• View in gallery

    Communities in Guna Yala region in Panama with sputum smear slide collected during 2012–2014. Accessibility to the number of samples are indicated in percentages. This figure appears in color at www.ajtmh.org.

  • 1.

    World Health Organization , 2019. Global Tuberculosis Report 2019. Geneva, Switzerland: WHO. Available at: https://www.who.int/tb/publications/global_report/en/. Accesed August 10, 2020.

  • 2.

    Guio H, Tarazona D, Galarza M, Borda V, Curitomay R , 2014. Genome analysis of 17 extensively drug-resistant strains reveals new potential mutations for resistance. Genome Announc 2: e00759-14.

    • Search Google Scholar
    • Export Citation
  • 3.

    Griffith DE , 2010. Nontuberculous mycobacterial lung disease. Curr Opin Infect Dis 23: 185190.

  • 4.

    Lopez-Roa P et al.2020. Epidemiology of non-tuberculous mycobacteria isolated from clinical specimens in Madrid, Spain, from 2013 to 2017. Eur J Clin Microbiol Infect Dis 39: 10891094.

    • Search Google Scholar
    • Export Citation
  • 5.

    Thomson RM , NTM working group at Queensland TB Control Centre and Queensland Mycobacterial Reference Laboratory, 2010. Changing epidemiology of pulmonary nontuberculous mycobacteria infections. Emerg Infect Dis 16: 15761583.

    • Search Google Scholar
    • Export Citation
  • 6.

    Victoria J, Botello AM, 2018. Ampliando el acceso a servicios de salud integrales, de calidad, centrados en las personas para el VIH y TB en Panama 2018. Organización Panamericana de la Salud 6: 3640.

  • 7.

    Whang J, Lee BS, Choi GE, Cho SN, Kil PY, Collins MT, Shin SJ , 2011. Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies. Diagn Microbiol Infect Dis 70: 6571.

    • Search Google Scholar
    • Export Citation
  • 8.

    Weerasekera DK, Magana-Arachchi DN, Madegedara D, Dissanayake N , 2014. Polymerase chain reaction - restriction fragment length polymorphism analysis for the differentiation of mycobacterial species in bronchial washings. Ceylon Med J 59: 7983.

    • Search Google Scholar
    • Export Citation
  • 9.

    Sinha P, Gupta A, Prakash P, Anupurba S, Tripathi R, Srivastava GN , 2016. Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments. BMC Infect Dis 16: 123.

    • Search Google Scholar
    • Export Citation
  • 10.

    Supply P et al.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
  • 11.

    Carniel F, Dalla Costa ER, Lima-Bello G, Martins C, Scherer LC, Rossetti ML , 2014. Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area. Braz J Med Biol Res 47: 10161020.

    • Search Google Scholar
    • Export Citation
  • 12.

    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
  • 13.

    Lee H, Park HJ, Cho SN, Bai GH, Kim SJ , 2000. Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol 38: 29662971.

    • Search Google Scholar
    • Export Citation
  • 14.

    Chimara E, Ferrazoli L, Ueky SY, Martins MC, Durham AM, Arbeit RD, Leao SC , 2008. Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65 patterns. BMC Microbiol 8: 48.

    • Search Google Scholar
    • Export Citation
  • 15.

    Ong CS, Ngeow YF, Yap SF, Tay ST , 2010. Evaluation of PCR-RFLP analysis targeting hsp65 and rpoB genes for the typing of mycobacterial isolates in Malaysia. J Med Microbiol 59: 13111316.

    • Search Google Scholar
    • Export Citation
  • 16.

    Zavala S, Rosas SCA, Sosa N, Henostroza G, Arauz Rodriguez AB , 2018. Characterization of non-tuberculous mycobacteria isolates in a national mycobacterial laboratory in Panama: 2012–2015. Open Forum Infect Dis 5 (Suppl 1 ):S280.

    • Search Google Scholar
    • Export Citation
  • 17.

