rpoB Gene Of Mycobacterium Tuberculosis Research Articles

Borderline rpoB mutations transmit at the same rate as common rpoB mutations in a tuberculosis cohort in Bangladesh.

The spread of multidrug-resistant tuberculosis (MDR-TB) is a growing problem in many countries worldwide. Resistance to one of the primary first-line drugs, rifampicin, is caused by mutations in the Mycobacterium tuberculosis rpoB gene. So-called borderline rpoB mutations confer low-level resistance, in contrast to more common rpoB mutations which confer high-level resistance. While some borderline mutations show lower fitness in vitro than common mutations, their in vivo fitness is currently unknown. We used a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, representing around 44 % of notified MDR-TB cases over 6 years, to look at differences in transmission clustering between isolates with borderline rpoB mutations and those with common rpoB mutations. We found a relatively low percentage of transmission clustering in the dataset (34.8 %) but no difference in clustering between different types of rpoB mutations. Compensatory mutations in rpoA, rpoB, and rpoC were associated with higher levels of transmission clustering as were lineages two, three, and four relative to lineage one. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster. Our findings show that although borderline rpoB mutations have lower in vitro growth potential this does not translate into lower transmission potential or in vivo fitness. Proper detection of these mutations is crucial to ensure they do not go unnoticed and spread MDR-TB within communities.

Molecular analysis of rpoB gene mutation in MTB detected isolates in a tertiary care centre (AGMC) of North-East, India

BackgroundRifampicin (RIF), one of the first line drug in treatment of tuberculosis. It acts on rpoB gene which encodes RNA polymerase β subunit. In 95% of RIF resistant cases, mutations are present in rpoB gene. Most of them are within 81bp RIF-resistance determining region (RRDR).Xpert MTB/RIF assay has been tremendously revolutionalised the diagnosis of tuberculosis (TB).Also sequencially detect bacteria and resistance to rifampicin (rif).Approximately 96% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide, showed mutations in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene. Here evaluation is made about frequent regions of amplification and mutation in various codons of 81bp of rpoB gene in rif sensitive and rif resistant cases. MethodsA total of 4116 samples were received at Mycobacteriology laboratory, AGMC and processed in CBNAAT.Data of MTB detected samples were collected & statistically analysed to detect frequency of amplification & no amplification in various regions of 81bp of rpoB genes. ResultsOut of 4116 samples, MTB was detected in 1323 samples. Among them 1291 (97.58%) cases were Rif sensitive (RS) and 32 (2.41%) cases were rif resistance (RR).Most of the MTBC detected samples showed amplification in probe A then in probe C.78.12% rif resistant cases showed mutation in either of the probe, commonest is probe E. Study also showed low bacillary loads in most of the RR cases. ConclusionStudy highlighted variations in amplification of different regions of 81bp of rpoB gene in MTBC detected cases. North-east India, like other part of world, also showed highest frequency of mutation in probe E in rif resistant cases.

Assessment of Haematological Parameters in Drug-Resistant TB

Mycobacterium tuberculosis causes tuberculosis (TB), the world's deadliest infectious illness. In addition to the lungs, bone marrow is also impacted by tuberculosis. Significant haematological abnormalities can be found in TB patients. This means that these haematological markers can be used to assess a patient's diagnosis, prognosis, and treatment outcome. The goals of this study are (a) to assess the hematological characteristics of TB patients, and (b) to examine the impact of rifampin (RIF) resistance on the prevalence of mutations in the whole rpoB gene of Mycobacterium tuberculosis. Fifty people participated in the study after being chosen through a systematic random sample process. From each participant, about 4 milliliters of blood were taken from a vein and estimated for the haematological parameters like Hb, WBC, Lymphocyes, neutrophils, Erythrocyte sedimentation rate and platelet count. Fifty tuberculosis isolates had their whole rpoB genes sequenced so that we could analyze the positions of the codons and the frequency with which they occurred. When compared to healthy controls, the values of hemoglobin and other blood indices were significantly lower or abnormal (p<0.05). ESR values were alarmingly increased in the subjects along with platelets (P value < 0.05). All of our 25 isolates exhibited four types of mutations at four RRDR positions (codons 510, 516, 522 and 526). We found codons 526 showed a high level (92%) of RIF resistance or mutations when compared to other positions. An easy and affordable way to forecast the progression of the illness and keep track of complications in underdeveloped nations is to test the haematological parameters of tuberculosis patients. RIF resistance is linked to certain mutations in the rpoB gene that may impact how RpoB and RIF interact. These results may be used to create new antibiotics and create cutting-edge diagnostic tools for TB medication resistance.

