5] pg/mL; P < .001). CONCLUSIONS: PDCD1 genetic polymorphisms were associated with the risk of MAC-LD in a sex-specific pattern, possibly through regulation of PD-1 expression.

M ycobacterium avium complex genomics and transmission in a London hospital.

BACKGROUND: Non-tuberculous mycobacteria (NTM) are environmental microorganisms and opportunistic pathogens in individuals with pre-existing lung conditions such as cystic fibrosis (CF) and non-CF bronchiectasis. While recent studies of Mycobacterium abscessus have identified transmission within single CF centres as well as nationally and globally, transmission of other NTM species is less well studied. METHODS: To investigate the potential for transmission of the Mycobacterium avium complex (MAC) we sequenced 996 isolates from 354 CF and non-CF patients at the Royal Brompton Hospital (London, UK; collected 2013-2016) and analysed them in a global context. Epidemiological links were identified from patient records. Previously published genomes were used to characterise global population structures. RESULTS: We identified putative transmission clusters in three MAC species, although few epidemiological links could be identified. For M. avium, lineages were largely limited to single countries, while for Mycobacterium chimaera, global transmission clusters previously associated with heater-cooler units (HCUs) were found. However, the immediate ancestor of the lineage causing the major HCU-associated outbreak was a lineage already circulating in patients. CONCLUSIONS: CF and non-CF patients shared transmission chains, although the lack of epidemiological links suggested that most transmission is indirect and may involve environmental intermediates or asymptomatic carriage in the wider population.