Comparison of capillary, venous and buffy coat blood samples in detecting Plasmodium species among malaria suspected patients attending at Hamusite health center. A cross-sectional study.

BACKGROUND: Both capillary and venous blood samples have been interchangeably used for the diagnosis of malaria in Ethiopia. However, Plasmodium parasites are thought to be more concentrated in capillary than in venous blood. Hence, selecting a sample source where parasites are more concentrated is indispensable approach in order to maximize the accuracy of blood film microscopy. Therefore, the present study aimed to compare the detection rate and the parasitemia level of Plasmodium species from conventional capillary and venous blood films, and buffy coat preparations. METHODS: A facility based cross-sectional study was conducted from Feburary to March 2020 among 210 febrile patients attending Hamusite health center, northwest Ethiopia. Capillary and venous blood samples were collected and buffy coat was prepared from each sample. Thin and thick blood films were prepared, stained, and examined microscopically following standard protocol. Data were analysed using Statistical Package for Social Sciences Software version 20 and Med-Calc software version 19.3. RESULTS: Capillary blood buffy coat (61/210, 29.0%) had significantly higher detection rate as compared to capillary (48/210, 22.9%) and venous (42/210, 20.0%) blood films (p < 0.001). However, no significant difference was observed between capillary and venous blood films (p = 0.070) in detecting Plasmodium species. The highest and the lowest mean asexual stage parasite counts were found in capillary blood buffy coat (4692.88) and venous blood (631.43) films, respectively showing significant variations (p < 0.001). Mean gametocyte count was also highest in capillary blood buffy coat (3958.44). As compared to capillary blood buffy coat, the sensitivity of venous blood buffy coat, capillary blood film and venous blood film were 73.8, 78.7, 68.9%, respectively. CONCLUSION: Capillary blood buffy coat samples showed the highest sensitivity in detecting and quantitating malaria parasites that its use should be promoted in clinical settings. However, conventional capillary and venous blood films could be used interchangeably.

Comparison of capillary and venous blood for malaria detection using two PCR-based assays in febrile patients in Sierra Leone.

BACKGROUND: Rapid and sensitive diagnostics are critical tools for clinical case management and public health control efforts. Both capillary and venous blood are currently used for malaria detection and while diagnostic technologies may not be equally sensitive with both materials, the published data on this subject are scarce and not conclusive. METHODS: Paired clinical samples of venous and capillary blood from 141 febrile individuals in Bo, Sierra Leone, were obtained between January and May 2019 and tested for the presence of Plasmodium parasites using two multiplexed PCR assays: the FilmArray-based Global Fever Panel (GFP) and the TaqMan-based Malaria Multiplex Sample Ready (MMSR) assay. RESULTS: No significant differences in Plasmodium parasite detection between capillary and venous blood for both assays were observed. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and Plasmodium falciparum) in both venous and capillary blood. CONCLUSIONS: No difference was found in malaria detection between venous and capillary blood using two different PCR-based detection assays. This data gives support for use of capillary blood, a material which can be obtained easier by less invasive methods, for PCR-based malaria diagnostics, independent of the platform.

Optical Coherence Tomography Angiography Analysis of Retinal and Choroidal Vascular Networks during Acute, Relapsing, and Quiescent Stages of Macular Toxoplasma Retinochoroiditis.

PURPOSE: To highlight the advantages of optical coherence tomography angiography (OCTA) in delineating the morphological features of the retinal and choroidal vascular network during acute, relapsing, and quiescent stages of macular toxoplasma retinochoroiditis. METHODS: This prospective study included patients presenting with both active and quiescent ocular toxoplasmoses. OCTA was obtained to diagnose and follow the subsequent vascular network changes at diagnosis and six months after acute presentation. RESULTS: Twenty-three eyes of 23 patients were included. In active lesions, OCTA showed extensive, well-delineated areas of intense hyposignal and perifoveal capillary arcade disruption in the parafoveal superficial capillary plexus (pSCP) and less extensive hyposignal in the parafoveal deep capillary plexus (pDCP). Signals of decreased deep capillary density and disorganization were also seen in the choroid. In nonactive lesions, OCTA demonstrated a homogenous and equally attenuated grayish hyposignal of the pSCP and pDCP and a partial restoration of the nonperfused choroidal areas. CONCLUSION: OCTA is a useful technique for vascular network analysis in toxoplasma retinochoroiditis. It allows the visualization of the different network changes and behaviors during the different stages of the infection.

