Absolute Concentration Method Research Articles

Molecular Characterization of Drug-Resistant <i>Mycobacterium tuberculosis</i> Isolates in Guangdong, China

The aim of this study was to characterize 160 clinical Mycobacterium tuberculosis isolates from Guangdong with respect to their drug susceptibility phenotypes to three common anti-tuberculosis drugs, isoniazid (INH), rifampin (RIF) and streptomycin (SM), and with respect to genetic mutations in the most commonly corresponding resistance genes (katG, rpoB and rpsL). The drug susceptibility profiles were evaluated by the absolute concentration method, and genetic mutations in the corresponding resistance genes were identified by DNA sequencing. Among these isolates, 33.1% (53/160) were drug-resistant. The percentages of isolates resistant to INH, RIF and SM were 21.9% (35/160), 16.9% (27/160) and 15.6% (25/160), respectively. Twenty-five of 35 (71%) INH-resistant isolates, 22 of 27 (81.5%) RIF-resistant isolates and 19 of 25 (76%) SM-resistant isolates were found to have mutations in the analyzed katG, rpoB and rpsL gene fragments. The most frequent mutation patterns for the three drugs were as follows: INH, Ser315Thr (68.6%) in katG; RIF, Ser531Leu (55.6%) in rpoB; and SM, Lys43Arg (72%) in rpsL. These findings provide useful data on the mutation types of drug-resistant genes in M. tuberculosis isolates from Guangdong province in China.

Study of MTT and resazurin assays for susceptibility test to first-line anti-tuberculosis drugs

Objecflve To evaluate the performance of two rapid and low-cost metheds(MTT test,and rosazurin mierotitre assay)for the detection of resistance to first-line drugs in Mycobacterium tuberculosis.Methods sixty-four Myeobaeterium tuberculosis clinical isohtes were tested by the MTT test and the rosazuxin microtitre assay(REMA)respectively,and the results were compared with those obtained with the absolute concentration method on L(o)wenstein Jensen medium.Results The MTT test and the resazurin microtitre assay showed a good agreement compared with the absolute concentration method for all first-line drugs tested.The sensitibity,specificity and accuracy of the MTT test were 94.8%,96.0%,95.3%,for RFP;93.8%,93.8%,93.8% for INH;92.9%,96.O%,95.3% for EMB,90.6%,87.5%,89.1% for SM,respectively.The sensitivity,specificity and accuracy of the resazurin microtitre assay were 92.3%,96.0%,93.8%,for RFP;90.6%,90.6%,90.6% for INH;92.9%,94.0%,93.8% for EMB,87.5%,87.5%,87.5% for SM,respectively.The Kappa value of the MTT test and the absolute concentration method for the detection of resistance to RFP,INH,EMB,SM were 0.857,0.831,0.714,0.792.respeedvely;The Kappa value of the regazurin mierotitre assay and the absolute concentration method for the detection of resistance to RFP,INH,EMB,SM were 0.871,0.826,0.826,0.750,respectively.The Kappa value of the MTT test and the resazurin microtitre assay for the detection of resistance to RFP,INH,EMB,SM wefe 0.889,0.875.0.787,0.844,respectively.Conclusions Both MTT test and the resazurin microtitre assays are simple,rapid,low-cost and sensitive for rapid detection of resistance to first-line drugs.They could be promising methods for susceptibility assay of the first-line antituberculosis drugs in low-resource countries. Key words: Mycobacterium tuberculosis; Antitubercular agents; Microbial susceptibility tests; Tetrazolium salts; Oxazines; Xanthenes

Clinical features and therapeutic efficacy in patients with pulmonary multidrug-resistant cavitary tuberculosis at Russian penitentiary system

