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A single-round multiplex PCR (mPCR) with species-specific primers (SSP) of three mitochondrial genes of Plasmodium, namely COX I, COX III and CYT B, was compared to microscopy and 18S rRNA semi-nested PCR, nested-PCR and Real Time PCRs (*PCRs). Each parasite has between 20 and 150 mitochondria and each mitochondria has one copy of each target gene, while 18S rRNA gene is repeated 4 to 8 times. The specificity of mPCR was assessed by testing Plasmodium from rodents and birds, parasites responsible for other endemic diseases in the country such as schistosomiasis, Chagas disease and leishmaniasis in addition to microorganisms that, like Plasmodium, can cause anemia (Bartonella henselae, Babesia vogeli, Rickettsia vini). No cross-reactions were detected. From a total of 149 specimens from suspected cases of malaria were tested, 97 were positive by microscopy (49 P. falciparum, 38 P. vivax, 6 P. malariae, 4 P. falciparum/P. vivax- mixed infections) and 52 were negative; 148 samples were positive by *PCRs (49 P. falciparum, 53 P. vivax, 7 P. malariae and 39 mixed infections) and one was negative; 146 were positive by mPCR (49 P. falciparum, 56 P. vivax, 9 P. malariae and 32 mixed infections) and three were negative. The comparison of groups found statistically significant differences between microscopy vs.*PCRs or vs. mPCR (p-values0.0001), but no difference was found between mPCR vs. *PCRs (p=0.946). The agreement in the identification of Plasmodium species was only regular, with Kappa indices of 0.407 (microscopy vs. *PCRs), 0.433 (microscopy vs. mPCR) and 0.558 (*PCRs vs. mPCR). In conclusion, the diagnostic performance of mPCR was comparable to those of *PCRs, and superior to microscopy, although the identification of Plasmodium species showed many disagreements. In conclusion, a sensitive and specific one-round SSP multiplex PCR, capable of simultaneously detecting and identifying P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum may be useful in resource-constrained countries where quantitative amplifications are not yet fully accessible.

Artemisinin resistance, presently confined to Southeast Asia and associated with mutations in the Plasmodium falciparum K13 (PfK13) propeller domain, represents a serious threat to global malaria control. This study aimed to provide baseline information for future artemisinin resistance surveillance, by analyzing the PfK13 propeller domain in P. falciparum field isolates collected from the Brazilian Amazon Basin between 1984 and 2011. A total of 152 P. falciparum mono-infections were assessed, of which 118 (78%) were collected before and 34 (22%) after the introduction of artemisinin-based combination therapy (ACT) in 2006. An 849-base pair fragment encoding the PfK13 propeller was amplified by nested polymerase chain reaction and sequenced in both directions. The sequences were compared with the reference sequence of P. falciparum 3D7. All samples showed wild-type sequences, thus, no mutations were observed. The results are in agreement with other recent reports and do not provide evidence for presence of PfK13 propeller domain polymorphisms associated with artemisinin resistance among P. falciparum field isolates in the Brazilian Amazon Basin neither before nor after the implementation of ACT.

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/μL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.

Made available in DSpace on 2019-10-06T16:00:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-01. Added 1 bitstream(s) on 2019-10-09T18:35:04Z : No. of bitstreams: 1 S1984-29612018000300363.pdf: 2770381 bytes, checksum: c15ec391d851104f6d38492a03ad8099 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum. PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples. Laboratório de Parasitologia Animal Curso de Medicina Veterinária Universidade Federal de Rondônia – UNIR Centro de Estudos da Malária Superintendência de Controle de Endemias – SUCEN Departamento de Saúde do Estado de São Paulo Instituto de Medicina Tropical de São Paulo – IMTSP Universidade de São Paulo – USP Laboratório de Patologia Veterinária Curso de Medicina Veterinária Universidade Federal de Rondônia – UNIR Laboratório de Imunoparasitologia Faculdade de Ciências Agrárias e Veterinárias – FCAV Universidade Estadual Paulista – UNESP Laboratório de Imunoparasitologia Faculdade de Ciências Agrárias e Veterinárias – FCAV Universidade Estadual Paulista – UNESP FAPESP: 10/12820-4 FAPESP: 12/03961-9

