Evaluation of prevalence's of pfdhfr and pfdhps mutations in Angola

<p>Abstract</p> <p>Background</p> <p>Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to <it>Plasmodium falciparum </it>infection are pregnant women and children under five years of age. The use of an intermittent preventive treatment (IPT) with sulphadoxine/pyrimethamine (SP) in pregnant women was introduced in Angola in 2006 by the National Malaria Control Programme, and currently this strategy has been considered to be used for children malaria control. Considering the previous wide use of SP combination in Angola, together to the reported cases of SP treatment failure it is crucial the evaluation of the prevalence of five mutations in <it>pfdhfr </it>and <it>pfdhps </it>genes associated to <it>P</it>. <it>falciparum </it>resistance to SP before the introduction of S/P IPT in children.</p> <p>Methods</p> <p>The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in <it>pfdhfr </it>and <it>pfdhps </it>genes was carried out in 452 <it>P</it>. <it>falciparum </it>blood samples by PCR RFLP.</p> <p>Results</p> <p>For <it>pfdhfr </it>gene, 90,3% of the samples carried the mutation 51<b>I</b>, with 7.5% of mixed infections; 51% carried wild type allele 59<b>C</b>, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 108<b>N</b>. Concerning, <it>pfdhps </it>gene, 83,1% were mutant type 437<b>G </b>with 11% mixed infections , while 87% of the studied isolates were wild type for codon 540.</p> <p>Discussion</p> <p>This is the first representative epidemiological study of the whole Angola country on the prevalence of the genotypes associated with SP chemoresistance. A high frequency of individual mutations in both genes (51<b>I </b>and 108<b>N </b>in <it>pfdhfr</it>, and 437<b>G </b>in <it>pfdhps</it>) was found, besides a low prevalence of the quintuple mutation.</p> <p>Conclusion</p> <p>The data showed that the implementation IPT using SP in children needs to be reviewed.</p

EMPODERAMENTO FEMININO ANGOLANO: O PENSAMENTO DAS MULHERES EM CARGOS DE PODER

Empoderamento é uma atitude que surge do indivíduo, quando faz parte de lugares privilegiados para aqueles que participam dos processos decisórios. O empoderamento feminino é a consciência coletiva, manifestada por atitudes e comportamentos que servem para incentivar a participação ativa das mulheres na sociedade e para praticar a igualdade de gênero. Ela insistiu em observar que as mulheres angolanas expressaram poder em suas atitudes quando se tornaram provedores de renda familiar e quando lutaram ao lado da independência nacional de Angola como forma de ajudar seus companheiros com suas demissões familiares. Perante isto, o estudo teve como objetivo conhecer o empoderamento das mulheres angolanas que trabalham em posições de poder. As mulheres, mesmo em posições de liderança que exigem muito e participam do seu cotidiano, predispõem-se às tarefas do lar, esse comportamento parte dos aspectos culturais relacionados aos deveres da mulher ao constituir sua própria família. Metodologicamente, caracterizou-se numa a pesquisa de campo, qualitativa. Quanto aos fins é descritiva e exploratória. A população alvo abrangeu mulheres executivas que ocupam cargos de liderança na sociedade angolana. A pesquisa preocupou-se em colher dados primários utilizando a técnica de entrevista com uso de gravador para auxiliar no registro. A pesquisa foi analisada de acordo como os dados coletados em campo.Palavras-chave: Empoderamento feminino. Submissão da mulher. Liderança

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

BACKGROUND: The increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem. METHODS: In the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP. RESULTS: The percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001) CONCLUSIONS: i) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.publishersversionpublishe

Extensive low-density Plasmodium falciparum reservoir in the island of Príncipe, an isolated malaria pre-elimination setting

