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Given the importance of the transducer elements in the performance of sensors for various applications, as well as the growing search for devices that are capable of providing the response in shorter time, in this work, titanium dioxide was examined as a candidate for application in an electrochemical biosensor. A TiO2 coating deposited by sol-gel method on a silicon wafer was obtained with an anatase crystalline structure, as an n-type semiconductor with donor density equal to 2.954 · 1017 cm-3. Its surface was functionalized to be tested as a biosensor to detect snake venom of the Bothrops genera, and each step of the functionalization was investigated using Electrochemical Impedance Spectroscopy (EIS) and Cyclic voltammetry. Despite being less sensitive than the reference method ELISA, the TiO2-based biosensor was also capable of detecting the analyte of interest at 20 µg mL-1, revealing an increase in its leakage resistance and phase shift after incubation in this solution. Furthermore, the total time for carrying out the biodetection with the TiO2-coated device (41.24 ± 0.05 min) was estimated to be approximately 80 % shorter than that required by the labelled standard assay, which indicates that TiO2 is a promising electrochemical transducer for biosensing applications. [ABSTRACT FROM AUTHOR]

Mosquitoes are important vectors of several pathogens and thereby contribute to the spread of diseases, with social, economic and public health impacts. Amongst the approximately 450 species of Anopheles, about 60 are recognized as vectors of human malaria, the most important parasitic disease. In Africa, Anopheles gambiae is the main malaria vector mosquito. Current malaria control strategies are largely focused on drugs and vector control measures such as insecticides and bed-nets. Improvement of current, and the development of new, mosquito-targeted malaria control methods rely on a better understanding of mosquito vector biology. An organism's transcriptome is a reflection of its physiological state and transcriptomic analyses of different conditions that are relevant to mosquito vector competence can therefore yield important information. Transcriptomic analyses have contributed significant information on processes such as blood-feeding parasite-vector interaction, insecticide resistance, and tissue- and stage-specific gene regulation, thereby facilitating the path towards the development of new malaria control methods. Here, we discuss the main applications of transcriptomic analyses in An. gambiae that have led to a better understanding of mosquito vector competence. [ABSTRACT FROM AUTHOR]

Malaria is one of the main human public health problems in the tropical world and is possibly becoming an emerging disease too in regions where it has been controlled. It has been an excellent model in the area of molecular studies, with scientific validation of techniques, application of data mainly in studies of parasite diversity and information on a number of different aspects associated with infection and disease. The transfer of the gathered knowledge and experience in malaria to other infections is of great use and we briefly review a number of molecular markers, methodologies and techniques mostly used for Plasmodium detection, as well as identification or characterization of parasite populations. Selection of appropriate techniques depends on the questions raised and the studies’ objectives – the antigen-coding genes, microsatellite loci and drug-resistance associated markers being the three most analysed classes of markers. The need of validation and standardization of laboratory protocols is addressed and discussed as it may determine the comparison of data between different studies and laboratories, with relevance in field-collected samples or studies. [ABSTRACT FROM AUTHOR]

Abstract The impact of a vector eradication programme, conducted in the 1980s, on Anopheles gambiae populations from the islands of São Tomé and Príncipe, was evaluated by microsatellite DNA analysis. Significant genetic differentiation was observed within and between the two islands and between the islands and a population from Gabon, suggesting a degree of isolation between them. Large estimates of long-term Ne suggested that the control programme did not affect the effective population size of the vector. Heterozygosity tests were also not consistent with a recent bottleneck. [ABSTRACT FROM AUTHOR]

Abstract Background A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. Method During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. Results On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children. Conclusions HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up.