The aim of this study was to analyze the course of the disease and efficacy of treatment of pulmonary multidrugresistant (MDR) cavitary tubercu losis in antituberculosis settings of Russian penitentiary system. Drug resistant was studied using the method of absolute concentrations. Among 190 males with MBTpositive cavitary pulmonary tuberculosis followedup in antituberculosis hospitals of Russian penitentiary system in Voronezh in 2003–2006, 79 patients (41.6 ± 3.4 %) were yielded MDR strains resistant to the firstline antimucobacterial drugs and 111 patients (58,4 ± 3,4 %) had strains resistant both to the firstline and the secondline drugs (isoniazid and rifampicin). Moreover, MBT strains resistant to cycloserine and quinolones were found in patients who have not been previously treated with these drugs. Both newly diagnosed patients with pulmonary tubercu losis and those with recurrent cavitary pulmonary tuberculosis had a high rate of primary and secondary MDR to the firstline and the secondline antimicobacterial drugs. Administration of I and IIA chemotherapeutic regimens in these patients resulted in occurrence of additional resistance to a wide spectrum of the firstline and the secondline antibiotics. Patients with chronic pulmonary tuberculosis are at high risk of MDR to combi nations of the firstline and the secondline drugs and need other treatment strategies including surgical interventions. Tuberculosis patients with MDR to combinations of the firstline and the secondline drugs were characterized by more severe chronic course of the disease with extended lesions and large cavities in the lungs. Adverse events to the secondline drugs were seen in 42.6 % of the patients with cavitary tuberculosis. Of them, serious adverse events requiring cancellation of the drug and change in chemotherapeutic regimen were noted in 17.9 % of cases.

Study of mycobacterium tuberculosis in patients with pulmonary tuberculosis by drug resistance gene detecting

Objective To investigate drug resistance of mycobacterium tuberculosis,compare detecting effect of two methods and evaluate their value of clinical application.Methods All of the strains of mycoba-cterium tuberculosis were tested for resistance to RFP,INH,SM,PZA and EMB by the absolute concentration method on lowenste in-jensen medium and the mutation of the rpoB,katG,rpsL,pncA and embB resistanc genes in mycobacterium.Tuberculosis was tested by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP).Results 400 cases of tuberculosis drug resistance in patients with conventional susceptibility test resistance 316 cases (79.8%);resistant strains resistant gene detection 288,the mutation rate(72.0%):the RFP cases of resistance and resistance was significantly higher than that of SM,PZA and the EMB(resistant cases:F=2.45,2.56,2.69,P＜0.05;ResNtance:F=2.55,2.66,2.79,P＜0.05);the mutants and mutation rate of rpoB was higher than that of katG,rpsL,rpoB and embB(mutant:F=2.28,2.46,3.19,3.33,P＜0.05～O.01;mutation rate:F=2.36,2.61,3.25,3.45,P＜0.05～0.01);the gene mutation rate was higher in strains isolated from high concentration resistance than those in strains isolated from low concentration resistance;the more irregular treatment was longer,the rate of drug-resistant was higher(resistance:F=2.77,2.88,P＜0.05;mutation rate:F=2.72,2.85,P＜0.05).Conclusion PCR-SSCP is a sensitive and specific method for rapid detecting rpoB,katG,rpsL,pncA and embB gene mutations of mycobacterium tuberculosis. Key words: Mycobacterium tuberculosis; Drugresistance; Drug resistancegene; Polymerase chain reaction

Evaluation of pyrosequencing for the detection of rpoB gene mutation in Mycobacterium tuberculosis

To detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates. Using the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC). Among the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively. Not only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

Comparison of Anti-mycobacterial Drug Susceptibility Test Results by Institutes and Methods

Background: The purposes of the current study were to evaluate the concordant rates of anti-mycobacterial drug susceptibility test (DST) results in different solid media performed in different institutes, and to determine reliable susceptible testing methods. Methods: One hundred and twenty two Mycobacterium tuberculosis strains were isolated from patients in A Hospital in 2005. DSTs were performed by the absolute concentration method using Lowenstein Jensen medium in both A Hospital (method A-1) and B Institute (method B-1) and by the proportion method using Middlebrook 7H10 agar in B Institute (method B-2). Nine drugs were used including isoniazid and rifampin. Sensitivity and specificity of each method were estimated by using the acceptable standard of 90% for isoniazid and rifampin and 80% for other drugs. The therapeutic outcomes of quinolone-administered patients were evaluated according to ofloxacin susceptibility results. Results: Method B-1 showed sensitivity and specificity levels over the acceptable standard levels for all drugs. Method B-2 showed specificity lower than the acceptable levels for rifampin and cycloserine. Method A-1 showed specificity lower than the acceptable levels for isoniazid, streptomycin, p-aminosalicylic acid, and ofloxacin and sensitivity lower than the acceptable levels for prothionamide and cycloserine. The concordance rates of therapeutic outcomes with method B-1, method B-2, and method A-1 were 77%, 74%, and 65%, respectively. Conclusion: The drug susceptibility results for some drugs were discordant between the testing laboratories and media, requiring an urgent application of quality control programs to raise the reliability of anti-mycobacterial DST. (Korean J Clin Microbiol 2008; 11:43-48)

