An adaptable, fit-for-purpose screening approach with high-throughput capability to determine speed of action and stage specificity of anti-malarial compounds.

A revamped in vitro compound identification and activity profiling approach is required to meet the large unmet need for new anti-malarial drugs to combat parasite drug resistance. Although compound hit identification utilizing high-throughput screening of large compound libraries is well established, the ability to rapidly prioritize such large numbers for further development is limited. Determining the speed of action of anti-malarial drug candidates is a vital component of malaria drug discovery, which currently occurs predominantly in lead optimization and development. This is due in part to the capacity of current methods which have low throughput due to the complexity and labor intensity of the approaches. Here, we provide an adaptable screening paradigm utilizing automated high content imaging, including the development of an automated schizont maturation assay, which collectively can identify anti-malarial compounds, classify activity into fast and slow acting, and provide an indication of the parasite stage specificity, with high-throughput capability. By frontloading these critical biological parameters much earlier in the drug discovery pipeline, it has the potential to reduce lead compound attrition rates later in the development process. The capability of the approach in its alternative formats is demonstrated using three Medicines for Malaria Venture open access compound "boxes," namely Pathogen Box (malaria set-125 compounds), Global Health Priority Box [Malaria Box 2 (80 compounds) and zoonotic neglected diseases (80 compounds)], and the Pandemic Response Box (400 compounds). From a total of 685 compounds tested, 79 were identified as having fast ring-stage-specific activity comparable to that of artemisinin and therefore of high priority for further consideration and development.

A qualitative study on determinants of the use of malaria rapid diagnostic test and anti-malarial drug prescription practices by primary healthcare workers in Ebonyi state, Nigeria.

BACKGROUND: The increased availability and use of malaria rapid diagnostic test (RDT) by primary healthcare (PHC) workers has made universal diagnostic testing before malaria treatment more feasible. However, to meaningfully resolve the problem of over-treatment with artemisinin-based combination therapy and the heightened risk of selection pressure and drug resistance, there should be appropriate response (non-prescription of anti-malarial drugs) following a negative RDT result by PHC workers. This study explored the determinants of the use of RDT and anti-malarial drug prescription practices by PHC workers in Ebonyi state, Nigeria. METHODS: Between March 2 and 10, 2020, three focus group discussions were conducted in English with 23 purposively-selected consenting PHC workers involved in the diagnosis and treatment of malaria. Data was analysed thematically as informed by the method by Braun and Clarke. RESULTS: The determinants of the use of RDT for malaria diagnosis were systemic (RDT availability and patient load), provider related (confidence in RDT and the desire to make correct diagnosis, PHC worker's knowledge and training, and fear to prick a patient), client related (fear of needle prick and refusal to receive RDT, and self-diagnosis of malaria, based on symptoms, and insistence on not receiving RDT), and RDT-related (the ease of conducting and interpreting RDT). The determinants of anti-malarial drug prescription practices were systemic (drug availability and cost) and drug related (effectiveness and side-effects of the drugs). The determinants of the prescription of anti-malarial drugs following negative RDT were provider related (the desire to make more money and limited confidence in RDT) and clients' demand while unnecessary co-prescription of antibiotics with anti-malarial drugs following positive RDT was determined by the desire to make more money. CONCLUSIONS: This evidence highlights many systemic, provider, client, and RDT/drug related determinants of PHC workers' use of RDT and anti-malarial drug prescription practices that should provide tailored guidance for relevant health policy actions in Ebonyi state, Nigeria, and similar settings.

Receipt of antimalarials among children aged 6-59 months in Nigeria from 2010 to 2021.

