scholarly journals Whole Genome Sequencing for the Analysis of Drug Resistant Strains of Mycobacterium tuberculosis: A Systematic Review for Bedaquiline and Delamanid

Antibiotics ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 133
Author(s):  
Luisa Maria Nieto Ramirez ◽  
Karina Quintero Vargas ◽  
Gustavo Diaz
Keyword(s):  
Systematic Review ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Drug Regimens ◽  
Resistant Strains ◽  
One Year ◽  
Drug Resistant Strains ◽  
Different Levels

Tuberculosis (TB) remains the deadliest Infectious disease worldwide, partially due to the increasing dissemination of multidrug and extensively drug-resistant (MDR/XDR) strains. Drug regimens containing the new anti-TB drugs bedaquiline (BDQ) and delamanid (DLM) appear as a last resort for the treatment of MDR or XDR-TB. Unfortunately, resistant cases to these drugs emerged just one year after their introduction in clinical practice. Early detection of resistant strains to BDQ and DLM is crucial to preserving the effectiveness of these drugs. Here, we present a systematic review aiming to define all available genotypic variants linked to different levels of resistance to BDQ and DLM that have been described through whole genomic sequencing (WGS) and the available drug susceptibility testing methods. During the review, we performed a thorough analysis of 18 articles. BDQ resistance was associated with genetic variants in Rv0678 and atpE, while mutations in pepQ were linked to a low-level of resistance for BDQ. For DLM, mutations in the genes ddn, fgd1, fbiA, and fbiC were found in phenotypically resistant cases, while all the mutations in fbiB were reported only in DLM-susceptible strains. Additionally, WGS analysis allowed the detection of heteroresistance to both drugs. In conclusion, we present a comprehensive panel of gene mutations linked to different levels of drug resistance to BDQ and DLM.

scholarly journals Drug susceptibility testing and mortality in patients treated for tuberculosis in high-burden countries

2018 ◽  
Author(s):  
Kathrin Zürcher ◽  
Marie Ballif ◽  
Lukas Fenner ◽  
Sonia Borrell ◽  
Peter M. Keller ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Reference Laboratory ◽  
Drug Resistant ◽  
Hiv Status ◽  
Resistant Tuberculosis ◽  
Resistant Strains ◽  
Drug Resistant Strains

ABSTRACTBackgroundDrug resistance and HIV co-infection are challenges for the global control of tuberculosis.MethodsWe collected Mycobacterium tuberculosis isolates from adult patients in Côte d’Ivoire, Democratic Republic of the Congo, Kenya, Nigeria, South Africa, Peru, and Thailand, stratified by HIV status and tuberculosis drug resistance. Molecular or phenotypic drug susceptibility testing (DST) was done locally and at the Swiss tuberculosis reference laboratory. We examined mortality during treatment according to DST results and treatment adequacy in logistic regression models adjusting for sex, age, sputum microscopy and HIV status.Findings634 tuberculosis patients were included; median age was 33.2 years, 239 (37.7%) were female, 272 (42.9%) HIV-positive and 69 (10.9%) patients died. Based on the reference laboratory DST, 394 (62.2%) strains were pan-susceptible, 45 (7.1%) mono-resistant, 163 (25.7%) multidrug-resistant (MDR-TB), and 30 (4.7%) had pre-extensive or extensive drug resistance (pre-XDR/XDR-TB). Results of reference and local laboratories were discordant in 121 (19.1%) cases, corresponding to a sensitivity of 84.3% and a specificity of 90.8%. In patients with drug-resistant tuberculosis, discordant results were associated with increased mortality (risk ratio 1.81; 95% CI 1.07-3.07). In logistic regression, compared to adequately treated patients with pan-susceptible strains, the adjusted odds ratio for death was 4.23 (95% CI 2.16-8.29) for adequately treated patients with drug-resistant strains and 21.54 (95% CI 3.36-138.1) for inadequately treated patients with drug-resistant strains. HIV status was not associated with mortality.InterpretationUsing a reference laboratory standard, inaccurate DST leading to inappropriate treatment of drug-resistant tuberculosis, but not HIV infection, contributed to mortality.

