ABSTRACT Malaria is the most important vector-borne disease in the world and a challenge for control programs. In Brazil, 99% of cases occur in the Amazon region. In the extra-Amazonian region, a non-endemic area, epidemiological surveillance focuses on imported malaria and on autochthonous outbreaks, including cases with mild symptoms and low parasitemia acquired in the Atlantic Forest biome. In this scenario, cases are likely to be underreported, since submicroscopic parasitemias are not detected by thick blood smear, considered the reference test. Molecular tests are more sensitive, detecting asymptomatic individuals and mixed infections. The aim of this study was to propose a more efficient alternative to detect asymptomatic individuals living in areas of low malaria endemicity, as they are reservoirs of Plasmodium that maintain transmission locally. In total, 955 blood samples from residents of 16 municipalities with autochthonous malaria outbreaks in the Sao Paulo State were analyzed; 371 samples were collected in EDTA tubes and 584 in filter paper. All samples were initially screened by a genus-specific qPCR targeting ssrRNA genes (limit of detection of 1 parasite/µL). Then, positive samples were subjected to a nested PCR targeting ssrRNA and dihydrofolate reductase-thymidylate synthase genes (limit of detection of 10 parasites/µL) to determine Plasmodium species. The results showed a statistically significant difference (K = 0.049; p < 0.0001) between microscopy positivity (6.9%) and qPCR (22.9%) for EDTA-blood samples. Conversely, for samples collected in filter paper, no statistical difference was observed, with 2.6% positivity by thick blood smear and 3.1% for qPCR (K = 0.036; p = 0.7). Samples positive by qPCR were assayed by a species-specific nested PCR that was in turn positive in 26% of samples (16 P. vivax and 4 P. malariae ). The results showed that molecular protocols applied to blood samples from residents in areas with autochthonous transmission of malaria were useful to detect asymptomatic patients who act as a source of transmission. The results showed that the genus-specific qPCR was useful for screening positives, with the subsequent identification of species by nested PCR. Additional improvements, such as standardization of blood plotting on filter paper and a more sensitive protocol for species determination, are essential. The qPCR-based algorithm for screening positives followed by nested PCR will contribute to more efficient control of malaria transmission, offering faster and more sensitive tools to detect asymptomatic Plasmodium reservoirs.

ABSTRACT Malaria is an infectious vector-borne disease with other important routes of transmission, such as blood transfusion and organ/tissue transplantation, due to asymptomatic reservoirs of Plasmodium presenting with low parasitemia. Reports of transfusion-transmitted malaria have shown that in immunosuppressed recipients, infections can be fatal if they are not diagnosed and timely treated. All Plasmodium species can survive on blood components at temperatures from 2 to 6 °C for some days or even weeks. This report describes two candidates for blood donation harboring Plasmodium, infected in an area considered non-endemic. Blood samples were collected from donors who attended a blood bank in Sao Paulo and tested by microscopy, qPCR for Plasmodium genus-specific amplification, targeting the parasite 18S ribosomal subunit gene and a multiplex qPCR based on mtDNA of the five species. Under microscopy, only structures resembling Plasmodium were observed. The qPCR whose standard curve tested parasites varying from 2 to 0.1 parasites/ µL, showed the presence of Plasmodium DNA in the two blood donors, as did the multiplex qPCR that revealed the presence of P. malariae. The prevalence of positive donors varies according to the level of transmission, ranging from 0.7 to 55% in endemic areas. In non-endemic regions, prevalences are lower, however, transfusion malaria can evolve to severe cases, due to the lack of suspicion of this transmission route. Asymptomatic donors from low transmission regions pose a risk to blood banks, with particular emphasis on those located in areas with malaria elimination goals.

Resumo Neste estudo objetivamos identificar Plasmodium spp. em amostras sangue de primatas não humanos (PNH) do estado do Maranhão, utilizando técnicas clássicas e alternativas para o exame da malária humana. Foram analisadas 161 amostras de sangue de PNH, sendo 141 de CETAS (cativeiro) e 20 de reserva particular (vida livre), utilizando microscopia, teste de diagnóstico rápido (RDT), imunofluorescência indireta (IFI) e técnicas moleculares (semi-nested PCR, PCR em tempo real quantitativo e LAMP). Dois métodos sorológicos (dot-ELISA e ELISA indireto) também foram padronizados com antígenos solúveis de roptrias de P. falciparum e P. berghei. Formas trofozoíticas de Plasmodium sp. foram identificadas em lâminas de cinco animais diferentes. Nenhuma amostra foi positiva em TDR e LAMP. Quatro amostras foram soropositivas para P. malariae na IFI. Os soros de PNH mostraram baixa reatividade pelo ELISA indireto. Plasmodium sp. foi detectado em 34,16% (55/161) das amostras utilizando a qPCR baseada no gene 18S rRNA. No sequenciamento, duas amostras mostraram identidade com P. malariae (100%), uma com Plasmodium sp. ZOOBH (97%) e uma com P. falciparum (99%). A PCR mostrou ser a técnica mais sensível para diagnósticos de Plasmodium em amostras de PNH.