Distribution and frequency of common mutations in rpoB gene of Mycobacterium tuberculosis detected by Xpert MTB/RIF and identification of residential areas of rifampicin resistant-TB cases: A first retrospective study from Mizoram, Northeast India

BackgroundRifampicin resistance (RR) is a surrogate marker of multidrug-resistant tuberculosis. The aim of this study is to determine the frequency of the commonly mutated probes for rpoB gene and locate the residential areas of the Rifampicin Resistant-TB (RR-TB) patients in a high endemic zone of Northeast India. MethodsArchived data of 13,454 Xpert MTB/RIF reports were evaluated retrospectively between 2014 and 2021. Socio-demographic and available clinical information were reviewed and analyzed for RR-TB cases only. Logistic Regression was used to analyze the parameters with respect to probe types. P-value ≤ 0.05 was considered to be statistically significant. Results2,894 patients had Mycobacterium tuberculosis infection out of which 460 were RR-TB, which was most prevalent among 25–34 years. The most common mutation occurred in probe A (25.9 %) followed by E (23.5 %), D (9.8 %), B (2.6 %) and the least in C (0.2 %). High prevalence (34.3 %) of probe mutation combinations were also found: probes AB (0.4 %), AD (32.8 %), AE (0.4 %), DE (0.2 %) and ADE (0.4 %). Seventeen patients (3.7 %) were found without any missing probes. RR-TB patients were mostly located in Aizawl North –III, South –II and East –II constituencies. ConclusionThis study provides genetic pattern of drug resistance accountable for RR over the past years in Mizoram which will help local clinicians in the initiation of correct treatment. Also, our findings provide a baseline data on the magnitude of RR-TB within the state and identification of the residential areas can help local health authorities in planning surveillance programs to control the spread of RR-TB.

RpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

ObjectiveTo investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.MethodsWe sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.ResultsResults revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.ConclusionSome mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.

False detection of rifampicin resistance using Xpert® MTB/RIF Ultra assay due to an A451V mutation in Mycobacterium tuberculosis.

BackgroundIn a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert® MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases.MethodsFurther laboratory investigations on the cultured isolates included GenoType MTBDRplus assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the rpoB gene was performed using Sanger sequencing and WGS.ResultsPhenotypic drug susceptibility tests determined the isolates to be rifampicin susceptible. Molecular investigations identified an A451V (codon 532) mutation in the Mycobacterium tuberculosis rpoB gene that has not previously been found to cause rifampicin resistance. Genome sequencing revealed that the three isolates’ genomes differed by ≤5 SNPs, indicating a high likelihood of recent transmission events. Furthermore, a cluster of six related M. tuberculosis isolates from our in-house typing database showed four were highly related; all were rifampicin susceptible and lacked this mutation.ConclusionsFalse detection of rifampicin resistance, albeit rare, should be considered possible with Xpert® MTB/RIF Ultra assay, particularly in low TB incidence settings. Confirmatory sequencing methods should be performed to prevent the unnecessary use of second-line anti-tuberculous drugs.

