Rapid Drug Susceptibility Testing Research Articles

Diagnostic accuracy of LiquidArray MTB-XDR VER1.0 for the detection of Mycobacterium tuberculosis complex, fluoroquinolone, amikacin, ethambutol, and linezolid susceptibility.

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.

Targeted next-generation sequencing of Mycobacterium tuberculosis from patient samples: lessons learned from high drug-resistant burden clinical settings in Bangladesh

ABSTRACT Lack of appropriate early diagnostic tools for drug-resistant tuberculosis (DR-TB) and their incomplete drug susceptibility testing (DST) profiling is concerning for TB disease control. Existing methods, such as phenotypic DST (pDST), are time-consuming, while Xpert MTB/RIF (Xpert) and line probe assay (LPA) are limited to detecting resistance to few drugs. Targeted next-generation sequencing (tNGS) has been recently approved by WHO as an alternative approach for rapid and comprehensive DST. We aimed to investigate the performance and feasibility of tNGS for detecting DR-TB directly from clinical samples in Bangladesh. pDST, LPA and tNGS were performed among 264 sputum samples, either rifampicin-resistant (RR) or rifampicin-sensitive (RS) TB cases confirmed by Xpert assay. Resistotypes of tNGS were compared with pDST, LPA and composite reference standard (CRS, resistant if either pDST or LPA showed a resistant result). tNGS results revealed higher sensitivities for rifampicin (RIF) (99.3%), isoniazid (INH) (96.3%), fluoroquinolones (FQs) (94.4%), and aminoglycosides (AMGs) (100%) but comparatively lower for ethambutol (76.6%), streptomycin (68.7%), ethionamide (56.0%) and pyrazinamide (50.7%) when compared with pDST. The sensitivities of tNGS for INH, RIF, FQs and AMGs were 93.0%, 96.6%, 90.9%, and 100%, respectively and the specificities ranged from 91.3 to 100% when compared with CRS. This proof of concept study, conducted in a high-burden setting demonstrated that tNGS is a valuable tool for identifying DR-TB directly from the clinical specimens. Its feasibility in our laboratory suggests potential implementation and moving tNGS from research settings into clinical settings.

Quantitative drug susceptibility testing for Mycobacterium tuberculosis using unassembled sequencing data and machine learning.

There remains a clinical need for better approaches to rapid drug susceptibility testing in view of the increasing burden of multidrug resistant tuberculosis. Binary susceptibility phenotypes only capture changes in minimum inhibitory concentration when these cross the critical concentration, even though other changes may be clinically relevant. We developed a machine learning system to predict minimum inhibitory concentration from unassembled whole-genome sequencing data for 13 anti-tuberculosis drugs. We trained, validated and tested the system on 10,859 isolates from the CRyPTIC dataset. Essential agreement rates (predicted MIC within one doubling dilution of observed MIC) were above 92% for first-line drugs, 91% for fluoroquinolones and aminoglycosides, and 90% for new and repurposed drugs, albeit with a significant drop in performance for the very few phenotypically resistant isolates in the latter group. To further validate the model in the absence of external MIC datasets, we predicted MIC and converted values to binary for an external set of 15,239 isolates with binary phenotypes, and compare their performance against a previously validated mutation catalogue, the expected performance of existing molecular assays, and World Health Organization Target Product Profiles. The sensitivity of the model on the external dataset was greater than 90% for all drugs except ethionamide, clofazimine and linezolid. Specificity was greater than 95% for all drugs except ethambutol, ethionamide, bedaquiline, delamanid and clofazimine. The proposed system can provide quantitative susceptibility phenotyping to help guide antimicrobial therapy, although further data collection and validation are required before machine learning can be used clinically for all drugs.

The Role of Rapid Molecular Tests, Whole-genome Sequencing, and Phenotypic Drug Susceptibility Testing in the Detection and Control of Drug-resistant Tuberculosis

The Role of Rapid Molecular Tests, Whole-genome Sequencing, and Phenotypic Drug Susceptibility Testing in the Detection and Control of Drug-resistant Tuberculosis

Rapid, antibiotic incubation-free determination of tuberculosis drug resistance using machine learning and Raman spectroscopy

