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.