Abstract
Background
In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit ...efficacies.
Methods
Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays.
Results
Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0–46% in 2018; 0–23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively).
Conclusions
We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.
The prevalence of genetic polymorphisms associated with antimalarial drug resistance in Plasmodium falciparum collected from 16 sites across Uganda was studied. Most concerning was increasing prevalence of mutations associated with resistance to antifolates and artemisinins.
Summary Background Intermittent preventive treatment (IPT) for malaria is used in infants, children, adults, and pregnant women. Dihydroartemisinin-piperaquine (DP) is an effective, well tolerated ...artemisinin-based combination therapy. The long half-life of piperaquine makes it attractive for IPT. We conducted a systematic review and meta-analysis to establish the efficacy and safety of repeated treatment with DP. Methods Following PRISMA guidelines, we searched multiple databases on Sept 1, 2016, with the terms: “human” AND “dihydroartemisinin-piperaquine” OR “DHA-PPQ”. Studies were eligible if they were randomised controlled trials (RCTs) or prospective cohort studies involving repeat exposures to standard 3-day courses of DP for either seasonal malaria chemoprevention, mass drug administration, or treatment of clinical malaria, conducted at any time and in any geographic location. Random-effects meta-analysis was used to generate pooled incidence rate ratios and relative risks, or risk differences. Findings 11 studies were included: two repeat treatment studies (one in children younger than 5 years and one in pregnant women), and nine IPT trials (five in children younger than 5 years, one in schoolchildren, one in adults, two in pregnant women). Comparator interventions included placebo, artemether-lumefantrine, sulfadoxine-pyrimethamine (SP), SP+amodiaquine, SP+piperaquine, SP+chloroquine, and co-trimoxazole. Of 14 628 participants, 3935 received multiple DP courses (2–18). Monthly IPT-DP was associated with an 84% reduction in the incidence of malaria parasitaemia measured by microscopy compared with placebo. Monthly IPT-DP was associated with fewer serious adverse events than placebo, daily co-trimoxazole, or monthly SP. Among 56 IPT-DP recipients (26 children, 30 pregnant women) with cardiac parameters, all QTc intervals were within normal limits, with no significant increase in QTc prolongation with increasing courses of DP. Interpretation Monthly DP appears well tolerated and effective for IPT. Additional data are needed in pregnancy and to further explore the cardiac safety with monthly dosing. Funding Bill & Melinda Gates Foundation and NIH.
The global malaria burden has fallen since 2000, sometimes before large-scale vector control programmes were initiated. While long-lasting insecticide-treated nets and indoor residual spraying are ...highly effective interventions, this study tests the hypothesis that improved housing can reduce malaria by decreasing house entry by malaria mosquitoes.
A systematic review and meta-analysis was conducted to assess whether modern housing is associated with a lower risk of malaria than traditional housing, across all age groups and malaria-endemic settings. Six electronic databases were searched to identify intervention and observational studies published from 1 January, 1900 to 13 December, 2013, measuring the association between house design and malaria. The primary outcome measures were parasite prevalence and incidence of clinical malaria. Crude and adjusted effects were combined in fixed- and random-effects meta-analyses, with sub-group analyses for: overall house type (traditional versus modern housing); screening; main wall, roof and floor materials; eave type; ceilings and elevation.
Of 15,526 studies screened, 90 were included in a qualitative synthesis and 53 reported epidemiological outcomes, included in a meta-analysis. Of these, 39 (74%) showed trends towards a lower risk of epidemiological outcomes associated with improved house features. Of studies assessing the relationship between modern housing and malaria infection (n=11) and clinical malaria (n=5), all were observational, with very low to low quality evidence. Residents of modern houses had 47% lower odds of malaria infection compared to traditional houses (adjusted odds ratio (OR) 0°53, 95% confidence intervals (CI) 0°42-0°67, p< 0°001, five studies) and a 45-65% lower odds of clinical malaria (case-control studies: adjusted OR 0°35, 95 % CI 0°20-0°62, p<0°001, one study; cohort studies: adjusted rate ratio 0°55, 95% CI 0°36-0°84, p=0°005, three studies). Evidence of a high risk of bias was found within studies.
Despite low quality evidence, the direction and consistency of effects indicate that housing is an important risk factor for malaria. Future research should evaluate the protective effect of specific house features and incremental housing improvements associated with socio-economic development.
The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, ...the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006-7 (n = 49) and from 2010-12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.
