The impact of imported malaria by gold miners in Roraima: characterizing the spatial dynamics of autochthonous and imported malaria in an urban region of Boa Vista

BACKGROUND The number of malaria cases in Roraima nearly tripled from 2016 to 2018. The capital, Boa Vista, considered a low-risk area for malaria transmission, reported an increasing number of autochthonous and imported cases. OBJECTIVES This study describes a spatial analysis on malaria cases in an urban region of Boa Vista, which sought to identify the autochthonous and imported cases and associated them with Anopheles habitats and the potential risk of local transmission. METHODS In a cross-sectional study at the Polyclinic Cosme e Silva, 520 individuals were interviewed and diagnosed with malaria by microscopic examination. Using a global positional system, the locations of malaria cases by type and origin and the breeding sites of anopheline vectors were mapped and the risk of malaria transmission was evaluated by spatial point pattern analysis. FINDINGS Malaria was detected in 57.5% of the individuals and there was a disproportionate number of imported cases (90.6%) linked to Brazilian coming from gold mining sites in Venezuela and Guyana. MAIN CONCLUSIONS The increase in imported malaria cases circulating in the west region of Boa Vista, where there are positive breeding sites for the main vectors, may represent a potential condition for increased autochthonous malaria transmission in this space.

First molecular detection of Haemoproteus spp. and Plasmodium spp. in eared doves (Zenaida auriculata) in Brazil / Primeira detecção molecular de Haemoproteus spp. e Plasmodium spp. em pombos (Zenaida auriculata) no Brasil

Abstract The aim of this study was to verify the presence and identify the species of haemosporidian parasites in eared doves (Zenaida auriculata) in Brazil. Two hundred and eleven male and female eared doves were trap-captured in four different regions of Londrina city, in southern Brazil. Whole blood was collected in EDTA tubes through heart puncture after euthanasia in a CO2 chamber. A nested PCR targeting the mitochondrial cytochrome b gene (cyt b) of Haemoproteus spp./Plasmodium spp. was performed, followed by an enzymatic digestion to identify the genus. Phylogenetic trees were constructed to determine the closely related species. Out of 211 eared doves, 209 (99.05%) were positive for Haemoproteus spp. and/or Plasmodium spp. RFLP analysis showed that 72.72% (152/209) of eared doves were positive only for Haemoproteus spp., 6.22% (13/209) were positive only for Plasmodium spp., and 21.05% (44/209) of eared doves had mixed infections. Genetic analysis found four samples that were homologous with Haemoproteus multipigmentatus and one that was homologous with Plasmodium sp. This is the first molecular study of hemoparasites from eared doves in Brazil, and it is also the first description of H. multipigmentatus and Plasmodium spp. infection in eared doves in Brazil.

Resumo O objetivo deste estudo foi verificar a presença e a identificação espécies de parasitas hemosporídeos em pombos (Zenaida auriculata) no Brasil. Duzentos e onze pombos machos e fêmeas foram capturados em quatro regiões diferentes de Londrina, sul do Brasil. Amostra de sangue foi coletada em tubos contendo EDTA por meio de punção cardíaca, após eutanásia em câmara de CO2. Uma nested PCR com alvo no gene mitocondrial citocromo b (cyt b) de Haemoproteus spp./Plasmodium spp. foi realizada, seguida de digestão enzimática para identificar o gênero. A árvore filogenética foi construída para determinar a relação com outras espécies. Das 211 pombas, 209 (99,05%) foram positivas para Haemoproteus spp./Plasmodium spp. A análise RFLP demonstrou que 72,72% (152/209) das pombas foram positivas somente para Haemoproteus spp.; 6,22% (13/209) foram positivas somente para Plasmodium e 21,05% (44/209) das pombas tiveram infecções mistas. A análise genética mostrou quatro amostras homólogas com H. multipigmentatus e uma com Plasmodium spp. Este é o primeiro estudo molecular de hemoparasitas em pombos no Brasil. E é também a primeira descrição da infecção por H. multipigmentatus e Plasmodium spp. em pombos Z. auriculata no Brasil.

