Detection of Gnathostoma spinigerum larva in the brain with complete follow-up after surgical treatment of human neurognathostomiasis.

Human gnathostomiasis is a food-borne zoonotic helminthic infection widely reported in Latin America, Asia, and Southeast Asia. Consuming raw, or under-cooked fresh-water fish is the leading cause of this helminthic infection, which is clinically characterized by signs of inflammation, itching sensation, or irritation with migratory swelling. Neurological symptoms resulting from neurognathostomiasis vary, and there is scant information due to the rareness of patient brain samples. This study aimed to demonstrate the first evidence of human neurognathostomiasis by the detection of Gnathostoma spinigerum larva in patient's brain during craniotomy, supported by histopathological, immunological and proteomic evidence. Clinical symptoms were obtained from medical history and physical examination with laboratory investigations, including magnetic resonance imaging (MRI), left temporal craniotomy, histopathology of brain tissue, and Western blot analysis, were performed to elucidate the causative pathogens for diagnosis. In addition, the host-parasite interaction of the parasite invading the patient's brain was characterized through proteomics. Histopathology revealed worms with the characteristic cuticular spines of G. spinigerum which were detected and identified. These histopathological findings were consistent with a positive Western blot showing a 24-kDa reactive-band for gnathostomiasis. Proteomic analysis revealed the presence of G. spinigerum serpin and serine protease in the patient's serum. Moreover, the leucine-rich alpha-2-glycoprotein was indicated as a systemic biomarker of early brain injury related to invasion by G. spinigerum. Therefore, our study provides the initial evidence of human neurognathostomiasis due to G. spinigerum larval invasion along with successful craniotomy and proven larval detection including complete follow-up, and the disease prognosis after surgical treatment.

Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera.

BACKGROUND: Endothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines. METHODS: Morphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ. RESULTS: The four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of "spiking pattern" of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ. CONCLUSIONS: ECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.

Efficacy of artesunate combined with Atractylodes lancea or Prabchompoothaweep remedy extracts as adjunctive therapy for the treatment of cerebral malaria.

BACKGROUND: Cerebral malaria is one of the most serious complications of Plasmodium infection and causes behavioral changes. However, current antimalarial drugs have shown poor outcomes. Therefore, new antimalarials with neuroprotective effects are urgently needed. This study aimed to evaluate the effects of selected extracts as monotherapy or adjunctive therapy with artesunate on antimalarial, anti-inflammatory, antioxidant, and neuroprotective properties in experimental cerebral malaria (ECM). METHODS: ECM was induced in male C57BL/6 mice by infection with Plasmodium berghei ANKA (PbA). Ethanolic extracts of Atractylodes lancea (a dose of 400 mg/kg) and Prabchompoothaweep remedy (a dose of 600 mg/kg) were evaluated as monotherapy and adjunctive therapy combined with artesunate at the onset of signs of cerebral malaria and continued for 7 consecutive days. Parasitemia, clinical scores, and body weight were recorded throughout the study. At day 13 post-infection, mouse brains were dissected and processed for the study of the inflammatory response, oxidative stress, blood-brain barrier (BBB) integrity, histopathological changes, and neurocognitive impairments. RESULTS: Ethanolic extracts of A. lancea and Prabchompoothaweep remedy alone improved cerebral malaria outcome in ECM, whereas artesunate combined with extracts of A. lancea or Prabchompoothaweep remedy significantly improved the outcome of artesunate and crude extracts alone. Using real-time PCR, PbA-infected mice that had received the combination treatment showed significantly reduced gene expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-10), chemokines (CXCL4 and CXCL10), and adhesion molecules (ICAM-1, VCAM1, and CD36). The PbA-infected mice that received the combination treatment showed a significantly decreased malondialdehyde level compared to the untreated group. Similarly, the Evans blue dye assay revealed significantly less dye extravasation in the brains of infected mice administered the combination treatment, indicating improved BBB integrity. Combination treatment improved survival and reduced pathology in the PbA-infected group. Additionally, combination treatment resulted in a significantly reduced level of cognitive impairment, which was analyzed using a novel object recognition test. CONCLUSIONS: This study demonstrated that artesunate combined with A. lancea or Prabchompoothaweep remedy extracts as adjunctive therapy reduced mortality, neuroinflammation, oxidative stress, BBB integrity protection, and neurocognitive impairment in the ECM.

Preclinical evaluation of antimalarial activity of CPF-1 formulation as an alternative choice for the treatment of malaria.