Funding Information: This work was supported by the Swedish Research Council Grant (Grant ref. 200075/2022-5), Conselho Nacional de Desenvolvimento Cient\u00EDfico e Tecnol\u00F3gico (CNPq), and GHTM IHMT. TNS is CNPq Research Productivity fellows. As previously mentioned, the samples were collected as part of the PNEP surveillance activities and were irreversibly anonymized before starting the study, with a new reference assigned. The samples were transferred from S\u00E3o Tom\u00E9 and Pr\u00EDncipe to Lisbon under an MTA signed between the Ministry of Health of S\u00E3o Tom\u00E9 and Pr\u00EDncipe and the Institute of Hygiene and Tropical Medicine. This study was approved by the ethics committee of the IHMT ITQB (ref 16.22). The authors thank all the patients who participated in this study and the S\u00E3o Tom\u00E9 and Pr\u00EDncipe National Malaria Control Program teams for their support in collecting samples and data. Study concept and design: DL and JPG. Statistical and data analysis: TNS, PCM, and IL. Writing the original draft: DL, JPG, and TNS. Project management: DL, JPG, and TNS. Data acquisition: PCM, IL, EV, AN, AP, AS, and MJT. Review of the final manuscript: DL, JPG, TNS, PCM, IL, EV, AN, AP, AS, and MJT. Funding Information: This work was supported by the Swedish Research Council Grant (Grant ref. 200075/2022-5), Conselho Nacional de Desenvolvimento Cient\u00EDfico e Tecnol\u00F3gico (CNPq), and GHTM IHMT. TNS is CNPq Research Productivity fellows. Publisher Copyright: © 2024 The AuthorsObjectives: The isolated Príncipe is at the malaria pre-elimination stage. Autochthonous clinical cases have been reported sporadically on the island, signaling the possibility of a sizable subpatent (i.e., rapid diagnostic test- and microscopy-negative and polymerase chain reaction [PCR]-positive) parasite reservoir. Methods: Asymptomatic low-density infections were detected by quantitative PCR (qPCR) targeting Plasmodium falciparum multicopy genes (pfr364 and varATS). Positivity rates were assayed for samples surveyed by active case detection (n = 112) and reactive case detection (n = 221) in 2022. Results: qPCR unveiled 70% of low parasitemia carriers, reaching >90% in reactive case detection. The high P. falciparum prevalence was confirmed by the two high-sensitivity qPCR protocols. Higher positivity rates were observed in the localities where most malaria cases were reported in 2022. Most parasitemias were very low (<2 Pf /µl). Conclusions: These findings suggest that pre-elimination surveillance can benefit from the routine application of highly sensitive tools to unveil otherwise invisible but potentially relevant parasite populations.publishersversionpublishe

Evaluation of Antiplasmodial activity of extracts and constituents from Ampelozizyphus amazonicus

PMID: 26664012 WOS: 000362879000003BACKGROUND: Ampelozizyphus amazonicus Ducke, a plant that is widely used by the population of the Amazonian region to prevent and treat malaria, was investigated in this work, which describes, for the first time, the antiplasmodial activity of its extracts and associates this activity with its isolated constituents. METHODS: Different extracts with solvents of increasing polarity (hexane, chloroform, ethanol, and water) were obtained of the root bark. This procedure resulted in extracts that were characterized for their constituents. The cytotoxicity and activity of the extracts against Plasmodium berghei (schizontocidal activity, liver stage) and Plasmodium falciparum (3D7 and Dd2 strains, erythrocyte stage) were assessed in vitro. RESULTS: Of the four extracts assayed against P. berghei, the chloroform extract showed the greatest activity, with an inhibitory concentration 50% (IC50) value of 30.1 µg/mL, followed by the aqueous extract (IC50 = 39.9 µg/mL). The chloroform extract exhibited the highest antiplasmodial activity in the erythrocyte stage of P. falciparum, with an IC50 value lower than 15 µg/mL. Fractionation of this more active extract led to the isolation and elucidation of pentacyclic triterpenes, lupeol, betulin and betulinic acid, which showed antiplasmodial activities with IC50 values ranging from 5.6 to 80.30 µM. The most active of these, betulinic acid, was further quantified in the extracts by high-performance liquid chromatography-photodiode array detector analyzes. The higher amount was found in the chloroform extract, which was the most active one against P. falciparum. CONCLUSION: The results obtained in this work may partly explain the popular intake of A. amazonicusas an antimalarial remedy in the Amazon region.publishersversionpublishe

Analysis of polymorphisms in Plasmodium falciparum genes related to drug resistance: a survey over four decades under different treatment policies in Brazil

