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| Diagnostic flowchart for presumptive multi-drug resistant tuberculosis in Vietnam (Vietnam's Ministry of Health) and perspectives. MTB, Mycobacterium tuberculosis; RIF, rifampicin; Hain 2, GenoType MTBDRsl (Hain Lifescience, Germany); MDR, multi-drug resistance; XDR, extensively drug resistance; NGS, next generation sequencing; TGS, targeted gene sequencing; DST, drug susceptibility testing; * If the result of consultation is non-TB, non-tuberculosis mycobacteria identification by culture is recommended; * * Culture and culture DST are performed when the patient is still suspected of having second-line drug resistant TB.
Source publication
Drug-resistant tuberculosis is a global health problem that hinders the progress of tuberculosis eradication programs. Accurate and early detection of drug-resistant tuberculosis is essential for effective patient care, for preventing tuberculosis spread, and for limiting the development of drug-resistant strains. Culture-based drug susceptibility...
Contexts in source publication
Context 1
... large-scale application of sequencing, especially in middle-and low-income countries is still difficult for the following reasons: (i) robust software and database tools need to be developed for the full exploitation of this technology in this specialized area of medicine; (ii) specialized personnel and bioinformatics facilities are required for the experiments, data acquisition and data analysis; (iii) the high cost of NGS platforms; (iv) need to determine whether new mutations confer anti-TB drug resistance; and (v) high amounts of high quality DNA are required for sequencing (Phelan et al., 2016;Dheda et al., 2017;Zignol et al., 2018). Moreover, the sample preparation procedure and DNA extraction method should be standardized for consistency (Zignol et al., 2018). ...
Context 2
... one sample should use several tests to get the complete drug-resistance genotype and to choose the most appropriate treatment for each patient. In Vietnam, a middleincome country, presumptive MDR-TB cases such as TB patients with treatment failure or HIV /TB co-infected patients are tested with Xpert MTB/RIF (Vietnam's Ministry of Health, 2018) (see Figure 1). Many steps are necessary from diagnosis to treatment, especially for the diagnosis of XDR-TB cases. ...
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Tuberculosis (TB), including multidrug-resistant (MDR; i.e., resistant to at least rifampicin and isoniazid)/rifampicin-resistant (MDR/RR) TB, is the most important opportunistic infection among people living with HIV (PLHIV). In 2005, Rwanda launched the programmatic management of MDR/RR-TB. The shorter MDR/RR-TB treatment regimen (STR) has been i...
Purpose
Multi-drug-resistant tuberculosis occurs when the tuberculosis bacteria develop resistance to at least the two most effective first-line anti-tuberculosis drugs, isoniazid and rifampicin. Sputum culture conversion is one of the indicators to monitor patients’ prognosis throughout the treatment. Hence, this study aimed to assess time to cult...
Background:
Household contacts of patients with drug-resistant tuberculosis are at high risk for being infected with Mycobacterium tuberculosis and for developing tuberculosis disease. To guide regimen composition for the empirical treatment of tuberculosis infection and disease in these household contacts, we estimated drug resistance profile con...
Tuberculosis is a major global health issue, with approximately 10 million people falling ill and 1.4 million dying yearly. One of the most significant challenges to public health is the emergence of drug-resistant tuberculosis. For the last half-century, treating tuberculosis has adhered to a uniform management strategy in most patients. However,...
Citations
... This pioneering assay can detect MTB and its drugresistant variants in sputum samples (Zhang et al. 2012). Microarray can detect six SNPs for lineage identification and gyrA, gyrB, rrs, eis, katG, inhA, rpoB, ahpC resistance genes (Nguyen et al. 2019). Although microarray can detect many genes at the same time, for simplicity of procedure it can be used in identifying mutations in the rpoB, gyrA, and pncA genes, predicting resistance to RIF, fluoroquinolones, and PZA, respectively (Havlicek et al. 2019; Wade et al. 2004). ...
... LPA utilizes a DNA strip technology with a cellulose acetate membrane strip. LPA relies on reverse hybridization (RH) of amplicons to immobilize membrane-bound probes, enabling the detection of mutations at frequently mutated codons (509 to 534) by using multiple probes (Mäkinen et al. 2006;Nguyen et al. 2019). In brief, the LPA process consists of three stages: DNA extraction, PCR, and RH. ...
