key: cord-0901386-7w7fffqk authors: Madi, Nada; Al‐Nakib, Widad; Mustafa, Abu Salim; Habibi, Nazima title: Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait date: 2017-11-11 journal: J Med Virol DOI: 10.1002/jmv.24984 sha: 72df5c13858e70a641dfa8b9d3b25efdc5632003 doc_id: 901386 cord_uid: 7w7fffqk A metagenomic approach based on target independent next‐generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next‐generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non‐respiratory viruses were detected in 14% of the clinical samples, while sequences of non‐human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real‐Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections. involved filtration, tissue culture, electron microscopy, and serology. 6 Among these, the standard gold method of virus detection was cell culture. However, many viruses cannot be easily cultivated, and two milestone innovations solved this problem; polymerase chain reaction (PCR) and DNA sequencing (Sanger method). 6 By using these methods, several important emerging viruses, such as hendra virus, 7 nipah virus, 8 menangle virus, 9, 10 melaka virus, 11 and reston ebola virus, 12 were discovered. Despite the sensitivity of PCR, it can only detect one virus at a time. Multiplex PCR, on the other hand, is used to identify multiple targets for the identification of more than one virus in a single test. However, it is often difficult to standardize the assay by using various primers. Hence, novel approaches that overcome the difficulties of viral detection with the conventional molecular methods are needed to discover novel human viruses. 13 Not surprisingly, virologists were the first to explore the use of sequence-independent, a metagenomic approach using Next Generation Sequencer (NGS) to detect humanassociated viruses. The genome of DNA and RNA viruses can be detected directly after extraction from samples through metagenomic sequencing. 14 Worldwide, respiratory tract infection (RTI) is an important cause of hospitalization among young children and elderly, with significantly high mortality and morbidity. 15 RTI is a group of diseases of both upper or lower respiratory tract. Upper respiratory tract infections (URTIs) include laryngitis, common cold, rhinitis, pharyngitis/tonsillitis, otitis media and rhinosinusitis/sinusitis. On the other hand, lower respiratory tract infections (LRTIs) include bronchitis, bronchiolitis, tracheitis and pneumonia. These respiratory tract infections increase the extent of the problem in patients with chronic comorbidities and asthma, 16 chronic obstructive pulmonary disease (COPD), 17 very young, elderly, 17 and immunocompromized patients. Respiratory viruses account for over 65% of all respiratory infections and 90% of URTIs. 18, 19 Bacteria, however, only represent 10% of all URTIs. 19 Despite the improvement in the molecular techniques for viral discovery, the viral aetiology of RTI is still unknown in a high number of cases either because the tests are ineffective or the virus is unrelated to any of the currently known respiratory viruses. Current diagnostic methods to detect respiratory viruses are mainly based on sequence-dependent molecular amplification techniques such as PCR, which identify a panel of known viruses, and therefore, new respiratory viruses are missed. To overcome the pitfalls associated with PCR approaches based on NGS techniques such as viral metagenomic will become a logical step as routine viral diagnostics on clinical samples. Broaden not only the detection range of viruses but also provide an additional characterization of the detected viruses such as genotypes and subtypes of viruses. However, the efficiency and viability of using such techniques in diagnostic setting require further study. Since there are no studies on the use of metagenomic approach for the identification of pathogens responsible for different diseases, the novelty of this study was to develop a metagenomic approach using NGS for the detection of known and unknown viruses associated with respiratory tract infections among patients in Kuwait. Fresh nucleic acids (RNA and DNA) were extracted from each respiratory sample processed for metagenomic analysis using the Illumina MiSeq (San Diego, CA) platform for NGS according to standard procedures. 20 Briefly, after nucleic acid extraction step, genomic and host DNA was removed from each sample using Ambion DNA-free (Life Technologies) according to manufacturer's instructions to obtain metagenome RNA. Then, 10 ng of DNA-free RNA was integrated into first-strand cDNA synthesis primed by random hexamers and then amplified using whole transcriptome amplification kit (Qiagen, The quality of the sequence data from a total of 86 Miseq runs was The data were analyzed using a computer software "Statistical Package The threshold cycle value (C T ) for each detected virus is shown in Table 1 . (1.01%) ( Table 1) . Table 1 showed that patients with respiratory tract infection due to dual viral infections were hospitalized significantly more often than those with respiratory disease due to a single virus. 25, 26 In agreement with our results, others did not demonstrate any correlation between the presence of multiple viruses and the severity of the respiratory diseases. [27] [28] [29] [30] Respiratory samples from the 86 patients were analyzed further for the presence of viruses by metagenomic sequencing using NGS. It should be noted that this study is the first of its kind to perform metagenomic analysis of the virome of respiratory tract specimens. In conclusion, we conducted a metagenomic sequencing analysis of the viruses in patients with RTI. The results presented in this study demonstrated that metagenomic approach is a promising diagnostic tool in clinical virology in which RT-Real-Time PCR couldn't detect many respiratory viruses that were detected by metagenomic approaches. Such sequence independent detection method will increase the chance to detect the causative agent of viral RTI and to gain information of the viruses that cannot be obtained by the current diagnostic tool. Although many obstacles to the routine use of metagenomic approaches do exist, which include cost, labor intensity and turnover time, it was proved to be highly sensitive in many studies, and it theoretically provides more information regarding virus species/ type for virus diagnosis with the ability of the detection of unknown viruses. The question is probably when metagenomics sequencing will become a routine test for detecting infectious viruses? This approach requires improvement in sample preparation, validated pipelines for read sorting and taxonomic assignation and soon will substitute the current diagnostic tools. Indeed, this novel study provided outstanding information on the association of different viruses with RTIs in Kuwait. Moreover, metagenomics queening approach will be used in the near future to study the viral diversity in other syndromes such as pyrexia of unknown origin and gastroenteritis. 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