Sterilizing activity of R207910 (TMC207)-containing regimens in the murine model of tuberculosis.

RATIONALE: The diarylquinoline R207910 (TMC207) has potent bactericidal activity in a murine model of tuberculosis (TB), but its sterilizing activity has not been determined. OBJECTIVES: To evaluate the sterilizing activity of R207910-containing combinations in the murine model of TB. METHODS: Swiss mice were intravenously inoculated with 6 log(10) of Mycobacterium tuberculosis strain H37Rv, treated with R207910-containing regimens, and followed for 3 months to determine relapse rates (modified Cornell model). MEASUREMENTS AND MAIN RESULTS: Quantitative lung and spleen colony-forming unit counts and bacteriological relapse rates 3 months after the end of therapy were compared for the following regimens: 2, 3, or 4 months of R207910 (J) and pyrazinamide (Z) combined with rifampin (R) or isoniazid (H) or both and 3 or 4 months of a moxifloxacin (M)-containing regimen and 6 months of the standard WHO regimen RHZ. All J-treated mice were culture negative after 4 months of therapy. The relapse rate in the group treated with 4 months of JHRZ was similar to that of mice treated for 6 months with the RHZ regimen (6 vs. 17%; P = 0.54) and lower than that of RMZ (6 vs. 42%; P = 0,03), a moxifloxacin-containing regimen that was the most active in mice on once-daily basis. CONCLUSIONS: Four months of treatment with some J-containing regimens was as effective as the 6-month standard regimen and more effective than 4 months of treatment with M-containing regimens. Supplementation of standard regimen (RHZ) with J or substitution of J for H may shorten the treatment duration needed to cure TB in patients.

A once-weekly R207910-containing regimen exceeds activity of the standard daily regimen in murine tuberculosis.

RATIONALE: R207910 (TMC207 or J) is a member of the diarylquinolines, a new family of antituberculous drugs with high bactericidal activity when given daily in the murine model of tuberculosis. R207910 exhibits a long half-life and thus is a good candidate for once-weekly therapy of tuberculosis. OBJECTIVES: To study the activity of once-weekly R207910 monotherapy and combinations of R207910 with other antituberculous agents (isoniazid, rifapentine, moxifloxacin, and pyrazinamide). METHODS: The established infection model of murine tuberculosis was used. Colony counts were determined in the lungs. MEASUREMENTS AND MAIN RESULTS: Eight weeks of monotherapy reduced the bacillary load by 3 to 4 log(10) for rifapentine and by 5 to 6 log(10) for R207910 (P < 0.05). The addition of rifapentine and isoniazid or moxifloxacin did not improve the bactericidal activity of R207910 monotherapy. In contrast, the triple combination of R207910 plus rifapentine plus pyrazinamide given once weekly for 2 months (i.e., a total of only eight administrations), was significantly (P < 0.05) more active than R207910 monotherapy or other R207910 combinations, and led to lung culture negativity in 9 of 10 mice, whereas all lungs were culture positive in the groups treated with other drug combinations. Moreover, R207910 plus rifapentine plus pyrazinamide given once weekly was more active than the current standard regimen of rifampin plus isoniazid plus pyrazinamide given five times per week. CONCLUSIONS: The unprecedented activity of the triple combination of R207910 plus rifapentine plus pyrazinamide suggests that it may be feasible to develop a fully intermittent once-weekly regimen.

An intervention programme for the management of multidrug-resistant tuberculosis in France.

