Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment.Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms.Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding.Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role.Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB, including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases.Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.

Extracellular vesicles (EVs) have demonstrated significant potential in drug delivery and anti-tumor therapy. Despite this promising strategy, challenges such as specific targeting, EVs purification persist. In this study, a personalized nanodrug delivery platform using patient-derived tumor EVs (PT-EVs) based on a microchip is presented. The microchip integrates multiple functions, including capture, enrichment, drug loading, and elution of PT-EVs. The isolation and drug-carrying procedures are completed within a 12 h timeframe, achieving a recovery rate of 65%, significantly surpassing the conventional ultracentrifuge (UC) method. Furthermore, PT-EVs derived from patient tumor models are first utilized as natural drug carriers, capitalizing on their inherent homing ability to precisely target homologous tumors. Lenvatinib and doxorubicin (DOX), two commonly utilized drugs in the clinical treatment of hepatocellular carcinoma (HCC), are loaded into PT-EVs and delivered to a matched in vitro tumor model that recapitulates original tumors for drug susceptibility testing. As is proven, PT-EVs exhibit robust tumor cell targeting and efficient receptor-mediated cellular uptake, and the efficacy of chemotherapeutic drugs is improved significantly. These results suggest that this platform could be a valuable tool for efficient isolation of PT-EVs and personalized drug customization, particularly when working with limited clinical samples, thus supporting personalized and precision medicine.

BACKGROUND: The whole-genome sequencing (WGS) is becoming an increasingly effective tool for rapid and accurate detection of drug resistance in Mycobacterium tuberculosis complex (MTBC). This approach, however, has still been poorly evaluated on strains from Central and Eastern European countries. The purpose of this study was to assess the performance of WGS against conventional drug susceptibility testing (DST) for the detection of multi-drug resistant (MDR) phenotypes among MTBC clinical strains from Poland and Lithuania.
METHODS: The study included 208 MTBC strains (130 MDR; 78 drug susceptible), recovered from as many tuberculosis patients in Lithuania and Poland between 2018 and 2021. Resistance to rifampicin (RIF) and isoniazid (INH) was assessed by Critical Concentration (CC) and Minimum Inhibitory Concentration (MIC) DST as well as molecular-based techniques, including line-probe assay (LPA) and WGS. The analysis of WGS results was performed using bioinformatic pipeline- and software-based tools.
RESULTS: The results obtained with the CC DST were more congruent with those by LPA compared to pipeline-based WGS. Software-based tools showed excellent concordance with pipeline-based analysis in prediction of RIF/INH resistance. The RIF-resistant strains demonstrated a relatively homogenous MIC distribution with the mode at the highest tested MIC value. The most frequent RIF-resistance conferring mutation was rpoB S450L. The mode MIC for INH was two-fold higher among double katG and inhA mutants than among single katG mutants. The overall rate of discordant results between all methods was calculated at 5.3%. Three strains had discordant results by both genotypic methods (LPA and pipeline-based WGS), one strain by LPA only, three strains by MIC DST, two strains by both MIC DST and pipeline-based WGS, and the remaining two strains showed discordant results with all three methods, compared to CC DST.
CONCLUSIONS: Considering MIC DST results, current CCs of the first-line anti-TB drugs might be inappropriately high and may need to be revised. Both molecular methods demonstrated 100% specificity, while pipeline-based WGS had slightly lower sensitivity for RIF and INH than LPA, compared to CC DST.

Drug susceptibility testing (DST) is essential for effectively starting people on effective tuberculosis (TB) regimens. No accuracy data exists for the new high-throughput LiquidArray MTB-XDR (LA-XDR) test, which detects Mycobacterium tuberculosis complex (MTBC) and susceptibility to the fluoroquinolones, amikacin, ethambutol, and linezolid (the latter two drugs have no rapid molecular DSTs available). We enrolled (n=720) people with presumptive TB who provided two sputa for Xpert MTB/RIF Ultra and culture (MTBC reference standard). Phenotypic DST and Sanger sequencing served as a composite reference standard. Manual FluoroLyse and automated GenoXtract-fleXT (fleXT) DNA extraction methods were compared. For MTBC, LA-XDR using fleXT-extracted or FluoroLyse-extracted DNA had similar sensitivities (85-87%; which improved upon eluate retesting) and specificities (99%). Drug susceptibility sensitivities varied: 94% (86, 98) for fluoroquinolones, 64% (45, 80) for amikacin, and 88% (79, 93) for ethambutol (specificities 97-100%). LA-XDR detected 86% (6/7) phenotypically resistant linezolid isolates. LA-XDR with fleXT had indeterminate proportions of 8% (21/251) for fluoroquinolones, 1% (2/251) for ethambutol, 25% (63/251) for amikacin, and 37% (93/251) for linezolid. In a hypothetical population of 100 smear-negative fluoroquinolones-resistant cases, 24% (24/100) could be missed due to an unsuccessful result (1 fleXT error and, for LA-XDR, 2 invalid results, 15 MTBC-negative, 6 fluoroquinolone-indeterminate, 1 false-susceptible). LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high sensitivity for fluoroquinolones and ethambutol resistance, moderate sensitivity for amikacin resistance, and promise for linezolid resistance, for which more data are needed. Improved MTBC detection would reduce missed resistance.

