BACTEC Mycobacteria Growth Indicator Tube Research Articles

In vitro, evaluation of anti-mycobacterial active plant extracts against Mycobacterium tuberculosis using BACTEC mycobacteria growth indicator tube 960 automated system

In vitro, evaluation of anti-mycobacterial active plant extracts against Mycobacterium tuberculosis using BACTEC mycobacteria growth indicator tube 960 automated system

Drug sensitivity of clinical isolates of Mycobacterium tuberculosis and its association with bacterial genotype in the Somali region, Eastern Ethiopia.

BackgroundDrug resistance is becoming a major bottleneck for tuberculosis (TB) control programs in countries with high TB burdens. Although several studies were conducted on the drug sensitivity of Mycobacterium tuberculosis (M. tuberculosis) in central Ethiopia, there is a lack of data on the drug sensitivity of M. tuberculosis in the peripheral regions of the country including in the Somali region. Therefore, the objective of this study was to evaluate the drug sensitivity of M. tuberculosis and its association with bacterial genotype and evaluate the performance of Xpert MTB/RIF (Xpert) in detecting resistance to rifampicin (RIF).MethodsA total of 302 M. tuberculosis were tested using the BD BACTEC-Mycobacteria Growth Indicator Tube 960 (MGIT 960) system for their drug sensitivity to the first-line anti-TB drugs. Besides, the drug sensitivity of 10 multidrug-resistant (MDR) M. tuberculosis isolates was evaluated for the second-line anti-TB drugs. Additionally, 177 of the 302 isolates were tested for genotypic drug resistance using Xpert. Chi-square and Fisher's exact tests were used for the evaluation of the association between variables and drug sensitivity.ResultsThe overall prevalence of resistance to at least one drug was 11.6% (95% CI: 7.9–15.2%), while the prevalence of MDR was 3.3% (95% CI: 1.3–5.3%). Two of the 10 MDR isolates were resistant to capreomycin. The spoligotype Shared International Type (SIT) 149 was significantly associated with either monoresistance or MDR (p < 0.05). Of the 177 isolates tested by Xpert, 6.2% (11/177) were RIF-resistant. Discordant between Xpert and MGIT 960 was observed in one isolate and linked with probe-binding delay (ΔCT max = 5.8). The sensitivity and specificity of the Xpert assay were 100 and 99.4%, respectively, while its positive and negative predictive values were 90.9 and 100%, respectively.ConclusionThe magnitude of MDR M. tuberculosis in the Somali region of Ethiopia was higher than the national prevalence of MDR-TB warranting the strengthening of the TB control program in the Somali region. Besides, drug resistance was associated with SIT 149 spoligotype (genotype). The Xpert assay was observed to have high sensitivity and specificity in detecting RIF-resistant M. tuberculosis, which is encouraging for its application widely.

Diagnostic Accuracy of Pyrazinamide Susceptibility Testing in Mycobacterium tuberculosis: A Systematic Review with Meta-Analysis.

Introduction: Pyrazinamide (PZA) susceptibility testing plays a critical role in determining the appropriate treatment regimens for multidrug-resistant tuberculosis. We conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of sequencing PZA susceptibility tests against culture-based susceptibility testing methods as the reference standard. Methods: We searched the MEDLINE/PubMed, Embase, and Web of Science databases for the relevant records. The QUADAS-2 tool was used to assess the quality of the studies. Diagnostic accuracy measures (i.e., sensitivity and specificity) were pooled with a random-effects model. All statistical analyses were performed with Meta-DiSc (version 1.4, Cochrane Colloquium, Barcelona, Spain), STATA (version 14, Stata Corporation, College Station, TX), and RevMan (version 5.3, The Nordic Cochrane Centre, the Cochrane Collaboration, Copenhagen, Denmark) software. Results: A total of 72 articles, published between 2000 and 2019, comprising data for 8,701 isolates of Mycobacterium tuberculosis were included in the final analysis. The pooled sensitivity and specificity of the PZA sequencing test against all reference tests (the combination of BACTEC mycobacteria growth indicator tube 960 (MGIT 960), BACTEC 460, and proportion method) were 87% (95% CI: 85-88) and 94.7% (95% CI: 94-95). The positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and the area under the curve estimates were found to be 12.0 (95% CI: 9.0-16.0), 0.17 (95% CI: 0.13-0.21), 106 (95% CI: 71-158), and 96%, respectively. Deek's test result indicated a low likelihood for publication bias (p = 0.01). Conclusions: Our analysis indicated that PZA sequencing may be used in combination with conventional tests due to the advantage of the time to result and in scenarios where culture tests are not feasible. Further work to improve molecular tests would benefit from the availability of standardized reference standards and improvements to the methodology.

