Growth on blood agar discriminates Mycobacterium avium and Mycobacterium intracellulare.

Mycobacterium intracellulare and Mycobacterium avium (MAC) require specialized culture and identification procedures. To simplify the diagnosis, we inoculated reference strains, and 85 M. avium and 12 M. intracellulare clinical isolates, on egg-based and sheep blood agar. After 5 days of culture, there were significantly more colonies on sheep blood than on egg-based agar for M. avium (ratio: 250.5 +/- 209) but not for M. intracellulare (ratio: 0.44 +/-0.11). Using a ratio > or = 20, the sensitivity of the identification of an MAC isolate as M. avium was 97.65%, the specificity was 100%, and the positive predictive value was 100%. Differential growth on egg-based and blood agar is an aid to the identification of MAC isolates.

Blood agar and Mycobacterium tuberculosis: the end of a dogma.

Incidental blood agar-based recovery of Mycobacterium tuberculosis led us to further investigate this routine medium for primary isolation and culture of M. tuberculosis. Fifteen respiratory tract and eight lymph node Ziehl-Neelsen-positive specimens were inoculated in parallel into tubes containing egg-based medium and 5% sheep blood agar. Colonies appeared sooner on this medium than on the egg-based medium, but this difference was not significant (P = 0.11, analysis of variance [ANOVA] test). Further experiments compared the growth of 38 respiratory and lymph node M. tuberculosis isolates when subcultured on the two media. After 6 days of incubation, 21 of 38 isolates had grown on blood agar, and the mean number of colonies was significantly greater on blood agar than on the egg-based medium (P < 0 0.001, ANOVA test). These results demonstrate that M. tuberculosis grows easily on blood agar within 1to 2 weeks, indicating that this basic medium is suitable for laboratory diagnosis of tuberculosis in addition to other media. Laboratories that routinely use prolonged incubations of blood plates, for example, for the recovery of Bartonella species, should consider the potential safety implications of encountering this highly infectious pathogen.

Isolation of blood-borne Mycobacterium avium by using the nonradioactive BACTEC 9000 MB system and comparison with a solid-culture system.

We conducted a 12-month prospective study comparing two approaches to the detection of Mycobacterium avium in the blood of human immunodeficiency virus type 1-infected patients, namely, a lytic centrifugation system combined with Middlebrook solid culture medium (the conventional procedure) and the nonradiometric BACTEC 9000 MB system. Species identification relied on 16S rRNA probe hybridization and cell wall fatty acids chromatography. M. avium was isolated in 17 of 345 (5%) blood specimens by the BACTEC 9000 MB automated system and in 14 of 345 (4%) blood specimens by the conventional procedure (nonsignificant, chi2 test). Detection time was 16 +/- 6 days by the BACTEC 9000 MB automated system and 27 +/- 3 days by the conventional procedure (P < 0.001, Student t test). Non-M. avium mycobacteria were not recovered during the study period. Contamination rate was 8% (30 specimens) by the BACTEC 9000 MB system and 0% by the conventional procedure, indicating the necessity of using an antibiotic mixture (PANTA, consisting of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin). Working time was 1 min 30 s by the BACTEC 9000 MB system and 8 min by the conventional procedure, which was 1.8 times more expensive than the BACTEC system. Use of the BACTEC 9000 MB system increased the sensitivity of M. avium detection and reduced detection time in blood culture.

[Use of the Bactec TB 460 method for the bacteriological diagnosis of tuberculosis. Results of a multicenter study]. / Utilisation de la méthode Bactec TB460 pour le diagnostic bactériologique de la tuberculose. Résultats d'une étude multicentrique.

