Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INH^r, 356 were RIF^r, 292 were MOX^r/OFX^r, 230 were AMK^r, 219 were CAP^r, and 286 were KAN^r. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INH^r. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIF^r. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOX^r/OFX^r. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMK^r/CAP^r but 71% sensitivity for KAN^r. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMK^r and to 91% for detecting KAN^r. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.     

Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis Isolates by Use of Real-Time-PCR-Based Melting Curve Analysis

The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions −17 to −8), inhA position 94, and the ahpC promoter (positions −44 to −30 and −15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2 × 10³ to 2 × 10⁴ bacilli/ml), reproducibility (standard deviation [SD], <0.4°C), and the lowest heteroresistance level (40%) all met the parameters preset by the kit. The assay could be run on five types of real-time PCR machines, with the shortest running time (105 min) obtained with the LightCycler 480 II. Clinical studies enrolled 1,096 clinical isolates collected from three geographically different tuberculosis centers, including 437 INH-resistant isolates and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing on Löwenstein-Jensen solid medium. The clinical sensitivity and specificity of the MeltPro TB/INH assay were 90.8% and 96.4%, respectively. DNA sequencing analysis showed that, except for the 5 mutants outside the detection range of the MeltPro assay, a concordance rate between the two methods of 99.1% (457/461) was obtained. Among the 26 mutation types detected, katG S315T (AGC→ACC), inhA −15C→T, katG S315N (AGC→AAC), and ahpC promoter −10C→T accounted for more than 90%. Overall, the MeltPro TB/INH assay represents a reliable and rapid tool for the detection of INH resistance in clinical isolates.     

Molecular evidence for heterogeneity of the multiple-drug-resistant Mycobacterium tuberculosis population in Scotland (1990 to 1997)

Multiple-drug-resistant Mycobacterium tuberculosis (MDR-MTB) has been well studied in hospitals or health care institutions and in human immunodeficiency virus-infected populations. However, the characteristics of MDR-MTB in the community have not been well investigated. An understanding of its prevalence and circulation within the community will help to estimate the problem and optimize the strategies for control and prevention of its development and transmission. In this study, MDR-MTB isolates from Scotland collected between 1990 and 1997 were characterized, along with non-drug-resistant isolates. The results showed that they were genetically diverse, suggesting they were unrelated to each other and had probably evolved independently. Several new alleles of rpoB, katG, and ahpC were identified: rpoB codon 525 (ACC→AAC; Thr525Asn); katG codon 128 (CGG→CAG; Arg128Gln) and codon 291 (GCT→CCT; Ala291Pro); and the ahpC synonymous substitution at codon 6 (ATT→ATC). One of the MDR-MTB isolates from an Asian patient had an IS6110 restriction fragment length polymorphism pattern very similar to that of the MDR-MTB W strain and had the same drug resistance-related alleles but did not have any epidemiological connection with the W strains. Additionally, a cluster of M. tuberculosis isolates was identified in our collection of 715 clinical isolates; the isolates in this cluster had genetic backgrounds very similar to those of the W strains, one of which had already developed multiple drug resistances. The diverse population of MDR-MTB in Scotland, along with a low incidence of drug-resistant M. tuberculosis, has implications for the control of the organism and prevention of its spread.     

Genotypic analysis of genes associated with isoniazid and ethionamide resistance in MDR-TB isolates from Thailand

Nucleotide sequences of genes conferring isoniazid resistance (katG, inhA, oxyR–ahpC and ndh) and ethionamide resistance (ethA) in 160 drug-resistant Mycobacterium tuberculosis clinical isolates from Thailand were analysed. Mutations in the katG gene were found in 129 isolates, predominantly at codon 315, which was mutated in 127 isolates. Twenty-two isolates had mutations in the inhA promoter and coding region. Mutations in the oxyR–ahpC intergenic region and in ndh were detected in four and one isolate(s), respectively. Of 24 ethionamide-resistant isolates, 13 had mutations in the ethA gene. However, these mutations were dispersed along the entire gene, with no codon predominating significantly.     

Evaluation of GenoFlow DR-MTB Array Test for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis

The aim of this study was to evaluate the GenoFlow DR-MTB array test (DiagCor Bioscience, Hong Kong) on 70 cultured isolates and 50 sputum specimens. The GenoFlow array test showed good sensitivity and specificity compared to the phenotypic Bactec 460TB. This array accurately detected mutations in rpoB, katG, and inhA associated with resistance to rifampin and isoniazid.     

