Development and preliminary evaluation toward a new tuberculosis treatment monitoring tool: the PATHFAST TB LAM Ag assay.

The PATHFAST TB LAM Ag assay is based on a chemiluminescent enzyme immunoassay to quantify lipoarabinomannan (LAM) in sputum within 1 h, and was developed as an alternative to conventional culture methods for monitoring tuberculosis (TB) treatment. This study aimed to evaluate the analytical performance and initial clinical feasibility of using five Mycobacterium tuberculosis variants, 178 non-tuberculous mycobacteria (NTM), 34 upper respiratory and oral cavity microorganisms, 100 sputum specimens from untreated patients, and potential interfering substances, including 27 drugs. The results reveled a single-site repeatability coefficient of variation (CV) of 5.2%-7.0%, and a multi-site reproducibility CV of 7.1%-8.4%. The limit of blank, limit of detection, and limit of quantification were 3.03 pg/mL, 6.67 pg/mL, and 7.44 pg/mL, respectively. Linearity was observed over the analytical measurement range (10.0 pg/mL-50,000 pg/mL), and no hook effect was observed. The assay tended to cross-react with slow-growing NTMs, but not with common upper respiratory and oral cavity microorganisms, except Nocardia asteroides, Nocardia farcinica, and Tsukamurella paurometabola. No interference was observed in the presence of mucin, blood, or major anti-TB, anti-HIV, and anti-pneumonia drugs. Regarding clinical performance, the assay had a sensitivity of 88.8% (95% CI: 80.0%-94.0%) and specificity of 100.0% (95% CI: 83.9%-100.0%) using mycobacterial culture as the reference standard, and a correlation (Spearman's r = -0.770) was observed between LAM concentration and time to detection of culture. These findings show, for the first time, that the PATHFAST TB LAM Ag assay has potential value for monitoring TB treatment.

Mycobacterium tuberculosis is less likely to acquire pathogenic mutations during latent infection than during active disease.

Most people infected with Mycobacterium tuberculosis (Mtb) are believed to be in a state of latent tuberculosis (TB) infection (LTBI). Although LTBI is asymptomatic and not infectious, there is a risk of developing active disease even decades after infection. Here, to characterize mutations acquired during LTBI, we collected and analyzed Mtb genomes from seven Japanese patient pairs, each pair consisting of two active TB patients whose starting dates of developing active disease were >3 years apart; one had a high suspicion of LTBI before developing active disease, whereas the other did not. Thereafter, we compared these genomes with those of longitudinal sample pairs within a host of chronic active TB infections combined with public data. The bacterial populations in patients with LTBI were genetically more homogeneous and accumulated single nucleotide polymorphisms (SNPs) slower than those from active disease. Moreover, the lower proportion of nonsynonymous SNPs indicated weaker selective pressures during LTBI than active disease. Finally, the different mutation spectrums indicated different mutators between LTBI and active disease. These results suggest that the likelihood of the acquisition of mutations responsible for antibiotic resistance and increased virulence was lower in the Mtb population from LTBI than active disease.IMPORTANCEControlling latent tuberculosis (TB) infection (LTBI) activation is an effective strategy for TB elimination, where understanding Mycobacterium tuberculosis (Mtb) dynamics within the host plays an important role. Previous studies on chronic active disease reported that Mtb accumulated genomic mutations within the host, possibly resulting in acquired drug resistance and increased virulence. However, several reports suggest that fewer mutations accumulate during LTBI than during the active disease, but the associated risk is largely unknown. Here, we analyzed the genomic dynamics of Mtb within the host during LTBI. Our results statistically suggest that Mtb accumulates mutations during LTBI, but most mutations are under low selective pressures, which induce mutations responsible for drug resistance and virulence. Thus, we propose that LTBI acts as a source for new TB disease rather than as a period for in-host genome evolution.

Ocean circulation contributes to genetic connectivity of limpet populations at deep-sea hydrothermal vents in a back-arc basin.

For endemic benthos inhabiting hydrothermal vent fields, larval recruitment is critical for population maintenance and colonization via migration among separated sites. The vent-endemic limpet, Lepetodrilus nux, is abundant at deep-sea hydrothermal vents in the Okinawa Trough, a back-arc basin in the northwestern Pacific; nonetheless, it is endangered due to deep-sea mining. This species is associated with many other vent species and is an important successor in these vent ecosystems. However, limpet genetic diversity and connectivity among local populations have not yet been examined. We conducted a population genetics study of L. nux at five hydrothermal vent fields (maximum geographic distance, ~545 km; depths ~700 m to ~1650 m) using 14 polymorphic microsatellite loci previously developed. Genetic diversity has been maintained among these populations. Meanwhile, fine population genetic structure was detected between distant populations, even within this back-arc basin, reflecting geographic distances between vent fields. There was a significant, positive correlation between genetic differentiation and geographic distance, but no correlation with depth. Contrary to dispersal patterns predicted by an ocean circulation model, genetic migration is not necessarily unidirectional, based on relative migration rates. While ocean circulation contributes to dispersal of L. nux among vent fields in the Okinawa Trough, genetic connectivity may be maintained by complex, bidirectional dispersal processes over multiple generations.

