Point-of-Care Strategies Applied to Malaria Diagnosis

Rapid and specific diagnosis of malaria remains one of the main strategies to fight the disease. The diagnosis is made primarily by the simple and low-cost thick drop technique, considered the gold standard test. However, the requirement for good quality microscopes and well-trained personnel often lead to inaccurate diagnosis, especially in cases of mixed infections or low parasitemia. Although PCR-based tests can help in these situations, this technique requires large and sensitive equipments, being unsuitable for point of care (POC) settings. A myriad of POC diagnostic tests have being developed in the last years, relying on molecular methods but also on novel strategies. New platforms, miniaturization techniques, and multiplexing possibilities promise great potential to improve disease diagnostics through fast and accurate detection of cases, even at remote places. Here, we will address the main POC strategies developed for the diagnosis of malaria, highlighting their strengths and weakness as POC applications

Distinctive genetic structure and selection patterns in Plasmodium vivax from South Asia and East Africa.

Despite the high burden of Plasmodium vivax malaria in South Asian countries, the genetic diversity of circulating parasite populations is not well described. Determinants of antimalarial drug susceptibility for P. vivax in the region have not been characterised. Our genomic analysis of global P. vivax (n = 558) establishes South Asian isolates (n = 92) as a distinct subpopulation, which shares ancestry with some East African and South East Asian parasites. Signals of positive selection are linked to drug resistance-associated loci including pvkelch10, pvmrp1, pvdhfr and pvdhps, and two loci linked to P. vivax invasion of reticulocytes, pvrbp1a and pvrbp1b. Significant identity-by-descent was found in extended chromosome regions common to P. vivax from India and Ethiopia, including the pvdbp gene associated with Duffy blood group binding. Our investigation provides new understanding of global P. vivax population structure and genomic diversity, and genetic evidence of recent directional selection in this important human pathogen

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 μM) and achieves artemisinin-like kill kinetics in vitro with a parasite clearance time of \u3c24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials

Antimalarial Activity and Mechanisms of Action of Two Novel 4-Aminoquinolines against Chloroquine-Resistant Parasites

Chloroquine (CQ) is a cost effective antimalarial drug with a relatively good safety profile (or therapeutic index). However, CQ is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of CQ-resistant strains, also reported for P. vivax. Despite CQ resistance, novel drug candidates based on the structure of CQ continue to be considered, as in the present work. One CQ analog was synthesized as monoquinoline (MAQ) and compared with a previously synthesized bisquinoline (BAQ), both tested against P. falciparum in vitro and against P. berghei in mice, then evaluated in vitro for their cytotoxicity and ability to inhibit hemozoin formation. Their interactions with residues present in the NADH binding site of P falciparum lactate dehydrogenase were evaluated using docking analysis software. Both compounds were active in the nanomolar range evaluated through the HRPII and hypoxanthine tests. MAQ and BAQ derivatives were not toxic, and both compounds significantly inhibited hemozoin formation, in a dose-dependent manner. MAQ had a higher selectivity index than BAQ and both compounds were weak PfLDH inhibitors, a result previously reported also for CQ. Taken together, the two CQ analogues represent promising molecules which seem to act in a crucial point for the parasite, inhibiting hemozoin formation

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Monitoramento da resistência do Plasmodium falciparum e P. vivax aos antimaláricos em Rondônia e busca de novos esquizonticidas sanguíneos e teciduais para controle e tratamento da doença

