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Carbon utilization tests have proven to be useful for the identification of some species of rapidly growing mycobacteria and have been described as one of the few tests useful for the differentiation of Mycobacterium mucogenicum from other rapid growers. We have found the carbon utilization tests to be unreliable for the identification of patient isolates of this species. In this study, using 28 isolates of rapidly growing mycobacteria, we examined several variables which might have an effect on results of citrate, inositol, and mannitol utilization: inoculum concentration, incubation temperature, and medium manufacturer. None of these variables affected results obtained for most species of rapid growers or for ATCC strains of M. mucogenicum. Results for patient isolates of M. mucogenicum were found to be inconsistent regardless of the methodology employed and resulted in an ambiguous identification of these isolates or an incorrect identification as Mycobacterium chelonae. Molecular or cell wall analysis may be the best technique to employ for accurate identification of M. mucogenicum.

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Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.

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The sodium chloride tolerance test is often used in the identification of rapidly growing mycobacteria, particularly for distinguishing between Mycobacterium abscessus and Mycobacterium chelonae. This test, however, is frequently unreliable for the identification of some species. In this study we examined the following variables: medium manufacturer, inoculum concentration, and atmosphere and temperature of incubation. Results show that reliability is improved if the test and control slants are inoculated with an organism suspension spectrophotometrically equal to a 1 McFarland standard. Slants should be incubated at 35 degrees C in ambient air and checked weekly for 4 weeks. Growth on control slants should be critically evaluated to determine the adequacy of the inoculum; colonies should number greater than 50. Salt-containing media should be examined carefully to detect pinpoint or tiny colonies, and colonies should number greater than 50 for a positive reaction. Concurrent use of a citrate slant may be helpful for distinguishing between M. abscessus and M. chelonae. Molecular methodologies are probably the most reliable means for the identification of rapidly growing mycobacteria and should be used, if possible, when unequivocal species identification is of particular importance.

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The use of antiretroviral agents and drugs for the treatment and prophylaxis of opportunistic infections has lengthened the survival of persons with AIDS. In the era of multidrug therapy, drug interactions are important considerations in designing effective and tolerable regimens. Clarithromycin has had a significant impact on the treatment of disseminated Mycobacterium avium complex infection, and zidovudine is the best-studied and one of the most widely used antiretroviral agents in this population. We conducted a study to determine the maximally tolerated dose of clarithromycin and the pharmacokinetics of clarithromycin and zidovudine individually and in combination. Mixing studies were conducted to simulate potential interaction in the gastric environment. The simultaneous administration of zidovudine and clarithromycin had little impact on the pharmacokinetics of clarithromycin or of its major metabolite. However, coadministration of zidovudine and clarithromycin at three doses (500 mg orally [p.o.] twice daily [b.i.d.], 1,000 mg p.o. b.i.d., and 2,000 mg p.o. b.i.d.) reduced the maximum concentration of zidovudine by 41% (P < 0.005) and the area under the concentration-time curve from 0 to 4 h for zidovudine by 25% (P < 0.05) and increased the time to maximum concentration of zidovudine by 84% (P < 0.05), compared with zidovudine administered alone. Mixing studies did not detect the formation of insoluble complexes due to chelation, suggesting that the decrease in zidovudine concentrations results from some other mechanism. Simultaneous administration of zidovudine and clarithromycin appears to decrease the levels of zidovudine in serum, and it may be advisable that these drugs not be given at the same time. Drug interactions should be carefully evaluated in persons with advanced human immunodeficiency virus infection who are receiving multiple pharmacologic agents.

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Mycobacterium kansasii isolates from two patients showed relatively slow growth in BACTEC 12B medium (12B) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) compared with the more rapid growth of these isolates on solid media. This finding prompted an evaluation of the effect of PANTA (Becton Dickinson) on the growth rate of these isolates. Suspensions of one isolate from each of these two patients (A and B), six additional isolates from six other patients (C through H), and one M. kansasii American Type Culture Collection isolate were inoculated into 12B with PANTA, 12B with reconstituting fluid only, and Middlebrook 7H11 agar plates (Remel, Lenexa, Kans.). For the isolates from patients A and B, the average times to detection for 12B with PANTA, 12B with reconstituting fluid, and Middlebrook 7H11 agar were 12.3, 7.4, and 9.0 days, respectively. For the remaining six patient isolates and the American Type Culture Collection strain, the average times to detection for these media were 9.2, 8.1, and 9.6 days. Susceptibility tests performed with the isolates from patients A and B with the individual component antibiotics of PANTA and testing of four of the other isolates with nalidixic acid alone suggested that nalidixic acid exerts some degree of inhibition on the growth of M. kansasii. The eight patient isolates were also inoculated onto Lowenstein Jensen medium (Remel) and onto a variety of selective mycobacterial media containing nalidixic acid and other antimicrobial agents. All isolates showed some degree of inhibition on at least one of these selective media.