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.

OBJECTIVE: Mycobacterium abscessus complex (MABC) infections are increasing worldwide. Furthermore, these infections have a low treatment success rate due to their resistance to many current antibiotics. This study aimed to determine the overall in vitro activity of the tetracyclines doxycycline (DOX), minocycline (MIN), and tigecycline (TGC) against MABC clinical isolates.
METHODS: A systematic review of PubMed/MEDLINE, Web of Science, and Embase was conducted up to August 28, 2023. Studies applying the drug susceptibility testing standards of the Clinical and Laboratory Standards Institute were considered. A random effects model was used to assess the total in vitro resistance rates of the MABC clinical isolates to DOX, MIN, and TGC. The I2 and Cochran\'s Q statistics were employed to evaluate the origins of heterogeneity. All analyses were conducted using CMA V.3 software.
RESULTS: Twenty-six publications (22, 12, and 11 studies on DOX, MIN, and TGC, respectively) were included. The pooled in vitro resistance rates of the MABC clinical isolates to DOX and MIN at the breakpoint of 8 μg/mL were 93.0 % (95 % CI, 89.2 %-95.5 %) and 87.2 % (95 % CI, 76.5 %-93.4 %), respectively. In the case of TGC, the breakpoints of 2, 4, and 8 μg/mL were associated with pooled resistance rates of 2.5 % (95 % CI, 0.5 %-11.6 %), 7.2 % (95 % CI, 4.0 %-12.5 %), and 16.8 % (95 % CI, 4.7 %-45.0 %), respectively.
CONCLUSIONS: Among the three examined tetracyclines, MABC exhibited extremely high resistance rates to DOX and MIN, thereby limiting their use in treating MABC infections. Conversely, MABC showed an increased susceptibility rate to TGC, highlighting TGC administration as a viable treatment option for patients with MABC infections.

UNASSIGNED: Pyrazinamide (PZA) is important for identification in multi-drug-resistant tuberculosis patients before starting therapy. PZA drug susceptibility testing (DST) is essential for the management of drug-resistant and susceptible TB patients.
UNASSIGNED: The degree of drug resistance among TB patients and discrepancy between DST results of the phenotype and genotype were assessed.
UNASSIGNED: Socio-demographic and clinical profiles of TB patients recruited in the study were documented. Sputum samples were processed for diagnosis using TrueNat Xpert MTB, TrueNat Xpert MTB Plus, and MGIT culture.
UNASSIGNED: Rifampicin (RIF) line probe assay (LPA) showed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100%, whereas isoniazid (INH) LPA testing showed a sensitivity of 85.7%, a specificity and PPV of 100%, and NPV of 94.8%. The gene mutation for RIF resistance was between the codon, 530-533 of rpoB gene, and that for INH resistance was at the codon, 315 of katG gene.
UNASSIGNED: Our findings demonstrated high prevalence of mono- and poly-drug resistance as well as pyrazinamide resistance.

