Another case of coincidental Giardia infection and pancreatic cancer.

Until now, few cases of coincidental giardiasis and pancreatic tumors have been described. Among these cases, three described giardiasis cases coincided with confirmed pancreatic cancer. We present another case of Giardia infection coexisting with pancreatic cancer in a 67-year-old man who suffered from stenosis of the distal ductus choledochus combined with a hypoechoic mass in the head of the pancreas. The diagnostic conclusion of suspicious adenocarcinoma was based on endoscopic ultrasound fine-needle aspiration (EUS-FNA) biopsy and confirmed by a partial duodenopancreatectomy. On bloody cytology smears prepared from the EUS-FNA specimen, trophozoites of Giardia intestinalis accompanying an inflammatory background and features that fulfilled the morphological criteria of malignancy were observed. In histological sections from the duodenopancreatectomy specimens, the parasites were observed attached to the epithelium, but individual Giardia parasites were also observed beneath the epithelial lining. According to conventional genotyping, the infecting Giardia belonged to sub-assemblage AII.

A natural zoonotic giardiasis: Infection of a child via Giardia cysts in pet chinchilla droppings.

Here, we report a case of direct zoonotic transmission of giardiasis between a pet chinchilla and a human. Microscopic and molecular examinations of stool samples from a child and samples of chinchilla droppings revealed cysts/DNA of Giardia intestinalis. The transmission from the chinchilla to the child has been confirmed as coprophagous after the 1-year-old toddler ingested pet chinchilla droppings. Molecular analysis of the gdh gene from both hosts classified the G. intestinalis cysts into the assemblage B genetic group, which has been previously shown to be characteristic of both human and chinchilla giardiasis. Both Giardia sub-assemblages BIII and BIV were present in the chinchilla droppings, whereas only the sub-assemblage BIV was isolated from the child's stool sample. To the best of our knowledge, this is the first report of a true zoonotic transmission of giardiasis, supporting the zoonotic potential of assemblage B.

The first multilocus genotype analysis of Giardia intestinalis in humans in the Czech Republic.

To date, genotyping data on giardiasis have not been available in the Czech Republic. In this study, we characterized 47 human isolates of Giardia intestinalis from symptomatic as well as asymptomatic giardiasis cases. Genomic DNA from trophozoites was tested by PCR-sequence analysis at three loci (ß-giardin, glutamate dehydrogenase and triose phosphate isomerase). Sequence analysis showed assemblages A and B in 41 (87.2%) and six (12.8%) isolates, respectively. Two of the 41 assemblage A samples were genotyped as sub-assemblage AI, and 39 were genotyped as sub-assemblage AII. Four previously identified multilocus genotypes (MLGs: AI-1, AII-1, AII-4 and AII-9) and six likely novel variations of MLGs were found. In agreement with previous studies, sequences from assemblage B isolates were characterized by a large genetic variability and by the presence of heterogeneous positions, which prevent the definition of MLGs. This study also investigated whether there was a relationship between the assemblage and clinical data (including drug resistance). However, due to the large number of genotypes and the relatively small number of samples, no significant associations with the clinical data were found.

Imported new world cutaneous leishmaniasis in a traveller from Slovakia.

We present a case of imported leishmaniasis in a 31-year-old woman from Slovakia, who visited the countries of South America for three months in 2011. On 29 and 31 August 2011, she was probably infected with Leishmania parasites in the jungles of Ecuador. Approximately one week after returning to Slovakia, a small papules appeared on patient's left leg. Another wound was found after two weeks. Both ulcers were enlarging. We proved amastigote forms of Leishmania spp. only in repeated dermal scrapings from the edge of the ulcer by Giemsa staining after negative results from examination of a wound scrape and biopsy specimen. We identified the species Leishmania (Viannia) panamensis as a causative agent by using the polymerase chain reaction (PCR) method and subsequent sequencing of the ITS region. Closure of wounds and scab formation were observed after 20 days of treatment with sodium stibogluconate. In the control microscopic examination after the end of the treatment, parasites were not present, and the PCR confirmed the negative result (Fig. 2, Ref. 31).

