Frontiers in parasitology最新文献

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most common nonviral neglected sexually transmitted disease worldwide. Biomarkers and therapeutic targets, including specific trichomonad cysteine proteases (CPs) and their endogenous inhibitors, have been identified to diagnose and treat this disease. Trichocystatin 2 (TC-2) was previously identified as one of the three endogenous inhibitors of the parasite's cathepsin L-like CPs, including TvCP39, which is involved in T. vaginalis cytotoxicity and is a potential therapeutic target. TC-2 contains five cysteines, including four located in the N-terminal sequence. These cysteines may be responsible for the formation of multimers of the recombinant protein expressed in E. coli. To determine whether these cysteines are responsible for the formation of TC-2 multimers and the effect of the N-terminus on CP inhibition, a recombinant TC-2 mutant was expressed, purified, characterized, and compared with the recombinant wild-type TC-2 protein. In silico and experimental analyses revealed that wild-type and mutant TC-2 proteins presented similar results in terms of secondary and tertiary structure prediction and high thermal stability. However, compared with that of wild-type TC-2, multimer formation was significantly reduced in the mutant lacking the four N-terminal cysteines, leading to a significant reduction in papain inhibition but not in trichomonal CP activity. These results support the hypothesis that the four cysteines located in the N-terminal region are responsible for aggregation, and their deletion affected the interaction of TC-2 with papain without affecting its inhibitory activity on homologous target proteases that are crucial for T. vaginalis virulence. Our results provide essential data supporting the use of TC-2 as a potential therapeutic target.

Introduction: Schistosomiasis is a public health concern and there is a need for reliable field-compatible diagnostic methods in endemic settings. The AiDx Assist, an artificial intelligence (AI)-based automated microscope, has shown promising results for the detection of Schistosoma haematobium eggs in urine. It has been further developed to detect Schistosoma mansoni eggs in stool.

Methods: In this study, we evaluated the performance of the AiDx Assist for the detection of S. mansoni eggs in stool samples and further validated the performance of the AiDx Assist for the detection of S. haematobium eggs in urine samples. Additionally, the potential of the AiDx Assist for the detection of other helminths in stool samples was explored. In total, 405 participants from an area endemic for both S. mansoni and S. haematobium provided stool and urine samples which were subjected to AiDx Assist (semi- and fully automated), while conventional microscopy was used as the diagnostic reference.

Results: Only samples with complete test results were included in the final analysis, resulting in 375 stool and 398 urine samples, of which 38.4% and 65.3% showed Schistosoma eggs by conventional microscopy. The collected images of the stool samples were retrospectively examined for other helminth eggs via manual analysis. For the detection of S. mansoni eggs, the sensitivity of the semi-automated AiDx Assist (86.8%) was significantly higher compared to the fully automated AiDx Assist (56.9%) while the specificity was comparable, with 81.4% and 86.8%, respectively. Retrospectively, eggs of Ascaris lumbricoides and Trichuris trichiura were visualized. For the examination of urine samples, a comparable sensitivity in the detection of S. haematobium eggs was found between the semi-and the fully automated modes of the AiDx Assist, showing 94.6% and 91.9%, respectively. Furthermore, the specificity was comparable, with 90.6%and 91.3% respectively.

Discussion: The AiDx Assist met the World Health Organization Target Product Profile criteria in terms of diagnostic accuracy for the detection of S. haematobium eggs in urine samples and performed modestly in the detection of S. mansoni eggs in stool samples. With some further improvements, it has the potential to become a valuable diagnostic tool for screening multiple helminth parasites in stool and urine samples.

Introduction: Cryptosporidium species are zoonotic protozoa responsible for cryptosporidiosis, a serious public health concern for humans and animals. These protozoa are recognized for their capacity to infect various hosts, resulting in outbreaks that can cause significant health and economic consequences. The One Health approach considers human, animal, and environmental health interconnectedness and is vital in understanding and controlling the spread of such zoonotic diseases. This study adopts this approach to evaluate the prevalence of Cryptosporidium in humans, companion animals, livestock, and environmental water sources in La Trinidad, Benguet.

Methods: A cross-sectional descriptive study was conducted from September 2020 to January 2022, adhering to research ethical standards approved by the Institutional Review Board (IRB) and following COVID-19 safety protocols such as social distancing, use of PPE, and regular sanitation of equipment and facilities. Stratified random sampling resulted in 314 participating households, which provided fecal samples from humans (up to two members), companion animals, and livestock. Samples were analyzed using microscopy (Sugar Flotation Technique, Formalin Ether Concentration Technique, and Kinyoun staining) and molecular methods, with genomic DNA extracted and nested PCR targeting the 18S rRNA gene. Water samples from 19 community sites underwent filtration and nested PCR analysis.

