2.1.1. Description of the Hospitals.

Mazandaran Province, with its administrative center in Sari, is one of the most important regions in northern Iran for the identification and treatment of respiratory virus cases. Patients are admitted to several medical centers, with Imam-Khomeini Hospital in Sari and Razi Hospital in Qaem-Shahr being the primary facilities for hospitalization and treatment of respiratory virus cases. The geographical coordinates of the hospital locations are as follows: Imam-Khomeini Hospital in Sari (36.5659° N, 53.0586° E) and Razi Hospital in Qaem-Shahr (36.4631° N, 52.8600° E).

At the time of sampling (between May and August 2022), Mazandaran Province was experiencing the fifth wave of the COVID-19 outbreak, classified as a “red zone” due to high transmission rates. Epidemiological investigations identified the Beta variant (lineage B.1.351) as the dominant strain in the region during this period.

Imam-Khomeini Hospital in Sari is a 410-bed facility with 25 wards. Wastewater from all wards is directed to Wastewater Treatment Plant A (WWTP A). Razi Hospital in Qaem-Shahr is a 253-bed facility with 15 wards, and its wastewater is treated at Wastewater Treatment Plant B (WWTP B). Both treatment plants operate using an extended aeration-activated sludge process. The treatment units at Imam-Khomeini Hospital include screening, aeration, sedimentation, and a chlorination basin, with a flow rate of 130 m³/day. Razi Hospital’s treatment plant consists of a selector pond, aeration, sedimentation, chlorination, and a digester basin, with a flow rate of 140 m³/day. Disinfection at both plants is achieved using perchlorate powder.

During the sampling period, 1,120 patients with suspected COVID-19 were hospitalized in Mazandaran Province based on clinical diagnosis, of which 198 were admitted to Intensive Care Units (ICUs) due to acute respiratory symptoms. Sari, with a population of 309,820, reported 30–58 COVID-19 cases, while Qaem-Shahr, with a population of 204,953, reported 47–68 cases during the same period.

This study did not involve human participants, human specimens, or personal data. All samples were collected from environmental sources (air, surfaces, and wastewater) in hospital settings. Therefore, ethics approval and consent to participate were not applicable.

2.1.2. Pre-sampling measures.

Before commencing each sampling session, all test kits, which had been previously autoclaved, were disinfected using a 70% alcohol solution in accordance with the protocol established by the Centers for Disease Control and Prevention (Atarod et al., 2022). Dulbecco’s Modified Eagle’s Medium (DMEM) liquid was then prepared in a sterile culture medium suitable for coronavirus growth and placed in impregnated glass tubes (Nagle et al., 2022). These prepared impingers, containing the culture medium, were stored at a temperature below 4 °C, and their lids were securely sealed until sampling began.

2.1.3. Collection, storage, and transfer of wastewater samples.

In this comprehensive study, a total of 72 grab wastewater samples (36 influent and 36 effluent) were collected through weekly sampling over an 18-week period (May to August 2022) from two hospital wastewater treatment plants, providing sufficient temporal coverage to capture fluctuations in viral shedding during the study period. As a precautionary measure, all collected wastewater samples were transferred into 100 mL autoclaved plastic bottles and transported to the laboratory within two hours using cold packs (4 °C). The samples were stored at −80 °C until analyzed using reverse transcription-polymerase chain reaction (RT-PCR). The electronegative membrane-vortex (EMV) method, as described by Haramoto et al. (2013) and adapted by Wilhelm et al. (2022) [20], was employed to concentrate viral particles from the wastewater samples. A 100 mL sample was spiked with 1 mL of 2.5 mol/L MgCl₂. The sample was then filtered through a cellulose-ester membrane (0.8 μm pore size, 47 mm diameter; Merck Millipore, Billerica, MA, USA). The membrane was placed in a plastic tube containing 10 mL of elution buffer and mixed vigorously using a ball-shaped stirrer. The eluate was collected in a new 50 mL plastic tube. This process was repeated with 5 mL of elution buffer until a final volume of approximately 15 mL was achieved. The eluate was clarified by centrifugation (10 minutes, 2000 × g, 4 °C), and the supernatant was further filtered through a 0.45 μm membrane filter. Finally, the filtrate was concentrated using Centriprep YM-50 ultrafiltration to achieve a higher viral concentration. All wastewater samples were analyzed in duplicate, and the mean Ct value was reported for positive samples. Samples with discordant results (positive/negative between duplicates) were re-analyzed in triplicate, and the consensus result was reported.

