key: cord-0702481-nsjqfdn3
authors: Chong, Yoong Min; Chan, Yoke Fun; Jamaluddin, Mohamad Fadhil Hadi; Hasan, M. Shahnaz; Pang, Yong Kek; Ponnampalavanar, Sasheela; Omar, Sharifah Faridah Syed; Voon, Kenny Gah Leong; Sam, I-Ching
title: Detection of respiratory viruses in adults with suspected COVID-19 in Kuala Lumpur, Malaysia
date: 2021-10-20
journal: J Clin Virol
DOI: 10.1016/j.jcv.2021.105000
sha: b34616e3cf4d832c2f7f225158268a719ba717d4
doc_id: 702481
cord_uid: nsjqfdn3

BACKGROUND: Reports of co-circulation of respiratory viruses during the COVID-19 pandemic and co-infections with SARS-CoV-2 vary. However, limited information is available from developing countries. OBJECTIVES: We aimed to investigate the incidence of respiratory viruses in adult patients with suspected COVID-19 in Kuala Lumpur, Malaysia. STUDY DESIGN: We collected 198 respiratory samples from adult patients hospitalized with suspected COVID-19 in a single teaching hospital in Kuala Lumpur in February-May 2020 and tested combined oro-nasopharyngeal swabs with the NxTAG Respiratory Pathogen Panel (Luminex) and Allplex RV Essential (Seegene) assays. Forty-five negative samples further underwent viral metagenomics analysis. RESULTS: Of the 198 samples, 74 (37.4%) had respiratory pathogens, including 56 (28.3%) with SARS-CoV-2 and 18 (9.1%) positive for other respiratory pathogens. There were five (2.5%) SARS-CoV-2 co-infections, all with rhinovirus/enterovirus. Three samples (6.7%; 3/45) had viruses identified by metagenomics, including one case of enterovirus D68 and one of Saffold virus genotype 6 in a patient requiring ICU care. Most of the COVID-19 patients (91.1%; 51/56) had mild symptoms but 5.4% (3/56) died. CONCLUSION: During the early COVID-19 period, common respiratory viruses other than SARS-CoV-2 only accounted for 9.1% of hospitalization cases with ARI and co-infections with SARS-CoV-2 were rare. Continued surveillance is important to understand the impact of COVID-19 and its associated public health control measures on circulation of other respiratory viruses. Metagenomics can identify unexpected or rare pathogens, such as Saffold virus, which is rarely described in adults.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS- 85 CoV-2) in December 2019, the coronavirus disease 2019 (COVID-19) pandemic has posed a 86 major public health threat and economic burden worldwide. As of September 2021, Malaysia 87 has had more than 2.1 million confirmed cases and 23,000 deaths [1] . A few studies have 88 reported decreases in common respiratory viral infection incidence and low rates of SARS- 89 CoV-2 co-infections with common respiratory viruses during the COVID-19 pandemic 90 period [2] [3] [4] . However, in developing tropical countries such as Malaysia, rapid testing for 91 SARS-CoV-2 has been the main focus among persons with respiratory symptoms and broad 92 testing for other respiratory viruses is performed inconsistently. Hence, there is little 93 information on co-circulating respiratory pathogens during COVID-19 in these countries. Over the last decade, viral metagenomics analysis has emerged as a sensitive pan-99 pathogen assay for diagnosis and identification of unknown or rare viral pathogens. 100 Metagenomics analysis can be used to detect undiagnosed pathogens in clinical samples that 101 are negative for or not assessed with routine diagnostic tests, [5] [6] [7] and would be of particular 102 interest for countries in Southeast Asia such as Malaysia, which are potential hotspots for 103 emerging infections. 104 Here we report the detection rate of SARS-CoV-2 co-infections and other respiratory 105 infections in suspected COVID-19 adult patients during the early wave of COVID-19 106 (February to May 2020) in Malaysia. We used multiplex molecular respiratory panels 107 including assays for select agents and viral metagenomics analysis to comprehensively 108 capture potential respiratory pathogens that might have been missed out by conventional 109 diagnostic approaches. [8] , SARS-CoV [9] and bacterial select agents (B. pseudomallei [10] , C. burnetii [11] , B. 132 anthracis [12] , F. tularensis [13] , and Y. pestis [14] ) were also tested using published real-133 time PCR assays with minor modifications and the primer and probes sequences as described 134 in Supplementary Material. Where collected, 5-10 ml of venous blood was cultured in 135 aerobic and anaerobic bottles for up to 5 days using the BacT/ALERT Virtuo system 136 (bioMérieux, France).

Of the 124 samples which were negative for all tested pathogens, forty-five cases 139 which either required ICU admission or mechanical ventilation, and/or had C-reactive protein 140 ≥ 10mg/L (which is associated with viral respiratory infection) [15] were randomly selected 141 for viral metagenomics analysis. These samples were subjected to a sequence-independent, Raw sequencing reads were trimmed to remove sequencing adapters and low quality 154 sequencing reads. Host sequences and NTC reads were filtered out before identifying 155 potential viral pathogens using IDseq portal (https://idseq.net) [17] . Detection of a virus was 156 reported if non-overlapping reads from ≥3 distinct genomic regions were identified [18] . The coverage of the corresponding viruses. PCR confirmatory testing of respiratory viral hits was Saffold virus), and the third was human papillomavirus type 4, usually found on skin (Table   222 2). Phylogenetic analysis of the partial VP1 of EV-D68 sequences revealed that our EV-D68 CoV-2 patients (Table 3) . However, none of these were found to be statistically significant 253 predictors following multivariable analysis. 

Chi-square test was used for categorical variables and Mann-Whitney U test was used for age. Significant p-269 values (p<0.05) are indicated with asterisks. However, none of these factors remained as significant independent 270 predictors on multivariable analysis.

Severe cases are those admitted to ICU, requiring ventilation or resulting in death.

Significant positive blood culture isolates including Escherichia coli ( to previously reported rates ranging from 1.8 -9.7% [4, [22] [23] [24] . The low rate of other 284 respiratory pathogens of 9.1% (18/198) we detected during this pandemic has been described 285 elsewhere [25] [26] [27] , and contrasts with pre-pandemic papers using multiplex PCR testing 286 reporting respiratory infection rates ranging from 22-63% [28] [29] [30] . Notably, influenza virus, 287 which usually accounts for 5-14% of respiratory infections in Malaysia [31, 32] , was much 288 reduced to only 0.5% (1/198) with COVID-19 and those with other respiratory pathogens.

Our study has some limitations. The study was limited to a single hospital and only 

Press statement from the Director-General of Health

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