... LPA can be performed using DNA extracted from clinical specimens or cultured samples. PCR is used to amplify the resistance determinant region of the related genes, followed by biotinylation of the PCR products and hybridization by probes and immobilized on a strip (Nguyen et al. 2019) (Fig. 4A). LPA is designed in three modules: module 1 identifies INH and RIF resistance by targeting the rpoB, katG, and inhA. ...
Mycobacterium tuberculosis, the causative agent of tuberculosis, is a pathogenic bacterium that has claimed millions of lives since the Middle Ages. According to the World Health Organization’s report, tuberculosis ranks among the ten deadliest diseases worldwide. The presence of an extensive array of genes and diverse proteins within the cellular structure of this bacterium has provided us with a potent tool for diagnosis. While the culture method remains the gold standard for tuberculosis diagnosis, it is possible that molecular diagnostic methods, emphasis on the identification of mutation genes (e.g., rpoB and gyrA) and single nucleotide polymorphisms, could offer a safe and reliable alternative. Over the past few decades, as our understanding of molecular genetics has expanded, methods have been developed based on gene expansion and detection. These methods typically commence with DNA amplification through nucleic acid targeted techniques such as polymerase chain reaction. Various molecular compounds and diverse approaches have been employed in molecular assays. In this review, we endeavor to provide an overview of molecular assays for the diagnosis of tuberculosis with their properties (utilization, challenges, and functions). The ultimate goal is to explore the potential of replacing traditional bacterial methods with these advanced molecular diagnostic techniques.
... The deployment of GeneXpert MTB/RIF in WA, allowing dual diagnosis of TB and rifampicin resistance within hours is commendable (116,117). The new GeneXpert MTB/XDR deployed in few reference labs in the sub-region work in concert with the GeneXpert MTB/RIF to give better resolution on MTBC susceptibility to RIF, INH, RQs and (118,119). Nevertheless, the GeneXpert is inapplicable as a point of care tool in rural areas. On the other hand, the newly introduced Seegene-Anyplex-II-MTB/MDR/XDR (120), a real-time PCR-based diagnostics may account for some limitations of the GeneXpert platform, but requires evaluation in WA the hot-spot of MTBC diversity to access its sensitivity and specificity (7,121,122). ...
Drug-resistant (DR) tuberculosis (TB) is a major public health concern globally, complicating TB control and management efforts. West Africa has historically faced difficulty in combating DR-TB due to limited diagnostic skills, insufficient access to excellent healthcare, and ineffective healthcare systems. This has aided in the emergence and dissemination of DR Mycobacterium tuberculosis complex (MTBC) strains in the region. In the past, DR-TB patients faced insufficient resources, fragmented efforts, and suboptimal treatment outcomes. However, current efforts to combat DR-TB in the region are promising. These efforts include strengthening diagnostic capacities, improving access to quality healthcare services, and implementing evidence-based treatment regimens for DR-TB. Additionally, many West African National TB control programs are collaborating with international partners to scale up laboratory infrastructure, enhance surveillance systems, and promote infection control measures. Moreso, novel TB drugs and regimens, such as bedaquiline and delamanid, are being introduced to improve treatment outcomes for DR-TB cases. Despite these obstacles, there is optimism for the future of DR-TB control in West Africa. Investments are being made to improve healthcare systems, expand laboratory capacity, and support TB research and innovation. West African institutions are now supporting knowledge sharing, capacity building, and resource mobilization through collaborative initiatives such as the West African Network for TB, AIDS, and Malaria (WANETAM), the West African Health Organization (WAHO), and other regional or global partners. These efforts hold promise for improved diagnostics, optimized treatment regimens, and provide better patient outcomes in the future where drug-resistant TB in WA can be effectively controlled, reducing the burden of the disease, and improving the health outcomes of affected individuals.
... Molecular resistance tests are routinely recommended for all positive samples and are necessary for individuals with risk factors for multidrug-resistant TB [4]. Regardless of molecular tests being carried out, phenotypic tests, cultural isolation of Mt, and subsequent antibiograms are mandatory and allow for the confirmation of both the diagnosis and the viability of the bacilli present in the biological sample. ...