2.1.4. Active air sampling in hospitals.

Active air sampling was conducted using impingers, a highly efficient method for collecting indoor air samples by impinging an air stream into a liquid medium [23]. This device included 1) SKC sampling pumps (AirChek® Serie) with suction power and flow rate of 5 L.min^-1; 2) an air flow calibrator (with CalChek Cat. No. 375-0550N); 3) the glass impinge; 4) the glass impinge trap; 5) the stainless steel holder for area sampling; and 6) trap sorbent to protect the pump from both organic and inorganic vapors. The sampling pumps were equipped with polytetrafluoroethylene (PTFE) membrane filters (37 mm diameter, 0.3 μm pore size), which were calibrated before each sampling session using an electronic calibrator (Bios Defender 510 H, USA) [2].

Air sampling was conducted in specialized wards for confirmed COVID-19 cases, COVID-19 ICUs, and administrative departments. The sampling pumps were positioned at a height of 1.5 to 2 meters above the ground, corresponding to the respiratory level of individuals, and at horizontal distances of 0.5, 1, 2, and 5 meters from the beds of confirmed COVID-19 patients. In the CT scan ward, sampling was performed at the head and body locations, as well as at a height of 1.5 meters above the ground.

During the sampling period, natural ventilation was absent, and mechanical ventilation systems were turned off. The sampling areas were not disinfected prior to sampling. To maintain indoor air temperature in the ICU during spring, a split air conditioner was installed at the entrance of the ward.

After identifying the sampling points, the inlet and outlet ports of the impingers were unsealed, and the inlet was connected to the SKC sampling pumps. The pumps, pre-adjusted and calibrated, were allowed to operate for 4 hours. Ambient temperature and humidity were measured using an HD50 hygrometer (KIMO, France), and particulate matter mass concentration (PMmass) in the indoor air was quantified using a portable Grimm 108 device (Grimm Aerosol Technik, Germany) for weighing and counting dust particles.

At the end of the 4-hour sampling period, the pumps were turned off, and the impingers containing the culture medium were disconnected. The inlet and outlet ports were resealed with sterile caps, and the samples were refrigerated at 4 °C before being promptly transferred to the virology reference laboratory for viral detection.

Air sampling at each distance and location was performed once due to logistical constraints associated with 4-hour sampling duration and limited impinger availability. However, the extended sampling time (4 hours) integrated viral particles over time, providing a representative measure of airborne contamination. Additionally, sampling was repeated on different days (three separate sessions) at selected locations to assess temporal variability.

2.1.5. Fomite sampling in hospitals.

In this study, the spread of the virus was measured in both Infection wards (housing confirmed COVID-19 cases) and non-Infection wards as part of a highly efficient sampling approach. Three Infection wards—dedicated to COVID-19 patients, including the special ward, ICU, and CT scan ward—and one non-infectious ward (the working room of the hospital’s environmental health department) were selected for sampling. High-risk environments and high-touch surfaces were identified as sampling locations in accordance with World Health Organization (WHO) guidelines [24].

Routine cleaning and disinfection of the sampling locations in both hospitals were performed twice daily using Septi Surface solution, which contains 500 mg/L chlorine (Behban Pharmed Lotus®, Iran). The active ingredients of this product—ethanol, chlorhexidine digluconate, and alkyl dimethyl benzyl ammonium chloride—are effective against a wide range of bacteria, viruses, and fungi. Cleaning rounds were conducted in the morning (9:00–10:30 AM) and afternoon (2:30–4:00 PM). Sampling was carried out before the morning cleaning round to avoid interference from disinfection activities.

A total of 92 samples (46 per hospital) were collected from high-touch surfaces and critical points in various parts of the hospitals. Sampling was performed using nylon flocked swabs, which were removed from their packaging and moistened with 2 mL of virus transport medium (serum-free DMEM, Biosera, UK). The swabs were rotated with sufficient pressure in multiple directions over a minimum surface area of 25 cm². The swabs were then transferred to tubes containing 200 mL of twice-distilled sterile water mixed with antibiotic, protein stabilizer, and buffer solution.

