A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid.

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain was Mycobacterium tuberculosis H37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 µg/ml), rifampin (0.03 to 0.25 µg/ml), ethambutol (0.25 to 2 µg/ml), levofloxacin (0.12 to 1 µg/ml), moxifloxacin (0.06 to 0.5 µg/ml), ofloxacin (0.25 to 2 µg/ml), amikacin (0.25 to 2 µg/ml), kanamycin (0.25 to 2 µg/ml), capreomycin (0.5 to 4 µg/ml), linezolid (0.25 to 2 µg/ml), and clofazimine (0.03 to 0.25 µg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against the M. tuberculosis H37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

A Multilaboratory, Multicountry Study To Determine Bedaquiline MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing.

The aim of this study was to establish standardized drug susceptibility testing (DST) methodologies and reference MIC quality control (QC) ranges for bedaquiline, a diarylquinoline antimycobacterial, used in the treatment of adults with multidrug-resistant tuberculosis. Two tier-2 QC reproducibility studies of bedaquiline DST were conducted in eight laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Agar dilution and broth microdilution methods were evaluated. Mycobacterium tuberculosis H37Rv was used as the QC reference strain. Bedaquiline MIC frequency, mode, and geometric mean were calculated. When resulting data occurred outside predefined CLSI criteria, the entire laboratory data set was excluded. For the agar dilution MIC, a 4-dilution QC range (0.015 to 0.12 µg/ml) centered around the geometric mean included 95.8% (7H10 agar dilution; 204/213 observations with one data set excluded) or 95.9% (7H11 agar dilution; 232/242) of bedaquiline MICs. For the 7H9 broth microdilution MIC, a 3-dilution QC range (0.015 to 0.06 µg/ml) centered around the mode included 98.1% (207/211, with one data set excluded) of bedaquiline MICs. Microbiological equivalence was demonstrated for bedaquiline MICs determined using 7H10 agar and 7H11 agar but not for bedaquiline MICs determined using 7H9 broth and 7H10 agar or 7H9 broth and 7H11 agar. Bedaquiline DST methodologies and MIC QC ranges against the H37Rv M. tuberculosis reference strain have been established: 0.015 to 0.12 µg/ml for the 7H10 and 7H11 agar dilution MICs and 0.015 to 0.06 µg/ml for the 7H9 broth microdilution MIC. These methodologies and QC ranges will be submitted to CLSI and EUCAST to inform future research and provide guidance for routine clinical bedaquiline DST in laboratories worldwide.

Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country.

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

Infection of cardiovascular implantable electronic devices: detection with sonication, swab cultures, and blood cultures.

BACKGROUND: The number of patients with cardiovascular implantable electronic devices (CIEDs) is increasing. The purpose of this study was to compare three methods (sonication, swab cultures, and blood cultures) to detect bacteria in infections of CIEDs. METHODS: Patients with pocket infection of CIEDs and/or CIED-associated infective endocarditis were prospectively included. Generators and leads were sonicated together. Swab cultures of the generator pocket and blood cultures were performed. RESULTS: Between January 2009 and September 2012, 18 patients with a CIED infection were recorded, including two definite and four possible CIED-IEs. In 17 patients, blood cultures were performed and the device was analyzed by sonication. A swab culture from the device pocket was performed in 16 cases. Ten (59%) patients received antibiotic therapy before the device was removed. In 15 of 16 cases (94%), bacteria were detected by sonication, while bacteria were detected in only nine (56%) and seven (41%) cases by swab-and blood cultures, respectively. In four cases (24%), sonication was the only method that detected bacteria. The following bacteria were detected by sonication: methicillin-sensitive Staphylococcus aureus (MSSA; n = 6), coagulase-negative staphylococci (CNS; n = 3), Propionibacterium acnes (n = 2), Staphylococcus lugdunensis (n = 1), MSSA/Pseudomonas aeruginosa (n = 1),Bacillus cereus (n = 1), MSSA/P. acnes (n = 1), andP. acnes/CNS (n = 1). All positive swab cultures and all positive blood cultures but one grew the same bacteria as sonication fluid cultures. CONCLUSION: Sonication can be more sensitive than swab cultures or blood cultures in detecting bacteria in infections of CIEDs.

Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter -15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection.

Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.

