Maternal SARS-CoV-2 Infection at Delivery Increases IL-6 Concentration in Umbilical Cord Blood.

BACKGROUND: SARS-CoV-2 infection in pregnant women may induce inflammation within the amniotic cavity and/or an increase in proinflammatory cytokines in fetal circulation. The aim was to investigate levels of IL-6 in maternal blood, umbilical cord blood, and amniotic fluid in pregnant women infected with SARS-CoV-2 at delivery. METHODS: A single-center prospective observational case-control study of pregnant women diagnosed with SARS-CoV-2 infection at delivery was conducted. A total of 48 infected and 42 healthy women had IL-6 concentrations measured in their blood, amniotic fluid, and umbilical cord blood. RESULTS: The concentrations of IL-6 in maternal blood and amniotic fluid were similar in the study and control groups, while umbilical cord blood concentrations were significantly higher in SARS-CoV-2-positive women. The umbilical cord blood IL-6 concentration was related to composite neonatal morbidity. CONCLUSIONS: Maternal SARS-CoV-2 infection in pregnant women at delivery increases umbilical cord blood IL-6 concentration. The correlation between maternal and umbilical blood concentrations indicates a possibility of passage of IL-6 through the placenta. Perinatal alterations resulting from maternal SARS-CoV-2 infection at delivery carry a risk of impacting the health of infants even in asymptomatic course of infection.

Ultrastructural Changes in Mitochondria in Patients with Dilated Cardiomyopathy and Parvovirus B19 Detected in Heart Tissue without Myocarditis.

Understanding the meaning of parvovirus B19 (PB19V) in an etiology of dilated cardiomyopathy (DCM) is difficult. Viruses change the dynamics of the mitochondria by interfering with the mitochondrial process/function, causing the alteration of mitochondrial morphology. In this study, the ultrastructural changes in the mitochondria in endomyocardial biopsy (EMB) samples from patients with DCM and PB19V were determined. METHODS: The PB19V evaluation was performed in EMB specimens by real-time PCR in 20 patients (age: 28 ± 6 years). The biopsy specimens were examined by histo- and immunohistochemistry to detect the inflammatory response. The ultrastructural features of the mitochondria were evaluated by electron microscopy. RESULTS: The presence of PB19V in the heart tissue without the presence of inflammatory process, defined according to Dallas and immunohistochemical criteria, was associated with ultrastructural changes in the mitochondria. Distinctive ultrastructural pathologies were indicated, such as the presence of mitochondria in the vicinity of the expanded sarcoplasmic reticulum with amorphous material, blurred structure of mitochondria, interrupted outer mitochondrial membrane and mitophagy. CONCLUSIONS: Extending diagnostics with ultrastructural analysis of biopsy samples provides new knowledge of the changes associated with the presence of PB19V in the heart tissue. The observed changes can be a basis for searching for the damage mechanisms, as well as for new therapeutic solutions.

Heat Shock Protein 90 Chaperones E1A Early Protein of Adenovirus 5 and Is Essential for Replication of the Virus.

Adenovirus infections tend to be mild, but they may pose a serious threat for young and immunocompromised individuals. The treatment is complicated because there are no approved safe and specific drugs for adenovirus infections. Here, we present evidence that 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 chaperone, decreases the rate of human adenovirus 5 (HAdV-5) replication in cell cultures by 95%. 17-AAG inhibited the transcription of early and late genes of HAdV-5, replication of viral DNA, and expression of viral proteins. 6 h after infection, Hsp90 inhibition results in a 6.3-fold reduction of the newly synthesized E1A protein level without a decrease in the E1A mRNA level. However, the Hsp90 inhibition does not increase the decay rate of the E1A protein that was constitutively expressed in the cell before exposure to the inhibitor. The co-immunoprecipitation proved that E1A protein interacted with Hsp90. Altogether, the presented results show, for the first time. that Hsp90 chaperones newly synthesized, but not mature, E1A protein. Because E1A serves as a transcriptional co-activator of adenovirus early genes, the anti-adenoviral activity of the Hsp90 inhibitor might be explained by the decreased E1A level.

