Intrathoracic non-tuberculous mycobacteriosis with endobronchial lesion in a child aged 11 with HIV infection diagnosed by bronchoscopic biopsy, EBUS-TBNA and confocal laser endomicroscopy.

The diagnosis of intrathoracic non-tuberculous mycobacteriosis (NTM) is challenging. We report a case of a pediatric pulmonary NTM with endobronchial lesion and lymphadenitis in a child with HIV infection diagnosed by bronchoscopic biopsy, EBUS-TBNA and probe-based confocal laser endomicroscopy (pCLE). The pCLE showed a large number of highly fluorescent cells and zones of density and disorganized elastin fibers at alveolar areas. A combination of diagnostic endoscopic procedures is required to establish the diagnosis of NTM.

Successful diagnosis of Mycobacterium marinum infection by mNGS in a patient with Human Immunodeficiency Virus: a case report.

INTRODUCTION: Mycobacterium marinum infection rarely occurs and has atypical symptoms. It is challenging to distinguish disseminated M. marinum infection from multifocal dermatosis caused by other factors clinically. CASE PRESENTATION: Herein, we reported a 68-year-old male patient with Human Immunodeficiency Virus (HIV) who presented redness and swelling in his left hand after being stabbed by marine fish for over 2 months. Mycobacterium tuberculosis infection was considered according to biochemical and pathological examinations, while empirical anti-infection treatment was ineffective. RESULTS: The metagenomic next-generation sequencing (mNGS) detected a large amount of M. marinum sequences, and the patient was finally diagnosed with M. marinum infection. After one month of combination therapy with ethambutol, rifabutin, moxifloxacin, and linezolid, the swelling disappeared significantly. In this case, the successful application of mNGS in diagnosing and treating M. marinum infection has improved the understanding of the microbe both in the laboratory and clinically, especially in patients with HIV. CONCLUSIONS: For diseases with atypical symptoms or difficulty in determining the pathogens, mNGS is suggested in clinical procedures for rapid and accurate diagnosis and treatment.

Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Identification of Mycobacterium abscessus using the peaks of ribosomal protein L29, L30 and hemophore-related protein by MALDI-MS proteotyping.

Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.

Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles.

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Identification of the Optimal Cultivation Period Required to Isolate Representatives of Mycobacterium abscessus Complex Isolated from Patients with Cystic Fibrosis.

BACKGROUND: In patients with cystic fibrosis (CF), representatives of the fast-growing Mycobacterium abscessus complex (MABSc) are often distinguished, but the culture of the material taken from such patients increases the growth time. We analyzed the terms of cultivation of MABSc representatives on dense nutrient media and also evaluated the productivity of a modified nutrient medium based on agar for the isolation of Burkholderia cepacia complex (BCC). METHODS: Sixty-four strains of MABSc isolated from patients with CF and suspected tuberculosis were analyzed. The material from the patients was cultured on a universal chromogenic medium, 5% blood agar, yolk-salt agar, selective medium for isolation of BCC, and Löwenstein-Jensen medium. The cultures were incubated for 5 days (37°C, aerobic conditions), after for 23 days (28°C, aerobic conditions). The productivity of the developed nutrient medium was evaluated by the number of cells that gave visible growth after culturing 0.1 mL of a bacterial suspension of 103 CFU/mL. RESULTS: 76.8% of the strains grew in a 2-week period, and 23.2% of the strains were obtained at a later date from 18 to 28 days (average: 21.23 days). The modified medium with a concentration of 240 mg of iron (III) polymaltose hydroxide proved to be the most optimal for the isolation of MABSc. CONCLUSION: When using a chromogenic medium for culture material from patients with CF, it is necessary to extend incubation up to 28 days to increase the probability of MABSc isolation. The modified BCC medium showed a good selectivity result but required further investigation.

Comparison of the sputum microbiome between patients with stable nontuberculous mycobacterial pulmonary disease and patients requiring treatment.

BACKGROUND: We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring antibiotic treatment and those requiring antibiotics. METHODS: We collected sputum samples from 21 clinically stable NTM-PD patients (stable group) and 14 NTM-PD patients needing antibiotic treatment (treatment group). We also obtained 13 follow-up samples from the stable group. We analyzed the 48 samples using 16S rRNA gene sequencing (V3-V4 region) and compared the groups. RESULTS: In the linear discriminant analysis effect size (LEfSe) analysis, the species Porphyromonas pasteri, Haemophilus parahaemolyticus, Prevotella nanceiensis, and Gemella haemolysans were significantly more prevalent in the sputum of the stable group compared to the treatment group. No taxa showed significant differences in alpha-/beta-diversity or LEfSe between the 21 baseline and 13 follow-up sputum samples in the stable group. In the stable group, the genus Bergeyella and species Prevotella oris were less common in patients who achieved spontaneous culture conversion (n = 9) compared to those with persistent NTM positivity (n = 12) (effect size 3.04, p = 0.039 for Bergeyella; effect size 3.64, p = 0.033 for P. oris). In the treatment group, H. parainfluenzae was more common in patients with treatment success (n = 7) than in treatment-refractory patients (n = 7) (effect size 4.74, p = 0.013). CONCLUSIONS: Our study identified distinct bacterial taxa in the sputum of NTM-PD patients based on disease status. These results suggest the presence of a microbial environment that helps maintain disease stability.

