The International Journal of Mycobacteriology

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 11  |  Issue : 3  |  Page : 268--272

The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe


Taeuk Kang, Da-Gyum Lee, Jihee Jung, Sungweon Ryoo 
 Clinical Research Center, Masan National Tuberculosis Hospital, Gyeongsangnam-do, South Korea

Correspondence Address:
Sungweon Ryoo
Masan National Tuberculosis Hospital, Gapo-ro 215, Masan Happo-gu, Changwon-si, Gyeongsangnam-do
South Korea

Abstract

Background: Tuberculosis (TB) is a severe public health challenge in Korea. Of all Mycobacterium tuberculosis (M. tb) strains, the Beijing genotype strain reportedly correlates with hypervirulence and drug resistance. Hence, an early identification of the Beijing genotype strain of M. tb plays a significant role in initial TB treatment. Kogenebiotech® (KoRT-polymerase chain reaction [PCR]) has developed a real-time PCR 17 18 kit to determine the Beijing genotype strain classified as M. tb. To determine the feasibility of the commercially produced KoRT-PCR kit in identifying the M. tb strain. Methods: We used 100 clinical isolates of M. tb and 100 non-M. tb samples for the assessment. We evaluated the overall concordance between the KoRT-PCR kit and the mycobacterial interspersed repetitive unite variable number tandem repeat typing kit (GenoScreen, Lille, France). Moreover, we measured the detection limits based on the chromosomal DNA copies for the KoRT-PCR kit. In addition, we determined the reproducibility among individual technicians using the KoRT-PCR. Results: The KoRT-PCR kit successfully discriminated all M. tb (confidence interval [CI]: 96.38%–100.00% for both sensitivity and specificity) and Beijing genotype strain (CI: 95.70%–100.00% for sensitivity and 96.87%–100.00% for specificity). We confirmed no significant deviation in the reproducibility between the technicians. Conclusions: The KoRT-PCR kit displayed sufficient capability of discriminating the Beijing genotype strain, which enabled the rapid identification of the Beijing genotype strain from the M. tb clinical isolates.



How to cite this article:
Kang T, Lee DG, Jung J, Ryoo S. The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe .Int J Mycobacteriol 2022;11:268-272


How to cite this URL:
Kang T, Lee DG, Jung J, Ryoo S. The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe . Int J Mycobacteriol [serial online] 2022 [cited 2022 Dec 2 ];11:268-272
Available from: https://www.ijmyco.org/text.asp?2022/11/3/268/355935


Full Text



 Introduction



Tuberculosis (TB) is a significant global health issue owing to rapid airborne transmission and high mortality.[1],[2] Mycobacterium tuberculosis (M. tb) with the Beijing genotype strain has recently emerged globally and caused outbreaks.[3],[4] The Beijing strain of M. tb is significant and could hasten the disease progression.[5] Moreover, this genotype is prone to severe clinical manifestation owing to hypervirulence and multidrug resistance.[6],[7],[8],[9],[10],[11] Researchers have frequently identified the Beijing strain among extensively drug-resistant TB strains.[12],[13],[14] TB, which predominantly occurs in East Asia, including Korea, occupies the genotype of the Beijing (East Asian) family.

Approximately 88.1% of the genotypes isolated from Korean patients with multidrug-resistant TB belong to the Beijing family.[15] The proportion of foreign patients with TB in their domestic counterparts has increased in Korea, and the majority of these foreign patients are from high TB burden countries.[16] This necessitates distinguishing the genotype of M. tb introduced from abroad and understanding the substantial role of the transmission route and the prevalence of the Beijing strain on National TB Control Programs. Researchers have developed several methods for distinguishing these strains. The most preferred strategies to recognize the Beijing genotype strains are IS6110 DNA fingerprinting and spoligotyping.[17],[18],[19] However, these methods are labor intensive; therefore, investigations on the Beijing strains of M. tb display slow progress. Hillemann et al. reported on a rapid real-time polymerase chain reaction (RT-PCR)-based method using the TaqMan probe.[20] They successfully applied PCR targeting IS6110 to identify the Beijing and non-Beijing strains.

