Association of apoptosis-related variants to malaria infection and parasite density in individuals from the Brazilian Amazon.

BACKGROUND: In malaria infection, apoptosis acts as an important immunomodulatory mechanism that leads to the elimination of parasitized cells, thus reducing the parasite density and controlling immune cell populations. Here, it was investigated the association of INDEL variants in apoptotic genes-rs10562972 (FAS), rs4197 (FADD), rs3834129 and rs59308963 (CASP8), rs61079693 (CASP9), rs4647655 (CASP3), rs11269260 (BCL-2), and rs17880560 (TP53)-and the influence of genetic ancestry with susceptibility to malaria and parasite density in an admixed population from the Brazilian Amazon. METHODS: Total DNA was extracted from 126 malaria patients and 101 uninfected individuals for investigation of genetic ancestries and genotypic distribution of apoptosis-related variants by Multiplex PCR. Association analyses consisted of multivariate logistic regressions, considering the following comparisons: (i) DEL/DEL genotype vs. INS/DEL + INS/INS; and (ii) INS/INS vs. INS/DEL + DEL/DEL. RESULTS: Individuals infected by Plasmodium falciparum had significantly higher African ancestry proportions in comparison to uninfected controls, Plasmodium vivax, and mixed infections. The INS/INS genotype of rs3834129 (CASP8) seemed to increase the risk for P. falciparum infection (P = 0.038; OR = 1.867; 95% CI 0.736-3.725), while the DEL/DEL genotype presented a significant protective effect against infection by P. falciparum (P = 0.049; OR = 0.446; 95% CI 0.185-0.944) and mixed infection (P = 0.026; OR = 0.545; 95% CI 0.281-0.996), and was associated with lower parasite density in P. falciparum malaria (P = 0.009; OR = 0.383; 95% CI 0.113-1.295). Additionally, the INS/INS genotype of rs10562972 (FAS) was more frequent among individuals infected with P. vivax compared to P. falciparum (P = 0.036; OR = 2.493; 95% CI 1.104-4.551), and the DEL/DEL genotype of rs17880560 (TP53) was significantly more present in patients with mono-infection by P. vivax than in individuals with mixed infection (P = 0.029; OR = 0.667; 95% CI 0.211-1.669). CONCLUSIONS: In conclusion, variants in apoptosis genes are associated with malaria susceptibility and parasite density, indicating the role of apoptosis-related genetic profiles in immune responses against malaria infection.

Association of cerebral malaria and TNF-α levels: a systematic review.

BACKGROUND: Cerebral malaria is the most severe form of infection with Plasmodium falciparum characterized by a highly inflammatory response. This systematic review aimed to investigate the association between TNF-α levels and cerebral malaria. METHODS: This review followed the Preferred Reporting of Systematic Review and Meta-analyses (PRISMA) guidelines. The search was performed at PubMed, LILACS, Scopus, Web of Science, The Cochrane Library, OpenGrey and Google Scholar. We have included studies of P. falciparum-infected humans with or without cerebral malaria and TNF-α dosage level. All studies were evaluated using a risk of bias tool and the GRADE approach. RESULTS: Our results have identified 2338 studies, and 8 articles were eligible according to this systematic review inclusion criteria. Among the eight articles, five have evaluated TNF- α plasma dosage, while two have evaluated at the blood and one at the brain (post-Morten). Among them, only five studies showed higher TNF-α levels in the cerebral malaria group compared to the severe malaria group. Methodological problems were identified regarding sample size, randomization and blindness, but no risk of bias was detected. CONCLUSION: Although the results suggested that that TNF-α level is associated with cerebral malaria, the evidence is inconsistent and imprecise. More observational studies evaluating the average TNF-alpha are needed.

Strategy to improve malaria surveillance system preventing transfusion-transmitted malaria in blood banks using molecular diagnostic.

