Mycobacterium terrae: case reports, literature review, and in vitro antibiotic susceptibility testing.

Mycobacterium terrae infection can cause debilitating disease that is relatively resistant to antibiotic therapy. Two cases are presented, and data from an additional 52 reports from the literature are reviewed. Tenosynovitis of the upper extremity, often following trauma, was the most commonly reported presentation (59% of cases), with pulmonary disease occurring in an additional 26% of cases. Underlying medical problems were absent (44%) or not reported (28%) in 72% of the cases. One-half of the patients with upper extremity tenosynovitis were treated with local or systemic corticosteroids, before microbiological identification. Only one-half of the patients with tenosynovitis who were followed up for 6 months had clinical improvement or were cured. The other one-half of the patients required repeated debridement, tendon extirpation, or amputation. The best antimicrobial therapy for M. terrae infection is unknown but might include a macrolide antibiotic plus ethambutol and one other effective drug for at least 12 months after clinical response. Parenteral treatment with an aminoglycoside and surgery may be useful in selected cases.

Drug susceptibility testing of Mycobacterium tuberculosis: a neglected problem at the turn of the century.

The prevailing opinion in most of the world is that drug susceptibility testing of Mycobacterium tuberculosis isolates may not be necessary, especially in countries where the diagnosis of tuberculosis is based predominantly on sputum smear examination without culture isolation, and even if it were functionally important it is too expensive to be practical. However, we believe that the spread of primary drug resistance calls for immediate action to prevent the real threat in a number of countries of an epidemic of incurable polyresistant tuberculosis. One step in this direction, among other necessary measures, is implementation of a system for timely detection of drug resistance in new patients. An overview of the currently available methods for drug susceptibility testing, and those under development, is presented in this article. Our aim is to stimulate discussion regarding where, when, and which methods can and should be implemented now, and which of them has the best potential for the future. This overview includes phenotypic approaches based on conventional cultivation and other techniques, as well as perspectives for the genotypic principle. Regardless of the methods chosen for now or to be implemented in the future, they are all laboratory methods, and we contend that dreams of a revolutionary method which can do away with the laboratory are unrealistic, at least for the time being. We argue that it is essential to create a system of direct, centralized laboratory services in order to make drug susceptibility testing reliable, practical and affordable. Such centralized laboratories would provide both more timely and trustworthy diagnosis of tuberculosis, and facilitate detection of primary drug resistance. Although such systems would require significant initial investment, we suggest that, over time, they would prove highly cost-effective for many countries.

Clarithromycin resistance and susceptibility patterns of Mycobacterium avium strains isolated during prophylaxis for disseminated infection in patients with AIDS.

A randomized, placebo-controlled trial was conducted to evaluate the efficacy of clarithromycin in the prevention of disseminated Mycobacterium avium complex (MAC) infection in patients with AIDS; special attention was given to the development of clarithromycin resistance. The median time to documented MAC bacteremia was 199 days for placebo-treated patients, 217 days for clarithromycin-treated patients infected with clarithromycin-susceptible MAC, and 385 days for clarithromycin-treated patients infected with clarithromycin-resistant MAC. Most of the patients with clarithromycin-resistant isolates (91%) had a baseline CD4 T-cell count of < 20/microL, while these low counts occurred in only 25% of patients having clarithromycin-susceptible breakthrough isolates. The emergence of clarithromycin resistance did not affect the total period of survival. Resistance to clarithromycin in breakthrough MAC isolates emerges most likely when the patient is extremely immunodeficient at the time of initiation of the preventative therapy.

Contribution of rpoB mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis.

The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the beta-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutant rpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoB mutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specific rpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

Susceptibilities of Mycobacterium malmoense determined at the growth optimum pH (pH 6.0).