Abstract\ud \ud Background\ud Anti-malarial resistance in Plasmodium falciparum remains an obstacle for malaria control. Resistance-associated genes were analysed in Brazilian samples over four decades to evaluate the impact of different treatment regimens on the parasite genetic profile.\ud \ud \ud Methods\ud Samples were collected on filter paper from patients infected in the Amazon region from 1984 to 2011. DNA was extracted with Chelex® 100 and monoinfection confirmed by PCR. SNPs in the pfcrt, pfmdr1, pfdhfr and pfdhps genes were assessed by PCR-RFLP. The pfmdr1 copy number was estimated using real time quantitative PCR with SYBR® Green. Parasite response was assessed ex vivo with seven concentrations of each anti-malarial. Patients were treated according to Brazilian guidelines: quinine plus tetracycline or mefloquine in period 1 and ACT in period 2.\ud \ud \ud Results\ud All 96 samples presented the pfcrt 76T mutant throughout the assessed periods. In addition, all isolates showed ex vivo chloroquine resistance. The pfmdr1 86Y was detected in 1.5% of samples in period 1, and in 25% in period 2. All samples presented the pfmdr1 1246Y. The analysis of pfmdr1 copy number showed amplification in 37.3% in period 1 and in 42% in period 2. Mutations in pfdhfr were shown as follows: 51I in all samples in period 1 and in 81.2% in period 2; 59R in 6.4% in period 2. The pfdhfr 108N and the pfdhps 437G were seen in all samples along time; the pfdhps 540E in 93.7% in period 1 and in 75% in period 2.\ud \ud \ud Conclusions\ud The 76T mutation associated to chloroquine resistance is still present in the parasite population, although this anti-malarial was withdrawn from the chemotherapy of P. falciparum in Brazil in the mid-1980s. All isolates assayed ex vivo for chloroquine showed resistant phenotype and 76T. No association was observed between pfmdr1 mutations and resistance to quinine, mefloquine and artemisinin derivatives. Additionally, the pfdhfr 108N mutation was detected in all samples throughout the evaluated periods, demonstrating fixation of the mutant allele in the parasite population. Changes in Brazilian national guidelines for the malaria chemotherapy in the last 27 years yielded a discreet genetic impact in the parasite population.We express our gratitude to all patients for agreeing to participate in this\ud study and to the staff of Núcleo de Estudos em Malária/SUCEN/IMTSP,\ud NACE-NUMETROP and Divisão de Endemias, 9° Centro Regional de Saúde de\ud Santarém/SESPA for the support in sample collection and hemoscopy and to\ud the staff of Instituto de Higiene e Medicina Tropical/UNL for laboratory\ud support.\ud This work was supported by grant #2011/07380-8, São Paulo Research\ud Foundation (FAPESP), Superintendência de Controle de Endemias (SUCEN),\ud Programa de Apoio à Pós-Graduação (PROAP)/Coordenação de\ud Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho\ud Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in Luanda, Angola

<p>Abstract</p> <p>Background</p> <p>Malaria is the infectious disease causing the highest morbidity and mortality in Angola and due to widespread chloroquine (CQ) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from CQ to artemisinin combination therapies (ACT) in adults, and sulphadoxine/pyrimethamine (S/P) in pregnant women. Loss of SP sensitivity is, however, progressing rapidly in Africa and, in this study, were investigated a number of molecular markers associated to CQ and S/P.</p> <p>Methods</p> <p>Blood samples were collected from 245 children with uncomplicated malaria, admitted at the Pediatric Hospital Dr. David Bernardino (HPDB), Angola, and the occurrence of mutations in <it>Plasmodium falciparum </it>was investigated in the <it>pfmdr1 </it>(N86Y) and <it>pfcrt </it>(K76T) genes, associated with CQ resistance, as well as in <it>pfdhfr </it>(C59R) and <it>pfdhps </it>(K540E), conferring SP resistance.</p> <p>Results</p> <p>The frequencies of <it>pfmdr1 </it>mutations in codon 86 were 28.6% N, 61.3% Y and 10.1% mixed infections (NY). The frequency of <it>pfcrt </it>mutations in codon 76 were 93.9% K, 5.7% T and 0.4% mixed infections (KT). For <it>pfdhfr </it>the results were in codon 59, 60.6% C, 20.6% R and 18.8% mixed infections (CR). Concerning <it>pfdhps</it>, 6.3% of the isolates were bearers of the mutation 540E and 5.4% mixed infections (K540E).</p> <p>Conclusion</p> <p>The results of this epidemiologic study showed high presence of CQ resistance markers while for SP a much lower prevalence was detected for the markers under study.</p

Plasmodium chabaudi chabaudi malaria parasites can develop stable resistance to atovaquone with a mutation in the cytochrome b gene

