A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria.

Background: P. ovale spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, P. ovale curtisi (Poc) and P. ovale wallikeri (Pow). These species are thought to differ in clinical symptomatology and latency, but existing diagnostic assays have limited ability to detect and distinguish them. In this study, we developed a new duplex assay for the detection and differentiation of Poc and Pow that can be used to improve our understanding of these parasites. Methods: Repetitive sequence motifs were identified in available Poc and Pow genomes and used for assay development and validation. We evaluated the analytical sensitivity and specificity of the best-performing assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), then validated its performance using 55 P. ovale spp. samples and 40 non-ovale Plasmodium samples from the DRC. Poc and Pow prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated. Results: The best-performing Poc and Pow targets had 9 and 8 copies within the reference genomes, respectively. Our duplex assay had 100% specificity and 95% confidence lower limits of detection of 4.2 and 41.2 parasite genome equivalents/µl for Poc and Pow, respectively. Species was determined in 80% of all P. ovale spp.-positive field samples and 100% of those with >10 parasites/µl. Most P. ovale spp. field samples from the DRC were found to be Poc infections. Conclusions: We identified promising multi-copy targets for molecular detection and differentiation of Poc and Pow and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density Pow infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of P. ovale spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.

Impact of malaria diagnostic choice on monitoring of Plasmodium falciparum prevalence estimates in the Democratic Republic of the Congo and relevance to control programs in high-burden countries.

Malaria programs rely upon a variety of diagnostic assays, including rapid diagnostic tests (RDTs), microscopy, polymerase chain reaction (PCR), and bead-based immunoassays (BBA), to monitor malaria prevalence and support control and elimination efforts. Data comparing these assays are limited, especially from high-burden countries like the Democratic Republic of the Congo (DRC). Using cross-sectional and routine data, we compared diagnostic performance and Plasmodium falciparum prevalence estimates across health areas of varying transmission intensity to illustrate the relevance of assay performance to malaria control programs. Data and samples were collected between March-June 2018 during a cross-sectional household survey across three health areas with low, moderate, and high transmission intensities within Kinshasa Province, DRC. Samples from 1,431 participants were evaluated using RDT, microscopy, PCR, and BBA. P. falciparum parasite prevalence varied between diagnostic methods across all health areas, with the highest prevalence estimates observed in Bu (57.4-72.4% across assays), followed by Kimpoko (32.6-53.2%), and Voix du Peuple (3.1-8.4%). Using latent class analysis to compare these diagnostic methods against an "alloyed gold standard," the most sensitive diagnostic method was BBA in Bu (high prevalence) and Voix du Peuple (low prevalence), while PCR diagnosis was most sensitive in Kimpoko (moderate prevalence). RDTs were consistently the most specific diagnostic method in all health areas. Among 9.0 million people residing in Kinshasa Province in 2018, the estimated P. falciparum prevalence by microscopy, PCR, and BBA were nearly double that of RDT. Comparison of malaria RDT, microscopy, PCR, and BBA results confirmed differences in sensitivity and specificity that varied by endemicity, with PCR and BBA performing best for detecting any P. falciparum infection. Prevalence estimates varied widely depending on assay type for parasite detection. Inherent differences in assay performance should be carefully considered when using community survey and surveillance data to guide policy decisions.

Neurocognitive outcomes in Malawian children exposed to malaria during pregnancy: An observational birth cohort study.

