• 1.

    WHO, 2012. The Global Tuberculosis Report 2012. Geneva: World Health Organization.

  • 2.

    Tarajia M, Goodridge A, 2014. Tuberculosis remains a challenge despite economic growth in Panama [Notes from the field]. Int J Tuberc Lung Dis 18: 286288.

    • Search Google Scholar
    • Export Citation
  • 3.

    MINSA, 2013. Evolución de la incidencia notificada de TBC y mortalidad general 1999–2011. Epidemiologia, ed. Panama: Ministerio de Salud.

    • Search Google Scholar
    • Export Citation
  • 4.

    Kean BH, 1946. The causes of death on the Isthmus of Panama; based on 14,304 autopsies performed at the Board of Health Laboratory, Gorgas Hospital, Ancon, Canal Zone, during the forty year period 1904–1944. Am J Trop Med Hyg 26: 733748.

    • Search Google Scholar
    • Export Citation
  • 5.

    Rosas S, Bravo J, Gonzalez F, de Moreno N, Sanchez J, Gavilan RG, Goodridge A, 2013. High clustering rates of multidrug-resistant Mycobacterium tuberculosis genotypes in Panama. BMC Infect Dis 13: 442.

    • Search Google Scholar
    • Export Citation
  • 6.

    Lanzas F, Karakousis PC, Sacchettini JC, Ioerger TR, 2013. Multidrug-resistant tuberculosis in panama is driven by clonal expansion of a multidrug-resistant Mycobacterium tuberculosis strain related to the KZN extensively drug-resistant M. tuberculosis strain from South Africa. J Clin Microbiol 51: 32773285.

    • Search Google Scholar
    • Export Citation
  • 7.

    Hricko A, 2012. Progress and pollution: port cities prepare for the Panama Canal expansion. Environ Health Perspect 120: A470A473.

  • 8.

    Kato-Maeda M, Metcalfe JZ, Flores L, 2011. Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies. Future Microbiol 6: 203216.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ferdinand S, Millet J, Accipe A, Cassadou S, Chaud P, Levy M, Theodore M, Rastogi N, 2013. Use of genotyping based clustering to quantify recent tuberculosis transmission in Guadeloupe during a seven years period: analysis of risk factors and access to health care. BMC Infect Dis 13: 364.

    • Search Google Scholar
    • Export Citation
  • 10.

    van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD, 1991. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 29: 25782586.

    • Search Google Scholar
    • Export Citation
  • 11.

    Banu S, Uddin MK, Islam MR, Zaman K, Ahmed T, Talukder AH, Rahman MT, Rahim Z, Akter N, Khatun R, Brosch R, Endtz HP, 2012. Molecular epidemiology of tuberculosis in rural Matlab, Bangladesh. Int J Tuberc Lung Dis 16: 319326.

    • Search Google Scholar
    • Export Citation
  • 12.

    Blackwood KS, Wolfe JN, Kabani AM, 2004. Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten? J Clin Microbiol 42: 50015006.

    • Search Google Scholar
    • Export Citation
  • 13.

    Cowan LS, Mosher L, Diem L, Massey JP, Crawford JT, 2002. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J Clin Microbiol 40: 15921602.

    • Search Google Scholar
    • Export Citation
  • 14.

    Savine E, Warren RM, van der Spuy GD, Beyers N, van Helden PD, Locht C, Supply P, 2002. Stability of variable-number tandem repeats of mycobacterial interspersed repetitive units from 12 loci in serial isolates of Mycobacterium tuberculosis. J Clin Microbiol 40: 45614566.

    • Search Google Scholar
    • Export Citation
  • 15.

    Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C, 2000. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 36: 762771.

    • Search Google Scholar
    • Export Citation
  • 16.

    Hunter PR, Gaston MA, 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26: 24652466.

    • Search Google Scholar
    • Export Citation
  • 17.

    Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, van Embden J, 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35: 907914.

    • Search Google Scholar
    • Export Citation
  • 18.

    Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, Allix C, Aristimuno L, Arora J, Baumanis V, Binder L, Cafrune P, Cataldi A, Cheong S, Diel R, Ellermeier C, Evans JT, Fauville-Dufaux M, Ferdinand S, Garcia de Viedma D, Garzelli C, Gazzola L, Gomes HM, Guttierez MC, Hawkey PM, van Helden PD, Kadival GV, Kreiswirth BN, Kremer K, Kubin M, Kulkarni SP, Liens B, Lillebaek T, Ho ML, Martin C, Mokrousov I, Narvskaia O, Ngeow YF, Naumann L, Niemann S, Parwati I, Rahim Z, Rasolofo-Razanamparany V, Rasolonavalona T, Rossetti ML, Rusch-Gerdes S, Sajduda A, Samper S, Shemyakin IG, Singh UB, Somoskovi A, Skuce RA, van Soolingen D, Streicher EM, Suffys PN, Tortoli E, Tracevska T, Vincent V, Victor TC, Warren RM, Yap SF, Zaman K, Portaels F, Rastogi N, Sola C, 2006. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6: 23.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kernodle DS, 2012. Warning: differences in the copy number of duplication unit 2 (DU2) within BCG Danish 1331 may influence findings involving genetically modified BCG Danish strains. Vaccine 30: 60136014, author reply 6015.

    • Search Google Scholar
    • Export Citation
  • 20.

    Sun YJ, Bellamy R, Lee AS, Ng ST, Ravindran S, Wong SY, Locht C, Supply P, Paton NI, 2004. Use of mycobacterial interspersed repetitive unit-variable-number tandem repeat typing to examine genetic diversity of Mycobacterium tuberculosis in Singapore. J Clin Microbiol 42: 19861993.

    • Search Google Scholar
    • Export Citation
  • 21.

    Lee AS, Tang LL, Lim IH, Bellamy R, Wong SY, 2002. Discrimination of single-copy IS6110 DNA fingerprints of Mycobacterium tuberculosis isolates by high-resolution minisatellite-based typing. J Clin Microbiol 40: 657659.

    • Search Google Scholar
    • Export Citation
  • 22.

    Mazars E, Lesjean S, Banuls AL, Gilbert M, Vincent V, Gicquel B, Tibayrenc M, Locht C, Supply P, 2001. High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci USA 98: 19011906.

    • Search Google Scholar
    • Export Citation
  • 23.

    Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, Savine E, de Haas P, van Deutekom H, Roring S, Bifani P, Kurepina N, Kreiswirth B, Sola C, Rastogi N, Vatin V, Gutierrez MC, Fauville M, Niemann S, Skuce R, Kremer K, Locht C, van Soolingen D, 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44: 44984510.

    • Search Google Scholar
    • Export Citation
  • 24.

    Cerezo I, Jimenez Y, Hernandez J, Zozio T, Murcia MI, Rastogi N, 2012. A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Bogota, Colombia. Infect Genet Evol 12: 657663.

    • Search Google Scholar
    • Export Citation
  • 25.

    Mendes NH, Melo FA, Santos AC, Pandolfi JR, Almeida EA, Cardoso RF, Berghs H, David S, Johansen FK, Espanha LG, Leite SR, Leite CQ, 2011. Characterization of the genetic diversity of Mycobacterium tuberculosis in Sao Paulo city, Brazil. BMC Res Notes 4: 269.

    • Search Google Scholar
    • Export Citation
  • 26.

    de Beer JL, Akkerman OW, Schurch AC, Mulder A, van der Werf TS, van der Zanden A, van Ingen J, van Soolingen D, 2014. Optimization of standard in-house 24-locus variable number of tandem repeat typing for Mycobacterium tuberculosis and its direct application to clinical material. J Clin Microbiol Epub ahead of print 5 February 2014. doi:10.1128/JCM.03436-13.

    • Search Google Scholar
    • Export Citation
  • 27.

    Driscoll JR, 2009. Spoligotyping for molecular epidemiology of the Mycobacterium tuberculosis complex. Methods Mol Biol 551: 117128.