    Silva RM, Bazzo ML, Chagas M , 2010. Quality of sputum in the performance of polymerase chain reaction for diagnosis of pulmonary tuberculosis. Braz J Infect Dis 14: 116120.

    • Search Google Scholar
    • Export Citation
  • 18.

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

  • 19.

    Acceso Global , 2017. Panama Transition Readiness Assessment Country Report. Available at: https://acesoglobal.org/work/2019-4-3-panama-transition-readiness-assessment-country-report/ . Accessed August 10, 2020.

  • 20.

    Mambuque ET, Abascal E, Venter R, Bulo H, Bouza E, Theron G, Garcia-Basteiro AL, Garcia-de-Viedma D , 2018. Direct genotyping of Mycobacterium tuberculosis from Xpert((R)) MTB/RIF remnants. Tuberculosis (Edinb) 111: 202206.

    • Search Google Scholar
    • Export Citation
  • 21.

    Fontes AN et al.2012. Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil. Mem Inst Oswaldo Cruz 107 (Suppl 1 ):143149.

    • Search Google Scholar
    • Export Citation
  • 22.

    Griffith DE et al. ATS Mycobacterial Diseases Subcommittee, American Thoracic Society, Infectious Disease Society of America, 2007. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175: 367416.

    • Search Google Scholar
    • Export Citation
 
 

 

 

 

 

 

 

Direct Molecular Characterization of Acid-Fast Bacilli Smear of Nontuberculosis Mycobacterium Species Causing Pulmonary Tuberculosis in Guna Yala Region, Panama

View More View Less
  • 1 Centro Regional de Salud de la Comarca Guna Yala, Ministerio de Salud (MINSA), Hospital Rural Inabaguinya, Sasardi, Tubuala, Guna Yala, Panamá;
  • | 2 Tuberculosis Biomarker Research Unit, Centro de Biología Molecular y Celular de Enfermedades (CBCME) del Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Ciudad del Saber, Panamá;
  • | 3 Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India;
  • | 4 Florida State University, Ciudad del Saber, Panamá;
  • | 5 Centro de Salud de Playon Chico, Ailigandi, Guna Yala, Panama;
  • | 6 Centro de Salud de Cartí Sugtupu, Narganá, Guna Yala, Panama;
  • | 7 Centro de Salud de Narganá, Narganá, Guna Yala, Panama

ABSTRACT.

Mycobacterium tuberculosis (MTB) stands out as the main causative agent of pulmonary tuberculosis (TB). However, nontuberculous mycobacteria (NTM) species also have the potential to infect and cause TB in susceptible individuals. The objective of this study was to identify NTM species that cause public health problems in remote areas. The study was carried out using 105 sputum smears obtained from patients from the Guna Yala Region of Panama with clinical signs suggestive of TB. DNA was extracted from sputum smears. Nontuberculous mycobacteria and MTB were characterized using polymerase chain reaction restriction analysis (hsp65, rpob) and an evaluation of 24-mycobacterial interspersed repetitive units–variable number of tandem repeats loci. Twenty-six Mycobacterium species were characterized; 19 (18%) were identified as MTB, and 7 (6.7%) were identified as NTM (four M. avium complex, two M. haemophilum, one M. tusciae). These results suggest that at least one in five cases of pulmonary TB among this population is caused by an NTM. Thus, identifying the bacteria causing pulmonary disease is key even in remote regions of the world where standard diagnosis and culture are not available. Strengthening the laboratory capacity within the Guna Yala Region is needed to identify NTM infections promptly.