[Febrile pancytopenia and hepatosplenomegaly as leading symptoms of visceral leishmaniasis]. / Febrilní pancytopenie a hepatosplenomegalie jako hlavní symptomy viscerální leishmaniózy.

The authors present a case report of a patient with febrile pancytopenia, hepatosplenomegaly and weight loss as main symptoms of visceral leishmaniasis. Standard treatment regimen with amphothericin B led to relapse of the disease after several weeks. The definitive cure of the disease was achieved with cytostatic miltefosin (Impavido©), which is not registered in the Czech Republic. The aim of this article is to point out this imported protozoan infection and its basic clinical and laboratory features.

[Visceral leishmaniasis (two case reports)]. / Viscerální leishmanióza (dve kazuistiky).

Two cases of imported visceral leishmaniasis are described. The first patient was a 32-year-old Czech man who developed leishmaniasis 5 months after a holiday in Italy (Bibione). The second patient was a 62-year-old Czech man who developed leishmaniasis 18 months after visiting Croatia (Makarska); the disease began after a course of chemotherapy due to metastasizing testicular tumor. Both patients were successfully treated with amphotericin B lipid complex (Abelcet). Difficulties in establishing the correct diagnosis of visceral leishmaniasis are discussed.

Nonpathogenic Entamoeba dispar quickly outgrows pathogenic Entamoeba histolytica in mixed xenic cultures.

Entamoeba histolytica and Entamoeba dispar are two microscopically indistinguishable amoebae living in the human colon. The former is a pathogen, whereas the latter is a nonpathogenic commensal. Using a model system of in vitro cocultures and PCR detection of the Entamoeba species, we found that the nonpathogenic species can rapidly outgrow the pathogen in xenic cultures.

[Pathogenic Entamoeba histolytica--a rare incidence in persons microscopically positive for cysts in faeces]. / Patogenní Entamoeba histolytica--vzácný výskyt u osob s mikroskopickým nálezem cyst ve stolici.

BACKGROUND: In the Czech Republic, 40 to 50 cases of Entamoeba histolytica/dispar infections are reported annually to the National Reference Laboratory for diagnostics of intestinal parasitoses. However, the actual number of patients with Entamoeba histolytica infection is unknown as diagnosis relies on cyst detection in faecal samples by microscopy, the method which cannot differentiate between pathogenic E. histolytica and nonpathogenic E. dispar. The aim of this study was (1) to evaluate the proportions between E.histolytica and E. dispar in patients, mainly travellers, using multiplex nested PCR technique, (2) to evaluate specificity of the technique for detection of these species. METHODS AND RESULTS: Faecal samples from 68 patients microscopically positive for cysts of E. histolytica/dispar were tested by PCR. Of these, 65 persons (95.6%) were positive for E. dispar, whereas only 3 patients (4.4%) were positive for E. histolytica. CONCLUSIONS: This study shows that the number of patients infected with pathogenic E. histolytica is very low in the Czech Republic and points to the necessity of differentiation of Entamoeba species for physician's decision in treatment of E. histolvtica- or E.dispar-infected persons.

[A case of Babesia microti imported into the Czech Republic from the USA]. / Prípad infekce Babesia microti importované do Ceské republiky z USA.

Human babesiosis is a zoonotic tick-borne protozoan infection caused by several species of the genus Babesia. It is a rare disease in Europe. Until recently, 31 cases have been described. Most of them were severe infections caused by a cattle parasite Babesia divergens in splenectomized patients. In contrast, a rodent species Babesia microti was responsible for hundreds of cases of human babesiosis in the U.S.A. In this report we describe the first case of human babesiosis in the Czech Republic. To our knowledge, it is also the first case of symptomatic B. microti infection imported to Europe from the United States.

[New drugs for treatment of parasitic infections]. / Nové léky proti parazitárním infekcím.

In this review we have summarized published data on two new compounds, which can represent important antiparasitic drugs in the near future, nitazoxanide for treatment of intestinal parasitic infections including cryptosporidiosis and miltefosine for oral treatment of visceral leishmaniasis.