Tuberculosis (TB) is the world's deadliest infectious disease, with over 1.5 million deaths and 10 million new cases reported anually. The causative organism Mycobacterium tuberculosis (Mtb) can take nearly 40 d to culture, a required step to determine the pathogen's antibiotic susceptibility. Both rapid identification and rapid antibiotic susceptibility testing of Mtb are essential for effective patient treatment and combating antimicrobial resistance. Here, we demonstrate a rapid, culture-free, and antibiotic incubation-free drug susceptibility test for TB using Raman spectroscopy and machine learning. We collect few-to-single-cell Raman spectra from over 25,000 cells of the Mtb complex strain Bacillus Calmette-Guérin (BCG) resistant to one of the four mainstay anti-TB drugs, isoniazid, rifampicin, moxifloxacin, and amikacin, as well as a pan-susceptible wildtype strain. By training a neural network on this data, we classify the antibiotic resistance profile of each strain, both on dried samples and on patient sputum samples. On dried samples, we achieve >98% resistant versus susceptible classification accuracy across all five BCG strains. In patient sputum samples, we achieve ~79% average classification accuracy. We develop a feature recognition algorithm in order to verify that our machine learning model is using biologically relevant spectral features to assess the resistance profiles of our mycobacterial strains. Finally, we demonstrate how this approach can be deployed in resource-limited settings by developing a low-cost, portable Raman microscope that costs <$5,000. We show how this instrument and our machine learning model enable combined microscopy and spectroscopy for accurate few-to-single-cell drug susceptibility testing of BCG.

Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.

Detection of a historic reservoir of bedaquiline/clofazimine resistance-associated variants in Mycobacterium tuberculosis

BackgroundDrug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug.MethodsWe compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence.ResultsWe identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation.ConclusionsThe presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

Rapid detection of clarithromycin resistance in clinical samples of nontuberculous mycobacteria by nucleotide MALDI-TOF MS

The multidrug resistance of nontuberculous mycobacteria (NTM) poses a significant therapeutic challenge. Rapid and reliable drug susceptibility testing is urgently needed for evidence-based treatment decision, especially for macrolides. This study evaluated the utility of nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (NMTMS) in detecting clarithromycin resistance. Sixty-four clinical isolates were identified to species by NMTMS, and mutations associated with clarithromycin resistance were detected. Twenty-three M. abscessus (MAB) isolates and 30 M. intracellulare isolates (including M. intracellulare alone and M. intracellulare in combination with other SGM species) were included for analysis. The predictive sensitivity of NMTMS in detecting clarithromycin resistance was 82.35% (95% CI, 56.57% to 96.20%), with an AUC of 0.89 (95% CI, 0.77 to 0.96) in all MAB and M. intracellulare (n = 53), and up to 93.33% (95% CI, 68.05% to 99.83%) in MAB alone (n = 23). The assay provides a rapid, high-throughput, and highly sensitive tool for detecting clarithromycin resistance in NTM, especially in MAB. Optimization of the panel is necessary to enhance diagnostic accuracy.

Why do patients with DR-TB do not complete their treatment? Findings of a qualitative study from Pakistan

BackgroundOne of the major reasons for unsuccessful treatment outcomes among patients with drug-resistant tuberculosis (DR-TB) is the high rate of loss to follow-up (LTFU). However, in Pakistan, no qualitative study...

Innovative laboratory methods for improved tuberculosis diagnosis and drug-susceptibility testing

The laboratory plays a vital role in the diagnosis of all clinical forms of tuberculosis (TB), from microbiological confirmation of Mycobacterium tuberculosis to drug-susceptibility testing (DST) and treatment monitoring. For many decades, laboratory diagnosis of tuberculosis was based on conventional methods such as smear microscopy, and culture-based methods. However, Mycobacterium tuberculosis is a slow-growing organism, requiring 2–4 weeks or longer for cultures to yield results. Therefore, the evaluation of novel and rapid diagnostic methods has been a priority for research and development. In the beginning of 1990s, molecular-based diagnostics became widely available providing rapid detection, identification, and DST of M. tuberculosis. In this paper, we review some of the new diagnostic methods introduced in the clinical laboratory for the diagnosis of tuberculosis. With the global goal of ending TB as a public health challenge by 2030, enhancing diagnostic capabilities for latent and active TB, along with improving DST, would improve identification and management of cases, reducing transmission rates and curbing the spread of drug-resistant strains. These innovations promise to transform TB control efforts, bringing us closer to eradicating this persistent global health threat.

Profile of gene mutations responsible for M.Tuberculosis resistance to antituberculosis drugs

The implementation of rapid diagnostic tests and drug susceptibility testing requires a complex approach to these methods. The pattern of gene mutations responsible for resistance to first-line antituberculosis drugs was studied in patients with resistant tuberculosis undergoing treatment in the inpatient department of the Phthisiopneumology Institute. The obtained results were compared with similar data from previous studies. It was determined that in the last 5 years the resistance to RIF is predominantly determined by the rpoB S531L mutation (82.9%), for the resistance to INH the katG S315T1 mutation (94.6%) is responsible.

Rapid Drug Susceptibility Testing to Preserve Antibiotics.

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.

