Valère J. Goossens

Intravenous immunoglobulin therapy for patients with idiopathic cardiomyopathy and endomyocardial biopsy-proven high PVB19 viral load.

BACKGROUND Parvovirus B19 (PVB19) persistence in the heart has been associated with progressive cardiac dysfunction and evolution to dilated cardiomyopathy. In the present study, we investigated whether immunomodulation with intravenous immunoglobulin (IVIg) in addition to conventional heart failure therapy is safe and achieves virus reduction. Such therapy might improve cardiac function in patients with chronic dilated cardiomyopathy (DCM) and a significant PVB19 viral load in the heart. METHODS PVB19 viral load was studied in 25 post-mortem cardiac samples of patients with a normal heart. Then, 17 consecutive patients (mean age 53 +/-3 years) with DCM and symptomatic heart failure for >1 year with a PVB19 viral load in endomyocardial biopsies of >250 copies/microg DNA were treated with a high dose of IVIg (2 g/kg). RESULTS The post-mortem cardiac samples revealed a PVB19 presence in 80% with a mean load of 131 +/-40 copies/microg DNA. In the treated patients, IVIg resulted in a significant decrease of PVB19 viral load from 1,420 +/-216 to 619 +/-200 copies/microg DNA (P=0.004) and significantly improved the ejection fraction from 33 +/-3% 6 months before treatment and 34 +/-3% at baseline to 41 +/-3% 6 months (P=0.001) after IVIg therapy. The New York Heart Association classification significantly improved from 2.5 +/-0.1 at baseline to 2.1 +/-0.1 at follow-up (P=0.004). No therapy-related complications were noted. CONCLUSIONS The present pilot study demonstrates that IVIg significantly reduces viral load and improves cardiac function in patients with DCM related to increased PVB19 viral load in the heart.

Early detection of human cytomegalovirus infection after kidney transplantation by nucleic acid sequence-based amplification.

BACKGROUND The early detection of human cytomegalovirus infection after organ transplantation is a prerequisite for effective antiviral therapy. We evaluated the diagnostic value of monitoring the viral immediate-early (IE) 1 mRNA expression in blood leukocytes by nucleic acid sequence-based amplification (NASBA). METHODS Nucleic acids were isolated from 489 blood samples collected from 42 kidney transplant recipients and subjected to amplification by IE NASBA. The IE NASBA results were compared to those from pp67 NASBA, pp65 antigenemia, cell culture (DEAFF and CPE), and serology. RESULTS IE NASBA proved to be the most sensitive assay which detected the onset of both primary and secondary cytomegalovirus infection significantly earlier than the other assays. CONCLUSIONS The early detection of cytomegalovirus infection with IE NASBA would enable the start of effective antiviral therapy at an early state of infection to prevent cytomegalovirus disease in patients at risk.

Early detection of cytomegalovirus in renal transplant recipients: comparison of PCR, NASBA, pp65 antigenemia, and viral culture

INFECTION with human cytomegalovirus (CMV) is common and usually subclinical. However, serious CMV infection is possible in immunocompromised individuals (eg, transplant recipients, HIV-infected patients, and pregnant women). After kidney or other solid organ transplantation, more frequent organ rejection and a higher mortality rate are seen in CMV-positive patients than in CMV-negative patients. The higher mortality rate as well as organ rejection can both be prevented in part by early antiviral treatment. However, early treatment requires early diagnosis. The purpose of this study was to select tests that allow for detection of CMV as early as possible after transplantation. We therefore compared the value of DNA detection by polymerase chain reaction (PCR), detection of immediate early 1 (IE1) mRNA and pp67 mRNA by nucleic acid sequence-based amplification (NASBA), detection of pp65 protein by antigenemia testing, and detection of CMV by culturing.

Diagnostic Value of Nucleic-Acid-Sequence- Based Amplification for the Detection of Cytomegalovirus Infection in Renal and Liver Transplant Recipients

To evaluate the diagnostic value of nucleic-acid-sequence-based amplification (NASBA) for the detection of cytomegalovirus (CMV) infection in transplant recipients, we compared immediate early 1 (IE1) and late pp67 mRNA detection by NASBA with the antigenemia assay, PCR and viral culture in 72 renal transplant (RTx) recipients and with antigenemia and serology in 25 liver transplant (LTx) recipients. Antigenemia, viral culture and pp67 NASBA were almost equivalent for the detection of CMV in RTx recipients. In LTx recipients, antigenemia detected more positive samples and more positive recipients compared to pp67 NASBA. In RTx recipients, PCR detected more positive samples and positive recipients compared to pp67 NASBA, antigenemia and viral culture. Also the first day of detection was slightly earlier for PCR. However, IE1 NASBA was the most sensitive test and detected 96% of all positive samples and positive transplant recipients. In addition, IE1 NASBA preceded PCR and all other positive results. This makes IE1 NASBA a very attractive screening test for the early detection of CMV infection.

