HIV infection and primary resistance to antituberculosis drugs in Abidjan, Côte d'Ivoire.

OBJECTIVE: To determine the prevalence of Mycobacterium tuberculosis resistance to antituberculosis drugs, and to relate this resistance to HIV serologic status. DESIGN: Cross-sectional prevalence study. SETTING: The two major outpatient tuberculosis clinics in Abidjan, Côte d'Ivoire, West Africa. PATIENTS: Sixty individuals with newly diagnosed pulmonary tuberculosis and sputum smears positive for acid-fast bacilli. MAIN OUTCOME MEASURES: HIV serologic status and in vitro testing for susceptibility of M. tuberculosis isolates to antituberculosis drugs. RESULTS: M. tuberculosis was isolated from 82% (49 out of 60) of sputum specimens. Thirty-five per cent (17 out of 49) were obtained from HIV-seropositive and 65% (32 out of 49) from HIV-seronegative patients. There was no statistically significant difference in the proportion of resistant isolates from HIV-seropositive versus HIV-seronegative patients, although the relatively small sample size limited power. Of the total number of isolates, 17% were resistant to isoniazid; resistance was less to streptomycin (7%), rifampin (2%), pyrazinamide (0%), and ethambutol (0%). Eighteen and 21% of mycobacterial isolates from HIV-seropositive and HIV-seronegative individuals, respectively, were resistant to one or more of these drugs. CONCLUSIONS: Surveys of this type are useful in planning and evaluating tuberculosis preventive therapy in individuals with dual infection.

A nosocomial pseudo-outbreak of Mycobacterium xenopi due to a contaminated potable water supply: lessons in prevention.

OBJECTIVES: To determine risk factors for Mycobacterium xenopi isolation in patients following a pseudo-outbreak of infection with the organism. DESIGN: Retrospective cohort analysis of mycobacteriology laboratory specimen records and frequency-matched case-control study of hospital patients. SETTING: General community hospital. PATIENTS: For the case-control study, 13 case patients and 39 randomly selected controls with mycobacterial cultures negative for M xenopi, frequency matched by specimen source, whose specimens were submitted from June 1990 through June 1991. RESULTS: Between June 1990 and June 1991, M xenopi was isolated from 13 clinical specimens processed at a midwestern hospital, including sputum (n = 6), bronchial washings (2), urine (4), and stool (1). None of the patients with M xenopi-positive specimens had apparent mycobacterial disease, although five received antituberculosis drug therapy for a range of one to six months. Specimens collected in a nonsterile manner were more likely to grow the organism than those collected aseptically (3.1% versus 0, relative risk = infinity, P = 0.003). M xenopi isolation was attributed to exposure of clinical specimens to tap water, including rinsing of bronchoscopes with tap water after disinfection, irrigation with tap water during colonoscopy, gargling with tap water before sputum collection, and collecting urine in recently rinsed bedpans. M xenopi was isolated from tap water in 20 of 24 patient rooms tested, the endoscopy suite, and the central hot water mixing tank, but not from water in the microbiology laboratory. The pseudo-outbreak occurred following a decrease in the hot water temperature from 130 degrees F to 120 degrees F in 1989. CONCLUSIONS: Maintenance of a higher water temperature and improved specimen collection protocols and instrument disinfection procedures probably would have prevented this pseudo-outbreak.

Evaluation of Syngene DNA-DNA probe assays for the identification of the Mycobacterium tuberculosis complex and the Mycobacterium avium complex.

Two hundred mycobacterial cultures were used to evaluate two alkaline-phosphatase-labeled DNA probe (SNAP) kits developed by Syngene (San Diego, CA) for identification of Mycobacterium tuberculosis complex and M. avium complex. The M. tuberculosis complex SNAP probe, when compared with standard biochemical identification tests, gave results that were in agreement at 100% sensitivity and 98.7% specificity. Ninety-nine M. avium complex strains that were previously tested by the Gen-Probe M. avium complex probe assays and mycolic acid analysis were included to evaluate the M. avium complex SNAP assay which contained three probes, A (avium), I (intracellulare), and X. Eight strains identified as members of the M. avium complex by biochemical tests did not react with the three SNAP probes. These strains were also negative by the Gen-Probe assays. However, 23 strains identified as M. avium complex by biochemical tests and mycolic acid analysis and negative with the Gen-Probe assays gave positive results with the X probe and negative results with the A and I probes of the SNAP assay.