SETTING: Pulmonary disease caused by Mycobacterium malmoense is increasing. Conventional in vitro antimicrobial susceptibilities correlate poorly with response to treatment for this organism. Radiometrically determined minimum inhibitory concentrations (MICs) allow quantitative susceptibility testing for non-tuberculous mycobacteria. The M. avium complex (MAC) has been investigated extensively with this approach, and clear interpretative criteria have been established at pH 6.8. However, there has been little work with the acidophilic M. malmoense, which grows poorly at pH 6.8. OBJECTIVE: To determine whether MICs at pH 6.0 provide results compatible with the interpretative criteria established for the MAC. DESIGN: MICs were performed in Middlebrook PZA medium (pH 6.0) and 7H12 medium (pH 6.8) for ten strains of M. malmoense. RESULTS: MICs can be determined at pH 6.0 for M. malmoense using the criteria adopted for the M. avium complex. CONCLUSION: The low optimal pH of M. malmoense suits this organism for growth in acid conditions. As with MAC, M. malmoense multiplies within macrophages in vivo, and MICs determined at pH 6.0 may reflect in vivo activity. The combination of radiometric MIC testing at optimal growth pH and interpretation based on pharmacokinetic parameters may be helpful in designing therapeutic regimens.

Once weekly azithromycin therapy for prevention of Mycobacterium avium complex infection in patients with AIDS: a randomized, double-blind, placebo-controlled multicenter trial.

We conducted a randomized, double-blind, placebo-controlled multicenter trial of azithromycin (1,200 mg once weekly) for the prevention of Mycobacterium avium complex (MAC) infection in patients with AIDS and a CD4 cell count of < 100/mm3. In an intent-to-treat analysis through the end of therapy plus 30 days, nine (10.6%) of 85 azithromycin recipients and 22 (24.7%) of 89 placebo recipients developed MAC infection (hazard ratio, 0.34; P = .004). There was no difference in the ranges of minimal inhibitory concentrations of either clarithromycin or azithromycin for the five breakthrough (first) MAC isolates from the azithromycin group and the 18 breakthrough MAC isolates from the placebo group. Of the 76 patients who died during the study, four (10.5%) of 38 azithromycin recipients and 12 (31.6%) of 38 placebo recipients had a MAC infection followed by death (P = .025). For deaths due to all causes, there was no difference in time to death or number of deaths between the two groups. Episodes of non-MAC bacterial infection per 100 patient years occurred in 43 azithromycin recipients and 88 placebo recipients (relative risk, 0.49; 95% confidence interval, 0.33-0.73). The most common toxic effect noted during the study was gastrointestinal, reported by 78.9% of azithromycin recipients and 27.5% of placebo recipients. Azithromycin given once weekly is safe and effective in preventing disseminated MAC infection, death due to MAC infection, and respiratory tract infections in patients with AIDS and CD4 cell counts of < 100/mm3.

Susceptibility to levofloxacin of Myocobacterium tuberculosis isolates from patients with HIV-related tuberculosis and characterization of a strain with levofloxacin monoresistance. Community Programs for Clinical Research on AIDS 019 and the AIDS Clinical Trials Group 222 Protocol Team.

OBJECTIVE: To characterize the susceptibility to levofloxacin of clinical isolates of Mycobacterium tuberculosis (MTB) obtained from patients with HIV-related tuberculosis and to characterize the molecular genetics of levofloxacin resistance. DESIGN AND METHODS: Isolates from culture-positive patients in a United States multicenter trial of HIV-related TB were tested for susceptibility to levofloxacin by minimum inhibitory concentration (MIC) determinations in Bactec 7H12 broth. Automated sequencing of the resistance determining region of gyrA was performed. RESULTS: Of the 135 baseline MTB isolates tested, 134 (99%; 95% exact binomial confidence interval, 95.9-99.9%) were susceptible to levofloxacin with an MIC < or = 1.0 microg/ml. We identified a previously unrecognized mis-sense mutation occurring at codon 88 of gyrA in a levofloxacin mono-resistant MTB isolate obtained from a patient with AIDS who had received ofloxacin for 8 months prior to the diagnosis of tuberculosis. CONCLUSIONS: Clinical MTB isolates from HIV-infected patients were generally susceptible to levofloxacin. However, the identification of a clinical isolate with mono-resistance to levofloxacin highlights the need for circumspection in the use of fluoroquinolones in the setting of potential HIV-related tuberculosis and for monitoring of rates of resistance of MTB isolates to fluoroquinolones.

