Molecular investigation of resistance to the antituberculous drug ethionamide in multidrug-resistant clinical isolates of Mycobacterium tuberculosis.

Ethionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active against Mycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded by ndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETH(r)) isolates, 24 ETH-susceptible (ETH(s)) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETH(Sip); defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETH(r) isolates, we found mutations in ethA, ethR, or inhA or its promoter, which mostly corresponded to new alterations in ethA and ethR. The 9 ETH(r) isolates without a mutation in these three genes (9/47, 19%) had no mutation in ndh, and a single isolate had a mutation in mshA. Of the 16 ETH(Sip) isolates, 7 had a mutation in ethA, 8 had no detectable mutation, and 1 had a mutation in mshA. Finally, of the 24 ETH(s) isolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in the inhA promoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETH(r) isolates, highlighting the complexity of the mechanisms of ETH resistance in M. tuberculosis.

Outbreak of tuberculosis in a migrants' shelter, Paris, France, 2002.

SETTING: An overcrowded 362-bed migrants' shelter in Paris, France. OBJECTIVES: To investigate an outbreak of tuberculosis (TB), to identify a common source of contamination and to prevent further transmission. METHODS: The outbreak was identified by radiographic screening and an active search for undeclared hospital treated cases, completed by strain phenotyping and a search for contact cases. RESULTS: Between October 2001 and October 2002, 56 cases of active TB were identified, 30 by radiological screening and 20 by contacting neighbouring hospitals. All cases involved men, with a median age of 30 years. Pulmonary involvement was present in 54% of cases, and nine patients were sputum smear-positive. Thirty-four of the 37 phenotyped strains clustered together. CONCLUSION: The grouping of the cases in time and place, the large number of cases with early-stage disease and the identical RFLP banding patterns of most of the isolates indicate that this outbreak results from transmission that occurred in France. This report underlines the need for public health departments in industrialised countries to maintain effective anti-tuberculosis control programmes.

Clarithromycin in the treatment of Mycobacterium avium lung infections in patients without AIDS. Clarithromycin Study Group of France.

Mycobacterium avium complex infections, common in patients with AIDS as either pulmonary or disseminated disease, are infrequent in patients without AIDS. Participants were 45 HIV-negative patients with lung disease and positive sputum cultures for M avium; 10 had documented immunocompromise, and 24 had preexisting lung disease. Clarithromycin dosage was 500 to 2,000 mg daily (mean +/- SD = 1,633 +/- 432 mg). The drug was administered either alone (n = 14) or in combination with rifampin (n = 8), aminoglycoside (n = 1), quinolone (n = 10), clofazimine (n = 18), isoniazid (n = 5), ethambutol (n = 9), pyrazinamide (n = 1), or minocycline (n = 6). At 3 months, 36 patients among 39 bacteriologically assessed had negative sputum cultures, 3 had positive culture, 3 were dead, and 3 discontinued treatment. At the end of treatment, 32 patients remained negative, 7 were positive. The success rate was 15 of 22 (64%) in patients previously treated with antimycobacterial drugs for M avium disease and 17 of 23 (74%) in new patients. Adverse effects included mild hearing loss (n = 4), increase in liver enzyme levels (n = 5), and gastrointestinal pain (n = 10, two of whom had to stop treatment). Patients stopped treatment after 300 +/- 186 days due to side effects (3), death (4), or the patient's (5) or physician's decision (33). During the follow-up, one patient suffered a relapse with peripheral lymph nodes. A daily dose of 30 mg/kg of clarithromycin in the treatment of M avium infections appears to be effective and safe. Concomitant drug therapy should be assessed for its ability to prevent relapse.

Mycobacterial lung disease in cystic fibrosis: a prospective study.

BACKGROUND: Patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual organisms, such as mycobacteria. The aim of the study was to determine the incidence and clinical picture of mycobacterial infection in CF children. METHODS: At least 2 acid-fast bacillus (AFB) smears and mycobacterial cultures were performed on a prospective basis on 682 sputum specimens from 106 patients during a 1-year period. RESULTS: Thirty-three percent of the cultures were contaminated with other bacteria. Seven children had at least one sputum culture positive for one mycobacterium. Five children had only one positive AFB culture. Their clinical status and lung function remained stable during follow-up. Two teenagers with severe lung disease had several positive AFB smears and cultures for Mycobacterium chelonae and Mycobacterium abscessus. The isolation of M. chelonae and M. abscessus was associated with a clinical and functional decline. Clarithromycin treatment resulted in temporary improvement with the disappearance of the mycobacteria after 6 months of treatment. This prospective study shows an incidence of 2.3% for positive cultures. The prevalence was 6.6% for mycobacterial colonization but only 1.9% for mycobacterial lung disease in our pediatric population. CONCLUSIONS: We recommend performing AFB smears and cultures in CF children with severe lung disease and/or during a lung exacerbation. In these patients persistence of M. chelonae or M. abscessus in sputum should lead to consideration of treatment with clarithromycin.

