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.

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.

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.

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.

[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.

T4 bacteriophage-mediated inhibition of adsorption and replication of human adenovirus in vitro.

AIM: The objective of this study was to evaluate the effects of T4 phage on adsorption and replication of human adenovirus (HAdV). MATERIALS & METHODS: Experiments were performed on cell lines A549 and HEK-293. Intracellular HAdV virions were released and titrated by Reed-Muench method. RESULTS: T4 significantly (p < 0.05) inhibited both the adsorption of HAdV and viral replication in a dose-dependent manner. Mean reductions in HAdV titer were within the range 0.634-2.12 and 0.238-1.88 log10 TCID50/ml for HAdV adsorption and replication, respectively. CONCLUSION: T4 phage can inhibit infections by HAdV-5 of target cells. Our results suggest that T4 might be considered a novel agent against HAdV and possibly other pathogenic viruses. Potential antiviral effects of other phages should also be investigated.

Incidence of adenoviral DNAemia in Polish adults undergoing allogeneic haematopoietic stem cell transplantation.

Human adenoviruses (HAdV) are important viral pathogens recognized increasingly in immunocompromised hosts, especially in allogeneic haematopoietic stem cell transplant recipients (alloHSCT). The clinical spectrum of HAdV disease ranges from asymptomatic viraemia and mild self-limiting disease to lower respiratory tract infection, multi-organ involvement and even death. Early detection and quantification of HAdV in peripheral blood using real-time PCR (qPCR) assay has been suggested as a useful monitoring tool, but is seldom used for regular surveillance of HAdV in haematology centers. A group of 112 alloHSCT recipients from two hospitals in Warsaw (Poland) was examined in the early post-transplant period using a quantitative qPCR assay. A total of 1,245 serum samples were evaluated for presence of HAdV DNA in patients where 66 (59 %) patients received grafts from unrelated donors whereas the other 46 (41 %) from sibling donors. HAdV sequences were detected in 64 (57 %) of the 112 patients. In 22 of all patients (20 %) HAdV DNA was detected only in a single positive sample, while 42 (37 %) had positive results in two or more subsequent sera. In total, DNAemia was present in 202 sera samples (16 %) with median time to observation of 47 days. Graft-versus-host disease (GvHD) was observed in 18 (28 %) adenovirus-infected transplant recipients and a significant correlation between HAdV infections and GvHD clinical presentation was found (p = 0.018). There is a high prevalence of HAdV infections in HSCT recipients in Poland during early post-transplant period. In consequence, we could only speculate if HAdV DNAemia could be also related to GvHD symptoms, enforcing the important pathogenic role of these viral infections in clinical complications post-alloHSCT.

[Usefulness of wide-range microbiological diagnostics proceedings in case of simultaneous infection with four herpesviruses after allogeneic haematopoietic stem cell transplantation--a case report]. / Przydatnosc wielokierunkowej diagnostyki mikrobiologicznej na przykladzie jednoczesnego zakazenia czterema herpeswirusami jako powiklania po allogenicznym przeszczepieniu komórek krwiotwórczych--opis przypadku.

INTRODUCTION: Infections caused with a variety of bacteria, fungi and viruses are still responsible for high level of mortality and morbidity in immunosupressed individuals. A case of fatal post-transplant reactivation with four herpesviruses in 49-year-old immunocompromised male with MDS-RAEB2, subjected to allogeneic haematopoietic stem cell transplantation was described. METHODS: Full microbiological examination of was performed in different types of clinical samples (whole blood, stool). Sera specimens were tested for the presence of different viral DNA using the real-time PCR assays. RESULTS AND CONCLUSIONS: DNA of HSV-1, VZV, HHV-6 and EBV in serum samples was detected using molecular biology techniques. Viral level of HSV-1 and VZV was constantly increasing despite routine applied oral acyclovir therapy. These findings underline the value of real-time PCR technique used in current therapeutic procedures and for monitoring of antiviral therapy with nucleoside analogs. We found that real-time PCR is a useful tool in detection and monitoring of disseminated herpesviral infection, especially for the detection of low-copy viraemia in clinical specimens.

Co-infections with cytomegalovirus and human herpesvirus type 7 in adult Polish allogeneic haematopoietic stem cell transplant recipients.

Human herpesvirus 7 (HHV-7) is widespread around the world and may also be a possible cofactor for cytomegalovirus (CMV) infection in haematopoietic stem cell transplant (HSCT) recipients. In case of viral diseases where specific treatment is available, real-time PCR assays constitute reliable diagnostic tools enabling timely initiation of appropriate therapy and rapid assessment of the efficacy of antiviral treatment strategies. The presence of CMV and HHV-7 was confirmed by the detection of viral DNA isolated from 1,027 plasma samples. A group of 69 allogeneic HSCT (alloHSCT) recipients was examined in early post-transplant period using quantitative real-time PCR methods. Within the study period, 62 % of patients had at least once CMV DNA-emia, while HHV-7 DNA was found in 43 % of subjects. Co-infection between these ß-herpesviruses was detected in the plasma samples collected from 18 patients (26 %). Patients with concomitant HHV-7 DNA-emia had significantly higher number of CMV DNA copies compared with those without HHV-7 infection (1986 vs. 432 copies/ml, p < 0.001) but there was no difference in duration of CMV DNA-emia between these groups. On the other hand, while the load of HHV-7 DNA was comparable between patients with CMV DNA-emia and without CMV DNA-emia, the duration of HHV-7 DNA-emia was significantly longer in the first group (38.5 vs. 14 days, p < 0.001). HHV-7 DNA-emia is very frequently detected in Polish alloHSCT recipients. In those, who have subsequent CMV reactivation, the coexistence of the viruses may negatively affect the kinetics of infection with either of them. Therefore the investigation of concomitant HHV-7 DNA-emia could affect the prognosis of post-transplant patients suffering from CMV reactivation.