MOLECULAR DRUG SUSCEPTIBILITY TEST FOR EARLY DIAGNOSIS FOR DRUG RESISTANT TUBERCULOSIS

The rise of drug-resistant tuberculosis poses a severe danger to the global effort to eradicate tuberculosis (DR-TB). Delays in the application of drug susceptibility testing (DST) based on culture led to extended durations of treatment failure and persistent tuberculosis transmission. This article's objective is to give a summary of the several rapid molecular drug susceptibility tests that have proven helpful in promptly identifying DR-TB cases, enabling prompt and efficient treatment of those individuals.

Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study

Bedaquiline is a life-saving tuberculosis drug undergoing global scale-up. People at risk of weak tuberculosis drug regimens are a priority for novel drug access despite the potential source of Mycobacterium tuberculosis-resistant strains. We aimed to characterise bedaquiline resistance in individuals who had sustained culture positivity during bedaquiline-based treatment. We did a retrospective longitudinal cohort study of adults (aged ≥18 years) with culture-positive pulmonary tuberculosis who received at least 4 months of a bedaquiline-containing regimen from 12 drug-resistant tuberculosis treatment facilities in Cape Town, South Africa, between Jan 20, 2016, and Nov 20, 2017. Sputum was programmatically collected at baseline (ie, before bedaquiline initiation) and each month to monitor treatment response per the national algorithm. The last available isolate from the sputum collected at or after 4 months of bedaquiline was designated the follow-up isolate. Phenotypic drug susceptibility testing for bedaquiline was done on baseline and follow-up isolates in MGIT960 media (WHO-recommended critical concentration of 1 μg/mL). Targeted deep sequencing for Rv0678, atpE, and pepQ, as well as whole-genome sequencing were also done. In total, 40 (31%) of 129 patients from an estimated pool were eligible for this study. Overall, three (8%) of 38 patients assessable by phenotypic drug susceptibility testing for bedaquiline had primary resistance, 18 (47%) gained resistance (acquired or reinfection), and 17 (45%) were susceptible at both baseline and follow-up. Several Rv0678 and pepQ single-nucleotide polymorphisms and indels were associated with resistance. Although variants occurred in Rv0676c and Rv1979c, these variants were not associated with resistance. Targeted deep sequencing detected low-level variants undetected by whole-genome sequencing; however, none were in genes without variants already detected by whole-genome sequencing. Patients with baseline fluoroquinolone resistance, clofazimine exposure, and four or less effective drugs were more likely to have bedaquiline-resistant gain. Resistance gain was primarily due to acquisition; however, some reinfection by resistant strains occurred. Bedaquiline-resistance gain, for which we identified risk factors, was common in these programmatically treated patients with sustained culture positivity. Our study highlights risks associated with implementing life-saving new drugs and shows evidence of bedaquiline-resistance transmission. Routine drug susceptibility testing should urgently accompany scale-up of new drugs; however, rapid drug susceptibility testing for bedaquiline remains challenging given the diversity of variants observed. Doris Duke Charitable Foundation, US National Institute of Allergy and Infectious Diseases, South African Medical Research Council, National Research Foundation, Research Foundation Flanders, Stellenbosch University Faculty of Medicine Health Sciences, South African National Research Foundation, Swiss National Science Foundation, and Wellcome Trust.

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia

BackgroundMultidrug-resistant tuberculosis (MDR–TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis.MethodsHere, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR–TB isolates. Overall, data for 12 anti-TB medications were analyzed.ResultsIn total, 63 MDR–TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR–TB cases in Latvia in 2012–2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates.ConclusionsWGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.

The Impact of Rapid Drug Susceptibility Tests on Gonorrhea Burden and the Life Span of Antibiotic Treatments: A Modeling Study Among Men Who Have Sex With Men in the United States.

Rapid point-of-care tests that diagnose gonococcal infections and identify susceptibility to antibiotics enable individualized treatment. This could improve patient outcomes and slow the emergence and spread of antibiotic resistance. However, little is known about the long-term impact of such diagnostics on the burden of gonorrhea and the effective life span of antibiotics. We used a mathematical model of gonorrhea transmission among men who have sex with men in the United States to project the annual rate of reported gonorrhea cases and the effective life span of ceftriaxone, the recommended antibiotic for first-line treatment of gonorrhea, as well as 2 previously recommended antibiotics, ciprofloxacin and tetracycline, when a rapid drug susceptibility test that estimates susceptibility to ciprofloxacin and tetracycline is available. The use of a rapid drug susceptibility test with ≥50% sensitivity and≥95% specificity, defined in terms of correct ascertainment of drug susceptibility and nonsusceptibility status, could increase the combined effective life span of ciprofloxacin, tetracycline, and ceftriaxone by at least 2 years over 25 years of simulation. If test specificity is imperfect, however, the increase in the effective life span of antibiotics is accompanied by an increase in the rate of reported gonorrhea cases even under perfect sensitivity.

Stable isotope labeling as a promising tool for rapid drug susceptibility testing in Neisseria gonorrhoeae.

