Significance of transcriptionally-active high-risk human papillomavirus in sinonasal squamous cell carcinoma: Case series and a meta-analysis.

Sinonasal cancers represent a highly heterogeneous group of head and neck cancers, for which etiological and prognostic significance of high-risk human papillomavirus (HPV) infections has not yet been conclusively established. We investigated the presence of transcriptionally-active high-risk HPV in a series of 34 sinonasal squamous cell cancer (SNSCC) cases and evaluated the effect of transcriptionally-active HPV on the overall survival. In addition, we performed a meta-analysis of previously published studies, including this study, to summarize the prevalence of HPV positivity across histological subtypes of SNSCC. The presence of transcriptionally-active HPV was detected by HPV mRNA using the polymerase chain reaction (PCR) or in situ hybridization (ISH). p16 expression was evaluated as a surrogate marker for transcriptionally-active HPV infection by immunohistochemistry (IHC), the presence of high-risk HPV DNA was tested by PCR and the HPV genotypes were determined by sequencing of PCR amplicons. Transcriptionally-active HPV infections were found in ~25% of the SNSCC cases. The role of HPV infection in keratinizing SNSCC may be higher than previously reported (~32% in our study vs. ~0-6.3% in all other studies). Patients with transcriptionally-active HPV-positive SNSCCs were more likely to be diagnosed at earlier stages (p<0.05) and displayed better mean overall survival, although the difference between HPV-positive and HPV-negative groups was not statistically significant. In contrast to other non-oropharyngeal squamous cell carcinomas (non-OPSCCs) of the head and neck, in SNSCCs, p16/IHC and p16/IHC+HPV DNA displayed high specificity as surrogate markers of transcriptionally-active HPV infections. However, p16/IHC may have significantly lower sensitivity as a surrogate marker of transcriptionally-active HPV in SNSCCs compared to OPSCCs. Furthermore, in our group of SNSCCs, all cases positive for high-risk HPV DNA by PCR were also transcriptionally-active (causative) infections with positive HPV mRNA by ISH. Our results imply a possible different role of HPV-mediated carcinogenesis of squamous cell epithelium in oropharyngeal and sinonasal sites with the latter displaying a lower proportion of causative HPV infections; nevertheless, most cases positive for high-risk HPV DNA, p16/IHC or combination thereof were also found positive for transcriptionally-active HPV. The prognostic significance of HPV status in SNSCCs remains inconclusive and future studies should investigate the presence of transcriptionally-active HPV by direct HPV testing.

Prediction of toxicities of prostate cancer radiotherapy.

UNLABELLED: We treated a cohort of 116 patients with prostate cancer with three-dimensional conformal hypofractionated radiotherapy to a total dose of 52.8 Gy in 16 fractions (3.3 Gy per fraction). The correlation between acute and late gastrointestinal (GI) and genitourinary (GU) toxicity and dose-volume parameters was analysed. Comparison of observed incidence of toxicity and normal tissue complication probability calculated from dose-volume histograms (DVH) of all patients using radiobiological Lyman-Kutcher-Burman model was performed. The results of our study suggest that acute gastrointestinal toxicity ≥ grade 2 (G2) is the significant predictor of late toxicity ≥ G2 (p=0.006). Observed incidence of acute and late GI toxicities ≥ G2 was 9.7% and 11.5%, respectively. NTCPs of acute and late GI complications ≥ G2 were 11.3% and 2.5%. Observed incidence of late GU toxicity ≥ G2 was 14.2%, NTCP was 0.8%. Comparison of calculated probability of acute GI toxicity ≥ G2 and observed incidence indicates that parameters of radiobiological models are set appropriately. Comparison of observed incidence of late GI and GU complications ≥ G2 and calculated NTCPs shows the need of refinement of LKB model parameters for acute and late GI and GU complications ≥ G2. KEYWORDS: prostate cancer, radiotherapy, acute and late toxicity, radiobiological modeling.

