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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 two major human malaria parasites, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv), following nationwide distribution of long-lasting insecticide-treated nets (LLINs) in Papua New Guinea (PNG). Parasite isolates from pre- (2005-2006) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially (East Sepik Province: Pf = 54.9%-8.5%, Pv = 35.7%-5.6%, Madang Province: 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 post-LLIN were positive indicators 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.

Chloroquine (CQ) is the first-line treatment for Plasmodium vivax malaria in most endemic countries. Monitoring P.vivax CQ resistance (CQR) is critical but remains challenged by the difficulty to distinguish real treatment failure from reinfection or liver relapse. Therapeutic efficacy of CQ against uncomplicated P.vivax malaria was evaluated in Gia Lai province, Vietnam. Sixty-seven patients were enrolled and followed-up for 42 days using microscopy and (RT)qPCR. Adequate clinical and parasitological response (ACPR) was 100% (66/66) on Day 28, but 75.4% (49/65) on Day 42. Eighteen recurrences (27.7%) were detected with a median time-to-recurrence of 42 days (IQR 35, 42) and blood CQ concentration 98% identity-by-descent to paired Day 0 samples. Primary infections leading to recurrence occurred in younger individuals (median age for ACPR=25 years [IQR 20, 28]; recurrences=18 [16, 21]; p=0.002), had a longer parasite clearance time (PCT for ACPR=47.5h [IQR 36.2, 59.8]; recurrences=54.2h [48.4, 62.0]; p=0.035) and higher pvcrt gene expression (median relative expression ratio for ACPR=0.09 [IQR 0.05, 0.22]; recurrences=0.20 [0.15, 0.56]; p=0.002), but there was no difference in ex vivo CQ sensitivity. This study shows that CQ remained largely efficacious to treat P.vivax in Gia Lai, i.e. recurrences occurred late (>Day 28) and in the presence of low blood CQ concentrations. However, the combination of WGS and gene expression analysis (pvcrt) with clinical data (PCT) allowed to identify potential emergence of low-grade CQR that should be closely monitored.

Continuous malaria control in Papua New Guinea has resulted in a marked decline of Plasmodium falciparum and P. vivax prevalence. Yet, an increasing proportion of submicroscopic infections, many of them carrying gametocytes, demands for novel strategies to target residual transmission., Background The scale-up of effective malaria control in the last decade has resulted in a substantial decline in the incidence of clinical malaria in many countries. The effects on the proportions of asymptomatic and submicroscopic infections and on transmission potential are yet poorly understood. Methods In Papua New Guinea, vector control has been intensified since 2008, and improved diagnosis and treatment was introduced in 2012. Cross-sectional surveys were conducted in Madang Province in 2006 (with 1280 survey participants), 2010 (with 2117 participants), and 2014 (with 2516 participants). Infections were quantified by highly sensitive quantitative polymerase chain reaction (PCR) analysis, and gametocytes were quantified by reverse-transcription qPCR analysis. Results Plasmodium falciparum prevalence determined by qPCR decreased from 42% in 2006 to 9% in 2014. The P. vivax prevalence decreased from 42% in 2006 to 13% in 2010 but then increased to 20% in 2014. Parasite densities decreased 5-fold from 2006 to 2010; 72% of P. falciparum and 87% of P. vivax infections were submicroscopic in 2014. Gametocyte density and positivity correlated closely with parasitemia, and population gametocyte prevalence decreased 3-fold for P. falciparum and 29% for P. vivax from 2010 to 2014. Conclusions Sustained control has resulted in reduced malaria transmission potential, but an increasing proportion of gametocyte carriers are asymptomatic and submicroscopic and represent a challenge to malaria control.

Monitoring the genetic structure of malaria parasite populations has been proposed as a novel and sensitive approach to quantify the impact of malaria control and elimination efforts. Here we describe the first population genetic analysis of sympatric 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 serial cross-sectional studies pre-(2005-6) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially in these communities (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. P. falciparum 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 between provinces. P. vivax 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, the emergence of clonal transmission and significant multilocus linkage disequilibrium (mLD) due to increased focal inbreeding post-LLIN was a strong indicator of impact on the parasite population using these markers. After eight years of intensive malaria control in PNG and substantial prevalence decline the impact on parasite population diversity and structure detectable by microsatellite genotyping was limited.