Made available in DSpace on 2018-12-11T17:11:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-04-26 Background: Considering the diversity of feeding habits that females of some species of anophelines present, it is important to understand which vertebrates are part of blood food sources and how important is the role of each in the ecoepidemiology of malaria. There are many vector species for Plasmodium spp. in the State of Maranhão, Brazil. In São Luís Island, Anopheles aquasalis is the main vector for human malaria; this species is abundant in areas with primates that are positive for Plasmodium. Anopheles aquasalis has natural exophilic and zoophilic feeding behavior, but in cases of high density and absence of animals, presents quite varied behavior, and feeds on human blood. In this context, the objective of the present study was to identify Plasmodium spp. and the blood meal sources of anophelines in two environmental reserves on São Luís Island, state of Maranhão, using molecular methods. Methods: Between June and July 2013, female anophelines were collected in the Sítio Aguahy Private Reserve, in the municipality of São José de Ribamar, and in the Sítio Mangalho Reserve, located within the Maracanã Environmental Protection Area, in the municipality of São Luís. CDC-type light traps, Shannon traps and protected human bait were used during three consecutive hours in peridomestic and wooded areas. Pools of anophelines were formed using mosquitoes of the same species that had been caught at the same site on the same date. A genus-specific amplification protocol based on the 18S rRNA gene was used for qPCR and cPCR. Results: A total of 416 anophelines were collected, of the following species: An. aquasalis (399), An. mediopunctatus (3), An. shannoni (1), An. nuneztovari (sensu lato) (1), An. goeldii (1), An. evansae (2) and An. (Nyssorhynchus) sp. (9), comprising 54 pools. Two pools were positive for Plasmodium (2/54) based on the 18S rRNA gene. In the phylogenetic analysis using the maximum likelihood method, based on a 240 bp fragment of the 18S rRNA gene, it was found that the sequences of Plasmodium sp. amplified from pools of An. aquasalis (pool 2) and An. nuneztovari (s.l.) (pool 10) were phylogenetically related to a clade of P. falciparum isolates from India, and to a clade of Plasmodium sp. isolates from psittacines in Brazil, respectively. Cat, dog and human DNA were identified in the blood meals of the anophelines sampled. Conclusion: The species An. aquasalis was the most abundant anopheline species in São Luís Island. Plasmodium spp. DNA was detected, thus confirming the importance of this species as the main vector on São Luís Island, Brazil. In addition, the presence of An. nuneztovari (s.l.) with DNA positive for Plasmodium spp. confirms its importance as a secondary vector. Immunoparasitology Laboratory School of Agrarian and Veterinary Sciences (FCAV) Universidade Estadual Paulista (UNESP) Jaboticabal Campus Center for Malaria Studies Superintendence of Control of Endemic Diseases State Secretariat of Health of São Paulo Institute of Tropical Medicine of São Paulo (IMT-SP) University of São Paulo (USP) Veterinary Pathology Laboratory Brazil University Descalvado Campus Immunoparasitology Laboratory School of Agrarian and Veterinary Sciences (FCAV) Universidade Estadual Paulista (UNESP) Jaboticabal Campus

Studies on autochthonous malaria in low-transmission areas in Brazil have acquired epidemiological relevance because they suggest continued transmission in what remains of the Atlantic Forest. In the southeastern portion of the state of São Paulo, outbreaks in the municipality of Juquitiba have been the focus of studies on the prevalence of Plasmodium, including asymptomatic cases. Data on the occurrence of the disease or the presence of antiplasmodial antibodies in pregnant women from this region have not previously been described. Although Plasmodium falciparum in pregnant women has been widely addressed in the literature, the interaction of Plasmodium vivax and Plasmodium malariae with this cohort has been poorly explored to date. We monitored the circulation of Plasmodium in pregnant women in health facilities located in Juquitiba using thick blood film and molecular protocols, as well as immunological assays, to evaluate humoural immune parameters. Through real-time and nested polymerase chain reaction, P. vivax and P. malariae were detected for the first time in pregnant women, with a positivity of 5.6%. Immunoassays revealed the presence of IgG antibodies: 44% for ELISA-Pv, 38.4% for SD-Bioline-Pv and 18.4% for indirect immunofluorescence assay-Pm. The high prevalence of antibodies showed significant exposure of this population to Plasmodium. In regions with similar profiles, testing for a malaria diagnosis might be indicated in prenatal care.

