Evaluation of Two Line Probe Assays for Rapid Detection of Mycobacterium tuberculosis,Tuberculosis (TB) Drug Resistance,and Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV⁺) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.     

Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.     

Multidrug-resistant tuberculosis: rapid detection of resistance to rifampin and high or low levels of isoniazid in clinical specimens and isolates

The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis.     

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.     

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.     

Characterization of rpoB Mutations in Rifampin-Resistant Clinical Mycobacterium tuberculosis Isolates from Greece

There is a geographic distribution of Mycobacterium tuberculosis strains with various rpoB gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoB mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect rpoB mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct rpoB mutations in codons Ser₅₃₁, His₅₂₆, and Asp₅₁₆ were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser₅₃₁ and His₅₂₆ accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosis strains can have subclones bearing distinct mutant rpoB alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosis before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.     

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.     

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.     

Feasibility of the GenoType MTBDRsl Assay for Fluoroquinolone,Amikacin-Capreomycin,and Ethambutol Resistance Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.The worldwide emergence of extensively drug-resistant tuberculosis (XDR TB, resistant at least to rifampin and isoniazid, a fluoroquinolone [FLQ], and one of the three injectable second-line drugs amikacin [AM], kanamycin [KM], and capreomycin [CM]) is a serious global health problem (20, 25). In the World Health Organization fourth global report on drug resistance (25), it was documented that more than 45 countries have reported XDR cases. The actual incidence could be underestimated, because second-line drug susceptibility testing (DST) is not available in many countries. To avoid a progressive development similar to that observed in multidrug-resistant TB (resistant at least to rifampin and isoniazid) worldwide, now having the highest rate ever at 5.3%, timely identification of resistant Mycobacterium tuberculosis complex (MTBC) strains is mandatory.Conventional DST for XDR strains is performed sequentially in a two-step procedure beginning with a culture and first-line drug testing, proceeding to further drug testing in the case of multidrug resistance. The time needed for testing, even with the most rapid liquid methods, is still around 1 week per test, constrained by the relatively slow growth of M. tuberculosis (15, 18). The required time can be shortened by fast molecular methods to 1 day per test (3, 8, 21). Since recently broad-based knowledge about mutations that cause resistance to ethambutol (EMB) and some second-line drugs is available. Resistance to FLQs, AM-CM, and EMB in M. tuberculosis is most frequently attributed to mutations in the gyrA, rrs, and embB genes, respectively. First investigations have shown that by targeting mutations in codons 90, 91, and 94 in the gyrA gene, approximately 70 to 90% of all FLQ-resistant strains can be correctly detected (2, 13, 24). Previous reports have linked mutations A1401G, C1402T, and G1484T in the rrs gene to AM, CM, and KAN resistance (1, 11, 12), each of them being responsible for a specific resistance pattern. Mutations G1484T and A1401G were found to cause high-level resistance to all drugs, whereas C1402T causes resistance to only CM and KAN.Furthermore, mutations at embB codon 306 are found in 30 to 68% of EMB-resistant clinical strains (16, 17, 26).PCR-based techniques provide new possibilities for the rapid diagnosis of first- and second-line drug resistance; however, not all mycobacterial laboratories have access to DNA-sequencing facilities. As an alternative, DNA strip assays for the detection of rifampin (INNO-LiPA Rif. TB; Innogenetics, Ghent, Belgium) or rifampin and isoniazid resistance of M. tuberculosis in a single assay (GenoType MTBDR; Hain Lifescience, Nehren, Germany) are now commercially available. These assays have been evaluated for M. tuberculosis cultures and specimens (3, 7, 8, 10, 21). The DNA strip assays are based on PCR or multiplex PCR in combination with reverse hybridization. The existence of a resistant strain is signaled either by the omission of a wild-type band or the appearance of bands representing specific mutations.In order to increase the capacity to detect further drug resistance in M. tuberculosis, the GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay was developed with a specific focus on the most prevalent gyrA, rrs, and embB gene mutations.The aim of the present study was to determine the sensitivity and specificity of the new MTBDRsl assay for the detection of FLQ, AM, CM, and EMB resistance-associated mutations in culture specimens and directly in smear-positive and -negative clinical specimens.     