Disseminated adenovirus disease in immunocompromised patient successfully treated with oral ribavirin: a case report.

In patients with immunological disorders, adenovirus infections are associated with significant rates of morbidity and mortality. Only few hematological units use molecular virological methods, such as polymerase chain reaction, for surveillance of adenovirus infection, and treatment strategies have never been evaluated in multicenter clinical trials. This report describes the detection and treatment of human adenovirus (HAdVs) disseminated disease in the case of a 46-year-old immunocompromised female having myelodysplastic syndrome with refractory cytopenia with multilineage dysplasia: International Prognostic Scoring System 1. Serum and urine samples were tested for the presence of adenoviral DNA using the quantitative real-time polymerase chain reaction (PCR) assay. For additional confirmation, sequencing of PCR products was also performed. With real-time PCR, we detected HAdV DNA in both serum and urine samples. The viral level constantly decreased with applied oral ribavirin therapy. As the result of sequencing, HAdVs type 11 was determined. Surveillance of adenovirus by real-time PCR is useful in detecting and monitoring disseminated HAdV infection; it is a potential standard diagnostic approach that could assist clinicians to decide whether antiviral therapy ought to be administered.

[Real-time PCR assay for detection and quantification of human adenoviruses in patients with haematological malignancies and symptoms of lower respiratory tract infection]. / Wykorzystanie ilosciowej reakcji Lanjcuchowej polimerazy w czasie rzeczywistym do wykrywania zakazen dolnych dróg oddechowych wywolanych adenowirusami u osób z chorobami nowotworowymi ukladu krwiotwórczego.

Human adenoviruses (HAdV) are one of the im-portant infectious etiological factors that affect immunocompromised patients. Because of the large number of HAdV serotypes and their genomic variations, they present a lot of difficulty in laboratory diagnostics. The recent introduction of real-time PCR (qPCR)-based assays has opened new ways to rapid, specific, and highly sensitive HAdV detection. For detection and quantification of HAdV DNA we retrospectively tested serum and bronchoalveolar lavage fluid (BALF) samples obtained from a cohort of 60 adult patients with haematological malignancies presenting clinical and radiological symptoms of lower respiratory tract infections. Human adenoviruses DNA was detected by qPCR method, using primers targeting a conserved region of the adenoviral hexon gene and a specific TaqMan probe. Adenovirus infection occurred with a high incidence in our study group patients. Using qPCR we found that a 21,7% and 15,0% of patients had adenoviral DNA in BALF and serum samples, respectively. The high level of sensitivity, specificity and accuracy provided by real-time PCR assay are favorable for the use in the detection of adenoviral DNA in clinical specimens, especially in immunocompromised patients.

Human herpesvirus 7 in allogeneic hemopoietic stem cell transplant recipients in the central clinical hospital in Warsaw: a three-year survey.

OBJECTIVES: Human herpesvirus 7 (HHV-7) is spread worldwide and has been described as a potential pathogen in immunosuppressed patients. Different clinical manifestations have been described including fever and skin rash; HHV-7 may also be a possible cofactor for cytomegalovirus disease in transplant recipients. MATERIALS AND METHODS: A retrospective review of a group of 58 adult recipients of allogeneic hemopoietic stem cell transplantation was made. Serum samples taken in the range of 0-180 days after transplant were examined for presence of specific HHV-7 sequences using the quantitative real-time PCR method. RESULTS: HHV-7 DNA was detected in plasma samples in 26 (45%) of the 58 recipients between day 20 and day 65 of transplantation. All of them developed fever of unknown origin; also HHV-5 DNA was detected in plasma samples collected from 11 HHV-7-positive patients. None of the described individuals died during detectable HHV-7 or HHV-5 viremia periods. CONCLUSIONS: There is a high frequency of detectable HHV-7 viral load in allogeneic stem cell transplant recipients in Poland. Limited availability and sensitivity of serological methods along with the necessity of rapid introduction of antiviral treatment has forced the development of molecular diagnostics. Furthermore, establishment of appropriate procedures for monitoring active HHV-7 infection is important to clarify the virus infection in transplant recipients.

[Detection of human enteroviruses with real-time PCR assay using TaqMan fluorescent probe]. / Zastosowanie metody real-time PCR do wykrywania RNA enterowirusów czlowieka.

Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.

Detection of ß-herpesviruses in Polish adult cord blood stem cell recipients by real-time PCR: single centre study.

Umbilical cord blood transplantation (UCBT) is known to be associated with increased risk of infections, compared to bone marrow or peripheral blood stem cell transplantation. In viral diseases for which specific treatment is available, real-time PCR assays are reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies. A retrospective review of samples from a group of seven adult cord blood stem cell recipients was made. Serum samples taken up to 180 days after transplantation were examined with quantitative real-time PCR for measurement of viral load (CMV, HHV-6, and HHV-7). Cytomegalovirus (CMV) DNA was detected in samples taken from four patients (57%) in the period of 20-80 days after transplantation. Products of amplification of human herpesvirus 6 (HHV-6) DNA were found in samples taken between days 25 and 37 following UCBT from only one patient (14%). On the other hand, the majority of patients (n = 6, 86%) had HHV-7 DNA detected in the period 15-58 days after transplantation. Co-infection with HHV-7 was demonstrated at onset of all episodes of microbiologically confirmed CMV or HHV-6 infection. Our observations indicate that real-time PCR is not only useful for monitoring herpesviral infections in transplant recipients, but is also a powerful method for clarifying the relationships between the viral load and clinical symptoms. Further investigation with a much larger group of patients will be needed to confirm these observations and translate them into a clinical approach.