Abstract Background Plasmodium falciparum and HIV-1 infection cause substantial morbidity and mortality in sub-Saharan Africa. Increasing evidence suggests these two pathogens interact negatively when infecting the same individual. Methods A cross-sectional study among HIV-1 infected and uninfected populations was recruited in Mocuba and Maputo, Mozambique to determine the prevalence of sub-clinical malarial parasitaemia using light microscopy and a nested PCR assay. Results The prevalence of sub-clinical P. falciparum parasitaemia was low in Maputo, whether determined by microscopy (0.4%) or PCR (1.9%), but substantially higher in Mocuba (7.6 and 14.7%, respectively). Nested PCR detected nearly 70% more cases of sub-clinical parasitaemia than microscopy, but differences occur by locality. HIV-1 infected persons were more likely to be sub-clinically parasitaemic than HIV-1 uninfected individuals recruited from the same geographic areas. Trimethoprim-sulphamethoxazole use did not substantially reduce sub-clinical parasitaemia. Conclusions Dried blood spots are a convenient and sensitive technique for detecting sub-clinical infection with P. falciparum by nested PCR. Prevalence of P. falciparum is substantially lower in Maputo where malaria control programmes have been more active than in the rural town of Mocuba. In Mocuba, among those presenting for HIV-1 counseling and testing, the prevalence of P. falciparum is substantially higher in those who test positive for HIV-1 than those without HIV-1 infection. The clinical implications of sub-clinical P. falciparum infection among HIV-1 infected persons warrant additional study.

Abstract Background Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. Methods Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. Results The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples). Conclusion This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola.

Abstract Background Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to Plasmodium falciparum 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 pfdhfr and pfdhps genes associated to P. falciparum resistance to SP before the introduction of S/P IPT in children. Methods The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in pfdhfr and pfdhps genes was carried out in 452 P. falciparum blood samples by PCR RFLP. Results For pfdhfr gene, 90,3% of the samples carried the mutation 51I, with 7.5% of mixed infections; 51% carried wild type allele 59C, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 108N. Concerning, pfdhps gene, 83,1% were mutant type 437G with 11% mixed infections , while 87% of the studied isolates were wild type for codon 540. Discussion 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 (51I and 108N in pfdhfr, and 437G in pfdhps) was found, besides a low prevalence of the quintuple mutation. Conclusion The data showed that the implementation IPT using SP in children needs to be reviewed.

Abstract Background There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission. Methods Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks. Results Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004

Abstract Background Plasmodium falciparum is the major cause of malaria infection in the island of São Tomé, in the Republic of São Tomé and Príncipe (STP), with an incidence of 40 - 50% before 2004. Since 2004, through the coordination of the Ministry of Health of STP and their Centro Nacional de Endemias (CNE), an integrated malaria control programme has been intensively deployed on the island of São Tomé. Malaria morbidity and mortality decreased by 95% after three years of effective intervention. In the low transmission settings, however, malaria seasonal fluctuation can be a potential problem directly related to epidemics if ongoing control measures are interrupted. Studies on a number of associated factors with malaria epidemics and the measures taken to respond to outbreaks are presented. Methods The integrated malaria control programme included indoor residual spraying (IRS), long-lasting insecticidal nets (LLINs), intermittent preventive therapy for pregnant women, as well as early diagnosis and prompt treatment with artemisinin-based combination therapy (ACT). Regular implementation of an island-wide IRS programme was carried out yearly in 2004-2007, and enhanced throughout the island in 2009. Malaria incidence and prevalence were estimated based on passive case detection and mass screening, respectively. Slide positivity rates were used for monitoring the beginning of a malaria epidemic or a seasonal peak. Results A steep decline of ca. 95% of malaria morbidity and mortality was observed between 2004 and 2008 with use of the combined control methods. Malaria incidence was 2.0%, 1.5%, and 3.0% for 2007, 2008, and 2009, respectively. In April 2008, a cross-sectional country-wide surveillance showed malaria prevalence of 3.5%, of which 95% cases were asymptomatic carriers. Only 50% of asymptomatic carriers were cured with ACT treatment, while 90% of the symptomatic patients were cured by ACT treatment as confirmed with a follow up study. Malaria morbidity increased by three-fold during the first half of 2009 as compared to the same period in 2008. Over this period of six months, severe malaria was also noted in all age groups and malaria mortality increased by two-fold in children less than five years old. After an emergency IRS was deployed, with increased use of LLINs, and an active search of asymptomatic carriers was followed and given complete ACT treatment, malaria incidence decreased to less than 1% in the second half of 2009. Conclusion At the initial stage of the integrated malaria control programme, IRS contributed to the visible effect on the rapid reduction of malaria morbidity and mortality, while this programme highlights an urgent demand for the improvement of other measures, particularly promotion of LLINs usage, with close monitoring of asymptomatic carriers and with ACT treatment in malaria transmission hotspots. In addition, both daily reports and a regular active surveillance to prevent malaria outbreaks should be established permanently, so that a fast response to epidemics can be effectively made when necessary.

