Conventional Drug Susceptibility Testing Research Articles

New dynamic microreactor system to mimic biofilm formation and test anti-biofilm activity of nanoparticles.

Microbial biofilms are composed of surface-adhered microorganisms enclosed in extracellular polymeric substances. The biofilm lifestyle is the intrinsic drug resistance imparted to bacterial cells protected by the matrix. So far, conventional drug susceptibility tests for biofilm are reagent and time-consuming, and most of them are in static conditions. Rapid and easy-to-use methods for biofilm formation and antibiotic activity testing need to be developed to accelerate the discovery of new antibiofilm strategies. Herein, a Lab-On-Chip (LOC) device is presented that provides optimal microenvironmental conditions closely mimicking real-life clinical biofilm status. This new device allows homogeneous attachment and immobilization of Pseudomonas aeruginosa PA01-EGFP cells, and the biofilms grown can be monitored by fluorescence microscopy. P. aeruginosa is an opportunistic pathogen known as a model for drug screening biofilm studies. The influence of flow rates on biofilms growth was analyzed by flow simulations using COMSOL® 5.2. Significant cell adhesion to the substrate and biofilm formation inside the microchannels were observed at higher flow rates > 100 µL/h. After biofilm formation, the effectiveness of silver nanoparticles (SNP), chitosan nanoparticles (CNP), and a complex of chitosan-coated silver nanoparticles (CSNP) to eradicate the biofilm under a continuous flow was explored. The most significant loss of biofilm was seen with CSNP with a 65.5% decrease in average live/dead cell signal in biofilm compared to the negative controls. Our results demonstrate that this system is a user-friendly tool for antibiofilm drug screening that could be simply applied in clinical laboratories.Key Points• A continuous-flow microreactor that mimics real-life clinical biofilm infections was developed.• The antibiofilm activity of three nano drugs was evaluated in dynamic conditions.• The highest biofilm reduction was observed with chitosan-silver nanoparticles.

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BackgroundThe current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed.MethodsTo evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results.ResultsThe susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST.ConclusionIn conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Evaluation of Five User-Friendly Whole Genome Sequencing Software for Mycobacterium tuberculosis in Clinical Application.

Whole genome sequencing (WGS) is an increasingly useful tool for tuberculosis (TB) diagnosis and disease management. In this study, we evaluated the utility of user-friendly WGS tools in reporting resistance profiles and identifying lineages of clinical TB isolates from South Korea. Forty clinical samples from TB patients showing discrepancies between their rapid molecular and conventional drug susceptibility tests were used in this study. Among these clinical isolates, 37 strains were successfully evaluated via WGS software, using the GenTB, TB Profiler, PhyResSE, CASTB, and Mykrobe. More accurate and faster susceptibility results could be obtained with isoniazid than with rifampin. Using the phenotypic test as the gold standard, the isoniazid concordance rate between phenotypic drug susceptibility test (DST) and WGS (GenTB: 45.9%, TB profiler: 40.5%, PhyResSE: 40.5%, CASTB: 48.6%, and Mykrobe: 43.2%) was much higher than between phenotypic DST and rapid molecular genotypic DST (18.9%) among the 37 strains. In contrast, the rifampin concordance rate between phenotypic DST and WGS and that between phenotypic DST and rapid molecular genotypic DST was similar (81.1-89.2%). We also found novel mutations associated with INH in katG and ahpC gene region, not covered by the line probe assay. In addition, lineage analysis identified 81.1% of these samples as L2 East Asian lineage strains, and 18.9% as L4 Euro-American lineage strains. WGS may play a pivotal role in TB diagnosis and the detection of drug resistance, genetic diversity, and transmission dynamics in the near future because of its accuracy, speed, and extensibility.

Evolution of Antibacterial Drug Screening Methods: Current Prospects for Mycobacteria.