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum</it>, has developed resistance to many of the drugs in use. The recommended treatment policy is now to use drug combinations. The atovaquone-proguanil (AP) drug combination, is one of the treatment and prophylaxis options. Atovaquone (ATQ) exerts its action by inhibiting plasmodial mitochondria electron transport at the level of the cytochrome bc1 complex. <it>Plasmodium falciparum in vitro </it>resistance to ATQ has been associated with specific point mutations in the region spanning codons 271-284 of the <it>cytochrome b </it>gene. ATQ -resistant <it>Plasmodium yoelii </it>and <it>Plasmodium berghei </it>lines have been obtained and resistant lines have amino acid mutations in their CYT <it>b </it>protein sequences. <it>Plasmodium chabaudi </it>model for studying drug-responses and drug-resistance selection is a very useful rodent malaria model but no ATQ resistant parasites have been reported so far. The aim of this study was to determine the ATQ sensitivity of the <it>P. chabaudi </it>clones, to select a resistant parasite line and to perform genotypic characterization of the <it>cytb </it>gene of these clones.</p> <p>Methods</p> <p>To select for ATQ resistance, <it>Plasmodium. chabaudi chabaudi </it>clones were exposed to gradually increasing concentrations of ATQ during several consecutive passages in mice. <it>Plasmodium chabaudi cytb </it>gene was amplified and sequenced.</p> <p>Results</p> <p>ATQ resistance was selected from the clone AS-3CQ. In order to confirm whether an heritable genetic mutation underlies the response of AS-ATQ to ATQ, the stability of the drug resistance phenotype in this clone was evaluated by measuring drug responses after (i) multiple blood passages in the absence of the drug, (ii) freeze/thawing of parasites in liquid nitrogen and (iii) transmission through a mosquito host, <it>Anopheles stephensi</it>. ATQ resistance phenotype of the drug-selected parasite clone kept unaltered. Therefore, ATQ resistance in clone AS-ATQ is genetically encoded. The Minimum Curative Dose of AS-ATQ showed a six-fold increase in MCD to ATQ relative to AS-3CQ.</p> <p>Conclusions</p> <p>A mutation was found on the <it>P. chabaudi cytb </it>gene from the AS-ATQ sample a substitution at the residue Tyr268 for an Asn, this mutation is homologous to the one found in <it>P. falciparum </it>isolates resistant to ATQ.</p

Evaluation of artemether-lumefantrine efficacy in the treatment of uncomplicated malaria and its association with pfmdr1, pfatpase6 and K13-propeller polymorphisms in Luanda, Angola

Background: Drug resistance in Plasmodium falciparum has posed an obstacle to effective treatment and challenges many malaria control programmes in endemic areas. In Angola, until 2003, chloroquine (CQ) was used as first-line therapy for uncomplicated malaria. It was replaced initially by amodiaquine and, in 2006, by artemisinin-based combination therapy (ACT) with artemether-lumefantrine (AL, Coartem®). Efficacy study of ACT, conducted in Angola between 2004 and 2005, showed a baseline efficacy of ≈99 %. Methods: 103 malaria patients were enrolled according to WHO proceedings. Patients were followed up with clinical and parasitological evaluations for 28 days, parasite density and identification was evaluated by microscopy, the pfmsp2 were genotyped by nested-PCR, to distinguish parasite recrudescence from new infections; the polymorphisms at codons 86 and 1246 of pfmdr1 gene, and 769 of pfatp6 gene were assessed by PCR-RFLP and sequencing for pfk13-propeller genotype. Results: The cure rate was 91.3 %. The obtained results showed that from 103 patients, 12.6 % (n = 13) still had parasitaemia 1 day after the treatment was finished. On day 0, of the 94 evaluated samples, wild-type alleles were identified in 73.4 % (n = 69) for pfmdr1 N86Y position and only one sample carried the mutant allele (Y) for pfmdr1 1246; 14 % of these samples showed increased pfmdr1 copy number; 100 % (n = 21) had wild-type allele of k13 gene in all the studied positions. Discussion: These results showed changes in parasite profile susceptibility to AL in comparison to the baseline data from 2002 to 2004 and on the genotyping characteristics; the clinical outcome after treatment with AL did not link a particular genotype with treatment failure; observed changes do not provide sufficient evidence for a treatment policy change, but they suggest that a carefully monitoring is needed in this area.publishersversionpublishe