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.

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.

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.

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.

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.

T. gondii Infection in Urban and Rural Areas in the Amazon: Where Is the Risk for Toxoplasmosis?

Toxoplasmosis, an infection caused by Toxoplasma gondii, which is found worldwide, can affect human and animal health in different ways. This study aimed to estimate the infection prevalence in humans and to determine risk factors related to urban and rural areas in a municipality in the Brazilian Amazon where an outbreak had been registered. Blood samples for serological analysis were obtained, and interviews were performed to fill out an epidemiological questionnaire. A total of 1140 individuals were included, of which 70.6% (804/1140; 95% CI: 67.9-73.2%) were positive for IgG anti-T. gondii antibodies. In rural areas, the prevalence was 62.6% (95% CI: 58.9-66.3%), while in urban areas, it was 81.9% (95% CI: 78.4-85.4%). The risk of becoming infected in urban areas was 2.7 times higher (95% CI = 2.0-3.6%) than that in rural areas. When comparing the prevalence in the age group from 1 to 10 years in both areas, the rate was 28.6% (42/147; 95% CI: 21.3-35.9%) for rural areas and 69.4% (61/88; CI 95%: 59.7-79.0%) for urban areas. Therefore, it is concluded that parasite exposure starts in the first years of life in urban areas and that disordered urban area expansion may cause an increase in exposure to the different strains of T. gondii present in the Amazon.

Binational burden of American cutaneous leishmaniasis in Oiapoque, Amapá State, Brazil, bordering French Guiana.

INTRODUCTION: American cutaneous leishmaniasis (ACL) is regarded as a public health problem in the Oiapoque basin, between Brazil and French Guiana. METHODS: Data on ACL occurrence/epidemiological profile and etiology were sourced from Brazilian health services and a reference laboratory. Rainfall correlation was also analyzed. RESULTS: Majority of the ACL cases were observed in adult men working as gold miners. ACL incidence peaks appeared to be linked to periods 2 months after the dry season. Migratory flow was found to be a non-negligible complicating factor in epidemiological surveillance. CONCLUSIONS: Binational strategies are required to minimize exposure for high-risk populations.

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

ABSTRACT 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.

Morphological identification of species of the Nuneztovari Complex of Anopheles (Diptera: Culicidae) from an area affected by a Brazilian hydroelectric plant.

The Nuneztovari Complex of Anopheles (Diptera: Culicidae) comprises four species: An. nuneztovari Gabaldon, An. goeldii Rozeboom Gabaldon, An. dunhami Causey and An. nuneztovari species A. This study aimed to identify morphologically the species of the Nuneztovari Complex that occur in the area of the Belo Monte hydroelectric dam. The morphological identification of adult males and male genitalia (aedeagus and ventral claspette) was performed. A statistical analysis of the difference in aedeagal leaflet length was done using the Mann-Whitney test. Of the 38 male genitalia of specimens of the Nuneztovari Complex examined, 33 were identified as An. goeldii/An. nuneztovari A and five as An. nuneztovari s.s. A statistically significant difference in aedeagal leaflet length was detected between the species: the mean length was 1.23 µm for An. goeldii/An. nuneztovari A and 9.18 µm for An. nuneztovari s.s. This is the first record of An. nuneztovari s.s.in areas of environmental modification in the Brazilian Amazon. This study provides a measurement tool that can identify and differentiate species of the complex in the region, which can be applied to the other species of the complex as well to other anopheline species; thus, fostering the acquisition of information about the role of each species in malaria transmission.

Leishmania DNA detection and species characterization within phlebotomines (Diptera: Psychodidae) from a peridomicile-forest gradient in an Amazonian/Guianan bordering area.

In the border region between Brazil and French Guiana, American cutaneous leishmaniasis is a worrisome public health issue, and entomological studies are required there to better identify classical and putative emerging transmission patterns. The present study aimed to detect and characterize Leishmania DNA in the phlebotomine population of Oiapoque (Amapá State, Brazil). Phlebotomines were captured in anthropized and wild environments in the outskirts of Oiapoque municipality, using CDC light traps installed in vertical (ground/canopy level) and horizontal (peridomicile/extradomicile/forest-edge/forest) strata. Captured specimens were identified according to their morphology. Females were processed for Leishmania DNA detection and characterization using a multiplex polymerase chain reaction targeting kinetoplast DNA (kDNA) and the phlebotomine cacophony gene. The kDNA positive samples were characterized by cloning and sequencing the Leishmania 234 bp-hsp70 gene. Among the 3957 phlebotomine specimens captured, 26 pooled female samples were positive for Leishmania (Viannia) spp. DNA. Sequencing analysis allowed species-specific identification of L. (V.) braziliensis DNA in Trichophoromyia ininii, Bichromomyia flaviscutellata, Nyssomyia umbratilis, and Evandromyia infraspinosa, and L. (V.) guyanensis DNA in Ny. umbratilis. A pooled sample of Ny. umbratilis was positive for both L. (V.) braziliensis and L. (V.) guyanensis DNA. The present study provided additional information regarding ACL ecology in Oiapoque, highlighting the presence of L. (V.) braziliensis DNA in different phlebotomine species. The epidemiological implications of these findings and the determinant incrimination of L. (V.) braziliensis as proven vectors in that region must be clarified. In this regard, studies on Leishmania spp. infection and suggestive anthropophilic behavior of associated phlebotomines need to be prioritized in entomological surveillance.

