We investigated the mitochondrial genetic diversity of Echinococcus multilocularis in red foxes from northern and northeastern Poland, and tested the hypothesis that the presence of Asian haplotypes in Poland stems from their introduction from Northeast Asia to Eastern Europe. We targeted the cytochrome c oxidase subunit 1 gene (cox1) of 263 tapeworms from 59 red foxes hunted in selected districts of Pomorskie, Warmińsko-Mazurskie, and Podlaskie Voivodships. We calculated several measures of genetic variation, and constructed median-joining haplotype networks and a RelTime timetree. In addition, a novel alignment-free computational method was applied: the 4D-Dynamic Representation of DNA/RNA Sequences combined with K-means clustering. PCR and sequencing were successful for 252 tapeworms. We identified six haplotypes, three of which were novel. The mismatch distribution was bimodal. The fixation index (FST) between the European and Asian haplogroups was 0.97. Our findings suggest secondary contact between two previously isolated metapopulations with distinct genetic lineages. Clustering patterns appear to support the hypothesis that Asian variants of E. multilocularis spread into northeastern Poland as a consequence of multiple introductions of raccoon dogs from the Soviet Far East to Eastern Europe.

Abstract Background Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite of human and animal hosts. The present study investigated and compared the assemblages of G. duodenalis-infected faecal samples in patients on corticosteroid therapy (POCT) and control patients-not on corticosteroid therapy (CONT) and differentiated its assemblages and/or sub-assemblages’ relationship with associated risk factors. Methods Utilizing multi-locus sequence typing (MLST) with three loci targeted—triosephosphate isomerase (tpi), ꞵ-giardin (bg), and glutamate dehydrogenase (gdh)—G. duodenalis isolated from POCT and CONT were analyzed. Risk factors linked with Giardia infection and its assemblages were investigated. Results In total, 52 G. duodenalis-infected patients were enrolled: 21 POCT and 31 CONT. The mean age was 12.3 years, the majority were male (59.6%), and 73.1% lived in rural areas. The POCT group was 36 times more likely than the CONT group to have a concurrent parasitic infection. About 73% (38/52) of Giardia samples were genotyped and/or sub-genotyped in at least one of the three loci. MLST identified sixteen isolates (42.0%) as assemblage B, ten isolates (26.3%) as assemblage A, and twelve isolates (31.6%) as a mixed infection of A + B and B + E. Most individuals of the POCT group were infected with G. duodenalis assemblage A while most of the CONT group were infected with assemblage B. Sub-assemblage AII was identified by phylogenetic analysis in the isolates of both groups under investigation. Conclusion This research advances giardiasis epidemiology in Arab Republic of Egypt (ARE) and reflects how corticosteroid-treated patients differ from those non-treated in Giardia assemblage pattern and their susceptibility to concomitant infection. Overall, Giardia assemblage patterns in this research populations reflect anthroponotic and zoonotic transmission, emphasizing the importance of public health policy and giardiasis prevention of illness transmission, particularly among those on corticosteroid therapy in ARE.

Knowing Echinococcus multilocularis prevalence in wild canids helps us assess the risk of zoonotic spillover. In this study, we updated the infection statistics among red foxes in northern and northeastern Poland since the last monitoring effort at the district (Polish: powiat) level (2001-2004). We collected faecal samples from 192 red foxes from the districts of Słupsk, Puck, Wejherowo, Kościerzyna, and Kartuzy (Pomorskie Voivodship); 200 individuals from the districts of Bartoszyce, Kętrzyn, Gołdap, Iława, and Węgorzewo (Warmińsko-Mazurskie Voivodship); and 47 individuals from the district of Augustów (Podlaskie Voivodship). Based on nested PCR testing confirmed by Sanger sequencing, we calculated the prevalence with a 95 % confidence interval and compared the results to previous reports. We recorded the highest proportion of infected red foxes in Gołdap District (21/40, 52.5 %, 95 % CI: 37.5 %-67.1 %). The shared prevalence in Puck, Wejherowo, Kościerzyna, and Kartuzy Districts was 10.4 % (19/182, 95 % CI: 6.7 %-15.8 %). One red fox tested positive in Iława District (1/48, 2.1 %, 95 % CI: -0.6 %-11.9 %). We found no statistically significant changes in prevalence in the sampled areas. Our results indicate that E. multilocularis prevalence has remained stable in red fox populations throughout the region over the past 20 years.