Concomitantly Diagnosed Disseminated M kansasii Infection and Hairy Cell Leukemia With Review of Pathophysiology.

The association between Hairy Cell Leukemia (HCL) and non-tuberculous mycobacterial infections (NTMs) is well described, most notably Mycobacterium kansasii. The exact pathophysiology is not known. We report a case of a 31-year-old male with concomitantly diagnosed HCL and disseminated M kansasii infection who presented with rash, pancytopenia, and bulky axillary lymphadenopathy. The M kansasii was initially diagnosed through use of cell-free DNA detection and confirmed by bone marrow and lymph node cultures. Hairy Cell Leukemia was diagnosed with peripheral flow cytometry and confirmed via the same bone marrow sample. His HCL was put into remission with a single course of cladribine and rituximab chemotherapy; however, his M kansasii infection persisted for 6 months despite aggressive antimicrobial and surgical therapy. It was finally controlled using high-dose rifampin in combination with azithromycin and ethambutol. This case highlights the known link between HCL and M kansasii. Furthermore, it hints at potential causes beyond chemotherapy-induced immunocompromise. Notable possibilities include HCL cells acting as sanctuary sites for M kansasii to evade the immune system, and subclinical M kansasii infections causing NLRP3 inflammasome overactivation to trigger the oncogenic transformation to HCL. More research into the pathophysiologic link between HCL and M kansasii infections would allow for more effective prevention, diagnosis, and treatment of these severe atypical infections which are the major cause of morbidity in the cladribine era of HCL treatment.

Clinical Characteristics and Treatment Outcomes of Pulmonary Diseases Caused by Coinfections With Multiple Nontuberculous Mycobacterial Species.

BACKGROUND: Coinfections with multiple nontuberculous mycobacterial (NTM) species have not been widely studied. We aimed to evaluate the clinical characteristics and treatment outcomes in patients with NTM-pulmonary disease (PD) caused by coinfection with multiple NTM species. METHODS: We retrospectively reviewed patients with NTM-PD at a tertiary referral hospital in Korea between March 2012 and December 2018. Coinfection was defined as two or more species of NTM pathogens isolated from the same respiratory specimen or different specimens within three months. RESULTS: Among 1,009 patients with NTM-PD, 147 (14.6%) NTM coinfections were observed (average age 64.7 years, 69.4% women). NTM species were identified more frequently (median 6 vs. 3 times, P < 0.001) in the coinfection group than in the single species group, and follow-up duration was also longer in the coinfection group (median 44.9 vs. 27.1 months, P < 0.001). Mycobacterium avium complex (MAC) and M. abscessus and M. massiliense (MAB) were the dominant combinations (n = 71, 48.3%). For patients treated for over six months in the MAC plus MAB group (n = 31), sputum culture conversion and microbiological cure were achieved in 67.7% and 41.9% of patients, respectively. We divided the MAC plus MAB coinfection group into three subgroups according to the target mycobacteria; however, no statistical differences were found in the treatment outcomes. CONCLUSION: In NTM-PD cases, a significant number of multiple NTM species coinfections occurred. Proper identification of all cultured NTM species through follow-up is necessary to detect multispecies coinfections. Further research is needed to understand the nature of NTM-PD in such cases.

Notes from the Field: Potential Outbreak of Extrapulmonary Mycobacterium abscessus subspecies massiliense Infections from Stem Cell Treatment Clinics in Mexico - Arizona and Colorado, 2022.

Mycobacterium abscessus is an intrinsically drug-resistant, rapidly growing, nontuberculous mycobacterium; extrapulmonary infections have been reported in association with medical tourism (1). During November-December 2022, two Colorado hospitals (hospitals A and B) treated patient A, a Colorado woman aged 30-39 years, for M. abscessus meningitis. In October 2022, she had received intrathecal donor embryonic stem cell injections in Baja California, Mexico to treat multiple sclerosis and subsequently experienced headaches and fevers, consistent with meningitis. Her cerebrospinal fluid revealed neutrophilic pleocytosis and grew M. abscessus in culture at hospital A. Hospital A's physicians consulted hospital B's infectious diseases (ID) physicians to co-manage this patient (2).