Exportado OPUSMade available in DSpace on 2019-08-13T13:43:39Z (GMT). No. of bitstreams: 1 anna_caroline_campos_aguiar.pdf: 18131346 bytes, checksum: e697015028a240875aae52673eafd379 (MD5) Previous issue date: 3A malária humana é a doença parasitária mais importante e um dos maiores problemas de saúde pública do mundo. Seu controle é dificultado pela falta de uma vacina eficaz, problemas no combate ao inseto, mas, sobretudo, pelo surgimento e disseminação de parasitos resistentes aos antimaláricos disponíveis. O tratamento espécie-específico permanece como principal estratégia visando a redução da morbidade e da mortalidade ocasionados pela doença. A busca de novos antimaláricos contra diferentes estágios do parasito torna-se essencial, tema abordado neste trabalho de tese. Na pesquisa de novos esquizonticidas sanguíneos, dois análogos da cloroquina (BAQ e MAQ) foram testados quanto a sua capacidade de inibir ex vivo o desenvolvimento de isolados de P. vivax e P. falciparum, coletados de pacientes diagnosticados em Porto Velho, Rondônia. Ambas as espécies foram sensíveis a MAQ e BAQ, com IC50 na ordem de nanomolar. Essa suscetibilidade ex vivo dos parasitos foi testada em paralelo contra os antimaláricos cloroquina (CQ), mefloquina (MQ) e artesunato (AS). A maioria dos isolados de P. vivax foi sensível à CQ (IC50 médio de 32nM), mas os isolados de P. falciparum apresentaram baixa sensibilidade ex vivo à CQ (IC50 médio de 70nM); todos os isolados foram sensíveis ao AS e à MQ. Polimorfismo de nucleotídeo único foi avaliado nos genes mdr1 e crt, ambos relacionados com a resistência dos parasitos aos antimaláricos. Mutações de troca foram encontradas no gene pfmdr1 nos códons 184 (100%), 1042 (100%) 1246 (100%) e 1034 (84%). Mutações no gene pfcrt, códons 72 e 76, foram encontradas em 100% dos isolados. No entando, os isolados de vivax não apresentaram mutações. Na busca de novos esquizonticidas teciduais, 15 tiazolidinonas derivadas da primaquina (PQTZs) foram avaliadas contra formas exoeritrocíticas (FEE) de P. berghei-ANKA in vitro inibindo o desenvolvimento dos esporozoítos nos hepatócitos, com redução significantemente na porcentagem de células infectadas e tamanho dos esquizontes. No entanto, somente o composto 4m foi ativo in vivo, reduzindo 95% o desenvolvimento hepático das FEE dos animais tratados e aumentando o período pré-patente da malária em 3 dias. Os PQTZs foram avaliados quanto à capacidade de bloquear a transmissão, nos modelos da malária aviária (P. gallinaceum e Aedes fluviatilis) e de roedores (Anopheles stephensi e P. berghei), após tratamento dos hospedeiros vertebrados com os compostos. Os compostos 4b, 4c, 4g, 4m, 4o, assim como a primaquina, inibiram 100% a infecção dos mosquitos anofelinos. O PQTZ 4m apresentou a melhor atividade contra FFE in vitro e in vivo e bloqueou 100% a transmissão, portanto, este composto deverá ser avaliado em modelos com malária pelo P. cynomolgi e/ou P. vivax, visando o desenvolvimento de um novo medicamento para o tratamento das recaídas da malária humana.Human malaria is the most important parasitic disease and a major public health problem. Its control remains difficult due to the lack of an effective vaccine, difficulties in the vector control and, especially, the emergency and spread of parasites resistant to most available antimalarial drugs. The specific drug treatment is a major strategy to reduce morbidity and mortality owed to malaria. The research for new antimalarials against different parasitic stages is essential and was studied in the present work. Two chloroquine analogs (BAQ and MAQ) were evaluated ex vivo against P. vivax e P. falciparum isolates from patients diagnosed in Porto Velho, Rondônia. The isolates were sensitive to MAQ and BAQ, presenting IC50 at nanomolar dose. The ex vivo sensitivity of those isolates was tested, in parallel, against the antimalarials chloroquine (CQ), mefloquine (MQ) and artesunate (AS). Most of P. vivax isolates were sensitive to CQ (IC50 of 32nM) whereas the P. falciparum isolates exhibited low sensitivity to CQ (IC50 of 70nM); all isolates were sensitive to AS and MQ. Single Nucleotide Polymorphisms were evaluated at mdr1 and crt genes, both related with antimalarial parasite resistance. Mutations were found in pfmdr1 gene at codons 184 (100%), 1042 (100%) and 1246 (100%), 1034 (84%). For the pfcrt gene, mutations were observed at codons 72 and 76 in all P. falciparum isolates. The P. vivax isolates did not present mutations. As tissue schizonticidals, fifteen primaquine derivatives (PQTZs) were evaluated against the exoritrocitic forms (EEF) of P. berghei-ANKA in vitro. The compounds inhibited the development of sporozoites into the hepatocytes, and reduced the number and the size of EEF in vitro. Only 4m was active in vivo reducing 95% the development of EEF and increasing the malaria pre-patent period in 3 days. The PQTZs were evaluated for their ability to block the malaria transmission in avian malaria (P. gallinaceum in chickens and Aedes fluviatilis) and rodent malaria (P. berghei in mice and Anopheles stephensi) using the vertebrate host treated with the compounds. The compounds 4b, 4c, 4g, 4m and 4o inhibited 100% infection of Anopheles mosquitoes by P. berghei, like primaquine, tested in parallel. The PQTZs 4m showed the best activity against EEF in vitro and in vivo and exhibited the best blocking transmission activity. This compound has yet to be evaluated against late EEF in models using P. cynomolgi malaria and/or P. vivax, aiming to develop a new drug to treat human malaria relapses