BACKGROUND: Electrophysiological cardiac devices are increasingly used. The frequency of subclinical infection is unknown. We investigated all explanted devices using sonication, a method for detection of microbial biofilms on foreign bodies. METHODS AND RESULTS: Consecutive patients in whom cardiac pacemakers and implantable cardioverter/defibrillators were removed at our institution between October 2007 and December 2008 were prospectively included. Devices (generator and/or leads) were aseptically removed and sonicated, and the resulting sonication fluid was cultured. In parallel, conventional swabs of the generator pouch were performed. A total of 121 removed devices (68 pacemakers, 53 implantable cardioverter/defibrillators) were included. The reasons for removal were insufficient battery charge (n=102), device upgrading (n=9), device dysfunction (n=4), or infection (n=6). In 115 episodes (95%) without clinical evidence of infection, 44 (38%) grew bacteria in sonication fluid, including Propionibacterium acnes (n=27), coagulase-negative staphylococci (n=11), Gram-positive anaerobe cocci (n=3), Gram-positive anaerobe rods (n=1), Gram-negative rods (n=1), and mixed bacteria (n=1). In 21 of 44 sonication-positive episodes, bacterial counts were significant (>or=10 colony-forming units/mL of sonication fluid). In 26 sterilized controls, sonication cultures remained negative in 25 cases (96%). In 112 cases without clinical infection, conventional swab cultures were performed: 30 cultures (27%) were positive, and 18 (60%) were concordant with sonication fluid cultures. Six devices and leads were removed because of infection, growing Staphylococcus aureus, Streptococcus mitis, and coagulase-negative staphylococci in 6 sonication fluid cultures and 4 conventional swab cultures. CONCLUSIONS: Bacteria can colonize cardiac electrophysiological devices without clinical signs of infection.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) in specimens from various body sites: performance characteristics of the BD GeneOhm MRSA assay, the Xpert MRSA assay, and broth-enriched culture in an area with a low prevalence of MRSA infections.

Universal surveillance upon patient admission is important in reducing the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and associated disease in hospitals. High costs for the health care system in conjunction with MRSA have promoted the development of rapid screening methods to detect MRSA carriers. This study compared two real-time PCR methods, the BD GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture to define their performance characteristics and rapidity in an area with low MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin from 425 patients were tested. Of those 425 patients, 378 had swabs from both the nose and groin in parallel. Two hundred thirty-one and 194 patients were randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In general, sensitivity, specificity, and negative predictive value (NPV) were high for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%, 98.2%, and 100%, respectively), irrespective of whether or not nasal and inguinal specimens were considered alone or combined. In contrast, the positive predictive value (PPV) was lower: before the resolution of discrepant results, the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%, and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA, respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3% and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA, respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were exclusively identified by nasal swabs and 2 of 13 were identified by inguinal swabs alone. Both PCR methods showed no significant difference in the number of discrepant results (odds ratio, 0.70 [P = 0.789]), but specimens from wounds and other body sites (axilla, vagina, and throat) produced discrepancies more often than nasal and groin specimens (odds ratios, 4.724 [P = 0.058] and 12.163 [P < 0.001], respectively). The facts that no false-negative PCR results were detected and increased PPVs were found after the resolution of discrepant results point to PCR as the actual gold standard. Since both sensitivity and NPV were exceptionally high for PCR, backup cultures may, therefore, be unnecessary in an area with low prevalence and with a preemptive isolation strategy but may still be useful for PCR-positive specimens because of the lower PPV for both methods and the possibility of susceptibility testing. The median time for analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time, including administration and specimen collection, the Xpert MRSA was faster than the BDGO (7 h 50 min versus 17 h).

Emergence of SCCmec type IV and SCCmec type V methicillin-resistant Staphylococcus aureus containing the Panton-Valentine leukocidin genes in a large academic teaching hospital in central Switzerland: external invaders or persisting circulators?

The hospital epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has changed in the past few years due to the encroachment of community-associated MRSA (CA-MRSA) strains into health care settings. MRSA strains that were isolated during a 2-year period from patients of the Luzerner Kantonsspital were analyzed to elucidate their epidemiology. Moreover, extended surveillance of individuals who were contacts of those patients was carried out for 6 months to identify the routes of spread and to assess the quality of the infection control measures used in our setting. Patient data were collected to distinguish CA-MRSA strains from health care-associated MRSA (HA-MRSA) strains by epidemiological criteria, as defined by the Centers for Disease Control and Prevention (CDC). On the basis of the CDC definition, the majority of the strains were considered to be HA-MRSA. However, 87% of them belonged to staphylococcal cassette chromosome mec (SCCmec) types IV and V, which are traditionally associated with CA-MRSA. Surprisingly, classical nosocomial SCCmec types I and II represented a minority, whereas SCCmec type III was completely absent. By PFGE analysis, four predominant clonal lineages and 21 highly variable sporadic genotypes were detected. Twenty-eight percent of the MRSA strains studied carried the genes encoding the Panton-Valentine leukocidin (PVL), of which 21% and 83% were associated with SCCmec types IV and V, respectively. Among 289 contact individuals screened for MRSA carriage throughout the extended surveillance, a single secondary patient was discovered. The possibility of nosocomial transmission could be excluded. The high proportions of SCCmec type IV and V strains as well as PVL-positive strains suggest strong infiltration of CA-MRSA into our institution. Moreover, the low endemic prevalence of MRSA demonstrates that current infection control measures are sufficient to limit its spreading and the emergence of large epidemic outbreaks.