GeneXpert MTB/RIF Based Detection of Rifampicin Resistance and Common Mutations in rpoB Gene of Mycobacterium Tuberculosis in Tribal Population of District Anuppur, Madhya Pradesh, India

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) continues to be one of the most significant causes of death in the developing countries. Development of Multi Drug Resistance (MDR) and Extremely Drug Resistance (XDR) strains of MTB has been recognised as a major threat. Rapid diagnosis along with drug sensitivity analysis is the prerequisite for effective treatment of TB, especially in rural and remote location settings. Aim: The goal of this study was to investigate the Rifampicin Resistance (RR) using GeneXpert MTB/Rifampicin (RIF) in tribal patients suffering from Pulmonary Tuberculosis (PTB) in District Anuppur, Madhya Pradesh, India. Materials and Methods: Sputum samples were obtained from 413 patients with symptoms of PTB, who visited District Hospital, Anuppur from April 2017- April 2018. Based on clinical symptoms and chest X-ray, GeneXpert MTB/RIF assay was performed for the confirmation of TB and detection of RR. The data was analysed and expressed in percentage. Results: Out of 413 samples, 104 (25.18%) were diagnosed with PTB. Out of 104 TB positive samples, RR was detected in 7(6.73%) samples. The most common mutations conferring RR were located in the region of Probe B (71.42%), followed by Probe C (14.28%) and Probe E (14.28%), while no mutations were found in the region of Probe A and Probe D. Conclusion: Possibly, this is the first report of RR and probe mutational analysis from this tribal region of India. High rate of mutation at Probe B locus may be the chief reason for RR development. Gene sequencing may be carried for understanding the higher rates of mutations at probe B locus.

Sequencing of the entire rpob gene and characterization of mutations in isolates of Mycobacterium tuberculosis circulating in an endemic tuberculosis setting.

To evaluate the use of a sequencing procedure for the entire rpoB gene of Mycobacterium tuberculosis to identify mutations pre-rifampicin resistance determining region (RRDR), within RRDR, and post-RRDR in isolates circulating in a region affected by tuberculosis (TB). Five primers were designed, with which five DNA fragments of rpoB were obtained, sequenced by Sanger, and analysed in silico in order to identify mutations over the entire rpoB gene in rifampicin-sensitive and rifampicin-resistant TB. It was possible to analyse the entire rpoB gene in five rifampicin-sensitive and 15 rifampicin-resistant isolates. Thirty-six mutations were identified. Two mutations were found pre-RRDR, nine within-RRDR and 25 post-RRDR. The most frequent mutations within RRDR were S531L (53%), followed by S512T (20%), all of which were found in rifampicin-resistant isolates. Of the 25 mutations found post-RRDR, 14 were only in resistant isolates, and the most frequent was D853N, which was present in 85% of isolates. Mutations E818K, D836N and T882P were observed in 80% of the rifampicin-resistant and rifampicin-sensitive isolates. The proposed sequencing method allowed identification of mutations in the entire rpoB gene. This procedure represents a useful tool for diagnosing rifampicin resistance. The number of mutations that were found raises new questions about the diversity of mutations in the rpoB gene and their role in rifampicin resistance in regions where TB is endemic.

Detection of Ser450Leu mutation in rpoB gene of Mycobacterium tuberculosis by allele-specific loop-mediated isothermal DNA amplification method

To identify genetic mutations a rather time-consuming and expensive method of polymerase chain reaction (PCR) is widely used. The aim of the present work was to evaluate the possibility of using the two schemes of the method of allele-specific isothermal loop amplification (LAMP) to detect the TCG/TTG (S450L) mutation in the rpoB gene of Mycobacterium tuberculosis. 48 clinical isolates of M. tuberculosis and 11 samples of sputum were used, randomized and obtained in the microbiological laboratory of the city of Novosibirsk from incident patients. It is shown that the use of an analysis scheme using the allele-specific primer FIP compared to F3 has the best resolution: the difference between the amplification time of the mutation and the wild type allele was 22 ± 2,4 versus 13 ± 4,1 minutes (p = 0,0011). When using 100 DNA genomic equivalents a true positive signal (amplification of the rpoB gene with a mutation using the corresponding allele-specific primer) was detected after 29,4 ± 3,4 minutes. A positive signal was visualized after adding SYBR Green I to the reaction, both when illuminated with daylight and when using a UV transilluminator. Using the developed method the DNA sample of 20 RIFR isolates from M. tuberculosis was analyzed containing the Ser450Leu mutation in the rpoB gene, 10 RIFR isolates containing other mutations in the rpoB gene and 18 RIFs isolates without any mutations; the presence of mutations in the samples was determined using classical Sanger sequencing. The sensitivity and specificity of LAMP for detecting a Ser450Leu mutation in the rpoB gene was 100%. This approach allows the use of crude lysates of mycobacteria as DNA, which reduces the total analysis time to 1,5 hour.

