Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Encephalitozoon cuniculi Microsporidia in Cerebrospinal Fluid from Immunocompetent Patients, Czech Republic.

We retrospectively analyzed of 211 frozen cerebrospinal fluid samples from immunocompetent persons in the Czech Republic and detected 6 Encephalitozoon cuniculi-positive samples. Microsporidiosis is generally underestimated and patients are not usually tested for microsporidia, but latent infection in immunodeficient and immunocompetent patients can cause serious complications if not detected and treated.

Host-specific Cryptosporidium, Giardia and Enterocytozoon bieneusi in shelter dogs from central Europe.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Genetic diversity of Cryptosporidium spp., Encephalitozoon spp. and Enterocytozoon bieneusi in feral and captive pigeons in Central Europe.

Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon spp. are the most common protistan parasites of vertebrates. The results show that pigeon populations in Central Europe are parasitised by different species of Cryptosporidium and genotypes of microsporidia of the genera Enterocytozoon and Encephalitozoon. A total of 634 and 306 faecal samples of captive and feral pigeons (Columba livia f. domestica) from 44 locations in the Czech Republic, Slovakia and Poland were analysed for the presence of parasites by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (18S rDNA), 60 kDa glycoprotein (gp60) and internal transcribed spacer (ITS) of SSU rDNA. Phylogenetic analyses revealed the presence of C. meleagridis, C. baileyi, C. parvum, C. andersoni, C. muris, C. galli and C. ornithophilus, E. hellem genotype 1A and 2B, E. cuniculi genotype I and II and E. bieneusi genotype Peru 6, CHN-F1, D, Peru 8, Type IV, ZY37, E, CHN4, SCF2 and WR4. Captive pigeons were significantly more frequently parasitised with screened parasite than feral pigeons. Cryptosporidium meleagridis IIIa and a new subtype IIIl have been described, the oocysts of which are not infectious to immunodeficient mice, whereas chickens are susceptible. This investigation demonstrates that pigeons can be hosts to numerous species, genotypes and subtypes of the studied parasites. Consequently, they represent a potential source of infection for both livestock and humans.

Pneumocystis jirovecii Colonization in Preterm Newborns With Respiratory Distress Syndrome.

We describe the prevalence of Pneumocystis jirovecii in mother-infant pairs of very low birth weight newborns <32 weeks gestation. Molecular and microscopic methods were used for detection of P. jirovecii in patients' specimens. Pneumocystis DNA was detected in 8 nasopharyngeal aspirates (14%) of 56 newborns and in 7 oral washes (21%) of 34 mothers. Pneumocystis detection immediately after birth suggests the possibility of its transplacental transmission. Compared to noncolonized infants, more frequent occurrence of bronchopulmonary dysplasia was seen in colonized infants (P = .02), suggesting a potential clinical importance of this pathogen in abnormal lung development.

Encephalitozoon cuniculi and Extraintestinal Microsporidiosis in Bird Owners.

We identified Encephalitozoon cuniculi genotype II parasites as a cause of extraintestinal microsporidiosis in 2 owners of birds also infected with E. cuniculi. Patients experienced long-lasting nonspecific symptoms; the disease course was more progressive in a patient with diabetes. Our findings suggest direct bird-to-human transmission of this pathogen.

Horse-Specific Cryptosporidium Genotype in Human with Crohn's Disease and Arthritis.

We identified an unusual subtype of a Cryptosporidium sp. horse genotype as the cause of cryptosporidiosis in a 13-year-old girl in Poland who was undergoing immunosuppressive treatment for juvenile rheumatoid arthritis and Crohn's disease. The same subtype was identified in a horse the girl had ridden.

Raw Goat's Milk, Fresh and Soft Cheeses as a Potential Source of Encephalitozoon cuniculi.

