Microbiological diagnosis of infections caused by the genus Mycobacterium. / Diagnóstico microbiológico de las infecciones causadas por el género Mycobacterium.

Mycobacterial culture has a high sensitivity and is the test of choice for the microbiological diagnosis of tuberculosis and nontuberculous mycobacterial infections. However, the results of this culture require at least 2-3 weeks to obtain positivity. Staining is rapid and can be used as a complementary study, although its sensitivity is low. Gene amplification tests have an intermediate sensitivity and obtain results in 1-2 days. These last tests are indicated in cases with moderate or high clinical suspicion. In HIV patients with severe immunodeficiency (<200 CD4), lipoarabinomannan antigen detection in urine may be useful. The identification of isolates from positive cultures is essential to evaluate the clinical significance of the culture results and consider the therapeutic options available. At present, there is a wide range of identification techniques available, which provide results within just 1-4 days. The future of diagnostic techniques in tuberculosis and nontuberculous mycobacterial infections lies in greater development of gene amplification techniques and promoting the search for biomarkers which enable a new approach to the diagnosis of these infections.

Growth of mycobacteria in urine determined by isothermal microcalorimetry: implications for urogenital tuberculosis and other mycobacterial infections.

OBJECTIVE: To overcome the limitations of current urine-based diagnostic assays of urogenital tuberculosis, we used isothermal microcalorimetry to detect the metabolic activity of Mycobacterium tuberculosis and other commonly neglected pathogenic mycobacteria in urine and accurately determine their growth parameters. METHODS: A microcalorimeter equipped with 48 channels was used. Detection was accomplished, and growth was monitored for 4 different Mycobacterium species in sterilized and modified urine at 37 °C by measuring metabolic heat flow (µW = µJ/s) as a function of time. These strains were M. smegmatis, M. phlei, M. kansasii, and M. tuberculosis. The data were integrated to perform curve fitting and extract the growth parameter from the raw data. RESULTS: In sterilized urine, M. smegmatis showed the fastest growth rate (0.089 ± 0.017 [h(-1)]), followed by M. phlei (0.072 ± 0.016 [h(-1)]) and M. kansasii (0.007 ± 0.001 [h(-1)]). No growth of M. tuberculosis was detected in sterilized urine. However, in serum-supplemented urine, growth of M. tuberculosis was observed within 3 weeks at a growth rate of 0.008 ± 0.001 [h(-1)]. Biofilm formation was enhanced in the serum supplemented urine. CONCLUSION: Isothermal microcalorimetry allows rapid and accurate detection of mycobacterial growth in urine. Given the absence of data on the mycobacterial growth in urine, isothermal microcalorimetry could be used to unravel key aspects of Mycobacterium physiology in the urinary tract and potentially contribute to improvement in the diagnosis and treatment of urogenital tuberculosis.

An evaluation of the recovery of mycobacteria from urine specimens using the automated Mycobacteria Growth Indicator Tube system (BACTEC MGIT 960).

The Mycobacteria Growth Indicator Tube (MGIT 960) system was evaluated against Lowenstein-Jensen (LJ) medium and the BACTEC 460 TB system for the recovery of mycobacteria from 1393 consecutive urine specimens. The MGIT had a sensitivity of 91.3% [95% confidence interval (CI), 83.2-99.4] when the combination of BACTEC 460 and LJ medium was used as the reference method. The mean time for positivity for MGIT and BACTEC 460 was 19.3 days and 20 days, respectively, while that for LJ medium was 35 days.The incidence of contamination was highest for LJ medium (n=148), followed by MGIT 960 (n=81), and BACTEC 460 had the lowest incidence of contamination (n=50). In conclusion, the isolation of mycobacteria from urine specimens by the MGIT 960 is comparable to that of the BACTEC 460 TB system and solid media.

Spectrum of mycobacterial infections: tuberculosis and Mycobacterium other than tuberculosis in dialysis patients.

