[Evaluation of molecular diagnosis of tuberculosis and resistance to rifampicin with GeneXpert® MTB/RIF in Algeria]. / Évaluation du GeneXpert® MTB/RIF dans le diagnostic moléculaire de la tuberculose et de la résistance à la rifampicine en Algérie.

Objective: 1) To evaluate the contribution of the GeneXpert® MTB/RIF (GX) test in the diagnosis of pulmonary and extra-pulmonary tuberculosis compared to culture. 2) To compare the rifampicin results resistance obtained by GX with the phenotypic sensitivity test. Materials and methods: Retrospective study carried out over a period of five years, from May 2017 to June 2022 at the microbiology laboratory of the Central army Hospital Mohamed Seghir Nekkache, Algiers (Algeria). The pulmonary and extrapulmonary clinical specimens were collected, cultivated, tested by GX PCR and direct examination by Ziehl-Neelsen staining. The study of sensitivity to antituberculosis drugs was performed according to the proportion method on liquid medium Bactec MGIT 960 (or on solid medium Lowenstein-Jensen at the Algerian Pasteur Institute). Results: 310 samples were included in the final analysis of the study, of which 156 were of pulmonary origin and 154 of extrapulmonary origin. Mycobacterium tuberculosis complex (MTBC) was detected in 95 samples from 88 tuberculosis patients (sex ratio 2,03 and middle age 37 years) with 49 cases of pulmonary tuberculosis and 39 cases of extra-pulmonary tuberculosis. For 2 cases, the GX was positive while the culture was negative and for 11 cases, the GX was negative while the culture was positive. Thus, in our study and compared to culture, GX showed an overall sensitivity of 88.2%, a specificity of 98.6%, a positive predictive value (PPV) of 96.4% and a negative predictive value (NPV) of 95.2%. The analysis of the data according to the type of samples, the sensitivity, specificity, PPV and NPV of GX for the pulmonary and extrapulmonary samples were 96.3% vs. 77.0%, 98.0% vs. 99.1%, 96.2% vs. 96.5% and 98.0% vs. 92.7% respectively. The sensitivity of GX for disco-vertebral, lymph node, meningeal and pleural tuberculosis were 100%, 90.0%, 71.4% and 57.1% respectively. The sensitivity of GX for pulmonary tuberculosis compared to microscopy was 96% vs. 68%. The comparison of the results of detection of resistance to rifampicin by GX and by phenotypic methods showed perfect agreement. Discussion and conclusion: A good sensitivity of GX compared to microscopy was revealed. The GX is a useful tool for the diagnosis of pulmonary tuberculosis, especially in smear-negative cases. The sensitivity of GX in extrapulmonary tuberculosis varied depending on the location of the infection. A negative result by GX does not exclude tuberculosis and cases of resistance to RIF detected by GX must be confirmed by phenotypic method.

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.

Drug resistance in drug-resistant tuberculosis patients with and without diabetes mellitus: a comparative analysis.

BACKGROUND: This dual burden of tuberculosis (TB) and diabetes mellitus (DM) has become a global public health concern. This study aims to compare drug resistance in drug-resistant tuberculosis (DR-TB) patients with and without DM and analyse the risk factors of multidrug-resistant tuberculosis (MDR-TB). METHODS: A total of 893 DR-TB patients were admitted to Wenzhou Central Hospital between January 2018 and December 2022. After excluding 178 cases with incomplete clinical and laboratory data, 715 patients were included in the study. These patients were then categorized into two groups based on the presence of type 2 DM: the DM group (160 cases) and the non-DM group (555 cases). Demographic information, baseline clinical characteristics, laboratory and imaging test results, clinical diagnoses, and other relevant data were collected for analysis. Statistical analysis was conducted on demographic information, clinical parameters, drug resistance spectrum, and risk factors for multidrug resistance. RESULTS: In both the DM and non-DM groups, the order of resistance to first-line anti-tuberculosis drugs is isoniazid, streptomycin, rifampicin, and ethambutol. There is no significant difference in the proportion of mono-resistant tuberculosis, polydrug-resistant tuberculosis, and multidrug-resistant tuberculosis between the two groups (P > 0.05). The prevalence of MDR-TB in both groups shows a downward trend between 2018 and 2022, but the trend is not statistically significant (P > 0.05). Among patients without DM, residence in rural areas, retreatment of tuberculosis, pulmonary cavity, and uric acid ≥ 346 µmol/L are identified as independent risk factors for MDR-TB. Among patients with DM, residence in rural areas, retreatment of tuberculosis, pulmonary cavity, and HbA1c ≥ 9.8% were identified as independent risk factors for MDR-TB. CONCLUSION: Isoniazid is the most resistant drug among DR-TB patients with and without DM. There is no statistically significant difference in drug resistance patterns between the two groups. Some progress has been made in the prevention and control of DR-TB in this area, but the effect is not very significant. There are differences in the risk factors of MDR-TB between patients with and without DM.