BACKGROUND: Nigeria has the highest malaria burden globally, and anti-malarials have been commonly used to treat malaria without parasitological confirmation. In 2012, Nigeria implemented rapid diagnostic tests (RDTs) to reduce the use of anti-malarials for those without malaria and to increase the use of artemisinin-based combination therapy (ACT) for malaria treatment. This study examined changes in anti-malarial receipt among children aged 6-59 months during a 12-year period of increasing RDT availability. METHODS: A cross-sectional analysis was conducted using the Nigeria Malaria Indicator Survey (NMIS) data from 2010 (before RDT implementation in 2012), 2015, and 2021. The analysis assessed trends in prevalence of malaria by survey RDT result, and fever and anti-malarial/ACT receipt in the 2 weeks prior to the survey. A multivariable logistic regression was used to account for the complex survey design and to examine factors associated with anti-malarial receipt, stratified by survey RDT result, a proxy for recent/current malaria infection. RESULTS: In a nationally-representative, weighted sample of 22,802 children aged 6-59 months, fever prevalence remained stable over time, while confirmed malaria prevalence decreased from 51.2% in 2010 to 44.3% in 2015 and 38.5% in 2021 (trend test p < 0.0001). Anti-malarial use among these children decreased from 19% in 2010 to 10% in 2021 (trend test p < 0.0001), accompanied by an increase in ACT use (2% in 2010 to 8% in 2021; trend test p < 0.0001). Overall, among children who had experienced fever, 30.6% of survey RDT-positive and 36.1% of survey RDT-negative children had received anti-malarials. The proportion of anti-malarials obtained from the private sector increased from 61.8% in 2010 to 80.1% in 2021 for RDT-positive children; most of the anti-malarials received in 2021 were artemisinin-based combinations. Factors associated with anti-malarial receipt for both RDT-positive and RDT-negative children included geographic region, greater household wealth, higher maternal education, and older children. CONCLUSION: From 2010 to 2021 in Nigeria, both malaria prevalence and anti-malarial treatments among children aged 6-59 months decreased, as RDT availability increased. Among children who had fever in the prior 2 weeks, anti-malarial receipt was similar between children with either positive or negative survey RDT results, indicative of persistent challenges in reducing inappropriate anti-malarials uptake.

Safety and efficacy of pyronaridine-artesunate paediatric granules in the treatment of uncomplicated malaria in children: insights from randomized clinical trials and a real-world study.

BACKGROUND: Children are particularly at risk of malaria. This analysis consolidates the clinical data for pyronaridine-artesunate (PA) paediatric granules in children from three randomized clinical trials and a real-world study (CANTAM). METHODS: An integrated safety analysis of individual patient data from three randomized clinical trials included patients with microscopically-confirmed Plasmodium falciparum, body weight ≥ 5 kg to < 20 kg, who received at least one dose of study drug (paediatric safety population). PA was administered once daily for 3 days; two trials included the comparator artemether-lumefantrine (AL). PCR-adjusted day 28 adequate clinical and parasitological response (ACPR) was evaluated. Real-world PA granules safety and effectiveness was also considered. RESULTS: In the integrated safety analysis, 63.9% (95% CI 60.2, 67.4; 426/667) of patients had adverse events following PA and 62.0% (95% CI 56.9, 66.9; 222/358) with AL. Vomiting was more common with PA (7.8% [95% CI 6.0, 10.1; 52/667]) than AL (3.4% [95% CI 1.9, 5.8; 12/358]), relative risk 2.3 (95% CI 1.3, 4.3; P = 0.004), occurring mainly following the first PA dose (6.7%, 45/667), without affecting re-dosing or adherence. Prolonged QT interval occurred less frequently with PA (3.1% [95% CI 2.1, 4.8; 21/667]) than AL (8.1% [95% CI 5.7, 11.4; 29/358]), relative risk 0.39 (95% CI 0.22, 0.67; P = 0.0007). In CANTAM, adverse events were reported for 17.7% (95% CI 16.3, 19.2; 460/2599) of patients, most commonly vomiting (5.4% [95% CI 4.6, 6.4; 141/2599]), mainly following the first dose, (4.5% [117/2599]), with all patients successfully re-dosed, and pyrexia (5.4% [95% CI 4.6, 6.3; 140/2599]). In the two comparative clinical trials, Day 28 ACPR in the per-protocol population for PA was 97.1% (95% CI 94.6, 98.6; 329/339) and 100% (95% CI 99.3, 100; 514/514) versus 98.8% (95% CI 95.7, 99.9; 165/167) and 98.4% (95% CI 95.5, 99.7; 188/191) for AL, respectively. In CANTAM, PA clinical effectiveness was 98.0% (95% CI 97.3, 98.5; 2273/2320). CONCLUSIONS: Anti-malarial treatment with PA paediatric granules administered once daily for 3 days was well tolerated in children and displayed good clinical efficacy in clinical trials, with effectiveness confirmed in a real-world study. Trial registration Clinicaltrials.gov: SP-C-003-05: identifier NCT00331136; SP-C-007-07: identifier NCT0541385; SP-C-021-15: identifier NCT03201770. Pan African Clinical Trials Registry: SP-C-013-11: identifier PACTR201105000286876.