BACKGROUND: Patients with end-stage renal disease are at high risk of mycobacterial infection. OBJECTIVES: To analyze the difficulties in reaching an accurate diagnosis of tuberculosis in dialysis patients. METHODS: We conducted a retrospective follow-up of patients who attended our peritoneal and hemodialysis units during the 10 year period 1995-2005. RESULTS: Our dialysis unit diagnosed 10 cases of tuberculosis caused by Mycobacterium tuberculosis and 9 cases of Mycobacterium other than tuberculosis. In the former group, five patients had Mycobacterium in the sputum, which was diagnosed by intraabdominal mass biopsy in one, culture of the gastric juices in one, and pleural fluid culture or pleural biopsy in three. One of these patients was suffering from pleural TB as well as Potts disease. Of the patients with Mycobacterium other than tuberculosis, five were diagnosed by sputum cultures, three by urine cultures and one in peritoneal fluid. Differences in treatment and outcome were also reviewed. CONCLUSIONS: The diagnosis of TB in dialysis patients should be approached with a high index of suspicion. It is clear that extensive diagnostic procedures are required to ensure an accurate diagnosis of the disease. Tuberculosis incurs a significant added burden due to the need for isolation of infected patients within the dialysis unit. Treatment of patients with Mycobacterium other than tuberculosis should be addressed individually.

A PCR-colorimetric microwell plate hybridization assay for detection of Mycobacterium tuberculosis and M. avium from culture samples and Ziehl-Neelsen-positive smears.

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

Accelerated detection and identification of mycobacteria with MGIT 960 and COBAS AMPLICOR systems.

An automated cultivation system for mycobacteria, the MGIT 960 system (MGIT system), was compared in the clinical routine with two variants of Löwenstein-Jensen (L-J) medium. A total of 152 isolates were recovered from 2,015 specimens: 139 (91%) with the MGIT system and 127 (84%) with L-J media (P = 0.05). These included 68 isolates of Mycobacterium tuberculosis, of which 88% grew in the MGIT system and 93% grew in L-J media (P = 0.389), and 84 isolates of mycobacteria other than M. tuberculosis (MOTT), of which 94% grew in the MGIT system and 76% grew in L-J media (P = 0.003). More M. avium complex isolates were detected in the MGIT system (n = 65) than in L-J media (n = 50) (P = 0.001). Growth in the MGIT system was detected in 2 weeks for 78% of the isolates, whereas growth was detected in the two L-J media for 17 and 25% of the isolates, respectively. The mean times to detection of M. tuberculosis were 12 days in the MGIT system and 20 days in L-J media, and for M. avium complex the mean times to detection were 8 and 22 to 25 days, respectively. The contamination rates were similar (8.7 to 8.9%) in all media. A commercial amplification system (COBAS AMPLICOR) was evaluated for its ability to rapidly identify M. tuberculosis, M. avium, and M. intracellulare directly from 393 samples in MGIT system broth. A correct PCR result, as evaluated by culture or clinical data, was obtained for 96% of the samples, with inhibition being detected for 2% of the samples. Of the 89 results positive for M. tuberculosis, 91% were regarded as true positive, 8% were regarded as inconclusive, and 2% were considered false positive. For results positive for M. avium and M. intracellulare, 97 and 79%, respectively, were regarded as true positive. Increased rapidity and enhanced isolation of MOTT were obtained with the MGIT system. COBAS AMPLICOR was suitable for rapid identification of these three common pathogens from MGIT system broth.

Characterization of an isolate belonging to the newly described species Mycobacterium hassiacum.

The isolation, from a urine sample, of a rapidly growing acid-fast mycobacterium assigned to the thermophilic species Mycobacterium hassiacum led to further insight into present knowledge of this newly described organism. Already known phenotypic traits of M. hassiacum were extended and its susceptibility to additional antimicrobials was investigated. The high-performance liquid chromatography pattern of mycolic acids is, for the first time, presented. So far, no clinical relevance was proved for our isolate; likewise for the one which led to the species' original description.