[This corrects the article DOI: 10.1016/j.onehlt.2023.100623.].

BACKGROUND:Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE). We explored the diversity of EmsB profiles of E. multilocularis from red foxes and humans in northern and northeastern Poland, with a particular focus on autochthonous Asian variants. METHODS:We investigated 263 adult tapeworms from 59 red foxes hunted in selected districts of three voivodships and ten metacestodes extracted from AE patients who never visited Asia. We conducted hierarchical clustering of the obtained EmsB profiles combined with a custom Asian reference dataset and interpreted the resulting phenogram by applying the standard genetic distance threshold (GDT) of 0.08 and Dynamic Tree Cut (DTC). RESULTS:The GDT divided the 273 profiles into six units, with Pol-B being the most frequent (220/273, 81 %) and widespread variant. DTC grouped the profiles into three phenons (PH). Eight out of ten people got infected with the predominant variant, PH-2/Pol-B. Among the 273 samples, thirty-six (13 %) matched the Asian reference set, including metacestodes from a Lithuanian patient and a Polish patient. CONCLUSIONS:Genetically extra-European variants reach as far north as Pomorskie Voivodship. They likely come from East Asia. The autochthonous AE case with an Asian profile confirms that such tapeworms have penetrated the synanthropic cycle.

Background: Biomonitoring is an essential activity for identifying possible vectors and reservoirs of pathogens and predicting potential outbreaks. Wild red foxes are present in both sylvatic and synanthropic environments, making them potential carriers of zoonotic pathogens. Experimental studies have shown that both coyotes and red foxes can transmit SARS-CoV-2. This study aimed to assess the prevalence and seroprevalence of SARS-CoV-2 in wild red foxes hunted in northern Poland. Methods: Oral swabs, blood clots or heat tissue samples were collected from 292 red foxes hunted in northern Poland. We used both molecular (RT-PCR) and serological (IFA) approaches to detect SARS-CoV-2 infections in the sampled animals. Results: We did not find any evidence of SARS-CoV-2 infection in the collected samples, using both molecular and serological methods. Conclusions: Despite foxes having frequent contact with humans, human waste, and other animals, they do not appear to participate in the circulation of the SARS-CoV-2 virus in our geographical region. Nevertheless, we believe that continuous biomonitoring should be performed to assess the SARS-CoV-2 epidemiological situation in the wild.

In this paper, an alignment-free bioinformatics technique, termed the 20D-Dynamic Representation of Protein Sequences, is utilized to investigate the similarity/dissimilarity between Baculovirus and Echinococcus multilocularis genome sequences. In this method, amino acid sequences are depicted as 20D-dynamic graphs, comprising sets of “material points” in a 20-dimensional space. The spatial distribution of these material points is indicative of the sequence characteristics and is quantitatively described by sequence descriptors akin to those employed in dynamics, such as coordinates of the center of mass of the 20D-dynamic graph and the tensor of the moment of inertia of the graph (defined as a symmetric matrix). Each descriptor unveils distinct features of similarity and is employed to establish similarity relations among the examined sequences, manifested either as a symmetric distance matrix (“similarity matrix”), a classification map, or a phylogenetic tree. The classification maps are introduced as a new way of visualizing the similarity relations obtained using the 20D-Dynamic Representation of Protein Sequences. Some classification maps are obtained using the Principal Component Analysis (PCA) for the center of mass coordinates and normalized moments of inertia of 20D-dynamic graphs as input data. Although the method operates in a multidimensional space, we also apply some visualization techniques, including the projection of 20D-dynamic graphs onto a 2D plane. Studies on model sequences indicate that the method is of high quality, both graphically and numerically. Despite the high similarity observed among the sequences of E. multilocularis, subtle discrepancies can be discerned on the 2D graphs. Employing this approach has led to the discovery of numerous new similarity relations compared to our prior study conducted at the DNA level, using the 4D-Dynamic Representation of DNA/RNA Sequences, another alignment-free bioinformatics method also introduced by us.