BACKGROUND: Annually 125 million pregnancies are at risk of malaria infection. However, the impact of exposure to malaria in pregnancy on neurodevelopment in children is not well understood. We hypothesized that malaria in pregnancy and associated maternal immune activation result in neurodevelopmental delay in exposed offspring. METHODS AND FINDINGS: Between April 2014 and April 2015, we followed 421 Malawian mother-baby dyads (median [IQR] maternal age: 21 [19, 28] years) who were previously enrolled (median [IQR] gestational age at enrollment: 19.7 [17.9, 22.1] weeks) in a randomized controlled malaria prevention trial with 5 or 6 scheduled assessments of antenatal malaria infection by PCR. Children were evaluated at 12, 18, and/or 24 months of age with cognitive tests previously validated in Malawi: the Malawi Developmental Assessment Tool (MDAT) and the MacArthur-Bates Communicative Development Inventories (MCAB-CDI). We assessed the impact of antenatal malaria (n [%] positive: 240 [57.3]), placental malaria (n [%] positive: 112 [29.6]), and maternal immune activation on neurocognitive development in children. Linear mixed-effects analysis showed that children exposed to antenatal malaria between 33 and 37 weeks gestation had delayed language development across the 2-year follow-up, as measured by MCAB-CDI (adjusted beta estimate [95% CI], -7.53 [-13.04, -2.02], p = 0.008). Maternal immune activation, characterized by increased maternal sTNFRII concentration, between 33 and 37 weeks was associated with lower MCAB-CDI language score (adjusted beta estimate [95% CI], -8.57 [-13.09, -4.06], p < 0.001). Main limitations of this study include a relatively short length of follow-up and a potential for residual confounding that is characteristic of observational studies. CONCLUSIONS: This mother-baby cohort presents evidence of a relationship between malaria in pregnancy and neurodevelopmental delay in offspring. Malaria in pregnancy may be a modifiable risk factor for neurodevelopmental injury independent of birth weight or prematurity. Successful interventions to prevent malaria during pregnancy may reduce the risk of neurocognitive delay in children.

Analysis of false-negative rapid diagnostic tests for symptomatic malaria in the Democratic Republic of the Congo.

The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen's kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

Interactions Between Antenatal Sulfadoxine-Pyrimethamine, Drug-Resistant Plasmodium falciparum Parasites, and Delivery Outcomes in Malawi.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is used as intermittent preventive therapy in pregnancy (IPTp) for malaria in sub-Saharan Africa. The resistance marker dhps A581G has been associated with reduced IPTp-SP efficacy and enhanced morbidity in SP recipients. METHODS: We measured SP-resistance allele frequencies in Malawian women participating in a trial comparing IPTp with SP against intermittent screening by rapid diagnostic tests (ISTp). We genotyped polymerase chain reaction-detected parasites using deep sequencing of SP-resistance alleles. RESULTS: Among 125 placental infections, A581G-bearing parasites were associated with reduced birth weight (mean difference [MD], 252 g; 95% confidence interval [CI], 46-457; P = .017). Relative to ISTp, IPTp-SP was associated with higher birth weights in women with wild-type parasites (MD, 116 g; 95% CI, -40 to 272; P = .142) and lower birth weights in women with A581G-bearing parasites (MD, 192 g; 95% CI, -264 to 648; P = .385) (Pinteraction = .033). Similar associations were noted on gestational age (Pinteraction = .075). Amongst only IPTp-SP recipients, relative to women who last received SP > 4 weeks before delivery, recent SP receipt was associated with lower birth weight in women with wild-type parasites (MD, 118 g; 95% CI, -376 to 139; P = .361) and higher birth weight in women with A581G-bearing parasites (MD, 783 g; 95% CI, -20 to 1586; P = .054) (Pinteraction = .005). CONCLUSIONS: The effectiveness in birth weight of IPTp-SP is compromised by A581G-bearing parasites, but there was no evidence that the adverse effects of these parasites are exacerbated by antenatal SP. ISRCTN REGISTRY: www.isrctn.com/ISRCTN69800930.

Minimal Impact by Antenatal Subpatent Plasmodium falciparum Infections on Delivery Outcomes in Malawian Women: A Cohort Study.

Antenatal malaria screening with a rapid diagnostic test (RDT) and treatment only of women with positive RDT findings may potentially prevent low birth weight resulting from malaria. The consequences of subpatent antenatal infections below the detection limit of RDTs are incompletely understood. In Malawi, pregnant women of any gravidity status were tested at each antenatal visit for Plasmodium falciparum, using an RDT and polymerase chain reaction analysis, and were followed until delivery. Associations between antenatal infections and delivery outcomes were assessed with Poisson regression or analysis of variance. Compared with women with no detected antenatal P. falciparum infection, women with positive RDT findings delivered babies with a lower mean birth weight (2960 vs 2867 g; mean difference, -93 g [95% confidence interval {CI}, -27 to -159]; P = .006); this was not observed among women with only subpatent infections (mean birth weight, 3013 g; mean difference, 54 [95% CI, -33-140]; P = .2268). These differences were apparent early in pregnancy, during the second trimester: compared with uninfected women, women with positive RDT findings delivered babies with a lower mean birth weight (mean difference, -94 g [95% CI, -31 to -156]; P = .003), but women with subpatent infections did not (mean difference, 36 g [95% CI, -49-122]; P = .409). Subpatent antenatal P. falciparum infections were not associated with adverse delivery outcomes. The association of patent infections at enrollment with low birth weight suggests the importance of preventing P. falciparum infection early in pregnancy.

