DISCUSSION
Parasite genetic diversity has been proposed as a key indicator of
malaria transmission dynamics to track control and elimination progress
(Stuckey et al., 2018). This demands an evaluation of whether genotyping
provides insights about changes in transmission following intensified
malaria control efforts. Here we have monitored the population genetics
of P. falciparum and P. vivax over an eight-year period of
LLIN-induced transmission decline in PNG, an area of high year round
transmission. Despite large reductions in parasite prevalence and
multiple infections from very high to low/moderate levels (Kattenberg et
al., 2020; Koepfli et al., 2017; Koepfli et al., 2015), we show that
population genetic changes were minimal with populations remaining
diverse and unstructured. P. falciparum diversity decreased
somewhat, though remained high relative to other malaria endemic areas
outside Africa (Anderson et al., 2000; Branch et al., 2011; Chenet et
al., 2012; Chenet et al., 2015; dalla Martha, Tada, Ferreira, da Silva,
& Wunderlich, 2007; Noviyanti et al., 2015; Orjuela-Sanchez et al.,
2009; Orjuela-Sanchez et al., 2013; Pava et al., 2017; Susomboon et al.,
2008), whereas P. vivax diversity was unchanged throughout the
study period. Surprisingly, P. falciparum populations that were
structured pre-LLIN (2005, 2006) (Jennison et al., 2015; Schultz et al.,
2010), were unstructured post-LLIN (2010, 2014), although a reduction in
multiple infections and an increase in multilocus LD due to clonal
haplotypes were detected. For P. vivax , there was also no
evidence of population structure after LLIN, however increasing pairwise
genetic differentiation within and between provinces was observed and
clonal transmission and inbreeding had emerged in at least one village.
These results demonstrate that declining transmission does not result in
an immediate decrease in overall population diversity nor an increase in
population structure. Sustained low transmission may be needed to
observe these changes using these small panels of microsatellite
markers. However, changes in multiple infection prevalence and
multilocus LD indicate increasing heterogeneity in transmission within
populations. These results have implications with respect to monitoring
changing transmission for any pathogen using population genetic
approaches.
Nkhoma and colleagues previously reported a limited impact on P.
falciparum diversity following a decrease from moderate to low
transmission on the Thai-Myanmar border over 10 years, however as we
have observed in PNG, there was an increase in multilocus LD which and
decreasing proportions of multiclonal infections (Nkhoma et al., 2013).
Daniels et al . have also reported decreasing multiclonal
infections, increasing proportions of repeated genotypes and multilocus
LD, and long-term persistence of particular clones in Senegal (Daniels
et al., 2013). These studies utilised 96 and 24 biallelic SNP markers
respectively, compared to our study using a small number of
microsatellite markers - similar panels of SNPs may reveal additional
insights in the PNG context. As for P. vivax , Gunawardena and
colleagues also reported sustained high P. vivax microsatellite
diversity and multiclonal infections during a five-year period as the
country progressed towards malaria elimination (Gunawardena et al.,
2014). Population genetic signals of declining transmission might take
longer to emerge for P. vivax due to new blood-stage infections
from the hypnozoite reservoir and could explain why we only observed mLD
in one village of East Sepik.
In PNG, the limited decline in P. falciparum diversity and loss
of parasite population structure after LLIN distribution indicates that
there may be increased gene flow between the sampled parasite
populations, which was unexpected. Population structure prior to
intensified control was thought to be the result of limited historical
human migration due to the rugged terrain and lack of direct transport
connections (Mueller, Bockarie, Alpers, & Smith, 2003; Riley, 1983) or
population bottlenecks due to past control efforts or emergence of drug
resistance (Anderson et al., 2000; Jennison et al., 2015; Schultz et
al., 2010; Tessema et al., 2015). Changes in first-line treatment
policies, for example the introduction of sulphadoxine/pyrimethamine
(SP) in the early 2000’s and artemether-lumefantrine in 2008, might have
played a role in shaping parasite population structure (Mu et al.,
2005). Chloroquine (CQ) and/or SP resistance (near fixation of resistantpfmdr1 and pfdhps resistant alleles were observed in the
same areas (Barnadas et al., 2015; Koleala et al., 2015; Mita et al.,
2018)) and may have contributed to the observed bottleneck and mLD in
pre-LLIN P. falciparum populations, with consequent reductions in
effective population size, while outcrossing due to high transmission
preserved within-population genetic diversity as the resistance mutation
spread (Mita et al., 2018). From 2000 to 2011 the PNG population
increased by over two million people (National Census Report, 2011), and
local observations suggest that large numbers of migrants from East
Sepik have moved into Madang in the last decade seeking employment
opportunities. As a result, post-LLIN, greater connectivity between
human populations may have broken down P. falciparum population
structure and maintained high gene flow between P. vivaxpopulations (Fola et al., 2018).