In vitro delayed response to dihydroartemisinin of malaria parasites infecting sickle cell erythocytes.

BACKGROUND: Decreased efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria has been previously reported in patients with sickle cell disease (SCD). The main purpose of this study was to investigate the in vitro susceptibility of isolates to dihydro-artemisinin (DHA) to provide a hypothesis to explain this treatment failure. METHODS: Isolates were collected from patients attending health centres in Abidjan with uncomplicated P. falciparum malaria. The haemoglobin type has been identified and in vitro drug sensitivity tests were conducted with the ring stage assay and maturation inhibition assay. RESULTS: 134 isolates were obtained. Parasitaemia and haemoglobin levels at inclusion were lower in patients with haemoglobin HbSS and HbSC than in patients with normal HbAA. After ex vivo RSA and drug inhibition assays, the lowest rate of parasitic growth was found with isolates from HbAS red cells. Conversely, a significantly higher survival rate of parasites ranging from 15 to 34% were observed in isolates from HbSS. Isolates with in vitro reduced DHA sensitivity correlate with lower RBC count and haematocrit and higher parasitaemia at inclusion compared to those with isolates with normal DHA sensitivity. However, this decrease of in vitro sensitivity to DHA was not associated with Kelch 13-Propeller gene polymorphism. CONCLUSION: This study highlights an in vitro decreased sensitivity to DHA, for isolates collected from HbSS patients, not related to the Pfkelch13 gene mutations. These results are in line with recent studies pointing out the role of the redox context in the efficacy of the drug. Indeed, SCD red cells harbour a highly different ionic and redox context in comparison with normal red cells. This study offers new insights into the understanding of artemisinin selective pressure on the malaria parasite in the context of haemoglobinopathies in Africa.

The isolation of sclerotinin A as an anti-malarial compound by utilization of a global secondary metabolism regulator, laeA gene.

Previously, we successfully introduced laeA gene into a fungal strain in order to significantly increase the production of a bioactive compound, allowing use to discover novel biological activity. To demonstrate the universal applicability of the laeA gene introduction strategy for taping the potential of fungal secondary metabolism, in this present study, we created a library of microorganisms which we had the laeA gene inserted, and from that library we aimed to isolate compounds which are produced at significantly greater quantities compared to the respective wild type strains. From this investigation, we were able to isolate sclerotinin A (1) from Pochonia sp. KTF-0504 strain. We revealed that 1 showed anti-malarial activity against Plasmodium falciparum parasite strains. On the other hands, 1 showed no anti-fungal activity against multidrug-sensitive budding yeast. Our study implies that the utilization of the laeA gene in fungi is a versatile method for the discovery of drug candidates.

Novel saturated 1,2,4-trioxanes and their antimalarial activity against multidrug resistant Plasmodium yoelii nigeriensis in Swiss mice model via oral route.