BACKGROUND: Kheaw Hom remedy is a traditional Thai medicine used to treat fever. Some plants used in the Kheaw Hom remedy show promising in vitro antimalarial activity. This study prepared novel formulations of plants from the Kheaw Hom remedy and evaluated their antimalarial and toxicological activities. METHODS: Seven new formulations were prepared by combining at least three herbs of six selected plants from the Kheaw Hom remedy, namely Mammea siamensis Kosterm., Mesua ferrea L., Dracaena loureiroi Gagnep., Pogostemon cablin (Blanco) Benth., Kaempferia galanga L, and Eupatorium stoechadosmum Hance. In vitro antimalarial activities of each formulation's aqueous and ethanolic extracts were evaluated using the parasite lactate dehydrogenase (pLDH) assay. Cytotoxicity in Vero and HepG2 cells was assessed using the MTT assay. An extract with good antimalarial potency and selectivity index (SI) was selected for in vivo antimalarial activity using Peter's 4-day suppressive test and acute oral toxicity test in mice. In addition, bioactive compounds were identified using Gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: Among the seven new formulations, ethanolic extracts of CPF-1 (Formulation 1) showed the highest activity with an IC50 value of 1.32 ± 0.66 µg/ml, followed by ethanolic extracts of Formulation 4 and Formulation 6 with an IC50 value of 1.52 ± 0.28 µg/ml and 2.48 ± 0.34 µg/ml, respectively. The highest SI values were obtained for the ethanolic extract of CPF-1 that was selected to confirm its in vivo antimalarial activity and toxicity. The results demonstrated a significant dose-dependent reduction in parasitemia. Maximum suppressive effect of the extract (72.01%) was observed at the highest dose administered (600 mg/kg). No significant toxicity was observed after the administration of 2000 mg/kg. Using GC-MS analysis, the most abundant compound in the ethanolic extract of CPF-1 was ethyl p-methoxycinnamate (14.32%), followed by 2-propenoic acid, 3-phenyl-, ethyl ester, (E)- (2.50%), and pentadecane (1.85%). CONCLUSION: The ethanolic extract of CPF-1 showed promising in vitro and in vivo antimalarial efficacy, with no toxic effects at a dose of 2000 mg/kg, suggesting that the ethanolic extract of CPF-1 may serves as a new herbal formulation for the treatment of malaria. Additional research is required for safety and clinical pharmacology studies.

Antimalarial efficacy and toxicological assessment of medicinal plant ingredients of Prabchompoothaweep remedy as a candidate for antimalarial drug development.

BACKGROUND: Drug resistance exists in almost all antimalarial drugs currently in use, leading to an urgent need to identify new antimalarial drugs. Medicinal plant use is an alternative approach to antimalarial chemotherapy. This study aimed to explore potent medicinal plants from Prabchompoothaweep remedy for antimalarial drug development. METHODS: Forty-eight crude extracts from Prabchompoothaweep remedy and its 23 plants ingredients were investigated in vitro for antimalarial properties using Plasmodium lactate dehydrogenase (pLDH) enzyme against Plasmodium falciparum K1 strain and toxicity effects were evaluated in Vero cells. The plant with promising antimalarial activity was further investigated using gas chromatography-mass spectrometry (GC-MS) to identify phytochemicals. Antimalarial activity in mice was evaluated using a four-day suppressive test against Plasmodium berghei ANKA at dose of 200, 400, and 600 mg/kg body weight, and acute toxicity was analyzed. RESULTS: Of the 48 crude extracts, 13 (27.08%) showed high antimalarial activity against the K1 strain of P. falciparum (IC50 <  10 µg/ml) and 9 extracts (18.75%) were moderately active (IC50 = 11-50 µg/ml). Additionally, the ethanolic extract of Prabchompoothaweep remedy showed moderate antimalarial activity against the K1 strain of P. falciparum (IC50 = 14.13 µg/ml). Based on in vitro antimalarial and toxicity results, antimalarial activity of the aqueous fruit extract of Terminalia arjuna (IC50 = 4.05 µg/ml and CC50 = 219.6 µg/ml) was further studied in mice. GC-MS analysis of T. arjuna extract identified 22 compounds. The most abundant compounds were pyrogallol, gallic acid, shikimic acid, oleamide, 5-hydroxymethylfurfural, 1,1-diethoxy-ethane, quinic acid, and furfural. Analysis of the four-day suppressive test indicated that T. arjuna extract at dose of 200, 400, and 600 mg/kg body weight significantly suppressed the Plasmodium parasites by 28.33, 45.77, and 67.95%, respectively. In the acute toxicity study, T. arjuna extract was non-toxic at 2000 mg/kg body weight. CONCLUSIONS: The aqueous fruit extract of T. arjuna exerts antimalarial activity against Plasmodium parasites found in humans (P. falciparum K1) and mice (P. berghei ANKA). Acute toxicity studies showed that T. arjuna extract did not show any lethality or adverse effects up to a dose of 2000 mg/kg.