Inhibitory Effects of Bacteriophage Preparations on Adenoviral Replication.

BACKGROUND/AIMS: Bacteriophages (phages) are viruses of bacteria. Escherichia coli phage (T4) can potentially interfere with adsorption of HAdV-5 to cellular integrins by its KGD motif, while staphylococcal A5/80 phage does not possess this structure. The objective of this study was to investigate the effects of T4 and A5/80 phage preparations on type 5 human adenovirus (HAdV-5) DNA synthesis and the expression of HAdV-5 genes. METHODS: Experiments were performed on the A549 cell line. HAdV-5 DNA synthesis was investigated with real-time PCR. Expression of HAdV-5 early (DBP) and late (hexon) genes was determined by quantitative real-time PCR in preincubation and coincubation experiments. RESULTS: While both phage preparations significantly reduced the expression of HAdV-5 genes, synthesis of HAdV-5 DNA was inhibited only by T4. CONCLUSION: Phage preparations show promise as novel antiviral agents. However, further studies are required to investigate their antiviral effects.

Evaluation of an Integrated Cell Culture-based and PCR Assay for Diagnosis of Genital Herpes in Women.

Diagnosis of genital herpes requires a combination of clinical presentation and laboratory studies. Laboratory diagnostics allow us to clearly establish the etiology (HSV-1 or HSV-2) in order to determine the course of infection and prognosis. Decisive factors in the selection of the appropriate test are: diagnostic goals, patient population, specimen type, and implementation of conditions for the specific method. In total, 187 samples collected during a routine gynecological examination from 120 women were examined for the presence of HSV-1 and HSV-2 in the genital area. Two methods were used to test swabs: cell culture isolation and PCR. HSV-1 was the dominant type of virus in both study groups. The cytopathic effect was observed in 67 (35.8%) cultures with clinical material. HSV-1 and HSV-2 DNA were detected by PCR in 73 (39.0%) cell cultures infected with clinical samples. We did not observe typical, virus related cytopathic changes in 13.7% DNA HSV positive cell cultures, but on the other hand we did not detect viral DNA in 6% of positive cell cultures. High values of the parameters, defining the usefulness of diagnostic tests (sensitivity, specificity, and predictive values) in both groups, are determined by previous viral replication in cell culture.

Sequence typing of human adenoviruses isolated from Polish patients subjected to allogeneic hematopoietic stem cell transplantation - a single center experience.

PURPOSE: Human adenoviruses (HAdV) from species A, B and C are commonly recognized as pathogens causing severe morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. The purpose of the present study was to determine HAdV types responsible for viremia in HSCT recipients at a large tertiary hospital in Poland. METHODS: Analysis of partial nucleotide sequences of HAdV hexon gene was used to type 40 clinical isolates of HAdV obtained from 40 HSCT recipients. RESULTS: We identified six different HAdV serotypes belonging to species B, C and E. We demonstrated high variability in sequences of detected HAdV types, and patients infected with the same HAdV types were not hospitalized at the same time, which suggests the low possibility of cross-infection. In almost all patients, anti-HAdV antibodies in IgG class were detected, which indicates a history of HAdV infection in the past. Clinical symptoms accompanying HAdV viremia were in 89%, and in 61.5% of individuals, HAdV was a sole pathogen detected. There were no cases with high-level HAdV viremia and severe systemic or organ infections. Graft-versus-host disease (GvHD) was present in patients infected with species B and C, but grade II of GvHD was observed only in patients infected with HAdV-B. CONCLUSIONS: The predominance of HAdV-C and common presence of anti-HAdV antibodies in IgG class may strongly suggest that most infections in the present study were reactivations of HAdV persisting into the patient's mucosa-associated lymphoid tissues. Variability of HAdV sequences suggests that cross-infections between patients were very rare. ABBREVIATIONS: GvHD: graft-versus-host disease; HAdV: human adenoviruses; HSCT: hematopoietic stem cell transplantation.