Eucoleus aerophilus is the causative agent of respiratory capillariosis in wild and domestic carnivores and has sporadically affected humans. To date, this parasite has been detected in 38 countries, confirming its worldwide distribution. The aim of this study was to evaluate the prevalence of E. aerophilus in red fox populations from Central Europe and to describe the sequence variations in the partial cox1 gene of this parasite recovered from stool samples. An investigation was carried out using 342 samples of red foxes faeces investigated via seminested PCR and coproscopy. PCR results confirmed the presence of E. aerophilus DNA in 230 samples and E. boehmi DNA in 14 samples. Molecular analysis of the retrieved sequences revealed 22 haplotypes of E. aerophilus, EaPL1-EaPL22, and 4 haplotypes of E. boehmi, EbPL1-EbPL4. Coproscopic examination revealed that eggs of the Capillariidae family were most prevalent in all regions, with a mean prevalence of 73%. Furthermore, the following eggs were detected: Taeniidae (21.3%), Toxocara spp. (26.3%), Toxascaris leonina (5.3%), Trichuris vulpis (1.5%), trematodes (23.4%), hookworms (2.3%) and Mesocestoides spp. (1.8%). This study evaluated the prevalence of E. aerophilus in red fox populations in selected regions of Poland. To the best of our knowledge, this is the first molecular study on E. aerophilus in Poland.

Rodents are recognized as reservoirs for Toxoplasma gondii, playing a crucial role in maintaining the parasite's presence in the environment. Biomonitoring was conducted to assess the role of sylvatic rodents in maintaining T. gondii, and to analyse the prevalence and seroprevalence of the parasite in seven wild rodent species. Rodents were collected in an open grassland study site located in northeastern Poland, and dissected. Brain, spleen, blood and serum samples were collected. Molecular (PCR assay, nested-PCR assay) and serological (ELISA and agglutination tests) methods were applied to indicate the best approach for application in the biomonitoring of T. gondii in small mammals. Samples were screened from 68 individuals using PCR assays but no T. gondii DNA were found. The agglutination test showed no signal. Antibodies against T. gondii were found in 5 sera samples out of 56 analysed (seroprevalence = 8.9% [4.4-16.8]). The results confirmed that rodents participate in the life cycle of T. gondii as reservoirs of this parasite in the sylvatic environment. However, for effective bio-monitoring of T. gondii in small mammals, the results suggest a preference for utilizing ELISA tests to detect T. gondii antigens, as opposed to relying solely on molecular methods.

The Grand Round concerns a 24-year-old man from Zimbabwe who was studying and living in Poland. The patient had been complaining of abdominal pain, fatigue, alternating diarrhoea and constipation, and presence of blood in his stool for 3 years. The patient had the following diagnostic tests: colonoscopy, CT scan, histopathology, and parasitological and molecular tests. Results of the examinations showed that the cause of the patient's complaints was chronic intestinal schistosomiasis due to the co-infection with Schistosoma intercalatum and Schistosoma mansoni. The patient had two cycles of praziquantel therapy (Biltricide) and responded well to the treatment. In the Grand Round, we describe full diagnostics as well as clinical and therapeutic management in the patient with S intercalatum and S mansoni co-infection. This case allows us to draw attention to cases of forgotten chronic tropical diseases (including rare ones) in patients from regions with a high endemic index staying in non-endemic regions of the world for a long time. Co-infection with S intercalatum and S mansoni should be considered as a very rare clinical case.