Apoptotic changes and aquaporin-1 expression in the choroid plexus of cerebral malaria patients.

BACKGROUND: Cerebral malaria (CM) is associated with sequestration of parasitized red blood cells (PRBCs) in the capillaries. Often, the association of CM with cerebral oedema is related with high mortality rate. Morphological changes of the choroid plexus (CP) and caspase-3 expression in CM have not been reported. In addition, limited knowledge is known regarding the role of aquaporin (AQP)-1 in CM. The present study evaluated changes in the CP, explored apoptotic changes and AQP-1 expression in CP epithelial cells (CPECs) in fatal CM patients. METHODS: CP from fatal Plasmodium falciparum malaria patients (5 non-CM [NCM], 16 CM) were retrieved and prepared for histopathological evaluation. Caspase-3 and AQP-1 expressions in CPECs were investigated by immunohistochemistry. RESULTS: Histologically, apoptotic changes in CPECs were significantly observed in the CM group compared with the NCM and normal control (NC) groups (p < 0.05). These changes included cytoplasmic and nuclear condensation/shrinkage of CPECs and detachment of CPECs from the basement membrane. The apoptotic changes were positively correlated with caspase-3 expression in the nuclei of CPECs. In addition, AQP-1 expression in CPECs was significantly decreased in the CM group compared with the NCM and NC groups (all p < 0.001). A negative correlation (rs = - 0.450, p = 0.024) was documented between caspase-3 expression in the nuclei of CPECs and AQP-1. CONCLUSIONS: Apoptotic changes and altered AQP-1 expression may contribute to CPEC dysfunction and subsequently reduce cerebrospinal fluid production, affecting the water homeostasis in the brains of patients with CM.

Antiplasmodial activity and cytotoxicity of plant extracts from the Asteraceae and Rubiaceae families.

The increasing resistance of parasites to antimalarial drugs and the limited number of effective drugs are the greatest challenges in the treatment of malaria. It is necessary to search for an alternative medicine for use as a new, more effective antimalarial drug. Therefore, this study aimed to evaluate the in vitro antimalarial activity and cytotoxicity of extracts from plants belonging to the Asteraceae and Rubiaceae families. The phytoconstituents of one hundred ten ethanolic and aqueous extracts from different parts of twenty-three plant species were analyzed. Evaluation of their antimalarial activities against the chloroquine (CQ)-resistant Plasmodium falciparum (K1) strain was carried out using the lactate dehydrogenase (pLDH) assay, and their cytotoxicity in Vero cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric method. A total of 40.91% of the extracts were active antimalarial agents. Three extracts (2.73%) exhibited high antiplasmodial activity (IC50 < 10 µg/ml), twenty-four extracts (21.82%) were moderately active with IC50 values ranging from 10-50 µg/ml, and eighteen extracts (16.36%) were mildly active with IC50 values ranging from 50-100 µg/ml. The ethanolic leaf extract of Mussaenda erythrophylla (Dona Trining; Rubiaceae) exhibited the highest activity against P. falciparum, with an IC50 value of 3.73 µg/ml and a selectivity index (SI) of 30.74, followed by the ethanolic leaf extract of Mussaenda philippica Dona Luz x M. flava (Dona Marmalade; Rubiaceae) and the ethanolic leaf extract of Blumea balsamifera (Camphor Tree; Asteraceae), with IC50 values of 5.94 and 9.66 µg/ml and SI values of 25.36 and >20.70, respectively. GC-MS analysis of these three plant species revealed the presence of various compounds, such as squalene, oleic acid amide, ß-sitosterol, quinic acid, phytol, oleamide, α-amyrin, sakuranin, quercetin and pillion. In conclusion, the ethanolic leaf extract of M. erythrophylla, the leaf extract of M. philippica Dona Luz x M. flava and the leaf extract of B. balsamifera had strong antimalarial properties with minimal toxicity, indicating that compounds from these plant species have the potential to be developed into new antiplasmodial agents.

Protective Effect of an Anti-HMGB-1 Neutralizing Antibody on Hemozoin-Induced Alveolar Epithelial Cell in a Model of Malaria Associated ALI/ARDS.