Nucleic Acid Sequence-Based Amplification: A New Technique for Monitoring Cytomegalovirus Infection in Transplant Recipients

HE MAJORITY of the adult human population is infected with cytomegalovirus (CMV), a member of the b-herpesvirus family. Normally, infection of immunocompetent individuals does not cause disease. In contrast, infection of immunocompromised individuals, such as neonates, patients with AIDS, and transplant recipients, can cause severe complications and can even lead to death. Therefore, early detection of active CMV infection is necessary to start effective antiviral treatment. Nucleic acid sequence-based amplification (NASBA) has been developed for the specific amplification of RNA. 1 We evaluated the diagnostic value of monitoring the expression of the CMV immediate early (IE) 1 mRNA using NASBA, in peripheral blood cells of kidney and liver transplant patients. The IE 1 mRNA is expressed directly after entrance of the virus into a cell. NASBA results were compared to the techniques which are routinely being used for the detection of CMV: pp65-antigenemia, virus culture, and serology.

Response to “Prevalence of Infection With Adenovirus-36 in Belgium and Holland and Association With Obesity”

TO THE EDITOR: We thank R.L. Atkinson for his Letter “Prevalence of Infection With Adenovirus-36 in Belgium and Holland and Association With Obesity” (1) as a supplement to our recent study concerning lack of evidence for the role of human adenovirus-36 in obesity in a European cohort (2). We are totally aware that our findings are in contrast to papers from different geographic regions outside Europe. In contrast, although only limited European data were available at the time of publication, all European ad-36 results were in concordance with our results. This is illustrated by a Danish study of Raben et al., with Atkinson as one of the co-authors, indicating an ad-36 seroprevalence of 5.7% in overweighted subjects and 10.0% in normal-weight subjects in Denmark and concluding that in that study, there was no evidence to support that infection by ad-36 is an important cause of the increasing prevalence of obesity in Denmark (3). During our study, we selected 25 samples from our cohort including negative (<2, or 2–4) and positive (≥8) results and these selected samples were send to Atkinson for confirmation. All our positive results were confirmed. Few negative results were discordant but sometimes difficult to interpret, e.g., two samples with undetectable titer (<2) in our hands were found doubtful by Atkinson, either “?8” in one sample and “<2/16” as duplo-results in a second sample. Therefore, rather than being a reason for concern, Atkinson’s results confirmed our analytical performance for discriminating positive and negative results. In our study, we used a species-specific adenovirus PCR described by Echavarria et al. (4). In our hands, this PCR is sensitive as described (2) and previously validated for clinical use in different kinds of patient samples. In addition, this PCR is appropriate for detection of a broad range of other adenovirus types including ad-5 and ad-37 (4). This is worthwhile because not only ad-36, but also ad-5, ad-9, ad-31, and ad-37 have been investigated in relation to obesity (5). Atkinson also comments on the amount of adipose tissue, e.g., 10 mg we used for DNA isolation, and he suggests the use of a much larger adipose tissue sample as described by Dhurandhar et al. (6). Within this manuscript, samples of about 1 g each were used for PCR after DNA isolation using a QIAamp Tissue Kit (cat. no. 29304; Qiagen, Valencia, CA) (6). However, according to the manufacturer instructions for this method, maximum DNA purify and yield are obtained with tissue samples of 20 mg or less (7). Larger samples are more difficult to handle because of their increased viscosity and do not lead to increased yields or purity (7). Finally, Dr Atkinson is correct in stating that our data only apply to Belgium and the Netherlands. Therefore, and in concordance with earlier work of Atkinson et al. in Denmark, an adapted conclusion might be that: adenovirus-36 does not play a role as a direct cause of BMI increase and obesity in humans in several countries in Western Europe.

Sensitive detection of cytomegalovirus infection in transplant recipients using nucleic acid sequence-based amplification

THE MAJORITY of the adult human population is seropositive for cytomegalovirus (CMV), a member of the b-herpesvirus family. Normally, infection of immunocompetent individuals does not cause disease. In contrast, infection of immunocompromised individuals, such as patients with AIDS and transplant recipients, can cause severe complications and can even lead to death. Therefore, early detection of active CMV infection is necessary to start effective antiviral treatment. Nucleic Acid Sequence-Based Amplification (NASBA) has been developed for the specific amplification of RNA. In previous studies, we evaluated the diagnostic value of monitoring the expression of the CMV immediate early (IE) 1 and late pp67 mRNA using NASBA, in peripheral blood cells of kidney transplant (Tx) patients. The IE 1 mRNA is expressed directly after entrance of the virus into a cell, while pp67 mRNA is expressed in a late stage in the replication cycle. NASBA results were compared to the techniques that are routinely being used for the detection of CMV: pp65-antigenemia, virus culture, and serology. From this study it was concluded that IE NASBA is a very sensitive assay that detects the onset of CMV infection significantly earlier than the other assays. This is especially valuable for patients that are at risk for symptomatic CMV infection, i.e., seronegative patients with an organ of a seropositive donor and/or intense immunesuppression. The sensitivity and specificity values of pp67 NASBA were comparable to those of the antigenemia assay. We set up a similar study to evaluate the diagnostic value of IE and pp67 NASBA for liver Tx patients. The liver Tx patients are usually severely ill, immunesuppression is more intense compared to the kidney Tx patients and they often have anti-rejection therapy. The results for IE NASBA are presented in this report. The evaluation of pp67 NASBA is still in progress.