Novel saturated 6-(4'-aryloxy phenyl) vinyl 1,2,4-trioxanes 12a(1-3)-12d(1-3) and 13a(1-3)-13d(1-3) have been designed and synthesized, in one single step from diimide reduction of 11a(1-3)-11d(1-3). All the newly synthesized trioxanes were evaluated for their antimalarial activity against multi-drug resistant Plasmodium yoelii nigeriensis via oral route. Cyclopentane-based trioxanes 12b1, 12c1 and 12d1, provided 100 % protection to the infected mice at 24 mg/kg × 4 days. The most active compound of the series, trioxane 12b1, provided 100 % protection even at 12 mg/kg × 4 days and 60 % protection at 6 mg/kg × 4 days. The currently used drug, ß-arteether provides only 20 % protection at 24 mg/kg × 4 days.

ANTI-MALARIAL ACTIVITY OF AMENTOFLAVONE ISOLATED FROM LEAF OF CALOPHYLLUM TOMENTOSUM WEIGHT.

Calophyllum tomentosum belonging to Clusiaceae family is an Indian medicinal plant used as folklore medicine to cure various kinds of diseases reported in Ayurveda, and the leaves of the plant are also used as an active ingredient for the preparation of a botanical medicine known as 'Punnaga', 'Surapunnaga' and 'Tamoil' among other common names. Chemical profiling of the methanol extract of the defatted leaf revealed the presence of amentoflavone as one of the constituents along with coumarins, terpenoids, steroids, and apetalic acids. Structural determination of these amentoflavone has been conducted by chemical, spectral, and spectrometric methods in comparison with spectral values available in the literature and confirmed by a single crystal X-ray diffraction study. Amentoflavone (1) and its derivative (2-5) tested to check the efficacy of anti-malarial activity against Plasmodium falciparum. Amongst them, only tetra methoxy amentoflavone, (2) exhibited moderate anti-malarial activity with IC50 value 1.99 ± 0.42 µM against Plasmodium falciparum in comparison with artemisinin as control, whereas the other products possessed almost negligible activity although their structural skeletons are identical with little variation of number and nature of substituents. The structure activity relationship (SAR) of the active constituent and its derivatives is reported herein.

Anti-Malarial and Multi-Bioactive Co (II), Cu (II) and Ni (II) Salen Complexes: Synthesis, Characterization and Computational Studies.

Herein, we report the anti-malarial, anti-bacterial and anti-inflammatory activities of the N2O2 donor tetradentate salen type ligand and its CoL, NiL, and CuL metal complexes. The synthesized compounds were characterized by various spectroscopic analytical methods. The in-vitro anti-malarial investigations revealed that the complex CuL exhibited equipotency with quinine drug having IC50 value 0.25 µg/mL. The compound L showed significant inhibition of bacterial spp. viz. E. Coli, P. Aeruginosa, and S. Aureus (MIC=12.5-50 µg/mL), while the compound CoL (MIC=12.5 µg/mL) exhibited potency against gram-positive bacteria. In the in-vitro anti-inflammatory study, the compound CuL displayed moderate activity than other tested compounds. The compound CuL showed the highest anti-malarial docking score with enzyme pLDH at -8.12 Kcal/mol. The DFT study also gives authentication of higher antimalarial activity of CuL due to high dipole moment. None of the potent compounds was found cytotoxic towards vero cell lines.

Anti-malarial drug use, appropriateness and associated factors among children under-five with febrile illnesses presenting to a tertiary health facility: a cross sectional study.