Comparison on simultaneous caillary and venous parasite density and genotyping results from children and adults with uncomplicated malaria: a prospective observational study in Uganda.

BACKGROUND: Blood smear microscopy remains the gold-standard method to diagnose and quantify malaria parasite density. In addition, parasite genotyping of select loci is the most utilized method for distinguishing recrudescent and new infections and to determine the number of strains per sample. In research settings, blood may be obtained from capillary or venous compartments, and results from these matrices have been used interchangeably. Our aim was to compare quantitative results for parasite density and strain complexity from both compartments. METHODS: In a prospective observational study, children and adults presenting with uncomplicated Plasmodium falciparum malaria, simultaneous capillary and venous blood smears and dried blood spots were collected over 42-days following treatment with artemether-lumefantrine. Blood smears were read by two microscopists, any discrepancies resolved by a third reader. Parasite DNA fingerprinting was conducted using six microsatellites. Bland Altman analysis and paired t-test/McNemar's test were used to assess the difference in density readings and measurements. RESULTS: Two hundred twenty-three participants were included in the analysis (177 children (35 HIV-infected/142 HIV-uninfected), 21 HIV-uninfected pregnant women, and 25 HIV-uninfected non-pregnant adults). Parasite density measurements did not statistically differ between capillary and venous blood smears at the time of presentation, nor over the course of 42-day follow-up. Characterization of merozoite surface protein-2 (MSP-2) genetic polymorphism demonstrated a higher level of strain diversity at the time of presentation in venous samples, as compared with capillary specimens (p = 0.02). There was a high degree of variability in genotype-corrected outcomes when pairs of samples from each compartment were compared using MSP-2 alone, although the variability was reduced with the use of multiple markers. CONCLUSIONS: Parasite density measurements do not statistically differ between capillary and venous compartments in all studied demographic groups at the time of presentation with malaria, or over the course of follow-up. More strains were detected by MSP-2 genotyping in venous samples than in capillary samples at the time of malaria diagnosis. The use of multiple polymorphic markers reduces the impact of variability in strain detection on genotype-corrected outcomes. This study confirms that both capillary and venous compartments can be used for sampling with confidence in the clinical research setting. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov under registration no. NCT01717885 .

Peripheral venous vs. capillary microfilariaemia in a dog co-infected with Dirofilaria repens and D. immitis: A comparative approach using triatomine bugs for blood collection.

Dirofilaria immitis and D. repens are mosquito-borne nematodes, primarily infecting dogs, but also other species of carnivores and even humans. Given their impact on animal and human health, the transmission of these filarioids has been widely studied. The microfilariaemia has been shown to have a circadian variation for both Dirofilaria species infecting dogs. Due to methodological difficulties, the periodicity was only studied using venous blood samples, while the mosquitoes feed, in fact, on capillary blood. In this context, the present study aimed to test the feasibility of using triatomine bugs for the collection of capillary blood and to comparatively evaluate the level of microfilariaemia and its circadian variation in capillary blood vs. peripheral venous blood in a dog naturally co-infected with D. immitis and D. repens. The results showed a feeding success of 50%, with variations in the blood meal volume that the bugs ingested. The relative values of microfilariaemia (mf/bug) were strongly correlated with the volume of blood recovered: the more blood recovered from each bug, the higher values of microfilariaemia in the evening samples while the opposite results were obtained for the morning samples. The counting of microfilariae revealed a dominance of D. immitis in all the samples, but with significantly higher microfilariaemia in the venous blood. Meanwhile, for D. repens, the situation was opposite, with higher counts in the capillary blood samples. Our study showed that triatomine bugs can be used as a model for the collection and study of microfilariaemia in the capillary blood in mammals.