Isolation of Mycobacteria on Loewenstein-Jensen medium lasts many weeks. The use of Radiometric method (Bactec TB 460) reduces the delays. Results of 79,064 cultures are reported from a multicentric study associating 16 laboratories. The average was 4.8% of positivity and 2.51% of contamination. The comparison of the results with conventional method previously obtained shows that radiometric method is more sensitive and contaminations are less numerous. Concerning hemocultures the Bactec method is very usefull. Among 11,277 tests performed 907 were positive (8.04%). Mycobacterium avium was identified in 89% of the cases. Identification test utilizes Biochemical and NAP tests, but also more and more Nucleic probes. The antibiotic sensitivity is performed in five days. The mean delay of analysis is about 25 days, lessening by half the conventional method delays. Nevertheless, Bactec method has the following inconveniences: syringe inoculation, use of radiolabelled products, expensive cost.

[Extra and intracellular activity of dirithromycin against Mycobacterium avium]. / Activité extra- et intra-cellulaire de la dirithromycine vis-à-vis de Mycobacterium avium.

The in vitro activity of dirithromycin alone and in combination with clofazimin, ethambutol and rifabutin was tested against thirty strains of Mycobacterium avium isolated from patients. Extracellular activity of dirithromycin was assessed by determining MICs using the radiometric methodology in 7H12 broth at two pHs 6.8 and 7.4. The MICs obtained at pH 7.4 were 3 to 4 more dilutions lower than those obtained at pH 6.8. Activity of pairs of antibiotics was measured using the FIC indices. Dirithromycin-clofazimin combination demonstrated the most important additive effects and even produced synergic effect against 5 of 30 strains. Studies of intracellular bacteria showed that the most effective bactericidal combination was dirithromycin, clofazimin and ethambutol together.

French multicenter study involving eight test sites for radiometric determination of activities of 10 antimicrobial agents against Mycobacterium avium complex.

The radiometric BACTEC 460-TB methodology has filled an increased need in the screening of a wide range of antimicrobial agents against Mycobacterium avium (MAC) isolates on a patient-to-patient basis. In this context, a multicenter study involving eight test sites across France was performed to determine the MICs of 10 antimicrobial agents for MAC organisms. The aim of the investigation was to compare the in vitro activities of D-cycloserine, ethambutol, ethionamide, rifampin, amikacin, streptomycin, ciprofloxacin, sparfloxacin, clofazimine, and clarithromycin against MAC isolates. All of the test sites received the same clinical isolates of MAC, and the MICs were determined by a common protocol. The overall interlaboratory reproducibility of the MICs within +/- 1 dilution of the modal MICs varied from 79.70 to 100% (mean, 95.2% +/- 2.1%), whereas overall agreement of the MICs among the test sites varied from a mean of 91% +/- 4.1% to a mean of 98 +/- 1.3%. We confirmed that the proposed methodology is easy, accurate, and sufficiently reproducible to be used routinely in a clinical laboratory. Despite variations in the MICs of the same drug among strains, no link between the origin of MAC isolates (from human immunodeficiency virus-positive or -negative patients) and their drug susceptibilities was established. On the basis of the MICs that inhibited 50 and 90% of isolates tested for the drugs used, clarithromycin, clofazimine, ethambutol, and streptomycin were the most uniformly active against MAC; this was followed by amikacin, rifampin, and sparfloxacin. On the other hand, ciprofloxacin, D-cycloserine, and ethionamide showed only marginal in vitro activities.

[Evaluation of the extra- and intracellular activity of clarithromycin against Mycobacterium chelonae]. / Evaluation de l'activité extra et intra-cellulaire de la clarithromycine vis-à-vis de Mycobacterium chelonae.

The in vitro activity of clarithromycin alone and in combination with amikacin, ethambutol and rifabutin was tested against twelve strains of M. chelonae abscessus and eight strains of M. chelonae chelonae isolated from patients. Extracellular activity of clarithromycin was assessed by determining MICs using the 1 p. cent proportion method in Middlebrook 7H11 agar media compared to the radiometric methodology in 7H12 broth at two pHs 6.8 and 7.4. The MICs obtained at pH 7.4 were 2 to 4 more dilutions lower than those obtained at pH 6.8. By both methods, clarithromycin appeared more active against isolates of M. chelonae chelonae than against isolates of M. chelonae abscessus. Clarithromycin- amikacin combination demonstrated the most important additive effect. The use of three drugs in association resulted in syngergistic effect. Studies of intracellular bacteria showed that the most effective bactericidal combination was clarithromycin amikacin and ethambutol together.

