Claudio Piersimoni

The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study

A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide. To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country. We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed. This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location. Species distribution among nontuberculous mycobacteria isolates from pulmonary specimens is geographically diverse http://ow.ly/npu6r

Clinical validation of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis

Extrapulmonary tuberculosis (EPTB) accounts for more than 20% of tuberculosis (TB) cases. Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, CA, USA) is a fully automated amplification system, for which excellent results in the diagnosis of pulmonary TB in highly endemic countries have been recently reported. We aimed to assess the performance of the Xpert system in diagnosing EPTB in a low incidence setting. We investigated with Xpert a large number of consecutive extrapulmonary clinical specimens (1,476, corresponding to 1,068 patients) including both paediatric (494) and adult samples. We found, in comparison with a reference standard consisting of combination of culture and clinical diagnosis of TB, an overall sensitivity and specificity of 81.3% and 99.8% for Xpert, while the sensitivity of microscopy was 48%. For biopsies, urines, pus and cerebrospinal fluids the sensitivity exceeded 85%, while it was slightly under 80% for gastric aspirates. It was, in contrast, lower than 50% for cavitary fluids. High sensitivity and specificity (86.9% and 99.7%, respectively) were also obtained for paediatric specimens. Although the role of culture remains central in the microbiological diagnosis of EPTB, the sensitivity of Xpert in rapidly diagnosing the disease makes it a much better choice compared to smear microscopy. The ability to rule out the disease still remains suboptimal.

Relevance of Commercial Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples

Mycobacteria are a group of acid-fast, aerobic, slow-growing organisms whose genus includes more than 90 different species. The causative agents of tuberculosis (TB), which is currently considered a global emergency, with more than 2 million people dying every year and 8 million new cases, belong to the Mycobacterium tuberculosis complex (MTB). Moreover, although of lesser public health importance, many other species referred to as nontuberculous mycobacteria (NTM) have also been associated with human disease with increasing frequency worldwide (65). As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Active TB is currently diagnosed by conventional laboratory procedures including specimen digestion and decontamination, microscopic examination for the presence of acid-fast bacilli (AFB), isolation by culture on solid and/or liquid media, and identification and drug susceptibility testing of the recovered isolate. Because of the slow growth of mycobacteria, the above-reported laboratory procedures may require turnaround times of 3 to 4 weeks or longer. During the last decade, several molecular methods have been developed for direct detection and identification of MTB in clinical specimens. These methods, being able to potentially reduce the diagnostic time from weeks to days, have been acquiring greater and greater relevance in the field of laboratory TB diagnosis. The basic principle of any molecular diagnostic test is the detection of a specific nucleic acid sequence by hybridization to a complementary sequence, a probe, followed by detection of the hybrid. However, the sensitivity of nucleic acid probe tests that do not involve amplification is much lower than that of amplified ones. Any portion of nucleic acid can be copied by using the specific polymerase, provided that some sequence data are known for the setup of appropriate primers. In general, amplification of target nucleic acid sequences is composed of three parts: denaturation, primer annealing, and primer extension. Discovery of PCRs in 1986 made this process reiterative, leading to an exponential increase in the production of the amplified target. Soon after, alternative amplification techniques were developed and patented by companies, which used different enzymes and strategies, but they are all based on reiterative reactions. Many different amplification targets including both DNA or RNA fragments have been proposed. The target most frequently amplified in MTB is the IS6110 (31) repetitive element, of which 10 to 16 copies are present in most clinical isolates. Numerous techniques for nucleic acid extraction have been proposed, as have different types of controls for monitoring the efficacy of nucleic acid extraction and amplification procedures. Currently, the U.S. Food and Drug Administration (FDA) requires that culture (still considered the “gold standard” for TB diagnosis) must be done in conjunction with the performance of each amplification-based test. In this paper, we review and discuss the currently available commercial methods which are capable of detecting MTB directly from clinical samples.

Extrapulmonary infections associated with nontuberculous mycobacteria in immunocompetent persons.

Incidence data are lacking, and diagnosis remains difficult.

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.

Pulmonary infections associated with non-tuberculous mycobacteria in immunocompetent patients

A decline in the prevalence of tuberculosis in the developed world over the past several years has been accompanied by an increase in the rate of mycobacterial disease caused by non-tuberculous mycobacteria. However, it is still unclear whether there is a real increase in prevalence or whether non-tuberculous mycobacterial disease is being recognised more frequently by clinicians in a variety of clinical settings, thus enhancing the competence of microbiologists to detect the more unusual and fastidious mycobacteria. The introduction of liquid media for isolation of mycobacteria coupled with more accurate methods for identification have allowed several new species associated with human disease to be recognised. Despite this progress, several issues related to non-tuberculous mycobacterial infections need to be addressed, including the timely and reliable identification of isolates, standardisation and clinical evaluation of susceptibility testing, and capability to distinguish disease-causing isolates from contaminant or saprophytic species. Treatment regimens for non-tuberculous mycobacterial disease are still largely undefined and outcome remains disappointing despite substantial upgrading in laboratory diagnosis and the availability of new antimicrobials. Treatment success is impaired by the long duration of regimens, side-effects, and drug interactions, which prevent patients from full compliance. We discuss the epidemiological features, clinical syndromes, and developments in the investigation, prevention, and treatment of pulmonary non-tuberculous mycobacterial infections.

Current Perspectives on Drug Susceptibility Testing of Mycobacterium tuberculosis Complex: the Automated Nonradiometric Systems

Until the beginning of the 1990s, it was quite common to learn that many patients with tuberculosis (TB) were treated for several months with a standard regimen including isoniazid (INH) and rifampin (RMP) without the physician knowing whether the causative organism was susceptible to these drugs. The main reason for such an attitude was that the probability of a Mycobacterium tuberculosis complex (MTC) initial isolate being resistant was negligible. Unfortunately, the situation has strikingly changed now. Not only does TB continue to represent one of the most relevant infectious diseases in the world, responsible for 8 million new cases and 2 to 3 million casualties occurring annually, but in low-prevalence countries, the rates of initial-drug- and multidrug-resistant (MDR) TB (e.g., resistant to both INH and RMP) (13) have been climbing, causing a worrying rise in morbidity and mortality. An additional factor is human immunodeficiency virus, which has significantly increased the incidence of TB, particularly in sub-Saharan Africa but also elsewhere. While the long-term solution is likely to come from the development of a better vaccine, for the near future, reliance on chemotherapy will have to be continued. This matter has refocused attention on the importance of MTC drug susceptibility testing (DST) and on the laboratorys key role in providing clinicians with timely, reliable, and comprehensive information (2). Currently, a number of automated, nonradiometric detection systems (NRS) that can also perform MTC susceptibility testing are commercially available to clinical laboratories (30). Not all the methods presented herewith have been cleared by the Food and Drug Administration. However, we performed a systematic review and meta-analysis to evaluate the performance of these systems in comparison with the radiometric BACTEC 460 TB (B460) system, currently regarded as a reference able to combine timely and reliable results (27, 36).

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.