Clinical consequences of submicroscopic malaria parasitaemia in Uganda.

BACKGROUND: Submicroscopic malaria parasitaemia is common in both high- and low-endemicity settings, but its clinical consequences are unclear. METHODS: A cohort of 364 children (0.5-10 years of age) and 106 adults was followed from 2011 to 2016 in Tororo District, Uganda using passive surveillance for malaria episodes and active surveillance for parasitaemia. Participants presented every 90 days for routine visits (n = 9075); a subset was followed every 30 days. Participants who presented with fever and a positive blood smear were treated for malaria. At all routine visits microscopy was performed and samples from subjects with a negative blood smear underwent loop-mediated isothermal amplification for detection of plasmodial DNA. RESULTS: Submicroscopic parasitaemia was common; the proportion of visits with submicroscopic parasitemia was 25.8% in children and 39.2% in adults. For children 0.5-10 years of age, but not adults, having microscopic and submicroscopic parasitaemia at routine visits was significantly associated with both fever (adjusted risk ratios [95% CI], 2.64 [2.16-3.22], 1.67 [1.37-2.03]) and non-febrile illness (aRR [CI], 1.52 [1.30-1.78], 1.26 [1.09-1.47]), compared to not having parasitaemia. After stratifying by age, significant associations were seen between submicroscopic parasitaemia and fever in children aged 2-< 5 and 5-10 years (aRR [CI], 1.42 [1.03-1.98], 2.01 [1.49-2.71]), and submicroscopic parasitaemia and non-febrile illness in children aged 5-10 years (aRR [CI], 1.44 [1.17-1.78]). These associations were maintained after excluding individuals with a malaria episode within the preceding 14 or following 7 days, and after adjusting for household wealth. CONCLUSIONS: Submicroscopic malaria infections were associated with fever and non-febrile illness in Ugandan children. These findings support malaria control strategies that target low-density infections

Characterizing microscopic and submicroscopic malaria parasitaemia at three sites with varied transmission intensity in Uganda.

BACKGROUND: Parasite prevalence is a key metric used to quantify the burden of malaria and assess the impact of control strategies. Most published estimates of parasite prevalence are based on microscopy and likely underestimate true prevalence. METHODS: Thick smear microscopy was performed in cohorts of children (aged 6 month to 10 years) and adults every 90 days over 2 years, at three sites of varying transmission intensity in Uganda. Microscopy-negative samples were tested for sub-microscopic parasitaemia using loop-mediated isothermal amplification (LAMP). Generalized estimating equation models were used to evaluate associations between age and parasitaemia, factors associated with sub-microscopic infection and associations between parasitaemia and haemoglobin. RESULTS: A total of 9260 samples were collected from 1245 participants. Parasite prevalence among children across the three sites was 7.4, 9.4 and 28.8 % by microscopy and 21.3, 31.8 and 69.0 % by microscopy plus LAMP. Parasite prevalence among adults across the three sites was 3.1, 3.0 and 5.2 % by microscopy and 18.8, 24.2 and 53.5 % by microscopy plus LAMP. Among those with parasitaemia, adults and persons recently treated with anti-malarial therapy had the highest prevalence of sub-microscopic infection. Children with sub-microscopic or microscopic parasitaemia had lower mean haemoglobin levels compared to children with no detectable parasites. CONCLUSIONS: Across a range of transmission intensities in Uganda, microscopy vastly underestimated parasite prevalence, especially among adults

Performance of Loop-Mediated Isothermal Amplification for the Identification of Submicroscopic Plasmodium falciparum Infection in Uganda.

