Potential application of body fluids autofluorescence in the non-invasive diagnosis of endometrial cancer.

BACKGROUND: Endometrial carcinoma (EC) is the most common cancer of the female reproductive tract in developed countries. The prognosis and 5-year survival rates are closely tied to the stage diagnosis. Current routine diagnostic methods of EC are either lacking specificity or are uncomfortable, invasive and painful for the patient. As of now, the gold diagnostic standard is endometrial biopsy. Early and non-invasive diagnosis of EC requires the identification of new biomarkers of disease and a screening test applicable to routine laboratory diagnostics. The application of untargeted metabolomics combined with artificial intelligence and biostatistics tools has the potential to qualitatively and quantitatively represent the metabolome, but its introduction into routine diagnostics is currently unrealistic due to the financial, time and interpretation challenges. Fluorescence spectral analysis of body fluids utilizes autofluorescence of certain metabolites to define the composition of the metabolome under physiological conditions. PURPOSE: This review highlights the potential of fluorescence spectroscopy in the early detection of EC. Data obtained by three-dimensional fluorescence spectroscopy define the quantitative and qualitative composition of the complex fluorescent metabolome and are useful for identifying biochemical metabolic changes associated with endometrial carcinogenesis. Autofluorescence of biological fluids has the prospect of providing new molecular markers of EC. By integrating machine learning and artificial intelligence algorithms in the data analysis of the fluorescent metabolome, this technique has great potential to be implemented in routine laboratory diagnostics.

NK cells not only in endometrium but also in ovulatory cervical mucus in patients with decreased fertility.

OBJECTIVE: An increased number of NK cells is associated with autoimmune disorder and is known to play a role in infertility. The aim of our research was to monitor the density of NK cells CD56+ and CD16+ in ovulatory cervical mucus (OCM) and in endometrium in infertile women as well as in connection with the actual status of antibodies against phospholipids, sperm and HHV-6 antibodies. TYPE OF STUDY: Original aticle. SETTING: Genetika - Plzeň. METHODS: Seventy-two randomly selected women aged 20-39 (mean age: 32.3) years old resulted in fifty-seven patients with repeated unexplained miscarriages, and fifteen fertile healthy women. The hormonal status was studied including ovulation, the humoral autoimmune responses to eight phospholipids, trombophilia, karyotyping, hysteroscopy, and endometrium immunohistology. Patients were without any clinical and laboratory symptoms of vaginitis at the time of OCM sampling and endometrium study. In one patient antiphospholipid syndrome was present, and in one woman diabetes mellitus was identified. Uterine NK cells CD56+ , CD16+ and NK cells in OCM were identified by immunocytochemistry, antiphospholipid antiboides by ELISA. We used indirect MAR-test for study of local spermagglutinating antibodies in OCM. Indirect immunofluorescent method was used for detection of serum and OCM IgM, IgG antibodies against HHV-6 levels at the time of ovulation. RESULTS: We found both high density of NK cells CD56+ and CD16+ in OCM and in endometrium in only two infertile women with repeated abortions. NK cells in OCM were missing in other samples of patients. The prevalence of high density of NK cells CD56+ in the endometrium was seen in twenty three (40%), NK cells CD16+ in eleven (19%), NK cells 56+ and NK cells 16+ together in eight (14%). Levels of serum and OCM IgG against HHV-6 in all examined patients were not elevated, no cervical sperm antibodies were found. CONCLUSION: We compared density of NK cells CD56+ and CD16+ in OCM and secretory endometrium in all infertile patients. Our results show that cell mucosal activity in the cervical area at the time of ovulation in two infertile patients was evident. We excluded the abnormal number of NK cells owing to local and general viral infection (HHV-6). But our question still remains - are cervical NK cells fixed or still migrating from endometrium into OCM? New research is planned.

[Enterovirus sequencing as a new approach to the laboratory diagnosis for clinical and epidemiological purposes]. / Sekvenace enteroviru jako nový prístup k laboratorní diagnostice pro klinické i epidemiologické úcely.