Results of the use of biochips to determine the drug resistance of Mycobacterium tuberculosis in the Novosibirsk and Tomsk regions

To evaluate the possibility of determining rifampicin resistance using a biochip developed at the Institute of Molecular Biology of the Russian Academy of Sciences (Moscow), 272 rifampicin-resistant isolates of M. tuberculosis, isolated from tuberculosis patients in the Novosibirsk and Tomsk regions in 2000–2005, were investigated. Rifampicin resistance was determined by the method of absolute concentrations on Lowenstein-Jensen medium; mutations in the rpoB gene were determined with the biochip and simultaneously by the SSCP method. Moreover, the variable region of the rpoB gene was sequenced in 60 strains of a randomized sample. In 86% of the cases, the results of the determination of rifampicin resistance by the method of absolute concentrations and with the biochip were in full agreement. The results differed in cases when cultures isolated from a patient contained a mixture of sensitive and resistant strains. Four main codons in which mutations can cause resistance to rifampicin were detected: S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). Resistance to isoniazid was also established in the region among 94% of the rifampicin-resistant strains, i.e., they were resistant to a number of drugs. Consequently, the biochip for the determination of rifampicin resistance can also be used to determine multiple-drug-resistant (MDR) strains. The use of the biochip to diagnose rifampicin resistance is significantly simplified if the two steps of the polymerase chain reaction (PCR) are replaced by one-step variant, in which a single-stranded labeled PCR fragment is synthesized according to a “nested PCR” scheme in one test tube.

Extensively Drug-ResistantMycobacterium tuberculosis, India

Extensively Drug-Resistant<i>Mycobacterium tuberculosis</i>, India

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong

ObjectivesTo characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA).MethodsThe MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products.ResultsResistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci.ConclusionsThese findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

P1643 Comparison of quality of drug susceptibility testing of Mycobacterium tuberculosis by absolute concentration method and BACTEC 460

P1643 Comparison of quality of drug susceptibility testing of Mycobacterium tuberculosis by absolute concentration method and BACTEC 460

The pattern of anti-tuberculosis drug resistance in pulmonary tuberculosis patients

Background: Tuberculosis is one of the major public health problems in Nepal. Drug resistant tuberculosis has posed a serious challenge in TB control program. Drug resistance tuberculosis is the most difficult for the treatment and cure of the patient. Objective: To find out the current situation of drug resistant pattern at national tuberculosis center, Nepal. Design: Central Department of Microbiology, Tribhuvan University in collaboration with NTC, STC, conducted a prospective study. Methods: One hundred ninety-nine Mycobacterium tuberculosis isolated from the sputumin national tuberculosis center (NTC) were studied to find out the drug resistant pattern by utilizing absolute concentration method. Results: Of the 199 culture positive strains were tested, 161from previously treated cases and 38 from new cases. Overall primary and acquired resistance were 36.82%and 75.05%respectively. The study showed that the Primary drug resistance (PDR) to one drug was found to be 23.67%, to two drugs was 10.52%, to three drugs was 0%, and four drugs was 2.63%where as acquired drug resistance (ADR) to one drug was 32.38%, to two drug was 19.19%, to three drugs was 11.78%, to four drugs was 11.80%. Primary and acquired multi-drug resistance (MDR)) was in 2.63%and 19.25% of the isolates respectively Conclusion: Overall, drug resistance of M. tuberculosis is high. It is concluded that regular monitoring of MDR–TB and making policy according to research finding is necessary as it is changing every year, which helps to control MDR-TB. Journal of Institute of Medicine Vol.27(3) 2005

