Simplified Microarray System for Simultaneously Detecting Rifampin,Isoniazid, Ethambutol,and Streptomycin Resistance Markers in Mycobacterium tuberculosis

We developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. In a blinded analysis of 153 clinical isolates, microarray sensitivity for first-line drugs relative to phenotypic DST (true resistance) was 100% for rifampin (RIF) (14/14), 90.0% for isoniazid (INH) (36/40), 70% for ethambutol (EMB) (7/10), and 89.1% (57/64) combined. Microarray specificity (true susceptibility) for first-line agents was 95.0% for RIF (132/139), 98.2% for INH (111/113), and 98.6% for EMB (141/143). Overall microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395). The overall positive and negative predictive values for RIF, INH, and EMB combined were 84.9% and 98.3%, respectively. For the second-line drug streptomycin (STR), overall concordance between the agar proportion method and microarray analysis was 89.5% (137/153). Sensitivity was 34.8% (8/23) because of limited microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130). All false-susceptible discrepant results were a consequence of DNA mutations that are not represented by a specific microarray probe. There were zero invalid results from 220 total tests. The simplified microarray system is suitable for detecting resistance-conferring mutations in clinical M. tuberculosis isolates and can now be used for prospective trials or integrated into an all-in-one, closed-amplicon consumable.     

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin,Isoniazid, and Fluoroquinolones

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.Rifampin (rifampicin), isoniazid, and the fluoroquinolones are the most important initial drug markers for extensively drug-resistant Mycobacterium tuberculosis strains, defined as multidrug-resistant (MDR) isolates (resistant to both isoniazid and rifampin) with additional resistance to a fluoroquinolone and to one of the injectable drugs (2). The fluoroquinolones have become an essential part of treatment regimens for MDR tuberculosis (7, 25). Due to their potency and safety, the new-generation fluoroquinolones are now even being evaluated as first-line medications for tuberculosis (3, 13, 20). Wang et al. further suggested that routine fluoroquinolone resistance testing may have a clinical impact by showing a significant correlation between development of fluoroquinolone and first-line M. tuberculosis drug resistance in an area in which resistant strains are highly endemic (28).The spontaneous acquisition of DNA sequence mutations is the primary genetic basis for the development of M. tuberculosis drug resistance (14). Since sequences are highly conserved, certain mutations correlate well with phenotypic resistance, and a limited number of mutations account for the majority of phenotypic resistance to the important antituberculosis medications, various methods of genotypic testing have successfully been used for the rapid detection of M. tuberculosis resistance (16, 22). The sites that most frequently contain mutations associated with phenotypic resistance, called resistance-determining regions (RDR), differ depending on the drug tested. Among rifampin-resistant isolates worldwide, 95 to 97% harbor mutations in the rifampin RDR, an 81-bp target encompassing codons 507 to 533 of the 3,519-bp rpoB gene (17). Isoniazid resistance has a more complex mechanism, involving several gene targets, the most important of which is codon 315 of the 2,223-bp katG gene, in which mutations are found in up to 50% of resistant isolates (18). Likewise, M. tuberculosis has a quinolone RDR which spans codons 88 to 94 of the 2,517-bp gyrA gene. Mutations in this region, particularly in codon 88, 90, 91, or 94, correlate with high-level resistance and are seen in 42 to 85% of resistant clinical isolates (6).Pyrosequencing, a method of DNA sequencing by synthesis, has been applied to the rapid detection of M. tuberculosis resistance to rifampin, isoniazid, and ethambutol (9, 29). Its main advantage is a much shorter turnaround time than that of conventional drug susceptibility testing, the latter taking 2 to 4 weeks from the time an isolate is obtained in pure culture.After isoniazid and rifampin, the next pivotal drug class in M. tuberculosis treatment is the fluoroquinolone class, as previously discussed (3, 7, 13, 20, 25, 28). Given that most resistance to the latter is determined by mutations that are generally limited to the quinolone RDR of the gyrA gene, it should be feasible and clinically more relevant to develop an assay for rapid resistance testing which includes fluoroquinolone resistance in addition to rifampin and isoniazid resistance.We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones, which we validated against the conventional submerged-disk proportion method. We also improved on the previously reported pyrosequencing assay by reducing the number of primers required to sequence for rifampin resistance (9).     

