BACTEC 12B Research Articles

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls.

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.

Evaluation of the LiPA MYCOBACTERIA assay for identification of mycobacterial species from BACTEC 12B bottles.

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacterium species and differentiates M. tuberculosis complex, M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonae group, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive for M. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive for Mycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

Evaluation of a fluorescence hybridisation assay using peptide nucleic acid probes for identification and differentiation of tuberculous and non-tuberculous mycobacteria in liquid cultures.

The performance was evaluated of a fluorescence in situ hybridisation assay using peptide nucleic acid probes (Dako Probe MTB Culture Confirmation Test; Dako, Denmark) for identification of Mycobacterium tuberculosis complex (MTC) organisms and differentiation between tuberculous and non-tuberculous mycobacteria (NTM) in material taken directly from Bactec 12B (Becton Dickinson, USA) and MB/BacT (Organon Teknika, USA) bottles. The test was applied to 129 smear-positive (Ziehl-Neelsen stain) clinical specimens, 48 previously identified clinical strains of mycobacteria (12 MTC and 36 NTM), and 51 reference strains (7 MTC and 44 NTM) which were all previously inoculated into Bactec 12B and MB/BacT bottles. The sensitivity and specificity of the assay for MTC-positive cultures was 87.6% and 100%, respectively, for Bactec 12B, and 100%, respectively, for MB/BacT. The sensitivity and specificity of the assay for NTM-positive cultures was 100% for both media.

Comparison of Recoveries of Mycobacterium tuberculosis Using the Automated BACTEC MGIT 960 System, the BACTEC 460 TB System, and Löwenstein-Jensen Medium

Using two different liquid media and one conventional solid medium, a total of 57 mycobacterial isolates (Mycobacterium tuberculosis, n = 55; nontuberculous mycobacteria, n = 2) were recovered from 377 clinical specimens. The rates of recovery of M. tuberculosis were 96.4% with the BACTEC MGIT 960 liquid medium, 92.7% with BACTEC 12B liquid medium, and 81.8% with the Löwenstein-Jensen (LJ) medium. The mean time to detection of M. tuberculosis in smear-positive specimens was 12.6 days for BACTEC MGIT 960 medium, 13.8 days for BACTEC 12B medium, and 20.1 days for LJ medium, and in smear-negative specimens it was 15.8 days for BACTEC MGIT 960 medium, 17.7 days for BACTEC 12B medium, and 42.2 days for LJ medium. The rates of contamination were 3.7, 2.9, and 1.2% for the BACTEC MGIT 960, BACTEC 12B, and LJ media, respectively. In conclusion, the nonradiometric, fully automated 7-ml BACTEC MGIT 960 system can be considered a viable alternative to the semiautomated, radiometric BACTEC 460 TB system.

Enhanced Amplified Mycobacterium Tuberculosis Direct Test for detection of Mycobacterium tuberculosis complex in positive BACTEC 12B broth cultures of respiratory specimens.

The reliability of the Gen-Probe enhanced Amplified Mycobacterium Tuberculosis Direct Test (MTD) for identification of Mycobacterium tuberculosis complex (MTBC) in BACTEC 12B broth cultures of respiratory specimens was evaluated by testing aliquots from 268 bottles with a growth index of >/=50. MTD results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen-Probe, Inc.) testing sediment from broth samples or colonies on a solid medium. For the first 134 cultures, from which 68 mycobacterial isolates (including 27 MTBC isolates) were recovered, both fresh and frozen aliquots were tested. MTD results for the frozen aliquots agreed with the identification by usual protocol in all cases, whereas there was one false-negative MTD result with fresh aliquots. For the remaining 134 cultures, only frozen aliquots were tested. Of the total 268 broth cultures (from 210 patients) evaluated, 137 (51.1%) grew mycobacteria, including 60 MTBC isolates. All 60 isolates were MTD positive, as was one additional culture that grew Mycobacterium gordonae. The latter culture was from a patient who was diagnosed with tuberculosis a few months earlier and was on therapy; therefore, the MTD result was considered a true positive. Sensitivity, specificity, and positive and negative predictive values of MTD were 100%. The mean times from specimen receipt to identification of MTBC were 15 (+/-1) days (range, 4 to 27 days) for BACTEC plus MTD and 19 (+/-1) days (range, 6 to 36 days) for the usual protocol (P < 0.001). These data indicate that the MTD is a rapid, reliable method for identification of MTBC in fresh or frozen aliquots of broth from positive BACTEC 12B cultures of respiratory specimens.

Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep.

Definitive diagnosis of Johne's disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium avium subsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. avium subsp. paratuberculosis from sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. avium subsp. paratuberculosis from sheep with Johne's disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne's disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the "gold standard" test for detection of ovine Johne's disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. avium subsp. paratuberculosis. The sensitivity of detection of M. avium subsp. paratuberculosis on solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. avium subsp. paratuberculosis in both liquid and solid media.