The aim of this work was to evaluate the effect of pasteurization and coagulation during goat cheese production on the infectivity of Encephalitozoon cuniculi spores for immunodeficient (SCID, CD4-/-, and CD8-/-) and immunocompetent (BALB/c and C57BL/6) mice. Goat milk and fecal samples were screened for the presence and quantity of microsporidial DNA using molecular methods. Experimentally produced cheese from E. cuniculi-enriched goat milk or goat cheese purchased from retail producers was fed with experimental mice susceptible to E. cuniculi infection. The mice were sacrificed in the presumed acute phase of infection and samples of their tissues were subject to molecular detection of specific E. cuniculi DNA. Specific DNA of E. cuniculi genotype II was detected in feces and milk of three out of 99 goats kept on 6 farms in the Czech Republic. Under experimental conditions, spores of E. cuniculi genotype II remained viable in artificially enriched fresh cheese and were able to cause infection in laboratory mice. E. cuniculi genotype I and II DNA were detected in eight of the nine goat cheeses purchased from various producers/breeders in the Czech Republic and these cheeses were able to develop infection in both immunodeficient and immunocompetent mice. The results of these experiments showed that spores of E. cuniculi genotype I and II are able to remain viable after cheese processing and thus fresh and soft cheeses should be considered a potential source of microsporidia.

Cryptosporidium baileyi Pulmonary Infection in Immunocompetent Woman with Benign Neoplasm.

Cryptosporidium baileyi, a bird-specific parasite, infects gastrointestinal, pulmonary, and urinary tracts of its host. We report on a C. baileyi infection associated with pulmonary hamartoma in an immunocompetent patient in Poland. Further work is needed to investigate the association between Cryptosporidium infections and tumors.

Prevalence and genotyping of Pneumocystis jirovecii in renal transplant recipients-preliminary report.

Pneumocystis jirovecii is an opportunistic fungus occurring in human lungs. The group at highest risk consists of HIV-infected and non-HIV-infected immunosuppressed individuals. In these patients, P. jirovecii infection may lead to Pneumocystis pneumonia; it may, however, persist also in an asymptomatic form. This study aimed to determine the prevalence of P. jirovecii and potential risk factors for infection in a group of renal transplant recipients and to characterize the genetic diversity of this fungus in the studied population. Sputum specimens from 72 patients were tested for presence of P. jirovecii using immunofluorescence microscopy, as well as nested PCR targeting the mtLSU rRNA gene. Genotyping involving analysis of four loci-mtLSU rRNA, CYB, DHPS, and SOD-was used to characterize the diversity of the detected organisms. Pneumocystis DNA was detected in eight (11.11%) patients. It has been shown that low eosinophil count and dual immunosuppressive treatment combining prednisone and calcineurin inhibitors are potential risk factors for colonization. Analysis of genotype distribution showed an association of the wild-type genotype of mtLSU rRNA with lower average age of patients and shorter time after kidney transplantation. Furthermore, CYB 2 genotype was detected only in patients with the ongoing prophylaxis regimen. In conclusion, renal transplant recipients are at risk of Pneumocystis colonization even a long time after transplantation. The present preliminary study identifies specific polymorphisms that appear to be correlated with certain patient characteristics and highlights the need for deeper investigation of these associations in renal transplant recipients.

Experimental Encephalitozoon cuniculi Infection Acquired from Fermented Meat Products.

This study describes the prevalence and concentration of Encephalitozoon cuniculi spores in pork meat and evaluates the effect of sausage fermentation on E. cuniculi infectivity for immunodeficient (severe combined immunodeficient) and immunocompetent (BALB/c and C57BL/6) mice. Using a nested polymerase chain reaction (PCR) approach, E. cuniculi genotype II was detected in the meat from 2 out of 50 pig carcasses at slaughter facilities, with 60-250 spores per gram detected by quantitative PCR. Under experimental conditions, 3000 E. cuniculi genotype II spores per gram of meat remained infective for mice following fermentation at 24°C for 48 h. Based on these findings, fermented meat products should be considered as a potential source of E. cuniculi infection in humans.

Disseminated Infection of Encephalitozoon cuniculi Associated With Osteolysis of Hip Periprosthetic Tissue.

