Detection of β-herpesviruses in allogenic stem cell recipients by quantitative real-time PCR
Introduction
The subfamily of the β-herpesviruses includes the human cytomegalovirus (CMV) and the two closely related roseoloviruses, human herpesvirus (HHV)-6, and HHV-7. All three β-herpesviruses are widespread pathogens with a high seroprevalence in the adult population (Tanaka-Taya et al., 1996). Primary infection appears commonly in early childhood (Okuno et al., 1989). It is usually subclinical, or may cause mononucleosis (CMV) or febrile illness, including exanthema subitum (HHV-6) (Yamanishi et al., 1988, Torigoe et al., 1995), resolving in the majority of cases without complications. After primary infection, the viruses persist in the infected individual through life. Like all other herpesviruses, the members of the β-subfamily can be reactivated from their state of latency in immunocompromised hosts, i.e. after stem cell or bone marrow transplantation, or in the course of HIV infection. In contrast to the self-limiting disease observed in immunocompetent patients, reactivation or reinfection in immunocompromised hosts causes severe clinical diseases (Ljungman, 2002). In CMV reactivation, several severe end-organ diseases like interstitial pneumonia, gastroenteritis, hepatitis, retinitis, and myelosuppression (Ljungman, 1996) are observed. In HHV-6 and HHV-7 reactivation, the clinical picture remains unclear (Chan et al., 1997). Sporadic case reports have shown an association of HHV-6 reactivation in immunosuppressed patients with complications like encephalitis, interstitial pneumonia, fever, myelosuppression, and a high-grade graft-versus-host-disease (GVHD) (Campadelli-Fiume et al., 1999). HHV-6 infections have also been reported to induce immunosuppresion by itself (Flamand et al., 1995) which leads in the case of co-infection with other pathogens, such as parvovirus B19, to severe complications (Rohayem et al., 2001). The pathogenicity of HHV-7 in immunocompromised hosts and in primary infection remains unclear. Interestingly, a potential increase in virulence of both roseoloviruses in the course of a simultaneous CMV reactivation has been postulated (Mendez et al., 2001), leading to a greater risk of CMV-associated disease. As described in previous studies, a direct association of CMV load in plasma with an increased risk for developing CMV-associated disease was observed (Spector et al., 1998). In this context, a rapid, sensitive and specific quantitation of human herpesvirus load in patient samples, particulary in the post-transplantation period, is of interest (Aritaki et al., 2001). For this purpose, conventional methods like polymerase chain reaction (PCR) or determination of pp65-antigenemia have been widely used. These methods have however disadvantages, requiring intensive handling (pp65-antigenemia) or being in comparison to more recent technologies like real-time PCR inaccurate. In past years, quantitation of CMV-load was routinely performed with a commercially available test (COBAS AMPLICOR™ CMV MONITOR Test, Roche) (Platzbecker et al., 2001, Caliendo et al., 2001). Recent developments have made use of the 5′-exonuclease assay (TaqMan), particularly for clinical routine testing.
In this study, three 5′-exonuclease assays were developed to assess the viral load of HHV-6 (types A and B), CMV, or HHV-7 in clinical samples. In addition, CMV DNA loads were validated in comparison to results generated in a restrospective setting using the COBAS AMPLICOR system.
Section snippets
Patients and clinical samples
Clinical samples were collected from day 5 to 392 post-transplantation for routine diagnostic purposes. In all patients, pp65-antigenemia, the seropositivity for CMV before and after transplantation (CMV-Enzygnost, Dade Behring), as well as the clinical status were assessed in the context of the management of the disease in the stem cell transplantation unit of the University Hospital of Dresden, Germany. End-organ diseases were diagnosed by X-ray imaging and pO2, pCO2, and pH values in the
Analytical sensitivity and inter-assay variability of the assays
The analytical sensitivity of the assays was assessed by amplifying a 10-fold serial dilution of standard plasmid DNA, followed by probit analysis as described by others (Smieja et al., 2001). As shown in Fig. 1, comparison of the 95% sensitivities of all assays showed similar results for HHV-7 and HHV-6 (17 GE/reaction), followed by CMV and Kanamycin (90% sensitivity with 20 GE/reaction, respectively). Interestingly, the CMV real-time PCR was able to amplify down to 5 GE/reaction (250 GE/ml) with
Discussion
In this study, two quantitative real-time PCRs for the detection of CMV and HHV-6, and an additional real-time PCR to differentiate between HHV-6 types A and B were developed on the LightCycler system. A quantitative HHV-7 real-time PCR was also adapted to the LightCycler system to cover the entire spectrum of human β-herpesviruses. All assays displayed similar sensitivities and could be run on one system with identical reagents and cycling conditions. This allows an optimized detection and
Conclusion
In conclusion, the results suggest that the detection and monitoring of CMV, HHV-6, and HHV-7 play an important role in the management of patients undergoing stem cell transplantation. In this context, sensitive, specific and quantitative real-time PCRs are of interest. As observed in other studies, the impact of the β-herpesviruses on stem cell transplantation recipients can be ranked from high risk pathogen (CMV) to low risk pathogen (HHV-7), HHV-6 being in-between a potential risk for
Acknowledgements
We are grateful to Axel Rethwilm for his support during the initial phase of the study. We are indebted to Enno Jacobs for his support. We thank Marc Cartellieri and Daniel Lüftenegger for technical assistance, as well as Wolfram Rudolph for review of the manuscript.
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