Leveraging deep learning for detecting red blood cell morphological changes in blood films from children with severe malaria anaemia.

In sub-Saharan Africa, acute-onset severe malaria anaemia (SMA) is a critical challenge, particularly affecting children under five. The acute drop in haematocrit in SMA is thought to be driven by an increased phagocytotic pathological process in the spleen, leading to the presence of distinct red blood cells (RBCs) with altered morphological characteristics. We hypothesized that these RBCs could be detected systematically and at scale in peripheral blood films (PBFs) by harnessing the capabilities of deep learning models. Assessment of PBFs by a microscopist does not scale for this task and is subject to variability. Here we introduce a deep learning model, leveraging a weakly supervised Multiple Instance Learning framework, to Identify SMA (MILISMA) through the presence of morphologically changed RBCs. MILISMA achieved a classification accuracy of 83% (receiver operating characteristic area under the curve [AUC] of 87%; precision-recall AUC of 76%). More importantly, MILISMA's capabilities extend to identifying statistically significant morphological distinctions (p < 0.01) in RBCs descriptors. Our findings are enriched by visual analyses, which underscore the unique morphological features of SMA-affected RBCs when compared to non-SMA cells. This model aided detection and characterization of RBC alterations could enhance the understanding of SMA's pathology and refine SMA diagnostic and prognostic evaluation processes at scale.

Dantu Blood Group Erythrocytes Form Large Plasmodium falciparum Rosettes Less Commonly.

Dantu erythrocytes, which express a hybrid glycophorin B/A protein, are protective against severe malaria. Recent studies have shown that Dantu impairs Plasmodium falciparum invasion by increasing erythrocyte membrane tension, but its effects on pathological host-parasite adhesion interactions such as rosetting, the binding of uninfected erythrocytes to P. falciparum-infected erythrocytes, have not been investigated previously. The expression of several putative host rosetting receptors-including glycophorin A (GYPA), glycophorin C (GYPC), complement receptor 1 (CR1), and band 3, which complexes with GYPA to form the Wrightb blood group antigen-are altered on Dantu erythrocytes. Here, we compare receptor expression, and rosetting at both 1 hour and 48 hours after mixing with mature trophozoite-stage Kenyan laboratory-adapted P. falciparum strain 11019 parasites in Dantu and non-Dantu erythrocytes. Dantu erythrocytes showed lower staining for GYPA and CR1, and greater staining for band 3, as observed previously, whereas Wrightb and GYPC staining did not vary significantly. No significant between-genotype differences in rosetting were seen after 1 hour, but the percentage of large rosettes was significantly less in both Dantu heterozygous (mean, 16.4%; standard error of the mean [SEM], 3.2) and homozygous donors (mean, 15.4%; SEM, 1.4) compared with non-Dantu erythrocytes (mean, 32.9%; SEM, 7.1; one-way analysis of variance, P = 0.025) after 48 hours. We also found positive correlations between erythrocyte mean corpuscular volume (MCV), the percentage of large rosettes (Spearman's rs = 0.5970, P = 0.0043), and mean rosette size (rs = 0.5206, P = 0.0155). Impaired rosetting resulting from altered erythrocyte membrane receptor expression and reduced MCV might add to the protective effect of Dantu against severe malaria.

Regulação da Septina 9 na síndrome do desconforto respiratório agudo associado à malária / The role of Septin 9 in regulating vascular permeability in malarial respiratory distress syndrome

