Attitudes and perceptions towards developing a health educational video to enhance optimal uptake of malaria preventive therapy among pregnant women in Uganda: a qualitative study involving pregnant women, health workers, and Ministry of health officials.

BACKGROUND: Malaria in pregnancy remains a major global public health problem. Intermittent prophylaxis treatment of malaria in pregnancy with Sulphadoxine-pyrimethamine and co-trimoxazole is efficacious for prevention of malaria in pregnancy HIV negative and positive women, respectively. However, uptake of the recommended doses of therapies has remained suboptimal in Uganda, majorly due to inadequate knowledge among pregnant women. Therefore, this study aimed to explore attitudes and perceptions towards developing an educational video for malaria preventive therapy. METHODS: We conducted an exploratory study with qualitative methods among pregnant women attending antenatal care at Kisenyi Health Center IV (KHCIV), health workers from KHCIV, and officials from the Ministry of Health. The study was conducted at KHCIV from October 2022 to March 2023. Focus group discussions (FGD) were conducted among purposively selected pregnant women and key informant interviews (KII) among health workers and Ministry of Health officials. Data were analyzed using inductive and deductive thematic methods in atlas ti.8. RESULTS: A total of five FGDs comprising of 7-10 pregnant women were conducted; and KIIs were conducted among four mid-wives, two obstetricians, and two Ministry of Health officials. Generally, all respondents mentioned a need for interventions to improve malaria preventive knowledge among pregnant women; were positive about developing an educative video for malaria preventive therapy in pregnancy; and suggested a short, concise, and edutaining video focusing both the benefits of taking and risks of not taking malaria preventive therapy. They proposed that women may be encouraged to view the video as soon as they conceive and throughout the pregnancy. It also was suggested that the video may be viewed on television sets in maternal and reproductive health clinics and homes, and on smart phones. CONCLUSION: Pregnant women, health workers, and Ministry of Health officials were positive about the development of a short edutaining video on malaria preventive therapy that focuses on both benefits of taking and risks of not taking the malaria preventive therapy in pregnancy. This information guided the video development and therefore, in the development of health educative videos, client and stakeholder inputs may always be solicited.

Determination of sulfadoxine and pyrimethamine in microvolume human plasma using ultra high performance liquid chromatography-tandam mass spectrometry.

To support the pharmacokinetic study of sulfadoxine (SD) and pyrimethamine (PM) in pregnant women and children, sensitive methods with small sample volume are desirable. Here we report a method to determine SD and PM with microvolume plasma samples: 5 µL plasma samples were cleaned up by protein precipitation with acetonitrile. The deuterated analytes were used as the internal standards. The samples after cleanup were injected onto an ACE Excel SuperC18 column (50 × 2.1 mm, 1.7 µm, Hichrom Limited) connected to a Waters I class UPLC coupled with a Sciex Triple Quad 6500+ Mass Spectrometer and eluted with water and acetonitrile both containing 0.1% formic acid in a gradient mode at 0.8mL/min. Detection utilized ESI+ as the ion source and MRM as the quantification mode. The precursor-to-product ion transitions m/z 311→245 for SD and 249→233 for PM were selected for quantification. The ion transitions for the corresponding internal standards were 315→249 for SD-d4 and 254→235 for PM-d3. The simplest linear regression weighted by 1/x was used for the calibration curves. The calibration ranges were 1-200 µg/mL SD and 2 - 1000ng/mL PM. The mean (± standard deviation) recoveries were 94.3±3.2% (SD) and 97.0±1.5% (PM). The validated method was applied to analysis of 1719 clinical samples, demonstrating the method is suitable for the pharmacokinetic study with samples collected up to day 28 post-dose.

The photo- and microbial degradation kinetics and pathways of sulfadoxine in seawater elucidated by liquid chromatography coupled with time-of-flight mass spectrometry.

Sulfadoxine (SDX) is a broad-spectrum veterinary antibiotic, which was used alone for the treatment of various infections in the past, and detected ubiquitously in the aqueous environment. However, understanding SDX's photo- and microbial degradation within the environment, especially in marine matrixes, remains limited. This research hones in on SDX's degradation dynamics in seawater. Photodegradation emerges as the dominant process, surpassing microbial degradation in speed and efficiency. Notably, 90% of SDX is photo-degraded within 12 h, while only 52% is removed via microbial degradation over two weeks. Time-of-flight mass spectrometry provides high-resolution molecular mass information on degradation products. The molecular structures of hydrolysis, photo-, and microbial degradation products are deduced from accurate precursor and fragment ion masses, alongside an integrated data processing workflow. Six hydrolysis products arise from the treatment, and photodegradation and microbial degradation yield nine and eighteen products, respectively. Molecular insights from these products inform plausible degradation pathways involving hydrolysis, photodegradation, and microbial degradation. Processes like bond cleavage, methylation, hydroxylation, oxidation, reduction, and methoxylation are identified and associated with degradation. This study presents a comprehensive workflow for acquiring and processing degradation product data linked to emerging organic pollutants. Moreover, it contributes to our comprehension of the environmental fate of veterinary drugs in marine ecosystems.

