Real‐time PCR with internal amplification control for detecting tuberculosis: method design and validation

Flores E, Rodríguez JC, Garcia‐Pachón E, Soto JL, Ruiz M, Escribano I, Royo G. Real‐time PCR with internal amplification control for detecting tuberculosis: method design and validation. APMIS 2009; 117: 592–7. Real‐time PCR has been a major development in the diagnosis of tuberculosis. However, most tests do not include an internal amplification control (IAC), which therefore limits it clinical application. In this study a new, easy to perform real‐time PCR test with IAC was designed and validated in clinical samples. The primers amplified a 163‐bp fragment of IS6110 of Mycobacterium tuberculosis and the IAC was designed with a fragment of a different microorganism (Chlamydia trachomatis). The interassay and intraassay variation of this test were very low (0.45–1.65% and 0.18–1.80%, respectively). The detection accuracy was validated in 50 samples (25 urine, 25 sputum) with different concentrations of M. tuberculosis, 18 clinical isolates of non‐tuberculous mycobacteria and 148 samples with clinical suspicion of pulmonary tuberculosis. The specificity was 100%. The detection limit of this PCR test without IAC was approximately 15 bacteria and with IAC approximately 32 bacteria. This real‐time PCR with IAC assay can improve the detection of M. tuberculosis and contribute to standardization of this diagnostic technique.     

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD₆₆₀) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n = 838) and nonrespiratory (n = 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens.     

Four-Year Experience of Use of the Cobas Amplicor System for Rapid Detection of<Emphasis Type="Italic"> Mycobacterium tuberculosis</Emphasis> Complex in Respiratory and Nonrespiratory Specimens in Greece

To evaluate the experience of a clinical microbiology laboratory with a DNA amplification assay for routine detection of Mycobacterium tuberculosis, the Cobas Amplicor Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) assay (Roche Diagnostics Systems, USA) was performed on 7,722 respiratory and 1,451 nonrespiratory specimens collected from 3,321 patients. The results were compared with those of culture in conventional Lowenstein-Jensen medium, culture in the MB/BacT system (Organon Teknika, France), and clinical investigations. A total of 240 of the 254 respiratory specimens culture positive for Mycobacterium tuberculosis were also positive in the PCR assay. Of the 7,300 culture-negative specimens, 45 (0.6%) were positive in the PCR. After detailed interpretation, the overall sensitivity, specificity, and positive and negative predictive values of the PCR assay were 84.5, 99.8, 94.1, and 99.4%, respectively, for respiratory specimens. The PCR assay was more sensitive for smear-positive respiratory specimens (97.1%) than for smear-negative respiratory specimens (48.6%). Of the 18 culture-positive (smear-negative) nonrespiratory specimens, 9 were positive in the PCR. None of the 1,384 culture-negative nonrespiratory specimens were positive in the PCR. The inhibition rates detected by the internal control of the test were 2.2% for respiratory specimens and 3.4% for nonrespiratory specimens. After resolving the discrepancies, the overall sensitivity, specificity, and positive and negative predictive values of the PCR assay were 82.5, 99.8, 94.3, and 99.4%, respectively, when compared to the results of diagnostic culture. In conclusion, the use of the Cobas Amplicor MTB-PCR assay might enable clinical microbiology laboratories with considerable previous experience in molecular biology testing to perform PCR and confirm tuberculosis infection immediately, leading to improved patient management.     

Evaluation of two tuberculosis PCR assays for routine use in a clinical setting of low population and low tuberculosis prevalence

Today, there are numerous different molecular diagnostic assays for the detection of tuberculosis (TB), allowing the optimization of rapid detection of TB according to the clinical need. In this study, two high‐throughput TB PCR assays with combined antimicrobial resistance detection, Anyplex? II MTB/MDR (Seegene) and RealTime MTB + RealTime MTB RIF/INH Resistance (Abbott Molecular), were evaluated for routine use in a clinical setting of low population and low TB prevalence in Finland. The RealTime MTB assay was 100% concordant (22/22 positive, n = 169) with the reference methods (culture and Xpert MTB/RIF PCR assay, Cepheid). However, with a limitation of four separate PCR cycles per kit, the routine use in a low TB‐prevalence setting would easily lead to wasting most of the RIF/INH Resistance reagents. The Anyplex? II MTB/MDR assay usability was more adaptive to suit the clinical setting but the assay sensitivity was considerably lower (86%, 19/22 positive, n = 76) being closer to the sensitivity of smear microscopy. The findings of this study suggest that the evaluated high‐throughput MTB/MDR assays are evidently suboptimal for routine use in a low population, low TB‐prevalence setting. In addition, neither of the two assays covers non‐tuberculous mycobacteria and could therefore not fully replace acid‐fast staining as the initial screening method.     

