The 7H11 Agar Medium Supplemented with Calf Bovine Serum for Susceptibility Testing of Mycobacterium tuberculosis Isolates Against Pyrazinamide.

Despite its importance, pyrazinamide (PZA) is a blind spot in drug susceptibility testing in tuberculosis laboratories. The aim of this study was to set up a reliable agar-based proportion method for detection of PZA-resistant phenotypes using Middlebrook 7H11 agar supplemented with calf bovine serum (CBS) compared with albumin/dextrose/catalase (ADC) enrichment and pncA/rpsA sequencing results. The 7H11 agar medium supplemented with 10% ADC or 10% CBS (pH 6.2) and 100 µg/mL PZA was used to detect PZA resistance among 64 Mycobacterium tuberculosis isolates. Sanger sequencing and whole-genome sequencing were performed to track mutations in the pncA, rpsA, and their upstream regions. A total of 43 rifampicin/multidrug-resistant, 20 drug-susceptible, and 1 isoniazid mono-resistant M. tuberculosis isolates were investigated. The 7H11+ADC and 7H11+CBS could detect 22 and 23 PZA-resistant strains, respectively. With the same specificity, the sensitivity and accuracy of 7H11+CBS was found to be a little greater than 7H11+ADC in PZA resistance detection compared with sequencing results. Twenty-four mutant strains were found to have different mutations in pncA-upstream, pncA and rpsA genes, in which Gly97Asp was the most dominant mutation. The results obtained from 7H11+CBS were comparable to the results of 7H11+ADC. Therefore, the 7H11 agar proportion method would be a less-expensive test using CBS and produces reliable results.

Efficacy Of Line Probe Assay In Detection Of Drug-Resistant Pulmonary Tuberculosis In Comparison With GeneXpert And Phenotypic Methods In Iran And Genetic Analysis Of Isolates By MIRU-VNTR.

BACKGROUND: Successful treatment of tuberculosis depends on early diagnosis and use of appropriate drug susceptibility testing in a timely manner. In the present study, LPA efficacy was assayed in detection and drug susceptibility testing of pulmonary tuberculosis in comparison to available methods in Iran and phylogenetic analyses of isolated cases carried out by MIRU-VNTR. METHODS: This study was conducted at the Tehran Regional Reference Laboratory for Tuberculosis. All sputum specimens were subjected to smear, culture, and drug susceptibility testing (DST), GeneXpert, and LPA. Finally, 15-locus-based MIRU-VNTR was used for molecular genotyping. RESULTS: From a total of 920 sputum specimens, 6.08% (n=56) were identified as MTBC by culture, 6.8% (n=63) by GeneXpert, and 6.5% (n=60) by LPA. Phenotype DST and LPA methods confirmed the resistance of 4 and 14 specimens to rifampin (RIF) and isoniazid (INH); two cases were considered as multidrug-resistant (MDR). Using GeneXpert, four cases were identified as RIF-resistant. Based on LPA results, inhA and katG mutations were detected in 100% and 21.4% of INH-resistant cases, respectively. All 56 culture positive Mycobacterium tuberculosis isolates were placed in 29 different clusters using MIRU-VNTR genotyping. Two MDR-TB, 2 RIF mono-resistant, and 12 INH mono-resistant cases were placed in different clusters. CONCLUSION: LPA is an appropriate method for early detection and accurate diagnosis of TB and drug-resistant cases that makes it possible to distinguish INH mono-resistant cases from MDR cases in Iran.

Low diagnostic accuracy of Xpert MTB/RIF assay for extrapulmonary tuberculosis: A multicenter surveillance.

Diagnostic accuracy of Xpert MTB/RIF assay for pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB) has not been investigated in Iran. This study was aimed to assess the diagnostic accuracy of Xpert MTB/RIF assay for both PTB and EPTB. A total of 2111 clinical samples (1218 pulmonary and 838 extra-pulmonary) were collected from 16 medical centers during the study period and were analyzed for detection of PTB and EPTB by both Xpert MTB/RIF assay and standard conventional methods (culture and direct smear microscopy). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Xpert MTB/RIF assay for PTB were found to be 95.5%, 96.7%, 83.8%, and 99.1% respectively. For EPTB, the sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay counted for 76.5%, 95.9%, 62%, and 97.9% respectively. Xpert MTB/RIF assay found to be highly sensitive, specific and comparable to standard conventional methods for the diagnosis of PTB. However, the sensitivity and specificity of Xpert MTB/RIF for EPTB specimens were highly variable; thus, Xpert MTB/RIF cannot be recommended to replace standard conventional tests for diagnosis of EPTB.

Direct drug susceptibility testing of Mycobacterium tuberculosis using the proportional method: A multicenter study.

INTRODUCTION: Conventional indirect drug susceptibility testing (DST) of Mycobacterium tuberculosis with solid media is inexpensive and reliable, but time-consuming. This study aimed to evaluate direct DST for testing sputum samples without culture to significantly reduce the time required to detect multidrug-resistant tuberculosis (MDR-TB). METHODS: Direct and indirect DST of isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) were performed on 334 sputum smear-positive specimens. RESULTS: There was full agreement between the results obtained from direct testing and after isolation of the bacteria by culture. Thus, the sensitivity and specificity were observed to be 100% for all three tested drugs when compared with indirect DST. In comparison with indirect DST, none of the samples with the direct method took >25days to report the DST (between 15-25days with a mean detection time of 20 days). CONCLUSIONS: Direct DST on solid media was shown to give reliable results at a much earlier stage than conventional phenotypic DST. The direct method was found to be more rapid, more accurate and simpler. In addition, it reduced the handling of pathogenic bacteria and thus reduced the bio hazards related to conventional DST.