BACKGROUND: Malaria is endemic in 95% of Uganda and constitutes the country's most significant public health problem-being the leading cause of morbidity and mortality, especially among children under five years of age. The current national malaria treatment policy is to use artemisinin-based combination therapy (ACT) as first-line treatment, and recommends parasitological confirmation of malaria before therapy. Adherence to this policy, however, remains suboptimal, with the self-initiated home-based therapy being common-posing undue exposures to, and pressure on the current artemisinin-based combinations, with the danger of emergence of drug resistance. The study evaluated the anti-malarial use and its appropriateness among febrile children under five presenting to a tertiary health facility in northern Uganda in light of the current malaria treatment policy. METHODS: This was a cross-sectional study in a tertiary health facility in northern Uganda between March and September 2021. Children aged 6-59 months with fever were selected using systematic random sampling. A pretested interviewer-administered questionnaire was used to collect clinical data from the caregivers. Data were analysed using SPSS version 23. Descriptive statistics and multiple logistic regression models were applied. P-value < 0.05 was considered for statistical significance. RESULTS: Seventy-two (34.3%) of the 210 children with fever in this study used anti-malarials prior to the hospital visit, 29.2% (21/72) of which were on a self-medication basis, 22.2% (16/72) were empiric prescriptions-all of which inappropriate, and only 48.6% (35/72) were prescribed based on a parasitological diagnosis of malaria. The most commonly used anti-malarials were artemether-lumefantrine 60/72 (88.3%), while a lesser proportion of quinine 7/72 (9.7%), artesunate 3/72 (4.2%) and dihydroartemisinin-piperaquine 2/72 (2.8%) were used. The factors independently associated with anti-malarial use among the children with febrile illnesses were duration of fever (p = 0.001); level of the nearest facility (p = 0.027), distance from the nearest health facility (p = 0.025), and caregivers' age (p = 0.038). CONCLUSIONS: Inappropriate use of anti-malarials for childhood febrile illnesses is prevalent in the study setting, facilitated by the ease of over-the-counter access, empiric prescription and use of leftover anti-malarials. This calls for a need to address communities' health-seeking behaviour and the health providers' practice alike.

Health workers' adherence to the malaria test, treat and track strategy during the COVID-19 pandemic in malaria high transmission area in Eastern Uganda.

BACKGROUND: Coronavirus disease 2019 (COVID-19) pandemic affected malaria control activities in sub-Saharan Africa (SSA) resulting in 690,000 excess deaths in the year 2021. The authors hypothesized that COVID-19 affected the World Health Organization (WHO) Test, Treat and Track (T3) strategy that has been implemented in Uganda since 2010. In this study, health worker's adherence to the T3 strategy during COVID-19 pandemic in Eastern Uganda was studied by assessing their knowledge, skills and practices. METHODS: A cross-sectional study utilizing mixed quantitative and qualitative data collections methods was conducted at Mbale Regional Referral Hospital in Eastern Uganda between November and December in 2020. Data were captured on demographics, knowledge, skills and practices for both health workers (HWs) and patients. Quantitative data were analysed using STATA 15.0 and reported as descriptive statistics, proportions and statistical associations. Moreover, qualitative data were collected via key informant interviews (KII) among purposively sampled study participants and analysed thematically using NVIVO software. Ethical approval was obtained prior to the study. RESULTS: A total of 436 study participants, of whom 103/436 (24%) and 333/436 (76%) were HWs and patients, respectively were studied. Among the HWs with mean age of 34 years (SD = 8.8 years), 81/103 (79%) had good practices, most 63/103 (61%) had good knowledge, and only 11/103 (10.7%) had good skills. Specifically, on the cadres, the laboratory personnel 19/103 (18%) had good knowledge 14/19 (74%) OR: 2.0 (95% CI 0.7-6) and were highly skilled OR: 4.6 (95% CI 1.2-18.1; P < 0.0150) compared to other cadres, respectively. Among the patients whose age ranged 3 months to 80 years (mean 17.8 years) and females 177/333 (53%); a majority 257/333 (77%) were tested, of whom 139/333 (42%) tested positive. Out of the positive cases, 115/333 (35%) were treated and tracked. About 75/333 (23%) were not tested but treated for malaria. Of the 168/239 (70.3%) patients tested, 115/168 (68.5%) were positive and treated, P = 0.0001. The KII revealed low level of In-service training, overwhelming number of patients and stock-out of supplies as a key factor for poor HW adherence to T3 strategy. CONCLUSIONS: During COVID-19 pandemic period HWs adherence to T3 initiative was low as 27% malaria patients did not receive treatment.

Rebound of multiple infections and prevalence of anti-malarial resistance associated markers following malaria upsurges in Dielmo village, Senegal, West Africa.