The increasing resistance of infectious agents to available drugs urges the continuous and rapid development of new and more efficient treatment options. This process, in turn, requires accurate and high-throughput techniques for antimicrobials’ testing. Conventional methods of drug susceptibility testing (DST) are reliable and standardized by competent entities and have been thoroughly applied to a wide range of microorganisms. However, they require much manual work and time, especially in the case of slow-growing organisms, such as mycobacteria. Aiming at a better prediction of the clinical efficacy of new drugs, in vitro infection models have evolved to closely mimic the environment that microorganisms experience inside the host. Automated methods allow in vitro DST on a big scale, and they can integrate models that recreate the interactions that the bacteria establish with host cells in vivo. Nonetheless, they are expensive and require a high level of expertise, which makes them still not applicable to routine laboratory work. In this review, we discuss conventional DST methods and how they should be used as a first screen to select active compounds. We also highlight their limitations and how they can be overcome by more complex and sophisticated in vitro models that reflect the dynamics present in the host during infection. Special attention is given to mycobacteria, which are simultaneously difficult to treat and especially challenging to study in the context of DST.

Characteristics of second-line anti-tuberculosis drug resistance and gene mutation of multidrug-resistant Mycobacterium tuberculosis in Ningbo

Objective To analyze the characteristics of second-line anti-tuberculosis (TB) drugs resistance and gene mutation of multidrug-resistant (MDR) Mycobacterium tuberculosis isolated in Ningbo. Methods In 1597 M. tuberculosis isolates collected in the first drug resistance survey in Ningbo between 2018 and 2019, 133 MDR isolates, including 31 isolated from elderly group and 102 isolated from young and middle age group, were used in the study. All the isolates were detected using RD105 deletion-targeted multiplex PCR for Beijing genotype identification. Gene sequencing with PCR was conducted to detect the mutations of rrs, tlyA, eis, gidB, gyrA and gyrB genes. The conventional drug susceptibility test was used to detect the resistances to 5 second-line anti TB drugs (kanamycin, amikacin, capreomycin, ofloxacin, levofloxacin). Results The multidrug resistance rate of M. tuberculosis isolates was 8.33% (133/1597) in Ningbo. The resistance rates of 133 MDR M. tuberculosis isolates to kanamycin, amikacin, capreomycin, ofloxacin and levofloxacin were 7.52% (10/133), 6.01% (8/133), 3.01% (4/133), 30.08% (40/133) and 29.32% (39/133). The rates of Pre-XDR and XDR of 133 MDR M. tuberculosis isolates were 28.57% (38/133) and 6.01% (8/133). The gene mutation rates of rrs, tlyA, eis, gidB, gyrA and gyrB of 133 MDR M. tuberculosis isolates were 6.77% (9/133), 7.52% (10/133), 1.50% (2/133), 3.76% (5/133), 34.59% (46/133) and 3.01% (4/133). There were 2 rrs mutations, 1 tlyA mutation, 2 eis mutations, 2 gidB mutations, 5 gyrA mutations and 4 gyrB mutations. Single base substitution was the main type. Conclusion The resistance of MDR M. tuberculosis to second-line anti-TB drugs was serious in Ningbo, especially the resistance to fluoroquinolone. It is necessary to strengthen the prevention and control of second-line anti TB drugs resistances of MDR M. tuberculosis.

High resolution melting assay as a reliable method for diagnosing drug-resistant TB cases: a systematic review and meta-analysis