Early bactericidal activity of isoniazid in pulmonary tuberculosis. Optimization of methodology. The DATRI 008 Study Group.

Early bactericidal activity (EBA) of antituberculosis drugs is the rate of decrease in the concentration of tubercle bacilli sputum during the initial days of therapy. The study reported here was designed to optimize the methodology for obtaining precise EBA measurements. The study compared the results with two versus five treatment days; overnight sputum collections with early morning collections; and quantitative smears for acid-fast bacilli (AFB) with quantitative cultures. Isoniazid (INH) was used as a model drug. Among 28 smear-positive patients enrolled in the study in five cities in the United States, 16 were evaluable (INH-susceptible tuberculosis [TB] and adequate sputum collections). The mean baseline bacterial load was 6.69 log10 cfu/ml (SE = 0.24). Quantitative culture of 10- or 12-h sputum collections obtained on two baseline days and treatment Day 5 was the optimal method for EBA measurement. The mean 5-d EBA was 0.21 log10 cfu/ml/d (SE = 0.03; p < 0.001) and the EBA appeared to be constant during the first five treatment days. On the basis of these data, multiarm studies of investigational drugs will require 25 evaluable subjects per arm to detect (80% power and two-tailed alpha of 0.05) an EBA at least 50% as large as the EBA of INH. In countries with a low incidence of TB, the usefulness of this methodology for rapidly assessing new antituberculosis agents may be limited by the relatively large number of subjects required to compare EBA values across treatment arms.

The mycobacteriology laboratory. Past, present, and future.

The role of the mycobacteriology laboratory in tuberculosis control programs has been underestimated in the past, but now laboratory services are proven to play an essential role if properly implemented. The importance and reliability of these services in the future will depend on their centralization in specialized mycobacteriology laboratories that will be able to put new technologies into practice. The shortest turnaround time and the greatest cost effectiveness can be achieved by direct submission of raw specimens to such laboratories, especially for the management of new tuberculosis patients.

Clinical mycobacteriology. Drug susceptibility testing.

An update on drug susceptibility testing is provided on methods for testing Mycobacteria tuberculosis, Mycobacteria avium, and rapidly growing mycobacteria. Emphasis is placed on the techniques that are currently available in clinical laboratories, and a critical overview is offered for some methods undergoing development.

Clarithromycin against Mycobacterium avium complex infections.

The turning point in antimicrobial therapy of Mycobacterium avium infections came with the development of two new macrolides, clarithromycin and azithromycin. Controlled clinical trials, the first ever conducted with any agent among patients with M. avium infection, indicated the high efficiency of clarithromycin, in either acquired immune deficiency syndrome (AIDS) patients having a disseminated infection or non-AIDS patients with localized pulmonary disease. Monotherapy with clarithromycin resulted in elimination of bacteremia in almost all patients with disseminated infection, which is inevitably followed by a relapse of bacteremia in patients who survived long enough to reach this event. The strains susceptible to clarithromycin isolated before therapy contained 10(-8) or 10(-9) resistant mutants, and the relapses of bacteremia were caused by multiplication of these pre-existing mutants. Clarithromycin-resistance was associated with a mutation in the 23S rRNA gene. Cross-resistance between clarithromycin and azithromycin was confirmed with laboratory mutants and clinical isolates. At least two methods for determining the susceptibility of the M. avium isolates to clarithromycin are available: one is minimum inhibitory concentration (MIC) determination on Mueller-Hinton agar (pH 7.4) supplemented with 10% Oleic acid-albumin-dextrose catalase, the other is MIC determination in 7H12 broth, also at pH 7.4. The breakpoints for 'susceptible' for these methods are < or = 8.0 micrograms/ml and < or = 2.0 micrograms/ml, respectively. The breakpoints for 'resistant' are > 128 micrograms/ml for the agar method and > 32.0 micrograms/ml for the broth method. The predictability value of MIC determination was confirmed by comparing the test results with the patients' clinical and bacteriological response to therapy. The remaining major problem in the therapy of the M. avium infections is a selection of companion drugs to be used in combination with clarithromycin (or azithromycin) to prevent the emergence of the macrolide-resistance. A number of clinical trials are now in progress to find a solution to this problem.

