Domenico Nista

Performance Assessment of New Multiplex Probe Assay for Identification of Mycobacteria

ABSTRACT A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii,Mycobacterium xenopi, Mycobacterium gordonae, the species of the Mycobacterium avium complex (MAC),Mycobacterium scrofulaceum, and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae, while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum, andMycobacterium malmoense, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.

Performance Assessment of Two Commercial Amplification Assays for Direct Detection of Mycobacterium tuberculosis Complex from Respiratory and Extrapulmonary Specimens

ABSTRACT The new BDProbeTec ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB) was compared with the enhanced M. tuberculosis Amplified Direct Test (AMTDII). The system is an automated walkaway system characterized by simultaneous DNA amplification (strand displacement amplification) and real-time fluorometric detection. It also contains an internal amplification control (IAC) designed to identify inhibition from the processed samples. The AMTDII assay amplifies rRNA by transcription-mediated amplification; it uses hybridization with a chemoluminescent probe as a detection system and is entirely manual. A total of 515 N-acetyl-l-cysteine-sodium hydroxide-decontaminated respiratory (n = 331) and extrapulmonary (n = 184) sediments (from 402 patients) were tested in parallel by both assays. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the “gold standard.” Culture results from the tested specimens were as follows: 121 Mycobacterium tuberculosis complex (MTB) (98 smear-positive), 46 nontuberculous mycobacteria (38 smear-positive), and 338 culture-negative results. After resolution of the discrepant results, the percent sensitivity, percent specificity, and positive and negative likelihood ratios for AMTDII were 88%, 99.2%, 110, and 0.11 for respiratory specimens and 74.3%, 100%, 740, and 0.26 for extrapulmonary specimens, respectively. The corresponding values for DTB were 94.5%, 99.6%, 235, and 0.05 for respiratory specimens and 92.3%, 100%, 920, and 0.07 for extrapulmonary specimens, respectively. The cumulative difference for all tuberculosis-positive extrapulmonary specimens was significant (P = 0.03). The overall inhibition rate for DTB was 5% (26 specimens). We conclude that both amplification assays proved to be rapid and specific for the detection of MTB in clinical samples and particularly feasible for a routine laboratory work flow. DTB combines a labor-intensive specimen preparation procedure with a completely automated amplification and detection. Finally, differences between AMTDII and DTB sensitivities were associated with the presence of inhibitory samples that the former assay, lacking IAC, could not detect.

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

ABSTRACT 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 Löwenstein-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 thep-nitro-α-acetylamino-β-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.

Direct Identification of Mycobacteria from MB/BacT Alert 3D Bottles: Comparative Evaluation of Two Commercial Probe Assays

ABSTRACT The new INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), a reverse-hybridization-based line probe assay, and the AccuProbe assay (Gen-Probe Inc., San Diego, Calif.) were applied to MB/BacT Alert 3D (MB/BacT) system (Organon Teknika, Boxtel, The Netherlands) culture bottles and evaluated for mycobacterial identification. From 2,532 respiratory and extrapulmonary specimens submitted for culture, 168 were flagged positive by the MB/BacT system and promptly evaluated for identification (within 24 h). Each of 163 vials grew one mycobacterial isolate, including Mycobacterium tuberculosis complex (n = 73), M. avium complex (n = 3), M. avium(n = 8), M. intracellulare(n = 5), M. kansasii (n = 15),M. gordonae (n = 8), M. malmoense (n = 3), M. chelonae(n = 13), M. abscessus (n= 2), M. xenopi (n = 11), M. scrofulaceum (n = 2), M. fortuitum(n = 7), M. terrae (n = 3),M. simiae (n = 2), M. celatum(n = 3), M. flavescens (n= 1), M. interjectum (n = 1), M. bohemicum (n = 1), and M. pulveris(n = 2). Five cultures yielded mixed growth of two mycobacterial species: M. tuberculosis complex plusM. gordonae (n = 2), M. tuberculosis complex plus M. chelonae(n = 1), M. tuberculosis complex plusM. xenopi (n = 1), and M. avium plus M. chelonae (n = 1). In testing of one-isolate vials, both systems showed excellent sensitivity and specificity for all species and complexes for which they are licensed (nine for INNO-LiPA Mycobacteria versus six for AccuProbe). There were minor discrepancies in results for two isolates identified by INNO-LiPA Mycobacteria as M. avium - M. intracellulare - M. scrofulaceum (MAIS) complex and by AccuProbe as M. intracellulare. In testing of two-isolate vials, INNO-LiPA Mycobacteria correctly identified all isolates, while the AccuProbe assay failed to identify three M. tuberculosiscomplex isolates and one M. avium isolate. The AccuProbe assay was completed within 2 h, while INNO-LiPA Mycobacteria required a 6-h period. In our opinion, INNO-LiPA Mycobacteria offers the following advantages: (i) it contains a genus-specific probe that, in addition to being used in genus identification, may be used as an internal control for both the amplification and hybridization steps; (ii) it simultaneously identifies M. tuberculosis complex, MAIS complex, and seven other mycobacterial species, even from mixed cultures; (iii) its mycobacterial DNA amplification ensures reliable results independent from the concentration of viable microorganisms; and (iv) it genotypically identifies M. kansasii andM. chelonae. In conclusion, even though INNO-LiPA Mycobacteria is considerably less easy to use than AccuProbe, requiring personnel skilled in molecular biology techniques, it represents an excellent approach for routine identification of frequently encountered mycobacteria.