Scheduled Intermittent Screening with Rapid Diagnostic Tests and Treatment with Dihydroartemisinin-Piperaquine versus Intermittent Preventive Therapy with Sulfadoxine-Pyrimethamine for Malaria in Pregnancy in Malawi: An Open-Label Randomized Controlled Trial.

BACKGROUND: In Africa, most plasmodium infections during pregnancy remain asymptomatic, yet are associated with maternal anemia and low birthweight. WHO recommends intermittent preventive therapy in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP). However, sulfadoxine-pyrimethamine (SP) efficacy is threatened by high-level parasite resistance. We conducted a trial to evaluate the efficacy and safety of scheduled intermittent screening with malaria rapid diagnostic tests (RDTs) and treatment of RDT-positive women with dihydroartemisinin-piperaquine (DP) as an alternative strategy to IPTp-SP. METHODS AND FINDINGS: This was an open-label, two-arm individually randomized superiority trial among HIV-seronegative women at three sites in Malawi with high SP resistance. The intervention consisted of three or four scheduled visits in the second and third trimester, 4 to 6 wk apart. Women in the IPTp-SP arm received SP at each visit. Women in the intermittent screening and treatment in pregnancy with DP (ISTp-DP) arm were screened for malaria at every visit and treated with DP if RDT-positive. The primary outcomes were adverse live birth outcome (composite of small for gestational age, low birthweight [<2,500 g], or preterm birth [<37 wk]) in paucigravidae (first or second pregnancy) and maternal or placental plasmodium infection at delivery in multigravidae (third pregnancy or higher). Analysis was by intention to treat. Between 21 July 2011 and 18 March 2013, 1,873 women were recruited (1,155 paucigravidae and 718 multigravidae). The prevalence of adverse live birth outcome was similar in the ISTp-DP (29.9%) and IPTp-SP (28.8%) arms (risk difference = 1.08% [95% CI -3.25% to 5.41%]; all women: relative risk [RR] = 1.04 [95% CI 0.90-1.20], p = 0.625; paucigravidae: RR = 1.10 [95% CI 0.92-1.31], p = 0.282; multigravidae: RR = 0.92 [95% CI 0.71-1.20], p = 0.543). The prevalence of malaria at delivery was higher in the ISTp-DP arm (48.7% versus 40.8%; risk difference = 7.85%, [95% CI 3.07%-12.63%]; all women: RR = 1.19 [95% CI 1.07-1.33], p = 0.007; paucigravidae: RR = 1.16 [95% CI 1.04-1.31], p = 0.011; multigravidae: RR = 1.29 [95% CI 1.02-1.63], p = 0.037). Fetal loss was more common with ISTp-DP (2.6% versus 1.3%; RR = 2.06 [95% CI 1.01-4.21], p = 0.046) and highest among non-DP-recipients (3.1%) in the ISTp-DP arm. Limitations included the open-label design. CONCLUSIONS: Scheduled screening for malaria parasites with the current generation of RDTs three to four times during pregnancy as part of focused antenatal care was not superior to IPTp-SP in this area with high malaria transmission and high SP resistance and was associated with higher fetal loss and more malaria at delivery. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201103000280319; ISRCTN Registry ISRCTN69800930.

The neurological assessment in young children treated with artesunate monotherapy or artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria.

