The undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children. From 17 August 2009 to 20 May 2010, 524 children aged 5–10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes. These results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission. ClinicalTrials.gov NCT02143934 Malaria is a mosquito-borne parasitic disease caused by Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. Although P. falciparum is responsible for most of the 600,000 malaria deaths that occur every year, P. vivax is the most common, most widely distributed cause of malaria. All malaria parasites have a complex life cycle. When infected mosquitoes bite people, they inject “sporozoites,” a parasitic form that replicates in the liver. After 8–9 days, the liver releases “merozoites,” which invade red blood cells, where they replicate rapidly before bursting out and infecting more red blood cells. This increase in the parasitic burden causes malaria’s recurring flu-like symptoms and can cause organ damage and death. Infected red blood cells also release “gametocytes,” which infect mosquitoes when they take a blood meal. In the mosquito, gametocytes multiply and develop into sporozoites, thus completing the parasite’s life cycle. Malaria can be prevented by controlling the mosquitoes that spread malaria and by avoiding mosquito bites. Treatment with anti-malarial drugs, which is essential to prevent potentially fatal complications, also decreases malaria transmission. Malaria control programs have greatly reduced the global malaria burden, but the ability of P. vivax and P. ovale to persist undetected in the liver as “hypnozoites” (another parasitic form) is hindering malaria control and elimination efforts. Hypnozoites can cause malaria relapses months or years after a primary infection and are not cleared by anti-malarial treatment unless the treatment includes primaquine, a drug that has to be given for 7–14 days and that causes hemolysis (red blood cell death) in people who have glucose-6-phosphate dehydrogenase (G6PD) deficiency. Here, the researchers determine the contribution that relapses make to the burden of P. vivax and P. ovale infection, illness, and transmission among Papua New Guinean children by undertaking a randomized placebo-controlled trial of a primaquine treatment regimen. The researchers also use mathematical modeling to investigate the effect of mass drug administration (MDA) on the occurrence of P. vivax relapses. A randomized placebo-controlled trial compares the outcomes of individuals randomly chosen to receive an active treatment or a dummy (placebo) treatment; MDA aims to control malaria by treating entire at-risk populations with anti-malarial drugs. The researchers enrolled 524 children aged 5–10 years (none of whom were G6PD deficient) living in a region of Papua New Guinea where P. falciparum and P. vivax are hyperendemic (always present at high levels). Half the children received a blood-stage plus liver-stage anti-malarial treatment regimen (primaquine arm); the rest received a blood-stage plus placebo anti-malarial treatment regimen (placebo arm). Compared to children in the placebo arm, children in the primaquine arm had a reduced risk of having at least one P. vivax or P. ovale infection detected using PCR (a highly sensitive molecular technique) during eight months of follow-up and a reduced risk of having at least one clinical P. vivax episode. Children in the primaquine arm were also less likely to carry P. vivax gametocytes. Finally, by feeding the trial data into a mathematical transmission model, the researchers predicted that MDA with blood-stage treatment alone, or mass screening and treatment with blood-stage treatment alone or blood- plus liver-stage treatment, would have only a transient effect on P. vivax transmission levels, whereas MDA that includes blood-plus liver-stage treatment would be an effective strategy for P. vivax elimination. From the trial results, the researchers estimate that, among children living in a malaria-hyperendemic region of Papua New Guinea, relapses cause about four of every five P. vivax infections and three of every five P. ovale infections. Thus, P. vivax and P. ovale relapses are important in sustaining malaria transmission in this population. Notably, mathematical modeling predicts that MDA campaigns that combine blood- and liver-stage treatment are likely to be a highly effective strategy for P. vivax elimination. These findings may not be generalizable to populations with lower malaria transmission levels. Also, because the trial used a 20-day drug regimen to maximize the clearance of hypnozoites and because people would need to be tested for G6PD deficiency before starting primaquine treatment, the chosen treatment regimen may not be applicable to real-world MDA programs. Nevertheless, these findings highlight the importance of developing new anti-hypnozoite drugs and MDA programs that target areas and risk groups with confirmed local transmission of P. vivax to achieve global malaria control and elimination. This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001891.

