Anti-tuberculosis Drug Susceptibility Testing Research Articles

Proficiency of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China, 2008-2021.

To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency testing performed DST, and the sensitivity, specificity, reproducibility, and accordance rate were calculated from data of 13 rounds proficiency testing results for DST from 2008 to 2021. A total of 30 and 20 strains of Mycobacterium tuberculosis with known susceptibility results were sent to each laboratory in 2008 to 2019, 2020 and 2021, respectively. The number of participating laboratories ranged from 30 in 2009 to 546 in 2021. L-J DST was the predominant method. The specificity presented relatively higher than sensitivity. Improvement of specificity were observed for all drugs through the years, while sensitivity did not show improvement for amikacin and capreomycin. Accordance rate of pyrazinamide and kanamycin and reproducibility of capreomycin and pyrazinamide were not significantly improved through the years. Most of the participating laboratories significantly improved the quality of their DST through the consecutive rounds of proficiency testing except for second-line injectable drugs and pyrazinamide. The results highlight the importance of developing novel and/or improving existing methods for phenotypic DST for certain drugs.

Comparative evaluation of critical concentrationsfor detecting borderline rifampin resistance in Mycobacterium tuberculosis

Background: Rifampin plays an important role in tuberculosis treatment. In recent years, the introduction of molecular testing techniques has enabled the rapid detection of rifampin resistance, leading to discrepancies with conventional methods. The World Health Organization (WHO) has analyzed mutations in the rpoB gene that induce rifampin resistance and identified certain mutations causing borderline resistance, which are often undetected using conventional tests. Consequently, the WHO has lowered the rifampin resistance criterion concentration from 1.0 to 0.5 μg/mL in 7H10 and MGIT 960. The present study aimed to evaluate the impact of this change in critical concentration on the detection of borderline rifampin resistance in Mycobacterium tuberculosis. Methods: Tuberculosis strains submitted for antituberculosis drug susceptibility testing from May 2021–2022 were analyzed. Three institutions participated; the Seoul Clinical Laboratories used the agar proportion method, whereas the Samsung Medical Center and Seoul National University Bundang Hospital utilized the MGIT 960 system to test both the original and revised concentrations. Mutations were confirmed through rpoB gene sequencing for strains showing discrepancies. Results: A total of 1,596 valid susceptibility tests were conducted during the study period. Rifampin resistance was detected in 35 cases (2.19%) at 1.0 μg/mL and in 38 cases (2.38%) at 0.5 μg/mL, whereas isoniazid resistance was observed in 158 cases (9.90%). Among the three rifampin discrepancy strains, one harbored an H445L mutation, whereas the remaining two exhibited an L452P mutation. These mutations were classified as borderline resistant. Conclusion: Applying the new rifampin critical concentration resulted in a 0.19% increase in resistance rate and an 8.57% increase in detection cases. Additionally, despite testing with large number of rifampin-susceptible strains, no false resistance results were obtained. Therefore, the application of the new critical concentration is considered beneficial for the management of rifampin-resistant tuberculosis.

Tuberculosis drug resistance in Southern Mozambique: results of a population-level survey in the district of Manhiça.

Mozambique, one of the world's high tuberculosis (TB) burden countries, has conducted only one national-level drug resistance survey, in 2007-2008. To determine the drug resistance patterns of laboratory-confirmed TB cases. This was a population-level survey conducted over a 1-year period in the district of Manhiça. All laboratory-confirmed cases were evaluated for first-line anti-tuberculosis drug susceptibility testing using liquid culture. Resistance to at least one first-line drug was observed in 44 of 276 isolates (15.9%). Prevalence of drug resistance to each of the five anti-tuberculosis drugs tested was 4.0% for streptomycin, 10.1% for isoniazid (INH), 6.2% for rifampicin, 3.6% for ethambutol and 1.1% for pyrazinamide. The overall prevalence of multidrug-resistant TB (MDR-TB) was 5.1%: 3.8% (95%CI 2.0-7.0) in new and 13.2% (95%CI 5.8-27.3) in retreatment cases. Respectively 4.6% and 2.6% of new and retreatment cases were INH-monoresistant. Previous history of anti-tuberculosis treatment was associated with having MDR-TB (OR 4.3, 95%CI 1.3-14.1). The prevalence of drug resistance in the district of Manhiça is slightly higher than, but still compatible with, previous national estimates. INH monoresistance was high, posing the risk of hidden monotherapy in the continuation phase.