Do the venous blood samples replicate malaria parasite densities found in capillary blood? A field study performed in naturally-infected asymptomatic children in Cameroon.

BACKGROUND: The measure of new drug- or vaccine-based approaches for malaria control is based on direct membrane feeding assays (DMFAs) where gametocyte-infected blood samples are offered to mosquitoes through an artificial feeder system. Gametocyte donors are identified by the microscopic detection and quantification of malaria blood stages on blood films prepared using either capillary or venous blood. However, parasites are known to sequester in the microvasculature and this phenomenon may alter accurate detection of parasites in blood films. The blood source may then impact the success of mosquito feeding experiments and investigations are needed for the implementation of DMFAs under natural conditions. METHODS: Thick blood smears were prepared from blood obtained from asymptomatic children attending primary schools in the vicinity of Mfou (Cameroon) over four transmission seasons. Parasite densities were determined microscopically from capillary and venous blood for 137 naturally-infected gametocyte carriers. The effect of the blood source on gametocyte and asexual stage densities was then assessed by fitting cumulative link mixed models (CLMM). DMFAs were performed to compare the infectiousness of gametocytes from the different blood sources to mosquitoes. RESULTS: Prevalence of Plasmodium falciparum asexual stages among asymptomatic children aged from 4 to 15 years was 51.8% (2116/4087). The overall prevalence of P. falciparum gametocyte carriage was 8.9% and varied from one school to another. No difference in the density of gametocyte and asexual stages was found between capillary and venous blood. Attempts to perform DMFAs with capillary blood failed. CONCLUSIONS: Plasmodium falciparum malaria parasite densities do not differ between capillary and venous blood in asymptomatic subjects for both gametocyte and trophozoite stages. This finding suggests that the blood source should not interfere with transmission efficiency in DMFAs.

First detection of Allobilharzia visceralis (Schistosomatidae, Trematoda) from Cygnus cygnus in Japan.

Adult schistosomes were detected in the veins or capillaries of the large intestine, mesentery, liver, and adrenal glands in eight of 13 whooper swans (Cygnus cygnus) examined in Iwate Prefecture, Japan. However, neither eggs nor severe tissue injuries were observed in any of the swans. The schistosomes were definitively identified as Allobilharzia visceralis based on the nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region. Allobilharzia visceralis infections have been reported in whooper swan in Iceland and tundra swan (Cygnus columbianus) in North America. These detections suggest that A. visceralis is distributed extensively along the swan flyways because the swans are migratory birds. To the best of our knowledge, this is the first report of A. visceralis infection in Asia.

Geometrical model for malaria parasite migration in structured environments.

Malaria is transmitted to vertebrates via a mosquito bite, during which rodlike and crescent-shaped parasites, called sporozoites, are injected into the skin of the host. Searching for a blood capillary to penetrate, sporozoites move quickly in locally helical trajectories, that are frequently perturbed by interactions with the extracellular environment. Here we present a theoretical analysis of the active motility of sporozoites in a structured environment. The sporozoite is modelled as a self-propelled rod with spontaneous curvature and bending rigidity. It interacts with hard obstacles through collision rules inferred from experimental observation of two-dimensional sporozoite movement in pillar arrays. Our model shows that complex motion patterns arise from the geometrical shape of the parasite and that its mechanical flexibility is crucial for stable migration patterns. Extending the model to three dimensions reveals that a bent and twisted rod can associate to cylindrical obstacles in a manner reminiscent of the association of sporozoites to blood capillaries, supporting the notion of a prominent role of cell shape during malaria transmission.

Pulmonary pathology in pediatric cerebral malaria.