Susceptibility of Mycobacterium tuberculosis Strains to Gatifloxacin and Moxifloxacin by Different Methods

Background: Considering the potentials of gatifloxacin and moxifloxacin in the treatment of tuberculosis, the present study was aimed to define resistance to both these drugs. Methods: Fifty Mycobacterium tuberculosis isolates, consisting of 30 ofloxacin-susceptible and 20 ofloxacin-resistant strains, were tested for their susceptibility to gatifloxacin and moxifloxacin using different susceptibility testing methods, namely the absolute concentration method on Lowenstein-Jensen medium (LJ), the proportion susceptibility testing method (PST) on LJ and 7H11 agar media, and the BACTEC radiometric method. Result: The minimal inhibitory concentration (MIC) of gatifloxacin and moxifloxacin was 1 µg/ml by the absolute concentration method on LJ. In the PST method on LJ and 7H11, using a criterion of ≧1% growth as resistant, there was 100% agreement with the absolute concentration method at a concentration of 0.5 µg/ml for gatifloxacin, and 96% agreement with the BACTEC method at a concentration of 0.25 µg/ml. For moxifloxacin, results by the PST method showed 96% agreement with the absolute concentration method on LJ at a concentration of 1 µg/ml and 92% agreement at a concentration of 0.5 µg/ml for both the absolute concentration method on 7H11 and the BACTEC method. Conclusions: The MICs of gatifloxacin and moxifloxacin were much lower than the MICs of other quinolones like ofloxacin and ciprofloxacin. Additionally, these two drugs have shown a low mean MIC and low concentration as a definition of resistance, which might help in treating the patients with low levels of quinolone resistance.

The Current Status of Multidrug-resistant Tuberculosis in Korea

Purpose : Multidrug-resistant tuberculosis (MDR-TB) is an emerging threat to human beings. However, there is little data on the current status of MDR-TB in Korea. This study investigated the current status of MDR-TB in Korea using a survey of all the data from drug susceptibility tests (DST) performed across the country over the last three years. Method : The DST results between Jan. 2000 and Dec. 2002 from 7 laboratories, which were in charge of all antituberculous DSTs across the country as of March 2002, were collected and analyzed to determine the actual number of drug-resistant or MDR-TB patients, annual trend, degree and pattern of resistance against anti-TB drugs, etc. Results : Six laboratories used the absolute concentration method for DST and one used the proportional method. 59, 940 tests had been performed over the 3 year study period. The number of DST performed annually was 18,071, 19,950, and 21,919 in 2000-2002, respectively. The number of resistant tuberculosis patients (resistant against at least one anti-TB drug) had increased by 16.9% from 6,338 in 2000 to 7,409 in 2002. The rate of resistant tuberculosis among all DST results was 35.1% in 2000, 34.5% in 2001, and 33.8% in 2002. The number of MDR-TB patients (resistant against at least both isoniazid and rifampin) showed an increasing trend (14.5%) from 3,708 in 2000 to 4,245 in 2002. Conclusion : Approximately 4,000 MDR-TB cases are newly identified by DST annually and the number is showing an increasing trend. This study suggests that in order to cope with the current MDR-TB situation, the DST methods will need to be standardized and more aggressive measures will be required.

Study on rapid detective technique for streptomycin resistance in Mycobacterium tuberculosis by phage amplified biologically assay

目的 建立结核分枝杆菌(结核菌) 链霉素耐药性的噬菌体快速检测技术,并探讨其临床应用价值.方法应用分枝杆菌噬菌体感染结核菌建立链霉素耐药性的噬菌体检测技术,并对最佳测定条件进行探讨;将所建立的方法用于38株世界卫生组织药敏质控株和372株临床分离株链霉素耐药性测定,并与绝对浓度法和Bactec 960药敏结果比较,对不符合菌株进行最低抑菌浓度(MIC)和rpsL耐药基因检测.结果链霉素终浓度5 μg /ml作用24 h、噬菌体108噬菌体形成单位/ml感染60 min、杀毒剂5%室温作用5 min为选择的检测条件.噬菌体法检测38株药敏质控株结果完全符合.372株临床分离株链霉素耐药性检测结果表明,如以绝对浓度法药敏结果为判断标准,则本法敏感性为97.4%、特异性为91.0%、阳性预测值为81.7%、阴性预测值为98.9%、准确性为92.9%;如以Bactec 960药敏结果为判断标准,则本法敏感性为97.2%、特异性为97.1%、阳性预测值为94.6%、阴性预测值为98.5%、准确性为97.1%.有22株噬菌体法药敏结果与常规药敏试验结果不符,其中的11株噬菌体法检测结果与MIC 和rpsL耐药基因检测结果相同.结论噬菌体法检测链霉素耐药性快速、简便,具有很高的敏感性和特异性,可作为结核菌链霉素耐药性的快速检测方法。