BackgroundTuberculosis (TB) is one of the most contagious infectious diseases worldwide. Currently, drug-resistant Mycobacterium tuberculosis (Mtb) isolates are considered as one of the main challenges in the global TB control strategy. Rapid detection of resistant strains effectively reduces morbidity and mortality of world’s population. Although both culture and conventional antibiotic susceptibility testing are time-consuming, recent studies have shown that high resolution melting (HRM) assay can be used to determine the types of antibiotic resistance. In the present meta-analysis, we evaluated the discriminative power of HRM in detecting all drug-resistance cases of TB.MethodsA systematic search was performed using databases such as Cochrane Library, Scopus, PubMed, Web of Science, and Google Scholar. Related studies on the effect of HRM in the diagnosis of drug-resistant (DR) TB cases were retrieved by April 2021. We used Meta-Disc software to evaluate the pooled diagnostic sensitivity and specificity of HRM for the detection of each type of drug-resistant cases. Finally, diagnostic value of HRM was characterized by summary receiver operating characteristic (SROC) curve and the area under the curve (AUC) method.ResultsOverall 47 studies (4,732 Mtb isolates) met our criteria and were included in the present meta-analysis. Sensitivity, specificity, and AUC of HRM were measured for antibiotics such as isoniazid (93%, 98%, 0.987), rifampin (94%, 97%, 0963), ethambutol (82%, 87%, 0.728), streptomycin (82%, 95%, 0.957), pyrazinamide (72%, 84%, 0.845), fluoroquinolones (86%, 99%, 0.997), MDR-TB (90%, 98%, 0.989), and pan-drug-resistant TB (89%, 95%, 0.973).ConclusionsThe HRM assay has high accuracy for the identification of drug-resistant TB, particularly firs-line anti-TB drugs. Therefore, this method is considered as an alternative option for the rapid diagnosis of DR-TB cases. However, due to heterogeneity of included studies, the results of HRM assays should be interpreted based on conventional drug susceptibility testing.

Ultra-Rapid Drug Susceptibility Testing for Klebsiella pneumoniae Clinical Isolates in 60 Min by SYBR Green I/Propidium Iodide Viability Assay.

BackgroundWe aimed to optimize and validate the drug susceptibility test (DST) assay by SYBR Green I/PI (SG-PI) method using a panel of 89 Klebsiella pneumoniae clinical isolates in comparison with the conventional DST method to three most important antibiotics used for treatment of this bacterial infection, including imipenem, cefmetazole, and gentamicin.MethodsBy staining with SYBR Green I and PI dyes, green fluorescence and red fluorescence, which linearly correlated with the percentages of live and dead or membrane damaged cells, respectively, were used to produce two standard curves to calculate the relative cell membrane impermeable rates for each log and stationary phase cultures. Stationary phase K. pneumoniae cells were used in imipenem and cefmetazole SG-PI DST assay whereas log phase cells were used in the gentamicin assay. The conventional broth microdilution method was used as a gold standard for DST for comparison.ResultsData showed that after antibiotic treatment for 30–60 min, the antibiotic-resistant K. pneumoniae strains had significantly higher numbers of surviving cells than the susceptible strains at different concentrations of imipenem, cefmetazole, and gentamicin, where the average relative membrane impermeable rates were 88.5, 92.5, and 103.8% for resistant clinical strains, respectively, and 9.1, 49.3, and 71.5% for susceptible strains, respectively. Overall, the total concordances between the ultra-rapid SG-PI method and conventional minimal inhibitory concentration assay in diagnosing imipenem, cefmetazole and gentamicin resistance were high and were 96.6% (86/89), 95.4% (83/87), and 95.5% (85/89), respectively.ConclusionWe demonstrate that our novel SG-PI assay can accurately and stably detect resistance to different antibiotics in clinical isolates of K. pneumoniae in an ultra-fast manner in 60–90 min.

Rifampicin resistance in Mycobacterium tuberculosis in Iran: a two-centre study

Multidrug-resistant tuberculosis remains a challenge. In this study, we investigated the incidence of rifampicin (RIF) resistance in Mycobacterium tuberculosis in a large number of pulmonary specimens.A two-center study in Tehran, the capital of Iran, was performed with 6624 pulmonary samples of patients with tuberculosis (TB) who were subjected to detection of RIF-resistant TB by GeneXpert MTB/RIF assay between May 2014 and July 2018. Conventional drug susceptibility testing was performed to confirm the results.Xpert MTB/RIF identified a total of 96 positives for M. tuberculosis, of which 5 (5.3%) samples were found to be RIF-resistant TB. All RIF-resistant and sensitive isolates detected by GeneXpert were phenotypically confirmed by drug susceptibility testing.These results indicated that the Xpert MTB/RIF test can be used as a rapid diagnostic method and can potentially decrease the morbidity associated with diagnostic delay and mistreatment.