Detection of rpoB,katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray

The emergence of multidrug-resistance Mycobacterium tuberculosis is an increasing threat to tuberculosis control programmes. Susceptibility testing of Mycobacterium tuberculosis complex isolates by traditional methods requires a minimum of 14 days. This can be reduced significantly if molecular analysis is used. DNA sequencing is a good method for detecting mutation, but cannot be used routinely because of its relatively high cost. A sensitive and specific microarray has been designed to detect mutations in the rifampin resistance determining region of rpoB and loci in katG and inhA associated with isoniazid (INH) resistance. A panel of Mycobacterium tuberculosis isolates containing 13 different rpoB genotypes, two mutation genotypes within codon 315 of katG and one mutation genotypes at inhA was used to validate the microarray. The results obtained indicate that 100% of rifampicin-resistant M. tuberculosis strains isolated in Chongqing had rpoB mutations, with 531-Ser and 526-His being the most common positions substituted. Of the total 50 INH resistant isolates, 82% had a katG315 mutation and 18% had an inhA mutation. All the mutations detected by the microarray method were also confirmed by conventional DNA sequencing. It is demonstrated that the microarray is an efficient, specialized technique and can be used as a rapid method for detecting rifampin and isoniazid resistance.     

Molecular genetic analysis of Isoniazid-resistant strains of Mycobacterium tuberculosis circulating in the Saratov region

The distribution and mutation spectrum of the katG, inhA, and ahpC genes encoding Isoniazid resistance have been studied in 257 patients with active lung tuberculosis in Saratov oblast. The tests were performed using biological microchips from TB-Biochip MDR pharmacological kits (Russia). The incidence rate of Isoniazid-resistant M. tuberculosis strains was found to be 55.7%. Isoniazid-resistant strains demonstrated a high rate (26.9%) of combined mutations in two and three genes simultaneously and predominance in gene katG Ser315 → Thr1 (91.1%) inducing high resistance to Isoniazid. The sensitivity and specificity of the TB-Biochip MDR test system were 88.2 and 91.3%, respectively. The results of this work show additional measures must be taken to reduce the incidence of Isoniazid-resistant strains.     

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.     

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Isolates

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position −15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.The rates of mortality and morbidity from tuberculosis (TB) remain high, despite intense worldwide efforts. One of the major factors sustaining the current TB epidemic is the increasing drug resistance of Mycobacterium tuberculosis strains (2). In the early 1990s, multidrug-resistant (MDR) TB cases that were resistant to at least rifampin (RIF) plus isoniazid (INH) arose (6). When the frequency and distribution of extensively drug-resistant (XDR) TB cases were assessed in 2004 by the U.S. Centers for Disease Control and Prevention and the World Health Organization, several cases of drug-resistant tuberculosis consistent with an XDR phenotype were found (7). This study revealed that 20% of the isolates met the MDR criteria; 2% of those were classifiable as XDR; and 4%, 15%, and 19% of the XDR TB cases were from the United States, South Korea, and Latvia, respectively (7). Thus, it is crucial that rapid drug susceptibility tests be developed to prevent the spread of MDR and XDR TB.Although drug susceptibility testing (DST) is a prerequisite for accurate results, such testing requires much time and labor (3). Therefore, several molecular techniques have been applied to detect mutations related to drug resistance (5, 10). Resistance to RIF and INH, the mainstays of antituberculosis treatment, is mainly attributable to mutations in genes encoding the drug target or drug-converting enzymes (8). Early studies demonstrated that 95% of the resistance to RIF is associated with mutation of the RIF resistance-determining region of rpoB, whereas mutations in katG and the regulatory zone of inhA are most frequently associated with INH resistance (11).The oligonucleotide chip method and real-time PCR have been used for detection of drug-resistant M. tuberculosis (17, 21, 27, 29). A novel method of high-resolution melting (HRM) curve analysis is an accurate and simple technique for analyzing the genotype without the need for specific probes. The dye LC Green, SYTO9, or Eva Green saturates amplified DNA, unlike SYBR green dye, during homogeneous melting curve analysis. Also, HRM curve analysis generates a difference plot curve, which analyzes nucleic acid sequences with high accuracy. Application of genotyping by HRM curve analysis has followed (13, 19). The aim of the study described here was to develop a useful molecular tool for the identification of drug resistance in M. tuberculosis in an accurate, rapid, and cost-effective manner.     

Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage,Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RD^Rio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.     