Brazil, a country of continental proportions, presents three profiles of malaria transmission. The first and most important numerically, occurs inside the Amazon. The Amazon accounts for approximately 60% of the nation’s territory and approximately 13% of the Brazilian population. This region hosts 99.5% of the nation’s malaria cases, which are predominantly caused by Plasmodium vivax (i.e., 82% of cases in 2013). The second involves imported malaria, which corresponds to malaria cases acquired outside the region where the individuals live or the diagnosis was made. These cases are imported from endemic regions of Brazil (i.e., the Amazon) or from other countries in South and Central America, Africa and Asia. Imported malaria comprised 89% of the cases found outside the area of active transmission in Brazil in 2013. These cases highlight an important question with respect to both therapeutic and epidemiological issues because patients, especially those with falciparum malaria, arriving in a region where the health professionals may not have experience with the clinical manifestations of malaria and its diagnosis could suffer dramatic consequences associated with a potential delay in treatment. Additionally, because the Anopheles vectors exist in most of the country, even a single case of malaria, if not diagnosed and treated immediately, may result in introduced cases, causing outbreaks and even introducing or reintroducing the disease to a non-endemic, receptive region. Cases introduced outside the Amazon usually occur in areas in which malaria was formerly endemic and are transmitted by competent vectors belonging to the subgenus Nyssorhynchus (i.e., Anopheles darlingi, Anopheles aquasalis and species of the Albitarsis complex). The third type of transmission accounts for only 0.05% of all cases and is caused by autochthonous malaria in the Atlantic Forest, located primarily along the southeastern Atlantic Coast. They are caused by parasites that seem to be (or to be very close to) P. vivax and, in a less extent, by Plasmodium malariae and it is transmitted by the bromeliad mosquito Anopheles (Kerteszia) cruzii. This paper deals mainly with the two profiles of malaria found outside the Amazon: the imported and ensuing introduced cases and the autochthonous cases. We also provide an update regarding the situation in Brazil and the Brazilian endemic Amazon.

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

This study investigated Plasmodium spp. infection in free-ranging neotropical primates from Brazilian Amazon regions under the impact of major anthropogenic actions. Blood samples from 19 new world primates were collected and analyzed with microscopic and molecular procedures. The prevalence of Plasmodium infection was 21.0% (4/19) and PCR positive samples were identified as P. brasilianum. Considering the social-economic changes that the Amazon is facing, the prevalence of P. brasilianum infection highlights the necessity to closely monitor the movement of both human and non-human primate populations, in order to mitigate pathogen exposure and the introduction of new agents into previously naïve areas.

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Leishmaniasis, Chagas disease, and malaria affect the poorest population around the world, with an elevated mortality and morbidity. In addition, the therapeutic alternatives are usually toxic or ineffective drugs especially those against the trypanosomatids. In the course of selection of new anti-protozoal compounds from Brazilian flora, the CH(2)C(l2) phase from MeOH extract obtained from the leaves of Pentacalia desiderabilis (Vell.) Cuatrec. (Asteraceae) showed in vitro anti-leishmanial, anti-malarial, and anti-trypanosomal activities. The chromatographic fractionation of the CH(2)Cl(2) phase led to the isolation of the bioactive compound, which was characterized as jacaranone [methyl (1-hydroxy-4-oxo-2,5-cyclohexandienyl)acetate], by spectroscopic methods. This compound showed activity against promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, and Leishmania (L.). amazonensis showing an IC(50) of 17.22, 12.93, and 11.86 μg/mL, respectively. Jacaranone was also tested in vitro against the Trypanosoma cruzi trypomastigotes and Plasmodium falciparum chloroquine-resistant parasites (K1 strain) showing an IC(50) of 13 and 7.82 μg/mL, respectively, and was 3.5-fold more effective than benznidazole in anti-Trypanosoma cruzi assay. However, despite of the potential against promatigotes forms, this compound was not effective against amastigotes of L. (L.) chagasi and T. cruzi. The cytotoxicity study using Kidney Rhesus monkey cells, demonstrated that jacaranone showed selectivity against P. falciparum (21.75 μg/mL) and a selectivity index of 3. The obtained results suggested that jacaranone, as other similar secondary metabolites or synthetic analogs, might be useful tolls for drug design for in vivo studies against protozoan diseases.

