Drug resistance and genotypes of strains of Mycobacterium tuberculosis isolated from human immunodeficiency virus-infected and non-infected tuberculosis patients in Bauru,São Paulo,Brazil

Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of S o Paulo. The objective of this study was to evaluate risk factors for transmission of Mycobacterium tuberculosis strains in this area. Strains were collected from patients attended at ambulatory services in the region and susceptibility towards the main first line antibiotics was determined and fingerprinting performed. A total of 57 strains were submitted to susceptibility testing: 23 (42.6%) were resistant to at least one drug while 3 (13%) were resistant against both rifampicin and isoniazide. Resistant strains had been isolated from patients that had not (n = 13) or had (n = 9) previously been submitted to anti-TB treatment, demonstrating a preoccupying high level of primary resistance in the context of the study. All strains were submitted to IS6110 restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element PCR (DRE-PCR). Using IS6110-RFLP, 26.3% of the strains were clustered and one cluster of 3 patients included 2 HIV-infected individuals that had been hospitalized together during 16 days; clustering of strains of patients from the hospital was however not higher than that of patients attended at health posts. According to DRE-PCR, 55.3% belonged to a cluster, confirming the larger discriminatory power of IS6110-RFLP when compared to DRE-PCR, that should therefore be used as a screening procedure only. No clinical, epidemiological or microbiological characteristics were associated with clustering so risk factors for transmission of TB could not be defined in the present study.     

Prediction of drug resistance in M. tuberculosis: molecular mechanisms,tools, and applications

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.     

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.     

Clinical data and molecular analysis of Mycobacterium tuberculosis isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.     

Whole Genome Sequencing Investigation of a Tuberculosis Outbreak in Port-au-Prince,Haiti Caused by a Strain with a “Low-Level” rpoB Mutation L511P – Insights into a Mechanism of Resistance Escalation

The World Health Organization recommends diagnosing Multidrug-Resistant Tuberculosis (MDR-TB) in high burden countries by detection of mutations in Rifampin (RIF) Resistance Determining Region of Mycobacterium tuberculosis rpoB gene with rapid molecular tests GeneXpert MTB/RIF and Hain MTBDRplus. Such mutations are found in >95% of Mycobacterium tuberculosis strains resistant to RIF by conventional culture-based drug susceptibility testing (DST). However routine diagnostic screening with molecular tests uncovered specific “low level” rpoB mutations conferring resistance to RIF below the critical concentration of 1 μg/ml in some phenotypically susceptible strains. Cases with discrepant phenotypic (susceptible) and genotypic (resistant) results for resistance to RIF account for at least 10% of resistant diagnoses by molecular tests and urgently require new guidelines to inform therapeutic decision making. Eight strains with a “low level” rpoB mutation L511P were isolated by GHESKIO laboratory between 2008 and 2012 from 6 HIV-negative and 2 HIV-positive patients during routine molecular testing. Five isolates with a single L511P mutation and two isolates with double mutation L511P&M515T had MICs for RIF between 0.125 and 0.5 μg/ml and tested susceptible in culture-based DST. The eighth isolate carried a double mutation L511P&D516C and was phenotypically resistant to RIF. All eight strains shared the same spoligotype SIT 53 commonly found in Haiti but classic epidemiological investigation failed to uncover direct contacts between the patients. Whole Genome Sequencing (WGS) revealed that L511P cluster isolates resulted from a clonal expansion of an ancestral strain resistant to Isoniazid and to a very low level of RIF. Under the selective pressure of RIF-based therapy the strain acquired mutation in the M306 codon of embB followed by secondary mutations in rpoB and escalation of resistance level. This scenario highlights the importance of subcritical resistance to RIF for both clinical management of patients and public health and provides support for introducing rpoB mutations as proxy for MICs into laboratory diagnosis of RIF resistance. This study illustrates that WGS is a promising multi-purpose genotyping tool for high-burden settings as it provides both “gold standard” sequencing results for prediction of drug susceptibility and a high-resolution data for epidemiological investigation in a single assay.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

The Rationale for Using Rifabutin in the Treatment of MDR and XDR Tuberculosis Outbreaks

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.     

The Burden of Drug-Resistant Tuberculosis in Papua New Guinea: Results of a Large Population-Based Survey

Background

Reliable estimates of the burden of multidrug-resistant tuberculosis (MDR-TB) are crucial for effective control and prevention of tuberculosis (TB). Papua New Guinea (PNG) is a high TB burden country with limited information on the magnitude of the MDR-TB problem.

Methods

A cross-sectional study was conducted in four PNG provinces: Madang, Morobe, National Capital District and Western Province. Patient sputum samples were tested for rifampicin resistance by the Xpert MTB/RIF assay and those showing the presence of resistance underwent phenotypic susceptibility testing to first- and second-line anti-TB drugs including streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamide, ofloxacin, amikacin, kanamycin and capreomycin.