Background: Among patients with hip joint endoprosthesis, periprosthetic osteolysis is the most common complication following primary arthroplasty, and subsequent implant loosening is the leading cause of arthroplasty revision. Causes of stability loss, though not always evident, can be mechanical, allergic, or infectious (bacterial and fungal agents) in nature. Microsporidia, widespread opportunistic fungal pathogens that infect most human tissues, are a potential infectious cause of stability loss. Infections caused by Encephalitozoon species-one of the most common microsporidial pathogens in humans-primarily localize to intestinal and respiratory tracts, but can disseminate to tissues throughout the body. Methods: We examined 53 immunocompetent patients, 23 after revision and 30 after primary hip arthroplasty, for infection by Encephalitozoon species. Periprosthetic tissue, urine sediments, and stool samples were tested by microscopic examination and genus-specific nested polymerase chain reaction followed by genotyping. Results: Ten patients had Encephalitozoon-positive periprosthetic tissues, 9 (39%) after revision and 1 (3.3%) after primary hip arthroplasty. Among the tissue-positive postrevision patients, 7 had a positive urine sample and 1 had a positive stool sample. Encephalitozoon cuniculi genotype II was identified in 88.8% (16/18) of samples. Two urine samples were positive for a novel Encephalitozoon species. Conclusions: Encephalitozoon cuniculi should be considered as a cause of osteolysis in hip periprosthetic tissue, leading to a loss of implant stability.

Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases.

Pneumocystis jirovecii is an opportunistic fungus causing Pneumocystis pneumonia primarily in immunosuppressed patients. However, immunocompetent individuals may become colonized and, as asymptomatic carriers, serve as reservoirs of the pathogen. Moreover, these asymptomatic carriers are at higher risk of developing pneumonia if favorable conditions occur. This study aimed to determine the prevalence of P. jirovecii in patients with various pulmonary diseases and to characterize the genetic diversity of organisms circulating in the studied population. Bronchial washing specimens from 105 patients were tested for presence of P. jirovecii using nested polymerase chain reaction (PCR) targeting the mtLSU rRNA gene, as well as immunofluorescence microscopy. Multilocus sequence typing involving analysis of three loci-mtLSU rRNA, CYB, and SOD-was used for genotyping analysis. P. jirovecii DNA was detected in 17 (16.2%) patients. Amplification of the SOD locus was successful only in five cases (29.4% of the positive patients), while mtLSU rRNA and CYB were genotyped in all positive samples. Therefore, combined genotypes were identified based only on mtLSU rRNA and CYB loci. Eight different genotypes were identified, with Pj 1 and Pj 2 being the most prevalent (29.4% of patients each). There was no statistical correlation between these genotypes and demographic or clinical data; however, we found that infection with mutant CYB strains occurred only in patients diagnosed with lung cancer. Of the potential predictors examined, only immunosuppressive treatment was significantly associated with colonization. In conclusion, patients with various respiratory diseases, especially when immunosuppressed, are at risk of Pneumocystis colonization.

Survey for Zoonotic Microsporidian Pathogens in Wild Living Urban Rooks (Corvus frugilegus).

Microsporidia are opportunistic pathogens in nature infecting all animal phyla. There is a potential risk of microsporidian spores transmission from urban rooks inhabiting some metropolitan cities to people through casual interactions. The aim of this study was to identify microsporidia species in the droppings of rooks in Wroclaw, Poland. A total of 15 collective sets of droppings were examined using nested-PCR method. Amplification of ITS rRNA gene revealed the presence of Enterocytozoon bieneusi D, Peru 6, and Encephalitozoon hellem 1A genotypes. This study indicates that excreta of urban rooks can be an important source of human infection with these pathogens.

Stray cats are more frequently infected with zoonotic protists than pet cats.

Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health.

Study of the impact of cranberry extract on the virulence factors and biofilm formation by Enterococcus faecalis strains isolated from urinary tract infections.

Drinking of cranberry fruit juice and application of commercial preparations containing the cranberry extracts are recommended in the prevention and treatment of urinary tract infections (UTIs), especially in women with recurrent UTIs. Many studies focus on the activity of cranberries against uropathogenic Escherichia coli (E. coli) strains. However, the knowledge of the cranberry effect on Gram-positive Enterococcus faecalis (E. faecalis) is limited. Therefore, the aim of our study was to establish the activity of commercial concentrated cranberry extract on the growth, virulence factors and biofilm formation of E. faecalis strains isolated from urine. Minimal inhibitory concentrations (MICs) of cranberry extract were determined by the broth microdilution method. Disc diffusion method was used to determine antimicrobial susceptibility. The impact of cranberry extract on bacterial survival, hydrophobicity, synthesis of lipase, lecithinase, DNase, hemolysin, gelatinase and biofilm mass was determined. Results show that cranberry extract inhibits the growth, enzymatic activities of bacteria and limits biofilm formation. The antibacterial activities of the studied cranberry extract confirm that it could be successfully used in prevention of UTIs caused by E. faecalis.