Malaria in pregnancy remains a substantial public health problem in malaria-endemic areas with detrimental outcomes for both the mother and the foetus. The placental changes that lead to some of these detrimental outcomes have been studied, but the mechanisms that lead to these changes are still not fully elucidated. There is some indication that imbalances in cytokine cascades, complement activation and angiogenic dysregulation might be involved in the placental changes observed. Nevertheless, the majority of studies on malaria in pregnancy (MiP) have come from areas where malaria transmission is high and usually restricted to Plasmodium falciparum, the most pathogenic of the malaria parasite species. We conducted a cross-sectional study in Cruzeiro do Sul, Acre state, Brazil, an area of low transmission and where both P. vivax and P. falciparum circulate. We collected peripheral and placental blood and placental biopsies, at delivery from 137 primigravid women and measured levels of the angiogenic factors angiopoietin (Ang)-1, Ang-2, their receptor Tie-2, and several cytokines and chemokines. We measured 4 placental parameters (placental weight, syncytial knots, placental barrier thickness and mononuclear cells) and associated these with the levels of angiogenic factors and cytokines. In this study, MiP was not associated with severe outcomes. An increased ratio of peripheral Tie-2:Ang-1 was associated with the occurrence of MiP. Both Ang-1 and Ang-2 had similar magnitudes but inverse associations with placental barrier thickness. Malaria in pregnancy is an effect modifier of the association between Ang-1 and placental barrier thickness., Author Summary Plasmodium infections during pregnancy represent a substantial health burden for mothers and their neonates leading to underweight babies, preterm deliveries, abortions, stillbirths, and even maternal mortality. The placenta, in its role as a bridge between mother and foetus, will either suffer or mediate the injuries which eventually lead to these adverse outcomes. Plasmodium infections during pregnancy can affect the metabolism of the placenta, its functions and even its architecture. These changes are well described in Sub-Saharan Africa, where P. falciparum infections predominate, but little is known in areas where P. vivax is predominant. The state of Acre in Brazil is one such area. We have recently shown that malaria in pregnancy is associated with placental pathology in this region. In the present study we sought to find out the association between the placental and peripheral levels of angiogenic factors, which are known and important players in the formation and function of the placenta, and the occurrence of placental pathology in Plasmodium-infected women. We found that, lower levels of Angiopoietin-1 in women who experienced malaria during pregnancy were associated with specific modifications occurring in the placenta. This provides a possible pathway through which these pathological changes occur.

In the search for new antimalarial agents, nine Brazilian plant species were selected, from the Annonaceae (6), Menispermaceae (2) and Siparunaceae (1) families naturally occurring at the cerrado and Atlantic rainforest regions, in order to investigate their in vitro antiplasmodial activity. The ethanol and the alkaloid extracts were tested against K1, chloroquine-resistant, and Palo Alto, chloroquine-sensitive, strains of Plasmodium falciparum. The majority of the alkaloid extracts were more active than the ethanol ones, with IC(50) ranging 0.3-8.2 microg/mL. The crude Guatteria australis alkaloids were the most active against K1 with an IC(50) = 0.3 microg/mL. The most promising total alkaloid fractions for further bioguided isolation are those with the IC(50)or = 5 microg/mL: G. australis, Cissampelos ovalifolia and Duguetia lanceolata.

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents. Verapamil foi ensaiado quanto ao efeito modulador em isolados brasileiros de Plasmodium falciparum resistentes à cloroquina. Outros agentes cardiovasculares, considerados como moduladores da resistência em malária e/ou em neoplasias multiresistentes a fármacos, como nifedipino, nitrendipino, diltiazem e propranolol foram ensaiados quanto ao mesmo efeito. Concentrações semelhantes às da terapia cardiovascular foram empregadas no ensaio de microtécnica de sensibilidade para fármacos antimaláricos. Atividade antiplasmódica intrínsica foi observada desde as menores concentrações, sem, entretanto, ocorrência de modulação significativa da resistência. Sob as mesmas condições experimentais, respostas semelhantes foram observadas para outros agentes moduladores conhecidos como o antipsicótico trifluoperazina e os antidepressivos desipramina e imipramina. Em conjunto, estes resultados sugerem alta sensibilidade e comportamento indiferente de cepas brasileiras ao efeito de agentes moduladores da resistência.

Made available in DSpace on 2018-12-11T17:13:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In the Brazilian Amazon region, malaria caused by Plasmodium malariae is considered to be a zoonosis because of cross-transfer of the parasite between humans and Neotropical primates. To contribute information on this issue, we investigated occurrences of natural infection with Plasmodium sp. among Neotropical primates in the Maranhense Amazon (Amazon region of the state of Maranhão), in the northeastern region of Brazil. Blood samples were collected from 161 Neotropical primates of six species that were caught in an environmental reserve (Sítio Aguahy) and from captive primates (CETAS—Wildlife Screening Center, municipality of São Luís), in Maranhão. Plasmodium sp. was diagnosed based on light microscopy, PCR, qPCR and LAMP for amplification of the 18S rRNA gene. Serum samples were also assayed by means of indirect immunofluorescence for IgG antibodies against P. malariae/P. brasilianum, P. falciparum and P. berghei. Parasites were detected through light microscopy on five slides from captive primates (four Sapajus spp. and one Callithrix jacchus). In the molecular tests, 34.16% (55/161) and 29.81% (48/161) of the animals sampled were positive in the qPCR and PCR assays, respectively. In the PCR, 47/48 animals were positive for P. malariae/P. brasilianum; of these, eight were free-living primates and 39 from CETAS, São Luís. One sample showed a band in the genus-specific reaction, but not in the second PCR reaction. Anti-P. malariae/P. brasilianum IgG antibodies were detected in four serum samples from Sapajus spp. in captivity. In this study, circulation of P. malariae/P. brasilianum in Neotropical primates was confirmed, with low levels of parasitemia and low levels of antibodies. The importance of these animals as reservoirs of human malaria in the region studied is still unknown. This scenario has an impact on control and elimination of malaria in this region. Laboratory of Immunoparasitology School of Agrarian and Veterinary Sciences (FCAV) UNESP Jaboticabal Campus Center for Malaria Studies Superintendency for Endemic Disease Control (SUCEN) Department of Health of the State of São Paulo Institute of Tropical Medicine of Sao Paulo (IMTSP) USP Laboratory of Veterinary Pathology Brazil University Descalvado Campus Laboratory of Immunoparasitology School of Agrarian and Veterinary Sciences (FCAV) UNESP Jaboticabal Campus FAPESP: 10/12820-4 FAPESP: 12/03961-9