Avaliação da atividade citotóxica e antimalárica de análogos da cloroquina

Exportado OPUSMade available in DSpace on 2019-08-09T12:28:18Z (GMT). No. of bitstreams: 1 19072011_disserta__o.pdf: 858276 bytes, checksum: 2057fb3b59f670e6fb2ceea98de82969 (MD5) Previous issue date: 19A malária humana, segundo a Organização Mundial da Saúde, é a doença parasitária mais importante e um dos maiores problemas de saúde pública do mundo. Seu controle é dificultado devido à resistência dos mosquitos aos inseticidas, à falta de uma vacina eficaz e, sobretudo, ao surgimento e disseminação de parasitos resistentes à maior parte dos fármacos disponíveis. O tratamento medicamentoso específico da malária permanece como principal estratégia na redução da morbidade e da mortalidade atribuídas à doença. No presente estudo, análogos da cloroquina e seus derivados metálicos foram avaliados, considerando que associar moléculas a metais parece umaestratégia promissora na busca de novos antimaláricos. Sete novos análogos da cloroquina foram avaliados quanto à: (i) sua atividade in vitro contra P. falciparum; (ii) sua toxicidade em duas linhagens celulares (hepatoma humano e célula renal de macaco) e contra hemácias humanas; (iii) sua atividade in vivo contra P. berghei; (iv) sua interação com a hematina dimérica (hemozoína sintética) in vitro e; (v) suainteração com a hematina dimérica no modelo molecular de docking. Todos os análogos da cloroquina apresentaram intensa atividade in vitro anti- P. falciparum (IC50 entre 0,04 e 0,55nM) nos ensaios de HRPII e de hipoxantina. Alguns dos análogos foram mais ativos que a cloroquina e sem efeito citotóxico nas duas linhagens celulares avaliadas. Além disso, os compostos estudados não causaram hemólise de hemáciashumanas normais. Os análogos da cloroquina atuaram inibindo a polimerização do heme, assim como atua a cloroquina. Dois dos compostos testados in vivo contra P. berghei foram também ativos causando intensa redução da parasitemia. Em conclusão, os análogos da cloroquina são moléculas promissoras, com atividade antimalárica eatuam em um alvo crucial para a sobrevivência do parasito, através da inibição da formação de hemozoínaHuman malaria, according to World Health Organization (WHO), remains the most important parasitic disease and a major public health problem. Their control remains difficult due to resistance of mosquitoes to insecticides, the lack of an effective vaccine, and, especially, the emergency and spread of resistant parasites to most available antimalarial drugs. The specific drug treatment remains a major strategy to reduce morbidity and mortality due to malaria. In this study, chloroquine-analogues in complex with metals were evaluated considering that such association to be believed to represent a promising strategy in the search for new drugs. Seven new analogues were evaluated: (i) in vitro for their activity against P. falciparum; (ii) for their toxicity against human erythrocytes and two cell lines, HepG2 (hepatoma) and BGM (a monkey basal kidney cells); (iii) in vivo against P. berghei; (iv) for possible interactions between the analogous and dimeric hematin in vitro; and, (v) through a molecular docking model, targeting the dimeric hematin. All chloroquine analogs showed intense in vitro anti-Pfalciparum activity (IC50 between 0.04 and 0.55 nM) as measured in animmunoenzimatic test using monoclonals to a parasite histidine rich protein (HRPII) and through radiotopic hypoxanthine incorporation. Some of these analogues were more active than chloroquine and none of them were cytotoxic to HepG2 and BGM cells or caused haemolysis to normal human erythrocytes. Like chloroquine, the chloroquine analogs inhibited heme polymerization in the in vitro as well as in the docking test. Inaddition, two of the compounds tested against P. berghei in experimentally infected mice caused a significant reduction of parasitemia. Taken together, the analogues evaluated in this study represent promising molecules and act on a crucial point for theparasite, by inhibition of hemozoine formatio

In vitro assessment for cytotoxicity screening of new antimalarial candidates

In antimalarial research there are no standard procedures to determine the toxicity of a drug candidate. Among the alternatives available,&nbsp;in vitro&nbsp;cytotoxicity assays are the most widely used to predict toxic effects of future therapeutic products. They have the advantage over the&nbsp;in vivo&nbsp;assays, in that they offer the possibility to restrain the number of experimental variables. The objective of the present study was to compare&nbsp;in vitro&nbsp;cytotoxic methods by testing various compounds currently used to treat malaria against different cell lines. Neutral red (NR) uptake and methylthiazoletetrazolium (MTT) colorimetric&nbsp;in vitro&nbsp;assays were used to determine preliminary toxicity of commercially available antimalarial drugs against tumor and non-tumor cells lines. Toxicity through brine shrimp lethality bioassay and hemolytic activity were also evaluated. Significant differences were observed in the tests measured by NR uptake. The tumor cell lines TOV-21G and HepG2 and non-tumor WI-26VA4 cells showed relatively uniform toxicity results, with TOV-21G being the most sensitive cell tested, presenting the lowest concentration to cause death to 50% of viable cells (CC50) values. The results of this study support the use of TOV-21G, HepG2 and WI-26VA4 cells lines as the choice for cytotoxicity tests to evaluate potential bioactive compounds