OBJECTIVE: Introducing enterovirus sequencing as an advanced approach to classify the viruses isolated according to the novel nomenclature and to characterize isolates in detail. MATERIAL AND METHODS: Seventy-five specimens collected from 64 patients in two hospitals, Liberec Regional Hospital, and Plzen University Hospital, were analyzed. The study patients' age ranged from four to 54 years, with a median of 15 years in males and 16 years in females. In most patients, the reasons for admission were intense headache, fever, vomiting, tiredness, meningeal symptoms, intestinal symptoms (in two patients), and skin symptoms (in one patient). The specimens collected were rectal and throat swabs, cerebrospinal fluid (CSF) and stool specimens. Molecular detection and typing were performed using the RT-PCR method. A segment of the 5´non-coding RNA was selected for typing. Specimens were amplified using single-step PCR with external primers and with the same primers extended to include M13 sequences (Generi-Biotech). The LASERGENE software (DIASTAR) was used in sequence editing, alignment, and quality check. The sequences obtained were checked against the central GenBank sequence database using the BLAST algorithm. RESULTS: The identification of the study isolates resulted in 61 ECHO viruses 30, three coxsackie viruses B1, one coxsackie virus B3, one coxsackie virus A9, one enterovirus 86, one enterovirus 71, Two ECHO viruses 13/coxsackie virus B5, one ECHO virus 7/30/coxsackie virus B4, one coxsackie virus B4/enterovirus B, one enterovirus 87/ECHO virus 30/enterovirus B, and one ECHO virus 3. All viruses isolated, except enterovirus 71 classified into group A, were of group B. CONCLUSION: The enteroviruses were identified unambigously, although the sequencing only targeted a short, conserved segment that showed considerable variability. The sequencing was an effective alternative to enterovirus identification by the neutralisation test and allowed for detailed characterization of the isolates. The predominance of ECHO 30 as the cause of aseptic meningitis is in accordance with the literature data.

[Cytological-energetic principle of the amniotic fluid examination on female patients with diagnosis of preterm premature rupture of membranes - up to now experiences and perspectives]. / Cytologicko-energetický princip vysetrování plodové vody po predcasném predtermínovém odtoku - dosavadní zkusenosti a perspektivy.

In our study we have dealt with the amniotic fluid examination on female patients with diagnosis of preterm premature rupture of membranes (PPROM). For one of the most important task in the feto-maternal medicine we consider the discovered method of amniotic fluid examination which is capable of the earliest possible detection in an intraamniotic inflammatory response. Unnoticed could harm or in the most serious cases threat the life of fetus or its mother. This was also the main reason why we chose this topic for our study. We have not been interested only in already known interleu-kin-6 (IL-6), but we have approached the examination comprehensively. We used the cytological-energetic principle supplemented by the examination of aspartate-amino-transferase (AST), in which already are long-term experiences in investigating other extracellular fluids. We have monitored values of C-reactive protein (CRP) and leucocytes in maternal serum, IL-6, AST and energetic score ratio (KEB) in amniotic fluid. Further we have investigated which imunocompetent cells have been dominant in amniotic fluid at different types of inflamantory reaction. According to the results of the examination of the control groups, it was necessary to correct the KEB limit in comparison to KEB limits of other extracellular fluids. Although our study includes untill now only 44 patients - 21 controls and 23 investigated persons, we have been already able to find out some trends of changes in observed parametres. As a very impotant discovery we consider the fact, that KEB, AST and cytological examination of amniotic fluid seem to be the methods for prediction of chorioamnionitis as reliable as the examination of IL-6. These methods are besides less expensive and therefore more suitable for the use in the small hospitals and developing countries. We continue in our study to confirm the previously found trends on the larger numbers of patients.

[Mumps outbreak in the Plzen Region in 2011]. / Epidemie príusnic v Plzenském kraji v roce 2011.

