Coexistence of Malaria and Thalassemia in Malaria Endemic Areas of Thailand

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and alpha-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and alpha-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.

Biomarkers for susceptibility to infection and disease severity in human malaria

Malaria remains a major infectious disease that affects millions of people. Once infected with Plasmodium parasites, a host can develop a broad range of clinical presentations, which result from complex interactions between factors derived from the host, the parasite and the environment. Intense research has focused on the identification of reliable predictors for exposure, susceptibility to infection and the development of severe complications during malaria. Although most promising markers are based on the current understanding of malaria immunopathogenesis, some are also focused more broadly on mechanisms of tissue damage and inflammation. Taken together, these markers can help optimise therapeutic strategies and reduce disease burden. Here, we review the recent advances in the identification of malarial biomarkers, focusing on those related to parasite exposure and disease susceptibility. We also discuss priorities for research in biomarkers for severe malaria.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0 percent oocyst rates were obtained, in comparison to the 86.67-100 percent oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0 percent sporozoite rates were obtained, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09 percent, 6.67 percent and 11.76 percent sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67 percent and 64.29 percent sporozoite rates were obtained, respectively, in comparison to 90 percent sporozoite rates recovered from An. cracens.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes and the infected placenta: a two-way pathway

Malaria is undoubtedly the world's most devastating parasitic disease, affecting 300 to 500 million people every year. Some cases of Plasmodium falciparum infection progress to the deadly forms of the disease responsible for 1 to 3 million deaths annually. P. falciparum-infected erythrocytes adhere to host receptors in the deep microvasculature of several organs. The cytoadhesion of infected erythrocytes to placental syncytiotrophoblast receptors leads to pregnancy-associated malaria (PAM). This specific maternal-fetal syndrome causes maternal anemia, low birth weight and the death of 62,000 to 363,000 infants per year in sub-Saharan Africa, and thus has a poor outcome for both mother and fetus. However, PAM and non-PAM parasites have been shown to differ antigenically and genetically. After multiple pregnancies, women from different geographical areas develop adhesion-blocking antibodies that protect against placental parasitemia and clinical symptoms of PAM. The recent description of a new parasite ligand encoded by the var2CSA gene as the only gene up-regulated in PAM parasites renders the development of an anti-PAM vaccine more feasible. The search for a vaccine to prevent P. falciparum sequestration in the placenta by eliciting adhesion-blocking antibodies and a cellular immune response, and the development of new methods for evaluating such antibodies should be key priorities in mother-child health programs in areas of endemic malaria. This review summarizes the main molecular, immunological and physiopathological aspects of PAM, including findings related to new targets in the P. falciparum var gene family. Finally, we focus on a new methodology for mimicking cytoadhesion under blood flow conditions in human placental tissue.

Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities.

BACKGROUND & OBJECTIVES: Plasmodium falciparum, the causative agent of the most serious form of malaria, infects about 5-10% of the world human population per year. It is well established that the erythrocytic stages of the malaria parasite rely mainly on glycolysis for their energy supply. In the present study, the glucose utilisation of erythrocyte population with parasitaemia levels similar to that of malaria patients was measured. The results allowed us to assess the effect of the parasites on the glucose utilisation of the vast majority of uninfected erythrocytes. METHODS: Using [2-13C]glucose and nuclear magnetic resonance (NMR) technique, the glucose utilisation in normal red blood cell (RBC) and P. falciparum infected red blood cell (IRBC) populations was measured. The IRBC population consisted of > 96% RBC and < 4% of parasite infected red blood cells (PRBC). The glycolytic enzymes were assayed to assess the effect of infected red cells on the enzymatic activities of uninfected ones. RESULTS: The rate of glucose utilisation by IRBC was considerably higher than that of RBC. Upon addition of 25% v/v conditioned culture medium (CM) of IRBC, RBCs exhibited a significant decrease in glucose utilisation. The CM could directly inhibit the activities of RBC glycolytic enzymes-phosphofructokinase (PFK) and pyruvate kinase (PK), without interfering with the activity of the pentose phosphate pathway enzyme-glucose-6-phosphate dehydrogenase (G-6-PD). INTERPRETATION & CONCLUSION: The present study showed that the clinical level of P. falciparum infected RBCs (< 4% parasitaemia) significantly enhance the glycolytic flux as well as down-regulate the glucose utilisation rate in the majority of uninfected RBC population. The mechanism of inhibition seems to be direct inhibition of the regulatory glycolytic enzymes-PFK and PK.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Quatro colônias desenvolvidas em laboratório, de duas formas cariotípicas de Anopheles aconitus i.e. forma B (cepa Chiang Mai e Phet Buri) e C (Cepa Chiang Mai e Mae Hong Son), foram infectadas experimentalmente com Plasmodium falciparum e P. vivax usando técnica de alimentação com membrana artificial e dissecados oito e 12 dias após alimentação da média de oocistos e esporozoitos, respectivamente. Os resultados revelaram que An. aconitus formas B e C foram suscetíveis ao P. falciparum e P. vivax isto é, forma B (cepa Chiang Mai e Phet Buri/P. falciparum e P. vivax) e forma C (cepa Chiang Mai e Mae Hong Son/P. vivax). Análises estatísticas comparativas das taxas de oocistos, número médio de oocistos por intestino médio infectado e taxas de esporozoitos entre todas as cepas de An. aconitus formas B e C ao grupo interno de vetores controles, An. minimus A e C, não exibiram nenhuma diferença significante, confirmando o alto potencial vetor das duas espécies de Plamodium. Os cristais semelhantes a esporozoitos encontrados no lobo médio das glândulas salivares que poderiam ser um fator enganoso na identificação de esporozoitos verdadeiros nas glândulas salivares foram encontrados em ambos An. aconitus formas B e C.

