Hemozoin Pigment: An Important Tool for Low Parasitemic Malarial Diagnosis

Low parasitemic condition in malaria remains a diagnostic challenge; as the available diagnostic methods failed to detect. Currently, hemozoin (Hz) pigment is gaining attention in the diagnosis of malaria. The major drawback is ease of detection of Hz in routine practice. A pilot study was conducted to evaluate the role of Hz pigment and to compare the performance of quantitative buffy coat assay (QBC) and PCR in such conditions. Clinically suspected cases of malaria were examined by both Giemsa stain and immunochromatographic test (ICT). Samples positive by ICT and negative by Giemsa stain were further examined by nested PCR targeting 18S rRNA and QBC for the presence of malaria parasites and pigments. Thirty blood samples fulfilled the inclusion criteria out of which 23 were Plasmodium vivax (Pv), 4 Plasmodium falciparum (Pf), and 3 mixed (Pv and Pf) by immunochromatographic test. Twenty-one out of 30 (70%) were positive by nested PCR in comparison to 25/30 (83%) by QBC. Samples containing both malaria parasites and Hz pigment by QBC completely showed concordance with the PCR result. However, 61% of total samples containing only Hz pigment were observed positive by PCR. Hz pigment remains an important tool for malaria diagnosis. Identification of leukocytes containing pigments by QBC not only indicates recent malarial infections but also puts light on severity of the disease. QBC assay is a rapid, highly sensitive, and cost-effective method to detect malaria parasites and Hz pigment especially in low parasitemic conditions.

Avaliação da atividade antimalárica de novos derivados quinolínicos / Evaluation of the antimalarial activity of new quinoline derivatives

A malária é uma doença causada por cinco espécies de parasitos do gênero Plasmodium que causa anualmente a morte de milhares de pessoas, principalmente em países pobres da África. Muito antiga, uma diversidade de fármacos já foram empregados na tentativa de erradicação da doença, entretanto o aparecimento de cepas resistentes, bem como efeitos adversos gerados pelo tratamento impossibilitou tal ação. Os quinolínicos configuram uma grande parte destes tratamentos, apresentando uma notável atividade antimalárica. Neste trabalho nós avaliamos o potencial antimalárico de três novos derivados quinolínicos BS 260, BS 318 e BS 373 em culturas de Plasmodium falciparum, cepa w2, cloroquina resistente. BS 373 apresentou melhor atividade contra culturas de Plasmodium falciparum e, assim como o BS 318, foi capaz de inibir a biocristalização de hemozoína pelos parasitos. A microscopia eletrônica de transmissão revelou uma desorganização celular, diminuição do tamanho e quantidade de cristais de hemozoína no vacúolo digestivo, bem como vacuolizações citoplasmáticas e presença de estruturas membranares no vacúolo digestivo, o que indica a ocorrência de um processo autofágico nas células tratadas com 10 LM e 20 LM do BS 373. A presença de cristais citoplasmáticos indica a ocorrência de autólise pela ruptura da membrana do vacúolo digestivo. Por fim, o efeito dos tratamentos se mostrou irreversível nos parasitos com 24 horas de tratamento para BS 318 e BS 373, enquanto que para BS 260 essa irreversibilidade só foi observada após 48 horas. Nossos dados mostram que os derivados quinolínicos testados são efetivos contra culturas de P. falciparum, configurando bons candidatos à novas moléculas antimaláricas.

Malaria is a disease caused by five Plasmodium species that cause deaths of thousands of people annually, mostly in poor countries of Africa. Very anccient, a variety of drugs have been used in an attempt to eradicate the disease, however the emergence of resistant strains, as well as adverse effects caused by treatment prevented such action. The quinoline are a large part of these treatments, presenting a remarkable antimalarial activity. In this paper we evaluate the antimalarial potential of three new quinoline derivative BS 260, BS 318 and BS 373 in Plasmodium falciparum chloroquine resistant, w2 strain, cultures. BS 373 showed the best activity against Plasmodium falciparum cultures, while and analogously to BS 318 was able to inhibit the hemozoin formation by parasites. The transmission electron microscopy revealed a cell disorganization, decreased size and amount of hemozoin crystals in the digestive vacuole, cytoplasmic vacuolization and presence of membrane structures in the digestive vacuole, which indicates an autophagic process in cells treated with 10 LM and 20 LM BS 373. Cytoplasmic being crystals indicate parasite cell autolusis caused by digestive vacuole membrane disrupture. Finally, the effect of treatment proved irreversible on parasites at 24 hours of treatment for BS 318 and BS 373, whereas for BS 260 this irreversibility was only observed after 48 hours. Our data show that the quinoline derivatives tested are effective against P. falciparum cultures, setting good candidates for new antimalarial molecules.