Fundamental gaps remain in our understanding of how immunity to malaria develops. We used detailed clinical and entomological data from parallel cohort studies conducted across the malaria ...transmission spectrum in Uganda to quantify the development of immunity against symptomatic
as a function of age and transmission intensity. We focus on: anti-parasite immunity (i.e. ability to control parasite densities) and anti-disease immunity (i.e. ability to tolerate higher parasite densities without fever). Our findings suggest a strong effect of age on both types of immunity, not explained by cumulative-exposure. They also show an independent effect of exposure, where children living in moderate/high transmission settings develop immunity faster as transmission increases. Surprisingly, children in the lowest transmission setting appear to develop immunity more efficiently than those living in moderate transmission settings. Anti-parasite and anti-disease immunity develop in parallel, reducing the probability of experiencing symptomatic malaria upon each subsequent
infection.
Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce ...precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual's recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86-0.93), whereas responses to six antigens accurately estimated an individual's malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.
Good house construction may reduce the risk of malaria by limiting the entry of mosquito vectors. We assessed how house design may affect mosquito house entry and malaria risk in Uganda.
100 ...households were enrolled in each of three sub-counties: Walukuba, Jinja district; Kihihi, Kanungu district; and Nagongera, Tororo district. CDC light trap collections of mosquitoes were done monthly in all homes. All children aged six months to ten years (n = 878) were followed prospectively for a total of 24 months to measure parasite prevalence every three months and malaria incidence. Homes were classified as modern (cement, wood or metal walls; and tiled or metal roof; and closed eaves) or traditional (all other homes).
A total of 113,618 female Anopheles were collected over 6,765 nights. 6,816 routine blood smears were taken of which 1,061 (15.6%) were malaria parasite positive. 2,582 episodes of uncomplicated malaria were diagnosed after 1,569 person years of follow-up, giving an overall incidence of 1.6 episodes per person year at risk. The human biting rate was lower in modern homes than in traditional homes (adjusted incidence rate ratio (IRR) 0.48, 95% confidence interval (CI) 0.37-0.64, p<0.001). The odds of malaria infection were lower in modern homes across all the sub-counties (adjusted odds ratio 0.44, 95%CI 0.30-0.65, p<0.001), while malaria incidence was lower in modern homes in Kihihi (adjusted IRR 0.61, 95%CI 0.40-0.91, p = 0.02) but not in Walukuba or Nagongera.
House design is likely to explain some of the heterogeneity of malaria transmission in Uganda and represents a promising target for future interventions, even in highly endemic areas.
The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed
genetic markers associated with drug sensitivity ...on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For
K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the
N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of
propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the
and
genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets
and
, 5 mutations previously associated with resistance were very common, and the
164L and
581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.
Long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are the primary vector control interventions used to prevent malaria in Africa. Although both interventions ...are effective in some settings, high-quality evidence is rarely available to evaluate their effectiveness following deployment by a national malaria control program. In Uganda, we measured changes in key malaria indicators following universal LLIN distribution in three sites, with the addition of IRS at one of these sites.
Comprehensive malaria surveillance was conducted from October 1, 2011, to March 31, 2016, in three sub-counties with relatively low (Walukuba), moderate (Kihihi), and high transmission (Nagongera). Between 2013 and 2014, universal LLIN distribution campaigns were conducted in all sites, and in December 2014, IRS with the carbamate bendiocarb was initiated in Nagongera. High-quality surveillance evaluated malaria metrics and mosquito exposure before and after interventions through (a) enhanced health-facility-based surveillance to estimate malaria test positivity rate (TPR), expressed as the number testing positive for malaria/number tested for malaria (number of children tested for malaria: Walukuba = 42,833, Kihihi = 28,790, and Nagongera = 38,690); (b) cohort studies to estimate the incidence of malaria, expressed as the number of episodes per person-year PPY at risk (number of children observed: Walukuba = 340, Kihihi = 380, and Nagongera = 361); and (c) entomology surveys to estimate household-level human biting rate (HBR), expressed as the number of female Anopheles mosquitoes collected per house-night of collection (number of households observed: Walukuba = 117, Kihihi = 107, and Nagongera = 107). The LLIN distribution campaign substantially increased LLIN coverage levels at the three sites to between 65.0% and 95.5% of households with at least one LLIN. In Walukuba, over the 28-mo post-intervention period, universal LLIN distribution was associated with no change in the incidence of malaria (0.39 episodes PPY pre-intervention versus 0.20 post-intervention; adjusted rate ratio aRR = 1.02, 95% CI 0.36-2.91, p = 0.97) and non-significant reductions in the TPR (26.5% pre-intervention versus 26.2% post-intervention; aRR = 0.70, 95% CI 0.46-1.06, p = 0.09) and HBR (1.07 mosquitoes per house-night pre-intervention versus 0.71 post-intervention; aRR = 0.41, 95% CI 0.14-1.18, p = 0.10). In Kihihi, over the 21-mo post-intervention period, universal LLIN distribution was associated with a reduction in the incidence of malaria (1.77 pre-intervention versus 1.89 post-intervention; aRR = 0.65, 95% CI 0.43-0.98, p = 0.04) but no significant change in the TPR (49.3% pre-intervention versus 45.9% post-intervention; aRR = 0.83, 95% 0.58-1.18, p = 0.30) or HBR (4.06 pre-intervention versus 2.44 post-intervention; aRR = 0.71, 95% CI 0.30-1.64, p = 0.40). In Nagongera, over the 12-mo post-intervention period, universal LLIN distribution was associated with a reduction in the TPR (45.3% pre-intervention versus 36.5% post-intervention; aRR = 0.82, 95% CI 0.76-0.88, p < 0.001) but no significant change in the incidence of malaria (2.82 pre-intervention versus 3.28 post-intervention; aRR = 1.10, 95% 0.76-1.59, p = 0.60) or HBR (41.04 pre-intervention versus 20.15 post-intervention; aRR = 0.87, 95% CI 0.31-2.47, p = 0.80). The addition of three rounds of IRS at ~6-mo intervals in Nagongera was followed by clear decreases in all outcomes: incidence of malaria (3.25 pre-intervention versus 0.63 post-intervention; aRR = 0.13, 95% CI 0.07-0.27, p < 0.001), TPR (37.8% pre-intervention versus 15.0% post-intervention; aRR = 0.54, 95% CI 0.49-0.60, p < 0.001), and HBR (18.71 pre-intervention versus 3.23 post-intervention; aRR = 0.29, 95% CI 0.17-0.50, p < 0.001). High levels of pyrethroid resistance were documented at all three study sites. Limitations of the study included the observational study design, the lack of contemporaneous control groups, and that the interventions were implemented under programmatic conditions.
Universal distribution of LLINs at three sites with varying transmission intensity was associated with modest declines in the burden of malaria for some indicators, but the addition of IRS at the highest transmission site was associated with a marked decline in the burden of malaria for all indicators. In highly endemic areas of Africa with widespread pyrethroid resistance, IRS using alternative insecticide formulations may be needed to achieve substantial gains in malaria control.
Studies of the association between malaria in pregnancy (MiP) and malaria during infancy have provided mixed results. A systematic review was conducted to evaluate available evidence on the impact of ...Plasmodium falciparum malaria infection during pregnancy, and intermittent preventive treatment of malaria during pregnancy (IPTp), on the risk of clinical malaria or parasitaemia during infancy.
MEDLINE, EMBASE, Global Health, and Malaria in Pregnancy Library databases were searched from inception to 22 May 2018 for articles published in English that reported on associations between MiP and malaria risk in infancy. Search terms included malaria, Plasmodium falciparum, pregnancy, placenta, maternal, prenatal, foetal, newborn, infant, child or offspring or preschool. Randomized controlled trials and prospective cohort studies, which followed infants for at least 6 months, were included if any of the following outcomes were reported: incidence of clinical malaria, prevalence of parasitaemia, and time to first episode of parasitaemia or clinical malaria. Substantial heterogeneity between studies precluded meta-analysis. Thus, a narrative synthesis of included studies was conducted.
The search yielded 14 published studies, 10 prospective cohort studies and four randomized trials; all were conducted in sub-Saharan Africa. Infants born to mothers with parasitaemia during pregnancy were at higher risk of malaria in three of four studies that assessed this association. Placental malaria detected by microscopy or histology was associated with a higher risk of malaria during infancy in nine of 12 studies, but only one study adjusted for malaria transmission intensity. No statistically significant associations between the use of IPTp or different IPTp regimens and the risk of malaria during infancy were identified.
Evidence of an association between MiP and IPTp and risk of malaria in infancy is limited and of variable quality. Most studies did not adequately adjust for malaria transmission intensity shared by mothers and their infants. Further research is needed to confirm or exclude an association between MiP and malaria in infancy. Randomized trials evaluating highly effective interventions aimed at preventing MiP, such as IPTp with dihydroartemisinin-piperaquine, may help to establish a causal association between MiP and malaria in infancy.
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