Comparative evaluation of two commercial assays for direct detection ofMycobacterium tuberculosis in respiratory specimens

Two commercial systems for the amplification and detection ofMycobacterium tuberculosis directly from respiratory samples were compared. The Roche Cobas Amplicor MTB Test and the Roche manual Amplicor MTB Test (Roche Diagnostic Systems, USA) were applied to 755 decontaminated respiratory specimens collected from 470 patients. Results were compared with those of acid-fast staining and culture. A total of 251 specimens were collected from 156 patients diagnosed with pulmonary tuberculosis, including 28 specimens corresponding to 13 patients that were receiving antituberculous treatment. Given the overall positivity rate of 33.2% (251/755), the sensitivity, specificity, and positive and negative predictive values were 92.4, 100, 100, and 96.5%, respectively, for the Cobas Amplicor MTB Test and 90.8, 100, 100, and 95.8%, respectively, for the Amplicor MTB Test. For 204 (81.3%) smear positive specimens and 47 (19.7%) smear negative specimens, the sensitivity values were 100 and 59.6%, respectively, for the Cobas Amplicor MTB Test and 100 and 51%, respectively, for the Amplicor MTB Test. There were no statistically significant differences in sensitivity or specificity between the two assays and culture (p>0.05). The overall results of both assays were concordant for 99.5% of the samples. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection ofMycobacterium tuberculosis complex in respiratory specimens, the Cobas Amplicor MTB Test appears to be slightly more sensitive than the Amplicor MTB Test when smear negative specimens are investigated.     

Comparison of the BDProbeTec ET System with the Cobas Amplicor PCR for Direct Detection of <Emphasis Type="Italic">Mycobacterium </Emphasis><Emphasis Type="Italic">tuberculosis</Emphasis> in Respiratory Samples

In the study presented here, the performance of the BDProbeTec ET system (Becton Dickinson, USA) was compared with the Roche Cobas Amplicor-PCR (Roche, Switzerland) to detect Mycobacterium tuberculosis complex (MTB) in clinical respiratory samples. The Bactec MGIT 960 liquid culture system (Becton Dickinson) was used as a reference method. A total of 411 samples were tested. Of the 93 culture-positive samples, both the BDProbeTec ET system and the Cobas Amplicor-PCR detected 87 (sensitivity, 93.5%). When only smear-negative samples were considered, the BDProbeTec ET exhibited a sensitivity of 50% and the Cobas Amplicor-PCR 60%. Specificity was 99.7% for the BDProbeTec ET system and 100% for the Cobas Amplicor-PCR. Percent agreement between the two nucleic amplification methods was 98.7%. Inhibition occurred in three (0.7%) samples in the BDProbeTec ET system. The high sensitivity and specificity of the BDProbeTec ET system suggest it is a useful method for the rapid and direct detection of MTB in smear-positive respiratory samples.     

Mycobacterium tuberculosis Rv1419 encodes a secreted 13 kDa lectin with immunological reactivity during human tuberculosis

In this study, we have identified a secreted 13 kDa lectin from Mtb (Mtb, Mycobacterium tuberculosis; sMTL‐13) by homology search of a non‐redundant lectin database. Bioinformatic analysis revealed that sMTL‐13 belongs to the ricin‐type β‐trefoil family of proteins containing a Sec‐type signal peptide present in Mtb complex species, but not in non‐tuberculous mycobacteria. Following heterologous expression of sMTL‐13 and generation of an mAb (clone 276.B7/IgG1κ), we confirmed that this lectin is present in culture filtrate proteins from Mtb H37Rv, but not in non‐tuberculous mycobacteria‐derived culture filtrate proteins. In addition, sMTL‐13 leads to an increased IFN‐γ production by PBMC from active tuberculosis (ATB) patients. Furthermore, sera from ATB patients displayed high titers of IgG Ab against sMTL‐13, a response found to be decreased following successful anti‐tuberculosis therapy. Together, our findings reveal a secreted 13 kDa ricin‐like lectin from Mtb, which is immunologically recognized during ATB and could serve as a biomarker of disease treatment.     