Abstract 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.

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.

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.

No Brasil a malária é endêmica na Amazônia, porém casos autóctones com baixas parasitemias ocorrem na área costeira de Mata Atlântica. De acordo com a legislação brasileira, não são obrigatórios testes para detecção de malária em doadores de sangue de áreas não-endêmicas; entretanto são excluídos por seis meses aqueles com relato de deslocamento para áreas de transmissão. Este trabalho descreve um caso de malária transfusional ocorrido em São Paulo, Brasil, em que um paciente recebeu sangue infectado, desenvolvendo a doença. Ele residia em São Paulo e não apresentava histórico de transfusão anterior ou deslocamentos para áreas endêmicas, incluindo as de baixa endemicidade, como a Mata Atlântica. A gota espessa revelou Plasmodium malariae. Os doadores eram residentes em São Paulo e um deles (045-0) apresentou hemoscopia e PCR positivos. Este era assintomático com PCR positiva para P. malariae e viagem para Juquiá, Mata Atlântica de São Paulo, onde são descritos casos esporádicos de malária autóctone. Amostras de soro do receptor e de todos os doadores foram ensaiadas por ELISA com antígenos de P. vivax e P. falciparum e RIFI com P. malariae. O doador 045-0 apresentou RIFI positiva para P. malariae. ELISA-P. vivax foi reagente no doador infectado (045-0) e em dois dos 44 doadores. Todos os soros foram negativos com antígeno de P. falciparum. Este caso aponta a necessidade de revisão dos critérios de triagem clínico-epidemiológica para evitar casos transfusionais e também adequar as estratégias de exclusão de doadores de sangue.

Malaria in Brazil is endemic in the Amazon region, but autochthonous cases with low parasitaemia occur in the Atlantic Forest area of the country. According to Brazilian legislation no test is mandatory for blood donors from non-endemic areas. However if they have traveled to malaria transmission regions they are deferred for six months before they can donate. This report describes a transfusion-transmitted malaria case in Sao Paulo, Brazil, where one recipient received infected blood and developed the disease. He lived in Sao Paulo and had no previous transfusion or trips to endemic areas, including those of low endemicity, such as Atlantic Forest. Thick blood smears confirmed Plasmodiummalariae. All donors lived in Sao Paulo and one of them (Donor 045-0) showed positive hemoscopy and PCR. This asymptomatic donor had traveled to Juquia, in the Atlantic Forest area of S ao Paulo State, where sporadic cases of autochthonous malaria are described. DNA assay revealed P. malariae in the donor's (Donor 045-0) blood. Serum archives of the recipient and of all blood donors were analyzed by ELISA using both P. vivax and P. falciparum antigens, and IFAT with P. malariae. Donor 045-0's serum was P. malariae IFAT positive and the P. vivax ELISA was reactive. In addition, two out of 44 donors' archive sera were also P. vivax ELISA reactive. All sera were P. falciparum ELISA negative. This case suggests the need of reviewing donor selection criteria and deferral strategies to prevent possible cases of transfusion-transmitted malaria.

A malária é complicação incomum após o transplante de células-tronco hematopoiéticas em países endêmicos. No entanto, candidatos a transplantes, receptores e doadores que vivem em regiões endêmicas frequentemente relatam episódios anteriores de malária. Este fato pode representar um risco importante para receptores imunossuprimidos, que podem desenvolver casos de malária grave. Relatamos um caso de transplante de células-tronco hematopoiéticas (TCTH) em que o doador teve história de malária anterior e um monitoramento por meio de exames parasitológicos e moleculares foi realizado antes e após o procedimento. O doador apresentou Plasmodium vivax na gota espessa um mês após o transplante e foi tratado de acordo com as orientações do Ministério da Saúde brasileiro. A reação em cadeia da polimerase (PCR) foi capaz de detectar a infecção por malária no doador uma semana mais cedo do que a gota espessa. Mesmo sem resultados positivos, o receptor foi preventivamente tratado com cloroquina, a fim de prevenir as formas sanguíneas assexuadas. Destacamos a importância do monitoramento de receptores e doadores em procedimentos de transplante, com o objetivo de reduzir o risco de transmissão da malária.