Performance of Wayne assay for detection of pyrazinamide resistance in Mycobacterium tuberculosis: a meta-analysis study

Conventional culture-based drug susceptibility testing (DST) of Mycobacterium tuberculosis to pyrazinamide (PZA) is time-consuming and difficult to perform. The current systematic review and meta-analysis was aimed to evaluate the diagnostic accuracy of Wayne assay against culture-based DSTs as the reference standard. We searched the MEDLINE/Pubmed, Embase, and Web of Science databases for the relevant records. The QUADAS-2 tool was used to assess the quality of the studies. Diagnostic accuracy measures (i.e., sensitivity and specificity) were pooled with a random-effects model. Statistical analyses were performed with STATA (version 14, Stata Corporation, College Station, TX, USA), RevMan (version 5.3; The Nordic Cochrane Centre, the Cochrane Collaboration, Copenhagen, Denmark), and Meta-DiSc (version 1.4, Cochrane Colloquium, Barcelona, Spain). A total of 31 articles comprising data for 2457 isolates of M. tuberculosis were included in the final analysis. The pooled sensitivity and specificity of the Wayne assay against all reference tests (the combination of BACTEC MGIT 960, BACTEC 460, and proportion method) were 86.6% (95% CI: 84.3-88.7) and 96.0% (95% CI: 94.8-97). The positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and the area under the curve (AUC) estimates were found to be 17.6 (95% CI: 10.5-29.3), 0.11 (95% CI: 0.06-0.20), 164 (95% CI: 83-320) and 97%, respectively. Deek's test result indicated no evidence for publication bias (P > 0.05).Although the current study shows that the Wayne test is sensitive and specific for detecting PZA resistance, it may be used in combination with conventional DSTs to diagnose PZA resistance accurately.

Comparative in vitro susceptibility of a novel fluoroquinolone antibiotic candidate WFQ-228, levofloxacin, and moxifloxacin against Mycobacterium tuberculosis

ObjectiveWFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. MethodsWe selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. ResultsMFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. ConclusionWFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.

Molecular characterization of multidrug-resistant tuberculosis against levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, and delamanid in southwest of China

ObjectivesTo explore the drug susceptibility of levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ) and delamanid (DLM) against multidrug resistant tuberculosis (MDR-TB) isolates from drug resistance survey of southwest China, and to illustrate the genetic characteristics of MDR-TB isolates with acquired drug resistance.MethodsA total of 339 strains were collected from smear-positive TB patients in the drug resistance survey of southwest China between January 2014 and December 2016. The MICs for the above mentioned drugs were determined for MDR-TB by conventional drug susceptibility testing. Genes related to drug resistance were amplified with their corresponding pairs of primers.ResultsMDR was observed in 88 (26.0%; 88/339) isolates. LFX had the highest resistance rate (50.0%; 44/88), followed by MFX (38.6%; 34/88). The resistance rate to LZD, CFZ, and DLM was 4.5% (4/88), 3.4% (3/88), and 4.5% (4/88), respectively, and the lowest resistance rate was observed in BDQ (2.3%; 2/88). Of the 45 isolates resistant to LFX and MFX, the most prevalent resistance mutation was found in gyrA with the substitution of codon 94 (34/45, 75.6%). Two strains with CFZ - BDQ cross resistance had a mutation in the Rv0678 gene. Of the four LZD resistant isolates, two carried mutations in rplC gene. For the four isolates resistant to DLM, one isolate had mutations in codon 318 of fbiC gene, and two isolates were with mutations in codon 81 of ddn gene.ConclusionThis study provided evidence of the usefulness of new anti-TB drugs in the treatment of MDR-TB in China.

