Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the disease COVID-19, and since 2019, many efforts have been made to determine the methods of transmission of this virus in society1. In Iran, the first person infected with this virus was identified in Qom city, and due to the high prevalence of this virus and the lack of information on how this virus spreads due to its emerging nature, the disease spread widely throughout the country2. So far, 7,562,446 people have been infected with this disease in Iran, of which 144,723 have died3,4. Due to the presence of this virus in the excreta of people suffering from this disease and the direct entry of sewage produced by the population into the wastewater collection systems in residential areas, the investigation of the RNA concentration of this virus in wastewater provides valuable information to measure its circulation in the entire population. In recent years, scientists have used wastewater microbial load analysis to investigate the overall status of a wide variety of water, food, and fecal-borne viruses that infected individuals typically excrete in high concentrations with feces5. They are discharged into the wastewater collection systems. Some studies have reported the presence of viral RNA in the feces of COVID-19 patients in percentages ranging from 16.5 to 100% at concentrations of up to 6,310,000 (gene copies/g) of feces6,7. So far, studies have been conducted in different countries such as America, Holland, France, Spain, and Australia to investigate the presence of SARS-CoV-2 RNA in treated and untreated wastewater8,9,10,11. Several studies from regions with similar climatic and socio-economic conditions have provided insights into the presence of SARS-CoV-2 RNA in untreated wastewater. For instance, studies in Middle Eastern countries have identified comparable RNA concentrations and highlighted similar challenges in wastewater management systems12,13. Adding these findings enriches the global context of SARS-CoV-2 wastewater surveillance and underscores the relevance of this research. These studies used different methods such as 100 kDa Centricon® Plus-70 centrifugal ultrafiltration device, adsorption-extraction using an electronegative membrane, aluminum hydroxide adsorption precipitation, and two-phase separation (PEG-dextran method) for the recovery of SARS. -CoV-2 has used wastewater. To date, no study has yet provided insight into the presence of SARS-CoV-2 in untreated municipal wastewater in Iran. This study, investigated the presence of SARS-CoV-2 RNA in the wastewater of southern Iran using RT-qPCR. This study reports the detection of SARS-CoV-2 RNA in urban wastewater collection systems in the southern regions of Iran, contributing to the growing body of evidence on WBE in the region.

Materials and methods

Study area

Behbahan County, located in Khuzestan Province, is one of the most populous regions in southwest Iran. From the beginning of the COVID-19 outbreak in late 2019 through February 2021, more than 2,300 confirmed infections and around 200 COVID-19-related deaths were reported in this County, highlighting the significant impact of the virus on the local population. The county generates approximately 21.5 million liters of municipal wastewater daily14, which is collected through an urban wastewater system and discharged into municipal wastewater treatment plant.

Sample collection

To survey the presence of SARS-CoV-2 RNA in the wastewater collection system, in January and February 2021, using sterile 1 L Nalgene bottles, 24 grab samples (1 L each) were collected at a single, fixed sampling port located in the influent channel of municipal wastewater treatment plant, which consolidates flow from the entire urban sewer network; no additional upstream or branch-specific sampling points were used. This approach ensured that each weekly 1 L sample represented the composite domestic and commercial effluent of the full catchment area, thereby providing a consistent, city‑wide indicator for SARS‑CoV‑2 RNA monitoring. All samples were taken on distinct dates under dry-weather conditions with no extreme weather events. Sampling was carried out every 2–3 days throughout the sampling period and no specific day of the week was consistently targeted. This schedule ensured a high-resolution temporal profile of SARS‑CoV‑2 RNA in the influent without any overlapping sampling events. Collected Samples were transported on ice to the virology reference laboratory to identify SARS-CoV-2 RNA15.