A malária é um problema mundial de saúde, com 249 milhões de casos de infecção, ocasionando 608 mil mortes no ano de 2022. Causada pelo gênero Plasmodium, são cinco principais espécies causadoras da malária no ser humano, o Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium vivax e Plasmodium falciparum sendo os dois últimos responsáveis pelo maior número de casos clínicos e mortes ao redor do mundo, transmitida pelo mosquito fêmea do gênero Anopheles durante o repasto sanguíneo. Sabe-se ainda que eritrócitos infectados por Plasmodium berghei ANKA causam alteração no citoesqueleto de actina, consequentemente levando a hiperpermeabiliade da barreira endotelial. Em experimentos in vitro, a imunofluorescência, foi observada alteração do citoesqueleto de actina em células estimuladas com eritrócitos parasitados por PbA (EP), em contrapartida, aquelas não estimuladas (NE) e estimuladas com eritrócitos não parasitados por PbA (EnP), não mostraram alterações no mesmo. Nos experimentos in vivo, ao observar dados coletados, sendo estes respiratórios (penh e frequência respiratória) e parasitemia coletados no 7º DPI, foi observado um mesmo padrão entre o experimento 1 e o experimento 2. Os animais infectados com 106 de eritrócitos infectados, foram alocados em dois grupos, sendo eles hiperparasitemia (HP) ou síndrome do desconforto respiratório agudo-associado a malária (SDRA/SDRA-MA) e comparados àqueles não infectados (NI). Os animais NI, não apresentam parasitemia, em contrapartida, os animais SDRA, tem maior parasitemia que os HP, visto que estes têm aumento em sua parasitemia após o 12º DPI, e assim seguem aumentando gradativamente até levar os animais a óbito.O penh tem o mesmo padrão que a parasitemia, os NI com penh mais baixa que os HPs e os SDRA, sendo dentre estes, o grupo SDRA o mais elevado. A frequência respiratória, por sua vez se apresenta mais elevada no grupo NI, sendo o grupo SDRAmenor que o HP, um achado tido como normal, visto que os pulmões de animais com SDRA sofrem maior dano que os HPs. Apesar de não apresentar um valor significativo, as imagens de gel SDS-PAGE (WB) mostram maior concentração da Septina 9 nos animais com SDRA em comparação com os HPs e com os NIs. O mesmo é observado na qRT-PCR, mesmo sem significância estatística, o valor mostrado nos gráficos temmaior concentração nos SDRA. Assim, a Septina 9 está presente nas CEPP, e, mesmo sem significância estatística, da mesma forma que está presente nas amostras de tecido pulmonar utilizadas no WB e qT-PCR. É hipotetizado ainda que esta proteína pode ser ativada e assim sofrer alteração em sua localização intracelular

Malaria is a global health problem, with 249 million cases of infection, causing 608 thousand deaths in the year 2022. Caused by the genus Plasmodium, there are five main species that cause malaria in humans, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium vivax and Plasmodium falciparum, the last two being responsible for the largest number of clinical cases and deaths around the world, transmitted by the female mosquito of the genus Anopheles during blood meal. It is also known that erythrocytes infected by Plasmodium berghei ANKA (PbA) cause changes in the actin cytoskeleton, consequently leading to hyperpermeability of the endothelial barrier. In in vitro experiments, immunofluorescence, changes in the actin cytoskeleton were observed in cells stimulated with erythrocytes parasitized by PbA (EP), in contrast, those not stimulated (NE) and stimulated with erythrocytes not parasitized by PbA (EnP), did not show changes the same. In the in vivo experiments, when observing collected data, these being respiratory (penh and respiratory frequency) and parasitemia collected on the 7th DPI, the same pattern was observed between experiment 1 and experiment 2. Animals infected with 106 infected erythrocytes were allocated into two groups, namely hyperparasitemia (HP) or malaria-associated acute respiratory distress syndrome (ARDS/ARDS-MA) and compared to those not infected (NI). NI animals do not present parasitemia, on the other hand, ARDS animals have greater parasitemia than HP animals, as the latter have an increase in their parasitemia after the 12th DPI, and thus continue to gradually increase until the animals die. the same pattern as parasitemia, NI with lower penh than HPs and ARDS, among these, the ARDS group being the highest. The respiratory rate, in turn, is higher in the NI group, with the ARDS group being lower than the HP, a finding considered normal, given that the lungs of animals with ARDS suffer greater damage than the HPs. Despite not showing a significant value, SDS-PAGE (WB) gel images show a higher concentration of Septin 9 in animals with ARDS compared to HPs and NIs. The same is observed in qRT-PCR, even without statistical significance, the value shown in the graphs has a higher concentration in ARDS. Thus, Septin 9 is present in CEPP, and, even without statistical significance, in the same way that it is present in lung tissue samples used in WB and qT-PCR. It is also hypothesized that this protein can be activated and thus undergo changes in its intracellular location

IL-27 produced during acute malaria infection regulates Plasmodium-specific memory CD4+ T cells.