Humoral Response against Native Plasmodium berghei Hemozoin and Synthetic Hemozoin in BALB/c Mice.

Aims: Malaria is a disease caused by protozoan parasites of the genus Plasmodium. One of the malaria mechanisms of adaptation to the host is the digestion of hemoglobin by the trophozoite stage. This mechanism provides the amino acids needed by the parasite and is carried out during the erythrocytics schozogony phase, which results in the formation of in soluble pigment crystals named hemozoin (Hz). Hz is responsible for many of the immune pathological complications of malaria, given that this pigment accumulates in various organs in severe cases of the disease. Here, we evaluated the humoral response in BALB/c mice against native Plasmodium berghei Hz (PbHz) and synthetic Hz (SHz). Place and Duration of Study: Laboratory of Immunology of Infection Diseases. Department of Cell Biology, Simón Bolívar University, Caracas, Venezuela. This study was performed between January 2012 and June 2012. Methodology: We determined the humoral response of SHz and PbHz by an enzyme linked immunosorbent assay (ELISA), using hyper-immune sera from mice experimentally infected with P. berghei or Plasmodium yoelii. In addition, SHz was evaluated as antigen by Western Blot and dot-ELISA. Results: When SHz was employed as antigen, we showed by indirect ELISA that the sera from mice immunized with SHz generated higher titers than sera obtained from mice infected with either Plasmodium species. Moreover, the sera from human infections also recognized SHz as antigen, but showed a better recognition by dot- ELISA or Western Blot than by indirect ELISA. Conclusion: In summary, our results indicated that SHz can be used as a rapid and successful diagnostic antigen for natural malaria infections by indirect ELISA, dot-ELISA and Western Blot techniques.

Effect of malaria components on blood mononuclear cells involved in immune response

During malaria infection, elevated levels of pro-inflammatory mediators and nitric oxide production have been associated with pathogenesis and disease severity. Previous in vitro and in vivo studies have proposed that both Plasmodium falciparum hemozoin and glycosylphosphatidylinositols are able to modulate blood mononuclear cells, contributing to stimulation of signal transduction and downstream regulation of the NF-κB signaling pathway, and subsequently leading to the production of pro-inflammatory cytokines, chemokines, and nitric oxide. The present review summarizes the published in vitro and in vivo studies that have investigated the mechanism of intracellular signal transduction and activation of the NF-κB signaling pathway in blood mononuclear cells after being inducted by Plasmodium falciparum malaria components. Particular attention is paid to hemozoin and glycosylphosphatidylinositols which reflect the important mechanism of signaling pathways involved in immune response.

¿La hemozoína, presente y futuro? / ¿Hemozoin present and future

La malaria es una enfermedad causada por parásitos del género Plasmodium estos parásitos tienen un ciclo intraeritocítico en el hospedador vertevrado. En el glóbulo rojo, el parásito ingiere la hemoglobina, obteniendo aminácidos y formando hemozoína. La hemozoína es un un material microcristalino oscuro, de color marrón amarillento, insoluble en agua, no tóxico, producido en la vacuola parasitófora del Plasmodium; este compuesto producido por el Plasmodium carece de la toxicidad que tiene el grupo hemo para el parásito. Asimismo se ha evidenciado que la hemozoína es una sustancia inmuno moduladora que tiene diversos efectos, como mediar la activación y migración de neutrófilos, incrementar la producción de óxido nítrico, inducir la activación de mataloproteínas 9, inducir la secreción de diferentes mediadores proinflamatorios, alterar las funciones de los monocitos y macrófagos humanos, tales como el estallido oxidativo, eliminación de bacterias, presentación de antígenos y la habilidad de diferenciarse a células dendríticas funcionales; por lo que la hemozína tiene efectos duales, tanto activadores como supresores de la respuesta inmune. Asimismo, la hemozoína es unblanco terapéutico potente, ya que los fármacos que inhiban su formación provocan toxicidad al parasíto e incluso la muerte del mismo.