For the post-LLIN East Sepik P. vivax population where prevalence
dropped to below 5%, significant mLD was observed resulting from very
closely related parasite strains circulating in a residual pocket of
relatively high transmission within a single village. This suggests
considerable inbreeding of parasites in that village, in a background of
high genetic diversity resulting in a lack of evidence of a bottleneck
at the population level. This paradoxical signature of significant mLD
with high diversity and a considerable proportion of multiple clone
infections of P. vivax appears to be a hallmark of lower
transmission areas (Barry et al., 2015; Batista et al., 2015; Chenet et
al., 2012; Delgado-Ratto et al., 2016; Delgado-Ratto et al., 2014;
Ferreira et al., 2007; Hong et al., 2016; Noviyanti et al., 2015;
Orjuela-Sanchez et al., 2013). Similar to P. falciparumpopulations though, there was a correlation between mLD and prevalence
of infection for P. vivax . This shows that reductions in
multiclonal infections and mLD is an earlier indicator of reduced
transmission than genetic diversity and population structure (for both
species).
Multilocus LD in post-LLIN P. vivax populations was explained by
both focal clonal transmission and inbreeding, as similarly observed in
other studies in Peru, Vietnam, and Papua Indonesia (Delgado-Ratto et
al., 2014; Hong et al., 2016; Noviyanti et al., 2015). The explanation
for this observation will most likely lie in unique P. vivaxcharacteristics related to hypnozoites, relapses and transmission
dynamics (Abdullah et al., 2013; Bright et al., 2014; Chen, Auliff,
Rieckmann, Gatton, & Cheng, 2007; Delgado-Ratto et al., 2014; Ferreira
et al., 2007; Fola et al., 2018; Iwagami et al., 2012; White, 2011). At
high transmission (e.g. pre-LLIN) with high prevalence and high
multiplicity of infection, these clusters of similar haplotypes might
also occur, but could be obscured due to sampling limitations and the
large number of different haplogroups circulating at the same time. As
transmission declines, infections have fewer clones and the diversity of
the hypnozoite reservoir decreases, resulting in fewer circulating
haplogroups, lower levels of recombination between distinct genomes and
more frequent clonal transmission and inbreeding, resulting in increased
mLD as in this and other studies (Barry et al., 2015; Batista et al.,
2015; Chenet et al., 2012; Delgado-Ratto et al., 2016; Delgado-Ratto et
al., 2014; Ferreira et al., 2007; Hong et al., 2016; Noviyanti et al.,
2015; Orjuela-Sanchez et al., 2013). These signatures are more likely to
be seen in a population with more sustained low transmission such as was
the case for the East Sepik Province. In this region, malaria
transmission is heterogeneous between villages. Besides the national
malaria control program, other initiatives were also distributing LLINs
in East Sepik Province prior to the nationwide distribution (Hetzel et
al., 2014; Hetzel et al., 2012; Hetzel et al., 2016) suggesting that
longer term sustained control efforts have been effective.
Considerable variance in the impact of the LLIN program was observed in
the two provinces. In Madang, whilst P. falciparum rates steadily
declined over the entire study period, there was a resurgence of
submicroscopic P. vivax infections in 2014 (Koepfli et al.,
2017). Although multiplicity of infection remained low, the lack of mLD
and population structure suggests that this is not due to an outbreak,
but more likely the residual P. vivax population was able to gain
a foothold once again despite the intensive control efforts. In
addition, an increase in the prevalence of pvdhfr triple and
quadruple mutants, related with SP resistance, were observed in Madang
province in 2010 (Barnadas et al., 2015), and a high proportion of
resistant parasites could be a possible explanation for the higher
infection prevalence by 2014. Different studies have shown that
selective pressure of drugs such as CQ and/or SP have had an impact on
shaping worldwide P. vivax populations in recent history (Hupalo
et al., 2016; Pearson et al., 2016). However, a population bottleneck as
seen in P. falciparum populations (Mita et al., 2018) did not
occur in P. vivax populations of PNG. Malaria control had a
greater impact on P. vivax prevalence in East Sepik and
population structure was observed in one village post-LLIN.