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The DAWN Model B laser light scattering instrument (Wyatt Technology Corporation, Santa Barbara, Calif.) was evaluated to assess its potential to provide rapid mycobacterial antimicrobial susceptibility test results. For Mycobacterium tuberculosis there was a clear separation between susceptible and resistant results with the isolates tested, and there was excellent correlation with reference laboratory results. For Mycobacterium avium there was no obvious breakpoint between susceptible and resistant results with the isolates tested, and correlation with reference laboratory results was less good than for M. tuberculosis. However, for M. avium there was also less agreement among reference laboratory results than for M. tuberculosis. Significant instrument design and software program changes would be required for the instrument to become a useful tool for mycobacterial susceptibility testing in the diagnostic laboratory.

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OBJECTIVE: To determine the safety, tolerance, pharmacokinetics, and antimycobacterial activity of orally administered clarithromycin in children with acquired immunodeficiency syndrome and disseminated Mycobacterium avium complex (MAC) infection. DESIGN: Phase I study with a 10-day pharmacokinetic phase followed by a 12-week continuation therapy phase. PATIENTS: Twenty-five patients with a median age of 8.3 years were enrolled. Ten were receiving zidovudine and 13 were receiving didanosine at the time of enrollment. INTERVENTION: Clarithromycin suspension was administered to each patient at one of three dose levels: 3.75, 7.5, and 15 mg/kg per dose every 12 hours. Clarithromycin and antiretroviral pharmacokinetics were measured during single-drug and concurrent-drug administration. Clinical and laboratory monitoring was performed biweekly. MEASUREMENTS AND MAIN RESULTS: Clarithromycin was well tolerated at all dose levels. Plasma clarithromycin concentrations increased proportionately with increasing doses, and significant pharmacokinetic interactions were not observed during concurrent administration with zidovudine or didanosine. Decreases in mycobacterial load in blood were observed only at the highest clarithromycin dose level. Decreased susceptibility to clarithromycin developed rapidly (within 12 to 16 weeks) in the majority of MAC strains isolated from study patients.

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Recent developments in diagnostic mycobacteriology are surveyed with emphasis on laboratory capabilities relevant to the more rapid and accurate detection and identification of mycobacterial pathogens. Some terminologic problems are reviewed, and some newly recognized mycobacterial pathogens are discussed. Newer methodologies presented in some detail include the radiometric technique for detection, identification, and susceptibility testing of mycobacteria, and the use of DNA probes for identification of mycobacterial species and species complexes. The utility of other developing methodologies, such as polymerase chain reaction technology, analysis of body fluids for tuberculostearic acid, and the use of ELISA are also assessed.

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As a result of several episodes of inter-bottle transfer of molds and mycobacteria in our BACTEC 460 TB System (Johnston Laboratories, Towson, MD), we designed some experiments to attempt to reproduce the transfer process. We demonstrated that organism transfer could occur with our instrument during routine use without any indication of instrument malfunction. Utilizing a redesigned A1B4 circuit board that extends the time of the needle heating cycle to 85 sec and increases the needle heater maximum temperature, we were not able to effect organism transfer even from bottles with very high growth indices. We also demonstrated that even with the redesigned A1B4 circuit board, the needles were not heated sufficiently to sterilize them after direct insertion into a mycobacterial suspension. Organism transfer also occurred while using a second redesigned A1B4 circuit board which extends the needle heating cycle to 75 sec. Users should be aware that organism transfer can occur with the BACTEC 460 under certain circumstances. Maintenance and testing recommendations from Johnston Laboratories should be scrupulously followed, and we suggest some additional procedures that might further reduce the possibility of organism transfer.

