DOI: 10.5061/dryad.37pvmcvfh
title: Monitoring Plasmodium falciparum and Plasmodium vivax using microsatellite markers indicates limited changes in population structure after substantial transmission decline in Papua New Guinea

description: Monitoring the genetic structure of pathogen populations may be an
 economical and sensitive approach to quantify the impact of control on
 transmission dynamics, highlighting the need for a better understanding of
 changes in population genetic parameters as transmission declines. Here we
 describe the first population genetic analysis of the major human malaria
 parasites, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv)
 populations following nationwide distribution of long-lasting insecticide
 treated nets (LLIN) in Papua New Guinea (PNG). Parasite isolates from pre-
 (2005-6) and post-LLIN (2010-2014) were genotyped using microsatellite
 markers. Despite parasite prevalence declining substantially (East Sepik:
 Pf=54.9-8.5%, Pv=35.7-5.6%, Madang: Pf=38.0-9.0%, Pv: 31.8-19.7%),
 genetically diverse and intermixing parasite populations remained. Pf
 diversity declined modestly post-LLIN relative to pre-LLIN (East Sepik: Rs
 = 7.1-6.4, He = 0.77-0.71; Madang: Rs= 8.2-6.1, He = 0.79-0.71).
 Unexpectedly, population structure present in pre-LLIN populations was
 lost post-LLIN, suggesting that more frequent human movement between
 provinces may have contributed to higher gene flow. Pv prevalence
 initially declined but increased again in one province, yet diversity
 remained high throughout the study period (East Sepik: Rs=11.4-9.3,
 He=0.83-0.80; Madang: Rs=12.2-14.5, He=0.85-0.88). Although genetic
 differentiation values increased between provinces over time, no
 significant population structure was observed at any time point. For both
 species, a decline in multiple infections and increasing clonal
 transmission and significant multilocus linkage disequilibrium (mLD)
 post-LLIN was a positive indicator of impact on the parasite population
 using microsatellite markers. These parameters may be useful adjuncts to
 traditional epidemiological tools in the early stages of transmission
 reduction.
description: Whole blood samples were collected from participants in
 cross-sectional studies conducted between 2005 and 2014 along the North
 Coast of PNG (Mueller et al., 2009; Schultz et al., 2010). In Madang
 Province (MAD), the same three catchment areas were studied in 2006
 (Schultz et al., 2010; Senn et al., 2012), 2010 (Koepfli et al., 2015) and
 2014 (Koepfli et al., 2017). For both species, a panel
 of 9-10 neutral microsatellite markers were amplified in the selected
 samples (Table S1) using a multiplex primary PCR followed by individual
 nested PCRs as previously described (Anderson et al., 2000; Jennison et
 al., 2015; Koepfli et al., 2013; Schultz et al., 2010). For <i>P.
 falciparum</i>, samples were genotyped at nine previously validated
 and commonly used, putatively neutral, microsatellite loci including
 <i>TA1, TAA60, Polya, ARA2, Pfg377, TAA87, PfPK2, TAA81</i>
 and <i>2490</i> (Anderson et al., 2000; Schultz et al., 2010).
 For <i>P. vivax</i>, 10 putatively neutral microsatellites
 were genotyped as previously described: <i>MS1, MS2, MS5, MS6, MS7,
 MS9, MS10, MS12, MS15</i>, and <i>MS20</i> (Jennison et
 al., 2015; Koepfli et al., 2013). All PCR products were sent to a
 commercial facility for fragment analysis on an ABI3730xl platform
 (Applied Biosystems) using the size standard LIZ500. Primers used were the
 same for all datasets (Jennison et al., 2015; Schultz et al., 2010). The
 electropherograms were analysed with Genemapper V4.0 (Applied Biosystems)
 with the same peak calling strategy as described previously (Jennison et
 al., 2015; Schultz et al., 2010). To avoid artefacts, precautions were
 taken to ensure allele calling was consistent (Jennison et al., 2015), and
 carefully reconstructing dominant haplotypes as per previously described
 methods (Anderson, Su, Bockarie, Lagog, & Day, 1999; Jennison et
 al., 2015; Schultz et al., 2010). For the Madang 2005 and Wosera 2006
 <i>P. falciparum</i> data, previously published cleaned and
 rounded microsatellite allele repeat numbers for <i>P. falciparum
 </i>single clone infections<i> </i>(Schultz et al.,
 2010) were converted back to allele sizes using the known number of
 nucleotides/repeat, whereas for <i>P. vivax</i> the raw data
 (allele calls) was available (Jennison et al., 2015). These data were
 combined with the newly generated MS data from the other studies before
 binning the alleles using the TANDEM software (Matschiner &
 Salzburger, 2009). Allele frequencies of the entire dataset (incl.
 previously genotyped datasets) were investigated and outlying alleles
 (most likely caused by PCR artefacts) were removed. Samples with missing
 data at six (60%) or more MS loci were excluded from further
 analysis.
description: Data provided as a tab delimited text file with haploid allele
 data sorted by population. Reconstructed dominant haplotypes are labelled
 with an ID starting with 'D_', whereas single clone haplotypes
 are labelled with 'S_'. We attempted to
 calibrate the <i>P. falciparum</i> data from pre-LLIN Madang
 2006 and Wosera (ESP1 2005) by converting rounded repeat numbers back to
 allele sizes, binning together with the newly generated data and removing
 outliers. However, there was strong population structure when compared to
 the new dataset, indicating experimental differences despite the use of
 the same protocols. Thus, we excluded direct comparisons between old and
 new datasets for <i>P. falciparum</i>.