Distribution of clinical isolates obtained from sterile body fluids: a four-year retrospective data

Aim: Infections of sterile body fluids (SBFs) require rapid and accurate diagnosis and treatment, since their morbidities and mortalities are high. To achieve this goal, definite epidemiologic data is absolutely required, since empiric and preemptive treatments are mainly based on this. The aim of this study was to evaluate infectious agents isolated from SBFs, susceptibility results and molecular analysis (PCR) data, retrospectively.&#x0D; Material and Method: Clinical samples of SBFs (Cerebrospinal, pleural, peritoneal, pericardial and synovial fluids) obtained from January 2017 to December 2020 in Atatürk City Hospital (tertiary center) were included. Identification of bacterial and fungal agents and antibiotic susceptibility were done by conventional and automated system (BD Phoenix™, Becton Dickinson Co., Sparks, MD, USA). Löwenstein-Jensen media and BACTEC Mycobacteria Growth Indicator Tube 960 (Becton Dickinson Co., Sparks, MD, USA) were used for mycobacterial analysis. Bosphore Viral Meningitis Panel Multiplex PCR Kit (Anatolia Geneworks, İstanbul, Turkey) were applied to detect HSV-1, HSV-2, VZV, Enterovirus and/or Parechovirus.&#x0D; Results: A total of 221 (9.74%) organisms were detected among 2269 samples. Particularly common gram negative bacterial agents covered the top of the list (Escherichia coli, Pseudomonas spp., Klebsiella spp. and Acinetobacter baumannii-Acinetobacter calcoaceticus complex). Staphylococcus aureus was the most frequent gram positive strain, followed by enteroccocci. Most of the A. baumannii isolates were multidrug resistant, Pseudomonas spp. showed over than 20% resistance rate to ceftazidime, cefepime and piperacillin-tazobactam. All enterococci were vancomycin-susceptible, one S. aureus strain was methicillin-resistant. All Mycobacterium tuberculosis complex isolates were found to be susceptible to first-line anti-tuberculosis drugs.&#x0D; Conclusions: Continuous laboratory surveillance even in local phase is important to guide clinicians. Even though our data did not show significant changes, improvements on laboratory capabilities and clinical awareness must be done. Isolation rates might be underestimated due to requirement of improvements in our laboratory, especially about sampling, anaerobe transportation and usage of blood culture vials.

Pattern of Drug Resistance in Primary Spinal Tuberculosis: A Single-Center Study From India.

Study Design:Retrospective observational analysis.Objectives:Spinal tuberculosis accounts for about 50% of cases among extra pulmonary osteoarticular tuberculosis. Resistance to drugs in spinal tuberculosis patients is on a rise and there is inadequate literature concentrating on the precise pattern of resistance in Indian subcontinent which harbors 24% of global prevalence. The aim was to study the pattern of drug resistance in spinal tuberculosis among first- and second-line drugs. Drug resistance is common in spinal tuberculosis and we intended to find the prevalence of various drug resistance patterns.Methods:Patients with spinal tuberculosis visiting a tertiary center were assessed. Samples were taken from the affected vertebrae and sent for BACTEC mycobacterium growth indicator tube (MGIT) 960 culture. Patients with a positive growth in MGIT were included in the study. All previously treated patients (relapse, treatment after failure, treatment after loss to follow-up and other previously treated patients) were excluded.Results:A total of 150 patients with a positive growth in MGIT report were included in the study, of whom 43 patients had some kind of drug resistance. Seven were multidrug resistant (MDR), 9 had preextensive drug resistance (pre-XDR), and 4 had extensive drug resistance (XDR). Seventeen patients had mono-drug resistance, which was most frequently for isoniazid. Resistance among second-line drugs was common in the fluoroquinolone group.Conclusion:Drug resistance in spinal tuberculosis was found to be 28.6%. Of these, MDR was in 16.2%, pre-XDR in 20.9%, and XDR in 9.3% patients.