Malaria infection elicits both protective and pathogenic immune responses, and IL-27 is a critical cytokine that regulate effector responses during infection. Here, we identified a critical window of CD4+ T cell responses that is targeted by IL-27. Neutralization of IL-27 during acute infection with Plasmodium chabaudi expanded specific CD4+ T cells, which were maintained at high levels thereafter. In the chronic phase, Plasmodium-specific CD4+ T cells in IL-27-neutralized mice consisted mainly of CD127+ KLRG1- and CD127- KLRG1+ subpopulations that displayed distinct cytokine production, proliferative capacity, and are maintained in a manner independent of active infection. Single-cell RNA-seq analysis revealed that these CD4+ T cell subsets formed independent clusters that express unique Th1-type genes. These IL-27-neutralized mice exhibited enhanced cellular and humoral immune responses and protection. These findings demonstrate that IL-27, which is produced during the acute phase of malaria infection, inhibits the development of unique Th1 memory precursor CD4+ T cells, suggesting potential implications for the development of vaccines and other strategic interventions.

Rhoptry neck protein 4 plays important roles during Plasmodium sporozoite infection of the mammalian liver.

During invasion, Plasmodium parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver in vivo. Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. In vitro infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, in vitro motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, Plasmodium berghei, we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver in vivo. Combined with in vitro investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in Plasmodium merozoites and Toxoplasma tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of Apicomplexa parasites.

Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique

Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

Vitamin C-rich juice co-administration with artemether-lumefantrine ameliorates oxido-inflammatory responses in Plasmodium berghei-infected mice.

This study investigated the effects of co-administration of a commercial juice rich in vitamin C (Vit C) on the antimalarial efficacy of artemether-lumefantrine (AL) in Plasmodium berghei-infected mice. Fifty Balb/c mice were infected with Plasmodium berghei NK65 strain from a donor mouse. Parasitemia was established after 72 h. Animals were grouped into 6 (n = 10) and treated daily for 3 days with normal saline, chloroquine, artemether-lumefantrine (AL), AL plus 50% commercial juice (CJ), and AL plus 50% Vit C supplementation in drinks ad libitum, respectively. Body weight, parasitemia levels, and mean survival time were determined. Tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), nitrite, malondialdehyde, reduced glutathione (GSH), catalase, and superoxide dismutase (SOD) were determined in the serum and liver tissues. Spleen histopathological changes were determined by H&E staining. Parasitemia was cleared by administration of AL and was not affected by Vit C and CJ supplementation. Vit C significantly prevented body weight reduction in AL-treated mice. CJ and Vit C supplementation to AL-treated mice significantly improved survival proportion compared with AL alone animals. Vit C and CJ supplementation significantly improved reduction of TNF-α, IL-6, and malondialdehyde, and increased GSH, CAT, and SOD in AL-treated mice. Spleen cell degeneration and presence of malaria pigment were reduced in AL-treated animals. The results suggest that ad libitum co-administration of commercial juice and vitamin C with artemether-lumefantrine does not impair its antimalarial efficacy but rather improved antioxidant and anti-inflammatory effects in mice.

The distinctive role of membrane fibrinogen-like protein 2 in the liver stage of rodent malaria infections.

Viral infection often induce the expression of murine fibrinogen-like protein 2 (mFGL2) triggering immune coagulation, which causes severe liver pathogenesis via increased fibrin deposition and thrombosis in the microvasculature. We aimed to investigate the role of mFGL2 in the liver stage of malaria infections. We reveal that infection with malaria sporozoites also induces increased expression of mFGL2 and that this expression is primarily located within the liver Kupffer and endothelial cells. In addition, we report that inhibition of FGL2 has no significant effect on immune coagulation but increases the expression of inflammatory cytokines in the livers of infected mice. Interestingly, FGL2 deficiency had no significant impact on the development of liver stage malaria parasites or the pathogenesis of the infected liver. In contrast to viral infections, we conclude that mFGL2 does not contribute to either parasite development or liver pathology during these infections, revealing the unique features of this protein in liver-stage malaria infections.