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in diverse clinical specimens by the BD GeneOhm MRSA assay and comparison with culture.

The efficacy of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay was assessed by analyzing nasal swabs and swabs from other body sites for the presence of MRSA in a low-prevalence area. From 681 patients with a high risk for MRSA carriage, 1,601 specimens were collected and transported in Amies agar. After discordant analysis, the sensitivity, specificity, positive predictive value, and negative predictive value of the BD GeneOhm MRSA assay were 84.3%, 99.2%, 88.4%, and 98.9%, respectively, compared to culture.

Pulmonary Coccidioides nodule in a Swiss patient with chronic lymphatic leukaemia.

The dimorphic fungus Coccidioides immitis, causative agent of coccidioidomycosis, is endemic in semi-arid regions of the Americas. The disease is rarely encountered in Europe. We describe the case of a 61-year-old Swiss patient with chronic lymphatic leukaemia who was eventually diagnosed with an initially unnoticed pulmonary coccidioidomycosis.

Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis.

This systematic review assesses the evidence for an association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease. We analysed 28 case-control studies comparing MAP in patients with Crohn's disease with individuals free of inflammatory bowel disease (IBD) or patients with ulcerative colitis. Compared with individuals free of IBD, the pooled odds ratio (OR) from studies using PCR in tissue samples was 7.01 (95% CI 3.95-12.4) and was 1.72 (1.02-2.90) in studies using ELISA in serum. ORs were similar for comparisons with ulcerative colitis patients (PCR, 4.13 [1.57-10.9]; ELISA, 1.88 [1.26-2.81]). The association of MAP with Crohn's disease seems to be specific, but its role in the aetiology of Crohn's disease remains to be defined.

Multilocus sequence typing scheme for the Mycobacterium abscessus complex.

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world.

Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials.

The BACTEC MGIT 960 system, a fully automated, nonradiometric, noninvasive system for detection and drug susceptibility testing of mycobacteria, was evaluated for the ability to test susceptibilities to second-line drugs. In this study, which was carried out in three phases (phase I, mostly susceptible strains; phase II, mostly resistant strains; phase III, final testing of the optimal drug concentrations found in phases I and II), we established the critical concentrations for seven drugs to be tested in the BACTEC MGIT 960 system compared to the BACTEC 460TB system. The critical concentrations for the seven drugs used in the MGIT 960 system are as follows: amikacin, 1.0 microg/ml; capreomycin, 2.5 microg/ml; ethionamide, 5.0 microg/ml; protionamide, 2.5 microg/ml; ofloxacin, 2.0 microg/ml; rifabutin, 0.5 microg/ml; linezolid, 1.0 microg/ml. Our results demonstrate that the BACTEC MGIT 960 system is an accurate method for rapid testing of the susceptibilities of Mycobacterium tuberculosis to second-line drugs.

Molecular characterization and drug resistance testing of Mycobacterium tuberculosis isolates from Chad.

The molecular characterizations of the first 40 Mycobacterium tuberculosis isolates from Chad revealed a high proportion of isolates of the Cameroon family (33%), of which one isolate showed a monodrug resistance. In total, 9/33 (27%) isolates were resistant to isoniazid. The implications of these findings are discussed.

Mycobacterium bovis isolates from tuberculous lesions in Chadian zebu carcasses.

This slaughterhouse study in Chad shows higher proportions of Mycobacterium bovis isolates among Mbororo than Arabe zebu cattle. Spoligotyping shows a homogenetic population structure for M. bovis and lack of spacer 30, as were found in neighboring Cameroon and Nigeria. This finding suggests transborder and ongoing transmission between cattle.

[Bone collected with a bone collector during dental implant surgery]. / Mit Knochenkollektor bei dentaler Implantation gewonnenes Knochenmaterial. Mikrobiologische Analyse.