Furthermore, Nagai et al. developed a multiplex loop-mediated isothermal amplification assay using a Beijing-specific single-nucleotide polymorphism on Rv0679c for detecting the Beijing lineage strains of M. tb.[21] However, the majority of reports are limited to experimental results for research purposes in the laboratory. There is a continuous need for M. tb and Beijing family diagnostic kits produced as standardized procedures and certificates by the licensing agency. In this study, we aimed to evaluate the KoRT-PCR kit composed of a primer and probe set capable of rapidly and accurately detecting M. tb and that of the Beijing strain concurrently. Currently, the accurate and simple RT-PCR-based diagnostic kit enables the rapid and appropriate detection of dnaA-IS6110, a specific target gene for M. tb of the Beijing strain, with a detection limit of 50 copies/uL of chromosomal DNA. The continuous monitoring of the pathogenesis and transmission of M. tb can contribute to managing patients with TB from neighboring countries and establishing an effective initial TB treatment program.

 Methods



DNA sample procurement

In this study, we procured 200 chromosomal DNAs from bacteria samples. Of these 200 DNA samples, 100 M. tb isolates were obtained from the biobank of the Masan National TB Hospital (MNTH), Korea, whereas the remaining 100 non-M. tb isolates were procured from biobanks and repositories, including the American Type Culture Collection (Virginia, USA), the Korean Collection for Type Culture (Jeongeup, Korea), Korea Veterinary Culture Collection (Gimcheon, Korea), the National Culture Collection for Pathogens (Cheongju, Korea), the Korea Bank for Pathogenic Viruses (Seoul, Korea), Vircell (Granada, Spain), and Promega (Wisconsin, USA) [Supplemental Table S1].[22] This study used the bacterial DNA similar to that in a previous publication.[23][INLINE:1]

We used M. tb H37Rv (ATCC 25618D-2) for an internal control.

The identification of M. tb and Beijing genotype strain

The 100 M. tb DNA isolates were extracted as described previously.[24] These clinically isolated M. tb samples were identified using the mycobacterial interspersed repetitive unite variable number tandem repeat (MIRU-VNTR) typing kit (GenoScreen, Lille, France) according to the manufacturer's protocol.

Briefly, the 24 loci of M. tb DNA were amplified via multiplex PCR and discriminated through capillary electrophoresis using 3500 Series Genetic Analyzers (Applied Biosystems, Massachusetts, USA).[25] We analyzed and interpreted the obtained data using the MIRU-VNTRplus database (URL: https://www.miru-vntrplus.org/MIRU/index.faces) to determine the M. tb strains.[26],[27],[28]

Designing and composing the KoRT-polymerase chain reaction kit

We sorted the sequences after searching for the M. tb and Beijing family gene sequences at the National Center for Biological Information (https://www.ncbi.nlm.nih.gov/). We obtained a database of 130 sequences for M. tb, including 30 Beijing families.

The obtained nucleotide sequences were analyzed by a multialignment method, in which several nucleotide sequences were bundled and aligned. Following the multialignment, we secured a conserved region comprising a nucleotide sequence without any mutations. We prepared the primer and probe sets by designing the nucleotide sequences within the conserved region [Supplemental Table S2].[INLINE:2]

This kit comprises a ×2 RT-PCR master mix, primer/probe mix, and positive control. A total of 15 μL PCR master mixes, 10 μL ×2 RT-PCR master mixes, 5 μL primer/probe mix, and 5 μL DNA templates were added to the prepared PCR master mix. The probe/primer mix comprises two fluorescence channels, namely the fluorescein amidites (FAM) fluorescence and victoria (VIC) fluorescence for detecting the M. tb and Beijing strain, respectively.

Limit of detection optimization in DNA concentration

We measured the limit of detection (LOD) of the KoRT-PCR kit using five different DNA concentrations of M. tb H37Rv (ATCC 25618D-2). We prepared quantified M. tb-specific IS6110 and Beijing family M. tb-specific dnaA-IS6110 DNA of each target gene by serial dilution with nuclease-free water (Applied Biosystems™, USA) to generate 200 copies/μL, 100 copies/μL, 50 copies/μL, 25 copies/μL, and 12.5 copies/μL. We performed multiple RT-PCR (ABI7500; Applied Biosystems 7500 RT-PCR Instrument System, Applied Biosystems, USA), and the results were analyzed using a detection software (ABI Version 2.3, USA) with the following conditions: one cycle of 2 min at 50°C for the initial preheating and 10 min at 95°C for denaturation, followed by 35 cycles of 15 s at 95°C for annealing and 1 min at 60°C for the final extension. We performed the experiment in triplicate.