Abstract Background Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) has evolved worldwide. In the archipelago of São Tomé and Principe (STP), West Africa, although SP resistance is highly prevalent the drug is still in use in particular circumstances. To address the evolutionary origins of SP resistance in these islands, we genotyped point mutations at P. falciparum dhfr and dhps genes and analysed microsatellites flanking those genes. Methods Blood samples were collected in July and December 2004 in three localities of São Tomé Island and one in Principe Island. Species-specific nested-PCR was used to identify P. falciparum infected samples. Subsequently, SNPs at the dhfr and dhps genes were identified through PCR-RFLP. Isolates were also analysed for three microsatellite loci flanking the dhfr gene, three loci flanking dhps and four loci located at putative neutral genomic regions. Results An increase of resistance-associated mutations at dhfr and dhps was observed, in particular for the dhfr/dhps quintuple mutant, associated with clinical SP failure. Analysis of flanking microsatellites suggests multiple independent introductions for dhfr and dhps mutant haplotypes, possibly from West Africa. A reduced genetic diversity and increased differentiation at flanking microsatellites when compared to neutral loci is consistent with a selective sweep for resistant alleles at both loci. Conclusions This study provides additional evidence for the crucial role of gene flow and drug selective pressures in the rapid spread of SP resistance in P. falciparum populations, from only a few mutation events giving rise to resistance-associated mutants. It also highlights the importance of human migration in the spread of drug resistant malaria parasites, as the distance between the islands and mainland is not consistent with mosquito-mediated parasite dispersal.

Abstract Background Immune responses to parasites, which start with pathogen recognition, play a decisive role in the control of the infection in mosquitoes. Peptidoglycan recognition proteins (PGRPs) are an important family of pattern recognition receptors that are involved in the activation of these immune reactions. Pathogen pressure can exert adaptive changes in host genes that are crucial components of the vector's defence. The aim of this study was to determine the molecular evolution of the three short PGRPs (PGRP-S1, PGRP-S2 and PGRP-S3) in the two main African malaria vectors - Anopheles gambiae and Anopheles arabiensis. Results Genetic diversity of An. gambiae and An. arabiensis PGRP-S1, PGRP-S2 and PGRP-S3 was investigated in samples collected from Mozambique and Tanzania. PGRP-S1 diversity was lower than for PGRP-S2 and PGRP-S3. PGRP-S1 was the only gene differentiated between the two species. All the comparisons made for PGRP-S1 showed significant P-values for Fst estimates and AMOVA confirming a clear separation between species. For PGRP-S2 and PGRP-S3 genes it was not possible to group populations either by species or by geographic region. Phylogenetic networks reinforced the results obtained by the AMOVA and Fst values. The ratio of nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) for the duplicate pair PGRP-S2 and PGRP-S3 was very similar and lower than 1. The 3D model of the different proteins coded by these genes showed that amino acid substitutions were concentrated at the periphery of the protein rather than at the peptidoglycan recognition site. Conclusions PGRP-S1 is less diverse and showed higher divergence between An. gambiae and An. arabiensis regardless of geographic location. This probably relates to its location in the chromosome-X, while PGRP-S2 and PGRP-S3, located in chromosome-2L, showed signs of autosomal introgression. The two short PGRP genes located in the chromosome-2L were under purifying selection, which suggests functional constraints. Different types of selection acting on PGRP-S1 and PGRP-S2 and S3 might be related to their different function and catalytic activity.

