Characteristic pattern of pleural effusion in electrical impedance tomography images of critically ill patients.

BACKGROUND: Electrical impedance tomography (EIT) is increasingly used for continuous monitoring of ventilation in intensive care patients. Clinical observations in patients with pleural effusion show an increase in out-of-phase impedance changes. We hypothesised that out-of-phase impedance changes are a typical EIT finding in patients with pleural effusion and could be useful in its detection. METHODS: We conducted a prospective observational study in intensive care unit patients with and without pleural effusion. In patients with pleural effusion, EIT data were recorded before, during, and after unilateral drainage of pleural effusion. In patients with no pleural effusion, EIT data were recorded without any intervention. EIT images were separated into four quadrants of equal size. We analysed the sum of out-of-phase impedance changes in the affected quadrant in patients with pleural effusion before, during, and after drainage and compared it with the sum of out-of-phase impedance changes in the dorsal quadrants of patients without pleural effusion. RESULTS: We included 20 patients with pleural effusion and 10 patients without pleural effusion. The median sum of out-of-phase impedance changes was 70 (interquartile range 49-119) arbitrary units (a.u.) in patients with pleural effusion before drainage, 25 (12-46) a.u. after drainage (P<0.0001) and 11 (6-17) a.u. in patients without pleural effusion (P<0.0001 vs pleural effusion before drainage). The area under the receiver operating characteristics curve was 0.96 (95% limits of agreement 0.91-1.01) between patients with pleural effusion before drainage and those without pleural effusion. CONCLUSIONS: In patients monitored with EIT, the presence of out-of-phase impedance changes is highly suspicious of pleural effusion and should trigger further examination.

Diffuse gastrointestinal bleeding and BK polyomavirus replication in a pediatric allogeneic haematopoietic stem cell transplant patient.

Patients undergoing haematopoietic stem cell transplantation (HSCT) are at high risk of severe gastrointestinal bleeding caused by infections, graft versus host disease, and disturbances in haemostasis. BK polyomavirus (BKPyV) is known to cause hemorrhagic cystitis, but there is also evidence of BKV shedding in stool and its association with gastrointestinal disease. We report putative association of BKPyV replication with high plasma viral loads in a pediatric HSCT patient developing hemorrhagic cystitis and severe gastrointestinal bleeding necessitating intensive care. The observation was based on chart review and analysis of BKPyV DNA loads in plasma and urine as well as retrospective BKPyV-specific IgM and IgG measurements in weekly samples until three months post-transplant. The gastrointestinal bleeding was observed after a >100-fold increase in the plasma BKPyV loads and the start of hemorrhagic cystitis. The BKPyV-specific antibody response indicated past infection prior to transplantation, but increasing IgG titers were seen following BKPyV replication. The gastrointestinal biopsies were taken at a late stage of the episode and were no longer informative of BK polyomavirus involvement. In conclusion, gastrointestinal complications with bleeding are a significant problem after allogeneic HSCT to which viral infections including BKPyV may contribute.

A case of primary JC polyomavirus infection-associated nephropathy.

A 15-year-old boy with a posterior urethral valve received a deceased donor kidney transplant (KT) in March 2011. Basiliximab induction followed by tacrolimus-based triple medication was used as immunosuppression. Eleven months after KT, the graft function deteriorated and the biopsy demonstrated interstitial nephritis suggestive of acute rejection. BK polyomavirus (BKPyV) surveillance in urine and plasma was negative. The patient received methylprednisolone pulses and anti-thymocyte globulin. Immunohistochemistry was positive for simian virus 40 (SV40) large T-antigen (LTag) in the biopsies, and quantitative polymerase chain reaction for JC polyomavirus (JCPyV) indicated high viral loads in urine and borderline levels in plasma. Immunosuppression was reduced and follow-up biopsies showed tubular atrophy and interstitial fibrosis. Two years after KT, antibody-mediated rejection resulted in graft loss and return to hemodialysis. Retrospective serologic work-up indicated a primary JCPyV infection with seroconversion first for IgM, followed by IgG, but no indication of BKPyV infection. In the SV40 LTag positive biopsies, JCPyV deoxyribonucleic acid (DNA) with archetype noncoding control region was detected, while BKPyV DNA was undetectable. To the best of our knowledge, this is the first reported case of primary JCPyV infection as the cause of PyV-associated nephropathy in KT.

Current characteristics and outcome of cytomegalovirus infections after kidney transplantation.