Accurately identifying and targeting the human reservoir of malaria parasitemia is critical for malaria control, and requires a reliable and sensitive diagnostic method. Loop-mediated isothermal amplification (LAMP) is increasingly used to diagnose submicroscopic parasitemia. Although most published studies report the sensitivity of LAMP compared with nested polymerase chain reaction (PCR) as ≥ 80%, they have failed to use a consistent, sensitive diagnostic as a comparator. We used cross-sectional samples from children and adults in Tororo, Uganda, a region with high but declining transmission due to indoor residual spraying, to characterize the sensitivity and specificity of pan-Plasmodium LAMP for detecting submicroscopic infections. We compared LAMP results targeting a mitochondrial DNA sequence conserved in all Plasmodium species, performed on DNA extracted from dried blood spots, to those of a gold standard quantitative PCR assay targeting the var gene acidic terminal sequence of Plasmodium falciparum (varATS qPCR), performed on DNA extracted from 200 µL of whole blood. Using LAMP and varATS qPCR increased the detection of parasitemia 2- to 5-fold, compared with microscopy. Among microscopy-negative samples, the sensitivity of LAMP was 81.5% for detecting infection ≥ 1 parasites/µL. However, low density infections were common, and LAMP failed to identify more than half of all infections diagnosed by varATS qPCR, performing with an overall sensitivity of 44.7% for detecting submicroscopic infections ≥ 0.01 parasites/µL. Thus, although the LAMP assay is more sensitive than microscopy, it missed a significant portion of the submicroscopic reservoir. These findings have important implications for malaria control, particularly in settings where low-density infections predominate

Persistent parasitemia despite dramatic reduction in malaria incidence after 3 rounds of indoor residual spraying in Tororo, Uganda.

Background:Indoor residual spraying of insecticide (IRS) has been associated with reductions in the incidence of malaria, but its impact on malaria parasitemia is unclear. Methods:We followed 469 participants from August 2011 to May 2016 in Tororo, Uganda, a historically high malaria transmission setting. Three rounds of IRS with bendiocarb were implemented from December 2014 to December 2015. Symptomatic malaria episodes were identified by passive surveillance. Parasitemia was identified by active surveillance every 1-3 months using microscopy and P.falciparum-specific loop-mediated isothermal amplification. Results:IRS was associated with a significant decline in the incidence of symptomatic malaria irrespective of age (episodes per person per year reduced from 3.98 to 0.13 in children <5 years, 2.30 to 0.15 in children 5 - 10 years, and 0.41 to 0 in adults, p<0.001 for all). IRS significantly reduced the prevalence of parasitemia, however, the prevalence remained high (58.5% to 11.3% in children <5, 73.3% to 23.7% in children 5 - 10 years, and 52.2% to 15.4% in adults, p<0.001 for all). Discussion:Although IRS was associated with significant reductions in the incidence of malaria and prevalence of parasitemia, a proportion of the population remained parasitemic, providing a potential reservoir for malaria transmission

The impact of gravidity, symptomatology and timing of infection on placental malaria.

BACKGROUND: Placental malaria is associated with increased risk of adverse perinatal outcomes. While primigravidity has been reported as a risk factor for placental malaria, little is known regarding the relationship between gravidity, symptomatology and timing of Plasmodium falciparum infection and the development of placental malaria. METHODS: The aim of this study was to investigate the relationship between the development of placental malaria and gravidity, timing of infection, and presence of symptoms. This is a secondary analysis of data from a double-blind randomized control trial of intermittent preventive therapy during pregnancy in Uganda. Women were enrolled from 12 to 20 weeks gestation and followed through delivery. Exposure to malaria parasites was defined as symptomatic (fever with positive blood smear) or asymptomatic (based on molecular detection of parasitaemia done routinely every 4 weeks). The primary outcome was placental malaria diagnosed by histopathology, placental blood smear, and/or placental blood loop-mediated isothermal amplification. Multivariate analyses were performed using logistic regression models. Subgroup analysis was performed based on the presence of symptomatic malaria, gravidity, and timing of infection. RESULTS: Of the 228 patients with documented maternal infection with malaria parasites during pregnancy, 101 (44.3%) had placental malaria. Primigravidity was strongly associated with placental malaria (aOR 8.90, 95% CI 4.34-18.2, p < 0.001), and each episode of malaria was associated with over a twofold increase in placental malaria (aOR 2.35, 95% CI 1.69-3.26, p < 0.001). Among multigravid women, the odds of placental malaria increased by 14% with each advancing week of gestation at first documented infection (aOR 1.14, 95% CI 1.02-1.27, p = 0.02). When stratified by the presence of symptoms, primigravidity was only associated with placental malaria in asymptomatic women, who had a 12-fold increase in the odds of placental malaria (aOR 12.19, 95% CI 5.23-28.43, p < 0.001). CONCLUSIONS: Total number of P. falciparum infections in pregnancy is a significant predictor of placental malaria. The importance of timing of infection on the development of placental malaria varies based on gravidity. In primigravidas, earlier asymptomatic infections were more frequently identified in those with placental malaria, whereas in multigravidas, parasitaemias detected later in gestation were associated with placental malaria. Earlier initiation of an effective intermittent preventive therapy may help to prevent placental malaria and improve birth outcomes, particularly in primigravid women

