Tina Ganzenmueller

Patient, Virus, and Treatment-Related Risk Factors in Pediatric Adenovirus Infection after Stem Cell Transplantation: Results of a Routine Monitoring Program

Human adenovirus (HAdV) infection after hematopoietic stem cell transplantation (HSCT) is associated with significant morbidity and mortality in children. The optimal surveillance and treatment strategies are under discussion. Here, we present data from 238 consecutive pediatric allogeneic HSCT recipients who underwent transplantation in a single center who were included in a prospective, weekly HAdV DNAemia monitoring program by quantitative PCR. HAdV loads >1000 copies/mL were detected in 15.5% of all patients. Despite a low mortality directly attributed to HAdV infection (2 patients, 0.84%), blood HAdV loads >10,000 copies/mL (6.7% of all patients) were significant and independent risk factors for poor survival. We searched for patient, virus, and treatment-related risk factors of HAdV DNAemia and disease. Detection of HAdV in blood before day 50 post transplantation was a major independent risk factor for the development of blood HAdV loads >10,000 copies/mL. HAdV typing revealed A31, C1, and C2 as the predominant pathogens among several other HAdV strains with type C species detected in most patients with severe HAdV disease. Stool HAdV loads were prospectively monitored in 111 patients and correlated with but did not significantly precede detection in blood. Treatment with cidofovir led to stable or reduced viral load in 70% of patients with blood HAdV loads >1000 copies/mL. Thus, early occurrence of HAdV-DNA in blood of pediatric HSCT recipients predisposes for development of high viral loads. Control of HAdV infections was attempted by preemptive cidofovir treatment of patients with high blood HAdV loads or with symptomatic organ infections and correlated with low HAdV-attributed mortality.

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.

Quantification of cytomegalovirus DNA levels in intestinal biopsies as a diagnostic tool for CMV intestinal disease

BACKGROUND CMV intestinal disease (CMV-ID) is a serious complication in immunocompromised patients and mainly diagnosed by clinical, endoscopic and histopathologic findings, whereas qualitative CMV-PCR in tissue samples is not recommended for diagnosis due to its low positive predictive value (PPV). OBJECTIVES To study the interpretation and diagnostic use of CMV-quantification by PCR in intestinal tissue biopsies to recognize CMV-ID. To develop cut-off intestinal CMV-loads attributing illness to CMV. STUDY DESIGN CMV-genome copies in 163 biopsies from the lower intestinal tract of immunocompromised patients were determined by quantitative real-time PCR, normalized to the cell number, and retrospectively compared to histopathological analysis, clinical findings and occurrence of CMV-antigenemia. Two cut-off intestinal CMV-loads, cut-off(histo) and cut-off(clin), were defined using histopathological or clinical criteria as gold standard, respectively. RESULTS CMV was detected in 32.5% of biopsies with a more than six log range of CMV-concentrations (1 x 10(-4)-1.4 x 10(2)copies/cell). Notably, biopsies with histopathologically or clinically confirmed CMV-ID had a significantly higher CMV-load (p<0.001). Cut-off(histo) and cut-off(clin) were defined at the intestinal CMV-load of 0.14 and 0.01 copies/cell, respectively, and improved the PPV. However, cut-off(histo) showed a decreased sensitivity for clinically defined CMV-ID cases. Interestingly, many patients with CMV-ID showed no concomitant CMV-antigenemia, suggesting a localized intestinal CMV-replication. CONCLUSIONS Quantification of CMV in intestinal biopsies is a useful diagnostic tool allowing the definition of cut-off values that can predict CMV-ID more accurate than qualitative PCR results. Further prospective studies have to clarify wether these cut-offs can improve diagnostics and treatment of CMV-ID in day-to-day clinical practice.