Clarithromycin therapy for bacteremic Mycobacterium avium complex disease. A randomized, double-blind, dose-ranging study in patients with AIDS. AIDS Clinical Trials Group Protocol 157 Study Team.

OBJECTIVE: To determine the antimicrobial activity and tolerability of clarithromycin for treating bacteremic Mycobacterium avium complex disease in patients with the acquired immunodeficiency syndrome (AIDS). DESIGN: A randomized, double-blind, dose-ranging study. SETTING: Outpatient clinics. PATIENTS: 154 patients with human immunodeficiency virus (HIV) infection and blood cultures positive for M. avium complex who had symptomatic disease. INTERVENTIONS: Random assignment to clarithromycin at dosages of 500 mg, 1000 mg, or 2000 mg twice daily for 12 weeks. MAIN OUTCOME MEASURE: Median number of colony-forming units of M. avium complex per milliliter of blood. RESULTS: Clarithromycin decreased mycobacterial CFUs from 2.7 to 2.8 log 10/mL of blood at baseline to less than 0 log 10/mL during follow-up (P < 0.0001). After 2 weeks, patients receiving 500 mg twice daily were less likely to be culture negative than were patients receiving 1000 or 2000 mg twice daily (11% compared with 33% or 29%; P = 0.08). At 6 weeks, the median number of CFUs of M. avium complex/mL of blood was 0 or 1 for all three groups. Clarithromycin-resistant isolates of M. avium complex developed in 46% of patients at a median of 16 weeks. Median survival was longer in patients assigned to 500 mg twice daily (median, 249 days) than in patients assigned to 1000 mg or 2000 mg. Death in the first 12 weeks was lowest in the 500-mg group (P = 0.007). CONCLUSIONS: Clarithromycin therapy acutely decreased M. avium complex bacteremia in patients with HIV infection by more than 99%. Clarithromycin, 500 mg twice daily, was well tolerated and associated with better survival. Emergence of clarithromycin-resistant organisms was an important problem.

[Radiometric determination of Mycobacterium avium-intracellulare and Mycobacterium xenopi sensitivity to antitubercular agents]. / Radiometrické urcování citlivosti Mycobacterium avium-intracellulare a Mycobacterium xenopi na antituberkulotika.

Using the macrodilution radiometric technique in a BACTEC 460 apparatus, the authors assessed in 25 strains of the M. avium complex and 20 strains of M. xenopi the MIC of the following chemotherapeutic agents: Ciprofloxacine, clofazimine, rifampin, cycloserine, kanamycin, etionamide, ethambutol and amikacin. In all instances wild strains isolated in the Czech Republic in 1991-1992 were involved. In strains of the M. avium complex in the majority of drugs a major predominance of slightly or completely resistant strains was found. However, in addition to resistant strains there were also sensitive strains and strains with a reduced sensitivity in the group. The highest ratio of sensitive strains was recorded in clofazimine and amikacin. Strains of M. xenopi had, as compared with avian mycobacteria, lower MIC values with the exception of cycloserine and ethambutol in all the remaining chemotherapeutic agents. The applied radiometric technique was evaluated as a suitable method for assessment of the sensitivity of mycobacteria to chemotherapeutic agents, in particular because it provides results within a short time, i.e. within 8 days after inoculation and because of the quantitative evaluation of MIC values. It was therefore recommended for centralized assessment of sensitivity in the Czech Republic.

Expedited detection of drug resistance in tuberculosis patients.