Abstract Background Plasmodium falciparum is the major species responsible for malaria transmission on the island of Príncipe, in the Republic of São Tomé and Príncipe (STP). Indoor residual spraying (IRS) has been intensively deployed on the island, since 2003. Other measures included intermittent preventive therapy (IPT), since 2004, as well as artemisinin-based therapy (ACT) and long-lasting insecticidal nets (LLINs) from 2005. The work was coordinated by the Ministry of Health of STP through their Centro Nacional de Endemias (CNE) and the impact of such an integrated control programme on the prevalence and epidemiology of malaria in Príncipe was evaluated. Methods The scaling-up of preventive strategies included IRS, LLINs, IPT for pregnant women, as well as early diagnosis and prompt treatment with ACT. Regular implementation of an island-wide IRS programme was carried out yearly in 2003-2005, and later in 2008. Malaria incidence and prevalence were estimated based on passive case detection and active case detection, respectively. Slide positivity rate (SPR) was used as an indicator of any increase of malaria cases during and after the control programme was initiated. Results Regular IRS achieved a coverage of 85-90% for each of the four annual cycles (2003-2005, annually and one spraying in 2008) while usage of LLINs was never superior to 50% from 2006-2009. Coverage of IPT steadily increased from 50% in 2004 to 80% in 2008. Since 2006, over 90% of uncomplicated malaria patients received ACT treatment. Severe malaria cases were hospitalized and treated with quinine. Monthly trends of SPR were constantly over 50% in 2003, but steadily decreased below 10% in 2006. SPR has been below 5% since 2007, but an increase to up to 15% was noted in June 2009 when 16 imported cases were detected. A steep decline by 99% of malaria incidence was observed between 2003 and 2008, with an incidence risk of the population of five per thousand, in 2008. No malaria mortality has been reported since 2005. Species shift from falciparum to non-falciparum malaria was noted after a five-year intensive control programme. Cross-sectional country-wide active surveillances showed malaria prevalences of 1.1%, 0.7%, and 0.9% in June 2006, Oct 2007, and July 2009, respectively, of which over 90% were asymptomatic. Conclusion The effective measures of the combination of four major control methods have produced a rapid decline in malaria morbidity and mortality on the island of Príncipe. The combination of IRS, IPT, and active surveillance with ACT treatment seemed to have played important roles to achieve a present status of low and stable malaria on the island. In low transmission settings, any increase of malaria morbidity indicates potential epidemics and assumes that current control strategies were interrupted. Active surveillance should be reinforced to follow and monitor all asymptomatic carriers and imported cases. Consolidation and a shift to elimination phase demands the sustainability of such integrated programmes.

Abstract Background In response to chloroquine (CQ) resistance, the policy for the first-line treatment of uncomplicated malaria in the Democratic Republic of East Timor (DRET) was changed in early 2000. The combination of sulphadoxine-pyrimethamine (SP) was then introduced for the treatment of uncomplicated falciparum malaria. Methods Blood samples were collected in two different periods (2003–2004 and 2004–2005) from individuals attending hospitals or clinics in six districts of the DRET and checked for Plasmodium falciparum infection. 112 PCR-positive samples were inspected for genetic polymorphisms in the pfcrt, pfmdr1, pfdhfr and pfdhps genes. Different alleles were interrogated for potential associations that could be indicative of non-random linkage. Results Overall prevalence of mutations associated with resistance to CQ and SP was extremely high. The mutant form of Pfcrt (76T) was found to be fixed even after five years of alleged CQ removal. There was a significant increase in the prevalence of the pfdhps 437G mutation (X2 = 31.1; p = 0.001) from the first to second survey periods. A non-random association was observed between pfdhfr51/pfdhps437 (p = 0.001) and pfdhfr 59/pfdhps 437 (p = 0.013) alleles. Conclusion Persistence of CQ-resistant mutants even after supposed drug withdrawal suggests one or all of the following: local P. falciparum may still be inadvertently exposed to the drug, that mutant parasites are being "imported" into the country, and/or reduced genetic diversity and low parasite transmission help maintain mutant haplotypes. The association between pfdhfr51/pfdhps437 and pfdhfr 59/pfdhps 437 alleles indicates that these are undergoing concomitant positive selection in the DRET.