A Complication of BCG Vaccine: A Case of Localized Cutaneous Abscess due toMycobacterium bovis

The attenuated bacille Calmette-Guérin (BCG) vaccine is administered to prevent tuberculosis. Complications of vaccination are uncommon. A case of cutaneous abscess due to BCG is presented in a 24-year-old woman. The abscess developed at the inoculation site four weeks after vaccination. Routine Gram stain and bacterial cultures of the pus were negative. The auramine stain was positive. Mycobacterial cultures were positive after 14 and 18 days, using the BACTEC 12B bottle and Löwenstein-Jensen media, respectively. The mycobacteria were identified as Mycobacterium bovis, vaccinal strain by high-performance liquid chromatography and DNA probe assays.

Cord formation in BACTEC medium is a reliable, rapid method for presumptive identification of Mycobacterium tuberculosis complex.

Serpentine cord formation in BACTEC 12B medium was evaluated as a rapid method for the presumptive identification of M. tuberculosis complex. Kinyoun acid-fast stained smears were prepared from 666 positive BACTEC 12B bottles and examined for the presence or absence of serpentine cording. Cord formation had a sensitivity, specificity, positive predictive value, and negative predictive value of 89.2, 99.2, 98.5, and 94.2%, respectively. The evaluation of the presence of cord formation in BACTEC 12B medium is reliable and permits the rapid presumptive reporting of M. tuberculosis.

Comparison of algorithms for selective use of nucleic-acid probes for identification of Mycobacterium tuberculosis from BACTEC 12B bottles

We retrospectively compared the sensitivity of two approaches, a time-to-detection algorithm and the presence of serpentine cords of acid-fast bacilli, for discriminating between BACTEC 12B cultures containing either Mycobacterium tuberculosis complex (MTB) or Mycobacterium avium complex (MAC). From January 1996 through March 1997 a total of 217 of 2089 respiratory specimens received in our laboratory were positive in the BACTEC 12B radiometric culture system for either MTB (120 specimens) or MAC (97 specimens). Use of a previously published time-to-positivity algorithm would have resulted in the correct use of the MTB probe on 109 of 120 cultures (91% sensitivity), and the MAC probe on 52 of 97 cultures (54% sensitivity). The presence of serpentine cords was detected in 58 of 120 cultures containing MTB (48%), and in 3 of 97 (3%) cultures containing MAC. Using a combination of time to positivity and cord formation to determine initial probe selection would have resulted in first use of the MTB probe in 116 of 120 (97%) instances in which MTB was present in the culture. In only 49 of 97 (51%) cultures, however, from which MAC was recovered would the correct probe have been selected. These results indicate that limiting the initial use of the MTB probe to those cultures that are either identified by the time-to-detection algorithm or demonstrate serpentine cords on acid-fast smear would eliminate a considerable amount of unnecessary probe use without compromising the efficiency of identification of isolates of MTB.

Rapid diagnosis and strain differentiation of Mycobacterium bovis in radiometric culture by spoligotyping

An assessment of spoligotyping for rapid detection and strain typing of Mycobacterium bovis isolates in radiometric culture was made. Spoligotyping was applied to BACTEC 12B broth cultures of 54 lesioned bovine lymph node specimens from 44 herds in Northern Ireland. A nucleic acid sequence capture technique was performed on BACTEC cultures at growth index points of approximately (∼) 60, ∼200, and 999. Definitive spoligotype patterns were obtained for 90.4% and 94.2% of all 52 BACTEC culture-positives at growth indexes ∼60 and ∼200, respectively. Within 10 days, definitive spoligotype patterns were obtained for 84.6% of the culture-positives. This technique, therefore, allowed earlier and more accurate diagnosis of M. bovis than traditional methodologies, as well as simultaneous strain differentiation. Application of this molecular tool to BACTEC cultures would be a significant advance in bovine tuberculosis eradication programmes. Seven distinct spoligotype patterns were identified in this study, 2 of which (ST21 and ST25), had not been identified previously in cattle from Northern Ireland. Two spoligotype patterns (ST1 and ST2) accounted for 80.7% of the culture-positives. These were found to have widespread geographic distribution, whereas 1 spoligotype pattern (ST14) had limited geographical distribution.

Laboratory media for the cultivation of tubercle bacillus

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 Löwenstein-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.