Background Anti-malarial resistance in Plasmodium falciparum remains an obstacle for malaria control. Resistance-associated genes were analysed in Brazilian samples over four decades to evaluate the impact of different treatment regimens on the parasite genetic profile. Methods Samples were collected on filter paper from patients infected in the Amazon region from 1984 to 2011. DNA was extracted with Chelex® 100 and monoinfection confirmed by PCR. SNPs in the pfcrt, pfmdr1, pfdhfr and pfdhps genes were assessed by PCR-RFLP. The pfmdr1 copy number was estimated using real time quantitative PCR with SYBR® Green. Parasite response was assessed ex vivo with seven concentrations of each anti-malarial. Patients were treated according to Brazilian guidelines: quinine plus tetracycline or mefloquine in period 1 and ACT in period 2. Results All 96 samples presented the pfcrt 76T mutant throughout the assessed periods. In addition, all isolates showed ex vivo chloroquine resistance. The pfmdr1 86Y was detected in 1.5% of samples in period 1, and in 25% in period 2. All samples presented the pfmdr1 1246Y. The analysis of pfmdr1 copy number showed amplification in 37.3% in period 1 and in 42% in period 2. Mutations in pfdhfr were shown as follows: 51I in all samples in period 1 and in 81.2% in period 2; 59R in 6.4% in period 2. The pfdhfr 108N and the pfdhps 437G were seen in all samples along time; the pfdhps 540E in 93.7% in period 1 and in 75% in period 2. Conclusions The 76T mutation associated to chloroquine resistance is still present in the parasite population, although this anti-malarial was withdrawn from the chemotherapy of P. falciparum in Brazil in the mid-1980s. All isolates assayed ex vivo for chloroquine showed resistant phenotype and 76T. No association was observed between pfmdr1 mutations and resistance to quinine, mefloquine and artemisinin derivatives. Additionally, the pfdhfr 108N mutation was detected in all samples throughout the evaluated periods, demonstrating fixation of the mutant allele in the parasite population. Changes in Brazilian national guidelines for the malaria chemotherapy in the last 27 years yielded a discreet genetic impact in the parasite population.

INTRODUCTION: Autochthonous malaria in the State of Sao Paulo is characterized by sporadic outbreaks in the western region and by persistent transmission in the eastern region, where oligosymptomatic cases with low parasitemia due to Plasmodium vivax occur. The objectives of this study were to assess the completeness of autochthonous malaria notification forms; to estimate the incidence trends of autochthonous cases in the State of Sao Paulo from 1980 to 2007; and to analyze the clinical and epidemiological patterns of cases in two autochthonous regions over this period. METHODS: This was a descriptive study that analyzed 18 variables on the notification form for malaria in the State of Sao Paulo, comparing these two regions over two periods (1980-1993 and 1994-2007). The data sources were SUCEN/SES/SP, SINAN/CVE/SES/SP and DATASUS. RESULTS: The completeness was greater than 85% for 11 variables. The incidence trend was decreasing. There were 821 autochthonous cases: 91.6% occurred in the eastern region, predominantly due to Plasmodium vivax. Asymptomatic infection had a higher percentage in the second period (p < 0.001). CONCLUSIONS: The completeness of the information was satisfactory. The clinical differences observed deserve attention from epidemiological surveillance agencies, which need to deal with the challenge of asymptomatic infection by Plasmodium.

Autochthonous malaria in the State of São Paulo is characterized by sporadic outbreaks in the western region and by persistent transmission in the eastern region, where oligosymptomatic cases with low parasitemia due to Plasmodium vivax occur. The objectives of this study were to assess the completeness of autochthonous malaria notification forms; to estimate the incidence trends of autochthonous cases in the State of São Paulo from 1980 to 2007; and to analyze the clinical and epidemiological patterns of cases in two autochthonous regions over this period.This was a descriptive study that analyzed 18 variables on the notification form for malaria in the State of São Paulo, comparing these two regions over two periods (1980-1993 and 1994-2007). The data sources were SUCEN/SES/SP, SINAN/CVE/SES/SP and DATASUS.The completeness was greater than 85% for 11 variables. The incidence trend was decreasing. There were 821 autochthonous cases: 91.6% occurred in the eastern region, predominantly due to Plasmodium vivax. Asymptomatic infection had a higher percentage in the second period (p0.001).The completeness of the information was satisfactory. The clinical differences observed deserve attention from epidemiological surveillance agencies, which need to deal with the challenge of asymptomatic infection by Plasmodium.

