Comparative genomic hybridization of cancer of the gastroesophageal junction: deletion of 14Q31-32.1 discriminates between esophageal (Barrett's) and gastric cardia adenocarcinomas

We evaluated the BACTEC MGIT 960 system, which is a fully automated, noninvasive system for the growth and detection of mycobacteria with a capacity to incubate and continuously monitor 960 7-ml culture tubes. We studied 3,330 specimens, 2,210 respiratory and 1,120 nonrespiratory specimens, collected from 2,346 patients treated at six sites. Processed specimens were inoculated into the BACTEC MGIT 960 and BACTEC 460 TB systems, as well as onto Lowenstein-Jensen slants and Middlebrook 7H11/7H11 selective plates. From all culture systems, a total of 362 isolates of mycobacteria were recovered; these were recovered from 353 specimens collected from 247 patients. The greatest number of isolates of mycobacteria (289, or 80% of the 362 isolates) was recovered with the BACTEC MGIT 960, followed by the BACTEC 460 TB (271, or 75%) and solid media (250, or 69%). From all culture systems a total of 132 isolates of Mycobacterium tuberculosis complex were recovered. The greatest number of isolates of M. tuberculosis complex was recovered when liquid medium was combined with conventional solid media; the number recovered with BACTEC 460 TB plus solid media was 128 (97%), that recovered with BACTEC MGIT 960 plus solid media was 121 (92%), that recovered with BACTEC 460 TB was 119 (90%) and that recovered with all solid media combined was 105 (79%). The recovery with BACTEC MGIT 960 alone was 102 (77%). The mean times to detection (TTD) for M. tuberculosis complex were 14.4 days for BACTEC MGIT, 15.2 days for BACTEC 460 TB, and 24.1 days for solid media. The numbers of isolates of Mycobacterium avium complex (MAC) recovered were 172 (100%) for all systems, 147 (85%) for BACTEC MGIT 960, 123 (72%) for BACTEC 460 TB, and 106 (62%) for all solid media combined. The TTD for MAC in each system were 10.0 days for BACTEC MGIT 960, 10.4 days for BACTEC 460 TB, and 25.9 days for solid media. Breakthrough contamination rates (percentages of isolates) for each of the systems were 8.1% for BACTEC MGIT 960, 4.9% for BACTEC 460 TB, and 21.1% for all solid media combined.     

Multicenter comparison of ESP Culture System II with BACTEC 460TB and with Lowenstein-Jensen medium for recovery of mycobacteria from different clinical specimens, including blood

The recently developed ESP Culture System II (AccuMed, Chicago, Ill.) was compared with radiometric BACTEC 460TB (Becton Dickinson, Towson, Md.) and with Lowenstein-Jensen medium for recovery of mycobacteria from over 2,500 clinical specimens both of respiratory and nonrespiratory origin, including blood. The majority of the 219 mycobacterial isolates (129) belonged to the Mycobacterium tuberculosis complex, followed by 37 isolates of the Mycobacterium avium complex (MAC) and 53 isolates of eight other mycobacterial species. Rates of recovery obtained with BACTEC, ESP, and Lowenstein-Jensen medium were 89, 79, and 64%, respectively, with such differences being statistically significant. Different media and systems appeared to behave differently when the more frequently detected organisms were considered: M. tuberculosis complex isolates grew better with BACTEC, and MAC isolates grew better with ESP. An analysis of the combinations of Lowenstein-Jensen medium with BACTEC and with ESP did not reveal significant differences in recovery rates. With regard to the times needed for the detection of positive cultures, they were significantly longer on Lowenstein-Jensen medium (average, 28 days) than with the remaining two systems, between which there was no difference (average, 18 days). We conclude, therefore, that the ESP system, when used in combination with a solid medium, performs as well as the thoroughly validated radiometric BACTEC system and offers the advantages of full automation and absence of radioisotopes.     