BACKGROUND: Mefloquine and artesunate combination therapy is the recommended first-line treatment for uncomplicated malaria throughout much of south-east Asia. Concerns have been raised about the potential central nervous system (CNS) effects of both drug components and there are no detailed reports in very young children. METHODS: Children, aged between three months and five years, with acute uncomplicated Plasmodium falciparum malaria were randomized to either 7 days of artesunate monotherapy or the same schedule of artesunate plus mefloquine on day 7 and 8. Neurological testing targeting coordination and behaviour was carried out at day 0, 7, 9, 10, 14 and 28. Non-febrile healthy control children from the same population were tested on days 0, 7, 14 and 28. RESULTS: From December 1994 to July 1997, 91 children with uncomplicated P. falciparum, 45 treated with artesunate monotherapy, 46 treated with mefloquine and artesunate combination therapy and 36 non-febrile controls, underwent neurological testing. Malaria and fever had a significant negative impact on testing performance. By contrast, the anti-malarial treatments were not associated with worsening performances in the various components of the test. Artesunate and mefloquine do not appear to have a significant influence on coordination and behaviour. Children treated with mefloquine were significantly less likely to suffer recurrent malaria infection during follow-up compared to those treated with artesunate alone (P = 0.033). CONCLUSION: In keeping with the results of randomized controlled trials in adults, mefloquine was not associated with a decrease in specific items of neurological performance. Likewise, children treated with artesunate did not perform significantly differently to control children. This study does not exclude subtle or rare treatment CNS effects of artesunate or mefloquine. Treatment of acute uncomplicated malaria results in a significant improvement on items of neurological performance.

Chloroquine pharmacokinetics in pregnant and nonpregnant women with vivax malaria.

PURPOSE: We compared the pharmacokinetics of chloroquine in pregnant and nonpregnant women treated for Plasmodium vivax malaria. METHODS: Twelve pregnant women and 15 nonpregnant women of child-bearing age with acute P. vivax malaria were treated with 25 mg chloroquine base/kg over 3 days on the northwestern border of Thailand. Blood concentrations of chloroquine and desethylchloroquine were measured using hydrophilic interaction liquid chromatography coupled with fluorescence detection. Twenty-five women completed the pharmacokinetic study. RESULTS: Although increasing gestational age was associated with reduced chloroquine AUC0-->infinity, there was no significant difference overall in the pharmacokinetics of chloroquine between pregnant and nonpregnant women. Fever was associated with lower chloroquine AUC0-->infinity values. Desethylchloroquine area under the curve (AUC) values were not significantly affected by pregnancy. CONCLUSIONS: Pregnancy did not significantly affect blood concentrations of chloroquine or its metabolite, desethylchloroquine, in women with P. vivax malaria.

An open label randomized comparison of mefloquine-artesunate as separate tablets vs. a new co-formulated combination for the treatment of uncomplicated multidrug-resistant falciparum malaria in Thailand.

BACKGROUND: Delivering drugs in a fixed combination is essential to the success of the strategy of artemisinin-based combination therapy. This prevents one drug being taken without the protection of the other, reducing the chance of emergence and spread of drug resistant strains of Plasmodium falciparum. A lower tablet burden should also facilitate adherence to treatment. A new fixed combination of mefloquine plus artesunate has been developed. This was compared with the conventional regimen of separate tablets for the treatment of uncomplicated multidrug-resistant falciparum malaria. METHODS: On the north-western border of Thailand 500 adults and children with uncomplicated falciparum malaria were randomized to receive either the new fixed combination or separate tablets. They were followed up weekly for 63 days. RESULTS: The day 63 polymerase chain reaction-adjusted cure rates were 91.9% (95% CI 88.2-95.6) in the fixed combination group and 89.2% (85.0-93.4) in the loose tablets group (P=0.3). There was a lower incidence of early vomiting in the group receiving the fixed combination. CONCLUSION: This new fixed combination of mefloquine and artesunate was efficacious, well tolerated and convenient to administer.

A randomized, controlled study of a simple, once-daily regimen of dihydroartemisinin-piperaquine for the treatment of uncomplicated, multidrug-resistant falciparum malaria.

