Detection and Molecular Characterization of Blastocystis Species in Polish Soldiers Stationed in the Republic of Kosovo.

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.

The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites.

The CRISPR/Cas9 system, a natural defence system of bacterial organisms, has recently been used to modify genomes of the most important protozoa parasites. Successful genome manipulations with the CRISPR/Cas9 system are changing the present view of genetics in parasitology. The application of this system offers a major chance to overcome the current restriction in culturing, maintaining and analysing protozoan parasites, and allows dynamic analysis of parasite genes functions, leading to a better understanding of pathogenesis. CRISPR/Cas9 system will have a significant influence on the process of developing novel drugs and treatment strategies against protozoa parasites.

The first detection of Toxoplasma gondii DNA in environmental air samples using gelatine filters, real-time PCR and loop-mediated isothermal (LAMP) assays: qualitative and quantitative analysis.

Toxoplasma gondii infections are acquired through the ingestion of oocysts present in the environment. However, there is no data about their occurrence in the air or about airborne transmission of these infections. In the present paper, we report on the identification of T. gondii using rapid molecular detection methods, supported by microscopic analysis, in environmental air samples. A total of 71 samples were collected, using gelatine filters, from kitchen gardens, recreational areas and sandpits located in northern and north-eastern Poland. Material recovered from the filters was analysed using real-time PCR and loop-mediated isothermal assays targeting the T. gondii B1 gene. Toxoplasma gondii DNA was found in two samples, as confirmed by both molecular assays. Genotyping at the SAG2 locus showed Toxoplasma SAG2 type I. Moreover, the presence of T. gondii oocysts was confirmed in one of the positive samples with the use of microscopy. The results showed that T. gondii may be present in environmental air samples and that respiratory tract infections may play a role in the high prevalence of toxoplasmosis in humans and animals. To the best of our knowledge, this is the first epidemiological evidence that oro-fecal and foodborne toxoplasmosis may be traceable to an airborne respiratory origin and that this may represent a new, previously unknown transmission route for this disease.

Detection of Giardia intestinalis in water samples collected from natural water reservoirs and wells in northern and north-eastern Poland using LAMP, real-time PCR and nested PCR.

Giardia intestinalis is a protozoan parasite, transmitted to humans and animals by the faecal-oral route, mainly through contaminated water and food. Knowledge about the distribution of this parasite in surface water in Poland is fragmentary and incomplete. Accordingly, 36 environmental water samples taken from surface water reservoirs and wells were collected in Pomerania and Warmia-Masuria provinces, Poland. The 50 L samples were filtered and subsequently analysed with three molecular detection methods: loop-mediated isothermal amplification (LAMP), real-time polymerase chain reaction (real-time PCR) and nested PCR. Of the samples examined, Giardia DNA was found in 15 (42%) samples with the use of LAMP; in 12 (33%) of these samples, Giardia DNA from this parasite was also detected using real-time PCR; and in 9 (25%) using nested PCR. Sequencing of selected positive samples confirmed that the PCR products were fragments of the Giardia intestinalis small subunit rRNA gene. Genotyping using multiplex real-time PCR indicated the presence of assemblages A and B, with the latter predominating. The results indicate that surface water in Poland, as well as water taken from surface wells, may be a source of Giardia strains which are potentially pathogenic for humans. It was also demonstrated that LAMP assay is more sensitive than the other two molecular assays.

First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens.

It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite's DNA and genotyping was performed using real-time PCR, specific to the ß-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

Fresh fruits, vegetables and mushrooms as transmission vehicles for Echinococcus multilocularis in highly endemic areas of Poland: reply to concerns.

Echinococcus multilocularis is a tapeworm that may cause alveolar echinococcosis (AE), one of the most dangerous parasitic zoonoses. As in the case of other foodborne diseases, unwashed fruits and vegetables, contaminated with dispersed forms of E. multilocularis, may serve as an important transmission route for this parasite. In this article, we reply to the incorrect interpretation of results of our study concerning the detection of E. multilocularis DNA in fresh fruit, vegetable and mushroom samples collected from the highly endemic areas of the Warmia-Masuria Province, Poland, to dispel any doubts. The accusations formulated by the commentators concerning our paper are unfounded; moreover, these commentators demand information which was beyond the purview of our study. Making generalisations and drawing far-reaching conclusions from our work is also unjustified. The majority of positive samples were found in only a few hyperendemic communities; this information corresponds with the highest number of both infected foxes and AE cases in humans recorded in this area. Our findings indicate that E. multilocularis is present in the environment and may create a potential risk for the inhabitants. These people should simply be informed to wash fruits and vegetables before eating. No additional far-reaching conclusions should be drawn from our data. We believe these commentators needlessly misinterpreted our results and disseminated misleading information. Nevertheless, we would like to encourage any readers simply to contact us if any aspects of our study are unclear.

Respiratory infections in travelers returning from the tropics.