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis: a Meta-Analysis

A rapid, simple, accurate, and affordable method for the detection of drug-resistant tuberculosis is very critical for the selection of antimicrobial therapy and management of patient treatment. High-resolution melting curve analysis has been used for the detection of rifampin resistance in Mycobacterium tuberculosis and has shown promise. We did a systematic review and meta-analysis of published studies to evaluate the accuracy of high-resolution melting curve analysis for the detection of rifampin resistance in clinical M. tuberculosis isolates. We searched the PubMed, BIOSIS Previews, and Web of Science databases to identify studies and included them according to predetermined criteria. We used the DerSimonian-Laird random-effects model to calculate pooled measures and applied Moses'' constant for linear models to fit the summary receiver operating characteristic curve. According to the selection criteria, most of the identified studies were excluded, and only seven studies were included in the final analysis. The overall sensitivity of the high-resolution melting curve analysis was 94% (95% confidence interval [CI], 92% to 96%), and the overall specificity was very high at 99% (95% CI, 98% to 100%). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 63.39 (95% CI, 30.21 to 133.00), 0.06 (95% CI, 0.04 to 0.09), and 892.70 (95% CI, 385.50 to 2,067.24), respectively. There was no significant heterogeneity across all included studies for the measurements we evaluated. The summary receiver operating characteristic curve for the same data shows an area of 0.99 and a Q* value of 0.97. High-resolution melting curve analysis has high sensitivity and specificity for the detection of rifampin resistance in clinical M. tuberculosis isolates. This method might be a good alternative to conventional drug susceptibility tests in clinical practice.     

Comparison of Two Rapid Colorimetric Methods for Determining Resistance of Mycobacterium tuberculosis to Rifampin, Isoniazid, and Streptomycin in Liquid Medium

 The usefulness of two colorimetric methods for the determination of the susceptibility or resistance of Mycobacterium tuberculosis to rifampin, streptomycin, and isoniazid in liquid medium based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was investigated. The agar proportion method was used as the reference method. Results obtained indicate that the sensitivity of the XTT reduction assay for the detection of rifampin resistance was comparable to that observed, and previously described, for the MTT assay. However, the reduction of XTT yields a water-soluble formazan that can be easily quantified without performing additional steps such as addition of lysing buffer and solubilization. Furthermore, the colorimetric assays, based on the reduction of XTT and MTT for the detection of isoniazid and streptomycin resistance in Mycobacterium tuberculosis, were standardized. The inhibition of MTT and XTT reduction after treatment with rifampin, streptomycin, or isoniazid was directly proportional to the reduction in the number of viable bacteria, and a strain of Mycobacterium tuberculosis could be reported as susceptible or resistant to rifampin, streptomycin, or isoniazid after 3, 6, or 8 days, respectively. The XTT and MTT reduction assays are rapid, reliable, and affordable and do not require the use of radioisotopes. Moreover, they can be performed with common laboratory equipment.     

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.     

Evaluation of a Bacteriophage-Based Assay (Phage Amplified Biologically Assay) as a Rapid Screen for Resistance to Isoniazid, Ethambutol, Streptomycin, Pyrazinamide, and Ciprofloxacin among Clinical Isolates of Mycobacterium tuberculosis

Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries.     

