Screening of mosquitoes for filarioid helminths in urban areas in south western Poland-common patterns in European Setaria tundra xenomonitoring studies.

In recent years, numerous studies screening mosquitoes for filarioid helminths (xenomonitoring) have been performed in Europe. The entomological monitoring of filarial nematode infections in mosquitoes by molecular xenomonitoring might serve as the measure of the rate at which humans and animals expose mosquitoes to microfilariae and the rate at which animals and humans are exposed to the bites of the infected mosquitoes. We hypothesized that combining the data obtained from molecular xenomonitoring and phenological studies of mosquitoes in the urban environment would provide insights into the transmission risk of filarial diseases. In our search for Dirofilaria spp.-infected mosquitoes, we have found Setaria tundra-infected ones instead, as in many other European studies. We have observed that cross-reactivity in PCR assays for Dirofilaria repens, Dirofilaria immitis, and S. tundra COI gene detection was the rule rather than the exception. S. tundra infections were mainly found in Aedes mosquitoes. The differences in the diurnal rhythm of Aedes and Culex mosquitoes did not seem a likely explanation for the lack of S. tundra infections in Culex mosquitoes. The similarity of S. tundra COI gene sequences found in Aedes vexans and Aedes caspius mosquitoes and in roe deer in many European studies, supported by data on Ae. vexans biology, suggested host preference as the most likely cause of the mosquito genus-biased infections. High diversity of the COI gene sequences isolated in the city of Wroclaw in south western Poland and the presence of identical or almost identical sequences in mosquitoes and roe deer across Europe suggests that S. tundra has been established in most of Europe for a very long time.

A cross-sectional study among Polish hunters: seroprevalence of hepatitis E and the analysis of factors contributing to HEV infections.

Hepatitis E virus (HEV) is known as zoonotic agent. The main reservoirs of HEV in Europe are pigs, wild boars, and deer. Hunting activity is considered to be a risk factor for HEV infection. We conducted a cross-sectional study among 1021 Polish hunters. To understand socio-demographic characteristics of this population and to gather information on potential exposures, all participants completed a questionnaire. Commercial immunoassays were employed to estimate seroprevalence anti-HEV. Samples with confirmed positive result of anti-HEV IgM were examined for HEV RNA. Anti-HEV IgG were identified in 227 people, 22.2% of the studied group. Seroprevalence among the studied hunters was associated with age ≥65 [adjusted prevalence ratio (aPR) 1.6, p = 0.037), living in a house (aPR 1.54, p = 0.013), professional contact with farm animals (aPR 1.09, p = 0.01), and consumption of stewed offal (aPR 1.61, p = 0.00). Washing hands after disembowelment was linked to lower seroprevalence (aPR 0.53; p = 0.00). Lower prevalence of anti-HEV IgG among hunters living in cities was associated with age: 35-49 (aPR 0.52, p = 0.011) and 50-64 (aPR 0.93, p = 0.58), living in a house (aPR 1.58, p = 0.002) and owning a cat (aPR 0.58, p = 0.042). Among hunters living in rural areas, seropositivity was associated with contact with farm animals (aPR 1.66, p = 0.013) and consumption of stewed offal (aPR 1.81; p = 0.001). Contrary to initial assumptions, it was concluded that hunting was of significantly lesser importance than other factors. Due to the high level of HEV seroprevalence identified, we recommend conducting a large-scale study in the general population of Poland.

High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

Genetic diversity of Blastocystis hominis sensu lato isolated from humans in Poland

INTRODUCTION AND OBJECTIVES: Blastocystis hominis s. l. is one of the most commonly detected protozoa in the human large intestine. The aim of the study was to determine the genetic subtypes of Blastocystis hominis s. l. occurring in humans in Poland. MATERIALS AND METHODS: Stool samples from patients diagnosed in the Laboratory of the Department of Parasitology, National Institute of Public Health ­ National Institute of Hygiene (NIZP-PZH) and in the Parasitology Laboratory of the Hospital for Infectious Diseases in Warsaw were examined. Blastocystis subtypes were assayed based on the fragment of small-subunit ribosomal RNA gene sequences (SSU rDNA). RESULTS: The examined isolates were classified into five Blastocystis subtypes (STs), fifteen of which belonged to ST3, three to ST1, two to ST2, two to ST6, and one isolate belonged to ST7. In three cases the subtype of isolate was not identified. DISCUSSION AND CONCLUSIONS: In Poland, the subtypes ST1, ST2, ST3, ST4, ST6 and ST7 have been reported in humans so far. The ST6 and ST7 subtypes are rarely detected in humans in Europe. In Poland, the ST6 subtype was previously described in chickens. On the basis of the studies, it was found that Blastocystis isolated from humans in Warsaw show high genetic diversity. In order to determine the possible pathogenic potential of individual Blastocystis subtypes, special epidemiological studies are required.

Is molecular xenomonitoring of mosquitoes for Dirofilaria repens suitable for dirofilariosis surveillance in endemic regions?