Alveolar echinococcosis is a life-threatening zoonotic disease caused by the larval stage of the cestode Echinococcus multilocularis. People are aberrant intermediate hosts accidentally infected with the parasite eggs via faecal-oral route, usually by the consumption of unwashed fruit and vegetable or direct contact with definitive hosts. The recently reported presence of Asian admixture in E. multilocularis tapeworms from Polish red foxes prompted the question of metacestode descent in the human population. In this study, a Maximum Likelihood tree based on partial sequences of E. multilocularis mitochondrial genes cox1, cob, and nad2 coupled with a hierarchical clustering analysis of microsatellite EmsB profiles and supplemented by Sammon's nonlinear mapping with k-means clustering revealed Asian genetic components, to date associated only with the sylvatic cycle, in two autochthonous samples from alveolar echinococcosis patients living in endemic Warmia-Masuria, north-eastern Poland. The red fox is the most likely source of contamination in the environment shared by people and wildlife that led to these infections. Our results confirm that Asian genetic variants participate in the synanthropic cycle in north-eastern Poland and indicate that they may be present in the human population in other areas where Asian genetic variants were detected in red foxes.

Blastocystis species (sp.) is one of the less well-understood water- and foodborne protozoa of medical and veterinary importance linked to different gastrointestinal disorders. Soldiers participating in military missions are particularly vulnerable to infection with this protozoa. The present study used molecular methods to detect, identify, and subtype (ST) Blastocystis sp. in Polish soldiers stationed in the Republic of Kosovo. Fecal samples were collected from 192 soldiers on arrival and after four months of stay. After DNA extraction, the barcoding region of the small subunit ribosomal RNA (SSU-rRNA) gene was amplified and sequenced. The DNA of Blastocystis sp. was detected in six (3.13%) and thirty (15.16%) samples in the first and second batch, respectively. Sequencing analysis revealed infections with ST 2, 3, 4, and 7. There was no statistical association between Blastocystis sp. infection and the parasite's ST or the age or rank of soldiers. The results indicate that the visit to a new environment and prolonged stay in the area of military operation in Kosovo resulted in a significant increase in both Blastocystis sp. infections and ST diversity among surveyed soldiers. This shows the need to undertake appropriate countermeasures to reduce Blastocystis infections in the military environment abroad.

Rodents are known to be reservoirs of Toxoplasma gondii and keep the parasite circulation in the environment. We conducted biomonitoring to assess the role of sylvatic rodents in maintaining T. gondii and to analyse the prevalence and seroprevalence of the parasite in seven wild rodent species. Rodents were collected in our study sites (woodland and open grasslands) located in northeastern Poland and dissected. We collected brain, spleen, blood and serum samples. We applied both molecular (PCR assay, nested-PCR assay) and serological (ELISA and agglutination tests) methods to indicate the best approach for application in the biomonitoring of T. gondii in small mammals. We screened samples from 95 individuals sing PCR assays and found no T. gondii DNA. The agglutination test showed no signal. We found antibodies against T. gondii in 6 sera samples out of 74 analysed (seroprevalence = 8.11% [4.0-17.1]). Our results confirm that rodents participate in the life cycle of T. gondii as reservoirs of this parasite in the sylvatic environment. However, biomonitoring should be performed with the ELISA tests to search for T. gondii antigens, rather than a molecular approach only.

Toxoplasma gondii is a protozoan parasite, causing one of the most prevalent parasitic infections in the world. In the present study water sources of the Qinghai-Tibet Plateau (QTP), China, where the hygienic infrastructure is still developing, were investigated. A total of 214 water samples of 10 L volume, were collected from wastewater treatment plants (WWTPs), a slaughterhouse and rivers. The samples were filtered and then analysed using real-time PCR and multilocus genotyping. T. gondii DNA was found in four (1.9%) samples representing T. gondii type I; in one of them T. gondii-like oocysts were also confirmed microscopically. The approximate level of contamination of positive samples ranged between 30 and 2300 T. gondii sporozoites. The results of this study confirmed that T. gondii is present in wastewater in the greater metropolitan area of Xining and a neighbouring county. Contamination of wastewater at this level constitutes rather a moderate source of Toxoplasma infections in humans and animals. It suggests, however, a link between environmental exposure of animals, meat processing facilities and WWTPs. To our knowledge, this is the first investigation describing T. gondii detection in wastewater and environmental water samples collected from the territory of P.R. China using sensitive molecular tools.