The swab samples were labeled, stored at 4 °C, and transported to the virology laboratory for analysis. Control samples were collected alongside experimental samples to ensure accuracy. After processing, the samples were tested using PCR, and results were reported as positive or negative for viral presence.

Each high-touch surface was sampled at a single time point during each weekly visit. However, to capture temporal variation in contamination levels, sampling was repeated weekly over the 18-week study period at selected high-touch surfaces (bed rails, doorknobs, bedside tables) in COVID-19 wards and ICUs.

2.1.6. RNA extraction and real-time PCR.

All collected samples were immediately immersed in viral transfer medium and stored at −80 °C until analysis. Viral RNA was extracted using a commercial viral RNA extraction kit (Viragene, Iran) following the manufacturer’s instructions. Briefly, 200 μL of sample was mixed with 400 μL of lysis buffer containing guanidine isothiocyanate and incubated at room temperature for 10 minutes. After adding 600 μL of binding buffer (ethanol-based), the mixture was passed through a silica membrane column by centrifugation at 8,000 × g for 1 minute. The column was washed twice with 500 μL of wash buffers, and RNA was eluted in 50 μL of RNase-free water by centrifugation at 10,000 × g for 2 minutes. The extracted RNA was stored at −80 °C until amplification.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect SARS-CoV-2RNA targeting two viral genes: the nucleocapsid (N) gene and the RNA-dependent RNA polymerase (RdRp) gene, as described by Atarod et al. (2022) [23] with minor modifications.

2.1.7. Quality control measures.

To ensure the reliability and validity of the results, several quality control measures were implemented throughout the sampling and analysis process. For each sampling session, field blank samples (sterile containers exposed to the sampling environment without sample collection) were transported alongside actual samples to monitor potential cross-contamination during handling and transport. A total of 10 field blanks (5 for each hospital) were collected and analyzed; all tested negative for SARS-CoV-2 RNA.

For RT-PCR analysis, nuclease-free water was used as a negative control in each PCR run to rule out reagent contamination. Additionally, for surface sampling, sterile swabs moistened with virus transport medium were processed as negative controls (one per sampling session). All negative controls consistently yielded negative results.

Known SARS-CoV-2 RNA standards (synthetic controls targeting N and RdRp genes) were included in each RT-PCR run to validate assay performance and confirm successful amplification. Positive controls consistently showed expected Ct values (range: 28–32).

A subset of samples was analyzed in duplicate to assess reproducibility: 10% of wastewater samples (7 samples), 15% of air samples (7 samples), and 10% of surface samples (9 samples). The coefficient of variation (CV) for Ct values ranged from 1.2% to 4.8%, indicating excellent reproducibility.

All sampling equipment was autoclaved or disinfected with 70% ethanol before use. Personnel wore appropriate personal protective equipment (PPE) including gloves, masks, and gowns, and changed gloves between sampling sites to prevent cross-contamination. Sampling was consistently performed before morning cleaning rounds to avoid interference from disinfection activities. The primer and probe sequences were as follows:

1. N gene assay:
   1. Forward primer: 5’-GGGGAACTTCTCCTGCTAGAAT-3’
   2. Reverse primer: 5’-CAGACATTTTGCTCTCAAGCTG-3’
   3. Probe: FAM-5’-TTGCTGCTGCTTGACAGATT-3’-BHQ1
2. RdRp gene assay:
   1. Forward primer: 5’-GTGARATGGTCATGTGTGGCGG-3’
   2. Reverse primer: 5’-CARATGTTAAASACACTATTAGCATA-3’
   3. Probe: VIC-5’-CAGGTGGAACCTCATCAGGAGATGC-3’-BHQ1

Each 20 μL reaction contained 5 μL of RNA template, 10 μL of 2X Master Mix (One-Step RT-PCR Master Mix, containing reverse transcriptase and Taq polymerase; Viragene, Iran), 0.5 μL of each primer (10 μM), 0.25 μL of probe (10 μM), and nuclease-free water to reach the final volume.

Thermal cycling was performed on a Rotor-Gene Q real-time PCR cycler (Qiagen, Germany) under the following conditions: reverse transcription at 50°C for 15 minutes, initial denaturation at 95°C for 3 minutes, followed by 45 cycles of denaturation at 95°C for 10 seconds and combined annealing/extension at 55°C for 30 seconds. Fluorescence signals (FAM for N gene, VIC for RdRp gene) were collected at the end of each annealing/extension step.