Clinical and molecular aspects of severe malaria

O ciclo eritrocítico do Plasmodium falciparum apresenta uma particularidade em relação às outras espécies de Plasmodium que infectam o homem. Trofozoítas maduros e esquizontes são seqüestrados da circulação periférica devido à adesão de eritrócitos infectados às células endoteliais. Modificações na superfície dos eritrócitos infectados, denominadas "knobs", permitem adesão ao endotélio e a outros eritrócitos. A adesão fornece uma melhor maturação na atmosfera venosa microaerofílica e permite que o parasita escape do clareamento pelo baço, que reconhece a perda de deformabilidade do eritrócito infectado. A adesão ao endotélio ou citoaderência, tem importante função na patogenicidade da doença, causando obstrução de pequenos vasos e contribuindo para danos em muitos órgãos. Citoaderência designa também a adesão de eritrócitos infectados a eritrócitos não infectados, fenômeno amplamente conhecido como "rosetting". Aspectos clínicos da malária grave bem como receptores do hospedeiro e ligantes do parasita envolvidos em citoaderência e "rosetting", são revisados aqui. A proteína de membrana do eritrócito 1 de P. falciparum (PfEMP1) parece ser o principal ligante adesivo dos eritrócitos infectados e será discutida em maiores detalhes. Uma melhor compreensão da função dos receptores do hospedeiro e dos ligantes do parasita no desenvolvimento de diferentes síndromes clínicas é urgentemente necessária para identificar alvos para vacinação visando diminuir as taxas de mortalidade desta doença.

Lack of association between CSF nitrate and sera nitrate in falciparum malaria infection.

Nitrate levels in CSF and sera from 16 coma and 19 noncoma falciparum malaria patients were determined using nitric oxide colorometric assay. The medians (range lower, upper limits) of nitrate in sera of comatose and noncomatose patients were 0.28 (0.11, 1.24) and 0.23 (0.05, 0.87) microM, respectively. The medians of nitrate level in CSF of coma and noncoma cases were 0.09 (0.01, 0.28) and 0.15 (0, 1.18) microM, respectively. There was no difference of nitrate level in sera and CSF from comatose or noncomatose patients compared to that in normal sera and CSF. The amount of nitrate in sera and CSF of both groups was not significantly correlated with coma depth, parasitemia, parasite clearance time and time to recovery. Contrast to our in vitro study using immunoperoxidase staining, we found inducible nitric oside synthase production by brain endothelial cells during 4-24 hours of coculturing with late stage of P. falciparum infected red blood cells. These results suggests that malaria severity can not be differentiated by nitrate level in body fluid.