As shown in a previous study, the outcome of multidrug-resistant tuberculosis (MDR-TB) in France is not satisfactory (41% success rate). To improve the management and outcome of patients with MDR-TB, a prospective intervention study was carried out in 1998-1999 by National Reference Centre (NRC) staff. NRC staff contacted (i) the microbiologists reporting MDR-TB patients in order to offer second-line drug testing and (ii) physicians to help tailor treatment regimens based on World Health Organization guidelines. A total of 45 MDR-TB patients were followed, including 33 for whom NRC staff successfully interacted with the physicians in charge and 12 patients managed without NRC collaboration. The crude proportion of favourable outcomes was 58%, and this proportion reached 70% for patients managed in collaboration with the NRC. Among the latter 33 patients, 32 were treated with at least three active drugs for a median duration of 12 months. In multivariate analysis, HIV-positive status was associated with treatment failure (hazard ratio (HR) 4.3), whereas NRC intervention was associated with a favourable outcome (HR 0.2). However, even in the NRC intervention group the median duration of treatment was shorter than planned (median 12 months) and 12% of patients were lost to follow-up. This intervention study provides encouraging results and should be continued until reference teams and a directly observed treatment strategy (DOTS) are implemented to improve patient management further.

[Modern diagnosis of tuberculosis]. / Diagnostic moderne de la tuberculose.

Microscopic examination of sputum smears (search for acid-fast bacilli or AFB) is the most rapid procedure for diagnosis of contagious tuberculosis. Gene amplification is not yet reliable for direct detection of M. tuberculosis in clinical specimens that are AFB smear-negative. Culture (3 to 8 weeks on Lowenstein-Jensen medium or 1 to 4 weeks in liquid media) remains essential to identify AFB and conduct antibiotic susceptibility testing. AFB from culture can be identified in a few hours by molecular approaches with specific DNA probes. Results of susceptibility testing, even in liquid media, are not available until 2 to 4 weeks after the recovery of specimens, although mutations of the rpoB and katG 315 genes, which confer resistance to rifampin and isoniazid, can be detected within hours by molecular hybridization with specific probes fixed on strips. Immunologic tests that measure the interferon gamma produced by sensitized lymphocytes are promising tools for the diagnosis of latent tuberculosis.

[Trends in tuberculosis treatment duration]. / Argumentation sur la durée des traitements antituberculeux.

The two principal characteristics of tuberculosis treatment are its length (several months) and the need to use several antibiotics simultaneously (multiple drug therapy). Multiple drug therapy is intended to prevent the selection of resistant mutants at the beginning of treatment, when the bacilli population is largest. The length of treatment is due to dormant bacilli, which are much more difficult for antibiotics to kill than actively multiplying bacilli are. Rifampin and pyrazinamide are the most potent drugs against these dormant bacilli. The so-called sterilizing activity of rifampin has reduced the duration of treatment from 18 to 9 months, and the contribution of pyrazinamide reduced this time still further, to 6 months. When one of these drugs cannot be used because of resistance or toxicity, duration of treatment increases to the earlier levels. In the extreme case of multidrug-resistant tuberculosis where neither isoniazid nor rifampin can be used, and sometimes even not pyrazinamide, treatment is recommended for 18 to 24 months. New antituberculosis drugs under development allow us to envision further reduction in the duration of treatment of both drug-resistant and drug-sensitive tuberculosis.

Mycobacterium szulgai infection in a captive population of African clawed frogs (Xenopus tropicalis).

A colony of captive Xenopus tropicalis became infected with Mycobacterium szulgai. Clinical signs, when observed, were lethargy, weight loss, and emaciation. Visceral granulomas were common findings at laparoscopy and necropsy. The diagnosis of mycobacteriosis was based on histologic appearance and Ziehl-Neelsen staining of tissues. The identification of M. szulgai organisms was based on comparison of the 16S rRNA gene sequence with several GenBank databases. There have been no reports of this mycobacterial species as the causative agent of naturally occurring disease in amphibians.

Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis: functional analysis of mutant enzymes.