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Three methods of concentrating 1 ml aliquots from BACTEC 13A bottles containing patient blood samples were evaluated for testing with the Gen-Probe Rapid Diagnostic System for Mycobacteria avium complex: 1. using no reagents, 2. using both lysing and wash reagents; and 3. using lysing reagent only. Aliquots from 13As containing human blood and seeded with eight mycobacterial species were also concentrated directly and using both reagents. Results for samples containing M. avium were as follows: 1. using the direct concentration technique 34 of 47 samples (72%) gave unequivocally positive results; 2. 43 of 47 samples (92%) concentrated using both reagents gave positive results; 3. the technique using lysing reagent only was not found useful. There were no false positives with any of the seeded specimens. We were also able to define the minimum Growth Index necessary to ensure un-equivocally positive results for each concentration technique. For those samples containing M. avium these values were 42 for the technique using both reagents and 86 for the direct technique. Direct or reagent concentration of 13A aliquots for testing with Gen-Probe DNA probes provides a rapid, sensitive, and specific means for the identification of M. avium complex bacteremia.

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Simultaneous infection with Mycobacterium avium and Mycobacterium intracellulare in an AIDS patient was suspected after direct analysis of two BACTEC 13A blood cultures with the Gen-Probe kit for M. avium complex. A mixed infection was confirmed by evaluating isolated colonies. The Gen-Probe kit may provide a simple technique for detecting mixed M. avium-M. intracellulare infections.

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BACTEC 13A medium (Johnston Laboratories, Towson, Md.) was compared with Isolator (Du Pont Co., Wilmington, Del.) concentrate for sensitivity, speed, and technical ease of isolation of mycobacteria from paired patient blood samples. Of 72 positive cultures, 63 were positive by both systems. Five positive cultures were detected by BACTEC 13A medium alone, and four were detected by Isolator alone. The median numbers of days to positivity were 12 for BACTEC 13A medium and 14 for Isolator concentrate. BACTEC 13A medium has an advantage over the Isolator in requiring less laboratory manipulation of the specimen but has the disadvantages of not providing isolated colonies or quantitation of organisms. Some technical problems with contamination in both systems are also discussed.

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Group A streptococci and enterococci can be differentiated from other streptococci by their ability to cleave pyrrolidonyl beta-naphthylamide (PYRase). We evaluated two PYRase systems [Strep-A-Chek (SAC), E-Y Laboratories, San Mateo, CA; Strep-A-Fluor (SAF), BioSpec, Inc., Dublin, CA) for the presumptive identification of enterococci. Initially, 40 enterococcal and 21 nonenterococcal streptococci were tested, retrospectively. A prospective comparison of SAC and SAF to bile-esculin reaction (BE) was then incorporated into our routine procedure for the identification of non-beta-hemolytic streptococcal colonies from cultures. All isolates were speciated using standard biochemical tests. We encountered 85 enterococcal and 26 nonenterococcal isolates. Tests were performed on colonies from primary plates whenever possible (77 isolates). Sensitivity and specificity of both SAC and SAF were greater than 96% in identifying enterococci from routine cultures. These PYRase tests were cost-effective, easily adaptable to work-flow, and yielded results within 30 min. Thus, PYRase testing appears to be a reasonable alternative for the identification of enterococci in the clinical laboratory.

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Group A streptococci can be rapidly identified by the Strep-A-Fluor system (Bio-Spec Inc., Dublin, CA), which uses a specific aminopeptidase to hydrolize L-pyrrolidonyl-beta-naphthylamide. In pure subcultured colonies and in isolated colonies from primary culture plates, the Strep-A-Fluor system had 100% sensitivity and 100% specificity compared with the results of Streptex latex agglutination (Wellcome Research Laboratories, Research Triangle Park, NC). However, in mixed culture from primary culture plates, the sensitivity and specificity were 73% and 95%, respectively. Furthermore, in a routine laboratory setting, the sensitivity was only 91% both from pure subcultured colonies and from isolated colonies from primary culture plates. Contamination with organism other than group A streptococci that are capable of hydrolyzing L-pyrrolidonyl-beta-naphthylamide may explain the low specificity in mixed culture. Deterioration of the buffer reagent due to prolonged use may account for the low sensitivity in a routine laboratory setting.

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