Tuberculosis Drug Susceptibility, Treatment, and Outcomes for Belarusian HIV-Positive Patients with Tuberculosis: Results from a National and International Laboratory.

Background To cure drug-resistant (DR) tuberculosis (TB), the antituberculous treatment should be guided by Mycobacterium tuberculosis drug-susceptibility testing (DST). In this study, we compared conventional DST performed in Minsk, Belarus, a TB DR high-burden country, with extensive geno- and phenotypic analyses performed at the WHO TB Supranational Reference Laboratory in Copenhagen, Denmark, for TB/HIV coinfected patients. Subsequently, DST results were related to treatment regimen and outcome. Methods Thirty TB/HIV coinfected patients from Minsk were included and descriptive statistics applied. Results Based on results from Minsk, 10 (33%) TB/HIV patients had drug-sensitive TB. Two (7%) had isoniazid monoresistant TB, 8 (27%) had multidrug-resistant (MDR) TB, 5 (17%) preextensive drug-resistant (preXDR) TB, and 5 (17%) had extensive drug-resistant (XDR) TB. For the first-line drugs rifampicin and isoniazid, there was DST agreement between Minsk and Copenhagen for 90% patients. For the second-line anti-TB drugs, discrepancies were more pronounced. For 14 (47%) patients, there were disagreements for at least one drug, and 4 (13%) patients were classified as having MDR-TB in Minsk but were classified as having preXDR-TB based on DST results in Copenhagen. Initially, all patients received standard anti-TB treatment with rifampicin, isoniazid, pyrazinamide, and ethambutol. However, this was only suitable for 40% of the patients based on DST. On average, DR-TB patients were changed to 4 (IQR 3-5) active drugs after 1.5 months (IQR 1-2). After treatment adjustment, the treatment duration was 8 months (IQR 2-11). Four (22%) patients with DR-TB received treatment for >18 months. In total, sixteen (53%) patients died during 24 months of follow-up. Conclusions We found high concordance for rifampicin and isoniazid DST between the Minsk and Copenhagen laboratories, whereas discrepancies for second-line drugs were more pronounced. For patients with DR-TB, treatment was often insufficient and relevant adjustments delayed. This example from Minsk, Belarus, underlines two crucial points in the management of DR-TB: the urgent need for implementation of rapid molecular DSTs and availability of second-line drugs in all DR-TB high-burden settings. Carefully designed individualized treatment regimens in accordance with DST patterns will likely improve patients' outcome and reduce transmission with drug-resistant Mycobacterium tuberculosis strains.

Value of routine whole genome sequencing for Mycobacterium tuberculosis drug resistance detection

Routine whole genome sequencing (WGS) of pathogens is becoming more feasible as sequencing costs decrease and access to benchtop sequencing equipment and bioinformatics pipelines increases. This study examined the added value gained from implementing routine WGS of all Mycobacterium tuberculosis isolates in New South Wales, Australia.Drug resistance markers inferred from WGS data were compared to commercial genotypic drug susceptibility testing (DST) assays and conventional phenotypic DST in all isolates sequenced between 2016 and 2019. Of the 1107 clinical M. tuberculosis isolates sequenced, 29 (2.6%) were multi-drug resistant (MDR); most belonged to Beijing (336; 30.4%) or East-African Indian (332; 30%) lineages. Compared with conventional phenotypic DST, WGS identified an additional 1% of isolates which were likely drug resistant, explained by mutations previously associated with treatment failure and mixed bacterial populations. However, WGS provided a 20% increase in drug resistance detection in comparison with commercial genotypic assays by identifying mutations outside of the classic resistance determining regions in rpoB, inhA, katG, pncA and embB genes. Gains in drug resistance detection were significant (p = 0.0137, paired t-test), but varied substantially for different phylogenetic lineages.In low incidence settings, routine WGS of M. tuberculosis provides better guidance for person-centered management of drug resistant tuberculosis than commercial genotypic assays.