Comparative morphometric evaluation of hepatic hemosiderosis in wild Magellanic penguins (Spheniscus magellanicus) infected with different Plasmodium spp. subgenera / Avaliação morfométrica comparativa da hemossiderose hepática em pinguins-de-Magalhães (Spheniscus magellanicus) de vida livre infectados por diferentes subgêneros de Plasmodium spp

Abstract Avian malaria is one of the most important diseases of captive penguins. We employed morphometric techniques to evaluate hepatic hemosiderosis in rehabilitating wild Magellanic penguins (Spheniscus magellanicus) that were negative (n = 9) or naturally infected by different subgenera of Plasmodium spp. (n = 24), according with: Plasmodium subgenera (Haemamoeba, Huffia, Other lineages, and Unidentified lineages), severity of Plasmodium histopathological lesions, and concurrent diseases, age class (juvenile or adult plumage), sex (male, female or not determined), body score (emaciated, thin, good, excellent, not available), molt, presence or absence of oil contamination upon admission, iron supplementation, and rehabilitation center. The percentage of the area occupied by hemosiderin was called 'Index of Hepatic Hemosiderosis (IHH)'. Plasmodium-positive females presented significantly higher IHH values (17.53 ± 12.95%) than males (7.20 ± 4.25%; p = 0.041). We observed higher levels of congestion (p = 0.0182) and pneumonia (p = 0.0250) severity between Unidentified lineages vs. Huffia. We believe that the hepatic hemosiderosis observed in this study was multifactorial, the result of pathological processes caused by malaria, molting, hemoglobin and myoglobin catabolism during migration, anemia, concomitant diseases, and iron supplementation, all possibly potentiated by decreased liver mass. Further studies are needed to clarify the mechanisms of these hypotheses.

Resumo Malária aviária é uma das mais relevantes doenças em pinguins cativos. Foram aplicadas técnicas morfométricas para avaliar a hemossiderose hepática em pinguins-de-Magalhães (Spheniscus magellanicus ) de vida livre em reabilitação negativos (n = 9) e naturalmente infectados por diferentes subgêneros de Plasmodium spp. (n = 24), quanto a: subgênero de Plasmodium (Haemamoeba , Huffia, Outras Linhagens, e Linhagens não identificadas), severidade das lesões histopatológicas causadas por Plasmodium e doenças concomitantes, faixa etária (plumagem juvenil ou adulta), sexo (macho, fêmea, indeterminado), condição corporal (emaciado, magro, bom, excelente, indisponível), muda, presença/ausência de óleo a admissão, suplementação de ferro, e centro de reabilitação. A porcentagem da área ocupada por hemossiderina foi denominada "Índice de Hemossiderose Hepática (IHH)". Fêmeas Plasmodium -positivas apresentaram IHH significativamente mais elevado que machos, respectivamente, 17,53 ± 12,95% e 7,20 ± 4,25% (p = 0,041). Níveis mais elevados de congestão (p = 0,0182) e pneumonia (p = 0,0250) foram observados entre Linhagens não identificadas vs. Huffia. Possivelmente, a hemossiderose hepática observada nesse estudo seja multifatorial, resultado de processos patológicos causados por malária, muda, catabolismo de hemoglobina e mioglobina durante a migração, anemia, doenças concomitantes e suplementação de ferro, potencialmente intensificados por massa hepática reduzida. Estudos complementares são necessários para esclarecer os mecanismos de tais hipóteses.

