Dinora Lopes

An estimation of the entomological inoculation rate for Ifakara: a semi-urban area in a region of intense malaria transmission in Tanzania.

Summary An entomological study on vectors of malaria and their relative contribution to Plasmodium falciparum transmission in the semi‐urban area of Ifakara, south‐eastern Tanzania, was conducted. A total of 32 houses were randomly sampled from the area and light trap catches (LTC) performed in one room in each house every 2 weeks for 1 year. A total of 147 448 mosquitoes were caught from 789 LTC; 26 134 Anopheles gambiae s.l., 615 A. funestus, 718 other anophelines and 119 981 culicines. More than 60% of the total A. gambiae s.l. were found in five (0.6%) LTCs, with a maximum of 5889 caught in a single trap. Of 505 A. gambiae s.l. speciated by polymerase chain reaction, 91.5% were found to be A. arabiensis. Plasmodium falciparum sporozoite enzyme‐linked immunosorbent assay tests were performed on 10 108 anopheles mosquitoes and 39 (0.38%) were positive. Entomological inoculation rate (EIR) estimates were generated using a standard method and an alternative method that allows the calculation of confidence intervals based on a negative binomial distribution of sporozoite positive mosquitoes. Overall EIR estimates were similar; 31 vs. 29 [95% confidence interval (CI): 19, 44] infectious bites per annum, respectively. The EIR ranged from 4 (95% CI: 1, 17) in the cool season to 108 (95% CI: 69, 170) in the wet season and from 54 (95% CI: 30, 97) in the east of the town to 15 (95% CI: 8, 30) in the town centre. These estimates show large variations over short distances in time and space. They are all markedly lower than those reported from nearby rural areas and for other parts of Tanzania.

Detection of atovaquone and Malarone resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb).

Clinical treatment failures of the hydroxynaphthoquinone atovaquone or its combination with proguanil (Malarone) in Plasmodium falciparum malaria has been recently documented. These events have been associated to single nucleotide polymorphisms (SNPs) in the parasite cytochrome b gene (cytb). In this report we describe a set of nest PCR-RFLP methods developed for the fast detection of all known cytb mutations associated to resistance to these drugs. The methods were successfully applied for the analysis of phenol-chloroform extracted DNA samples from patients not cured by Malarone, and from an established parasite clone. Further, the protocol for the detection of the A803C mutation was applied to 164 DNA field samples extracted through crude methanol-based protocols, originated from several malaria settings. The PCR-RFLP methods here presented can be used as a valuable for the clinical detection and study of Malarone and atovaquone P. falciparum resistance.

Malaria in São Tomé and Príncipe: parasite prevalences and vector densities.

A cross-sectional survey was carried out in 16 localities on the island of São Tomé and three on the island of Príncipe, at the end of the rainy season of 1997, to determine malaria prevalence and vector densities. Blood samples from 664 inhabitants of all ages were examined by optical microscopy (OM) and PCR. Mosquito collections were made by outdoor landing captures from 21:00-23:00 h. Great differences were found between OM and PCR readings. OM had a sensitivity of 66%, a specificity of 79% and failed to reveal any mixed-infections. Overall prevalence, determined by PCR, was higher in São Tomé (53%) than in Príncipe (35%). It was highest in children below 16 years-old. All four human Plasmodium species occurred in São Tomé but P. ovale was not detected in Príncipe. The human population was largely asymptomatic. Bednet users had lower prevalence than did non-users. The FOREST form of Anopheles gambiae s.s., identified by PCR and cytogenetics, was the only vector on the islands. The sporozoite rate in São Tomé, assessed by ELISA, was 0.5%. Parasite prevalence and vector densities were positively correlated in São Tomé, where malaria transmission must occur predominantly in the more populated coastal areas.

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

BackgroundThe 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.MethodsIn 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.ResultsThe 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)Conclusionsi) 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.

Plasmodium falciparum: diversity studies of isolates from two Colombian regions with different endemicity.

The population structure of Plasmodium falciparum has been widely studied in diverse epidemiological contexts, but emphasis has been made in regions with high and stable transmission. In order to establish the genetic structure of P. falciparum in areas of Colombia with different degree of endemicity, we studied 100 samples from malaria patients of two different municipalities. The frequency of multiclonal infection in these areas and the correlation with the endemicity were carried out by comparison of the amplified products from polymorphic segments of MSP-1, MSP-2, and GLURP genes. We found low size polymorphism of the studied genes: 1 MSP-1 allele, 3 MSP-2 alleles, and 4 GLURP alleles. We conclude that the P. falciparum population in the regions studied is genetically homogeneous.

In vitro assessment of artesunate, artemether and amodiaquine susceptibility and molecular analysis of putative resistance-associated mutations of Plasmodium falciparum from São Tomé and Príncipe

Objective  To evaluate the basal in vitro responses of Plasmodium falciparum isolates collected in The Democratic Republic of São Tomé and Príncipe to artemether (ATH), artesunate (ATN) and amodiaquine (AMQ).