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.     

Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources.     

Evaluation of a commercial probe assay for detection of rifampin resistance inMycobacterium tuberculosis directly from respiratory and nonrespiratory clinical samples

A commercial assay (Inno-Line Probe Assay; Innogenetics, Belgium) was evaluated to determine its ability to detect rifampin resistance inMycobacterium tuberculosis directly from clinical specimens. Fifty-nine selected specimens (42 respiratory and 17 nonrespiratory) culture positive forMycobacterium tuberculosis were tested along with their corresponding isolates in culture. The results were compared with those obtained by in vitro susceptibility testing. The results of the line probe assay to detect rifampin resistance inMycobacterium tuberculosis present in clinical specimens and in cultured isolates were concordant for 58 of 59 (98.3%) isolates (95% confidence limits = 90.9–99.9%). The line probe assay failed only once, when a fecal specimen was tested; no amplification was observed due to the presence of inhibitory compounds. The most frequently observed mutation was His₅₂₆Asp (58.7%), followed by the His₅₂₆Tyr (23.9%); together, they represented 82.6% of rifampin-resistant samples. In conclusion, the Inno-Line Probe Assay is a rapid, useful method for detecting the presence ofMycobacterium tuberculosis complex and its resistance to rifampin directly from clinical specimens and culture. Moreover, since rifampin resistance is a potential marker for multidrug resistance inMycobacterium tuberculosis, this assay may constitute an important tool for the control of tuberculosis.     

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.     

Development and evaluation of a molecular assay for detection of nontuberculous mycobacteria by use of the cobas amplicor platform

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.     

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.     

Evaluation of the MTBDRsl Test for Detection of Second-Line-Drug Resistance in Mycobacterium tuberculosis

