Plasmodium falciparum gametocyte dynamics after pyronaridine-artesunate or artemether-lumefantrine treatment.

BACKGROUND: Artemisinin-based combinations differ in their impact on gametocyte prevalence and density. This study assessed female and male gametocyte dynamics after treating children with uncomplicated Plasmodium falciparum malaria with either pyronaridine-artesunate (PA) or artemether-lumefantrine (AL). METHODS: Kenyan children with uncomplicated Plasmodium falciparum malaria were included and randomly assigned to PA or AL treatment. Filter paper blood samples were collected as a source of RNA for quantitative reverse-transcription PCR (qRT-PCR) and nucleic acid sequence based amplification (QT-NASBA) to detect female gametocytes (targeting Pfs25 mRNA). Male gametocytes were detected by qRT-PCR (targeting PfMGET mRNA). Duration of gametocyte carriage, the female and male gametocyte response and the agreement between qRT-PCR and QT-NASBA were determined. RESULTS: The mean duration of female gametocyte carriage was significantly longer for PA (4.9 days) than for AL (3.8 days) as estimated by QT-NASBA (P = 0.036), but this difference was less clear when determined by Pfs25 qRT-PCR (4.5 days for PA and 3.7 for AL, P = 0.166). qRT-PCR based female gametocyte prevalence decreased from 100% (75/75) at baseline to 6.06% (4/66) at day 14 in the AL group and from 97.7% (83/85) to 13.9% (11/79) in the PA group. Male gametocyte prevalence decreased from 41.3% (31/75) at baseline to 19.7% (13/66) at day 14 in the AL group and from 35.3% (30/85) to 22.8% (18/79) in the PA group. There was good agreement between Pfs25 qRT-PCR and QT-NASBA female gametocyte prevalence (0.85, 95% CI 0.82-0.87). CONCLUSIONS: This study indicates that female gametocyte clearance may be slightly faster after AL compared to PA. Male gametocytes showed similar post-treatment clearance between study arms. Future studies should further address potential differences between the post-treatment transmission potential after PA compared to AL. Trial registration This study is registered at clinicaltrials.gov under NCT02411994. Registration date: 8 April 2015. https://clinicaltrials.gov/ct2/show/NCT02411994?term=pyronaridine-artesunate&cond=Malaria&cntry=KE&rank=1.

Pyronaridine-artesunate and artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Kenyan children: a randomized controlled non-inferiority trial.

BACKGROUND: Pyronaridine-artesunate is a novel artemisinin-based combination therapy. The efficacy and safety of pyronaridine-artesunate were compared with artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in children. METHODS: This phase III open-label randomized controlled non-inferiority trial was conducted in Western Kenya. Children aged 6 months to ≤ 12 years with a bodyweight > 5 kg and microscopically confirmed P. falciparum malaria were randomly assigned in a 1:1 ratio to orally receive pyronaridine-artesunate or artemether-lumefantrine, dosed according to bodyweight, for 3 days. RESULTS: Of 197 participants, 101 received pyronaridine-artesunate and 96 received artemether-lumefantrine. The day-28 adequate clinical and parasitological response in the per-protocol population, PCR-corrected for reinfections, was 98.9% (93/94, 95% CI 94.2-99.8) for pyronaridine-artesunate and 96.4% (81/84, 95% CI 90.0-98.8) for artemether-lumefantrine. Pyronaridine-artesunate was found to be non-inferior to artemether-lumefantrine: the treatment difference was 2.5% (95% CI - 2.8 to 9.0). Adverse events occurred in 41.6% (42/101) and 34.4% (33/96) of patients in the pyronaridine-artesunate group and the artemether-lumefantrine group, respectively. No participants were found to have alanine or aspartate aminotransferase levels > 3 times the upper limit of normal. CONCLUSIONS: Pyronaridine-artesunate was well tolerated, efficacious and non-inferior to artemether-lumefantrine for the treatment of uncomplicated P. falciparum malaria in Kenyan children. Results are in line with previous reports and inclusion of pyronaridine-artesunate in paediatric malaria treatment programmes should be considered. This study is registered at clinicaltrials.gov under NCT02411994. Registration date: 8 April 2015. https://clinicaltrials.gov/ct2/show/NCT02411994?term=pyronaridine-artesunate&cond=Malaria&cntry=KE&rank=1.

Molecular malaria diagnostics: A systematic review and meta-analysis.