 
 
 

 

 

 

 

 

 

Mycobacterium tuberculosis Isolates from Single Outpatient Clinic in Panama City Exhibit Wide Genetic Diversity

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  • Tuberculosis Biomarker Research Unit, Centro de Biología Celular y Molecular de las Enfermedades (CBCME) del Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), Ciudad del Saber, Panamá; Department of Biotechnology, Acharya Nagarjuna University, Guntur, India; Laboratorio Clínico, Complejo Hospitalario Metropolitano Dr. Arnulfo Arias Madrid, Caja de Seguro Social (CHMDrAAM-CSS), Panamá, Panamá; Departamento de Ciencias de Laboratorio Clínico, Facultad de Medicina, Universidad de Panamá, Panamá, Panamá

Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities.

Tuberculosis (TB) affects nearly 8.7 million people worldwide.1 In 2011, most TB cases were reported in Asia (59%) and Africa (26%), although cases were reported to a lesser extent in the Eastern Mediterranean Region (7.7%), the European Region (4.3%), and the Americas Region (3%). Panama stands as the country with the highest TB mortality rate in Central America.2 In 2012, more than 1,500 TB cases were reported in Panama, for an average incidence rate of 43.1 cases per 100,000 inhabitants.3 Areas located at the Pacific and Atlantic entries of the Panama Canal have harbored the highest numbers of TB cases since the Canal's construction.4 Despite sanitation improvements in terminal port cities, recent studies have revealed elevated TB transmission as a result of a high clustering rate among multidrug-resistant TB cases.5,6 However, data on the transmission of drug-susceptible TB within the general population remain scarce and have not been updated to reflect a second wave of immigration connected with Panama Canal expansion activities.7

Mycobacterium tuberculosis genotyping has proven to be the most important laboratory tool in understanding TB transmission.8 In addition to studies on patient contacts; information on molecular epidemiology is useful for evaluating TB control program results. Genotyping also assists in monitoring molecular markers associated with virulence, immunogenicity, and drug resistance9; among the genotyping tools available, the IS6110-restriction fragment length polymorphism (RFLP) reference standard method is based on the number of repetitions of the IS6110 sequence along the M. tuberculosis genome.10 This tool discriminates between clonally related and unrelated isolates. On the other hand, Spoligotyping focuses on detecting 43 spacer sequences in the direct repeat region of the M. tuberculosis genome. Unfortunately, the IS6110–RFLP method is a complex and laborious procedure, whereas Spoligotyping is faster and simpler but less discriminating.11,12 The study of mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) is an alternative to genotyping M. tuberculosis isolates.13,14 Our study aimed to characterize the genetic diversity of M. tuberculosis isolates in one outpatient clinic using a combination of 12 loci MIRU-VNTR.

A total of 62 clinical isolates were collected at the Social Security Clinical Laboratory of the Complejo Hospitalario Metropolitano Dr. Arnulfo Arias Madrid between January and December of 2005. The strain collection was performed as part of the Panamanian standard of patient care for TB diagnosis and control in Panama City. These isolates accounted for 16.3% of all pulmonary TB cases reported in Panama City in 2005. The DNA extraction was performed using a method described previously.10 A total of 12 MIRU-VNTR loci were amplified according to a modified protocol described by Cowan and colleagues.13 The amplification products were analyzed by electrophoresis on an agarose gel. The number of MIRU-VNTR alleles was determined according to the sizes proposed by Cowan and colleagues, which allocate the number of alleles by the fragment size.13 The allelic diversity for each MIRU-VNTR was calculated using the number of alleles at each locus.15 The ability to detect the number of allelic repetitions of each allele for every MIRU-VNTR was then classified as high, moderate, or low. We used the Hunter-Gaston discriminatory index (HGDI) to determine the discriminating power of possessing all 12 MIRU -VNTR loci in our study.16 Spoligotyping was performed on genomic DNA using the standard method described by Kamerbeek and colleagues.17 The family label and Spoligotype octal code numbers were obtained from the SPOLDB4.0.18