AIM: The mumps outbreak in the Plzen Region in 2011 was analysed retrospectively using the epidemiological, clinical, and laboratory data. Vaccine efficacy analysis was also conducted in various population groups. MATERIAL AND METHODS: The routine procedure and standard form were used by the epidemiologists to collect data on the age, sex, place of residence, presence in collectivities, date of disease onset, type of complications and date of their onset, hospital admission, vaccination, and results of laboratory analyses. Based on the records of general practitioners for children and adolescents, data on the vaccination of children born in the previous three years have been provided to the epidemic control departments every year by 30 June since 1989. To estimate the vaccination coverage rate, the numbers of single-dose or two-dose recipients are related to the number of children registered in a given year. The first year of vaccine recipients were children born in 1986 who were aged 25 in 2011. The data collected on the population of the Plzen Region were used for the primary analyses. To estimate the efficacy of the mumps vaccine, age and vaccine coverage cohort analysis was performed using the screening method. To analyse categories, the chi-square test with Yates correction was applied at a significance level of p = 0.05 % (EPIINFO version 6.04d). RESULTS: In 2011, 721 mumps cases were reported in the Plzen Region (incidence: 126.1 cases per 100 000 population). The average patient age was 19.4 years, with a median of 18 years (age range 1-77 years). Four hundred and seventeen (57.8%) patients were males. Biological specimens from 375 (52.0%) patients were investigated serologically in the virology laboratory and mumps were laboratory confirmed in 316 (43.8%) of them - in 222 patients, one blood specimen was analysed. The most afflicted area was the Klatovy district with the incidence of 449.3/100 000 population. The most affected age group were 15-19 year-olds with the incidence of 1008.2/100 000 population. Forty-two (5.8%) patients were diagnosed with complications and 68 (9.4%) patients were admitted to the hospital. No statistically significant difference was found in the incidence of complications between the vaccinated and non-vaccinated patients. The assessment of hospitalisation risk showed a statistically significant difference between the vaccinated and non-vaccinated persons in both the whole study cohort and subcohort of patients who received the mumps vaccine within the childhood immunization schedule (p = 0.000 and p = 0.004, respectively). The average age of the vaccinated patients was 16.1 years (median of 17) and that of the non-vaccinated patients was, 29.3 years (median of 27). At the six to ten-month interval, as laid down by the legislation, the second dose of the vaccine was only given to 83.6 % of the patients. The screening method showed a high efficacy of the vaccine in the age group 20-25 years (97.6% in two-dose recipients), declining to 29.6% in the most affected age group of 15-19 years. CONCLUSIONS: Mumps outbreak may has been a result of the accumulation of high numbers of susceptible individuals in the population. The most affected area was the Klatovy district where the last epidemic outbreak occurred 23 years ago. To make the favourable epidemiological situation with occasional local outbreaks continue, the high two-dose vaccine coverage rate needs to be maintained.

The diagnosis of norovirus infections in hospitalized children and adolescents with acute gastroenteritis: a study from Pilsen, Czech Republic.

BACKGROUND: Norovirus-associated sporadic gastroenteritis is an important cause of illness in Western Europe. However, at present, little information on the role of norovirus in sporadic gastroenteritis in Central Europe is available. Our study aimed at providing an assessment of their significance in hospitalized children and adolescents with acute gastroenteritis using ELISA test at the time of their introduction. METHODS: A prospective hospital based study of the etiology of acute gastroenteritis was undertaken in a total of 618 patients (mean age 39.8 months, range 0-228), who were hospitalized at the Charles University Hospital in Pilsen. All subjects were monitored in six fragmented periods during the years 2003 and 2004, 2006 and 2007. Clinical and laboratory data were processed, norovirus antigens in stools were detected using the EIA kits IDEIA Norovirus, DakoCytomation. RESULTS: A norovirus infection was confirmed in 62 cases, i.e., 10.0% of all patients with acute gastroenteritis. Vomiting was the most common symptom, recorded in 95.2 % of all the patients with norovirus infection. No severe extragastrointestinal complications were detected. The average interval between initial symptoms and the beginning of hospitalization was considerably shorter in patients with norovirus infection (1.3 days) compared with patients with rotavirus infections (2.4 days). The frequency of Salmonella spp., rotavirus, Campylobacter spp. and enteric adenovirus was 15.4%, 11.2%, 3.9%, 3.6%, respectively. CONCLUSIONS: Our findings confirm the clinical importance of noroviruses as a causative agent of acute gastroenteritis in children and teenagers in the region of a Central European country. Identification of norovirus infection should be included in the routine screenings of sporadic cases of acute gastroenteritis.

Valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation.

