Reduction of Thymoglobuline from 7.5 mg/kg to 6 mg/kg in conditioning regimen extended time to the first cytomegalovirus detection after allogenic haematopoietic stem cell transplantation.

INTRODUCTION: The optimal dosage of anti-thymocyte globulin (ATG) may influence the outcome of patients after allogenic haematopoietic stem cell transplantation (HSCT). The aim of our study was to analyse human cytomegalovirus (CMV) infection data, incidence of graft-versus-host disease and other clinical endpoints comparing two patients cohorts that were administered two different Thymoglobuline Genzyme doses as part of the HSCT conditioning regimen. MATERIALS AND METHODS: Total of 65 adult patients received ATG (7.5 mg/kg or 6 mg/kg) as a part of the fludarabine/busulfan/ATG conditioning regimen. CMV DNAemia was monitored after HSCT using quantitative real-time PCR and preemptive treatment was started for viral loads above 1000 cp/ml. RESULTS: The mild ATG dose reduction extended the time to the first CMV detection after transplantation (28 days for 7.5 mg/kg dose vs. 40 days for 6 mg/kg dose, p = 0.04). But it did not reduce the incidence or influence first anti-CMV treatment onset, the initial viral load, peak viral load in whole blood or the antiviral therapy parameters (all p 0.18). No impact of ATG dose reduction on incidence of graft-versus-host-disease, relapse of underlying disease or mortality within first year after transplantation (all p 0.32) were observed. CONCLUSIONS: The reduced ATG dosages can allow lower toxicity of conditioning regimen while keeping the performance.

Diagnosis and treatment of Bartonella endocarditis.

The Bartonella genus comprises more than 20 species of Gram-negative rods which are difficult to culture. These are facultative intracellular bacteria. Humans are reservoir hosts for B. quintana and B. bacilliformis or accidental hosts for other species. Bartonella is a cause of zoonosis. Bartonella infection can be completely asymptomatic or can be linked to various conditions. Our experience with Bartonella endocarditis from 2012-2017 is presented. The most effective diagnostic method for Bartonella endocarditis is PCR detection of DNA of the pathogen from excised valve tissue. The European Society of Cardiology (ESC) in the guidelines from 2015 recommends the combination doxycycline gentamycin for the treatment of Bartonella endocarditis.

[Nonculture techniques of microorganisms determination in amniotic fluid in patients with preterm prelabor rupture of membranes]. / Nekultivacní stanovení mikroorganismu v plodové vode u pacientek s predcasným odtokem plodové vody.

OBJECTIVE: The preterm prelabor rupture of membranes is a serious obstetric complication that is frequently complicated by the presence of microorganisms in amniotic fluid. The aim of our work is to characterize current status of nonculture detection of microbial invasion into the amniotic cavity and the experience with the technique performed in University Hospital in Hradec Kralove. DESIGN: Original survey article. SETTING: Institute of Clinical Biochemistry and Diagnostics - molecular biology department, University Hospital Hradec Kralove. CONCLUSION: Application of nonculture techniques of microorganisms determination in amniotic fluid in patients with preterm prelabor rupture of membranes is currently available. According to the detection of genital mycoplasmas as the dominant pathogens in the amniotic fluid this technique should be regarded as the standard examination method in these patients.

[Ganciclovir treatment failure in adult allogeneic hematopoietic stem cell transplant recipients with cytomegalovirus infection--a single centre experience]. / Klinická rezistence lidského cytomegaloviru pri lécbe gancyklovirem u pacientu po alogenní transplantaci hematopoetických bunek--zkusenosti jednoho centra.

OBJECTIVE: To determine the incidence of infection with ganciclovir-resistant cytomegalovirus (CMV) in adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. Clinical resistance or treatment failure was defined as persistent DNAemia or increasing viral load in peripheral blood after 2 weeks of virostatic treatment. The association between the treatment failure and viral resistance was analysed. The presence of ganciclovir-resistant CMV strains was confirmed by genotypic testing able to detect mutations conferring resistance. METHODS: In 2012 and 2014, 40 patients who underwent allogeneic HSCT for hematologic malignancies and were treated for human CMV reactivation/disease were followed up prospectively. In patients with treatment failure, CMV DNA was isolated and analysed by nucleotide sequence analysis of the UL 97 and UL 54 genes conferring resistance to the virostatic agent. RESULTS: The treatment failure occurred in seven patients, but ganciclovir resistance conferring mutations were only detected in two of them (mutations L595F and M460I in the UL 97 gene). Another mutation in the UL 97 gene (N510S) was found in a patient with recurrent CMV replication who needed to be retreated but did not meet the criteria for treatment failure. CONCLUSION: The low incidence of genetically confirmed ganciclovir-resistant CMV isolates in HSCT recipients with relatively common clinical treatment failure suggests that the mechanism underlying slower viral clearance is often other than mutations conferring ganciclovir resistance to the virus.