Respiratory signs are common in African children where malaria is highly endemic, and thus, parsing the role of pulmonary pathology in illness is challenging. We examined the lungs of 100 children from an autopsy series in Blantyre, Malawi, many of whom death was attributed to Plasmodium falciparum malaria. Our aim was to describe the pathologic manifestations of fatal malaria; to understand the role of parasites, pigment, and macrophages; and to catalog comorbidities. From available patients, which included 55 patients with cerebral malaria and 45 controls, we obtained 4 cores of lung tissue for immunohistochemistry and morphological evaluation. We found that, in patients with cerebral malaria, large numbers of malaria parasites were present in pulmonary alveolar capillaries, together with extensive deposits of malaria pigment (hemozoin). The number of pulmonary macrophages in this vascular bed did not differ between patients with cerebral malaria, noncerebral malaria, and nonmalarial diagnoses. Comorbidities found in some cerebral malaria patients included pneumonia, pulmonary edema, hemorrhage, and systemic activation of coagulation. We conclude that the respiratory distress seen in patients with cerebral malaria does not appear to be anatomic in origin but that increasing malaria pigment is strongly associated with cerebral malaria at autopsy.

Loss of neurovirulence is associated with reduction of cerebral capillary sequestration during acute Babesia bovis infection.

BACKGROUND: Severe neurological signs that develop during acute infection by virulent strains of Babesia bovis are associated with sequestration of infected erythrocytes in cerebral capillaries. Serial passage of virulent strains in cattle results in attenuated derivatives that do not cause neurologic disease. We evaluated whether serial passage also results in a loss of cerebral capillary sequestration by examining brain biopsies during acute disease and at necropsy. FINDINGS: Cerebral biopsies of spleen intact calves inoculated intravenously with a virulent or attenuated strain pair of B. bovis were evaluated for capillary sequestration at the onset of babesiosis and during severe disease. In calves infected with the virulent strain, there was a significant increase in sequestration between the first and second biopsy timepoint. The attenuated strain was still capable of sequestration, but at a reduced level, and did not change significantly between the first and second biopsy. Necropsy examination confirmed the second biopsy results and demonstrated that sequestration identified at necropsy reflects pathologic changes occurring in live animals. CONCLUSIONS: Loss of neurovirulence after serial in vivo passage of the highly virulent T2Bo strain of B. bovis in splenectomized animals is associated with a significant reduction of cerebral capillary sequestration. Previous genomic analysis of this and two other strain pairs suggests that this observation could be related to genomic complexity, particularly of the ves gene family, rather than consistent gene specific differences. Additional experiments will examine whether differential gene expression of ves genes is also associated with reduced cerebral sequestration and neurovirulence in attenuated strains.

Massive Plasmodium falciparum visceral sequestration: a cause of maternal death in Africa.

Sequestration of Plasmodium falciparum-infected erythrocytes (PfIE) in the capillaries of the central nervous system (CNS) is the pathognomonic feature of cerebral malaria, a condition frequently leading to death. Sequestration of PfIE in the placental intervillous spaces is the characteristic feature of malaria in pregnancy and is associated with low birthweight and prematurity. Although both patterns of sequestration are thought to result from the expression of different parasite proteins involved in cytoadhesion to human receptors, scant information exists on whether both conditions can coexist and whether this can lead to death. We conducted a prospective autopsy study including all consecutive pregnancy-related deaths in a tertiary-level referral hospital in Maputo, Mozambique, between October 2002 and December 2006. Extensive sampling of all major viscera was performed. All cases showing parasites in any of the viscera were included in the analysis. From 317 complete autopsies PfIEs were identified in ten women (3.2%). All cases showed massive accumulation of PfIE in small capillaries of the CNS but also in most visceral capillaries (heart, lung, kidney, uterus). Placental tissue, available in four cases, showed a massive accumulation of maternal PfIE in the intervillous space. Coma (six women) and dyspnoea (five women) were the most frequent presenting clinical symptoms. In conclusion, massive visceral sequestration of PfIE with significant involvement of the CNS is an infrequent but definite direct cause of maternal death in endemic areas of Africa. The PfIE sequestered in cerebral capillaries and the placenta coexist in these fatal cases.

Plasmodium falciparum intercellular adhesion molecule-1-based cytoadherence-related signaling in human endothelial cells.