Human papillomavirus and Epstein-Barr virus in nasopharyngeal carcinoma in a non-endemic eastern european population.

Nasopharyngeal carcinoma (NPC) is a rare malignancy in the Czech Republic and Slovakia, with the standardized incidence rate of < 1:100000 person-years. Viral status of NPC in these non-endemic Eastern European regions is currently unknown. In a retrospective study, we evaluated the presence of EBV and HPV in 62 NPC cases. EBV status was determined by the use of in situ hybridization (ISH) for EBV encoded small RNA 1 (EBER1). HPV status was examined with p16 immunohistochemistry, DNA ISH and DNA polymerase chain reaction. Sixty-one studied cases showed non-keratinizing morphology and one was keratinizing squamous cell carcinoma. Only one NPC with non-keratinizing morphology was scored as p16-positive (nuclear and cytoplasmic staining ≥ 70% of tumor cells). This case was positive for high-risk HPV by ISH and the DNA PCR confirmed the presence of HPV18 type. At the same time, this case was found negative for EBV. Remaining sixty-one cases that were scored as p16-negative were all found HPV-negative by ISH and the DNA PCR. EBV was detected in 85.5% (53/62) of cases and 9 cases were EBV-negative, including the case of keratinizing NPC. In contrast with previous reports on the prevalence of EBV-positivity in Caucasian patients with NPC, the majority of patients coming from this non-endemic region show EBV-positivity; therefore, they may be candidates for novel EBV-targeting therapies. Conversely, HPV-positive NPC is very rare and HPV does not seem to play a significant role in the etiopathogenesis of NPC in these Eastern European populations.

Molecular diagnosis of Toxoplasma gondii in pregnant women.

BACKGROUND: Novel approaches in the diagnostics of T. gondii have enabled a detection of the parasite in the amniotic fluid or blood of pregnant women. OBJECTIVE: The high titres of IgM and IgG antibodies against T. gondii are not always indicative of the presence of this parasite in pregnant women, therefore the molecular assays can be used to diagnose and genetically characterise T. gondii in amniotic fluids and blood samples. METHODS: The study analysed 15 samples of amniotic fluid and 1 sample of the blood from pregnant women suspected for toxoplasmosis. The serological ELISA test was used for the immunological study and molecular analyses, PCR at SAG2 locus followed by RFLP analysis were used for Toxoplasma gondii genotyping. CONCLUSION: Using PCR assay with TGR1E gene we have confirmed the presence of T. gondii in the blood of a pregnant woman. The parasite was typed as genotype I, belonging to virulent strains (Tab. 1, Fig. 2, Ref. 30).

Concurrent weekly cisplatin and simultaneous integrated boost intensity-modulated radiotherapy of locally advanced squamous cell carcinoma of the head and neck.

BACKGROUND: Radiotherapy of locally advanced head and neck cancer represents a major clinical challenge. Any treatment intensification aiming at improved treatment outcomes poten-tially results in a higher toxicity. The search for optimal treatment schedule involving conventional or altered fractionation of radiotherapy and the frequency and dose of concomitant cisplatin or other systemic agents has been spanning over several decades. PURPOSE: To evaluate long-term outcomes and toxicity of accelerated chemoradiotherapy of locally advanced squamous cell carcinoma of the head and neck (LA SCCHN). PATIENTS AND METHODS: Forty patients with stage III and IVA (TNM, 7th Ed.) LA SCCHN were treated with accelerated radiotherapy with a total dose of 67.5 Gy in 6 weeks delivered with simultaneous integrated boost intensity-modulated radiotherapy (SIB IMRT) and concomitant weekly cisplatin 40mg/m2. Five-year outcomes and early and late toxicity were evaluated. RESULTS: With the median follow-up of 47.8 months, a 5-year locoregional control rate (LCR) was 56.5%, distant control rate (DCR) was 87% and 5-year progression-free survival (PFS) and overall survival (OS) were 37 and 45%, respectively. Cisplatin cumulative dose of 200mg/m2 was administered in 83% of patients. Grade 2 late toxicity with dietary change was observed in 21 (53%) patients. Human papillomavirus (HPV) status determined by p16 immunohistochemistry was the only significant factor in 5-year treatment outcomes analysis with LCR 100 vs. 41% (P < 0.01), DCR 100 vs. 78% (P = 0.154), PFS 80 vs. 23% (P = 0.01) and OS 80 vs. 34% (P = 0.03) for HPV positive oropharyngeal cancer (OPC) and other HPV negative LA SCCHN. CONCLUSION: High proportion of patients with LA SCCHN received an adequate cumulative dose of concurrent cisplatin with accelerated radiotherapy with SIB IMRT. This study demonstrated that chemoradiotherapy with weekly cisplatin resulted in favorable local control rate and survival in patients with HPV+ OPC.