Detection of Plasmodium in faeces of the New World primate Alouatta clamitans

Abstract Plasmodium falciparum and Plasmodium vivax have evolved with host switches between non-human primates (NHPs) and humans. Studies on the infection dynamics of Plasmodium species in NHPs will improve our understanding of the evolution of these parasites; however, such studies are hampered by the difficulty of handling animals in the field. The aim of this study was to detect genomic DNA of Plasmodium species from the faeces of New World monkeys. Faecal samples from 23 Alouatta clamitans from the Centre for Biological Research of Indaial (Santa Catarina, Brazil) were collected. Extracted DNA from faecal samples was used for molecular diagnosis of malaria by nested polymerase chain reaction. One natural infection with Plasmodium simium was identified by amplification of DNA extracted from the faeces of A. clamitans. Extracted DNA from a captive NHP was also used for parasite genotyping. The detection limit of the technique was evaluated in vitro using an artificial mixture of cultured P. falciparum in NHP faeces and determined to be 6.5 parasites/µL. Faecal samples of New World primates can be used to detect malaria infections in field surveys and also to monitor the genetic variability of parasites and dynamics of infection.

Plasmodium simium/Plasmodium vivax infections in southern brown howler monkeys from the Atlantic Forest

Blood infection by the simian parasite, Plasmodium simium, was identified in captive (n = 45, 4.4%) and in wild Alouatta clamitans monkeys (n = 20, 35%) from the Atlantic Forest of southern Brazil. A single malaria infection was symptomatic and the monkey presented clinical and haematological alterations. A high frequency of Plasmodium vivax-specific antibodies was detected among these monkeys, with 87% of the monkeys testing positive against P. vivax antigens. These findings highlight the possibility of malaria as a zoonosis in the remaining Atlantic Forest and its impact on the epidemiology of the disease.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Variation in haematological parameters in children less than five years of age with asymptomatic Plasmodium infection: implication for malaria field studies

During the season of high malaria transmission, most children are infected by Plasmodium, which targets red blood cells (RBCs), affecting haematological parameters. To describe these variations, we examined the haematological profiles of two groups of children living in a malaria-endemic area. A cross-sectional survey was conducted at the peak of the malaria transmission season in a rural area of Burkina Faso. After informed consent and clinical examination, blood samples were obtained from the participants for malaria diagnosis and a full blood count. Of the 414 children included in the analysis, 192 were not infected with Plasmodium, whereas 222 were asymptomatic carriers of Plasmodium infection. The mean age of the infected children was 41.8 months (range of 26.4-57.2) compared to 38.8 months (range of 22.4-55.2) for the control group (p = 0.06). The asymptomatic infected children tended to have a significantly lower mean haemoglobin level (10.8 g/dL vs. 10.4 g/dL; p < 0.001), mean lymphocyte count (4592/µL vs. 5141/µL; p = 0.004), mean platelet count (266 x 103/µL vs. 385 x 103/µL; p < 0.001) and mean RBC count (4.388 x 106/µL vs. 4.158 x 106/µL; p < 0.001) and a higher mean monocyte count (1403/µL vs. 1192/µL; p < 0.001) compared to the control group. Special attention should be applied when interpreting haematological parameters and evaluating immune responses in asymptomatic infected children living in malaria-endemic areas and enrolled in vaccine trials.

Apoptose em eritrócitos: envolvimento na patogênese da anemia da malária / Apoptosis in erythrocytes: involvement in the pathogenesis of malarial anemia

