Commercial kits were at least as sensitive as in-house tests and potentially easier to use in the field than the latter. This qualitative comparison of real-time reverse transcription PCR assays may serve as a basis for the design of future Ebola virus disease diagnostics.
Geneva University Hospitals, Swiss Office of Public Health, Swiss Agency for Development and Cooperation, and Swiss National Science Foundation.
bRhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5=-untranslated regions (5=UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5=UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ؎10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible. Human rhinoviruses (HRVs) are small nonenveloped viruses containing a positive-strand RNA genome. They belong to the Enterovirus genus, which is part of the Picornaviridae family. HRVs are important human pathogens and the most frequent cause of respiratory infections. Their tropism is not restricted to the upper respiratory tract, and they can cause complications in the lower respiratory tract, especially in children, the elderly, and immunocompromised patients (5-7, 15, 16). Many HRVs, in particular those belonging to the newly discovered HRV-C species, do not grow under traditional cell culture conditions. Therefore, molecular diagnostic tools, such as real-time reverse transcription-PCR (RT-PCR), are the methods of choice for diagnosing HRVs.Thanks to numerous clinical studies, data have been gathered regarding implication of HRV in the exacerbation of underlying diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease (1, 3,7,8,10,17,18,20,21,25), as well as concerning the possible impact of a given HRV species on disease severity (9, 11-13, 23). However, there are inconsistent data about whether viral load is correlated with disease severity, and the threshold cycle (C T ) values from real-time RT-PCR are converted too frequently into viral copies per milliliter without any validation. This is particularly important because many published assays include primers with degenerate nucleotides or probe sequences that are biased toward a given genotype or species (4,14,22,24).We validated previously a two-step real-time RT-...
Background Viral infections are common complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients with steroid-refractory/dependent graft-versus-host disease (GvHD) are highly immunosuppressed and are more vulnerable to infections with weakly pathogenic or commensal viruses. Here, twenty-five adult allo-HSCT recipients from 2016 to 2019 with acute or chronic steroid-refractory/dependent GvHD were enrolled in a prospective cohort at Geneva University Hospitals. We performed metagenomics next-generation sequencing (mNGS) analysis using a validated pipeline and de novo analysis on pooled routine plasma samples collected throughout the period of intensive steroid treatment or second-line GvHD therapy to identify weakly pathogenic, commensal, and unexpected viruses. Results Median duration of intensive immunosuppression was 5.1 months (IQR 5.5). GvHD-related mortality rate was 36%. mNGS analysis detected viral nucleotide sequences in 24/25 patients. Sequences of ≥ 3 distinct viruses were detected in 16/25 patients; Anelloviridae (24/25) and human pegivirus-1 (9/25) were the most prevalent. In 7 patients with fatal outcomes, viral sequences not assessed by routine investigations were identified with mNGS and confirmed by RT-PCR. These cases included Usutu virus (1), rubella virus (1 vaccine strain and 1 wild-type), novel human astrovirus (HAstV) MLB2 (1), classic HAstV (1), human polyomavirus 6 and 7 (2), cutavirus (1), and bufavirus (1). Conclusions Clinically unrecognized viral infections were identified in 28% of highly immunocompromised allo-HSCT recipients with steroid-refractory/dependent GvHD in consecutive samples. These identified viruses have all been previously described in humans, but have poorly understood clinical significance. Rubella virus identification raises the possibility of re-emergence from past infections or vaccinations, or re-infection.
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