Tuberculosis (TB) remains a significant public health challenge; 23% of the world population is estimated to have a latent TB infection; these individuals are at risk of developing active TB disease during their lifetime.1,2 Although Mycobacterium tuberculosis is the most important causative agent of TB, nontuberculous mycobacteria (NTM) may play a important role in the etiology of TB-like syndromes. The number of mycobacterial species recognized as potential pathogens is increasing, and in the past year, an increase in NTM isolation rates has been observed worldwide.3,4 Nontuberculous mycobacteria are opportunistic pathogens that are acquired from environmental reservoirs (water, soil) and often represent an important cause of morbidity and mortality in immunocompromised humans.3,5

In Central America, Panama stands as the country with the highest TB rate (52 cases per 100,000 population),1 and the Guna Yala Region (GYR) is the area with the highest incidence (191.8 per 100,000 population).6 The GYR is located in northeastern Panama and is characterized by its lengthy Caribbean coastline inhabited by indigenous people (Figure 1). Determining the incidence and prevalence of NTM lung disease in this area remains problematic primarily because disease reporting is not mandatory and because individual NTM isolates must be assessed to determine their clinical significance. This is in contrast to M. tuberculosis, for which each isolate is assumed to be associated with true disease. Therefore, the prevalence of NTM isolates is not known with certainty.

Figure 1.
Figure 1.

Communities in Guna Yala region in Panama with sputum smear slide collected during 2012–2014. Accessibility to the number of samples are indicated in percentages. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 3; 10.4269/ajtmh.21-0096

Molecular epidemiology based on polymerase chain reaction (PCR) to genotype M. tuberculosis complex (MTBC) is widely used to understand TB transmission and outbreaks. Polymerase chain reaction restriction fragment length polymorphism analysis (PRA) is preferred because it offers an easy and rapid method to characterize Mycobacterium species by applying several targets (16S rDNA, hsp65, IS6110, and rpoB) to a single copy of DNA.79 Currently, mycobacterial interspersed repetitive units–variable number of tandem repeats (MIRU-VNTR) is the reference approach for genotyping MTBC with good specificity and discrimination, which improves the cost-benefit and reduces workload.10

The application of molecular tests in routine TB diagnosis using slides already prepared for microscopy could be a useful option, especially at sites that are far from reference centers and have poor biosafety infrastructure, making safe sample transport a significant challenge.

For example, a study performed by Carniel et al.11 in the Amazonian area of northern Brazil applied PCR-IS6110 directly to sputum smears stained with Zielh Neelsen (ZN), and they were able to detect TB cases with an accuracy of 91%. This approach offers an affordable strategy for accurate TB diagnosis in remote areas where culture is not available. For these reasons, analyzing DNA extracted from sputum smears offers a good strategy for characterizing Mycobacterium species responsible for TB cases in areas with limited laboratory capacity. The objective of this study was to identify possible atypical mycobacterial species that cause TB using a DNA fingerprinting technique on DNA extracted from sputum smear slides.

A retrospective collection of 105 acid-fast sputum smears collected in GYR (November 2012–February 2014) were analyzed. The slide collection corresponded to samples taken at Centro Regional de Salud de la Comarca Guna Yala as part of the TB Control Program of the Ministerio de Salud of Panama. The 23 Guna Yala (GY) communities included in this study are represented in Figure 1. The ZN method of acid-fast staining was used for detection of acid-fast bacilli (AFB) in smears and examined microscopically. All sputum smears (two slides per sample) were sent to the Tuberculosis Biomarker Research Unit, in the Centro de Biologia Celular y Molecular de las Enfermedades laboratory of the INDICASAT-AIP in City of Knowledge.

The DNA extraction was performed using a method previously described by Van Soolingen et al.12 Briefly, a slice of the smear was removed and transferred to a microtube containing 1X TE buffer. The material was centrifugated and heated to 90°C for 10 minutes. Cellular lysis was performed with the addition of 40 µL of lysozyme (20 mg/mL) and Proteinase K (10 mg/mL) followed by incubation at 37°C; then the sample was agitated at 65°C. After that, 100 µL of the CTAB was added, and the sample was incubated for 30 minutes. The material was centrifuged at 7,000 rpm for 5 minutes, and phenol-chloroform-isoamyl alcohol (24:24:1) solution was added to the supernatant to remove interfering agents. Absolute ethanol and 3M sodium acetate was used to precipitate the DNA. The extracted DNA was stored at 4°C.