In-depth analysis of multi- and extensively drug-resistant mycobacterium tuberculosis isolates in the capital of iran

Aims and objectives: The emergence and spread of multidrug resistant (MDR) Mycobacterium tuberculosis (M. tuberculosis) strains is a critical global health problem. In order to shorten the course of treatment and its effectiveness, it is essential to gain an in-depth insight into the genetic diversity, drug resistance mechanisms and host-pathogen interactions of drug resistant M. tuberculosis. Methods: Between 2014 and 2018, 606 MTBC strains were isolated from 13,892 suspected pulmonary tuberculosis (TB) patients in Tehran, Iran, including 16 MDR-TB cases. A combination of phenotypic (conventional drug susceptibility testing, MIC, Verapamil effectiveness) and genotypic methods (whole-genome sequencing and qPCR) was employed for the identification of additional drug resistances, the role of efflux pumps and strain-to-strain genetic distances as a marker for recent transmission events. Finally, the consequences of mutations in genes encoding the beta subunit of RNA polymerase (rpoB) for host-pathogen interactions was investigated by means of THP-1-derived macrophages infection and Cytokines and Chemokines Real-Time RT2 Profiler PCR Array. Results: MDR and extensively drug-resistant (XDR) TB cases were almost exclusively infected by lineage 2/Beijing strains (14/16, P < 0.001). Out of the 16 MDR/pre-XDR/XDR isolates, 6 (37.5%) and 3 (18.8%) isolates demonstrated a significant decrease in rifampicin (RIF) MIC and isoniazid (INH) MIC due to the Verapamil exposure (64 μg/mL), respectively. More importantly, it was found that the administration of Verapamil profoundly decreased the MIC of Bedaquiline (BDQ) among most of the studied clinical isolates. Moreover, the efflux pump genes of Rv2938, Rv2936, Rv1145, Rv1146, Rv933, Rv1250, Rv876, Rv2333, Rv2459, Rv849, and Rv1819 were overexpressed in the presence of anti-TB drugs, showing the contribution of these efflux pumps to the overall resistance phenotype. Finally, the macrophages immune responses induced in response to infection with drug-resistant M. tuberculosis (RMR strain) may not be substantially different from drug susceptible M. tuberculosis (H37Rv strain), but the severity of the responses was different in some cases specially IL-6, IL-8, CNTF, IL-13, TNFSF10 and CD40LG in response to RMR and H37Rv strains. In addition, RMR strain showing significantly higher intracellular bacillary growth compared to H37Rv strain. Conclusions: The crucial finding is that almost all of our MDR/pre-XDR/XDR strains are of the Beijing lineage (14/16) and more than half of them are probably from recent transmission (8/14). Our results clearly showed that efflux systems, besides spontaneous mutations, play a role in the development of INH/RIF resistance. In addition, Verapamil was very successful at the phenotypic level specially in response to BDQ. Finally, the results initially demonstrate that the M. tuberculosis carrying the rpoB-S450L can modulate macrophage responses to mediate bacterial survival.

Genome Research for Asian Tuberculosis (GReAT)

Aims and objectives: To establish a Mycobacterium tuberculosis genome database in collaboration with Asian countries, and to utilise the data for genotypic drug susceptibility and testing molecular epidemiology Methods: M. tuberculosis isolates collected in each participating country based on drug resistance and/or prevalence survey was collected as bacteria or genomic DNA, and whole genome sequencing was performed using Illumina sequencers. As a meta-data, the minimum inhibitory concentration (MIC) and conventional drug susceptibility testing (DST) data were collected for each isolate. Results: Approximately 5,000 genome data has been collected from Japan, China, Korea, Taiwan, Philippines, Vietnam, and Mongolia. Approximately 75% of isolates belonged to lineage 2.2.1. Base on the MIC and mutations/indels data, some confident mutations/indels were identified. With the data, the sensitivity of genetic DST for pyrazinamide was increased from 60.1% to 97.2% without losing specificity. Conclusions: Genome database will be a basis of genotypic DST and molecular epidemiology. However, the confidence of meta-data including MIC and other clinical information will be the key factor of genome database. In addition, genome data solution programme/software will be of great importance.