The MPB64 immunochromatography assay: an analysis of doubtful results.

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth indicator tube (MGIT), endorsed by the World Health Organization (WHO), is widely used. Further identification of a positive culture is done with the help of an immunochromatography assay, which often shows faint bands that are difficult to interpret. We analysed 125 BACTEC MGIT culture positive results, of which 11/16 (68.7%) of the doubtful assays, analysed by MGIT™ TBc Identification test (TBcId), were positive for Mycobacterium tuberculosis complex (MTBC), the remaining being non-tuberculous mycobacteria as determined by an in-house duplex polymerase chain reaction and line probe assay. Guidelines on faint or doubtful bands in immunochromatography assays are important so as not to overlook true-positive cases of TB.

The role of mini-bronchoalveolar lavage fluid in the diagnosis of pulmonary tuberculosis in critically ill patients

BackgroundThe detection of Mycobacterium tuberculosis (MTB) in the intensive care unit (ICU) presents several challenges, mainly associated to the clinical state of the patient. The presence of HIV infection further aggravates this scenario, requiring a reliable collection method, with better performance in the microbiological/molecular techniques to be used. We evaluated the performance of two methods for sample collection, mini bronchoalveolar lavage (Mini-BAL) and endotracheal aspirate (ETA), for diagnosis of pulmonary tuberculosis (PTB) in critically ill patients.MethodsThis prospective study involved 26 HIV positive ICU internalized patients, with presumptive PTB who required mechanical ventilation. Two samples were obtained prospectively from 26 HIV ICU patients with presumptive PTB by Mini-BAL and ETA. The samples were processed for smear microscopy, Löwenstein-Jensen medium and the BACTEC Mycobacteria Growth Indicator Tube 960 system®. We define as confirmed PTB patients with positive MTB culture. Furthermore, all samples obtained through the Mini-BAL were analyzed by Xpert® MTB/RIF.ResultsOur results demonstrated that the respiratory samples obtained by Mini-BAL were able to increase MTB detection in critically ill patients with presumptive PTB. The Mini-BAL allowed 30% increased recovery and guaranteed enough sample volume for processing in all methods. In addition, the larger volume of the samples obtained with this technique enabled the Xpert® MTB/RIF molecular test for diagnosis of TB.ConclusionsThe Mini-BAL showed be an acceptable alternative to ETA in this population, since these critically ill and often-immunocompromised patients are more likely to develop complications related to invasive procedures.

The Outcome and Hepatic Function of an Extended Category II Treatment in a HIV-Seronegative Suspected Extensively Drug-Resistant TB Patient

A 25 year old HIV- seronegative client was diagnosed with pulmonary tuberculosis and placed on Category I (CAT I) treatment. After two months, acid fast bacilli (AFB) persisted in sputum. Rifampicin (Rif) resistance was detected with the GeneXpert MTB-Rif Assay® (Xpert; Cepheid, Sunnyvale, CA, USA), leading to a change in treatment to CAT II after completing CAT I in the third month. Continuous detection of Rif resistant species and persisting ‘Scanty’ results (1-9 AFB found in 100 microscopic fields) from sputum smear microscopy (SSM) after one (1) year was suggestive of treatment failure to CAT II [possible extensively drug-resistant (XDR) species]. Category II treatment was continued for the next eight months as we anticipated the arrival of XDR medicines. Before commencement of XDR medication, SSM and GeneXpert were repeated. The SSM result was negative (no AFB seen), while Rif resistance persisted. The controversy was eventually clarified with the Bactec Mycobacteria Growth Indicator Tube (MGIT) liquid culture, which indicated no growth for M. tuberculosis. Since the rapid molecular GeneXpert will persistently detect Rif resistance even after months of treatment, we support the use of MGIT to confirm when treatment should be halted. This case study also showed tolerance to prolonged treatment with CAT I and II drugs and confirms evidence in existing literature.