A anemia grave é sem dúvida uma das complicações mais comuns durante a infecção malárica e é uma das principais responsáveis pela elevada taxa de mortalidade de crianças e mulheres grávidas em regiões hiperendêmicas africanas. Embora os mecanismos que desencadeiam a anemia grave da malária ainda não tenham sido completamente elucidados, sabe-se que o aumento da destruição dos eritrócitos não parasitados tem importante contribuição na gênese dessa manifestação clínica. Frente a esse fato e tendo em vista a já descrita susceptibilidade dos eritrócitos ao processo de apoptose, que inclusive parece estar relacionado com a anemia de diversas patologias e pode ser induzida por diferentes tipos de drogas, decidimos estudar a apoptose eritrocitária e sua participação na anemia no decurso da infecção malárica. Primeiramente, avaliamos in vitro a capacidade da cloroquina, mefloquina, artemisinina e quinina em induzir apoptose em eritrócitos humanos. Nesta primeira investigação, observamos que os antimaláricos não tiveram efeito proapoptótico nestas células e, portanto, parecem não participar da anemia da malária através da indução de apoptose eritrocitária. Em seguida, estudando a infecção experimental de camundongos Balb/c pelo Plasmodium yoelii 17XL, constatamos um aumento nos níveis basais de apoptose de eritrócitos não parasitadas e sugerimos que este aumento poderia estar contribuindo com a anemia neste modelo experimental de malária. Porém, quando posteriormente examinamos em paralelo os níveis de apoptose e o grau de anemia, não foi possível observar relação entre estes dois eventos, mas sim entre a carga parasitária e os níveis de apoptose detectados na fase tardia da infecção. A possível influência da parasitemia nos níveis de apoptose eritrocitária parece ter encontrado suporte quando estudamos pacientes infectados pelo P. vivax. De fato, nessa infecção, que cursa com baixa parasitemia, não detectamos alteração nos níveis de apoptose eritrocitária. Como a apoptose além de ter propriedades patogênicas, também está envolvida com a limitação da propagação de parasitas intracelulares, avaliamos então in vitro a capacidade do P. falciparum de infectar eritrócitos apoptóticos. Neste último estudo, verificamos que estas células apoptóticas são refratárias à infecção plasmodial e, portanto, poderiam participar no controle da parasitemia na malária.

Malária autóctone notificada no Estado de São Paulo: aspectos clínicos e epidemiológicos de 1980 a 2007 / Autochthonous malaria notified in the State of São Paulo: clinical and epidemiological characteristics from 1980 to 2007

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 por cento em 11 variáveis. A tendência da incidência foi decrescente. Foram notificados 821 casos autóctones, 91,6 por cento 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 percent for 11 variables. The incidence trend was decreasing. There were 821 autochthonous cases: 91.6 percent 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.

Epidemiological and ecological aspects related to malaria in the area of influence of the lake at Porto Primavera dam, in western São Paulo State, Brazil / Aspectos epidemiológicos e ecológicos relacionados à malária na área de influência do lago da Represa de Porto Primavera, região oeste do Estado de São Paulo, Brasil

A study was carried out in the area of influence of the Porto Primavera Hydroelectric Power Station, in western São Paulo State, to investigate ecological and epidemiological aspects of malaria in the area and monitor the profile of the anopheline populations following the environmental changes brought about by the construction of the lake. Mosquitoes captured were analyzed by standardized indicator species analysis (ISA) before and during different flooding phases (253 m and 257 m elevations). The local human population was studied by means of parasitological (thin/thick blood smears), molecular (PCR) and serological tests. Serological tests consisted of Enzyme Linked Immunosorbent Assay (ELISA) with synthetic peptides of the circumsporozoite protein (CSP) from classic Plasmodium vivax, P. vivax variants (VK247 and "vivax-like"), P. malariae and P. falciparum and Indirect Immunofluorescence Assay (IFA) with asexual forms of P. vivax, P. malariae and P. falciparum. The results of the entomological survey indicated that, although the Anopheles darlingi population increased after the flooding, the population density remained very low. No malaria, parasite infection or DNA was detected in the inhabitants of the study area. However, there was a low frequency of antibodies against asexual forms and a significant prevalence of antibodies against P. vivax, P. vivax variants, P. falciparum and P. malariae; the presence of these antibodies may result from recent or less recent contact with human or simian Plasmodium (a parallel study in the same area revealed the existence of a sylvatic cycle) ...