Risk factors associated with multidrug-resistant tuberculosis in Espírito Santo, Brazil.

ABSTRACTOBJECTIVE To analyze the prevalence and factors associated with multidrug-resistant tuberculosis in Espírito Santo, Brazil.METHODS This is a cross-sectional study of cases of tuberculosis tested for first-line drugs (isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin) in Espírito Santo between 2002 and 2012. We have used laboratory data and registration of cases of tuberculosis – from the Sistema Nacional de Agravos de Notificação and Sistema para Tratamentos Especiais de Tuberculose. Individuals have been classified as resistant and non-resistant and compared in relation to the sociodemographic, clinical, and epidemiological variables. Some variables have been included in a logistic regression model to establish the factors associated with resistance.RESULTS In the study period, 1,669 individuals underwent anti-tuberculosis drug susceptibility testing. Of these individuals, 10.6% showed resistance to any anti-tuberculosis drug. The rate of multidrug resistance observed, that is, to rifampicin and isoniazid, has been 5%. After multiple analysis, we have identified as independent factors associated with resistant tuberculosis: history of previous treatment of tuberculosis [recurrence (OR = 7.72; 95%CI 4.24–14.05) and re-entry after abandonment (OR = 3.91; 95%CI 1.81–8.43)], smoking (OR = 3.93; 95%CI 1.98–7.79), and positive culture for Mycobacterium tuberculosis at the time of notification of the case (OR = 3.22; 95%CI 1.15–8.99).CONCLUSIONS The partnership between tuberculosis control programs and health teams working in the network of Primary Health Care needs to be strengthened. This would allow the identification and monitoring of individuals with a history of previous treatment of tuberculosis and smoking. Moreover, the expansion of the offer of the culture of tuberculosis and anti-tuberculosis drug susceptibility testing would provide greater diagnostic capacity for the resistant types in Espírito Santo.

Sensititre® MYCOTB MIC plate for drug susceptibility testing of Mycobacterium tuberculosis complex isolates.

National Tuberculosis Clinical Laboratory, China. To evaluate the accuracy and feasibility of the MYCOTB MIC plate in anti-tuberculosis drug susceptibility testing. MYCOTB testing of Mycobacterium tuberculosis isolates, the Löwenstein-Jensen (LJ) proportion method and the resurazine microtitre assay (REMA), which is extensively used for in-house assay for minimal inhibition concentration (MIC) testing, were performed simultaneously for comparison. A total of 126 clinical isolates were tested using both MYCOTB and the LJ proportion method against 12 anti-tuberculosis drugs; 80 were also tested using REMA. Categorical agreement between MYCOTB and the LJ proportion method was 99.2% for rifampicin, ofloxacin, amikacin, kanamycin and cycloserine, and 98.4% for isoniazid and para-aminosalicylic acid; ethambutol (EMB) had the lowest agreement (86.5%). The overall categorical agreement between MYCOTB and REMA ranged between 98.8% and 100%. MYCOTB outcomes, interpreted on day 10 and 21, were stable for all drugs except EMB. MYCOTB is a rapid, convenient, quantitative and accurate method for testing both first- and second-line anti-tuberculosis drugs.