New antimalarials with a triterpenic scaffold from Momordica balsamina

Bioassay-guided fractionation of the methanol extract of Momordica balsamina led to the isolation of three new cucurbitane-type triterpenoids, balsaminols C-E (1-3). Their structures were elucidated on the basis of spectroscopic methods including 2D NMR experiments (COSY, HMQC, HMBC and NOESY). Balsaminols C-E, together with ten cucurbitacins isolated from the same plant (4-13), were evaluated for their antimalarial activity against the Plasmodium falciparum chloroquine-sensitive strain 3D7 and the chloroquine-resistant clone Dd2. Most of the compounds displayed antimalarial activity. Compounds 9 and 12 revealed the highest antiplasmodial effects against both strains (IC50 values: 4.6, and 7.4 microM, 3D7, respectively; 4.0, and 8.2 microM, Dd2, respectively). Structure-activity relationships are discussed. Furthermore, the preliminary toxicity toward human cells of compounds 1-5 and 9 was investigated in breast cancer cell line (MCF-7). Compounds were inactive or showed weak toxicity (IC50 values>19.0).

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

BackgroundMalaria 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.MethodsBlood 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 Plasmodium falciparum was investigated in the pfmdr1 (N86Y) and pfcrt (K76T) genes, associated with CQ resistance, as well as in pfdhfr (C59R) and pfdhps (K540E), conferring SP resistance.ResultsThe frequencies of pfmdr1 mutations in codon 86 were 28.6% N, 61.3% Y and 10.1% mixed infections (NY). The frequency of pfcrt mutations in codon 76 were 93.9% K, 5.7% T and 0.4% mixed infections (KT). For pfdhfr the results were in codon 59, 60.6% C, 20.6% R and 18.8% mixed infections (CR). Concerning pfdhps, 6.3% of the isolates were bearers of the mutation 540E and 5.4% mixed infections (K540E).ConclusionThe 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.

A review of antimalarial plants used in traditional medicine in communities in Portuguese-Speaking countries: Brazil, Mozambique, Cape Verde, Guinea-Bissau, São Tomé and Príncipe and Angola

The isolation of bioactive compounds from medicinal plants, based on traditional use or ethnomedical data, is a highly promising potential approach for identifying new and effective antimalarial drug candidates. The purpose of this review was to create a compilation of the phytochemical studies on medicinal plants used to treat malaria in traditional medicine from the Community of Portuguese-Speaking Countries (CPSC): Angola, Brazil, Cape Verde, Guinea-Bissau, Mozambique and São Tomé and Príncipe. In addition, this review aimed to show that there are several medicinal plants popularly used in these countries for which few scientific studies are available. The primary approach compared the antimalarial activity of native species used in each country with its extracts, fractions and isolated substances. In this context, data shown here could be a tool to help researchers from these regions establish a scientific and technical network on the subject for the CPSC where malaria is a public health problem.

Triterpenoids as inhibitors of erythrocytic and liver stages of Plasmodium infections

Bioassay-guided fractionation of the methanol extract of Momordica balsamina led to the isolation of two new cucurbitane-type triterpenoids, balsaminol F (1) and balsaminoside B (2), along with the known glycosylated cucurbitacins, cucurbita-5,24-diene-3β,23(R)-diol-7-O-β-D-glucopyranoside (3) and kuguaglycoside A (4). Compound 1 was acylated yielding two new triesters, triacetylbalsaminol F (5) and tribenzoylbalsaminol F (6). The structures were elucidated based on spectroscopic methods including 2D-NMR experiments (COSY, HMQC, HMBC and NOESY). Compounds 1-6, were evaluated for their antimalarial activity against the erythrocytic stages of the Plasmodium falciparum chloroquine-sensitive strain 3D7 and the chloroquine-resistant clone Dd2. Assessment of compounds (1-3 and 5, 6) activity against the liver stage of Plasmodium berghei was also performed, measuring the luminescence intensity in Huh-7 cells infected with a firefly luciferase-expressing P. berghei line, PbGFP-Luc(con). Active compounds were shown to inhibit the parasites intracellular development rather than its ability to invade hepatic cells. Toxicity of compounds (1-3 and 5, 6) was assessed on the same cell line and on mouse primary hepatocytes through the fluorescence measurement of cell confluency. Furthermore, toxicity of compounds 1-6 towards human cells was also investigated in the MCF-7 breast cancer cell line, showing that they were not toxic or exhibited weak toxicity. In blood stages of P. falciparum, compounds 1-5 displayed antimalarial activity, revealing triacetylbalsaminol F (5) the highest antiplasmodial effects (IC(50) values: 0.4μM, 3D7; 0.2μM, Dd2). The highest antiplasmodial activity against the liver stages of P.berghei was also displayed by compound 5, with high inhibitory activity and no toxicity.