Reductions in malaria in pregnancy and adverse birth outcomes following indoor residual spraying of insecticide in Uganda.

BACKGROUND: Indoor residual spraying of insecticide (IRS) is a key intervention for reducing the burden of malaria in Africa. However, data on the impact of IRS on malaria in pregnancy and birth outcomes is limited. METHODS: An observational study was conducted within a trial of intermittent preventive therapy during pregnancy in Tororo, Uganda. Women were enrolled at 12-20 weeks of gestation between June and October 2014, provided with insecticide-treated bed nets, and followed through delivery. From December 2014 to February 2015, carbamate-containing IRS was implemented in Tororo district for the first time. Exact spray dates were collected for each household. The exposure of interest was the proportion of time during a woman's pregnancy under protection of IRS, with three categories of protection defined: no IRS protection, >0-20 % IRS protection, and 20-43 % IRS protection. Outcomes assessed included malaria incidence and parasite prevalence during pregnancy, placental malaria, low birth weight (LBW), pre-term delivery, and fetal/neonatal deaths. RESULTS: Of 289 women followed, 134 had no IRS protection during pregnancy, 90 had >0-20 % IRS protection, and 65 had >20-43 % protection. During pregnancy, malaria incidence (0.49 vs 0.10 episodes ppy, P = 0.02) and parasite prevalence (20.0 vs 8.9 %, P 20-43 % IRS protection (1.6 %, P = 0.006). Compared to women with no IRS protection, those with >20-43 % IRS protection had a lower risk of LBW (20.9 vs 3.1 %, P = 0.002), pre-term birth (17.2 vs 1.5 %, P = 0.006), and fetal/neonatal deaths (7.5 vs 0 %, P = 0.03). CONCLUSION: In this setting, IRS was temporally associated with lower malaria parasite prevalence during pregnancy and at delivery, and improved birth outcomes. IRS may represent an important tool for combating malaria in pregnancy and for improving birth outcomes in malaria-endemic settings. Trial Registration Current Controlled Trials Identifier NCT02163447

Relationships between infection with Plasmodium falciparum during pregnancy, measures of placental malaria, and adverse birth outcomes.