BACKGROUND: Malaria can be transmitted by blood transfusion through donations collected from asymptomatic or parasitic donors. The parasites are released into the bloodstream during its life cycle and will therefore be present in donated blood by infected individuals. All cases of transfusion-transmitted malaria (TTM) notified since 2005 in Brazil were fatal. A good screening tool for Plasmodium spp. detection in blood units must have a high detection threshold, and the prevention of TTM relies entirely on the exclusion of potentially infected donors. However, in Brazilian blood banks, the screening test relies on blood thick smears examination. METHODS: The molecular diagnostic based on mitochondrial DNA (mtDNA) using real time PCR (mt-qPCR) was improved to detect Plasmodium falciparum, Plasmodium vivax, and standardized for use in Plasmodium malariae. The analytic sensitivity of this mt-qPCR methodology was performed using a sample of P. vivax. RESULTS: The mt-qPCR was highly efficient, and the analytic sensitivity for P. vivax was determined (0.000006 parasites/µL). This method was tested to detect P. vivax and P. falciparum in individuals from two malaria-endemic areas in Brazil, Amazon region (Pará and Rondônia states), the samples were collected in 10 reference units of two blood banks (Pará/nine cities and Rondônia/Porto Velho), and parasites mtDNA were detected in 10 of 2224 potential blood donors (0.45%). In all 10 positive samples, only P. vivax was detected. CONCLUSION: Molecular diagnostic using mt-qPCR was effective in revealing infected potential donors with good perspectives to be applied as screening routine of asymptomatic carriers for preventing transfusion-transmitted malaria in blood banks.

Immunogenetic markers associated with a naturally acquired humoral immune response against an N-terminal antigen of Plasmodium vivax merozoite surface protein 1 (PvMSP-1).

BACKGROUND: Humoral immune responses against proteins of asexual blood-stage malaria parasites have been associated with clinical immunity. However, variations in the antibody-driven responses may be associated with a genetic component of the human host. The objective of the present study was to evaluate the influence of co-stimulatory molecule gene polymorphisms of the immune system on the magnitude of the humoral immune response against a Plasmodium vivax vaccine candidate antigen. METHODS: Polymorphisms in the CD28, CTLA4, ICOS, CD40, CD86 and BLYS genes of 178 subjects infected with P. vivax in an endemic area of the Brazilian Amazon were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The levels of IgM, total IgG and IgG subclasses specific for ICB2-5, i.e., the N-terminal portion of P. vivax merozoite surface protein 1 (PvMSP-1), were determined by enzyme-linked immuno assay. The associations between the polymorphisms and the antibody response were assessed by means of logistic regression models. RESULTS: After correcting for multiple testing, the IgG1 levels were significantly higher in individuals recessive for the single nucleotide polymorphism rs3116496 in CD28 (p = 0.00004). Furthermore, the interaction between CD28 rs35593994 and BLYS rs9514828 had an influence on the IgM levels (p = 0.0009). CONCLUSIONS: The results of the present study support the hypothesis that polymorphisms in the genes of co-stimulatory components of the immune system can contribute to a natural antibody-driven response against P. vivax antigens.

Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon.

BACKGROUND: Several studies have recently demonstrated that the immune responses against malaria is governed by different factors, including the genetic components of the host. The IL-4 gene appears to be a strong candidate factor because of its role in the regulation of the Th2 response. The present study investigated the role of IL-4 polymorphisms in the development of IgG antibodies against PvAMA-1 and the IL-4 levels in individuals infected with Plasmodium vivax in a malaria endemic area in the Brazilian Amazon. METHODS: The study sample included 83 patients who were diagnosed with P. vivax infection using thick smear and confirmed by nested-PCR. The IL-4 -590C>T and IL-4 -33C>T polymorphisms were genotyped by PCR-RFLP, and the intron 3 VNTR was genotyped by PCR. A standardised ELISA protocol was used to measure the total IgG against PvAMA-1. The cytokine/chemokine levels were measured using a Milliplex multiplex assay (Millipore). All of the subjects were genotyped with 48 ancestry informative markers to determine the proportions of African, European and Amerindian ancestry using STRUCTURE software. RESULTS: Of the 83 patients, 60 (73%) produced IgG antibodies against PvAMA-1. A significant decrease in the percentage of respondents was observed among the primo-infected individuals. No significant differences were observed in the frequencies of genotypes and haplotypes among individuals who were positive or negative for IgG antibodies against PvAMA-1. Furthermore, no significant correlation was observed between the IL-4 polymorphisms, antibody levels, IL-4 levels, and parasitemia. CONCLUSIONS: This study indicated that the polymorphisms identified in the IL-4 gene are not likely to play a role in the regulation of the antibody response against PvAMA-1 and IL-4 production in vivax malaria.