Ameliorating effects of ethanol extract of root bark of Salacia nitida on blood electrolyte and renal perturbations in Plasmodium berghei-infected mice

Abstract Malaria, a disease of public health concern is a known cause of kidney failure, and dependence on herbal medicines for its treatment is increasing due to the high cost of drugs. So this study is designed to evaluate the ameliorating effect of ethanol extract from Salacia nitida root bark on electrolyte and renal perturbations in Plasmodium berghei-infected mice. Thirty malariainfected mice divided into five groups of six mice each and another group of six uninfected mice were used for the study. 280, 430, and 580 mg/kg of extract were given to infected mice in groups B, C, and D, 4 mg/kg of artesunate given to group E mice, and 4 ml/kg of physiological saline given to group A and uninfected group F mice for five days. Serum Na+, K+, HCO3, Cl-, TB, urea, creatinine, BUN concentrations, and BUN/creatinine ratio were determined using standard methods. Results showed significant increases (p < 0.05) in Na+, K+, and HCO3 and decreases in Cl-, TB, urea, creatinine, BUN, and BUN/creatinine ratio in the infected treated mice in groups B - E. This study showed that ethanol extract of S. nitida root bark is efficient in the treatment of renal disorders and blood electrolyte perturbations

Using location history data from cell phones of infectious patients for disease surveillance / Usando dados do histórico de localização de telefones celulares de pacientes infecciosos para vigilância de doenças

Infectious diseases significantly contribute to global morbidity and mortality, highlighting the critical need for robust disease surveillance systems. The rapid and accurate identification of infection hotspots is crucial for effective disease control and eliminating vector reservoirs. Traditional methods, reliant on patient-reported data, are vague, slow, and non-integrative, presenting substantial barriers to fully understanding the underlying causes of infection transmission. The widespread usage of smartphones presents a unique opportunity to access, analyze, and monitor digital data. Particularly, location data can offer potential insights into infectious disease dynamics, which has remained largely unexplored. Firstly, the present study leverages location history data from smartphones of malaria patients in Manaus, Amazonas region, to pinpoint mosquito-breeding sites. Upon quantifying the location data, the primary transmission hotspots were identified to be concentrated on the outskirts of the city of Manaus. Additionally, the quantification and hotspot validation confirmed that newly visited locations during the exposure period were potential sources of infection transmission. Secondly, the current study also employs a novel digital contact investigation method for a human-to-human transmission infection such as tuberculosis to measure the exposure risk between the active index cases and their close contacts. The digital contact investigation revealed varied exposure durations between the recruited paired index and close contact participants based on the outcome of close contact. To summarize, the present study determines distinct mobility patterns associated with both these infectious diseases, potentially aiding in drafting targeted public health strategies and policies for digital epidemiological surveillance

As doenças infecciosas são um dos principais contribuintes para a morbidade e a mortalidade globais, enfatizando a necessidade crítica de sistemas robustos de vigilância de doenças. A identificação rápida e precisa dos pontos críticos de infecção é fundamental para o controle eficaz de doenças e a eliminação de reservatórios de vetores. Os métodos tradicionais, que dependem de dados relatados por pacientes, são vagos, lentos e não integrativos, apresentando barreiras significativas para a compreensão total das causas subjacentes da transmissão de infecções. O uso generalizado de dispositivos móveis apresenta uma oportunidade única de acessar, analisar e monitorar dados digitais. Especialmente, dados de localização podem oferecer informações úteis sobre a dinâmica de doenças infecciosas, que permanecem em grande parte inexploradas. Primeiramente, o presente estudo utiliza dados de histórico de localização de smartphones de pacientes com malária em Manaus, na região do Amazonas, para identificar locais de reprodução de mosquitos. Ao quantificar os dados de localização, identificaram-se os principais pontos de transmissão concentrados nos arredores da cidade de Manaus. Além do mais, a quantificação e a validação em campo confirmaram que os locais recém-visitados durante o período de exposição eram potenciais fontes de transmissão da infecção. Em segundo lugar, o estudo atual também emprega um inovador método de investigação digital de contato para uma infecção por transmissão de humano para humano, como a tuberculose, a fim de medir o risco por exposição entre os casos índice ativos e seus contatos próximos. A investigação digital de contato revelou períodos de exposição variados entre os participantes recrutados em pares de casos índice e contatos próximos, com base no resultado do contato próximo. Em resumo, o presente estudo identifica padrões distintos de mobilidade associados a ambas essas doenças infecciosas, auxiliando potencialmente na elaboração de estratégias e políticas de saúde pública direcionadas para a vigilância epidemiológica digital

ACUTE KIDNEY DAMAGE AS A COMPLICATION OF MALARIA CAUSED BY PL. MALARIA AND PL. FALCIPARUM: CLINICAL CASES.