Since 1997, the World Health Organization (WHO) publishes the Global Tuberculosis Report every year, which contains the most up-to-date and accurate statistical data on tuberculosis (TB) worldwide. TB is an infectious disease that is one of the leading causes of human morbidity. It ranks among the top 10 most common causes of death worldwide and is more likely than other pathogens, such as the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS), to result in a fatal outcome. After the introduction of the TB vaccine, the situation improved significantly, but the disease was not defeated. In this context, we present the clinical case of a male patient who was admitted to the Emergency Department (ED) due to a productive cough, dyspnea, and weight loss. After a complementary study, he was diagnosed with extensive diffuse pulmonary TB with a bacterial infection.
... MTB drug and multi-drug resistance primarily stem from spontaneous single nucleotide polymorphisms (SNPs) [25]. These SNPs are linked to mutations in various genes associated with resistance (inhA, rpoB, pncA, embB, rrs, gyrA, gyrB, katG, ahpC, ndh, furA, rpsL) to specific antibiotics (pyrazinamide, rifampicin, isoniazid, ethambutol, streptomycin, capreomycin, and fluoroquinolone) [26][27][28][29]. Although fluctuation tests have been conducted for these antibiotics, the mutations arise spontaneously at rates ranging from 10 − 6 to 10 − 9 , depending on the antibiotic. ...
Antimicrobial resistance is a global health threat. Misuse and overuse of antimicrobials are the main drivers in developing drug-resistant bacteria. The emergence of the rapid global spread of multi-resistant bacteria requires urgent multisectoral action to generate novel treatment alternatives. Combination therapy offers the potential to exploit synergistic effects for enhanced antibacterial efficacy of drugs. Understanding the complex dynamics and kinetics of drug interactions in combination therapy is crucial. Therefore, this review outlines the current advances in antibiotic resistance's evolutionary and genetic dynamics in combination therapies-exposed bacteria. Moreover, we also discussed four pivotal future research areas to comprehend better the development of antibiotic resistance in bacteria treated with combination strategies.
... Correspondingly, the missed 4.2 million cases can lead to new chains of transmission in the community, thus constituting new challenges in preventing and controlling TB. These challenges are further complicated by the deadly rise of drug-resistant TB, especially multidrug-resistant/rifampicin-resistant tuberculosis (MDR/ RR-TB), considering that the spread of MDR/RR-TB is more severe relative to drug-susceptible TB [6,7]. Consequently, continuous molecular surveillance of TB provides valuable insights for the outbreak management and the dynamics of currently circulating subtypes, which is of great importance to formulate effective interventions [8]. ...
In this study, we conducted this population-based study to evaluate the genetic diversity and clustering rate of Mycobacterium tuberculosis (MTB) strains using the whole-genome sequencing (WGS), to better understand its transmission in Ordos.
All patients with culture-positive TB notified in Ordos from January 2021 to December 2022 were recruited. WGS was performed to analyze single-nucleotide polymorphism (SNP) and to identify genotypic drug susceptibilities of MTB isolates.
Overall, a total of 186 patients were included in the present study, of whom 35 (18.8%) had no symptoms suggestive of active TB. Lineage 2 was the predominant MTB sublineage, accounting for 186 of isolates tested. When the pairwise SNP difference ≤ 12 was used as the cutoff for WGS-based clusters, we identified 17 genotypic clusters, and 38 isolates belonged to these 17 clusters, resulting in a clustering rate of 20.4%. The Beijing genotype was an independent factor associating with genomic-clustering (adjusted OR 4.219, 95% CI 0.962–18.502). The overall sensitivity on WGS-based resistance prediction was 85.7% for rifampicin, 73.1% for isoniazid, 60.0% for Ethambutol, 72.7% for streptomycin, and 72.7% for fluoroquinolones.
To conclude, the present study demonstrates the extensive recent transmission of Beijing genotype strains in the community of Ordos. The failure to provide a comprehensive pattern of transmission indicated the missed diagnosis of active TB within the community. A substantial proportion of subclinical TB cases are recognized in the bacteria-positive cases, emphasizing that we must interrupt transmission by finding people with active TB before they infect others.