Differential identification of Mycobacterium kansasii and Mycobacterium marinum.

This report deals with the differential diagnosis between Mycobacterium marinum and M. kansasii. We found that the two species could be differentiated by using six main tests, namely, the nitrate reduction test, the arylsulfatase test, the ability to grow in the presence of 10.0 mug of amithiazone per ml, the ability to grow in the presence of 5.0 mug of kanamycin per ml, the temperature-ratio test, and the rate of growth on solid medium. In contrast to M. kansasii, considerable variation was observed among strains of M. marinum. However, the evidence obtained was not considered sufficient to justify the conclusion that more than one species was represented among the strains identified as M. marinum.

Identification of clinically significant Mycobacterium fortuitum complex isolates.

Recent outbreaks of nosocomial infections caused by organisms identified as the Mycobacterium fortuitum complex suggest that species and subspecies identification is epidemiologically important. In a study of 170 strains, M. fortuitum was differentiated from M. chelonei by nitrate reduction and iron uptake. M. fortuitum was further divided into biovariant fortuitum, biovar peregrinum, and an unnamed third biovar by inositol and mannitol utilization. M. chelonei was further divided into subsp. chelonei, subsp. abscessus, and an unnamed subspecies by tolerance to 5% sodium chloride, utilization of mannitol and sodium citrate, and uptake of iron.

Rapidly growing mycobacteria: testing of susceptibility to 34 antimicrobial agents by broth microdilution.

A total of 18 strains of Mycobacterium fortuitum, 15 strains of M. chelonei, and 31 strains of M. chelonei-like organisms were tested by both broth microdilution and agar dilution to determine their susceptibility to 34 antimicrobial agents. All strains grew well enough in cation-supplemented Mueller-Hinton broth for endpoints to be read after 72 h of incubation. Some strains of M. chelonei did not grow on Mueller-Hinton agar. A few discrepancies were noted between the broth and agar procedures. For M. fortuitum, doxycycline, minocycline, amikacin, sulfamethoxazole, and sulfamethoxazole-trimethoprim were the most active agents. For M. chelonei, amikacin, sisomicin, tobramycin, and erythromycin were the most active agents. The M. chelonei-like organisms were most susceptible to ampicillin, doxycycline, minocycline, amikacin, erythromycin, sulfamethoxazole, and sulfamethoxazole-trimethoprim. Broth microdilution appears to be a reliable method for susceptibility testing of rapidly growing mycobacteria, although clinical studies are needed to determine how well in vitro results correlate with therapeutic in vivo outcome.

Pulmonary disease due to Mycobacterium malmoense.

A case of pulmonary disease due to Mycobacterium malmoense was recently diagnosed in a 43-year-old man from Virginia. This organism was isolated from sputum and bronchial washings. This is the first case of documented human infection due to this organism in the United States

Human disease due to Mycobacterium smegmatis.

Mycobacterium smegmatis is a rapidly growing environmental species not considered a human pathogen. We identified 22 human isolates of M. smegmatis from Australia and the southern United States: 19 were from skin or soft-tissue infections, and none were from urine or the male genital tract. These isolates closely resembled Mycobacterium fortuitum, except for a negative three-day arylsulfatase test; growth at 43-45 C; a low semiquantitative catalase test; and, in 50% of isolates, a late-developing, yellow-to-orange pigment. The isolates were biochemically identical to four reference strains and the type strain of M. smegmatis. Isolates were resistant to isoniazid and rifampin but susceptible to ethambutol, doxycycline, sulfamethoxazole, ciprofloxacin, imipenem, and amikacin. Eleven patients treated on the basis of in vitro susceptibility tests responded well to therapy. The similarity of M. smegmatis to M. fortuitum and the failure to recognize that the former is an environmental species may have contributed to previous failures to recognize it as a human pathogen.