An algorithm for the rational choice of sodium profile during hemodialysis

Two systems, the newly developed Mycobacteria Growth Indicator Tube (MGIT) and biphasic Septi-Chek AFB based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. The difference in the rates of isolation of bacteria between the two groups of media was more remarkable with smear-negative specimens. The isolation of the Mycobacterium tuberculosis complex by MGIT occurred 8 days previous to the isolation by the conventional Ogawa method. The mean time for detecting M. tuberculosis complex by Septi-Chek AFB was similar to those of the Ogawa method. A greater difference in isolation time was observed for mycobacteria other than M. tuberculosis (MOTT) isolates. These results indicate that the MGIT and Septi-Chek AFB systems based on liquid media are efficient for the recovery of mycobacteria. PCR and other nucleic acid amplification methods are widely used for the detection of M. tuberculosis in clinical specimens. Although the sensitivities of the Gen-Probe Amplified Mycobacteria Direct Test (MTD) and Amplicor Mycobacteria for the detection of the M. tuberculosis complex appear to be similar to the sensitivity of the culture method using the Septi-Chek AFB, the two methods should be quite useful for rapid detection of M. tuberculosis infections. On the other hand, two cooperative blind studies conducted between 6 to 9 laboratories to estimate the reliability and reproducibility of these two commercially available kits revealed the necessity of good laboratory practice and development of reference reagents to monitor the performance of the whole assay, including pretreatment of clinical specimens. Considerable progress has been made in recent years toward understanding the molecular basis of the resistance to antituberculosis drugs, isoniazid (katG, inhA, ahpC), rifampin (rpoB), pyrazinamide (pncA), streptomycin (rpsL, rrs), ethambutol (embB), and fluoroquinolones (gyrA). Most cases of resistance are related usually to simple nucleotide substitutions rather than to acquisition of new genetic elements. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. The basis of resistance is not able to be explained yet in a substantial percentage of strains (> 90%) for other antituberculosis drugs than rifampin. Further studies are required to fully understand the molecular mechanisms of resistance.     

Clinical evaluation of a new non-radiometric automatic system for the rapid diagnosis of tuberculosis

BACKGROUND: Tuberculosis (TB) is again a public health problem un many countries and is considered a re-emerging disease. The fastest possible diagnosis in our patients is essential for TB control programs. ESP is a non-radioactive, totally automated, continuously monitored system designed to detect mycobacteria. METHODS: Clinical evaluation of this new system for the rapid diagnosis of tuberculosis. During 1997 a total of 1,022 clinical sputum specimens were investigated. Specimens were processed in triplicate for ESP, BACTEC 460 TB and L?wenstein-Jensen systems. The validity, isolates of Mycobacterium tuberculosis and time required for detecting M. tuberculosis by the three systems were determined. RESULTS: The sensitivity, specificity, positive predictive and negative predictive values of the new systems were 98%, 99.8%, 98% and 99.8%, respectively. No significant differences were found between the recovery rates by the three systems. The mean time for detection was 10 days (range: 7-13 days) for specimens with positive bacilloscopy and 14 days (range: 10-28 days) for specimens with negative bacilloscopy. The difference was statistically significant between ESP and L?wenstein-Jensen, but not between ESP and BACTEC. CONCLUSIONS: The new system proved to have an excellent sensitivity and specificity, which along with its total automation renders it a system of great clinical interest for the rapid diagnosis of TB and an alternative method for radiometric systems.     

Early detection of Mycobacterium tuberculosis in BACTEC cultures by ligase chain reaction

The LCx Mycobacterium tuberculosis ligase chain reaction system (Abbott Diagnostic Division, Abbott Park, Ill.) was used to detect M. tuberculosis in 150 consecutive BACTEC vials on the day on which a positive growth index (GI) was recorded. By LCx, M. tuberculosis DNA was detected in BACTEC vials on average 2.6 days before the presence of acid-fast bacilli could be confirmed by microscopic examination. A total of 106 of 108 M. tuberculosis isolates were detected without centrifugation from bottles presenting very low GIs (average, 70; median, 33). No false-positive result was obtained from nontuberculous mycobacteria or from isolates with contaminants.     