Pentacyclic triterpenes combined with ciprofloxacin help to eradicate the biofilm formed in vitro by Escherichia coli.

BACKGROUND & OBJECTIVES: Ciprofloxacin is commonly used in clinical practice for the treatment of recurrent urinary tract infections caused by Escherichia coli. However, very often these recurrent infections are due to a failure in a complete eradication of the microorganisms colonizing the urinary tract, especially in catheterized patients. To enhance the bactericidal activity of ciprofloxacin against biofilm-forming uropathogenic E. coli (UPECs), we examined its effect in combination with two pentacyclic triterpenes - asiatic and ursolic acids. METHODS: The anti-biofilm activity of ciprofloxacin and pentacyclic triterpenes - asiatic acid (AA) and ursolic acid (UA), as well as their synergistic effect were tested on two types of surfaces - polystyrene microtiter plates and silicone catheters. It was investigated using the time-killing and biofilm assays. RESULTS: a0 nti-biofilm activity of ciprofloxacin was not observed on microtiter plates or on the catheters. Ciprofloxacin combined with ursolic acid inhibited the biofilm formation on microtitre plates. This mixture, however, did not express such a strong activity against the synthesis of biofilm on the surface of catheters. Ciprofloxacin combined with asiatic acid had very weak inhibiting effect on the synthesis of biofilm mass on microtitre plates as well as on the catheters. Despite this, both mixtures - ciprofloxacin and asiatic acid, as well as ciprofloxacin and ursolic acid, exhibited strong and significant impact on the eradication of mature biofilm (P < 0.05). INTERPRETATION & CONCLUSIONS: Although ciprofloxacin is recommended in the treatment of urinary tract infections caused by UPECs, but its efficacy is arguable. Subinhibitory concentrations of ciprofloxacin did not inhibit the formation of biofilm. Pentacyclic triterpenes used in combination with ciprofloxacin enhanced its anti-biofilm effectiveness. However, this anti-biofilm activity was found to depend on the type of surface on which biofilm was formed.

Pneumocystis jirovecii--from a commensal to pathogen: clinical and diagnostic review.

Pneumocystis pneumonia is an opportunistic disease caused by invasion of unicellular fungus Pneumocystis jirovecii. Initially, it was responsible for majority of morbidity and mortality cases among HIV-infected patients, which later have been reduced due to the introduction of anti-retroviral therapy, as well as anti-Pneumocystis prophylaxis among these patients. Pneumocystis pneumonia, however, is still a significant cause of mortality among HIV-negative patients being under immunosuppression caused by different factors, such as transplant recipients as well as oncologically treated ones. The issue of pneumocystosis among these people is particularly emphasized in the article, since rapid onset and fast progression of severe symptoms result in high mortality rate among these patients, who thereby represent the group of highest risk of developing Pneumocystis pneumonia. In contrast, fungal invasion in immunocompetent people usually leads to asymptomatic colonization, which frequent incidence among healthy infants has even suggested the possibility of its association with sudden unexpected infant death syndrome. In the face of emerging strains with different epidemiological profiles resulting from genetic diversity, including drug-resistant genotypes, the colonization phenomenon desires particular attention, discussed in this article. We also summarize specific and sensitive methods, required for detection of Pneumocystis invasion and for distinguish colonization from the disease.

Concurrent infection of the urinary tract with Encephalitozoon cuniculi and Enterocytozoon bieneusi in a renal transplant recipient.

A urinary tract coinfection, caused by Encephalitozoon cuniculi genotype II and Enterocytozoon bieneusi genotype D, was identified in an HIV-seronegative renal transplant recipient kept under lifelong immunosuppression. To our knowledge, this is the first report describing concurrent infection with these two microsporidia species in organ transplant recipients.

Gastroenteritis caused by the Cryptosporidium hedgehog genotype in an immunocompetent man.

The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses, was identified as the cause of the diarrheal disease cryptosporidiosis in an immunocompetent man in the Czech Republic. This is the first report of human illness caused by the Cryptosporidium hedgehog genotype.