Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

Metagenomic Analysis of Respiratory RNA Virome of Children with and without Severe Acute Respiratory Infection from the Free State, South Africa during COVID-19 Pandemic Reveals Higher Diversity and Abundance in Summer Compared with Winter Period.

Viral respiratory infections contribute to significant morbidity and mortality in children. Currently, there are limited reports on the composition and abundance of the normal commensal respiratory virome in comparison to those in severe acute respiratory infections (SARIs) state. This study characterised the respiratory RNA virome in children ≤ 5 years with (n = 149) and without (n = 139) SARI during the summer and winter of 2020/2021 seasons in South Africa. Nasopharyngeal swabs were, collected, pooled, enriched for viral RNA detection, sequenced using Illumina MiSeq, and analysed using the Genome Detective bioinformatic tool. Overall, Picornaviridae, Paramoxyviridae, Pneumoviridae, Picobirnaviridae, Totiviridae, and Retroviridae families were the most abundant viral population in both groups across both seasons. Human rhinovirus and endogenous retrovirus K113 were detected in most pools, with exclusive detection of Pneumoviridae in SARI pools. Generally, higher viral diversity/abundance was seen in children with SARI and in the summer pools. Several plant/animal viruses, eukaryotic viruses with unclear pathogenicity including a distinct rhinovirus A type, were detected. This study provides remarkable data on the respiratory RNA virome in children with and without SARI with a degree of heterogeneity of known viruses colonizing their respiratory tract. The implication of the detected viruses in the dynamics/progression of SARI requires further investigations.

Pathogen Profile of Children Hospitalised with Severe Acute Respiratory Infections during COVID-19 Pandemic in the Free State Province, South Africa.

Severe acute respiratory infections (SARI) contribute to mortality in children ≤5 years. Their microbiological aetiologies are often unknown and may be exacerbated in light of coronavirus disease 19 (COVID-19). This study reports on respiratory pathogens in children ≤5 years (n = 84) admitted with SARI during and between the second and third waves of COVID-19 infection in South Africa. Nasopharyngeal/oropharyngeal swabs collected were subjected to viral detection using QIAstat-Dx® Respiratory SARS-CoV-2 Panel. The results revealed viral positivity and negativity detection rates of 88% (74/84) and 12% (10/84), respectively. Of the 21 targeted pathogens, human rhinovirus/enterovirus (30%), respiratory syncytial virus (RSV; 26%), and severe acute respiratory syndrome coronavirus 2 (24%) were mostly detected, with other viruses being 20% and a co-infection rate of 64.2% (54/84). Generally, RSV-positive samples had lower Ct values, and fewer viruses were detected during the third wave. Changes in the circulation patterns of respiratory viruses with total absence of influenza virus could be attributed to measures against COVID-19 transmission, which may result in waned immunity, thereby increasing susceptibility to severe infections in the following season. High viral co-infection rate, as detected, may complicate diagnosis. Nonetheless, accurate identification of the pathogens may guide treatment decisions and infection control.

A decontamination strategy for resolving SARS-CoV-2 amplicon contamination in a next-generation sequencing laboratory.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) amplicon contamination was discovered due to next-generation sequencing (NGS) reads mapping in the negative controls. Environmental screening was undertaken to determine the source of contamination, which was suspected to be evaporation during polymerase chain reaction (PCR) assays while using the coronavirus disease 2019 (COVID-19) ARTIC protocol. A decontamination strategy is hereby documented to assist laboratories that may experience similar amplicon contamination. Routine molecular laboratory environmental screening as a quality control is highly recommended.