Prevalence of Malaria Infection in Pregnant Women Attending Antenatal Clinics in Southern Senegal.

Despite marked progress in Senegal, three regions in the southeast part continue to have a high burden of malaria, but there have been no recent studies assessing the prevalence of malaria associated with pregnancy. This study aimed to determine the prevalence of malaria infection in pregnant women attending antenatal clinics in Senegal. During the malaria transmission season of 2019, pregnant women attending 11 health care facilities for a scheduled visit and those presenting unwell with signs of malaria were invited to participate in a malaria screening study. A finger prick blood sample was taken for malaria diagnosis by rapid diagnosis test (RDT) and polymerase chain reaction (PCR). A total of 877 pregnant women were enrolled, 787 for a scheduled antenatal consultation and 90 for an unscheduled consultation with signs of malaria. The prevalence of Plasmodium falciparum among the first group was 48% by PCR and 20% by RDT, and that among the second group was 86% by PCR and 83% by RDT. RDT sensitivity in capturing asymptomatic, PCR-positive infections was 9.2% but ranged from 83% to 94% among febrile women. The prevalence of infection by PCR in women who reported having received at least three doses of sulfadoxine pyrimethamine (SP) was 41.9% compared with 58.9% in women who reported they had not received any SP doses (prevalence ratio adjusted for gravidity and gestational age, 0.54; 95% CI, 0.41-0.73). The burden of P. falciparum infections remains high among pregnant women, the majority of which are not captured by RDT. More effective measures to prevent malaria infection in pregnancy are needed.

Increasing the uptake of Intermittent Preventive Treatment of malaria in pregnancy using Sulfadoxine-Pyrimethamine (IPTp-SP) through seasonal malaria chemoprevention channel delivery: protocol of a multicenter cluster randomized implementation trial in Mali and Burkina Faso.

BACKGROUND: The uptake of Intermittent Preventive Treatment of malaria in pregnancy using Sulfadoxine-Pyrimethamine (IPTp-SP) remains unacceptably low, with more than two-thirds of pregnant women in sub-Saharan Africa still not accessing the three or more doses recommended by the World Health Organisation (WHO). In contrast, the coverage of Seasonal Malaria Chemoprevention (SMC), a more recent strategy recommended by the WHO for malaria prevention in children under five years living in Sahelian countries with seasonal transmission, including Mali and Burkina-Faso, is high (up to 90%). We hypothesized that IPTp-SP delivery to pregnant women through SMC alongside antenatal care (ANC) will increase IPTp-SP coverage, boost ANC attendance, and increase public health impact. This protocol describes the approach to assess acceptability, feasibility, effectiveness, and cost-effectiveness of the integrated strategy. METHODS AND ANALYSIS: This is a multicentre, cluster-randomized, implementation trial of IPTp-SP delivery through ANC + SMC vs ANC alone in 40 health facilities and their catchment populations (20 clusters per arm). The intervention will consist of monthly administration of IPTp-SP through four monthly rounds of SMC during the malaria transmission season (July to October), for two consecutive years. Effectiveness of the strategy to increase coverage of three or more doses of IPTp-SP (IPTp3 +) will be assessed using household surveys and ANC exit interviews. Statistical analysis of IPT3 + and four or more ANC uptake will use a generalized linear mixed model. Feasibility and acceptability will be assessed through in-depth interviews and focus group discussions with health workers, pregnant women, and women with a child < 12 months. DISCUSSION: This multicentre cluster randomized implementation trial powered to detect a 45% and 22% increase in IPTp-SP3 + uptake in Mali and Burkina-Faso, respectively, will generate evidence on the feasibility, acceptability, effectiveness, and cost-effectiveness of IPTp-SP delivered through the ANC + SMC channel. The intervention is designed to facilitate scalability and translation into policy by leveraging existing resources, while strengthening local capacities in research, health, and community institutions. Findings will inform the local national malaria control policies. TRIAL REGISTRATION: Retrospectively registered on August 11th, 2022; registration # PACTR202208844472053. Protocol v4.0 dated September 04, 2023. Trail sponsor: University of Sciences Techniques and Technologies of Bamako (USTTB), Mali.