The MTBDRsl assay (Hain Lifescience GmbH, Germany) is a new line probe assay for the detection of extensively drug-resistant tuberculosis (XDR TB). The test simultaneously detects resistance to ethambutol, aminoglycosides/cyclic peptides, and fluoroquinolones through detection of mutations in the relevant genes. The assay format is identical to the MTBDR Hain assay. The assay was evaluated for the detection of second-line-drug resistance in Vietnamese isolates using two sample sets from the microbiology department of Pham Ngoc Thach Hospital, Ho Chi Minh City, Viet Nam, with existing conventional phenotypic drug susceptibility results for second-line drugs: 41 consecutive fluoroquinolone-resistant isolates and 21 consecutive multidrug-resistant but fluoroquinolone-sensitive isolates. The sensitivity for detection of fluoroquinolone resistance was 75.6% (31/41) (95% confidence interval [95% CI], 59.7% to 87.6%), and for kanamycin resistance, the sensitivity was 100% (5/5) (95% CI, 47.8% to 100%). The sensitivity of the test for detection of ethambutol resistance was low, consistent with previous reports, at 64.2% (34/53) (95% CI, 49.8% to 76.9%). The specificity of the test was 100% for all three drugs. These data suggest that the MTBDRsl assay is a rapid, specific test for the detection of XDR TB but should not be used exclusively to “rule out” second-line-drug resistance. Further operational evaluation is required and should be integrated with evaluations of the MTBDR test.The World Health Organization (WHO) has estimated that 5% of all tuberculosis (TB) cases globally are now multidrug-resistant tuberculosis (MDRTB) (resistance to at least rifampin [RIF] and isoniazid [INH]), based on data acquired since 2000 from more than 100 countries (14). Every year, an estimated 490,000 new cases of MDRTB occur, causing more than 130,000 deaths (14). In 2006, the documentation of a rapidly fatal TB outbreak among hospitalized HIV patients in Kwa Zulu Natal, South Africa (5) led to the definition of extensively drug-resistant tuberculosis (XDR TB) as TB resistant to a fluoroquinolone and injectable second-line drug (amikacin, capreomycin, or kanamycin) in addition to isoniazid and rifampin. XDR TB has subsequently been reported from over 50 countries by WHO (14). It is likely that the majority of XDR TB cases worldwide remain undetected due to the lack of second-line-drug testing in most high-burden settings. There are an estimated 40,000 new cases of extensively drug-resistant tuberculosis each year (15).The recognition of XDR TB worldwide has made timely identification of XDR TB cases to achieve effective disease management and to prevent their spread a priority (3, 8).Significant challenges exist; although standard protocols exist for second-line-drug susceptibility testing, strong evidence is lacking on many factors, such as the reproducibility and reliability of results, applicability of MIC to clinical outcomes, and intermethod variability. Proficiency testing for second-line-drug susceptibility testing has only recently been integrated into the supranational reference laboratory panel in an effort to improve standardization of second-line drugs across the WHO reference laboratory network.Conventional drug resistance testing takes more than 2 weeks to return a result even after a positive culture has been isolated. Rapid commercial liquid-based culture systems, such as Bactec MGIT 960 testing (Becton Dickinson), for second-line drugs are not yet formally FDA/WHO approved but are reported to be accurate, widely used in developed settings, and reduce turnaround times to approximately 8 days (7). In recent years, many second-line-drug susceptibility testing methods have been developed. The most rapid results are achieved by direct testing of patient specimens by molecular methods; however, in addition to the high cost of such tests, the sensitivity remains suboptimal, and rigorous contamination control is required to maintain accuracy. The majority of high-burden settings currently lack the resources to implement such tests effectively. Two commercial DNA strip assays, INNO-LiPA RifTB (Innogenetics, Zwijndrecht, Belgium) and MTBDRplus (Hain Lifescience GmbH, Germany), targeting the rpoB plus katG and inhA genes have been extensively evaluated for use with Mycobacterium tuberculosis culture and directly on sputum to identify MDR TB cases (2, 10). The Foundation for Innovative Diagnostics (FIND) demonstration projects in South Africa of the GenoType MTBDRplus assay resulted in the recommendation of commercial line probe assays for use in high-burden settings by WHO (16). This assay is based on a multiplex PCR in combination with reverse hybridization. Either the absence of wild-type bands or the appearance of bands targeting specific mutations indicates the presence of a resistant strain. MDR TB cases can be detected within 1 or 2 days of sputum sampling using this assay.In order to rapidly detect second-line-drug resistance, the MTBDRsl test (Hain Lifescience GmbH, Germany) has been developed. This assay can detect mutations in gyrA, rrs, and embB genes, detecting resistance to the fluoroquinolones (FQ), aminoglycosides/cyclic peptides, and ethambutol (EMB), respectively, with a single assay. A previous evaluation study in Germany showed that this assay has a high accuracy for FQ and amikacin-capreomycin resistance testing in clinical strains and sputum samples (6). EMB detection was specific (100%), but its sensitivity (69.2%) was low.The MTBDRsl assay contains 22 probes, including 16 probes for gene mutation detection and 6 probes for the control of the test procedure. The six control probes include a conjugate control (CC), an amplification control (AC), a Mycobacterium tuberculosis complex control (TUB), and three locus probes (gyrA, rrs, and embB) for gene amplification control. The remaining probes detect FQ resistance (gyrA), amikacin/capreomycin resistance (rrs), and ethambutol resistance (embB). The probes contained in the assay do not detect all mutations in these genes but are targeted to the most commonly occurring mutations.The aim of the present study was to determine the accuracy of this assay for detection of FQ, kanamycin, and ethambutol resistance against conventional phenotypic testing as the gold standard on Vietnamese isolates of M. tuberculosis.     

GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples

The purpose of this study was to evaluate the GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 62 clinical strains characterized with the Bactec 460TB system were included. For the INH-resistant strains, the MIC was measured and sequencing was performed. Sixty-five clinical samples from 28 patients (39 smear-positive samples and 26 smear-negative samples) were also tested directly. The corresponding isolates of the clinical specimens were studied with the Bactec 460TB system. The overall rates of concordance of the MTBDRplus assay and the Bactec 460TB system for the detection of RIF and INH susceptibility in clinical strains were 98.3% (61/62) and 79% (49/62), respectively. The rate of concordance between the Bactec 460TB system and the MTBDRplus test for the detection of INH resistance in the group of 27 strains with low-level resistance was 62.9% (17/27), and that for the detection of INH resistance in the group of 21 strains with high-level resistance was 85.71% (18/21). Valid test results were obtained for 78.45% (51/65) of the clinical samples tested. The rates of concordance between both assays for the detection of drug resistance in these samples were 98% (50/51) for RIF and 96.2% (49/51) for INH. Taking into account only one sample per patient, the overall rate of concordance between both tests was 92.85% (26/28). The GenoType MTBDRplus assay is easy to perform and is a useful tool for the management of tuberculosis, as it allows the detection of resistance to RIF and INH in M. tuberculosis strains and also in clinical samples.     

Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, cartridge-based real-time PCR assay designed to detect Mycobacterium tuberculosis and rifampin resistance within 2 h. The performance of the Xpert assay has been evaluated in various clinical settings. However, there are few data comparing the Xpert assay to the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), one of the most widely utilized molecular assays for M. tuberculosis detection. In this prospective study, 320 consecutive respiratory specimens were processed simultaneously using acid-fast bacillus (AFB) staining, mycobacterial cultures with both solid and liquid media, and the Cobas and Xpert assays. The Xpert assay was performed with direct respiratory specimens, while the Cobas assay was done with decontaminated concentrated specimens. Based on the culture as a reference method, the overall sensitivities of the Cobas and Xpert assays were 71.4% and 67.9%, respectively. When AFB smear results were taken into consideration, the sensitivities of the Cobas assay for smear-positive and -negative specimens were 87% and 54%, while those of the Xpert assay were 67% and 69%, respectively. The Cobas assay showed 100% specificity and 100% positive predictive value (PPV) regardless of smear results, while the Xpert assay showed 100% specificity and 100% PPV for smear-positive specimens but 98% specificity and 60% PPV for smear-negative specimens. In conclusion, the Xpert assay showed performance that was slightly inferior to that of the Cobas assay but seems useful for the rapid detection of M. tuberculosis, considering that it was performed without laborious and time-consuming decontamination and concentration procedures.     

Direct Detection of Mycobacterium tuberculosis in Clinical Specimens Using Nucleic Acid Amplification Tests

Tuberculosis continues to be a significant cause of morbidity and mortality worldwide. Rapid detection of Mycobacterium tuberculosis directly in clinical specimens using nucleic acid amplification tests enables infected patients to be placed on appropriate therapy much sooner than when results of conventional culture methods are used. The availability of rapid results also facilitates infection control measures to interrupt transmission of tuberculosis in healthcare settings. The era of commercially available molecular diagnostics for detection of M. tuberculosis began 25 years ago and now encompasses multiple assays that can detect organisms in the M. tuberculosis complex. Several assays also detect chromosomal mutations associated with antimicrobial resistance to further refine therapeutic strategies early in the course of disease. NAATs have revolutionized the diagnosis of TB across the globe and have become the standard of care in many high-burden developing countries.     

Phenotypic and genotypic characteristics of drug resistance in Mycobacterium tuberculosis isolates from pediatric population of Chennai,India

Purpose: Multidrug-resistant TB (MDR-TB) has been reported in almost all parts of the world. Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. Good data on the burden of all forms of TB among children in India are not available. Objective: To study the drug sensitivity pattern of tuberculosis in children aged from 3 months to 18 years and the outcome of drug-resistant tuberculosis by BACTEC culture system and PCR-based DNA sequencing technique. Materials and Methods: This is a retrospective study. One hundred and fifty-nine clinical specimens were processed for Ziehl-Neelsen stain, Mycobacterial culture by BACTEC method, phenotypic DST for first-line drugs for Mycobacterium tuberculosis (M. tuberculosis) isolates and PCR-based DNA sequencing was performed for the M. tuberculosis isolates targeting rpoB, katG, inhA, oxyR-ahpC, rpsL, rrs and pncA. Results and Conclusion: Out of the 159 Mycobacterial cultures performed during the study period, 17 clinical specimens (10.7%) were culture positive for M. tuberculosis. Among the 17 M. tuberculosis isolates, 2 were multidrug-resistant TB. PCR-based DNA sequencing revealed the presence of many novel mutations targeting katG, inhA, oxyR-ahpC and pncA and the most commonly reported mutation Ser531Leu in the rpoB gene. This study underlines the urgent need to take efforts to develop methods for rapid detection and drug susceptibility of tubercle bacilli in the pediatric population.