Detection ofMycobacterium tuberculosis complex in clinical specimens by a commercial polymerase chain reaction kit

A total of 722 respiratory and 86 nonrespiratory specimens obtained from 456 patients were tested for detection ofMycobacterium tuberculosis complex by a commercial polymerase chain reaction (PCR) kit (Amplicor, Roche Diagnostic Systems) and the results compared with those of microscopy and culture (solid and radiometric media). Respiratory and nonrespiratory specimens were analysed separately. Of the respiratory specimens, 54 were positive forMycobacterium tuberculosis complex both in the PCR and in culture, five were positive in the PCR but negative in culture, and eight were positive in culture but negative in the PCR. Four cultures were positive for mycobacteria other thanMycobacterium tuberculosis; none of these gave a positive result in the commercial test. Resolution of discrepant results was performed by analysis of patients' clinical data. For respiratory specimens the sensitivity of the commercial test was 87.6%, the specificity 99.6%, the positive predictive value 96.6%, and the negative predictive value 98.7%. For nonrespiratory specimens the sensitivity was 60%, whereas the specificity ranged as high as 98.6%. For this group the positive predictive value was 85.7% and the negative predictive value 94.9%. When respiratory specimens are used, the commercial PCR test for detection ofMycobacterium tuberculosis complex, with its high sensitivity and specificity, is a good complementary diagnostic tool for rapid diagnosis of bronchopulmonary tuberculosis in a routine mycobacterial laboratory.     

Early Identification of Mycobacterium tuberculosis and Mycobacterium avium Using the Polymerase Chain Reaction on Samples Positive by a Rapid Commercial Culture System

 A combination of two methods – a rapid culture method [Mycobacteria Growth Indicator Tube (MGIT); Becton-Dickinson, USA] and a double polymerase chain reaction (PCR) assay – was assessed for the detection and identification of Mycobacterium tuberculosis and Mycobacterium avium from clinical samples. The aim of the study was to evaluate the ability of the system to offer rapid and accurate diagnosis of mycobacterial infections. After decontamination, clinical samples (n=554) were stained and cultured in parallel on solid media and in MGITs following standard procedures. The performance of the two culture systems was compared. Positive MGITs were tested for the presence of Mycobacterium tuberculosis and Mycobacterium avium by PCR of IS6110 (Mycobacterium tuberculosis) and the 16S rRNA gene (Mycobacterium avium). A total of 41 mycobacteria – 27 Mycobacterium tuberculosis isolates, eight Mycobacterium avium isolates, and six other species of mycobacteria – were isolated by one or both culture media. The MGIT system recovered 36 (87.8%) mycobacteria and the solid media 33 (80.4%). The mean time to detection by the two culture systems did not differ overall, but the mean time to detection of Mycobacterium avium from smear-positive specimens was shorter in MGITs than in solid media (5.25 days vs. 16.25 days, P<0.05). The double PCR assay performed on the 36 positive MGITs correctly identified all 24 Mycobacterium tuberculosis-positive MGITs and all six Mycobacterium avium-positive vials. Therefore, application of the PCR assay to positive MGITs may mean that Mycobacterium tuberculosis and Mycobacterium avium can be identified at an earlier stage than with current methods.     

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.     

Evaluation of the Xpert MTB/RIF test accuracy for diagnosis of tuberculosis in areas with a moderate tuberculosis burden

The Xpert MTB/RIF assay (Xpert) is a molecular assay used for direct detection of Mycobacterium tuberculosis (MTB) in clinical specimens. In this study, we aimed to assess the accuracy of the Xpert assay for the diagnosis of tuberculosis (TB) in TB suspected patients from the northern region of Iran. The obtained results were compared with the culture method. The sputum specimens were examined using the Xpert assay, smear microscopy, and solid culture media as a reference diagnostic tool. Among 293 presumptive TB cases, 92 (31.4%) were positive according to the culture method. The Xpert method detected 88 (95.7%) cases that were positive according to the culture method, compared with 78 (84.8%) positive cases according to smear microscopy. The overall sensitivity and specificity of the Xpert method for TB diagnosis were 95.7% and 99%, respectively. Also, the sensitivity and specificity for smear microscopy were 84.8% and 97.5%, respectively. The Xpert assay showed high overall sensitivity and specificity; thus, it can be effectively used for the early and accurate diagnosis of MTB in TB endemic areas. In addition, the agreement between semi‐quantitative results of Xpert and smear microscopy assays could be helpful in evaluating transmission potential in TB patients.     

Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85

A polymerase chain reaction (PCR) assay was developed for detection of mycobacteria using amplification of a 162 bp region of the genes coding for the mycobacterial antigen 85 complex. Strains belonging to theMycobacterium tuberculosis complex were further differentiated from non-tuberculous mycobacteria by hybridization of the PCR derived Southern blot with an internal oligonucleotide probe and washing under stringent conditions. The method allowed rapid and sensitive detection of mycobacterial DNA in uncultured clinical samples. PCR results obtained forMycobacterium tuberculosis in 206 specimens from 180 untreated patients gave a sensitivity of 93.9% and a specificity of 94.3% compared with the culture. PCR detected DNA fromMycobacterium tuberculosis in seven samples from patients with clinically evident tuberculosis in whom culture was negative. The results suggest that this PCR assay could be used for early and specific diagnosis of tuberculosis.     

Rapid and specific detection of Mycobacterium tuberculosis from acid-fast bacillus smear-positive respiratory specimens and BacT/ALERT MP culture bottles by using fluorogenic probes and real-time PCR

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.     

The value of microscopic‐observation drug susceptibility assay in the diagnosis of tuberculosis and detection of multidrug resistance

Inexpensive, rapid, and reliable tests for detecting the presence and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are urgently needed to control the transmission of tuberculosis. In this study, we aimed to assess the accuracy and speed of the microscopic‐observation drug susceptibility (MODS) assay in the identification of MTBC and detection of multidrug resistance. Sputum samples from patients suspected to have tuberculosis were simultaneously tested with MODS and conventional culture [Löwenstein‐Jensen (LJ) culture, BACTEC MGIT? 960 (MGIT) system], and drug susceptibility testing (MGIT system) methods. A total of 331 sputum samples were analyzed. Sensitivity and specificity of MODS assay for detection of MTBC strains were 96% and 98.8%, respectively. MODS assay detected multidrug resistant MTBC isolates with 92.3% sensitivity and 96.6% specificity. Median time to culture positivity was similar for MGIT (8 days) and MODS culture (8 days), but was significantly longer with LJ culture (20 days) (p < 0.0001 for both comparisons). Median time to availability of the susceptibility results was significantly (p < 0.0001) shorter with MODS assay (8 days) than MGIT system (20 days). In conclusion, MODS is an inexpensive and rapid test with good performance characteristics for direct diagnosis of tuberculosis and detection of multidrug resistance.     

Evaluation of a whole‐blood chemiluminescent immunoassay of IFN‐γ, IP‐10, and MCP‐1 for diagnosis of active pulmonary tuberculosis and tuberculous pleurisy patients

The study explored the use of IP‐10, MCP‐1, and IFN‐γ as biomarkers to improve the diagnoses of active pulmonary tuberculosis and tuberculous pleurisy. We enrolled 267 individuals, including 134 TB patients, 93 patients with non‐tuberculous pulmonary diseases, and 40 healthy controls. Whole bloods were stimulated in vitro with rCFP‐10/ESAT‐6 protein antigen of Mycobacterium tuberculosis. The levels of IFN‐γ, IP‐10, and MCP‐1 in cultured supernatants of whole bloods were detected by a chemiluminescence immunoassay. A receiver operating characteristic (ROC) curve was drawn to determine the cutoff value for diagnosing TB and to evaluate the diagnostic efficacies of the IFN‐γ, IP‐10, and MCP‐1 for TB. The antigen‐specific release of each cytokine, IFN‐γ, IP‐10, and MCP‐1, was significantly higher in the TB groups than in either the non‐tuberculous pulmonary disease group (p < 0.001) or the healthy control group (p < 0.001). The ROC curves indicated cutoff values for IFN‐γ, IP‐10, and MCP‐1 at 147.8, 160.4, and 496.4 pg/mL, respectively. The sensitivity, specificity, PPV, NPV, and diagnostic efficiency for IFN‐γ were 85.8%, 70.7%, 74.7%, 83.2%, and 78.3%, respectively; for IP‐10 were 72.4%, 75.9%, 75.2%, 73.2%, and 74.2%, respectively; and for MCP‐1 were 90.3%, 97.0%, 96.8%, 90.8%, and 93.6%, respectively. IFN‐γ combined MCP‐1 improved the sensitivity to 97.8% compared with IFN‐γ (p < 0.001). Our findings indicate high sensitivity and specificity of MCP‐1 as novel biomarkers for the diagnosis of active pulmonary tuberculosis and tuberculous pleurisy.     