BACKGROUND: We aimed to determine whether neutralizing high mobility group box-1 (HMGB-1) prevents the release of HMGB-1 and proinflammatory cytokines on hemozoin (Hz)-induced alveolar epithelial cell in a model of malaria associated ALI/ARDS. METHODS: This study was conducted in the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand in 2020. Human pulmonary alveolar epithelial cells (HPAEpiCs) were exposed to medium alone or 20 µM Hz for 24 h and incubated with different concentrations (1, 5, and 10 µg/ml) of anti-HMGB-1 monoclonal antibody (mAb) for various times (0, 4, 12, 24, and 48 h). The levels of HMGB-1, TNF-α and IFN-γ in the supernatants were measured by ELISA. The mRNA expression of RAGE, TLR-2 and TLR-4 were analyzed by real-time PCR. RESULTS: The HPAEpiCs treated with 10 µg/ml anti-HMGB-1 mAb showed a significant reduction in HMGB-1 release into the supernatant compared with those treated with 1 and 5 µg/ml anti-HMGB-1 mAb. The levels of TNF-α and IFN-γ were significantly decreased in the supernatant of HPAEpiCs treated with 1, 5, and 10 µg/ml anti-HMGB-1 mAb for 4, 12, 24, and 48 h compared with those stimulated with Hz alone. The mRNA expression levels of RAGE, TLR-2, and TLR-4 were significantly decreased after 24 h of anti-HMGB-1 antibody treatment at all concentrations. CONCLUSION: An anti-HMGB-1 antibody could be an effective agent for inhibiting the release of HMGB-1, TNF-α and IFN-γ. Furthermore, a neutralizing anti-HMGB-1 antibody could be applicable for the treatment of malaria-associated ALI/ARDS.

In Vivo Antimalarial Activity and Toxicity Study of Extracts of Tagetes erecta L. and Synedrella nodiflora (L.) Gaertn. from the Asteraceae Family.

OBJECTIVE: To investigate the antimalarial effects and toxicity of the extracts of the flowers of Tagetes erecta L. and the leaves of Synedrella nodiflora (L.) Gaertn. in a mouse model. METHODS: To determine the in vivo antimalarial activity of the extracts, mice were intraperitoneally injected with the Plasmodium berghei ANKA strain and then administered T. erecta or S. nodiflora extract daily for 4 days. Parasitemia was observed by light microscopy. For the detection of acute toxicity, the mice received a single dose of T. erecta or S. nodiflora extract and were observed for 14 days. Biochemical parameters of liver and kidney function and the histopathology of liver and kidney tissues of the acute toxicity group were then examined. RESULTS: T. erecta and S. nodiflora crude extracts at a dose of 600 mg/kg body weight significantly suppressed parasitemia in malaria-infected mice by 65.65% and 62.65%, respectively. Mice treated with 400 mg/kg T. erecta and S. nodiflora crude extracts showed 50.82% and 57.67% suppression, and mice treated with 200 mg/kg displayed 26.33% and 38.57% suppression, respectively. Additionally, no symptoms of acute toxicity were observed in the T. erecta- and S. nodiflora-treated groups. Moreover, no significant alterations in the biochemical parameters of liver and kidney function and no histological changes in the liver or kidney tissues were observed. CONCLUSIONS: This study revealed that both T. erecta and S. nodiflora extracts have antimalarial properties in vivo with less toxic effects. Further studies are needed to elucidate the mechanisms of the active compounds from both plants.

Antimalarial properties and molecular docking analysis of compounds from Dioscorea bulbifera L. as new antimalarial agent candidates.