Evaluation of the GenoType® MTBDRsl assay in Korean patients with MDR or XDR tuberculosis

Background This study used the GenoType® MTBDRsl assay (MTBDRsl, Hain Lifescience, Nehren, Germany), an assay for anti-tuberculosis drug susceptibility testing, for the detection of resistance of 40 tuberculosis strains to fluroquinolones (FLQ), injectable drugs (amikacin or capreomycin) and ethambutol to identify multi-drug resistant and extensively drug-resistant tuberculosis patients previously identified by solid drug susceptibility test (DST). Methods These tuberculosis strains were analysed by both DNA sequencing and conventional drug susceptibility testing on solid medium. Results The overall agreement rates of the MTBDRsl assay and phenotypic drug susceptibility testing for the detection of ofloxacin (OXF), moxifloxacin (MXF), amikacin (AMK), capreomycin (CPM) and ethambutol (EMB) susceptibility in clinical strains were 87.5% (35/40), 87.5% (35/40), 97.5% (39/40), 60.0% (24/40) and 65.0% (26/40), respectively. Conclusions This study reconfirmed 37 extensively drug-resistant tuberculosis strains using the MTBDRsl assay and these strains were verified by phenotypic drug susceptibility testing. After identification with the MTBDRsl assay, the results were analysed by DNA sequencing with specific primers. From the various mutations identified in the DNA sequencing results, this study found new mutations that characterised EMB-resistant strains, namely, R507K and G406T mutations, which had not been previously reported.

Sensititre MycoTB plate compared to Bactec MGIT 960 for first- and second-line antituberculosis drug susceptibility testing in Tanzania: a call to operationalize MICs.

MIC testing for Mycobacterium tuberculosis is now commercially available. Drug susceptibility testing by the MycoTB MIC plate has not been directly compared to that by the Bactec MGIT 960. We describe a case of extensively drug-resistant tuberculosis (XDR-TB) in Tanzania where initial MIC testing may have prevented acquired resistance. From testing on archived isolates, the accuracy with the MycoTB plate was >90% for important first- and second-line drugs compared to that with the MGIT 960, and clinically useful quantitative interpretation was also provided.

A summary of tuberculosis drug resistance in Canada, 2003−2013

Drug-resistant strains of tuberculosis (TB) pose a serious threat to prevention and control efforts. In response to this growing worldwide concern, the Public Health Agency of Canada (PHAC) established and maintains the Canadian Tuberculosis Laboratory Surveillance System (CTBLSS) in partnership with the Canadian Tuberculosis Laboratory Technical Network (CTLTN) and participating laboratories. To report on national trends and patterns in anti-tuberculosis drug resistance in Canada for the years 2003 to 2013. At the beginning of each calendar year, participating laboratories submit to PHAC reports on the results of anti-tuberculosis drug susceptibility testing for all isolates tested during the preceding year. These data are then analyzed by PHAC and the results are validated by supplying laboratories. The results are published annually as the Tuberculosis Drug Resistance in Canada series. In 2013, anti-tuberculosis drug susceptibility test results for 1,380 isolates were reported to PHAC. Of these, 762 (54%) were reported as Mycobacterium tuberculosis complex (MTBC) where the species was known. Two thirds (68%) of all the reported isolates originated from the three largest provinces, British Columbia, Ontario and Quebec. Overall, of the laboratory results received, 112 (8.1%) showed resistance to at least one first-line drug and, of these, the majority (93 or 83%) were monoresistant. TB drug resistance observed in Canada remains well below the global average. Over the last 10 years, the percentage of isolates with resistance to one or more of the first-line medications has decreased from 10.5% in 2003 to 8.1% in 2013.

Second-line drug resistance patterns among patients with multidrug-resistant tuberculosis of Gujarat, western India