Radical external beam reirradiation of recurrent head and neck cancer.

BACKGROUND: Recurrent head and neck carcinomas represent a therapeutic challenge for the surgeons as well as for the treating radiation oncologists. Despite advances in the treatment of newly dia-gnosed head and neck carcinomas, relapses occur very frequently and contribute significantly to patient mortality. External beam reirradiation is still considered to be a controversial therapeutic intervention. Historically, in the era of 2- and 3-dimensional conformal radiotherapy, its narrow therapeutic ratio has long led to a general concern that the potential benefit could not sufficiently outweigh the high risk associated with the treatment. The unfavorable therapeutic ratio, however, could be positively influenced by the use of modern techniques of intensity-modulated radiation therapy (IMRT), volumetric-modulated arc therapy (VMAT), stereotactic body radiation therapy (SBRT) and heavy-ion radiotherapy (protons, carbon ions). PURPOSE: The purpose of this review is to create a practical tool for clinical decision-making on the type of patient suitable for reirradiation and the exact type of radiotherapy technique with respect to its expected therapeutic effect, toxicity profile and availability throughout the institutions. In the introductory section, selection of patients is defined with an emphasis on factors that significantly affect overall survival and may be particularly useful in shared decision-making approach within multidisciplinary teams. Furthermore, the text deals with the individual types of external beam radiotherapy techniques (IMRT, SBRT, heavy-ion radiotherapy) with a clear summary of therapeutic outcomes and toxicity from published scientific evidence that had a significant impact on clinical practice.

Dirofilaria infections in working dogs in Slovakia.

A monitoring programme aimed at the diagnosis of subcutaneous dirofilariasis and heartworm disease in working (police and military) dogs in Slovakia has been performed during the period of September 2007 to February 2008. In co-operation with the Ministry of the Interior and the Ministry of Defence, in total, 710 dogs (591 police dogs and 119 military dogs) were investigated for the presence of microfilariae in blood. All police and military dogs in active service held on the territory of Slovakia were included. Microfilariae were detected in 118 (20.0%) police dogs and 10 (8.4%) military dogs. The most infected individuals originated from southern parts of Slovakia (Trnava region 53.6% and Nitra region 39.6%); the prevalence was low in northern regions (Zilina 3.1% and Presov 6.6%). In several districts of southern Slovakia, the prevalence of subcutaneous dirofilariasis in working dogs exceeded 40%. In all infected animals, the autochthonous origin of the disease was confirmed; however, due to the frequent movement of working dogs, it was not possible to identify the exact locality of infection. At present, a dog living in Nemsová village in Trencín district (north-western part of the country) is regarded as the northernmost localized autochthonous case of subcutaneous dirofilariasis in Slovakia. In three dogs, co-infection of Dirofilaria repens and Dirofilaria immitis was detected. High prevalence rates in working dogs and the zoonotic characteristic of the disease represent an undoubtedly important veterinary and medical problem that requires the urgent introduction of prophylactic and control measures.

Neospora caninum, potential cause of abortions in dairy cows: the current serological follow-up in Slovakia.