Both preemptive therapy and universal prophylaxis are used to prevent cytomegalovirus (CMV) disease after transplantation. Randomized trials comparing both strategies are sparse. Renal transplant recipients at risk for CMV (D+/R-, D+/R+, D-/R+) were randomized to 3-month prophylaxis with valacyclovir (2 g q.i.d., n = 34) or preemptive therapy with valganciclovir (900 mg b.i.d. for a minimum of 14 days, n = 36) for significant CMV DNAemia (>/=2000 copies/mL by quantitative PCR in whole blood) assessed weekly for 16 weeks and at 5, 6, 9 and 12 months. The 12-month incidence of CMV DNAemia was higher in the preemptive group (92% vs. 59%, p < 0.001) while the incidence of CMV disease was not different (6% vs. 9%, p = 0.567). The onset of CMV DNAemia was delayed in the valacyclovir group (37 +/- 22 vs. 187 +/- 110 days, p < 0.001). Significantly higher rate of biopsy-proven acute rejection during 12 months was observed in the preemptive group (36% vs. 15%, p = 0.034). The average CMV-associated costs per patient were $5525 and $2629 in preemptive therapy and valacyclovir, respectively (p < 0.001). However, assuming the cost of $60 per PCR test, there was no difference in overall costs. In conclusion, preemptive valganciclovir therapy and valacyclovir prophylaxis are equally effective in the prevention of CMV disease after renal transplantation.

G-serotypes of group A rotaviruses in Pilsen region (Czechia).

A total of 260 feces samples from children with confirmed rotavirus infection collected during 1999-2002 were serotyped, using enzymoimmunoassay with VP7 specific monoclonal antibodies for G1-G4 serotypes. The serotypes were identified in 185 feces, i.e. 71.2 %. Individual serotypes occurred in 43, 2, 16 and 2 %; 8 % samples reacted with 2 type-specific monoclonal antibodies. The G1 serotype was prevalent over the whole period. The G3 type occurred with a statistically higher significance in children of up to 36 months (chi2 = 4.6, p = 0.028). In 4 children a different serotype was demonstrated in the first and second, or in the second and third stools, respectively. No dominant serotype was found in children with nosocomial infection.

First experience with diagnosing astroviral infections in children hospitalized in Pilsen (Czechia).

Between the years 2000-2002 astroviral infection was confirmed in 0.8 % of 2606 children admitted for a diarrheal disease to the University Hospital in Pilsen (Czechia). Astrovirus infections most frequently occurred in the age group between 49-60 months, and 77.3 % of the infections occurred in winter. When the incidence was calculated for these most susceptible age groups, the positive rates were 2.4 % (between 49-60 months) and 1.2 % (between 7-12 months) respectively. On average the hospitalization lasted for 5 d, the diarrhea 3.7 d, vomiting 1.1 d, and 40.9 % of infected children had a raised temperature. In 7 out of 872 stool samples (0.8 %), astrovirus antigen has appeared in the course of the monitored period, i.e. nosocomial astrovirus infection was recorded. In view of the rarity of detecting astroviral infections it is probably not effective to include the diagnosis of these infections in the routine panel when examining the children admitted for gastrointestinal infections. The diagnosis of these infections should, however, be performed within the framework of epidemic incidence and in nosocomial infections.

[Diagnosis of rotavirus infections--comparison of various methods]. / Diagnostika rotavirových infekcí--porovnání ruzných metod.

In 179 samples of faeces from patients hospitalized with diarrhoeal diseases rotavirus infections were examined concurrently by three different sets--the latex agglutination test Diarlex Rota-Adeno (Orion Diagnostica) and two immunoenzyme tests (EIA)--EIA Rotavirus (Test-Line, CR) and IDEIA Rotavirus (Dako Ltd.) By the latex agglutination reaction the rotavirus infection was proved 31 times (17.3%), by the Test-Line test 87 times (48.6%), by the Dako test 96 times (53.6%). During the interval of 0-3 days after the onset of the disease the positivity assessed by different tests was 22.8%, 48.6% and 54.3%, on the 4th-7th day 12.8%, 51.1% and 57.4%, on the 8th and subsequent days 3.7%, 44.4% and 44.4%. Comparison of sets Orion Diagnostica and Dako revealed the sensitivity of the latex agglutination test only as 30.2% and specificity 97.6%. After the 9th day from the onset of the disease the infection was no longer confirmed by the latex agglutination test, while both EIA tests proved the rotavirus antigen up to the 16th day from the onset of the disease.

