Metagenomic Next-Generation Sequencing of Nasopharyngeal Swabs in Acute Febrile Illness in Cambodia

ABSTRACT

Background. There is growing recognition of metagenomic next-generation sequencing (mNGS) as a valuable diagnostic tool capable of providing unbiased pathogen detection, but data on performance in low-resource settings remains scant. Here, we use mNGS of nasopharyngeal (NP) swabs taken from subjects in Cambodia to identify potential pathogens causing acute febrile illness. Methods. Febrile subjects aged 2 months to 65 years were enrolled in a crosssectional study conducted across 4 tertiary hospitals in Cambodia. NP swabs were collected at hospital presentation. Depending on reported symptom constellations, sera was also taken in a subset of subjects for comparison of mNGS results. RNA was isolated from biosamples, converted to cDNA libraries, and sequenced on a NextSeq2000 (Illumina). Raw sequence reads were stripped for host reads and aligned to NCBI nucleotide and protein databases using a cloud-based bioinformatics platform (CZID). Results. NP swabs were collected from 97 subjects between April 2020 and June 2021. Subjects were predominantly male (53.6%) and young (median age 3 years [IQR 1-25]). Pathogens were identified in 42 (43.2%) NP swabs;of these, 26 (61.9%) were respiratory viruses including 9 rhinovirus, 7 coronavirus (1 SARS-CoV-2), and 5 respirovirus cases. Co-infection was identified in 3 subjects with coronavirus and respirovirus (N=2) and coronavirus and rhinovirus (N=1). Of subjects with paired sera and NP samples (N=61), 18 (29.5%) had positive NP swabs but negative sera, 7 (11.5%) had negative NP swabs but positive sera, 12 (19.7%) had positive NP swabs and sera, and 24 (39.3%) had negative NP swabs and sera. Pathogen hits correlated in NP swabs and sera in 10 of 12 subjects, including six subjects with chikungunya. Conclusion. mNGS can be successfully implemented in low-resource settings to identify emerging pathogens and common respiratory pathogens, including coinfecting pathogens, from NP swabs of febrile patients. mNGS may also be able to detect chikungunya from NP swab alone, raising the possibility of non-invasive diagnostics for infections associated with high viremic states.