Neosporosis is considered to be a contributing risk factor for abortions in dairy cows and other farm animals and has negative economic impact on their breeding. In respect of the rapid spread of neosporosis in herds throughout the world, our aim was to detect the prevalence of anti-Neospora antibodies in cows post-abortion (PA) (PA Group n=716) and in cows without any reproduction problems (Control Group n=247) on large breeding farms from Eastern Slovakia. The overall mean seropositivity in PA Group (20.1%) was significantly higher (p<0.0001) in comparison with the Control Group (2.3%), and this demonstrates the causal dependency of abortions on neosporosis. On farms from south-east region, 15.6% of cows were seropositive on average ranging from 7.8% to 25.8% in different districts. In the northern region, significantly (p=0.0002) higher 26.2% mean seropositivity was detected. The seroprevalence in different districts ranged from 2.9% to 39.4%. In the PA Group a high seropositivity to infectious bovine rhinotracheitis (41.5%) and sporadic occurrence of Q-fever, leptospirosis and chlamydiosis was also detected. From 11 Toxoplasma-seropositive cows, only one animal was simultaneously positive to Neospora. The relatively high prevalence rate in cows warrants the attention and a need of surveillance in Slovak herds. A better knowledge of epidemiology of this etiological agent and the mechanisms of its transmission may help in the introduction of more effective preventive and control measures.

Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country.

A sample of 22 Echinococcus granulosus isolates collected from 12 sheep and ten humans from a focus of cystic echinococcosis in western Turkey was examined by DNA sequencing of four mitochondrial genes (cox1, atp6, nad1, rrnS). Results demonstrated the presence of two species of E. granulosus complex, E. granulosus sensu stricto and E. canadensis. Of E. granulosus sensu stricto, the G1 genotype (including three microvariants) was found in 17 isolates from humans and sheep, the G3 genotype and an intermediate form G1/G3 in one isolate each (both from sheep). Of E. canadensis, the pig strain G7 was found in three isolates from sheep and human. This is the first report of this strain in Turkey. Its presence has implications for local control programs due to its shorter maturation rate in dogs compared with E. granulosus sensu stricto. Goat and/or wild boar are likely reservoirs for G7 in the region. We provided further data on the pattern and frequency of nucleotide substitutions within the G1/G3 cluster. Based on our results and GenBank records, G2 (Tasmanian sheep strain) is not considered as a discrete genotypic unit, as its sequences at polymorphic sites conform to microvariants of both G1 and (more often) G3.

First case of cutaneous human dirofilariosis in Slovak Republic.

Dirofilariosis is a zoonotic disease caused by several species of the genus of Dirofilaria. The causative agent initiates a dangerous canine disease reported from many parts of the globe. The parasites are transmitted by arthropods, which act either as a vector or intermediate hosts. In humans the parasites do not usually reach the adult stage but microfilaremia is absent because of mating impossibility. Human dirofilariosis is caused by D. immitis or D. repens and has been reported from many parts of the world including European countries, namely Italy, France, Spain and Greece. Sporadically, this parasitosis is detected in Central European countries such as Hungary and Switzerland. The presented paper reports the first case of human cutaneous dirofilariosis in Slovakia. The clinical manifestation was a typical subcutaneous granuloma with the adult worm in the center. The identification of Dirofilaria repens was made based on morphological appearance of the parasite. The patient 60 years old, lives in the area where in 2005 six cases of canine dirofilariosis caused by the same species were reported (Fig. 2, Ref. 16).

Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes.