Viral Heart Disease and Acute Coronary Syndromes - Often or Rare Coexistence?

BACKGROUND: Clinical presentation of viral myocarditis can mimic acute coronary syndrome and making diagnosis of viral heart disease (VHD) may be challenging. The presence of coronary artery disease (CAD) does not always exclude VHD and these entities can coexist. However, the incidence of co-occurrence of CAD and VHD is not precisely known. Moreover, inflammatory process caused by viruses may result in atherosclerotic plaque destabilization. METHODS: The goal of this work is to summarize the current knowledge about co-occurrence of VHD and CAD. This article presents the importance of inflammatory process in both diseases and helps to understand pathophysiological mechanisms underlying their coexistence. It provides information about making differential diagnosis between these entities, including clinical presentation, noninvasive imaging features and findings in endomyocardial biopsy. Although currently there are no standard therapy strategies in coexistence of VHD and CAD, we present some remarkable aspects of treatment of patients, in whom VHD co-occurs with CAD. RESULTS: Viral heart disease may occur both in patients without and with atherosclerotic plaques in coronary arteries. Destabilization of atherosclerotic plaques in coronary arteries can be facilitated by inflammatory process. Increased inflammatory infiltrates in the coronary lesions of patients with VHD can lead to plaques' instability and consequently trigger acute coronary syndrome. CONCLUSION: In this article we attempted to present that co-occurrence of VHD and CAD may have therapeutic implications and as specific antiviral treatment is currently available, proper diagnosis and treatment can improve patient's condition and prognosis.

Perspectives of Phage Therapy in Non-bacterial Infections.

While the true value of phage therapy (PT) in human bacterial infections still awaits formal confirmation by clinical trials, new data have been accumulating indicating that in the future PT may be applied in the treatment of non-bacterial infections. Thus, "phage guests" may interact with eukaryotic cells and such interactions with cells of the immune system may protect human health (Guglielmi, 2017) and cause clinically useful immunomodulatory and anti-inflammatory effects when administered for therapeutic purposes (Górski et al., 2017; Van Belleghem et al., 2017). Recently, a vision of how these effects could translate into advances in novel means of therapy in a variety of human pathologies secondary to immune disturbances and allergy was presented (Górski et al., 2018a). In this article we present what is currently known about anti-microbial effects of phage which are not directly related to their antibacterial action and how these findings could be applied in the future in treatment of viral and fungal infections.

Viral Infection of the Heart: Pathogenesis and Diagnosis.

Viral infections of the heart cause serious clinical problems, either as infectious myocarditis, which usually is a consequence of acute infection or as idiopathic dilated cardiomyopathy, resulting rather from a chronic infection. This minireview presents an up-to-date view on pathomechanisms of viral infection of the heart tissues, the role of immune system in controlling infectious process at its various stages and current possibilities of recognizing viral infection of the heart with use of both cardiological and virological methods. Our goal was to present the variety of known viral agents causing heart infection, level of complexity in mutual virus-cell interactions, and consequent clinical scenarios.

[Serological and molecular methods in diagnostics of primary acute hepatitis caused with human herpesvirus type 6 - a case report].

INTRODUCTION: HHV-6 has been identified as the etiologic agent of exanthema subitum in infants and acute febrile illness in young children, although primary HHV-6 infection in immunocompetent adolescents and adults is very rare. We report the case of acute hepatitis in an 18-year-old man without any immunological dysfunctions. MATERIAL AND METHODS: Full virological examination of sera samples was performed. Specimens were tested for the presence of specific anti-EBV and anti-HHV-6 antibodies and also for the presence of DNA of other herpesviruses, using qPCR assays. RESULTS: Obtained results demonstrated the presence of HHV-6 DNA in all examined sera samples and IgM-class antibodies against HHV-6 in the second specimen. No other etiologic agents were found that are known to induce hepatitis. Hence, patient was diagnosed as having acute hepatitis triggered by HHV-6. CONCLUSIONS: We found that qPCR is a very useful tool for the detection of different herpesvirus infections, especially with low-copy viraemia in clinical specimens.

