Evaluation of a New Commercial Assay for Diagnosis of Pulmonary and Nonpulmonary Tuberculosis

A new commercial assay for the diagnosis of tuberculosis, the BDProbeTec ET Direct Detection assay (Becton Dickinson, USA), was evaluated using 351 respiratory and 372 nonrespiratory specimens. The results were compared to detection of Mycobacterium tuberculosis complex (MTC) by conventional culture. Among the 351 respiratory specimens, MTC bacteria were identified in 150, of which 85 were positive by both microscopy and the assay. Sixty-five specimens culture positive for MTC were microscopy negative; of these, 39 were positive in the assay. All 26 specimens culture positive for nontuberculous mycobacteria (NTM) were negative by the assay. Of 175 specimens culture negative for MTC, 3 were falsely positive by the assay and 1 yielded inhibition. The overall sensitivity and specificity values were 82.7% and 98.5%, respectively. The sensitivity for microscopy-positive and -negative respiratory specimens was 100% and 60%, respectively. After correction for discrepancies, the specificity was 99% compared with notification data. The BDProbeTec ET assay detected 66 of 67 microscopy-positive and 50 of 125 microscopy-negative nonrespiratory specimens. The result for one specimen was inconclusive. All nine specimens containing NTM were negative by the assay. Of 171 specimens culture negative for MTC, 6 were falsely positive by the assay. The overall sensitivity and specificity values obtained with nonrespiratory specimens were 60.7% and 96.7%, respectively. After examining discrepancies by reviewing the patients' histories, the specificity was 98.9%. The sensitivity was 98.5% in microscopy-positive specimens and 40.3% in microscopy-negative specimens. The overall inhibition rate was 0.3%. The BDProbeTec ET assay is a fast, effective, and user-friendly system that can be used for rapid detection of MTC bacteria in respiratory and microscopy-positive nonrespiratory specimens as an important supplement to smear and culture. Electronic Publication     

Protein-coding housekeeping gene Rv2461c can be used as an amplification target in loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis in sputum samples

The study is to explore the potential of the conserved Rv2461c gene as a biomarker for Tuberculosis (TB) diagnosis. The conservation of the hypothetical genes was evaluated in this study using multiple sequence alignment and phylogenetic analysis. The conservation of Rv2461c coding gene was evaluated by polymerase chain reaction using six reference strains of M. tuberculosis complex (MTC), 156 M. tuberculosis clinical isolates, 25 species of non-tuberculosis mycobacteria (NTM), and 10 non-mycobacterial species. A total of 126 clinical sputum specimens were collected from patients with respiratory symptoms, including 79 specimens from suspected TB patients, and 47 specimens from patients with respiratory diseases other than TB. Genomic DNAs were extracted and subject to polymerase chain reaction for nucleic acid amplification test. In addition, we successfully developed loop-mediated isothermal amplification (LAMP) technology for rapid detection of M. tuberculosis in sputum specimens. The sensitivity and specificity of LAMP assay were evaluated for the detection of M. tuberculosis. Phylogenetic analysis of the clpP sequences revealed that the Mycobacterium strains were split into two major clusters: i) MTC; ii) NTM strains and M. leprae. During the evaluation of the conservation of Rv2461c coding gene, all MTC strains yielded positive results, and no false-positive results were observed in NTM or other bacterial species. LAMP analysis showed high sensitivity and specificity (84.8% and 95.7%, respectively) for the detection of M. tuberculosis from sputum. Our result indicated that Rv2461c coding gene was an efficient and promising alternative nucleic acid amplification test target for the detection of M. tuberculosis.     

Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.     

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.     

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.     

Epidemiology and Risk Factors Associated with NTM Pulmonary and Extrapulmonary Infections in a High Tuberculosis Endemic Region

Introduction: Non-tuberculous mycobacteria, although identified as pathogenic to humans long time ago, are emerging as the new threat in the past two decades. Even in tuberculosis endemic country such as India, they are being isolated from the clinical specimens more often than previously. This change in trend is of concern, because they are often misdiagnosed as Mycobacterium tuberculosis or even as drug-resistant tuberculosis. Objectives: A prospective, observational study was planned to identify the frequency and risk factors associated with pulmonary and extrapulmonary non-tuberculous mycobacterial (NTM) infections. Agreement between two commercially available molecular systems, namely GenoType Mycobacteria CM assay and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) used in the identification of mycobacterial species is also analysed. Materials and Methods: NTM isolated from pulmonary and extrapulmonary clinical specimens over a period of 1½ year was included in the study. Patient demographics were collected, and the risk factors associated with NTM infections were analyzed. NTM grown on culture was speciated using GenoType Mycobacteria CM assay (HAIN Life Sciences, Germany) and MALDI TOF MS (bioMerieux, France). Drug-susceptibility tests were done for rapid-growing NTM using E-test (bioMerieux, France). Results: Eight hundred and fifty-four mycobacteria were isolated from 5009 specimens processed during the study period. Out of the mycobacteria grown, 74 (8.7%) were NTM and 780 (91.3%) were Mycobacterium tuberculosis complex. The NTM isolated from pulmonary specimens were 46 (62.16%) and from extrapulmonary sources were 28 (37.84%). The most common species isolated from pulmonary specimens was Mycobacterium intracellulare and from extrapulmonary specimens was Mycobacterium abscessus. Concordance between the two commercial assays used for the identification was 96.49%. The most common risk factor associated with pulmonary NTM was previous lung pathology, while with extrapulmonary NTM infection was previous surgical intervention. Drug-susceptibility tests for rapid growers showed amikacin and clarithromycin as the most active drugs in vitro. Conclusions: NTM plays a significant role in causing pulmonary and extrapulmonary infections even in our part of the country with high endemicity of tuberculosis. NTM has emerged as important pathogens even in the immunocompetent patients. There is a need for rapid diagnosis and susceptibility testing of NTM to aid physicians administer timely and appropriate treatment to the patients.     

Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory specimens

A total of 217 specimens submitted for routine smear and culture from three different sites within the western United States were used to evaluate the GeneXpert MTB/RIF assay (for research use only) (Cepheid, Sunnyvale, CA). Overall agreement compared to culture was 89% (98% for smear positives and 72% for smear negatives) for detection of Mycobacterium tuberculosis.     

Evaluation of a Fluorescence Hybridisation Assay Using Peptide Nucleic Acid Probes for Identification and Differentiation of Tuberculous and Non-Tuberculous Mycobacteria in Liquid Cultures

 The performance was evaluated of a fluorescence in situ hybridisation assay using peptide nucleic acid probes (Dako Probe MTB Culture Confirmation Test; Dako, Denmark) for identification of Mycobacterium tuberculosis complex (MTC) organisms and differentiation between tuberculous and non-tuberculous mycobacteria (NTM) in material taken directly from Bactec 12B (Becton Dickinson, USA) and MB/BacT (Organon Teknika, USA) bottles. The test was applied to 129 smear-positive (Ziehl-Neelsen stain) clinical specimens, 48 previously identified clinical strains of mycobacteria (12 MTC and 36 NTM), and 51 reference strains (7 MTC and 44 NTM) which were all previously inoculated into Bactec 12B and MB/BacT bottles. The sensitivity and specificity of the assay for MTC-positive cultures was 87.6% and 100%, respectively, for Bactec 12B, and 100%, respectively, for MB/BacT. The sensitivity and specificity of the assay for NTM-positive cultures was 100% for both media.     

Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR

Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.     

Proficiency testing of clinical microbiology laboratories using modified decontamination procedures for detection of nontuberculous mycobacteria in sputum samples from cystic fibrosis patients. The Nontuberculous Mycobacteria in Cystic Fibrosis Study Group.

An improved decontamination method has been demonstrated to reduce overgrowth of mycobacterial media by Pseudomonas aeruginosa and allow the successful recovery of nontuberculous mycobacteria (NTM) from cystic fibrosis (CF) patients. Twenty microbiology laboratories participating in a multicenter investigation designed to determine the significance of NTM in CF patients were required to demonstrate proficiency in the incorporation of this improved method; this was accomplished by successful decontamination and culture workup of a panel of simulated sputum samples seeded with P. aeruginosa and various NTM. All laboratories successfully recovered NTM from samples with acid-fast bacillus (AFB) smear scores of 3+/4+ (i.e., 2 to 18 or > 18 organisms/field). Low-inoculum samples (1+/2+ AFB smears [2 to 18 organisms in 100 or 10 fields]) were problematic in that processed specimens were often smear and/or culture negative.     

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.     

Development of a Novel PCR Restriction Analysis of the hsp65 Gene as a Rapid Method To Screen for the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in High-Burden Countries

The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.     

Does Neutralization of Gastric Aspirates from Children with Suspected Intrathoracic Tuberculosis Affect Mycobacterial Yields on MGIT Culture?

The microbiological confirmation of pulmonary tuberculosis in children relies on cultures of gastric aspirate (GA) specimens. Conventionally, GAs are neutralized to improve culture yields of mycobacteria. However, there are limited data to support this practice. To study the utility of neutralization of GAs with sodium bicarbonate in children with intrathoracic tuberculosis, a total of 116 children of either sex, aged 6 months to 14 years (median age, 120 months; interquartile range [IQR], 7 to 192 months), underwent gastric aspiration on 2 consecutive days. Gastric aspirates were divided into two aliquots, and only one aliquot was neutralized with 1% sodium bicarbonate. Both aliquots were processed for smear and culture examinations. Out of the 232 gastric aspirates, 12 (5.17%) were acid-fast bacilli (AFB) smear positive. There were no differences in smear positivity rates from samples with or without neutralization. The yield of Mycobacterium tuberculosis on a Bactec MGIT 960 culture system was significantly lower in the neutralized samples (16.3% [38/232]) than in the nonneutralized samples (21.5% [50/232]) (P = 0.023). There was no significant difference between the neutralized and the nonneutralized samples in time to detection using the MGIT 960 system (average, 24.6 days; IQR, 12 to 37 days) (P = 0.9). The contamination rates were significantly higher in the neutralized samples than in the nonneutralized samples (17.2% [40/232] versus 3.9% [9/232]) (P = 0.001). The agreement for positive mycobacterial culture between the two approaches was 66.5% (P = 0.001). Hence, we recommend that gastric aspirate samples not be neutralized with sodium bicarbonate prior to culture for M. tuberculosis.     