Echinococcus multilocularis parasitizes the small intestine of red foxes (Vulpes vulpes) and other carnivores, and has a wide distribution throughout the northern hemisphere. This cestode is the causative agent of human alveolar echinococcosis, a life-threatening helminth zoonosis. In 2000-2002, 2130 red foxes were examined for its presence in Slovakia, with a total prevalence of 30.7%. The data on occurrence were obtained by the combination of necropsy of small intestines from red foxes and coproantigen detection in faecal samples. The correlation between the number of detected specimens and the value of optical density of copro-ELISA test was found. When worm burdens were low (1-25 specimens) the sensitivity of the method was 31.3+/-8.64%, when worm burdens were >50 specimens, 81.8+/-0.66%, and with high worm burdens (>1000 specimens) the sensitivity reached 100+/-0.34%. E. multilocularis presence was detected using the nested PCR method from the eggs in the faecal samples with a 100% specificity. In epidemiological surveys of this zoonosis, it is of crucial importance to detect animals with a high level of infection, which are responsible for the bulk of environmental contamination. The advantage of copro-ELISA test lies in allowing the intravital diagnostics to be employed within the epidemiological survey of E. multilocularis occurrence in the protected and urban areas.

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus.

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.

Toxocara canis: ultrastructural aspects of larval moulting in the maturing eggs.

The morphology of the surface of Toxocara canis larvae, developing in the eggs to reach infectivity, has been studied for the first time at an electron microscopical level. In most 11-day and some 15-day eggs, the larvae are surrounded by two shed cuticles. The outer first shed cuticle is composed of two layers. the inner second shed cuticle is much thicker than the outer one. The presence of both shed cuticles indicates that the larva has undergone two developmental stages in the maturing egg. The larvae in most 15-day eggs are surrounded by one shed cuticle composed of outer electron-dense and inner layers. This cuticular sheath is identical with the described inner second shed cuticle, except for its apparently reduced thickness. The infective larvae inside the 30-day eggs are enveloped by one cuticular sheath, derived from the second moulted cuticle, and consisting only of a single layer. The findings are discussed with respect to data concerning the moulting process in other nematode species.

Prevalence of echinococcosis in pigs and sheep in the Slovak Republic.

The prevalence of echinococcosis in slaughtered animals has been monitored in the Slovak Republic between 1971 and 1990. In pigs, the prevalence dropped almost constantly from 3.85% to 0.13%, by as much as 15% per year. The dynamics of prevalence in sheep differs substantially from that in pigs. Significant 12 year, 6 year and 3 year periodicities predominate over a nonsignificant and slight decrease of prevalence by only 1% per year. Therefore, the prevalence fluctuates in the period of observations between 0.49 and 3.72%. No significant correlations or cross-correlations have been found between the prevalences in the two groups of animals.

Circulation of Toxocara spp. in suburban and rural ecosystems in the Slovak Republic.

Toxocara spp., the common roundworms of domestic and wild animals, are the causative agents of larval toxocarosis in humans. The migration of Toxocara larvae in men causes clinical syndrome, called larva migrans visceralis or larva migrans ocularis. The objective of the present work was to investigate the prevalence of toxocarosis in dogs, red foxes (Vulpes vulpes), and small mammals in the Slovak Republic. T. canis infection was diagnosed in 16.6% out of 145 dogs examined. Young animals showed significantly higher positivity (50.0%) than adults (12.4%). Coprological investigation of 310 red foxes showed 8.1% prevalence of T. canis, with higher occurrence in animals from rural conditions (8.6%) than from suburban environment (5.6%). Out of 710 small mammals examined, anti-Toxocara antibodies were detected in 7.7% of the animals. The most frequently seropositive species was Apodemus agrarius (15.9%). Seropositivity of small mammals from suburban localities was higher (10%) compared with rural areas (5.8%), with adult animals exhibiting higher seropositivity (8.0%) than subadults (6.8%). Our results have confirmed the importance of dogs, red foxes and small mammals in circulation of this serious helminthozoonosis in suburban and rural ecosystems.

Some precursors of glycogen in Ascaris suum females.