The increase in disease-causing organisms resistant to standard drug therapy has captured the attention of the medical community and the lay press. Drug resistance in rapidly growing bacteria can be detected within a short period. However, in the case of Mycobacterium tuberculosis, detection may take weeks. This paper examines the current methods available to determine drug susceptibility in M tuberculosis. These laboratory methods can be used as a model to assist the clinician in making rational decisions when managing patients with potentially resistant bacterial infections.

Antimycobacterial drugs.

This review consists of the following three sections: (1) General principles in selecting antimycobacterial drugs for the treatment regimens, (2) The antimicrobial activity in vitro with an emphasis on inhibitory and bactericidal potency of various agents, and (3) Drug susceptibility testing including methodology and interpretation of the test results. Each of these sections addresses three groups of antimycobacterial agents: (1) against tuberculosis, (2) against Mycobacterium avium complex infections, and (3) against infections caused by M. fortuitum and M chelonae. The following are the drugs examined in the sections and subsections of this review: isoniazid, ethionamide, thiacetazone, rifampin, rifabutin, rifapentine, KRM-1648, pyrazinamide, streptomycin, kanamycin, amikacin, capreomycin, gentamicin, tobramycin, ethambutol, para-aminosalicylic acid, D-cycloserine, ofloxacin, levofloxacin, ciprofloxacin, sparfloin, clofazimine, clarithromycin, azithromycin, erythromycin, cefoxitin, cefmetazole, imipenem, sulfamethoxazole, sulfisoxazole, sulfadiazine, sulfathiazole, trimethoprim, and doxycycline.

Rapid broth macrodilution method for determination of MICs for Mycobacterium avium isolates.

A multicenter study was done to investigate the accuracy and reproducibility of a method for determining the MICs of antimicrobial agents against the Mycobacterium avium complex in 7H12 broth with the BACTEC system. In phase I, with eight drugs and 10 strains, intralaboratory reproducibility was 95.7 to 100%, allowing a 1-dilution difference upon repeat testing. The results of phase II testing with 41 additional strains were consistent with those obtained in phase I, with good interlaboratory reproducibility. The radiometric method was validated by sampling and plating of the same broth cultures and determining, by the number of CFU per milliliter, the lowest drug concentration that inhibited more than 99% of the initial bacterial population. Three test concentrations of each drug and the tentative interpretation of results are proposed. Radiometric MIC determination has the potential to become the method of choice for clinical microbiology laboratories and evaluation of new agents for the treatment of M. avium infections, both pulmonary and disseminated.

Bacteriostatic and bactericidal activities of gentamicin alone and in combination with clarithromycin against Mycobacterium avium.

The inhibitory activity of gentamicin against Mycobacterium avium depended on the pH of the medium, and the broth-determined MICs for 90% of strains were 5.0 micrograms/ml at pH 7.4, 9.5 micrograms/ml at pH 6.8, and greater than 16.0 micrograms/ml at pH 5.0. The MBCs were two- to eightfold higher than the MICs. The combined effect of gentamicin and clarithromycin was additive, and the MICs and MBCs of each drug were either the same as those in the single-drug tests or reduced twofold.

Clarithromycin minimal inhibitory and bactericidal concentrations against Mycobacterium avium.

Minimal inhibitory and bactericidal concentrations (MIC and MBC) of clarithromycin were determined with 49 Mycobacterium avium strains isolated from patients with acquired immunodeficiency syndrome. The inhibitory activity depended on the pH of the medium: the drug was more active at pH 7.4 and less active at pH 5.0, with activity at pH 6.8 in an intermediate position. The broth-determined MIC found at pH 7.4 were 0.25 and 0.5 micrograms/ml for most strains. The agar-determined MIC for most strains ranged from 1.0 to 4.0 micrograms/ml. The MBC of the drug were 8- to 64-fold higher than the MIC, which indicates that the efficacy of clarithromycin can be associated with its inhibitory rather than its bactericidal activity.