Infections due to alphaherpesviruses in early post-transplant period after allogeneic haematopoietic stem cell transplantation: Results of a 5-year survey.

BACKGROUND: Infections caused by human α-herpesviruses usually have a benign course with recurrencies. However, they may become dangerous in immunocompromised hosts. In this case, molecular methods constitute a reliable diagnostic tool enabling rapid assessment of the efficacy of antiviral treatment strategies. OBJECTIVES: We estimated the frequency of alphaherpesviral DNAemia and the viral load during early post-transplantation period after alloHSCT; we also analyzed association of the DNAemia and chosen parameters of the patients. STUDY DESIGN: A cohort of 190 alloHSCT recipients from two hospitals in Warsaw, Poland, was examined weekly during 100-day early post-transplantation period using quantitative real time PCR assays. A total of 2475 sera samples were evaluated for the presence of α-herpesviral DNA in patients, of whom 117 (62%) received unrelated grafts, while the remaining 73 (38%) received grafts from sibling donors. All patients received standard antiviral prophylaxis with acyclovir. In the examined group, anti-HSV-1, anti-HSV-2 and anti-VZV IgGs were examined prior to transplantation, RESULTS: Within the study period, DNA of α-herpesviruses was detected in 44 patients (23.2%). Most patients tested positive for HSV-1 DNA (43 patients, 22.6%), single patient for HSV-2, and no patient positive for VZV. Clinical symptoms such as pneumonia, skin changes, elevated levels of aminotransferases were observed in five patients, four of these patients presented symptoms of GvHD at the same time. (2,6%). Statistics shows that GvHD (P<0.001) and matched unrelated donor as a source of HSCT (P=0.048) are associated with the development of HSV-1 DNAemia. CONCLUSIONS: Although our data demonstrate frequent reactivation of HSV-1 in the early post-transplant period, the rate of symptomatic infections was low. We did not find association between HSV-1 viremia and mortality, but significant association with GvHD and donor source was observed.

Analysis of the shedding of three ß-herpesviruses DNA in Polish patients subjected to allogeneic hematopoietic stem cell transplantation: Six-year follow up.

BACKGROUND: Infections with human ß-herpesviruses are common worldwide and are still frequent in patients after hematopoietic stem cell transplantation. Some data suggest that HHV-6 and HHV-7 could take part in CMV reactivation from latency and/or progression of CMV disease in immunosupressed patients. OBJECTIVES: The aims of this study were: (1) to summarise retrospectively the results of ß-herpesviruses DNA detection in a large group of adult allogeneic haematopoietic stem cell transplant recipients; and (2) to find a potential correlation between viruses belonging to this subfamily. STUDY DESIGN: AlloHSCT recipients (N=142) were examined in the early post-transplant period (median=89 days). The presence of CMV, HHV-6 and HHV-7 was confirmed through detection and quantification of viral DNA, isolated from 1679 sera samples. RESULTS: CMV DNA alone was detected in 23.9% of patients, while single HHV-6 and HHV-7 were detected in 14.8% and 9.9% of individuals, respectively. The reactivation of more than one virus was identified in 31% of analysed patients. In cases of concurrent infection, HHV-7 was detected at the same time as HHV-6, and both of them were usually reactivated before CMV. The kinetics of virus reactivation and measured viral load may suggest a potential role of HHV-6 and HHV-7 as co-factors in CMV reactivation. CONCLUSIONS: The observed kinetics of virus reactivation may strongly suggest a potential role of HHV-6 and/or HHV-7 as co-factors of CMV reactivation. The co-infection with these ß-herpesviruses could predispose patients after hematopoietic stem cell transplantation to a longer and more severe CMV infection.

Interferon-α and inosine pranobex-mediated inhibition of reploication of human RNA viruses in vitro.