In-vitro evaluation of clarithromycin, temafloxacin, and ethambutol in combination against Mycobacterium avium complex.

The in-vitro activity of clarithromycin, temafloxacin, and ethambutol were assessed by MIC, FIC and intra-macrophage killing determinations alone and in various combinations against ten pigmented and ten non-pigmented strains of Mycobacterium avium complex bacteria. Alone, either clarithromycin or temafloxacin were found to be active against M. avium, but the best results were obtained from combinations of drugs. Clarithromycin and temafloxacin together were found to have an additive effect against two of ten pigmented variants and one of ten non-pigmented variants. Clarithromycin and ethambutol demonstrated a synergistic effect against two pigmented and one non-pigmented strain, and an additive effect against five and three pigmented and non-pigmented strains, respectively. For temafloxacin and ethambutol, additive effects were observed in four and two pigmented and non-pigmented strains, respectively. In cultures of macrophages both clarithromycin and temafloxacin alone reduced the numbers of bacteria growing intracellularly after six days, but the most effective bactericidal combination was clarithromycin, temafloxacin, and ethambutol together.

Detection and identification of Mycobacterium tuberculosis, M. bovis/BCG, and M. avium by two-step polymerase chain reaction. Comparison with ELISA using A60 antigen.

We propose a rapid two-step PCR to amplify a 767-bp sequence present in the gene coding for the 65-kD antigen of mycobacteria. The high G+C content (80%) permitted annealing to occur at 70 degrees C, enhancing the specificity. The amplified fragment contains a restriction site for differentiation between M. tuberculosis, M. bovis/BCG, and M. avium. Complete diagnosis can be achieved in less than four hours without labelled probe or nucleic acid transfer.

Serological response of tuberculosis patients to antigen 60 of BCG.

Two ELISA tests (IgG and IgM) for the serodiagnosis of tuberculosis, both based on antigen 60 (A60) of M. bovis BCG, were applied to 1,644 controls and patients to analyse the immune response in different forms of this infectious disease. Out of 200 healthy individuals, 148 being tuberculin--positive BCG-vaccinated adults, only 10 contacts--nurses of the pneumology department and laboratory technicians of the mycobacterial laboratory--were found positive for anti-A60 IgG. One quarter of hospitalized patients affected by non-tuberculous pneumopathies (194 in total) were found weakly positive for anti-A60 IgG. We suppose that these positive cases have suffered from inapperant infections and are in a "persistent state". Out of 344 cases of primary pulmonary tuberculosis, 88% were positive for anti-A60 IgG and 75% for the corresponding IgM. Among 97 cases of primary extra-pulmonary tuberculosis, 94% were found IgG positive and 33% IgM positive. The difference between active and inactive post-primary (chronic) tuberculosis was striking: about 100% of both pulmonary and extra-pulmonary cases (367 altogether) had high titers of anti-A60 IgG but IgM positivity was observed in only 15% of the cases, whereas in inactive and quiescent noncavitary tuberculosis (442 cases), 57% of the patients were weakly positive for anti-A60 IgG and none were positive for IgM. Kinetics of synthesis of anti-A60 IgG and IgM were analysed in primary and post-primary (chronic) active tuberculosis. The IgM tracing immune response to A60 was shorter and lower during primary tuberculosis as compared to post-primary tuberculosis. Our findings point to the high prognostic value of the A60- ELISA test for tuberculosis. Anti-A60 IgM mark initial stages of the disease or reactivation processes whereas anti-A60 IgG last longer than IgM and provide an evaluation of the intensity of the infectious process. Repeated serological tests allow monitoring of the course of the infection and the efficacy of therapy. The test is negative in healthy BCG-vaccinated persons (tuberculin-positive) and healed tuberculous infection cases. The combined use of both IgG and IgM tests helps in the correct diagnosis of "false positive" cases.