Apramycin has high in vitro activity against Mycobacterium tuberculosis.

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.

Diagnostic accuracy of the Xpert® MTB/XDR assay for detection of Isoniazid and second-line antituberculosis drugs resistance at central TB reference laboratory in Tanzania.

INTRODUCTION: Early diagnosis of tuberculosis (TB) and universal access to drug-susceptibility testing (DST) are critical elements of the WHO End TB Strategy. Current rapid tests (e.g., Xpert® MTB/RIF and Ultra-assays) can detect rifampicin resistance-conferring mutations, but cannot detect resistance to Isoniazid and second-line anti-TB agents. Although Line Probe Assay is capable of detecting resistance to second-line anti-TB agents, it requires sophisticated laboratory infrastructure and advanced skills which are often not readily available in settings replete with TB. A rapid test capable of detecting Isoniazid and second-line anti-TB drug resistance is highly needed. METHODS: We conducted a diagnostic accuracy study to evaluate a new automated Xpert MTB/XDR 10-colour assay for rapid detection of Isoniazid and second-line drugs, including ethionamide, fluoroquinolones, and injectable drugs (Amikacin, Kanamycin, and Capreomycin). Positive Xpert MTB/RIF respiratory specimens were prospectively collected through routine diagnosis and surveillance of drug resistance at the Central TB Reference Laboratory in Tanzania. Specimens were tested by both Xpert XDR assay and LPA against culture-based phenotypic DST as the reference standard. FINDINGS: We analysed specimens from 151 TB patients with a mean age (SD) of 36.2 (12.7) years. The majority (n = 109, 72.2%) were males. The sensitivity for Xpert MTB/XDR was 93.5% (95% CI, 87.4-96.7); for Isoniazid, 96.6 (95% CI, 92.1-98.6); for Fluoroquinolone, 98.7% (95% Cl 94.8-99.7); for Amikacin, 96.6%; and (95% CI 92.1-98.6) for Ethionamide. Ethionamide had the lowest specificity of 50% and the highest was 100% for Fluoroquinolone. The diagnostic performance was generally comparable to that of LPA with slight variations between the two assays. The non-determinate rate (i.e., invalid M. tuberculosis complex detection) of Xpert MTB/XDR was 2·96%. CONCLUSION: The Xpert MTB/XDR demonstrated high sensitivity and specificity for detecting resistance to Isoniazid, Fluoroquinolones, and injectable agents. This assay can be used in clinical settings to facilitate rapid diagnosis of mono-isoniazid and extensively drug-resistant TB.

New generation fluoroquinolone sitafloxacin could potentially overcome the majority levofloxacin and moxifloxacin resistance in multidrug-resistant Mycobacterium tuberculosis.