INTRODUCTION: Interferon- a (IFN-a), produced by immune cells, exhibits pleiotropic anti- viral activity. Inosine pranobex (PI), a synthetic derivative of a purine, shows direct anti- viral activity, and also acts indirectly, by activation of immune cells. The aim of this study was to evaluate an in vitro inhibition of Coxackievirus A16 (CAI6), enterovirus 71 (EV71) and human parainfluenza virus 4 (HPIV-4) replication by PI in combination with IFN-a. MATERIALS AND METHODS: In the present study we evaluated an in vitro effect of interferon-a and inosine pranobex on replication ofRNAviruses: CA-16, EV71, HPIV-4. Antiviral effects of IFN-a and IPwere assessed by phenotypic assays. The yield reduction assay (YRA), which evaluates the ability of the compounds to inhibit virus multiplication in cell cultures, was ap- plied. The Reed-Muench statistical method was used to determine the 50% end point (IC51). RESULTS: Our studies have shown that combination of IFN-a and inosine pranobex dis- play higher efficacy than treatment with either compound alone, and suggest syn- ergy that may increase therapeutic effectiveness. The reduction of the average viral ti- ters of EV71, CA-16 and HPIV-4 in A549 cell culture after applying 400 Ig/mL Ip and IFN-a (1000 IU/mL), in comparison to the viral titer in the control was reduced by 1,76 log,, TCID,,/ml, o 3,00 log, TCID50/ml , and 1,60 log,( TCID50/ml respec- tively. The antiviral activity of the tested compounds was also analyzed on the basis of IC., values. Application of 1000 IU/ml IFN-a, with PI after infection of A549 cells with mention above viruses reduced the IC,, by 3,5%, 41,3% and 29% respectively. CONCLUSIONS: Our study demonstrated that enhanced antiviral activity was observed when cells infected with RNA viruses were treated with combination of IFN-a and IP. The ef- fectiveness of IFN-a and IP under these conditions has not been previously reported. CA16 virus turned out to be the most sensitive to the action of used inhibitors.

Viral Nucleic Acids in the Serum Are Dependent on Blood Sampling Site in Patients with Clinical Suspicion of Myocarditis.

BACKGROUND: The meaning of viral nucleic acids in the myocardium in many cases is difficult for clinical interpretation, whereas the presence of viral nucleic acids in the serum is a marker of active infection. We determined the diagnostic value of viral nucleic acids in ventricular serum and peripheral serum samples in comparison with endomyocardial biopsy (EMB) specimens in patients with clinically suspected myocarditis. METHODS: The viral nucleic acid evaluation was performed in serum samples and EMB specimens by real-time PCR in 70 patients (age: 47 ± 16 years). The biopsy specimens were examined by histo- and immunohistochemistry to detect inflammatory response. RESULTS: The viral nucleic acids were detected in ventricular and peripheral serum, and EMB samples of 10 (14%), 14 (20%), and 32 (46%) patients, respectively. Notably, viral nucleic acids of the same virus as in the EMB sample were present more often in ventricular than in peripheral serum (60 vs. 7%, p = 0.01). A significant concurrence was observed between the positive and the negative results of viral nucleic acids present in EMB and ventricular serum (p = 0.0001). CONCLUSIONS: The detection of the same viral nucleic acid type in the myocardium and in ventricular serum being significantly more frequent than in the peripheral serum may suggest that the site of the blood collection is important for more precise and reliable confirmation of the active viral replication in the heart.