BACKGROUND: Simultaneous implantation and augmentation using autogenous bone collected during implant surgery is a well-established procedure in oral implantology. The aims of this study were (1) to identify any bacterial contamination of bone obtained with a bone collector, and (2) to verify the antimicrobial effect of rinsing the bone collector with a 0.1% chlorhexidine solution prior to augmentation. MATERIAL AND METHODS: A total of 39 patients undergoing a simultaneous implantation and augmentation procedure were examined. All patients rinsed their mouths with a 0.1% chlorhexidine solution for 2 min prior to surgery. Bone was collected with the Osseous Coagulum Trap, while saliva was collected with a separate suction tip. Once bone collection was complete a microbiological swab was taken from the bone collector (sample 1); before the bone was taken from it 200 ml of a 0.1% chlorhexidine solution was aspirated into the collector, after which the bone was removed and the collector sieve was (sample 2) sent for microbiological analysis which included aerobic and anaerobic cultivation of microorganisms and their identification and semiquantitative assessment of microbial growth. RESULTS: Before the collector was rinsed with chlorhexidine microbial contamination was found in 34 (82.7%) of the 39 samples, and 37 different microbial species were identified in cultures. When the collector had been rinsed with 200 ml 0.1% chlorhexidine a significantly lower rate of microbial contamination was found: 66.7% of the samples were sterile. CONCLUSIONS: Despite separate suction techniques for bone dust and saliva and preoperative use of a chlorhexidine mouthwash, bacterial contamination of bone obtained from the mouth with a bone collector has to be anticipated. Not only the physiological bacteria of the oral flora, but also the microorganisms frequently associated with implant failure can be found. Rinsing the bone collector with 200 ml of a 0.1% chlorhexidine solution significantly reduces microbial contamination. The effects on bone vitality must be studied before routine rinsing with chlorhexidine can be recommended.

[Ranking of the polymerase chain reaction (PCR) and of other amplification methods in the diagnosis of tuberculosis]. / Stellenwert der Polymeraseketten-Reaktion (PCR) und anderer Amplifikationsverfahren in der Diagnostik der Tuberkulose.

Worldwide, tuberculosis (TB) remains one of the leading infectious diseases, accounting for nearly 3 million deaths and more than 8 million new cases annually. Among them are 3.2 % multidrug-resistant strains of Mycobacterium tuberculosis. In the control of the spread of TB clinical mycobacteriology laboratories play an essential role providing the clinicians with timely detection, isolation, identification, and drug susceptibility testing results for M. tuberculosis. In the past twenty years numerous molecular techniques have been introduced to comply with the obvious needs for a reliable and rapid diagnosis of the disease. Nucleic acid amplification-based assays, in particular, the polymerase chain reaction (PCR), allow (i) direct detection of M. tuberculosis complex in clinical specimens; (ii) identification of mycobacteria; (iii) detection of resistance of M. tuberculosis to antimicrobial agents; and (iv) DNA typing to answer questions such as reactivation of disease or exogenous reinfection, and to track transmission and internal laboratory contaminations.

Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide with the nonradiometric BACTEC MGIT 960 system.

The reliability of the novel BACTEC MGIT 960 pyrazinamide (PZA) kit (Becton Dickinson Microbiology Systems, Sparks, Md.) was assessed for testing of susceptibility of Mycobacterium tuberculosis to PZA. Results generated by the BACTEC MGIT 960 system (Becton Dickinson) were compared with those obtained with the BACTEC 460TB system. Extensive proficiency testing (phase I) and reproducibility testing (phase II) as well as susceptibility testing of blinded strains of M. tuberculosis from the Centers for Disease Control and Prevention (phase III) were performed prior to testing 58 strains isolated from clinical specimens (phase IV). After resolution of discrepant results obtained by the two BACTEC methods by two other laboratories which acted as independent arbiters (phase V), overall agreement of the BACTEC MGIT 960 system with the BACTEC 460TB system for PZA testing of phase IV strains was 96.6%. Between the two systems there was no statistically significant difference in time until results were obtained, i.e., 6.8 days (BACTEC MGIT 960) versus 5.4 days (BACTEC 460TB), the latter not counting the time required for a subculture with a growth index of 200, however. The new BACTEC MGIT PZA susceptibility testing procedure works equally well for inocula prepared from liquid (MGIT) and solid (Löwenstein-Jensen) cultures. PZA MGIT medium in plastic tubes yielded results equivalent to medium dispensed in glass tubes.