Detection of novel mutations associated with independent resistance and cross-resistance to isoniazid and prothionamide in Mycobacterium tuberculosis clinical isolates

ObjectivesProthionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tuberculosis (MDR-TB). Both drugs have a common target InhA, so prothionamide can be ineffective against isoniazid-resistant (INH^R) Mycobacterium tuberculosis. We aimed to investigate the prevalence of mutations in katG, ethA, ndh, ethR, mshA, inhA and/or its promoter associated with independent resistance and cross-resistance to INH^R and/or prothionamide-resistant (PTO^R) M. tuberculosis isolates.MethodsWe sequenced the above genes in 206 M. tuberculosis isolates with susceptibility testing against ten drugs.ResultsOf the 173 INH^R PTO^R isolates, 170 (98.3%) harboured mutations in katG, 111 (64.2%) in ethA, 58 (33.5%) in inhA or its promoter, 5 (2.9%) in ndh, 3 (1.7 %) in ethR and 2 (1.2%) in mshA. Among the 18 INH^R PTO^S isolates, mutations in katG were found in all of them; one had a mutation in the inhA promoter and another in ndh. Of the five INH^S PTO^R isolates, four showed mutations in ethA and two in the inhA promoter. Notably, 55 novel non-synonymous mutations were found in them and 20.2% of the PTO^R M. tuberculosis isolates harboured no known mutations.ConclusionsThis is the first report to investigate cross-resistance between INH^R and/or PTO^R isolates. Among INH^R (94.4% MDR-TB) M. tuberculosis isolates, the high diversity of mutations for independent resistance and cross-resistance with prothionamide highlight the importance of both phenotypic susceptibility and genotypic diagnosis when using it to treat patients with INH^R-TB. The high proportion (one-fifth) of PTO^R M. tuberculosis isolates showed no known mutation related to PTO^R genes, so uncovered resistance mechanism(s) of prothionamide exist.     

Unequal distribution of resistance-conferring mutations among Mycobacterium tuberculosis and Mycobacterium africanum strains from Ghana

Isoniazid (INH) and rifampicin (RMP) resistance in Mycobacterium tuberculosis complex (MTC) isolates are mainly based on mutations in a limited number of genes. However, mutation frequencies vary in different mycobacterial populations. In this work, we analyzed the distribution of resistance-associated mutations in M. tuberculosis and M. africanum strains from Ghana, West Africa. The distribution of mutations in katG, fabG1-inhA, ahpC, and rpoB was determined by DNA sequencing in 217 INH-resistant (INH^r) and 45 multidrug-resistant (MDR) MTC strains isolated in Ghana from 2001 to 2004. A total of 247 out of 262 strains investigated (94.3%) carried a mutation in katG (72.5%), fabG1-inhA (25.1%), or ahpC (6.5%), respectively. M. tuberculosis strains mainly had katG 315 mutations (80.1%), whereas this proportion was significantly lower in M. africanum West-African 1 (WA1) strains (43.1%; p < 0.05). In contrast, WA1 strains showed more mutations in the fabG1-inhA region (39.2%, p < 0.05) compared to M. tuberculosis strains (20.9%). In 44 of 45 MDR strains (97.8%) mutations in the 81-bp core region of the rpoB gene could be verified. Additionally, DNA sequencing revealed that 5 RMP-susceptible strains also showed mutations in the rpoB hotspot region. In conclusion, although principally the same genes were affected in INH^rM. tuberculosis and M. africanum strains, disequilibrium in the distribution of mutations conferring resistance was verified that might influence the efficiency of molecular tests for determination of resistance.     

Predictive Value of Molecular Drug Resistance Testing of Mycobacterium tuberculosis Isolates in Valle del Cauca,Colombia

Previous evaluations of the molecular GenoType tests have promoted their use to detect resistance to first- and second-line antituberculosis drugs in different geographical regions. However, there are known geographic variations in the mutations associated with drug resistance in Mycobacterium tuberculosis, and especially in South America, there is a paucity of information regarding the frequencies and types of mutations associated with resistance to first- and second-line antituberculosis drugs. We therefore evaluated the performance of the GenoType kits in this region by testing 228 M. tuberculosis isolates in Colombia, including 134 resistant and 94 pansusceptible strains. Overall, the sensitivity and specificity of the GenoType MTBDRplus test ranged from 92 to 96% and 97 to 100%, respectively; the agreement index was optimal (Cohen''s kappa, >0.8). The sensitivity of the GenoType MTBDRsl test ranged from 84 to 100% and the specificity from 88 to 100%. The most common mutations were katG S315T1, rpoB S531L, embB M306V, gyrA D94G, and rrs A1401G. Our results reflect the utility of the GenoType tests in Colombia; however, as some discordance still exists between the conventional and molecular approaches in resistance testing, we adhere to the recommendation that the GenoType tests serve as early guides for therapy, followed by phenotypic drug susceptibility testing for all cases.     