In Vitro Anti-mycobacterial Activity of Three Medicinal Plants of Lamiaceae Family.

BACKGROUND: Mycobacterium tuberculosis as an intracellular pathogen causes Tuberculosis (TB). Due to the long time required for treatment, hepatotoxicity of drugs and also emergence of Multidrug-Resistant (MDR) and Extremely Drug Resistant (XDR) strains, TB is currently a major public health problem. Some medicinal plants possess remarkable activity against Mycobacterium. Among them, Lamiaceae family are of pharmaceutical interest because of their potential antimicrobial properties. The aim of the study was to evaluate the in vitro activities of Satureja rechingeri, Satureja khuzestanica and Zataria multiflora against MDR M. tuberculosis and two Non-Tuberculous Mycobacteria (NTM). METHODS: The essential oils were prepared by the standard method. The confirmed strains were obtained from the microbial collection of Tehran University of Medical Sciences. Minimum Inhibitory Concentrations (MICs) of essential oils of plants against mycobacterial strains were determined using standard broth microdilution method. RESULTS: MDR M. tuberculosis was completely inhibited by Z. multiflora at 78µg/ml concentration. S. rechingeri and S. khuzestanica also showed same anti-mycobacterial activity against MDR M. tuberculosis with MICs of 156 µg/ml. The MICs of the essential oils against M. tuberculosis H37Rv, M. kansasii and M. fortuitum were in the range from 39 to 156 µg/ml. CONCLUSION: The studied medicinal plants showed notable effects against mycobacterial strains. Our results indicated that utilization of Lamiaceae family can be helpful for treatment of mycobacterial infections.

Feasibility of Colon Cancer Screening by Fecal Immunochemical Test in Iran.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in Iran, where there is no mass screening for the disease yet. We aimed to measure the feasibility of a pilot CRC screening program based on fecal immunochemical test (FIT) in Iranian population and the implications for scaling-up at the national level. METHODS: A single quantitative FIT was offered by health navigators to individuals aged between 45 and 75 years in primary health centers in rural and urban areas in Tehran. Participants who had a positive FIT were referred for colonoscopy. RESULTS: A total of 1044 asymptomatic average-risk individuals were enrolled. The mean age (SD) was 54.1 ± 7.0 years and nearly 63.0% (n = 657) were female. Only a small fraction of the participants had a prior screening practice (2.2%) and were aware of colon cancer (13.7%). In sum, 1002 returned the FIT kit, of whom the stool sample was unsatisfactory for testing in six participants (0.6%). The FIT uptake was 96.0%, positivity rate was 9.1% and the detection rates were 11.9% for adenomas and 7.1% for advanced adenomas. No cancer was detected. The positive predictive value (PPV) of the FIT was about 17% for any colonic neoplasms. CONCLUSION: This is the first study that reports minimal quality metrics within a CRC screening process. FIT modality as a test of choice for colon cancer screening in average-risk people is a safe and highly acceptable method of screening in Iranian people. The results of the current study may not be limited to Iranians, and could have implications to other developing countries with similar trends of CRC epidemic.

Molecular characterization of Mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping.

INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

Molecular characterization of mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping

Abstract: INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

Evaluation of current guthrie TSH cut-off point in Iran congenital hypothyroidism screening program: a cost-effectiveness analysis.

BACKGROUND: The threshold of thyroid-stimulating hormone (TSH) in current screening for congenital hypothyroidism (CH) from the heel prick test is 5 mU/l. This study uses cost-effective analysis to evaluate increasing the threshold to minimize false-positive results and recall rates. METHODS: Cost of screening, diagnosis and treatment, education, and care of mentally retarded patients were gathered from the Ministry of Health State Welfare Organization and Department of Education in Tehran. Screening data were obtained from 34,007 neonates in the Central Health Laboratory of Tehran University of Medical Sciences in 2009. Sensitivity analysis and calculation of confidence interval for incremental costs and effects (gained disability adjusted life years - DALYs) and incremental cost-effectiveness ratios (ICER) were performed by Monte Carlo simulation with Ersatz software. RESULTS: ICER for screening programs with different TSH cut-off points versus no screening was similar, and approximately -4.5 ± 0.2 thousand US dollars per gained DALY. In the proposed cohort (10,000 neonates), gained DALYs were 316 ± 50 for a cut off point of 5 mU/l, 251 ± 40 for 10 mU/l, 146 ± 23 for 15 mU/l, and 113 ± 18 for a cut-off point of 20 mU/l. Sensitivity analysis showed that the model remained the same when the input parameters were changed. CONCLUSION: This study demonstrates that the current threshold of TSH in the national CH screening program in terms of cost-effectiveness is the most appropriate threshold. However, more studies are needed to examine new strategies and methods to reduce recall rates and related consequences such as repeated thyroid testing in neonates.