The 4D-Dynamic Representation of DNA/RNA Sequences, an alignment-free bioinformatics method recently developed by us, has been used to study the genetic diversity of Echinococcus multilocularis in red foxes in Poland. Sequences of three mitochondrial genes, i.e., NADH dehydrogenase subunit 2 (nad2), cytochrome b (cob), and cytochrome c oxidase subunit 1 (cox1), are analyzed. The sequences are represented by sets of material points in a 4D space, i.e., 4D-dynamic graphs. As a visualization of the sequences, projections of the graphs into 3D space are shown. The differences between 3D graphs corresponding to European, Asian, and American haplotypes are small. Numerical characteristics (sequence descriptors) applied in the studies can recognize the differences. The concept of creating descriptors of 4D-dynamic graphs has been borrowed from classical dynamics; these are coordinates of the centers or mass and moments of inertia of 4D-dynamic graphs. Based on these descriptors, classification maps are constructed. The concentrations of points in the maps indicate one Polish haplotype (EmPL9) of Asian origin.

INTRODUCTION:Malaria remains a diagnostic and therapeutic challenge in many endemic regions of sub-Saharan Africa. It is one of the most important causes of morbidity and mortality, especially in children <5 years. Plasmodium falciparum is responsible for the majority of severe malaria cases in sub-Saharan Africa, but is not the exclusive one.OBJECTIVE:The objective of the study was to assess the prevalence of Plasmodium spp. in BaAka Pygmies with clinical symptoms of malaria, and define the percentage distribution of infections caused by species other than P. falciparum in order to assess the need for diversification of malaria treatment protocols.MATERIAL AND METHODS:The study was conducted during the dry and rainy seasons in 2018 and involved a group of 540 symptomatic BaAka Pygmies, patients of both genders, aged 1-75-years-old. Two diagnostic methods for detecting Plasmodium in the bloodstream were used: RDTs targeting HRP2-protein specific for P. falciparum, and PCR assays aimed at detecting P. falciparum, P. vivax, P. ovale, P. malariae species.RESULTS:Only 40.5% of symptomatic patients tested with RDTs for P. falciparum infections were positive. Molecular tests (PCR) confirmed P. falciparum in 94.8% of the samples and also revealed the genetic material of P. malariae (11.1%), P. ovale (9.8%), and P. vivax (0.7%). BaAka Pygmies aged <5 years of age dominated in patients with positive results; the common clinical symptoms reported by the sick individuals were fever, shivers and fatigue.CONCLUSIONS:The study suggests the need for introducing accurate diagnostic methods for the diagnosis of malaria and the revision of malaria treatment protocols. Assessment of the Pfhrp2/Pfhrp3 deletions is necessary for evaluating malaria epidemiology in Central Africa.

According to the World Health Organization 94% of global malaria cases and 94% of global malaria deaths have been reported from Africa. Unfortunately, it is difficult to determine the exact prevalence of disease in some African countries due to a large number of asymptomatic cases. The aim of this study was to assess the prevalence of malaria infections in seemingly healthy children living in the Central African Republic (CAR). CareStartTM Malaria HRP2 rapid diagnostic test (RDT) targeting Plasmodium falciparum was used to test a group of 500 asymptomatic children aged 1-15 years old (330 settled Bantu and 170 semi-nomadic BaAka Pygmies) inhabiting the villages in the Dzanga Sangha region (south-west CAR) in March 2020. In total, 32.4% of asymptomatic Bantu and 40.6% of asymptomatic Pygmy children had a positive result of malaria RDT. Our findings allowed us to demonstrate the high prevalence of asymptomatic malaria infections in south-west CAR. RDTs seem to be a useful tool for the detection of Plasmodium falciparum in areas with limited possibilities of using other diagnostic methods, such as light microscopy and molecular biology.