Significance ofahpC promoter mutations for the prediction of isoniazid resistance inMycobacterium tuberculosis

To determine the value ofahpC promoter mutations for the rapid prediction of isoniazid resistance, this genomic region was characterized in 50 isoniazid-resistant and 12 isoniazid-sensitiveMycobacterium tuberculosis isolates. Of the resistant isolates, 12 hadahpC promoter mutations, but only one possessed both anahpC promoter mutation and akatG codon 315 substitution, although the latter was found in the majority (54%) of the isoniazid-resistant isolates investigated. This investigation presents empirical evidence that the central portion of theahpC promoter is the most valuable genetic locus to complementkatG codon 315 characterizations in order to increase the sensitivity of molecular tests for the prediction of isoniazid resistance.     

Molecular and phenotypic characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to kanamycin,amikacin, and capreomycin in China

Although second-line anti-tuberculosis (TB) injectable drugs have been widely used to improve treatment outcomes of multidrug-resistant TB (MDR-TB), little is known about the prevalence and mechanism of second-line injectable drug resistance among MDR Mycobacterium tuberculosis isolates in China. Here, we found that 12.7 % (20/158) of isolates showed resistance to at least one second-line injectable drug among 158 MDR isolates. At the same time, there were 16 (10.1 %) strains resistant to kanamycin (KAN), 9 (5.7 %) to amikacin (AMK), and 12 (7.6 %) to capreomycin (CAP). In addition, our data revealed no significant difference in the drug resistance patterns for Beijing versus non-Beijing genotype strains (p?>?0.05). The most frequently observed mutation was A-to-G substitution at position 1401 of the rrs gene, conferring high-level resistance to KAN and AMK, but had varying minimum inhibitory concentrations (MICs) for CAP. The mutations in the eis promoter and tlyA gene were responsible for low-level resistance to CAP. 83.3 % of A1401G substitutions in the rrs gene was observed in Beijing genotype strains, while the difference was not significant (p?=?0.157). Our data demonstrated that the hot-spot regions localized in the rrs gene serve as excellent markers for AMK, but is not a sensitive marker for KAN and CAP. In addition, the cross-resistance patterns and MICs differed among different genetic mutation types, which challenge the practice in China of generalizing resistance to AMK and CAP based on the resistance to KAN alone. Our findings suggested that the individualized drug susceptibility to three major second-line injectable drugs is essential in order to generate more effective treatment regimens for MDR patients.     

Frequency of occurrence and types of mutations in the rpoB, katG, inhA , and ahpC genes associated with rifampicin and isoniasid resistance in M. tuberculosis strains circulating in the Kyrgyz Republic

The availability of an MDR TB-Biochip for the express detection of Mycobacterium tuberculosis (MBT) strains resistant to rifampicin and isoniasid has been studied in 940 patients with pulmonary tuberculosis. The rpoB gene of rifampicin-resistant MBT strains circulating in the Kyrgyz Republic was shown to have substitutions at codons 531, 526, 516, 511, 513, 512, 533, and 522. MBT strains with mutations at codons 531 and 526 are prevalent. Among all rifampicin-resistant strains, MBT strains with the mutation of Ser531 → Leu occurred most frequently (59.7%). Isoniasid resistance is determined by mutations in the katG (94.5%), inhA (3.5%), and ahpC (1%) genes. Among isoniasid-resistant MBT strains, those with the mutation of Ser315 → Thr315 (94%) in the katG gene codon are predominant. Original Russian Text ? Zh.T. Isakova, 2008, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 2008, No. 4, pp. 36–38.     

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis

Background: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.     

Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType^® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.     

Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis by Use of Real-Time PCR and High-Resolution Melt Analysis