Introduction. Pre-existing fluoroquinolones (FQs) resistance is a major threat in treating multidrug-resistant (MDR) tuberculosis. Sitafloxacin (Sfx) is a new broad-spectrum FQ.Hypothesis. Sfx is more active against drug-resistant Mycobacterium tuberculosis (Mtb) isolates.Aim. To determine whether there is cross-resistance between Sfx and ofloxacin (Ofx), levofloxacin (Lfx) and moxifloxacin (Mfx) in MDR Mtb.Methods. A total of 106 clinical Mtb isolates, including 23 pan-susceptible and 83 MDR strains, were analysed for Sfx, Lfx and Mfx resistance using MIC assay. The isolates were also subjected to whole-genome sequencing to analyse drug-resistant genes.Results. Sfx exhibited the most robust inhibition activity against Mtb clinical isolates, with a MIC50 of 0.0313 µg ml-1 and MIC90 of 0.125 µg ml-1, which was lower than that of Mfx (MIC50 = 0.0625 µg ml-1, MIC90 = 1 µg ml-1) and Lfx (MIC50 = 0.125 µg ml-1, MIC90 = 2 µg ml-1). We determined the tentative epidemiological cut-off values as 0.5 µg ml-1 for Sfx. Also, 8.43% (7/83), 43.37% (36/83), 42.17% (35/83) and 51.81% (43/83) MDR strains were resistant to Sfx, Mfx, Lfx and Ofx, respectively. Cross-resistance between Ofx, Lfx and Mfx was 80.43% (37/46). Only 15.22% (7/46) of the pre-existing FQs resistance isolates were resistant to Sfx. Among the 30 isolates with mutations in gyrA or gyrB, 5 (16.67%) were Sfx resistant. The combination of Sfx and rifampicin could exert partial synergistic effects, and no antagonism between Sfx and six clinically important anti-Mtb antibiotics was evident.Conclusion. Sfx exhibited superior activity against MDR isolates comparing to Lfx and Mfx, and could potentially overcome the majority pre-existing FQs resistance in Mtb strains.

Performance evaluation of the Xpert MTB/XDR test for the detection of drug resistance to Mycobacterium tuberculosis among people diagnosed with tuberculosis in South Africa.

Drug-resistant tuberculosis (TB) poses a significant public health concern in South Africa due to its complexity in diagnosis, treatment, and management. This study assessed the diagnostic performance of the Xpert MTB/XDR test for detecting drug resistance in patients with TB by using archived sputum sediments. This study analyzed 322 samples collected from patients diagnosed with TB between 2016 and 2019 across South Africa, previously characterized by phenotypic and genotypic methods. The Xpert MTB/XDR test was evaluated for its ability to detect resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolones (FLQ), and second-line injectable drugs (SLIDs) compared with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS). Culture, Xpert MTB/RIF Ultra, and Xpert MTB/RIF (G4) tests were performed to determine sensitivity and agreement with this test for TB detection. The sensitivities using a composite reference standard, pDST, and sequencing were >90% for INH, FLQ, amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) resistance, meeting the WHO target product profile criteria for this class. A lower sensitivity of 65.9% (95% CI: 57.1-73.6) for ETH resistance was observed. The Xpert MTB/XDR showed a sensitivity of 98.3% (95% CI: 96.1-99.3) and specificity of 100% (95% CI: 86.7-100) compared with culture, a positive percent agreement (PPA) of 98.8% (95% CI: 93.7-99.8) and negative percent agreement (NPA) of 100.0% (95% CI: 78.5-100.0) compared with G4, and a PPA of 99.5% (95% CI: 97.3-99.9) and NPA of 100.0% (95% CI: 78.5-100.0) compared with Xpert MTB/RIF Ultra for detecting Mycobacterium tuberculosis. The test offers a promising solution for the rapid detection of drug-resistant TB and could significantly enhance control efforts in this setting.

Strain identification of Mycobacterium tuberculosis in multidrug-resistant tuberculosis (MDR-TB) patients at Undata Hospital, Palu, Central Sulawesi.