Sensitivity and specificity analysis

We determined the analytical specificity of the KoRT-PCR kit through real-time multiplex PCR using a primer and probe set for the simultaneous detection of the M. tb and Beijing family M. tb with two channels, namely dnaA-IS6110 and IS6110 for the Beijing M. tb and M. tb target gene, respectively. Multiple RT-PCR were performed under similar LOD optimization method conditions.

Evaluating interpersonal reproducibility

Duplicate aliquots of each DNA were created for each technician with three DNA concentrations of 50/μL, 100/μL, and 200/μL copies using the identical protocol to assess the reproducibility.

We calculated the cycle threshold (Ct), standard deviation (SD), and the coefficient of variance (CV) based on the RT-PCR results. A CV value <5% was considered reproducible.

 Results



The identification of M. tb strain

We assessed the 100 clinical M. tb samples using a MIRU-VNTR typing kit to determine their specific strain. Of these samples, 84 samples were identified as Beijing strains. The remaining samples were identified as non-Beijing strains, including eight, six, two, and one isolate of NEW-1, Uganda and EAI, Turkey, and Haarlem, respectively. Intriguingly, the non-Beijing strains were initially isolated from geographically distant regions, except the EAI strain.

Limit of detection optimization in DNA concentration

We determined the DNA concentration to optimize the concentration for KoRT-PCR. This experiment selected five different DNA concentrations, namely 12.5 copies, 25 copies, 50 copies, 100 copies, and 200 copies per μL. Of five different DNA concentrations, those equal to or above 50 copies per μL displayed a 100% amplification rate, whereas concentrations of 12.5 copies and 25 copies per μL displayed an amplification rate of 66.7% and 0%, respectively. Thus, we selected 50 copies per μL DNA concentration for the subsequent experiment and analysis as this concentration was optimized.

Sensitivity and specificity analysis

Upon conducting KoRT-PCR on 200 samples, 100 clinical M. tb samples were amplified for VIC fluorescence. Of these samples, only 84 Beijing strains were amplified for FAM fluorescence. The non-M. tb samples were amplified neither for VIC nor FAM fluorescence. We calculated the sensitivity and specificity [Table 1]. For M. tb discrimination, the KoRT-PCR kit displayed 100% sensitivity and specificity (95% confidence interval [CI]: 96.38%–100%). Simultaneously, both sensitivity and specificity for M. tb with Beijing strain discrimination was 100% (CI: 95.70%–100% and 96.87%–100%). In other words, the KoRT-PCR kit displayed good discriminatory power for the M. tb and Beijing strain.{Table 1}

Evaluating interpersonal reproducibility

We performed the interpersonal reproducibility test to evaluate and determine any deviation in reproducibility between the performing technicians. In this experiment, we selected two technicians with comparable research capabilities. They performed KoRT-PCR using three different DNA concentrations, namely 1X LOD, 2X LOD, and 4X LOD, once per day for 10 days. We conducted an analysis based on the Ct value, SD, and CV.

In general, a CV value <5% was considered nonsignificant. We obtained CV values of 0.73%, 0.64%, and 0.73% for 1X LOD, 2X LOD, and 4X LOD, respectively [Table 2]. Thus, the KoRT-PCR kit was less likely to be influenced by the DNA concentration or human-derived factors.{Table 2}

 Discussion



Each strain of M. tb has a unique feature, and specific strains are strongly associated with particular patterns of drug resistance and hypervirulence.[8],[29],[30] Despite the significant role of strain identification, it is not easily conducted in epidemiological and medical fields. This is because M. tb strain identification requires a complicated protocol combined with in silico analysis and takes prolonged time for data interpretation. Previously, IS6110-restriction fragment length polymorphism (IS6110-RFLP) was the available option for strain identification; however, it was unsuitable for Beijing strain-specific typing. Moreover, the IS6110-RFLP requires an average of 44 days until the result is released for interpretation.[31] Considering our failure to perform rapid strain identification, the consideration of the strain feature of M. tb during initial treatment remains challenging.[32] The KoRT-PCR kit was developed following the high demand for Beijing strain-specific diagnostic tools.