Influenza in seasons 2009-2013 in the Faculty Hospital Hradec Kralove, East Bohemia.

PURPOSE: The evaluation of four post-pandemic influenza seasons 2009-2013 in the Faculty hospital Hradec Králové and comparison of used rapid antigen tests (RATs) with real time RT-PCR. MATERIAL AND METHODS: Between November 2009 and June 2013 were examined 3845 samples from patients with respiratory tract infections by RATs (Influenza A/B 2 Panel Test (GECKO® Pharma, Germany), Rapid VIDITEST Influenza A+B Card (VIDIA®, Czech Republic) and BinaxNOW Influenza A&B Test (ALERE®, USA) or real time RT-PCR (RTR InfA/H1N1 Detection Set (Roche®), RealStar® Influenza S and T RT-PCR Kit 3.0 (Altona ®, Germany). RESULTS: A totally 1059 samples were examined simultaneously by RAT and real time RT-PCR. The overall sensitivity and specificity of RATs compared with real time RT-PCR were 32,2 % and 98,1 % for influenza A and 17,6 % and 99,4 % for influenza B. Higher sensitivity of RATs were in children (66,6 %) compared with adults (14,3 - 40,0 %). In the first three post-pandemic seasons were continuously decrease of positive samples from 23,5 % in season 2009-2010 to 3,3% in season 2011-2012, but in season 2012-2013 were rapidly in-crease of positive results, to 31,5%, with high share of influenza -A/H1N1/2009 (79,6%). CONCLUSION: Our results shown insufficient sensitivity of all used RATs and necessity of having other confirmatory test, like RT-PCR. It was also shown unexplained increase of case and influenza severity in season 2012-2013.

Viral gastroenteritis in Eastern Bohemia Region of the Czech Republic.

PURPOSE: Acute gastroenteritis is one of the most common diseases in humans worldwide and represents a significant cause of mortality and morbidity. The majority of cases are of viral aetiology, evidence for which has been increasing in the past decade. Several studies on the prevalence in European countries of viral aetiology of gastroenteritis have been published in the last decade, but none from the Czech Republic. MATERIAL AND METHODS: In total 107 faeces samples obtained from patients hospitalised in the University Hospital in Hradec Králové were examined by immunochromatographic tests using ROTA-ADENO Card Rapid-Viditest (VIDIA, Czech Republic) and RidaQuick Norovirus (R-Biopharm, Germany), and by an in-house Real-time PCR panel. RESULTS: Overall findings of viruses detected by PCR in the tested faeces samples were: rotaviruses in 29.9%, noroviruses in 14.0% and adenoviruses in 5.0%. Immunochromatographic antigen detection performed at lower sensitivity compared with PCR: rotaviruses in 28.0%, noroviruses in 4.7% and adenoviruses in 2.0%. Our findings demonstrate even lower sensitivity of the used immunochromatographic tests compared with manufacturers data. CONCLUSION: Our study has revealed limitations in immunochromatographic tests, especially in their sensitivity and the necessity for using another confirmatory method. We have set up real-time PCR in routine diagnosis of viral gastroenteritis in our hospital.

[Evaluation of the importance of a ready-made, gentamicin-impregnated spacer in relation to bacteriological findings in patients with periprosthetic joint infections]. / Zhodnocení významu ready-made spaceru s gentamicinem ve vztahu k bakteriologickým nálezum u pacientu s infekcí kloubní náhrady.