Metabolic profiles of multidrug resistant and extensively drug resistant Mycobacterium tuberculosis unveiled by metabolomics

Although treatable with antibiotics, tuberculosis is a leading cause of death. Mycobacterium tuberculosis antibiotic resistance is becoming increasingly common and disease control is challenging. Conventional drug susceptibility testing takes weeks to produce results, and treatment is often initiated empirically. Therefore, new methods to determine drug susceptibility profiles are urgent. Here, we used mass-spectrometry-based metabolomics to characterize the metabolic landscape of drug-susceptible (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis. Direct infusion mass spectrometry data showed that DS, MDR, and XDR strains have distinct metabolic profiles, which can be used to predict drug susceptibility and resistance. This was later confirmed by Ultra-High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry, where we found that levels of ions presumptively identified as isoleucine, proline, hercynine, betaine, and pantothenic acid varied significantly between strains with different drug susceptibility profiles. We then confirmed the identification of proline and isoleucine and determined their absolute concentrations in bacterial extracts, and found significantly higher levels of these amino acids in DS strains, as compared to drug-resistant strains (combined MDR and XDR strains). Our results advance the current understanding of the effect of drug resistance on bacterial metabolism and open avenues for the detection of drug resistance biomarkers.

GeneXpert: A new tool for the rapid detection of rifampicin resistance in mycobacterium tuberculosis

Background: present study was planned to evaluate the diagnostic accuracy of GeneXpert assay for the detection of rifampicin resistance in mycobacterium tuberculosis using conventional drug susceptibility testing as gold standard. Methods and materials: This cross-sectional study was conducted at Jinnah Hospital, Lahore,/Allama Iqbal Medical College, Lahore, Pakistan, from January 2012 to December 2014, and comprised clinically and radiologically diagnosed tuberculosis suspected cases. Pulmonary and extra-pulmonary specimens were collected from strong tuberculosis suspects. All specimens were processed for Ziehl Neelsen staining, Lowenstein-Jensen culture and GeneXpert assay. All mycobacterium tuberculosis positive cases on Lowenstein-Jensen culture were further processed for drug susceptibility testing. Results: Of the 2,200 cases, 840(49.46%) were positive for mycobacterium tuberculosis on GeneXpert assay. Of them, 134(15.6%) cases showed rifampicin resistance on GeneXpert assay. The sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert assay for rifampicin resistance were 127(98.3%), 704(99.1%), 127(94.7%) and 704(99.4%), respectively, by comparing the results with drug susceptibility testing. Conclusion: GeneXpert assay was an extremely helpful diagnostic tool for the detection of rifampicin resistance in tuberculosis suspects with fairly high sensitivity and specificity along with short turnout time

Interpretation of Discordant Rifampicin Susceptibility Test Results Obtained Using GeneXpert vs Phenotypic Drug Susceptibility Testing.