[Hemophagocytic lymphohistiocytosis as a manifestation of visceral leishmaniasis]. / Hemofagocytující lymfohistiocytóza jako manifestace viscerální leishmaniózy.

A 7-year-old previously healthy Czech boy was admitted due to fever, hepatosplenomegaly and pancytopenia. Aspiration of bone marrow revealed no signs of hemoblastosis (nor hemophagocytosis). He was treated with antibiotics and virostatics without effect. Progression of hepatosplenomegaly and pancytopenia induced suspicion of hemophagocytic lymphohistiocytosis (HLH). Five weeks later, bone marrow hemophagocytosis of erythrocytes, nuclear elements and platelets was detected. He was given corticoids and intravenous immunoglobulins and transferred to our haematology department. Laboratory findings of mild pancytopenia, hypofibrinogenaemia, hyperlipidaemia and elevated levels of ferritin, LDH and immunoglobulins were compatible to the diagnosis of HLH. Immunologic evaluation revealed T-lymphocyte activation. Appropriate immunosuppressive treatment with Dexamethasone, etoposide and Cyclosporine A was launched, followed by transient subside of fever and improvement of peripheral blood count, but not regression of hepatosplenomegaly. Four weeks later, relapse of fever and deterioration of blood count led to intensification of immunosuppression. However, no effect was evident. Moreover, hypertrophic cardiomyopathy with ventricular arrhythmia occurred. Treatment with antilymphocytic globulin for resistant course of HLH was planned. Before that, a fifth bone marrow aspiration was performed. Surprisingly, many Leishmania amastigotes were observed within marrow macrophages. Leishmania infection was confirmed by positive serology. Immunosuppressive treatment was withdrawn and changed for causal treatment with liposomal Amphotericin B. Positive clinical effect with subside of fever was evident in ten days, splenomegaly gradually resolved during three weeks, restoration of normal blood count lasted six weeks. No relapses of HLH nor leishmaniasis occurred. In control bone marrow aspirate performed three months later, the parasites were not detected. Ten months after the event, the patient is in complete remission of HLH with normal immunologic parameters. Most probably, he contracted visceral leishmaniasis during a visit of a Neapol area in Italy 3 months before the onset of the disease.

Salivary proteins and glycoproteins in phlebotomine sandflies of various species, sex and age.

Salivary gland proteins were studied in sandflies (Diptera: Psychodidae: Phlebotominae) by electrophoretic techniques. In Phlebotomus duboscqi Neveu-Lemaire the protein concentration was about 30 times higher in females than in males. SDS PAGE revealed eight major bands of 29-62 kDa in salivary gland extracts (SGE) from females, whereas only one band of 57kDa was detected in males. The number of protein components in SGE gradually increased with the age of females. In P. papatasi (Scopoli) the typical electrophoretic pattern was reached in 3-5 days after imago emergence, depending on the temperature at which females were maintained. All major protein components of the female SGE were present in the content of glands. Female SGE were compared in seven colonies of five Phlebotomus and Lutzomyia species; electrophoretic profiles distinguished between species and even between colonies of different geographical origin. In general, the highest variability of major protein components was observed in the 38-48kDa region. Four colonies of the subgenus Phlebotomus (P. duboscqi and P. papatasi) possessed common mobility polypeptides, the highest similarity was found between two colonies of P. papatasi. Other species tested significantly differed, specific prominent bands of 33, 35 and 38kDa were found in P. halepensis Theodor, P. perniciosus Newstead and Lutzomyia longipalpis (Lutz & Neiva), respectively. Glycoproteins in SGE of Lu. longipalpis and P. duboscqi females were identified and analysed using blotting with five lectin conjugates. Specific reaction of lectins ConA and WGA revealed the complex type of N-glycans in the 48 and 53-54kDa glycoproteins present in both species. Similar glycosylation was detected in species-specific bands of the 57-60 and 65-67 kDa in P. duboscqi and Lu. longipalpis, respectively. The high mannose type of glycosylation was found in the 20 and 39 kDa polypeptides of Lu. longipalpis and the 40-42 kDa polypeptides of P. duboscqi. Innate lectin activity specific for aminosugars was detected in SGE of P. duboscqi females using haemagglutination tests with rabbit erythrocytes.