BACKGROUND: Periprosthetic infection is a serious complication in total hip and knee arthroplasty. The complex therapeutic approach within two-stage reimplantation includes the use of antibiotic-impregnated spacers (temporary joint replacements). The aim of this paper was to evaluate bacteriological findings in selected patients with periprosthetic infection in whom a ready-made gentamicin-impregnated spacer was used to treat the infection. MATERIALS AND METHODS: Between 2008 and 2012, a ready-made, gentamicin-impregnated cement spacer was used in 24 patients to treat periprosthetic hip or knee infection within two-stage reimplantation. All components of the prosthesis and periprosthetic tissue samples were sent for microbiological examination at the first revision surgery, while at the second revision surgery, the spacer and surrounding tissue samples were sent in. In six patients with an inserted knee spacer, the level of gentamicin in the joint fluid was measured. Subsequently, the patients were regularly monitored. RESULTS: Twenty-two (92%) of 24 patients were bacteriologically positive by culture. The most commonly detected causative agents were coagulase-negative staphylococci. Other isolates were Staphylococcus aureus, Corynebacterium, anaerobic bacteria, and Salmonella serotype Enteritidis. Nineteen (76%) of 25 primary pathogens were gentamicin sensitive. Spacers from two patients were culture positive for coagulase-negative staphylococci that tested resistant to gentamicin. During the follow-up of at least two years, none of the patients developed another periprosthetic infection. CONCLUSION: The success rate of two-stage-reimplantation total hip and knee arthroplasty using ready-made, gentamicin-impregnated spacers was 100 % in our cohort of patients; no other periprosthetic infection was reported during the follow-up of at least two years. From the bacteriological results, it appears that the ready-made, gentamicin-impregnated spacer only covers 76 % of the range of the causative agents. The solution would be to use a spacer impregnated with a combination of vancomycin and gentamicin that would be effective against all cultured species.

[The use of IgG antibody avidity assays in the diagnosis of cytomegalovirus infection]. / Vyuzití testu avidity IgG protilátek v diagnostice cytomegalovirové infekce.

Human cytomegalovirus (CMV) is the most common cause of congenital infection. Primary CMV infection can lead to severe disease and complications in patients immunocompromised as a result of disease or therapy. IgG antibody avidity assays make it possible to differentiate between primary infection and reactivation of latent infection or reinfection. The study objective was to determine CMV IgG avidity by enzyme-linked immunosorbent assay (ELISA) with denaturation of IgG antibody binding to the antigen and by chemiluminiscent microparticle immunoassay (CMIA) on an Abbott Architect analyzer. Both methods yielded comparable CMV IgG avidity results. In some cases, the Abbott test was superior in reflecting IgG antibody maturation during primary infection to microplate ELISA using antigen-antibody complex dissociation by a denaturing agent.

Long-term protection against hepatitis B after newborn vaccination: 20-year follow-up.

BACKGROUND: Hepatitis B vaccination in children born to hepatitis B surface antigen (HBsAg)-positive mothers considerably decreases the risk of vertical transmission. However, whether this protection against carriage of hepatitis B virus is maintained into early adulthood is as yet unknown. PATIENTS AND METHODS: A combined passive-active immunization programme for newborns of HBsAg-positive mothers was initiated in the north-eastern part of the Czech Republic in 1988. The number of immunized newborns had reached 665 newborns by the end of 2006. All mothers of immunized infants were HBsAg-positive during pregnancy, and 34 (5%) were also hepatitis B e antigen (HBeAg)-positive. The immunization programme consists of providing newborns with protection at birth with hepatitis B immunoglobulin, followed by three 10-µg doses of plasma-derived or, since 1990, recombinant vaccine administered at 0, 1 and 6 months of life. Only 29 children of HBeAg-positive mothers received vaccine at 0, 1 and 2 months of life. Blood samples were obtained after immunization, at 2 years of age, and biennially thereafter. Samples were tested for HBsAg and hepatitis B surface and core antibodies (anti-HBs, anti-HBc). RESULTS: The immunization schedules were completed in 640 children. A protective anti-HBs level after immunization was proven in 574 of 620 children (93%). Persistence of protective anti-HBs antibodies was detected in 70, 40 and 25% of children at 5, 10 and 15 years of age. Vertical transmission with chronic HBsAg carrier status was detected in two infants. Anti-HBc seroconversion was proven in ten children from 3 to 15 years of age. Natural boosting with an anti-HBs increase was detected in 38 children (twice in one child). CONCLUSION: Our results show that combined active-passive immunization of newborns against hepatitis B provides persistent protection up to adolescence despite a frequent waning of anti-HBs antibodies, suggesting there is no need for booster vaccination during adolescence.

[The use of molecular genetics techniques in clinical microbiology--final report from the workshop of the Molecular Microbiology Working Group TIDE]. / Aplikace metod molekulární genetiky v klinické mikrobiologii: zpráva z jednání Pracovní skupiny molekulárni mikrobiologie--TIDE.