BackgroundThe 3-month difference in turnaround time between Xpert and conventional phenotypic drug susceptibility testing (pDST) causes patient treatment challenges when pDST rifampin (RIF) susceptibility results and earlier Xpert results disagree, resulting in unnecessary tuberculosis (TB) patient exposure to toxic second-line drugs. Here, the prevalence of discordant RIF susceptibility test results, specifically Xpert (resistant) vs pDST (susceptible) results, was determined.MethodsTuberculosis patients enrolled between January 2015 and June 2018 at Beijing Chest Hospital who consecutively tested positive for RIF resistance using Xpert then negative using pDST were studied. DNA sequences and minimal inhibitory concentration (MIC) results provided insights for understanding discordant results.ResultsOf 26 826 patients with suggestive TB symptoms undergoing Xpert MTB/RIF testing, 728 diagnosed as RIF-resistant were evaluated. Of these, 118 (16.2%) exhibiting Xpert RIF resistance and phenotypic RIF susceptibility yielded 104 successfully subcultured isolates; of these, 86 (82.7%) harbored rpoB gene RIF resistance–determining region mutations and 18 (17.3%) did not. The Leu511Pro (25.0%) and Leu533Pro (17.3%) mutations were most frequently associated with discordant RIF susceptibility test results. Of the 86 isolates with rpoB mutations, 42 (48.8%) with MICs ≤1.0 mg/L were assigned to the RIF-susceptible group, with Leu511Pro being the most common mutation observed. Isolates with a very low bacterial load were most frequently misdiagnosed as RIF-resistant by Xpert.ConclusionsApproximately one-sixth of RIF-resistant TB isolates identified via Xpert yielded discordant pDST results due to questionable interpretation of specific “disputed” mutations. Thus, a diagnostic flowchart should be used to correctly interpret Xpert RIF resistance results to best guide patient treatment.

Specific gyrA Gene Mutations Correlate with High Prevalence of Discordant Levofloxacin Resistance in Mycobacterium tuberculosis Isolates from Beijing, China

Although rapid, highly sensitive molecular diagnostics tests are useful for diagnosing fluoroquinolone-resistant tuberculosis (TB), results of molecular testing versus conventional sequential phenotypic drug susceptibility testing (pDST) are frequently discordant. This article determined the discordance rate of levofloxacin (LFX) resistance results, obtained via MeltPro TB molecular testing, versus pDST of clinical TB isolates collected in Beijing, China, between January and December 2018. Isolates with discordant results were further subjected to LFX minimal inhibitory concentration (MIC) determinations and DNA sequence analysis to explore causes of discordance. Of 571 total TB cases, 126 (22.1%) were identified as LFX resistant using the MeltPro TB assay. However, 34 of these 126 LFX-resistant isolates yielded LFX-susceptible test results using pDST, for an overall discordance rate of 27.0%. LFX MICs mainly clustered around the critical LFX concentration, with 7 (21.2%) and 13 (39.4%) of isolates exhibiting MICs of 2.0 and 4.0 mg/L, respectively. The most prevalent LFX resistance mutations associated with discordant results were involved in DNA gyrase subunit A amino acid substitutions Ala90Val (13, 39.4%) and Asp94Ala (11, 33.3%). Notably, more than one-quarter of isolates deemed LFX resistant via the MeltPro assay were scored as LFX susceptible on the basis of pDST results. Ultimately, highly discordant LFX-resistance test results were associated with specific gyrA mutations in isolates with MICs approaching the critical LFX concentration.

A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay.

Rapid and accurate diagnosis of multidrug-resistant tuberculosis (MDR-TB) is important for timely and appropriate therapy. In this study, a rapid and easy-to-perform molecular test that integrated polymerase chain reaction (PCR) amplification and a specific 96-well microplate hybridization assay, called PCR-ELISA (enzyme-linked immunosorbent assay), were developed for detection of mutations in rpoB, katG, and inhA genes responsible for rifampin (RIF) and isoniazid (INH) resistance and prediction of drug susceptibility in Mycobacterium tuberculosis clinical isolates. We evaluated the utility of this method by using 32 multidrug-resistent (MDR) isolates and 22 susceptible isolates; subsequently, we compared the results with data obtained by conventional drug susceptibility testing and DNA sequencing. The sensitivity and specificity of the PCR-ELISA test were 93.7% and 100% for detecting RIF resistance, and 87.5% and 100% for detecting INH resistance, respectively. These results were comparable to those yielded by commercially available molecular tests such as the GenoType MTBDRplus assay. Based on the aforementioned results, we conclude that the PCR-ELISA microplate hybridization assay is a rapid, inexpensive, convenient, and reliable test that will be useful for rapid diagnosis of MDR-TB, for improved clinical care.