BACKGROUND: Dihydroartemisinin-piperaquine (DP) is a fixed-combination antimalarial drug increasingly deployed in Southeast Asia. The current regimen involves 4 doses given over 3 days. Simplification of the dose regimen should facilitate treatment adherence and thereby increase effectiveness. METHODS: In a randomized, controlled, 3-arm trial conducted along the northwestern border of Thailand, the standard 4-dose course of DP (DP4) was compared to an equivalent dose given as a once-daily regimen (DP3) and to the standard treatment of mefloquine-artesunate (MAS3). RESULTS: A total of 499 patients were included in the study. Times to fever and parasite clearance were similar in all groups. The PCR genotyping-adjusted cure rates at day 63 after treatment initiation were 95.7% (95% confidence interval [95% CI], 92.2%-98.9%) for MAS3, 100% for DP4, and 99.4% (95% CI, 98.1%-100%) for DP3. The DP4 and DP3 cure rates were significantly higher than that for MAS3 (P=.008 and P=.03, respectively). All regimens were well tolerated. There were 3 deaths (1 in the MAS3 group and 2 in the DP3 group), all of which were considered to be unrelated to treatment. Rates of other adverse events were comparable between the groups, except for diarrhea, which was more common in the DP4 group (P=.05 vs. the MAS3 group). CONCLUSIONS: A once-daily, 3-dose regimen of DP is a highly efficacious treatment for multidrug-resistant falciparum malaria. This simple, safe, and relatively inexpensive fixed combination could become the treatment of choice for falciparum malaria.

Seasonal variation in hyperparasitaemia and gametocyte carriage in patients with Plasmodium falciparum malaria on the Thai-Burmese border.

Between January 2000 and December 2002 monthly rainfall was correlated with the proportion of patients with hyperparasitaemic Plasmodium falciparum malaria and with the proportion of patients with P. falciparum gametocytes. During the observation period 6953 cases of P. falciparum malaria were treated at the Shoklo Malaria Research Unit in Maela refugee camp on the Thai-Burmese border. Three hundred and seventy-five of these patients had >/=4% of parasitized red blood cells. Although there were more monthly malaria cases in the rainy season, rainfall was negatively correlated with the proportion of patients with hyperparasitaemia (Spearman's rho = -0.59, P < 0.001 ), and the proportion of gametocyte carriers among P. falciparum cases, (Spearman's rho = -0.39, P = 0.018). After controlling for age and the origin of the patient, the odds ratio for developing hyperparasitaemia during the dry season was 1.6 (95% CI 1.14-2.2; P = 0.006). The adjusted odds ratio for gametocyte carriage during the dry season was 1.3 (95% CI 1.03-1.6; P = 0.02). Migrations, changes in transmission patterns, the haematological burden of cumulative infections, and ultraviolet immunosuppression are discussed as potential explanations for these observations.

The effects of quinine and chloroquine antimalarial treatments in the first trimester of pregnancy.

Quinine (n = 246) was used to treat uncomplicated Plasmodium falciparum and chloroquine (n = 130) was used to treat P. vivax, in a total of 376 episodes of malaria in the first trimester of pregnancy, in 300 Karen women (Thailand, 1995-2000). Parasites were still present on day 6 or 7 in 4.7% (11/234) of episodes treated with quinine. The overall 28 day parasite reappearance rate following quinine was 28.7% (60/209) for primary treatments and 44% (11/25) for re-treatments. Quinine treatment resulted in a high rate of gametocyte carriage: person-gametocyte-weeks = 42.5 (95% CI 27.8-62.1) per 1000 woman-weeks. For P. vivax, the reappearance rate for all episodes by day 28 was 4.5% (5/111). Significantly more women complained of tinnitus following quinine treatment compared to on admission: 64.5% (78/121) vs 31.6% (59/187), P < 0.001. Using survival analysis, the community rate of spontaneous abortion in women who never had malaria in pregnancy, 17.8% (16.5-19.0), did not differ significantly from rates in women treated with quinine: 22.9% (95% CI 15.5-30.3), or chloroquine: 18.3% (95% CI 9.3-27.3), P = 0.42. Pregnancies exposed to quinine or chloroquine and carried to term did not have increased rates of congenital abnormality, stillbirth or low birthweight. These results suggest that therapeutic doses of quinine and chloroquine are safe to use in the first trimester of pregnancy.