In the last decade, there has been a rapid development in the use of molecular genetics methods in clinical microbiology. Novel technologies bring new knowledge and approaches to various disciplines of microbiology--taxonomy, identification of microbes, clinical diagnosis, epidemiology of infectious diseases and antibiotic resistance. This article summarizes the conclusions from the workshop of the Molecular Microbiology Working Group TIDE held during the Second Annual Meeting of the Society for Medical Microbiology of the J. E. Purkyne Czech Medical Association.

BAL fluid analysis in the identification of infectious agents in patients with hematological malignancies and pulmonary infiltrates.

The present study aims to evaluate the diagnostic yield of bronchoalveolar lavage (BAL) fluid in patients with hematological malignancies and describe the most common pathogens detected in BAL fluid (BALF.) An analysis of 480 BALF samples was performed in patients with hematological malignancies over a period of 7 years. The results of culture methods, PCR, and immunoenzymatic sandwich microplate assays for Aspergillus galactomannan (GM) in BALF were analyzed. Further, the diagnostic thresholds for Aspergillus GM and Pneumocystis jiroveci were also calculated. Microbiological findings were present in 87% of BALF samples. Possible infectious pathogens were detected in 55% of cases; 32% were classified as colonizing. No significant difference in diagnostic yield or pathogen spectrum was found between non-neutropenic and neutropenic patients. There was one significant difference in BALF findings among intensive care units (ICU) versus non-ICU patients for Aspergillus spp. (22% versus 9%, p = 0.03). The most common pathogens were Aspergillus spp. (n = 86, 33% of BAL with causative pathogens) and Streptococcus pneumoniae (n = 46, 18%); polymicrobial etiology was documented in 20% of cases. A quantitative PCR value of > 1860 cp/mL for Pneumocystis jirovecii was set as a diagnostic threshold for pneumocystis pneumonia. The absorbance index of GM in BALF of 0.5 was set as a diagnostic threshold for aspergillosis. The examination of BAL fluid revealed the presence of pathogen in more than 50% of cases and is, therefore, highly useful in this regard when concerning pulmonary infiltrates.

[Amniotic fluid heat shock protein 70 concentration in preterm premature rupture of membranes]. / Heat shock protein 70 v plodové vode u pacientek s predcasným odtokem plodové vody.

OBJECTIVE: The purpose of this study was to determinate the changes of amniotic fluid HSP 70 concentrations in patiens with preterm premature rupture of the membranes, and in the presence of intraamniotic infection and histological changes of inflammations. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology Medical Faculty Charles University Hradec Králové. METHODS: We studied 30 women between 24 and 36 weeks of gestation with preterm premature rupture of the membranes. Samples of amniotic fluid were collected by transabdominal amniocentesis. These patients were divided into 2 groups. In group 1 were patiens with intraamniotic infection. In group 2 were patiens without intraamniotic infection. Among 76% (35/30) patients placenta were collected and assessed for presence or absence acute inflammatory lesions. HSP70 concentration in amniotic fluid were determined using a sensitive and specific diagnostic kit Hsp 70- ELISA kit manufactered Assay Desings, USA. RESULTS: There was no significant difference in the median amniotic fluid HSP70 concentration between patients with preterm rupture of the membranes with IAI and without IAI (patients with IAI: median 5.12 ng/ml, range 3.01-90.37 ng/ml vs. patients without IAI: median 4.68 ng/ml, range 0.58-84.28 ng/ml; p = 0.56). There was no significant difference in the median amniotic fluid HSP70 concentration between patients with preterm rupture of the membranes with presence and absence histological of acute inflammatory lesions in the placenta and membranes (patients with presence: median 6.97 ng/ml, range 2.61-90.37 ng/ml vs. patients with absence: median 4.63 ng/ml, range 0.58-84.28 ng/ml; p = 0.68). CONCLUSION: Intraamniotic levels HSP70 were not associated with intraamniotic infection and acute inflammatory lessions in the placenta and membranes.

[Amniotic fluid interleukin 6 levels in preterm premature rupture of membranes]. / Interleukin 6 v plodové vode pacientek s predcasným odtokem plodové vody.