[Prevalence of CI-HHV-6 in patients with hematological diseases and healthy donors in Poland - preliminary study].

of specific anti-HHV-6 antibodies in IgM and IgG classes and viral DNA. Quantitative real-time PCR assay was also used to determine viral load in alloHSCT recipients in plasma samples. Results: All individuals from studied group have not IgM antibodies against-HHV-6 prior transplantation. Specific IgG-class anti-HHV-6 antibodies were detected in 38 of 54 (70%) donors and in 47 of 54 recipients before HSCT (870/o), respectively. High load of HHV-6 DNA (>1x10A6 copies/ml) was detected in plasma samples taken only from one person (1,9%) of the 54 recipients. Conclusions: There is a high frequency specific anti-HHV-6 antibody in studied Polish patients; otherwise CI-HHV-6 was rare detected. Nonetheless, we urge careful observation of individuals with hematological malignances supposed to have CI-HHV-6. Further research on larger study group is needed to determine the clear role of CI-HHV-6 in alloHSCT recipients.

[Comparison of three multiplex real-time PCR assays for the detection of herpes simplex viruses type 1 and type 2 DNA].

INTRODUCTION: Herpes simplex viruses type 1 and type 2 (HSV-1 and HSV-2) cause widespread infection worldwide with different course and intensity. Although the disease caused by this viruses mainly concern healthy children and adults, the HSV infections are much more dangerous for people with immunodeficiencies. The aim of this work was to compare the diagnostic value of two qPCR methods for detection HSV-1/2 DNA, based on TaqMan* and HybProbes chemistry with commercial HSV-1/2 Qual Kit. METHODS: DNA from 51 clinical samples was tested for presence of HSV-1/2 sequences on LightCycler 2.0 thermocycler, using two ,,in-house" developed multiplex real-time PCR assays and commercial test using SCORPIONSTM primers. RESULTS: The results showed high specificity and sensitivity of all molecular biology tests used. Statistically, there was no significant difference in the sensitivity of real-time PCR assays when using TaqMan* and HybProbes' chemistry and when using the commercial SCORPIONSTM based method (P>0.05). CONCLUSIONS: Obtained results show that all tested methods are highly specific and can possibly be used to simultaneously detect and differentiate HSV-1/2 DNA in clinical samples. The high detection rate and short duration of qPCR assayas has great importance for immunocompromised patients where quick application of effective and safe treatment is necessary. It is also important in the event of amorphous form of the infection and the occurrence of nonspecific and generalized symptoms.

[Frequency of detection of soluble antigens of Candida spp. and Aspergillus spp. using serological assays in body fluids of patients of Central Clinical Hospital in Warsaw].

INTRODUCTION: Immunodeficient patients, e.g. transplant recipients, patients treated with corticosteroids, people with AIDS and individuals undergoing prolonged antibiotic therapy are at high risk of invasive fungal infections, especially invasive aspergillosis. Basic method for detection of organ/systemic fungal infection is serological monitoring in body fluids, first of all in serum, bu also in broncho-alveolar lavages (BALF). Proven invasive fungal infection should be diagnosed by culture of the pathogen or histopathological examination of infected tissues, however the detection of soluble fungal antigens in body fluids gives enough information for diagnosis of probable fungal infection, according to European Organization for Research and Treatment of Cancer recommendations, what allows introduction of antifungal therapy. Aim of the study was to asses the frequency of detection of circulation soluble fungal antigens with use of immunoenzymatic techniques in patients hospitalized between 2010 and 2015 in Independent Public Central Clinical Hospital (IPCCH) in Warsaw. Methods: In IPCCH, between 2010 and 2015, 6475 serum samples, taken from 2096 patients, was tested for Candida spp. mannan antigen, and 7745 sera from 2243 patients were tested for Candida spp. mannan antigen, and 7745 sera from 2243 patients were tested for galactomannan antigen of Aspergillus spp, as well as 64 samples of BALF. Material was collected mainly from haematopoietic stem cell transplant recipients, hospitalized in Haematology and Oncology Clinics, during their routine pos-transplant monitoring. Testing was performed with use of quantitative (Candida antigen) or semiquantitative (Aspergillus antigen) immunoenzymatic methods (BioRad-Platelia), according to respective protocols. Results: During examined period, increase in number of examinations was observed, starting from 1311 tests performed in 2010, up to 3052 examination in 2015. In 2015 testing for Aspergillus antigen in BALF samples was also introduced, resulting in 64 samples tested. Candida spp. antigen was detected in 171 samples (2,7% of all tested samples), and Aspergillus galactomannan was detected in 645 serum samples (8,4%) and 8 BALF samples (12,5%). Majority of examinations was performed for patients hospitalized in Haematology and Oncology Clinics (72,7%), Blood Vessel Surgery and Transplantology Clinics (3,8%), as well as in patients under care of post-transplantation (8,3%) and haematology (4,2%) out-patients clinics. Conclusions: (i) In the 2015-2015 visible increase in number of fungal antigens examinations was observed, (ii) significant number of examinations was performed in onco-haematological patients (88,7%), what also indicates main risk group, (iii) 8,3% of fungal antigen testing was performed in solid organ transplant recipients, the second risk group for invasive fungal infection.