Our findings show high genetic diversity of M. tuberculosis clinical isolates obtained from outpatients from a clinic in Panama City. Our results reveal that a total of 45 (72.5%) M. tuberculosis isolates showed unique MIRU-VNTR patterns (Table 1). Moreover, a total of 13 (20%) isolates were grouped into four clusters. Cluster A (225326-133323) included six isolates, and three isolates were grouped into cluster B (224326-153321). The other two clusters were composed of two M. tuberculosis isolates each: cluster C (223325-153324) and cluster D (123237-253227). Four (6.4%) isolates showed only two alleles simultaneously. The presence of two alleles may suggest infection with two or more M. tuberculosis strains in these patients. Alternatively, the presence of these strains could be a result of triploid polyclones, similar to that reported for Mycobacterium bovis Bacillus Calmette Guérin.19 These findings indicate the genetic diversity of M. tuberculosis circulating in patients with drug-susceptible pulmonary TB in an outpatient clinic in Panama City. Further detailed studies are needed to determine the connection between patients with isolates in the same clusters.

Table 1

MIRU-VNTR genotypes for M. tuberculosis clinical isolates with a unique genotype recovered from an outpatient clinic in Panama City (2005)*

IsolateMIRU-VNTRIsolateMIRU-VNTRIsolateMIRU-VNTR
12243251533231622432515322131223326153321
22264251533221722432514332432223325173533
32262261533231822432514332433223325153323
42253352533231922432215332334223236253323
52253352333222022431514332435225325153324
62253261333242122431514332336224335253323
72253251533212222421625232137224325153322
82253251531232322352614332238225326133323
92253251433232422342514332439124326154326
102253251433212522333525332440123336253222
112252261633212622333525332141123326153326
122244251735332722332625332142123326163326
132243362533232822332615332143224325153323
142243352533232922332615332144123326153326
152243261333133022332615332145223326153321

MIRU-VNTR = mycobacterial interspersed repetitive units-variable number of tandem repeats.

The 12 MIRU-VNTR loci we used showed high discriminatory power for M. tuberculosis clinical isolates from the studied outpatient clinic. The discriminatory power of each MIRU facilitated allelic diversity assessment. In our study, the MIRU-VNTR 10, 23, 26, 31, and 40 were highly discriminating. The MIRU 16, 20, and 24 showed low discriminatory power. Thus, our 12 MIRU-VNTR loci showed high discriminatory power, similar to previous reports using this marker set.14,20 This allelic diversity allowed us to reach an HGDI of 0.988. Thus, we confirmed the discriminatory power of this set of 12 MIRU-VNTR loci for analyzing M. tuberculosis isolate samples in Panama City. This feature will be useful in tracking outbreak episodes, relapses, or cross-contamination of M. tuberculosis in community-based studies.2123 In contrast, the Spoligotyping analysis identified 93% of the clinical M. tuberculosis isolates (Table 2). We identified 16 M. tuberculosis family Spoligotypes including Latin American-Mediterranean, Harlem, and Beijing. Only four (7%) of the M. tuberculosis clinical isolates were not annotated in the SPOLDB4.0 database.

Table 2

Spoligotype family genotypes of M. tuberculosis clinical isolates recovered from an outpatient clinic in Panama City (2005)

Octal Spoligotyping coden (%) of M. tuberculosis isolatesFamily label
7777777777607317 (13.0)T2
7761774000001716 (11.1)U (LAM3?)
7777777777207716 (11.1)H3
7777776077607715 (9.3)LAM9
0000767777606713 (5.6)LAM5
7040033477604713 (5.6)T4_CEU1
7777777777607713 (5.6)T1
0000000000037712 (3.7)BEIJING
7761776077607712 (3.7)LAM3
7777367760000712 (3.7)unknown
7777777437607712 (3.7)LAM10_CAM
0000000075607711 (1.9)T1
4661774000001711 (1.9)unknown
4761774000001711 (1.9)unknown
7776776077607711 (1.9)LAM9
7777367777607711 (1.9)X1
7777677777607311 (1.9)T1
7777767777606011 (1.9)X2
7777774774000011 (1.9)U
7777777640207711 (1.9)H1
7777777700000001 (1.9)U (likely H)
7777777700001711 (1.9)unknown
7777777740207311 (1.9)H1
7777777740207711 (1.9)H1