The Beijing strain of M. tb is a specific type of genotypic lineage strain prevalent worldwide, particularly in the Eastern Asian region.[33],[34] In Korea, approximately 82.4% of the total patients with TB among foreigners had reported the Beijing strain.[35] The significance this strain is not only its high prevalence but also its clinical manifestation. The Beijing strain exhibits a more significant correlation with antitubercular drugs, such as rifampicin, ofloxacin, and multidrug resistance.[36],[37] In addition, this strain and specific drug resistance can act as a critical risk factor for treatment failure because of increased spreading capability via an intervening febrile and other immunological responses.[38],[39],[40],[41],[42]

For this study, we procured 100 clinical M. tb isolates from MNTH biobank. MNTH is dedicated to infectious diseases, particularly TB. It is an appropriate institution for using and evaluating the Beijing strain identification kit.[22]

We recommend developing a convenient and accurate multiplex PCR system to discriminate different M. tb genotypes. In addition, well-characterized clinical information and background could help us anticipate the goal, thus enabling the optimization of personalized medical treatment.

 Conclusion



In summary, M. tb and Beijing strain-specific RT-PCR were handled and evaluated for this study. The KoRT-PCR kit displayed sufficient discriminative capability for the M. tb with Beijing strain. Using the KoRT-PCR kit in a practical setting for clinical and epidemiological purposes will likely enable the rapid identification of the Beijing strain and contribute to TB control and treatment.[41],[43],[44],[45],[46] Moreover, it could contribute to understanding the dynamics of the TB transmission landscape in Korea, which will eventually provide greater insights into the mechanism of M. tb dissemination.

Ethical statement

Public Institutional Review Board designated by the Ministry of Health and Welfare approved this study (Approval No. P01-201908-33-001).

Financial support and sponsorship

This work was supported by the Tuberculosis Clinical Research Program (Grant number: 4600-4631-304) of the Clinical Research Center, Masan National Tuberculosis Hospital, and was funded by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (grant No. 2018R1A5A2021242).

Conflicts of interest

There are no conflicts of interest.