BACKGROUND: At present, the emergence and spread of antimalarial drug resistance has become a significant problem worldwide. There has been a challenge in searching for natural products for the development of novel antimalarial drugs. Therefore, this study aims to evaluate compounds from Dioscorea bulbifera responsible for antimalarial properties and investigate potential interactions of the compounds with Plasmodium falciparum lactate dehydrogenase (PfLDH), an essential glycolytic enzyme in the parasite's life cycle. METHODS: An in vitro study of antimalarial activity against chloroquine (CQ)-resistant Plasmodium falciparum (K1 strain) and CQ-sensitive P. falciparum (3D7 strain) was performed using the 3H-hypoxanthine uptake inhibition method. The cytotoxic effects of the pure compounds were tested against Vero cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The interactions of the compounds with the PfLDH active site were additionally investigated using a molecular docking method. RESULTS: Quercetin (6) exhibited the highest antimalarial activity against the P. falciparum K1 and 3D7 strains, with IC50 values of 28.47 and 50.99 µM, respectively. 2,4,3',5'-Tetrahydroxybibenzyl (9), 3,5-dimethoxyquercetin (4) and quercetin-3-O-ß-D-galactopyranoside (14) also possessed antimalarial effects against these two strains of P. falciparum. Most pure compounds were nontoxic against Vero cells at a concentration of 80 µg/ml, except for compound 9, which had a cytotoxic effect with a CC50 value of 16.71 µM. The molecular docking results indicated that 9 exhibited the best binding affinity to the PfLDH enzyme in terms of low binding energy (- 8.91 kcal/mol) and formed strong hydrogen bond interactions with GLY29, GLY32, THR97, GLY99, PHE100, THR101 and ASN140, amino acids as active sites. In addition, 6 also possessed remarkable binding affinity (- 8.53 kcal/mol) to PfLDH by interacting with GLY29, ILE31, ASP53, ILE54, THR97 and THR101. CONCLUSION: Quercetin is a major active compound responsible for the antimalarial activity of D. bulbifera and is an inhibitor of PfLDH. These findings provide more evidence to support the traditional use of D. bulbifera for malaria treatment. Structural models of its interactions at the PfLDH active site are plausibly useful for the future design of antimalarial agents.

High mobility group box-1 (HMGB-1) and its receptors in the pathogenesis of malaria-associated acute lung injury/acute respiratory distress syndrome in a mouse model.

The DNA-binding protein high mobility group box-1 (HMGB-1) mediates proinflammatory cytokines that contribute to acute lung injury (ALI). Although ALI is a frequent complication of malaria infection, the contribution of HMGB-1 and its receptors to the pathogenesis of malaria-associated ALI/acute respiratory distress syndrome (MA-ALI/ARDS) has not been investigated in a mouse model. Here, the malaria-infected mice were divided into two groups according to lung injury score: the ALI/ARDS and non-ALI/ARDS groups. The expression of HMGB-1 and its receptors (RAGE, TLR-2 and TLR-4) in lung tissues was investigated by using immunohistochemical staining and real-time polymerase chain reaction (PCR). Additionally, HMGB-1 and proinflammatory cytokine (TNF-α, IFN-γ, IL-1 and IL-6) levels in plasma and lung tissues were quantified by using enzyme-linked immunosorbent assays. Cellular expression of both HMGB-1 and its receptors (RAGE, TLR-2 and TLR-4) was significantly increased in the lung tissues of the ALI/ARDS group compared with those in the non-ALI/ARDS and control groups. The levels of HMGB-1, TNF-α, IFN-γ, IL-1 and IL-6 were significantly increased in both plasma and lung tissues of the ALI/ARDS group compared with those in the non-ALI/ARDS and control groups, which were similar to the results obtained by real-time PCR. Increased mRNA expression of RAGE, TLR-2 and TLR-4 was found in the lung tissues of the ALI/ARDS group. Furthermore, the plasma HMGB-1 level was positively correlated with TLR-4 mRNA expression in the ALI/ARDS group. HMGB-1 levels were significantly increased in plasma and lung tissues of MA-ALI/ARDS mice and were related to the upregulated expression of HMGB-1 and proinflammatory cytokines. In conclusion, this study demonstrates that HMGB-1 is an important mediator of MA-ALI/ARDS pathogenesis and may represent a target for therapeutic malaria interventions with ALI/ARDS.

Prevalence of soil-transmitted helminth infections and associated risk factors among elderly individuals living in rural areas of southern Thailand.

BACKGROUND: Soil-transmitted helminth (STH) infection is a neglected tropical disease affecting approximately 1.5 billion people worldwide. In past decades, most studies focused on STH infection in preschool-aged and school-aged children in different regions of Thailand. However, little is known about the prevalence and intensity of STH infection in the elderly population. Therefore, the aim of this study was to determine the current prevalence and intensity of STH infections and to identify associated risk factors among the elderly population. METHODS: A cross-sectional study was conducted from July to November 2019 to assess the prevalence of STH infections and associated risk factors among elderly populations living in five subdistricts of Thasala District, Nakhon Si Thammarat Province, Thailand. A total of 439 elderly individuals were selected using a random sampling technique. Each fresh stool sample was examined using the formalin ethyl acetate concentration technique (FECT), Kato-Katz thick smears and agar plate culture (APC). A structured questionnaire was used to obtain relevant information regarding associated risk factors for STH infection. RESULTS: The overall prevalence of STH infection was 15.7%. Hookworms (10.9%, 48/439) were the most prevalent STH species, followed by Strongyloides stercoralis (3.4%, 15/439) and Trichuris trichiura (2.1%, 9/439). Most elderly individuals infected with hookworms or T. trichiura had light-intensity infections. A higher prevalence of STH infection was observed among individuals aged older than 80 years (23.4%) than among those aged between 70 and 79 years (15.2%) and 60-69 years (14.5%). Males were 1.85-times more likely to present with STH infections than females. Not washing vegetables before eating increased the risk of STH infection by 3.19 times, while defecation in an open field increased the risk of STH infection by 2.65 times. CONCLUSIONS: The findings suggested that STH infection is prevalent, and that hookworms are the most common STH species among elderly populations in southern Thailand. Personal hygiene and deworming programs should be implemented among the elderly population to reduce the risk and prevent the spread of STH infections.