Uptake of intermittent preventive treatment of malaria in pregnancy and risk factors for maternal anaemia and low birthweight among HIV-negative mothers in Dschang, West region of Cameroon: a cross sectional study.

BACKGROUND: Approximately 32 million pregnant women are at risk of malaria with up to 10,000 maternal deaths and 200,000 neonates at risk annually. Intermittent Preventive Treatment (IPT) with sulfadoxine-pyrimethamine (SP) is recommended by the World Health Organization (WHO) to reduce disease in pregnancy and adverse maternal and newborn outcomes. At least three doses of SP should be taken by pregnant women during antenatal consultation (ANC) beginning from the thirteenth week of pregnancy till parturition. The aim of this study was to assess uptake of IPT during pregnancy and risk factors for maternal anaemia and infant birth weight in Dschang, West region of Cameroon. METHODS: A total of 380 consenting pregnant women at delivery were recruited in a cross- sectional prospective survey between January to December 2021. Data on ANC attendance, total dose of IPT and history of malaria were abstracted from hospital ANC records while socio-demographic characteristics, bed net use and obstetrics history of each participant were also recorded through an interview. Further, blood samples were collected from the intervillous space for assessment of maternal anaemia and microscopic parasitology. Nested PCR based on amplification of the Plasmodium 18S sRNA was carried out to detect submicroscopic infection. IPTp coverage was calculated per WHO recommendation and the prevalence of anaemia and low birth weight were estimated as proportions in the total sample of pregnant women and live births, respectively. Crude and adjusted odds ratios and their 95% confidence intervals were used to estimate associations between pregnancy outcomes considered and risk factors in specific and general models. A p < 0.05 was considered significant. The R software (V4.1.4) was used for all analyses. RESULTS: A majority of pregnant women was aged between 24 and 34 years old (59.2%) and had secondary education (58.8%). Uptake of ≥ 3 IPTp was 64.99% with 77.20% of all who received at least one IPTp doses taking a mix of SP and DP or DP alone in successive ANC contacts. Those with four or more ANC contacts (73.42%) were more likely to have received at least one IPTp. Furthermore, 13.9% of live births had low birthweights (BW < 2500 g) and one in four parturient women with moderate anaemia by WHO criteria. Microscopy (blood smear examination) and PCR-based diagnosis revealed between 0% and 1.57% of parasite-infected placental samples, respectively. Reported malaria in pregnancy predicted maternal anaemia at birth but not birth weight. Only gestational age (< 37 weeks) and bed net use (< 5 months) significantly predicted infant birth weight at delivery. CONCLUSION: The uptake of WHO recommended IPT doses during pregnancy was moderately high. Reported malaria in pregnancy, poor bed net coverage, gestational age less than 37 weeks adversely affect maternal haemoglobin levels at birth and infant birth weight. Asymptomatic and submicroscopic placental parasite infections was found at low prevalence. Together these results highlight the importance of maintaining aggressive measures to prevent malaria in pregnancy and protect the health of mother and baby.

Identification of terpenoids as dihydropteroate synthase and dihydrofolate reductase inhibitors through structure-based virtual screening and molecular dynamic simulations.

Bacterial infections are rising, and antimicrobial resistance (AMR) in bacteria has worsened the scenario, requiring extensive research to find alternative therapeutic agents. Terpenoids play an essential role in protecting plants from herbivores and pathogens. The present study was designed to focus on in silico evaluation of terpenoids for their affinity towards two necessary enzymes, i.e. DHFR and DHPS, which are involved in forming 5, 6, 7, 8-tetrahydrofolate, a key component in bacterial DNA synthesis proteins. Additionally, to account for activity against resistant bacteria, their affinity towards the L28R mutant of DHFR was also assessed in the study. The structure-based drug design approach was used to screen the compound library of terpenes for their interaction with active sites of DHFR and DHPS. Further, compounds were screened based on their dock score, pharmacokinetic properties, and binding affinities. A total of five compounds for each target protein were screened, having dock scores better than their respective standard drug molecules. CNP0169378 (-8.4 kcal/mol) and CNP0309455 (-6.5 kcal/mol) have been identified as molecules with a higher affinity toward the targets of DHFR and DHPS, respectively. At the same time, one molecule CNP0298407 (-5.8 kcal/mol for DHPS, -7.6 kcal/mol for DHFR, -6.1 kcal/mol for the L28R variant), has affinity for both proteins (6XG5 and 6XG4). All the molecules have good pharmacokinetic properties. We further validated the docking study by binding free energy calculations using the MM/GBSA approach and molecular dynamics simulations.Communicated by Ramaswamy H. Sarma.

