Abstract
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)*
| Isolate | MIRU-VNTR | Isolate | MIRU-VNTR | Isolate | MIRU-VNTR |
|---|---|---|---|---|---|
| 1 | 224325153323 | 16 | 224325153221 | 31 | 223326153321 |
| 2 | 226425153322 | 17 | 224325143324 | 32 | 223325173533 |
| 3 | 226226153323 | 18 | 224325143324 | 33 | 223325153323 |
| 4 | 225335253323 | 19 | 224322153323 | 34 | 223236253323 |
| 5 | 225335233322 | 20 | 224315143324 | 35 | 225325153324 |
| 6 | 225326133324 | 21 | 224315143323 | 36 | 224335253323 |
| 7 | 225325153321 | 22 | 224216252321 | 37 | 224325153322 |
| 8 | 225325153123 | 23 | 223526143322 | 38 | 225326133323 |
| 9 | 225325143323 | 24 | 223425143324 | 39 | 124326154326 |
| 10 | 225325143321 | 25 | 223335253324 | 40 | 123336253222 |
| 11 | 225226163321 | 26 | 223335253321 | 41 | 123326153326 |
| 12 | 224425173533 | 27 | 223326253321 | 42 | 123326163326 |
| 13 | 224336253323 | 28 | 223326153321 | 43 | 224325153323 |
| 14 | 224335253323 | 29 | 223326153321 | 44 | 123326153326 |
| 15 | 224326133313 | 30 | 223326153321 | 45 | 223326153321 |
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.21–23 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 code | n (%) of M. tuberculosis isolates | Family label |
|---|---|---|
| 777777777760731 | 7 (13.0) | T2 |
| 776177400000171 | 6 (11.1) | U (LAM3?) |
| 777777777720771 | 6 (11.1) | H3 |
| 777777607760771 | 5 (9.3) | LAM9 |
| 000076777760671 | 3 (5.6) | LAM5 |
| 704003347760471 | 3 (5.6) | T4_CEU1 |
| 777777777760771 | 3 (5.6) | T1 |
| 000000000003771 | 2 (3.7) | BEIJING |
| 776177607760771 | 2 (3.7) | LAM3 |
| 777736776000071 | 2 (3.7) | unknown |
| 777777743760771 | 2 (3.7) | LAM10_CAM |
| 000000007560771 | 1 (1.9) | T1 |
| 466177400000171 | 1 (1.9) | unknown |
| 476177400000171 | 1 (1.9) | unknown |
| 777677607760771 | 1 (1.9) | LAM9 |
| 777736777760771 | 1 (1.9) | X1 |
| 777767777760731 | 1 (1.9) | T1 |
| 777776777760601 | 1 (1.9) | X2 |
| 777777477400001 | 1 (1.9) | U |
| 777777764020771 | 1 (1.9) | H1 |
| 777777770000000 | 1 (1.9) | U (likely H) |
| 777777770000171 | 1 (1.9) | unknown |
| 777777774020731 | 1 (1.9) | H1 |
| 777777774020771 | 1 (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.
Disclaimer: Authors declare no conflicting interests.
Footnotes
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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