Foi realizada pesquisa na área de influência do lago da Usina Hidrelétrica de Porto Primavera, região oeste do Estado de São Paulo, para estudar aspectos ecológicos e epidemiológicos da malária na localidade e acompanhar o perfil das populações de anofelinos frente às mudanças decorrentes do impacto ambiental pela formação do lago. Mosquitos capturados foram analisados pelo Índice de Abundância de Espécies Padronizado (IAEP), antes e durante o enchimento do reservatório (cotas 253 e 257 m). A população humana local foi estudada por meio de teste parasitológico (gota espessa e esfregaço sangüíneo), testes moleculares (PCR) e testes sorológicos. A sorologia consistiu na reação de ELISA com peptídeos sintéticos correspondentes à porção repetitiva da proteína circumsporozoíta (CSP) de Plasmodium vivax clássico, e suas variantes VK247 e "vivax-like", P. malariae e P. falciparum; e reação de Imunofluorescência Indireta (RIFI) com formas assexuadas de P. vivax, P. malariae e P. falciparum. Os resultados do estudo entomológico indicaram que, embora a população de Anopheles darlingi tenha aumentando após o enchimento, permaneceu em baixa densidade. Não foi detectada malária nem a presença de parasitos ou de DNA parasitário nos habitantes estudados. No entanto, foi observada baixa freqüência de anticorpos contra formas assexuadas e significativa prevalência de anticorpos contra esporozoítos de P. vivax e suas variantes, P. falciparum e P. malariae, que poderiam decorrer de contatos prévios, recentes ou não, com plasmódios humanos ou símios (o ciclo silvestre foi evidenciado em estudo paralelo realizado na mesma área)...

Presencia de Plasmodium brasilianum (Apicomplexa, Plasmodidae) en el mono congo (Alouatta palliata, Primates: Cebidae) de Costa Rica: importancia epidemiológica en relación con el ser humano / Plasmodium brasilianum (Apicomplexa, Plasmodidae) in the congo monkey Alouatta brasilianum (Primate: Cebidae) of Costa Rica: epidemiological importance related to human

La especie productora de malaria en primates, Plasmodium brasilianum, fue encontrada por primera vez en Costa Rica en 6 de 104 ejemplares de monos congo o aulladores (Alouatta palliata). Los animales fueron capturados y anestesiados por medio de dardos que contenían hidrocloruro de tiletamina y zolazepam (Zoletil®) combinados en partes iguales. Para estudiar estos animales por parásitos sanguíneos, se prepararon frotis sanguíneos que luego se tiñeron y se estudiaron en el laboratorio, encontrándose las formas de trofozoitos jóvenes o avanzados así como gametocitos y esquizontes. La morfología característica de algunos estados evolutivos, como por ejemplo, las formas en banda de trofozoitos avanzados y los esquizontes en forma de margarita o "rosetta" permitieron el diagnóstico de la especie. Puesto que se han encontrado casos humanos infectados con este organismo y éste es casi indiferenciable de Plasmodium malariae, una especie parásita del ser humano, se discute el hallazgo de este parásito desde un punto de vista epidemiológico en el área de la salud.

Management of malaria: recent trends.

In the present day scenario of resurgence of infectious diseases, malaria compounded with problems of multi drug resistance, assumes paramount importance. A combination of artemisinine derivatives with other effective anti-malarial drug remains the most effective form of treatment against the falciparum malaria which is most lethal form of disease. Oral chloroquin in the dose of 25 mg base/kg over 48 hours is effective in infections due to P. vivax, P. ovale P. malariae and chloroquine sensitive P. Falciparum. For chloroquine resistant P. vivax and multidrug resistant falciparum malaria, a combination of Quinine with doxycycline or clindamycin for 5-7 days, Quinine with singlt dose sulfadoxine-pyrimethamine combination. Mefloquine with artemeter or artesunate for 3 days, artesunate with doxycycline or clindamycin for 7 days and Otovaquin with proguanil for 3 days have been found to be effective. Primiquin as a hypnozoticide for 5-10 days is mandatory for preventing relapse in cases of P. vivax, P. Ovale and P. malariae. Death due to complicated malaria can be as high as 75% if case diagnosis is delayed or the patient arrives late. The artemisinine based rectal suppositories can be very effective in home/village setting in patients who can not be given oral anti malarial, though not yet approved for use in our country. In ICU settings, properly administered loading dose of quinine has proved to be effective and safe in almost all therapeutic trials including our study on Indian patients. Frequent blood glucose monitoring is mandatory. Parentral artemisinine with oral mefloquine is an effective alternative to quinine based therapy. The cerebral malaria management in the ICU setting includes monitoring fluid and electrolyte balance so as to maintain a CVP of 5 cm of water and pulmonary arterial occlusive pressure at less than 15 mm of mercury. In renal failure haemofiltration is ideal. Mefloquine is safe in second and third trimester of pregnancy. Exchange transfusion, haemopheresis and plasmapheresis are new techniques in the treatment of gravely ill patients with PF malaria especially when parasitemia exceeds 10%.