Murine Malaria Model: Ketoconazole Prevented Malaria while Proguanil and Sulfadoxine/Pyrimethamine Protected against Malaria-associated Anemia and Kidney Damage.

BACKGROUND: The concern about the global spread of resistant malaria has made the researchers not focus only on the treatment of established infections but relatively more on the prevention of the disease. OBJECTIVE: This study evaluates the chemopreventive activity of ketoconazole in a murine malarial model. METHOD: Five out of seven groups of mice were pretreated for five days with proguanil (PRG), sulfadoxine/ pyrimethamine (SP), 10, 20, and 40 mg/kg body weight (b.w) of ketoconazole (KET10, KET20, and KET40), before being infected (on the sixth day) with Plasmodium berghei. Two other groups were infected-not-treated (INT) and not-infected-nor-treated (NINT). At 72 hours postinfection, five out of ten mice in each group were sacrificed to assess parasitemia, chemoprevention, hematologic, hepatic, and renal parameters. The remaining mice were observed for 28 days to determine their mean survival day post-infection (SDPI). RESULTS: All ketoconazole groups, except KET10, demonstrated 100% chemoprevention and significantly higher mean SDPI (p<0.001) in relation to INT (negative control). There was no significant difference in the mean SDPI observed in KET20 in relation to PRG or NINT (healthy control). A dose-related increase (p<0.01) in the mean plasma urea was observed when ketoconazole groups were compared to one another: KET10 versus KET20 (p<0.01) and KET20 versus KET40 (p<0.01). Sulfadoxine/pyrimethamine demonstrated significantly reduced mean plasma urea (p<0.001) and creatinine (p<0.05) in relation to INT and NINT, respectively. While PRG demonstrated significantly higher mean red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT) in relation to INT. CONCLUSION: Ketoconazole possesses prophylactic antimalarial activity with associated dose-related renal impairment. Sulfadoxine/pyrimethamine demonstrated renoprotective potentials, while PRG prevented malaria-associated anemia.

Randomized Field Trial to Assess the Safety and Efficacy of Dihydroartemisinin-Piperaquine for Seasonal Malaria Chemoprevention in School-Aged Children in Bandiagara, Mali.

BACKGROUND: Owing to the increased cases of malaria in older children, the World Health Organization has recently recommended extending seasonal malaria chemoprevention (SMC) to children >5 years of age and using other effective drugs for malaria. In this study, we report the safety and efficacy of dihydroartemisinin-piperaquine (DHA-PQ) for SMC in school-aged children in Mali. METHOD: This randomized, controlled trial included 345 participants aged 6-15 years randomized to receive DHA-PQ, sulfadoxine-pyrimethamine plus amodiaquine (SP-AQ), or no chemoprevention (albendazole) at a 1:1:1 ratio. Four rounds of SMC were conducted from September to December 2021. The participants were assessed 7 days after each round for safety and efficacy of the interventions. RESULTS: Abdominal pain (11.8% vs 29.2%), headache (11.2% vs 19.2%), and vomiting (5.7% vs 15.2%) were frequently reported in the DHA-PQ and SP-AQ arms. On Day 120 of follow up, the incidence of clinical malaria was 0.01 episodes/person-month in the DHA-PQ and SP-AQ arms and 0.17 episodes/person-month in the control arm (P < .0001). Gametocytes were detected in 37 participants in all arms. CONCLUSIONS: Children in DHA-PQ arm reported less adverse events compared to the SP-AQ arm. Both drugs were effective against clinical malaria and infection.

Systematic Review and Meta-Analysis of Seasonal Malaria Chemoprevention.

