Discussion
Due to its key location at the isthmus between North and South America, Panama has undergone great demographic and population changes of diverse geographic origins, which has probably affected the composition of circulating MTB strains. Few and discontinuous efforts have been made to genotypically characterize MTB isolates, mainly on convenience samples (Rosas et al., 2013; Sambrano et al., 2014).
To help solve the lack of a systematic molecular epidemiology program in Panama, we introduced (Dominguez et al., 2019) a novel strategy as follows: i) fast MIRU-VNTR-based snapshots of the most prevalent strains, ii) targeted WGS on these major clones, and iii) tailored PCRs to identify marker SNPs for prevalent strains (Dominguez et al., 2019). This allowed us to determine that six strains were responsible for 50% of MTB cases in Panama City and Colon. These six strains were unequally distributed between the two settings, and remarkably, the Beijing lineage was mainly restricted to Colon with an incidence of 21.5%. As the Beijing strain found in Colon is pansusceptible it went unnoticed in a study carried out in Panama that focused only on multidrug-resistant (MDR) TB cases (Rosas et al., 2013). A single previous study carried out in Panama City, in which genotypic data were obtained from non-selected strains from one outpatient clinic reported a much lower percentage (3.7%) of the Beijing lineage (Sambrano et al., 2014).
The purpose of this study was to perform a more in-depth analysis of Beijing strain in the province of Colon. A standard characterization by MIRU-VNTR on a small retrospective sample of isolates alerted us that the true figures were higher than those observed from our previous convenience sample. In fact, over 40% of Colon´s isolates in 2018 were Beijing A strains. Furthermore, additional prospective labelling efforts with strain-specific PCRs, confirmed its high representativeness (44.1%) in incident cases in 2019 in the same city and its absence in a distant region in the same country. Fortunately, most of Beijing A isolates were pansusceptible with 4.8% rifampicin-resistant isolates.
Our first interpretation of the results was that the high presence of Beijing A strains was due to uncontrolled recent transmissions in Colon. Several studies have described the Beijing lineage as responsible for major outbreaks (Bifani et al., 2002; Iwamoto et al., 2012; Johnson et al., 2006; Perez-Lago et al., 2019). Colon has elevated rates of poverty, overcrowded and substandard quality housing, and high HIV coinfection, all factors associated to active MTB transmission. However, WGS analysis of 26 Beijing A isolates revealed higher-than-expected SNP-based diversity, beyond diversity thresholds (12 SNPs) (Walker et al., 2013) for determining transmission clusters, with few cases showing SNP based distances consistent with recent transmissions.
Our findings confirm the homoplasy described before in MIRU-VNTR typing, particularly for the Beijing lineage (Alaridah et al., 2019; Gurjav et al., 2016; Nikolayevskyy, Kranzer, Niemann, & Drobniewski, 2016), revealed by the high discriminatory power of WGS, as described for other settings (Gardy et al., 2011; Stucki et al., 2016). Moreover, our data allows to suggest that the Beijing A strain has probably been present for a long time, with only a minority of cases due to recent transmission.
This new long-term status for the Beijing A strain, as revealed by WGS data, led us to redirect our efforts from our initial molecular epidemiological purpose, namely, to track recent transmissions for their control to another phylogenetically-oriented interest, i.e. to try to explain the reasons behind the predominance of the Beijing A strain in Colon. For this, we first needed to identify the geographical origin for this strain.
Comparisons of the MIRU-VNTR and SNP profiles against global datasets placed the Beijing A strain to the Asian African 3 branch of the modern Beijing sublineage (L2.2.5), which is mainly reported in East Asia (Luo et al., 2015; Merker et al., 2015; Shitikov et al., 2017). The modern Beijing sublineage is considered more virulent and with a higher mutation rate than the ancestral Beijing (Yang et al., 2012). Further detailed analysis of our WGS data positioned the Beijing A representatives close to isolates from a sublineage isolated in Vietnam, with strong bootstrap support (96%). This suggests that the origin of the Colon Beijing A strain may be traced in Southeast-Asia, probably from a non-predominant strain, as scarce L2.2.5 (0.7%) presence in studies with good strain representativeness from Vietnam (Mestre et al., 2011) has been reported. The estimated substitution rate per genome and year for the Beijing A strain is 1.6, which is high when considering the average substitution rates in MTB. Eldholm et al . determined a slightly lower substitution rate in a similar dataset (85 Beijing isolates), still consistent with the normally elevated substitution rates observed throughout the Beijing lineage (Eldholm et al., 2016).
Global spread of the Beijing lineage results from decades of migration events. The introduction from Liberia to the island of Gran Canaria (Spain) in 1993, responsible for its fast subsequent spread in the Canary Islands (Perez-Lago et al., 2019) and its importation to the USA from Asia (Bifani et al., 2002), are examples of recent events. In Peru, the Beijing strain is detected in 9% of TB cases in certain settings; its introduction in this Latin American country probably occurred during Chinese immigration in the mid-19th century for economic and political reasons (Iwamoto et al., 2012). Similarly, the most obvious historical event that may explain the entry of an inedit MTB lineage in Panama is the massive migration from abroad to assure the workload required for the construction of the Panama Canal, although migrants were mainly from France, China and India (”Sanitation and the Panama Canal,” 1905), rather than from Vietnam. In addition, our analysis indicates that the Beijing A strains converged at a time point between 2000 – 2012, while the most recent ancestor between the Panama and Vietnam strains has a time interval of 1987 – 1995. Thus, the Beijing A strain must have been introduced into the region sometime after 2000 (most likely just before 2012), far more recently than the construction of the Canal in the 19th century, and before the more recent Canal expansion in 2015.
Panama´s geographical location at the isthmus between North and South America draws people from many different origins. Most (~85%) people of the Panamanian population have Asian, European and American descendants/ancestors (Arias TC, 2002). Immigration to Panama from Asian countries has been increasing over the years. Between 2000 and 2012 over 50,000 passengers were reported to enter Panama from Asian countries every year (”Instituto Nacional de Estadísticas y Censo, Republica de Panamá (INEC). Movimiento internacional de Pasajeros (2000-2012),”). Thus, the introduction of the Beijing A strain may have easily occurred in any of these multiple opportunities rather than during a previous major historical event.
In summary, to overcome the lack of a systematic molecular epidemiology program in Panama we used a combined MIRU-VNTR and strain-specific PCR approach that allowed us to identify high presence of the Beijing lineage in a specific Panamanian setting. The analysis based on WGS data were key to reveal the long-term nature for this lineage and rule out a role uncontrolled recent transmission. Phylogenetic analysis suggests a not too remote importation from Southeast Asia of a not well represented isolate form the Modern Beijing sublineage. The precise reasons behind the introduction of this Beijing strain and spread throughout the province of Colon requires classical epidemiology and in-depth-omic analysis of both M. tuberculosis and human population.