Monitoring for cytomegalovirus infection in organ transplant recipients: Analysis of pp65 antigen, DNA and late mRNA in peripheral blood leukocytes

Abstract

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA-polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1-2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load > 100/2 x 10(5) positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow-up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse.

Clinical Utility of Viral Load in Management of Cytomegalovirus Infection after Solid Organ Transplantation

“…However, some studies have demonstrated that a patient with a small number of pp65-positive cells may still develop CMV disease, while some patients with larger numbers of positive cells resolve their infection spontaneously, without antiviral therapy (106). These divergent observations and the lack of a defined standardized threshold are likely due to the various risks among SOT recipients (highest among lung recipients and lower among kidney recipients), their preexisting CMV-specific immunity (highest among CMV Dϩ/RϪ patients compared to others), and the severity of pharmacologic immunosuppression (highest with lymphocyte-depleting drugs) (83,84,88,(106)(107)(108)(109).…”

New Strategies for Prevention and Therapy of Cytomegalovirus Infection and Disease in Solid-Organ Transplant Recipients

“…The presence of a small number of antigen-positive cells following solid-organ transplantation generally indicates asymptomatic infection, whereas a large number implies an increased likelihood of CMV disease; the exact cutoffs for this assay, however, remain to be defined and may vary among different types of transplant recipients and among individual assays (142,266,311,323,466,505). While antigenemia is highly sensitive and specific for the diagnosis of CMV infec- tion, both the specificity and positive predictive value of the test for the diagnosis of CMV disease are less impressive; that is, patients with asymptomatic infection are frequently antigenemia positive (313,505).…”

“…Although PCR is extremely sensitive and specific in detecting the presence of CMV DNA, there is the concern that a positive signal resulting from the presence of very few DNA copies may not differentiate between replicating and latent viruses (91,142,157,507). On the other hand, negative CMV detection by PCR strongly advocates against CMV infection (33,278,347,448).…”

Quantitation of the spliced late gene of human cytomegalovirus and its kinetics during experimental infection

“…This time course is in concordance with the onset of viral DNA replication [16]. RT-PCR assays of different target sequences from IE to late stages of transcription have been developed for clinical diagnosis of hCMV disease [1,4,5,13]. However, with the exception of IE transcripts all mRNAs tested are unspliced (pp150, pp65, pp67).…”

“…Immediately early (IE) gene transcription is independent of protein synthesis, early transcription (E) is independent of viral DNA synthesis, and late gene transcription corresponds to viral DNA replication. Reactivation of hCMV from the latent state is associated with the transcription of late genes encoding phosphoproteins (pp150), glycoproteins (gB), proteins of unknown functions and mRNAs with unknown translation products [2,4]. As the majority of hCMV genes are unspliced, discrimination of viral DNA and mRNA using polymerase-chain-reaction (PCR) primers spanning exon/exon boundaries is complicated.…”