Identification Of Mycobacterium Tuberculosis Complex Research Articles

Retrospective and prospective evaluation of the FluoroType®-Mycobacteria VER 1.0 assay for the identification of mycobacteria from cultures in a French center.

Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER1.0kit. The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER1.0kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER1.0database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER1.0database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER1.0kit. The good performance of the FluoroType®-Mycobacteria-VER1.0kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level.

Diagnostic performance of two commercial MPT64 assays for the identification of Mycobacterium tuberculosis complex from liquid culture

BackgroundTuberculosis (TB) is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC). Early diagnosis of TB is the most essential component to control TB. In this study, we evaluated the diagnostic performance of SD Bioline TB Ag MPT64 (SD Bioline) and BD MGIT TBc Identification (BD TBc ID) kits for the detection of MTBC from MGIT (Mycobacteria Growth Indicator Tube) 960 culture tube, positive for acid-fast bacilli (AFB). MethodsA total of 198 AFB positive MGIT cultures [MTBC, 150 and non-tuberculous mycobacteria (NTM), 48] were collected during the study period (April 2021 to March 2023) and subjected to SD Bioline and BD TBc ID assays, respectively. All the 150 MTBC positive cultures were confirmed by multiplex PCR targeting MPB64, protein B and IS6110 genes, and the 48 NTM cultures were speciated by using line probe assays technique- Genotype Mycobacterium CM assay. ResultsSD Bioline and BD TBc ID kits detected all 150 MTBC strains correctly from MGIT positive cultures and no false negative was observed. However, there was one false positive for M. abscessus by SD Bioline and two false positive results for M. abscessus and M. intracellulare each by BD TBc ID. Overall, the sensitivity of both SD Bioline and BD TBc ID tests was 100 % and specificity was 97.9 % and 95.8 %, respectively. ConclusionOur results highlighted that SD Bioline and BD TBc ID assays played a promising role in the earlier identification of MTBC present in liquid culture and may act as a potential alternative to biochemical and expensive molecular techniques.

Clinical Whole-Genome Sequencing Assay for Rapid Mycobacterium tuberculosis Complex First-Line Drug Susceptibility Testing and Phylogenetic Relatedness Analysis.

The global rise of drug resistant tuberculosis has highlighted the need for improved diagnostic technologies that provide rapid and reliable drug resistance results. Here, we develop and validate a whole genome sequencing (WGS)-based test for identification of mycobacterium tuberculosis complex (MTB) drug resistance to rifampin, isoniazid, pyrazinamide, ethambutol, and streptomycin. Through comparative analysis of drug resistance results from WGS-based testing and phenotypic drug susceptibility testing (DST) of 38 clinical MTB isolates from patients receiving care in Los Angeles, CA, we found an overall concordance between methods of 97.4% with equivalent performance across culture media. Critically, prospective analysis of 11 isolates showed that WGS-based testing provides results an average of 36 days faster than phenotypic culture-based methods. We showcase the additional benefits of WGS data by investigating a suspected laboratory contamination event and using phylogenetic analysis to search for cryptic local transmission, finding no evidence of community spread amongst our patient population in the past six years. WGS-based testing for MTB drug resistance has the potential to greatly improve diagnosis of drug resistant MTB by accelerating turnaround time while maintaining accuracy and providing additional benefits for infection control, lab safety, and public health applications.

Rapid identification of Mycobacterium tuberculosis in cultures by molecular and immunological methods

Objective : The study aimed to evaluate molecular and immunological methods and to propose a workflow using them for tuberculosis (TB) diagnosis routine. Methods : A cross-sectional retrospective study was performed, including 121 liquid cultures from a TB laboratory located in the extreme south of Brazil. All cultures were positive for Mycobacterium tuberculosis complex (MTBC) by in-house Polymerase Chain Reaction (PCR) using DNA extracted by the CTAB method (PCR-CTAB) for IS6110 detection. These cultures were subjected to faster tests than this one, the immunological MPT64 assay and the PCR using DNA extracted by thermal lysis method (PCR-TL), and these were evaluated for MTBC identification using PCR- CTAB as a reference method. Results :The sensitivity of MPT64 assay and PCR-TL to identify MTBC in positive cultures by PCR-CTAB were 73.6% (89/121) and 98.3% (119/121), respectively. We proposed a workflow based on the use of MPT64 assay in liquid cultures suggestive of MTBC, and in case of a negative result, we suggest the performance of PCR-TL. The PCR-CTAB is suggested only if faster tests are negative. Conclusions : Methods capable of confirming MTBC in cultures should continue to be standardized, tested, and optimized to meet the ideal requirements of simplicity, quickness, and effectiveness. The molecular and immunological methods evaluated have differences in the execution and detection of MTBC in cultures, but they are rapid tools for laboratory TB diagnosis.