Key Points: In P falciparum–infected anemic children, immature gametocytes are more prevalent and abundant in bone marrow than in peripheral blood. P falciparum–infected anemic children are gametocyte carriers that can potentially contribute to malaria transmission.

Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer–derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. Significance: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201

Intermittent preventive treatment in pregnancy has not been evaluated outside of Africa. Low birthweight (LBW, <2,500 g) is common in Papua New Guinea (PNG) and contributing factors include malaria and reproductive tract infections. From November 2009 to February 2013, we conducted a parallel group, randomised controlled trial in pregnant women (≤26 gestational weeks) in PNG. Sulphadoxine-pyrimethamine (1,500/75 mg) plus azithromycin (1 g twice daily for 2 days) (SPAZ) monthly from second trimester (intervention) was compared against sulphadoxine-pyrimethamine and chloroquine (450 to 600 mg, daily for three days) (SPCQ) given once, followed by SPCQ placebo (control). Women were assigned to treatment (1:1) using a randomisation sequence with block sizes of 32. Participants were blinded to assignments. The primary outcome was LBW. Analysis was by intention-to-treat. Of 2,793 women randomised, 2,021 (72.4%) were included in the primary outcome analysis (SPCQ: 1,008; SPAZ: 1,013). The prevalence of LBW was 15.1% (305/2,021). SPAZ reduced LBW (risk ratio [RR]: 0.74, 95% CI: 0.60–0.91, P = 0.005; absolute risk reduction (ARR): 4.5%, 95% CI: 1.4–7.6; number needed to treat: 22), and preterm delivery (0.62, 95% CI: 0.43–0.89, P = 0.010), and increased mean birthweight (41.9 g, 95% CI: 0.2–83.6, P = 0.049). SPAZ reduced maternal parasitaemia (RR: 0.57, 95% CI: 0.35–0.95, P = 0.029) and active placental malaria (0.68, 95% CI: 0.47–0.98, P = 0.037), and reduced carriage of gonorrhoea (0.66, 95% CI: 0.44–0.99, P = 0.041) at second visit. There were no treatment-related serious adverse events (SAEs), and the number of SAEs (intervention 13.1% [181/1,378], control 12.7% [174/1,374], P = 0.712) and AEs (intervention 10.5% [144/1,378], control 10.8% [149/1,374], P = 0.737) was similar. A major limitation of the study was the high loss to follow-up for birthweight. SPAZ was efficacious and safe in reducing LBW, possibly acting through multiple mechanisms including the effect on malaria and on sexually transmitted infections. The efficacy of SPAZ in the presence of resistant parasites and the contribution of AZ to bacterial antibiotic resistance require further study. The ability of SPAZ to improve pregnancy outcomes warrants further evaluation. ClinicalTrials.gov NCT01136850 (06 April 2010).

Naturally acquired humoral immunity to the malarial parasite Plasmodium falciparum can protect against disease, although the precise mechanisms remain unclear. Although antibody levels can be measured by ELISA, few studies have investigated functional antibody assays in relation to clinical outcomes. In this study we applied a recently developed functional assay of antibody-mediated opsonisation of merozoites, to plasma samples from a longitudinal cohort study conducted in a malaria endemic region of Papua New Guinea (PNG). Phagocytic activity was quantified by flow cytometry using a standardized and high-throughput protocol, and was subsequently evaluated for association with protection from clinical malaria and high-density parasitemia. Opsonising antibody responses were found to: i) increase with age, ii) be enhanced by concurrent infection, and iii) correlate with protection from clinical episodes and high-density parasitemia. Stronger protective associations were observed in individuals with no detectable parasitemia at baseline. This study presents the first evidence for merozoite phagocytosis as a correlate of acquired immunity and clinical protection against P. falciparum malaria.