INTRODUCTION: The clinical course of cytomegalovirus (CMV) infections in the current era is poorly described. We characterized the symptoms and outcome of all CMV infections in a large cohort of kidney transplant recipients. Among 1129 kidney transplant recipients transplanted between 2004 and 2011 in Charité Universitätsmedizin Berlin and Helsinki University Hospital, 297 patients with CMV infection were characterized. RESULTS: CMV disease occurred in 217/1129 patients (19.2%), and CMV infection in 297/1129 (26.3%). Gastrointestinal symptoms were recorded in 58% and fever in 47% patients with primary CMV disease, compared to 46% and 27% patients with symptomatic CMV reactivation, whereas leukopenia or thrombocytopenia were seen in only 17-28% patients, and malaise in 9-10%. Tissue-invasive CMV gastroenteritis was confirmed in 11% and CMV pneumonia in only 1% of patients with CMV disease. Only 1 patient died because of CMV infection (mortality 0.3%). Virus-related factors or the use of secondary prophylaxis did not predict the risk of recurrence, which occurred in 33% patients. CONCLUSION: In conclusion, CMV disease remains a common problem after kidney transplantation. Gastrointestinal symptoms were common, especially in patients with primary CMV infection, whereas bone marrow suppression, hepatopathy, or malaise were seen less frequently.

[Assessment of the inter- and intraobserver variability for sonographical measurement of intestinal wall thickness in dogs without gastrointestinal diseases]. / Inter- und Intraobserver-Variabilität bei der sonographischen Bestimmung der Darmwanddicke von Hunden ohne gastrointestinale Erkrankungen.

OBJECTIVE: The aim of the present study was to determine inter- and intraobserver agreement for transabdominal ultrasonographic measurements of the intestinal wall in dogs without gastrointestinal diseases. MATERIAL AND METHODS: This prospective study included 30 dogs diagnosed with a non-gastrointestinal disease and were evaluated using a transabdominal ultrasound scan in our clinic. Transverse ultrasonographic images for each segment (duodenum, jejunum, colon descendens) were obtained. These images were masked, randomized and imported as DICOM files in the OsiriX® version 5.0 program for Mac Os X. Two observers independently determined the intestinal wall thicknesses using the software inherent measurement tools. The measurements were repeated five times for each segment in all patients on 4 consecutive days. Therefore, each observer performed 1800 measurements, and 3600 measurements in total were analyzed. RESULTS: The mean values for each intestinal segment were comparable to those in the literature. The statistical analyses showed a significant positive correlation (p  <  0.01) for the inter- and intraobserver measurements at all intestinal locations. There was very high intraobserver repeatability for the measurements, with deviations of <  10%. In addition, the study displayed good interobserver reproducibility for the measurements of all intestinal segments, with variances of <  20% for the duodenum and jejunum, and <  50% for the colonic wall thickness. Even with these variances the interobserver variability for all segments was much less than the mean deviance between normal and diseased dogs. CONCLUSION AND CLINICAL RELEVANCE: Transabdominal ultrasonography is a practicable tool to assess intestinal wall thickness and integrity in small animal medicine. Our results are comparable to established reference ranges for the normal canine intestinal wall thickness. In addition, we found a good inter- and intraobserver agreement for the measurements of the canine wall thicknesses in dogs without gastrointestinal diseases.

Detection of HHV-6-specific mRNA and antigens in PBMCs of individuals with chromosomally integrated HHV-6 (ciHHV-6).

After inheritance of chromosomally integrated HHV-6 (ciHHV-6), viral DNA is found in every nucleated cell. The prevalence of ciHHV-6 is estimated to be 0.2-5% of humans. There are conflicting data on the potential for replication, possibly leading to clinical implications. We analysed peripheral blood mononuclear cells (PBMCs) from individuals with ciHHV-6 proven by fluorescence in situ hybridization (FISH) for HHV-6-specific mRNA (U94, U42, U22) and antigens by means of reverse transcription PCR and an indirect immunoperoxidase staining. U94 transcripts indicative of latent infection were detected in six (54.5%) out of 11 individuals at least once. Transcripts indicative of lytic infection (i.e. U42 and U22) were detected in four (36.4%) out of 11 individuals at least once. HHV-6 antigen was detected in seven (70%) out of 10 individuals at least once. The presence of viral mRNA and proteins supports virus gene expression from ciHHV-6, which may lead to virus replication. Considering the properties of active HHV-6 infection together with obvious replicative activity in individuals with ciHHV-6, pathophysiological effects leading to clinical consequences of chromosomally integrated viral DNA might be considered.

