key: cord-1022682-oaed60zr authors: Habibi, N.; Uddin, S.; Al‐Salameen, F.; Al‐Amad, S.; Kumar, V.; Al‐Otaibi, M.; Razzack, N. Abdul; Shajan, A.; Shirshikar, F. title: SARS‐CoV‐2, other respiratory viruses and bacteria in aerosols: Report from Kuwait's hospitals date: 2021-06-14 journal: Indoor Air DOI: 10.1111/ina.12871 sha: 6350c49b264ef7f64778a6aef7081d2d639bbf29 doc_id: 1022682 cord_uid: oaed60zr The role of airborne particles in the spread of severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) is well explored. The novel coronavirus can survive in aerosol for extended periods, and its interaction with other viral communities can cause additional virulence and infectivity. This baseline study reports concentrations of SARS‐CoV‐2, other respiratory viruses, and pathogenic bacteria in the indoor air from three major hospitals (Sheikh Jaber, Mubarak Al‐Kabeer, and Al‐Amiri) in Kuwait dealing with coronavirus disease 2019 (COVID‐19) patients. The indoor aerosol samples showed 12–99 copies of SARS‐CoV‐2 per m(3) of air. Two non‐SARS‐coronavirus (strain HKU1 and NL63), respiratory syncytial virus (RSV), and human bocavirus, human rhinoviruses, Influenza B (FluB), and human enteroviruses were also detected in COVID‐positive areas of Mubarak Al Kabeer hospital (MKH). Pathogenic bacteria such as Mycoplasma pneumonia, Streptococcus pneumonia and, Haemophilus influenza were also found in the hospital aerosols. Our results suggest that the existing interventions such as social distancing, use of masks, hand hygiene, surface sanitization, and avoidance of crowded indoor spaces are adequate to prevent the spread of SARS‐CoV‐2 in enclosed areas. However, increased ventilation can significantly reduce the concentration of SARS‐CoV‐2 in indoor aerosols. The synergistic or inhibitory effects of other respiratory pathogens in the spread, severity, and complexity of SARS‐CoV‐2 need further investigation. The transmission of SARS-CoV-2 is believed to be via humanto-human contact, touching infected surfaces, and inhalation of the exhaled virus in respiratory droplets. [4] [5] [6] Recent studies have also provided evidences of aerosol-mediated spread of SARS-CoV-2, 7-16 mostly from confined spaces in hospitals and quarantine camps. 15, [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] Thus, it becomes an issue of preeminent importance to understand the microbial assemblage in indoor aerosols to ensure the safety of healthcare personnel and uninfected people involved in the care of COVID patients. Drawing similarities with the previous SARS epidemic in Hong Kong, the airborne spread was suggested as the primary transmission pathway of the type I coronavirus. 29 The understanding of the presence and concentration of SARS-CoV-2 in aerosols becomes challenging, because of the difficulties in sampling virus-containing aerosols in real-world settings and problems in their quantification at low concentrations. 4, 7 Van Doremalen, et al. 2020 30 showed the viability of SARS-CoV-1 and SARS-CoV-2 in aerosols and on surfaces like plastic, stainless steel, copper, and cardboard. Both SARS-CoV-1 and SARS-CoV-2 remained viable in the aerosol after 3 h. However, there was a reduction in infectious titer from 10 4 . 3 to 10 3.5 TCID 50 /L of air for SARS-CoV-1, and 10 3.5 to 10 2.7 TCID 50 /L of air for SARS-CoV-2. It has been reported that the viral load is high in the respiratory tract of infected persons and can be transmitted through droplets via coughing or sneezing, 31 and it is a widely accepted fact. Still, there is no information reported on whether there are viral loads in the exhaled air that probably adds to indoor air. Besides, in closed environments such as hospital wards, droplets can remain suspended for more than 10 min and cover long distances, potentially maintaining their ability to transmit disease. [32] [33] [34] The information on the levels and concentrations of SARS-CoV-2 and its spread in indoor scenarios is still in its infancy. To the best of our knowledge, there has been no assessment made so far on SARS-CoV-2 concentration in the indoor air of the hospitals in Kuwait. A recent study has suggested the need for investigations on adsorption, survival, and behavior of the SARS-CoV-2 virus within the aerosol community to understand its spread and its interaction with other viral communities 6 that are possibly having a cumulative effect on virulence and infectivity. The clinical presentation of the illness caused by SARS-CoV-2 appears to range from mild or asymptomatic to severe and fatal respiratory illness. 35 The clinical manifestation of COVID-19 fatalities is reported to be heterogeneous. 36, 37 One of the risk factors is the presence of other co-infections that require management and treatment. 38 The frequency of respiratory failure and mortality among patients with COVID-19 and co-infections is high, 36 similar to influenza, 39 SARS-CoV-1, 40 Respiratory Syndrome coronavirus (MERS-CoV). 