In Vitro Activity of Fluoroquinolones against Mycobacterium tuberculosis

The In Vitro activity of fluoroquinolones, including lomefloxacin, ofloxacin, ciprofloxacin, sparfloxacin, moxifloxacin and gatifloxacin, was evaluated against 55 clinical isolates of Mycobacterium tuberculosis by absolute concentration method on Lowenstein-Jensen (L-J) and Middlebrook's 7H11 media. Both ofloxacin susceptible and ofloxacin resistant strains of M. tuberculosis isolates were tested. The in vitro activities of these fluoroquinolones on the M. tuberculosis isolates were in the order: lomefloxacin<ciprofloxacin≤ofloxacin<sparfloxacin<moxifloxacin=gatifloxacin. Gatifloxacin and moxifloxacin showed low minimal inhibitory oncentrations (MIC) for both ofloxacin resistant and ofloxacin susceptible strains even though some cross resistances were observed.

Drug resistance in tuberculosis

A drug-resistant strain of Mycobacterium tuberculosis is defined as one differing from the tight distribution of wild strains that have not come into contact with the drug concerned. Sensitivity tests are performed by the absolute concentration method, the resistance ratio method or the proportion method. The hypothesis underlying the proportion method is that there are appreciable differences in inoculum size so that there should be an association between the proportion on drug-free medium and the proportion on drug-containing medium. This hypothesis was not supported by a study on ethionamide-resistant strains. It indicated that variation in the proportion on drug-free medium was due to clumping of the bacilli in the inoculum rather than to differences in the number of bacilli. Hence, the use of the proportion method introduces errors in susceptibility testing. While the method can produce reliable results, it is more time consuming than a minimal inhibitory concentration determination, and should not be adopted as a standard method.

The Proportion of Rifabutin-susceptible Strains among Rifampicin-resistant Isolates and Its SpecificrpoBMutations

Background : Rifabutin (ansamycin) is a spiro-piperidyl rifamycin, which is highly active against Mycobacterium tuberculosis. It has been found that some clinical isolates of tubercle bacilli that are resistant to rifampicin are susceptible to rifabutin, with some patients with multi-drug resistant pulmonary tuberculosis having shown favorable clinical and bacteriological responses to the rifabutin. This study was conducted to find the proportion of rifabutin-susceptible strains among rifampicin-resistant isolates from Korean MDR-TB patients, and investigate the presence of specific rpoB mutations, which may confer resistance to rifampicin, but not to rifabutin. Methods : 201 rifampicin-resistant and 50 pan-susceptible M. tuberculosis isolates were randomly selected for this study. The isolates were retested at rifampicin and rifabutin concentrations of 0, 20, 40 and , respectively. The isolates that grew at and/or over a rifabutin concentration of were judged rifabutin-resistant. The rpoB gene was extracted from the isolates, and then amplified for direct sequencing to investigate specific rpoB mutations that conferred rifabutin- susceptibility but rifampicin-resistance. Results : Out of the 201 rifampicin-resistant M. tuberculosis, 41 strains (20.4%) were susceptible to rifabutin using the absolute concentration method on Lowenstein-Jensen media. The rpoB mutation types that showed susceptibility to rifabutin were Leu511Pro, Ser512Arg, Gln513Glu, Asp516Ala, Asp516Gly, Asp516Val, Asp516Tyr, Ser522Leu, His526Asn, His526Leu, His526Cys, Arg529Pro and Leu533Pro. A reverse hybridization technique was able to detect 92.5% of the rifabutin-susceptible isolates, with a specificity of 96.1% among 195 M. tuberculosis isolates with the rpoB mutation. Conclusions : Around 20% of the rifampicin-resistant isolates in Korea showed susceptibility to rifabutin, which was associated with some specific mutations of rpoB. Rifabutin could be used for the treatment of MDR-TB patients, especially when drug susceptibility testing reveals susceptibility to rifabutin.