A sample was considered positive if it met both of the following criteria: (1) cycle threshold (Ct) value ≤ 38 for at least one target gene, and (2) exponential amplification curves with typical sigmoidal shape. Samples with Ct values between 38 and 40 were re-analyzed in duplicate; if at least one replicate showed Ct ≤ 40 with exponential amplification, the sample was reported as weakly positive. Samples with Ct > 40 or no amplification were considered negative. For quality assurance, each run included no-template controls (nuclease-free water) and positive controls (synthetic SARS-CoV-2 RNA).

Given the exploratory and descriptive nature of this environmental surveillance study, descriptive statistics (frequencies and percentages) were used to summarize the detection rates of SARS-CoV-2 RNA across different sample types and locations.

2.2.1. Systematic review methodology.

A systematic review was conducted to identify and synthesize studies reporting the persistence of enveloped viruses in air, surfaces, and wastewater environments. The review was performed in accordance with the PRISMA 2020 guidelines to ensure transparent and comprehensive reporting. The study selection process is presented in Fig. 1 (PRISMA flow chart), detailing the number of records identified, screened, included, and excluded with corresponding reasons.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 1. PRISMA flow diagram.

https://doi.org/10.1371/journal.pone.0345644.g001

2.2.2. Inclusion and exclusion criteria.

Studies were included if they investigated the persistence, survival, or detection of enveloped viruses such as SARS-CoV-2, influenza viruses, or other coronaviruses in environmental matrices (air, surfaces, or wastewater), were published in English between 1995 and September 2025, and reported experimental or field-based data. Studies were excluded if they were review articles, conference abstracts without full data, modeling-only studies without empirical validation, or lacked quantitative information on environmental persistence.

2.2.3. Information sources.

The literature search was conducted across PubMed, Scopus, ScienceDirect, and Web of Science databases, complemented by manual screening of reference lists and relevant gray literature. The last search was completed on September, 2025.

2.2.4. Search strategy.

Search strategies were constructed using combinations of MeSH terms and keywords related to enveloped viruses and environmental persistence. A representative search strategy used for PubMed was as follows: (“enveloped virus” OR “SARS-CoV-2” OR “influenza virus” OR “coronavirus” OR “HcoV” OR “TGEV” OR “MHV” OR “Enterovirus” OR “Rhinovirus” OR “Respiratory Syncytial Virus” OR “Hepatitis” OR “HIV” OR “Adenovirus”) AND (“environmental persistence” OR “surface contamination” OR “airborne transmission” OR “wastewater-based epidemiology” OR “viral stability”). Equivalent strategies were adapted for PubMed, Scopus, ScienceDirect, and Web of Science using Boolean operators and database-specific syntax. Filters were applied to restrict results to English-language studies published between 1995 and 2025.

2.2.5. Study selection and data extraction.

All identified records were imported into a reference management software, and duplicates were removed prior to screening. Two reviewers independently screened titles and abstracts for relevance, followed by full-text assessments to confirm eligibility. Discrepancies were resolved through discussion and consensus. Extracted data included virus type, environmental matrix (air, surface, or wastewater), detection method (RT-qPCR, culture-based assay, or plaque assay), persistence duration, environmental parameters such as temperature, humidity, and pH, and key study findings.

2.2.6. Outcomes.

The primary outcome of interest was viral persistence, expressed as survival time or reduction in viral titer within different environmental matrices. Secondary outcomes included environmental and physicochemical factors influencing persistence such as temperature, humidity, organic matter, and pH as well as the detection and quantification methods used in each study.

2.2.7. Risk of bias assessment.

The methodological robustness and potential sources of bias in the included studies were systematically evaluated using a modified version of the Joanna Briggs Institute (JBI) Critical Appraisal Checklist for Systematic Reviews and Research Syntheses (Aromataris and Munn, 2020). This tool comprises eleven criteria designed to assess the rigor of the study design, reliability of data collection, and transparency of reporting. Each item was rated as “Yes” (2 points), “Unclear” (1 point), or “No” (0 points), producing an overall quality score ranging from 0 to 22.