Evaluation of a nonradiometric system (BACTEC 9000 MB) for detection of mycobacteria in human clinical samples

This study was carried out to evaluate the rate of recovery and time required for detection of mycobacteria from pulmonary and extrapulmonary human clinical samples, by using a fluorescence-quenching-based oxygen sensor (BACTEC 9000 MB; Becton Dickinson Microbiology Systems, Sparks, Md.). The results were compared with those obtained by microscopy, conventional culture in Lowenstein-Jensen (LJ) medium, and a BACTEC radiometric system (BACTEC 460 TB; Becton Dickinson). Of the 779 clinical samples processed, 364 from pulmonary sites and 415 from extrapulmonary sites, 62 (7.9%) were positive for mycobacterial isolates; of the positive samples, 59 (95.1%) were detected with the fluorescent BACTEC 9000 MB system, 57 (91.9%) were detected with the radiometric system (BACTEC 460 TB), and 43 (69.3%) were detected with LJ conventional culture. The mean times to detection of all mycobacteria with BACTEC 9000 MB and BACTEC 460 TB were similar (10.3 and 10.0 days, respectively). The results obtained indicate that the nonradiometric BACTEC (BACTEC 9000 MB) system is as efficient as Bactec 460 TB and significantly more efficient than LJ for the rapid recovery of mycobacteria from both pulmonary and extrapulmonary clinical specimens. Though the BACTEC 9000 MB system is recommended for respiratory specimens, we demonstrated that it can be successfully used also for recovery of mycobacteria from clinical specimens from various extrapulmonary sites.     

Hydraulic analog for simultaneous representation of pharmacokinetics and pharmacodynamics: application to vecuronium

A variety of different media for the cultivation of mycobacteria have been described but a few of them are in use today. Those currently used can be characterized by three basic types. The first is egg-based media represented by Ogawa and L?wenstein-Jensen. The second type is agar-based media; the most common one are Middlebrook 7H10 and 7H11. The third type is liquid media such as Middlebrook 7H9. Several weeks of incubation may be required for the isolation of M. tuberculosis on solid media. Substantial improvement in the time to detection and the recovery rate was realized by using broth-based culture system such as the BACTEC 460TB, Septi-Chek AFB, MGIT and BACTEC 9000. In the BACTEC 460TB system, the mycobacteria is detected radiometrically. The processed specimen is added to a modified 7H9 medium (BACTEC 12B) containing 14C-labeled palmitic acid and an antibiotic complex, PANTA. Mycobacterial growth can be ascertained by the liberation of 14CO2 and detected by BACTEC 460TB instrument. The Septi-Chek AFB is a biphasic medium which combines broth and solid media. The liquid medium is a modified Middlebrook 7H9 in a carbon-dioxide-enriched culture bottle. After inoculation of the sample, the bottle is capped with a slide consisting of three solid media; a non-selective Middlebrook 7H11 agar, an egg-based medium, and chocolate agar. A novel system is the MGIT, which is a nonradiometric broth method for the detection of mycobacteria from clinical specimens. The MGIT consists of a modified Middlebrook 7H9 broth and a sensor embedded in silicone on the bottom of a tube. The appearance of orange-colored fluorescence in the sensor when excited indicates the growth of mycobacteria. MB Redox is a modified, serum-supplemented Kirchner medium containing p-indonitrotetrazolium violet (INT) as an indicator of microbial growth. The INT is reduced by the redox system of the mycobacteria to deep violet-colored formazan. This substance is water insoluble and is reduced to the cell surface, by which bacterial clamps can be easily detected by their violet color. At present, the egg-based media are the first choice for the culture of clinical samples. However, there are advantages to each type of medium and not all strains of mycobacteria can be recovered on a single medium. Therefore, it is recommended that one representative of each type of medium be used for primary isolation; one example in Japan may be Ogawa egg medium in combination with Middlebrook 7H11 and MGIT.     

Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation

To explore a simple, rapid, and inexpensive way to identify Mycobacterium tuberculosis complex culture, dot blot hybridization using IS6110 as the marker was performed against 2788 known clinical isolates of mycobacteria including M. tuberculosis (n = 721), M. kansasii (177), M. marinum (10), M. avium complex (700), M. terrae complex (203), M. fortuitum (476), M. chelonae (439), and other nonpigmented Runyon's Group IV mycobacteria (62). We found that the sensitivity and specificity of the test were 94.3% and 100%, respectively. When this method was evaluated in a laboratory blind study of 1253 initially unknown clinical isolates, its sensitivity and specificity were 91.2% and 100%, respectively. Because this identification test is technically simple, rapid, and can be done in batches, together with its high sensitivity and specificity, it is a cost-effective method for routine identification of M. tuberculosis complex in laboratories of areas with a high incidence of tuberculosis.     

Safe determination of susceptibility of Mycobacterium tuberculosis to antimycobacterial agents by flow cytometry

We showed previously that susceptibility testing for Mycobacterium tuberculosis labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of M. tuberculosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.     

Evaluation of the MB/BacT system and comparison to the BACTEC 460 system and solid media for isolation of mycobacteria from clinical specimens

The MB/BacT automated system is designed for the isolation of mycobacteria from clinical specimens. It utilizes a colorimetric sensor and reflected light to continuously monitor the CO2 concentration in the culture medium. We compared its performance to that of the BACTEC 12B media for the radiometric BACTEC 460 instrument and that of solid culture media. Respiratory specimens and urine samples were decontaminated with 4% NaOH. The vials of the two instruments were inoculated with 500 microl of sample and two solid egg-based media at 200 microl each. All vials were incubated at 37 degrees C for 6 weeks. A total of 1,078 specimens (633 respiratory specimens, 78 cerebrospinal fluid specimens, 177 other body fluid specimens, 87 urine specimens, and 103 other types of specimens) were cultured in parallel. Mycobacteria could be identified from 73 (6.8%) specimens: 67 M. tuberculosis, 3 M. kansasii, 1 M. xenopi, 1 M. terrae, and 1 mixed M. avium with M. scrofulaceum. Of these, 63 (86.3%) specimens were positive with the MB/BacT system, 67 (91.8%) were positive with the BACTEC 460 instrument, and 58 (79.5%) were positive with the two egg-based media. MB/BacT cultures were positive on average after 17.5 (+/-6.4) days, BACTEC cultures with a growth index of >20 (mean, 200) were positive after 14.3 (+/-8.2) days, and egg-based media were positive after 24.2 (+/-7.5) days. Microorganisms other than mycobacteria contaminated 46 (4.3%) MB/BacT cultures and 31 (2.9%) BACTEC cultures, which had to be discarded. The MB/BacT system is a well-automated system for the detection of M. tuberculosis in clinical specimens without using radioactive reagents. Further trials are required to determine whether it is suitable for the culture of nontuberculous mycobacteria.     