The wide genetic diversity of drug-susceptible M. tuberculosis clinical isolates collected from a single outpatient clinic is a limited reflection of population dynamics throughout the Panamanian Isthmus. During the early 20th century, the Panama Canal construction attracted a worldwide workforce, especially laborers from the Caribbean and Europe. As a result, Panama and Colon Cities comprised a wide variety of ethnic backgrounds that possibly harbored various M. tuberculosis genotypes. The high diversity of M. tuberculosis strains from a single outpatient clinic in our study is one example of this hypothesis. A similar effect of migration on M. tuberculosis diversity has been shown in other cosmopolitan cities in the Americas.24,25 These studies have associated the great genetic diversity of M. tuberculosis clinical isolates with the mixture of city inhabitants. A century later, Panama is currently expanding the Panama Canal and attracting a new labor force that could introduce new M. tuberculosis strains.7 Detailed surveillance studies using larger data sets are urgently required to monitor and understand the spread of M. tuberculosis among Panamanian and immigrant TB case contacts. Such studies would help improve TB control measures to decrease the mortality rate. Prompt genotyping of clinical isolates using state-of-the-art polymerase chain reaction-based tools, such as 24-MIRU-VNTR and Spoligotyping, should be implemented to determine epidemiological relationships and infections with two or more M. tuberculosis strains.26,27 Such a strategy would allow the identification of genotypes that are sustaining the disease burden and provoking death. This approach would also determine if there is any specific M. tuberculosis subpopulations related to higher TB transmission within the country.

ACKNOWLEDGMENTS

We thank Jorge Jordan for his collaboration on the DNA extraction and PCR amplification procedures. We also thank the colleagues of Laboratorio de Microbiología of Complejo Hospitalario Metropolitano of Caja de Seguro Social for providing the M. tuberculosis isolates collection. We also thank Jose Loaiza, Ricardo Lleonart, and Colleen Goodridge for critically reviewing this manuscript.

  • 1.

    WHO, 2012. The Global Tuberculosis Report 2012. Geneva: World Health Organization.

  • 2.

    Tarajia M, Goodridge A, 2014. Tuberculosis remains a challenge despite economic growth in Panama [Notes from the field]. Int J Tuberc Lung Dis 18: 286288.

    • Search Google Scholar
    • Export Citation
  • 3.

    MINSA, 2013. Evolución de la incidencia notificada de TBC y mortalidad general 1999–2011. Epidemiologia, ed. Panama: Ministerio de Salud.

    • Search Google Scholar
    • Export Citation
  • 4.

    Kean BH, 1946. The causes of death on the Isthmus of Panama; based on 14,304 autopsies performed at the Board of Health Laboratory, Gorgas Hospital, Ancon, Canal Zone, during the forty year period 1904–1944. Am J Trop Med Hyg 26: 733748.

    • Search Google Scholar
    • Export Citation
  • 5.

    Rosas S, Bravo J, Gonzalez F, de Moreno N, Sanchez J, Gavilan RG, Goodridge A, 2013. High clustering rates of multidrug-resistant Mycobacterium tuberculosis genotypes in Panama. BMC Infect Dis 13: 442.

    • Search Google Scholar
    • Export Citation
  • 6.

    Lanzas F, Karakousis PC, Sacchettini JC, Ioerger TR, 2013. Multidrug-resistant tuberculosis in panama is driven by clonal expansion of a multidrug-resistant Mycobacterium tuberculosis strain related to the KZN extensively drug-resistant M. tuberculosis strain from South Africa. J Clin Microbiol 51: 32773285.

    • Search Google Scholar
    • Export Citation
  • 7.

    Hricko A, 2012. Progress and pollution: port cities prepare for the Panama Canal expansion. Environ Health Perspect 120: A470A473.

  • 8.