Detection of ser450leu mutation in rpoB gene of mycobacterium tuberculosis by allele-specific loop-mediated isothermal dna amplification method

Для выявления генетических мутаций широко используют достаточно трудоемкий и дорогостоящий метод полимеразной цепной реакции (ПЦР). Целью работы было оценить возможность применения двух схем метода аллель-специфичной изотермической петлевой амплификации (loop-mediated isothermal amplification, LAMP) для выявления мутации TCG/TTG (S450L) в гене rpoB Mycobacterium tuberculosis. Использовали 48 клинических изолятов M. tuberculosis и 11 образцов мокроты, выбранных случайным образом и полученных в микробиологической лаборатории г. Новосибирска от пациентов с впервые выявленным заболеванием. Показано, что применение схемы анализа с использованием аллель-специфичного праймера FIP по сравнению с F3 имеет лучшую разрешающую способность: разница между временем амплификации мутации и аллеля дикого типа составила 22 ± 2,4 против 13 ± 4,1 мин (p = 0,0011). При использовании 100 геном-эквивалентов ДНК истинно положительный сигнал (амплификация гена rpoB с мутацией при использовании соответствующего аллель-специфического праймера) детектировался после 29,4 ± 3,4 мин. Положительный сигнал визуализировался после добавления в реакцию SYBR Green I, как при освещении дневным светом, так и при использовании трансиллюминатора с УФ-излучением. С помощью разработанного нами метода была проанализирована выборка ДНК 20 RIFR изолятов M. tuberculosis, несущих мутацию Ser450Leu в гене rpoB, 10 RIFR изолятов, несущих другие мутации в гене rpoB, а также 18 RIFs изолятов без мутаций; наличие мутаций в образцах было определено с помощью классического секвенирования по Сенгеру. Чувствительность и специфичность LAMP для выявления мутации Ser450Leu в гене rpoB составили 100%. Данный подход позволяет использовать в качестве ДНК грубые лизаты микобактерий, что сокращает тотальное время анализа до 1,5 ч.

RpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates.

Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

MUTATIONS IN THE RPOB GENE OF MYCOBACTERIUM TUBERCULOSIS IDENTIFIED BY SEQUENCING METHOD

ABSTRACTObjective: To identify the mutation in the rpoB gene of Mycobacterium tuberculosis (MTB), using by sequencing method from pulmonary specimensof presumptive TB patients belonging to the districts of Tamil Nadu.Methods: A total of 8697 clinical specimens of presumptive MTB patients were collected from various districts of Tamil Nadu. Smear microscopy wasperformed by light emitting diode fluorescent microscopy and all the smear positive samples were tested using line probe assay (LPA) to detect thepercentage of drug resistance pattern and to identify the missing mutation in LPA by the sequencing method.Results: Among 4897 smear positives subjected to LPA method; 407 (8.3%) MTB was not detected and 16 (0.3%) showed invalid result; 4473 (91.4%)strains showed MTB positive; 3695 (82.6%) were sensitive for both rifampicin (RIF) and isoniazid (INH) drugs; 502 (11.2%) were resistance for INH;73 (1.6%) resistant for RIF; 203 (4.5%) were resistance for RIF and INH. Totally, 52 (1.2%) strains results cannot be confirmed by LPA and reportedas sensitive for RIF, because of the faint and the missing bands in both wild type and mutation. These strains were sequenced and 39 (75%) strainsshowed resistant to RIF.Conclusion: Hence LPA may be the molecular technology for the rapid, feasible and reliable method for the detection of multidrug resistant mutationbut few confusion bands cannot be reported as resistance, which should be confirmed by either conventional phenotypic drug susceptibility testingor by sequencing method.Keywords: Line probe assay, Sequencing, Mutation, Multidrug resistant tuberculosis.