Two independent reviewers performed the assessment to minimize subjective bias, and any differences in scoring were resolved through mutual discussion and consensus. Studies achieving a total score of 18 or above were classified as high quality (low risk of bias), those scoring between 12 and 17 as moderate quality (moderate risk of bias), and those below 12 as low quality (high risk of bias).

The results of this evaluation provided a comprehensive overview of the methodological soundness of the included studies and helped identify areas where bias may have affected the reliability of the findings. A detailed summary of the quality assessment for all included studies is provided in the Supplementary Materials (S1 Table).

3.1.1. Wastewater based epidemiology (WBE).

The detection of SARS-CoV-2 RNA in hospital wastewater indicates the presence of the virus in the digestive system and feces of infected patients [21,25]. Based on Table 1, out of 72 wastewater samples collected from two WWTPs, 47 (65.28%) tested positive for SARS-CoV-2 RNA. Positive ratios were 61.11% (22/36) for WWTP A and 69.44% (25/36) for WWTP B. All influent samples from both WWTPs were positive during the sampling period (May-August 2022), while effluent samples showed variable detection rates. In WWTP A, four effluent samples tested positive (two in May and two in June), but none were detected in July or August, indicating improved treatment efficiency. In WWTP B, effluent samples were positive only in May (27.78% or 5/18) and late August (11.11% or 2/18), with no detections between June and early August.

Download:

• PNG
  larger image
• TIFF
  original image

Table 1. Detection of new coronavirus RNA in wastewater samples along with characteristics of hospital wastewaters.

https://doi.org/10.1371/journal.pone.0345644.t001

The persistence of SARS-CoV-2 RNA in wastewater is influenced by factors such as organic pollutants, pH, temperature, and disinfection practices [26]. In our study, positive samples were observed at temperatures ranging from 20–28°C, suggesting that temperature alone did not significantly affect viral RNA detection. However, other studies have highlighted the role of temperature in viral stability, with lower temperatures favoring longer persistence [27,28].

Chlorination was used as a pre-treatment step before wastewater discharge. Residual chlorine levels were critical; when absent, effluent samples tested positive for SARS-CoV-2 RNA. Zhang et al., (2020) demonstrated that free chlorine at 6.5 mg/L with 1.5 hours of contact time effectively eliminated SARS-CoV-2 RNA (Zhang et al., 2020). However, RNA persistence in chlorinated wastewater may be due to its protection within fecal particles or adsorption to suspended solids [29,30].

As shown in Table 1, the absence of residual chlorine in the effluent of both hospitals correlated with the detection of SARS-CoV-2 RNA in wastewater. This persistence of viral RNA, even after chlorination, can be attributed to the high stability of RNA, which is often protected within fecal particles. Viruses, particularly those with sizes ranging from 1 to 200 nm, tend to adsorb onto suspended solids in wastewater, further stabilized by fat or protein layers. Within wastewater treatment plants (WWTPs), activated sludge acts as a microbial community composed of microorganisms and non-living organic and inorganic matter [31]. Enveloped viruses, such as coronaviruses, adsorb to solid materials in wastewater [32], which can shield them from disinfection by free chlorine [33,34]. For instance, Forés et al. (2021) reported that approximately 23% of coronaviruses in wastewater were attached to solid materials, highlighting their resistance to disinfection processes [35].

noted that the detection of viral RNA in wastewater is often associated with chemical oxygen demand (COD) and flow rates [34]. In this study, elevated levels of COD and biochemical oxygen demand (BOD) coincided with higher detection rates of SARS-CoV-2 RNA (Table 1). While biological processes like activated sludge can remove over 80% of BOD, COD, and total suspended solids (TSS), the presence of residual organic contaminants necessitates tertiary treatments such as chlorination, ozonation, and UV disinfection. Moreover, the persistence of antibiotic-resistant microorganisms, including viruses, often requires higher doses of disinfectants for effective inactivation [36].

