First molecular epidemiological study of tuberculosis in Benin.

OBJECTIVES: To assess the diversity of Mycobacterium tuberculosis strains in Cotonou, Benin, and the risk factors associated with clustering. METHODS: We analysed one sputum sample from 194 consecutive new pulmonary tuberculosis (TB) cases using two genotyping methods: spoligotyping and the 12 loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR). The data obtained were compared to the SpolDB4.0 database. RESULTS: We have found that spoligotype 61, highly predominant in West Africa, was also the most prevalent strain in Cotonou. We observed that the Beijing family represented 10.3% of strains and was associated with resistance to streptomycin. We also confirmed that combining spoligotyping and MIRU-VNTR provided a higher discriminatory power than the two techniques used individually. CONCLUSION: Spoligotype 61 and Beijing genotype are the most prevalent genotypes of M. tuberculosis in Cotonou.

High levels of resistance to second-line anti-tuberculosis drugs among prisoners with pulmonary tuberculosis in Georgia.

SETTING: Penitentiary system of Georgia. OBJECTIVE: To determine the prevalence of resistance to second-line drugs among prisoners with pulmonary tuberculosis (PTB). DESIGN: Retrospective evaluation of resistance to second-line drugs in tuberculosis (TB) patients treated from 2001 to 2003. RESULTS: The overall observed prevalence of multidrug-resistant TB (MDR-TB) was 14.4% (39/270). The lowest resistance was found for ofloxacin (OFX), which was 2.2% (6/270) overall and 5.1% (2/39) among MDR patients. Isolates from four non-MDR patients who had never received anti-tuberculosis treatment were found to be resistant to OFX. Resistance to kanamycin and capreomycin occurred simultaneously only among MDR patients and was observed in 17/39 cases (43.6%). High rates of resistance to > or =2 second-line drugs (18/39, 46.2%) and > or =3 second-line drugs (10/39, 25.6%) were observed among all MDR-TB patients, reaching respectively 59.3% and 29.6% among previously treated MDR-TB cases. Only one patient was found to be resistant to four second-line drugs. No extensively drug-resistant TB (XDR-TB) according to the latest definition was detected. CONCLUSION: Our findings reveal a serious threat to the TB control efforts in the study population.

Low levels of second-line drug resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Rwanda.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) has become a therapeutic problem in many parts of the world, necessitating the inclusion of second-line anti-tuberculosis drugs in specific treatment regimens. METHODS: We studied the susceptibility of 69 MDR Mycobacterium tuberculosis isolates from Rwanda to second-line drugs by the BACTEC 460 method. RESULTS: The results showed that 62 (89.9%) were resistant to rifabutin while a low rate (4.3%) of resistance was registered for ofloxacin; there was one case (1.4%) of resistance each for para-aminosalicylic acid, kanamycin, ethionamide, and clarithromycin. CONCLUSIONS: This information is important for devising an appropriate treatment regimen for MDR-TB patients in order to stop the spread of MDR strains and contain the acquisition of additional drug resistance in Rwanda.

Molecular investigation of recurrent tuberculosis in patients from Rwanda.

SETTING: Pulmonary tuberculosis (TB) patients enrolled in four provinces of Rwanda. OBJECTIVE: To determine the cause of recurrent TB. DESIGN: Serial Mycobacterium tuberculosis isolates obtained from patients with recurrent TB from January 2002 to September 2005 were genotyped by spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Drug resistance was determined by phenotypic susceptibility testing and sequencing of rpoB, katG, inhA and embB genes. RESULTS: Among 710 culture-positive TB patients enrolled in the study, initial drug susceptibility testing results were available for 638. Sixty-nine of these had multidrug-resistant (MDR) TB and 569 were non-MDR-TB. Among the MDR-TB patients, 22 had follow-up isolates after cure (n = 12) or chronic infection (n = 10). The DNA patterns of sequential isolates from 4 of the 12 previously cured MDR-TB patients were different, indicating re-infection. DNA patterns of isolates from the remaining 8 previously cured and 10 chronic MDR-TB patients were identical, suggesting reactivation and treatment failure, respectively. Among the non-MDR-TB patients, disease recurrence was observed in one case; this was determined to be due to reactivation after initial mixed infection. CONCLUSION: These results document a high treatment failure/reactivation rate for MDR-TB and suggest that re-infection within 2 years may not be a common cause of recurrent TB in this setting.