Aspidosperma species as sources of anti-malarials: uleine is the major anti-malarial indole alkaloid from Aspidosperma parvifolium (Apocynaceae).

BACKGROUND: Several species of the genus Aspidosperma (Apocynaceae) are used for the treatment of human malaria in Brazil and other meso- and South American countries. METHODS: Ethanol extract from Aspidosperma parvifolium trunk bark was submitted to acid-base extractions leading to alkaloid and neutral fractions. The alkaloid fraction was chromatographed over a silica gel column. Ethanol extract, fractions and uleine were analysed by HPLC-DAD, UPLC-ESI-MS/MS and HPLC-ESI-MicroTOF-MS. The anti-malarial activity was assayed against resistant and sensitive chloroquine Plasmodium falciparum strains by microscopic, [(3)H]-hypoxanthine incorporation and HRPII techniques. Cytotoxicity (CC50) was evaluated against Vero and HepG2 cell lines by the MTT technique; selectivity indexes (SI = CC50/IC50) were calculated. RESULTS: The major peak in the HPLC-DAD chromatograms of the ethanol extract, alkaloid and neutral fractions suggested the presence of uleine that was isolated from the alkaloid fraction by column chromatography and was characterized by spectroscopic methods. A total of 15 alkaloids, besides uleine, were identified in the alkaloid fraction by UPLC-DAD-ESI-MS/MS and HPLC-ESI-MicroTOF-MS. The ethanol extract from Aspidosperma parvifolium and the neutral fraction were moderately active against P. falciparum strains. The alkaloid fraction and uleine disclosed high anti-malarial activity against chloroquine-resistant P. falciparum strain (IC50 < 1 µg/mL). The ethanol extract, neutral fraction and uleine showed low cytotoxicity against Vero and HepG2 cell lines (CC50 > 300 µg/mL). The alkaloid fraction showed moderate cytotoxicity to HepG2 cell line (CC50 = 74.4 µg/mL). High SI values (>10) were determined for all samples. CONCLUSION: Ethanol extract from Aspidosperma parvifolium trunk bark afforded uleine that is the major constituent of the alkaloid fraction and disclosed a good in vitro anti-malarial activity. Moreover, 15 other indole alkaloids have been identified along with uleine.

The evolutionary history of Plasmodium vivax as inferred from mitochondrial genomes: parasite genetic diversity in the Americas.

Plasmodium vivax is the most prevalent human malaria parasite in the Americas. Previous studies have contrasted the genetic diversity of parasite populations in the Americas with those in Asia and Oceania, concluding that New World populations exhibit low genetic diversity consistent with a recent introduction. Here we used an expanded sample of complete mitochondrial genome sequences to investigate the diversity of P. vivax in the Americas as well as in other continental populations. We show that the diversity of P. vivax in the Americas is comparable to that in Asia and Oceania, and we identify several divergent clades circulating in South America that may have resulted from independent introductions. In particular, we show that several haplotypes sampled in Venezuela and northeastern Brazil belong to a clade that diverged from the other P. vivax lineages at least 30,000 years ago, albeit not necessarily in the Americas. We propose that, unlike in Asia where human migration increases local genetic diversity, the combined effects of the geographical structure and the low incidence of vivax malaria in the Americas has resulted in patterns of low local but high regional genetic diversity. This could explain previous views that P. vivax in the Americas has low genetic diversity because these were based on studies carried out in limited areas. Further elucidation of the complex geographical pattern of P. vivax variation will be important both for diversity assessments of genes encoding candidate vaccine antigens and in the formulation of control and surveillance measures aimed at malaria elimination.

Ground-dwelling invertebrates from an Urban park in the Brazilian Amazon, with particular reference to Phlebotominae (Diptera: Psychodidae).