MDR-TB is a tuberculosis disease resistant to the two most effective anti-TB drugs, rifampin and isoniazid. MDR-TB is a threat to TB control. This study aims to identify the Mycobacterium tuberculosis strain in MDR-TB patients at Undata Hospital in Palu, Central Sulawesi. This type of research is descriptive and observational with a cross-sectional design. The study was conducted in Palu City, Donggala Regency, and Sigi Regency from April-June 2021. The sample in this study consisted of 22 patients who had undergone MDR-TB treatment from 2019 to 2020. The results showed that 55% of the patients with MDR-TB lived in Palu City, 27% in Donggala Regency, and 18% in Sigi Regency. Out of the 22 patients, 13 were men (59%) and nine were women (41%). Based on the results of the examination of Mycobacterium tuberculosis culture, three samples from patients with MDR-TB were positive for M. tuberculosis. The results of the spoligotyping examination showed that the strain belonged to the Beijing family. Identifying the type of M. tuberculosis strain through spoligotyping examination should be carried out in TB patients who have not undergone MDR-TB treatment or those who have failed treatment and are still found to be positive for M. tuberculosis bacteria.

Pattern and characteristics of mutations conferring resistance to second line drugs in Mycobacterium tuberculosis isolates of pulmonary and extrapulmonary TB samples.

BACKGROUND & OBJECTIVES: The purpose of present study is to analyse the distribution and pattern of genetic mutations in PRE-XDR-TB and extensive drug resistant Mycobacterium tuberculosis (XDR-TB) using second-line line probe assay and to compare them with different parameters. METHOD: Sputum, Lymph node aspirate and cold accesses from patients with rifampicin resistant Tuberculosis were subjected to first line and second line Probe Assay (Genotype MTBDRsl by Hain Life Science, Germany) to assess additional drug resistance to fluroquinolones (Levofloxacin & Moxifloxacin) and Aminoglycosides (Amikacin, Ofloxacin and Kanamycin). The genetic mutation pattern was analysed and compared with demographic, clinical and other parameters. RESULTS: The final study population included 123 fluoroquinolone resistant isolates including 14 isolates with additional second line aminoglycosides drug resistance. The most frequent mutation observed among Gyr A drug resistance mutation was D94G (Gyr A MUT3C, 50/123,40%) corresponding to high level resistance to levofloxacin and moxifloxacin. The most frequent wild type mutant among Gyr A gene locus was WT 3 (85/123,69%). The most common mutation among second line aminoglycoside resistant isolates was at eis WT2 (7/14,50%) followed by rrs MUT 2 (4/14,29%). CONCLUSIONS: GyrA MUT3C (Asp94Gly) was the most common mutation in Gyr A gene locus in M. tuberculosis causing high level levofloxacin and moxifloxacin resistance. Patients with Asp94Gly mutation was significantly associated with underweight body mass index (p = 0.026). This study also observed that history of anti-tuberculosis therapy is a risk factor for FQ drug resistance mutations (p < 0.001).

In-silico transcriptome analysis of antibiotic-treated Mycobacterium tuberculosis identifies novel antibiotic resistance factors.

The emergence of drug resistant Mycobacterium tuberculosis strains increases the burden on the treatment of tuberculosis. In this study, through in-silico transcriptome analysis of drug-treated M. tuberculosis samples, novel drug targets for the treatment of drug resistance in tuberculosis were identified. Gene expression datasets of tuberculosis patients samples treated with different antibiotics (Isoniazid, Rifampicin, Pyrazinamide, Bedaquiline and Linezolid) were considered in this study. DESeq2 was used to identify the differentially regulated genes. Novel genes which were up-regulated during antibiotic treatment were identified which could be antibiotic resistance factors. Further, to understand the resistance mechanism of the novel genes, we performed gene ontology and gene network analysis for the differentially up-regulated genes. Thus, the in-silico transcriptome analysis paves way for a deeper understanding of the antibiotic resistance in M. tuberculosis.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh.

BACKGROUND: Multi-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear-positive patients using Line Probe Assay (LPA). METHODS: A cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. RESULTS: The overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30-59 years (43.8 %) and predominantly in males. CONCLUSION: Tuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Clinical outcomes of multidrug-resistant tracheobronchial tuberculosis receiving anti-tuberculosis regimens containing bedaquiline or delamanid.