Seasonal malaria chemoprevention (SMC) for children under 5 years of age for up to four monthly cycles during malaria transmission season was recommended by the WHO in 2012 and has been implemented in 13 countries in the Sahel, reaching more than 30 million children annually. Malaria control programs implementing SMC have asked the WHO to consider expanding the age range or number of monthly cycles. We conducted a systematic review and meta-analysis of SMC among children up to 15 years of age and up to six monthly cycles. Twelve randomized studies were included, with outcomes stratified by age (< 5/≥ 5 years), by three or four versus five or six cycles, and by drug where possible. Drug regimens included sulfadoxine-pyrimethamine + amodiaquine, amodiaquine-artesunate, and sulfadoxine-pyrimethamine + artesunate. Included studies were all conducted in Sahelian countries in which high-grade resistance to sulfadoxine-pyrimethamine was rare and in zones with parasite prevalence ranging from 1% to 79%. Seasonal malaria chemoprevention resulted in substantial reductions in uncomplicated malaria incidence measured during that transmission season (rate ratio: 0.27, 95% CI: 0.25-0.29 among children < 5 years; rate ratio: 0.27, 95% CI: 0.25-0.30 among children ≥ 5 years) and in the prevalence of malaria parasitemia measured within 4-6 weeks from the final SMC cycle (risk ratio: 0.38, 95% CI: 0.34-0.43 among children < 5 years; risk ratio: 0.23, 95% CI: 0.11-0.48 among children ≥ 5 years). In high-transmission zones, SMC resulted in a moderately reduced risk of any anemia (risk ratio: 0.77, 95% CI: 0.72-0.83 among children < 5 years; risk ratio: 0.70, 95% CI: 0.52-0.95 among children ≥ 5 years [one study]). Children < 10 years of age had a moderate reduction in severe malaria (risk ratio: 0.53, 95% CI: 0.37-0.76) but no evidence of a mortality reduction. The evidence suggests that in areas in which sulfadoxine-pyrimethamine and amodiaquine remained efficacious, SMC effectively reduced malaria disease burden among children both < 5 and ≥ 5 years old and that the number of cycles should be commensurate with the length of the transmission season, up to six cycles.

Meta-analysis on Plasmodium falciparum sulfadoxine-pyrimethamine resistance-conferring mutations in India identifies hot spots for genetic surveillance.

BACKGROUND: India is on track to eliminate malaria by 2030 but emerging resistance to first-line antimalarials is a recognised threat. Two instances of rapid development, spread, and natural selection of drug-resistant mutant parasites in India (chloroquine across the country and artesunate + sulfadoxine-pyrimethamine [AS+SP] in the northeastern states) translated into drug policy changes for Plasmodium falciparum malaria in 2010 and 2013, respectively. Considering these rapid changes in the SP drug resistance-conferring mutation profile of P. falciparum, there is a need to systematically monitor the validated mutations in Pfdhfr and Pfdhps genes across India alongside AS+SP therapeutic efficacy studies. There has been no robust, systematic countrywide surveillance reported for these parameters in India, hence the current study was undertaken. METHODS: Studies that reported data on WHO-validated SP resistance markers in P. falciparum across India from 2008 to January 2023 were included. Five major databases, PubMedⓇ, Web of ScienceTM, ScopusⓇ, EmbaseⓇ, and Google Scholar, were exhaustively searched. Individual and pooled prevalence estimates of mutations were obtained through random- and fixed-effect models. Data were depicted using forest plots created with a 95% confidence interval. The study is registered with PROSPERO (CRD42021236012). RESULTS: A total of 37 publications, and 533 Pfdhfr and 134 Pfdhps National Centre of Biotechnology Information (NCBI) DNA sequences were included from >4000 samples. The study included information from 80 districts, 21 states and 3 union territories (UTs) from India. The two PfDHFR mutations, C59R (62%) and S108N (74%), were the most prevalent mutations (pooled estimates 61% and 71%, respectively) and appeared to be stabilised/fixed. Although rarest overall, the prevalence of I164L was observed to be as high as 32%. The PfDHFR double mutants were the most prevalent overall (51%; pooled 42%). The prevalence of triple and quadruple mutations was 6% and 5%, respectively, and is an immediate concern for some states. The most prevalent PfDHPS mutation was A437G (39%), followed by K540E (25%) and A581G (12%). There was a low overall prevalence of PfDHFR/PfDHPS quintuple and sextuple mutations but surveillance for these mutations is critical for some areas. CONCLUSION: The analyses span the two critical policy changes, highlight the areas of concern, and guide policymakers in strategising and refining the anti-malaria drug policy for malaria elimination. The results of the analyses also highlight the SP-resistance hot spots, critical gaps and challenges, and indicate that focal and local malaria genetic surveillance (including drug-resistance markers) is needed until malaria is successfully eliminated.

Use of Supervision Data to Improve Quality of Care for Malaria in Pregnancy: Experience in Six African Countries.