Iron and Mycobacterium tuberculosis infection.

BACKGROUND: iron is known to play a role in the susceptibility to and outcome of several infections. In view of the increasing worldwide problem of tuberculosis, it may be important to ascertain whether this is also the case with this infection. OBJECTIVES: (1) to review studies conducted in vitro, in experimental animals, and in humans that provide evidence that iron status may influence the occurrence and outcome of tuberculosis. (2) To perform an in vivo study in mice, examining the effect of iron loading on experimental infection caused by a virulent strain of Mycobacterium tuberculosis. RESULTS: we studied the effect of iron loading on the growth in spleen and lungs of a virulent strain of M. tuberculosis, injected i.v. in female Balb/C mice. At sacrifice on day 42 after the experimental infection, the iron-loaded mice presented a significantly enhanced multiplication of M. tuberculosis in both the spleen and the lungs, when compared to the mice without iron loading. CONCLUSION: Most of the studies, including our experimental study in mice, tend to suggest that an excess of iron may enhance the growth of M. tuberculosis and worsen the outcome of human tuberculosis.

Mycobacterium avium complex common-source or cross-infection in AIDS patients attending the same day-care facility.

To delineate the epidemiology of Mycobacterium avium complex (MAC) infection in acquired immunodeficiency syndrome patients, we studied 32 case patients with disseminated MAC infection who attended the same daycare facility during a period of 13 months. Pulsed-field gel electrophoresis analysis showed very low similarity between MAC strains, suggesting that, despite close contacts between the patients, nosocomial cross-transmission or exposure to a common source of MAC did not occur.

Rapid, simple, and culture-independent detection of rpsL codon 43 mutations that are highly predictive of streptomycin resistance in Mycobacterium tuberculosis.

The substitution of codon 43 in the gene rpsL is the single most common mutation found in streptomycin-resistant Mycobacterium tuberculosis. The characterization of this mutation has been hampered by the need for prior cultivation of the mycobacteria, the need for DNA sequencing, or both. In this report we describe a simple and culture-independent technique to detect this mutation directly from sputum samples, requiring little more than a polymerase chain reaction (PCR) machine and a simple agarose minigel. There is no need for labeled probes or DNA sequencing. In a preliminary test of feasibility, interpretable results were obtained from all of 16 smear-positive and 1 of 4 smear-negative, culture-positive samples. Two of two samples containing M. tuberculosis with rpsL codon 43 mutations were correctly identified.

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis.

A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.

Surveillance of Mycobacterium tuberculosis drug resistance in France, 1995-1997. AZAY Mycobacteria Study Group.

OBJECTIVE: To measure the rate of primary and secondary drug resistance of Mycobacterium tuberculosis on an ongoing basis. DESIGN: Data on all culture-positive tuberculosis were collected prospectively from 1995 through 1997 from a microbiological laboratory network of 19 university hospitals throughout France, and submitted quarterly to the National Reference Centre for Surveillance of Mycobacterial Diseases. RESULTS: A total of 2998 patients were included in the study. Among the 2333 (78%) previously untreated patients, 8.6% had isolates resistant to any drug, 4.8% to streptomycin (SM) alone, 1.2% to isoniazid (INH) alone, 1.8% to SM + INH, and 0.3% to INH + rifampicin (RMP) or multidrug resistance (MDR). Foreign birth was independently associated with a higher risk of primary resistance to any drug (odds ratio [OR] 1.5). Among the 268 (9%) previously treated patients, 20.9% had isolates resistant to any drug, 6.3% to SM alone, 3.4% to INH alone, 4.1% to SM + INH, and 3.7% to INH + RMP. Foreign birth (OR = 2.3), and human immunodeficiency virus positive status (OR = 4.4) were independently associated with a higher risk of secondary resistance to any drug. CONCLUSION: During the last 30 years there has been no increase in resistance to any drug among previously untreated patients. As expected, secondary resistance was highly associated with foreign birth. MDR-TB remains a rare event in France.

Twenty-five years of tuberculosis in a French university hospital: a laboratory perspective.