[Extracellular and intracellular activity of sparfloxacin against Mycobacterium avium complex and Mycobacterium xenopi]. / Activité extracellulaire et intracellulaire de la sparfloxacine vis-à-vis du complexe Mycobacterium avium et de Mycobacterium xenopi.

Activity of the new fluoroquinolone sparfloxacin against 30 strains of M. avium complex and 25 strains of M. xenopi was tested in vitro. Sparfloxacin was used alone (determination of MICs and MBCs) and in combination with ethambutol and rifabutin. Synergy studies with determination of the FIC and FBC indices showed that the sparfloxacin-ethambutol combination was synergistic against 10 M. avium complex strains and 12 M. xenopi strains. With the three-drug combination (sparfloxacin-ethambutol-rifabutin), synergy was found against 12 M. avium and 14 M. xenopi strains. Studies of intracellular bacteria showed that the decrease in viable bacteria with the three-drug combination was 1 log for M. avium and 2 log for M. xenopi.

[Identification of Mycobacterium tuberculosis and Mycobacterium avium by non-radioactive probes: evaluation of the Snap Syngene system]. / Identification de Mycobacterium tuberculosis et des mycobactéries aviaires par sondes non radioactives: évaluation du système Snap Syngène.

We evaluated an alkaline phosphatase-labeled oligonucleotide probe for the rapid identification of Mycobacterium tuberculosis and mycobacteria belonging to the M avium and M intracellulare complex (MAIS). Sixty-two strains of mycobacteria and eight strains belonging to related genera were studied. All M tuberculosis strains hybridized with the tuberculosis probe. All M avium and M intracellulare gave a strong signal with their probes. However the 3 M xenopi strains tested hybridized with all probes for MAIS complex.

[Activity of antibiotics against pigmented and unpigmented variants of Mycobacterium avium-intracellulare]. / Action des antibiotiques sur des variants pigmentés ou non pigmentés de mycobacterium avium-intracellulare.

Efficacy of various antibiotics: amikacin, rifampicin, rifabutin, ciprofloxacin, temafloxacin, erythromycin and clarithromycin was evaluated against pigmented and unpigmented variants of Mycobacterium avium complex isolated from patients with acquired immunodeficiency syndrome. The minimal inhibitory concentrations of antibiotics against unpigmented variants were multiplied by 2 to 16 compared to those of pigmented variants. Observations with intracellularly growing bacteria showed that unpigmented variants were more resistant to antibiotics than variants with a difference of approximately 0.25 a 0.3 log 10 in decrease of viable or bacterial counts. The combinations of temafloxacin or ciprofloxacin with rifabutin and amikacin were most effective against unpigmented variants.

[Intra-macrophagic activity of antibiotics combinations against Mycobacterium marinum]. / Mesure de l'activité intra-macrophagique d'associations d'antibiotiques vis-à-vis de Mycobacterium marinum.

Fourteen strains of Mycobacterium marinum were isolated from patients with granulomatous skin lesion. All the strains were resistant to isoniazid and pyrazinamid and five resistant to rifampicin. The following antibiotics are used alone or in combination: ethambutol, rifabutin, ciprofloxacin, temafloxacin, erythromycin, clarithromycin, minocyclin. Minimal inhibitory concentrations are evaluated using agar dilution method. After what, activity of antibiotics against Mycobacterium marinum strains within human macrophages in investigated. After treating the monolayers at 24 and 96 h, a bactericidal effect is observed with rifabutin, ciprofloxacin, temafloxacin, clarithromycin and minocyclin. Combinations of ciprofloxacin, temafloxacin or clarithromycin with ethambutol and rifabutin produced synergistic effect.