Comparison of LJ Medium and BACTEC MGIT 960 Culture System for the Diagnosis of Tuberculosis

Introduction: Sputum negative pulmonary Tuberculosis (TB) is a major public health problem. So, the emergence of new techniques for a more precise and rapid microbiological identification of Mycobacterium tuberculosis in clinical samples is of great importance to improve the management of TB. Aim: To determine and compare the sensitivity and turnaround time for Mycobacterium tuberculosis detection by the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system, Lowenstein Jensen (LJ) medium and Ziehl-Neelsen (ZN) staining. Materials and Methods: An Institution based, observational, cross-sectional study was conducted at Rajendra Institute of Medical Sciences (RIMS), Ranchi, Jharkhand, India, from July 2013-March 2016. Sputum, pericardial fluid, pleural fluid, peritoneal fluid, pus and endometrial tissue samples were collected from 80 patients of suspected TB cases. All were Acid-Fast stained by ZN staining method and cultured on solid culture LJ medium and on liquid medium (MGIT). Data was analysed using Statistical Package for the Social Sciences (SPSS) software, Version 20.0 (SPSS Inc, Chicago, IL, USA). Fisher’s-Exact test was used to show association of categorical variables. Non-parametric Mann-Whitney U test was used to show median difference of non-normally distributed continuous variables of two groups. Results: Out of the 80 samples, 41 cases were positive by either of the all methods. The positive specimen for ZN staining, LJ media and MGIT were 21, 29 and 41 cases respectively. The mean Time To Detection (TTD) was shorter for MGIT system than LJ media. Both LJ medium and MGIT 960 detected all cases of sputum smear positive cases and in addition significantly higher number than ZN stain in sputum smear negative cases. MGIT 960 detected significantly higher number of cases of sputum negative cases than LJ Media. The mean TTD was also significantly shorter in case of smear positive cases than the smear negative cases by both the solid and liquid culture mediums. Conclusion: The use of liquid media (MGIT) is more accurate and rapid method for the diagnosis of TB. The combination of more than one method is also highly recommended for rapid detection and early treatment of TB.

MDR/XDR-TB Colour Test for drug susceptibility testing of Mycobacterium tuberculosis, Northwest Ethiopia

BackgroundAppropriate technology tests are needed for Mycobacterium tuberculosis drug-susceptibility testing (DST) in resource-constrained settings. This study was performed to evaluate the MDR/XDR-TB Colour Test (a colour platethin-layer agar test; TB-CX) for M. tuberculosis DST by directly testing sputum at University of Gondar Hospital. MethodsSputum samples were each divided into two aliquots. One aliquot was mixed with disinfectant and applied directly to the TB-CX quadrant petri-plate containing culture medium with and without isoniazid, rifampicin, or ciprofloxacin. Concurrently, the other aliquot was decontaminated with sodium hydroxide, centrifuged, and cultured on Lӧwenstein–Jensen medium; the stored M. tuberculosis isolates were then sub-cultured in BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 for reference DST. ResultsThe TB-CX test yielded DST results for 94% (123/131) of positive samples. For paired DST results, the median number of days from sputum processing to DST was 12 for TB-CX versus 35 for LJ-MGIT (p<0.001). Compared with LJ-MGIT for isoniazid, rifampicin, and multidrug-resistant tuberculosis, TB-CX had 59%, 96%, and 95% sensitivity; 96%, 94%, and 98% specificity; and 85%, 94%, and 98% agreement, respectively. All ciprofloxacin DST results were susceptible by both methods. ConclusionThe TB-CX test was simple and rapid for M. tuberculosis DST. Discordant DST results may have resulted from sub-optimal storage and different isoniazid concentrations used in TB-CX versus the reference standard test.

Isoniazid Resistance in Mycobacterium tuberculosis Is a Heterogeneous Phenotype Composed of Overlapping MIC Distributions with Different Underlying Resistance Mechanisms.

MIC testing using the Bactec mycobacteria growth indicator tube system 960 of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing explained most of the levels of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration endorsed by the Clinical and Laboratory Standards Institute to detect low-level resistance to isoniazid and is, consequently, likely not optimally set.

Efficacy and role of Xpert® Mycobacterium tuberculosis/rifampicin assay in urinary tuberculosis.