The ground-dwelling invertebrate fauna from an urban park in Belém, Amazonian Brazil, with particular reference to the subfamily Phlebotominae (Diptera: Psychodidae), were characterized. Sampling was performed from March 2022 to May 2023, with 10 emergence traps installed in 2 microhabitats, 5 in each 1: "M1," which included surrounding (up to 1.5 m) trees with tabular roots, and "M2," which included no trees with tabular roots. Invertebrates trapped in adhesive papers were assessed on 2 occasions/cycles (D21/D42). During 10 sampling cycles (10 traps/cycle = 100 samples), 6,490 invertebrates were captured (M1, n = 4,203; M2, n = 2,287) and classified into 5 classes and 21 orders, with Diptera (n = 2,309; 35.6%) being the most abundant. Twenty-nine specimens of the following phlebotomine species were captured: Nyssomyia antunesi (M1, n = 10; M2, n = 3), Trichophoromyia ubiquitalis (M1, n = 6; M2, n = 1), Th. brachipyga (M1, n = 0; M2, n = 2), Bichromomyia flaviscutellata (M1, n = 2; M2, n = 1) and 4 unidentified specimens (M1, n = 2; M2, n = 2). The male/female ratio was 1.08. Fractional vegetation cover was compared, and the physiochemical characteristics of the soil were compared between the microhabitats. Only temperature showed significant differences. A weak positive correlation was found between phlebotomines and other dipterans and between temperature and the amount of organic matter in the soil. Both sampled microhabitats were shown to be suitable for the development and maintenance of different invertebrates, mainly dipterans. The composition of ground-emerging phlebotomine species was similar to that previously surveyed with light traps, including species associated with the transmission of Leishmania spp.

Experimental Susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis (Psychodidae: Phlebotominae) to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi (Trypanosomatidae: Leishmaniinae).

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi-Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis-parasite combinations, as well with Lu. longipalpis-uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.

Malaria in a vulnerable population living in quilombo remnant communities in the Brazilian Amazon: a cross-sectional study from 2005-2020.

Quilombo remnant communities are areas officially recognized by the Brazilian government as historical communities founded by formerly enslaved individuals. These communities are mostly located in the endemic areas of malaria in the Brazilian Amazon. We retrospectively described the prevalence of malaria among individuals living in 32 recognized quilombo remnant communities in the Baiao and Oriximina municipalities located in the Para State. The number of malaria cases and the Annual Parasitic Incidence (API) recorded by the Brazilian malaria surveillance system (SIVEP-Malaria) from January 2005 to December 2020 were analyzed. We found that all communities registered at least one case over the 16-year period, the most frequent parasitic species being Plasmodium vivax (76.1%). During this period, 0.44% (4,470/1,008,714) of the malaria cases registered in Para State were reported in these quilombo remnant communities, with frequencies of 10.9% (856/7,859) in Baiao municipality and 39.1% (3,614/9,238) in Oriximina municipality, showing that individuals living in these rural communities are exposed to malaria. These data indicate that effective surveillance requires improved measures to identify malaria transmission among vulnerable populations living in quilombo remnant communities in the Brazilian Amazon.

Physical durability and insecticidal activity of long-lasting insecticidal nets in Cruzeiro do Sul, Brazil.

Vector control is one of the principal strategies used for reducing malaria transmission. Long-lasting insecticidal bed nets (LLINs) are a key tool used to protect populations at risk of malaria, since they provide both physical and chemical barriers to prevent human-vector contact. This study aimed to assess the physical durability and insecticidal efficacy of LLINs distributed in Cruzeiro do Sul (CZS), Brazil, after 4 years of use. A total of 3000 LLINs (PermaNet 2.0) were distributed in high malaria risk areas of CZS in 2007. After 4 years of use, 27 'rectangular' LLINs and 28 'conical' LLINs were randomly selected for analysis. The evaluation of physical integrity was based on counting the number of holes and measuring their size and location on the nets. Insecticidal efficacy was evaluated by cone bioassays, and the amount of residual insecticide remaining on the surface of the LLINs was estimated using a colorimetric method. After 4 years of use, physical damage was highly prevalent on the rectangular LLINs, with a total of 473 holes detected across the 27 nets. The upper portion of the side panels sustained the greatest damage in rectangular LLINs. The overall mosquito mortality by cone bioassay was < 80% in 25/27 rectangular LLINs, with panel A (at the end of the rectangular bednet) presenting the highest mortality (54%). The overall mean insecticide concentration was 0.5 µg/sample, with the bednet roof containing the highest average concentration (0.61 µg/sample). On the conical LLINs, 547 holes were detected, with the bottom areas sustaining the greatest damage. The cone bioassay mortality was < 80% in 26/28 of the conical LLINs. The mean insecticide concentration was 0.3 µg/sample. After 4 years of use, the insecticidal efficacy of the LLINs was diminished to below acceptable thresholds.

