Comparison of the Automated Mycobacteria Growth Indicator Tube System (BACTEC 960/MGIT) with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens

We examined whether the BACTEC/Mycobacteria Growth Indicator Tube (MGIT) system alone could supplant the use of a supplemental L?wenstein-Jensen (LJ) slant for routine recovery of Mycobacterium species from clinical specimens. A total of 6,062 specimens were included in the study. Of these, 273 specimens were positive for 278 mycobacterial isolates while 15 specimens were smear positive but culture negative using both media. Further analysis showed that 143 (51.4%) of the 278 total isolates were recovered from both the MGIT and LJ media. An additional 106 isolates (38.1%) were recovered from the MGIT only, while 29 (10.4%) isolates grew only on the LJ slant. The overall sensitivities of the MGIT and LJ media were 86.5% and 59.7%, respectively, for the recovery of mycobacteria from clinical materials. This study shows that although the MGIT system demonstrates better sensitivity for the recovery of mycobacteria from clinical specimens, both media types are necessary to maximize the sensitivity of detection.     

Comparison of the Automated Nonradiometric Bactec MGIT 960 System with Löwenstein-Jensen, Coletsos, and Middlebrook 7H11 Solid Media for Recovery of Mycobacteria

The aim of this study was to evaluate the sensitivity of as well as the time to detection of mycobacteria by three procedures: solid media with traditional reading, microscopy on solid media, and liquid culture using the automated nonradiometric Bactec MGIT 960 system. A total of 2832 respiratory specimens were tested, 315 of which were positive for mycobacteria. The most frequently isolated species was Mycobacterium tuberculosis (201 isolates). One hundred twenty mycobacteria other than tuberculosis were isolated, 72 of which were Mycobacterium xenopi strains. Sensitivity of each of the different media compared to all media combined for growth of Mycobacterium tuberculosis was 93%, 76.1%, 79.6%, and 75.1% for Bactec MGIT 960, Middlebrook 7H11 plates, Löwenstein-Jensen, and Coletsos, respectively. Sensitivity of the Bactec MGIT 960 for detection of all mycobacterial isolates was 75.1%. When this automated system was supplemented with visual inspection, the sensitivity increased to 89.4%. The sensitivity of Middlebrook 7H11 plates, Löwenstein-Jensen, and Coletsos was 50.8%, 60.7%, and 52.3%, respectively. Time to detection of Mycobacterium tuberculosis using the Bactec MGIT 960 system and Middlebrook 7H11 plates with microscopic reading was 12.7 and 13 days, respectively; using the traditional Löwenstein-Jensen and Coletsos media, time to detection was 22.8 and 22.7 days, respectively.     

Comparison of MB/Bact alert 3D system with radiometric BACTEC system and Löwenstein-Jensen medium for recovery and identification of mycobacteria from clinical specimens: a multicenter study

The MB/BacT ALERT 3D System (MB/BacT) (Organon Teknika, Boxtel, The Netherlands) is a fully automated, nonradiometric system with a revised antibiotic supplement kit designed for the recovery of mycobacteria from clinical specimens. In a multicenter study, the recovery rate of acid-fast bacilli (AFB) and the mean time to their detection from clinical specimens was determined by using the MB/BacT system. Data were compared to those assessed by the radiometric BACTEC 460 system (B460) and by culture on L?wenstein-Jensen (L-J) solid medium. A total of 2,859 respiratory and extrapulmonary specimens were processed by the N-acetyl-L-cysteine (NALC)-NaOH method using two different concentrations of sodium hydroxide; 1.5% was adopted in study design A (1,766 specimens), and 1.0% was used in study design B (1,093 specimens). The contamination rates for MB/BacT were 4.6% (study design A) and 7.1% (study design B). One hundred seventy-nine mycobacterial isolates were detected by study design A, with 148 Mycobacterium tuberculosis complex (MTB) isolates and 31 nontuberculous mycobacteria (NTM) isolates. Overall recovery rates were 78.8% for MB/BacT (P = 0.0049), 64.2% for L-J (P < 0.0001), and 87.1% for B460, whereas they were 84.5, 70.9, and 91.2%, respectively, for MTB alone. A total of 125 mycobacteria were detected by study design B, with 46 MTB and 79 NTM. Overall recovery rates by the individual systems were 57.6% (P = 0.0002), 56.8% (P = 0.0001), and 80% for MB/BacT, L-J, and B460, respectively, whereas the rates were 91.3, 78.3, and 97.8% for MTB alone. By study design A, the mean times to detection of smear-positive MTB, smear-negative MTB, and NTM were 11.5, 19.9, and 19.6 days, respectively, with the MB/BacT; 8.3, 16.8, and 16.6 days, respectively, with the B460; and 20.6, 32.1, and 27.8 days, respectively, with L-J medium. By study design B, the mean times were 15.1, 26.7, and 26 days with the MB/BacT; 11.7, 21.3, and 24.8 days with the B460; and 20.4, 28.7, and 28.4 days with L-J medium. Identification was attempted by probing (Accuprobe) MB/BacT-positive bottles within the first working day following instrument positive flag. Results were compared to those obtained in the B460 positive vials by the p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test (study design A) or by the Accuprobe assay (study design B). About 90% of MTB and 100% of NTM could be identified, showing turnaround times closely related to those obtained by combining B460 and the NAP test or the Accuprobe assay. In conclusion, even though recovery rates were shown to be lower than B460, especially for NTM, and contaminants were somewhat higher, MB/BacT represents a valuable alternative to the radiometric system, especially in those laboratories where disposal of radioactive waste is restricted. Finally, when AFB are cultured in nonradiometric liquid media, our data (detection times and bacterial overgrowth rates) suggest that decontamination with 1.5% NaOH may be more suitable than the standard NALC-NaOH.     

Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium

Background: Mycobacterial antigen MPB64 is a secretory protein specific for Mycobacterium tuberculosis complex. A lateral flow immunochromatographic assay (ICA) is a method used for the rapid differentiation of M. tuberculosis complex. Aim: We aimed to evaluate the performance of ICA in rapid differentiation of M. tuberculosis complex from 97 Mycobacterium species other than tuberculosis (MOTT), which are grown in Löwenstein-Jensen and TK-selective (SLC) medium. Materials and Methods: The study was performed in our laboratory between January 2009 and January 2010. A total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different MOTT bacilli, 7 Mycobacterium bovis BCG substrains and total 256 clinical Mycobacterium isolates were tested by ICA, which is based on anti-MPB64 monoclonal antibodies. All the strains were inoculated onto a TK-SLC (selective) medium and Löwenstein-Jensen medium. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. Results: The growth of mycobacterial strains was observed in 10–12 days on TK-SLC medium. ICA test was performed in 15 minutes. All strains belonging to M. tuberculosis complex group were found positive and all MOTT species were found negative on ICA slides. The results were confirmed with nucleic acid amplification by polymerase chain reaction (PCR) using primers specific for M. tuberculosis complex. Conclusion: With the additive effect of growth on TK-SLC medium in 10–12 days, the mycobacterial antigen MPB64 is a very useful and specific tool in rapid differentiation of M. tuberculosis and MOTT grown in culture.     

Comparative evaluation of Löwenstein-Jensen proportion method, BacT/ALERT 3D system, and enzymatic pyrazinamidase assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis

Pyrazinamide (PZA) is an important first-line antituberculosis drug because of its sterilizing activity against semidormant tubercle bacilli. In spite of its very high in vivo activity, its in vitro activity is not apparent unless an acidic environment is available, which makes PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of the colorimetric BacT/ALERT 3D system and compare the results with those from conventional tests, i.e., the L?wenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all of these tests was 89.71% after the first round of testing and reached 92.52% after resolution of the discordant results by retesting. Prolonged incubation of the PZase tube for up to 10 days was found to increase the specificity of the PZase test. The concordances between LJ proportion and BacT/ALERT 3D, LJ proportion and the PZase assay, and BacT/ALERT 3D and the PZase assay were found to be 99.06%, 93.46%, and 92.52%, respectively. Using the LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and the PZase assay were 100 and 82.85%, respectively, while the specificity was 98.61% for both of the tests. The difference between the sensitivities of BacT/ALERT 3D and the PZase assay was significant (P = 0.025). The mean turnaround times for the detection of resistant and susceptible results by BacT/ALERT 3D were 8.04 and 11.32 days, respectively. While the major limitations associated with the PZase assay and the LJ proportion method are lower sensitivity in previously treated patients and a longer time requirement, respectively, the BacT/ALERT 3D system was found to be rapid, highly sensitive, and specific.     