Abstract Background 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. Methods 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 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. Results The 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). Conclusion 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.

Abstract Background Plasmodium vivax is estimated to affect 75 million people annually. It is reportedly absent, however, from west and central Africa due to the high prevalence of the Duffy negative phenotype in the indigenous populations. Despite this, non-African travellers consistently return to their own countries with P. vivax malaria after visiting this region. An attempt was made, therefore, to detect the presence of P. vivax parasites in blood samples collected from the indigenous populations of west and central Africa. Methods Parasite species typing (for all four human malaria parasites) was carried out by PCR on 2,588 blood samples collected from individuals from nine African malaria-endemic countries. Results Most infections (98.5%) were Plasmodium falciparum, Plasmodium malariae was identified in 8.5% of all infections, and Plasmodium ovale in 3.9%. The prevalence of both parasites varied greatly by country. Only one case of P. vivax was detected from Sao Tome, an island off the west coast of Africa, confirming the scarcity of this parasite in Africa. Conclusion The prevalence of P. vivax in local populations in sub-Saharan Africa is very low, despite the frequent identification of this parasite in non-African travellers.

Abstract Background Patterns of genetic structure among mosquito vector populations in islands have received particular attention as these are considered potentially suitable sites for experimental trials on transgenic-based malaria control strategies. In this study, levels of genetic differentiation have been estimated between populations of Anopheles gambiae s.s. from the islands of Bioko and Annobón, and from continental Equatorial Guinea (EG) and Gabon. Methods Genotyping of 11 microsatellite loci located in chromosome 3 was performed in three island samples (two in Bioko and one in Annobón) and three mainland samples (two in EG and one in Gabon). Four samples belonged to the M molecular form and two to the S-form. Microsatellite data was used to estimate genetic diversity parameters, perform demographic equilibrium tests and analyse population differentiation. Results High levels of genetic differentiation were found between the more geographically remote island of Annobón and the continent, contrasting with the shallow differentiation between Bioko island, closest to mainland, and continental localities. In Bioko, differentiation between M and S forms was higher than that observed between island and mainland samples of the same molecular form. Conclusion The observed patterns of population structure seem to be governed by the presence of both physical (the ocean) and biological (the M-S form discontinuity) barriers to gene flow. The significant degree of genetic isolation between M and S forms detected by microsatellite loci located outside the "genomic islands" of speciation identified in A. gambiae s.s. further supports the hypothesis of on-going incipient speciation within this species. The implications of these findings regarding vector control strategies are discussed.

Abstract Background The anti-malarial chloroquine can modulate the outcome of infection during the Plasmodium sporogonic development, interfering with Plasmodium gene expression and subsequently, with transmission. The present study sets to identify Plasmodium genes that might be regulated by chloroquine in the mosquito vector. Methods Differential display RT-PCR (DDRT-PCR) was used to identify genes expressed during the sporogonic cycle that are regulated by exposure to chloroquine. Anopheles stephensi mosquitoes were fed on Plasmodium yoelii nigeriensis-infected mice. Three days post-infection, mosquitoes were fed a non-infectious blood meal from mice treated orally with 50 mg/kg chloroquine. Two differentially expressed Plasmodium transcripts (Pyn_chl091 and Pyn_chl055) were further characterized by DNA sequencing and real-time PCR analysis. Results Both transcripts were represented in Plasmodium EST databases, but displayed no homology with any known genes. Pyn_chl091 was upregulated by day 18 post infection when the mosquito had a second blood meal. However, when the effect of chloroquine on that transcript was investigated during the erythrocytic cycle, no significant differences were observed. Although slightly upregulated by chloroquine exposure the expression of Pyn_chl055 was more affected by development, increasing towards the end of the sporogonic cycle. Transcript abundance of Pyn_chl055 was reduced when erythrocytic stages were treated with chloroquine. Conclusion Chloroquine increased parasite load in mosquito salivary glands and interferes with the expression of at least two Plasmodium genes. The transcripts identified contain putative signal peptides and transmembrane domains suggesting that these proteins, due to their location, are targets of chloroquine (not as an antimalarial) probably through cell trafficking and recycling.