... 55 The incidence of drug-resistant TB is increasing worldwide, making MDR TB treatment difficult and costly, with many adverse effects on patients. Furthermore, drug resistance is a significant barrier to efficient prevention and control of TB. 56 Thus, TB is a global public health problem and one of the top ten mortality diseases worldwide. 57 Previously, WHO recommended a universal policy for controlling TB called the directly observed treatment short-course (DOTS) approach. ...
Tuberculosis (TB) is a significant public health challenge, especially in developing nations. Developing a TB eradication strategy is hampered by the global health concern of drug-resistant (DR) TB. Effective patient treatment, preventing TB transfer and avoiding the upsurge of DR strains depend primarily on the timely and accurate identification of DR TB. Due to inadequate sensitivity, the necessity of trained laboratory personnel, the sluggish growth pattern of Mycobacterium bacilli in culture, and the small number of bacilli that are usually found in extrapulmonary TB samples, TB diagnosis is still tricky in clinical practice. Although mycobacterial culture is the gold standard to identify TB and determine drug resistance, it takes 2 to 8 weeks to develop. Despite their high cost, nucleic acid amplification tests (NAATs) and whole-genome sequencing (WGS) are the commonly employed molecular-based methods for diagnosing and identifying TB. The WHO suggested the GeneXpert MTB/RIF to identify TB and detect resistance to rifampicin. In comparison, numerous molecular techniques were developed, including allele-specific PCR (MAS-PCR), solid-phase hybridization, real-time PCR (RT-PCR) and droplet digital PCR-based technique (DDPCR). This manuscript is intended to overview the current approaches for the phenotypic and genotypic diagnosis of TB disease and identifying resistance to antitubercular drugs depending on recently published articles, WHO and CDC reports, and commercially available diagnostic tools.
... The Xpert system has been recommended by the WHO, which is a common molecular technology used in clinical practice, including Xpert MTB/RIF and Xpert Ultra [6]. Several molecular diagnostic methods have been designed for the timely and accurate diagnosis of DR-TB, including Abbott RealTime MTB RIF/INH, BD MAX MDR-TB, Cobas MTB RIF/INH, and FluoroType MTBDR [7][8][9]. However, these assays detect a limited number of mutation sites and do not detect the heteroresistance of MTB strains. ...
Purpose
We aimed at evaluating the diagnostic efficacy of a nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF–MS) assay to detect drug resistance of Mycobacterium tuberculosis.
Methods
Overall, 263 M. tuberculosis clinical isolates were selected to evaluate the performance of nucleic MALDI-TOF–MS for rifampin (RIF), isoniazid (INH), ethambutol (EMB), moxifloxacin (MXF), streptomycin (SM), and pyrazinamide (PZA) resistance detection. The results for RIF, INH, EMB, and MXF were compared with phenotypic microbroth dilution drug susceptibility testing (DST) and whole-genome sequencing (WGS), and the results for SM and PZA were compared with those obtained by WGS.
Results
Using DST as the gold standard, the sensitivity, specificity, and kappa values of the MALDI-TOF–MS assay for the detection of resistance were 98.2%, 98.7%, and 0.97 for RIF; 92.8%, 99%, and 0.90 for INH; 82.4%, 98.0%, and 0.82 for EMB; and 92.6%, 99.5%, and 0.94 for MXF, respectively. Compared with WGS as the reference standard, the sensitivity, specificity, and kappa values of the MALDI-TOF–MS assay for the detection of resistance were 97.4%, 100.0%, and 0.98 for RIF; 98.7%, 92.9%, and 0.92 for INH; 96.3%, 100.0%, and 0.98 for EMB; 98.1%, 100.0%, and 0.99 for MXF; 98.0%, 100.0%, and 0.98 for SM; and 50.0%, 100.0%, and 0.65 for PZA.
Conclusion
The nucleotide MALDI-TOF–MS assay yielded highly consistent results compared to DST and WGS, suggesting that it is a promising tool for the rapid detection of sensitivity to RIF, INH, EMB, and MXF.