No human virus sequences detected by next-generation sequencing in benign verrucous skin tumors occurring in BRAF-inhibitor-treated patients

Patients treated with BRAF inhibitors (e.g. vemurafenib), a novel targeted therapy for advanced melanoma harbouring certain BRAF mutations, develop numerous adverse cutaneous side effects, including skin tumors such as squamous cell carcinoma or non‐malignant verruciform keratinocyte proliferations, termed ‘BRAF‐inhibitor‐associated verrucous keratosis (BAVK) lesions’. These keratinocyte proliferations are believed to be caused by paradoxical hyperactivation of the MAPK pathway in cells with wild‐type BRAF, but mutated RAS. However, due to the clinical and histological verruca‐like appearance of these lesions, additional aetiologic cofactors, such as infectious agents (i.e. oncogenic viruses), might be suspected. Therefore, we performed 454 high‐throughput sequencing of BAVK lesions from vemurafenib‐treated patients on the transcript level to identify actively transcribed viral sequences of known [e.g. human papilloma viruses (HPV)] or even yet‐unknown viruses. Next‐generation sequencing did not identify transcripts of any human viruses out of 1 595 161 reads obtained from BAVK lesions of four patients. Nevertheless, all controls were recognized correctly, and the detection of sequences derived from the cutaneous microbiome (e.g. skin commensals and bacterial phages) confirmed the validity and sensitivity of the sequencing data. Our results are consistent with preliminary histological and immunohistochemical findings recently reported by others, who also failed to detect the expression of HPV proteins in BAVK. Although the patient number is limited and we cannot exclude the possibility of having missed a viral transcript of very low abundance, our study argues against a viral aetiology of BRAF‐inhibitor‐associated verruciform keratoses occurring under vemurafenib.

Next‐generation sequencing fails to identify human virus sequences in cutaneous squamous cell carcinoma

Nonmelanoma skin cancer (NMSC) shows a strongly increased incidence in solid organ transplant recipients (OTRs) and AIDS patients, suggesting an infectious etiology. The role of certain viruses, i.e., cutaneous human papillomaviruses (HPVs), in NMSC in immunosuppressed patients remains controversial. Merkel cell polyomavirus (MCPyV), which was recently identified using high‐throughput sequencing, has been linked to cutaneous proliferations. Here, we aimed to identify novel or known viral sequences at the transcript level in cutaneous squamous cell carcinomas (SCCs) from OTR by using 454 high‐throughput pyrosequencing, which can produce long reads (∼400 bp) and thus is better suited for the analysis of unknown sequences than other sequencing platforms. cDNA libraries from three OTR SCC biopsies were generated and submitted to next‐generation sequencing using a 454 platform. Bioinformatic analysis included digital transcriptome subtraction and—in parallel—reference mapping as an alternative way for depleting human sequences. All control sequences introduced for bioinformatics analysis were recovered correctly. Among 717,029 454‐sequenced transcripts, nearly all identified viral reads were derived from phages. Bacterial sequences originated from the skin flora or environmental sources. Our study did not reveal any transcripts of known oncogenic or related unknown human viruses. These findings suggest that there is no abundant expression of known human viruses, or viruses with a high degree of homology to known human viruses, in cutaneous SCCs of OTR. Further studies are required to exclude the presence of viruses in NMSC, which cannot easily be identified on the basis of sequence homology to known viruses.

Adenoviral load diagnostics by quantitative polymerase chain reaction: techniques and application

Human adenoviruses (HAdV) can cause fatal complications such as disseminated disease especially in a post‐transplant setting. With conventional methods, disseminated HAdV disease could only be diagnosed with delay. Quantification of the HAdV load by real‐time PCR in peripheral blood promised to solve this diagnostic dilemma. Here we review the development, applications and significance of quantitative HAdV PCR.

Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation.

With the establishment of routine virus load (DNAemia) screening for Human adenovirus (HAdV) and Cytomegalovirus (CMV) in post‐transplant care quality standards for quantitative PCR‐assays are increasing. Established real‐time PCR assays were improved with a fully automated DNA‐extraction and with a competitive internal control DNA packaged into a lambda phage which serves as an extraction and amplification control in each sample. HAdV and CMV DNA were detected and quantified simultaneously in various types of diagnostic samples like blood, feces or respiratory tract materials. Inhibition was observed in 0.33–0.66% of over 14,000 diagnostic samples, an infrequent but nevertheless not negligible event, which is observed mainly in stool samples. CMV viral load in broncho‐alveolar lavage fluid (BALF) ranged between positive but below the quantitation limit of 1,000 copies/ml up to 1.8 × 107 copies/ml with a median of 6.0 × 103 copies/ml. Forty‐one (4.7%) BALF samples had a viral load above 5.0 × 105 copies/ml, which was proposed as a threshold for the diagnosis of pneumonia. HAdV viral loads ranged between positive but below the quantitation limit of 1,000 copies/ml to a very high concentration of 1.3 × 1011 copies/ml in stool and BALF samples. A HAdV‐DNAemia of >104 copies/ml was found only in patients with stool viral load of above 105 copies/ml. These data support the hypothesis that quantitation in diagnostic materials other than blood may give valuable diagnostic information and that further evaluation of this approach is reasonable. J. Med. Virol. 84:890–896, 2012.