scholarly journals Non-pncAGene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

2017 ◽  
Vol 55 (6) ◽  
pp. 1920-1927 ◽  
Author(s):  
Jim Werngren ◽  
Erik Alm ◽  
Mikael Mansjö
Keyword(s):  
Susceptibility Testing ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Molecular Techniques ◽  
Content Type ◽  
Resistant Strains ◽  
Clear Majority

ABSTRACTPyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB).pncAgene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-STB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncAgene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panDorrpsAmutations identified by whole-genome sequencing (WGS) (n= 7), heteroresistance (n= 8), and mixed populations withMycobacterium avium(n= 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-Rclinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncAmutation (Thr87Met), whereas a nonsynonymousrpsAmutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanDmutation, while all 15 of the MDRPZA-Risolates harbored a nonsynonymouspncAmutation. Our findings indicate that it is necessary to consider the occurrence ofpanDmutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsAmutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsAin PZA resistance.

scholarly journals rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199759
Author(s):  
Mei-Chun Zeng ◽  
Qing-Jun Jia ◽  
Lei-Ming Tang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rural Areas ◽  
Gene Mutations ◽  
Gene Sequencing ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Extensively Drug Resistant

Objective The aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China. Methods We prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing. Results A total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR. Conclusions The frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

scholarly journals Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

2011 ◽  
Vol 56 (3) ◽  
pp. 1253-1257 ◽  
Author(s):  
J. Werngren ◽  
E. Sturegård ◽  
P. Juréen ◽  
K. Ängeby ◽  
S. Hoffner ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Wild Type ◽  
First Line ◽  
Content Type ◽  
Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

scholarly journals Light Forge: A Microfluidic DNA Melting-based Tuberculosis Test

2020 ◽  
Vol 5 (3) ◽  
pp. 440-453
Author(s):  
Ian M Mbano ◽  
Tawanda Mandizvo ◽  
Jerome Rogich ◽  
Tafara T R Kunota ◽  
Jared S Mackenzie ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Low Income ◽  
Thermodynamic Model ◽  
Nearest Neighbor ◽  
Low Cost ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Initial Development ◽  
Resistant Strains ◽  
Drug Resistant Strains

Abstract Background There is a well-documented lack of rapid, low-cost tuberculosis (TB) drug resistance diagnostics in low-income settings across the globe. It is these areas that are plagued with a disproportionately high disease burden and in greatest need of these diagnostics. Methods In this study, we compared the performance of Light Forge, a microfluidic high-resolution melting analysis (HRMA) prototype for rapid low-cost detection of TB drug resistance with a commercial HRMA device, a predictive “nearest-neighbor” thermodynamic model, DNA sequencing, and phenotypic drug susceptibility testing (DST). The initial development and assessment of the Light Forge assay was performed with 7 phenotypically drug resistant strains of Mycobacterium tuberculosis (M.tb) that had their rpoB gene subsequently sequenced to confirm resistance to Rifampin. These isolates of M.tb were then compared against a drug-susceptible standard, H37Rv. Seven strains of M.tb were isolated from clinical specimens and individually analyzed to characterize the unique melting profile of each strain. Results Light Forge was able to detect drug-resistance linked mutations with 100% concordance to the sequencing, phenotypic DST and the “nearest neighbor” thermodynamic model. Researchers were then blinded to the resistance profile of the seven M.tb strains. In this experiment, Light Forge correctly classified 7 out of 9 strains as either drug resistant or drug susceptible. Conclusions Light Forge represents a promising prototype for a fast, low-cost diagnostic alternative for detection of drug resistant strains of TB in resource constrained settings.

scholarly journals Morphologic characterization of Mycobacterium tuberculosis circulating strains in a Lisbon hospital.