European perspective on human polyomavirus infection, replication and disease in solid organ transplantation.

Human polyomaviruses (HPyVs) are a growing challenge in immunocompromised patients in view of the increasing number of now 12 HPyV species and their diverse disease potential. Currently, histological evidence of disease is available for BKPyV causing nephropathy and haemorrhagic cystitis, JCPyV causing progressive multifocal leukoencephalopathy and occasionally nephropathy, MCPyV causing Merkel cell carcinoma and TSPyV causing trichodysplasia spinulosa, the last two being proliferative skin diseases. Here, the current role of HPyV in solid organ transplantation (SOT) was reviewed and recommendations regarding screening, monitoring and intervention were made. Pre-transplant screening of SOT donor or recipient for serostatus or active replication is currently not recommended for any HPyV. Post-transplant, however, regular clinical search for skin lesions, including those associated with MCPyV or TSPyV, is recommended in all SOT recipients. Also, regular screening for BKPyV replication (e.g. by plasma viral load) is recommended in kidney transplant recipients. For SOT patients with probable or proven HPyV disease, reducing immunosuppression should be considered to permit regaining of immune control. Antivirals would be desirable for treating proven HPyV disease, but are solely considered as adjunct local treatment of trichodysplasia spinulosa, whereas surgical resection and chemotherapy are key in Merkel cell carcinoma. Overall, the quality of the clinical evidence and the strength of most recommendations are presently limited, but are expected to improve in the coming years.

The long-term outcomes of patients transplanted due to acute liver failure with hepatic human herpesvirus-6 infection.

Human herpesvirus (HHV)-6, comprised of HHV-6A and HHV-6B, belongs to the betaherpesviruses that infect 95%-100% of humans. Primary infection, known as exanthema subitum, occurs in early childhood. Reactivations of latent HHV-6, mostly HHV-6B, are common after liver transplantation. The vast majority of them are asymptomatic; in a minority of cases, the virus may infect the liver transplant, causing graft dysfunction or hepatitis. An association between hepatic HHV-6 infection and indeterminate acute liver failure (ALF) has been shown, but the causality is not clear because of the ubiquitous nature of HHV-6. We have previously observed HHV-6B antigens in the explanted livers of most patients (80%, n = 32) transplanted with ALF of unknown cause, whereas it was not observed among those with ALF of known cause. After transplantation, half of the patients with pretransplant HHV-6 infection (9/18) developed recurrences. The aim of this study was to investigate their long-term course (9-14 years). Half of the patients with pretransplant HHV-6 developed recurrences. Two also showed cytomegalovirus (CMV) hepatitis, whereas none of the other patients demonstrated intrahepatic CMV. During the 9 years or more of follow-up, 1 graft and 2 patients were lost in both groups (HHV-6 recurrence/HHV-6-negative patients). The reasons for graft loss were hepatic arterial thrombosis and portal venous thrombosis. In addition 2 patients died in the HHV-6 recurrence group, one because of rethrombosis of hepatic artery (day 460) and one with a functioning transplant (4.5 years after transplantation). In the control group 1 patient died at 1.5 years and 1 at 10 years after liver transplantation because of pneumonia. HHV-6 relapse was common in ALF patients after transplantation. However, HHV-6 did not cause liver failure and had no significant long-term effect on survival.

Primary CMV infections are common in kidney transplant recipients after 6 months valganciclovir prophylaxis.

Prolonging cytomegalovirus (CMV) prophylaxis in CMV seronegative recipients of a kidney from CMV seropositive donor (D+/R-) may reduce the incidence of late infections. We analyzed late-onset primary CMV infections after 6 months valganciclovir prophylaxis. Data from all CMV D+/R- kidney transplant recipients between January 2004 and December 2008 at our center were analyzed. Patients with a functioning graft at 6 months after transplantation who received 6 months of valganciclovir prophylaxis 900 mg once daily were included (N = 127). CMV was diagnosed with quantitative PCR. Prophylaxis was completed in 119 patients. Prophylaxis was stopped at 3-5 months due to leukopenia or gastrointestinal side effects in eight patients. Late-onset primary CMV infection developed in 47/127 (37%) patients median 244 days after transplantation (range 150-655) and median 67 days after the cessation of prophylaxis (range 1-475). Four infections were asymptomatic. In others, symptoms included fever (N = 28), gastrointestinal symptoms (nausea, vomiting, diarrhea) (N = 24), respiratory tract symptoms (N = 12), and hepatopathy (N = 6). Median peak viral load was 13500 copies/mL (range 400-2,831,000). Recurrent CMV infection developed in 9/47 (19%) patients. No significant risk factors for CMV infection were identified. Symptomatic primary CMV infections were commonly detected also after prolonged valganciclovir prophylaxis.

