Astrovirus VA1 identified by next-generation sequencing in a nasopharyngeal specimen of a febrile Tanzanian child with acute respiratory disease of unknown etiology

Dear Editor, Over the past decade, next-generation sequencing (NGS) has become a useful tool to identify novel or emerging infectious agents.1 Although NGS is not yet used as a routine diagnostic tool, it is increasingly used as a second line of investigation for select clinical cases, particularly when routine molecular and serological assays fail to identify a causative pathogen. We developed a NGS-based pipeline (ezVIR) to process NGS data for the identification of viruses in humans2 with the aim of identifying divergent or unexpected viruses in biological specimens of subjects with unexplained clinical syndromes.2,3 We used ezVIR to analyze 30 nasopharyngeal swab specimens collected in a previous study that examined the causes of fever in Tanzanian children4 who were suffering from respiratory symptoms of any type but for whom an infectious agent was not detected (except colonizing bacteria in the nasopharynx) despite extensive investigations, including viral, bacterial and parasitic molecular and serological assays.4 The specimens were pooled in groups of five and assessed according to the specific RNA and DNA library preparation protocols as previously described2 for NGS analysis (paired-end sequencing using the 100-bp protocol with indexing on a HiSeq 2500 (Illumina, San Diego, CA, USA). Unexpectedly, an astrovirus VA1 was identified in pool 1 (Figure 1; Supplementary Table S1). Each specimen in pool 1 (patients T32, T794, T359, T560 and T524) was analyzed individually using the astrovirus VA1-specific real-time reverse-transcription polymerase chain reaction (RT-PCR) targeting the capsid region (forward primer 5′-CCA TCA GCA GTT ACY GGG TCT GT-3′; reverse primer 5′-CGT GGC TCC AGG TGA YTG T-3′; probe 5′-FAM-TTT CCG CAT ATC CC-MGB_NFQ-3′) under the following cycling conditions: 50 °C for 30 min; 95 °C for 15 min; 45 cycles of 15 s at 94 °C and 1 min at 55 °C. Patient T359 was confirmed positive for astrovirus VA1 (Ct value= 29.6; Figure 1). The initial T359 nasopharyngeal specimen was reanalyzed individually by NGS. NGS data were analyzed with ezVIR and completed by performing a de novo analysis. As shown in the ezVIR phase 2 report, 91.5% coverage of the astrovirus VA1 genome was obtained (305 mapped reads), with a maximum coverage depth of 11-fold. Phylogenetic analysis of the capsid fragment consensus sequence (1077 nucleotides long) obtained from de novo assembly5 shows the closest nucleotide homology (97.8%; Figure 1) with the novel human astrovirus (HAstV) VA1/HMO-C-UK1 sequence (KM358468, corresponding to nucleotides 5137− 6213). The presence of a parainfluenza virus type 4 (PIV-4) was also detected in the ezVIR phase 2 report, with lower signals for both parameters (174 mapped reads, 38.5% genome coverage, maximum coverage depth of sevenfold). However, using the FTD Respiratory pathogens 21 commercial assay (Fast-track Diagnostics, Sliema, Malta), the PIV-4 genome was not detected in the specimen, suggesting a viral load below the limit of detection of this assay (the limit of positivity was Ct values ⩽ 37). By contrast, in agreement with the ezVIR phase 2 report for pool 1 (Figure 1), patients T32 and T794 were confirmed positive for PIV-2 (Ct value= 21.6) and PIV-4 (Ct value = 31), respectively, by specific real-time RT-PCR. This finding suggests that the PIV-4-negative result in patient T359 reflects a very low viral load rather than potential mismatches between the assay target and the viral genome sequences of some PIV-4 circulating in this specific area during the study. For this reason, PIV-4 is considered unlikely to be responsible for the respiratory symptoms in this patient. Furthermore, an intrinsic consequence of highly sensitive NGS technology is the increased detection of interspecimen contamination. Therefore, the presence of