BACKGROUND: Thanks to the scale up of malaria control interventions, the malaria burden in Senegal has decreased substantially to the point that the National Malaria Control Programme plans to achieve malaria elimination by 2030. To guide such efforts, measuring and monitoring parasite population evolution and anti-malarial drugs resistance is extremely important. Information on the prevalence of parasite mutations related to drug resistance can provide a first signal of emergence, introduction and selection that can help with refining drug interventions. The aim of this study was to analyse the prevalence of anti-malarial drug resistance-associated markers before and after the implementation of artemisinin-based combination therapy (ACT) from 2005 to 2014 in Dielmo, a model site for malaria intervention studies in Senegal. METHODS: Samples from both malaria patients and Plasmodium falciparum asymptomatic carriers were analysed with high resolution melting (HRM) technique to genotype P. falciparum chloroquine resistance transporter (Pfcrt) gene haplotypes and multidrug-resistant protein 1 (Pfmdr1) gene at codons N86 and Y184. RESULTS: Among the 539 samples analysed, 474, 486, and 511 were successfully genotyped for Pfmdr1 N86, Y184, and Pfcrt, respectively. The prevalence of drug resistance markers was high, particularly during the malaria upsurges. Following the scale-up in bed net distribution, only the mutant (86F-like) variant of Pfmdr1 86 was present while during the malaria upsurges the predominance of two types 86Y-86N (43%) and 86F-like (56%) were observed. Most infections (87%) carried the wild type Y-allele at Pfmdr1 184 during the period of nets scale-up while during the malaria upsurges only 16% of infections had wild type and 79% of infections had mixed (mutant/wild) type. The frequency of the mixed genotypes SVMNT-like_CVMNK and SVMNT-like_CVIET within Pfcrt gene was particularly low during bednet scale up. Their frequency increased significantly (P < 0.001) during the malaria upsurges. CONCLUSION: This data demonstrated the effect of multiple interventions on the dynamics of drug resistance-associated mutations in the main malaria parasite P. falciparum in an endemic village in Senegal. Monitoring drug resistance markers should be conducted periodically to detect threats of emergence or resurgence that could compromise the efficacy of anti-malarial drugs.

Drug rechanneling: A novel paradigm for cancer treatment.

Cancer continues to be one of the leading contributors towards global disease burden. According to NIH, cancer incidence rate per year will increase to 23.6 million by 2030. Even though cancer continues to be a major proportion of the disease burden worldwide, it has the lowest clinical trial success rate amongst other diseases. Hence, there is an unmet need for novel, affordable and effective anti-neoplastic medications. As a result, a growing interest has sparkled amongst researchers towards drug repurposing. Drug repurposing follows the principle of polypharmacology, which states, "any drug with multiple targets or off targets can present several modes of action". Drug repurposing also known as drug rechanneling, or drug repositioning is an economic and reliable approach that identifies new disease treatment of already approved drugs. Repurposing guarantees expedited access of drugs to the patients as these drugs are already FDA approved and their safety and toxicity profile is completely established. Epidemiological studies have identified the decreased occurrence of oncological or non-oncological conditions in patients undergoing treatment with FDA approved drugs. Data from multiple experimental studies and clinical observations have depicted that several non-neoplastic drugs have potential anticancer activity. In this review, we have summarized the potential anti-cancer effects of anti-psychotic, anti-malarial, anti-viral and anti-emetic drugs with a brief overview on their mechanism and pathways in different cancer types. This review highlights promising evidences for the repurposing of drugs in oncology.

High prevalence of Pfcrt 76T and Pfmdr1 N86 genotypes in malaria infected patients attending health facilities in East Shewa zone, Oromia Regional State, Ethiopia.

BACKGROUND: Plasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine-pyrimethamine (SP) and subsequently in 2004 from SP to artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia. METHODS: Finger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively. RESULTS: Of 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86). CONCLUSION: Although CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria.

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019.

BACKGROUND: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine-pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. METHODS: From July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. RESULTS: Out of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72-76 SVMNT haplotype associated with resistance to amodiaquine was not detected. CONCLUSION: The study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ-resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region.