Malaria in pregnancy (MiP) intervention coverage, especially intermittent preventive treatment in pregnancy (IPTp), lags behind other global malaria indicators. In 2020, across Africa, only 32% of eligible pregnant women received at least three IPTp doses, despite high antenatal care attendance. We conducted a secondary analysis of data collected during Outreach Training and Supportive Supervision visits from 2019 to 2020 to assess quality of care and explore factors contributing to providers' competence in providing IPTp, insecticide-treated nets, malaria case management, and respectful maternity care. Data were collected during observations of provider-patient interactions in six countries (Cameroon, Cote d'Ivoire, Ghana, Kenya, Mali, and Niger). Competency scores (i.e., composite scores of supervisory checklist observations) were calculated across three domains: MiP prevention, MiP treatment, and respectful maternity care. Scores are used to understand drivers of competency, rather than to assess individual health worker performance. Country-specific multilinear regressions were used to assess how competency score was influenced by commodity availability, training, provider gender and cadre, job aid availability, and facility type. Average competency scores varied across countries: prevention (44-90%), treatment (78-90%), and respectful maternity care (53-93%). The relative association of each factor with competency score varied. Commodity availability, training, and access to job aids correlated positively with competency in multiple countries. To improve MiP service quality, equitable access to training opportunities for different cadres, targeted training, and access to job aids and guidelines should be available for providers. Collection and analysis of routine supervision data can support tailored actions to improve quality MiP services.

Direct therapeutic effect of sulfadoxine-pyrimethamine on nutritional deficiency-induced enteric dysfunction in a human Intestine Chip.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) antimalarial therapy has been suggested to potentially increase the birth weight of infants in pregnant women in sub-Saharan Africa, independently of malarial infection. Here, we utilized female intestinal organoid-derived cells cultured within microfluidic Organ Chips to investigate whether SP could directly impact intestinal function and thereby improve the absorption of essential fats and nutrients crucial for fetal growth. METHODS: Using a human organ-on-a-chip model, we replicated the adult female intestine with patient organoid-derived duodenal epithelial cells interfaced with human intestinal endothelial cells. Nutrient-deficient (ND) medium was perfused to simulate malnutrition, resulting in the appearance of enteric dysfunction indicators such as villus blunting, reduced mucus production, impaired nutrient absorption, and increased inflammatory cytokine secretion. SP was administered to these chips in the presence or absence of human peripheral blood mononuclear cells (PBMCs). FINDINGS: Our findings revealed that SP treatment effectively reversed multiple intestinal absorptive abnormalities observed in malnourished female Intestine Chips, as validated by transcriptomic and proteomic analyses. SP also reduced the production of inflammatory cytokines and suppressed the recruitment of PBMCs in ND chips. INTERPRETATION: Our results indicate that SP could potentially increase birth weights by preventing enteric dysfunction and suppressing intestinal inflammation. This underscores the potential of SP as a targeted intervention to improve maternal absorption, subsequently contributing to healthier fetal growth. While SP treatment shows promise in addressing malabsorption issues that can influence infant birth weight, we did not model pregnancy in our chips, and thus its usefulness for treatment of malnourished pregnant women requires further investigation through clinical trials. FUNDING: The Bill and Melinda Gates Foundation, and the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the HDDC Organoid Core of the P30 DK034854.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India.

BACKGROUND: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. METHODS: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. RESULTS: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. CONCLUSION: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Geographical emergence of sulfadoxine-pyrimethamine drug resistance-associated P. falciparum and P. malariae alleles in co-existing Anopheles mosquito and asymptomatic human populations across Cameroon.

Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase (dhfr) and dihydropteroate-synthase (dhps) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5-15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A16I51R59N108I164. The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V431A436G437K540A581A613 alleles (20.8%). Similarly, the Pmdhfr N50K55L57R58S59S114F168I170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps- S436A437occured at 37.2% frequency. The combined quadruple N50K55L57R58S59S114F168I170_ S436G437K540A581A613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems.

Antiplasmodial and Antileishmanial Activities of a New Limonoid and Other Constituents from the Stem Bark of Khaya senegalensis.