The PRA method based on PCR analysis was performed for the target hsp65 and rpob genes according to the methods described in Lee et al.13 Target segments of the hsp65 and rpob genes were amplified by specific primers and viewed following agarose gel electrophoresis. The PCR products were analyzed using the restriction enzymes BstEII, HaeIII, and MspI. The presence of amplified products was confirmed in 4% agarose gel electrophoresis. The mediate pattern size was compared with a previously described study to confirm NTM species and compared with the international PRASITE database (http://app.chuv.ch/prasite/index.html).1315

Genotyping based on 24-MIRU-VNTR loci was performed to characterize MTBC. The amplification products were analyzed by capillary electrophoresis (Applied Biosystems, Foster City, CA). The number of MIRU-VNTR alleles was determined according to a protocol described by Supply et al.10

Among the communities included in this study, Playon Chico (31.5%), Ustupu (13.5%), Nargana (7.9%), Gardi Sugdup (6.7%), and Soledad Mandiga (6.7%) provided the most AFB sputum slides.

A total of 26 sputum smear slides were characterized as Mycobacterium spp.; 19 were identified as MTBC, and seven were identified as NTM species (Table 1). The main NTM species were characterized as M. avium complex (N = 4), M. haemophilum (N = 2), and M. tusciae (N = 1). Polymerase chain reaction for both hsp65 and rpoB did not result in amplification for 79 (75.2%) DNA samples extracted from the sputum smears. All MTBC (N = 19) were genotyped according to MIRU-VNTR and compared with the MIRU-VNTR plus database (https://www.miru-vntrplus.org/MIRU/index.faces). Our findings show five genotypes with 24 complete loci; the obtained samples GY-117 and GY-120 showed a change in a single-locus variant (MIRU 10 [4/3] and VNTR 48 [2/1]); the rest of the DNA samples (GY-1, GY-69, and GY-81) had more than four single locus variants or incomplete MIRU-VNTR (N =14) (see Table 2).

Table 1

Characterization of mycobacteria species by hsp65-rpob restriction fragment length polymorphism methods

BacilloscopyN (%)
1+2 (1.9)
2+2 (1.9)
3+5 (4.8)
AFB positive17 (16.1)
AFB negative79 (75.2)
Species ID
 M. tuberculosis Complex19 (18.0)
 Nontuberculosis7 (6.7)
 M. avium complex4 (3.8)
M. haemophilum2 (1.9)
 M. tusciae1 (0.9)
No amplification product79 (75.2)

AFB = acid-fast bacillus.

Table 2

The 24-loci MIRU-VNTR genotyping of M. tuberculosis Complex from sputum smear slide

IDMIRU 02VNTR 42VNTR 43MIRU 04MIRU 40MIRU 10MIRU 16VNTR 1955MIRU 20VNTR QUB11bVNTR ETR-AVNTR 46VNTR 47VNTR 48MIRU 23MIRU 24MIRU 26MIRU 27VNTR 49MIRU 31VNTR 52VNTR QUB-26VNTR 53MIRU 39
GY-1223235330434425153321232
GY-59223232332434433153233752
GY-82223243342234425143334242
GY-117244234322424226133131232
GY-120244233322424216133131232
H37Rv*224313222534236133335522

ETR = exact tandem repeats; MIRU-VNTR = mycobacterial interspersed repetitive units–variable number of tandem repeats; QUB = Queens University of Belfast.

Control, Mycobacterium tuberculosis American Type Culture Collection (ATCC) strain H37Rv.