Rapid Identification of Mycobacterium tuberculosis Complex Using Mass Spectrometry: A Proof of Concept.

Mycobacteria that form the Mycobacterium tuberculosis complex are responsible for deadly tuberculosis in animals and patients. Identification of these pathogens at the species level is of primary importance for treatment and source tracing and currently relies on DNA analysis, including whole genome sequencing (WGS), which requires a whole day. In this study, we report the unprecedented discrimination of M. tuberculosis complex species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), with WGS as the comparative reference standard. In the first step, optimized peptide extraction applied to 36 isolates otherwise identified in five of the 11 M. tuberculosis complex variants by WGS yielded 139 MALDI-TOF spectra, which were used to identify biomarkers of interest that facilitate differentiation between variants. In a second step, 70/80 (88%) other isolates were correctly classified by an algorithm based on specific peaks. This study is the first to report a MALDI-TOF-MS method for discriminating M. tuberculosis complex mycobacteria that is easily implemented in clinical microbiology laboratories.

Clinical Validation of the Xpert MTB/RIF Test for Identification of the Mycobacterium tuberculosis Complex in Acid-Fast Bacillus Smear-Positive MGIT Broth Cultures.

The identification of the Mycobacterium tuberculosis complex (MTBC) from smear-positive broth cultures can be achieved using several methods, including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from acid-fast bacilli (AFB) smear-positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampin resistance. The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear-positive MGIT 960 broth cultures (Becton, Dickinson, Sparks, MD). We sought to validate the Xpert MTB/RIF test for use with AFB smear-positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false-positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampin resistance from AFB smear-positive MGIT 960 broth cultures.

Highly specific and sensitive detection of the Mycobacterium tuberculosis complex using multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor.

Tuberculosis (TB) is the deadliest infectious caused by Mycobacterium tuberculosis complex (MTBC). Because most TB cases occur within low-income populations, developing a specific, sensitive, cost-saving, and rapid point-of-care test for the early diagnosis of TB is important for achieving the WHO's End Tuberculosis Strategy. In the current study, a novel nucleic acid detection strategy that includes multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor (mLAMP-LFB) was used to detect MTBC. The two sets of LAMP primers specific to the IS6110 and gyrB genes of MTBC were successfully designed and validated for the detection of MTBC. The preferred reaction conditions for this assay were confirmed to be 65°C for 40min, and the amplification products could be visually identified through LFB within 2min. The full assay process, including genomic DNA template extraction, LAMP reaction, and product detection, could be completed in 80min. The limit detection of the assay was 100fg of DNA in pure culture. The specificity of the assay was 100%, and it had no cross-reactions to other strains. Thus, the m-LAMP-LFB technology established in the present study was an objective, rapid, simple, and sensitive assay for MTBC identification, which could be applied in a clinical setting, especially in resource-constrained regions of the world.

Performance assessment of SD Bioline TB MPT64 assay for the diagnosis of Mycobacterium tuberculosis complex in Lagos, Nigeria

ABSTRACT This study assessed the performance of SD Bioline MPT64 immunochromatographic test for the identification of Mycobacterium tuberculosis complex (MTBC) in Nigeria. A total of 157 mycobacterial isolates, comprising 120 (76.4%) MTBC (M. tuberculosis, 112; M. africanum, 5; M. bovis, 3) and 37 (23.6%) non-tuberculous mycobacteria (NTM) isolates from patients attending six DOTS centers in Lagos between June 2012 and July 2014 were analyzed. All the isolates were grown on Bactec MGIT960 liquid media and identified in parallel by the conventional method and MPT64 immunochromatographic test. Discrepant results were resolved using the line probe assay. The comorbid disease rates for HIV and type 2 diabetes were 20.9% and 8.2%, respectively. Compared to the conventional method, SD Bioline MPT64 identified 117 MTBC isolates correctly, producing a sensitivity of 97.5% (95% CI, 92.9–99.2) at a shorter growing median time of 11 days compared to 26 days by the conventional method. The three undetected MTBC were confirmed by the line probe assay to be M. tuberculosis strains. The test also identified all the NTM correctly producing a specificity of 100% (95% CI, 90.7–100). This study supports the integration of SD Bioline TB MPT64 antigen test into diagnostic workflow for rapid MTBC case identification in Nigeria.