Growth Inhibition of Mycobacterium tuberculosis After Single-Pulsed Exposures to Streptomycin, Ethambutol, and Rifampin

It was reported earlier that the degree of growth inhibition of tubercle bacilli after single-pulsed isoniazid exposures is a function of the time-concentration product (TCP) of exposure rather than of either time or concentration alone (TCP = hr exposure × μg of drug/ml). In the present investigation, these time and concentration studies have been extended to streptomycin (SM), ethambutol (EMB), and rifampin (RMP). Mycobacterium tuberculosis H37Ra was grown in Sautons liquid medium at 37 C with aeration. Rapidly growing cells were harvested and resuspended at 0.5 to 0.6 mg (dry wt)/ml in fresh medium; incubation was continued in the presence of various concentrations of the appropriate drug. At time intervals, samples were diluted 1:200 into drug-free medium for turbidimetric growth assay. Minimal inhibitory concentrations of EMB, SM, and RMP were approximately 0.2, 0.02, and 0.002 μg/ml, respectively. When cells were pulsed with 0.0125 to 0.0625 μg of RMP per ml at time intervals of up to 9.5 hr, the degree of subsequent growth inhibition appeared to be a function of TCP. A similar relationship was observed when SM was tested over a range of 0.125 to 1.0 μg/ml and various time intervals of up to 8 hr. In contrast, inhibition of tubercle bacilli after EMB exposures was dependent primarily on exposure time and was affected only slightly by concentration. At any particular exposure time between 3 and 16 hr, 1.25 to 7.5 μg of EMB per ml produced similar levels of inhibition, but marked inhibition did not occur unless the exposure time exceeded 10 hr. Relationships of these latter findings to the mode of action of EMB and the potential clinical significance of the RMP, SM, and EMB data are discussed.     

Evaluation of Agar-Based Medium with Sheep Sera for Testing of Drug Susceptibility of Mycobacterium tuberculosis to Isoniazid,Rifampin, Ethambutol,and Streptomycin

The performance of sheep sera instead of sheep blood in agar-based media was investigated for susceptibility testing of Mycobacterium tuberculosis against primary drugs. The levels of agreement between agar-based medium supplemented with sheep sera and the proportion method on Middlebrook 7H11 agar as the reference method for determining susceptibility to isoniazid (INH), rifampin (RIF), ethambutol (EMB), and streptomycin (STR) were 98.4, 98.4, 95.3, and 100%, respectively.     

Detection of Ofloxacin Resistance by Nitrate Reductase Assay in Mycobacterium tuberculosis Isolates from Extrapulmonary Tuberculosis

Context: Increased use of fluoroquinolones to treat community-acquired infections has led to the decreased susceptibility to Mycobacterium tuberculosis. There is a paucity of data on ofloxacin (OFX) resistance detection by nitrate reductase assay (NRA). Hence, the present study was carried out to find the efficacy of NRA for detection of OFX resistance in M. tuberculosis isolated from extrapulmonary tuberculosis (EPTB) cases. Aims: (1) To compare sensitivity, specificity and median time required to obtain results by NRA with economic variant proportion method (PM) for detection of OFX resistance.(2) To determine the extent of OFX resistance in clinical isolates of M. tuberculosis. Settings and Design: Seventy-three M. tuberculosis isolates from cases of EPTB were subjected to economic variant of PM for isoniazid, rifampicin and OFX. NRA was done for detection of OFX resistance. Subjects and Methods: Seventy-three isolates from clinical samples of suspected EPTB received in the Department of Microbiology were included in the study. Drug susceptibility test was performed on Lowenstein– Jensen medium with and without drugs. Statistical Analysis Used: Of turnaround time was done by Mann–Whitney test on SPSS (version 19, released in 2010, IBM Corp, Armonk NY), P < 0.05. Results: OFX resistance was seen in nine isolates. The sensitivity and specificity of OFX resistance by NRA was 100% and 96.87%, respectively. Median time required to obtain results by NRA was 10 days as compared to 28 days by PM. Conclusions: NRA is a specific and sensitive method for detection of OFX resistance in resource-restricted settings.     

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.     