The world is heading towards an era of intractable and impending untreatable N. gonorrhoeae, thereby underlining the significance of rapid and accurate prediction of drug resistance as an indispensable need of the hour. In the present study, we optimized and evaluated a stable isotope labeling-based approach using the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) for rapid and reliable detection of ciprofloxacin and azithromycin resistance in N. gonorrhoeae. All the isolates were cultured under three varied condition setups viz. medium supplemented with normal lysine, heavy lysine (isotope), and heavy lysine along with the antibiotics (ciprofloxacin/azithromycin), respectively. After incubation, spectra were acquired using the MALDI-TOF MS which were further screened for unique patterns (media-specific spectra) to differentiate drug-susceptible and resistant isolates. The results of the stable isotope labeling assay were comparable to the results of phenotypic methods used for susceptibility testing.

Evaluation of two colorimetric microplate microtiter tests for susceptibility testing of clinical isolates of Mycobacterium tuberculosis to first-line antituberculosis drugs.

Drug-resistance tuberculosis (TB) is one of the most important global public health problems. Accurate and rapid drug-susceptibility testing is critical for the effective treatment of TB patients. Various colorimetric methods are used for anti-TB drug-susceptibility testing (DST) and minimum inhibitory concentration (MIC) determination. This study was conducted to evaluate the resazurin microtiter assay (REMA) and malachite green decolorization assay (MGDA). A total of 65 Mycobacterium tuberculosis strains isolated from patients with suspected TB using REMA and malachite green microtiter assay methods were tested against streptomycin (SM), isoniazid (INH), rifampicin (RIF), and ethambutol (ETB). The Mycobacterial Growth Indicator Tube 960 DST method was accepted as the gold standard in the evaluation of test results. The sensitivity of REMA and MGDA tests was found to be 87.5% and 62.5% for INH, respectively. RIF and SM sensitivity for both tests was 100%. While ETB sensitivity was 81.8 for the REMA test, this rate was 60% for the MGDA test. Specificity of both tests varied between 92.5% and 98.2% according to the drug types. REMA and MGDA are a simple, rapid, and low cost. They can be used as an alternative test for drug-susceptibility testing and MIC determination. Extensive studies and standardization are needed for the routine use of both tests.

Pre-extensively drug-resistant tuberculosis among multidrug-resistant tuberculosis patients in Ethiopia: a laboratory-based surveillance study

BackgroundThe rise of drug-resistant tuberculosis (DR-TB) has presented a substantial challenge to the national tuberculosis (TB) control program. Understanding the epidemiology of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) could help clinicians to adapt MDR-TB treatment regimens at an earlier stage. This study aimed to assess second-line anti-TB drug resistance among MDR-TB patients in Ethiopia using routine laboratory-based data. MethodsLaboratory-based cross-sectional data were collected from the national TB reference laboratory and seven regional tuberculosis culture laboratories in Ethiopia from July 2019 to March 2022. The required data, such as drug-susceptibility testing (DST) results and sociodemographics, were collected on a structured checklist from laboratory registration books and electronic databases. Data were entered into a Microsoft Excel spreadsheet and analyzed using SPSS version 23. Descriptive statistics were performed to show the distribution and magnitude of drug resistance. ResultsSecond-line drugs (SLDs) susceptibility testing was performed for 644 MDR isolates, of which 19 (3%) were found to be pre-XDR-TB cases. Of the total MDR-TB isolates, 19 (3%) were resistant to at least one fluoroquinolone drug, while 11 (1.7%) were resistant to at least one injectable second-line drug. Of the 644 MDR-TB isolates, 1.9% (5/261) pre-XDR were from new MDR-TB cases, while 3.7% (14/383) were from previously treated MDR-TB patients. The most frequently identified mutations, based on MTBDRsl results, were in codon A90V of the gyrA gene (77.3%) and A1401G of the rrs gene (45.5%). ConclusionThe overall prevalence of pre-XDR-TB in Ethiopia is considerable. The majority of SLD resistance mutations were in the gyrA gene at position A90V. Modern, rapid DST is necessary to enable identification of pre-XDR-TB and XDR-TB in supporting proper regimen administration for patients.

Single-cell raman spectroscopy: a powerful diagnostic tool for identification and drug susceptibility testing of Mycobacterium tuberculosis

Close to one quarter of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) is hard to treat and causes 3,800 deaths each day. Multidrug-resistant TB (MDR-TB) is a global public health threat. Rapid identification and drug susceptibility testing (DST) of Mtb is key to prevent the spread of MDR-TB. We studied whether single-cell Raman spectroscopy is suitable to provide reliable species identification (ID) and DST results while significantly accelerating the time-to-result (TTR) compared to the standard of care (SoC), that is PCR-based species ID within one day and phenotypic DST results at 7-10 days of culture-positivity.