Value of ELISA using A60 antigen in the diagnosis of active pulmonary tuberculosis.

This investigation was undertaken to assess the effectiveness of an enzyme-linked immunosorbent assay (ELISA) using A60 antigen in ascertaining diagnosis in hospitalized patients suspected to have pulmonary tuberculosis (TB) but with negative sputum stains. Cultures were performed to confirm active or inactive disease. IgG and IgM antibody activity was determined by adding a 1:100 dilution of serum to plates coated with A60 antigen. After addition of peroxidase-conjugated antihuman IgG or IgM and color development, optical density (OD) was determined. A total of 83 patients was studied, taking into account their current disease status and prior history. Using as a cutoff value the mean value +/- 2 SD measured in the negative culture, no TB history group, that is, OD = 0.50 for IgG measurements and 0.43 for IgM measurements, the sensitivity, specificity, and positive predictive value of IgG measurements were equal to 48, 71, and 50%, respectively. Using IgM measurements, these parameters were equal to 76, 98, and 95%, respectively. Combining the results of IgG and IgM measurements, sensitivity, specificity, and positive predictive value were equal to 68, 100, and 100%, respectively. Thus, the ELISA described here can greatly facilitate the diagnosis of TB in patients with negative smears.

[Activity of azithromycin and roxithromycin alone or in combination against Mycobacterium avium and Mycobacterium xenopi]. / Etude de l'activité de l'azithromycine et de la roxithromycine seules et en associations vis-à-vis de Mycobacterium avium et de Mycobacterium xenopi.

The effect of two new macrolides, azithromycin and roxithromycin used either alone or in combination with amikacin rifabutine and 1.25 (HO)2 vitamin D3 was examined in vitro. Macrophage monolayers infected with M. avium complex or M. xenopi were treated with antibiotics or 1.25 (OH)2 vitamin D3 by using different protocols: antibiotics or 1.25 (OH)2 vitamin D3 was added to the macrophage monolayers immediately after infection and released by washing out after 24 h; antibiotics or 1.25 (OH)2 vitamin D3 was replenished daily for 4 days; or infected macrophage monolayers were treated with antibiotics plus vitamin D3 for 4 consecutive days. Treatment for 24 h resulted in an inhibition of growth. After treating the macrophage monolayers with the four antibiotics alone or in combination for 4 consecutive days intracellular killing of mycobacteria was observed and the combinations were significantly more lethal than antibiotics alone. The mycobactericidal effect was enhanced when vitamin D3 was added to the culture.

Heat and moisture exchangers and vaporizing humidifiers in the intensive care unit.

A prospective, randomized, controlled study was undertaken to compare the Pall Ultipor breathing circuit filter (PUBCF), a heat-and-moisture exchanger, and heated hot water systems (HHWSs) in ICU patients submitted to controlled mechanical ventilation. Humidification of inspired gas and bacterial contamination of breathing circuits were evaluated. During the study, there were six episodes of tracheostomy tube (TT) occlusion in six patients included in the PUBCF group. No patient out of 42 included in the HHWS group experienced this complication (p less than 0.01). There were 4 percent of days with thick and tenacious bronchial secretions in the PUBCF group and no case in the HHWS group (p less than 0.02). In the PUBCF group, 23 percent of days with hypothermia were noted as opposed to 12 percent in the HHWS group (p less than 0.01). Fewer breathing circuits were found to be contaminated in the PUBCF group (11 percent) than in the HHWS group (54 percent, p less than 0.01). In patients with an organism growing in bronchial specimens, the same organism was found to contaminate the breathing circuit in 10 percent of cases in the PUBCF group and 77 percent of cases in the HHWS (p less than 0.01). We conclude that, in the conditions of this study, the PUBCF did not provide sufficient humidification of inspired gas in ICU patients. Protection against contamination of breathing circuits was effective, but 10 percent of patients remained at risk for this complication.