Comparison of the mycobacteria growth indicator tube with MB redox, Löwenstein-Jensen, and Middlebrook 7H11 media for recovery of mycobacteria in clinical specimens

The rate of recovery and the mean time to detection of mycobacteria in clinical specimens were evaluated with two nonradiometric broth-based systems, the Mycobacteria Growth Indicator Tube (MGIT) and MB Redox systems. The data obtained for each system were compared with each other and with those obtained with the L?wenstein-Jensen (LJ) and Middlebrook 7H11 reference media. A total of 117 mycobacterial isolates (Mycobacterium tuberculosis, n = 112; nontuberculous mycobacteria, n = 5) were detected in 486 clinical specimens. The recovery rates for M. tuberculosis were 91 of 112 (81.3%) isolates with MGIT and 81 of 112 (72.3%) isolates with MB Redox. The combination of MGIT plus MB Redox recovered 104 of the 112 (92.9%) M. tuberculosis isolates. MGIT plus LJ plus Middlebrook 7H11 recovered 106 of the 112 (94.6%) isolates, MB Redox plus LJ plus Middlebrook 7H11 recovered 99 of the 112 (88.4%) isolates, and LJ plus Middlebrook 7H11 recovered 84 of the 112 (75. 0%) isolates. The mean time to detection of M. tuberculosis in smear-positive specimens was 7.2 days with MGIT, 6.9 days with MB Redox, 20.4 days with LJ, and 17.6 days with Middlebrook 7H11. The mean time to detection of M. tuberculosis in smear-negative specimens was 19.1 days with MGIT, 15.5 days with MB Redox, 25.8 days with LJ, and 21.6 days with Middlebrook 7H11. The contamination rates were 4.4, 3.8, 2.1, and 2.7% for MGIT, MB Redox, LJ, and Middlebrook 7H11, respectively. In conclusion, MGIT and MB Redox can be viable tools in the routine mycobacteriology laboratory.     

Mycobacterial Growth Indicator Tube Versus the Proportion Method on Löwenstein-Jensen Medium for Antibiotic Susceptibility Testing of Mycobacterium tuberculosis

The Mycobacterial Growth Indicator Tube, a reliable system for detection of mycobacterial growth, was compared with the reference proportion method on Löwenstein-Jensen medium for antibiotic susceptibility testing of Mycobacterium tuberculosis. A total of 62 clinical strains and four reference strains of Mycobacterium tuberculosis were tested for susceptibility to streptomycin, isoniazid, rifampicin and ethambutol. Of these, 36 were susceptible to all four antibiotics and 30 were resistant to at least one of them. Tests were repeated in cases of discrepant results. When each drug/strain combination was considered separately, the overall agreement between the two methods was 96.5% (98.4% for streptomycin, 95.3% for isoniazid, 96.9% for rifampicin and 95.3% for ethambutol) with regard to the initial testing and 98.8% (100, 98.5, 98.5 and 98.4%, respectively) after repeated testing. When the results were considered strain by strain, the agreement was 86% after the initial testing and 95% after repeated testing. The results were obtained after a mean time of 9.5 days. These results suggest that the Mycobacterial Growth Indicator Tube is a reliable method for testing susceptibility of mycobacterial strains to first-line antituberculous drugs.     

Comparison of gas exchange data using the Aquatrainer® system and the facemask with Cosmed K4b2 during exercise in healthy subjects

The aim of this study was to determine the level of agreement between the new Aquatrainer^® system and the facemask in the assessment of submaximal and maximal cardiopulmonary responses during exercise performed on ergocycle. Twenty-six physically active healthy subjects (mean age: 41 ± 14 years) performed a submaximal constant work test followed by maximal incremental exercise test on ergocycle, one with cardiopulmonary responses measured using the Cosmed K4b2 facemask, the other using the Cosmed K4b2 Aquatrainer^®. Using the Aquatrainer^®, the gas exchange variables at 100 W were significantly lower for VO₂ (1,483 ± 203 vs. 1,876 ± 204 ml min^?1, P < 0.0001), VCO₂ (1,442 ± 263 vs. 1,749 ± 231 ml min^-1, P < 0.0001), VE (38 ± 5 vs. 44 ± 6 l min^?1, P < 0.0001), and VT (1.92 ± 0.47 vs. 2.18 ± 0.41 l, P < 0.0001) relative to facemask. The bias ±95% limits of agreement (LOA) for VO₂ was 393 ± 507 ml min^?1 for the submaximal constant work test at 100 W and 495 ± 727 ml min^?1 for VO_2max. At maximal intensity, cardiopulmonary responses measured with the Aquatrainer^® system were significantly lower for: VO₂ (2,799 ± 751 vs. 3,294 ± 821 ml min^?1, P < 0.0001), VCO₂ (3,426 ± 836 vs. 3,641 ± 946 ml min^?1, P = 0.012), VE (98 ± 21 vs. 108 ± 26 l min^?1, P = 0.0009) relative to facemask. A non-constant measurement error [interaction effect: (facemask or aquatrainer) × power] was noted from 60 to 270 W for VO₂ (ml min^?1), VCO₂ (ml min^?1), ventilation (l min^?1) (P < 0.0001) and VT (l, P = 0.0001). Additional studies are required to detect the main sources of error that could be physical and/or physiological in nature. Due to the significant measurement error, the new Aquatrainer^® system should be used with extreme caution in filed testing conditions of swimmers.     