The treatment of multidrug-resistant tracheobronchial tuberculosis poses challenges, and research investigating the efficacy of bedaquiline or delamanid as treatment for this condition is limited. This retrospective cohort study was conducted from 2017 to 2021. The study extracted data of patients with multidrug-resistant tracheobronchial tuberculosis from medical records and followed up on prognoses. Participants were divided into three groups: the bedaquiline, delamanid, and control group. Clinical outcomes and the risk factors associated with early culture conversion were analyzed. This study included 101 patients, with 32, 25, and 44 patients in the bedaquiline, delamanid, and control groups respectively. The differences in the treatment success rates among the three groups did not show statistical significance. Both the bedaquiline and delamanid groups had significantly higher culture conversion rates compared to the control after 2 or 6 months of treatment, with significantly shorter median times to culture conversion (bedaquiline group: 2 weeks, delamanid group: 2 weeks, control group: 12 weeks, P < 0.001). Treatment with bedaquiline or delamanid were identified as independent predictors of culture conversion at 2 months (bedaquiline group: aOR = 13.417, 95% CI 4.067-44.260, delamanid group: aOR = 9.333, 95% CI 2.498-34.878) or 6 months (bedaquiline group: aOR = 13.333, 95% CI 3.379-52.610, delamanid group: aOR = 5.000, 95% CI 1.357-18.426) of treatment through multivariable logistic regression analyses. The delamanid group showed better improvement in lumen stenosis compared to bedaquiline. Regimens containing bedaquiline or delamanid may accelerate the culture conversion during the early treatment phase in multidrug-resistant tracheobronchial tuberculosis, and delamanid appears to have the potential to effectively improve airway stenosis.

The Impact of the Mycobacterium tuberculosis RDRio Subfamily on Multidrug-Resistant Tuberculosis in Latin America: A Comprehensive Review.

Latin American region is a high-burden setting for tuberculosis where multidrug-resistant tuberculosis (MDR-TB) is among the main challenge to move forward the End TB Strategy goals. It has been shown that MDR-TB is associated to certain Mycobacterium tuberculosis (MTB) lineages like L2-Beijing sublineage or L4-LAM. Although L2-Beijing is present in South America, the L4 lineage is the most prevalent with values ranging from 75% to 99% depending on the country. Within L4, Latin American-Mediterranean (LAM) family is the most prevalent. Moreover, within LAM, RDRio subfamily is present in high prevalence in several countries in South America like Venezuela or Brazil. RDRio has been associated to MDR-TB in several studies in Brazil but more epidemiological information is needed for South America. Here we discuss the problem of MDR-TB in Latin America and the potential threat that RDRio could represent. At this time, more molecular epidemiology studies are necessary to improve TB surveillance programs in Latin America by tracking MTB strains potentially responsible for MDR-TB spread.

Potential of Pan-Tuberculosis Treatment to Drive Emergence of Novel Resistance.

New tuberculosis (TB) drugs with little existing antimicrobial resistance enable a pan-TB treatment regimen, intended for universal use without prior drug-susceptibility testing. However, widespread use of such a regimen could contribute to an increasing prevalence of antimicrobial resistance, potentially rendering the pan-TB regimen ineffective or driving clinically problematic patterns of resistance. We developed a model of multiple sequential TB patient cohorts to compare treatment outcomes between continued use of current standards of care (guided by rifampin-susceptibility testing) and a hypothetical pan-TB approach. A pan-TB regimen that met current target profiles was likely to initially outperform the standard of care; however, a rising prevalence of transmitted resistance to component drugs could make underperformance likely among subsequent cohorts. Although the pan-TB approach led to an increased prevalence of resistance to novel drugs, it was unlikely to cause accumulation of concurrent resistance to novel drugs and current first-line drugs.

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.

Whole Genome Sequencing of Mycobacterium tuberculosis under routine conditions in a high-burden area of multidrug-resistant tuberculosis in Peru.