The importance of Anopheles albitarsis E and An. darlingi in human malaria transmission in Boa Vista, state of Roraima, Brazil

In several districts of Boa Vista, state of Roraima, Brazil we found Anopheles (Nyssorhynchus) albitarsis E to be the primary vector of human malaria parasites, and during 2001-2002 it was significantly more abundant than An. darlingi (p < 0.001). Other species sampled were An. (Nys.) braziliensis, An. (Ano.) peryassui, An. (Nys.) nuneztovari, An. (Nys.) oswaldoi s.l., and An. (Nys.) triannulatus. As determined by the ELISA technique An. darlingi had a higher overall infection rate (2.1 percent) compared with An. albitarsis E (1.2 percent). However, a marginally higher proportion of An. albitarsis E was infected with Plasmodium vivax compared with An. darlingi, and the An. albitarsis E biting index was also much higher. These results suggest the importance of An. albitarsis E in malaria transmission in a savannah ecoregion of northern Amazonian Brazil, and reconfirm the importance of An. darlingi even if at lower abundance.

Fluorometría; un método rápido y sencillo para evaluar la Actividad Antipalúdica / Fluorometers; a rapid and simple method for evaluating the Antimalarial Activity

La Malaria es una enfermedad que oscila entre 250 y 300 millones de casos y aproximadamente dos millones de muertes anualmente1. Esta enfermedades causada por cuatro especies de Plasmodium (P.falciparum, P. vivax, P.ovale y P. malariae) que son transmitidas a los humanos por picadura de las hembras del mosquito Anopheles. El aumento de la resistencia del parásito al tratamiento con agentes antimaláricos conocidos como la cloroquina se convierte en uno de los aspectos responsables del crecimiento de esta enfermedad. Además de este problema también se cuenta con la resistencia delvector a los insecticidas y la limitación de una potencial vacuna antimalárica, todo esto contribuye a la necesidad urgente de encontrar nuevos agentes para el tratamiento de la malaria, en particular agentes efectivos contra P. falciparum, que es la responsable de la forma más severa de malaria.

Malaria: the value of the automated depolarization analysis.

This retrospective and descriptive study was carried out in the University of Malaya Medical Center (UMMC) from January to September, 2004. This study aimed to evaluate the diagnostic utility of the Cell-Dyn 4000 hematology analyzer's depolarization analysis and to determine the sensitivity and specificity of this technique in the context of malaria diagnosis. A total of 889 cases presenting with pyrexia of unknown origin or clinically suspected of malaria were examined. Sixteen of these blood samples were found to be positive; 12 for P. vivax, 3 for P. malariae, and 1 for P. falciparum by peripheral blood smear as the standard technique for parasite detection and species identification. Demographic characteristics showed that the majority of patients were in the age range of 20-57 with a mean of 35.9 (+/- SD) 11.4 years, and male foreign workers. Of these, 16 positive blood samples were also processed by Cell-Dyne 4000 analyzer in the normal complete blood count (CBC) operational mode. Malaria parasites produce hemozoin, which depolarizes light and this allows the automated detection of malaria during routine complete blood count analysis with the Abbot Cell-Dyn CD4000 instrument. The white blood cell (WBC) differential plots of all malaria positive samples showed abnormal depolarization events in the NEU-EOS and EOS I plots. This was not seen in the negative samples. In 12 patients with P. vivax infection, a cluster pattern in the Neu-EOS and EOS I plots was observed, and appeared color-coded green or black. In 3 patients with P. malariae infection, few random depolarization events in the NEU-EOS and EOS I plots were seen, and appeared color-coded green, black or blue. While in the patient with P. falciparum infection, the sample was color-coded green with a few random purple depolarizing events in the NEU-EOS and EOS I plots. This study confirms that automated depolarization analysis is a highly sensitive and specific method to diagnose whether or not a patient has malaria. This automated approach may prove to be particularly useful in situations where there is little or no clinical suspicion of malaria.