One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX? PCR system

A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra? MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l'Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters (n?=?419) and other Gram stain forms (n?=?361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4?% (158/159), 100.0?% (9/9), and 99.3?% (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8?%. The lowest detectable amount of viable cells in the blood culture sample was 4 × 10(4)?CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX? instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.     

Protostrongylus pulmonalis (Frölich, 1802) and P. oryctolagi Baboš, 1955 (Nematoda: Protostrongylidae), parasites of the lungs of European hare (Lepus europaeus L.) in France: morphological and molecular approaches

Pulmonary protostrongyliasis of hare is a parasitic disease caused by nematodes belonging to the genus Protostrongylus (Nematoda, Protostrongylidae). During survey of wildlife disease in the South-East of France, pathologic examination of lungs from European hares found dead or hunter-killed between 2009 and 2012 was performed. Adult male worms were morphologically characterized and the identification confirmed by molecular biology (D2 domain of the 28S and ITS2 of rDNA). Two different species were identified: the first one, Protostrongylus pulmonalis, is identical with the haplotype previously deposited in GenBank. Based on morphological criteria of copulatory bursa of adult male worms (especially length of spicules and gubernaculum structure), we identified a second species found in France as Protostrongylus oryctolagi. This is the first report of P. oryctolagi in France from European hare and rabbit. P. oryctolagi was isolated from 248 hares and 3 rabbits in the South of France. P. pulmonalis was isolated from four hares found dead in the Northern France and from one hare in the South, which was co-parasitized by P. oryctolagi and P. pulmonalis. It’s the first coinfection observed with these two species from a lung of hare in France.     

Performance comparison of the 4th generation Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA on the EVOLIS™ automated system versus Abbott ARCHITECT HIV Ag/Ab Combo,Ortho Anti-HIV 1 + 2 EIA on Vitros ECi and Siemens HIV-1/O/2 enhanced on Advia Centaur

BackgroundA multisite study was conducted to evaluate the performance of the Bio-Rad 4th generation GS HIV Combo Ag/Ab EIA versus Abbott 4th generation ARCHITECT HIV Ag/Ab Combo. The performance of two 3rd generation EIAs, Ortho Diagnostics Anti-HIV 1 + 2 EIA and Siemens HIV 1/O/2 was also evaluated.ObjectiveStudy objective was comparison of analytical HIV-1 p24 antigen detection, sensitivity in HIV-1 seroconversion panels, specificity in blood donors and two HIV false reactive panels.Study designAnalytical sensitivity was evaluated with International HIV-1 p24 antigen standards, the AFFSAPS (pg/mL) and WHO 90/636 (IU/mL) standards; sensitivity in acute infection was compared on 55 seroconversion samples, and specificity was evaluated on 1000 negative blood donors and two false reactive panels.ResultsGS HIV Combo Ag/Ab demonstrated better analytical HIV antigen sensitivity compared to ARCHITECT HIV Ag/Ab Combo: 0.41 IU/mL versus 1.2 IU/mL (WHO) and 12.7 pg/mL versus 20.1 pg/mL (AFSSAPS); GS HIV Combo Ag/Ab EIA also demonstrated slightly better specificity compared to ARCHITECT HIV Ag/Ab Combo (100% versus 99.7%). The 4th generation HIV Combo tests detected seroconversion 7–11 days earlier than the 3rd generation HIV antibody only EIAs.ConclusionBoth 4th generation immunoassays demonstrated excellent performance in sensitivity, with the reduction of the serological window period (7–11 days earlier detection than the 3rd generation HIV tests). However, GS HIV Combo Ag/Ab demonstrated improved HIV antigen analytical sensitivity and slightly better specificity when compared to ARCHITECT HIV Ag/Ab Combo assay, with higher positive predictive values (PPV) for low prevalence populations.