Clinical value of the MeltPro MTB assays in detection of drug-resistant tuberculosis in paraffin-embedded tissues / 中华病理学杂志

Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.

Evaluation of the commercial kit SIRE nitratase for detecting resistant mycobacterium tuberculosis in Brazil

Abstract INTRODUCTION: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility of clinical Mycobacterium tuberculosis isolates. METHODS: The accuracy of the Kit SIRE Nitratase was evaluated by examining the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. RESULTS: The detection accuracy for streptomycin, isoniazid, rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. CONCLUSIONS: The exceptional accuracy demonstrated by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems / Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems.

The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.

Evaluation of mycobacteria growth indicator tube [MGIT] for drug susceptibility testing of mycobacterium tuberculosis

The importance of rapid availability of Mycobacterium tuberculosis [M. tuberculosis] antimicrobial susceptibility testing results is universally acknowledged. This study aimed to evaluate the performance and practicability of manual Mycobacteria Growth Indicator Tube [MGIT] in performing indirect susceptibility testing of M. tuberculosis. The reliability of manual MGIT for testing susceptibilities of 318 M. tuberculosis isolates to streptomycin [SM], isoniazid [INH], rifampin [RIF] and ethambutol [EMB] was evaluated in comparison with conventional indirect method of proportion [MOP]. MGIT detected overall drug resistance of; 50%, 59.4%, 40.3% and 37.1% as well as resistances among new cases of; 22%, 26.7%, 15.5% and 15.4% and resistances among treated cases of; 28%, 32.7%, 24.8% and 21.7% for SM, INH, RIF and EMB, respectively. Multi-drug resistant tuberculosis [MDR-TB] among new cases was 3.3% and that among treated cases was 12.4%. MGIT showed very good agreement with MOP susceptibility testing results [Kappa ranged from 0.7 to 0.82]. For MDR-TB detection there is 100% agreement between the two methods. The turnaround times [TAT] "from specimen processing to reporting of the antimicrobial susceptibility testing [AST] results" ranged between 10 and 30 days [mean=17.4] by the indirect MGIT method and 31 and 73 days [mean=48.9] by the indirect MOP. Manual MGIT appears to be a reliable, rapid and convenient method for performing indirect DSTs of M. tuberculosis in low-resource settings

Susceptibilidad de M. tuberculosis a drogas antituberculosas, determinada por dos técnicas, en el estado Sucre, Venezuela / usceptibility of M. tuberculosis to antituberculosis drugs as determined by two methods, in Sucre state, Venezuela

El objetivo de este trabajo fue evaluar la resistencia a isoniacida (INH), rifampicina (RIF), estreptomicina (STR) y etambutol (EMB) de 59 cepas de Mycobacterium tuberculosis, aisladas en el período agosto 2005-diciembre 2006, en el estado Sucre, Venezuela, empleando el método de proporciones de Canetti y de nitrato reductasa. Se encontró 6,3% de resistencia primaria y 14,3% de adquirida. Una cepa fue considerada MDR, al presentar resistencia a RIF e INH. Se comparó la prueba de nitrato reductasa con el método de las proporciones, encontrándose 100% de concordancia entre los resultados de los dos métodos para INH, RIF y EMB, y 95,65% para STR. Además, la prueba nitrato reductasa produjo resultados en 10 a 14 días, comparado con 42 días para el método de proporciones, por lo que la primera se postula como una alternativa muy valiosa para acortar el tiempo de respuesta en la valoración de la susceptibilidad de M. tuberculosis. La secuencia del gen rpoB en la cepa resistente a RIF demostró la presencia de una mutación no descrita anteriormente en la región hipervariable de 81 pares de bases, donde se ha reportado el mayor número de mutaciones de cepas resistentes a RIF. Esta mutación produjo un cambio en el codón 456 de TCG > CAG. Al comparar nuestros resultados con los hallados en el último estudio de prevalencia de resistencia realizado en el estado, se demuestra una disminución en la circulación de cepas resistentes en la zona de estudio.

The objective of this study was to evaluate the resistance to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (EMB), with the Canetti’s proportions method (PM) and the nitrate reductase assay (NRA) of 59 clinical strains of Mycobacterium tuberculosis, isolated in the period of august 2005 to december 2006, in Sucre state, Venezuela. Primary and acquired drug resistance was 6.3% and 14.3%, respectively. Only one strain was found to be multidrug resistant (MDR). The overall agreement between the NRA and PM was 100% for INH, RIF and EMB, and 96% for STR. The time to obtain results was 10 to 14 days for the NRA, compared to 42 days for the PM. The NRA was easy to perform and therefore represents a useful tool for rapid and accurate determination of drug-resistant M. tuberculosis. The sequence of the rpoB gene of the RIF resistant strain demonstrated a never described mutation (change in the codon 456; TCG > CAG) in the hypervariable region of 81 base pairs where most of the mutations of the RIF resistant strains have been reported. Comparison of our results with those of the last resistance prevalence study carried out in the years 1998-1999, shows a decrease in the studied area.