41 This baseline study demonstrates the presence of viral and pathogenic bacterial species present in the aerosols, including the SARS-CoV-2 in the indoor atmosphere of hospitals dealing with COVID-19 patients in Kuwait. A custom-designed sampler was used, the details of which have been described elsewhere, 7 and the culture-independent quantitative polymerase chain reaction (qPCR) method was employed for identification and quantification. This study was conducted to identify and characterize the novel coronavirus, associated respiratory viruses, and pathogenic bacteria in indoor aerosols of three major hospitals in Kuwait. Samples were collected using a specialized sampler developed for this purpose 7 ; it is efficient in capturing the entire aerosol load and safe for use, as it lyses the captured microbes (bacteria, fungi, and viruses) during sampling itself. This sampling device utilizes a variable speed suction pump (Tisch, Environmental International) with a flow controller that allows air to pass through gas wash bottles. Three glass gas wash bottles attached in series were filled with 100 ml of TRIzol ® (APB Biosciences) (henceforth mentioned as Trizol). The sampler was deployed and the air was pumped through this sampling setup for 2 hours @ 30 L per min. In total, 3.6 m 3 (3600 L) of air was collected from four, seven, and two locations in Sheikh Jaber Hospital (SJH), Mubarak Al Kabeer hospital (MKH), and Al Amiri hospital (AMH), respectively, in Kuwait (Table 1) . With ongoing restrictions imposed due to the COVID-19 pandemic, prior approval of the Ministry of Health (MOH), Kuwait, was obtained for sample collection. In this study, only the locations approved by the MOH within the hospital premises were covered. Samples were also collected from five areas within the Kuwait Institute for Scientific Research (KISR) to see the aerosol microbial load in a non-hospital setting. Two ambient air samples from an outdoor site in a residential area were collected as control samples for this study. The standard procedure of RNA isolation from Trizol was followed to purify total RNA. 42 The highsensitivity Qubit HS ssRNA kit was used for fluorometric estimation 43 of isolated RNA on a Qubit 4 fluorometer (Thermo Scientific). All these steps were performed under BSL2 cabinets. • This study utilized a simplified and safe sampling technique that can be employed even in labs that fall short of biosafety level III compliance. • The RNA-based culture-independent approach used in this study quantifies only the viable bacterial cells that have direct health implications in indoor hospital air. The hospitals in Kuwait are constructed as per the Ministry of Health Kuwait hospital design guidelines. The design parameters ensure protection of outside air intakes; use of a variable-air-volume system to minimize the need of air exchange while ensuring that there is no stagnation of air inside the wards. The ventilation is met by central air conditioning system, with filtration and humidification provisions. The fresh air intakes are located at least 7.62 m from the exhaust outlets of ventilating systems, combustion vents (including those serving rooftop air handling equipment), medical-surgical vacuum systems, plumbing vents, or areas that may collect vehicular exhaust or other noxious fumes; however, this is not applicable to relief air. The air conditioning design parameter also accounts for the prevailing wind directions and proximity to other structures ensuring appropriate clearances. The plumbing vents are at least 3.05 meters above the intake, while the bottom of outdoor air intakes serving central systems shall be 0.91 m above roof level, as most of the central air conditioning systems are located above the roof. The air exchange in the wards used for patient care is 6-10 times per hour. To maintain asepsis control, airflow supply, and exhaust, the air movement is generally controlled to ensure movement of air from "clean" to "less clean" areas. 44 The culture-independent RNA-based 16sRNA gene amplification was used to estimate the live bacterial load of the collected samples. 