References

1Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: Estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 1999;282:677-86.
2Pearson ML, Jereb JA, Frieden TR, Crawford JT, Davis BJ, Dooley SW, et al. Nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis. A risk to patients and health care workers. Ann Intern Med 1992;117:191-6.
3Glynn JR, Whiteley J, Bifani PJ, Kremer K, van Soolingen D. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: A systematic review. Emerg Infect Dis 2002;8:843-9.
4Jiménez P, Calvopiña K, Herrera D, Rojas C, Pérez-Lago L, Grijalva M, et al. Identification of the Mycobacterium tuberculosis Beijing lineage in Ecuador. Biomedica 2017;37:233-7.
5de Jong BC, Hill PC, Aiken A, Awine T, Antonio M, Adetifa IM, et al. Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in The Gambia. J Infect Dis 2008;198:1037-43.
6Aguilar D, Hanekom M, Mata D, Gey van Pittius NC, van Helden PD, Warren RM, et al. Mycobacterium tuberculosis strains with the Beijing genotype demonstrate variability in virulence associated with transmission. Tuberculosis (Edinb) 2010;90:319-25.
7Manca C, Tsenova L, Freeman S, Barczak AK, Tovey M, Murray PJ, et al. Hypervirulent M. tuberculosis W/Beijing strains upregulate type I IFNs and increase expression of negative regulators of the Jak-Stat pathway. J Interferon Cytokine Res 2005;25:694-701.
8Ribeiro SC, Gomes LL, Amaral EP, Andrade MR, Almeida FM, Rezende AL, et al. Mycobacterium tuberculosis strains of the modern sublineage of the Beijing family are more likely to display increased virulence than strains of the ancient sublineage. J Clin Microbiol 2014;52:2615-24.
9Ramazanzadeh R, Sayhemiri K. Prevalence of Beijing family in Mycobacterium tuberculosis in world population: Systematic review and meta-analysis. Int J Mycobacteriol 2014;3:41-5.
10Appelgren A, Morquin D, Dufour S, Le Moing V, Reynes J, Lotthé A, et al. Investigation of pre-XDR Beijing Mycobacterium tuberculosis transmission to a healthcare worker in France, 2016. J Hosp Infect 2017;97:414-7.
11Panaiotov S, Bachiyska E, Yordanova S, Atanasova Y, Brankova N, Levterova V, et al. Beijing lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007-2011. Emerg Infect Dis 2014;20:1899-901.
12Mlambo CK, Warren RM, Poswa X, Victor TC, Duse AG, Marais E. Genotypic diversity of extensively drug-resistant tuberculosis (XDR-TB) in South Africa. Int J Tuberc Lung Dis 2008;12:99-104.
13Igor M, Viacheslav S, Polina K, Alena G, Ekaterina C, Natalia S. Genomic analysis of new pre-XDR/XDR cluster of Mycobacterium tuberculosis Beijing genotype emerging in Russia. Eur Respir J 2020;56. doi: 10.1183/13993003.congress-2020.4593.
14Hu Y, Mathema B, Zhao Q, Chen L, Lu W, Wang W, et al. Acquisition of second-line drug resistance and extensive drug resistance during recent transmission of Mycobacterium tuberculosis in rural China. Clin Microbiol Infect 2015;21:1093.e9-18.
15Young Mi K, Jeong Seob L, Dong Hyeok K, Junyoung K, Jae Il Y, Seung-Eun S. Analysis of the genotypic characteristics of multidrug-/rifampicin-resistant tuberculosis in 2020 in Korea. KCDC Public Health Wkly Rep 2021;14: 3621-3.
16The Korea National Tuberculosis Association. A Study for the Molecular Typing of Mycobacterium tuberculosis Isolated in Korea Using IS6110 and MIRU-VNTR. Seoul, Korea: The Korea National Tuberculosis Association; 2015.
17Bifani PJ, Mathema B, Kurepina NE, Kreiswirth BN. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol 2002;10:45-52.
18Moss AR, Alland D, Telzak E, Hewlett D Jr., Sharp V, Chiliade P, et al. A city-wide outbreak of a multiple-drug-resistant strain of Mycobacterium tuberculosis in New York. Int J Tuberc Lung Dis 1997;1:115-21.
19Narvskaya O, Otten T, Limeschenko E, Sapozhnikova N, Graschenkova O, Steklova L, et al. Nosocomial outbreak of multidrug-resistant tuberculosis caused by a strain of Mycobacterium tuberculosis W-Beijing family in St. Petersburg, Russia. Eur J Clin Microbiol Infect Dis 2002;21:596-602.
20Hillemann D, Warren R, Kubica T, Rüsch-Gerdes S, Niemann S. Rapid detection of Mycobacterium tuberculosis Beijing genotype strains by real-time PCR. J Clin Microbiol 2006;44:302-6.
21Nagai Y, Iwade Y, Nakano M, Akachi S, Kobayashi T, Nishinaka T. Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay. Microbiol Immunol 2016;60:459-67.
22Hwang Y, Kim J, Park S, Ryoo S. Biobank for multidrug-resistant tuberculosis research: Importance of sequential samples. Pathog Dis 2021;79:ftab011.
23Jung J, Kang T, Hwang Y, Ryoo S. Validation and comparative analysis of kogene mycobacterial interspersed repetitive unit-variable number of tandem repeat typing kit and its application on clinically isolated Mycobacterium tuberculosis samples from national tuberculosis hospital, Republic of Korea. Int J Mycobacteriol 2022;11:23-9.