Mutations in the DNA gyrase GyrA2GyrB2 complex are associated with resistance to quinolones in Mycobacterium tuberculosis. As fluoroquinolones are being used increasingly in the treatment of tuberculosis, we characterized several multidrug-resistant clinical isolates of M. tuberculosis carrying mutations in the genes encoding the GyrA or GyrB subunits associated with quinolone resistance or hypersusceptibility. In addition to the reported putative quinolone resistance mutations in GyrA, i.e., A90V, D94G, and D94H, we found that the GyrB N510D mutation was also associated with ofloxacin resistance. Surprisingly, several isolates bearing a novel combination of gyrA T80A and A90G changes were hypersusceptible to ofloxacin. M. tuberculosis GyrA and GyrB subunits (wild type [WT] and mutants) were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. Mutant proteins were produced similarly from engineered gyrA and gyrB alleles by mutagenesis. MICs, enzyme inhibition, and drug-induced DNA cleavage were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. Mutant gyrase complexes bearing GyrA A90V, D94G, and D94H and GyrB N510D were resistant to quinolone inhibition (MICs and 50% inhibitory concentrations [IC50s] at least 3.5-fold higher than the concentrations for the WT), and all, except the GyrB mutant, were less efficiently trapped as a quinolone cleavage complex. In marked contrast, gyrase complexes bearing GyrA T80A or A90G were hypersusceptible to the action of many quinolones, an effect that was reinforced for complexes bearing both mutations (MICs and IC50s up to 14-fold lower than the values for the WT). This is the first detailed enzymatic analysis of hypersusceptibility and resistance in M. tuberculosis.

Combinations of R207910 with drugs used to treat multidrug-resistant tuberculosis have the potential to shorten treatment duration.

The objective of the present study was to identify the optimal R207910-containing regimen to administer to patients who cannot receive rifampin (RIF) and isoniazid (INH) because of multidrug-resistant tuberculosis (MDR-TB), concomitant use of antiretroviral drugs, or toxicity. Mice were infected intravenously with 5 x 10(6) CFU of the H37Rv strain and treated five times per week with R207910 alone or various combinations of R207910 with the second-line drugs amikacin (AMK), pyrazinamide (PZA), moxifloxacin (MXF), and ethionamide (ETH). All R207910-containing regimens were significantly more active than the non-R207910-containing regimens after 1 month of therapy. When given for 2 months, R207910 alone was more active than the WHO standard first-line regimen RIF-INH-PZA. When R207910 was combined with second-line drugs, the combinations were more active than the currently recommended regimen of MDR-TB AMK-ETH-MXF-PZA, and culture negativity of both the lungs and spleen was reached after 2 months of treatment in almost every case.

Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance.

We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the katG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in katG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG protein for 7 strains, a C-->T mutation in position -15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.

Hip prosthesis infection due to Mycobacterium wolinskyi.

Mycobacterium wolinskyi, first described in 1999, is a rapidly growing mycobacterium related to the Mycobacterium smegmatis group. Only eight cases of infection due to this microorganism have been reported, including three cases of bone infection. Here, we present the first case of a joint prosthesis infection cured with the combination of surgery and prolonged antibiotic therapy. The microorganism was identified by biochemical tests and 16S rRNA and Hsp65 gene sequence analysis.

Functional analysis of DNA gyrase mutant enzymes carrying mutations at position 88 in the A subunit found in clinical strains of Mycobacterium tuberculosis resistant to fluoroquinolones.

We investigated the enzymatic efficiency and inhibition by quinolones of Mycobacterium tuberculosis DNA gyrases carrying the previously described GyrA G88C mutation and the novel GyrA G88A mutation harbored by two multidrug-resistant clinical strains and reproduced by site-directed mutagenesis. Fluoroquinolone MICs and 50% inhibitory concentrations for both mutants were 2- to 43-fold higher than for the wild type, demonstrating that these mutations confer fluoroquinolone resistance in M. tuberculosis.

A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.

The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific drugs have been discovered in 40 years. We identified a diarylquinoline, R207910, that potently inhibits both drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 mug/ml). In mice, R207910 exceeded the bactericidal activities of isoniazid and rifampin by at least 1 log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis treatment regimen (rifampin, isoniazid, and pyrazinamide) with R207910 accelerated bactericidal activity, leading to complete culture conversion after 2 months of treatment in some combinations. A single dose of R207910 inhibited mycobacterial growth for 1 week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.

Fluoroquinolone-containing third-line regimen against Mycobacterium tuberculosis in vivo.