[A case of nontuberculous mycobacterium presenting as a mass and atelectasis with mediastinal and hilar lymph node enlargement].

With the development of testing technology, the diagnosis of nontuberculous mycobacterium (NTM) lung disease has gradually increased in recent years. Because the clinical characteristics of NTM are not typical, and its imaging manifestations are diverse and nonspecific, missed diagnosis and misdiagnosis are common. Etiological investigation is necessary for diagnosis. Conventional etiological investigations are very limited for the diagnosis of NTM. We reported a case of NTM lung disease presenting with a mass and atelectasis with mediastinal and hilar lymph node enlargement that resembled malignant tumors. The literature on this condition was reviewed to improve the clinician's understanding and broaden clinical thinking.

Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

The diagnostic impact of fractional exhaled nitric oxide for asthmatic cough in nontuberculous mycobacterial pulmonary disease.

BACKGROUND: Measurement of exhaled nitric oxide (FeNO) is a potentially useful diagnostic test for asthma. However, no study has explored the relationship between FeNO and respiratory symptoms of nontuberculous mycobacterial pulmonary disease (NTM-PD) complicated with asthma. The objective of this study was to assess the utility of measuring FeNO levels in patients with NTM-PD complicated by asthma. METHODS: In this single-center retrospective cohort study, 140 NTM-PD patients with FeNO measured were enrolled. We selected NTM-PD patients who complicated with asthma as the NTM+BA group, defined using the following criteria: NTM patients with symptoms consistent with asthma, and NTM patients with symptomatic improvement after diagnostic therapy with ICS ± a long-acting beta 2-agonist (LABA). We then calculated a diagnostic cutoff point to distinguish between the NTM+BA groups and the NTM groups (all others). High-resolution computed tomography (HRCT) images were evaluated using the CT scoring system and their association with FeNO was examined. RESULTS: A total of 89 patients were included in the study. (31 in the NTM+BA group and 58 in the NTM group). Compared with the NTM group, the NTM+BA group had higher rates of allergic disease (51.6% vs. 22.4%; p=0.0085) and higher FeNO values (median, 23 [interquartile range {IQR}, 15.0-43.0] ppb vs. median, 17 [IQR, 11.8-23.0] ppb; p=0.015). With diagnostic asthma care using mainly ICS/LABA with reference to the FeNO, most patients (91.0%, 20/22) in the NTM-preceding subgroup in the NTM+BA group demonstrated a prompt improvement of their symptoms and AFB culture findings did not worsen (Culture positive rate (%): Pre-treatment: 59.1% vs. Post-treatment: 40.9%, p=0.3660) at 6 months after starting diagnostic therapy. The optimal diagnostic cutoff point of FeNO to distinguish between the two groups was calculated as 21.5 ppb by the ROC curve (sensitivity 75%, specificity 71.93%, p<0.0001; area under the curve: 0.7989). No significant correlation was observed between FeNO and the severity of CT images in the patients. CONCLUSIONS: A certain number of patients with NTM-PD showed exacerbated respiratory symptoms due to asthmatic complications. Elevated FeNO levels suggest asthma complications, even in patients with NTM.

In vitro activity of cefoxitin, imipenem, meropenem, and ceftaroline in combination with vaborbactam against Mycobacterium abscessus.

Mycobacterium abscessus (MAB) infections pose a growing public health threat. Here, we assessed the in vitro activity of the boronic acid-based ß-lactamase inhibitor, vaborbactam, with different ß-lactams against 100 clinical MAB isolates. Enhanced activity was observed with meropenem and ceftaroline with vaborbactam (1- and >4-fold MIC50/90 reduction). CRISPRi-mediated blaMAB gene knockdown showed a fourfold MIC reduction to ceftaroline but not the other ß-lactams. Our findings demonstrate vaborbactam's potential in combination therapy against MAB infections.

An early and trustable indicator suggestive of non-tuberculosis mycobacteria isolation in a high tuberculosis burden setting.