OBJECTIVE: To determine the impact of recent changes in the epidemiology of tuberculosis in France and other industrialised countries on the primary trends of tuberculosis case rates in a French university hospital. DESIGN: Descriptive study of all 4549 culture-positive tuberculosis cases hospitalised at Pitié-Salpêtrière Hospital between 1972 and 1996. RESULTS: From 1972, there was a decline of 5% per year in the tuberculosis case rate, which levelled off in 1983. The proportion of tuberculosis patients who were human immunodeficiency virus (HIV) positive increased from 2% in 1983 to 28% in 1990, and thereafter remained stable. The proportion of foreign-born tuberculosis patients also increased, from 40% in 1972 to 55% in 1985. These two changes affected drug resistance patterns. Drug resistance was more common among foreign-born than among French-born patients, whether previously treated or not. Resistance to rifampicin and multidrug resistance among previously untreated patients was highly related to HIV co-infection. Extrapulmonary sites of tuberculosis were more often smear-positive in HIV-positive than in non-HIV-positive patients (22.8% vs 12.6%), and bacteraemia was diagnosed almost exclusively in HIV-positive patients. CONCLUSION: The changes in clinical and bacteriological tuberculosis patterns at the hospital level over the last 25 years have paralleled those observed at national and international level in industrialised countries, including a slowing in the decrease in the case rate, due in part to the HIV epidemic, a higher proportion of foreign-born patients and an increase in drug resistance.

Impact of iron loading on the activity of isoniazid or ethambutol in the treatment of murine tuberculosis.

OBJECTIVE: To assess the impact of iron loading on the activity of isoniazid and ethambutol in the treatment of murine tuberculosis. DESIGN: Iron-loaded and iron-normal female Balb/C mice infected with 1.5 x 10(7) colony forming units of Mycobacterium tuberculosis were treated with either isoniazid or ethambutol for 28 days. RESULTS: For both treatments, the outcome was impaired by the iron loading: bactericidal activity of isoniazid was partially but significantly reduced and ethambutol bactericidal activity was totally inhibited. CONCLUSION: The treatment of tuberculosis in patients with iron loading should be longer than for normal patients or should contain an additional drug.

A national survey of human Mycobacterium bovis infection in France. Network of Microbiology Laboratories in France.

OBJECTIVE: To evaluate the role of Mycobacterium bovis in the epidemiology of human tuberculosis in France. DESIGN: A national survey in France in 1995 using a questionnaire mailed to all French microbiological laboratories performing mycobacteria cultures. RESULTS: M. bovis was isolated in 38 out of 7075 cases of bacteriologically confirmed tuberculosis (0.5%) notified to the National Reference Centre (CNR) in 1995, resulting in an incidence of 0.07 per 100,000 population. Incidence rates increased with age, and were the highest among patients of 75 years or more (range 0.02-0.33/100,000). Two cases of tuberculosis due to M. bovis were reported in foreign-born children who had come to France for treatment of their disease. No cases were reported among French-born children. The site of tuberculosis was pulmonary in 17 cases, extra-pulmonary in 14, both pulmonary and extra-pulmonary in one, and unknown in six. Extra-pulmonary sites were more frequent in older patients, and pulmonary sites more frequent in younger patients. Two patients were coinfected with the human immunodeficiency virus. Occupational exposure was identified in 13 cases and ingestion of non pasteurised milk in three. In addition, 11 patients had a possible risk of exposure related to their country of birth, family contact or occupation. CONCLUSION: In France, the 0.5% proportion of human tuberculosis due to M. bovis is similar to that of other developed countries. The higher incidence of the disease among older people is likely to reflect the efficacy of the control measures for tuberculosis in cattle.

Use of DNA fingerprinting for primary surveillance of nosocomial tuberculosis in a large urban hospital: detection of outbreaks in homeless people and migrant workers.

SETTING: A large urban teaching hospital in the southeast of Paris. OBJECTIVE: Primary surveillance of nosocomial transmission of tuberculosis (TB) by systematic restriction fragment length polymorphism analysis (RFLP) of isolates (n = 161) recovered from smear-positive pulmonary TB patients identified from 1 March 1993 to 28 February 1994, and from all TB patients (with any form of tuberculous infection) identified from 1 March 1994 to 30 April 1995. RESULTS: Systematic RFLP analysis revealed 12 clusters of patients (n = 40) infected by strains of Mycobacterium tuberculosis showing matching RFLP patterns. None of the isolates were multidrug-resistant. Compared with non-clustered patients, clustered patients were more likely to be homeless (55% vs 19%, P < or = 0.001), or Africans living in hostels for migrant workers (20% vs 6%, P = 0.01), and had fewer previous admissions to hospital (12% vs 28%, P = 0.05). Further epidemiological investigations showed that the clustered TB cases actually resulted not from nosocomial transmission, but from transmission in the community, very likely in homeless shelters and hostels for migrant workers. CONCLUSION: No nosocomial transmission of TB was identified among the patients included during the study period. Systematic RFLP analysis using hospital-based sampling can detect the spread of TB in specific environments in the community where transmission is occurring.