The objective of the present study was to compare the activities of a third-line regimen recommended by the World Health Organization (WHO) and two derivatives of that regimen with the activity of the standard combination of isoniazid, rifampin, and pyrazinamide as a positive control against Mycobacterium tuberculosis in a murine model. The WHO regimen combines ofloxacin (OFX), ethionamide, amikacin, and pyrazinamide; in the two derivatives of this regimen, OFX was replaced by levofloxacin (LVX) or moxifloxacin (MXF). The four drugs, a fluoroquinolone (either OFX, LVX, or MXF), ethionamide, pyrazinamide, and amikacin, were administered for the first 2 months (initial phase); and two drugs, a fluoroquinolone (either OFX, LVX, or MXF) and ethionamide, were administered for the following 10 months (continuation phase). After 6 months of treatment, only the spleens and lungs of mice treated with the standard regimen became culture negative. From 9 months onward, all of the organs of mice treated with the MXF-containing third-line regimen were culture negative. The majority of organs from mice treated with the OFX-containing regimen continued to be culture positive, and the mean CFU counts remained unchanged for as long as 12 months. The results for mice treated with the LVX-containing regimen fell between those for the groups receiving the MXF- and OFX-containing regimens. In conclusion, the activity of the OFX-containing third-line regimen against M. tuberculosis was rather weak in vivo, whereas when OFX was replaced by MXF, 9 months of treatment with a modified third-line regimen displayed bactericidal activity comparable to that of 6 months of treatment with the standard regimen in mice. The MXF-containing third-line regimen seems to be a powerful alternative for the treatment of tuberculosis (TB) when isoniazid and rifampin cannot be used, which is the main feature of multidrug-resistant TB.

Identification of mycobacterial species by PCR sequencing of quinolone resistance-determining regions of DNA gyrase genes.

The determination of the amino acid sequence of quinolone resistance-determining regions (QRDRs) in the A and B subunits of DNA gyrase is the molecular test for the detection of fluoroquinolone resistance in mycobacteria. We looked to see if the assignment of mycobacterial species could be obtained simultaneously by analysis of the corresponding nucleotide sequences. PCR sequencing of gyrA and gyrB QRDRs was performed for 133 reference and clinical strains of 21 mycobacterial species commonly isolated in clinical laboratories. Nucleotide sequences of gyrA and gyrB QRDRs were species specific, regardless of fluoroquinolone susceptibility.

Impacts of interleukin-12 on multiplication of Mycobacterium tuberculosis and Mycobacterium avium complex in mice.

OBJECTIVE: To evaluate the potential impacts of exogenous administration of murine recombinant interleukin-12 (IL-12) on multiplication of Mycobacterium tuberculosis and M. avium complex (MAC) in murine models. METHODS: Swiss or beige mice were infected intravenously with M. tuberculosis H37Rv or MAC respectively, and were treated by subcutaneous injection with various doses of IL-12, either alone or in combination with chemotherapy. Effectiveness of treatment was assessed by the enumeration of CFUs in the spleens and lungs, together with other indicators. RESULTS: Multiplication of M. tuberculosis was reduced by IL-12 in a dose-dependent manner if the treatment began at day 1, whereas no statistically significant suppression was observed if the treatment began at day 14. Combination with IL-12 did not enhance the bactericidal activity of antituberculosis chemotherapy. The growth curves of MAC in IL-12-treated mice were almost identical to those of untreated controls, indicating that IL-12 did not affect the multiplication of MAC in beige mice. In both experiments, the dosing of IL-12 approached levels of severe toxicity for the mouse strains used. CONCLUSIONS: IL-12 had a positive affect on early multiplication of M. tuberculosis. It had no effect on early multiplication of M. avium complex.

Disseminated infection with a mycobacterium related to Mycobacterium triplex with central nervous system involvement associated with AIDS.

We report on a case of disseminated infection with central nervous system involvement due to an atypical mycobacterium related to Mycobacterium triplex in a severely immunodepressed human immunodeficiency virus-infected man.