High lethality of human adenovirus disease in adult allogeneic stem cell transplant recipients with high adenoviral blood load.

BACKGROUND: Human adenoviruses (HAdV) can cause disseminated disease as a severe complication after haematopoietic stem cell transplantation (SCT) and may originate from the reactivation of latent infections. However, data about the clinical relevance of HAdV DNAaemia and disease in adults are scarce. OBJECTIVES: To retrospectively analyse the outcome of adult allogeneic SCT recipients with high HAdV loads in peripheral blood. STUDY DESIGN: Our diagnostic database was screened for allogeneic SCT recipients with peak HAdV DNAaemia above 1.0×10(4)copies/ml (tested by quantitative real-time PCR) and medical records were reviewed retrospectively. RESULTS: From 1674 adult allogeneic SCT recipients 539 (32.2%) received HAdV DNAaemia testing. In twenty-seven of these HAdV blood loads above 1.0×10(4) (range: 1.6×10(4)-1.8×10(9))copies/ml were observed. Seven of these 27 succumbed to HAdV disease and their median peak HAdV DNAaemia was significantly higher than in patients without HAdV-associated death (1.0×10(8) vs. 3×10(5)copies/ml, p<0.001). T-cell depletion was a risk factor for fatal HAdV disease. HAdV of species C predominated (66.7%) and were of high virulence (6 of 7 fatal cases). HAdV of species B were observed more frequently (n=6) in our study than reported for paediatrics, indicating a different pattern of HAdV reactivation in adults. CONCLUSIONS: The presence of several HAdV-associated deaths in adult SCT recipients with high-level HAdV DNAaemia confirmed the clinical relevance of HAdV DNAaemia testing in adults. Quantitative HAdV DNAaemia testing is a promising tool to predict the outcome of HAdV disease.

Rapid routine detection of enterovirus RNA in cerebrospinal fluid by a one-step real-time RT-PCR assay.

BACKGROUND AND OBJECTIVES: This study provides a one-step transcription/real-time (TaqMan probe) PCR assay (TM-PCR) with new consensus primer and probe sequences for generic detection of human pathogenic enteroviruses including difficult to detect ones like for instance Echovirus 30. The amplicon included parts of domain IV and V of the highly conserved internal ribosomal entry site. Generic detection was confirmed by testing a panel of 41 prototypes representing all five human enterovirus/poliovirus species. STUDY DESIGN AND RESULTS: The 95% detection limit was found to be 100 copies per run using in vitro transcribed coxsackievirus B3 RNA. TM-PCR was compared to an in house nested-PCR assay implemented in detecting enterovirus RNA from CSF samples of patients suffering from meningitis and encephalitis. Concordant results were obtained in all samples (11 positive, 101 negative). Specificity was confirmed with laboratory strains of other neurotropic viruses, and by testing 76 CSF samples of patients with encephalomyelitis disseminata, which all gave negative results. CONCLUSIONS: The new TM-PCR is a convincing alternative to conventional PCR protocols for the diagnosis of enterovirus meningitis. The one-step strategy limits hands on time and cross contamination risk combined with accelerated assay procedure of only 100 min.

Molecular identification of adenovirus sequences: a rapid scheme for early typing of human adenoviruses in diagnostic samples of immunocompetent and immunodeficient patients.

Precise typing of human adenoviruses (HAdV) is fundamental for epidemiology and the detection of infection chains. As only few of the 51 adenovirus types are associated with life- threatening disseminated diseases in immunodeficient patients, detection of one of these types may have prognostic value and lead to immediate therapeutic intervention. A recently published molecular typing scheme consisting of two steps (sequencing of a generic PCR product closely adjacent to loop 1 of the main neutralization determinant epsilon, and for species HAdV-B, -C, and -D the sequencing of loop 2 [Madisch et al., 2005]) was applied to 119 clinical samples. HAdV DNA was typed unequivocally even in cases of culture negative samples, for example in immunodeficient patients before HAdV causes high virus loads and disseminated disease. Direct typing results demonstrated the predominance of HAdV-1, -2, -5, and -31 in immunodeficient patients suggesting the significance of the persistence of these viruses for the pathogenesis of disseminated disease. In contrast, HAdV-3 predominated in immunocompetent patients and cocirculation of four subtypes was demonstrated. Typing of samples from a conjunctivitis outbreak in multiple military barracks demonstrated various HAdV types (2, 4, 8, 19) and not the suspected unique adenovirus etiology. This suggests that our molecular typing scheme will be also useful for epidemiological investigations. In conclusion, our two-step molecular typing system will permit the precise and rapid typing of clinical HAdV isolates and even of HAdV DNA in clinical samples without the need of time-consuming virus isolation prior to typing.

A rapid quantitative PCR-based assay for testing antiviral agents against human adenoviruses demonstrates type specific differences in ribavirin activity.

Human adenovirus (HAdV) infections are increasingly frequent and potentially fatal as a disseminated disease in highly immunocompromised patients. Determining the in vitro sensitivity of HAdV to antiviral agents is not an easy task because HAdV CPE reduction assays are difficult to interpret and may take more than 1 week. We developed a phenotypic assay for testing the antiviral activity during the first round of replication using HAdV DNA concentration as an objective readout within 30 h. After evaluating the assay with cidofovir, we focused on determining the antiviral of ribavirin against different HAdV serotypes because clinical response of HAdV infections towards ribavirin treatment varied considerably. Several HAdV prototypes (1, 2, 5, 11, 31, 34, 48) associated with disseminated infections and clinical isolates were tested. Predominating HAdV of species C were more sensitive to ribavirin (HAdV-2 and -5: EC(50)<10 microM, EC(99) 111 and 104 microM, respectively) than HAdV of other species, for example HAdV-31 (EC(50) 56 microM, EC(99)>500 microM). Differential ribavirin sensitivity of HAdV types may contribute to the variable outcome of ribavirin therapy. Rapid screening of antiviral agents with the rapid qPCR-based assay against a multitude of HAdV serotypes may also facilitate development of future antiviral agents.

Phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy.

Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization epsilon determinant (loops 1 and 2) and the hemagglutination gamma determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. epsilon and gamma determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of epsilon determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (gamma determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (epsilon determinant) and a speculative, not-yet-isolated HAdV prototype (gamma determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the epsilon determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, gamma determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.

Rapid and quantitative detection of human adenovirus DNA by real-time PCR.

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was