Case Report: The First Direct Evidence of Gnathostoma spinigerum Migration through Human Lung.

Gnathostomiasis is a helminthic infection caused by the third-stage larvae of nematodes of the genus Gnathostoma. The life cycle in humans starts with an enteric phase, with the worm perforating the gastric or intestinal mucosa to reach the peritoneal cavity and migrating through the human body. Subsequent penetration through the diaphragm may produce pleuropulmonary symptoms. We herein present a previously healthy 56-year-old Thai man from Southern Thailand who was an ex-smoker presented with chronic dry cough progressing to hemoptysis after consuming grilled swamp eels and freshwater fish. Chest computed tomography showed consolidation at the lingular segment, and the differential diagnosis was primary lung cancer and pulmonary tuberculosis. The lung tissue biopsied during bronchoscopy displayed segments of organisms with the phenotypic characteristics of Gnathostoma spp., and abundant eosinophils were seen in the alveolar tissue. Gnathostoma spinigerum infection was confirmed by a Western blot assay for G. spinigerum-specific 24-kDa reactive band. The patient received albendazole, and a follow-up chest radiograph revealed improvement in the consolidation in the lung and reduction in hemoptysis. We report the first direct evidence including pathology and immunohistochemistry of Gnathostoma invasion via the human lung, with clinical and radiographic presentations mimicking either malignancy or chronic infection.

M1 macrophage features in severe Plasmodium falciparum malaria patients with pulmonary oedema.

BACKGROUND: Pulmonary oedema (PE) is a serious complication of Plasmodium falciparum malaria which can lead to acute lung injury in severe cases. Lung macrophages are activated during malaria infection due to a complex host-immune response. The molecular basis for macrophage polarization is still unclear but understanding the predominant subtypes could lead to new therapeutic strategies where the diseases present with lung involvement. The present study was designed to study the polarization of lung macrophages, as M1 or M2 macrophages, in the lungs of severe P. falciparum malaria patients, with and without evidence of PE. METHODS: Lung tissue samples, taken from patients who died from severe P. falciparum malaria, were categorized into severe malaria with PE and without PE (non-PE). Expression of surface markers (CD68+, all macrophages; CD40+, M1 macrophage; and CD163+, M2 macrophage) on activated lung macrophages was used to quantify M1/M2 macrophage subtypes. RESULTS: Lung injury was demonstrated in malaria patients with PE. The expression of CD40 (M1 macrophage) was prominent in the group of severe P. falciparum malaria patients with PE (63.44 ± 1.98%), compared to non-PE group (53.22 ± 3.85%, p < 0.05), whereas there was no difference observed for CD163 (M2 macrophage) between PE and non-PE groups. CONCLUSIONS: The study demonstrates M1 polarization in lung tissues from severe P. falciparum malaria infections with PE. Understanding the nature of macrophage characterization in malaria infection may provide new insights into therapeutic approaches that could be deployed to reduce lung damage in severe P. falciparum malaria.

The activation of BAFF/APRIL system in spleen and lymph nodes of Plasmodium falciparum infected patients.

Previous studies have reported activation of the B cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) system in T independent immunity against malaria infection. Plasmodium falciparum (P. falciparum) infected animal model is not feasible. Therefore, little is known about the occurrence of BAFF/APRIL system and changes in falciparum lymphoid tissues. This study aimed to investigate the expression of BAFF/APRIL system components in lymphoid tissues from P. falciparum infected patients. Spleen and lymph node samples from 14 patients were collected at autopsy. Normal spleens and bacterially infected tonsils served as controls. The protein and/or mRNA expression of BAFF/APRIL and their cognate receptors, BAFF-R, TACI and BCMA, were determined by immunohistochemistry and RT-qPCR, respectively. The spleens of the patients exhibited significantly higher BAFF-R protein expression than normal spleens. Although without appropriate control, BCMA protein was markedly observed only in the lymph nodes. BAFF and BCMA mRNA levels were also significantly elevated in the spleen tissues of the patients compared with normal spleens. The overall BAFF-R protein levels in the lymphoid tissues of the patients correlated positively with parasitaemia. These findings are the first to confirm that BAFF/APRIL system activation in lymphoid tissues and is positively correlated with the parasitaemia levels in falciparum malaria.