Abstract Background Plasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate. Methods A total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa. Results The frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/108N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/108N + 437G/540E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected. Conclusion The observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.

Abstract Background Malaria has come near eradication at archipelago of Cabo Verde in 1970. Infections are now only observed in Santiago, where outbreaks occur. In these islands, malaria is considered by the international community as being of limited risk and, therefore, no prophylaxis is recommended. Since the understanding of factors that determine malaria outbreaks are crucial for controlling the disease, the present study aimed to investigate if the malaria infections observed in Santiago Island are maintained in isolated foci and in asymptomatic individuals. Methods The occurrence of asymptomatic carriers in villages with history of malaria as well as the level of exposure of these populations were investigated using PCR and serological analyses. Results Results indicate that malaria is maintained as asymptomatic and sub-patent infections and that the majority of the circulating parasite populations harbour chloroquine-resistant mutations. Conclusion These observations highlight the alarming prospect of malaria to become a serious public health problem and underscore the need for a tighter surveillance.

Abstract Background The fixed dose combination atovaquone-proguanil is a recently introduced antimalarial for treatment and prophylaxis of Plasmodium falciparum malaria. It is highly effective with a good tolerability profile and a convenient prophylactic regimen. Nevertheless, cases of treatment failure have already been reported, which have been associated to mutations in the cytochrome b gene of the Plasmodium (pfcytb). The presence of atovaquone-proguanil in vivo resistance conferring mutations in pfcytb gene in Luanda, Angola, was investigated, in order to make recommendations on prescribing this antimalarial as prophylaxis for travellers. Methods Two hundred and forty nine blood samples from children hospitalized at Luanda Pediatric Hospital for malaria were studied. The PCR-RFLP methodology was used in order to identify pfcytb wild type codon 268 and two point mutations: T802A and A803C. Results All samples were identified as wild type for pfcytb gene at codon 268. In the studied population, no mutations associated to atovaquone-proguanil treatment failure were found. Prevalence of the studied mutations in the region was estimated to be less than 0.77% (99% significance level). Conclusion Atovaquone-proguanil can be recommended for use by travellers to Luanda with expected high efficacy. This represents an improvement compared to other currently used prophylatic antimalarials in this region. However, it is imperative to continue surveillance.

Abstract Background Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked. Methods The ΔΔCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses. Results Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa. Conclusion The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.

Abstract Background In many parts of continental Africa house construction does not appear to impede entry of malaria vectors and, given their generally late biting cycle, the great majority of transmission takes place indoors. In contrast, many houses in São Tomé, 140 km off the coast of Gabon, are raised on stilts and built of wooden planks. Building on stilts is a time-honoured, but largely untested, way of avoiding mosquito bites. Exposure may also be affected by mosquito activity times and age composition of host-seeking females. A study was therefore undertaken on the island of São Tomé to determine if exposure to Anopheles gambiae, the only vector on the island, varied with house construction or time of the night. Methods A series of all-night landing collections were undertaken out of doors at ground level, inside houses at ground level, on the verandas of, and inside houses built on stilts. The gonotrophic age of an unselected sample of insects from the first three hours of landing collection (18:00–21:00) was determined by dissection. In addition, 1,149 miniature light-trap collections were obtained from 125 houses in the study area. Numbers collected were related to house construction. Results Biting of An. gambiae took place primarily outside at ground level. Less than one third of biting occurred inside houses. Houses built on stilts had half the number of An. gambiae in them compared to those built at ground level. Conversely houses with an eaves gap had more An. gambiae in them than houses without such a gap. Gonotrophic age did not affect house entry rates in An. gambiae. House construction affected Culex quinquefasciatus less than An. gambiae. Mean density per house, derived from a series of 1,490 randomly assigned light-trap collections, was over-dispersed with 18% of houses having 70% of the vectors. Conclusion House construction plays an important role in determining exposure to malaria vectors in São Tomé. Neighbours can have very different exposure levels. Recommendations for improvement in control are given.