VK210 and VK247 genotypes of Plasmodium vivax in anopheline mosquitoes from Brazilian Amazon.

Plasmodium vivax sporozoites are differenced by circumsporozoite protein. Studies on the circulation of P. vivax VK210 and P. vivax VK247 in anopheline mosquitoes are important to verify the adaptability of these parasites on mosquitoes in different locations and periods. This study aimed to describe and compare the distribution of these genotypes in anopheline mosquitoes from four states of the Brazilian Amazon. Epidemiological databases about CSP infections on mosquitoes from Pará (2000-2015), Amapá (2000-2010), Roraima (2000-2003 and 2009-2011) and Acre States (2012-2015) were used for analysis. A total of 895 specimens were found infected mainly by P. vivax VK210. We showed that the distribution of P. vivax VK247 changed over time in the main malaria vectors on the Brazilian Amazon. We note that A. darlingi was abundant in certain localities while A. albitarsis s.l. in anothers, which highlights the importance of entomological studies for the control of human malaria.

Ecological aspects of Phlebotomines (Diptera: Psychodidae) and the transmission of American cutaneous leishmaniasis agents in an Amazonian/ Guianan bordering area.

BACKGROUND: An entomological study was conducted in the municipality of Oiapoque (lower Oyapock River Basin) in the Brazilian side bordering French Guiana to gain information on the transmission pattern of American cutaneous leishmaniasis (ACL) in that region, presumed to reflect the classical Amazonian/Guianan enzootic scenario. METHODS: Three ecologically isolated forested areas near urban environments were surveyed during the rainy and dry seasons of 2015 and 2016, using a multi-trapping approach comprising ground-level and canopy light traps, black and white colored cloth Shannon traps and manual aspiration on tree bases. Female phlebotomines were dissected to find infections and isolate flagellates from Leishmania spp. The strains were characterized by restriction fragment length polymorphism analysis and compared with those of local ACL cases and World Health Organization reference strains. RESULTS: Nyssomyia umbratilis, Trichopygomyia trichopyga and Evandromyia infraspinosa were the most frequently found species. Findings on relative abundance, spatiotemporal vector/ACL congruence, natural infections and anthropophilic insights strengthened the Guianan classical transmission of Leishmania (Viannia) guyanensis by Ny. umbratilis and suggested further investigations for Ev. infraspinosa. Nyssomyia umbratilis showed an eclectic feeding habit, including bird blood. Ecological data and literature reports also included Psychodopygus squamiventris maripaensis and Bichromomyia flaviscutellata on the list of suspected vectors. CONCLUSIONS: These findings contributed to understanding ACL ecoepidemiology in the Amazonian/Guianan scenario. Local studies are required to better comprehend the Leishmania spp. enzootic mosaic in specific ecotopes.

Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil.

Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loop-mediated isothermal amplification-based molecular tool (LAMP), facilitates rapid target amplification at a single temperature setting, reducing the need for sophisticated equipment. We evaluated the performance of a field-adapted RealAmp assay for malaria diagnosis in Cruzeiro do Sul, Acre State, Brazil, a remote area in Brazil with limited laboratory capabilities. We enrolled 1,000 patients with fever (axillary temperature ≥ 37.5 C) or history of fever in last 24 h presenting for malaria diagnosis from February through June 2015. DNA was extracted from dried blood spots using a boil and spin method (heat treatment) at the sample processing site, and also using commercial kits at a Brazilian national reference laboratory. RealAmp was performed for Plasmodium genus, P. falciparum, and P. vivax identification. In addition, Giemsa-stained blood smears were prepared and examined by two independent well-trained study microscopists. A combination of Real-time PCR and nested PCR was used as reference test. The sensitivity and specificity of RealAmp in the field site laboratory were 94.1% (95% confidence interval [CI]: 90.1-96.8) and 83.9% (95% CI: 81.1-86.4), respectively. The sensitivity and specificity of local microscopy were 87.7% (95% CI: 82.6-91.7) and 98.9% (95% CI: 97.8-99.4), respectively, while study microscopy showed sensitivity of 96.4% (95% CI: 93.0-98.4) and specificity of 98.2% (95% CI: 97.0-99.0). None of the three tests detected 20 P. falciparum and P. vivax mixed infections identified by the reference test. Our findings highlight that it is possible to implement simple molecular tests in facilities with limited resources such as Cruzeiro do Sul in Brazil. RealAmp sensitivity was similar to that of microscopy performed by skilled professionals; both RealAmp and study microscopy performed poorly in detection of mixed infection. Attempts to develop and evaluate simpler molecular tools should continue, especially for the detection of malaria infection in remote areas.

A new polymerase chain reaction/restriction fragment length polymorphism protocol for Plasmodium vivax circumsporozoite protein genotype (VK210, VK247, and P. vivax-like) determination.

For the molecular diagnosis of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) using DNA amplification procedures in the laboratory, the choice of rapid and inexpensive identification products of the 3 different genotypes is an important prerequisite. We report here the standardization of a new polymerase chain reaction/restriction fragment length polymorphism technique to identify the 3 described P. vivax circumsporozoite protein (CSP) variants using amplification of the central immunodominant region of the CSP gene of this protozoan. The simplicity, specificity, and sensitivity of the system described here is important to determine the prevalence and the distribution of infection with these P. vivax genotypes in endemic and nonendemic malaria areas, enabling a better understanding of their phylogeny.