Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow.

BACKGROUND: In recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking. METHODS: A total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7). RESULTS: The optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses). CONCLUSIONS: Although the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

[Functional immunoassays in the setting of infectious risk and immunosuppressive therapy of non-HIV immunocompromised patients]. / Place des tests immunitaires fonctionnels dans la prise en charge du risque infectieux et de la gestion des thérapies immunosuppressives chez les patients immunodéprimés non-VIH.

The holistic approach of the human immune system is based on the study of its components collectively driving a functional response to an immunogenic stimulus. To appreciate a specific immune dysfunction, a condition is mimicked ex vivo and the immune response induced is assessed. The application field of such assays are broad and expanding, from the diagnosis of primary and secondary immunodeficiencies, immunotherapy for cancer to the management of patients at-risk for infections and vaccination. These assays are immune monitoring tools that may contribute to a personalised and precision medicine. The purpose of this review is to describe immune functional assays available in the setting of non-HIV acquired immune deficiency. First, we will address the use of theses assays in the diagnosis of opportunistic infections such as viral reactivation. Secondly, we will report the usefulness of these assays to assess vaccine efficacy and to manage immunosuppressive therapies.

Serologic and genetic characterization of HIV type 1 subtypes on Reunion Island.

The aim of this study was to determine the HIV subtypes present on Reunion Island, a French island located in the Indian Ocean, where the first case of AIDS was diagnosed in 1987. Paired sera and blood samples were collected between September 1996 and September 1997 from 53 HIV-1-positive patients. Subtyping was performed by serotyping with a previously described subtype-specific enzyme immunoassay (SSEIA) and by genotyping with the heteroduplex mobility assay (HMA). When samples gave uninterpretable results with either of the methods, or discordant results, the V3 env region was sequenced and genetic subtypes were determined by phylogenetic analysis. Genetic subtyping showed that 48 of 53 patients were infected with HIV-1 subtype B (90.5%). This high prevalence of subtype B on Reunion Island is probably due to the regular exchanges with metropolitan France. The other five patients were infected with subtype A (9.5%); they had been directly linked to African populations. Of the 48 subtype B samples, 44 (91.7%) were correctly subtyped by SSEIA and 43 (89.6%) by HMA. However, the SSEIA did not allow the subtyping of A strains in three of five patients. Thus, the SSEIA could be an alternative routine technique for screening subtype B versus nonsubtype B HIV-1 strains.

DNA arrays, electronic noses and tongues, biosensors and receptors for rapid detection of toxigenic fungi and mycotoxins: a review.

This paper presents an overview of how microsystem technology tools can be applied to the development of rapid, out-of-laboratory measurement capabilities for the determinations of toxigenic fungi and mycotoxins in foodstuffs. Most of the topics discussed are all under investigation within the European Commission-sponsored project Good-Food (FP6-IST). These are DNA arrays, electronic noses and electronic tongues for the detection of fungal contaminants in feed, and biosensors and chemical sensors based on microfabricated electrode systems, antibodies and novel synthetic receptors for the detection of specific mycotoxins. The approach to resolution of these difficult measurement problems in real matrices requires a multidisciplinary approach. The technology tools discussed can provide a route to the rapid, on-site generation of data that can aid the safe production of high-quality foodstuffs.

Transient infection of astrocytes with HIV-1 primary isolates derived from patients with and without AIDS dementia complex.

We have studied the replication capacity of primary HIV-1 isolates obtained from four AIDS patients in astrocytes. Two patients (P1 and P2) had neurological manifestations without AIDS Dementia Complex (ADC). The other two patients (P3 and P4) had ADC. Two astrocytoma cell lines and normal fetal astrocytes were inoculated with each of these four viral isolates. Viral DNA and mRNA synthesis and also protein accumulation were followed at various times after infection. We found that tumoral as well as fetal astrocytes were susceptible to HIV-1 infection. Three of four viral isolates (P2, P3, P4) were able to infect astrocytes. Both ADC viral isolates (P3, P4) infected astrocytes with identical transcriptional patterns: rev, nef and unspliced mRNAs were expressed for 2 days after infection. The non-ADC patient (P2) with the isolate leading to viral replication in astrocytes had an HIV-1 associated multifocal demyelinating neuropathy. In this case, only nef and unspliced mRNAs were detected a few days after virus inoculation. In all cases, infection of astrocytes was transient and the level of unspliced mRNAs in infected astrocytes was lower than in chronically HIV-1 infected T cells. More extensive work would allow a better understanding of the role of astrocytes in ADC.

Influence of three successive antiviral treatments on viral heterogeneity in nonresponder chronic hepatitis C patients.

The purpose of the present study was to assess the viral diversity of hepatitis C virus (HCV) in six nonresponder patients during three unsuccessful treatments. These patients were treated successively with IFN-alpha2a (IFN-alpha) at a posology of 3.10(6) units (MIU) three times a week, 10 MIU three times a week, and a combination of IFN-alpha (3 MIU) plus ribavirin (1,000 mg/day). However, only two chronically infected patients could be included in the study due to the persistence of HCV RNA during the three successive treatments. The viral diversity was analysed by cloning and sequencing the HVR-1 region. The treatment of the two nonresponder patients was associated with the persistence of a wide diversity in the viral population and with the emergence of new or minor variants. Under the influence of standard doses of IFN-alpha, a rearrangement of the quasispecies present was observed at this time point. No significant change in viral load or in the complexity of the quasispecies was observed. A second treatment with a high dose of IFN-alpha induced a significant decrease in the associated viral load and, in one case, resulted in a radical change of the viral diversity. Administration of a combination of IFN-alpha and ribavirin did not affect the evolution of the variants but was followed by the emergence of various multiple variants. These results reinforce the hypothesis of the presence of preexisting quasispecies best adapted to the host environment, and therefore resistant to any current therapy.

Detection of human and animal rotavirus sequences in drinking water.

Reverse transcription-PCR analysis of drinking water in the homes of 56 children suffering from rotaviral gastroenteritis has shown the presence of the rotavirus genome in four samples. These strains were different from human rotaviruses detected in the children's feces, as determined by sequencing of the VP7-amplified fragments-three of them of animal origin (porcine or bovine) and one of human origin.