A review on synthetic account of 1,2,4-oxadiazoles as anti-infective agents.

Most of the currently marketed drugs consist of heterocyclic scaffolds containing nitrogen and or oxygen as heteroatoms in their structures. Several research groups have synthesized diversely substituted 1,2,4-oxadiazoles as anti-infective agents having anti-bacterial, anti-viral, anti-leishmanial, etc. activities. For the first time, the present review article will provide the coverage of synthetic account of 1,2,4-oxadiazoles as anti-infective agents along with their potential for SAR, activity potential, promising target for mode of action. The efforts have been made to provide the chemical intuitions to the reader to design new chemical entity with potential of anti-infective activity. This review will mark the impact as the valuable, comprehensive and pioneered work along with the library of synthetic strategies for the organic and medicinal chemists for further refinement of 1,2,4-oxadiazole as anti-infective agents.

Identification of first-in-class plasmodium OTU inhibitors with potent anti-malarial activity.

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

Is there evidence of anti-malarial multidrug resistance in Burkina Faso?

Recently, Gansané and colleagues published an article on inadequate efficacy of two different forms of artemisinin-based combination therapy (ACT) in Burkina Faso. The development of Plasmodium falciparum resistance to different ACT partner drugs at levels that could affect the efficacy of two ACT would both be startling and a cause for great concern. In reviewing the available data collected since 2008 on ACT efficacy in Burkina Faso, the analysis shows that the reported efficacy of the tested ACT varies greatly. Most of the studies have considerable methodological deviations and challenges, especially in PCR correction done to distinguish between recrudescence and re-infection, and in the failure to omit re-infections in the calculation of efficacy rates. So far, there is no convincing evidence in the articles reviewed that multidrug resistance has emerged in Burkina Faso. However, the potential consequence of failing ACT means that the results published by Gansané et al. urgently need to be confirmed. Furthermore, articles reporting on efficacy data need to include an examination of the potential consequences of any methodological deviations.

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018-2019.

BACKGROUND: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. METHODS: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. RESULTS: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed. CONCLUSION: No molecular markers currently known to be associated with resistance to the first-line ACT in use were detected in isolates of P. falciparum from treatment failure patients. Regular monitoring through in vivo drug efficacy and molecular studies must continue to ensure the effectiveness of malaria treatment in Democratic Republic of Congo.

Analysing the essential proteins set of Plasmodium falciparum PF3D7 for novel drug targets identification against malaria.

BACKGROUND: Plasmodium falciparum is an obligate intracellular parasite of humans that causes malaria. Falciparum malaria is a major public health threat to human life responsible for high mortality. Currently, the risk of multi-drug resistance of P. falciparum is rapidly increasing. There is a need to address new anti-malarial therapeutics strategies to combat the drug-resistance threat. METHODS: The P. falciparum essential proteins were retrieved from the recently published studies. These proteins were initially scanned against human host and its gut microbiome proteome sets by comparative proteomics analyses. The human host non-homologs essential proteins of P. falciparum were additionally analysed for druggability potential via in silico methods to possibly identify novel therapeutic targets. Finally, the PfAp4AH target was prioritized for pharmacophore modelling based virtual screening and molecular docking analyses to identify potent inhibitors from drug-like compounds databases. RESULTS: The analyses identified six P. falciparum essential and human host non-homolog proteins that follow the key druggability features. These druggable targets have not been catalogued so far in the Drugbank repository. These prioritized proteins seem novel and promising drug targets against P. falciparum due to their key protein-protein interactions features in pathogen-specific biological pathways and to hold appropriate drug-like molecule binding pockets. The pharmacophore features based virtual screening of Pharmit resource predicted a lead compound i.e. MolPort-045-917-542 as a promising inhibitor of PfAp4AH among prioritized targets. CONCLUSION: The prioritized protein targets may worthy to test in malarial drug discovery programme to overcome the anti-malarial resistance issues. The in-vitro and in-vivo studies might be promising for additional validation of these prioritized lists of drug targets against malaria.