... Resistance to SLIDs in M. tuberculosis has been linked to mutations in the rr gene, which encodes the 16S rRNA component of the 30S small subunit of the bacterial ribosome [9]. The identification of drug-resistant strains relies on validating the isolates' drug susceptibility pattern using phenotypic techniques or locating altered drug target genes using genotypic technologies [10]. The use of genotypic techniques for the rapid screening of patients at risk for MDR-TB was supported by the WHO based on evidence and professional opinion [11]. ...
Introduction:
The persistence of tuberculosis (TB) infection in some patients after treatment has highlighted the importance of drug susceptibility testing (DST). This study aimed to determine the drug susceptibility patterns of Mycobacterium tuberculosis (M. tuberculosis) isolates from pulmonary TB (PTB) patients in Central and Southern Ethiopia.
Methods:
A health institution-based cross-sectional study was conducted between July 2021 and April 2022. Sputum samples were collected from newly diagnosed smear microscopy and/or Xpert MTB/RIF-positive PTB patients. The samples were processed and cultivated in Lowenstein-Jensen (LJ) pyruvate and glycerol medium. M. tuberculosis isolates were identified using polymerase chain reaction (PCR) based region of difference 9 (RD9) deletion typing. Phenotypic DST patterns of the isolates were characterized using the BACTEC MGIT™ 960 instrument with SIRE kit. Isoniazid (INH) and Rifampicin (RIF) resistant M. tuberculosis isolates were identified using the GenoType® MTBDRplus assay.
Results:
Sputum samples were collected from 350 PTB patients, 315 (90%) of which were culture-positive, and phenotypic and genotypic DST were determined for 266 and 261 isolates, respectively. Due to invalid results and missing data, 6% (16/266) of the isolates were excluded, while 94% (250/266) were included in the paired analysis. According to the findings, 14.4% (36/250) of the isolates tested positive for resistance to at least one anti-TB drug. Gene mutations were observed only in the rpoB and katG gene loci, indicating RIF and high-level INH resistance. The GenoType® MTBDRplus assay has a sensitivity of 42% and a specificity of 100% in detecting INH-resistant M. tuberculosis isolates, with a kappa value of 0.56 (95%CI: 0.36-0.76) compared to the BACTEC MGIT™ DST. The overall discordance between the two methods was 5.6% (14/250) for INH alone and 0% for RIF resistance and MDR-TB (resistance to both INH and RIF) detection.
Conclusion:
This study reveals a higher prevalence of phenotypic and genotypic discordant INH-resistant M. tuberculosis isolates in the study area. The use of whole-genome sequencing (WGS) is essential for gaining a comprehensive understanding of these discrepancies within INH-resistant M. tuberculosis strains.
... A similar observation was made in a recent systematic review, where only two (0.7%) of 290 mutations could be statistically associated with BDQ resistance 8 . The insufficiency of data highlighted by these studies directly impacts the potential accuracy and development of molecular diagnostics to detect BDQ resistance 9 . Alternative methods to determine the molecular mechanisms of BDQ resistance are needed. ...
Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25–0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.
... For example, the rifampicin resistance in Mtb (rpoB gene) is analyzed using this method. This is a very sensitive test with a high specificity of 88-100% [86,87]. ...
Tuberculosis has remained a global concern for public health affecting the lives of people for ages. Approximately 10 million people are affected by the disease and 1.5 million succumb to the disease worldwide annually. The COVID-19 pandemic has highlighted the role of early diagnosis to win the battle against such infectious diseases. Thus, advancement in the diagnostic approaches to provide early detection forms the foundation to eradicate and manage contagious diseases like tuberculosis. The conventional diagnostic strategies include microscopic examination, chest X-ray and tuberculin skin test. The limitations associated with sensitivity and specificity of these tests demands for exploring new techniques like probe-based assays, CRISPR-Cas and microRNA detection. The aim of the current review is to envisage the correlation between both the conventional and the newer approaches to enhance the specificity and sensitivity. A significant emphasis has been placed upon nanodiagnostic approaches manipulating quantum dots, magnetic nanoparticles, and biosensors for accurate diagnosis of latent, active and drug-resistant TB. Additionally, we would like to ponder upon a reliable method that is cost-effective, reproducible, require minimal infrastructure and provide point-of-care to the patients.
