Soolingen, D. van

Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array (Article)

2012-08-01T00:00:00Z

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Isolation of non-tuberculous mycobacteria at three rural settings in Zambia; a pilot study (Article)

2010-08-01T00:00:00Z

To assess the role of non-tuberculous mycobacteria (NTM) as a cause of tuberculosis-like diseases in Zambia, 167 chronically ill patients, hospitalized in three rural hospitals in Katete, Sesheke and Chilonga, were examined by microscopy and liquid culture for the presence of NTM. The percentages of patients with a positive culture for Mycobacterium tuberculosis complex were similar in the three geographical locations (19-25%). In contrast, the percentage of NTM ranged from 78% in Katete and 65% in Sesheke to 21% in Chilonga. Furthermore, the distribution of NTM species was different at the three geographical sites. In seven patients, true NTM-associated disease was suspected: five with Mycobacterium lentiflavum and two with Mycobacterium intracellulare. Analysis of possible risk factors indicated that the OR for NTM culture-positive sputum was significantly higher for patients living in Katete and Sesheke. Female gender and chest X-ray appearances of tuberculosis were independently associated with NTM culture-positive sputum. NTM colonization and disease in hospitalized, chronically ill patients in rural Zambia appear to be common. © 2010 The Authors. Journal Compilation

Misdiagnosis of tuberculosis and the clinical relevance of non-tuberculous mycobacteria in Zambia (Article)

2010-05-01T00:00:00Z

Objective: To determine the accuracy of TB diagnosis of TB in Zambia in the era of increasing HIV prevalence. Methods: Sputum of the clinically diagnosed TB cases was additionally subjected to liquid culture and molecular identification. This study distinguished between TB cases confirmed by positive Mycobacterium tuberculosis (M. tuberculosis) cultures and mycobacterial disease caused by non-tuberculous mycobacteria (NTM). Results: Only 49% of the 173 presumptively diagnosed TB cases was M. tuberculosis cultured, while in 13% (22) cases, a combination of M. tuberculosis and NTM was found. In 18% of the patients only NTM were cultured. In 28%, no mycobacteria was cultivable. HIV positive status was correlated with the isolation of NTM (P <0.05). Conclusions: The diagnosis of tuberculosis based on symptoms, sputum smear and/or chest X-ray leads to significant numbers of false-positive TB cases in Zambia, most likely due to the increased prevalence of HIV. The role of NTM in tuberculosis-like disease also seems relevant to the false diagnosis of TB in Zambia.

Nontuberculous Mycobacterium infection and tumor necrosis factor-α antagonists (Article)

2009-10-01T00:00:00Z

Nontuberculous mycobacteria, Zambia (Article)

2009-02-01T00:00:00Z

Clinical relevance of nontuberculous mycobacteria (NTM) isolated from 180 chronically ill patients and 385 healthy controls in Zambia was evaluated to examine the contribution of these isolates to tuberculosis (TB)-like disease. The proportion of NTM-positive sputum samples was significantly higher in the patient group than in controls; 11% and 6%, respectively (p<0.05). NTM-associated lung disease was diagnosed for 1 patient, and a probable diagnosis was made for 3 patients. NTM-positive patients and controls were more likely to report vomiting and diarrhea and were more frequently underweight than the NTM-negative patients and controls. Chest radiographs of NTM-positive patients showed deviations consistent with TB more frequently than those of controls. The most frequently isolated NTM was Mycobacterium avium complex. Multiple, not previously identified mycobacteria (55 of 171 NTM) were isolated from both groups. NTM probably play an important role in the etiology of TB-like diseases in Zambia.

Rapid identification of mycobacteria by Raman spectroscopy (Article)

2008-03-01T00:00:00Z

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting. Copyright

Isolation of nontuberculous mycobacteria in Zambia: eight case reports. (Article)

2005-12-01T00:00:00Z

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens. Mycobacterium lentiflavum was isolated from four patients, and Mycobacterium goodii was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for M. lentiflavum was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of M. lentiflavum and M. goodii infections in Africa.

Antimycobacterial agents differ with respect to their bacteriostatic versus bactericidal activities in relation to time of exposure, mycobacterial growth phase, and their use in combination. (Article)

2005-06-01T00:00:00Z

A number of antimycobacterial agents were evaluated with respect to their bacteriostatic activity (growth inhibition) versus the bactericidal activity against a clinical isolate of Mycobacterium avium (Mycobacterium avium complex [MAC] strain 101) in relation to the time of exposure and the growth phase of the mycobacteria. In terms of growth inhibition the MAC in the active phase of growth was susceptible to clarithromycin, ethambutol, rifampin, amikacin, and the quinolones moxifloxacin, ciprofloxacin, and sparfloxacin. In terms of bactericidal activity in relation to the time of exposure these agents differed substantially with respect to the killing rate. An initial high killing capacity at low concentration was observed for amikacin, which in this respect was superior to the other agents. The bactericidal activity of clarithromycin and ethambutol was only seen at relatively high concentrations and increased with time. Killing by rifampin was concentration dependent as well as time dependent. The bactericidal activity of moxifloxacin was marginally dependent on the concentration or the time of exposure. The activity of clarithromycin in combination with ethambutol was not significantly enhanced compared to single-agent exposure. Only an additive effect was observed. The addition of rifampin or moxifloxacin as a third agent only marginally effected increased killing of MAC. However, by addition of amikacin the activity of the clarithromycin-ethambutol combination was significantly improved. The combination of amikacin and amoxicillin-clavulanic acid exhibited synergistic antimycobacterial activity. Towards MAC at low growth rates, only the quinolones exhibited a bactericidal effect.

Analysis of rate of change of IS6110 RFLP patterns of Mycobacterium tuberculosis based on serial patient isolates (Article)

1999-12-01T00:00:00Z

The rate of change of IS6110 restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis was determined in serial isolates from 544 patients. In 25 patients (4.6%), the RFLP patterns of the follow-up isolates differed from the initial isolates. Patients with different follow-up strains were less likely to cluster with patients whose strains had indistinguishable RFLP patterns. Changes in RFLP patterns were more common for persons with extrapulmonary disease and for those who had both pulmonary and extrapulmonary isolates. Based on serial isolates spanning for the most part <3 months, the half-life was extrapolated to be 3.2 years (95% confidence interval, 2.1-5.0). The main implication of this study is that the rate of change of IS6110-based RFLP of M. tuberculosis supports the use of IS6110 typing in epidemiologic studies of recent transmission of tuberculosis.