key: cord-0891269-425g88vl authors: Quintão, Tatyane de Souza Cardoso; Slavov, Svetoslav Nanev; de Oliveira, Pâmela Maria; Bezerra, Rafael dos Santos; Cassemiro, Évelin Mota; Alves, Priscilla Pedrette de Melo; Gontijo, Carolina Carvalho; Martins, Fabiano dos Anjos Pereira; Gurgel, Helen da Costa; Noronha, Elza Ferreira; Ramalho, Walter Massa; de Araújo, Wildo Navegantes; Pereira, Alex Leite; Haddad, Rodrigo title: Viral metagenomics in nasopharyngeal swabs of Brazilian patients negative for SARS-CoV-2 unveils the presence of Chikungunya virus infection date: 2021-12-05 journal: J Infect DOI: 10.1016/j.jinf.2021.12.001 sha: 576bad9b3029f3bd59e9a821818819a1d7d70f83 doc_id: 891269 cord_uid: 425g88vl nan . The application of metagenomics is useful for the management of COVID-19 patients and gives essential information for the presence of co-infections which additionally can worsen the clinical prognosis. The authors of the above-cited study tested exclusively patients with a SARS-CoV-2 confirmed infection. A syndromic surveillance data obtained in Brasilia, Brazil showed that only 33% of patients were SARS-CoV-2 positive. We therefore applied viral metagenomics on SARS-CoV-2 negative samples in order to characterize the co-circulating respiratory viruses in the Federal District of Brazil. Currently, pan-pathogen assays based on viral metagenomics uncover a variety of fungus, bacterial and viral co-infections in COVID-19 patients [2, 3] which justifies the application of metagenomics in order to detect other circulating respiratory viruses. Moreover, there is no information on which respiratory viruses most commonly cocirculate in the examined Brazilian region. In this study, samples were obtained from residents of the Cidade Estrutural, Federal The metagenomic analysis of 160 NS samples assembled into 20 pools revealed Chikungunya virus (CHIKV) genomic reads into two pools (namely, 11 and 18). These two pools generated a total abundance of 10,654,714 reads from which just 3357 were classified as viral (0.03%), which was normal as viruses did not make part of the normal microbial composition of the nasopharynx. We classified as CHIKV 46 reads in pool 11 and 92 reads in pool 18. The identification of CHIKV reads in NS puzzled us and therefore we proceeded to test samples from the CHIKV-positive pools using Reverse Transcription PCR (RT-PCR). RT-PCR assays were carried out with two previously described sets of primers detecting both circulating in Brazil CHIKV genotypes [5] in Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System. Two samples of the NS showed RT-PCR positive results for CHIKV, one sample from each CHIKVpositive pool. The amplification threshold (Ct) of both samples demonstrated well-presented viral load (Cts were 28 and 26 respectively) (Fig. 1B) . The positive samples belonged to patients with suspicion of SARS-CoV-2 infection. One of them was a female, 33 years old patient who reported fever, headache, jaundice, myalgia and earache. The other patient was male individual with 41 years of age, who reported fever, myalgia, retro-orbital pain, cough, headache, algesia, jaundice, anosmia, hyporexia and consciousness changes. Here, the potential of metagenomics to detect unsuspected viral agents in any type of clinical sample has been demonstrated, while it was also revealed that arthropod-borne viruses (arboviruses) were largely neglected during the SARS-CoV-2 pandemic. While the NS is a common procedure to diagnose influenza, SARS, MERS-CoV, COVID-19 and other respiratory infections, the sampling of blood is the procedure of choice for arboviral diagnosis [6] . Although saliva has also been a suitable clinical sample for CHIKV RNA detection during the first week after symptoms onset [7, 8] , to our knowledge this is the first report showing the metagenomic evaluation and confirmation of CHIKV RNA in NS samples. The detection of CHIKV RNA was only possible due to the application of metagenomics, since CHIKV infection was not suspected as a causative agent of the reported symptoms. The presence of multiple viruses co-circulating in a symptomatic population may hide the presence of less-expected viral agents, mainly during the COVID-19 pandemic, which was our case. Therefore, viral metagenomics is a powerful diagnostic tool not only for analysis of the viral diversity in clinical samples but also provides important information regarding epidemiological surveillance and circulating viruses [9] . In support of this, we performed phylogenetic analysis of the obtained viral contigs, which identified the circulation of ECSA CHIKV genotype, the most widely spread CHIKV genotype in Brazil (Fig. 1C) . In summary, our study demonstrates the use of viral metagenomics for identification of unsuspected viral agents in NS of patients showing respiratory symptoms, but negative for SARS-CoV-2 RNA. This investigation draws the attention to the circulation of viruses, which are clinically important but have been largely neglected/unsuspected during the SARS-CoV-2 pandemic but must be included in the differential diagnosis of the patients. Despite the significant advantages of the metagenomics for virus identification, some issues like costefficiency, need of high-cost equipment and laboratory expertise must be carefully analyzed in regards to metagenomic application for diagnostic purposes especially in resource-limited countries. and positive sample from pool 11 (3). C) Approximate maximum likelihood tree of the obtained Chikungunya virus (CHIKV) contigs during the metagenomic analysis of nasopharyngeal swabs (2 contigs of 655 and 718 bp belonging to the nonstructural polyprotein). In the phylogenetic reconstruction 397 complete CHIKV genomes obtained from the GenBank were used under the GTR+G4+F nucleotide substitution model with a statistical support of 1000 bootstrap replicates. The phylogenetic tree showed 3 major clades comprising the CHIKV genotypes. Our samples (red dots) were clustered along the CHIKV East-Central-South African genotype, which by far is the most common genotype in Brazil. SARS-CoV-2 and co-infections detection in nasopharyngeal throat swabs of COVID-19 patients by metagenomics Metagenomic Analysis Reveals Clinical SARS-CoV-2 Infection and Bacterial or Viral Superinfection and Colonization Co-infections in people with COVID-19: a systematic review and meta-analysis Detection of Influenza A(H3N2) Virus RNA in Donated Blood Chikungunya virus in US travelers returning from India Find the right sample: A study on the versatility of saliva and urine samples for the diagnosis of emerging viruses Detection of chikungunya virus in saliva and urine Infectious Chikungunya Virus in the Saliva of Mice, Monkeys and Humans The Potential Role of Clinical Metagenomics in Infectious Diseases: Therapeutic Perspectives The authors declare that there is no conflict of interest.