Malaria: a 10-year (1994-2003) retrospective study at University Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia.

Malaria is still one of the most important vector-borne diseases in Malaysia, particularly in remote areas. This retrospective study was carried out to find the prevalence of malaria among patients admitted to UMMC Kuala Lumpur, from 1994-2003. A total of 86 malaria cases were analyzed. Most cases occurred among foreigners [57% (49 cases)] while Malaysians constituted 43% (37 cases). Among foreigners, Indonesians constituted the most [57% (28 cases)]. Among Malaysians, most cases occurred among the Chinese [35% (13 cases)] followed by the Malays [30% (11 cases)]. Males [70%(60 cases)] were more commonly affected. The majority of cases were within the 20-39 year age group (69%). Three species of malaria parasites were reported, of which Plasmodium vivax constituted the most [55%( 47 cases)], followed by Plasmodium falciparum [29% (25 cases)], and only four cases (5%) of Plasmodium malariae. Nine percent (8 cases) were mixed infections. In this study, 12%(10 cases) developed chloroquine resistance: 7 cases of P. falciparum, and 3 cases of P. vivax. The most common complications were jaundice and anemia [77% (23 cases)], followed by blackwater fever [13% (4 cases)] and cerebral malaria [10% (3 cases)]. Most of the complications were due to P. falciparum [43% (13 cases)]. There were no reported deaths. This new source of malaria coming from foreigners must be given serious attention, as it has great potential of increasing malaria cases in urban Malaysia.

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients.

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.

Identification of human malaria parasites and detection of mixed infection in Thai patients by nested PCR.

The species-specific nested PCR previously described by Snounou and others, for detecting the four species of human malaria parasites, is evaluated in the current study testing 40 blood samples from malaria patients admitted during July-September, 2003, at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand. Parasite DNA of each blood sample was extracted and purified by QIAamp. DNA mini kit. Nested PCR was performed using genus-specific primers for the first PCR cycle and species-specific primer for the second cycle. Thin and thick smears were also made, stained with Giemsa, and examined by expert microscopists. Only one of 40 samples (2.5%) was identified as Plasmodium malariae infection by both microscopy and nested PCR. Twenty blood samples (50%) were identified as Plasmodium falciparum infections by both methods. However, 19 blood samples (47.5%) were reported as Plasmodium vivax infections by microscopic methods, whereas nested PCR could detect a mixed infection of Plasmodium vivax and Plasmodium falciparum in one sample taken from a young girl with 8 ameboid trophozoites of P. vivax per 200 white blood cells. These results demonstrated that the nested PCR assay surpasses microscopy and also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment, but also for the study of malaria epidemiology.

Malaria species and Southeast Asian ovalocytosis defined by a 27-bp deletion in the erythrocyte band 3 gene.

To evaluate the resistance of SAO against species specific malaria infection, relationships between parasite species and the 27-bp deletion in the band 3 gene were studied in malaria endemic Sumba Island, eastern Indonesia. Thick blood films were prepared from patients with malaria symptoms (n=129) and healthy controls (n=231). Species of Plasmodium was identified by microscopic observation. The 27-bp deletion was screened by the PCR method. Among 231 healthy controls, 29 (12.6%) had the 27-bp deletion, whereas 14 (10.9%) among 129 patients confirmed with malaria infection harbored the 27-bp deletion. No significant difference was observed in the prevalence of the 27-bp deletion between controls and patients (p>0.8). There was no significant difference in the frequency of the 27-bp deletion between P. vivax and P. falciparum infected subjects at 5% level by Fisher's exact test. The present result showing no correlation between the presence of the 27-bp deletion and infected parasite species is consistent with the post-invasion resistance hypothesis that may involve not a single malaria species.