Distinct population structure for co-occurring Anopheles goeldii and Anopheles triannulatus in Amazonian Brazil.

To evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in Anopheles triannulatus, larval habitat characteristics across the Brazilian states of Roraima and Pará and genetic sequences were examined. A comparison with Anopheles goeldii was utilised to determine whether high genetic diversity was unique to An. triannulatus. Student t test and analysis of variance found no differences in habitat characteristics between the species. Analysis of population structure of An. triannulatus and An. goeldii revealed distinct demographic histories in a largely overlapping geographic range. Cytochrome oxidase I sequence parsimony networks found geographic clustering for both species; however nuclear marker networks depicted An. triannulatus with a more complex history of fragmentation, secondary contact and recent divergence. Evidence of Pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography. We hypothesise that niche partitioning is a driving force for diversity, particularly in An. triannulatus.

Polycystic echinococcosis in the state of Acre, Brazil: contribution to patient diagnosis, treatment and prognosis.

The lack of knowledge regarding polycystic hydatid disease results in delayed or even incorrect diagnosis. The lack of systematic information regarding treatment also makes it difficult to assess the results and prognosis in patients with peritoneal and hepatic lesions caused by Echinococcus vogeli. Here we describe the clinical features of patients, propose a radiological classification protocol and describe a therapeutic option for the treatment of hydatid disease that previously had only been used for cases of cystic echinococcosis (Echinococcus granulosus). A prospective cohort study was initiated in 1999 and by 2009 the study included 60 patients. These patients were classified according to the PNM classification (parasite lesion, neighbouring organ invasion and metastases) and placed in one of three therapeutic modalities: (i) chemotherapy with albendazole at a dose of 10 mg/kg/day, (ii) surgical removal of cysts or (iii) percutaneous puncture of the cysts via puncture, aspiration, injection and re-aspiration (PAIR). The results were stratified according to therapeutic outcome: "cure", "clinical improvement", "no improvement", "death" or "no information". The PNM classification was useful in indicating the appropriate therapy in cases of polycystic hydatid disease. In conclusion, surgical therapy produced the best clinical results of all the therapies studied based on "cure" and "clinical improvement" outcomes. The use of PAIR for treatment requires additional study.

Morphological atypia and molecular profile of Plasmodium vivax: Findings from an outbreak in the Brazilian Amazon.

This study aimed to perform morphological and molecular analyses of parasites isolated from the blood of malaria-infected individuals during an outbreak in the Microregion of Cametá, State of Pará, Brazilian Amazon. A total of 260 positive samples were identified by microscopy as Plasmodium vivax; however, in three samples, forms considered unusual for the species were found and defined as morphological atypia of P. vivax. Single P. vivax infection was confirmed by qPCR in all samples. Among 256 genotyped samples, the VK247 genotype alone was identified in 255 samples, and the VK210 genotype was found in only one. The study showed that this malaria outbreak was caused by the etiological agent P. vivax, and for the first time, morphological atypia was described in isolates circulating in Brazil. Likewise, for the first time, the VK247 genotype was detected predominantly in single infections in an area of the State of Pará, which may suggest a greater circulation of the genotype in the region.