Background: Lack of laboratory diagnostic capacity is a crucial barrier preventing an effective response to early and appropriate case detection and treatment of multidrug-resistant tuberculosis (MDR-TB). To date, limited representative drug resistance data are available from India on second-line drug (SLD) resistance. Objectives: To study SLD resistance patterns in MDR-TB patients of Gujarat, western India. Materials and Methods: Antituberculosis drug susceptibility testing of 50 MDR-TB isolates particularly for SLDs, namely p-aminosalicylic acid, ciprofloxacin, amikacin, D-cycloserine, kanamycin, and ethionamide, was conducted on solid Lowenstein–Jensen medium using the 1% proportion sensitivity method. Results: Of 50 MDR-TB isolates tested, 24 (24/50, 48%) were found to be resistant to one or more SLDs. Of 50 MDR-TB isolates, 18 (18/50, 36%) showed resistance to ethionamide, 13 (13/50, 26%) to D-cycloserine, 11 (11/50, 22%) to ciprofloxacin, 7 (7/50, 14%) to kanamycin, 6 (6/50, 12%) to p-aminosalicylic acid, and 5 (5/50, 10%) to Amikacin. Conclusion: High levels of SLD resistance were found to be present in this study. There is an urgent need for more control and rational use of the widely available fluoroquinolones and other SLDs outside of the Revised National Tuberculosis Control Programme, by both the public and the private health sectors in India.

Poor treatment outcomes among multidrug-resistant tuberculosis patients in Gomel Region, Republic of Belarus.

Tuberculosis (TB) health facilities in the Gomel Region, Republic of Belarus-settings with a high burden of multidrug-resistant TB (MDR-TB) and human immunodeficiency virus (HIV) infection. To determine treatment outcomes among MDR-TB patients diagnosed in 2009-2010 and factors associated with unsuccessful outcomes (death, failure and loss to follow-up). Retrospective cohort study involving a review of an electronic patient database maintained under the National Tuberculosis Control Programme. Of 517 patients diagnosed, 78 (15%) did not start treatment. Among 439 patients who started treatment (84% males, median age 45 years, 15% HIV-infected), 291 (66%) had unsuccessful outcomes (35% deaths, 18% treatment failure and 13% lost to follow-up). Multivariate regression analysis showed that patients aged ⩾45 years (aRR 1.2, 95%CI 1.1-1.3), HIV-infected patients and those not receiving antiretroviral therapy (ART) (aRR 1.5, 95%CI 1.4-1.6) and those with a previous history of anti-tuberculosis treatment (aRR 1.2, 95%CI 1.1-1.4) had significantly higher risk of unsuccessful outcomes. Treatment outcomes among MDR-TB patients were poor, with high rates of death, failure and loss to follow-up (including pre-treatment loss to follow-up). Urgent measures to increase ART uptake among HIV-infected MDR-TB patients, improved access to second-line anti-tuberculosis drug susceptibility testing and comprehensive patient support measures are required to address this grim situation.

Increase in anti-tuberculosis drug resistance in Botswana: results from the fourth National Drug Resistance Survey

Although approximately 0.5 million cases of multidrug-resistant tuberculosis (MDR-TB) occur globally each year, surveillance data are limited. Botswana is one of the few high TB burden countries to have carried out multiple anti-tuberculosis drug resistance surveys (in 1995-1996, 1999 and 2002). In 2007-2008, we conducted the fourth national survey of anti-tuberculosis drug resistance in Botswana to assess anti-tuberculosis drug resistance, including trends over time. In the previous survey, 0.8% (95%CI 0.4-1.5) of new patients and 10.4% (95%CI 5.6-17.3) of previously treated patients had MDR-TB. During the survey period, eligible specimens from all new sputum-smear positive TB patients and from all TB patients with history of previous anti-tuberculosis treatment underwent mycobacterial culture and anti-tuberculosis drug susceptibility testing (DST). Of 924 new TB patients and 137 with previous anti-tuberculosis treatment with DST results, respectively 23 (2.5%, 95%CI 1.6-3.7) and 9 (6.6%, 95%CI 3.3-11.7) had MDR-TB. The proportion of new TB patients with MDR-TB has tripled in Botswana since the previous survey. Combatting drug-resistant TB will require the scale-up of MDR-TB diagnosis and treatment to prevent the transmission of MDR-TB and strengthening of general TB control to prevent the emergence of resistance.