Duplex detection of the Mycobacterium tuberculosis complex and medically important non‐tuberculosis mycobacteria by real‐time PCR based on the rnpB gene

A duplex real‐time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non‐tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non‐respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB‐PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non‐tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay.     

UV-Fixed-Thick-Blotch Preparation Improves Sensitivity of Auramine Staining

We describe here a very simple modification of the auramine staining procedure based on preparation of a UV-fixed thick blotch which allowed us to reach an overall sensitivity of 0.82 (592 acid-fast bacillus [AFB]-positive specimens/722 initial respiratory specimens with positive mycobacterial culture) and sensitivities of 0.93 (526 AFB-positive specimens/564 culture-positive specimens) for Mycobacterium tuberculosis complex and 0.42 (66 AFB-positive specimens/158 culture-positive specimens) for nontuberculous mycobacteria.     

Evaluation of a semi-automated Seegene PCR workflow with MTB,MDR, and NTM detection for rapid screening of tuberculosis in a low-prevalence setting

In areas of low tuberculosis (TB) prevalence, laboratory diagnosis of TB may essentially cover non-tuberculous mycobacteria (NTM) in addition to Mycobacterium tuberculosis (MTB). In this study, a semi-automated PCR workflow distinguishing MTB and NTM (Anyplex™ MTB/NTMe, Seegene) and subsequently detecting MTB isoniazid/rifampicin resistance (Allplex™ MTB/MDRe, Seegene) was evaluated for replacing smear microscopy of acid-fast bacilli as the rapid screening method for TB. With 279 clinical samples, 47 cultures positive for MTB and 76 for NTM, the Anyplex™ MTB/NTMe assay and smear microscopy showed equal sensitivities (49.6% vs 50.8%, respectively) but Anyplex™ MTB/NTMe was more sensitive for MTB (63.8% vs 25.6%) than for NTM (40.8% vs 64.5%). Allplex™ MTB/MDRe showed a slightly higher sensitivity of 68.1% for MTB (32/47 positive, n = 222). Antibiotic resistance profiles were correctly identified for all MTB isolates (one MDR isolate). Specificity was 100% for both assays. Anyplex™ MTB/NTMe detected all the 18 NTM species present in the study. The analytical performance of the evaluated high-throughput workflow was relatively weak compared to culture but potentially adequate as a rapid screening method analogous to smear microscopy with additional differentiation between TB, MDR-TB, and NTM.     

Sensitivity of Fluorochrome Microscopy for Detection of Mycobacterium tuberculosis versus Nontuberculous Mycobacteria

The results for 6,532 consecutive mycobacterial respiratory specimens collected from 1,040 patients from 1993 to 1995 in a Texas hospital were studied to determine the sensitivity of fluorescence microscopy for detection of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Smears were positive for acid-fast bacilli (AFB) in 63% (677 of 1,082) of specimens growing M. tuberculosis and 56% (638 of 1,148) of specimens growing the four most common species of NTM. Smear positivity by species was 58% (446 of 776) for M. avium complex, 51% (154 of 300) for rapidly growing mycobacteria (98% were M. abscessus), 78% (29 of 37) for M. kansasii, and 26% (9 of 35) for M. gordonae. Definite or probable disease by clinical criteria was present in 79% of patients with M. avium complex, 93% of patients with rapidly growing mycobacteria, 100% of patients with M. kansasii, and 0% of patients with M. gordonae. Patients with M. avium complex had a low incidence of AIDS (7%), and approximately 50% of non-AIDS patients had upper-lobe cavitary disease and 50% had nodular bronchiectasis. Only 23 of 6,532 (0.35%) of AFB smears were positive with a negative culture excluding patients on therapy for established mycobacterial disease. These studies suggest that NTM are as likely as M. tuberculosis to be detected by fluorescent microscopy in specimens from patients from areas endemic for NTM lung disease and at low risk for AIDS.     

Rapid identification of mycobacteria by Raman spectroscopy

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.     

Evaluation of the New MB Redox System for Detection of Growth of Mycobacteria

We evaluated a new mycobacterial culture system, MB Redox, for recovery rate and time to detection of mycobacteria from 742 consecutive respiratory specimens and compared the results to those found with L?wenstein-Jensen (LJ) medium. Twenty specimens (2.7%) were positive for M. tuberculosis: 17 on LJ medium and 19 in MB Redox, with 16 specimens positive in both media. In addition, 24 specimens (3.2%) were positive for nontuberculous mycobacteria (NTM), 20 on LJ medium, 18 in MB Redox, and 14 in both media. For M. tuberculosis, the mean times to detection were 28.9 days on LJ medium and 23.6 days in MB Redox, and for NTM, the mean times to detection were 40.6 days on LJ medium and 32.3 days in MB Redox.     

Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.