Novel mutations in emb B gene of ethambutol resistant isolates of Mycobacterium tuberculosis: a preliminary report.

BACKGROUND & OBJECTIVE: Ethambutol (EMB) resistance, thought to be occurring due to mutations in embB gene of Mycobacterium tuberculosis on the rise is a cause of grave concern. The present study was planned to investigate the presence of EMB resistance in M. tuberculosis isolates and to look for prevalent mutations in embB gene. METHODS: A total of 591(283 from new and 308 from previously treated cases) sputum samples from the same number of pulmonary tuberculosis cases were cultured. Isolates were tested by 1 per cent proportion method for resistance to isoniazid, rifampicin streptomycin and ethambutol. Minimum inhibitory concentration (MIC) of EMB was measured by absolute concentration method. Ten randomly selected isolates were subjected to single strand conformational polymorphism (SSCP) and direct DNA sequencing to look for mutation in 364 bp segments of embB gene. RESULTS: Of 353 isolates of M. tuberculosis from 591 sputum samples, 62 (17.58%) were resistant to EMB, of which, 16 (25.8%) showed initial resistance and 46 (74.2%) acquired. Mono resistance to EMB was rare. Only two isolates showed resistance to EMB alone. From 62 EMB resistant isolates, 88.7 per cent (55) were resistant to INH, 82.2 per cent (51) to rifampicin and 61.2 per cent (38) were resistant to streptomycin. Co-resistance to isoniazid and rifampicin (multidrug resistant, MDR-TB) with EMB resistance was seen in 41(66.1%) isolates. High level of EMB resistance was seen in 16.5 per cent isolates. SSCP showed altered mobility in 8 of 10 isolates tested. Among the 8 mutants, 4 had known mutations at codon Met 306 being replaced by Val/ Leu. The second most frequent mutation encountered was at codon Phe 287 being replaced by Val, Cys or Leu (novel mutations). Sequence analysis revealed 10 novel mutations in codon 221, 225, 227, 271, 272, 281, 282, 287, 293 and 294 within embB gene. INTERPRETATION & CONCLUSION: Presence of high frequency of EMB resistance, occurrence of high level EMB resistance, co-existence of MDR-TB with EMB resistance and novel mutations in emb B gene of M. tuberculosis clinical isolates reported highlight the need to work on larger samples to identify the diagnostic marker of EMB resistance in mycobacteria.

Progression of chronic pulmonary tuberculosis in mice intravenously infected with ethambutol resistant Mycobacterium tuberculosis.

Purpose: Ethambutol (EMB) is an important first line drug, however little information on its molecular mechanism of resistance and pathogenicity of resistant isolates is available. Present work was designed to study virulence of the EMB resistant M. tuberculosis strains and the host responses in-vivo on infection of EMB resistant M. tuberculosis using Balb/c mouse model of infection. Methods: Three groups of Balb/c mice (female, age 4-6 wk; 21 mice in each group) were infected intravenously with 106 CFU of M. tuberculosis H37Rv and two EMB resistant clinical isolates. Age and sex matched control animals were mock inoculated with Middlebrook 7H9 broth alone. At 10, 20, 30, 40, 50, 60, and 70 days post-infection three animals from each group were sacrificed by cervical dislocation and lung tissue was collected for further analysis. Results: Infection with EMB resistant M. tuberculosis led to progressive and chronic disease with significantly high bacillary load (p=0.02). Massive infiltration and exacerbated lung pathology with increased expression of IFN-gamma and TNF-alpha was observed in lungs of mice infected with EMB resistant strains. The present study suggests that infection with EMB resistant M. tuberculosis leads to chronic infection with subsequent loss of lung function, bacterial persistence with elevated expression of TNF-alpha resulting in increased lung pathology. Conclusion: These findings highlight that EMB resistant M. tuberculosis regulates host immune response differentially and its pathogenicity is different from drug sensitive strains of M. tuberculosis.

Comparative evaluation of BACTEC 460TB system and Lowenstein-Jensen medium for the isolation of M. tuberculosis from cerebrospinal fluid samples of tuberculous meningitis patients.