We analyzed a conventional method AFB and simple PRA analysis to identify and characterize NTM. Specifically, of the 79 samples with AFB-negative results according to the microscopic test, 12.6% (10/79) were positive with hsp65-rpob restriction fragment length polymorphism. Under usual practices these cultures would have been discarded, but molecular screening alerted us to the presence of mycobacteria (six MTBC and four NTM) in the samples. A previous study by Carniel et al.11 applied a combination method using ZN/PCR and IS6110-PCR. They reported that the ZN/PCR combination identified seven (24.14%) additional cases, whereas IS6110-PCR showed positivity in 26% of samples that were AFB negative. In our analysis, the sensitivity and specificity of AFB staining were 24.7% and 75.2%, respectively, whereas the sensitivity and specificity of the PRA analysis were 61.5% and 87.4%, respectively. The combined ZN/PCR analysis showed a sensitivity of 61.5% and a specificity of 81.3%. The performance of this strategy with sputum samples with low bacillary load has not been determined.

The proportions of NTM and MTBC found in our study were similar to those reported by Zavala et al.16 In this study, the National Mycobacterial Laboratory in Panama analyzed 4,257 sputum samples collected between 2012 and 2015 to confirm their identification. This study identified 65.3% (2,783/4,257) as M. tuberculosis and 18% (769/4,257) as NTM. Among the NTM, M. avium complex was the most frequent NTM strain, accounting for 22.3% of the samples. In contrast, our study showed the most frequent NTM isolate was M. avium complex in four (15.3%) samples, followed by M. haemophilum in two samples (7.6%) and M. tusciae in one sample (3.8%). Together these findings highlight the importance of NTM and the clinical significance in patients with suspected TB.

The number of NTM characterized in GYR is consistent with the increasing rates reported worldwide in recent decades. A multicenter epidemiology study (2013–2017) of NTM in Spain described the frequency and diversity of 65.5% NTM and 34.5% MTB species.4

For this type of analysis, the quality of the sputum sample significantly influences the performance of AFB and PRA analyses. High-quality sputum smaples resulted in an improved AFB or PRA analysis. Low-quality sputum samples represent a challenge in routine diagnosis. Approximately 25% of sputum samples are not adequate for culture, which leads to high variability in results. The PCR analysis is rapid and sensitive but presents two major problems: 1) false-negative reactions caused by inhibitors and false-positive reactions due to contamination with DNA and amplicons and 2) the influence of respiratory tract microbiota on the performance of AFB/PRA analysis.17 Culture remains the gold standard for laboratory confirmation of NTM infection. Thus, a direct correlation between molecular and culture results from a sputum specimen (not prepared as an AFB smear) is needed to validate this strategy.

We studied a convenience sputum smear sample set. The true NTM proportion among the GYR inhabitants remains to be determined. We hypothesize that the lifestyle in GY could account for the high proportion of NTM among people with tuberculosis. For example, the GY population harbor a closed cultural behavior, and it is difficult to access their traditions. The population prefers to use traditional medicine at home and does not seek medical attention at government or private health centers. This leads to increased infection rates and difficulty in following up patients. In addition, there is only one AFB laboratory in the GYR, which serves people with limited accessibility and frequent interruptions in anti-tuberculosis therapies.18

GeneXpert was introduced in GY in 2017 by international support from Global Fund to Combat HIV, Tuberculosis and Malaria.19 This technology is used specifically for MTBC diagnosis and does not identify NTM. Our results indicate that collecting microscopy slides and applying PCR detection and genotyping methods is one option for the GYR. To genotype MTBC, we support the option to save GeneXpert positive cartridges and use the GeneXpert remnants for further MTBC genotyping, as previously reported.11,1921

The American Thoracic Society and Infectious Diseases Society of America have provided guidelines to assist in the accurate diagnosis of lung disease caused by NTM.22 These microbiologic, radiographic, and clinical criteria are considered equally important and must be met to make a proper NTM diagnosis. Specifically, the American Thoracic Society recommends having positive culture results from at least two separate expectorated sputum samples. We agree that the same criteria should be applied for NTM molecular diagnosis. At least two sputum smear slides should have positive molecular test results before initiating treatment with antibiotics. On the other hand, the presence of NTM in sputum samples indicates a need for attention in the GYR; the high prevalence of TB and poor access to the health system need to be addressed urgently. Because NTM are not reported infections, the extent and prevalence data are limited. However, there is evidence of their increase not only in low-income countries but also in developed countries.4