45 (Table S1 ). We performed a multiplex qPCR to detect the presence of sixteen air- Respective targets of each organism are given in Table S2 . The respiratory panel III kit 46,47 from VIASURE (CerTest, Biotec) was used to set up the multiplex qPCR reaction. As per the manufacturer's instruction, the PCR reaction was assembled in a volume of 20 µl by adding 5µl of isolated RNA to each well to the reconstituted master mix. 46 The PCR was performed on the CFX96TM Deep Well (BioRad) thermal cycler. The cycling conditions of the reaction included the steps of reverse transcription (15 min at 45°C) followed by initial denaturation (2 min at 95°C) and then 45 cycles of denaturation (10 s at 95°C), annealing/ extension (50 s at 60°C). Fluorogenic data were collected during the extension step through the FAM, ROX, Cy5, and HEX channels as per the respective targets (Table S2 ). The sample was considered positive if the Ct value was ≤40 after baseline subtraction and fluorescence drift correction. All the wells were checked for amplification of internal controls, positive controls, and non-amplification in negative controls. All the reactions were done in duplicate and the Ct values were estimated by the CFX MaestroTM software (BioRad). The qPCR was conducted on aerosol samples to detect and quantify the SARS-CoV-2 on the isolated RNA samples. The VIASURE based on two targets that are ORF1ab and N genes were used for this purpose. 4 The PCR reaction was assembled as per the kit instructions. A reaction volume of 20 µl was assembled by adding 5 µl of isolated RNA to the rehydrated master mix. 48 (Table S3) . Samples exhibiting Ct above 40 were excluded from the analysis. The culture-independent RNA-based bacterial quantification is recently being used for the detection of metabolically active bac- The respiratory panel PCR revealed some common (frequently detected at most sampling locations) and unique (not so frequently de- In addition to these common viruses, few unique viruses were de- The indoor aerosols showed positive amplification of N-gene of the SARS-CoV-2 at three, two, and one location at MKH, SJH, and F I G U R E 2 Common and unique respiratory viruses including SARS-CoV-2 and pathogenic bacteria detected by the qPCR in three major hospitals of Kuwait (MKH, SJH &AMH). KISR represents samples collected from a non-hospitalized setting and OUT signifies aerosols in ambient air from a residential area used as a control in the present study AMH, respectively ( Table 2 ). In contrast, negligible amplification of Nosocomial aerosols have made news highlights, especially the notion that SARS-CoV-2 also follows an airborne transmission. The reality is that COVID-19 is just the latest episode of a century-long attack on the human race by zoonotic respiratory viruses and a much longer assault by several respiratory bacterial pathogens. 49, 50 The co-inhalation of respiratory viruses and bacterial pathogens has been hypothesized to influence the respiratory stress induced by the SARS-CoV-2 virus. 6 Hospital-acquired infections cause one of the most severe complications in COVID patients admitted in ICU and among the immunosuppressed people. 6 The results from this study provide an insight into the viral and bacterial load in the indoor aerosols in hospitals in Kuwait serving to combat COVID-19. Infectious bacteria-laden aerosols are generated in hospitals by COVID-19 and other patients through breathing, talking, coughing, and sneezing. 49 According to a risk assessment study, hospital staff, and people having frequent encounters with healthcare facilities, a risk ratio (RR) of 2.5 was established for acquiring viral or bacterial infections. 51, 52 In the current investigation, we have used the RNAbased qPCR approach to quantify viable bacterial cells in aerosol samples. The technique has been applied in determining live bacterial cells in water 45 and biofilms. 53 We report an average of 1. The highest number of live bacterial cells were recorded in outdoor air, but none was pathogenic. Among the viral communities only, rhinovirus (Average Ct-23.16) was found in outdoor air. This was very perplexing to find rhinovirus in outdoor air, even in higher concentration than indoor air. However, in congruence with our findings, The susceptibility of acquiring an infectious agent largely de- 85 The minimum infective dose for SARS-CoV-2 via inhalation of droplets was estimated to be 300 particles using computational simulations of the nasopharynx. 86 Other studies on transgenic mice, 87 cynomolgus macaques (Macaca fascicularis), 88 and African green monkeys, 88 CoV-2 at these low concentrations within the aerosols will lead to inhalation by individuals in hospital (healthcare professionals and non-COVID patients) who might become susceptible to infection. It poses a question that requires further investigation, that is, will such a low level of exposure lead to the development of antibodies against COVID-19 in these individuals? It is evident from our findings that common respiratory viruses exist in the indoor air of hospitalized settings in a higher frequency as We are thankful to Dr. Scott W. Fowler for editing the manuscript. The authors explicitly state that there was no conflict of interest. The peer review history for this article is available at https://publo ns.com/publo n/10.1111/ina.12871. The entire dataset used in this study is available as supplementary data files S1, S2, and S3 to this submission. S. 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