PURPOSE: To evaluate the role of the radiometric BACTEC 460TB system and the conventional Lowenstein-Jensen (LJ) medium for isolation of M. tuberculosis from cerebrospinal fluid (CSF) samples of tuberculous meningitis (TBM) patients. METHODS: CSF specimens (n=2325) from suspected TBM patients were processed for isolation of mycobacteria by inoculating BACTEC 12B medium and the LJ medium. The isolation of mycobacteria in both media was confirmed by microscopy and biochemical identification. Drug sensitivity testing for the anti-TB drugs was carried out by BACTEC radiometric method. RESULTS: Among the total 2325 CSF specimens processed by both methods, M. tuberculosis was isolated from 256 specimens. The isolation rates were 93% and 39% for the BACTEC system and LJ medium respectively. Both the media supported growth in 32% of the culture-positive specimens. BACTEC system alone yielded growth in 61% and LJ alone in 7%, of the culture-positive specimens. Among 205 isolates tested for drug susceptibility 81% were sensitive to all the drugs tested and 19% were resistant. CONCLUSIONS: The BACTEC 460TB system provides a highly sensitive and rapid tool for the isolation and drug susceptibility testing of M. tuberculosis, from CSF of TBM patients. Use of a solid medium in conjunction with the BACTEC 12B medium is essential for optimal recovery for M. tuberculosis from CSF specimens.

Declining prevalence of drug-resistant tuberculosis among HIV/tuberculosis co-infected patients receiving antiretroviral therapy.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) is a serious threat in developing countries where the prevalence of both HIV and TB are high. Antiretroviral therapy (ART) has been more accessible in these countries. The present study aimed to determine the impact of ART on the prevalence of DR-TB among HIV/TB co-infected patients. MATERIAL AND METHOD: A retrospective cohort study was conducted among HIV-infected patients with culture-proved TB from 1999 to 2004. Susceptibilities of Mycobacterium tuberculosis to antituberculous drugs and rate ofART use were studied. RESULTS: There were 225 patients, mean age 35.8 years, 72.4% male and median CD, 44 cells/mm(3). Patients who had received ART increased from 18.5% in 1999 to 92.1% in 2004 (p<O. 001). The prevalence of DR-TB in the years 1999 and 2004 were 48% and 7.9%, respectively (p<O.001). The prevalence of isoniazid- and rifampicin-resistance significantly declined in 2004 when compared with those in 1999 (p<O. 05). CONCLUSION: The declines in the prevalence of DR-TB, INH- and RFP-resistance in HIV/TB co-infected patients are possibly attributable to the use of ART In addition to the survival benefit from ART in HIV-infected patients, increasing use of ART among HIV-infected patients may eliminate DR-TB in this population.

Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT, NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90 percent. With most of the assays, sensitivity for ethambutol was low (62-87 percent) whereas for streptomycin, sensitivity values ranged from 84 to 100 percent; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff. MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.

Simultaneous ethambutol & isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVE: There is a need to understand the nature of drug resistance patterns and predictors of emergence of drug resistance in Mycobacterium tuberculosis. There could be common factors/mechanisms for resistance to the drugs, isoniazid and ethambutol, both acting on cell wall. The present study was conducted to analyze the antimycobacterial susceptibility patterns of M. tuberculosis isolates to determine the minimum inhibitory concentrations (MICs) of ethambutol for M. tuberculosis; and to find out possible association of ethambutol resistance with isoniazid resistance. METHODS: A total of 380 M. tuberculosis isolates were tested for their susceptibilities to ethambutol at 2, 4, 6 microg/ml, isoniazid at 1 microg/ml and rifampicin at 64 microg/ml using MIC method. RESULTS: 44.21, 24.73 and 14.21 per cent isolates were resistant to ethambutol at concentrations of 2, 4 and 6 microg/ml respectively. At 6 microg/ml of ethambutol concentration, 85.18 per cent ethambutol resistant isolates were resistant to isoniazid also. At the same ethambutol concentration a fraction of 28.75 per cent isoniazid resistant isolates were ethambutol resistant. INTERPRETATION & CONCLUSION: Ethambutol resistance was accompanied with isoniazid resistance in a large percentage of isolates whereas ethambutol resistance was weakly linked with multidrug resistance. On the other hand, association between isoniazid and ethambutol resistance was weak showing one way linkage.

E-Test: an alternative method for susceptibility testing of mycobacterium tuberculosis

The purpose of this study was to compare the agar proportion method with the E-test method for susceptibility testing of Mycobacterium tuberculosis. Materials and A total of 100 isolates were tested for isoniazid, rifampin, streptomycin and ethambutol susceptibility using an indirect-proportion method as well as the E-test method. Categorical agreement between the methods was 100% for isoniazid, rifampin, streptomycin, and ethambutol. The E-test method appears to be an alternative method to agar proportion for testing the susceptibility of M. tuberculosis isolates to the first-line antituberculous agents

Bioavailability of rifampicin following concomitant administration of ethambutol or isoniazid or pyrazinamide or a combination of the three drugs.