Characterization of an isolate of the newly described species Mycobacterium interjectum.

The phenotypic features of a clinical isolate of the new species Mycobacterium interjectum, identified on the basis of its 16S rRNA gene sequence, are compared with those of the type strain. The differentiation of M. interjectum from Mycobacterium gordonae or Mycobacterium scrofulaceum is not achievable on the basis of phenotypic traits usually tested for mycobacterial speciation, but it can be reached by 16S rRNA gene sequencing or by high performance liquid chromatography (HPLC) of cell wall mycolic acids. The former reveals sequence identity with the signature region of the type species, and the latter yields a profile which is easily differentiated from those of the other two species. The unique HPLC profile of M. interjectum is reported here for the first time and so are the MICs of a wide spectrum of drugs.

Multicentre evaluation of a biphasic culture system for recovery of mycobacteria from clinical specimens.

A new biphasic system (MB Check, Roche) for isolation of mycobacteria from clinical specimens was evaluated by eight different microbiological laboratories in comparison with methods routinely used in the respective laboratories. Altogether 1125 clinical specimens were processed; pretreatment, if performed, was by a variety of methods. Mycobacteria were recovered from 167 specimens with the biphasic system and 165 specimens with the other methods. The average time required for isolation of Mycobacterium tuberculosis was 22.6 days with the biphasic system and 24.7 days with egg-based media; for other mycobacterial species it was 23.5 versus 20.8 days. The inclusion of a chocolate agar section in the biphasic system facilitated the early detection of contaminants, while the NAP-containing section appeared unable to differentiate Mycobacterium tuberculosis from other mycobacterial species. The biphasic system, which enables recovery of mycobacteria in small laboratories without specialized equipment, is more practical than conventional methods and at least as sensitive.

Effect of relative centrifugal force and centrifugation time on sedimentation of mycobacteria in clinical specimens.

Optimum relative centrifugal force (RCF) and centrifugation time to concentrate mycobacteria in clinical specimens were determined by processing split samples of sputa and urines containing mycobacteria with combinations of different RCFs and centrifugation times. Although individual test results showed considerable variation in the recovery rates of mycobacteria in the sediment, the data indicated that higher recovery rates occurred as centrifugation speed and time were increased. With a 15- to 20-min centrifugation time, on the average, 67 to 71% of mycobacteria were recovered at an RCF of 2,074 X g, and 76 to 80% were recovered at 3,005 or 3,895 X g at maximum radius. The remainder of mycobacteria was mostly recovered from the supernatant, but culturing of supernatant was not profitable. Increasing RCF had a negligible effect on acid-fast bacillus smear sensitivity. The smear sensitivity for about 25,000 clinical specimens processed with an RCF of 3,800 X g for 20 min was 71% compared with 69% as determined for over 30,000 specimens processed in a similar manner but an RCF of 2,000 X g. An RCF of 3,000 X g applied for 15 min, or an RCF of about 2,000 to 2,500 X g applied for 20 min, is considered adequate to concentrate mycobacteria in clinical specimens.

[The importance of Scotochromogenous mycobacteria in the urine]. / La importancia de las micobacterias escotocromógenas en la orina. Análisis de 155 casos

In order to find the significance of the presence of Scotochromogenous Mycobacteria in the urine, 155 cases with this finding and without other Mycobacteria were analyzed. Most had urinary symptoms (91.6%), abnormal urine analysis (62.5%), negative urine culture (71.7%), and changes in intravenous pyelograms (89.5%). Clinical and laboratory improvement was noticed in 70% of those who received antituberculous drugs and in 30% of those who did not receive them (P less than 0.01). All of the foregoing characteristics were present in each 23 patients with repeated isolations of Scotochromogens in the urine, and could be cases of urinary-tract disease due to Scotochromogens. In other patients this Mycobacteria could coexist with M. tuberculosis, or be a contaminant, although Scotochromogens were not found in the urine of 100 control subjects, a significant difference to these patients (P less than 0.0001).