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

Susceptibility to Leishmania major infection in mice: multiple loci and heterogeneity of immunopathological phenotypes.

Susceptibility as opposed to resistance of mouse strains (e.g., BALB/c vs C57BL/6) to Leishmania major has been attributed to a defective Th1 and a predominant Th2-response, resulting in increased IL-4 and IgE production, and decreased interferon gamma (IFN gamma) production, macrophage activation and elimination of parasites. Here we report dissection of genetic and functional aspects of susceptibility to leishmaniasis using two contrasting inbred strains BALB/cHeA (susceptible) and STS/A (resistant) and a resistant Recombinant Congenic (RC) Strain, CcS-5/Dem, which carries a random set of 12.5% of genes from the strain STS and 87.5% genes from the susceptible strain BALB/c. Linkage analysis of F2 hybrids between the resistant RC strain CcS-5 and the susceptible strain BALB/c revealed five loci affecting the response to the infection, each apparently associated with a different combination of pathological symptoms and immunological reactions. The correlation between Th2-type immune reactions and the disease in the F2 mice was either absent, or it was limited to mice with specific genotypes at loci on chromosomes 10 and 17. This suggests that the resistance vs susceptibility is influenced by mechanisms additional to the postulated antagonistic effects of Th1 and Th2 responses, and that the host's genotype affects the development of leishmaniasis in a complex way.

Amebic encephalitis caused by Balamuthia mandrillaris in a Czech child: description of the first case from Europe.

We describe a fatal case of amebic encephalitis caused by Balamuthia mandrillaris in a 3-year-old Czech boy who had never traveled abroad. This is the first such infection reported in Europe. The diagnosis was established by brain biopsy, in which abundant trophozoites and a few round amebic cysts were identified. The presence of multiple nucleoli in some trophozoites suggested the organism to be Balamuthia mandrillaris and this was confirmed by indirect immunofluorescence. The amebae invaded brain tissue, including neurons, and blood vessel walls, causing thrombovasculitis. The tissue reaction was a subacute necrotizing and granulomatous encephalitis (GAE) with an infiltrate of CD4- and CD8-positive T-lymphocytes, B-lymphocytes, plasma cells and macrophages. The child, in whom no underlying immunodeficiency was demonstrated, died after 45 days. The mode of infection was not established. Postmortem examination of the brain revealed massive areas of necrosis and microscopic findings like those in the surgical specimen. In vitro isolation of B. mandrillaris was unsuccessful.

Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes.

Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture or growth in suckling mice, were compared genetically by analysis of DNA amplified from loci encoding variant surface proteins or the enzyme glutamate dehydrogenase and by allozyme analysis. The isolates were heterogeneous, but all showed affinity with genetic Assemblage A--one of two major assemblages defined previously by analysis of Giardia from humans. Three distinct genotypes were evident. Ten isolates (eight axenic and two established in suckling mice) from an alpaca, pig, horse, cattle and sheep were indistinguishable from human-derived G. intestinalis belonging to a previously designated genetic group (Group I). This genotype seems to have broad host specificity, including a zoonotic potential for humans. Five isolates (two axenic and three established in suckling mice) from an alpaca, a horse and sheep had close affinity with human-derived Group I and Group II G. intestinalis genotypes. The other 16 isolates (comprising both axenic and suckling mouse-propagated cultures derived from cattle, sheep, alpaca, a goat and pigs in Australia and Europe) differed from all other Giardia with "duodenalis" morphology that have been examined by these methods and they segregated as a highly distinct sublineage (referred to herein as 'Novel livestock') within genetic Assemblage A. The predominance of 'Novel livestock' genotypes in the test panel and their apparent exclusive association with artiodactyl hosts indicates that they may be confined to this group of mammals. Assemblage B genotypes, which are prevalent in humans and some other animal species, were not detected.

A light microscopic study of the course of cellular division of Giardia intestinalis trophozoites grown in vitro.

The cellular division of trophozoites of Giardia intestinalis group was followed microscopically in stained preparations from axenic cultures. Eleven successive stages were described, documented by photomicrographs and their survival times calculated.