Aspergillus infection poses a major clinical challenge, particularly in immunocompromised individuals, with invasive diseases associated with high mortality. While Aspergillus fumigatus remains the predominant species causing human infections, recent studies highlight the growing clinical significance of lesser-known and cryptic Aspergillus species, which often exhibit reduced susceptibility to standard antifungal therapies. In this study, we analyzed 196 clinical Aspergillus isolates from 107 patients treated at the NIH Clinical Center between 2019 and 2022. A total of 38 Aspergillus species across 11 taxonomic sections were identified, with non-fumigatus and cryptic species accounting for 77.1% of all isolates. The most frequently recovered species were A. fumigatus sensu stricto (22.9%), A. sydowii (8.7%), A. calidoustus (7.1%), A. nidulans (6.6%), A. tanneri (6.1%), and A. terreus (5.6%). Species-level identification was achieved in 43% of isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In contrast, PCR sequencing confirmed species identity in over 88% of isolates by targeting the internal transcribed spacer (ITS) region of rDNA, 81% using the β-tubulin gene, and 68% using the calmodulin gene. The most common underlying clinical conditions among patients were bronchiectasis (35%), chronic granulomatous disease (22%), and pulmonary non-tuberculous mycobacterial infection (17%). Out of 107 patients, eight died (8/107, 7.5%); six of these deaths occurred in patients with chronic granulomatous disease (CGD) and two in patients with RAG1 deficiency. Antifungal susceptibility testing showed that olorofim had the lowest minimal inhibitory concentrations across species. In contrast, the activity of triazoles and amphotericin B was variable, particularly against A. tanneri, A. calidoustus, and A. sydowii. This study presents one of the largest species-level data sets of Aspergillus isolates to date, underscoring the diversity, pathogenic potential, and resistance profiles of non-fumigatus species. Accurate species identification plays an important role in guiding appropriate antifungal therapy and improving clinical outcomes, although further studies are needed to elucidate its direct impact on treatment decisions.

Thymomas are thymic epithelial-derived, most common primary anterior mediastinal masses. Non-tuberculous mycobacteria (NTM) are species that do not cause leprosy and belong to species outside the Mycobacterium tuberculosis complex. With the clinical application of targeted next-generation sequencing (tNGS), we promptly confirmed a case of NTM infection combined with NTM infection after thymoma surgery, and we performed a joint literature analysis of the two diseases to improve clinicians' understanding and recognition of lung infections after thymoma surgery. Chest CT of both lungs showed multiple hyperdense shadows. Sputum bacterial culture and characterization detected Neisseria Dryad and Streptococcus Grass Green. The presence of Mycobacterium abscessus infection was confirmed by alveolar lavage fluid sent for second-generation macro gene sequencing. The body's immune function decreases after thymoma surgery. When empirical anti-infection treatment for recurrent pneumonia in the lungs is ineffective, we should be alerted to the possibility of the presence of pulmonary non-tuberculous mycobacterial infection, and next-generation sequencing should be performed promptly to arrive quickly at a diagnosis.

A 65-year-old Japanese woman repeatedly withdrew and resumed antibiotics against pulmonary non-tuberculous mycobacterial infection caused by Mycobacterium intracellulare for more than 10 years. Although she continued to take medications, her respiratory symptoms and chest computed tomography indicated an enlarged infiltrative shadow in the lingular segment of the left lung that gradually worsened over the course of a year or more. Bronchoscopy was performed and mycobacterial culture of the bronchial lavage fluid was negative, whereas Exophiala dermatitidis was detected. After administration of oral voriconazole was initiated, the productive cough and infiltrative shadow resolved. There are no characteristic physical or imaging findings of E. dermatitidis, and it often mimics other chronic respiratory infections. Thus, when confronting refractory non-tuberculous mycobacterial cases, it might be better to assume other pathogenic microorganisms, including E. dermatitidis, and actively perform bronchoscopy.

There is an increasing prevalence of nontuberculous mycobacteria pulmonary disease (NTM-PD) in the US. Treatment of NTM-PD typically requires multiple medications, which can be associated with unpleasant morbidity and eradication of infection is difficult. Therefore, there is a critical need for novel effective and well-tolerated therapies. Recent in vitro data and case reports have suggested that nitric oxide, inhaled as a gas (gNO), has antimicrobial activity against NTM. We sought to investigate the effect of gNO in patients with NTM-PD in an open-label proof of concept trial. Eligible participants had NTM-PD with persistently positive respiratory cultures for NTM even if on antibiotic treatment. Participants were treated with gNO for 50 min three times daily, five days per week, for three weeks (total of 15 treatment days). Ten participants, of whom nine were on long-term NTM antibiotic therapy, were enrolled. All participants completed the regimen without interruption or discontinuation. Small increases in methemoglobin were noted during treatment, and all resolved to baseline within 2 h. Four participants (40%) met the primary outcome measure of negative sputum cultures after three weeks of therapy. Following treatment discontinuation, three of these participants were again culture positive during the 3-month post-treatment monitoring period, although with measures suggesting low bacterial burden. Patients tolerated a 3-week regimen of gNO without safety concerns, and despite highly refractory disease four individuals completed the study with negative cultures, although three were again positive in subsequent months. These data support further investigation of gNO as a potential therapy for NTM-PD.

Clofazimine (CFZ) is used to treat pulmonary non-tuberculous mycobacterial (NTM) infection; however, its pharmacokinetics remain unexplored in patients with pulmonary NTM, and the relationship between CFZ serum concentration and adverse effects has not been investigated. The objectives of this study were to characterize the pharmacokinetics of CFZ in pulmonary NTM disease treatment and to investigate the relationship between the steady-state CFZ serum concentration and adverse effects. A prospective observational study was conducted on 45 patients with pulmonary NTM treated with CFZ (UMIN000041053). A maximum of five serum samples per patient were taken at the CFZ trough, and serum concentration was measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pharmacokinetics of CFZ were analyzed using a nonlinear mixed effect model. The relationships among steady-state CFZ serum concentration and adverse effects, pigmentation, and heart rate-corrected QT (QTc) interval were investigated. Twenty-six patients had M. avium or M. intracellulare infection and nineteen had M. abscessus infection. The primary CFZ dosage was 50 mg/day. The estimated apparent CFZ clearance, apparent volume of distribution, and half-life were 2.4 L/h, 2,960 L, and 36 days, respectively. The combined use of rifampicin and CFZ significantly reduced CFZ exposure by 22%. Although there was no relationship between CFZ serum concentration and pigmentation intensity, the QTc interval was significantly correlated with CFZ serum concentration. The estimation of accurate pharmacokinetics for CFZ required approximately 5 months of monitoring. The relationship between the serum concentration and specific adverse effects of CFZ confirmed that CFZ serum concentration was not associated with pigmentation but did affect the QTc interval.