Sensible Functional Linear Discriminant Analysis Effectively Discriminates Enhanced Raman Spectra of Mycobacterium Species.

Tuberculosis caused by Mycobacterium tuberculosis complex (MTBC) is one of the major infectious diseases in the world. Identification of MTBC and differential diagnosis of nontuberculous mycobacteria (NTM) species impose challenges because of their taxonomic similarity. This study describes a differential diagnosis method using the surface-enhanced Raman scattering (SERS) measurement of molecules released by Mycobacterium species. Conventional principal component analysis and linear discriminant analysis methods successfully separated the acquired spectrum of MTBC from those of NTM species but failed to distinguish between the spectra of different NTM species. A novel sensible functional linear discriminant analysis (SLDA), projecting the averaged spectrum of a bacterial specie to the subspace orthogonal to the within-species random variation, thereby eliminating its influence in applying linear discriminant analysis, was employed to effectively discriminate not only MTBC but also species of NTM. The successful demonstration of this SERS-SLDA method opens up new opportunities for the rapid differentiation of Mycobacterium species.

MfloDx® - an innovative technology for MDR-TB antibiotic resistance profiling

Aims & objectives: The aim of this work was to evaluate the performance of mfloDx® MDR-TB assay kit on DNA extracted from 100 pulmonary sputum samples. mfloDx® MDR-TB is a qualitative test that can identify bacteria belonging to the Mycobacterium tuberculosis complex (MTC) as well as robustly identify single-nucleotide variants of the most common mutations in multidrug resistant M. tuberculosis DNA. The current assay can detect the most prevalent mutations causing resistance to Rifampicin (RIF) and Isoniazid (INH). Methods: Genes of interest were selectively enriched and targeted using amplifiable padlock probes (PLP). PLPs that recognize mutation or wild type were ligated upon specific recognition of the correct target sequence. Once PLPs were circularized, they underwent rolling circle amplification. Polymers resulting from this amplification were monomerized and subsequently visualized on disposable cassettes. The developed bands allowed for accurate genotyping of eleven resistance-related mutations, as well as respective wild type alleles and MTC identification. Results: From 100 samples; 81 were successfully genotyped, 16 were negative for MTC, two were inconclusive and one ran out during analysis. Among the genotyped samples; 46 were resistant to both RIF and INH, eight samples were resistant only to INH, three samples were resistant only to RIF. The remaining samples were susceptible to both drugs. Clinical sensitivity and specificity were calculated based on phenotypic drug susceptibility test results (MGIT culture) as reference. Sensitivity was 89.6% for RIF and 92% for INH resistance. Specificity was 95.5% for RIF and 89.5% for INH susceptibility. Conclusions: In conclusion, mfloDx® MDR-TB offers a reliable and affordable rapid drug-susceptibility testing for MDR-TB with potential implications in early detection of drug-resistance in resource-limited, high burden TB and MDR-TB settings. It enables clinicians to ensure the timely administration of effective drug therapy which will not only reduce the risk of treatment failure but also limit the further development and spread of dug resistant TB.

Identification of mycobacteria species through mass spectrometry (MALDI-TOF)

Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.

Mycobacterium tuberculosis kompleks ve Rifampisin Direncinin Saptanmasında Hızlı Moleküler Yöntemle Konvansiyonel Yöntemlerin Karşılaştırılması

Mycobacterium tuberculosis kompleks ve Rifampisin Direncinin Saptanmasında Hızlı Moleküler Yöntemle Konvansiyonel Yöntemlerin Karşılaştırılması

Sandwich antibody-based biosensor system for identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria

ABSTRACTMycobacterial infection, leading to pulmonary disease, remains a world health problem. Clinical symptoms of pulmonary disease caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) are very similar. A rapid method for the differentiation of MTBC and NTM infection is essential for appropriate therapy. In this study, we aim to establish an antibody-based biosensor system for the identification of MTBC and NTM infection. Monoclonal antibodies (mAbs) specific for Ag85B proteins of mycobacteria were generated and characterized. The generated anti-Ag85B mAb clones AM85B-5 and AM85B-8 reacted to Ag85B of Mycobacterium spp.; in contrast, clone AM85B-9 specifically reacted to Ag85B of MTBC. By employing the produced mAbs, single and sandwich antibody-based biosensors using bio-layer interferometry were established for determination of Ag85B proteins. The sandwich antibody-based biosensor system was demonstrated to be suitable for detection of Ag85B protein and identification of MTBC and NTM. Using anti-Ag85B mAbs AM85B-8 and AM85B-9 as immobilized antibodies on sensor chips and using mAb AM85B-5 as secondary antibody, the established sandwich antibody-based biosensor could discriminate MTBC and NTM. The developed biosensor system can be used for culture confirmation of mycobacteria and speciation to MTBC and NTM.