Em virtude da existência de poucas informações, devidamente registradas, sobre freqüência e épocas de recaídas de malária por Plasmodium vivax, contraída no Brasil, foi analisada casuística observada em região não endêmica e constituída por pacientes corretamente tratados. O índice de recaídas documentadas em São Paulo, foi alto (24,5%), com desenvolvimento precoce na maioria das oportunidades, ou seja, em tempo inferior a três meses. Very few well-established information is available about the frequency and timeliness of relapses in cases of Plasmodium vivax malaria acquired in Brazil. So, we analysed a series of correctly treated patients observed out of endemic areas. The rate of relapses seen in São Paulo, which may represent that of the parasitosis in the whole country, was high, ranging from 7.5% to 24.5%, and early in most cases, i.e. appearing by three months, what anticipates a high endemicity.

We examined the extent of variation of the 3′ region of the Circumsporozoite gene among Plasmodium falciparum isolates through amplification of a selected DNA fragment followed by DNA sequencing. A total of 32 isolates were analyzed, of which 24 were from Amazon endemic areas in Brazil and 8 from widely separated geographical regions in the world. Among Brazilian isolates only 2 variants were detected: 19 displayed the same sequence of strain 7G8 whereas the 4 remaining isolates differed from the 7G8 strain at five nucleotide positions which also led to amino acid changes. Variation was restricted to one of the T-helper epitopes while the sequence identified as a cytotoxic T cell epitope was conserved in all Brazilian isolates. P. falciparum samples from other geographical regions in the world showed sequences distinct from those of Brazilian isolates. However, some constancy could be observed within that variation. For instance, the most frequent nucleotide substitutions, from A to C at nucleotide positions 1015 and 1024, were the same in all isolates.

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.

The present study was carried out to evaluate the Malar-CheckTM Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-CheckTMwere compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm³ (TBS and Malar-CheckTM, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-CheckTMyielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-CheckTMperformed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-CheckTM should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-CheckTM for early diagnosis could avoid disease evolution to severe cases. Este trabalho avaliou o Malar-CheckTM Pf test, ensaio imunocromatográfico que detecta a proteína rica em histidina de Plasmodium falciparum, dispensa uso de equipamentos, é rápido e de fácil execução. Ensaios de diluição com o isolado Palo Alto ou sangue de pacientes com P. falciparum, foram realizados para testar a sensibilidade em diferentes densidades do parasita. Malar-CheckTM foi comparado à gota espessa (GE), padrão ouro para diagnóstico de malária. A média do limiar de sensibilidade para cada técnica em três experimentos independentes foi de 12 e 71 parasitas/mm³ (GE e Malar-CheckTM, respectivamente). Em ensaios de campo, amostras foram coletadas de pacientes febris de áreas endêmicas. Comparado à GE, Malar-CheckTM foi verdadeiramente positivo em 38 pacientes, falso positivo em 3, verdadeiramente negativo em 23 e falso negativo em um. Malar-CheckTMrealizado com sangue de pacientes com P. falciparum após tratamento mostrou persistência do antígeno durante 30 dias. Malar-CheckTM pode ser útil no diagnóstico de P. falciparum em áreas remotas e auxiliar a rotina diagnóstica, mesmo quando a microscopia está disponível. Deve ser considerada infecção pregressa por P. falciparum, que pode determinar testes positivos em indivíduos curados. A rapidez do Malar-CheckTM para o diagnóstico precoce pode evitar evolução para casos graves.