OBJECTIVE: The purpose of this study was to determinate the changes of amniotic fluid interleukin 6 (IL-6) concentrations in patients with preterm premature rupture of the membranes (PPROM), and in the presence of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA). The aim was to examine amniotic fluid IL-6 in relation to MIAC and HCA. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology Medical Faculty Charles University Hradec Králové. METHODS: We studied 37 women between 24 and 36 weeks of gestation with PPROM. Samples of amniotic fluid were collected by transabdominal amniocentesis. Polymerase chain reaction for the genital mycoplasmas and culture for aerobic and anaerobic bacteria were performed. Twenty-eight of 37 patients placentas were collected and assessed for presence or absence HCA. IL-6 concentration in amniotic fluid were determined using a sensitive and specific diagnostic kit Human IL-6 Quantikine ELISA manufactured R&D Systems, USA. RESULTS: There was significant difference in the median amniotic fluid IL-6 concentration between patients with preterm rupture of the membranes with and without MIAC and HCA (patients with MIAC and HCA: median 915 pg/ml, range 651-1854 pg/ml vs. patients without MIAC and HCA: median 780 pg/ml, range 184-1059 pg/ml; p=0.047). There was no significant difference in the median amniotic fluid IL-6 concentration between patients with preterm rupture of the membranes with and without MIAC (patients with MIAC: median 915 pg/ml, range 195-1854 pg/ml vs. patients without MIAC: median 792 pg/ml, range 184-1993 pg/ml; p=0.53). There was no significant difference in the median amniotic fluid IL-6 concentration between patients with preterm rupture of the membranes with and without HCA (patients with HCA: median 829 pg/ml, range 195-1992 pg/ml vs. patients without HCA: median 768 pg/ml, range 184-1890 pg/ml; p = 0.31). CONCLUSION: Amniotic fluid IL-6 concentrations patients with PPROM with presence HCA and MIAC were significantly higher than IL-6 concentration patients without HCA and MIAC.

[Difficulties in laboratory diagnosis of cerebral toxoplasmosis]. / Uskalí laboratorní diagnostiky mozkové toxoplazmózy.

Toxoplasmosis is the most wide-spread parasitic disease in the Czech Republic. According to the results of serological studies, about 25-50% of its population come in contact with this protozoan. A serious form of the disease may develop in severely immunocompromised patients. In these patients, problems with diagnosing toxoplasmosis may occur, especially in the case of its rare but serious cerebral form. The aim of the case report is to present potential difficulties in the diagnosis of cerebral toxoplasmosis.

[Mumps--a reemerging infection? The current incidence of mumps in the East Bohemian region in the Czech Republic]. / Príusnice--znovu se vracející infekce? Soucasná situace ve výskytu príusnic ve východoceském regionu.

A review of mumps outbreaks among both non-vaccinated and vaccinated children and young adults in the East Bohemian region in 2003-2005 is presented. A significant increase in mumps cases was observed over this period. The clinical diagnosis was confirmed serologically by ELISA detection of IgM antibodies and/or IgG seroconversion and increased levels of IgG antibodies. A reverse transcriptase nested PCR was introduced for direct detection of mumps virus RNA from clinical specimens (nasopharyngeal secretion, saliva, CSF and serum). The isolated RNA will be stored for further analysis and mumps virus genotyping attempts, helpful in tracing the virus circulation in the East Bohemia region. Possible causes of the recent significant increase in mumps cases among the vaccinated population in the Czech Republic are discussed.

Polymerase chain reaction for detection of Toxoplasma gondii in human biological samples.

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).

Laboratory diagnosis of leptospirosis.

Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999-2003) was 0.38-4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii and L. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5-10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.

Dyserythropoietic changes and sideroblastic anemia in patients with hairy cell leukemia before and after therapy with 2-chlorodeoxyadenosine.

Active hairy cell leukemia is associated with an increase of RDW (Red Cell Distribution Width) which normalizes after successful therapy with 2-chlorodeoxyadenosine (2-CdA). To clarify this phenomenon, bone marrow films performed before therapy with 2-CdA and after its successful completion were subjected to careful evaluation. Dyserythropoietic changes were present in 5 out of 17 patients before the therapy with 2-CdA. In 2 patients the changes were only slight, characterized by irregularities of the shape of nucleus and nuclear contour, in the remaining 3 patients the changes were marked, represented by nuclear lobulation, karyorrhexis and binuclearity, with the presence of ringed sideroblasts in one of them. After therapy with 2-CdA complete hematologic remission was achieved in 4 patients with disappearance of dyserythropoietic changes and normalization of RDW values. In the last patient with ringed sideroblasts despite complete remission with disappearance of tumoral cells in the bone marrow as demonstrated by immunohistochemical analysis of trephine bone marrow biopsy with monoclonal antibody DBA 44 the condition deteriorated, RDW remained unchanged, the sideroblastic anemia progressed.