Investigating Bacteria as co-infecting agent in Cerebral Malaria and as an underlying cause of coma of undetermined aetiology in children using 16s rRNA sequencing.. Cause of coma of undetermined aetiology in children using 16s rRNA sequencing.

Background: In malaria endemic areas, Cerebral Malaria (CM) is likely overestimated mainly due to misdiagnosis and comorbidity. Asymptomatic malaria parasitemia can be common and the presence of parasites may imply that a patient fulfils the diagnostic criteria for CM while in fact another infectious cause such as bacteria or virus exists. Children admitted with non-traumatic coma of unknown etiology (UE) may also have an underlying bacterial or viral infection. Here, we investigated whether bacteria are a co-infecting agent in CM and whether it is an underlying cause of coma in children clinically classified as UE using 16s rRNA sequencing.Methods: The V4 region of the 16S rRNA gene was PCR amplified and sequenced using the ion torrent platform from DNA extracted from archived matched plasma and CSF samples from children admitted with culture-confirmed ABM (n=43), CM (n =26), and UE (n =13). Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance) and all genomic analysis done in Quantitative Insights into Microbial Ecology (QIIME ™) software. Hierarchical clustering based on population profiles of most common and abundant taxa was performed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering on the distance matrix of OTU abundance. For each sample, the dominant bacterium was determined by the largest proportion of reads at the genus and species level. Results: There was a strong concordance between CSF bacterial culture results and 16S rRNA sequence data in 76.74% (33/43) of ABM samples. CSF results showed that two patients diagnosed with CM had underlying bacterial infection (Haemophilus influenzae n=1 and Streptococcus pneumoniae n=1(2 of 26 in CM; 7.6%)). Additionally, 2 UE patients also had bacterial infection (H. influenzae n=1 and S. pneumoniae n=1(2 of 13 in UE; 15%)).