[Monitoring cytomegalovirus infection using the 2-step nested polymerase chain reaction in patients with allogenic bone marrow transplantation]. / Monitorace aktivity cytomegalovirové infekce u nemocných po alogenní transplantaci kostní drene metodou dvoustupnové "nested" polymerázové retezové reakce.

Cytomegalovirus infections and their sequelae are the most serious complications in patients after allogenic bone marrow transplantations. Therefore in recent years quite rightly attention is paid to new diagnostic methods which make earlier and more sensitive detection of incipient CMV infection possible. The method of the two-stage polymerase chain reaction (PCR) was used for early detection of CMV infection and its possible further monitoring in 38 recipients of allogenic bone marrow. A positive result was recorded in 15 (39%) patients whereby in 10 (13%) repeated positivity was involved. In three in this group of patients (8%) CMV disease developed, always preceded by PCR positivity. Sensitivity, specificity, positive and negative predictive CMV PCR values were 100, 66, 20 and 100%. Concomitant comparison with serological examination (antibody titre class IgM/seroconversion) revealed the small contribution of serology (corresponding values are 67, 54, 11 and 95%) in patients after bone marrow transplantations. The presented results provide evidence of the contribution of PCR in the diagnosis of CMV in those patients where due to their high sensitivity and excellent predictive value it is possible to avoid the toxicity of unnecessary antiviral treatment.

[Herd immunity and the role of Chlamydia pneumoniae in the etiology of respiratory diseases in the Czech Republic]. / Promorenost a úloha Chlamydia pneumoniae v etiologii respiracních onemocnení v Ceské republice.

The objective of the investigation was to assess the seroprevalence of IgG in the population of the Czech Republic and to assess the etiological role of Chlamydia pneumoniae in respiratory diseases. The sera were examined by specific tests-genus by ELISA and species by the microimmunofluorescent test. The examination of sera from 1974-1975 for the serological survey was selected to make the results comparable with the results of examinations planned for 1996. Chlamydia IgG were detected already in the age group from 1-4 years with a marked rise in youth, and in adult age the seroprevalence was between 72.0 and 86.2% (mean 80.1%). In the seropositivity of Chlamydia IgG participated mainly immunoglobulin C. pneumoniae, which was present already in child and school age (seroprevalence 7.7-88.2%), culminating in adult age (92.0-100%). IgG C. trachomatis were detected practically only in adult age, IgG C. psittaci were found only rarely in serum. In 16 subjects (of 216 examined) serological tests revealed acute (in 27 suspect), in 6 subjects recent (in 17 suspect) infection with C. pneumoniae and 53 to 63 subjects had the infection in the past.

[Serologic overview of rotavirus antibodies in children under 2 years of age]. / Sérologický prehled rotavirových protilátek u detí do dvou let veku.

A serological investigation in 120 children under two years confirmed the elevated prevalence of rotavirus antibodies after the age of nine months. In children above one year antibodies were detected in 30%. The results are consistent with those of similar studies where different laboratory methods were used.

[Long-term follow-up of nosocomial rotavirus infections at the Infectious Diseases Clinic of the Medical School Hospital in Plzen (1987-1994)]. / Dlouhodobé sledování nozokomiálních rotavirových infekcí na Infekcní klinice Fakultní nemocnice v Plzni (1987-1994).

The authors evaluated in 1987-1994 the incidence of nosocomial rotavirus infections during hospitalization in 1718 junior children, 450 senior children and adults admitted with diarrhoeal diseases. Rotavirus infection was revealed in 6.2 and 1.3% of the patients resp. During hospitalization the rotaviruses were the most frequent causal agent of nosocomial infections. Hospital infection was contracted regardless of the initial diagnosis most frequently by patients aged 0-12 months. The majority of nosocomial rotavirus infections was associated with symptoms of diarrhoeal disease, on average symptomatic infections prolonged the hospitalization period by 4.2 days. The authors discuss the possibility to influence the incidence of these infections.