We have described two clinical cases of severe malaria caused by different pathogens: Pl. falciparum and Pl. malaria, common to which there was a severe course, complicated by acute renal failure and hemolytic anemia. In a detailed analysis of both clinical cases, Patient 1 had acute kidney damage arose after the increase of anemia and thrombocytopenia, in combination with hemoglobinuria. This shows that the leading mechanism of kidney injure in this case is acute tubular necrosis, due to the toxic effects of free hemoglobin and sequestration in the capillaries of the glomerulus. A Patient 2 had a significant increase of anemia after appears of acute kidney damage; there was no hemoglobinuria, however, significant leukocytosis was observed. It seems, that the leading mechanism in this case is immune-mediated kidney injure or due to hypoperfusion of kidney tubules with the development of acute interstitial nephritis or immune complex glomerular injure with the development of glomerulonephritis, or a combination of them. A detailed analysis of the described two clinical cases of severe malaria caused by Pl. falciparum and Pl. malaria, respectively, and complicated by acute renal failure and hemolytic anemia, suggests that the pathogenetic mechanisms and severity of kidney damage depend on the type of malaria.

Effect of black seeds (Nigella sativa) on inflammatory and immunomodulatory markers in Plasmodium berghei-infected mice.

Nigella sativa, a core dietary supplement and food additive in folklore is one of the most broadly studied seed plants in the global nutraceutical sector. Malaria infection impairs the ability of principal cells of the immune system to trigger an efficient inflammatory and immune response. Ninety-six mice, weighing 20-25 g, were grouped into 12 consisting of 8 animals each. The mice were infected with standard inoculum of the strain NK65 Plasmodium berghei (chloroquine sensitive) and the percentage parasitemia suppression were evaluated. The individual effect of black seed supplemented diet and its combinatory effect with chloroquine (CQ) were investigated on reactive oxygen species (ROS), glutathione peroxidase (GPx), reduced glutathione (GSH), glutathione-S-transferase (GST), serum immunoglobulins (IgG and IgM), and the hematological parameters (hemoglobin, packed cell volume, and red blood cell count) in P. berghei infected mice. The inflammatory cytokines, tumor necrosis factor (TNF-α), interleukin (IL-6 and IL-10), as well as IgG and IgM were assayed in the serum. The mice temperature and behavioral changes were observed. Infected mice treated with the dietary supplementation of black seed with a percentage inclusion (2.5%, 5%, 10%) showed significantly decreased parasitemia and ROS levels (p < 0.05) compared with the untreated mice. The result demonstrated a significant suppression in the pro-inflammatory cytokines (TNF-α, IL-6) levels and a notable elevation in the anti-inflammatory cytokine (IL-10), antioxidant markers as well as the immunoglobulin levels of the P. berghei-infected mice treated with black seed. The study revealed that black seed enhanced host antioxidant status, modulated inflammatory and immune response by regulating some inflammatory cytokines and immunomodulatory mediators. PRACTICAL APPLICATIONS: Black seed (Nigella sativa) has been a dietary supplement and natural remedy for many centuries. Inflammatory and immune diseases are the most notable cause of mortality in the world and more than 50% of deaths have been attributed to it. However, there is paucity of information on the effect of N. sativa on anti-inflammatory and immunomodulatory ability during malaria infection. The result suggests that N. sativa produced antioxidant, anti-inflammatory, and immunomodulatory effect in Plasmodium berghei-infected mice via the participation of glutathione antioxidant system, serum antibodies, and some inflammatory cytokines.

Experimental Models to Study the Pathogenesis of Malaria-Associated Acute Respiratory Distress Syndrome.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is increasingly gaining recognition as a severe malaria complication because of poor prognostic outcomes, high lethality rate, and limited therapeutic interventions. Unfortunately, invasive clinical studies are challenging to conduct and yields insufficient mechanistic insights. These limitations have led to the development of suitable MA-ARDS experimental mouse models. In patients and mice, MA-ARDS is characterized by edematous lung, along with marked infiltration of inflammatory cells and damage of the alveolar-capillary barriers. Although, the pathogenic pathways have yet to be fully understood, the use of different experimental mouse models is fundamental in the identification of mediators of pulmonary vascular damage. In this review, we discuss the current knowledge on endothelial activation, leukocyte recruitment, leukocyte induced-endothelial dysfunction, and other important findings, to better understand the pathogenesis pathways leading to endothelial pulmonary barrier lesions and increased vascular permeability. We also discuss how the advances in imaging techniques can contribute to a better understanding of the lung lesions induced during MA-ARDS, and how it could aid to monitor MA-ARDS severity.