The diagnosis of MTBC or NTM in remote areas presents major challenges. The main difficulties include late arrival to the reference laboratory in Panama City due to inefficient sample transport mechanisms, resulting in lost or deteriorated specimens that cannot be analyzed. Our study shows that we are able to characterize mycobacteria directly from AFB smears and simultaneously genotype Mycobacterium species that most affect rural regions in Panama, such as GY. This is particularly important to provide an affordable alternative to other remote areas, including Ngäbe Buglé and Bocas del Toro, where TB rates are double or triple the national average. These areas demonstrate social determinants for TB disease, including high levels of poverty, internal migrants moving from rural to urban areas, and poor access to health services. Sputum slides can be easily transported at room temperature, making their conservation easier. This would facilitate the characterization of NTM within Mycobacterium species in a shortened period and represents an alternative where culture remains challenging in this remote area. Furthermore, we recommend strengthening laboratory diagnosis within the GYR to identify infections with NTM in a timely manner.

ACKNOWLEDGMENTS

We thank the health care workers in Guna Yala for providing access to data and sputum smears. We also thank Colleen Goodridge for critically reviewing this manuscript.

References

  • 1.

    World Health Organization , 2019. Global Tuberculosis Report 2019. Geneva, Switzerland: WHO. Available at: https://www.who.int/tb/publications/global_report/en/. Accesed August 10, 2020.

  • 2.

    Guio H, Tarazona D, Galarza M, Borda V, Curitomay R , 2014. Genome analysis of 17 extensively drug-resistant strains reveals new potential mutations for resistance. Genome Announc 2: e00759-14.

    • Search Google Scholar
    • Export Citation
  • 3.

    Griffith DE , 2010. Nontuberculous mycobacterial lung disease. Curr Opin Infect Dis 23: 185190.

  • 4.

    Lopez-Roa P et al.2020. Epidemiology of non-tuberculous mycobacteria isolated from clinical specimens in Madrid, Spain, from 2013 to 2017. Eur J Clin Microbiol Infect Dis 39: 10891094.

    • Search Google Scholar
    • Export Citation
  • 5.

    Thomson RM , NTM working group at Queensland TB Control Centre and Queensland Mycobacterial Reference Laboratory, 2010. Changing epidemiology of pulmonary nontuberculous mycobacteria infections. Emerg Infect Dis 16: 15761583.

    • Search Google Scholar
    • Export Citation
  • 6.

    Victoria J, Botello AM, 2018. Ampliando el acceso a servicios de salud integrales, de calidad, centrados en las personas para el VIH y TB en Panama 2018. Organización Panamericana de la Salud 6: 3640.

  • 7.

    Whang J, Lee BS, Choi GE, Cho SN, Kil PY, Collins MT, Shin SJ , 2011. Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies. Diagn Microbiol Infect Dis 70: 6571.

    • Search Google Scholar
    • Export Citation
  • 8.

    Weerasekera DK, Magana-Arachchi DN, Madegedara D, Dissanayake N , 2014. Polymerase chain reaction - restriction fragment length polymorphism analysis for the differentiation of mycobacterial species in bronchial washings. Ceylon Med J 59: 7983.

    • Search Google Scholar
    • Export Citation
  • 9.

    Sinha P, Gupta A, Prakash P, Anupurba S, Tripathi R, Srivastava GN , 2016. Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments. BMC Infect Dis 16: 123.

    • Search Google Scholar
    • Export Citation
  • 10.

    Supply P et al.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
  • 11.

    Carniel F, Dalla Costa ER, Lima-Bello G, Martins C, Scherer LC, Rossetti ML , 2014. Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area. Braz J Med Biol Res 47: 10161020.

    • Search Google Scholar
    • Export Citation
  • 12.

    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
  • 13.

    Lee H, Park HJ, Cho SN, Bai GH, Kim SJ , 2000. Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol 38: 29662971.

    • Search Google Scholar
    • Export Citation
  • 14.

