Although infections with the novel pandemic 2009 influenza A (H1N1) virus (A/H1N1/2009) appeared to be relatively mild during the first summer of circulation ('off season'), there has been significant morbidity and hospitalization and several fatal cases. Thus, rapid detection of A/H1N1/2009 is crucial for efficient treatment and infection control measures. In contrast to seasonal influenza, where point-of-care (POC) rapid antigen tests and direct fluorescent antibody (DFA) staining ensure rapid detection, diagnosis of A/H1N1/2009 has so far been based on RT-PCR. This study retrospectively compared the performance of the Quidel QuickVue POC test, DFA staining and virus isolation with that of RT-PCR for A/H1N1/2009 detection in 526 respiratory specimens collected during the first wave of the outbreak from May to September 2009. A/H1N1/2009 was detected in 9.1 % (48/526) of samples. One hundred and thirty-seven of the A/H1N1/2009 PCR-negative samples were additionally tested using a RealAccurate Respiratory RT-PCR panel, revealing other respiratory viruses (mainly entero/ rhino-and adenoviruses) in 42.3 % (58/137). All methods analysed detected A/H1N1/2009 with excellent specificity but different sensitivities (POC test: 18.2 %; DFA staining: 38.7 %; virus isolation: 45.7 %). Therefore, the POC test was not suitable for diagnosis, detecting A/H1N1/2009 only if present in high concentrations (corresponding median C t value519.0; range516.5-21.4). DFA staining was also able to detect A/H1N1/2009 in specimens with a lower virus concentration (median C t value524.0; range516.5-29.8). Virus isolation, which was positive after a median time of 7.5 days, was too time-consuming. In summary, DFA staining is superior to POC testing and may be appropriate for patients expected to have a rather high level of virus replication. Nevertheless, in DFA-negative specimens, A/H1N1/2009 should be excluded by RT-PCR.

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 nextgeneration 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.Nonmelanoma skin cancer (NMSC) including cutaneous squamous cell carcinoma (SCC) is the most common skin cancer among solid organ transplant recipients (OTRs) and a significant cause of morbidity and mortality in these patients.1,2 The NMSC rates are markedly increased in OTR and AIDS patients compared to the general population. 3,4 Furthermore, OTRs with NMSC tend to have multiple tumor lesions, a higher recurrence rate and the tumors are more likely to metastasize. 5-7Established risk factors for NMSC include exposure to ultraviolet radiation, fair color of the skin and immunosuppressive treatment.1,2 The question whether the increased NMSC rate in immunocompromised individuals-in particular OTR-could reflect a viral involvement in skin carcinogenesis has been investigated intensively. Although there is evidence that human papillomaviruses (HPVs), especially from species beta and gamma, could play a role in the development of NMSC, their etiologic role is discussed controversially. 8,9 Consistent with this, a recent study investigating the cancer transcriptome of 31 cutaneous SCCs by high-throughput sequencing (Illumina) failed to detect active HPV expression in any of the tumor samples.10 This suggests that HPV may not play role in the continuous proliferation of SCC cells.Massive parallel sequencing has opened ...

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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. © 2012 Wiley Periodicals, Inc.

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.