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25 pages, 392 KiB

Open AccessReview

Diagnostic Approach to Pneumonia in Immunocompromised Hosts

by Nadir Ullah, Ludovica Fusco, Luigi Ametrano, Claudia Bartalucci, Daniele Roberto Giacobbe, Antonio Vena, Malgorzata Mikulska and Matteo Bassetti

J. Clin. Med. 2025, 14(2), 389; https://doi.org/10.3390/jcm14020389 - 9 Jan 2025

Cited by 1 | Viewed by 4402

Abstract

In immunocompromised patients, pneumonia presents a diagnostic challenge due to diverse etiologies, nonspecific symptoms, overlapping radiological presentation, frequent co-infections, and the potential for rapid progression to severe disease. Thus, timely and accurate diagnosis of all pathogens is crucial. This narrative review explores the latest advancements in microbiological diagnostic techniques for pneumonia in immunocompromised patients. It covers major available microbiological tools for diagnosing both community-acquired and hospital-acquired pneumonia, encompassing a wide spectrum of pathogens including bacterial, viral, fungal, and parasitic. While traditional culture methods remain pivotal in identifying many pneumonia-causing etiologies, their limitations in sensitivity and time to results have led to the rise of non-invasive antigen tests and molecular diagnostics. These are increasingly employed alongside cultures and microscopy for more efficient diagnosis, mainly in viral and fungal infections. Lastly, we report the future of pneumonia diagnostics, exploring the potential of metagenomics and CRISPR/Cas13a for more precise and rapid pathogen detection in immunocompromised populations. Full article

(This article belongs to the Special Issue Update on Acute Severe Respiratory Infections)

26 pages, 1444 KiB

Open AccessArticle

Assessment of Rapid MinION Nanopore DNA Virus Meta-Genomics Using Calves Experimentally Infected with Bovine Herpes Virus-1

by Gaelle Esnault, Bernadette Earley, Paul Cormican, Sinead M. Waters, Ken Lemon, S. Louise Cosby, Paula Lagan, Thomas Barry, Kate Reddington and Matthew S. McCabe

Viruses 2022, 14(9), 1859; https://doi.org/10.3390/v14091859 - 24 Aug 2022

Cited by 8 | Viewed by 3846

Abstract

Bovine respiratory disease (BRD), which is the leading cause of morbidity and mortality in cattle, is caused by numerous known and unknown viruses and is responsible for the widespread use of broad-spectrum antibiotics despite the use of polymicrobial BRD vaccines. Viral metagenomics sequencing on the portable, inexpensive Oxford Nanopore Technologies MinION sequencer and sequence analysis with its associated user-friendly point-and-click Epi2ME cloud-based pathogen identification software has the potential for point-of-care/same-day/sample-to-result metagenomic sequence diagnostics of known and unknown BRD pathogens to inform a rapid response and vaccine design. We assessed this potential using in vitro viral cell cultures and nasal swabs taken from calves that were experimentally challenged with a single known BRD-associated DNA virus, namely, bovine herpes virus 1. Extensive optimisation of the standard Oxford Nanopore library preparation protocols, particularly a reduction in the PCR bias of library amplification, was required before BoHV-1 could be identified as the main virus in the in vitro cell cultures and nasal swab samples within approximately 7 h from sample to result. In addition, we observed incorrect assignment of the bovine sequence to bacterial and viral taxa due to the presence of poor-quality bacterial and viral genome assemblies in the RefSeq database used by the EpiME Fastq WIMP pathogen identification software. Full article

(This article belongs to the Section Animal Viruses)

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11 pages, 2554 KiB

Open AccessArticle

Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms

by Vanja Miljanić, Jernej Jakše, Denis Rusjan, Andreja Škvarč and Nataša Štajner

Viruses 2022, 14(5), 921; https://doi.org/10.3390/v14050921 - 28 Apr 2022

Cited by 2 | Viewed by 3237

Abstract

Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked. Full article

(This article belongs to the Special Issue Plant Virus Surveillance and Metagenomics)

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16 pages, 6767 KiB

Open AccessArticle

HTS-Based Diagnostics of Sugarcane Viruses: Seasonal Variation and Its Implications for Accurate Detection

by Martha Malapi-Wight, Bishwo Adhikari, Jing Zhou, Leticia Hendrickson, Clarissa J. Maroon-Lango, Clint McFarland, Joseph A. Foster and Oscar P. Hurtado-Gonzales

Viruses 2021, 13(8), 1627; https://doi.org/10.3390/v13081627 - 17 Aug 2021

Cited by 17 | Viewed by 4153

Abstract

Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses. Full article

(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)

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15 pages, 2149 KiB

Open AccessArticle

Two Years of Viral Metagenomics in a Tertiary Diagnostics Unit: Evaluation of the First 105 Cases

by Verena Kufner, Andreas Plate, Stefan Schmutz, Dominique L. Braun, Huldrych F. Günthard, Riccarda Capaul, Andrea Zbinden, Nicolas J. Mueller, Alexandra Trkola and Michael Huber

Genes 2019, 10(9), 661; https://doi.org/10.3390/genes10090661 - 29 Aug 2019

Cited by 45 | Viewed by 6703

Abstract

Metagenomic next-generation sequencing (mNGS) can capture the full spectrum of viral pathogens in a specimen and has the potential to become an all-in-one solution for virus diagnostics. To date, clinical application is still in an early phase and limitations remain. Here, we evaluated the impact of viral mNGS for cases analyzed over two years in a tertiary diagnostics unit. High throughput mNGS was performed upon request by the treating clinician in cases where the etiology of infection remained unknown or the initial differential diagnosis was very broad. The results were compared to conventional routine testing regarding outcome and workload. In total, 163 specimens from 105 patients were sequenced. The main sample types were cerebrospinal fluid (34%), blood (33%) and throat swabs (10%). In the majority of the cases, viral encephalitis/meningitis or respiratory infection was suspected. In parallel, conventional virus diagnostic tests were performed (mean 18.5 individually probed targets/patients). mNGS detected viruses in 34 cases (32%). While often confirmatory, in multiple cases, the identified viruses were not included in the selected routine diagnostic tests. Two years of mNGS in a tertiary diagnostics unit demonstrated the advantages of a single, untargeted approach for comprehensive, rapid and efficient virus diagnostics, confirming the utility of mNGS in complementing current routine tests. Full article

(This article belongs to the Special Issue Viral Diagnostics Using Next-Generation Sequencing)

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