O presente trabalho teve como objetivo estabelecer critérios adequados à triagem de doadores de sangue de regiões com características epidemiológicas distintas, para malária. Foram estudados 3 locais com critérios de seleção diferentes: São Paulo, SP (sem transmissão vetorial), Belém (baixa transmissão ativa), Matupá, Belém, PA e Peixoto de Azevedo, MT (alta transmissão ativa). A pesquisa de plasmódios foi realizada por gota espessa, QBC Test® e imunofluorescência para pesquisa de antígenos, tendo sido todas as amostras negativas. Houve grande variação na positividade para anticorpos antiplasmodiais por IFI-IgG anti P. vivax e P. falciparum entre doadores aptos nos 3 locais de estudo e entre doadores aptos e inaptos em São Paulo (aptos 1,98%, inaptos 22,3%, p < 0.01) e Belém (aptos 17,2%, inaptos 58,3%, p < 0.01), o que atesta a validade da triagem clínico-epidemiológica realizada. Em Matupá e Peixoto de Azevedo não houve doadores inaptos e a positividade foi de 80,6%. Consideramos que em bancos de sangue a triagem deve seguir critérios clínico-epidemiológicos adequados à situação de cada região. Os métodos laboratoriais de triagem, devem ser para detecção de plasmódios (gota espessa/QBC Test® ou detecção de antígenos parasitários. The aim of the present work was to establish appropriate criteria for screening of donor blood from regions with distinct Malaria epidemiological characteristics. Three locations with different screening criteria were studied: São Paulo, SP (with no vectorial transmission), Belém, PA (with low active transmission) and Matupá and Peixoto de Azevedo, MT (with high active transmission). The Malaria parasite3/4Plasmodium sp3/4was searched for by "thick film", QBC Test( and antigen Immunofluorescence test, and was not detected in any of the samples. There was, however, a great variation in the positivity of anti-plasmodial antibodies, as determined by IIF-IgG anti-P. vivax and -P. falciparum, between accepted donors in the 3 studied locations and between rejected and accepted donors in São Paulo (1.98% accepted, 22.3% rejected - p < 0.01) and Belém (17.2% accepted, 58.3% rejected - p < 0.01). These data endorse the use of the applied clinical and epidemiological screening. In Matupá and Peixoto de Azevedo, where there was no rejected donor, the serological positivity was 80.6%. We, therefore, consider that the Malaria screening in blood banks should follow clinical and epidemiological criteria suitable to each region. The laboratorial screening techniques should then detect either the parasites (thick film/ QBC Test® or the parasite antigens.

The states that make up the Legal Amazon Region, which include the state of Maranhão, account for 99% of registered cases of human malaria in Brazil. It is also believed that transmission of malaria from nonhuman primates (NHP) to humans occurs in this region, because of current reports of seroepidemiological results from samples from humans and NHP coexisting in the same areas. This study aimed to make morphological, serological and molecular diagnoses of Plasmodium spp. in neotropical primates on the island of São Luís, state of Maranhão, Brazil. The diagnostic techniques used were optical microscopy, the polymerase chain reaction (PCR) and the indirect immunofluorescence assay (IFA). From June 2009 to April 2010, 70 NHP were sampled: 50 at the Wild Animal Screening Center (CETAS), located in the municipality of São Luís and 20 free-living individuals that were caught in a private reserve located in the municipality of São Jose de Ribamar, state of Maranhão. Under an optical microscope, 140 slides (two from each animal) were evaluated and five animals (7.1%) were found to be positive. IFA did not detect anti-Plasmodium spp. From PCR on the 70 animals sampled, amplified Plasmodium spp. products were observed in 13 samples, of which eight (61.5%) were from free-living animals and five (38.5%) were from animals at CETAS. Os Estados que compõem a Amazônia Legal, entre eles o Estado do Maranhão, respondem a 99% dos casos registrados de malária humana no Brasil. Também se acredita que nessa região ocorra a transmissão de malária de primatas não humanos (PNH) para humanos, devido a relatos atuais de resultados soroepidemiológicos de amostras de humanos e PNH que coexistem nas mesmas áreas. O presente estudo objetivou realizar o diagnóstico morfológico, sorológico e molecular de Plasmodium spp. em primatas neotropicais na Ilha de São Luís, Estado do Maranhão, Brasil. Foram utilizadas como técnicas de diagnóstico: a microscopia de luz, a reação em cadeia pela polimerase (PCR) e a imunofluorescência indireta (RIFI). No período de junho de 2009 a abril de 2010, foram amostrados 70 PNH, sendo 50 provenientes do Centro de Triagem de Animais Silvestres (CETAS), localizado no município de São Luís, e 20 de vida livre, capturados em reserva particular localizada no município de São José de Ribamar, Estado do Maranhão. Foram avaliadas pela microscopia de luz 140 lâminas (duas de cada animal), das quais cinco animais (7,1%) foram positivos. Pela RIFI não se detectou anticorpos anti-Plasmodium spp. Pela PCR, dos 70 animais amostrados, foi possível observar produtos amplificados para Plasmodium spp. em 13 amostras, das quais oito (61,5%) eram de animais de vida livre e cinco (38,5%) de CETAS.