Real-Time PCR Analysis of Chimerism in T Cell Subsets as an Early Predictor of Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation.

BACKGROUND: Graft-versus-host-disease (GvHD) is the major cause of morbidity and mortality after stem cell transplantation. The development of early prediction methods is therefore of importance. Our aim was to analyze the usefulness of early donor chimerism monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in T cells and in CD4+ and CD8+ (lineage chimerism) for GvHD prediction. MATERIAL AND METHODS: Chimerism was analyzed in 76 consecutive adult patients using RQ-PCR TaqMan technology on DNA extracted from Pan T, CD4+, and CD8+ cell subsets on Day 5, 10, 15 and 30 after allo-HSCT. RESULTS: The threshold of chimerism predictive for GvHD was the same for all tested cell subsets. In acute myeloid leukemia (AML) patients treated with myeloablative conditioning (MAC), the threshold predictive for acute graft versus host disease was 95% and 99% for Day 10 and Day 15, respectively. In patients treated with reduced intensity conditioning (RIC), the threshold predictive for chronic graft versus host disease was 98% on Day 10. The differences were statistically significant. CONCLUSIONS: Chimerism analysis in T cell subsets by RQ-PCR on Day 10 and Day 15 after transplantation is useful for prediction of aGvHD (AML patients after MAC) and cGvHD (patients after RIC). However, there was no difference in the results between chimerism in the T cell subsets. Our RQ-PCR protocol was highly sensitive and proved effective for analysis of lineage chimerism.

[Development of TaqMan-based real-time PCR assay for detection of Epstein-Barr virus DNA]. / Zaprojektowanie i optymalizacja metody PCR z detekcja w czasie rzeczywistym do wykrywania DNA wirusa Epsteina-Barr z wykorzystaniem fluorescencyjnej sondy typu TaqMan.

INTRODUCTION: Epstein-Barr virus (EBV) is a human herpesvirus which infects almost all of the world's population subclinically during childhood and thereafter remains for a life. Immunocompromised persons often show active EBV infection, which may progress to virus-associated lymphoproliferative disorders. Many clinical researches show a strong role for viral load measurement in predicting and monitoring EBV-associated diseases, especially in immunosuppressed patients. The aim of this work was to design and to optimize novel real-time PCR assay for detection and quantification of Epstein-Barr virus DNA. MATERIALS AND METHODS: In described experiment TaqMan chemistry-based primers and probes were designed to specific EBV sequence of BALF5 viral gene. To test laboratory utility of the designed method, 80 sera samples, positive for EBV DNA in routine investigations, were also analyzed and 1st International WHO WBV Standard was applied for recalculation of the results to international units. RESULTS: Developed real-time PCR assay gave positive result only in the samples containing genetic material of EBV. Mean viral load of the 80 clinical samples tested was 2,838 and 3,241 log10 copies/ml for analyzed and reference method, respectively. Correction with EBV Standard led to equalization of these results (3,229 and 3,244 log10 international units/ml respectively). CONCLUSIONS: The obtained data indicate that this TaqMan-based qPCR assay is accurate, rapid and reliable method for the diagnosis and monitoring of EBV DNAemia in clinical samples, coming from immunosuppressed individuals.