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.     

Polymerase chain reaction for detection ofMycobacterium tuberculosis in sputum

The polymerase chain reaction (PCR) was evaluated in a trial which, with respect to the positive-to-negative ratio, approximated the situation of a diagnostic laboratory in a tuberculosis-endemic area. Three hundred sputum samples were included in the study, of which one-third were known to contain mycobacteria as judged by direct microscopy. The repetitive insertion sequence IS6110/IS986 ofMycobacterium tuberculosis was used as a target. The samples were spiked with DNA from a modified IS6110/IS986 sequence, which gives rise to PCR products easily distinguished from PCR products amplified from chromosomalMycobacterium tuberculosis DNA. This allowed identification of samples that contained substances inhibitory to theTaq polymerase. The detection limit of the assay was 0.05 pg to 0.5 pg of purifiedMycobacterium tuberculosis DNA, corresponding to 10 to 100 organisms. The sensitivity and specificity of the PCR was compared with that of conventional microscopy and culture. It was concluded that this method is fast and sensitive, but that culture currently is crucial for assessing viability and thus infectivity.     

Limits of Detection of Mycobacterium tuberculosis in Spiked Cerebrospinal Fluid Using the Polymerase Chain Reaction in Tuberculous Meningitis

 The limit of detection of Mycobacterium tuberculosis in spiked cerebrospinal fluid (CSF) using polymerase chain reaction (PCR) was compared to that of a radiometric liquid culture. Serial dilutions of clinical isolates of Mycobacterium tuberculosis were prepared in CSF (n=3) or broth (n=11) with estimated concentrations of 0–550 cfu/ml. Each dilution was examined concurrently by PCR and radiometric culture. PCR and radiometric culture detected Mycobacterium tuberculosis DNA in all dilutions with an estimated 2 cfu/ml in the CSF. At lower concentrations (estimated <2 cfu/ml), PCR and radiometric culture were positive in three of five (60%) and five of five (100%) CSF samples, respectively. In comparison to PCR in broth dilutions, no evidence of inhibition or interference was noted. These results imply that PCR can provide a rapid and reliable diagnosis of tuberculous meningitis, although there is a potential for false-negative results to occur in samples containing very few organisms (<2 cfu/ml).     

Potential cross‐reactivity of monoclonal antibodies against clinically relevant mycobacteria

Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTb). In 2011, global mortality due to tuberculosis was 1·4 million individuals. The only available vaccine is the attenuated M. bovis [bacillus Calmette–Guérin (BCG)] strain, which confers variable protection against pulmonary tuberculosis. Some widely distributed non‐tuberculous mycobacteria (NTM), such as M. avium and M. arupense, are also potential pathogens for humans. This work aimed to produce and characterize monoclonal antibodies against the M. bovis BCG Mexico strain of the MTb, M. avium subs. hominissuis and the M. arupense strain from NTM. Hybridomas were produced from splenocytes of BALB/c female mice immunized with radiation‐inactivated mycobacteria, and the immunoglobulin (Ig)G2a antibody‐producing clones with the highest antigenic recognition were selected. The selected clones, Mbv 2A10 for M. bovis BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense, were used in further studies. Enzyme‐linked immunosorbent assay (ELISA) and immune proteomics analyses characterized the clones as having the highest cross‐reactivity with mycobacteria. Using mass spectrometry, a number of proteins recognized by the monoclonal antibody (mAb) clones were identified. These proteins had roles in metabolic processes, hypoxia, cell cycle and dormancy. In addition, a Clustal W and Immune Epitope Database (IEDB) in‐silico analysis was performed in protein sequences that result in the conserved regions within probability epitopes that could be recognized for Mbv2A10 and Mav3H1 clones.