Detección y expresión de una proteína de unión a calmodulina durante el ciclo celular asexual de Plasmodium falciparum / Detection of Giardia lamblia Colombian strains antigens in eluates from human faeces using the specific anti-31, 65 and 170 kDa parasitic policlonal antibodies by ELISA

Los organismos vivos han establecido varios mecanismos con los cuales traducen las señales extracelulares a un mensaje intracelular que puede ser entendido por la maquinaria bioquímica de la célula. En eucariotes la calmodulina juega un papel central en el procesamiento de la señal de calcio, disparando respuestas bioquímicas altamente específicas que se ejercen a través de las proteínas de unión a calmodulina PUCaM, proteínas que se convierten realmente en las moléculas efectoras de una cascada de eventos donde la respuesta final depende de la PUCaM activada. En Plasmodium falciparum se ha reportado la presencia de calmodulina y se sabe que el calcio juega un papel determinante en diferentes eventos del parásito, sin embargo aun no se ha establecido el compromiso de las PUCaMs. En el grupo de bioquímica del INS se aislaron 9 PUCaMs, de las cuales se escogió una de 30 kDa para este estudio. Se produjo un anticuerpo monoclonal contra la PUCaM de 30 kDa que nos permitió detectar, localizar y establecer el patrón de expresión de la proteína durante el ciclo asexual del parásito. Mediante Western blot se detectó la proteína de interés en extractos de parásito como una banda a la altura de 30 kDa y adicionalmemte el anticuerpo presentó reacción cruzada con las cadenas de la espectrina sugeriendo la presencia de un epítope similar o una estructura estrechamente relacionada en ambas proteínas. Los estudios de localización celular se realizaron por inmunofluorescencia indirecta y éstos mostraron una señal fluorescente dispersa únicamente en el citoplasma del parásito. Ni la membrana, ni el citoplasma de los eritrocitos infectados y los eritrocitos sanos presentaron fluorescencia, corroborando el origen plasmodial de la proteína de 30 kDa. La cinética de expresión reveló que esta proteína es expresada de una manera estado específica (en estadios maduros de 40 y 48 horas) lo que lleva a pensar que la PUCaM de 30 kDa es regulada a lo largo del desarrollo del parásito- Este estudio preliminar confirma la presencia de una proteína de union a calmodulina de 30 kDa en Plasmodium falciparum y abre grandes interrogantes que plantean la necesidad de continuar con estudios futuros para determinar con exactitud la identidad de la proteína y llegar a establecer su papel en los diferentes procesos del parásito, así como entender la fisiología de la señal de calcio en este organismo

Aislamiento y caracterización de proteínas de unión a calmodulina en el Plasmodium falciparum

Se detectaron en el citoplasma de P. falciparum, por Cromatografía de afinidad a calmodulina-agarosa en presencia de 0.2 mM calcio, nueve proteínas de unión a calmodulina de 135.0, 81.5, 66.0, 58.6, 45.0, 41.0, 36.5, 30.0 y 24.5 kDa que fueron eluidas con 1mM EGTA. Las mismas proteínas fueron identificadas independientemente por prueba de immunocoprecipitación de un extracto del parásito marcado con superíndice 35S-Met, usando calmodulina bovina y un anticuerpo monoclonal contra ésta. Cuando las nueve proteínas fueron sometidas a SDS-PAGE y transferidas a membrana de PVDF, ocho de ellas mantuvieron la habilidad de unirse a calmodulina biotinilada. Las proteínas fueron aisladas por Cromatografía de afindad, seguida por electroforesis preparativa e inoculadas en ratones Balb-C para producir anticuerpos monoclonales con los que se estudió su localización por Inmunofluorescencia e Inmunomicroscopia electrónica en los diferentes estadios del parásito. Igualmente se detectaron por inmunocoprecipitación dos bandas con peso mol superior a 205.0 kDa con capacidad de unión a un anticuerpo anti-espectrina. Una de las proteínas de unión a CaM (36.500) fue aislada y sometida a ruptura enzimática con tripsina, diseñandose a partir del extremo N-terminal de los fragmentos obtenidos, oligonucleótidos degenerados con los que se hizo amplificación del genoma del parásito por PCR, lográndose aislar un fragmento de 554 pb, que presentó una alta homología con la glutamato sintasa, proteína altamente relacionada con cloroplastos de algas y plantas, planteándose la probable importancia que esta proteína tiene tanto en la historia evolutiva de los aplicomplexas, como en el diseño de fármacos que permitan nuevas alternativas para elcontrol de las enfermedades ocasionadas por éste grupo de organismos