Heterogeneity among isolates of rapidly growing mycobacteria responsible for infections following augmentation mammaplasty despite case clustering in Texas and other southern coastal states.

Thirty-seven cases of rapidly growing mycobacterial wound infections following augmentation mammaplasty were identified between 1979 and 1988. The infections were usually unilateral and had a narrow geographic distribution: almost 60% were from Texas and 92% from five southern coastal states. In Texas a seasonal incidence was observed; 45% of all previously reported and current patients had undergone mammaplasty in April, May, or June. Although these findings suggested a possible common source, analysis of 35 available isolates revealed 19 different phenotype patterns. Five different taxonomic groups were represented, although most isolates (70%) were Mycobacterium fortuitum biovar fortuitum. Plasmid bands were identified in 10 of 15 strains studied, with nine different profiles. An additional 11 cases of breast infection due to rapidly growing mycobacteria not associated with augmentation were also identified, of which nine came from the same states that contributed mammaplasty cases. Rapidly growing mycobacterial infections of the breast are endemic in Texas and other southern coastal states, and the heterogeneity of the isolates suggests that most cases are unrelated.

Differential identification of Mycobaterium szulgai and other scotochromogenic mycobateria.

Strains of scotochromogenic mycobacteria were studied by using numerical taxonomy methods in an attempt to more clearly define Mycobacterium szulgai and to find tests useful in identifying the species. In this study all strains of M. szulgai were strong reducers of nitrate, were slow in hydrolyzing Tween 80, and gave a high semiquantitative catalase reaction. Results obtained indicate that the use of increased pigmentation after 1 h of light exposure at 25 C and that the use of arylsulfatase activity are of questionable diagnostic value in separating the species from other scotochrompgenic mycobacteria.

Treatment of nonpulmonary infections due to Mycobacterium fortuitum and Mycobacterium chelonei on the basis of in vitro susceptibilities.

One hundred twenty-three patients with nonpulmonary infections due to Mycobacterium fortuitum or Mycobacterium chelonei were treated by wound debridement and with chemotherapy on the basis of in vitro susceptibilities of the organism. Of 76 patients with infections caused by M. fortuitum, 13 required no therapy or were adequately treated with surgery alone. Patients with active localized disease received single drug therapy (usually with a sulfonamide) for a mean period of 10.6 weeks for cellulitis and seven months for osteomyelitis. Patients with extensive disease received amikacin or amikacin plus cefoxitin (mean, four weeks) followed by a sulfonamide (mean, six months). The 47 patients with infections caused by M. chelonei received no therapy or were treated with surgery alone (6); with amikacin (10), erythromycin (6), doxycycline (3), or cefoxitin (1); or with amikacin plus cefoxitin followed by cefoxitin alone for a total of 10-12 weeks (20); or other multiple-drug regimens (1). Surgery was performed on 74 (60%) patients. Schlichter tests or serum drug levels were determined for 81 (66%) patients. Response to therapy was excellent; 68 (90%) infections with M. fortuitum and 34 (72%) with M. chelonei were successfully treated. Cultures became negative within six weeks of chemotherapy, except for sternal osteomyelitis, for which cultures were not negative until up to 14 weeks. Follow-up for a mean period of 12 months following therapy was possible in 80% of cases. Relapses were rare except in patients with disseminated disease, and drug resistance developed in only one patient. These studies demonstrate the value of routine susceptibility testing of these mycobacterial species and the benefit of chemotherapy on the basis of in vitro susceptibilities.

Bacteremia caused by a previously unidentified species of rapidly growing Mycobacterium successfully treated with vancomycin.

Bacteremia caused by rapidly growing mycobacteria are usually due to Mycobacterium fortuitum or M. chelonei. Other rapidly growing mycobacteria generally are considered to be nonpathogenic. We report the case of a patient with bacteremia due to an unidentified, rapidly growing, scotochromogenic mycobacteria that was detected by a radiometric blood culture system. Results of in-vitro susceptibility testing indicated that the organism was susceptible to vancomycin and other antimicrobial agents, and the patient was successfully treated with vancomycin. We believe that this is the first report of successful use of vancomycin therapy for a mycobacterial infection.