Mycobacterium celatum Pulmonary Infection in the Immunocompetent: Case Report and Review

Mycobacterium celatum has been shown to cause disease in immunocompromised patients. We report a case of serious pulmonary infection caused by M. celatum in an apparently immunocompetent patient and review the characteristics of two other reported cases. Clinical and radiologic symptoms and signs included cough, malaise, and weight loss associated with cavitary lesions and pulmonary infiltrates. Although M. celatum is easy to detect in clinical specimens by liquid and solid media, it may be misidentified as a member of the M. tuberculosis complex or as M. xenopi. M. celatum pulmonary infection appears to respond to antimycobacterial chemotherapy, particularly with clarithromycin.

Multicenter Evaluation of the MB-Redox Medium Compared with Radiometric BACTEC System, Mycobacteria Growth Indicator Tube (MGIT), and Lowenstein-Jensen Medium for Detection and Recovery of Acid-Fast Bacilli

MB-Redox is a new manual culture system designed for the recovery of mycobacteria from clinical specimens. It consists of a liquid medium (modified Kirchner medium) containing a redox indicator, a colorless tetrazolium salt, which is reduced to colored formazan by actively growing mycobacteria. Acid fast bacilli (AFB) are easily detected in the medium as pink to purple pinhead-sized particles. We report the results of a multicenter study (involving four Italian microbiology laboratories processing 2370 clinical specimens) aiming to evaluate the recovery rates of AFB and time required for their detection by using the MB-Redox medium. Two different protocols were set up: in Protocol A (1580 specimens) the performance of MB-Redox was compared with those of the radiometric BACTEC 460 TB system (B460) and Löwenstein-Jensen medium (L-J), whereas in Protocol B (790 specimens) it was compared with those of the Mycobacteria Growth Indicator Tube (MGIT) and L-J. A total of 213 mycobacteria were recovered, including 172 Mycobacterium tuberculosis complex (MTB) isolates and 41 nontuberculous mycobacteria (NTM) isolates. In Protocol A, recovery rates were 81% for MB-Redox system, 84% for B460 system, and 77% for L-J. In Protocol B the recovery rates by individual system were 87, 83, and 76% for MB-Redox, MGIT, and L-J, respectively. Differences in both the protocols were not statistically significant. The MB-Redox system plus L-J (Combination 1) recovered 94% of the isolates in Protocol A and 93% in Protocol B, while B460 plus L-J (Combination 2) and MGIT plus L-J (Combination 3) detected 91 and 89% of all mycobacteria isolates respectively. No statistically significant differences were found among the combinations. The mean time to detection of mycobacteria was 16.3 days in Protocol A and 19.1 days in Protocol B with the MB-Redox system, 22.4 and 25.9 days with L-J, 13.2 days with B460, and 18.2 days with MGIT. The contamination rates were 2.1, 2.0, 1.9, and 3.6 for MB-Redox, B460, MGIT, and L-J respectively. The MB-Redox is a reliable, nonradiometric system for growth and detection of mycobacteria. When used in combination with a solid medium it proved to be an effective replacement for B460. The MB Redox system is a labor-intensive method requiring much handling during the visual reading procedures.

Mycobacterium lentiflavum as an Emerging Causative Agent of Cervical Lymphadenitis

ABSTRACT A lymph node excision was performed on a 45-year-old woman with left cervical swelling. The disorder which developed after the patient had undergone oral surgery for a severe periodontal disease failed to respond to antimicrobial chemotherapy. A mycobacterial strain subsequently identified by high-performance liquid chromatography analysis of cell wall mycolic acids as Mycobacterium lentiflavum grew from the excised specimen. This case and previously published reports highlight the relevance of M. lentiflavum as an emerging causative agent of mycobacterial cervical lymphadenitis.

Unreliable detection of Mycobacterium xenopi by the nonradiometric Bactec MGIT 960 culture system.

From June 2006 to December 2007, 3,648 clinical specimens consecutively received for mycobacterial culture were investigated. Each processed sample was inoculated into Bactec MGIT 960 liquid medium and a Löwenstein-Jensen slant. Tubes that were flagged as positive by the instrument as well as those determined to be negative after 42 days of incubation were removed, visually inspected for growth, and checked for the presence of acid-fast bacilli. Three hundred sixty-nine mycobacterial strains were recovered; of the 44 Mycobacterium xenopi isolates recovered by MGIT medium, only 13 were detected by the instrument (P < 0.0001). Most tubes yielding M. xenopi exhibited a peculiar pattern of growth characterized by a scant number of round, yellow-pigmented granules instead of the fine, evenly dispersed clumps usually observed for mycobacteria. It is suggested to check all individual tubes discarded by the MGIT 960 system at the end of the incubation period to prevent a significant amount of previously undetected growth from being missed.

Mycobacterium triplex Pulmonary Disease in Immunocompetent Host

Mycobacterium triplex, a recently described, potentially pathogenic species, caused disease primarily in immunocompromised patients. We report a case of pulmonary infection due to this mycobacterium in an immunocompetent patient and review the characteristics of two other cases. In our experience, Mycobacterium triplex pulmonary infection is unresponsive to antimycobacterial chemotherapy.

Evolution of Resistance to Erythromycin, Norfloxacin, and Tetracycline in Thermophylic Campylobacters

Thermophylic campylobacters are important enteric pathogens and have emerged as one of the leading causes of bacterial diarrhoea worldwide. Campylobacter enteritis is usually a mild to moderate self-limited disease and does not require antibiotic treatment except in severe cases when serious symptoms, relapses or a prolonged course of infection occur.