Intensity of incorporation of the labelled carbon from glucose, glucosamine and leucine into the glycogen of somatic cells and reproductive organs of Ascaris suum females has been studied. Glucose was most intensively incorporated into the growing oocytes and into the uterine epithelial cells, whereas its incorporation into muscle glycogen was found to result in lowest rates. Radiocarbons from glucosamine and leucine were incorporated into tissue glycogen of female worms much less than glucose. Incorporation of isotopes into glycogen of the eggs depended on the formation of their primary shells. The ways of incorporation of isotopes into glycogen of the eggs after formation of all the shell layers are discussed.

Inhibition of lymphoproliferative response and its restoration with a glucan immunomodulator in mice with experimental larval toxocarosis.

A high infective dose of Taxocara canis eggs (2,500 eggs per mouse) induced a partial immunosuppression in mice, manifested by inhibition of the proliferative response of splenic T and B cells to polyclonal activators. A glucan immunomodulator given to infected animals at the beginning of the experiment showed a marked stimulative and restorative effect on the parasite-suppressed lymphoproliferative response. The ability of T. canis to migrate in the host was reduced in glucan-treated animals by 27%.

Comparison of the sensitive screening kit with two ELISA sets for detection of anti-Toxocara antibodies.

Three serological tests (ELISA NOVUM, ELISA PU and ToxocaraCHEK) were compared using excretory-secretory (ES) antigen of second stage larvae of Toxocara canis on two sets of sera from patients aged 1-15 years, living in town and in the country, suspected of having larval toxocarosis. Of 60 serum samples examined for the presence of anti-Toxocara IgM and IgG antibodies 35 (58.3%) were positive with ELISA NOVUM, 30 (50.0%) with ELISA PU and 37 (61.6%) with ToxocaraCHEK. A significantly higher seropositivity with all the tests was detected in group of patients from the village (test chi2, p < 0.005) when compared with patients from the town. The highest sensitivity (100%) was detected for a rapid qualitative ToxocaraCHEK. Compared with this screening test, both ELISA sets showed higher specificity but a lower sensitivity. Consistent findings (+;-) with ELISA NOVUM and ELISA PU were 88.3%; ELISA NOVUM and ToxocaraCHEK 96.7% and ELISA PU and ToxocaraCHEK 86.7%. This comparison indicates the suitability of ToxocaraCHEK set for screening for its simplicity and rapidity in detecting anti-Toxocara antibodies.

Estimating the genetic divergence and identification of three Trichinella species by isoenzyme analysis.

Isoenzyme-based approach was applied to compare Trichinella spiralis, T. britovi and T. pseudospiralis species. Among 13 enzyme systems examined, esterase (EST), malic enzyme (ME) and phosphoglucomutase (PGM) have been found as fully diagnostic, with no common allele in species studied. Adenosine deaminase (ADA), adenylate kinase (AK), hexokinase (HK), peptidase leucyl-alanine (PEP-C) and fructose-bis-phosphatase (FBP) have been capable of distinguishing the two species from resulting profiles. In addition, ADA, AK and PGM displayed the enzyme expression in the lowest amounts of muscle larvae in systems tested (100 larvae/100 microliters of extracts). Based on allozyme data, T. pseudospiralis has been found as the most distinct species within the group of taxa. Only a subtle genetic variability was recorded for T. pseudospiralis in which solely phosphoglucomutase exhibited variant patterns. In addition to the study of reference isolates, T. spiralis from lowland fox in Eastern Slovakia has been evidenced by use of genetic markers. This finding has proved that T. britovi is not the exclusive species parasitizing in the sylvatic ecosystem of the Slovak region.

Trichinellosis in the Slovak Republic.

The occurrence of trichinellosis in farm or wildlife animals in some areas pose the permanent risk of infection in humans. In Slovakia, where only the sylvatic trichinellosis exists, the humans trichinellosis is sporadic. The most common cause of infection is wild boar meat. Unusual local eating customs may also favour an outbreak of disease in humans. The outbreak of trichinellosis in Slovakia in 1998, when 336 people were affected, was of the same kind. Trichinella britovi was the agent of the infection.