Background In 2009, the Papua New Guinea (PNG) Department of Health adopted artemether–lumefantrine (AL) and dihydroartemisinin–piperaquine (DHA-PPQ) as the first- and second-line treatments for uncomplicated malaria, respectively. This study was conducted to assess the efficacy of both drugs following adoption of the new policy. Methods Between June 2012 and September 2014, a therapeutic efficacy study was conducted in East Sepik and Milne Bay Provinces of PNG in accordance with the standard World Health Organization (WHO) protocol for surveillance of anti-malarial drug efficacy. Patients ≥ 6 months of age with microscopy confirmed Plasmodium falciparum or Plasmodium vivax mono-infections were enrolled, treated with AL or DHA-PPQ, and followed up for 42 days. Study endpoints were adequate clinical and parasitological response (ACPR) on days 28 and 42. The in vitro efficacy of anti-malarials and the prevalence of selected molecular markers of resistance were also determined. Results A total of 274 P. falciparum and 70 P. vivax cases were enrolled. The day-42 PCR-corrected ACPR for P. falciparum was 98.1% (104/106) for AL and 100% (135/135) for DHA-PPQ. The day-42 PCR-corrected ACPR for P. vivax was 79.0% (15/19) for AL and 92.3% (36/39) for DHA-PPQ. Day 3 parasite clearance of P. falciparum was 99.2% with AL and 100% with DHA-PPQ. In vitro testing of 96 samples revealed low susceptibility to chloroquine (34% of samples above IC50 threshold) but not to lumefantrine (0%). Molecular markers assessed in a sub-set of the study population indicated high rates of chloroquine resistance in P. falciparum (pfcrt SVMNT: 94.2%, n = 104) and in P. vivax (pvmdr1 Y976F: 64.8%, n = 54). Conclusions AL and DHA-PPQ were efficacious as first- and second-line treatments for uncomplicated malaria in PNG. Continued in vivo efficacy monitoring is warranted considering the threat of resistance to artemisinin and partner drugs in the region and scale-up of artemisinin-based combination therapy in PNG. Electronic supplementary material The online version of this article (10.1186/s12936-018-2494-z) contains supplementary material, which is available to authorized users.

Objectives To evaluate persistence of several Plasmodium antigens in pregnant women after treatment and compare diagnostics during treatment follow-up. Methods Thirty-two pregnant women (N = 32) with confirmed malaria infection by a histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) and microscopy were followed for 28 days after artemisinin-based combination therapy (ACT). A Plasmodium lactate dehydrogenase (pLDH)-based RDT and two ELISAs based on the detection of dihydrofolate reductase–thymidylate synthase (DHFR-TS) and haeme detoxification protein (HDP) were compared with each other and to RT-PCR at each visit. Results The mean visit number (95% confidence interval) on which the HRP2-based RDT was still positive after treatment was 3.4 (2.7–4.1) visits with some patients still positive at day 28. This is significantly later than the pLDH-based RDT [0.84 (0.55–1.1)], microscopy (median 1, range 1–3), DHFR-TS-ELISA [1.7 (1.1–2.3)] and RT-PCR (median 2, range 1–5) (P

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP),dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, K-D) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have K-D values of 0.10 nM +/- 0.014 (D5) and 0.068 +/- 0.015 nM (D6) for DHFR-TS mAbs, 0.10 +/- 0.022 nM (H16) and 0.21 +/- 0.022 nM (H18) for HDP mAbs and 0.11 +/- 0.028 nM (G23) and 0.33 +/- 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (K-D of 0.16 +/- 0.13 nM for PTL3 and 1.0 +/- 0.049 nM for C1-13), making them promising reagents for further test development

BACKGROUND: The diagnosis of malaria during pregnancy is complicated by placental sequestration, asymptomatic infection, and low-density peripheral parasitaemia. Where intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine is threatened by drug resistance, or is inappropriate due to low transmission, intermittent screening and treatment (ISTp) with rapid diagnostic tests for malaria (RDT) could be a valuable alternative. Therefore, the accuracy of RDTs to detect peripheral and placental infection was assessed in a declining transmission setting in Papua New Guinea (PNG). METHODS: The performance of a combination RDT detecting histidine-rich protein-2 (HRP-2) and Plasmodium lactate dehydrogenase (pLDH), and light microscopy (LM), to diagnose peripheral Plasmodium falciparum and Plasmodium vivax infections during pregnancy, were assessed using quantitative real-time PCR (qPCR) as the reference standard. Participants in a malaria prevention trial in PNG with a haemoglobin 50 % of these infections. CONCLUSIONS: In PNG, HRP-2/pLDH RDTs may be useful to diagnose peripheral P. falciparum infections in symptomatic pregnant women. However, they are not sufficiently sensitive for use in intermittent screening amongst asymptomatic (anaemic) women. These findings have implications for the management of malaria in pregnancy. The adverse impact of infections undetected by RDT or LM on pregnancy outcomes needs further evaluation.