BACKGROUND: In South Africa, Xpert® MTB/RIF Ultra (Ultra) is the recommended diagnostic assay for TB with line-probe assays for first- (LPAfl) and second-line drugs (LPAsl) providing additional drug susceptibility testing (DST) for samples that were rifampicin-resistant (RR-TB). To guide implementation of the recently launched Xpert® MTB/XDR (MTB/XDR) assay, a cost-outcomes analysis was conducted comparing total costs for genotypic DST (gDST) for persons diagnosed with RR-TB considering three strategies: replacing LPAfl/LPAsl (centralised level) with MTB/XDR vs. Ultra reflex testing (decentralised level). Further, DST was performed using residual specimen following RR-TB diagnosis.
METHODS: The total cost of gDST was determined for three strategies, considering loss to follow-up (LTFU), unsuccessful test rates, and specimen volume.
RESULTS: For 2019, 9,415 persons were diagnosed with RR-TB. A 35% LTFU rate between RR-TB diagnosis and LPAfl/LPAsl-DST was estimated. Unsuccessful test rates of 37% and 23.3% were reported for LPAfl and LPAsl, respectively. The estimated total costs were $191,472 for the conventional strategy, $122,352 for the centralised strategy, and $126,838 for the decentralised strategy. However, it was found that sufficient residual volume for reflex MTB/XDR testing is a limiting factor at the decentralised level.
CONCLUSIONS: Centralising the implementation of XDR testing, as compared to LPAfl/LPAsl, leads to significant cost savings.
BACKGROUND: En Afrique du Sud, Xpert® MTB/RIF Ultra (Ultra) est le test de diagnostic recommandé pour la TB avec des tests par sonde de ligne pour les médicaments de première (LPAfl) et de deuxième ligne (LPAsl) fournissant des tests de sensibilité aux médicaments (DST) supplémentaires pour les échantillons résistants à la rifampicine (RR-TB). Afin d\'orienter la mise en œuvre du test Xpert® MTB/XDR (MTB/XDR) récemment lancé, une analyse coûts-résultats a été réalisée en comparant les coûts totaux de la DST génotypique (gDST) pour les personnes diagnostiquées avec une RR-TB en tenant compte de trois stratégies : remplacer le LPAfl/LPAsl (niveau centralisé) par le MTB/XDR par rapport au test Ultra reflex (niveau décentralisé). De plus, l\'heure d\'été a été réalisée à l\'aide d\'un échantillon résiduel après le diagnostic de RR-TB.
UNASSIGNED: Le coût total de la gDST a été déterminé pour trois stratégies, en tenant compte de la perte de suivi (LTFU), des taux d\'échec des tests et du volume d\'échantillons.
UNASSIGNED: En 2019, 9 415 personnes ont reçu un diagnostic de RR-TB. Un taux de LTFU de 35% entre le diagnostic de RR-TB et le diagnostic de LPAfl/LPAsl-DST a été estimé. Des taux d\'échec de 37% et de 23,3% ont été signalés pour LPAfl et LPAsl, respectivement. Les coûts totaux estimés étaient de 191 472 dollars pour la stratégie conventionnelle, de 122 352 dollars pour la stratégie centralisée et de 126 838 dollars pour la stratégie décentralisée. Cependant, il a été constaté qu\'un volume résiduel suffisant pour les tests réflexes MTB/XDR est un facteur limitant au niveau décentralisé.
CONCLUSIONS: La centralisation de la mise en œuvre des tests XDR, par rapport à LPAfl/LPAsl, permet de réaliser d\'importantes économies.

Antibiotic resistance is a global challenge likely to cost trillions of dollars in excess costs in the health system and more importantly, millions of lives every year. A major driver of resistance is the absence of susceptibility testing at the time a healthcare worker needs to prescribe an antimicrobial. The effect is that many prescriptions are unintentionally wasted and expose mutable organisms to antibiotics increasing the risk of resistance emerging. Often simplistic solutions are applied to this growing issue, such as a naïve drive to increase the speed of drug susceptibility testing. This puts a spotlight on a technological solution and there is a multiplicity of such candidate DST tests in development. Yet, if we do not define the necessary information and the speed at which it needs to be available in the clinical decision-making progress as well as the necessary integration into clinical pathways, then little progress will be made. In this chapter, we place the technological challenge in a clinical and systems context. Further, we will review the landscape of some promising technologies that are emerging and attempt to place them in the clinic where they will have to succeed.

BACKGROUND: The emergence of multidrug-resistant tuberculosis (MDR-TB) or rifampicin-resistant (RR) TB poses a significant challenge for TB control initiatives on a global scale. This study\'s aim was to estimate the incidence of MDR-/RR-TB and identify the risk factors associated with their incidence in four provinces in northern Iran.
METHODS: Drug susceptibility testing was conducted using the proportion method on Lowenstein-Jensen media. The demographic and clinical data were collected from the Iranian TB registry.
RESULTS: Among 1083 individuals diagnosed with TB, 27 (2.5%) were identified as having MDR-/RR-TB, while 73 cases (6.7%) were any drug resistant (ADR). The statistical analysis revealed a significant association between marital status and MDR-/RR-TB (p=0.003). In addition, significant associations were observed between ADR-TB and gender (p=0.035) and previous treatment for TB (p=0.02).
CONCLUSIONS: Our findings provide important information on the drug resistance pattern of Mycobacterium tuberculosis strains, as well as risk factors in northern Iran. Given the identified risk factors, creative approaches to promote treatment adherence in TB patients, particularly divorced/widowed women and individuals with a previous history of TB treatment, are required.