Whole Genome Sequencing (WGS) is a promising tool in the global fight against tuberculosis (TB). The aim of this study was to evaluate the use of WGS in routine conditions for detection of drug resistance markers and transmission clusters in a multidrug-resistant TB hot-spot area in Peru. For this, 140 drug-resistant Mycobacterium tuberculosis strains from Lima and Callao were prospectively selected and processed through routine (GenoType MTBDRsl and BACTEC MGIT) and WGS workflows, simultaneously. Resistance was determined in accordance with the World Health Organization mutation catalogue. Agreements between WGS and BACTEC results were calculated for rifampicin, isoniazid, pyrazinamide, moxifloxacin, levofloxacin, amikacin and capreomycin. Transmission clusters were determined using different cut-off values of Single Nucleotide Polymorphism differences. 100% (140/140) of strains had valid WGS results for 13 anti-TB drugs. However, the availability of final, definitive phenotypic BACTEC MGIT results varied by drug with 10-17% of invalid results for the seven compared drugs. The median time to obtain results of WGS for the complete set of drugs was 11.5 days, compared to 28.6-52.6 days for the routine workflow. Overall categorical agreement by WGS and BACTEC MGIT for the compared drugs was 96.5%. Kappa index was good (0.65≤k≤1.00), except for moxifloxacin, but the sensitivity and specificity values were high for all cases. 97.9% (137/140) of strains were characterized with only one sublineage (134 belonging to "lineage 4" and 3 to "lineage 2"), and 2.1% (3/140) were mixed strains presenting two different sublineages. Clustering rates of 3.6% (5/140), 17.9% (25/140) and 22.1% (31/140) were obtained for 5, 10 and 12 SNP cut-off values, respectively. In conclusion, routine WGS has a high diagnostic accuracy to detect resistance against key current anti-TB drugs, allowing results to be obtained through a single analysis and helping to cut quickly the chain of transmission of drug-resistant TB in Peru.

Trends of Mycobacterium tuberculosis and rifampicin resistance at the Ho Teaching Hospital in Ghana.

BACKGROUND: Tuberculosis remains a major public health threat worldwide, causing significant morbidity and mortality, particularly in low- and middle-income countries. In recent years, efforts to combat tuberculosis have focused on strengthening healthcare systems and increasing access to diagnostics and treatment services. There is scarcity of data on the prevalence of Mycobacterium tuberculosis and rifampicin-resistant tuberculosis in the Volta region of Ghana. Therefore, the aim of this study was to determine the trends of Mycobacterium tuberculosis and rifampicin resistance in a major teaching hospital in Ghana spanning a six-year period. METHODOLOGY: A retrospective cross-sectional hospital study was conducted at Ho Teaching Hospital, Ho, Ghana. Study data included archived results on tuberculosis testing using GeneXpert from 2016-2021. Archived data on tuberculosis testing were collected and entered using Microsoft Excel 2019. IBM SPSS (v26) was used for a statistical analysis of the prevalence of tuberculosis. P-value <0.05 was considered statistically significant. RESULTS: The study included 5128 presumptive tuberculosis cases from 2016 to 2021, of which 552 were positive, revealing an overall prevalence of 10.76%. Males exhibited a significantly higher prevalence of tuberculosis (14.20%) compared to females (7.48%), with a male-to-female ratio of 2:1. The burden of tuberculosis varied significantly between age groups, with those aged 30-45 years and 46-60 years facing twice the risk compared to those under 15 years (p<0.001). Rainy seasons correlated with heightened tuberculosis occurrences (12.12%) compared to dry seasons (8.84%) (p = 0.008). Rifampicin-resistant tuberculosis was prevalent at 3.45%, slightly higher in women, particularly in the 45-59 age group (5.97%). In particular, tuberculosis prevalence exhibited fluctuations, peaking in 2016 (17.1%) and 2020 (11.5%), with a trough in 2019 (4.6%). CONCLUSION: The overall prevalence of laboratory confirmed tuberculosis was 10.76%, and resistance to rifampicin, 3.45%, indicating high infection and possible treatment failure. Considering its infectious nature, this calls for concerted efforts to curb the spread of the infection.