Title: Atypie morphologique et profil moléculaire de Plasmodium vivax : résultats issus d'une épidémie en Amazonie brésilienne. Abstract: Cette étude visait à effectuer des analyses morphologiques et moléculaires de parasites isolés du sang d'individus infectés par le paludisme lors d'une épidémie dans la microrégion de Cametá, État du Pará, Amazonie brésilienne. Au total, 260 échantillons positifs ont été identifiés par microscopie comme Plasmodium vivax mais dans trois échantillons, des formes considérées comme inhabituelles pour l'espèce ont été trouvées et définies comme des atypies morphologiques de P. vivax. Une infection simple à P. vivax a été confirmée par qPCR dans tous les échantillons. Sur 256 échantillons génotypés, le génotype VK247 seul a été identifié dans 255 échantillons, et le génotype VK210 a été trouvé dans un seul échantillon. L'étude a montré que cette épidémie de paludisme était causée par l'agent étiologique P. vivax et, pour la première fois, une atypie morphologique a été décrite dans des isolats circulant au Brésil. De même, pour la première fois, le génotype VK247 a été détecté principalement dans des infections uniques dans une zone de l'État de Pará, ce qui peut suggérer une plus grande circulation du génotype dans la région.

Implementation of basic quality control tests for malaria medicines in Amazon Basin countries: results for the 2005-2010 period.

BACKGROUND: Ensuring the quality of malaria medicines is crucial in working toward malaria control and eventual elimination. Unlike other validated tests that can assess all critical quality attributes, which is the standard for determining the quality of medicines, basic tests are significantly less expensive, faster, and require less skilled labour; yet, these tests provide reproducible data and information on several critical quality attributes, such as identity, purity, content, and disintegration. Visual and physical inspection also provides valuable information about the manufacturing and the labelling of medicines, and in many cases this inspection is sufficient to detect counterfeit medicines. The Promoting the Quality of Medicines (PQM) programme has provided technical assistance to Amazon Malaria Initiative (AMI) countries to implement the use of basic tests as a key screening mechanism to assess the quality of malaria medicines available to patients in decentralized regions. METHODS: Trained personnel from the National Malaria Control Programmes (NMCPs), often in collaboration with country's Official Medicine Control Laboratory (OMCL), developed country- specific protocols that encompassed sampling methods, sample analysis, and data reporting. Sampling sites were selected based on malaria burden, accessibility, and geographical location. Convenience sampling was performed and countries were recommended to store the sampled medicines under conditions that did not compromise their quality. Basic analytical tests, such as disintegration and thin layer chromatography (TLC), were performed utilizing a portable mini-laboratory. RESULTS: Results were originally presented at regional meetings in a non-standardized format that lacked relevant medicines information. However, since 2008 information has been submitted utilizing a template specifically developed by PQM for that purpose. From 2005 to 2010, the quality of 1,663 malaria medicines from seven AMI countries was evaluated, mostly collected from the public sector, 1,445/1,663 (86.9%). Results indicate that 193/1,663 (11.6%) were found not to meet quality specifications. Most failures were reported during visual and physical inspection, 142/1663 (8.5%), and most of these were due to expired medicines, 118/142 (83.1%). Samples failing TLC accounted for 27/1,663 (1.6%) and those failing disintegration accounted for 24/1,663 (1.4%). Medicines quality failures decreased significantly during the last two years. CONCLUSIONS: Basic tests revealed that the quality of medicines in the public sector improved over the years, since the implementation of this type of quality monitoring programme in 2005. However, the lack of consistent confirmatory tests in the quality control (QC) laboratory, utilizing methods that can also evaluate additional quality attributes, could still mask quality issues. In the future, AMI countries should improve coordination with their health authorities and their QC lab consistently, to provide a more complete picture of malaria medicines quality and support the implementation of corrective actions. Facilities in the private and informal sectors also should be included when these sectors constitute an important source of medicines used by malaria patients.

In vitro antimalarial activity of six Aspidosperma species from the state of Minas Gerais (Brazil).

Ethnomedicinal informations point to some Aspidosperma species (Apocynaceae) as antimalarial plants in Brazil and have motivated the evaluation of six species which were collected in the state of Minas Gerais: A. cylindrocarpon Müll. Arg., A. parvifolium A. DC., A. olivaceum Müll. Arg., A. ramiflorum Müll. Arg., A. spruceanum Benth. ex Müll. Arg. and A. tomentosum Mart.. A total of 23 extracts of different plant parts in different solvents were assayed in vitro against chloroquine-resistant (W2) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum. All the extracts were shown to be active with IC50 values in the range of 5.0 ± 0 2.8 µg/mL to 65.0 ± 4.2 µg/mL. TLC profile of the extracts revealed the presence of alkaloids in the six species assayed. These results seem to confirm the popular use of Aspidosperma species to treat human malaria in Brazil and seem point to alkaloids as the putative active compounds of the assayed species.