    Kato-Maeda M, Metcalfe JZ, Flores L, 2011. Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies. Future Microbiol 6: 203216.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ferdinand S, Millet J, Accipe A, Cassadou S, Chaud P, Levy M, Theodore M, Rastogi N, 2013. Use of genotyping based clustering to quantify recent tuberculosis transmission in Guadeloupe during a seven years period: analysis of risk factors and access to health care. BMC Infect Dis 13: 364.

    • Search Google Scholar
    • Export Citation
  • 10.

    van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD, 1991. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 29: 25782586.

    • Search Google Scholar
    • Export Citation
  • 11.

    Banu S, Uddin MK, Islam MR, Zaman K, Ahmed T, Talukder AH, Rahman MT, Rahim Z, Akter N, Khatun R, Brosch R, Endtz HP, 2012. Molecular epidemiology of tuberculosis in rural Matlab, Bangladesh. Int J Tuberc Lung Dis 16: 319326.

    • Search Google Scholar
    • Export Citation
  • 12.

    Blackwood KS, Wolfe JN, Kabani AM, 2004. Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten? J Clin Microbiol 42: 50015006.

    • Search Google Scholar
    • Export Citation
  • 13.

    Cowan LS, Mosher L, Diem L, Massey JP, Crawford JT, 2002. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J Clin Microbiol 40: 15921602.

    • Search Google Scholar
    • Export Citation
  • 14.

    Savine E, Warren RM, van der Spuy GD, Beyers N, van Helden PD, Locht C, Supply P, 2002. Stability of variable-number tandem repeats of mycobacterial interspersed repetitive units from 12 loci in serial isolates of Mycobacterium tuberculosis. J Clin Microbiol 40: 45614566.

    • Search Google Scholar
    • Export Citation
  • 15.

    Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C, 2000. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 36: 762771.

    • Search Google Scholar
    • Export Citation
  • 16.

    Hunter PR, Gaston MA, 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26: 24652466.

    • Search Google Scholar
    • Export Citation
  • 17.

    Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, van Embden J, 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35: 907914.

    • Search Google Scholar
    • Export Citation
  • 18.

    Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, Allix C, Aristimuno L, Arora J, Baumanis V, Binder L, Cafrune P, Cataldi A, Cheong S, Diel R, Ellermeier C, Evans JT, Fauville-Dufaux M, Ferdinand S, Garcia de Viedma D, Garzelli C, Gazzola L, Gomes HM, Guttierez MC, Hawkey PM, van Helden PD, Kadival GV, Kreiswirth BN, Kremer K, Kubin M, Kulkarni SP, Liens B, Lillebaek T, Ho ML, Martin C, Mokrousov I, Narvskaia O, Ngeow YF, Naumann L, Niemann S, Parwati I, Rahim Z, Rasolofo-Razanamparany V, Rasolonavalona T, Rossetti ML, Rusch-Gerdes S, Sajduda A, Samper S, Shemyakin IG, Singh UB, Somoskovi A, Skuce RA, van Soolingen D, Streicher EM, Suffys PN, Tortoli E, Tracevska T, Vincent V, Victor TC, Warren RM, Yap SF, Zaman K, Portaels F, Rastogi N, Sola C, 2006. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6: 23.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kernodle DS, 2012. Warning: differences in the copy number of duplication unit 2 (DU2) within BCG Danish 1331 may influence findings involving genetically modified BCG Danish strains. Vaccine 30: 60136014, author reply 6015.

    • Search Google Scholar
    • Export Citation
  • 20.

    Sun YJ, Bellamy R, Lee AS, Ng ST, Ravindran S, Wong SY, Locht C, Supply P, Paton NI, 2004. Use of mycobacterial interspersed repetitive unit-variable-number tandem repeat typing to examine genetic diversity of Mycobacterium tuberculosis in Singapore. J Clin Microbiol 42: 19861993.

    • Search Google Scholar
    • Export Citation
  • 21.

    Lee AS, Tang LL, Lim IH, Bellamy R, Wong SY, 2002. Discrimination of single-copy IS6110 DNA fingerprints of Mycobacterium tuberculosis isolates by high-resolution minisatellite-based typing. J Clin Microbiol 40: 657659.