Dirofilaria repens is a parasite of animals and humans, transferred by mosquitoes. The assessment of the presence of D. repens-infected vertebrate hosts in the investigated area can be performed by xenomonitoring­detection of the parasite in blood-feeding arthropods. Our study aimed to evaluate PCR xenomonitoring of mosquitoes as a tool for dirofilariosis surveillance in Poland. We were also interested whether inter-study comparisons at the international level would be possible. Mosquitoes were collected in a single locality in Mazowsze province in Poland, in which between 12 and 20% of dogs were infected with D. repens and autochthonous human dirofilariosis was confirmed. All captured female mosquitoes were divided into pools; alternatively, single mosquitoes were analyzed; DNA was isolated and subjected to PCR and real-time PCR for detection of D. repens. The estimations of infection rates of mosquitoes with D. repens, based on PCR results, varied from 0 to 1.57% even between assays for detection of distinct fragments of the same marker­cytochrome oxidase subunit one gene. Polymorphisms of the DNA sequence within binding sites of the primers used in D. repens xenomonitoring assays, applied in European studies, were identified. Non-specific amplification of Setaria tundra (Nematoda: Onchocercidae) DNA occurred. Surveillance of dirofilariosis by PCR mosquito xenomonitoring is possible; however, the efficiency of the approach on territories where the prevalence of the disease among definitive hosts is lower than 12% remains unknown. Furthermore, mosquito infection rate estimations can be PCR assay dependent, which makes inter-study comparisons difficult. The results obtained in independent European xenomonitoring studies were contradictory. International collaboration would be required to establish a standardized set of assays for sensitive and specific xenomonitoring-based dirofilariosis surveillance.

Human babesiosis.

Babesiosis is an emerging parasitic, anthropo-zoonotic tick-borne disease, seldom diagnosed in humans. Caused by Protozoa, Babesia (also called Piroplasma) intraerytrocytic piriform microorganism. Infection of vertebrates is transmitted by ticks. Out of more than 100 Babesia species/genotypes described so far, only some were diagnosed in infected humans, mostly B. microti, B. divergens and B. venatorum (Babesia sp. EU1). Infection in humans is often asymptomatic or mild but is of a particular risk for asplenic individuals, those with congenital or acquired immunodeficiencies, and elderly. Infections transmitted with blood and blood products raise concerns in hemotherapy. Epidemiological situation of babesiosis varies around the world. In Europe, no increase in the number of cases was reported, but in the USA its prevalence is increasing and extension of endemic areas is observed. The aim of this publication is to describe the problems connected with the current epidemiological situation, diagnosis and treatment of human babesiosis with regard to clinical status of patients.

Evaluation of the frequency of Pneumocystis jirovecii occurrence in a group of children hospitalized for acute respiratory infections.

Primary infection with Pneumocystis jirovecii in small children may cause inflammation of the respiratory tract which requires hospitalization. Lack of characteristic clinical symptoms makes it impossible to recognize P. jirovecii infections without performing laboratory analyses. Nasopharyngeal swabs from 70 children with respiratory tract infections were screened for fragments of the P. jirovecii genome. Pneumocystis DNA was found in swabs taken from two (2.9%) of the tested children: a newborn who was infected in the hospital and a six month old baby admitted to hospital two days after pneumonia was diagnosed. The obtained results confirm that primary P. jirovecii infections may occur in the form of acute respiratory tract inflammations suggesting a viral infection. In differential diagnosis of Pneumocystis infections in children molecular methods are useful as their high sensitivity makes it possible to analyze samples obtained in a non-invasive way.

[Cases of Toxoplasma gondii infection in foetus's and toxoplasmic encephalitis in immunosupressed patients confirmed by PCR method in NIPH - NIH, 2009-2010]. / Potwierdzenie przypadków toksoplazmozy plodu oraz toksoplazmozy centralnego ukladu nerwowego za pomoca metody PCR w badaniach wykonanych w NIZP-PZH w latach 2009-2010.

The results of molecular tests for Toxoplasma gondii infection carried out in NIPH-NIH were presented in the light of current data on diagnostics and epidemiology of toxoplasmosis. Between January 2009 and December 2010 four cases of active toxoplasmosis were confirmed using PCR targeting the 529-bp repeat element of Toxoplasma gondii. Intrauterine infection was found in 3 out of 180 (1.7%) examined women. Toxoplasmic encephalitis was confirmed in one patient who was a liver transplant recipient. T. gondii was not detected in cerebrospinal fluid of 19 out of 20 examined patients with encephalitis. The cases of intrauterine toxoplasmosis diagnosed in 2009-2010 make 57.1% of all cases diagnosed in the last decade. In 2001-2010 toxoplasmic encephalitis was detected in two patients out of 45 examined. The choice of tests and interpretation of the results are important elements of T. gondii infection diagnosis and require cooperation between clinicians and laboratory diagnosticians. Establishing reference center for toxoplasmosis in Poland would contribute towards the improvement of standards of diagnostics and treatment of cases of congenital infections and the infection in immunosuppresed patients.