Quantitative HHV-6B antigenemia test for the monitoring of transplant patients.

Human herpesvirus-6 (HHV-6) infection, mostly caused by variant B, is common after transplantation. Here, we report a new modified method using an HHV-6B glycoprotein IgG antibody, OHV-3, and attempt to quantify the HHV-6 antigenemia after liver transplantation. Twenty-four liver transplant recipients were frequently monitored by the HHV-6 antigenemia test, which detects the HHV-6B virion protein in peripheral blood mononuclear cells (PBMC). HHV-6B antigens were now retrospectively demonstrated using a glycoprotein OHV-3 IgG antibody in the immunoperoxidase staining from the same specimens and quantified as positive cells/10,000 PBMC. The results were confirmed and quantified by DNA hybridization in situ. Altogether, 206 blood specimens were analyzed. During the first six months, HHV-6 antigenemia was detected in 17/24 (71%) recipients by using the HHV-6B virion antibody. In total, 37% (77/206) of specimens were positive with the virion antibody and 39% (78/201) by the OHV-3 antibody. The peak number of OHV-3-positive cells in the PBMC varied from 5 to 750/10,000 (mean 140/10,000). The OHV-3 antibody was useful to quantify the HHV-6B antigenemia. The findings of the HHV-6B quantitative antigenemia using the OHV-3 antibody correlated well with the previous qualitative HHV-6 antigenemia assay, and can be used as an alternative quantitative method in the monitoring of HHV-6 in transplant patients.

Case report: human herpesvirus 6 reactivation associated with colitis in a lung transplant recipient.

Whereas human herpesvirus 6 (HHV-6) reactivation is frequent in solid organ transplant recipients, symptomatic disease is rare. A case of colitis associated with HHV-6B reactivation was observed in a lung transplant recipient. This case report suggests that symptomatic HHV-6 infection may occur in the absence of detectable viremia.

Inhibition of semicarbazide-sensitive amine oxidases decreases lymphocyte infiltration in the early phases of rat liver allograft rejection.

Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation in vitro and in vivo. VAP-1 is also an ectoenzyme with semicarbazide-sensitive amine oxidase (SSAO) activity. In this study we investigated whether inhibition of SSAO influences the inflammatory infiltration in acute rat liver allograft rejection. BN recipients of DA liver allografts were treated with 50 mg/kg/d semicarbazide, an inhibitor of SSAO, or similar volumes of saline. 10 rats/group were followed for graft survival, and 10 rats/group were sacrificed on day 7 post-transplantation for histology and T-lymphocyte isolation. The area percentage of portal inflammatory infiltrates in the grafts was assessed from digital photomicrographs. The proportion of CD4-, CD8- and IL2-receptor positive lymphocytes in the graft was quantified with flow cytometry. On day 7, semicarbazide treatment significantly decreased the inflammatory infiltrate area in the grafts. CD4-, CD8- and IL2-receptor positive cells were equally affected. However, animal survival was not affected. Blockade of the enzymatic activity of VAP-1 has a significant effect on lymphocyte infiltration early in acute liver rejection. Later, activation of other adhesion pathways can by-pass the blockade caused by VAP-inhibition.

STAT-1 decoy oligonucleotide improves microcirculation and reduces acute rejection in allogeneic rat small bowel transplants.