Phenotypic and Genotypic Characters of Mycobacteria Isolated from Extrapulmonary Tuberculosis Patients in Sohag Governorate

Tuberculosis is a major global health problem. ExtrapulmonaryTB(EPTB) refers to TB involving organs other than the lungs. It shoulddrag more attention after increasing numbers of immunocompromised individualswith higher risk of developing EPTB. Aim: This study aimed to detectEPTB in clinically suspected cases and identificationof mycobacteria species by phenotypic and genotypicmethods. Materials and Methods: A total of 100 clinical specimens were collected during the study period from September 2016 to January 2018 at Sohag University from patients suspected to have EPTB. All samples subjected to ZN staining & culture on LJ media then all positive culture were subjected for identification of mycobacteria speciesby two methods, phenotypically by biochemical tests and genotypically by conventional PCR for detection of mycobacterium Tuberculosis Complex ( MTC).Immunochromatographic tests were done for all strains with negative PCR in addition to some strains with positive PCR for confirmation and for comparison between immunochromatographic test and both biochemical test and PCR in identification of MTC. Results: Mycobacteria were isolated from 66 samples of which the highest number of isolates were obtained from urine samples followed by stool samples then other extrapulmonary samples. By using different methods of identification, 53 isolates were identified as MTC and 13 isolates were identified as nontuberculus mycobacteria (NTM). Conclusion: Our results suggest that due attention should be given for EPTB and rapid differentiation between MTC and NTM is very important for the proper management of patients with mycobacterial infections.

Use of an immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex clinical isolates in routine diagnosis.

Accurate identification of Mycobacterium tuberculosis complex (MTBC) isolates is essential for tuberculosis (TB) control, especially in a high-burden country such as Brazil. Conventional identification methods are laborious and time-consuming, while rapid molecular methods are expensive and require skilled personnel and appropriate physical laboratory infrastructure. Immunochromatographic assays (ICAs) have been shown to provide a rapid and reliable TB diagnosis at a low cost. The use of the SD Bioline TB Ag MPT64 ICA (MPT64 assay) for rapid identification of MTBC clinical isolates in the routine diagnosis of a large-volume reference TB laboratory was evaluated. We analysed 375 isolates on solid and liquid media concurrently with conventional phenotypic methods, the PRA-hsp65 molecular technique and the MPT64 assay. The sensitivity, specificity and accuracy of the ICA were 97.7, 100 and 98.1 %, respectively. The MPT64 assay yielded rapid and accurate results, enabling the treatment to be initiated early and also impacting on TB control.

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 (8days) and MODS culture (8days), but was significantly longer with LJ culture (20days) (p<0.0001 for both comparisons). Median time to availability of the susceptibility results was significantly (p<0.0001) shorter with MODS assay (8days) than MGIT system (20days). In conclusion, MODS is an inexpensive and rapid test with good performance characteristics for direct diagnosis of tuberculosis and detection of multidrug resistance.

Laboratory evaluation of the Anyplex™ II MTB/MDR and MTB/XDR tests based on multiplex real-time PCR and melting-temperature analysis to identify Mycobacterium tuberculosis and drug resistance

We evaluated the performance of two multiplex, real-time PCR tests (Anyplex II MTB/MDR and MTB/XDR; Seegene, Seoul, Korea), designed to detect the Mycobacterium tuberculosis complex (MTC) and drug-resistance mutations associated with isoniazid, rifampicin, fluoroquinolones, and second-line injectable drugs. We analyzed 122 clinical isolates with the Anyplex II MTB/MDR test, 68 of which were also tested with the Anyplex II MTB/XDR test. The Anyplex II MTB/MDR and MTB/XDR tests showed the following respective sensitivities and specificities: 68.8% and 100% for detecting isoniazid resistance, 93.8% and 100% for rifampicin, 82.8% and 100% for levofloxacin, 75.0% and 100% for kanamycin, and 92.6% and 100% for MTC identification. These kits correctly identified 61.8% of multi-drug resistant M. tuberculosis isolates and 64.7% of extensively drug-resistant M. tuberculosis isolates, and enabled semi-automatic detection of drug-resistant MTC in 3 hours. The Anyplex II kits could be useful as rule-in tests for detecting MTC and drug resistance.