First proficiency testing of second-line anti-tuberculosis drug susceptibility testing in 12 provinces of China

To evaluate the performance of drug susceptibility testing (DST) against the main second-line (SL) anti-tuberculosis drugs in tuberculosis (TB) laboratories in China. The supranational TB reference laboratory issued 30 Mycobacterium tuberculosis isolates to the participating laboratories. Each participating laboratory performed DST against kanamycin (KM), amikacin (AMK), capreomycin (CPM) and ofloxacin (OFX) using the proportion method in Löwenstein-Jensen medium per World Health Organization recommendations. Reported results were checked and compared with the judicial results. The main performance indicators for the four anti-tuberculosis drugs evaluated (KM, AMK, CPM, OFX) were as follows: accordance rates: 91.62%, 99.16%, 96.93% and 96.37%; reproducibility: 99.16%, 99.16%, 94.96% and 94.12%; specificity: 99.12%, 99.64%, 98.00% and 98.41%; sensitivity: 78.03%, 97.62%, 94.44% and 91.51%. The accordance rates and sensitivity values of the four drugs showed statistically significant differences, while specificities showed no significant differences. Eight (66.7%) participating laboratories met the set requirement criteria; however, DST in four (33.3%) laboratories requires greater attention. Of the four drugs tested, the results for KM were lower than those for the other drugs. External quality assessment can lead to effective evaluation of laboratory performance in SL DST.

Extrapulmonary tuberculosis among adults: Experience at Chris Hani Baragwanath Academic Hospital, Johannesburg, South Africa

Extrapulmonary tuberculosis (EPTB) occurs in 15 - 20% of immunocompetent and 20 - 70% of HIV-infected patients with tuberculosis. There are few recent incidence data for EPTB. Adults (N=2 963) with culture-proven EPTB seen over 2 years at Chris Hani Baragwanath Academic Hospital, the main referral hospital serving Soweto, Johannesburg, South Africa, were retrospectively studied for pattern and incidence. The commonest sites of EPTB were the pleura (39.1%), lymph nodes (31.0%), blood (21.8%), meninges (7.3%), and peritoneum (2.9%). Disseminated tuberculosis occurred in 25.0%. The median age was 33 years (range 18 - 87 years). Males comprised 53.2% overall, with a female majority in the peritonitis group. For Soweto, the incidence of adult EPTB was 88.6/100 000 population, rising to 139.4/100 000 and 125.7/100 000 in the 25 - 34-year and 35 - 44-year age groups, respectively. There was no secondary peak in the elderly (17.9/100 000). This retrospective cohort showed a high incidence of EPTB, most marked in the 25 - 44-year age group. Culture of extrapulmonary sites is of importance to confirm diagnosis of tuberculosis and to ensure antituberculosis drug susceptibility testing.

167 Application of isothermal microcalorimetry for rapid mycobacterial detection and anti-tuberculosis drug susceptibility testing – implication for urogenital tuberculosis and other mycobacterial infections

167 Application of isothermal microcalorimetry for rapid mycobacterial detection and anti-tuberculosis drug susceptibility testing – implication for urogenital tuberculosis and other mycobacterial infections

Endogenous reactivation followed by exogenous re-infection with drug resistant strains, a new challenge for tuberculosis control in Saudi Arabia

Endogenous reactivation and exogenous reinfection of tuberculosis were studied for the first time in Saudi Arabia after enrolling a total of 39 patients with multiple episodes of tuberculosis between 2009 and 2010. All of the primary and subsequent isolates enrolled were subjected to spoligotyping, 24 loci based MIRU-VNTR typing and first line anti-tuberculosis drug susceptibility testing. The primary episode isolates from patients born outside Saudi Arabia were dominated by lineages which are prevalent in their country of origin (e.g. Ghana, Cameroon, Uganda-I, among African patients/Delhi/CAS and EAI among Asian patients). On the other hand, in Saudi Arabian patients, (median age of 67 years) Delhi/CAS, TUR and Slineages were predominant. The second episode of infection was mainly caused by the lineages Delhi/CAS, EAI, Uganda-I, Haarlem, and LAM which are currently disseminating in the country. Surprisingly, all of the first episode isolates were pan-susceptible, while 35.9% of the re-infected cases were drug resistant. Reactivation of a remote infection eventually followed by an exogenous reinfection was confirmed among patients, particularly those of African origin. Immediate actions to break the cycle of transmission of drug resistant tuberculosis are greatly needed in Saudi Arabia.