Role of sphingosine kinase and sphingosine-1-phosphate receptor in the liver pathology of mice infected with Plasmodium berghei ANKA.

Decreased serum sphingosine 1-phosphate (S1P) has been reported in severe malaria patients, but the expression of receptors and enzymes associated with S1P has not been investigated in the liver of malaria patients. Therefore, this study aimed to investigate the expression of sphingosine kinase (SphK) and S1P receptors (S1PRs) in the liver of malaria-infected mice. C57BL/6 male mice were divided into a control group (n = 10) and a Plasmodium berghei (PbA)-infected group (n = 10). Mice in the malaria group were intraperitoneally injected with 1×106 P. berghei ANKA-infected red blood cells, whereas control mice were intraperitoneally injected with normal saline. Liver tissues were collected on Day 13 of the experiment to evaluate histopathological changes by hematoxylin and eosin staining and to investigate SphK and S1PR expression by immunohistochemistry and real-time PCR. Histological examination of liver tissues from the PbA-infected group revealed sinusoidal dilatation, hemozoin deposition, portal tract inflammation and apoptotic hepatocytes, which were absent in the control group. Immunohistochemical staining showed significant increases in the expression of SphK1 and SphK2 and significant decreases in the expression of S1PR1, S1PR2, and S1PR3 in the endothelium, hepatocytes, and Kupffer cells in liver tissue from the PbA-infected group compared with the control group. Real-time PCR analysis showed the upregulation of SphK1 and the downregulation of S1PR1, S1PR2, and S1PR3 in the liver in the PbA-infected group compared with the control group. In conclusion, this study demonstrates for the first time that SphK1 mRNA expression is upregulated and that S1PR1, S1PR2, and S1PR3 expression is decreased in the liver tissue of PbA-infected mice. Our findings suggest that the decreased levels of S1PR1, S1PR2, and S1PR3 might play an important role in liver injury during malaria infection.

A novel murine model of post-implantation malaria-induced preterm birth.

Despite major advances made in malaria treatment and control over recent decades, the development of new models for studying disease pathogenesis remains a vital part of malaria research efforts. The study of malaria infection during pregnancy is particularly reliant on mouse models, as a means of circumventing many challenges and costs associated with pregnancy studies in endemic human populations. Here, we introduce a novel murine model that will further our understanding of how malaria infection affects pregnancy outcome. When C57BL/6J (B6) mice are infected with Plasmodium chabaudi chabaudi AS on either embryonic day (E) 6.5, 8.5, or 10.5, preterm birth occurs in all animals by E16.5, E17.5, or E18.5 respectively, with no evidence of intrauterine growth restriction. Despite having the same outcome, we found that the time to delivery, placental inflammatory and antioxidant transcript upregulation, and the relationships between parasitemia and transcript expression prior to preterm birth differed based on the embryonic day of infection. On the day before preterm delivery, E6.5 infected mice did not experience significant upregulation of the inflammatory or antioxidant gene transcripts examined; however, peripheral and placental parasitemia correlated positively with Il1ß, Cox1, Cat, and Hmox1 placental transcript abundance. E8.5 infected mice had elevated transcripts for Ifnγ, Tnf, Il10, Cox1, Cox2, Sod1, Sod2, Cat, and Nrf2, while Sod3 was the only transcript that correlated with parasitemia. Finally, E10.5 infected mice had elevated transcripts for Ifnγ only, with a tendency for Tnf transcripts to correlate with peripheral parasitemia. Tumor necrosis factor deficient (TNF-/-) and TNF receptor 1 deficient (TNFR1-/-) mice infected on E8.5 experienced preterm birth at the same time as B6 controls. Further characterization of this model is necessary to discover the mechanism(s) and/or trigger(s) responsible for malaria-driven preterm birth caused by maternal infection during early pregnancy.

Liver Environment-Imposed Constraints Diversify Movement Strategies of Liver-Localized CD8 T Cells.

Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate Ag either in the lymph node or in infected tissue. Although quantitative details of T cell movement strategies in some tissues such as lymph nodes or skin have been relatively well characterized, we still lack quantitative understanding of T cell movement in many other important tissues, such as the spleen, lung, liver, and gut. We developed a protocol to generate stable numbers of liver-located CD8 T cells, used intravital microscopy to record movement patterns of CD8 T cells in livers of live mice, and analyzed these and previously published data using well-established statistical and computational methods. We show that, in most of our experiments, Plasmodium-specific liver-localized CD8 T cells perform correlated random walks characterized by transiently superdiffusive displacement with persistence times of 10-15 min that exceed those observed for T cells in lymph nodes. Liver-localized CD8 T cells typically crawl on the luminal side of liver sinusoids (i.e., are in the blood); simulating T cell movement in digital structures derived from the liver sinusoids illustrates that liver structure alone is sufficient to explain the relatively long superdiffusive displacement of T cells. In experiments when CD8 T cells in the liver poorly attach to the sinusoids (e.g., 1 wk after immunization with radiation-attenuated Plasmodium sporozoites), T cells also undergo Lévy flights: large displacements occurring due to cells detaching from the endothelium, floating with the blood flow, and reattaching at another location. Our analysis thus provides quantitative details of movement patterns of liver-localized CD8 T cells and illustrates how structural and physiological details of the tissue may impact T cell movement patterns.

Awareness and perception of malaria and dengue at school and college level in the district of Multan.

The purpose of this study is to examine the awareness and perception of malaria and dengue fever in Multan Punjab, Pakistan while taking into account the important role of government policies and other variables. The goal of this study is to examine the awareness of students in Multan, Pakistan on malaria and dengue. This study is based on a quantitative approach of secondary evidence from scientific journals and questionnaire surveys. It is also based on observational evidence gathered in Multan Punjab Pakistan, in a field study. The survey with school children, teachers and healthcare professionals were both formal and semi-structuralize. Studies have found that malaria and dengue mainly affect children's schooling through their absence, but can also induce brain loss and cognitive disability. In questionnaires, students were seen to have different understanding of the illness, but also to be able to serve as agents of health reform only through teachers. A sample size of 500 respondents has been selected from different colleges of district Multan Punjab, Pakistan. Correlation technique is used for the data analysis. According to our results it is concluded that the students at college level are aware of malaria and dengue diseases, but they are not capable of engaging and serving as agents for health reform. On the basis of results it is recommended that students must teach about epidemics diseases regarding how to handle these diseases.

Nek2 Kinase Signaling in Malaria, Bone, Immune and Kidney Disorders to Metastatic Cancers and Drug Resistance: Progress on Nek2 Inhibitor Development.

Cell cycle kinases represent an important component of the cell machinery that controls signal transduction involved in cell proliferation, growth, and differentiation. Nek2 is a mitotic Ser/Thr kinase that localizes predominantly to centrosomes and kinetochores and orchestrates centrosome disjunction and faithful chromosomal segregation. Its activity is tightly regulated during the cell cycle with the help of other kinases and phosphatases and via proteasomal degradation. Increased levels of Nek2 kinase can promote centrosome amplification (CA), mitotic defects, chromosome instability (CIN), tumor growth, and cancer metastasis. While it remains a highly attractive target for the development of anti-cancer therapeutics, several new roles of the Nek2 enzyme have recently emerged: these include drug resistance, bone, ciliopathies, immune and kidney diseases, and parasitic diseases such as malaria. Therefore, Nek2 is at the interface of multiple cellular processes and can influence numerous cellular signaling networks. Herein, we provide a critical overview of Nek2 kinase biology and discuss the signaling roles it plays in both normal and diseased human physiology. While the majority of research efforts over the last two decades have focused on the roles of Nek2 kinase in tumor development and cancer metastasis, the signaling mechanisms involving the key players associated with several other notable human diseases are highlighted here. We summarize the efforts made so far to develop Nek2 inhibitory small molecules, illustrate their action modalities, and provide our opinion on the future of Nek2-targeted therapeutics. It is anticipated that the functional inhibition of Nek2 kinase will be a key strategy going forward in drug development, with applications across multiple human diseases.

Impact of Galectin-Receptor Interactions on Liver Pathology During the Erythrocytic Stage of Plasmodium berghei Malaria.