Limited fluoroquinolone resistance among Mycobacterium tuberculosis isolates from Rwanda: results of a national survey.

OBJECTIVES: There is an increasing interest in the possible role of fluoroquinolone antibiotics for the treatment of tuberculosis (TB), but widespread use of these antibiotics for the treatment of other bacterial infections may select for fluoroquinolone-resistant Mycobacterium tuberculosis strains. METHODS: We evaluated fluoroquinolone susceptibility using the proportion method (ofloxacin, critical concentration 2.0 mg/L) in isolates from patients enrolled in a national drug resistance survey in Rwanda from November 2004 to February 2005. RESULTS: Of the 701 M. tuberculosis isolates studied, 617 (88%) were susceptible to all first-line drugs, 32 (4.6%) were multidrug-resistant (MDR) and 52 (7.4%) were resistant to one or more first-line drugs but not MDR. Ofloxacin resistance was found in four (0.6%) of the isolates; three of them being MDR and one susceptible to all first-line drugs. Mutations in the gyrA gene were found in all ofloxacin-resistant strains at codons 80 and 94. CONCLUSIONS: Our finding is not alarming for Rwanda, but highlights the general risk of producing resistance to fluoroquinolones, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the programmatic management of drug-resistant TB and creating incurable TB strains.

Evidence of 'amplifier effect' in pulmonary multidrug-resistant tuberculosis: report of three cases.

INTRODUCTION: A cluster of three related cases of tuberculosis (TB) with primary multidrug resistance was investigated at the Centre Hospitalier Universitaire of Kigali (CHUK) in Rwanda. The patients were HIV-1/2 seronegative. Patients 1 and 2 were hospitalized in the same room of CHUK for one month. Patient 3 was a younger sibling of patient 2. METHODS: Drug susceptibility of two consecutive Mycobacterium tuberculosis isolates from each patient was tested by the BACTEC 460 radiometric method. DNA fingerprinting was performed using spoligotyping and mycobacterial interspersed repetitive units of variable numbers of tandem repeats (MIRU-VNTR) analysis. All patients initially received the World Health Organization category I regimen. RESULTS: The isolates collected during the first TB episode were resistant to isoniazid, rifampin and ethambutol. After subsequent retreatment regimens with rifampin, isoniazid, streptomycin, pyrazinamide (8 months) and rifampin, isoniazid, streptomycin, pyrazinamide, ciprofloxacin (21 months), patients 1 and 2 developed additional resistance to streptomycin and quinolones. Patient 3 received only the category I regimen and consecutive isolates retained the initial drug susceptibility pattern. All isolates were genetically indistinguishable by spoligotyping and MIRU-VNTR, indicating the same origin. CONCLUSIONS: These observations highlight the risk of nosocomial transmission of multidrug-resistant (MDR) TB and the possible selection of secondary resistance to second-line drugs if a single new drug is added at the time of retreatment of MDR TB patients.

Molecular genetic methods for diagnosis and antibiotic resistance detection of mycobacteria from clinical specimens.

Mycobacteria comprise a diverse group of bacteria that are widespread in nature, some of which cause significant disease in humans. Members of the Mycobacterium tuberculosis complex (MTBC) are the most important human pathogens of the genus Mycobacterium. Traditional methods for detection and identification of mycobacteria include microscopy, culture and phenotypic tests. These methods either lack sensitivity, specificity, or are time consuming. Advances in the field of molecular biology have provided rapid diagnostic tools that have reduced the turnaround times for detecting MTBC and drug resistance in cultures and directly in clinical specimens from weeks to days. This review discusses the molecular genetic techniques for detecting and identifying MTBC as well as drug resistance of mycobacteria in clinical specimens.

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity.

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.