Acta Parasitologica最新文献

Introduction and Objective

Cutaneous leishmaniasis (CL) is an endemic disease in tropical and subtropical regions which caused by Leishmania parasites. Rapid and accurate diagnosis of this disease is crucial for prognosis, control, and prevention. Considering the importance of accurate diagnosis of this disease and preventing possible errors in common methods, we aimed to design an effective method using the quantitative real time PCR (qPCR) for accurate, and feasible diagnosis of this disease.

Materials and Methods

Total of 109 samples were included: 89 negative smears from patients clinically suspected of CL from endemic areas in Semnan and Golestan provinces, 10 positive samples with varying parasite loads, and 10 samples from patients with other skin diseases mimicking CL to assess test specificity. DNA was extracted using the phenol-chloroform method. The in-house qPCR was performed using designed primers targeting the kinetoplast DNA of Leishmania. In addition, conventional ITS-PCR was used as a complementary method. PCR-positive samples were submitted for Sanger sequencing, and results were compared with patients’ clinical data.

Results

After re-examination of the negative sears, 4 cases (4.49%) were found positive using microscopic method. The qPCR identified 9 positive cases (10.58%) among the 85 negative samples. This technique demonstrated the ability to detect very low parasite loads (grade 1+) in clinical conditions. The limit of detection the assay under laboratory conditions was determined 0.001 ng/µl. The conventional ITS-PCR identified 6 positive cases among the qPCR-positive samples. Sequencing analysis confirmed that all isolates belonged to Leishmania major.

Discussion and Conclusion

The findings of this study indicate that in-house qPCR is a highly sensitive and specific tool for diagnosing CL, even in negative smears. It can serve as a valuable complementary and reliable method to microscopic, leading to earlier and more accurate diagnosis and prevention of misdiagnosis or mistreatment. Furthermore, the identification of L. major in all positive cases supports the existing epidemiological data in the studied regions.

Background

Enterocytozoon bieneusi is an obligate intracellular microsporidian pathogen. It primarily causes diarrhea and weight loss in infected humans and animals, resulting in substantial economic losses to the livestock industry. Therefore, establishing a highly sensitive and specific detection method for E. bieneusi is critical for its prevention and control.

Methods

crRNA and recombinase polymerase amplification (RPA) primers were designed based on partial sequences of the 18 S ribosomal RNA gene and the internal transcribed spacer (ITS) of E. bieneusi. DNA extracted from fecal samples was amplified using RPA or nested polymerase chain reaction (PCR). PCR amplicons were treated with a Tris-saturated phenol-chloroform-isoamyl alcohol mixture to obtain purified target DNA, which was subsequently introduced into the CRISPR–Cas12a reaction system. Post-reaction detection was performed via qPCR instrumentation, fluorescence visualization, and lateral flow strip (LFS) assays. The operational parameters for E. bieneusi detection were subsequently optimized using RPA/CRISPR–Cas12a or nested PCR/CRISPR–Cas12a platforms. The aforementioned methodology was concurrently validated using 50 clinical specimens with known E. bieneusi infection status.

Results

The limits of detection were 7.13 copies/µL for RPA/CRISPR–Cas12a and 2.35 × 10^− 2 copies/µL for nested PCR/CRISPR–Cas12a. When the concentration of unamplified DNA in the CRISPR–Cas12a reaction system reached ≥ 1 × 10^− 4 µg/µL, the single-stranded DNA reporter was efficiently cleaved, resulting in a detectable fluorescence signal. The nested PCR/CRISPR–Cas12a technology was used to analyze 50 fecal samples with confirmed E. bieneusi–positive or –negative status. The results obtained from instrument-based detection, fluorescence observation, and lateral flow test strip detection were completely consistent.

Conclusions

We established the first integration of nested PCR with CRISPR–Cas12a for the detection of E. bieneusi. and were also the first to quantitatively explore the detection limit of Cas12a using non-amplified E. bieneusi DNA. This approach offers a rapid, specific, and highly sensitive diagnostic method. Furthermore, the wide selection of appropriate visualization methods facilitates adaptation to various laboratory conditions and sample template concentrations, enabling accurate result interpretation.

Purpose Enterocytozoon bieneusi is an obligate intracellular eukaryotic parasite of the phylum Microsporidia that infects a wide range of hosts, including humans. In Algeria, epidemiological information on microsporidian infections caused by E. bieneusi is scarce, with only three molecular studies conducted in equines, cattle, and dromedary camels. Here, we investigated the prevalence and genotypic diversity of E. bieneusi in cattle (n = 288), including cows (n = 182) and their calves (n = 106), from 54 farms in central Algeria.

Materials and methods The initial screening of cattle faecal samples was conducted using a nested PCR targeting a partial sequence of the internal transcribed spacer (ITS) region.