Respiratory tract infections (RTIs), beside diarrheas, skin lesions, and fevers of unknown origin, are one of the most common health problems acquired by travelers going to tropical and subtropical countries. Visitors to African, Asian, or South American destinations, typically characterized by harsh environmental conditions and poor sanitation standards, are at risk of exposure to a large number of pathogens causing infectious diseases. The infections are transmitted from contaminated food and water, through the air, direct contact, or by insects. The main modes of RTIs transmission include droplet infection and direct contact. The clinical spectrum of RTIs in travelers is broad, from upper respiratory tract infections, pharyngitis, bronchitis, pneumonia, to influenza-like illness. The spectrum of microbial agents causing respiratory infections include numerous viruses and bacteria, rarely fungi, and parasites. Most travelers complain of mild infections, only a small minority seek medical assistance and report to health care facilities. Because of the risk of importing pathogens into Europe or North America and transferring them onto the local population, it is important to present the scale of the problem in relation to rapid development of tourism industry and an increasing number of intercontinental journeys. The aim of the study was to discuss the occurrence of travel-related respiratory infections among representatives of temperate climate traveling to and returning from the tropics.

The first detection of Echinococcus multilocularis DNA in environmental fruit, vegetable, and mushroom samples using nested PCR.

The aim of this study was to estimate the presence of Echinococcus multilocularis DNA in fruits, vegetables, and mushrooms in rural areas of Varmia-Masuria Province, Poland, which is the region with the highest number of human alveolar echinococcosis (AE) cases in this country. Recovery tests showed that E. multilocularis DNA is detectable in samples contaminated with at least 100 eggs of this tapeworm. In total, 103 environmental fruit, vegetable, and mushroom samples collected in forests, plantations, and kitchen gardens were analyzed using nested PCR assay based on the mitochondrial 12S ribosomal RNA (rRNA) gene. The parasite DNA was detected in 23.3% of the samples. Sequencing confirmed that the obtained PCR products represented E. multilocularis. This study is the first environmental survey of the presence of E. multilocularis DNA in fruits, vegetables, and mushrooms intended for consumption. The results clearly demonstrate that it may be a direct source of human infections and shows the need to educate the public about the threat, especially people living in at-risk areas.

Detection and quantification of Anaplasma phagocytophilum and Babesia spp. in Ixodes ricinus ticks from urban and rural environment, northern Poland, by real-time polymerase chain reaction.

Anaplasma phagocytophilum and Babesia spp. are emerging tick-borne pathogens which can threaten human health. A duplex real-time PCR and qPCRs with primers and probes targeting 97 and 116 bp fragments of 16S rRNA and 18S rRNA genes, respectively, were used for qualitative and quantitative detection of both pathogens in Ixodes ricinus ticks. Altogether 1875 ticks (1084 adults and 791 nymphs) were collected from rural and urban habitats in northern Poland. Of them, at least 0.9% were found to be infected with A. phagocytophilum while 2.5% with Babesia spp. A comparison of the infection rates by the tick stage, the type of area, the collection site, habitats of different tick density and by the month of collection was done. The prevalence of pathogens was significantly lower in nymphs than in adult ticks (p = 0.02) and in rural areas than in urban areas (p = 0.007). Four different 16S rRNA gene variants of A. phagocytophilum were determine, however none of them showed 100% identity with compared sequences isolated from human patients. The dominant Babesia species was B. venatorum. Results of qPCRs with circular and linearized forms of plasmids used as the standards showed significant difference in the pathogen loads (p = 0.001). The copy numbers of A. phagocytophilum and Babesia spp. estimated from the linear plasmids were 28.7 and 5.1 times lower, respectively, when compared with their circular forms, and were accepted as more reliable. The average number of copies of 16S rRNA gene of A. phagocytophilum in the positive I. ricinus samples were 3.39 × 10(5) ± 6.09 × 10(5). The mean copy number of 18S rRNA gene of Babesia spp. was ~2.55 × 10(5) ± 1.04 × 10(6). We confirmed the presence of A. phagocytophilum and Babesia spp. in I. ricinus in both rural and urban environments. The determined low infection rates suggests, however, that the risk for local population and tourists to acquire infection is also low. Moreover, we confirmed recent findings that serious overestimation by circular plasmid DNA makes it less suitable as a standard and that the linear standards should be recommended for qPCR.

Epidemiology of intestinal parasitic infections in school children in Ghazni Province, eastern Afghanistan.

OBJECTIVE: To estimate the prevalence of intestinal parasites and their species in Afghan school children and to establish appropriate treatment methods for detected pathogens. METHODS: Parasitological examination of stool samples collected from 1369 children aged 8-18, students of the Jahan Malika High School in Ghazni Province in eastern Afghanistan, was conducted in the period November 2013-April 2014. Three stool samples were collected from each patient every second day; the samples were fixed in 10% formalin and tested by light microscopy using the methods of direct smear in Lugol's solution, decantation in distilled water, and Fülleborn's flotation. RESULTS: Of 535 examined children (39.1% of the study group) were infected with nematodes (n=324), cestodes (n=118), trematodes (n=12), and protozoa (n=228), 132 were diagnosed with co-infections (mainly ascariasis+giardiasis, ascariasis+hymenolepiasis) and received single or combined therapy. CONCLUSIONS: The Afghan community is an example of population characterized by a high rate of parasitic infections. Owing to high prevalence of multiple infections among inhabitants of Afghanistan, it seems that a mass deworming campaign with a single-dose chemotherapy may prove ineffective in eradicating intestinal parasites in the local population.