In a randomized controlled trial Tim Davis and colleagues investigate Artemisinin-naphthoquine versus artemether-lumefantrine for the treatment of P. falciparum and P. vivax malaria. Please see later in the article for the Editors' Summary, Background Artemisinin combination therapies (ACTs) with broad efficacy are needed where multiple Plasmodium species are transmitted, especially in children, who bear the brunt of infection in endemic areas. In Papua New Guinea (PNG), artemether-lumefantrine is the first-line treatment for uncomplicated malaria, but it has limited efficacy against P. vivax. Artemisinin-naphthoquine should have greater activity in vivax malaria because the elimination of naphthoquine is slower than that of lumefantrine. In this study, the efficacy, tolerability, and safety of these ACTs were assessed in PNG children aged 0.5–5 y. Methods and Findings An open-label, randomized, parallel-group trial of artemether-lumefantrine (six doses over 3 d) and artemisinin-naphthoquine (three daily doses) was conducted between 28 March 2011 and 22 April 2013. Parasitologic outcomes were assessed without knowledge of treatment allocation. Primary endpoints were the 42-d P. falciparum PCR-corrected adequate clinical and parasitologic response (ACPR) and the P. vivax PCR-uncorrected 42-d ACPR. Non-inferiority and superiority designs were used for falciparum and vivax malaria, respectively. Because the artemisinin-naphthoquine regimen involved three doses rather than the manufacturer-specified single dose, the first 188 children underwent detailed safety monitoring. Of 2,542 febrile children screened, 267 were randomized, and 186 with falciparum and 47 with vivax malaria completed the 42-d follow-up. Both ACTs were safe and well tolerated. P. falciparum ACPRs were 97.8% and 100.0% in artemether-lumefantrine and artemisinin-naphthoquine-treated patients, respectively (difference 2.2% [95% CI −3.0% to 8.4%] versus −5.0% non-inferiority margin, p = 0.24), and P. vivax ACPRs were 30.0% and 100.0%, respectively (difference 70.0% [95% CI 40.9%–87.2%], p, Editors' Summary Background Malaria is a mosquito-borne parasitic disease that kills more than 600,000 people (mainly young children in sub-Saharan Africa) every year. Plasmodium falciparum causes most of these deaths, but P. vivax is the most common and most widely distributed cause of malaria outside sub-Saharan Africa. Infection with malaria parasites causes recurring flu-like symptoms and must be treated promptly with antimalarial drugs to prevent the development of anemia and potentially fatal damage to the brain and other organs. In the past, malaria was treated with “monotherapies” such as chloroquine, but the parasites quickly developed resistance to many of these inexpensive drugs. The World Health Organization now recommends artemisinin combination therapy (ACT) for first-line treatment of malaria in all regions where there is drug-resistant malaria. In ACT, artemisinin derivatives (fast-acting antimalarial drugs that are rapidly cleared from the body) are used in combination with a slower acting, more slowly eliminated partner drug to prevent reemergence of the original infection and to reduce the chances of the malaria parasites becoming resistant to either drug. Why Was This Study Done? Because falciparum and vivax malaria respond differently to antimalarial drugs, wherever there is transmission of both types of malaria but limited facilities for species-specific malaria diagnosis—as in Papua New Guinea—compromises have to be made about which ACT should be used for the treatment of malaria. Thus, Papua New Guinea's national guidelines recommend artemether-lumefantrine, which is effective against the more deadly P. falciparum, for first-line treatment of uncomplicated (mild) malaria even though this ACT is ineffective against the more common P. vivax. In this open-label randomized trial (a study in which participants are randomly assigned to receive different drugs but know which drug they are being given), the researchers ask whether an alternative ACT might be preferable for the treatment of uncomplicated malaria in young children in Papua New Guinea by comparing outcomes after treatment with artemether-lumefantrine versus artemisinin-naphthoquine (an ACT that should be more effective against vivax malaria than artemether-lumefantrine because naphthoquine stays in the body longer than lumefantrine). Specifically, the researchers test the non-inferiority of artemisinin-naphthoquine compared to artemether-lumefantrine for the treatment of falciparum malaria (whether artemisinin-naphthoquine is not worse than artemether-lumefantrine) and the superiority of artemisinin-naphthoquine compared to artemether-lumefantrine for the treatment of vivax malaria (whether artemisinin-naphthoquine is better than