Pan-lineage Mycobacterium tuberculosis reference genome for enhanced molecular diagnosis.

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, 'MtbRf'. We assembled 'unmapped' reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 coding sequences [CDSs]). We constructed MtbRf through: (1) searching for contig homologues among genome database that precipitate results uniquely within Mycobacteria genus; (2) comparing genomes with H37Rv ('lift-over') to define 18 insertions; and (3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60-0.75). MtbRf thus unveils previously hidden genomic information and lays the foundation for lineage-specific molDST-WGS.

Pharmacokinetics of anti-Mycobacterium avium-intracellulare disease drugs in silkworms.

Pulmonary Mycobacterium avium-intracellulare complex (MAC) disease is a typical non-tuberculous mycobacterial infection. The incidence of pulmonary MAC is increasing worldwide. This study aimed to clarify the pharmacokinetic parameters of anti-pulmonary MAC disease drugs in silkworms. The pharmacokinetic parameters investigated included maximum concentration, area under the concentration-time curve, total clearance, and volume of distribution at steady-state. In addition, protein-binding rates, fat body transferability, and drug-drug interactions were examined. Antibiotic concentrations were measured using a validated high-performance liquid chromatography-mass spectrometry method. Among the antibiotics investigated, amikacin was not eliminated from silkworms during the 48-h observation period. In contrast, dose-proportional pharmacokinetics were observed in silkworms for all antibiotics tested, except for amikacin. Protein-binding rates in hemolymph for clarithromycin, azithromycin, rifampicin, ethambutol, and amikacin were 39.6 ± 3.0%, 39.5 ± 4.3%, 76.3 ± 3.2%, 20.9 ± 4.2%, and 73.1 ± 4.7%, respectively (mean ± standard deviation). The distribution of antibiotics in the fat bodies of silkworms was related to drug lipophilicity. No drug-drug interactions were observed in the silkworms. The pharmacokinetics of these drugs in silkworms differed significantly from those in humans. Therefore, while it is challenging to predict the pharmacokinetics of these drugs in humans based on silkworm data, the silkworm infection model has facilitated a comprehensive assessment of the relationship between antibiotic exposure and efficacy.

Beyond Symptoms: Radiologic identification of asymptomatic Mycobacterium avium complex pulmonary infections.

BACKGROUND: Although international nontuberculous mycobacterial pulmonary disease (NTM-PD) guidelines highlight symptom presence at diagnosis, the clinical characteristics of asymptomatic Mycobacterium avium complex pulmonary infection (MAC-PI) patients remain understudied. We clarified the clinical characteristics and course of asymptomatic MAC-PI patients. METHODS: We retrospectively analyzed 200 consecutive patients with MAC-PIs and adequate available data who newly met the microbiological and radiological criteria for NTM-PD at Fukujuji Hospital from January 2018 to June 2020. We compared the clinical characteristics and course of asymptomatic patients with symptomatic patients and evaluated factors influencing treatment initiation through multivariate analysis. RESULTS: 111 patients were symptomatic and 89 were asymptomatic at diagnosis. While the proportion was significantly lower than that in the symptomatic group (28.8 %), 15.7 % of asymptomatic group patients had cavitary lesions (P = 0.042). In the asymptomatic group, treatments were initiated in 38 (42.7 %) patients, and cavitary lesions, a positive acid-fast bacilli smear, and younger age were independent risk factors for treatment initiation. Among 22 (57.9 %) patients who experienced disease progression necessitating treatment during follow-up, 13 (34.2 %) displayed radiological progression without any worsening of symptoms. Agents used for treatment were consistent across the groups, with no significant differences in culture conversion, microbiological recurrence rates, or spontaneous culture conversion rates. CONCLUSION: Routine health checkups and radiological examinations can detect clinically important MAC-PIs even in the absence of symptoms. Considering that the clinical course of asymptomatic MAC-PI patients is largely similar to that of symptomatic patients, timely and appropriate management and intervention are essential for all MAC-PI patients.

Clofazimine serum concentration and safety/efficacy in nontuberculous mycobacterial pulmonary disease treatment.

BACKGROUND: Clofazimine (CFZ) has shown promising effects against Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD) and Mycobacterium abscessus species pulmonary disease (MABS-PD). However, the optimal CFZ dose remains unknown. We aimed to explore the relationship between steady-state CFZ concentration and its safety and efficacy in MAC-PD and MABS-PD. METHODS: This prospective observational study focused on patients with MAC-PD and MABS-PD treated with CFZ (UMIN 000041053). To understand the safety and efficacy profile of CFZ and elucidate its optimal concentration, we analyzed CFZ-induced pigmentation grade, QTc interval, and culture conversion outcomes in relation to serum CFZ concentration using Student's t-test, a concentration-QTc model, and multivariable logistic regression analysis, respectively. In total, 64 patients (34 with MAC-PD; 30 with MABS-PD) were included. RESULTS: The steady-state concentration of CFZ was higher in the moderate-to-severe pigmentation group than in the none-to-light pigmentation group (P < 0.001). At a CFZ concentration of 1 mg/L, the QTc interval was prolonged by 17.3 ms (95 % confidence interval [CI], 3.9-25.4) from baseline. Culture conversion was achieved in 33 (51.6 %) patients. The only significant predictor of culture conversion was surgery (adjusted odds ratio, 5.4; 95 % CI, 1.3-38.0). CFZ concentration and MIC of CFZ less than 0.25 mg/L were not associated with culture conversion in this study. CONCLUSION: CFZ-induced pigmentation and QT interval prolongation are associated with serum CFZ concentrations. CFZ dosage may be optimized by monitoring serum CFZ concentration.