Plasmodium falciparum and Leishmania sp. resistance to antiparasitic drugs has become a major concern in malaria and leishmaniasis control. These diseases are public health problems with significant socioeconomic impacts, and mostly affect disadvantaged populations living in remote tropical areas. This challenge emphasizes the need to search for new chemical scaffolds that preferably possess novel modes of action to contribute to antimalarial and antileishmanial research programs. This study aimed to investigate the antimalarial and antileishmanial properties of a methanol extract (KS-MeOH) of the stem bark of the Cameroonian medicinal plant Khaya senegalensis and its isolated compounds. The purification of KS-MeOH led to the isolation of a new ordered limonoid derivative, 21ß-hydroxybourjotinolone A (1a), together with 15 known compounds (1bc-14) using a repeated column chromatography. Compound 1a was obtained in an epimeric mixture of 21α-melianodiol (1b) and 21ß-melianodiol (1c). Structural characterization of the isolated compounds was achieved with HRMS, and 1D- and 2D-NMR analyses. The extracts and compounds were screened using pre-established in vitro methods against synchronized ring stage cultures of the multidrug-resistant Dd2 and chloroquine-sensitive/sulfadoxine-resistant 3D7 strains of Plasmodium falciparum and the promastigote form of Leishmania donovani (1S(MHOM/SD/62/1S). In addition, the samples were tested for cytotoxicity against RAW 264.7 macrophages. Positive controls consisted of artemisinin and chloroquine for P. falciparum, amphotericin B for L. donovani, and podophyllotoxin for cytotoxicity against RAW 264.7 cells. The extract and fractions exhibited moderate to potent antileishmanial activity with 50% inhibitory concentrations (IC50) ranging from 5.99 ± 0.77 to 2.68 ± 0.42 µg/mL, while compounds displayed IC50 values ranging from 81.73 ± 0.12 to 6.43 ± 0.06 µg/mL. They were weakly active against the chloroquine-sensitive/sulfadoxine-resistant Pf3D7 strain but highly potent toward the multidrug-resistant PfDd2 (extracts, IC50 2.50 ± 0.12 to 4.78 ± 0.36 µg/mL; compounds IC50 2.93 ± 0.02 to 50.97 ± 0.37 µg/mL) with selectivity indices greater than 10 (SIDd2 > 10) for the extract and fractions and most of the derived compounds. Of note, the limonoid mixture [21ß-hydroxylbourjotinolone A (1a) + 21α-melianodiol (1b) + 21ß-melianodiol (1c)] exhibited moderate activity against P. falciparum and L. donovani. This novel antiplasmodial and antileishmanial chemical scaffold qualifies as a promising starting point for further medicinal chemistry-driven development of a dually active agent against two major infectious diseases affecting humans in Africa.

Prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine before and after community delivery of intermittent preventive treatment of malaria in pregnancy in sub-Saharan Africa: a multi-country evaluation.

BACKGROUND: The effectiveness of community delivery of intermittent preventive treatment (C-IPT) of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine has been evaluated in selected areas of the Democratic Republic of the Congo, Madagascar, Mozambique, and Nigeria. We aimed to assess the effect of C-IPTp on the potential development of Plasmodium falciparum resistance to sulfadoxine-pyrimethamine, since it could threaten the effectiveness of this strategy. METHODS: Health facility-based cross-sectional surveys were conducted at baseline and 3 years after C-IPTp implementation in two neighbouring areas per country, one with C-IPTp intervention, and one without, in the four project countries. Dried blood spots from children under five years of age with clinical malaria were collected. Sulfadoxine-pyrimethamine resistance-associated mutations of the P falciparum dhfr (Asn51Ile/Cys59Arg/Ser108Asn/Ile164Leu) and dhps (Ile431Val/Ser436Ala/Ala437Gly/Lys540Glu/Ala581Gly/Ala613Ser) genes were analysed. FINDINGS: 2536 children were recruited between June 19 and Oct 10, 2018, during baseline surveys. Endline surveys were conducted among 2447 children between July 26 and Nov 30, 2021. In the Democratic Republic of the Congo, the dhfr/dhps IRNI/ISGEAA inferred haplotype remained lower than 10%, from 2% (5 of 296) at baseline to 8% (24 of 292) at endline, and from 3% (9 of 300) at baseline to 6% (18 of 309) at endline surveys in intervention and non-intervention areas respectively with no significant difference in the change between the areas. In Mozambique, the prevalence of this haplotype remained stable at over 60% (194 [64%] of 302 at baseline to 194 [64%] of 303 at endline, and 187 [61%] of 306 at baseline to 183 [61%] of 301 in endline surveys, in non-intervention and intervention areas respectively). No isolates harbouring the dhps ISGEAA genotype were found in Nigeria. In Madagascar, only five isolates with this haplotype were found in the non-intervention area (2 [>1%] of 300 at baseline and 3 [1%] of 300 at endline surveys). No isolates were found carrying the dhps ISGEGA genotype. INTERPRETATION: C-IPTp did not increase the prevalence of molecular markers associated with sulfadoxine-pyrimethamine resistance after three years of programme implementation. These findings reinforce C-IPTp as a strategy to optimise the control of malaria during pregnancy, and support the WHO guidelines for prevention of malaria in pregnancy. FUNDING: UNITAID [2017-13-TIPTOP].

