Comparative evaluation of Xpert MTB/RIF and the new Xpert MTB/RIF ultra with respiratory and extra-pulmonary specimens for tuberculosis case detection in a low incidence setting.

BACKGROUND: The Xpert MTB/RIF assay (Xpert) is an automated molecular test for the detection of tuberculosis and rifampin resistance (RIF-R), but it lacks sensitivity in smear-negative samples and some limitations in determination of RIF-R have also been reported. The new Xpert MTB/RIF Ultra (Ultra) was developed to overcome these limitations. We aimed to compare Ultra and Xpert diagnostic accuracy setting culture and drug susceptibility testing as reference standards. METHODS: A retrospective analysis was performed on 359 consecutive, respiratory (269) and extrapulmonary (90) specimens collected from 340 patients investigated for TB along a two-year period. Patients presenting at primary health-care centres and hospitals were recruited on the basis of symptoms and abnormal X-ray imaging. One-hundred seventy-four subjects were identified to have active tuberculosis by culture and 2 were MDR. FINDINGS: Sensitivities of Ultra and Xpert were 87% and 75% for the 48 individuals with smear-negative and culture-positive respiratory TB (difference of 12%, 95% CI 3 to 21); 95% and 72% for the 40 individuals with smear-negative and culture-positive extrapulmonary disease (22%, 95% CI 10 to 34); and 95% and 86%, respectively, across all 174 individuals with culture-positive samples (8.5%, 95% CI 4.5 to 12.5). Specificities of Ultra and Xpert for tuberculosis case detection were 98% and 100% (-2.0%, 95% CI -4.3 to +0.3). Ultra and Xpert performed equal in detecting RIF-R. INTERPRETATION: Sensitivity of Ultra was superior to that of Xpert in all categories of clinical samples. However, improved sensitivity was associated with a modest reduction in specificity.

Revisiting problems and solutions to decrease Mycobacterium tuberculosis pyrazinamide false resistance when using the Bactec MGIT 960 system.

Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.

Treating Primary Arthroprosthesis Infection Caused by Mycobacterium abscessus subsp. abscessus.

Prosthetic joint infections (PJI) caused by nontuberculous mycobacteria are very rare, and results of treatment can be unpredictable. A 72-year-old female underwent hip replacement after an accidental fall in a local hospital in Santo Domingo. The postoperative period was uneventful except for a traumatic wound near the surgical scar. PJI caused by Mycobacterium abscessus subsp. abscessus was diagnosed 6 months later. A two-stage reimplantation was performed after a 3-month period of aetiology-directed therapy, including amikacin, imipenem, and clarithromycin. M. abscessus isolate was reported to be resistant to clarithromycin when incubation was protracted for 14 days and to harbour the gene erm(41). The patient manifested major side effects to tigecycline. At reimplant, microbiologic investigations resulted negative. Overall, medical treatment was continued for a 7-month period. When discontinued and at 6-month follow-up, the patient was clinically well, inflammatory markers were normal, and the radiography showed well-positioned prosthesis. Mycobacterium abscessus subsp. abscessus is a very rare cause of PJI, yet it must be included in the differential diagnosis, especially when routine bacteria cultures are reported being negative. Further investigations are needed to determine any correlations between clinical results and in vitro susceptibility tests, as well as the clinical implications of M. abscessus subsp. abscessus harbouring the functional gene erm(41). Moreover, investigations are needed for determine optimal timings of surgery and lengths of medical therapy to improve patient outcome.

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.

Prevention of false resistance results obtained in testing the susceptibility of Mycobacterium tuberculosis to pyrazinamide with the Bactec MGIT 960 system using a reduced inoculum.

The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.

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.

Tuberculosis in kindergarten and primary school, Italy, 2008-2009.

An outbreak of tuberculosis (TB) in Italy involved 19 schoolchildren with active TB and 43 with latent infection. The source of the outbreak was a school assistant born in Italy who had a family history of TB. This outbreak highlights the need for maintaining clinical and public health expertise in countries with low TB incidence.