Proteínas de unión a actina en Plasmodium falciparum

La invasión del Plasmodium falciparaum, el agente causante de la malaria en humanos, implica un movimiento de penetración del parásito a los eritrocitos. Con el objetivo de determinar proteínas de unión a actina, se utilizaron dos estrategias diferentes: la primera consistió en preparar columnas de afinidad con actina polimerizada (actina F), para cromatografiar extractos de parásito marcados radioactivamente con L-[Superíndice 35S] metionina, y seleccionar proteínas de unión a actina que posteriormente, fueron identificadas por medio de anticuerpos específicos. En la segunda se utilizó la Reacción en Cadena de la Polimerasa (PCR) y oligonucleótidos generados, diseñados con base en secuencias consenso de miosinas, para buscar un fragmento de este gen en el ADN genómico (ADNg) del parásito y posteriormente clonarlo y secuenciarlo. Los resultados obtenidos sugieren la existencia de un motor molecular basado en actina-miosina, en la invasión del parásito a los eritrocitos. Esta afirmación esta sustentanda en primer lugar por el hallazgo de un gen de miosina en P. falciparum, que por homología de su secuencia parcial se clasifica dentro de la clase I de las miosinas no convencionales, caracterizadas por su capacidad de generar movimientos sobre filamentos de actina en diferentes células. Adicionalmente por medio de Western Blot y anticuerpos específicos, anti alfa actina, tripomiosina, espectrina, miosina y actina se determinó la exitencia de estas proteínas en el parásito

Susceptibility of Thai zoophilic Anophelines and suspected malaria vectors to local strains of human malaria parasites.

Wild caught zoophilic Anopheles and suspected malaria vector species collected in northwest Thailand were experimentally infected with local human malaria parasites using a membrane feeding. One week post-feeding a number of mosquitos were dissected for oocyst examination. The remainder were kept for another one week or more, and then the salivary glands were examined for the presence of sporozoites. The results revealed that An. vagus, An. kochi and An. annularis were susceptible to both Plasmodium falciparum and P. vivax whereas An. barbirostris and An. sinensis were susceptible to only P. vivax. The non-susceptibility to P. falciparum of these two mosquito species may indicate their poor vector status of this malaria species in the field.

Falciparum malaria in naturally infected Saudi patients: fine structure of intraerythrocytic gametocytes and their effects on host cell alterations: a comparison with those from various geographic regions

The fine structure of intraerythrocytic mature gametocytes [MG] was described in naturally infected Saudi patients [SP] from Tihama Valley, Asir Province, the most endemic region of falciparum malaria in the kingdom of Saudi Arabia and compared with those previously reported in vivo and in vitro MG from various geographic regions. In addition, the ultrastructural alterations of host cells infected with MG were also considered. The most striking finding in this study was that the MG cell wall from Saudi patients consisted of only a single plasma membrane, i.e. monolayer, without subpellicular microtubules [Sp Mt], while it was triand pentalaminar with Sp Mt in MG from other geographic regions. Unlike other MG, mitochondria, Golgi bodies and vacuoles have not been observed in parasites found in SP. As in some MG, the malaria pigment particles found in parasites from SP were embedded directly in the cytoplasm, while in other MG, they were enclosed in sizable vacuoles. The alterations induced in erythrocytes by MG from SP were similar to those found in MG from various geographic regions and include clefts, laveran's "bibs", a multilaminar membranous system and condensation of the host cell cytoplasm

Respuesta inmune celular contra el Plasmodium falciparum

Se presenta una revisión sobre el papel de las células del sistema inmune contra el Plasmodium falciparum. Parece ser que los polimorfonucleres ejercen una acción importante en el control de los parasitos al inicio de la infección; después las células importantes son los linfocitos T que a través de linfoquinaspueden activar otras células que en última instancia son las efectoras contra los globulos rojos y muy posiblemente las células hepáticas infectadas con el parasito. Estas células efectoras pueden ser linfocitos T citotoxicos, células NK y células del sistema monocitomacrófago, siendo tal vez estas últimas las más involucradas en la destrucción del parasito; pero, al mismo tiempo, participando en la patogénesis de la enfermedad sobre todo en malaria cerebral, a través de mediadores como el factor de nécrosis tumoral y los radicales del oxígeno.