Where chloroquine still works: the genetic make-up and susceptibility of Plasmodium vivax to chloroquine plus primaquine in Bhutan

Bhutan has made substantial progress in reducing malaria incidence. The national guidelines recommend chloroquine (CQ) and primaquine (PQ) for radical cure of uncomplicated Plasmodium vivax, but the local efficacy has not been assessed. The impact of cases imported from India on the genetic make-up of the local vivax populations is currently unknown.Patients over 4\ua0years of age with uncomplicated P. vivax mono-infection were enrolled into a clinical efficacy study and molecular survey. Study participants received a standard dose of CQ (25\ua0mg/kg over 3\ua0days) followed by weekly review until day 28. On day 28 a 14-day regimen of PQ (0.25\ua0mg/kg/day) was commenced under direct observation. After day 42, patients were followed up monthly for a year. The primary and secondary endpoints were risk of treatment failure at day 28 and at 1\ua0year. Parasite genotyping was undertaken at nine tandem repeat markers, and standard population genetic metrics were applied to examine population diversity and structure in infections thought to be acquired inside or outside of Bhutan.A total of 24 patients were enrolled in the clinical study between April 2013 and October 2015. Eight patients (33.3\ua0%) were lost to follow-up in the first 6\ua0months and another eight patients lost between 6 and 12\ua0months. No (0/24) treatment failures occurred by day 28 and no (0/8) parasitaemia was detected following PQ treatment. Some 95.8\ua0% (23/24) of patients were aparasitaemic by day 2. There were no haemolytic or serious events. Genotyping was undertaken on parasites from 12 autochthonous cases and 16 suspected imported cases. Diversity was high (H E 0.87 and 0.90) in both populations. There was no notable differentiation between the autochthonous and imported populations.CQ and PQ remains effective for radical cure of P. vivax in Bhutan. The genetic analyses indicate that imported infections are sustaining the local vivax population, with concomitant risk of introducing drug-resistant strains

Neighbour-joining tree illustrating the genetic relatedness between the <i>P</i>. <i>vivax</i> isolates across 9 loci.

<p>Only isolates with complete genotyping data across all 9 loci are presented. The two isolates highlighted with black dots, 6J and 9J, exhibited differing <i>pvmdr1</i> multi-locus genotypes from the other isolates: L953-Y976-N1010-<u>F</u>0176 in 6J and 9J versus L953-Y976-N1010-<u>L</u>1076 in the other isolates. The dotted grey outlines illustrate the isolates with high ancestry to <i>K</i>1 and <i>K</i>2 at <i>K</i> = 2.</p

Complexity of infection and population diversity.

<p>Complexity of infection and population diversity.</p

Pair-wise differentiation between sites.

<p><b><i>F</i></b>_<b>ST</b> (<i>P-value</i>) in lower left triangle. <b><i>F’</i></b>_<b>ST</b> in upper right triangle.</p><p>Pair-wise differentiation between sites.</p

Patient sampling details.

<p>Patient sampling details.</p

<i>P</i>. <i>vivax</i> prevalence maps.

<p>These maps were generated by the Malaria Atlas Project, University of Oxford. The colour scales reflect the model-based geostatistical point estimates of the annual mean <i>P</i>. <i>vivax</i> parasite rate in the 1–99 year age range (<i>Pv</i>PR_1-99) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166124#pone.0166124.ref017" target="_blank">17</a>] within the stable spatial limits of transmission in 2010. The approximate locations of the study sites described here are indicated with numbered open circles in the Iran panel: Bandar Lengeh County (1), Qeshm County (2), Minab County (3), Rudan County (4) and Jask County (5). All MAP maps are available to users under the CCAL 3.0. <a href="http://www.map.ox.ac.uk/about-map/open-access/" target="_blank">http://www.map.ox.ac.uk/about-map/open-access/</a>.</p

<i>P</i>. <i>vivax</i> prevalence map for Ethiopia illustrating the location of the study sites.

<p>This map was generated by the Malaria Atlas Project, University of Oxford. The colour scale reflects the model-based geostatistical point estimates of the annual mean <i>P</i>. <i>vivax</i> parasite rate in the 1–99 year age range (<i>Pv</i>PR) within the stable spatial limits of <i>P</i>. <i>vivax</i> transmission in 2010 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140780#pone.0140780.ref039" target="_blank">39</a>]. The approximate locations of the study sites are indicated with numbered black dots: Shone Health Center, Badawacho (1), Guba Health Center, Halaba (2), Adare Hospital, Hawassa (3), Millenium Health Center, Hawassa (4), Arbaminch Hospital, Arbaminch (5), and Shele Health Center, Arbaminch (6). All MAP maps are available to users under the CCAL 3.0. <a href="http://www.map.ox.ac.uk/about-map/open-access/" target="_blank">http://www.map.ox.ac.uk/about-map/open-access/</a>.</p

Summary of Pvmdr1 haplotypes.

<p>Summary of Pvmdr1 haplotypes.</p

Population structure.

<p>Bar plots illustrating the population structure at <i>K</i> = 2, <i>K</i> = 3, <i>K</i> = 4 and <i>K</i> = 5. Each vertical bar represents an individual sample and each colour represents one of the <i>K</i> clusters (sub-populations) defined by STRUCTURE. For each sample, the predicted ancestry to each of the K sub-populations is represented by the colour-coded bars. <i>K</i>1 = dark green, <i>K</i>2 = light green, <i>K</i>3 = red, <i>K</i>4 = orange, and <i>K</i>5 = white.</p

Complexity of infection and population diversity.

<p>Complexity of infection and population diversity.</p