The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.The World Health Organization (WHO) estimates that approximately one-third of the world''s population is infected with Mycobacterium tuberculosis, with an estimated 9.27 million new cases reported in 2007 (20). In that year alone, an estimated 1.77 million people died from this treatable disease. Despite this significant burden, only a limited number of tests have been developed and implemented for the rapid diagnosis of tuberculosis (TB). Further, since the majority of TB disease burden occurs in underdeveloped and resource-limited settings, the need for a cost-efficient method is paramount.The emergence of drug-resistant strains of M. tuberculosis is one of the most critical issues facing TB researchers and clinicians today. Multidrug-resistant (MDR) M. tuberculosis is defined as being resistant to the two best first-line drugs used to treat TB: rifampin (RIF) and isoniazid (INH). Extensively drug-resistant (XDR) M. tuberculosis is defined as having additional resistance to a fluoroquinolone (ciprofloxacin, moxifloxicin, etc.) and an injectable drug (kanamycin, capreomycin, or amakacin), the two best classes of second-line drugs. The WHO estimates that 5% of new TB cases are MDR, with approximately 10% of those actually being XDR (20). Compounding this problem is the fact that no new drugs have been developed and approved for the treatment of TB in the past 30 years (16). The limited number of antibiotics available to treat TB necessitates rapid diagnosis not only to reduce the spread of drug-resistant strains but also to monitor and limit the emergence of newly resistant strains.While RIF and INH are very effective in the treatment of susceptible strains of M. tuberculosis, drug resistance can emerge quickly, in part due to patient nonadherence to the multidrug regimen or noncontinuous treatment. The molecular basis of resistance to these drugs is well documented. The target of RIF is the β-subunit of bacterial DNA-dependent RNA polymerase, which is encoded by the rpoB gene. At the genetic level, the majority of RIF resistance is due to the accumulation of mutations within an 81-bp region of rpoB, termed the rifampicin resistance determinant region (RRDR). Mutations within this region account for up to 98% of the RIF resistance observed (15). The strong correlation between genotypic changes in this region resulting in phenotypic resistance makes the RRDR an optimal target for the design of rapid molecular diagnostics.There are two described mechanisms that account for the majority of INH resistance. The most common mechanism involves mutations within the katG gene, which encodes a catalase peroxidase whose activity is required for the activation of INH (9). Nucleotide changes resulting in amino acid substitutions at codon 315 of katG account for up to 50% of the clinical resistance to INH (15). Another less common mutation occurs in the promoter region of the inhA gene, which encodes enoyl-ACP reductase, which is required for mycolic acid biosynthesis (18). Mutations at this locus account for up to 34% of the clinical INH resistance observed and are typically found in combination with additional mutations in katG (15).The vast majority of mutations that occur within rpoB, katG, and the inhA promoter regions are due to accumulation of single-nucleotide polymorphisms (SNPs), of which there are four classes (8). Class I SNPs, also called transitions, are changes in which a purine is exchanged for a purine (A/G→G/A) or a pyrimidine is exchanged for a pyrimidine (C/T→T/C) (8). Class II, III, and IV SNP changes are collectively referred to as transversions, and all involve the change of a purine to a pyrimidine, or vice versa (17). Class II changes result in A/C→C/A or T/G→G/T transversions, class III changes result in C/G→G/C transversions, and class IV changes result in A/T→T/A changes (8). These genetic mutations often result in phenotypic changes, such as RIF and INH resistance observed in M. tuberculosis, and are excellent targets for rapid molecular diagnostics.A significant obstacle in controlling TB is the amount of time required to reach a diagnosis. Due to the slow growth rate of M. tuberculosis, the initial diagnosis can take up to 6 weeks, with up to an additional 12 weeks to obtain drug susceptibility profiles for clinical isolates, depending on the techniques available to the laboratory. These labor-intensive methods can cause significant delays in identifying MDR or XDR cases, adjusting treatment regimens, and initiating epidemiological investigations. Recently, attention has shifted toward the development of dependable, molecular-based assays that can rapidly detect drug resistance. The development of new methodologies could potentially reduce the time required to diagnose drug resistance so that effective treatment regimens can be established. Direct sequencing of genes known to have a role in antibiotic resistance is one method that is currently used. However, while reliable, it is costly and may not be readily available. Another rapid method, the GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany), has made substantial contributions to the area of rapid diagnostics but still requires approximately 8 h to complete the assay and additional training to ensure that results are interpreted correctly (7). High-resolution melt (HRM) analysis is a molecular technique that can be used for detecting subtle genetic changes, such as SNPs conferring drug resistance in M. tuberculosis. By slowly melting the DNA amplicon products of a real-time PCR assay, slight genetic differences can be visualized by changes in dissociation profiles.The current study describes the use of multiple real-time PCR chemistries and HRM technology to detect RIF, INH, and more importantly, MDR strains of M. tuberculosis. This novel assay design is also capable of distinguishing M. tuberculosis complex bacteria (MTC) from nontuberculous mycobacterium (NTM) strains. This assay provides a rapid, robust, and inexpensive way to identify MDR TB that could result in numerous advantages over current molecular and culture-based techniques.