Accurate diagnosis of malaria is essential for identification and subsequent treatment of the disease. Currently, microscopy and rapid diagnostic tests are the most commonly used diagnostics, next to treatment based on clinical signs only. These tests are easy to deploy, but have a relatively high detection limit. With declining prevalence in many areas, there is an increasing need for more sensitive diagnostics. Molecular tools may be a suitable alternative, although costs and technical requirements currently hamper their implementation in resource limited settings. A range of (near) point-of-care diagnostics is therefore under development, including simplifications in sample preparation, amplification and/or read-out of the test. Accuracy data, in combination with technical characteristics, are essential in determining which molecular test, if any, would be the most promising to be deployed. This review presents a comprehensive overview of the currently available molecular malaria diagnostics, ranging from well-known tests to platforms in early stages of evaluation, and systematically evaluates their published accuracy. No important difference in accuracy was found between the most commonly used PCR-based assays (conventional, nested and real-time PCR), with most of them having high sensitivity and specificity, implying that there are no reasons other than practical ones to choose one technique over the other. Loop-mediated isothermal amplification and other (novel) diagnostics appear to be highly accurate as well, with some offering potential to be used in resource-limited settings.

Effect of Daily Antenatal Iron Supplementation on Plasmodium Infection in Kenyan Women: A Randomized Clinical Trial.

IMPORTANCE: Anemia affects most pregnant African women and is predominantly due to iron deficiency, but antenatal iron supplementation has uncertain health benefits and can increase the malaria burden. OBJECTIVE: To measure the effect of antenatal iron supplementation on maternal Plasmodium infection risk, maternal iron status, and neonatal outcomes. DESIGN, SETTING, AND PARTICIPANTS: Randomized placebo-controlled trial conducted October 2011 through April 2013 in a malaria endemic area among 470 rural Kenyan women aged 15 to 45 years with singleton pregnancies, gestational age of 13 to 23 weeks, and hemoglobin concentration of 9 g/dL or greater. All women received 5.7 mg iron/day through flour fortification during intervention, and usual intermittent preventive treatment against malaria was given. INTERVENTIONS: Supervised daily supplementation with 60 mg of elemental iron (as ferrous fumarate, n = 237 women) or placebo (n = 233) from randomization until 1 month postpartum. MAIN OUTCOMES AND MEASURES: Primary outcome was maternal Plasmodium infection at birth. Predefined secondary outcomes were birth weight and gestational age at delivery, intrauterine growth, and maternal and infant iron status at 1 month after birth. RESULTS: Among the 470 participating women, 40 women (22 iron, 18 placebo) were lost to follow-up or excluded at birth; 12 mothers were lost to follow-up postpartum (5 iron, 7 placebo). At baseline, 190 of 318 women (59.7%) were iron-deficient. In intention-to-treat analysis, comparison of women who received iron vs placebo, respectively, yielded the following results at birth: Plasmodium infection risk: 50.9% vs 52.1% (crude difference, -1.2%, 95% CI, -11.8% to 9.5%; P = .83); birth weight: 3202 g vs 3053 g (crude difference, 150 g, 95% CI, 56 to 244; P = .002); birth-weight-for-gestational-age z score: 0.52 vs 0.31 (crude difference, 0.21, 95% CI, -0.11 to 0.52; P = .20); and at 1 month after birth: maternal hemoglobin concentration: 12.89 g/dL vs 11.99 g/dL (crude difference, 0.90 g/dL, 95% CI, 0.61 to 1.19; P < .001); geometric mean maternal plasma ferritin concentration: 32.1 µg/L vs 14.4 µg/L (crude difference, 123.4%, 95% CI, 85.5% to 169.1%; P < .001); geometric mean neonatal plasma ferritin concentration: 163.0 µg/L vs 138.7 µg/L (crude difference, 17.5%, 95% CI, 2.4% to 34.8%; P = .02). Serious adverse events were reported for 9 and 12 women who received iron and placebo, respectively. There was no evidence that intervention effects on Plasmodium infection risk were modified by intermittent preventive treatment use. CONCLUSIONS AND RELEVANCE: Among rural Kenyan women with singleton pregnancies, administration of daily iron supplementation, compared with administration of placebo, resulted in no significant differences in overall maternal Plasmodium infection risk. Iron supplementation led to increased birth weight. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01308112.

Diagnostic utility of zinc protoporphyrin to detect iron deficiency in Kenyan pregnant women.