In vitro effects of the new oral ß-lactamase inhibitor xeruborbactam in combination with oral ß-lactams against clinical Mycobacterium abscessus isolates.

Non-tuberculosis mycobacteria (NTM), particularly Mycobacterium abscessus subsp. abscessus (M. abscessus), are increasingly being recognized as etiological agents of NTM pulmonary disease. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics, including ß-lactams. M. abscessus produces a class A ß-lactamase, whose activity is inhibited by cyclic boronic acid ß-lactamase inhibitors. We aimed to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid ß-lactamase inhibitor, against M. abscessus when combined with five ß-lactams (amoxicillin, tebipenem, cefdinir, cefuroxime, and cefoxitin). The drug susceptibilities of 43 M. abscessus clinical isolates obtained from 43 patients between August 2005 and May 2014 were tested. The MIC results for each ß-lactam with or without 4 µg/mL xeruborbactam were examined. Xeruborbactam lowered the MIC90 values of tebipenem, amoxicillin, cefuroxime, and cefdinir by 5, ≥4, 3, and 3 dilutions, respectively. The MIC90 values of cefoxitin without xeruborbactam were 32 µg/mL and did not change upon the addition of xeruborbactam. The lowest MIC90 value was obtained for tebipenem with xeruborbactam. Almost all isolates had an MIC of 4 µg/mL; one isolate had an MIC of 2 µg/mL. With respect to the susceptibility to the same family drug, the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%) for tebipenem with xeruborbactam. Combining tebipenem and xeruborbactam could be considered an effective all-oral regimen that benefits outpatient treatment of M. abscessus pulmonary disease. IMPORTANCE: Mycobacterium abscessus subsp. abscessus (M. abscessus) disease is treated in two phases; injectable drugs for initial followed by others for continuation. There is a need to develop all-oral treatment methods for M. abscessus infection, especially in the continuation phase. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics. This is the first report to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid ß-lactamase inhibitor capable of inhibiting the class A ß-lactamase produced by M. abscessus, against 43 M. abscessus clinical isolates when combined with five ß-lactam antibiotics. Xeruborbactam lowered the MIC90 values of tebipenem by five dilutions, and the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%). We showed that the tebipenem-xeruborbactam combination might be of interest to explore further as a potentially effective oral regimen for outpatient treatment of M. abscessus pulmonary disease.

Use of the Lung Flute ECO to assist in sputum collection for tuberculosis testing: a randomised crossover trial.

The Lung Flute ECO, a self-powered, low-cost oscillatory positive expiratory pressure device, assisted people with presumptive tuberculosis to produce an adequate sputum volume for diagnostic testing and was well tolerated https://bit.ly/47sDq8W.

Prospective multicentre accuracy evaluation of the FUJIFILM SILVAMP TB LAM test for the diagnosis of tuberculosis in people living with HIV demonstrates lot-to-lot variability.

There is an urgent need for rapid, non-sputum point-of-care diagnostics to detect tuberculosis. This prospective trial in seven high tuberculosis burden countries evaluated the diagnostic accuracy of the point-of-care urine-based lipoarabinomannan assay FUJIFILM SILVAMP TB LAM (FujiLAM) among inpatients and outpatients living with HIV. Diagnostic performance of FujiLAM was assessed against a mycobacterial reference standard (sputum culture, blood culture, and Xpert Ultra from urine and sputum at enrollment, and additional sputum culture ≤7 days from enrollment), an extended mycobacterial reference standard (eMRS), and a composite reference standard including clinical evaluation. Of 1637 participants considered for the analysis, 296 (18%) were tuberculosis positive by eMRS. Median age was 40 years, median CD4 cell count was 369 cells/ul, and 52% were female. Overall FujiLAM sensitivity was 54·4% (95% CI: 48·7-60·0), overall specificity was 85·2% (83·2-87·0) against eMRS. Sensitivity and specificity estimates varied between sites, ranging from 26·5% (95% CI: 17·4%-38·0%) to 73·2% (60·4%-83·0%), and 75·0 (65·0%-82·9%) to 96·5 (92·1%-98·5%), respectively. Post-hoc exploratory analysis identified significant variability in the performance of the six FujiLAM lots used in this study. Lot variability limited interpretation of FujiLAM test performance. Although results with the current version of FujiLAM are too variable for clinical decision-making, the lipoarabinomannan biomarker still holds promise for tuberculosis diagnostics. The trial is registered at clinicaltrials.gov (NCT04089423).