Disk diffusion testing with polymyxin and amikacin for differentiation of Mycobacterium fortuitum and Mycobacterium chelonei.

Disk diffusion is one method of susceptibility testing of the Mycobacterium fortuitum complex to antibacterial agents. We utilized disks of polymyxin B (300 U), amikacin, and kanamycin to determine whether they could also be used for species identification when compared with standard biochemical methods. With the polymyxin disk, 100% of 75 M. fortuitum strains produced zones of inhibition, whereas none (0%) of 58 Mycobacterium chelonei subspecies abscessus and chelonei strains had any zone of inhibition. With the amikacin disk, 99% of M. fortuitum biovariant fortuitum had zones of greater than or equal to 30 mm compared with 6% of M. chelonei. The rare M. chelonei-like organisms gave variable results, and 42% of the unnamed "third group" biovariant of M. fortuitum exhibited an unusual but diagnostic pattern of small zone sizes to amikacin and no zone to kanamycin. The kanamycin disk was otherwise not helpful, although it resulted in larger zone sizes for M. chelonei than did amikacin. Thus, disk diffusion susceptibilities which include these disks (especially polymyxin) will provide presumptive evidence of species as well as susceptibility data.

Antimicrobial susceptibility of five subgroups of Mycobacterium fortuitum and Mycobacterium chelonae.

Broth microdilution MICs were determined for 258 clinical isolates of Mycobacterium fortuitum (3 biovariants) and M. chelonae (2 subspecies) with amikacin, tobramycin, cefoxitin, doxycycline, erythromycin, and sulfamethoxazole-trimethoprim and with several new beta-lactams and aminoglycosides and ciprofloxacin. Variations in susceptibility by and within species subgroups confirm the need for susceptibility testing against clinically important strains.

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.

Susceptibility of mycobacteria to rifampin.

The Mycobacterium species M. tuberculosis, M. avium-intracellulare, M. kansasii, M. marinum, M. scrofulaceum, M. fortuitum, M. terrae, and M. gordonae were analyzed for their susceptibility to rifampin. M. tuberculosis, M. kansasii, and M. marinum were susceptible to the antibiotic, and resistant populations developed as a result of the interplay of mutation and selection. The mutation rates (susceptibility --> resistance) were calculated to be 4.9 x 10(-10) and 1.7 x 10(-9) mutations per bacterium per generation in, respectively, M. kansasii and M. marinum. M. fortuitum was found to be naturally resistant to the antibiotic, whereas the nature of resistance in the other species was unclear and is discussed.

Preparation of a stable mycobacterial tween hydrolysis test substrate.

A Tween 80 hydrolysis test substrate was found to be stable at room temperature for at least 6 months.

Spectrum of disease due to rapidly growing mycobacteria.

One hundred twenty-five cases of disease due to rapidly growing mycobacteria were observed over a four-year period. Cutaneous infections accounted for 74 cases (59%). Of these, 40 followed surgical procedures (especially augmentation mammaplasty or median sternotomy), and 34 were due to accidental penetrating trauma. Among the 24 patients with pulmonary disease, the mean age was approximately 60 years, the majority of patients (63%) were women, and most had unilateral noncavitary disease. Other infections included disseminated disease with multiple nodular skin lesions and positive blood cultures, cervical lymphadenitis, keratitis, and endocarditis associated with a prosthetic valve. Infected tissues showed mixed acute and granulomatous inflammation; acid-fast bacilli, when present, occurred in extracellular clumps within microabscesses. Mycobacterium fortuitum and Mycobacterium chelonei were encountered with approximately equal frequency; 80% of isolates of M. chelonei were subspecies abscessus, and 83% of isolates of M. fortuitum were biovariant fortuitum. The outcome in these infections was generally good, although 9% of the patients, including all those with endocarditis, died. Infections due to M. fortuitum and M. chelonei are probably markedly under-diagnosed, and these organisms are capable of causing a wide spectrum of clinical disease.

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.