BACKGROUND: Cytoadherence of Plasmodium falciparum-infected erythrocytes to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying disease are poorly understood. METHODS: We compared parasite lines varying in their binding properties to human endothelial cells for their ability to stimulate signaling activity. RESULTS: In human umbilical vein endothelial cells (HUVECs), which rely on adhesion to intercellular adhesion molecule (ICAM)-1 for binding, signaling is related to the avidity of the parasite line for ICAM-1 and can be blocked either through the use of anti-ICAM-1 monoclonal antibodies or HUVECs with altered ICAM-1 binding properties (i.e., ICAM-1(Kilifi)). Human dermal microvascular endothelial cells (HDMECs), which can bind infected erythrocytes via ICAM-1 and CD36, have a more complex pattern of signaling behavior, but this is also dependent on adhesive interactions rather than merely contact between cells. CONCLUSIONS: Signaling via apposition of P. falciparum-infected erythrocytes with host endothelium is dependent, at least in part, on the cytoadherence characteristics of the invading isolate. An understanding of the postadhesive processes produced by cytoadherence may help us to understand the variable pathologies seen in malaria disease.

Lung injury in vivax malaria: pathophysiological evidence for pulmonary vascular sequestration and posttreatment alveolar-capillary inflammation.

BACKGROUND: The mechanisms underlying lung injury in vivax malaria are not well understood. Inflammatory responses to Plasmodium falciparum and P. vivax, to our knowledge, have not previously been compared at an organ level. METHODS: Respiratory symptoms and physiological aspects were measured longitudinally in Indonesian adults with uncomplicated vivax (n=50) and falciparum (n=50) malaria. Normal values were derived from 109 control subjects. Gas transfer was partitioned into its alveolar-capillary membrane (D(M)) and pulmonary capillary vascular (V(C)) components, to characterize the site and timing of impaired gas transfer. RESULTS: Mean baseline V(C) volume was significantly reduced in vivax and falciparum malaria, improving with treatment in each species. Baseline D(M) function was not impaired in either species. The progressive deterioration in D(M) function after treatment was statistically significant in vivax malaria but not in uncomplicated falciparum malaria. Oxygen saturation deteriorated after treatment in vivax but improved in falciparum malaria. CONCLUSIONS: The baseline reduction in V(C) volume but not in D(M) function suggests encroachment on V(C) volume by parasitized erythrocytes and suggests that P. vivax-infected erythrocytes may sequester within the pulmonary microvasculature. Progressive alveolar-capillary dysfunction after treatment of vivax malaria is consistent with a greater inflammatory response to a given parasite burden in P. vivax relative to that in P. falciparum.

Impairment of functional capillary density but not oxygen delivery in the hamster window chamber during severe experimental malaria.

Microcirculatory changes and tissue oxygenation were investigated during Plasmodium berghei-induced severe malaria in the hamster window chamber model, which allows chronic, noninvasive investigation of the microvasculature in an awake animal. The main finding was that functional capillary density, a parameter reflecting tissue viability independent of tissue oxygenation, was reduced early during the course of disease and continued to decline to approximately 20% of baseline of uninfected controls on day 10 after infection. Parasitized red blood cells and leukocytes adhered to arterioles and venules but did not affect overall blood flow, and there was little evidence of complete obstruction of blood flow. According to the sequestration hypothesis, obstruction of blood flow by adherent parasitized erythrocytes is the cause of tissue hypoxia and, eventually, cell death in severe malaria. Tissue oxygen tensions were lower on day 10 of infection when the animals were moribund compared with uninfected controls, but this level was markedly higher than the lethal threshold. No necrotic cells labeled with propidium iodide were detected in moribund animals on day 10 after infection. We therefore conclude that loss of functional capillaries rather than tissue hypoxia is a major lethal event in severe malaria.

Studies on the glycoprotein modification in erythrocyte membrane during experimental cerebral malaria.