Evaluation of the MycoAKT Latex Agglutination Test for Rapid Diagnosis of Mycobacterium avium Complex Infections

Rapid diagnosis of Mycobacterium avium complex (MAC) bacteremia is important for management of patients with the acquired immunodeficiency syndrome who have disseminated MAC. The purpose of this study was to determine the reliability of the MycoAKT latex agglutination test for direct detection of MAC in positive mycobacterial blood cultures. First, colonies of isolates of previously identified mycobacteria, including 35 MAC, were tested. Of the 55 isolates evaluated, 33 were identified as MAC by the latex test, including 31 of the known MAC and 2 M. chelonae (sensitivity, 88.6%; specificity, 90.0%). Second, broth from 20 ESP II and 20 BACTEC 12B bottles seeded with isolates of MAC were tested. Aliquots from 19 (95%) ESP II cultures and 16 (80%) 12B cultures were positive by the latex test. In phase 3, broth from 115 signal-positive ESP II blood cultures were tested by latex agglutination. Forty-three subcultures from these bottles grew mycobacteria (41 MAC and 2 Mycobacterium tuberculosis complex); the remainder grew no organisms. Broth from 40 of the blood cultures (39 that grew MAC and 1 from which no organisms were recovered) were latex positive; thus, the sensitivity, specificity, and positive and negative predictive values of the latex test for direct identification of MAC in ESP II blood cultures were 95.1, 98.6, 97.5, and 97.3%, respectively. The mean time to detection of MAC was 14.6 days (range, 6–34 days) with the direct latex test, compared with 18.3 days (range, 9–36 days) with subculture and probe (p < 0.05).

Detection and identification of mycobacteria by amplification of RNA and DNA in pretreated blood and bone marrow aspirates by a simple lysis method.

A sodium dodecyl (lauryl) sulfate method was evaluated for the preparation of blood specimens and bone marrow aspirates for use in two amplification procedures (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTDT] and Roche Amplicor M. avium/M. intracellulare [MAI] Test) for the detection and identification of Mycobacterium tuberculosis and M. avium and M. intracellulare, respectively. The AMTDT is based on amplification of rRNA, whereas the Amplicor MAI Test amplifies a specific DNA region of the 16S rRNA gene. The results of amplification techniques were compared with those of standard culture and culture in BACTEC 13A and BACTEC 12B liquid media. A total of 121 blood specimens and 15 bone marrow aspirates were collected from 136 AIDS patients. Mycobacterial growth was recovered for 103 specimens; 35 yielded M. tuberculosis, 62 yielded M. avium, 5 yielded M. genavense, and 1 yielded M. kansasii. The values of sensitivity and specificity in pretreated specimens for detection of M. tuberculosis by the AMTDT were 94.3 and 100%, respectively, and those for detection of M. avium by the Amplicor MAI Test were 91.9 and 100%, respectively. The simple lysis method described in the present work allows the recovery of mycobacteria from blood specimens and bone marrow aspirates and may be used in combination with the AMTDT and the Amplicor MAI Test to detect and identify different members of the genus Mycobacterium. This method might also be applicable for the identification of mycobacteria from blood culture fluids with acridinium-ester-labeled DNA probes.

Radiometric culture of Mycobacterium avium paratuberculosis from the feces of tule elk.

To determine if Mycobacterium avium paratuberculosis has persisted in tule elk (Cervus elaphus nannodes) at Point Reyes National Seashore (California, USA), 100 fresh fecal samples were collected. Feces were cultured on a modified BACTEC 12B radiometric medium for detection of M. avium paratuberculosis. Four samples, coming from two separate groups of elk tested positive for M. avium paratuberculosis. Thus, a noninvasive technique was used to document the continued presence of M. avium paratuberculosis in elk at Point Reyes National Seashore. These findings document persistence of this organism for a period of at least 13 yr in a free ranging herd of elk, with a 6 yr absence of observed clinical signs.

Comparison of BACTEC 12B vs solid media for the recovery of Mycobacterium avium complex from blood cultures in AIDS patients

We compared liquid (BACTEC 12B) and solid culture media for the diagnosis of Mycobacterium avian complex (MAC) bacteremia among 258 AIDS patients with a positive blood culture. Neither culture media alone had adequate sensitivity; BACTEC 12B detected growth earlier. Use of both liquid and solid media may improve the yield of mycobacterial blood culture.

Detection of Mycobacterium tuberculosis in BACTEC 12B broth cultures by the Roche Amplicor PCR assay.