24van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for a standardized methodology. J Clin Microbiol 1993;31:406-9.
25Philip S. Multilocus Variable Number Tandem Repeat Genotyping of Mycobacterium tuberculosis Technical Guide. Lille, France: Institut Pasteur de Lille; 2005.
26Pedersen MK, Andersen AB, Folkvardsen DB, Rasmussen EM, Svensson E, Lillebaek T, et al. Set-up and validation of mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) analysis of Mycobacterium tuberculosis using BioNumerics software. PLoS One 2018;13:e0205336.
27de Beer JL, Kremer K, Ködmön C, Supply P, van Soolingen D, Global Network for the Molecular Surveillance of Tuberculosis 2009. First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains. J Clin Microbiol 2012;50:662-9.
28Applied Biosystems, Applied Biosystems 3500/3500xL Genetic Analyzer User Guide. https://tools.thermofisher.com/content/sfs/manuals/4401661.pdf.
29Mathuria JP, Srivastava GN, Sharma P, Mathuria BL, Ojha S, Katoch VM, et al. Prevalence of Mycobacterium tuberculosis Beijing genotype and its association with drug resistance in North India. J Infect Public Health 2017;10:409-14.
30Shanmugam S, Selvakumar N, Narayanan S. Drug resistance among different genotypes of Mycobacterium tuberculosis isolated from patients from Tiruvallur, South India. Infect Genet Evol 2011;11:980-6.
31The Editors. Dealing with variation in molecular typing of Mycobacterium tuberculosis: Low-intensity bands and other challenges. J Med Microbiol 2001;50:749-51.
32De Beer JL, Kodmon C, van der Werf MJ, van Ingen J, van Soolingen D, ECDC MDR-TB Molecular Surveillance Project Participants. Molecular surveillance of multi- and extensively drug-resistant tuberculosis transmission in the European Union from 2003 to 2011. Euro Surveill 2014;19:20742.
33Wada T, Iwamoto T, Maeda S. Genetic diversity of the Mycobacterium tuberculosis Beijing family in East Asia revealed through refined population structure analysis. FEMS Microbiol Lett 2009;291:35-43.
34van Soolingen D, Qian L, de Haas PE, Douglas JT, Traore H, Portaels F, et al. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J Clin Microbiol 1995;33:3234-8.
35Jimin H, Jaeil Y, Donghyeok K, Kyujam H. Molecular epidemiology of Mycobacterium tuberculosis isolated from foreigners among the contact investigations in Korea, 2017-2018. Pub Heal Week Rep KCDA 2020;13.
36Pang Y, Zhou Y, Zhao B, Liu G, Jiang G, Xia H, et al. Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China. PLoS One 2012;7:e32976.
37Park YK, Shin S, Ryu S, Cho SN, Koh WJ, Kwon OJ, et al. Comparison of drug resistance genotypes between Beijing and non-Beijing family strains of Mycobacterium tuberculosis in Korea. J Microbiol Methods 2005;63:165-72.
38Sun YJ, Lim TK, Ong AK, Ho BC, Seah GT, Paton NI. Tuberculosis associated with Mycobacterium tuberculosis Beijing and non-Beijing genotypes: A clinical and immunological comparison. BMC Infect Dis 2006;6:105.
39van Crevel R, Nelwan RH, de Lenne W, Veeraragu Y, van der Zanden AG, Amin Z, et al. Mycobacterium tuberculosis Beijing genotype strains associated with febrile response to treatment. Emerg Infect Dis 2001;7:880-3.
40Parwati I, Alisjahbana B, Apriani L, Soetikno RD, Ottenhoff TH, van der Zanden AG, et al. Mycobacterium tuberculosis Beijing genotype is an independent risk factor for tuberculosis treatment failure in Indonesia. J Infect Dis 2010;201:553-7.
41Lan NT, Lien HT, Tung le B, Borgdorff MW, Kremer K, van Soolingen D. Mycobacterium tuberculosis Beijing genotype and risk for treatment failure and relapse, Vietnam. Emerg Infect Dis 2003;9:1633-5.
42Hanekom M, van der Spuy GD, Streicher E, Ndabambi SL, McEvoy CR, Kidd M, et al. A recently evolved sublineage of the Mycobacterium tuberculosis Beijing strain family is associated with an increased ability to spread and cause disease. J Clin Microbiol 2007;45:1483-90.
43Buu TN, van Soolingen D, Huyen MN, Lan NT, Quy HT, Tiemersma EW, et al. Increased transmission of Mycobacterium tuberculosis Beijing genotype strains associated with resistance to streptomycin: A population-based study. PLoS One 2012;7:e42323.
44Holt KE, McAdam P, Thai PV, Thuong NT, Ha DT, Lan NN, et al. Frequent transmission of the Mycobacterium tuberculosis Beijing lineage and positive selection for the EsxW Beijing variant in Vietnam. Nat Genet 2018;50:849-56.
45Kong Y, Cave MD, Zhang L, Foxman B, Marrs CF, Bates JH, et al. Association between Mycobacterium tuberculosis Beijing/W lineage strain infection and extrathoracic tuberculosis: Insights from epidemiologic and clinical characterization of the three principal genetic groups of M. tuberculosis clinical isolates. J Clin Microbiol 2007;45:409-14.
46Parwati I, van Crevel R, van Soolingen D. Possible underlying mechanisms for successful emergence of the Mycobacterium tuberculosis Beijing genotype strains. Lancet Infect Dis 2010;10:103-11.