This study has some limitations related to sampling and the genotyping
approach used. The samples were collected in serial cross-sectional
surveys over a period of malaria control initiated at different times in
the two provinces. Fewer years of sustained control pressure compared to
East Sepik might explain why, despite substantial prevalence decline in
Madang Province by 2010, we did not observe any signature of changing
population structure. Whilst, in East Sepik 2012, a cluster of closely
related parasites was observed in one village suggesting more focal
transmission than previous years. The microsatellite panels were
selected as these have been the gold standard genotyping tool for
large-scale malaria population genetic studies for many years (Anderson
et al., 1999; Imwong et al., 2006; Karunaweera et al., 2006). However,
they are limited in number (less than one per chromosome), rapidly
evolving and prone to error. Although these markers are extremely useful
for measuring parasite population structure on local and global scales
(Auburn & Barry, 2017; Barry et al., 2015; Koepfli & Mueller, 2017;
Sutton, 2013), they are not ideal for cross-study comparisons due to the
difficultly in calibrating alleles from different data sources. The lack
of raw data from the previously published P. falciparum study
(Schultz et al., 2010), prevented the direct comparison of haplotypes
and thus the analysis of population structure between the earlierP. falciparum time points for the East Sepik II (Wosera) and
Madang populations. Furthermore, dominant haplotypes derived from
multiple clone infections can be reconstructed incorrectly, thus
inflating diversity values (Messerli, Hofmann, Beck, & Felger, 2017).
However, only haplotype-based analyses such as multilocus LD and
phylogenetic analysis were vulnerable to these possible artefacts, and
would result in an overestimate of diversity and underestimate of LD.
All other analyses were conducted using mean values across markers or
allele frequencies and thus limit the impact of such errors. Moreover,
the dataset included a large proportion of single infection haplotypes
in all populations, and the detected clones included dominant haplotypes
suggesting that allele calling was reliable. Finally, the highly
polymorphic and rapidly evolving nature of microsatellite markers
(Ellegren, 2004) may limit the resolution of the population genetic
parameters such as population level diversity and population structure
in high transmission areas (Branch et al., 2011). This may both lead to
false assignment of unrelated parasites (e.g. from different provinces)
as related and reduce the detection of truly related parasites
(identical by descent), both of which would result in a lack of
population structure. Other more stable markers, such as SNPs (Baniecki
et al., 2015; R. Daniels et al., 2008) or whole genome sequencing
(Hupalo et al., 2016; Miotto et al., 2013; Mu et al., 2005; Pearson et
al., 2016; Volkman et al., 2007) may be more sensitive and specific to
detect changes in parasite population structure.
According to the data presented, in two high transmission provinces of
PNG, recent reductions in malaria transmission result in limited changes
in parasite population diversity and structure. The high parasite
diversity and lack of population structure are consistent with both
species maintaining a large and evolutionarily fit population
immediately after prevalence decline suggesting the gains made are
fragile. However, fewer multiclonal infections, and the emergence of
significant mLD for both species indicates there is focally interrupted
transmission and suggests that these parameters may be useful markers
for measuring control impact and early changes in parasite population
structure with intervention. The results were in contrast to our
expectations of decreasing diversity and increasing population structure
(Jennison et al., 2015) and show that long term sustained control
efforts may need to be maintained to observe significant change in
population structure, at least as measured by the microsatellite panels
used in this study. The PNG national malaria control program has made an
impact on the malaria burden through the substantial reductions in
infections circulating in the community (Hetzel et al., 2016; Kattenberg
et al., 2020; Koepfli et al., 2017), however it appears that this has
not been sustained long enough for the underlying parasite population to
fragment. Finally, this study demonstrates how parasite population
genetics can inform whether malaria intervention has reduced and
fragmented the parasite population or if significantly more control
effort will be required to do so.