Results: From the 493 human, 363 animal, and 19 water samples analyzed, microscopic analysis revealed that 151 samples tested positive for Cryptosporidium oocysts, and molecular confirmation identified 135 (15.77%) as Cryptosporidium parvum. Livestock exhibited the highest prevalence (37.27%), followed by companion animals (18.58%) and humans (9.33%), indicating significant zoonotic transmission risks and highlighting the need for improved biosecurity measures. All water samples were negative.

Discussion: The high burden of Cryptosporidium in livestock presents significant risks for zoonotic transmission and reflects major shortcomings in biosecurity and sanitation. In contrast, the low human prevalence of COVID-19 suggests that enhancing hygiene practices combined with social restraint may help control infectious events. Further research is required to confirm this relationship. These results highlight the need for targeted public health interventions to reduce transmission risks.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis, is a severe zoonotic disease in humans. One of the major metacestode antigens of E. multilocularis is the Em2 or Em2(G11) native purified antigen. The Em2 antigen is used for the serological and histopathological diagnosis of AE in humans and plays an important role in parasite-host interactions. As the Em2(G11) antigen is a mucin-type and glycosylated protein, the protein backbone has not been identified yet. We have targeted the protein backbone identification through mass spectrometry (LC-MS/MS) analysis of the Em2(G11) antigen. As a result, we evidenced that the Em2(G11) antigen consists of 33 unique protein candidates of which the most abundant was ''EmuJ_001105600.1''. This protein (889 amino acids) had 427 predicted glycosylation sites. Amino acid composition comparison was in agreement with earlier studies and further confirmed the candidate of interest as the most likely Em2(G11) protein backbone. NCBI BLAST revealed no other known protein homologues in related Echinococcus species nor helminths. After successfully producing this protein recombinantly (Em2rec), a monoclonal antibody (mAbEm2rec) was raised against it. Immunohistochemical stainings of liver tissue sections of AE patients showed that the mAbEm2rec reacts specifically with E. multilocularis antigens solely after deglycosylation with an O-glycosidase cocktail. Similarly, in ELISA, the mAbEm2rec recognized the recombinant and native antigens of E. multilocularis after deglycosylation. These results reveal the nature of this highly glycosylated and specific protein, where mucins are covering the proteomic backbone. For antibody detection in human patients, the native Em2(G11) antigen was superior compared to the Em2rec antigen, indicating the importance of glycosylated epitopes in this immuno-dominant antigen. Of note is the second most abundant protein in the Em2(G11) antigen, namely phosphoenolpyruvate carboxykinase (PEPCK; EmuJ_000292700.1). PEPCK is known to play an important part in the metabolic pathway of gluconeogenesis in E. multilocularis. However, whether this co-eluted protein has any functional importance in the parasite-host interplay of nutrients, growth, and diagnostic significance, is not explored. By combining various approaches, we were able to uncover and confirm the protein backbone of the diagnostic Em2(G11) antigen of E. multilocularis.

Parasites have a severe impact on animal and human health. Parasites like worms, ticks, mites, fleas, biting flies, mosquitoes, and pathogenic protozoa affect humans and their pets as well as their livestock globally, both in terms of severity and numbers. Parasitic infections are a global phenomenon, and they can be associated with severe or mild symptoms but represent a continuous risk of severe diseases for animals and humans. Therefore, effective treatment options and the prevention of infection are key for the wellbeing of pets, livestock, and humans, including the reduction of zoonotic risk of infection. The effective control of parasites in animals can greatly improve their quality of life and is also beneficial for humans; this is threatened by drug-resistant parasite populations. Today's key areas for improvement of parasite control are as follows: a) convenience of prevention and treatment, b) effectiveness against drug-resistant parasites, c) availability and reduced costs of treatment, and d) control measurements that are environmentally friendly.

Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent neglected parasitic sexually transmitted infection worldwide. Cysteine peptidases (CPs) are the most abundant proteins in the parasite degradome. Some CPs are virulence factors involved in trichomonal pathogenesis, cytoadherence, hemolysis, and cytotoxicity. Few are immunogenic and are found in the vaginal secretions of patients with trichomoniasis. Legumains are CPs of the C13 family of clan CD. T. vaginalis has 10 genes encoding legumain-like peptidases, and TvLEGU-1 and TvLEGU-2 have been characterized. Both are immunogenic and found in the vaginal secretions of patients with trichomoniasis that could be considered as potential biomarkers. Thus, our goal was to evaluate the effects of glucose on the proteolytic activity and secretion processes of TvLEGU-1 and TvLEGU-2. We performed in vitro secretion assays using different glucose concentrations, examined the presence and proteolytic activity of secreted legumains by Western blot and spectrofluorometry assays, and analyzed the localization of TvLEGU-1 and TvLEGU-2 in the parasites by indirect immunofluorescence. Our results show that TvLEGU-1 and TvLEGU-2 were secreted in vitro in a time-dependent manner and had legumain-like proteolytic activity that could contribute to parasite pathogenesis, supporting their relevance during infection and potential as trichomoniasis biomarkers.