In this study, SARS-CoV-2 RNA was detected in effluent samples when total and fecal coliform levels exceeded 1100 MPN/100 mL. The highest number of positive effluent samples was observed in May, coinciding with a surge in COVID-19 cases. This trend suggests that increased viral loads in influent wastewater can overwhelm treatment processes, leading to incomplete removal of viral RNA. In Razi Hospital, where the patient load was higher than in Imam Hospital, the elevated viral concentration in wastewater further challenged the treatment plant’s efficiency. Tandukar et al. (2022) similarly reported that high viral RNA concentrations in influent samples often correlate with peaks in COVID-19 transmission. [37]. Additionally, Serra-Compte et al. (2021) found SARS-CoV-2 RNA in both primary and secondary treatment stages, as well as in treated sludge after dewatering and anaerobic digestion, indicating that conventional treatment processes may not fully eliminate viral RNA. The tendency of enveloped viruses to adsorb to solid particles further contributes to their retention in sludge, as noted by Balboa et al. (2021). Consequently, the findings of this study suggest that WWTPs may release SARS-CoV-2 RNA into the environment, particularly in cases of high viral loads [38].

The high detection rate of SARS-CoV-2 RNA in hospital wastewater effluents underscores the potential of wastewater-based epidemiology (WBE) as a tool for disease surveillance and control. WBE can serve as an early warning system for COVID-19 outbreaks, especially in developing countries where wastewater treatment infrastructure may be inadequate [39]. However, it is important to note that the presence of viral RNA in wastewater does not necessarily indicate infectivity. Fecal marker-normalized SARS-CoV-2 RNA concentrations have been shown to correlate with clinical case numbers, suggesting that fecal indicators can help monitor fluctuations in viral loads and predict near-term trends in COVID-19 cases [37].

3.1.2. Airborne transmission of SARS-CoV-2.

This study investigated the presence of SARS-CoV-2 RNA in hospital air, focusing on parameters such as temperature, relative humidity (RH), particulate matter (PM) concentrations, and distance from patients. Out of 23 air samples collected from Imam-Khomeini Hospital, 5 (21.74%) tested positive for SARS-CoV-2 RNA, with 4 positives in the COVID-19 patient ward and 1 in the ICU. In Razi Hospital, 7 out of 23 samples (30.43%) were positive, with detections in the COVID-19 ward, ICU, and CT scan areas. These findings align with Grimalt et al. (2022), who reported higher viral RNA concentrations in hospital corridors than in patient rooms, likely due to virus dispersion from rooms to common areas [2]. Stern et al. (2021) also found that non-COVID-19 wards occasionally showed high viral RNA levels, potentially reflecting asymptomatic infections among staff or broader community transmission [40]: The detailed results of SARS-CoV-2 RNA detection in hospital air samples are presented in Table 2.

Download:

• PNG
  larger image
• TIFF
  original image

Table 2. Details of Sampling Sites and Detection of SARS-CoV-2 in the air of Imam-Khomeini Hospital.

https://doi.org/10.1371/journal.pone.0345644.t002

The detection of SARS-CoV-2 RNA in air samples underscores the importance of airborne transmission in healthcare settings. While close contact and respiratory droplets remain primary transmission routes, aerosols containing viral particles can travel beyond 2 meters, particularly in poorly ventilated spaces. This study detected viral RNA at distances of 0.5–5 meters from patients, consistent with findings by Lednicky et al. (2020) [17] and Santarpia et al. (2020) [41], who identified SARS-CoV-2 in airborne aerosols ≥2 meters from infected individuals. However, the results contrast with studies by Faridi et al. (2020) [42] and Masoumbeigi et al. (2020) [43], who reported no positive air samples, likely due to differences in ventilation systems or sampling methods. Effective ventilation and air filtration, such as HEPA systems, are critical to reducing airborne viral loads and mitigating transmission risks [44,45].

The study also measured PM concentrations (PM₁₀, PM_2.5, and PM₁) in hospital air. PM_2.5 and smaller particles (≤2.5 μm) are particularly concerning, as they can remain suspended in the air and carry viral particles, potentially depositing deep into the respiratory tract [46,47]. In Imam-Khomeini Hospital, PM₁₀, PM_2.5, and PM₁ levels ranged from 23.02–37.15, 7.025–15.25, and 6.75–8.10 μg/m³, respectively. In Razi Hospital, PM levels were slightly higher, with PM₁₀ reaching 42.12 μg/m³. While these values are below the WHO 24-hour average guidelines (PM_2.5: 25 μg/m³; PM₁₀: 50 μg/m³), they exceeded the stricter American Conference of Governmental Industrial Hygienists (ACGIH) standards for 8-hour exposure (PM_2.5: 3 μg/m³; PM₁₀: 10 μg/m³) [48]. Elevated PM levels in hospital wards may result from re-suspension of particles due to indoor activities, emphasizing the need for regular cleaning to minimize PM accumulation.