Epidemiology of Rifampicin Resistant Tuberculosis and Common Mutations in rpoB Gene of Mycobacterium tuberculosis: A Retrospective Study from Six Districts of Punjab (India) Using Xpert MTB/RIF Assay.

Background:Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in <2 h. The RIF resistance pattern in Malwa region of Punjab, India, is not documented. Here, we report the epidemiology of RIF-resistant TB and mutations in rpoB gene of Mycobacterium tuberculosis (MTB).Materials and Methods:A total of 1612 specimens received between October 2013 and February 2015 were tested by Xpert MTB/RIF assay following manufacturer's instructions. The results thus obtained were analyzed using SPSS version 20.0.0 (SPSS Inc., Chicago, IL, USA) statistical software.Result:RIF resistance was statistically higher in previously treated patients in comparison to the new patients (P = 0.006) and in patients with acid fast-Bacilli (AFB) positive smears to AFB-negative smears (P = 0.048). RIF resistance mutations in 130 specimens revealed frequency of E 73/130 (56%), B 28/130 (21.5%), D 18/130 (13.8%), A 11/130 (8.4%), and C 1/130 (0.7%) while in one specimen, mutation combination, i.e., mutations associated with more than one probe (A and B both) was present.Conclusion:Xpert MTB/RIF assay is a user-friendly screening tool for detection of MTB and RIF resistance from suspected TB/MDR cases in a shorter period of time. It could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of RIF resistance in MTB isolates.

E-DNA sensor of Mycobacterium tuberculosis based on electrochemical assembly of nanomaterials (MWCNTs/PPy/PAMAM).

Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose.

Development of impedimetric DNA biosensor for selective detection and discrimination of oligonucleotide sequences of the rpoB gene of Mycobacterium tuberculosis

Impedimetric DNA biosensor for the detection of oligonucleotide sequences of the rpoB gene of Mycobacterium tuberculosis, mutations of which cause resistance to anti-tuberculosis drug rifampicin, is developed. A bioselective element of the biosensor was formed on the gold electrode surface by immobilization of thiolated single-stranded DNA probes P2, which reproduce a corresponding fragment of the rpoB gene, and by passivation of the electrode surface (to reduce the non-specific adsorption) using 6-mercapto-1-hexanol. High selectivity of this biosensor has been shown: at injection of 1nM complementary oligonucleotides T2, the charge transfer resistance (Rt) was significantly higher than that of 50nM non-complementary oligonucleotides TC (632Ω and 121Ω, respectively). Moreover, a high level of discrimination between complementary oligonucleotides T2 and TN with single-base mismatch was achieved by the increasing hybridization stringency (reducing the ionic strength of the hybridization buffer solution)

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.

Rifampicin resistance mutations in the 81bp RRDR of rpoB gene in Mycobacterium tuberculosis clinical isolates using Xpert® MTB/RIF in Kampala, Uganda: a retrospective study.