BACKGROUND & OBJECTIVES: Poor bioavailability of rifampicin (R) in combination with other anti-tuberculosis drugs such as isoniazid (H), pyrazinamide (Z), and ethambutol (E) is a subject of much concern for the last few decades. This could be due to an interaction between R and other drugs. An investigation was therefore undertaken to examine the bioavailability of R in the presence of H, Z and E or a combination of the three drugs. METHODS: The study included eight healthy volunteers, each being investigated on four occasions at weekly intervals once with R alone and with three of the four combinations on the three remaining occasions. A partially balanced incomplete block design was employed and the allocation of R or the drug combinations was random. Plasma concentrations of R at intervals up to 12 h were determined by microbiological assay using Staphylococcus aureus as the test organism. The proportion (%) dose of R as R plus desacetyl R (DR) in urine excreted over the periods 0-8 and 8-12 h was also determined. Bioavailability was expressed as an index (BI) of area under time concentration curve (AUC) calculated from the plasma concentrations or proportion of dose of R excreted as R plus DR in urine with the combinations to that with R alone. RESULTS: The bioavailability indices based on AUC were 0.96 with RE, 0.76 with RH, 1.08 with RZ and 0.65 with REHZ. The indices based on urine estimations (0-8 h) were similar, the values being 0.94, 0.84, 0.94 and 0.75, respectively. A second investigation revealed that the decrease of bioavailability of R with H was not due to the excipients present in H tablets. INTERPRETATION & CONCLUSION: Isoniazid alone or in combination with E and Z reduces the bioavailability of R. Urinary excretion data offer a simple and non invasive method for the assessment of bioavailability of R.

Prevalence of initial drug resistance to M. tuberculosis in new sputum positive RNTCP patients.

There is an increased prevalence of drug resistant M. tuberculosis strains and of these, multi drug resistant organisms are of particular concern. With the implementation of Revised National Tuberculosis Control Programme (RNTCP) allover the state of Delhi, Initial drug resistance (IDR) to Isoniazid and Rifampicin assumes great importance and needs to be monitored on a regular basis. We undertook to study the IDR against the first line essential drugs i.e. Isoniazid (H), Rifampicin (R), Ethambutol (E) and Streptomycin (S) from April 1999 to March 2000 in newly diagnosed sputum positive cases of pulmonary tuberculosis attending TB clinics under RNTCP in Delhi. A total of 157 consecutive new smear positive patients attending TB clinics under RNTCP were taken into the study. All sputum samples were subjected to culture and drug sensitivity tests on LJ medium after decontamination of samples by Petroff's method. Resistance was expressed as the percentage of colonies that grow on critical concentration of the drugs. To determine the proportion of resistance, the number of colonies on the control and the number of colonies on the drug medium were determined. A total of 94.77% samples were sensitive to the four first line essential drugs and IDR to any drug was 5.22%. The resistance to Rifampicin alone was nil but the resistance to Isoniazid alone was 2.24%. Combined resistance to both Rifampicin and Isoniazid was 2.98 %. The incidence of resistance to first line drugs in tuberculosis is not very high among new sputum positive patients attending TB clinics under RNTCP.

Drug resistance in tuberculosis in Delhi: a 2 year profile (2001--2002).

234 isolates of Mycobacterium tuberculosis obtained from 1000 suspected cases of tuberculosis reporting at National Institute of Communicable Disease, Delhi for laboratory investigation between Jan 2001 to August 2002 were subjected to invitro drug sensitivity test against the first line drugs (Isoniazid, Streptomycin, Rifampicin, Ethambutol and Thiacetazone) by proportion method using Lowenstein Jensen (LJ) media. Out of 234 isolates of Mycobacterium tuberculosis, 142 were from cases of untreated tuberculosis, whereas only 92 isolates were from treated cases of tuberculosis. An initial drug resistance of 21.83% was seen against INH, 9.85% against Streptomycin, 15.49% against Rifampicin, 4.22% against ethambutol and 2.11% to thiacetazone. Multidrug resistance (MDR-i.e. Resistance to both INH and Rifampicin) was seen in 11.97% of isolates. 4(2.8%) isolates were found to be resistant to all drugs tested. A much higher level of acquired resistance was seen the figures being 61.95% for INH, 53.36% for rifampicin, 35.86% for streptomycin, 20.65% for ethambutol and 10.86% for thiacetazone. Avery high acquired MDR to the tune of 42.39% was seen. 24(26%) isolates were found to be resistant to all drugs tested. No significant difference were observed in the drug resistance pattern between pulmonary and extrapulmonary cases of tuberculosis in both initial and acquired drug resistance category.

Reference to the Mutations in the embB Locus among Korean Clinical Isolates of Mycobacterium tuberculosis Resistant to Ethambutol

NO Abstract Available

Mutations in the embB Locus among Korean Clinical Isolates of Mycobacterium tuberculosis Resistant to Ethambutol

Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y322C, D331Y double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.

Standartization of broth microdilution method for Mycobacterium tuberculosis

Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100 per cent for all drugs and specifities of 91, 100, 96, 98 and 85 per cent were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days) than the proportional method (20-28 days). The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.