2013 ◽  
Vol 19 (S4) ◽  
pp. 11-12
Author(s):  
C. Silva ◽  
E. Alverca ◽  
A.P. Alves de Matos ◽  
P.A. Carvalho ◽  
I. Portugal ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Drug Susceptibility ◽  
Drug Resistant ◽  
Lowenstein Jensen ◽  
Resistant Strains ◽  
Drug Resistant Strains ◽  
Surface Figure

Tuberculosis (TB) is one of the major causes of mortality and morbidity worldwide accounting for 3.1 million deaths per year. This disease, caused by Mycobacterium tuberculosis (M. tuberculosis) made a deadly comeback, during the 1990’s, triggered mainly by the emergence of acquired immunodeficiency syndrome (AIDS). More recently, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis strains, uncovered the most freighting face of this disease an incurable infection with the currently available therapeutic tools. Although Portugal is considered a medium incidence setting, annually are reported MDR and even XDR TB cases. The majority of these cases occur in the Lisbon area and the strains involved are genetically related being known as Lisboa family.In the present work a group of 283 M. tuberculosis isolates collected in a Lisbon hospital during a two years period (2008-2009) were studied. The morphology of colonies grown on Lowenstein-Jensen slants was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using previously described procedures. The aim of the study was the establishment of a link between mycobacteria drug susceptibility and structure. In the first part of the study approximately 20 isolates, with different drug susceptibility profiles ranging from pan-susceptible to XDR, were grown on Lowenstein-Jensen slants and their morphology was compared. Although all mycobacteria originated rough colonies their size differ with the drug susceptibility profile. The pan-susceptible strains generated larger colonies than drug resistant strains as shown in figure 1.These colonies were then processed for SEM analysis. The results obtained show that mycobacteria surface are distinct in susceptible and drug resistant strains as shown in figure 2.A and B. While drug susceptible mycobacteria have a homogenous surface (Figure 1A), drug resistant bacteria present a heterogeneous surface (Figure 2B) with small protrusions (Fig. 2B inset). In order to evaluate the existence of differences in the ultrastructure of circulating M. tuberculosis strains the colonies were processed and analysed by TEM. For this approach were selected only two isolates: the pan-susceptible R188/09 and the XDR HPV108/09.The results obtained by the analysis of at least 300 bacteria present in non consecutive sections show that mycobacteria cell width (0 350 nm) is similar for both bacteria (Table 1). Nevertheless, their cell length and cell envelope width are significantly different. The XDR strain is shorter (p=0.009) and has a ticker cell envelope (p=0.004) than the pan-susceptible strain. These results are in agreement with those published in the literature.Altogether our data clearly shows the existence of a link between mycobacteria ultrastructure and drug susceptibility. In order to better evaluate these differences a larger number of isolates must be studied. The use of other electron microscopy techniques, such as CEMOVIS, will avoid the formation of undesirable artefacts (e.g. mesosome) produced by dehydration and room temperature sectioning allowing a better characterization of mycobacteria ultrastructure.The authors acknowledge the funding by Fundação para a Ciência e Tecnologia (SFRH/BD/73579/2010, C2008-C2008_P2 and PEst-OE/CTM-UI0084/2011 grants.)

scholarly journals The contribution of microfluidics to the fight against tuberculosis

2021 ◽  
Vol 11 (1) ◽  
pp. 40-54
Author(s):  
Marina Cañadas-Ortega ◽  
Clara Gómez-Cruz ◽  
Juan José Vaquero ◽  
Arrate Muñoz-Barrutia
Keyword(s):  
Low Income ◽  
Drug Testing ◽  
Basic Research ◽  
Microfluidic Devices ◽  
Drug Susceptibility ◽  
Drug Resistant ◽  
Resistant Strains ◽  
Novel Approaches ◽  
On Chip ◽  
Drug Resistant Strains

Abstract The high mortality associated with tuberculosis brings forward the urgency of developing new therapies and strategies against the disease. With the advance of drug-resistant strains, traditional techniques have proven insufficient to manage the disease appropriately. Microfluidic devices have characteristics that can enhance treatment prescription and significantly advance our knowledge about the disease and its interaction within the human body. In addition, microfluidic systems provide advantages in terms of time and costs, which are particularly important in countries with low income and resources. This review will highlight how microdevices can help bridge the gaps in disease management, including their use for drug testing and development, drug susceptibility, basic research, and novel approaches to anti-TB vaccines and organ-on-chip studies.

scholarly journals Molecular characterization of rpoB gene mutations in isolates from tuberculosis patients in Cubal, Republic of Angola