Overexpression of Sphingosine Kinase-1 and Sphingosine-1-Phosphate Receptor-3 in Severe Plasmodium falciparum Malaria with Pulmonary Edema.

Pulmonary edema (PE) is a major cause of pulmonary manifestations of severe Plasmodium falciparum malaria and is usually associated with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The sphingosine kinase-1 (SphK-1)/sphingosine-1-phosphate receptor-3 (S1PR-3) pathway has recently been reported to affect the pathogenesis of lung injury, but the expression of these proteins in the lungs of severe P. falciparum malaria patients has not been investigated. The cellular expression of SphK-1 and S1PR-3 in lung tissues from autopsied patients with P. falciparum malaria was investigated using immunohistochemistry (IHC). Lung tissues from patients who died of severe P. falciparum malaria were classified into two groups based on histopathological findings: those with PE (18 patients) and those without PE (non-PE, 19 patients). Ten samples of normal lung tissues were used as the control group. The protein expression levels of SphK-1 and S1PR-3 were significantly upregulated in endothelial cells (ECs), alveolar epithelial cells, and alveolar macrophages (AMs) in the lungs of severe P. falciparum malaria patients with PE compared to those in the non-PE and control groups (all p < 0.001). In addition, the SphK-1 and S1PR-3 expression levels were significantly positively correlated in pulmonary ECs (r s = 0.922, p < 0.001), alveolar epithelial cells (r s = 0.995, p < 0.001), and AMs (r s = 0.969, p < 0.001). In conclusion, both the SphK-1 and S1PR-3 proteins were overexpressed in the lung tissues of severe P. falciparum malaria patients with PE, suggesting that SphK-1 and S1PR-3 mediate the pathogenesis of PE in severe malaria. Targeting the regulation of SphK-1 and/or S1PR-3 may be an approach to treat pulmonary complications in severe P. falciparum patients.

Effects of Gnathostoma spinigerum infective stage larva excretory-secretory products on NK cells in peripheral blood mononuclear cell culture: focused on expressions of IFN-γ and killer cell lectin-like receptors.

Human gnathostomiasis is mainly caused by third-stage larvae of Gnathostoma spinigerum (G. spinigerum L3). Excretory-secretory products (ES) released from infective helminthic larvae are associated with larval migration and host immunity modulation. Natural killer (NK) cells have important immune functions against helminth infection. Currently, the effects of ES from G. spinigerum L3 (G. spinigerum ES) on NK cell activity are unclear. This study investigated whether G. spinigerum ES affected human NK cells. Human normal peripheral blood mononuclear cell (PBMC) cultures were used to mimic immune cells within the circulation. PBMC were co-cultured with G. spinigerum ES (0.01-0.05 µg/ml) for 5 or 7 days. Levels of IFN-γ in cultured supernatants were measured by enzyme-linked immunosorbent assay. The expressions of mRNA encoding NK cell receptors, especially the C type killer cell lectin-like family (KLR; NKG2A, NKG2C, and NKG2D) and IFN-γ in ES induced PBMC were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). ES induced PBMC markedly decreased the levels of IFN-γ and increased the expressions of NKG2A and NKG2D on NK cells. In conclusion, low amounts of G. spinigerum ES modulated NK cells by downregulating the transcription of IFN-γ and upregulating the expressions of KLR (NKG2A and NKG2D receptors) during the 7-day observation period. These findings indicate more in-depth studies of NK cell function are required to better understand the mechanism involved in immune evasive strategies of human gnathostomiasis.