To evaluate the ability of the Amplicor MTB Assay to detect Mycobacterium tuberculosis complex (MTBC) organisms in BACTEC 12B broth cultures, 249 cultures with a growth index (GI) of > or = 20 from 160 patients were tested retrospectively. Specimens were processed by standard methods, and then BACTEC 12B vials and Middlebrook 7H11/7H115 plates were inoculated, incubated, and interpreted in accordance with the manufacturer's instructions and laboratory protocol. From 12B vials with a GI of > or = 20, and aliquot of broth was removed and frozen at -20 degrees C until assayed by PCR. PCR results were compared to those obtained by the usual laboratory protocol, whereby MTBC organisms were identified by a DNA probe assay performed on broth from 12B vials with a GI or > or = 300 or on colonies from solid medium. Of the 249 broth cultures evaluated, 142 contained mycobacteria, including 44 that contained MTBC organisms. Of these 44 cultures, 41 were PCR positive; the 3 that were PCR negative were blood specimens collected in an Isolator tube. All 98 cultures with nontuberculous mycobacteria and the 107 that did not contain mycobacteria were PCR negative. Thus, the sensitivity and specificity of PCR were 93 and 100%, respectively. For those culture sin which MTBC organisms were identified by both the DNA probe and PCR assays, the mean time from specimen inoculation to detection and identification of MTBC organisms was 16 (range, 4 to 26) days for the PCR and 28 (range, 13 to 43) days for the DNA probe assay (P < 0.0001). In summary, PCR is a rapid, reliable method for detection of MTBC organisms in BACTEC 12B broth cultures with a GI of > or = 20.

Isolator component responsible for inhibition of Mycobacterium avium-M. intracellulare in BACTEC 12B medium.

We previously reported an inhibitory effect on Mycobacterium avium-M. intracellulare (MAI) when blood collected and processed with the Isolator system was placed in BACTEC 12B bottles for radiometric monitoring. We sought to identify the specific component(s) of the Isolator lysis-anticoagulant reagent (LAR) responsible for the inhibitory effect. We added the three components of the LAR, saponin, polyanetholesulfonate, and polypropylene glycol (PPG), to triplicate sets of BACTEC bottles separately and in various combinations. These bottles were then seeded with 10(2) CFU of MAI. The growth index (GI) was observed over a 42-day period and was compared with the GI for a growth control bottle set with no reagents and with the GI for a bottle set containing an equivalent volume of LAR. Growth in all growth control bottles and those bottles containing no PPG reached the maximum GI (GI = 999) within 8 to 10 days. Growth in bottles containing PPG never reached the maximum GI before day 14. In addition, the GIs of one bottle containing all three reagent components as well as all of the bottles containing actual Isolator LAR failed to reach the maximum within 42 days. Our data suggest that PPG is the main cause of the inhibitory effect, but that a secondary synergistic interaction between all three of the reagents may also be present.

Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media.

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics.

Drug-susceptibility pattern of mycobacterium tuberculosis among pulmonary tuberculosis patients in Riyadh, Saudi Arabia

To identify the pattern of drug-susceptibility of newly diagnosed pulmonary tuberculosis patients in Riyadh, we conducted a study on all Mycobacterium tuberculosis positive-culture patients admitted to Sahari Chest Hospital from January 1994 to April 1995. Demographic data, antituberculous therapy and drug-susceptibility testing results of each patient were reviewed from patients' hospital records. The samples were cultured on Lowenstein-Jensen media and drug susceptibility was tested by Bactec 12B (Middlebrook 7H12) media against selected antituberculous drugs Drug susceptibility was performed on 362 (91%) of the sputum positive-cultures. The overall initial resistance rate (1 or more drugs) was 12.4%. Initial. resistance was more common with a single drug (9.4%), followed by two drugs (2.3%) and then three drugs (0.3%). Resistance to isoniazid was most common (10.4%), followed by streptomycin (2.7%), rifampicin (1.9%) and ethambutol (0.6%). Single isoniazid resistance was 60%, followed by two drugs: streptomycin and isoniazid (13.3%). Resistance to multiple drugs is not yet a significant problem in Riyadh. A continuous monitoring of drug resistance is important for planning and assessing the national TB control program. Timely and complete reporting is essential to identify the problem as and when it begins.

Use of a PCR method based on IS6110 polymorphism for typing Mycobacterium tuberculosis strains from BACTEC cultures.

Two PCR typing methods, based on polymorphism of the insertion sequence IS6110, were compared with Mycobacterium tuberculosis strains by using a single primer complementary to the inverted repeats of IS6110. Total M. tuberculosis DNA either was amplified directly (IS6110-PCR) or was amplified following digestion and ligation (IS6110-inverse-PCR). Both PCR techniques showed a similar degree of discrimination. Because of its simplicity, IS6110-PCR was chosen to confirm that a single M. tuberculosis strain was responsible for an outbreak of tuberculosis in a secondary school. IS6110-PCR was used to study the degree of differentiation in 85 clinical M. tuberculosis isolates from BACTEC 12B broth cultures. Results were consistent with those of the standardized IS6110 restriction fragment length polymorphism (RFLP) analysis method, showing identical PCR types for identical RFLPs, although the degree of discrimination was greater by RFLP analysis. The study concludes that due to its simplicity, IS6110-PCR is a good screening method when quick differentiation between M. tuberculosis strains is needed because BACTEC cultures may be used directly.