Introduction:The aim was to study the accuracy of Xpert® (Cepheid Inc., Sunnyvale, CA, USA) Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay as compared to a composite gold standard (urine culture, imaging, and biopsy) and to asses its utility as the initial test compared to smear microscopy to diagnose urinary tuberculosis.Methods:This prospective study included adult patients suspected to have urinary tuberculosis from March 2014 to December 2017. Three urine samples were collected from each patient and were subjected to Xpert MTB/RIF assay, acid-fast bacillus (AFB) smear microscopy, and liquid media (BACTEC Mycobacteria Growth Indicator Tube [MGIT] 960) culture. Imaging and tissue biopsies were performed as clinically indicated. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the bootstrap method for 95% confidence intervals for the Xpert assay.Results:Xpert MTB/RIF assay was found to be superior to the currently best available light-emitting diode fluorescent smear microscopy as the initial test for urinary tuberculosis (sensitivity of 69.09% vs. 32.72%). The Xpert MTB/RIF polymerase chain reaction test was found to have a moderate sensitivity (69.09%) and high specificity (100%) as compared to the composite reference standard. The sensitivity of liquid AFB culture MGIT 960 as compared to the reference standard was 90.32%.Conclusions:Xpert MTB/RIF assay on an early morning first void urine specimen can replace smear microscopy as the initial diagnostic test for urinary tuberculosis.

Genetic diversity and distribution of Mycobacterium tuberculosis genotypes in Limpopo, South Africa

BackgroundTuberculosis remains a major health problem and knowledge of the diversity of Mycobacterium tuberculosis strains in specific geographical regions can contribute to the control of the disease. This study describes the genetic profile of M. tuberculosis in five districts of Limpopo Province.MethodsA total 487 isolates were collected from the National Health Laboratory Services from all regions/districts of Limpopo Province. Only 215 isolates were confirmed to be M. tuberculosis by Bactec Mycobacterium Growth Indicator Tube 960® and Rhodamine-Auramine staining. Isolates were subcultured on Löwenstein-Jensen medium agar slants to validate purity. They were spoligotyped and data analysed using the international spoligotyping database 4 (SpolDB4).ResultsOf the 215 isolates, 134 (62.3%) were genotyped into 21 genotype families while 81 (37.7%) were orphans. The 81 orphans were further subjected to resolution employing SpolDB3/RIM. Overall, the study revealed a high diversity of strains of 32 predominantly the non-Beijing lineages: the LAM- LAM3 (9.8%), LAM9 (4.7%) and LAM11- ZWE (3.3%), the T-T1(15.0%), T2 (0.9%), T2-T3 (1.4%), the CAS-CAS1-Delhi 5 (1.9%) and CAS1-KILI (1.4%) the MANU2 (1.4%), U (0.5%), X-X1(1.4%), X3 (1.9%), S (9.8%), CAS (1.4%), LAM7(0.9%), T3(0.5%), LAM8(4.7%), T4(1.4%), X2(0.4%), AI5(1.9%), LAM1(0.5%), FAMILY33 (1.9%), EAI4(1.4%), M. microti (1.9%). The Beijing and Beijing-like families were (14.9%) and (0.9%), respectively. A total of 28(13%) clusters and 77(36%) unique cases were identified. Beijing strain (SIT 1) formed the biggest cluster constituting 14%, followed by LAM3 (SIT 33), T1 (SIT 53) and LAM4 (SIT 811) with 7%, 5.1% and 2.8%, respectively. The Beijing family was the only genotype found in all the five districts and was predominant in Mopani (18.8%), Sekhukhune (23.7%) and Vhembe (23.3%). Dominant genotypes in Capricorn and Waterberg were LAM3 (11.9%) and T1 (13.3%), respectively.ConclusionA wide diversity of lineages was demonstrated at district level. A high number of clusters per district provided evidence of on-going transmission in this Province.

Combination of MGIT liquid culture and Xpert MTB/RIF test in the rapid diagnosis and drug resistance detection in Mycobacterium tuberculosis