    Chimara E, Ferrazoli L, Ueky SY, Martins MC, Durham AM, Arbeit RD, Leao SC , 2008. Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65 patterns. BMC Microbiol 8: 48.

    • Search Google Scholar
    • Export Citation
  • 15.

    Ong CS, Ngeow YF, Yap SF, Tay ST , 2010. Evaluation of PCR-RFLP analysis targeting hsp65 and rpoB genes for the typing of mycobacterial isolates in Malaysia. J Med Microbiol 59: 13111316.

    • Search Google Scholar
    • Export Citation
  • 16.

    Zavala S, Rosas SCA, Sosa N, Henostroza G, Arauz Rodriguez AB , 2018. Characterization of non-tuberculous mycobacteria isolates in a national mycobacterial laboratory in Panama: 2012–2015. Open Forum Infect Dis 5 (Suppl 1 ):S280.

    • Search Google Scholar
    • Export Citation
  • 17.

    Silva RM, Bazzo ML, Chagas M , 2010. Quality of sputum in the performance of polymerase chain reaction for diagnosis of pulmonary tuberculosis. Braz J Infect Dis 14: 116120.

    • Search Google Scholar
    • Export Citation
  • 18.

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

  • 19.

    Acceso Global , 2017. Panama Transition Readiness Assessment Country Report. Available at: https://acesoglobal.org/work/2019-4-3-panama-transition-readiness-assessment-country-report/ . Accessed August 10, 2020.

  • 20.

    Mambuque ET, Abascal E, Venter R, Bulo H, Bouza E, Theron G, Garcia-Basteiro AL, Garcia-de-Viedma D , 2018. Direct genotyping of Mycobacterium tuberculosis from Xpert((R)) MTB/RIF remnants. Tuberculosis (Edinb) 111: 202206.

    • Search Google Scholar
    • Export Citation
  • 21.

    Fontes AN et al.2012. Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil. Mem Inst Oswaldo Cruz 107 (Suppl 1 ):143149.

    • Search Google Scholar
    • Export Citation
  • 22.

    Griffith DE et al. ATS Mycobacterial Diseases Subcommittee, American Thoracic Society, Infectious Disease Society of America, 2007. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175: 367416.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Amador Goodridge, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Ciudad del Saber, Panamá. E-mail: agoodridge@indicasat.org.pa †These authors contributed equally to this article.

These authors contributed equally to this article.

Financial support: This research was funded partially by the National Secretariat of Science and Technology of Panama (SENACYT) through the Sistema Nacional de Investigadores de Panamá (SNI), Programa de Inserción de Talento Especializado Grant No. ITE-11-020 and by the Programa de Beca Doctoral IFARHU-SENACYT (grant no. 270-2016-293) to F.A.

Authors’ addresses: Arístides López, Florentino Arias, Baudilio Escobar, Porfirio Ortis, and Fidel Adames, Ministerio de Salud Panama, Centro Regional de Salud de la Comarca Guna Yala, Ailigandí, Guna Yala, Panama, E-mails: alopez@minsa.gob.pa, farias@minsa.gob.pa, bescobar@minsa.gob.pa, portis@minsa.gob.pa, and fadames@minsa.gob.pa. Fermin Acosta, INDICASAT AIP, Centro de Biología Molecular de las Enfermedades, Panama City, Panama, E-mail: fermin2819@gmail.com. Dilcia Sambrano and Amador Goodridge, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología INDICASAT-AIP, Centro de Biología Celular y Molecular de las Enfermedades, City of Knowledge, Panama, E-mails: ludy25305@gmail.com and amadorgj@yahoo.com. Musharaf Tarajia, INDICASAT-AIP, Tuberculosis Biomarker Research Unit at Centro de Biología Molecular y Celular de Enfermedades, Ciudad del Saber, Panama City, Panama, E-mail: drtarajia@me.com. Sophia Navajas, Florida State University, Panama City, Biology, City of Knowledge, Panama, E-mail: sn13@my.fsu.edu.

Save