Background: Malaria remains a diagnostic and therapeutic challenge in many endemic regions of sub-Saharan Africa. The objective of the study was to assess the prevalence of Plasmodium spp. in BaAka Pygmies with clinical symptoms of malaria, inhabitants of the rural Dzanga Sangha region in Central African Republic. Additionally, the research aimed to define the percentage distribution of infections caused by other than P. falciparum species in order to assess the need for diversification of malaria treatment protocols in the region of Central Africa. Material and methods: The study was conducted during dry and rainy season in 2018 and involved a group of 540 symptomatic BaAka Pygmies, patients of both sexes, aged 1–75 years old. Two diagnostic methods for detecting Plasmodium in the bloodstream were used: RDTs targeting HRP2–protein specific for P. falciparum and PCR assays aimed at detecting P. falciparum, P. vivax, P. ovale, P. malariae species. Results: Only 40.5% of symptomatic patients tested with RDTs for P. falciparum infections were positive. While molecular tests (PCR) confirmed P. falciparum in 94.8% of the samples and also revealed the genetic material of P. malariae (11.1%), P. ovale (9.8%), and P. vivax (0.7%). BaAka Pygmies aged <5 years old dominated in patients with positive results; the common clinical symptoms reported by the sick individuals were fever, shivers and fatigue. Conclusions: The presented study suggest the need for introducing more accurate diagnostic methods for the diagnosis of malaria and the revision of malaria treatment protocols. The assessment of the Pfhrp2/Pfhrp3 deletions is necessary for evaluating malaria epidemiology in Central Africa.

According to the World Health Organization 94% of global malaria cases and 94% of global malaria deaths have been reported from Africa. Unfortunately, it is difficult to determine the exact prevalence of disease in some African countries due to a large number of asymptomatic cases. The aim of this study was to assess the prevalence of malaria infections in seemingly healthy children living in the Central African Republic (CAR). CareStartTM Malaria HRP2 Pf Ag RDT targeting Plasmodium falciparum was used to test a group of 500 asymptomatic children aged 1-15 years old (330 settled Bantu and 170 semi-nomadic BaAka Pygmies) inhabiting the villages in the Dzanga Sangha region (south-west CAR) in March 2020. 32.4% of asymptomatic Bantu and 40.6% of asymptomatic Pygmy children had a positive result of malaria RDT. The mean age of the study participants with RDT (+) was 8.7 in Bantu and 7.0 years in Pygmies; the mean body temperature was 36.8oC in both groups; the mean haemoglobin level was 10.6 g/dL and 10.1 g/dL, respectively. Our findings allowed us to demonstrate the high prevalence of asymptomatic malaria infections in south-west CAR. RDTs seem to be a useful tool for the detection of Plasmodium falciparum in areas with limited possibilities of using other diagnostic methods, such as light microscopy and molecular biology.

Echinococcus multilocularis is a parasite that causes a dangerous zoonosis, alveolar echinococcosis (AE). Its presence in water sources, however, has scarcely been studied heretofore. Accordingly, 222 samples of different origin including wastewater from wastewater treatment plants (WWTPs) (n = 137), slaughterhouse (n = 49) as well as water from rivers (n = 26) and a cattle farm (n = 10) were collected from Xining City and a rural area in Qinghai-Tibet Plateau (QTP), an endemic area. Material obtained after processing of 10 L volume samples was subsequently analysed using three molecular detection methods: nested PCR, real-time PCR and LAMP. E. multilocularis DNA was found in 13 (5.85%) water samples; including 8 (5.8%), 3 (6%), 2 (20%) and 0 positive samples found in WWTPs, a slaughterhouse, a cattle farm and rivers, respectively. All three (LAMP, PCR, RT-PCR) molecular tools displayed high agreement and effectiveness in their ability of detecting the parasite’s DNA in environmental material. This is the first investigation describing E. multilocularis detection in wastewater samples, using three sensitive molecular diagnostic tools. Results indicate the role of wastewater in dissemination of E. multilocularis and the risk of contamination of water sources.