Home healthcare nurses as leaders

This article presents a novel, qualitative approach for detecting airborne M. tuberculosis. Culturing or sample purification is not required. A DNA diagnostic method involving the polymerase chain reaction (PCR) coupled to an enzymatically generated color reaction was used for direct detection of M. bovis BCG (Bacillus of Calmette-Guérin), a surrogate for pathogenic M. tuberculosis. Fewer than 10 mycobacteria were detected with no culturing using this bioanalytical method. Analysis was completed in 1 to 1.5 days, in contrast to traditional culturing methods requiring a minimum of 2-3 weeks. To evaluate an air sampling method coupled to a PCR bioanalytical method, liquid cultures of the surrogate were aerosolized and collected for PCR analyses using 37-mm filter cassettes containing polytetrafluoroethylene filters. An Andersen six-stage (viable) particle sizing sampler was employed as a reference sampler. Aerosolized BCG impacted onto Andersen agar plates required incubation periods of 6-8 weeks before small colony forming units could be detected and enumerated. Although the BCG mean length of the rod-shaped particles was 8.3 microns, the airborne BCG particles were collected predominantly on the Andersen 4-6 stages, representing aerodynamic diameters 0.7 to 3.3 microns. Approximately 25 mycobacteria were detected without culturing using the PCR-filter cassette method. This approach could be used to detect airborne mycobacterial species of the M. tuberculosis complex and could permit the early detection of contaminated indoor air. Also, the efficacy of environmental controls could be evaluated and monitored. This approach could also be used to study the expulsion of infectious particles from patients and may permit risk assessment in regard to personal respiratory protection.     

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.     

Comparison of PCR, culture, and histopathology for diagnosis of tuberculous pericarditis

Nucleic acid amplification techniques for the diagnosis of tuberculosis (TB) are rapidly being developed. Scant work, however, has focused on pericardial TB. Using cryopreserved specimens from a prior study of pericarditis, we compared PCR to culture and histopathology for the diagnosis of tuberculous pericarditis in 36 specimens of pericardial fluid and 19 specimens of pericardial tissue from 20 patients. Fluid and tissue were cultured on Lowenstein-Jensen and Middlebrook solid media and in BACTEC radiometric broth. Tissue specimens were stained with hematoxylin-eosin, Ziehl-Neelsen, auramine O, and Kinyoun stains and were examined for granuloma formation and acid-fast bacilli. PCR was performed with both fluid and tissue with IS6110-based primers specific for the Mycobacterium tuberculosis complex by published methods. Sixteen of the 20 patients had tuberculous pericarditis and 4 patients had other diagnoses. TB was correctly diagnosed by culture in 15 (93%) patients, by PCR in 13 (81%) patients, and by histology in 13 of 15 (87%) patients. PCR gave one false-positive result for a patient with Staphylococcus aureus pericarditis. Considering the individual specimens as the unit of analysis, M. tuberculosis was identified by culture in 30 of 43 specimens (70%) from patients with tuberculous pericarditis and by PCR in 14 of 28 specimens (50%) from patients with tuberculous pericarditis (P > 0.15). The sensitivity of PCR was higher with tissue specimens (12 of 15; 80%) than with fluid specimens (2 of 13; 15%; P = 0.002). In conclusion, the overall accuracy of PCR approached the results of conventional methods, although PCR was much faster. Therefore, PCR merits further development in this regard. The sensitivity of PCR with pericardial fluid was poor, and false-positive results with PCR remain a concern.     

Comparative activity of azithromycin against clinical isolates of mycobacteria

Azithromycin exhibited in-vitro activity against 20 clinical isolates of Mycobacterium avium complex for which the MIC90 was 32 mg/L and 22 clinical isolates of other mycobacteria but showed no activity against 20 isolates of Mycobacterium tuberculosis (MIC90 > 128 mg/L) nor against the single isolate of Mycobacterium marinum tested (MIC 128 mg/L). These results suggest that the drug may prove useful for the prophylaxis and treatment of infections due to non-tuberculous mycobacteria, including M. avium complex in patients with AIDS.     

Assessment of use of the COBAS AMPLICOR system with BACTEC 12B cultures for rapid detection of frequently identified mycobacteria

The use of the COBAS AMPLICOR System (Roche Molecular Diagnostics, Basel, Switzerland), the only automated system for PCR testing, was evaluated for a rapid identification of mycobacteria with positive BACTEC 12B cultures. Two hundred ninety-six specimens with a growth index of >/=30 were analyzed for the presence of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare. Compared to traditional methods and provided that samples with PCR inhibition are retested at a 1:10 dilution, the sensitivity and specificity of the COBAS AMPLICOR System with BACTEC 12B cultures were 100 and 98%, respectively. The COBAS AMPLICOR method is rapid and reliable for identifying the most common mycobacteria in cultures.     