BACKGROUND: Iron-deficient erythropoiesis results in excess formation of zinc protoporphyrin (ZPP), which can be measured instantly and at low assay cost using portable haematofluorometers. ZPP is used as a screening marker of iron deficiency in individual pregnant women and children, but also to assess population iron status in combination with haemoglobin concentration. We examined associations between ZPP and disorders that are common in Africa. In addition, we assessed the diagnostic utility of ZPP (measured in whole blood and erythrocytes), alone or in combination with haemoglobin concentration, in detecting iron deficiency (plasma ferritin concentration <15 µg/L). METHODS: Single blood samples were collected from a population sample of 470 rural Kenyan women with singleton pregnancies, gestational age 13 to 23 weeks, and haemoglobin concentration ≥90 g/L. We used linear regression analysis to assess associations between ZPP and iron markers (including anaemia), factors known or suspected to be associated with iron status, inflammation markers (plasma concentrations of C-reactive protein and α 1-acid glycoprotein), infections (Plasmodium infection, HIV infection), and other disorders (α(+)-thalassaemia, plasma concentrations of total bilirubin, and lactate dehydrogenase). Subsequently, in those without inflammation, Plasmodium infection, or HIV infection, we used logistic discriminant analysis and examined receiver operating characteristics curves with corresponding area-under-the-curve to assess diagnostic performance of ZPP, alone and in combination with haemoglobin concentration. RESULTS: Individually, whole blood ZPP, erythrocyte ZPP, and erythrocyte protoporphyrin had limited ability to discriminate between women with and without iron deficiency. Combining each of these markers with haemoglobin concentration had no additional diagnostic value. Conventional cut off points for whole blood ZPP (>70 µmol/mol haem) resulted in gross overestimates of the prevalence of iron deficiency. CONCLUSIONS: Erythrocyte ZPP has limited value to rule out iron deficiency when used for screening in conditions with a low prevalence (e.g., 10%). ZPP is of unreliable diagnostic utility when discriminating between pregnant women with and without iron deficiency. Based on these findings, guidelines on the use of ZPP to assess iron status in individuals or populations of pregnant women need review. TRIAL REGISTRATION: NCT01308112 (2 March 2011).

Molecular Detection of Residual Parasitemia after Pyronaridine-Artesunate or Artemether-Lumefantrine Treatment of Uncomplicated Plasmodium falciparum Malaria in Kenyan Children.

Artemisinin resistance is rapidly rising in Southeast Asia and may spread to African countries, where efficacy estimates are currently still excellent. Extensive monitoring of parasite clearance dynamics after treatment is needed to determine whether responsiveness to artemisinin-based combination therapies (ACT) is changing in Africa. In this study, Kenyan children with uncomplicated falciparum malaria were randomly assigned to pyronaridine-artesunate (PA) or artemether-lumefantrine (AL) treatment. Parasite clearance was evaluated over 7 days following the start of treatment by quantitative polymerase chain reaction (qPCR) and direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA), a simplified molecular malaria diagnostic. Residual parasitemia at day 7 was detected by qPCR in 37.1% (26/70) of AL-treated children and in 46.1% (35/76) of PA-treated participants (P = 0.275). Direct-on-blood PCR nucleic acid lateral flow immunoassay detected residual parasites at day 7 in 33.3% (23/69) and 30.3% (23/76) of AL and PA-treated participants, respectively (P = 0.692). qPCR-determined parasitemia at day 7 was associated with increased prevalence and density of gametocytes at baseline (P = 0.014 and P = 0.003, for prevalence and density, respectively) and during follow-up (P = 0.007 and P = 0.011, respectively, at day 7). A positive db-PCR-NALFIA outcome at day 7 was associated with treatment failure (odds ratio [OR]: 3.410, 95% confidence interval [CI]: 1.513-7.689, P = 0.003), but this association was not found for qPCR (OR: 0.701, 95% CI: 0.312-1.578, P = 0.391). Both qPCR and db-PCR-NALFIA detected substantial residual submicroscopic parasitemia after microscopically successful PA and AL treatment and can be useful tools to monitor parasite clearance. To predict treatment outcome, db-PCR-NALFIA may be more suitable than qPCR.

Plasmodium Detection and Differentiation by Direct-on-Blood PCR Nucleic Acid Lateral Flow Immunoassay: Development, Validation, and Evaluation.

Decreasing malaria transmission warrants the search for highly sensitive point-of-care diagnostics, especially in resource-limited settings. The direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA) is a simplified PCR-based technique with a lateral flow readout that does not require sample preparation. Two duplex db-PCR-NALFIAs were developed: a pan-Plasmodium/Plasmodium falciparum (pan/P. falciparum) and a pan-Plasmodium/Plasmodium vivax (pan/P. vivax) assay. Confirmed positive samples (n = 61) and negative controls (n = 40) were used for laboratory validations. A prospective field evaluation of the pan/P. falciparum assay was performed in Kenya (n = 300). In the laboratory validation, sensitivity and specificity of the pan/P. falciparum assay were 100% (95% CI, 94.1%-100%) and 100% (95% CI, 91.2%-100%), respectively. Sensitivity and specificity of the pan/P. vivax assay were 100% (95% CI, 94.1%-100%) and 97.5% (95% CI, 86.8%-99.9%), respectively. In Kenya, sensitivity of the pan/P. falciparum db-PCR-NALFIA was 97.2% (95% CI, 93.0%-99.2%) and specificity was 74.2% (95% CI, 67.0%-81.0%) compared with reference standard microscopy. When using real-time quantitative PCR as a reference standard, sensitivity was 84.5% (95% CI, 78.7%-89.3%) and specificity was 85.4% (95% CI, 77.1%-91.6%). Db-PCR-NALFIA is a sensitive, specific, and easy method for the detection and species differentiation of Plasmodium. This test is especially of interest for malaria control or elimination programs in low-transmission settings that require accurate detection of low parasite densities.