Results Enterocytozoon bieneusi was identified in 16.0% (46/288) of faecal samples and in 42.6% (23/54) of sampled farms. Infection rates varied from 10.9 to 17.7% across the study areas. Significant differences in prevalence were observed according to cattle age, sex, and the presence/absence of other livestock species within cattle farms. Conversely, study area, production system, and presence/absence of diarrhoea showed no significant associations. Out of 46 E. bieneusi-positive cattle, genotyping was successful for 56.5% (26/46) of isolates. Phylogenetic analyses revealed seven known E. bieneusi genotypes clustering within the zoonotic Group 1: Type IV (42.3%, 11/26), D (7.7%, 2/26), and Peru6 (3.8%, 1/26), and the ruminant-adapted Group 2: J (19.2%, 5/26), I (15.4%, 4/26), BEB6 (7.7%, 2/26), and CM7 (3.8%, 1/26).

Conclusions The present study provides valuable molecular data on E. bieneusi infections in cattle in Algeria. It highlights the important role that cattle can play as reservoirs of zoonotic E. bieneusi genotypes.

China has achieved remarkable progress toward eliminating schistosomiasis, yet potential risks persist due to residual snail habitats, animal reservoirs, and uneven surveillance coverage. This paper synthesizes current surveillance frameworks and recent literature to identify key challenges and propose actionable directions for sustaining elimination achievements. It emphasizes the need to distinguish serological evidence of past exposure from active infection when interpreting surveillance results. To consolidate elimination outcomes, we recommend a phased, evidence-based strategy: (1) strengthen integrated human–animal–environment surveillance systems, (2) enhance data sharing and cross-sectoral coordination under the One Health framework, and (3) develop long-term mechanisms for resource allocation and emergency response in re-emergence areas. These recommendations aim to guide policy refinement and prioritize resource investment for sustainable schistosomiasis control in the post-elimination era.

Background

Trypanosoma evansi (T. evansi) is the causative agent of surra, and it remains a serious threat to livestock health and productivity. This study aimed was to evaluate the trypanocidal potential of amodiaquine (AQ), alone and in combination with isometamidium chloride (IC), against T. evansi, using both in-silico and in-vitro testing.

Methods

Computational testing involved induced-fit docking of AQ which trypanothione reductase (TR), followed by molecular dynamics (MD) simulations, and downstreampost-MD simulation analyses, intended to guide mechanistic hypotheses. In-vitro assays included drug sensitivity profiling across ten T. evansi isolates, checkerboard synergy testing, time–dependent kinetics, and qRT-PCR–based transcriptional analysis. Statistical tests included non-linear regression for IC90 estimation, one-way ANOVA and two-way ANOVA.

Results

Molecular docking revealed favorable AQ–TR interactions (Glide gscore –9.59 kcal/mol), further stabilized in MD simulations (RMSD 1.04 Å, RMSF 0.64 Å). MM-GBSA analysis yielded a favorable binding free energy (ΔG bind –27.52 kcal/mol). In-vitro, AQ had the IC90 values ranging from 4.27 to 69.29 µg/mL, whereas that of IC ranged from 4.49 to 31.01 ng/mL. Combination treatment significantly reduction in effective concentrations (AQ 8 µg/mL + IC 8 ng/mL vs. AQ 69.29 µg/mL, IC 31.01 ng/mL), with a synergistic FICI of 0.37 and complete clearance in high-dose time–kill assays. Upregulation of TR, TS, and TH under drug pressure was confirmed through qRT-PCR, which was the most pronounced with combination regimens (p < 0.05).

Conclusion

Collectively, these findings revealed AQ–IC co-administration is a mechanistically rational and pharmacodynamically synergistic strategy against drug-resistant T. evansi.

Purpose

Taenia hydatigena is a globally distributed cestode causing cysticercosis in livestock, particularly in small ruminants. In North Africa, data on its distribution and prevalence are scarce, with no prior studies addressing its molecular epidemiology. This study aimed to investigate the prevalence and genetic diversity of T. hydatigena in sheep and goats from Algeria.

Methods

A total of 1,002 animals (939 sheep and 63 goats) were examined for the presence of T. hydatigena cysts at the municipal slaughterhouse of Djelfa province between October 2021 and July 2022. Sixty-nine isolates were randomly selected for molecular analysis using the complete cytochrome c oxidase subunit 1 gene (cox1; 1,620 bp).

Results

Taenia hydatigena cysts were identified in 17% of sheep and 32% of goats, predominantly located in the mesentery and omentum. Sixty-one isolates were genetically characterized, revealing 48 distinct haplotypes, four of which were shared between the two host species. Haplotype network analysis showed no host-specific clustering, and population genetic indices indicated no significant differentiation between the sheep and goat sub-populations of T. hydatigena. Comparative analysis with sequences from Ghana, Nigeria, Saudi Arabia, and China revealed a star-like haplotype network with limited genetic overlap and generally high haplotype diversity.

Conclusion

The present study provides valuable data on the genetic diversity of T. hydatigena in North Africa. The findings underscore the need for more extensive molecular epidemiological studies to elucidate transmission dynamics and dispersal pathways.

Purpose

To describe the therapeutic efficacy of intravitreal faricimab in a refractory case of macular neovascularization (MNV) secondary to toxoplasmic chorioretinitis.

Methods

Case report.