Plasmodium berghei ANKA (Pb ANKA) is a lethal strain of malaria that causes experimental cerebral malaria (ECM) in rodent models. Pathology of the disease is associated with the sequestration of the infected rbc (irbc) in the micro vessels of brain. In the present study, we analyzed the nature of the glycoprotein modification occurring in irbc membrane during erythrocytic stages of Pb ANKA infection. Titration of naturally occurring glycoproteins with concanavalin A (Con A) and wheat germ agglutinin (WGA) lectins revealed an enhanced lectin binding ability for the irbc membrane preparations. Partial characterization of the Con A specific determinants (alpha-d-methyl mannoside specificity) by lectin affinity chromatography followed by 2D electrophoresis and WGA specific determinants (sialic acid specificity) by Western analysis revealed the association of novel lectin specific determinants in irbc membrane. To correlate the biochemical changes with the morphological changes, SEM of irbc, and TEM of sequestered irbc were performed. These ultra structural studies revealed variable and irregular surface protrusions and deep surface indentations on irbc. These observations implicate that altered glycoprotein profiles may lead to cytoarchitectural changes in irbc membrane and such changes may be essential to establish contact with the host endothelial cells. These observations may be central to the microvascular sequestration and pathology of ECM.

Attenuation of cytoadherence of Plasmodium falciparum to microvascular endothelium under flow by hemodilution.

The cytoadherence of Plasmodium falciparum-infected erythrocytes (IRBCs) to endothelium is mediated by adhesion molecules within the physical constraints of a viscous fluid containing mostly erythrocytes. The volume fraction of erythrocytes (hematocrit) and their physical properties, such as deformability, are important properties of blood that affect cell recruitment to the vascular wall. In the present study, we examined the effect of hematocrit on IRBC rolling and adhesion on human microvascular endothelial cells in a flow chamber system in vitro. We found hematocrit to be a major determinant of IRBC/endothelial cell interactions. There was a 5-fold and 12-fold increase in IRBC rolling and adhesion, respectively, when hematocrit increased from 10% to 30%, as a result of changes in shear rate. Similar effects were seen in the presence of less deformable erythrocytes, serum proteins, and on endothelium stimulated with tumor necrosis factor-alpha. The results indicate that hemorheologic variations are an important determinant of the degree of cytoadherence.

Differentiating the pathologies of cerebral malaria by postmortem parasite counts.

To study the pathogenesis of fatal cerebral malaria, we conducted autopsies in 31 children with this clinical diagnosis. We found that 23% of the children had actually died from other causes. The remaining patients had parasites sequestered in cerebral capillaries, and 75% of those had additional intra- and perivascular pathology. Retinopathy was the only clinical sign distinguishing malarial from nonmalarial coma. These data have implications for treating malaria patients, designing clinical trials and assessing malaria-specific disease associations.

A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-microm widths were readily traversed by the 8 microm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-microm channels; some that passed through the 4-microm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-microm channels, trophozoites mimicked "pitting," a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-microm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-microm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.

A myxozoan-like parasite causing xenomas in the brain of the mole, Talpa europaea L., 1758 (Vertebrata, Mammalia).

Light and transmission electron microscopy revealed pericytes of brain capillaries of moles (Talpa europaea L., 1758) as parasitized intracellularly. These host cells were enlarged and of globular or ellipsoid shape, and incorporated a cell-within-cell sequence of primary, secondary and, rarely found, tertiary developmental stages of an eukaryotic organism. Other stages like spores were not discovered either in brain or in other organs. Due to the vertebrate host, and the parasitic cells showing the enveloped state this parasite can be classified as belonging to the Myxozoa rather than Paramyxea. Since spores, which would allow an exact identification of the parasite, could not be detected and mammals are very unusual hosts for Myxozoa, the parasite was designated a myxozoan-like organism.

Cloned lines of Babesia bovis differ in their ability to induce cerebral babesiosis in cattle.

Clones of a Babesia bovis isolate known to cause particularly severe cerebral babesiosis were tested for virulence phenotype by inoculation of cattle. Clones were selected for phenotyping by two criteria - rate of growth in culture and hybridization of a virulence-related probe to Southern blots. Largely on the basis of associated mortality, B. bovis clones were judged to vary in their pathogenic potential.