First isolation of Mycobacterium microti (Llama-type) from a dog.

We report the first isolation of Mycobacterium microti from a dog with lesions of acute peritonitis. The isolate was demonstrated to be M. microti of Llama-Type by spoligotyping. Epidemiological implications of the isolation of this possibly zoonotic agent from a dog are discussed.

[Study of the minimal inhibitory concentration of rifabutine (Ansamycin LM 427) for Mycobacterium tuberculosis, Mycobacterium xenopi and Mycobacterium avium-intracellulare]. / Etude des concentrations minima inhibitrices de rifabutine (Ansamycine LM 427) pour Mycobacterium tuberculosis, Mycobacterium xenopi et Mycobacterium avium-intracellulare.

Ansamycin LM 427 (or rifabutine) is a spiropiperidyl-rifamycin which has been proposed as a treatment for tuberculosis and mycobacterial infections resistant to rifampicin. The minimal inhibitory concentration (MCI) of rifabutine and of rifampicin for 10 strains of M. tuberculosis sensitive to rifampicin, 15 strains of M. tuberculosis resistant to rifampicin, 10 strains of M. xenopi and 15 of M. avium-intracellulare have been measured in a comparative fashion on gel DIFCO 7H11 medium and also on a Loewenstein-Jensen medium. The MCI of rifampicin and of rifabutine inhibiting 90% of the strains (MCI 90) of M. tuberculosis sensitive to rifampicin have been 0.06 mg/l on 7H11 media and 1 mg/l on Loewenstein-Jensen media respectively. For strains of M. tuberculosis resistant to rifampicin they were respectively 256 and 8 mg/l on 7H11 and of 1024 and 128 Loewenstein-Jensen, for the strains of M. xenopi they were 8 and 0.5 on 7H11 and 64 and 4 mg/l on Loewenstein-Jensen respectively, and finally for strains of M. avium-intracellulare they were 64 and 2 mg on 7H11 and 256 and 16 on Loewenstein-Jensen media. With rare exceptions for proven mycobacterial infections there was a link of the order of 20:1 between the values for CMI of rifampicin and rifabutine measured on Loewenstein-Jensen medium and on 7H11; the MCIs were ten times more elevated on the first than on the second and the liaison between the MCI of rifampicin and the MCI of rifabutine tended to show that these two antibiotics have the same mode of action.(ABSTRACT TRUNCATED AT 250 WORDS)

[The bacteriology of tuberculosis and non-tuberculosis mycobacterial infections]. / Bactériologie de la tuberculose et des infections à mycobactéries non tuberculeuses.

Changing incidence and nature of mycobacterial infections subsequent to the historical regression of tuberculosis and the acquired human immunodeficiency syndrome (AIDS) epidemic, as well as the development of new technical tools for molecular biology, have profoundly modified the methods used for the bacteriological diagnosis of mycobacteria infections. Although microscopic search for acid-fast bacilli, culture and antibiotic resistance tests on Löwenstein-Jensen medium remain the reference methods, more rapid and sophisticated methods are now available. Culture on radiolabeled media using the Bactec system has shortened the delay for positive culture and interpretable antibiotic sensitivity tests. Molecular techniques allow: 1) rapid identification of the most frequently isolated mycobacteria strains, including the most frequent laboratory contaminant M. gordonae, with genome probes; 2) genome typing of M. tuberculosis strains to trace interhuman transmission, detect recurrence or exogenous reinfection or demonstrate laboratory contamination; 3) rapid detection of rifampicin resistance; and 4) direct detection of M. tuberculosis and M. avium in pathological specimens. The role of mycobacteria in the environment causing opportunistic infections, atypical mycobacteria or non-tuberculosis mycobacteria (NTM), particularly the aviaire complex, has grown considerably. Isolation and identification relies on methods used to detect bacilli as well as blood cultures and analysis of fecal matter. NTM are naturally resistant to most of the antituberculosis antibiotics but are sometimes sensitive to aminoglycosides, fluoroquinolones or new macrolides.