Objective To evaluate the feasibility of Mycobacteria growth indicator tube (MGIT) liquid culture combined with rifampin resistance test real-time PCR (Xpert MTB/RIF test) in the diagnosis of tuberculosis. Methods 652 cases of suspected pulmonary tuberculosis from October 2014 to March 2015 in Quzhou People's Hospital were enrolled. The morning sputum samples were collected for acid-fast staining, Lowenstein-Jensen (L-J) culture, BACTEC MGIT culture and Xpert MTB/RIF test. Samples with positive results of MGIT liquid culture were subjected to strain identification and drug sensitivity test with fluid method.Methodological comparison between four methods was made and the performance of MGIT liquid culture combined with Xpert MTB/RIF test in the rapid diagnosis and drug resistance detection in Mycobacterium tuberculosis was evaluated by chi-square test. Results Among the samples from 399 confirmed tuberculosis patients, the diagnostic sensitivity of the 4 methods (acid-fast staining, L-J culture, MGIT culture and Xpert MTB/RIF test) were 17.0% (68/399), 23.8% (95/399), 37.8% (151/399) and 37.3% (149/399) respectively. The sensitivity and specificity of MGIT culture combined with Xpert MTB/RIF test was 39.8% (159/399) and 94.8% (240/253). The sensitivity of MGIT culture combined with Xpert MTB/RIF test was significantly higher than acid-fast staining (χ2=50.9, P<0.01) and L-J culture (χ2=23.7, P<0.01). The average detection time of MGIT culture was 7.5 days (smear positive and Xpert MTB/RIF positive), 13.4 days (smear negative and Xpert MTB/RIF positive) and 16.9 days (smear negative and Xpert MTB/RIF negative). The sensitivity and specificity of Xpert MTB/RIF test in rifampin resistance detection were 9/9 and 97.3% (129/132) respectively. The average detection time of fluid method for drug sensitivity test was 8.3 days. Conclusions MGIT liquid culture combined with Xpert MTB/RIF test can detect Mycobacterium tuberculosis and the drug sensitivity rapidly. The method is highly sensitive and specific. It is important for clinical diagnosis and treatment of tuberculosis.(Chin J Lab Med, 2016, 39: 272-276) Key words: Mycobacterium tuberculosis; Rifampin; Tuberculosis, multidrug-resistant; Polymerase chain reaction; Microbial sensitivity tests

Molecular characterisation and associated drug susceptibility patterns of Mycobacterium tuberculosis isolates from South African children

According to the World Health Organization, South Africa is ranked the third highest country (0.4-0.6 million), after India and China, with a tuberculosis burden, coupled with drug resistance that is threatening tuberculosis control efforts. Understanding the genetic diversity and drug resistance patterns of Mycobacterium tuberculosis is crucial. The purpose of this study was to describe the molecular characterisation and associated drug susceptibility patterns of M. tuberculosis isolates from South African children. M. tuberculosis strains from children from four South African provinces were characterised and the drug resistance patterns determined. One hundred and four isolates that were culture positive in the Bactec Mycobacterium Growth Indicator Tube 960® and positively identified by AccuProbe® assay as M. tuberculosis were used. To ensure purity, the samples were subcultured on Lowenstein-Jensen medium agar slants. Spoligotyping was performed according to the manufacturer's instructions. The interna...

Macrocolonies (Granules) Formation as a Cause of False-Negative Results in the MGIT 960 System: Cause Analysis and Correlation with Mycobacterial Species.

Background. The viable mycobacterial bacilli can sometimes form granules in the Mycobacterium Growth Indicator Tube (MGIT) to produce instrument-negative outcomes when BACTEC MGIT 960 culture is performed. The cause of this phenomenon has never been analyzed. Methods. Thirty-one instrument-negative, granule presenting MGIT vials were collected for conducting acid-fast staining and also liquid and solid subculture. Species identification and drug susceptibility test were performed with the recovered strains. Cultivation test was done by inoculating small amount of bacilli into the MGIT vials. Results. Twenty-four and twenty-nine of the tested MGIT vials were smear and culture positive, respectively. In total, 18, 4, and 7 of the cultivated strains were identified as Mycobacterium tuberculosis complex, M. intracellulare, and M. xenopi, respectively. When a limited amount of bacilli was inoculated, the granule formation was observed for M. xenopi strains in the MGIT system. Conclusions. The granules observed in the instrument-negative MGIT vials consisted of viable bacilli, which emphasized the need of visual inspection to increase recovery rate. Limited bacterial load and specific species might be the cause of granule forming.

Use of amplified Mycobacterium tuberculosis direct test in respiratory samples from HIV-infected patients in Brazil