Extrapulmonary infections associated with nontuberculous mycobacteria in immunocompetent persons.

Over the past several years, the prevalence of human disease caused by nontuberculous mycobacteria (NTM) has increased. Whether the increase in cases is real or whether more cases are being recognized remains unclear. Despite a considerable increase in knowledge about NTM infections, they still represent a diagnostic and therapeutic challenge for several reasons: 1) pathogenic isolates may be indistinguishable from contaminant or saprophytic isolates; 2) timely and reliable identification of isolates may depend on proper communication between clinicians and laboratory staff; 3) lack of standardized susceptibility testing makes adoption of tailored therapies unrealistic; and 4) lack of treatment guidelines exposes patients to toxic drugs and disappointing outcomes. Laboratory research and multicenter controlled trials are needed to improve diagnosis and treatment of these infections.

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.

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.

Validation of the agar proportion and 2 liquid systems for testing the susceptibility of Mycobacterium tuberculosis to moxifloxacin.

Moxifloxacin (MOX), an 8-methoxyquinolone compound, is now widely used for the treatment of bacterial infections and also accepted as 2nd-line drug for the treatment of multidrug-resistant (MDR) tuberculosis. To tentatively correlate the clinical outcome with in vitro results, we sought to set up susceptibility test conditions for Mycobacterium tuberculosis against MOX by using the reference agar proportion method, the BACTEC 460 radiometric system, and the recently validated nonradiometric BACTEC MGIT 960 system. Our aim was to determine the critical MOX test concentration to be used with the abovementioned methods for routine susceptibility testing. MICs were determined for 20 pan-susceptible strains, 10 MDR strains, and 10 fluoroquinolone-resistant strains with defined gyrA mutations. MOX MICs resulted in a bimodal pattern with values for gyrA mutants considerably higher than those for pan-susceptible and MDR strains. Our data showed that a concentration of 0.5 microg/mL allowed a clear-cut separation between susceptible and resistant strains when tested by all the studied methods. Confirmatory test with a subset of pan-susceptible and MDR isolates appeared to validate the selected critical concentration. The MOX-resistant strains were almost isolated from patients with prior fluoroquinolone exposure.

Clinical suspicion as a primary guidance to use commercial amplification tests for rapid diagnosis of pulmonary tuberculosis.

The Abbott LCx (Abbott Park, IL) Mycobacterium tuberculosis complex is a commercial amplification assay discontinued from the European market in 2002. A prospective clinical study was carried out to evaluate the clinical utility of the above test as applied by specialists for the rapid diagnosis of active pulmonary tuberculosis (PTB). According to the physician's clinical judgment, patients were classified into 3 groups (low, intermediate, and high) aiming to estimate the probability of active disease. The gold standard for final diagnosis was based on microbiologic and clinical information including data from a 6-month follow-up period. Sensitivities and specificities of rapid microbiologic tests were compared with those based on an integrated approach including clinical evaluation plus the above tests. The incidence of PTB in 214 patients was 13.1%. The basis for initial treatment of PTB was smear-positive results in 46%, positive LCx results in 29%, and clinical suspicion in 18%. For the remaining 7%, therapy was started upon receipt of culture results. The sensitivity, specificity, and positive and negative predictive values of the LCx assay were 68%, 99%, 95%, and 95%, respectively. In comparison, they were 93%, 99%, 96%, and 99%, respectively, for the combination of clinical evaluation plus the LCx test. It is concluded that in patients with high-to-moderate pretest probabilities, the combination of clinical judgment and amplification results strongly enhances a rapid and correct diagnosis of PTB.

Drug resistance evolution of a Mycobacterium tuberculosis strain from a noncompliant patient.

The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.

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.

Mycobacterium lentiflavum as an emerging causative agent of cervical lymphadenitis.

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.