Results

Among 1,182 TB patients enrolled in the study, MDR-TB was detected in 20 new (2.7%; 95% confidence intervals [CI] 1.1–4.3%) and 24 previously treated (19.1%; 95%CI: 8.5–29.8%) TB cases. No case of extensively drug-resistant TB (XDR-TB) was detected. Thirty percent (6/20) of new and 33.3% (8/24) of previously treated cases with MDR-TB were detected in a single cluster in Western Province.

Conclusion

In PNG the proportion of MDR-TB in new cases is slightly lower than the regional average of 4.4% (95%CI: 2.6–6.3%). A large proportion of MDR-TB cases were identified from a single hospital in Western Province, suggesting that the prevalence of MDR-TB across the country is heterogeneous. Future surveys should further explore this finding. The survey also helped strengthening the use of smear microscopy and Xpert MTB/RIF testing as diagnostic tools for TB in the country.     

Detection of Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates Using Four Molecular Methods in China

To evaluate the relationship between mutations in rpsL or rrs genes and streptomycin (SM) resistance, we compared four molecular methods for their clinical value in the detection of SM resistance. Genotypic analysis of SM resistance in 167 M. tuberculosis clinical strains isolated from Chinese patients was performed by direct DNA sequencing, SSCP, RFLP, and reverse dot-blot hybridization (RDBH) assays. Of the 98 SM-resistant isolates, 78 (79.6%) had missense mutations in codon 43 or 88 of rpsL resulting in a Lys to Arg substitution, 6 (6.1%) had mutations of the rrs gene at positions 513 A to C or T or 516 C to T, and 14 (14.3%) had the wild-type sequence. None of the 69 SM-susceptible isolates examined had alterations in rpsL or rrs. The results of the SSCP, RFLP, and RDBH analyses for these mutations and wild-type sequences were completely consistent with DNA sequencing data. Five distinct single-nucleotide substitutions in codon 43 or 88 of rpsL gene or in position 513 or 516 of rrs gene were correctly identified in 84 of 98 (85.7%) phenotypically SM-resistant isolates by RDBH assay. Molecular analyses of the rpsL and rrs genes are useful for rapid prediction of SM resistance in most clinical strains of M. tuberculosis. Reverse dot-blot hybridization assay is a rapid, simple, and reliable method for the detection of drug resistance.     

New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.     

Estimating fitness by competition assays between drug susceptible and resistant Mycobacterium tuberculosis of predominant lineages in Mumbai, India

Background

Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages.

Methodology

Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system.

Conclusion/Significance

While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains.     

Treatment of tuberculosis in a region with high drug resistance: outcomes, drug resistance amplification and re-infection

Introduction

Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries.

Methods

We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment.

Results

At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome.

Conclusion

In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.     

Interaction of wild type, G68R and L125M isoforms of the arylamine-N-acetyltransferase from Mycobacterium tuberculosis with isoniazid: a computational study on a new possible mechanism of resistance

Isoniazid (INH) is a front-line drug used in the treatment of tuberculosis (TB), a disease that remains a major cause of death worldwide. Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase-peroxidase (CP) enzyme. Recent studies have suggested that acetylation of INH by the arylamine-N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) may be a possible cause of inactivation of the drug thus resulting in resistant strains. In this study, computational techniques were applied to investigate the binding of isoniazid to three TBNAT isoforms: wild type, G68R and L125M. Since there is no experimental structure available, molecular dynamics (MD) simulations were initially used for the refinement of TBNAT homology models. Distinct conformations of the models were selected during the production stage of MD simulations for molecular docking experiments with the drug. Finally, each mode of binding was refined by new molecular MD simulations. Essential dynamics (ED) analysis and linear interaction energy calculations (LIE) were used to evaluate the impact of amino acid substitutions on the structural and binding properties of the enzymes. The results suggest that the wild type and the G68R TBNATs have a similar pattern of affinity to INH. On the other hand, the calculated enzyme-INH dissociation constant (KD) was estimated 33 times lower for L125M isoform in comparison with wild type enzyme. This last finding is consistent with the hypothesis that isolated mutations in the tbnat gene can produce M. tuberculosis strains resistant to isoniazid.     

Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.     

Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout

Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.     

Spontaneous emergence of multiple drug resistance in tuberculosis before and during therapy

The emergence of drug resistance in M. tuberculosis undermines the efficacy of tuberculosis (TB) treatment in individuals and of TB control programs in populations. Multiple drug resistance is often attributed to sequential functional monotherapy, and standard initial treatment regimens have therefore been designed to include simultaneous use of four different antibiotics. Despite the widespread use of combination therapy, highly resistant M. tb strains have emerged in many settings. Here we use a stochastic birth-death model to estimate the probability of the emergence of multidrug resistance during the growth of a population of initially drug sensitive TB bacilli within an infected host. We find that the probability of the emergence of resistance to the two principal anti-TB drugs before initiation of therapy ranges from 10(-5) to 10(-4); while rare, this is several orders of magnitude higher than previous estimates. This finding suggests that multidrug resistant M. tb may not be an entirely "man-made" phenomenon and may help explain how highly drug resistant forms of TB have independently emerged in many settings.