OBJECTIVE: To compare the accuracy of the amplified Mycobacterium tuberculosis direct (AMTD) test with reference methods for the laboratory diagnosis of tuberculosis in HIV-infected patients. METHODS: This was a study of diagnostic accuracy comparing AMTD test results with those obtained by culture on Löwenstein-Jensen (LJ) medium and by the BACTEC Mycobacteria Growth Indicator Tube 960 (BACTEC MGIT 960) system in respiratory samples analyzed at the Bioassay and Bacteriology Laboratory of the Oswaldo Cruz Foundation Evandro Chagas Clinical Research Institute in the city of Rio de Janeiro, Brazil. RESULTS: We analyzed respiratory samples collected from 118 patients, of whom 88 (74.4%) were male. The mean age was 36.6 ± 10.6 years. Using the AMTD test, the BACTEC MGIT 960 system, and LJ culture, we identified M. tuberculosis complex in 31.0%, 29.7%, and 27.1% of the samples, respectively. In comparison with LJ culture, the AMTD test had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.5%, 89.4%, 75.7%, and 95.0%, respectively, for LJ culture, whereas, in comparison with the BACTEC MGIT 960 system, it showed values of 88.6%, 92.4%, 83.8%, and 94.8%, respectively. CONCLUSIONS: The AMTD test showed good sensitivity and specificity in the population studied, enabling the laboratory detection of M. tuberculosis complex in paucibacillary respiratory specimens.

Detonation Nanodiamonds for Rapid Detection of Clinical Isolates of Mycobacterium tuberculosis Complex in Broth Culture Media

Routinely used molecular diagnostic methods for mycobacterium identification are expensive and time-consuming. To tackle this problem, we develop a method to streamline identification of Mycobacterium tuberculosis complex (MTBC) in broth culture media by using detonation nanodiamonds (DNDs) as a platform to effectively capture the antigen secreted by MTBC which is cultured in BACTEC MGIT 960, followed by the analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). The 5 nm DNDs can capture the MTBC secretory antigen without albumin interference. With on diamond digestion, we confirm the DND captured antigen is cell filtrate protein 10 (CFP-10) because its Mascot analysis shows a score of 68. The dot blotting method further verifies a positive reaction with anti-CFP-10, indicating that CFP-10 is secreted in the medium of mycobacterium growth indicator tube (MGIT) and captured by DNDs. The minimal CFP-10 protein detection limit was 0.09 μg/mL. Furthermore, our approach can avoid the false-positive identification of MTBC by immunological methods due to cross-reactivity. Five hundred consecutive clinical specimens subjected to routine mycobacteria identification in hospital were used in this study, and the sensitivity of our method is 100% and the specificity is 98%. The analysis of each MTBC sample from culture solution can be finished within 1 h and thus shortens the turnaround time of MTBC identification of gold standard culture methods. In sum, DND MALDI-TOF MS for the detection of MTBC is rapid, specific, safe, reliable, and inexpensive.

Usefulness of the BACTEC MGIT 960 System for Mycobacterial Culture and TB Ag MPT64 Immunochromatographic Assay to IdentifyMycobacterium tuberculosis

Background: This study was conducted to evaluate the usefulness of the BACTEC MGIT (Mycobacterium Growth Indicator Tube) 960 system for mycobacteria culture and immunochromatographic assay to identify Mycobacterium tuberculosis (MTB) in positive MGIT culture. Methods: Mycobacteria-culture-positive cases were retrospectively analyzed from December 2010 to July 2011. The detection rates and the recovery times of the mycobacteria between the Ogawa media and the MGIT were compared. An immunochromatographic assay (ICA) (SD BIO-LINE) was also performed in the positive MGIT culture for identification, and the results were compared with those of the Ogawa media in the Korea National Tuberculosis Association. Results: Among the 261 patients (M:F, 168:93; mean age, 61.6±17.16 yrs), 450 specimens (sputa, 365; bronchial washing, 61; and pleural effusion, 24) were found positive with mycobacteria. Mycobacteria were grown both on the MGIT and Ogawa media in 310 cases (68.9%); only on the MGIT in 115 cases (22.6%); and only on the Ogawa media in 25 cases (5.5%) (p<0.05).The recovery time was 28.2±8.9 days in the Ogawa media and 11.1±5.8 days in the MGIT (p<0.05). Among the 127 cases from the positive MGIT culture, all 92 cases that were confirmed as MTB cases bythe Korea National Tuberculosis Association were identified as MTB by ICA, with 100% sensitivity. Conclusion: MGIT increases the detection rate and shortens the recovery time of mycobacteria in clinical respi- ratory specimens, and the TB Ag MPT64 kit using ICA is useful in identifying MTB in a positive MGIT culture.

Humans as Source ofMycobacterium tuberculosisInfection in Cattle, Spain

Humans as Source of<i>Mycobacterium tuberculosis</i>Infection in Cattle, Spain