Clinical evaluation of the BDProbeTec strand displacement amplification assay for rapid diagnosis of tuberculosis

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96. 2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.     

Diminished adherence and/or ingestion of virulent Mycobacterium tuberculosis by monocyte-derived macrophages from patients with tuberculosis

The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [3H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and 3H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls.     

PHLS mycobacteriology reference services in England and Wales

Tuberculosis (TB) is the most important cause of infectious disease in the world, with eight million new cases and three million deaths each year. The increasing incidence of TB in the developed and the developing world, increasing drug resistance, and the occurrence of nosocomial outbreaks of drug sensitive as well as drug resistant TB has led the PHLS to establish TB as a priority area. This article reviews the enhanced reference services for mycobacteriology provided by the PHLS in England and Wales. These include microscopy and culture on solid and liquid media, rapid culture systems, identification of mycobacteria using macroscopic, microscopic, growth, and biochemical characteristics, and molecular DNA analysis. The Mycobacterium Reference Unit (MRU) provides rapid molecular DNA amplification techniques to identify Mycobacterium tuberculosis in specimens. All four PHLS Regional Centres test isolates for drug susceptibility. This work is quality controlled by MRU, which is one of the World Health Organisation's reference centres for global surveillance on drug resistance in tuberculosis. National data on drug resistance are collated through 'Mycobnet', a surveillance scheme run through the collaboration of PHLS and other UK reference centres and the PHLS Communicable Disease Surveillance Centre.     

Evaluation of the ESP culture system II for testing susceptibilities of Mycobacterium tuberculosis isolates to four primary antituberculous drugs

The reliability of the ESP Culture System II (herein referred to as ESP II) for testing susceptibilities of Mycobacterium tuberculosis isolates to isoniazid, rifampin, ethambutol, and streptomycin was evaluated by comparing results to those of the method of proportion (MOP), which was considered the reference method, for 20 clinical isolates and 30 challenge strains provided by the Centers for Disease Control and Prevention (CDC). Clinical isolates also were tested with the BACTEC TB 460 system; these results agreed with those obtained by the MOP for all isolates and all drugs, except the high concentration of isoniazid, for which agreement was 95%. After resolution of discrepancies, levels of agreement between ESP II and MOP for the clinical isolates were 95 and 100%, respectively, for the low and high concentrations of isoniazid, 100% for rifampin and ethambutol, and 95% for streptomycin. For the 30 challenge isolates, ESP II results for both concentrations of isoniazid agreed with the expected results in all cases, whereas agreement was 93% for both rifampin and streptomycin and 90% for ethambutol. All discrepancies with the CDC isolates were due to failure of ESP II to correctly classify resistant strains. By testing isolates yielding discrepant ethambutol and streptomycin results with a lower concentration of both drugs in the ESP II system, agreement increased to 93% for ethambutol and 100% for streptomycin. For the clinical isolates, the times to an ESP II result of susceptible (means +/- standard errors of the means) were 8.47 +/- 0.12 days (range, 7 to 10 days) and 8.73 +/- 0.29 days (range, 5 to 11 days) when the inoculum was prepared from a McFarland equivalent and from a seed bottle, respectively. The time to an ESP II result of resistant varied by drug and method of inoculum preparation, ranging from 5.50 +/- 0.22 days for ethambutol with the inoculum prepared from a McFarland standard to 8. 0 days for ethambutol with the inoculum prepared from a seed bottle. These data suggest that the ESP II system is a rapid and reliable method for testing susceptibilities of M. tuberculosis isolates to isoniazid and rifampin. Performance, however, may be suboptimal for ethambutol and streptomycin. Testing additional ethambutol-resistant and streptomycin-resistant strains with two concentrations of both drugs is necessary.     

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".