[Prevention of resistance to rifampicin by pyrazinamide in experimental tuberculosis in the mouse]. / Prévention de la résistance à la rifampicine par le pyrazinamide dans la tuberculose expérimentale de la souris.

The capacity of Pyrazinamide (PZA) to prevent the selection of Rifampicin (RMP) resistant mutants was tested by the intravenous innoculation of 160 mice with 2 X 10(6) c.f.u. of M. tuberculosis, and two weeks later, the treatment of the mice either with 10 mg/kg/day RMP alone or with RMP + PZA 150 mg/kg/day. After three months of treatment, lungs and spleen were culture positive in respectively 95% and 90% of the mice treated with RMP alone and in 37% and 24% of the mice treated by RMP + PZA (p less than 10(-5). The strains isolated from lungs and spleen were resistant to Rifampicin in respectively 87% and 75% of the mice treated with RMP alone and in none of those treated with RMP + PZA.

[Mycobacterium fortuitum skin infection occurring after a facelift]. / Infection cutanée à Mycobacterium fortuitum après lifting.

INTRODUCTION: Mycobacterium fortuitum skin infections are rare and usually iatrogenic. We report a case with cervical involvement following a facelift. OBSERVATION: A 65 year-old woman, without past history, underwent bilateral surgical facelift, complicated by cutaneous necrosis and treated with directed healing at home. Six weeks later, an abscessed nodule appeared under the left maxillary and was drained surgically. Then other pre-auricular and left cervical inflammatory nodules appeared without adenopathy or fever. M. fortuitum was isolated in bacteriological samples. The initially probabilistic antibiotherapy with carithromycin, subsequently adapted with amikacine and cirprofloxacine and then imipeneme for a total duration of 3 months, led to the clinical cure. DISCUSSION: Mycobacterium fortuitum is a rapidly growing, ubiquitous, mycobacteria responsible for nosocomial infections in immunocompetent patients, notably following plastic surgery. Contamination occurs where there has been a rupture in the skin barrier through contact with a vector (water, surgical material, antiseptic.). Treatment, which is not codified, consists in the association of surgery and antibiotics for several months.

[Bacteriological diagnosis of tuberculosis]. / Le diagnostic bactériologique de la tuberculose.

Microscopic examination and culture are still today essential elements of the bacteriological diagnosis of tuberculosis. Microscopic examination of a Ziehl fuchsin or auramine stained specimen allows detection of most strains in less than an hour. Culture on Löwenstein-Jensen medium is more sensitive than the microscopic examination and is required for identification and to measure sensitivity to antibiotics. Mycobacterium colonies, generally the causal agent in tuberculosis, usually grow within 28 days and are easily recognized by their "cauliflower" aspect. The niacin test is used for formal identification. Currently, radiometric respirometry allows detection of M. tuberculosis growth and provides antibiotic sensitivity results more rapidly, usually within 10 days. Use of this technique is however limited because the culture medium contains radioactive carbon. Genetic probes are on the other hand quite easy to use and allow identification of cultured bacteria in only a few hours. After polymerization chain reaction gene amplification, M. tuberculosis strains can be detected directly in the specimen within 2 or 3 hours, but in practice, this method has not become a routine laboratory technique, particularly due to lack of sufficient specificity and sensitivity. No other serologic tests are currently reliable enough for the diagnosis of tuberculosis. For cases with low-count specimens, there still is no reliable "on-the-spot" diagnostic test.

Impact of a 14-year screening programme on tuberculosis transmission among the homeless in Paris.

OBJECTIVE: To evaluate the impact of an active case-finding programme on tuberculosis (TB) transmission in homeless shelters in Paris, France. DESIGN: Between 1994 and 1997, an active case-finding programme was implemented in homeless shelters using a mobile radiological screening unit, and continued from 1997 to 2007. During these periods, the strains isolated from TB cases diagnosed in the homeless were genotyped by restriction fragment length polymorphism analysis using the insertion sequence IS6110 as a probe. RESULTS: Between 1994 and 2007, 313 new TB cases were diagnosed among the homeless population: 179 through the programme among shelter users, and 134 among homeless people not using shelters. Half of the strains were clustered in 35 distinct patterns (2-48 cases/cluster). The clustering of TB cases steadily decreased in shelters during the 13 years of the survey, from 14.3 to 2.7 related cases per year (P < 0.01) and from 75% to 30% of related cases among all TB cases (P < 0.01). In contrast, there was only a slight trend towards a decrease in homeless people not using shelters. CONCLUSION: Active case finding in homeless shelters resulted in a decrease in case clustering, mainly in shelter users. Genotyping contributed to confirming the positive impact of the intervention.