    • Search Google Scholar
    • Export Citation
  • 22.

    Mazars E, Lesjean S, Banuls AL, Gilbert M, Vincent V, Gicquel B, Tibayrenc M, Locht C, Supply P, 2001. High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci USA 98: 19011906.

    • Search Google Scholar
    • Export Citation
  • 23.

    Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, Savine E, de Haas P, van Deutekom H, Roring S, Bifani P, Kurepina N, Kreiswirth B, Sola C, Rastogi N, Vatin V, Gutierrez MC, Fauville M, Niemann S, Skuce R, Kremer K, Locht C, van Soolingen D, 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44: 44984510.

    • Search Google Scholar
    • Export Citation
  • 24.

    Cerezo I, Jimenez Y, Hernandez J, Zozio T, Murcia MI, Rastogi N, 2012. A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Bogota, Colombia. Infect Genet Evol 12: 657663.

    • Search Google Scholar
    • Export Citation
  • 25.

    Mendes NH, Melo FA, Santos AC, Pandolfi JR, Almeida EA, Cardoso RF, Berghs H, David S, Johansen FK, Espanha LG, Leite SR, Leite CQ, 2011. Characterization of the genetic diversity of Mycobacterium tuberculosis in Sao Paulo city, Brazil. BMC Res Notes 4: 269.

    • Search Google Scholar
    • Export Citation
  • 26.

    de Beer JL, Akkerman OW, Schurch AC, Mulder A, van der Werf TS, van der Zanden A, van Ingen J, van Soolingen D, 2014. Optimization of standard in-house 24-locus variable number of tandem repeat typing for Mycobacterium tuberculosis and its direct application to clinical material. J Clin Microbiol Epub ahead of print 5 February 2014. doi:10.1128/JCM.03436-13.

    • Search Google Scholar
    • Export Citation
  • 27.

    Driscoll JR, 2009. Spoligotyping for molecular epidemiology of the Mycobacterium tuberculosis complex. Methods Mol Biol 551: 117128.

Author Notes

* Address correspondence to Amador Goodridge, Tuberculosis Biomarker Research Unit, Centro de Biología Molecular y Celular de Enfermedades (CBCME) Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), P.O. Box 0843-01103, Ciudad del Saber, Republic of Panamá. E-mail: agoodridge@indicasat.org.pa

Financial support: This study was partially funded by the Network for Research and Training in Tropical Diseases in Central America (NeTropica) Grant nos. 09-R-2003 and 05-N-2005.

Authors' addresses: Dilcia Sambrano, Centro de Biología Celular y Molecular (CBCME) del Instituto de Investigaciones Científicas y Servicios de Alta Tecnología INDICASAT-AIP, Ciudad del Saber, Panamá, Panamá, E-mail: dsambrano@indicasat.org.pa. Ricardo Correa, Centro de Biología Celular y Molecular (CBCME) del Instituto de Investigaciones Científicas y Servicios de Alta Tecnología INDICASAT-AIP, Ciudad del Saber, Panamá, Panamá Department of Biotechnology, Acharya Nagarjuna University, Guntur, India, E-mail: rcorrea@indicasat.org.pa. Pedro Almengor, Laboratorio de Microbiología del Complejo Hospitalario Dr. Arnulfo Arias Madrid de la Caja de Seguro Social CHMDrAAM-CSS, Panamá, Panamá. Amada Domínguez, Departamento de Ciencias de Laboratorio Clínico, Facultad de Medicina, Universidad de Panamá, Panamá, Panamá, E-mail: laboratorioup@yahoo.com. Silvio Vega, Laboratorio de Microbiología del Complejo Hospitalario Dr. Arnulfo Arias Madrid de la Caja de Seguro Social CHMDrAAM-CSS, Panamá, Panamá, E-mail: silviove@yahoo.com. Amador Goodridge, Centro de Biología Celular y Molecular (CBCME) del Instituto de Investigaciones Científicas y Servicios de Alta Tecnología INDICASAT-AIP, Ciudad del Saber, Panamá, Panamá, E-mail: agoodridge@indicasat.org.pa.

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