MmpR5 protein truncation and bedaquiline resistance in Mycobacterium tuberculosis isolates from South Africa: a genomic analysis.

BACKGROUND: The antibiotic bedaquiline is a key component of new WHO regimens for drug-resistant tuberculosis; however, predicting bedaquiline resistance from bacterial genotypes remains challenging. We aimed to understand the genetic mechanisms of bedaquiline resistance by analysing Mycobacterium tuberculosis isolates from South Africa. METHODS: For this genomic analysis, we conducted whole-genome sequencing of Mycobacterium tuberculosis samples collected at two referral laboratories in Cape Town and Johannesburg, covering regions of South Africa with a high prevalence of tuberculosis. We used the tool ARIBA to measure the status of predefined genes that are associated with bedaquiline resistance. To produce a broad genetic landscape of M tuberculosis in South Africa, we extended our analysis to include all publicly available isolates from the European Nucleotide Archive, including isolates obtained by the CRyPTIC consortium, for which minimum inhibitory concentrations of bedaquiline were available. FINDINGS: Between Jan 10, 2019, and July, 22, 2020, we sequenced 505 M tuberculosis isolates from 461 patients. Of the 64 isolates with mutations within the mmpR5 regulatory gene, we found 53 (83%) had independent acquisition of 31 different mutations, with a particular enrichment of truncated MmpR5 in bedaquiline-resistant isolates resulting from either frameshift mutations or the introduction of an insertion element. Truncation occurred across three M tuberculosis lineages, and were present in 66% of bedaquiline-resistant isolates. Although the distributions overlapped, the median minimum inhibitory concentration of bedaquiline was 0·25 mg/L (IQR 0·12-0·25) in mmpR5-disrupted isolates, compared with 0·06 mg/L (0·03-0·06) in wild-type M tuberculosis. INTERPRETATION: Reduction in the susceptibility of M tuberculosis to bedaquiline has evolved repeatedly across the phylogeny. In our data, we see no evidence that this reduction has led to the spread of a successful strain in South Africa. Binary phenotyping based on the bedaquiline breakpoint might be inappropriate to monitor resistance to this drug. We recommend the use of minimum inhibitory concentrations in addition to MmpR5 truncation screening to identify moderate increases in resistance to bedaquiline. FUNDING: US Centers for Disease Control and Prevention.

Applying whole genome sequencing to predict phenotypic drug resistance in Mycobacterium tuberculosis: leveraging 20 years of nationwide data from Denmark.

Infection with Mycobacterium tuberculosis remains one of the biggest causes of death from a single microorganism worldwide, and the continuous emergence of drug resistance aggravates our ability to cure the disease. New improved resistance detection methods are needed to provide adequate treatment, such as whole genome sequencing (WGS), which has been used increasingly to identify resistance-conferring mutations over the last decade. The steadily increasing knowledge of resistance-conferring mutations increases our ability to predict resistance based on genomic data alone. This study evaluates the performance of WGS to predict M. tuberculosis complex resistance. It compares WGS predictions with the phenotypic (culture-based) drug susceptibility results based on 20 years of nationwide Danish data. Analyzing 6,230 WGS-sequenced samples, the sensitivities for isoniazid, rifampicin, ethambutol, and pyrazinamide were 82.5% [78.0%-86.5%, 95% confidence interval (CI)], 97.3% (90.6%-99.7%, 95% CI), 58.0% (43.2%-71.8%, 95% CI), and 60.5% (49.0%-71.2%, 95% CI), respectively, and specificities were 99.8% (99.7%-99.9%, 95% CI), 99.8% (99.7%-99.9%, 95% CI), 99.4% (99.2%-99.6%, 95% CI), and 99.9% (99.7%-99.9%, 95% CI), respectively. A broader range of both sensitivities and specificities was observed for second-line drugs. The results conform with previously reported values and indicate that WGS is reliable for routine resistance detection in resource-rich tuberculosis low-incidence and low-resistance settings such as Denmark.