The Study of the Susceptibility of M. Tuberculosis to Rifampin by Micro-well Phage Replication Assay

目的 应用并评价微孔噬菌体扩增法(micro-well phage replication assay,MPRA)快速检测临床结核分枝杆菌分离株对利福平的敏感性.方法将适当浓度的利福平溶液作用于结核分枝杆菌临床分离株,一定时间后,加入噬菌体悬液.应用MPRA法检测利福平药物的敏感性.结果应用绝对浓度法对利福平敏感的42 株菌株中38株MPRA法敏感;绝对浓度法对利福平耐药的131株菌株中124株MPRA法耐药.MPRA法与绝对浓度法的符合率为93.6%(162/173),与绝对浓度法相比,MPRA 法的敏感度为94.7%(124/131),特异度为90.5%(38/42).结论 MPRA法不失为一种快速检测结核分枝杆菌对利福平敏感性的较好方法。

Detection of Pyrazinamide Resisitance in Mycobacterium Tuberculosis by Sequencing of pncA Gene

Background : Examining the biological susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) in vitro is very difficult as PZA is inactive under normal culture conditions. The biological susceptibility test, an enzyme assay for Pzase activity, and a genetic test for pncA gene mutations, were performed in order to predict PZA resistance. Methods : 28 cultured clinical isolates of Mycobacterium tuberculosis were tested. The biological susceptibility was performed by the absolute concentration method using Lowenstein-Jensen media. The PZase activity was tested by means of Wayne's method. A 710-bp region includes the entire open reading frame of pncA was amplified and sequenced. Results : All six strains with positive PZase activity exhibited no pncA mutations with one strain showing a false resistance in the biological susceptibility test. Among the 22 strains with no PZase activity, 21 exhibited showed pncA mutations. In the biological susceptibility test, 20 strains were resistant, and one was susceptible, and the other flied to test. The mutation types varied with ten missense, one silent and one nonsense mutation 1 slipped-strand mispairing, and 6 frameshift mutations. Three strains had an adenine to guanine mutation at position -11 upstream of the start codon. Conclusion : The mutation at the pncA promotor region is frequent at -11 upstream position. Automatic sequencing of pncA is a useful tool for rapid and accurate detection of PZA resistant M. tuberculosis, and for demonstrating the epidemiological relatedness of the PZA resistant M. tuberculosis strains.

Comparison of the in vitro activities of rifapentine and rifampicin against Mycobacterium tuberculosis complex.

The in vitro activity of rifapentine for 44 clinical isolates of Mycobacterium tuberculosis complex was compared with that of rifampicin using the Bactec radiometric method and the absolute concentration method for susceptibility testing. Twenty-nine M. tuberculosis, 11 Mycobacterium bovis and four Mycobacterium africanum strains were studied. Control tests showed that rifapentine was stable for 14 days in 7H9 broth and for 3 weeks in 7H10 agar medium. The 44 M. tuberculosis complex strains were more susceptible to rifapentine than to rifampicin, irrespective of the testing method. In the radiometric system, the MIC50 and MIC90 of rifapentine for M. tuberculosis complex strains were one or two two-fold dilutions lower than those of rifampicin (0.06-0.125 mg/L versus 0.25 mg/L, respectively). By the absolute concentration method, the MIC50 and MIC90 of rifapentine for M. tuberculosis complex strains were two two-fold dilutions lower than those of rifampicin (0.125-0.25 mg/L versus 0.5-1 mg/L, respectively). The MIC90 of rifapentine for the 44 M. tuberculosis complex strains was always 0.25 mg/L, irrespective of the method used, but the radiometric method was more reliable and more reproducible than the agar 7H10 method.