BACKGROUND: Distinguishing between nontuberculous mycobacterial (NTM) lung infections and pulmonary tuberculosis becomes challenging due to their similar clinical manifestations and radiological images. Consequently, instances of delayed diagnosis or misdiagnosis are highly frequent. A feasible and reliable indicator of the existence of NTM in the early stages of the disease would help to solve this dilemma. METHODS: In this study, we evaluated the potential of smear-positive and Xpert assay (Cepheid, USA) negative outcomes as an early indicator of possible NTM infection in a high TB-burden setting retrospectively and prospectively. RESULTS: During the study period, 12·77% (138/1081) of the smear-positive cases yielded negative outcomes with the simultaneous Xpert assay. From the 110 patients who yielded smear-positive/Xpert-negative outcomes and cultivated strain as well, 105 (95·45%) were proved to have NTM isolated. By incorporating an additional criterion of a negative result from the Interferon-gamma release assay, the accuracy of the screening method reached 100%. Regarding the NTM presence prediction value, smear-positive/Xpert-negative has a sensitivity of 24·86% (45/181) in all NTM isolated cases but 93·75-96·55% accuracy in retrospective study or 93·75% accuracy in prospective study in smear-positive NTM isolated cases. In addition, the specificity was ∼99·47% (943/948) in smear-positive tuberculosis cases. CONCLUSION: The clue of the presence of NTM could be obtained on the first day of the hospital visit due to the point of care (POC) feature of smear testing and Xpert assay. About one-fourth of the NTM-isolated patients would benefit from this rapid, convenient, and reliable screening strategy in the given circumstance. Smear-positive/Xpert-negative outcome is an early, trustable indicator that is indicative of NTM isolation.

Pulmonary infections with nontuberculous mycobacteria.

This review focuses on the treatment of nontuberculous pulmonary disease caused by Mycobacterium avium complex and M. abscessus. It covers treatment indications, antibiotic choice, resistance and side effects. Treatment of nontuberculous pulmonary disease is complex, lengthy, and fraught with side effects. Increased attention on this disease is needed in order to alleviate the severe consequences of this growing disease. Cooperation between pulmonologists and infectious disease specialists is needed to ensure uniform treatment, and to account for the heterogeneity seen in patients and mycobacteria alike.

Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases and cutaneous infections. However, treatment of M. abscessus infections remains particularly challenging, largely due to intrinsic resistance to a wide panel of antimicrobial agents. New therapeutic alternatives are urgently needed. Herein, we show that, upon limited irradiation with a blue-light source, newly developed porphyrin-peptide cage-type photosensitizers exert a strong bactericidal activity against smooth and rough variants of M. abscessus in planktonic cultures and in biofilms, at low concentrations. Atomic force microscopy unraveled important morphological alterations that include a wrinkled and irregular bacterial surface. The potential of these compounds for a photo-therapeutic use to treat M. abscessus skin infections requires further evaluations.IMPORTANCEMycobacterium abscessus causes persistent infections and is extremely difficult to eradicate. Despite intensive chemotherapy, treatment success rates remain very low. Thus, given the unsatisfactory performances of the current regimens, more effective therapeutic alternatives are needed. In this study, we evaluated the activity of newly described porphyrin-peptide cage-type conjugates in the context of photodynamic therapy. We show that upon light irradiation, these compounds were highly bactericidal against M. abscessus in vitro, thus qualifying these compounds for future studies dedicated to photo-therapeutic applications against M. abscessus skin infections.

Loss of LpqM proteins in Mycobacterium abscessus is associated with impaired intramacrophage survival.

Mycobacterium abscessus, an emerging pathogen responsible for severe pulmonary infections in cystic fibrosis patients, displays either a smooth (S) or a rough (R) morphotype. Infections with M. abscessus R are associated with increased pathogenicity in animal models and humans. While the S-to-R transition correlating with reduced glycopeptidolipid (GPL) production is well-documented, the recent screening of a transposon library revealed additional gene candidates located outside of the GPL locus involved in this transition. These genes include MAB_1470c, encoding the putative lipoprotein peptidase LpqM. However, experimental confirmation of the implication of this gene in the morphotype switch is lacking. Herein, we re-examined the role of MAB_1470c, and its homolog MAB_1466c, in colonial morphotype changes by generating unmarked deletion mutants in M. abscessus S. Our results indicate that the morphotype of these mutants stayed smooth in different media. Unexpectedly, the intracellular growth of ΔMAB_1470c and ΔMAB_1466c in THP-1 macrophages was significantly reduced as compared to the parental S strain, and these defects were rescued upon complementation with their corresponding genes. Strikingly, the intracellular survival defect was further exacerbated in a mutant lacking both MAB_1470c and MAB_1466c genes. This implies that, despite their primary sequence relatedness, the two proteins are not functionally redundant. Collectively, this suggests that these two LpqM-related lipoproteins are unlikely to be involved in the S-to-R transition but are key players for intramacrophage survival of M. abscessus. IMPORTANCE: Mycobacterium abscessus causes persistent infections in patients with underlying pulmonary diseases, resulting in progressive lung function deterioration. The rough (R) morphotype is well-established as associated with chronic and more aggressive infections in patients. In this study, we individually and simultaneously deleted the MAB_1470c and MAB_1466c genes in M. abscessus S, without observing changes in colony morphotypes. However, these mutants exhibited a severe impairment in their ability to survive within human macrophages, highlighting the critical role of these two lipoproteins in M. abscessus virulence.