The first genotype determination of Acanthamoeba potential threat to human health, isolated from natural water reservoirs in Poland.

Different species of amoebae belonging to the genus Acanthamoeba are widely distributed in many parts of the world and known as free-living organisms. Some strains of the protozoans may exist as parasites and cause risk to human health as causative agents of serious human diseases. Currently, in Poland, there is no sufficient information about the distribution of Acanthamoeba strains and their genotypes in the environment. Therefore, 20 environmental surface water samples were collected from different sites located at five water reservoirs in Gdynia, Sopot, and Gdansk (northern Poland). The material was cultured to obtain Acanthamoeba isolates that were then specifically analyzed with both PCR and real-time PCR assays. Of the 20 samples examined, Acanthamoeba DNA was found in 13 samples tested with the use of real-time PCR; in 10 of them, DNA of the amoeba was also detected using PCR technique. The comparison with sequences available in the GenBank confirmed that the PCR products are fragments of Acanthamoeba 18S rRNA gene and that isolates represent T4 genotype, known as the most common strains related to AK cases. This is the first investigation in Poland describing Acanthamoeba detection in environmental water samples with molecular techniques and genotyping. The results indicate that surface water in Poland may be a source of acanthamoebic strains potentially pathogenic for humans.

Chronic intestinal schistosomiasis caused by co-infection with Schistosoma intercalatum and Schistosoma mansoni.

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.

Detection of Asian genetic components in autochthonous human Echinococcus multilocularis infections from endemic Warmia-Masuria (north-eastern Poland).

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.

4D-Dynamic Representation of DNA/RNA Sequences: Studies on Genetic Diversity of Echinococcus multilocularis in Red Foxes in Poland.

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.

Investigation of Toxoplasma gondii in wastewater and surface water in the Qinghai-Tibet Plateau, China using real-time PCR and multilocus genotyping.

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.

Prevalence of Asymptomatic Malaria Infections in Seemingly Healthy Children, the Rural Dzanga Sangha Region, Central African Republic.

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.

Prevalence of Plasmodium spp. in symptomatic BaAka Pygmies inhabiting the rural Dzanga Sangha region of the Central African Republic.

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.

Contamination of wastewater with Echinococcus multilocularis - possible implications for drinking water resources in the QTP China.

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.

Usefulness of PCR method for detection of Leishmania in Poland.

Leishmania parasites are the etiological agents of leishmaniosis, with severe course and often fatal prognosis, and the global number of cases has increased in recent decades. The gold standards for the diagnosis of leishmaniosis are microscopic examinations and culture in vitro of the different clinical specimens. The sensitivity of these methods is insufficient. Recent development in specific and sensitive molecular methods (PCR) allows for detection as well as identification of the parasite species (subspecies). The aim of the study was to estimate the usefulness of molecular methods (PCR) for detection of Leishmania species and consequently for the implementation of such methods in routine diagnostics of leishmaniosis in Polish patients returning from endemic areas of the disease. In our investigations we used 54 known Leishmania positive DNA templates (from culture and clinical specimens) received from the CDC (Atlanta, GA, USA). Moreover, 25 samples of bone marrow, blood or other tissues obtained from 18 Polish individuals suspected of leishmaniosis were also examined. In PCR we used two pairs of primers specific to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircle (13A/13B and F/R). Using these primers we obtained amplicons in all DNA templates from the CDC and in three Polish patients suspected for Leishmania infection. In one sample from among these cases we also obtained positive results with DNA isolated from a blood specimen which was previously negative in microscopic examinations.

First molecular detection of Toxoplasma gondii in vegetable samples in China using qualitative, quantitative real-time PCR and multilocus genotyping.

Toxoplasma gondii infection is becoming increasing problem in China but there is no data concerning contamination of vegetables intended for consumption with this parasite. The aim of the present study was to investigate fresh vegetables originated from open markets located in the Xining City, the Qinghai-Tibet Plateau (QTP), P.R. China for their contamination with T. gondii. A total of 279 fresh vegetable samples were collected and analysed using real-time PCR assay targeting B1 gene and multilocus genotyping. T. gondii DNA was found in 10 (3.6%) samples tested; eight of them represented T. gondii type I and remaining two T. gondii type II. The approximate level of contamination of positive vegetables samples, estimated based on quantitative real-time PCR (qPCR), ranged between less than one and 27000 T. gondii oocysts per sample, with majority not exceeding several oocysts per sample. The results of the study confirmed that T. gondii is present in vegetables offered in open markets in the Qinghai province, P.R. China; eating them unwashed and raw may therefore pose a threat to consumers. This is the first investigation describing T. gondii detection in fresh vegetables intended for consumption collected from the territory of P.R. China using sensitive molecular tools.