Evaluation of the SD Bioline TB Ag MPT64 test for identification of Mycobacterium tuberculosis complex from liquid cultures in Southwestern Uganda.

BackgroundTo confirm presence of Mycobacterium tuberculosis complex, some tuberculosis culture laboratories still rely on para-nitrobenzoic acid (PNB), a traditional technique that requires sub-culturing of clinical isolates and two to three weeks to give results. Rapid identification tests have improved turnaround times for mycobacterial culture results. Considering the challenges of the PNB method, we assessed the performance of the SD Bioline TB Ag MPT64 assay by using PNB as gold standard to detect M. tuberculosis complex from acid-fast bacilli (AFB) positive cultures.ObjectivesThe aim of this study was to determine the sensitivity, specificity and turnaround time of the SD MPT64 assay for identification of M. tuberculosis complex, in a setting with high prevalence of tuberculosis and HIV.MethodsA convenience sample of 690 patients, with tuberculosis symptoms, was enrolled at Epicentre Mbarara Research Centre between April 2010 and June 2011. The samples were decontaminated using NALC-NaOH and re-suspended sediments inoculated in Mycobacterium Growth Indicator Tubes (MGIT) media, then incubated at 37 °C for a maximum of eight weeks. A random sample of 50 known negative cultures and 50 non-tuberculous mycobacteria isolates were tested for specificity, while sensitivity was based on AFB positivity. The time required from positive culture to reporting of results was also assessed with PNB used as the gold standard.ResultsOf the 138 cultures that were AFB-positive, the sensitivity of the SD MPT64 assay was 100.0% [95% CI: 97.3 – 100] and specificity was 100.0% (95% CI, 96.4 – 100). The median time from a specimen receipt to confirmation of strain was 10 days [IQR: 8–12] with SD MPT64 and 24 days [IQR: 22–26] with PNB.ConclusionThe SD MPT64 assay is comparable to PNB for identification of M. tuberculosis complex and reduces the time to detection.

Predictable repeatability issues with GeneXpert-Xpert MTB/RIF (version 4) derived rifampicin resistant tuberculosis results from South India: Appreciating the limits of a technological marvel!

Background: GeneXpert MTB/RIF (Xpert), the fully automated cartridge-based nucleic acid amplification test for simultaneous identification of Mycobacterium tuberculosis complex and rifampicin resistance (RR), directly from samples is considered as a game changer for tuberculosis (TB) control programs worldwide. Methods: We are reporting serious issues with repeatability among a subgroup of Xpert (Version 4) identified RR results from South Indian state recently switched to Xpert by the National TB control program. Results: We have demonstrated that poor repeatability is frequently associated with those Xpert derived RR results, identified by detection of delayed amplification of any probe in the presence of positive analyte results for all probes. Another significant contributing factor was found to be lower bacterial loads in samples. The repeat tests were done by Xpert and/or by line probe assay depending on smear positivity. The finding is worrying as Xpert is recommended over other tests due to its reportedly better performance among low bacterial load samples such as pediatric, extra-pulmonary, HIV-TB co-infected, and smear negative pulmonary TB and the same samples, it seems are more likely to cause error prone RR results. Conclusions: We recommend for additional genotypic tests with specific mutant probes for detecting mutations at rpoB hot sites and growth based tests for all Xpert derived RR-TB cases identified by the above algorithm for confirmation of the presence of mutation, based on our available data.

Raman spectroscopic identification of Mycobacteriumtuberculosis.

In this study, Raman microspectroscopy has been utilized to identify mycobacteria to the species level. Because of the slow growth of mycobacteria, the per se cultivation-independent Raman microspectroscopy emerges as a perfect tool for a rapid on-the-spot mycobacterial diagnostic test. Special focus was laid upon the identification of Mycobacterium tuberculosis complex (MTC) strains, as the main causative agent of pulmonary tuberculosis worldwide, and the differentiation between pathogenic and commensal nontuberculous mycobacteria (NTM). Overall the proposed model considers 26 different mycobacteria species as well as antibiotic susceptible and resistant strains. More than 8800 Raman spectra of single bacterial cells constituted a spectral library, which was the foundation for a two-level classification system including three support vector machines. Our model allowed the discrimination of MTC samples in an independent validation dataset with an accuracy of 94% and could serve as a basis to further improve Raman microscopy as a first-line diagnostic point-of-care tool for the confirmation of tuberculosis disease.