BackgroundIntroduction of Xpert® MTB/RIF assay has revolutionalised the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (rif), a surrogate marker for multi-drug resistant TB (MDR-TB) as well as one of the principal first-line anti-tuberculosis drugs. In general, rpoB mutations can be found in 96.1% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide and these mutations usually are located in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, which is referred to as Rifampicin-resistance-determining region (RRDR). In this study, we determined the frequency of MDR-TB in Kampala using Xpert® MTB/RIF in comparison with the agar proportion method using Middlebrook 7H11and further determined the frequency of probes for different rpoB gene mutations using Xpert® MTB/RIF assay in the 81 bp RRDR.MethodsA total of 1501 specimens received at Mycobacteriology laboratory, Makerere University for Xpert testing between May 2011 and May 2014 were analysed by Xpert® MTB/RIF assay. Specimens that were positive for both MTB and rifampicin resistance were further subjected to a complete first line anti-mycobacterial drug susceptibility testing using Middlebrook 7H11 agar proportion method (APM).ResultsXpert® MTB/RIF assay detected 313 MTB positive specimens and out of which 12 specimens had both MTB and rifampicin- resistance conferred by four different rpoB gene mutations in the 81 bp-RRDR of MTB, further one (1/12), specimen was found to be rifampicin mono-resistant on APM while the 11 were found to be MDR-TB. Probes associated with the observed rif- resistance were as follows: E (7/12), B (3/12), A (1/12), D (1/12) and no rif-resistance was associated with probe C. No specimen yielded rif-resistance associated with more than one probe failure (mutation combinations). Probe D was associated with rifampicin mono-resistant.ConclusionsMDR-TB was at 3.5% in the studied population. Mutations associated with Probe E (58%) also known as codons 531and 533 are the commonest rpoB gene mutation identified by Xpert® MTB/RIF assay in this setting and mutations identified by probe E of the assay, turned out to be MDR-TB strains by agar proportion method antimicrobial susceptibility testing. No mutation was detected in the codon 522.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-481) contains supplementary material, which is available to authorized users.

Isothermal DNA amplification coupled to Au-nanoprobes for detection of mutations associated to Rifampicin resistance in Mycobacterium tuberculosis

BackgroundTuberculosis accounted for 8.7 million new cases in 2011 and continues to be one of the leading human infectious diseases. Burdensome is the increasing rate of multi-drug resistant tuberculosis (MDRTB) and the difficulties created for treatment and public health control programs, especially in developing countries. Resistance to rifampicin (RIF), a first line antibiotic, is commonly associated with point mutations within the rpoB gene of Mycobacterium tuberculosis (Mtb) whose detection is considered the best early molecular predictor for MDRTB. Gold nanoparticles functionalized with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for Mtb and single base alterations associated with antibiotic resistance, namely in rpoB gene associated to RIF resistance.ResultsWe developed a strategy based on the isothermal amplification of sample DNA (LAMP) coupled to specific Au-nanoprobes capable of identifying members of the Mtb complex (MTBC) and discriminating specific mutations within the rpoB gene. Integration of LAMP and Au-nanoprobe assay allowed to detect MTBC member and identify mutations linked to RIF resistance. A total of 12 biological samples were tested and a 100% specificity and sensitivity was attained.ConclusionsThere is an increasing demand for simple, fast and cheap methods for the molecular identification of Mtb and for the detection of molecular tags associated to drug resistance suitable for use at point-of-need. Here we describe such a method, that as the potential to get molecular diagnostic of tuberculosis to remote environments.

Discrimination of single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis using a surface plasmon resonance biosensor

Single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis, the mutations of which cause drug resistance of the infectious agent, were detected and discriminated using a surface plasmon resonance biosensor system. Thiol-modified oligonucleotides of the selected sequence (the probe) and 1-mercapto-6-hexanol were immobilized on a gold sensor surface. Hybridization between immobilized probe P2 and perfectly matched target T2 as well as a single base mismatched target TN was investigated in buffer solutions of various stringencies. Discrimination of perfectly matched and single base mismatched targets is achieved due to a difference in the level of their hybridization with the immobilized probe depending on stringency of the buffer solution. In 0.5×SSC buffer solution (7.5 mM sodium citrate, pH 7, containing 75 mM NaCl), sensor response at T2 injection into the measuring sensor cell was 16 times that at TN injection. The experimental results on surface hybridization between the studied oligonucleotides demonstrated a good correlation with theoretical calculations of thermodynamic parameters of these interactions in the solution. The described approach could be proposed as a basis for creating a biosensor for real-time label-free diagnostics of drug-resistant tuberculosis.