Studies have linked high PM_2.5 concentrations to increased COVID-19 hospitalization and mortality rates [49–51]. Fine particles can carry viruses and exacerbate respiratory infections, reducing the body’s ability to combat pathogens [52]. Van Doremalen et al. (2020) demonstrated that SARS-CoV-2 remains viable on surfaces and in aerosols for extended periods, highlighting the role of PM in viral transmission [14].

Although this study found no direct correlation between temperature, RH, and viral RNA detection, other research suggests that low temperatures and low RH favor SARS-CoV-2 stability. For instance, Aboubakr et al. (2021) [53] and Matson et al. (2020) [54] reported that SARS-CoV-2 persists longer in cooler, drier conditions. At 20°C and 80% RH, the virus exhibited a half-life of 3 hours in aerosols [55]. Low RH can also compromise the respiratory tract’s defense mechanisms by drying mucus and impairing cilia function, increasing susceptibility to viral infections [56].

3.1.3. Detection of SARS-CoV-2 on inanimate surfaces.

The study examined high-touch surfaces in Imam-Khomeini and Razi Hospitals, focusing on areas such as patient rooms, nursing stations, ICUs, CT scan wards, and administrative departments. Based on Table 3, a total of 92 surface samples were collected, with 26.09% (12/46) testing positive for SARS-CoV-2 RNA in Imam-Khomeini Hospital and 32.61% (15/46) in Razi Hospital. In Imam-Khomeini Hospital, positive samples were primarily found in the special ward (50%), ICU (25%), and CT scan ward (25%). In Razi Hospital, 66.66% of positive samples were from the special ward, 20% from the ICU, and 6.67% each from the CT scan and administrative areas. High-touch surfaces such as bed rails, bedside tables, computer keyboards, and medical equipment were frequently contaminated, highlighting the risk of fomite transmission.

Download:

• PNG
  larger image
• TIFF
  original image

Table 3. Details of Sampling Sites and Detection of SARS-CoV-2 on High-touch Surfaces of Imam-Khomeini Hospital.

https://doi.org/10.1371/journal.pone.0345644.t003

Environmental conditions during sampling varied, with temperatures ranging from 24–28°C and relative humidity (RH) between 45–50% in Imam-Khomeini Hospital, and 24–27°C and 46–52% RH in Razi Hospital. These conditions, while not directly correlated with viral RNA detection in this study, are known to influence viral stability. For instance, higher temperatures and RH can reduce droplet size, facilitating longer airborne transmission [57,58].

The persistence of SARS-CoV-2 on high-touch surfaces underscores the importance of rigorous cleaning and disinfection protocols. Studies have shown that SARS-CoV-2 can survive on surfaces for hours to days, depending on material type and environmental conditions [59,60]. For example, Moore et al. (2021) [61]detected high viral RNA concentrations on door handles and bed rails, emphasizing the risk of hand-to-surface transmission. Jiang et al. (2020) [62] similarly found viral RNA on sheets, pillows, and counterpanes in quarantine rooms, reinforcing the need for frequent disinfection of high-touch surfaces.

In this study, contamination in ICUs and nursing stations was particularly concerning. The presence of viral RNA on patient clothing, pillows, and medical devices suggests that healthcare workers and visitors may inadvertently spread the virus if proper hygiene practices are not followed. Disposable gloves and personal protective equipment (PPE) are essential but must be used correctly to prevent surface contamination [63,64]. Regular glove changes and hand hygiene are critical to minimizing transmission risks.

The study revealed variability in surface contamination between the two hospitals. For instance, while SARS-CoV-2 RNA was detected on multiple surfaces in the CT scan ward of Imam-Khomeini Hospital, only the floor tested positive in Razi Hospital. This discrepancy may be attributed to differences in cleaning protocols or surface materials. Additionally, the absence of viral RNA in the administrative department of Imam-Khomeini Hospital suggests that strict hygiene measures, such as shoe covers and clothing changes, can effectively reduce contamination in non-clinical areas. However, the detection of viral RNA on the administrative floor in Razi Hospital highlights the need for consistent implementation of such measures.