PCR-Single-Strand Conformational Polymorphism Method for Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis: Systematic Review and Meta-Analysis

The reference standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as culture on Lowenstein-Jensen or Middlebrook 7H10/11 medium, are very slow to give results; and due to the emergence of multidrug-resistant M. tuberculosis and extensively drug-resistant M. tuberculosis, there is an urgent demand for new, rapid, and accurate drug susceptibility testing methods. PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been proposed as a rapid method for the detection of resistance to rifampin, but its accuracy has not been systematically evaluated. We performed a systematic review and meta-analysis to evaluate the accuracy of PCR-SSCP analysis for the detection of rifampin-resistant tuberculosis. We searched the Medline, Embase, Web of Science, BIOSIS, and LILACS databases and contacted authors if additional information was required. Ten studies met our inclusion criteria for rifampin resistance detection. We applied the summary receiver operating characteristic (SROC) curve to perform the meta-analysis and to summarize diagnostic accuracy. The sensitivity of PCR-SSCP analysis for the rapid detection of rifampin-resistant tuberculosis was 0.79 (95% confidence interval [CI], 0.75 to 0.82), the specificity was 0.96 (95% CI, 0.94 to 0.98), the positive likelihood ratio was 16.10 (95% CI, 5.87 to 44.13), the negative likelihood ratio was 0.20 (95% CI, 0.10 to 0.40), and the diagnostic odds ratio was 100.93 (95% CI, 31.95 to 318.83). PCR-SSCP analysis is a sensitive and specific test for the rapid detection of rifampin-resistant M. tuberculosis. Additional studies in countries with a high prevalence of multidrug-resistant M. tuberculosis and also cost-effectiveness analysis are required in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in M. tuberculosis.Tuberculosis (TB) is a major global health problem. Early detection of drug resistance in patients with tuberculosis allows the use of appropriate treatment regimens for the patient, which has an important impact for the better control of the disease. The development of rapid methods for drug susceptibility testing is very important due to the increasing rates of multidrug-resistant tuberculosis (MDR-TB) worldwide and the recently described extensively drug-resistant tuberculosis (XDR-TB). The World Health Organization (WHO) urgently called for expanded access to culture and drug susceptibility testing in response to the spread of MDR-TB and XDR-TB, declared serious emerging threats to public health (28). This poses significant challenges for TB laboratory capacity and the need for faster drug susceptibility testing methods.Conventional culture methods using egg- or agar-based media are still the most commonly used approaches in many countries. To test for drug resistance, the standard methods using Lowenstein-Jensen (LJ) medium include the proportion method, the absolute concentration method, and the resistant ratio method, which are well standardized with clinical isolates, at least for the major antituberculosis drugs. The proportion method was developed in the 1960s and is still the “gold standard” method used in many laboratories, especially in developing countries, because it is an inexpensive method easily accessible in these settings (1, 2). In recent years, due to the long turnaround times of conventional drug susceptibility testing methods, several new approaches have been proposed for the faster detection of MDR Mycobacterium tuberculosis, including both genotypic and phenotypic methods (23, 24). Among the genotypic methods proposed, PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been an important tool for detecting point mutations underlying genetic diseases since it was established in 1989 (9, 21). Under nondenaturing conditions, single-stranded DNA (ssDNA) has a folded structure that is determined by intramolecular interactions and, therefore, by its sequence. Even a single point mutation can alter the conformation of ssDNA, so that the altered conformation affects the migration of ssDNA, which can be detected as abnormal bands on a nondenaturing gel. Thus, SSCP analysis has a high resolving power to distinguish most conformational changes caused by subtle sequence differences even in a several-hundred-base fragment (9). The PCR-SSCP assay is relatively simple and was initially promising. Recently, the PCR-SSCP assay has been used in some studies for the rapid detection of rifampin-resistant M. tuberculosis (3, 11, 12, 17). However, individually, these trials found that the sensitivity and specificity were statistically inconsistent. The purpose of the present study was to perform a systematic review and meta-analysis to synthesize all available literature on the PCR-SSCP assay for the rapid detection of rifampin-resistant M. tuberculosis and to evaluate the overall accuracy of this method for the detection of rifampin resistance in isolates and in sputum samples.     