Release of endothelial activation markers in lungs of patients with malaria-associated acute respiratory distress syndrome.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an understudied complication of malaria and is characterized by pulmonary inflammation and disruption of the alveolar-capillary membrane. Its pathogenesis remains poorly understood. Since endothelial activation plays an important role in other malarial complications, the expression of two endothelial activation markers, von Willebrand factor (VWF) and angiopoietin-2 (ANG-2), was investigated in the lungs of patients with MA-ARDS. METHODS: Post-mortem lung sections of Plasmodium falciparum-infected patients without alveolar oedema (NA), P. falciparum-infected patients with alveolar oedema (MA-ARDS), and uninfected people who died accidentally with no pathological changes to the lungs (CON) were immunohistochemically stained for VWF and ANG-2, and were evaluated with semi-quantitative analysis. RESULTS: Alveolar oedematous VWF and ANG-2 and intravascular VWF staining were significantly increased in patients with MA-ARDS versus infected and uninfected control groups. The levels of VWF in the alveolar septa and endothelial lining of large blood vessels of patients with MA-ARDS was significantly decreased compared to controls. ANG-2 expression was increased in the alveolar septa of malaria patients without alveolar oedema versus control patients, while ANG-2+ leukocytes were increased in the alveoli in both infected patient groups. CONCLUSIONS: This study documents a high level of VWF and ANG-2, two endothelial activation markers in the oedematous alveoli of post-mortem lung sections of Thai patients with MA-ARDS. Decreased detection of VWF in the endothelial lining of blood vessels, in parallel with an increased presence of intravascular VWF staining suggests marked endothelial activation and Weibel-Palade body release in the lungs of patients with MA-ARDS.

Expression of sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 in malaria-associated acute lung injury/acute respiratory distress syndrome in a mouse model.

This study aimed to investigate the expression of sphingosine kinase 1 (SphK-1) and sphingosine 1-phosphate receptor 3 (S1PR-3) in a mouse model of malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS). DBA/2 mice were infected with Plasmodium berghei ANKA to generate an experimental model of malaria-associated ALI/ARDS. The infected mice were divided into 2 groups based on the histopathological study of lung tissues: those with and those without ALI/ARDS. The expression of the SphK-1 and S1PR-3 proteins in the lung tissues was investigated using immunohistochemical staining and Western blot analysis. In addition, the S1P level was quantified in plasma and lung tissues using an enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the cellular expression of the SphK-1 and S1PR-3 proteins was significantly upregulated in endothelial cells, alveolar epithelial cells and alveolar macrophages in the lung tissues of malaria-infected mice with ALI/ARDS compared with those in the control groups. The increased expression of the SphK-1 and S1PR-3 proteins was confirmed using Western blot analysis. The concentration of S1P in plasma and lung tissues was significantly decreased in malaria-infected mice with ALI/ARDS compared with non-ALI/ARDS and control mice. Furthermore, increased expression of the SphK-1 and S1PR-3 proteins significantly correlated with lung injury scores and S1P concentrations in malaria-infected mice with ALI/ARDS. These findings highlight increased expression of SphK-1 and S1PR-3 in the lung tissues of malaria-infected mice with ALI/ARDS.

Surfactant Protein D Is Altered in Experimental Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome.

Surfactant protein D (SP-D) is in the collectin family of C-type lectins and plays an important role in the regulation of inflammation and the innate immune defense against pathogens. This protein has been proposed as a biomarker for acute lung injury. However, the expression of SP-D in the lung and the circulating levels of SP-D during malaria infection have received limited attention. Therefore, the aim of this study was to determine the location and expression of the SP-D protein in lung tissue and to measure the plasma level of SP-D in experimental malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Malaria-infected mice induced by Plasmodium berghei ANKA were classified into two groups, namely, the ALI/ARDS and non-ALI/ARDS groups, according to lung histopathology. The lungs of uninfected mice were used as a control group. The location and expression of SP-D in the lung tissues were investigated by immunohistochemical staining and Western blot analysis. In addition, the level of SP-D in plasma and lung homogenate was measured by an enzyme-linked immunosorbent assay. Immunohistochemical staining of SP-D was significantly increased in the lung tissues of the malaria-infected mice in the ALI/ARDS group compared with that in the malaria-infected mice in the non-ALI/ARDS group and the mice in the control group (p < 0.05). The levels of SP-D in the plasma and lung homogenate were significantly increased in the malaria-infected mice in the ALI/ARDS group compared with those in the malaria-infected mice in the non-ALI/ARDS group and the mice in the control group (p < 0.05). There was a significant positive correlation between SP-D in the plasma and SP-D in the lung homogenate (r s = 0.900, p = 0.037). In conclusion, this study demonstrated increased expression levels of SP-D in the lung tissue and high levels of plasma SP-D in the malaria-infected mice with ALI/ARDS compared with those in the mice in the other groups. The current study supports that the elevation of the plasma SP-D level may provide useful biological confirmation of the diagnosis of ALI/ARDS during malaria infection.