The findings align with previous research demonstrating widespread surface contamination in healthcare settings. Bloise et al. (2020) [65] identified SARS-CoV-2 RNA on frequently touched objects like telephones and keyboards, underscoring the role of fomites in viral transmission. Similarly, Ding et al. (2021) [66]and Ye et al. (2020) [24] reported contamination in hospital wards, though they noted that widespread infection was unlikely if proper disinfection protocols were followed. These studies collectively emphasize the importance of targeted cleaning strategies, particularly in high-risk areas like ICUs and patient rooms.

3.2.1. Wastewater environment.

Wastewater-based epidemiology (WBE) has emerged as a valuable tool for monitoring viral outbreaks, particularly in healthcare settings. Based on Table 4, in our study, SARS-CoV-2 RNA was detected in 65.28% of wastewater samples collected from hospital WWTPs. Specifically, in Imam-Khomeini Hospital, 61.11% of influent samples tested positive for SARS-CoV-2 RNA, while only 11.11% of effluent samples were positive. Similarly, in Razi Hospital, 69.44% of influent samples were positive, compared to 16.67% of effluent samples. These findings align with Mohan et al. (2021), who demonstrated that enveloped viruses such as SARS-CoV-1 and HCoV-229E can remain viable in water for 2–100 days, depending on temperature, with longer survival at lower temperatures [67]. This confirms that temperature is a key factor in the persistence of viruses in aquatic environments. Additionally, La Rosa et al. (2020) reported that coronaviruses have low stability in water and are sensitive to oxidants like chlorine, which aligns with our observations of reduced viral RNA in effluent samples with residual chlorine [68]. Furthermore, Dalziel et al. (2016) showed that low pathogenic influenza A virus (LPAIV) can persist in water for hours to days, with factors such as temperature, pH, and salinity significantly influencing its survival. These results highlight the critical role of environmental conditions in the persistence of viruses in wastewater [69].

Download:

• PNG
  larger image
• TIFF
  original image

Table 4. Persistence and Detection of Viruses in Wastewater, Air and Surface: A Comprehensive Analysis.

https://doi.org/10.1371/journal.pone.0345644.t004

In our study, SARS-CoV-2 RNA was detected in 65.28% of hospital wastewater samples, consistent with the findings of Adelodun et al. (2021). They reported that SARS-CoV-2 RNA in wastewater indicates the potential for fecal-oral transmission [70]. Similarly, Mohan et al. (2021) demonstrated that SARS-CoV-2 RNA persists in feces, and enveloped viruses inactivate faster in wastewater than non-enveloped viruses. These findings emphasize that hospital wastewater can serve as an important source for monitoring disease outbreaks [67]. Moreover, Williams et al. (2024) reported that SARS-CoV-2 RNA is detected in wastewater year-round, with higher concentrations in winter [71], which aligns with our observations of increased viral loads during colder periods.

The persistence of SARS-CoV-2 in wastewater has been further elucidated by studies such as Corpuz et al. (2020), who found that the virus can remain detectable for 2 days at 20°C and up to 14 days at 4°C, underscoring the potential of wastewater surveillance for monitoring community spread [72]. Similarly, Mohapatra et al. (2021) highlighted that SARS-CoV-2 RNA can persist for several days in untreated wastewater and up to 7 days in tap water, with coagulation, filtration, and chlorination proving effective in removing or inactivating the virus [73].

In addition to SARS-CoV-2, other viruses such as influenza and enteroviruses have also been detected in wastewater. Williams et al. (2024) found that influenza RNA was detected in 75% of wastewater samples, with higher detection rates in winter. Enteroviruses were detected in all wastewater samples, although only one sample tested positive using RT-qPCR [71]. These findings indicate that wastewater can serve as a valuable tool for monitoring the spread of various viruses. In conclusion, integrating wastewater surveillance with advanced treatment methods, such as membrane bioreactors (MBR), is essential for mitigating the risks associated with viral persistence in aquatic environments and preventing environmental transmission.

3.2.2. Air and surface environment.

The persistence and detection of enveloped viruses in air and surface environments, particularly in healthcare settings, have been extensively studied. In this section, the findings from the case study conducted at Imam Khomeini and Razi Hospitals are compared with the results of a systematic review to provide a comprehensive understanding of the persistence of these viruses in various environments.