A human adenovirus species B subtype 21a associated with severe pneumonia

Between 2005 and 2013 six severe pneumonia cases (all requiring mechanical ventilation, two fatal outcomes) caused by human adenovirus type 21 (HAdV-B21) were observed in Germany. So far, HAdV-B21 was mainly associated with non-severe upper and lower respiratory tract infections. However, a few highly virulent HAdV types, e.g. HAdV-B14p1, were previously associated with severe, fatal pneumonia. Complete genomic sequences of the German HAdV-B21 pneumonia isolates formed a single phylogenetic cluster with very high sequence identity (≥ 99.897%). Compared to the HAdV-B21 prototype (only 99.319% identity), all isolates had a unique 15 amino acid deletion and a 2 amino acid insertion in the RGD loop of the penton base which may affect binding to the secondary receptor on the host cells. Moreover, a recombinant E4 gene region derived of HAdV-B3 was identified by bootscan analysis. Thus, the highly virulent, pneumotropic HAdV-B21 was denominated as subtype 21a. Surprisingly, there was 99.963% identity with agent Y/SIBU97 (only 13.4 kb available in GenBank of the 35.4 kb genome) which was associated with 10 fatalities due to cardiopulmonary failure in Sarawak, Malaysia, in 1997. In conclusion, a HAdV-B21 subtype (21a) associated with severe pneumonia in Germany was phylogenetically linked to an adenovirus isolated in Malaysia.

Generation of high-titre virus stocks using BrK.219, a B-cell line infected stably with recombinant Kaposi's sarcoma-associated herpesvirus.

Kaposis sarcoma-associated herpesvirus (KSHV) is a gamma-2-lymphotropic human oncogenic herpesvirus associated with Kaposis sarcoma (KS) and two B-cell lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castlemans disease (MCD). KSHV establishes latency soon after infection in vivo and in vitro. Consequently, it is technically difficult to generate high-titre virus stocks required for infection experiments in tissue culture. Currently used methods of KSHV stock production involve induction of the lytic/productive cycle in PEL cell lines or in adherent cell lines harbouring recombinant KSHV genomes. In this study, the BJAB-derived B-cell line BrK.219, which is infected latently with a recombinant KSHV (rKSHV.219), is used to produce high-titre virus stocks. BrK.219 cells enter the lytic KSHV replication cycle upon cross-linking of B-cell receptors (BCRs) with anti-IgM antibodies without the need for additional, potentially toxic chemical inducers. High cell concentrations can be cultured and induced easily in spinner flasks, saving time and resources. The established protocol allows the generation of KSHV virus stocks with titres of up to 10(6) IU/ml in unconcentrated culture supernatants, representing a 10(3)-10(4)-fold improvement compared to conventional methods.

Varicella Zoster Virus Meningitis in a Young Immunocompetent Adult without Rash: A Misleading Clinical Presentation

Meningitis caused by varicella zoster virus (VZV) is rare in healthy population. Predominantly immunocompromised patients are affected by reactivation of this virus with primary clinical features of rash and neurological symptoms. Here we report a young otherwise healthy man diagnosed with a VZV meningitis without rash. He complained of acute headache, nausea, and vomiting. The clinical examination did not show any neurological deficits or rash. Cerebrospinal fluid (CSF) analysis revealed a high leukocyte cell count of 1720 cells/µL and an elevated total protein of 1460 mg/L misleadingly indicating a bacterial infection. Further CSF analyses, including polymerase chain reaction (PCR) and detection of intrathecal synthesis of antibodies, showed a VZV infection. Clinical and CSF follow-up examinations proved the successful antiviral treatment. In conclusion, even young immunocompetent patients without rash might present with VZV meningitis. CSF examination is a key procedure in the diagnosis of CNS infections but in rare cases the standard values cell count and total protein might misleadingly indicate a bacterial infection. Thus, virological analyses should be considered even when a bacterial infection is suspected.