Malaria is a disease caused by parasites of the genus Plasmodium. These parasites have intraerythrocytic cycle in the vertbrate host. In the red cell, the parasite ingests hemoglobin, obtaining amino acids and formin hemozoin. The microcrystaline material hemozoin is a dark, yellowish brown, insoluble in water, nontoxic, produced in the Plasmodium parasitophorous vacuole, this compound produced by Plasmodiun lacks the toxicity that has heme to the parasite. It has also been shown that hemozoin is an immune modulating substance that has different ffects, mediating the neutrophils activation and migration, increased nitric oxide production, induce activation of metallproteinase-9, induce the secretion of various proinflammatory mediators, alter the funcions of human monocytes and macrophages such as oxidative burst, removing bacteria, antigen presentation and the ability to differentiate into functional dendritic cells, so the hemozoin has dual effects, both activators and suppressors of the immune response. Also, the hemozoin is a potent therapeutic target, since rugs that inhibit their formation causes toxicity to the parasite and even death itself.

Three Cases of Pseudoeosinophilia Associated with Malaria Determined in the Sysmex XE-2100 Automated Hematology Analyzer / 대한진단검사의학회지

In Korea, the incidence of malaria has been increasing in the civilian population and in the areas previously considered as noninfected. Malaria can be suspected based on the patient's symptoms and the physical findings at examination. However, for a definitive diagnosis to be made, the malaria parasites or their components must be demonstrated by laboratory tests, which will take time and require expertise. Since general screening tests, such as a complete blood cell count, are always done for patients with a fever, it can be expected that the attention of laboratory hematologists drawn to any abnormalities found in automated hematology analyzers can help reduce delays in the diagnosis of malaria even if such a diagnosis was not initially considered. We report three cases of malaria that had thrombocytopenia and pseudoeosinophilia shown in the Sysmex XE-2100 (TOA Medical Electronics, Kobe, Japan) automated hematology analyzer. It is feasible that the pseudoeosinophilia presented as a result of hemozoin-containing white blood cells may contribute to the diagnosis of malaria, especially for patients unsuspected of the disease.

Conditional Inhibition of Hemozoin Formation by Chloroquine in vitro / 中国寄生虫学与寄生虫病杂志

Objective To study the characteristics of inhibition on hemozoin formation by chloroquine under in vitro condition.Methods Under different concentrations(0.5-2 mol/L) of sodium acetate(NaAc) and at the pH range of 4.0-5.0, chloroquine was tested for inhibition of ?-hematin(hemozion) formation by using the HPIA(heme polymerization inhibitory activity) assay.The morphology of ?-hematin crystals was determined by light microscopy.Ultraviolet spectrophotometry was employed to measure ?-hematin content, and the size of ?-hematin crystal was analyzed by X-ray diffraction(XRD) .Results Chloroquine exhibited varied effect on ?-hematin formation, depending on pH value and Na+ concentration.When the NaAc concentration increased from 0.5 mol/L(pH 4.2) to 2 mol/L(pH 4.8), the chloroquine inhibitory effect also increased.Results suggested that there exists a threshold pH, below which the ?-hematin formation escalates and chloroquine inhibition declines, and at or above which chloroquine exerts a stronger inhibitory effect on ?-hematin formation.With the increase of pH from 4.4 to 4.8, the crystallinity and the size of crystal changed from 6.93% and 357  to 6.32% and 264 , respectively.When pH reached to 5, no more ?-hematin formed.Chloroquine could reduce the crystallinity and crystal size of ?-hematin at same pH value.Morphology analysis on the samples was consistent with the above results.Conclusion Chloroquine inhibits hemozoin formation only when the pH value is at or above threshold pH.