artemether-lumefantrine). What Did the Researchers Do and Find? The researchers assigned nearly 250 children (aged 0.5 to 5 years) with falciparum malaria, vivax malaria, or both types of malaria to receive six doses of artemether-lumefantrine over three days or three daily doses of artemisinin-naphthoquine. They then followed the children to see how many children in each treatment group and with each type of malaria were free of malaria 42 days after treatment (an “adequate clinical and parasitological response”). Among the patients originally infected with P. falciparum, 97.8% of those treated with artemether-lumefantrine and 100% of those treated with artemisinin-naphthoquine were clear of their original P. falciparum infection (though some had acquired a new P. falciparum infection) 42 days after treatment. By contrast, among the patients infected with P. vivax, 30% of those treated with artemether-lumefantrine and 100% of those treated with artemisinin-naphthoquine were clear of P. vivax infection 42 days after treatment. Both ACTs were safe and well tolerated. What Do These Findings Mean? These findings indicate that artemisinin-naphthoquine was non-inferior to artemether-lumefantrine for the treatment of uncomplicated falciparum malaria among young children in Papua New Guinea and had greater efficacy than artemether-lumefantrine against vivax malaria. The accuracy of these findings may be limited by several aspects of the study design. For example, not all the artemether-lumefantrine doses were directly observed, so some children may not have received the full treatment course. Moreover, because all the study participants lived in coastal communities in Papua New Guinea where malaria is highly endemic, treatment responses among children living in areas with lower levels of malaria transmission might be different. Nevertheless, these findings suggest that artemisinin-naphthoquine should be considered alongside other ACTs for the treatment of uncomplicated malaria in regions where there is transmission of multiple Plasmodium species and that artemisinin-naphthoquine may be better than artemether-lumefantrine for the treatment of uncomplicated malaria in young children in regions where P. vivax predominates. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001773. Information is available from the World Health Organization on malaria (in several languages); the World Malaria Report 2013 provides details on the current global malaria situation, including information on malaria in Papua New Guinea; the World Health Organization's Guidelines for the Treatment of Malaria is available The US Centers for Disease Control and Prevention provides information on malaria (in English and Spanish), including personal stories about malaria Information is available from the Roll Back Malaria Partnership on the global control of malaria, including information about malaria in Papua New Guinea, malaria in children, and ACTs The Malaria Vaccine Initiative has a fact sheet on Plasmodium vivax malaria MedlinePlus provides links to additional information on malaria (in English and Spanish) More information about this trial is available

Rapid diagnostics tests (RDTs) detect malaria specific antigen(s) in the circulation, even when parasites are sequestered in the placenta and not visible by microscopy. However, research on their diagnostic accuracy during pregnancy is limited. Pregnant women (n = 418) were screened for malaria during routine antenatal care by using two RDTs that detect histidine-rich protein 2 (HRP2) or Plasmodium lactate dehydrogenase, and enzyme-linked immunosorbent assays with antibodies that detect dihydrofolate reductase–thymidylate synthase or heme-detoxification protein, and compared with real-time polymerase chain reaction (RT-PCR) and microscopy for evaluation of their diagnostic accuracy. Prevalence of malaria infection was high (53% by PCR). The RT-PCR and the HRP2 RDT detected most cases of malaria during pregnancy, whereas microscopy, the Plasmodium lactate dehydrogenase RDT, and enzyme-linked immunosorbent assays for dihydrofolate reductase–thymidylate synthase and heme-detoxification protein antibodies did not detect several low-density infections. Therefore, the HRP2 RDT could be a useful tool in high-transmission areas for diagnosis of malaria in asymptomatic pregnant women.

Background During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. Methods MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. Results The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. Conclusion The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy.

Background: In northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013. Methods: A validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay. Results: CQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were