Safety and tolerability of repeated doses of dihydroartemisinin-piperaquine for intermittent preventive treatment of malaria in pregnancy: a systematic review and an aggregated data meta-analysis of randomized controlled trials.

BACKGROUND: Malaria infection during pregnancy is an important cause of maternal and infant mortality and morbidity with the greatest effect being concentrated in sub-Saharan Africa. In areas of moderate to high malaria transmission, the World Health Organization (WHO) recommends the administration of intermittent preventive treatment of malaria in pregnancy (IPTp) using sulfadoxine-pyrimethamine (SP) to be given to all pregnant women at each scheduled antenatal care visit at monthly intervals. However, there is concern that increased resistance has compromised its effectiveness. This has led to a need for evaluation of alternatives to SP for IPTp with dihydroartemisinin-piperaquine (DP) emerging as a very promising candidate. Thus, this systematic review and aggregated data meta-analysis was conducted to establish the safety and tolerability of repeated doses with DP in IPTp. METHODS: A systematic review and aggregated data meta-analysis of randomized controlled trials (RCTs) was performed by searching electronic databases of PubMed, Science Direct, ClinicalTrials.gov and Google Scholar. RCTs comparing IPTp DP versus recommended standard treatment for IPTp with these outcome measures were analyzed; change in QTc interval, serious adverse events (SAE), grade 3 or 4 adverse events possibly related to study drug and vomiting within 30 min after study drug administration. The search was performed up to 24th June 2023. Data was extracted from eligible studies and an aggregated data meta-analysis was carried out with data pooled as risk ratio (RR) with a 95% confidence interval (CI), using RevMan software (5.4). This study is registered with PROSPERO, CRD42022310041. RESULTS: Six RCTs involving 7969 participants were included in this systematic review and aggregated data meta-analysis. The pooled analysis showed that DP was associated with a change from baseline of the QTc interval although this change was not associated with cardiotoxicity. There was no statistically significant difference in the risk of occurrence of SAEs among participants in both treatment groups (RR = 0.80, 95% CI [0.52-1.24], P = 0.32). However, significant difference was observed in grade 3 or 4 AEs possibly related to study drug where analysis showed that subjects on IPT DP were statistically significantly more likely to experience an AE possibly related to study drug than subjects on IPT SP (RR = 6.65, 95% CI [1.18-37.54], P = 0.03) and in vomiting within 30 min after study drug administration where analysis showed that the risk of vomiting is statistically significantly higher in subjects receiving IPT DP than in subjects receiving IPT SP (RR = 1.77, 95% CI [1.02-3.07], P = 0.04). CONCLUSION: DP was associated with a higher risk of grade 3 or 4 AEs possibly related to study drug and a higher risk of vomiting within 30 min after study drug administration. However, these were experienced in a very small percentage of women and did not affect adherence to study drugs. DP was also better tolerated in these studies as compared to most alternatives that have been proposed to replace SP which have proved to be too poorly tolerated in IPTp use.

Peripheral and Placental Prevalence of Sulfadoxine-Pyrimethamine Resistance Markers in Plasmodium falciparum among Pregnant Women in Southern Province, Rwanda.

Intermittent preventive therapy during pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended in areas of moderate to high malaria transmission intensity. As a result of the increasing prevalence of SP resistance markers, IPTp-SP was withdrawn from Rwanda in 2008. Nonetheless, more recent findings suggest that SP may improve birthweight even in the face of parasite resistance, through alternative mechanisms that are independent of antimalarial effects. The prevalence of single nucleotide polymorphisms in Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes associated with SP resistance among 148 pregnant women from 2016 to 2018 within Rwanda's Southern Province (Huye and Kamonyi districts) was measured using a ligase detection reaction-fluorescent microsphere assay. The frequency of pfdhps K540E, A581G, and the quintuple (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E) and sextuple (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E + A581G) mutant genotypes was 90%, 38%, 75%, and 28%, respectively. No significant genotype difference was seen between the two districts, which are approximately 50 km apart. Observed agreements for matched peripheral to placental blood were reported and found to be 207 of 208 (99%) for pfdhfr and 239 of 260 (92%) for pfdhps. The peripheral blood sample did not miss any pfdhfr drug-resistant mutants or pfdhps except at the S436 loci. At this level of the sextuple mutant, the antimalarial efficacy of SP for preventing low birthweight is reduced, although overall SP still exerts a nonmalarial benefit during pregnancy. This study further reveals the need to intensify preventive measures to sustain malaria control in Rwanda to keep the overall incidence of malaria during pregnancy low.