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.

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.

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Since the COVID-19 outbreak emerged, SARS-CoV-2 has continuously evolved into variants with underlying mutations associated with increased transmissibility, potential escape from neutralizing antibodies, and disease severity. The SARS-CoV-2 pandemic in South Africa has been characterized by periods of infections with four major epidemic waves. To determine whether the variants driving the epidemic waves at the national level were also driving the epidemic waves at the local level, we performed analysis of a total of 1287 samples from qPCR confirmed SARS-CoV-2 positive individuals. The samples were subjected to viral RNA extraction, genomic amplification, and sequencing. Variant assignment of the viral sequences and mutation identification were conducted using PANGOLIN and SARS-CoV-2 genome annotator, respectively. Our analysis revealed that during the initial part of the first wave, B.1, B.1.1, B.1.1.53, B.1.1.448 and B.1.237 circulated in the Free State province, followed by Beta variant, B.1.351 later in the wave. Although most of the initially detected variants disappeared during the second wave, the Beta variant, B.1.351, persisted. Early in the third wave, the Beta variant, B.1.351, predominated but was replaced by the Delta sub-lineage, AY.45. The fourth wave was characterized by unique emergence of the Omicron sub-variant, BA.1. The data further indicates that SARS-CoV-2 variants driving the epidemic waves in the Free State at the local level correlated with the ones driving the epidemic waves at the national level. Findings from this study highlight the importance of continued genomic surveillance and monitoring of the circulating SARS-CoV-2 variants to inform public health efforts and ensure adequate control of the ongoing pandemic.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics nextgeneration 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.