In order to investigate whether gentian violet exhibited "in vitro" inhibitory activity against Plasmodium falciparum, the Authors have carried out 20 sensitivity tests according to the microtechnique described by RIECK MANN et al.5. Results have shown inhibition of schizonts'maturation at the following concentration: 1/1000; 1/1500; 1/2000; 1/2500; 1/3000 and 1/4000, thus demonstrating inhibitory activity of the tested dye against asexual blood parasites. The present data suggest gentain violet may be possibly used in the prophylaxis of transfusion-acquired malaria. Utilizando a técnica de RIECKMANN e col.5, os autores realizaram 20 microtestes de sensibilidade, procurando verificar a capacidade da violeta de genciana em impedir, na cultura "in vitro", o desenvolvimento habitual do Plasmodium falciparum. Os resultados mostraram que houve inibiçáo da evolução do protozoário nas concentrações de 1/1000, 1/1500, 1/2000, 1/2500, 1/3000 e 1/4000, significando que nas condições da experiência o corante atuou sobre as formas sangüíneas assexuadas do protozoário. Estas verificações sugerem que a violeta de genciana poderia ser usada na profilaxia da malária transfusional.

In order to study the chemoresistance of Plasmodium falciparum to commonly used antimalarial drugs in Brazil the authors have studied ten patients with falciparum malaria, acquired in the Brazilian Amazon region. Patients were submitted to in vivo study of drug sensitivity, after chemotherapy with either 4-aminoquinolines (chloroquine or amodiaquine) or quinine. Adequate drug absorption was confirmed by standard urine excretion tests for antimalarials. Eight patients could be followed up to 28 days. Among these in vivo resistance (R I and R II responses) was seen in all patients who received 4-amino-quinolines. One patient treated with quinine exhibited a R III response. Peripheral blood samples of the same patients were submitted to in vitro microtests for sensitivity to antimalarials. Out of nine successful tests, resistance to chloroquine and amodiaquine was found in 100% and resistance to quinine in 11.11% of isolates. Probit analysis of log dose-response was used to determine effective concentrations EC50, EC90 and EC99 to the studied drugs. Good correlation between in vivo and in vitro results was seen in six patients. The results emphasize high levels of P. falciparum resistance to 4- aminoquinolines and suggest an increase in resistance to quinine in the Brazilian Amazon region, reinforcing the need for continuous monitoring of drug sensitivity to adequate chemotherapy according to the most efficacious drug regimensResistência in vivo e in vitro do Plasmodium falciparum à cloroquina, amodiaquina e quinino na Amazônia Brasileira. Com o propósito de avaliar a resistência do Plasmodium falciparum às drogas antimaláricas, rotineiramente empregadas no Brasil, os autores acompanharam dez pacientes com malária falciparum adquirida na Amazônia brasileira. Os pacientes foram submetidos a estudo in vivo de sensibilidade a drogas, após tratamento com derivados 4-aminoquinoleínicos (cloroquina e amodiaquina) ou quinino. A absorção das drogas foi verificada através de testes padronizados de excreção urinária de antimaláricos. Oito pacientes puderam ser seguidos por 28 dias. Dentre eles detectou-se resistência in vivo (em nível de R I e R II) em todos os pacientes tratados com 4-aminoquinoleínas. Um paciente tratado com quinino exibiu padrão de resistência R III ao tratamento. Alíquotas de sangue periférico dos mesmos pacientes foram ainda submetidas a microtestes in vitro de sensibilidade a antimaláricos. Em nove microtestes houve desenvolvimento satisfatório de esquizontes. Destes detectou-se resistência in vitro à cloroquina e amodiaquina em 100% e ao quinino em 11,11% das amostras. Através da análise de probitos calcularam-se as concentrações de cloroquina, amodiaquina e quinino, capazes de inibir o crescimento in vitro de esquizontes (Cl50, Cl90 e Cl99). Em seis pacientes observou-se concordância entre os achados in vivo e in vitro. Os resultados ressaltam o alto grau de resistência do P. falciparum às 4-aminoquinoleínas e indicam um aumento na resistência do parasita ao quinino na Amazônia brasileira. Reitera-se, assim, a necessidade de monitoração contínua da sensibilidade a antimaláricos, com o intuito de recomendar esquemas mais eficazes na terapêutica da malária por P. falciparum

A study searching for Plasmodium vivax and Plasmodium falciparum DNA among blood donors from the non-endemic area in Brazil reported a rate of 7.41%. This number is at least three times higher than what has been observed in blood donors from the Amazon, an endemic area concentrating >99% of all malaria cases in Brazil. Moreover, the majority of the donors were supposedly infected by P. falciparum, a rare finding both in men and anophelines from the Atlantic forest. These findings shall be taken with caution since they disagree with several publications in the literature and possibly overestimate the actual risk of malaria transmission by blood transfusion in Sao Paulo city.