Malaria is an unusual complication after hematopoietic stem cell transplantation in non-endemic countries. However, transplant candidates, recipients and donors living in endemic regions frequently report previous episodes of malaria. This fact could represent an important risk for immunosuppressed recipients that could develop severe malaria cases. We report a case of hematopoietic stem cell transplant (HSCT) in which the donor had a history of previous malaria, and close monitoring was performed before and after procedure by parasitological and molecular tests. The donor presented Plasmodium vivax in thick blood smears one month after transplant and was treated according to Brazilian Health Ministry guidelines. The polymerase chain reaction (PCR) was able to detect malaria infection in the donor one week earlier than thick blood film. Even without positive results, the recipient was pre-emptively treated with chloroquine in order to prevent the disease. We highlight the importance of monitoring recipients and donors in transplant procedures with the aim of reducing the risk of malaria transmission.

INTRODUÇÃO: A malária autóctone no Estado de São Paulo caracteriza-se por surtos esporádicos na região oeste e transmissão persistente na região leste onde ocorrem casos oligossintomáticos com baixa parasitemia pelo Plasmodium vivax. Os objetivos deste estudo foram: analisar a completitude das fichas de notificação de malária autóctone; estimar a tendência da incidência de casos autóctones no ESP de 1980 a 2007; analisar o comportamento clínico e epidemiológico dos casos em duas regiões de autoctonia neste período. MÉTODOS: Foi realizado um estudo descritivo com 18 variáveis das FIN de malária do ESP, analisadas em duas regiões e em dois períodos (1980-1993 e 1994-2007). Fontes de dados: SUCEN/SES/SP, SINAN/CVE/SES/SP e DATASUS. RESULTADOS: A completitude foi superior a 85% em 11 variáveis. A tendência da incidência foi decrescente. Foram notificados 821 casos autóctones, 91,6% na região leste, predominando Plasmodium vivax. A infecção assintomática teve maior porcentagem no segundo período (p<0,001). CONCLUSÕES: A completitude das informações foi considerada satisfatória. As diferenças clínicas encontradas merecem atenção da vigilância epidemiológica que deve lidar com o desafio da infecção assintomática por Plasmodium.

INTRODUCTION: 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. METHODS: 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. 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.

Embora a gota espessa corada por Giemsa (GTS) permaneça o padrão ouro para o diagnóstico de malária, métodos moleculares são mais sensíveis e específicos para detectar parasitas e podem ser utilizados em centros de referência para avaliar o desempenho da microscopia. A descrição das seqüências dos genes ssrRNA de Plasmodium falciparum, P. vivax, P. malariae e P. ovale permitiu o desenvolvimento de uma reação em cadeia da polimerase (PCR) que tem sido utilizada para diferenciar as quatro espécies. O objetivo deste estudo foi determinar as espécies de Plasmodium através de PCR em 190 lâminas positivas de pacientes para verificar a qualidade do diagnóstico realizado no Laboratório de Malária da SUCEN. Considerando somente os 131 resultados positivos em ambas as técnicas, GTS detectou 4,6% de infecções mistas e 3,1% de P. malariae enquanto o PCR identificou 19,1% e 13,8%, respectivamente.

Although the Giemsa-stained thick blood smear (GTS) remains the gold standard for the diagnosis of malaria, molecular methods are more sensitive and specific to detect parasites and can be used at reference centers to evaluate the performance of microscopy. The description of the Plasmodium falciparum, P. vivax, P. malariae and P. ovale ssrRNA gene sequences allowed the development of a polymerase chain reaction (PCR) that had been used to differentiate the four species. The objective of this study was to determine Plasmodium species through PCR in 190 positive smears from patients in order to verify the quality of diagnosis at SUCEN's Malaria Laboratory. Considering only the 131 positive results in both techniques, GTS detected 4.6% of mixed and 3.1% of P. malariae infections whereas PCR identified 19.1% and 13.8%, respectively.

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.

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.

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.

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 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.