Artificial blood-feeding of phlebotomines (Diptera: Psychodidae: Phlebotominae): is it time to repurpose biological membranes in light of ethical concerns?

BACKGROUND: The aims of the present study were to evaluate and compare the efficacy of blood-feeding in phlebotomines through industrially processed membranes from the small intestine of pigs (used for the production of commercial sausages) and the skin of euthanized chicks. METHODS: Laboratory-bred Lutzomyia longipalpis and different field-caught phlebotomine species were subjected to the artificial feeding systems under similar conditions. Paired tests were performed using the control (skin from euthanized chicks) and test membranes (pig small intestine). The feeding rates were compared by paired t-test, and Pearson correlation was used to examine the relationship between the thickness of the membranes and feeding rate. RESULTS: The feeding rate was greater with the test membrane than with the control membrane for L. longipalpis (t-test, t = -3.3860, P = 0.0054) but not for the most frequent field-caught species, Nyssomyia antunesi (t-test, t = 0.7746, P = 0.4535). The average thicknesses of the control and test membranes were 184 ± 83 µm and 34 ± 12 µm, respectively (Mann-Whitney U-test, U = 0.00, Z = 2.8823, P = 0.0039); however, there was no correlation between feeding rate and membrane thickness. A moderate positive correlation was observed between the number of phlebotomines that fed and the total number of phlebotomines in the cage for each type of membrane and for each species. CONCLUSIONS: The test membrane is a viable alternative for the artificial blood-feeding of phlebotomines, and is thus a potential substitute for the skin of animals that are euthanized for this purpose. Feeding rate was independent of membrane thickness.

Phylogeny of Anopheles darlingi (Diptera:Culicidae) based on the antimicrobial peptide genes cecropin and defensin.

Cecropins and defensins are the main classes of antimicrobial peptides in the mosquito innate immune system, acting against bacteria, fungi and protozoa. There is a knowledge gap concerning these peptide genes in anopheline mosquitoes from the Brazilian Amazon. Thus, this work aimed to describe molecular techniques for detecting the genes encoding the antimicrobial peptides cecropin A (CecA) and defensin in Anopheles darlingi mosquitoes and to perform molecular phylogeny of the sequenced genes using the maximum likelihood method and Bayesian inference with other species from different geographic areas. Our results show, for the first time, a molecular biology method for detecting CecA and defensin in Anopheles darlingi that allows for the use of these molecular markers for phylogenetic analysis in anopheline species, separating the species into single and monophyletic clades.

Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex.

Many vectors of human malaria belong to complexes of morphologically indistinguishable cryptic species. Here we report the analysis of the newly sequenced complete mitochondrial DNA molecules from six recognized or putative species of one such group, the Neotropical Anopheles albitarsis complex. The molecular evolution of these genomes had been driven by purifying selection, particularly strongly acting on the RNA genes. Directional mutation pressure associated with the strand-asynchronous asymmetric mtDNA replication mechanism may have shaped a pronounced DNA strand asymmetry in the nucleotide composition in these and other Anopheles species. The distribution of sequence polymorphism, coupled with the conflicting phylogenetic trees inferred from the mitochondrial DNA and from the published white gene fragment sequences, indicates that the evolution of the complex may have involved ancient mtDNA introgression. Six protein coding genes (nad5, nad4, cox3, atp6, cox1 and nad2) have high levels of sequence divergence and are likely informative for population genetics studies. Finally, the extent of the mitochondrial DNA variation within the complex supports the notion that the complex consists of a larger number of species than until recently believed.

Increased interleukin-10 and interferon-γ levels in Plasmodium vivax malaria suggest a reciprocal regulation which is not altered by IL-10 gene promoter polymorphism.

BACKGROUND: In human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed. METHODS: The serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). RESULTS: The levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma. CONCLUSIONS: This study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.