BACKGROUND: Malaria in pregnancy has been associated with maternal morbidity, placental malaria, and adverse birth outcomes. However, data are limited on the relationships between longitudinal measures of malaria during pregnancy, measures of placental malaria, and birth outcomes. METHODS: This is a nested observational study of data from a randomized controlled trial of intermittent preventive therapy during pregnancy among 282 participants with assessment of placental malaria and delivery outcomes. HIV-uninfected pregnant women were enrolled at 12-20 weeks of gestation. Symptomatic malaria during pregnancy was measured using passive surveillance and monthly detection of asymptomatic parasitaemia using loop-mediated isothermal amplification (LAMP). Placental malaria was defined as either the presence of parasites in placental blood by microscopy, detection of parasites in placental blood by LAMP, or histopathologic evidence of parasites or pigment. Adverse birth outcomes assessed included low birth weight (LBW), preterm birth (PTB), and small for gestational age (SGA) infants. RESULTS: The 282 women were divided into three groups representing increasing malaria burden during pregnancy. Fifty-two (18.4%) had no episodes of symptomatic malaria or asymptomatic parasitaemia during the pregnancy, 157 (55.7%) had low malaria burden (0-1 episodes of symptomatic malaria and < 50% of samples LAMP+), and 73 (25.9%) had high malaria burden during pregnancy (≥ 2 episodes of symptomatic malaria or ≥ 50% of samples LAMP+). Women with high malaria burden had increased risks of placental malaria by blood microscopy and LAMP [aRR 14.2 (1.80-111.6) and 4.06 (1.73-9.51), respectively], compared to the other two groups combined. Compared with women with no malaria exposure during pregnancy, the risk of placental malaria by histopathology was higher among low and high burden groups [aRR = 3.27 (1.32-8.12) and aRR = 7.07 (2.84-17.6), respectively]. Detection of placental parasites by any method was significantly associated with PTB [aRR 5.64 (1.46-21.8)], and with a trend towards increased risk for LBW and SGA irrespective of the level of malaria burden during pregnancy. CONCLUSION: Higher malaria burden during pregnancy was associated with placental malaria and together with the detection of parasites in the placenta were associated with increased risk for adverse birth outcomes. Trial Registration Current Controlled Trials Identifier NCT02163447

Dihydroartemisinin-Piperaquine for the Prevention of Malaria in Pregnancy.

BACKGROUND: Intermittent treatment with sulfadoxine-pyrimethamine is widely recommended for the prevention of malaria in pregnant women in Africa. However, with the spread of resistance to sulfadoxine-pyrimethamine, new interventions are needed. METHODS: We conducted a double-blind, randomized, controlled trial involving 300 human immunodeficiency virus (HIV)-uninfected pregnant adolescents or women in Uganda, where sulfadoxine-pyrimethamine resistance is widespread. We randomly assigned participants to a sulfadoxine-pyrimethamine regimen (106 participants), a three-dose dihydroartemisinin-piperaquine regimen (94 participants), or a monthly dihydroartemisinin-piperaquine regimen (100 participants). The primary outcome was the prevalence of histopathologically confirmed placental malaria. RESULTS: The prevalence of histopathologically confirmed placental malaria was significantly higher in the sulfadoxine-pyrimethamine group (50.0%) than in the three-dose dihydroartemisinin-piperaquine group (34.1%, P=0.03) or the monthly dihydroartemisinin-piperaquine group (27.1%, P=0.001). The prevalence of a composite adverse birth outcome was lower in the monthly dihydroartemisinin-piperaquine group (9.2%) than in the sulfadoxine-pyrimethamine group (18.6%, P=0.05) or the three-dose dihydroartemisinin-piperaquine group (21.3%, P=0.02). During pregnancy, the incidence of symptomatic malaria was significantly higher in the sulfadoxine-pyrimethamine group (41 episodes over 43.0 person-years at risk) than in the three-dose dihydroartemisinin-piperaquine group (12 episodes over 38.2 person-years at risk, P=0.001) or the monthly dihydroartemisinin-piperaquine group (0 episodes over 42.3 person-years at risk, P<0.001), as was the prevalence of parasitemia (40.5% in the sulfadoxine-pyrimethamine group vs. 16.6% in the three-dose dihydroartemisinin-piperaquine group [P<0.001] and 5.2% in the monthly dihydroartemisinin-piperaquine group [P<0.001]). In each treatment group, the risk of vomiting after administration of any dose of the study agents was less than 0.4%, and there were no significant differences among the groups in the risk of adverse events. CONCLUSIONS: The burden of malaria in pregnancy was significantly lower among adolescent girls or women who received intermittent preventive treatment with dihydroartemisinin-piperaquine than among those who received sulfadoxine-pyrimethamine, and monthly treatment with dihydroartemisinin-piperaquine was superior to three-dose dihydroartemisinin-piperaquine with regard to several outcomes. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development; ClinicalTrials.gov number, NCT02163447.)