Introduction: Trypanosomatids are parasites widely distributed in nature, parasitizing several host species in single or co-infections. Campo Grande (CG), capital of Mato Grosso do Sul State, is characterized by several green areas and forest fragments where wild mammals have been reported infected by diverse trypanosomatid species. In this study, we evaluated the parasitism by trypanosomatids in the non-human primates (NHP) Sapajus cay and Alouatta caraya sampled in three different areas of CG.

Material and methods: For the detection of infections and identification of trypanosomatid species, we made hemoculture, blood smears, molecular and serological tests.

Results: We detected trypanosomatids in 37/55 (67.3%) of sampled animals, all by the molecular test. DNA sequencing analyzes were performed on 32 samples, resulting in the following species identification: Trypanosoma cruzi, T. minasense, T. rangeli, Leishmania (L.) infantum and L. (L.) amazonensis (species already recorded in primates in Latin America), and for the first time T. lainsoni, a parasite related to small mammals, and Trypanosoma sp. DID, originally reported in marsupials Didelphis sp.

Discussion: The detection of trypanosomatids of public health importance as L. infantum, L. amazonensis and T. cruzi (genotypes TcI, TcII/TcVI and TcIV) indicates the enzootic character of these species in the studied area. Also, the presence of T. cruzi TcIV and T. minasense in the conservation area supports previous studies that these parasites would be associated with the arboreal stratum. We conclude that (i) the NHP at CG participate in a complex reservoir system for parasites of great importance for Public Health in the studied area, such as L. infantum, L. amazonensis and T. cruzi, and (ii) there is a great diversity of trypanosomatids circulating in the urban area of this city located in the Brazilian Midwest.

Background: Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing.

Methods: Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (Pf-SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach.

Results: We evaluated Pf-SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf-SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf-SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed.

Conclusion: Overall, Pf-SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible.

Background: Malaria is the most important parasitic illness causing morbidity and mortality with high prevalence in tropical regions.

Objective: This study was aimed at evaluating the 7-year malaria trend and community awareness at Jawi Health Center and primary Hospital in Northwest Ethiopia.

Methods: A retrospective and cross-sectional or prospective design were used for the study. The data was analyzed using SPSS version 22 software. The findings were considered significant at P < 0.05.

Results: Among 62,624 blood films between 2015 and 2021 at Jawi Health Center, 40.9% were positive. Plasmodium falciparum accounted for 85.8%. Women had more mixed infections (P. falciparum and P. vivax) (X² = 8.9, df = 2, P = 0.011) than men. A greater proportion (20.6%) of malaria cases was observed within the under 5 years age group and the number of malaria cases was higher in September, October, and June. The overall prevalence of malaria was found to be 25.2% and June had the highest proportion (75.6%). In total, 335 (80.9%) respondents recognized mosquito bites as the cause and fever (50%) as a clinical symptom of malaria. More than half of the respondents (60.1%) never sleep under mosquito nets.

Conclusion: Thus, these findings have substantial implications for the trend of malaria prevalence and patient awareness of the disease which support the existing malaria control efforts.

Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus sensu lato, the metacestode of a tapeworm parasite of high medical importance. Infection of the parasite leads to the development of echinococcal cysts, and the spleen is a rarely infected organ. A 46-year-old woman who was born and who resides in Sardinia, Italy, was referred to the Echinococcosis outpatient clinic at the University Hospital of Sassari (Sardinia, Italy) for a pain in the left flank. She used to live in the countryside, in contact with several animals, and for 2 years, she had been working in a family garden, growing vegetables as a hobby. Ultrasounds and X-ray were performed, which evidenced a rounded formation in the upper third of the spleen, while a CT scan confirmed a parasitological cyst. Immunological examinations on serum samples did not detect specific antibodies against Echinococcus spp. Following surgical exportation, the whole spleen with the cystic lesion was delivered to the World Organisation for Animal Health (WOAH) and the National Reference Laboratory for Echinococcosis for further laboratory analyses. Moreover, characterization of the cyst fluid resulted dense and shiny. Observation under a light microscope at ×400 magnification revealed the formation of rectangular crystals and aggregates attributable to cholesterol molecules. Subsequently, through parasitological investigation, molecular biology investigations confirmed E. granulosus sensu stricto G1. We describe cholesterol crystals in a splenic echinococcal cyst for the first time. There is no clear explanation of how the cholesterol crystals formed in this case, but this was attributed to multifactorial causes, including atherosclerosis, chronic inflammation, parasite metabolism, and host responses.