Flow Cytometric Testing of Susceptibilities of Mycobacterium tuberculosis Isolates to Ethambutol, Isoniazid, and Rifampin in 24 Hours

Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.     

Comparison of Three Molecular Assays for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is an emerging problem of great importance to public health, with higher mortality rates than drug-sensitive TB, particularly in immunocompromised patients. MDR-TB patients require treatment with more-toxic second-line drugs and remain infectious for longer than patients infected with drug-sensitive strains, incurring higher costs due to prolonged hospitalization. It is estimated that 90% of United Kingdom rifampin-resistant isolates are also resistant to isoniazid, making rifampin resistance a useful surrogate marker for multidrug resistance and indicating that second- and third-line drugs to which these isolates are susceptible are urgently required. Resistance in approximately 95% of rifampin-resistant isolates is due to mutations in a 69-bp region of the rpoB gene, making this a good target for molecular genotypic diagnostic methods. Two molecular assays, INNO-LiPA Rif.TB (Innogenetics, Zwijndrecht, Belgium) and MisMatch Detect II (Ambion, Austin, Tex.), were performed on primary specimens and cultures to predict rifampin resistance, and these methods were compared with the resistance ratio method. A third method, the phenotypic PhaB assay, was also evaluated in comparison to cultures in parallel with the genotypic assays. In an initial evaluation 16 of 16, 15 of 16, and 16 of 16 rifampin-resistant cultures (100, 93.8, and 100%, respectively), were correctly identified by line probe assay (LiPA), mismatch assay, and PhaB assay, respectively. Subsequently 38 sputa and bronchealveolar lavage specimens and 21 isolates were received from clinicians for molecular analysis. For the 38 primary specimens the LiPA and mismatch assay correlated with culture and subsequent identification and susceptibility tests in 36 and 38 specimens (94.7 and 100%), respectively. For the 21 isolates submitted by clinicians, both assays correlated 100% with routine testing.     

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand,Near-Patient Technology

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System''s MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay''s buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.An alarming increase in the global incidence of drug-resistant Mycobacterium tuberculosis infection has created a critical need for methods that can rapidly detect M. tuberculosis and identify drug-resistant cases (53). Failure to quickly and effectively recognize and treat patients with drug-resistant tuberculosis (TB), particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, leads to increased mortality, nosocomial outbreaks, and resistance to additional antituberculosis drugs (14, 37). However, MDR and XDR tuberculosis can be effectively treated if properly identified (35). A number of new diagnostic approaches have brought incremental improvements in detection and drug susceptibility testing (2, 9, 19, 24, 37, 41, 46); however, none can realistically provide actionable information within the time frame of a single office or clinic visit. Thus, despite technical advances, rapid diagnostics have not yet been able to have an impact on critical initial decisions regarding hospitalization, isolation, and the choice of treatment regimens for suspected tuberculosis patients.Previously, we showed that direct molecular detection of M. tuberculosis and rifampin resistance could be accomplished simultaneously (27); more recently, our group developed a single-tube, molecular beacon-based real-time PCR assay for the detection of rifampin-resistant M. tuberculosis (42, 43). Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene, which occur in 95 to 98% of all rifampin-resistant strains (and which are almost invariably absent in rifampin-susceptible strains), were detected by five overlapping molecular beacons (34). The assay proved to be simple, rapid, specific, and highly sensitive in tests on isolates of M. tuberculosis from New York City, Madrid (42), India, and Mexico (51). As most rifampin-resistant isolates are also resistant to isoniazid, rifampin resistance can be used as a marker for MDR M. tuberculosis (36, 44, 49). However, like all nucleic acid amplification-based assays for M. tuberculosis detection (15), this assay was too complex and too prone to operator errors, sample cross-contamination, and biohazards for rapid near-patient use.The Cepheid GeneXpert System (Sunnyvale, CA), a single-use sample-processing cartridge system with integrated multicolor real-time PCR capacity (45), has the potential to greatly simplify nucleic acid amplification tests. Here, we utilized this new technology to develop an on-demand, near-patient PCR assay that employs a novel six-color dye set to detect M. tuberculosis and identify rifampin resistance as a surrogate for MDR directly from a patient''s sputum in less than 2 h. The many features of this system, including sample decontamination, hands-free operation, on-board sample processing, and ultrasensitive hemi-nested PCR, enabled us to create a low-complexity assay with a sensitivity that approached certain culture methods. This type of assay may prove to be useful in the initial management of suspected tuberculosis cases in both the United States and the world at large.     

Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting

In a head-to-head comparison of the MTBDRplus version 2.0 (Hain Lifescience), the Xpert MTB/RIF (Cepheid), and the Anyplex MTB/NTM (Seegene) assays, we demonstrated equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampin resistance with further analysis of discordances. The Xpert assay does not detect isoniazid resistance while the Anyplex assay showed high false positivity.     

Point-of-Care System for Detection of Mycobacterium tuberculosis and Rifampin Resistance in Sputum Samples

Early detection of Mycobacterium tuberculosis complex (MTBC) and markers conveying drug resistance can have a beneficial impact on preventive public health actions. We describe here a new molecular point-of-care (POC) system, the Genedrive, which is based on simple sample preparation combined with PCR to detect MTBC and simultaneously detect mutation markers in the rpoB gene directly from raw sputum sample. Hybridization probes were used to detect the presence of the key mutations in codons 516, 526, and 531 of the rpoB gene. The sensitivities for MTBC and rpoB detection from sputum samples were assessed using model samples spiked with known numbers of bacteria prepared from liquid cultures of M. tuberculosis. The overall sensitivities were 90.8% (95% confidence interval [CI], 81, 96.5) for MTBC detection and 72.3% (95% CI, 59.8, 82.7) for rpoB detection. For samples containing ≥1,000 CFU/ml, the sensitivities were 100% for MTBC and 85.7% for rpoB detection, while for samples containing ≤100 CFU/ml, the sensitivities were 86.4% and 65.9% for MTBC and rpoB detection, respectively. The specificity was shown to be 100% (95% CI, 83.2, 100) for MTBC and rpoB. The clinical sputum samples were processed using the same protocol and showed good concordance with the data generated from the model. Tuberculosis-infected subjects with smear samples assessed as scanty or negative were detectable by the Genedrive system. In these paucibacillary patients, the performance of the Genedrive system was comparable to that of the GeneXpert assay. The characteristics of the Genedrive platform make it particularly useful for detecting MTBC and rifampin resistance in low-resource settings and for reducing the burden of tuberculosis disease.     

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.     

Detection of Rifampin Resistance in Mycobacterium tuberculosis in a Single Tube with Molecular Beacons

Current clinical assays for determining antibiotic susceptibility in Mycobacterium tuberculosis require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. tuberculosis in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene, rpoB. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the rpoB gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible M. tuberculosis is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148 M. tuberculosis clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.     

噬菌体生物扩增法测定痰标本结核分枝杆菌利福平耐药性

目的建立快速测定痰标本中结核分枝杆菌利福平（RFP）耐药性的噬菌体生物扩增法，探讨其在临床应用的可行性。方法噬菌体生物扩增法测定362份涂阳痰标本结核分枝杆菌对RFP的耐药性，并与罗氏绝对浓度法结果进行比较，对不符合的样本，用基因芯片的方法分析rpoB基因的突变情况。结果362份涂阳痰标本，培养阳性360份，菌种初步鉴定有17份样本为非结核分枝杆菌，噬菌体生物扩增法RFP耐药为123份，敏感的196份，两法结果一致的为88．6％，如以绝对浓度法药敏结果为标准，则噬菌体生物扩增法测定利福平耐药的敏感性为93-3％，特异性为87．9％，阴性预测值为96．4％，阳性预测值为78．9％，准确性为89．7％，涂阳等级（1＋～3＋）与实验的有效性相关。结论噬菌体生物扩增法测定痰标本利福平的耐药性只需2d，可作为快速筛诜方法府用千临床．