Results

A 29-year-old woman presented with decreased vision in her right eye. She had been diagnosed with ocular toxoplasmosis four years earlier and subsequently developed secondary MNV, which was treated with intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections. Previous intravitreal bevacizumab and aflibercept treatments resulted in minimal functional and anatomical improvement. On examination, best-corrected visual acuity was counting fingers at 2 m in the right eye and 1.0 in the left. Anterior segment findings were unremarkable. Fundus evaluation of the right eye revealed a subfoveal MNV with juxtafoveal hemorrhage and subretinal fluid, confirmed by optical coherence tomography (OCT) and OCT angiography. Due to persistent activation despite prior therapy, a single intravitreal faricimab injection was administered. At three-month post-faricimab injection, visual acuity improved to 0.2, and OCT demonstrated complete resolution of subretinal fluid with a marked decrease in central macular thickness.

Conclusion

In the present case, intravitreal faricimab provided a remarkable anatomical and functional improvement in MNV secondary to toxoplasmic chorioretinitis that had been resistant to previous anti-VEGF treatments. Given its dual inhibition of VEGF-A and Angiopoetin-2, faricimab may offer an additional therapeutic advantage in managing inflammatory neovascular lesions.

Purpose

Entamoeba spp. are common zoonotic intestinal protozoa with a broad host range, posing a potential threat to public health. The purpose of this study was to investigate the prevalence, conduct genotyping, and evaluate the genetic diversity of Entamoeba spp. in cattle within Hebei Province, China. This research aims to enhance our understanding of the infection dynamics and associated risk factors.

Methods

A total of 2746 fecal samples were collected from cattle across 11 cities in Hebei Province. PCR amplification and sequencing of the 18S rRNA gene were performed, and subtype identification, sequence characterization, genetic distance, and diversity index analyses were conducted using MEGA, PhyloSuite, and PopART software.

Results

The overall prevalence of Entamoeba spp. in cattle was 51.06% (1402/2746). Infection rates were significantly influenced by geographical location (OR = 0.009, P < 0.0001, breed (OR = 0.255, P < 0.01), feeding practices (OR = 11.811, P < 0.0001), and age (OR = 0.295, P < 0.0001). The prevalence in southern Hebei (97.39%) was markedly higher than that in northern Hebei (24.48%), and higher infection rates were observed in older cattle (59.31%), intensively farmed cattle (56.83%), and dairy cattle (65.68%) compared with under one year of age (30.06%), free-range cattle (10.03%), and beef cattle (10.03%), respectively. Five species were identified in the cattle population: Entamoeba bovis (E. bovis), Entamoeba moshkovskii (E. moshkovskii), Entamoeba spp. RL4, Entamoeba spp. RL2, and Entamoeba spp. MG107/BEL. The overall nucleotide diversity index (π) was 0.077, and the haplotype diversity index (Hd) was 0.986. Both indices indicated high genetic diversity among Entamoeba spp. species, with E. bovis exhibiting the highest level of intraspecific diversity.

Conclusion

In conclusion, the conclusions of this regional study from Hebei Province should be interpreted within its inherent limitations, including its sample size, single-region focus, and cross-sectional nature. Entamoeba spp. are not only widely distributed in cattle but also display considerable host and genetic diversity. These findings enhance our understanding of the infection status and potential impacts on cattle health, and provide valuable data for the assessment of public health significance.

Purpose

Leishmania major and Trichomonas vaginalis infections pose a significant global health burden, while current treatments are limited by toxicity, resistance, and restricted accessibility. This study aimed to evaluate the in vitro and ex vivo antileishmanial effects of Artemisia vulgaris and Berberis vulgaris extracts against L. major, as well as their in vitro antitrichomonal activity against T. vaginalis trophozoites.

Methods

Ethanolic extracts of A. vulgaris and B. vulgaris were tested against L. major promastigotes, intracellular amastigotes, and T. vaginalis trophozoites. Parasite viability was determined by CellTiter-Glo^®, microscopy, and rescue–transformation assays, and selectivity indices (SI) were calculated. Amphotericin B and metronidazole served as reference drugs.

Results

Both extracts exhibited low cytotoxicity in THP-1 macrophages (A. vulgaris CC₅₀ = 465.2 µg/mL; B. vulgaris = 357.7 µg/mL). Against L. major, B. vulgaris showed greater potency (IC₅₀ = 76.8 µg/mL; SI = 4.7 for amastigotes) than A. vulgaris (IC₅₀ = 179.7 µg/mL; SI = 2.6). Both extracts reduced intracellular parasite burden in a dose-dependent manner, achieving complete clearance at non-cytotoxic concentrations (≥ 300 µg/mL). In T. vaginalis, the extracts induced concentration-dependent inhibition, with IC₅₀ values of 68.9 µg/mL (B. vulgaris, SI = 5.2) and 104.4 µg/mL (A. vulgaris, SI = 4.5).

Conclusion

Both extracts exhibited selective, dose-dependent antiprotozoal activity, with B. vulgaris showing superior efficacy, particularly against intracellular L. major and T. vaginalis. These results highlight their potential as natural antiprotozoal sources, warranting further studies on active constituents, mechanisms, and in vivo efficacy.