During acute rejection leukocyte-endothelial cell interaction fuelled by costimulatory molecules such as the CD40/CD154 receptor/ligand dyad disrupts microcirculation of the small bowel. Downregulating endothelial CD40 expression by employing a decoy oligonucleotide (dODN) neutralizing the transcription factor signal transducer and activator of transcription-1 (STAT-1) may protect the graft. Therefore allogenic small bowel transplantation was performed in the Brown Norway to Lewis rat model. Graft vessels were pretreated with STAT-1 dODN, mutant control ODN (20 microM) or vehicle (n=8). CD40 antisense ODN and scrambled control ODN-treated transplants served as target control (n=3 each). Intravital microscopy, histology, immunohistochemistry and Western blot analyses were performed 7 days later. Functional capillary density, red blood cell velocity and perfusion index in STAT-1 dODN and CD40 antisense ODN-treated transplants were improved whereas stasis index was reduced. Leukocyte-endothelial cell interaction showed no difference. Histological parameters of rejection, infiltrating CD3-positive cells and apoptotic bodies were also reduced in STAT-1 dODN and CD40 antisense ODN-treated transplants 7 days post-transplantation. CD40 protein abundance was reduced to less than 10% of control in STAT-1 dODN-treated grafts. STAT-1 dODN blockade of CD40 expression improves mucosal perfusion, reduces graft rejection, T-cell infiltration and apoptosis in rat small bowel allografts during acute rejection.

Cytomegalovirus and proliferative signals in the vascular wall of CABG patients.

OBJECTIVE: To further elucidate the mechanism by which cytomegalovirus (CMV) may promote atherosclerosis, we studied the expression pattern of cellular inflammatory and proliferative signals in the aortic wall of CMV(+) and CMV(-) patients undergoing coronary artery bypass grafting (CABG). METHODS: Aortic biopsies and blood samples of 68 CABG patients were investigated for CMV-DNA by PCR and IN SITU hybridisation. Expression of pp65 antigen, adhesion molecules (ICAM-1, VCAM-1, E-selectin), growth factors (PDGF-AA, TGF-beta), and the cellular proliferation factor Ki-67 was studied by immunohistochemistry. Logistic regression was used to test the correlation between the presence of CMV, vascular inflammation, and traditional noninflammatory risk factors for atherosclerosis. RESULTS: CMV-DNA was detected in the aortic tissue of 52 (76%) patients, and was localised predominantly in vascular smooth muscle cells. In CMV(+) patients, the expression of adhesion molecules and growth factors in the aortic endothelium was increased compared with CMV(-) patients. A positive correlation of elevated CRP, the induction of adhesion molecules and growth factors and CMV(+) was found. Female gender, smoking, and hyperlipidaemia were identified as risk factors for CMV(+). CONCLUSIONS: CMV-DNA in smooth muscle cells induces local growth factor expression as well as endothelial activation, both of which can promote the progression of atherosclerosis. Since traditional atherogenic risk factors increase the likelihood of aortic CMV manifestation, we suggest that CMV plays a crucial role in mediating the progression of atherosclerosis.

Cytomegalovirus infection of the liver transplant: virological, histological, immunological, and clinical observations.

The most common organ-specific manifestation of cytomegalovirus (CMV) infection after liver transplantation is hepatitis. Here we retrospectively describe the detailed virological, histological, immunological, and clinical findings associated with CMV infection in 229 consecutive adult liver transplantation patients. CMV infection was diagnosed by pp65 antigenemia. From 439 liver biopsies, CMV antigens were demonstrated by immunohistochemistry and CMV DNA by hybridization. The Banff criteria were used for histology. The expression of various adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], endothelial leukocyte adhesion molecule-1 [ELAM-1]), their ligands (leukocyte function antigen-1 [LFA-1], very late antigen-4 [VLA-4], Sialyl-LewisX-molecule [SLeX]), and lymphoid activation markers (major histocompatibility complex [MHC] Class II, interleukin-2-receptor [IL-2R]) was demonstrated by immunohistochemistry. CMV infection of the transplant occurred in 26 patients (11% of all 229 patients and 17% of the 151 patients with liver biopsy). The incidence was higher among seronegative (26%) than in seropositive recipients (9%), but most cases 18/26 (70%) were reactivations. The CMV pp65 antigenemia levels were usually high in primary infections (893+/-1069, range 50-3000 pp65+cells), but varied widely in reactivations (388+/-740, range 3-3000). The histological Banff score was slightly increased (2.3+/-0.9). Microabscesses, lymphocytic infiltration, Kupffer cell reaction, and parenchymal alterations were common but viral inclusions rare. CMV significantly (P<0.05) increased ICAM-1 and VCAM-1 expression and the number of LFA-1, VLA-4, and Class II-positive lymphocytes in the graft. All CMV infections were successfully treated with antivirals. Intragraft CMV infection had no influence on the long-term outcome, but biliary complications were common. In conclusion, CMV infection of the liver transplant occurred both in primary infection and in reactivation, and also in the cases with low pp65 antigenemia levels. Microabscesses and other histological alterations were common but viral inclusions rare. Increased adhesion molecule expression was associated with lymphocyte infiltration. Successfully treated CMV hepatitis had no influence on the long-term clinical outcome.

Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients.

Cytomegalovirus (CMV) infection is a significant problem in transplantation. In this study, a quantitative PCR test was compared with the CMVpp65 antigenemia assay not only in the diagnosis CMV infections but especially in the monitoring of viral loads during ganciclovir treatment of CMV disease in individual renal transplant patients. Altogether 342 blood specimens were obtained from 116 patients. Blood specimens were used for Cobas Amplicor Monitor plasma PCR and for the pp65 assay. Also shell vial culture was performed. The patients with a positive pp65 finding were monitored for CMV weekly during ganciclovir treatment and/or until the antigenemia subsided. CMV was detected in 31/116 (27%) patients, of whom 14 (12%) developed CMV disease and were treated with ganciclovir. CMV was found by shell vial culture in 13/14 cases, but by PCR and pp65 test in all 14 patients. CMV was detected in 156 (45%) samples; by PCR in 121/156 (range 344-103,000 copies/ml) and by pp65 test in 138/156 (range 1-1,000 positive cells/50,000 leukocytes) and by culture in 59/156 (38%) only. The peak viral loads were significantly (P<0.0001) higher in CMV disease than in untreated infections (19,650 vs. 379 copies/ml, and 100 vs. 5pp65 positive cells). In the monitoring of individual patients, the time-related CMV-DNAemia and pp65 antigenemia correlated well during the treatment of CMV disease. In conclusion, Cobas Amplicor Monitor plasma PCR and CMVpp65 antigen assays can be equally used in the diagnosis CMV infection and in the monitoring of viral load during antiviral treatment.

DNA microarray-based gene expression profiles of cytomegalovirus infection and acute rejection in liver transplants.

An association between cytomegalovirus (CMV) infection and alloresponse has been suggested. CMV increases inflammation and adhesion molecule expression in graft, and induces cytokines and growth factors, linked with transplant vasculopathy and chronic rejection. We have investigated the gene expression of various inflammatory factors in the CMV-associated immune response and compared this with the immune response of acute rejection in liver transplants by using DNA microarray technology. Gene expression was studied at mRNA level in biopsies from liver transplant patients experiencing CMV infection or acute rejection. RNA extracted from liver grafts after reperfusion was used as control material. Among the strongly upregulated genes in the specimens obtained from liver transplants during CMV infection were IFN-gamma, caspases 1 and 3, granzymes A and B, TGF-beta receptors II and III, IL-10 receptor alpha, VCAM-1, TNF receptor, IL-4, TNF-alpha, IL-10, IL-2 receptor beta, IL-1beta, PDGF-receptor beta, vascular adhesion protein-1, TGF-beta2, and ICAM-1. In biopsies with acute liver allograft rejection, the most significantly upregulated genes were MHC class II, IFN-gamma, caspases 1 and 3, IL-2R beta and gamma, granzymes A and B, VLA-4, L-selectin, E-selectin, VCAM-1, and IL-1beta. Upregulated genes common for CMV and alloresponse were granzyme A and B, E-selection, IFN-gamma, VCAM-1, VLA-4, TNF, caspases 1, 3, and 8, and PDGF. Microarray analysis defined different entities in the immune responses of CMV infection and acute rejection. The differences and similarities of the gene expression profiles related to those in CMV infection and rejection may help to understand the intragraft immunologic events.

HHV-6-DNAemia related to CMV-DNAemia after liver transplantation.