Distribution characteristics of resistance to other first line anti-tuberculosis drugs among 1034 rifampin-resistant mycobacterium tuberculosis strains

Objective To explore rifampicin-resistant status of mycobacterium tuberculosis isolates in Guangzhou during the recent seventeen years, and their distribution characteristics of susceptibility to other first line anti-tuberculosis drugs. Methods The results of first line anti-tuberculosis drug susceptibility testing performed during the period of February 1992 to July 2008 in clinic patients were retrospectively analyzed. Results Of 10 167 mycobacterium tuberculosis isolates, 1034 were rifampin-resistant, 2214 isoniazid-resistant, 1073 streptomycinresistant, and 622 ethambutol-resistant; with a total drug resistance rate of 25.80%. Of 1034 rifampin-resistant isolates, 927 also developed isoniazid resistance, being 89.65% for the total number of rifampin-resistant isolates but only 41.87% for 2214 isoniazid-resistant isolates. Meanwhile, the number of rifampin- and streptomycin -resistant(502 strains)and rifampin- and ethambutol-resistant(237 strains)isolates was significantly smaller than that of rifampin- and isoniazid-resistant isolats. Conclusions Most of rifampin-resistant isolates also develop resistance to isoniazid, but not vice versa. The existing mechanisms of drug resistance are difficult to completely explain this phenomenon. Wether mycobacterium tuberculosis has other mechanisms of drug resistance requires further research. Key words: Mycobacterium tuberculosis; Rifampin; Isoniazid; Drug resistance

Validación de la reacción en cadena de la polimerasa basada en generador universal de heterodúplex para la identificación de Mycobacterium tuberculosis resistente a rifampicina y multirresistente en Lima, Perú

To validate the polymerase chain reaction-based universal heteroduplex generator (PCR UHG-Rif) assay for identifying rifampin-resistant and multidrug-resistant (MDR, resistant to isoniazid and rifampin) Mycobacterium tuberculosis in patients with pulmonary tuberculosis from communities in Lima (Peru) with a high incidence of resistant tuberculosis. To compare the results of antituberculosis drug susceptibility testing in clinical samples performed by the proportion method with those obtained by the PCR UHG-Rif assay, with the aim of analyzing the diagnostic capability of PCR UHG-Rif. Concordance for the identification of antituberculosis drug susceptibility was 0.95 (kappa = 0.899; P < .05), with a sensitivity and specificity of 0.973 and 0.922 (P < .05), respectively. The positive predictive value was 0.939 (95% CI: 0.879-0.970) and the negative predictive value was 0.965 (95% CI: 0.902-0.988). Nevertheless, the probability for MDR prediction was 0.981 (P < .05). PCR UHG-Rif allows the detection of mixed populations; discordant results can be explained by the presence of point mutations, missense mutations and mutations outside the rpoB "hot" region associated with rifampin-resistance. The PCR UHG-Rif assay detects mutations in the rpoB gene with excellent sensitivity and specificity, and suitable predictive values when compared with the standard method for determining susceptibility to antituberculosis drugs. This test can be considered an excellent tool that can contribute to tuberculosis control by correctly identifying patients infected with resistant and MDR bacilli, leading to a reduction in the cases of tuberculosis and resistant tuberculosis.

Isothermal RNA sequence amplification method for rapid antituberculosis drug susceptibility testing of Mycobacterium tuberculosis.

RNA transcript quantification by an isothermal sequence amplification reaction was evaluated for susceptibility testing of 15 Mycobacterium tuberculosis strains. Agreement with the proportion method on Ogawa egg medium and the BACTEC MGIT 960 system was 100 and 87% for rifampin, 93 and 100% for isoniazid, 60 and 53% for ethambutol, and 80 and 80% for streptomycin, respectively.