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ariadna Rando-Segura ◽  
María Luisa Aznar ◽  
María Milagros Moreno ◽  
Mateu Espasa Soley ◽  
Elena Sulleiro Igual ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Resistance Pattern ◽  
Tuberculosis Patients ◽  
High Incidence ◽  
Resistant Strains

Abstract Background The importance of Mycobacterium tuberculosis strains with disputed rpoB mutations remains to be defined. This study aimed to assess the frequency and types of rpoB mutations in M. tuberculosis isolates from Cubal, Angola, a country with a high incidence of tuberculosis. Methods All isolates included (n = 308) were analyzed using phenotypic drug susceptibility testing and GenoType MTBDRplus assay. DNA sequencing of the rpoB gene and determination of rifampicin MIC by macrodilution method were additionally performed on isolates yielding discordant results (n = 12) and those in which the mutation detected was not characterized (n = 8). Results In total, 85.1% (74/87) of rifampicin-resistant strains had undisputed rpoB mutations -S450L (49), D435V (15), H445D (3), H445Y (2), Q432ins (1), L449M plus S450F (1), S450F (1), S450W (1) and S450Y (1)-; 10.3% (9/87) had disputed rpoB mutations—L430P plus S493L (1), N437del (1), H445L (3), D435Y (2), L452P (2)-, 2.3% (2.3%) showed no rpoB mutations and 2.3% (2/87) showed heteroresistance—D435Y plus L452P and L430P plus S493L-. Conclusion Disputed rpoB mutations were common, occurring in 10.3% of rifampicin resistant isolates. Current phenotyping techniques may be unable to detect this resistance pattern. To increase their sensitivity, a lower concentration of RIF could be used in these tests or alternatively, rpoB mutations could be screened and characterized in all M. tuberculosis strains.

scholarly journals QUANTITATIVE ANALYSIS OF GYRA AND GYRB KNOWN MUTATIONS IN DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS TREATED WITH OFLOXACIN

2020 ◽  
pp. 86-89
Author(s):  
PUSHPARAJU RAMASAMY ◽  
VIGNESH SOUNDERRAJAN ◽  
SHAKILA HARSHAVARDHAN
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Gene Mutations ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Phenotypic Resistance ◽  
Potential Association ◽  
Proportion Method ◽  
Resistant Strains ◽  
Different Levels

Objective: The aim of the study is to measure the minimum inhibitory concentrations (MICs) of ofloxacin antibiotic from gyrA and gyrB mutations present in fluoroquinolones (FQs) resistant strains of Mycobacterium tuberculosis (MTB) and to further concentrate the potential association between gene mutations and phenotypic resistance based on their MICs. Methods: Different levels of ofloxacin MICs levels were detected in 31 archived multi drug-resistant MTB isolates showing gyrA mutations in codon at A90V, S91P, D94A, D94N/Y, D94 G, and D94H and two gyrB probes (N538D and E540V). The MIC determinations were made using the 1% proportion method. Results: Genotypic assay detected 32 mutations in the gyrA (n=29) and gyrB (n=3) genes among the 31 FQs resistant isolates. Most frequently seen in gyrA mutations at codon D94G (n=16; 50%), these mutations had a clearly elevated MIC level from 2 to 16 μg/ml, that was phenotypically resistant. The A90V mutation region consistently exhibited the lowest levels of ofloxacin resistance, with three out of eight (37.50%) of these isolates had a MIC of <2 μg/ml. In addition, a further strain of S91P mutations for MIC was determined to be less than the critical concentration (CC). These low levels of resistance have been detected in a phenotypic manner which is noticeable in the study. Furthermore, fewer mutations in codons at D94A, D94N/Y were identified. Three wild-type absent isolates from gyrB QRDR were identified and the MIC of those strains for ofloxacin was lower than the critical cutoff value. Conclusion: Based on the results, it is shown that different resistance mutations were associated with different levels of MICs and the current concentration for MGIT will be lowered from 2 μg/ml to 1 μg/ml for the ofloxacin drug.

Sign in / Sign up

Export Citation Format

Share Document