In addition to cytomegalovirus (CMV), activation of other betaherpesviruses, especially human herpesvirus 6 (HHV-6), has been reported in liver transplant patients. The purpose of this study was to investigate the posttransplant HHV-6-DNAemia in relation to CMV-DNAemia in liver transplant patients. Thirty-one adult liver allograft recipients were regularly monitored for CMV and HHV-6 during the first 3 months after transplantation. For the diagnosis of CMV infections, pp65-antigenemia assay and quantitative DNA-PCR were used. HHV-6 was demonstrated by using quantitative DNA-PCR and HHV-6 antigenemia test. Altogether 253 blood specimens of 31 recipients were analyzed. In addition, CMV and HHV-6 specific antigens were demonstrated by immunohistochemistry in liver biopsy specimens in the case of graft dysfunction. Thirteen patients (40%) developed a clinically significant CMV infection, at a mean of 33 days (range 5 to 62 days) after transplantation and were treated with intravenous ganciclovir. The peak viral loads of these symptomatic CMV infections were high (CMV-DNA 34210 +/- 37557 copies/mL plasma). Six additional asymptomatic patients demonstrated significantly lower CMV-DNAemia levels (1020 +/- 1008 copies/mL, P < .05), and were not treated. Concurrently with CMV, HHV-6 DNAemia and antigenemia were detected in 17 of 19 patients, mean 11 days (range 6 to 24 days) after transplantation. HHV-6 appeared prior to CMV in most cases (12 of 17). However, the peak viral loads were low (HHV-6-DNA <1500 copies/mL blood), even in the five patients who demonstrated HHV-6 antigens on liver biopsy. All CMV infections were successfully treated with ganciclovir and the CMV DNAemia/antigenemia subsided. HHV-6 also responded to the antiviral treatment, but more slowly and less clearly. In conclusion, HHV-6 activations were common and usually associated with CMV infection in liver transplant patients. Further investigation of the clinical significance of HHV-6 DNAemia/antigenemia is necessary.

Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients.

BACKGROUND: Quantitative PCR assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. However, usually these tests are still quite time-consuming and labor-intensive which diminishes their utility of these tests in routine diagnostic laboratories. OBJECTIVES: The objective of this study was to develop a quantitative CMV PCR test which is time-saving and easy to perform for the detection and monitoring of CMV infection of transplant patients. STUDY DESIGN: The quantitative real time CMV PCR assay using TaqMan chemistry and an automated sample preparation system, MagNA Pure LC, was developed. The designed quantitative CMV test was compared to commercial quantitative PCR test, Cobas Amplicor Monitor, in the determination of CMV DNA loads in plasma samples of liver and kidney transplant patients. The results were also correlated with the CMV pp65-antigenemia test. The clinical material of 270 blood specimens of transplant patients were tested using these two PCR methods and pp65-antigenemia test in parallel. Plasma samples were used for PCR assays and leucocytes for the antigenemia test. RESULTS: The TaqMan assay described was easy to perform, it was rapid (3-4 h) and hands-on time needed for performing the test was short. The detection limit of the assay was 250 copies/ml (cps/ml) plasma and the linear range up to 25,000,000 cps/ml. TaqMan assay was the most sensitive test detecting 92% of the CMV positive findings. Cobas Monitor detected 80% and pp65 test 88% of the positive findings. The correlations between TaqMan and antigenemia assays, and between Cobas Amplicor and antigenemia were statistically significant and high, R = 0.84 (P < 0.0001) and R = 0.80 (P < 0.0001), respectively. Also correlation between two PCR tests was statistically significant (R = 0.64, P < 0.0001). Of the 27 patient studied, 19 demonstrated CMV antigenemia and DNAemia in their blood during the post transplant monitoring. Thirteen of these patients developed a symptomatic CMV infection and were treated with ganciclovir. The peak viral loads of symptomatic patients were statistically higher by all three methods than those of asymptomatic patients. CONCLUSIONS: The developed real time TaqMan assay was rapid and easily performed and could be the best alternative for the diagnosis of CMV infection and monitoring of liver and kidney transplant patients.

Detection of CMV-DNA in peripheral blood leukocytes of liver transplant patients after ganciclovir treatment.

Cytomegalovirus (CMV) infections are common after transplantation, but usually successfully treated with antivirals. In this study, the detection of CMV-DNA in peripheral blood leukocytes was monitored and compared with CMVpp65-antigenemia in liver transplant patients receiving ganciclovir treatment. Twenty adult liver transplant recipients were frequently monitored for CMV up to 6 months after transplantation. CMV infections were diagnosed by pp65-antigenemia and the same specimens were used for CMV-DNA in situ hybridization. Altogether 202 blood specimens were analyzed. During the first 6 months, 14/20 patients developed CMV antigenemia and 11 were treated with ganciclovir. In all patients, CMV-DNA was detected before antigenemia (mean 15 days earlier). All patients responded to ganciclovir and pp65-antigenemia disappeared. However, 8/11 demonstrated persistence of CMV-DNA for up to 6 months. Recurrences appeared in 6/11 patients. In conclusion, detection of CMV-DNA preceded pp65-antigenemia. Persistence of CMV-DNA demonstrates that the virus is not eliminated by ganciclovir and recurrences can be expected.