Hepatopathy is frequently observed in patients with severe malaria but its pathogenesis remains unclear. Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses, and exhibit pivotal roles during Plasmodium spp. infection. Here, we analyzed the impact of blockage of galectin-receptor interactions by treatment with alpha (α)-lactose on liver immunopathology during the erythrocytic stage of malaria in mice infected with Plasmodium berghei ANKA (PbANKA). Our results found that compared with PbANKA-infected mice (malarial mice), blockage of galectin-receptor interactions led to decreased host survival rate and increased peripheral blood parasitemia; exacerbated liver pathology, increased numbers of CD68+ macrophages and apoptotic cells, and increased parasite burden in the livers on days 5 and 7 post infection (p.i.) as well as increased mRNA expression levels of galectin-9 (Gal-9) and its receptor, the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), interferon (IFN)α, IFNγ, and the triggering receptor expressed on myeloid cells (TREM)-1 in the livers or spleens of PbANKA-infected mice on day 7 p.i. Observed by transmission electron microscopy, the peritoneal macrophages isolated from malarial mice with α-lactose treatment had more pseudopodia than those from malarial mice. Measured by using quantitative real-time reverse transcription-polymerase chain reaction assay, the mRNA expression levels of Gal-9, IFNα, IFNß, IFNγ, and TREM-1 were increased in the peritoneal macrophages isolated from malarial mice with α-lactose treatment in comparison of those from malarial mice. Furthermore, significant positive correlations existed between the mRNA levels of Gal-9 and Tim-3/IFNγ/TREM-1 in both the livers and the peritoneal macrophages, and between Gal-9 and Tim-3/TREM-1 in the spleens of malarial mice; significant positive correlations existed between the mRNA levels of Gal-9 and IFNγ in the livers and between Gal-9 and IFNα in the peritoneal macrophages from malarial mice treated with α-lactose. Our data suggest a potential role of galectin-receptor interactions in limiting liver inflammatory response and parasite proliferation by down-regulating the expressions of IFNα, IFNγ, and TREM-1 during PbANKA infection.

Cost-effectiveness of point-of-care C-Reactive Protein test compared to current clinical practice as an intervention to improve antibiotic prescription in malaria-negative patients in Afghanistan.

BACKGROUND: Antimicrobial resistance (AMR) is a global health problem requiring a reduction in inappropriate antibiotic prescribing. Point-of-Care C-Reactive Protein (POCCRP) tests could distinguish between bacterial and non-bacterial causes of fever in malaria-negative patients and thus reduce inappropriate antibiotic prescribing. However, the cost-effectiveness of POCCRP testing is unclear in low-income settings. METHODS: A decision tree model was used to estimate cost-effectiveness of POCCRP versus current clinical practice at primary healthcare facilities in Afghanistan. Data were analysed from healthcare delivery and societal perspectives. Costs were reported in 2019 USD. Effectiveness was measured as correctly treated febrile malaria-negative patient. Cost, effectiveness and diagnostic accuracy parameters were obtained from primary data from a cost-effectiveness study on malaria rapid diagnostic tests in Afghanistan and supplemented with POCCRP-specific data sourced from the literature. Incremental cost-effectiveness ratios (ICERs) reported the additional cost per additional correctly treated febrile malaria-negative patient over a 28-day time horizon. Univariate and probabilistic sensitivity analyses examined the impact of uncertainty of parameter inputs. Scenario analysis included economic cost of AMR per antibiotic prescription. RESULTS: The model predicts that POCCRP intervention would result in 137 fewer antibiotic prescriptions (6%) with a 12% reduction (279 prescriptions) in inappropriate prescriptions compared to current clinical practice. ICERs were $14.33 (healthcare delivery), $11.40 (societal), and $9.78 (scenario analysis) per additional correctly treated case. CONCLUSIONS: POCCRP tests could improve antibiotic prescribing among malaria-negative patients in Afghanistan. Cost-effectiveness depends in part on willingness to pay for reductions in inappropriate antibiotic prescribing that will only have modest impact on immediate clinical outcomes but may have long-term benefits in reducing overuse of antibiotics. A reduction in the overuse of antibiotics is needed and POCCRP tests may add to other interventions in achieving this aim. Assessment of willingness to pay among policy makers and donors and undertaking operational trials will help determine cost-effectiveness and assist decision making.