SARS-CoV-2 RNA detection

To determine the viral load, based on the manufacturer’s protocol, total RNA was isolated from 200 µL of each wastewater sample using the Sinaclon RNA extraction kit (Iran)13. The collected samples were stored at -80 °C until further analysis. Viral load measurement was conducted using the COVID-19 real-time PCR kit (Pishtaz Teb). A LightCycler real-time PCR system (Roche, Germany) was applied to detect viral RNA. The kit’s probe and primer mixture were designed using a dual-target gene strategy, simultaneously targeting conserved sequences in the RdRp and N regions14. Moreover, in the kit, there was a solution with an internal control probe and primer for the RNase P gene. To identify the RdRp region, N region, and RNase P gene, the FAM, HEX, and ROX channels were used, respectively. Real-time PCR amplification was conducted at an initial reverse transcription step at 50 °C for 20 min, followed by cDNA denaturation at 95 °C for 3 min. This was followed by 45 amplification cycles, each consisting of a 10-second denaturation step at 94 °C and a 40 s annealing, extension, and fluorescence measurement step at 55 °C. The process was conducted with a cooling step at 25 °C for 10 s. All RT-qPCR assays were conducted in technical triplicate for each wastewater sample. The average viral RNA concentration was calculated along with the standard error of the mean (SEM) to reflect the variability between replicates. A sample was considered positive if all three replicates crossed the Ct threshold (< 40) with a typical sigmoidal amplification curve.

Statistical analysis

Descriptive statistics were used to summarize SARS-CoV-2 RNA concentrations in wastewater samples. To assess the relationship between viral RNA levels and confirmed COVID-19 case numbers in Behbahan County, Pearson correlation analysis was performed using SPSS version 22. Epidemiological data on weekly confirmed COVID-19 cases in Behbahan County during the sampling period were obtained from the official records of Jundishapur University of Medical Sciences, Ahvaz, Iran. A significance threshold of p < 0.05 was applied.

Results and discussion

SARS-CoV-2 RNA concentration in untreated wastewater

The information of Sampling date, SARS-CoV-2 RNA concentration, and environmental data for untreated municipal wastewater are presented in Table 1.

Table 1 Sampling date, SARS-CoV-2 RNA mean concentration, standard error, and environmental data for untreated municipal wastewater.
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As shown in Table 1, SARS-CoV-2 RNA was detected in 75% of untreated wastewater samples and tested positive with RT-qPCR assays. Additionally, environmental factors such as temperature and precipitation were considered as potential modifiers of RNA persistence in wastewater, though their effects require further validation. During the ongoing COVID-19 pandemic from 2019, several research have been conducted using various methods for the detection of SARS-CoV-2 RNA concentration in untreated wastewater. E Cuevas and his colleagues reported 140,000–340,000 (gene copies/L) RNA Concentration range for positive samples in untreated wastewater in Spain using an aluminum hydroxide adsorption-precipitation method16. Haramoto et al. used the N Sarbeco RT-qPCR assay and found SARS-CoV-2 RNA concentration in the none detectable range in untreated wastewater samples of Japan17. Medema and his colleagues reported the SARS-CoV-2 RNA Concentration range in untreated wastewater samples in the Netherlands between 2600 and 2,200,000 (gene copies/L) for positive samples using three CDC N1, N2, and N3 assays18. A similar study conducted by Ahmed and his colleagues, in South Africa observed SARS-CoV-2 RNA Concentration among 19–120 (gene copies/L) in untreated wastewater19. This study is the first to investigate the presence of SARS-CoV-2 RNA in untreated municipal wastewater in Iran. The findings align with previous international research, which highlights the role of human wastewater as an indicator of viral transmission. Randazzo and his colleagues reported SARS-CoV-2 RNA concentrations ranging from 140,000 to 3.40000 (gene copies/L) in untreated wastewater using an aluminum hydroxide adsorption-precipitation method. These values are significantly higher than those observed in the present study. The differences might stem from factors such as population density, infection prevalence, or environmental conditions. The findings from previous studies emphasize the importance of integrating wastewater surveillance into public health decision-making, particularly in resource-constrained regions. Authorities can leverage this approach for early detection of outbreaks, reducing the reliance on clinical testing. Additionally, the study underscores the necessity of upgrading wastewater management systems to mitigate environmental and health risks associated with untreated sewage, especially during pandemics. Six of the 24 grab samples returned no detectable SARS‑CoV‑2 RNA (Ct ≥ 40). These intermittent non‑detects can arise from the inherent variability of grab sampling since viral shedding into wastewater fluctuates daily and the assay’s detection limit (~ 200 gene copies/L). On these sampling dates, wastewater temperature, pH, and flow rate remained within normal operational ranges, and no significant RT‑qPCR inhibition was observed based on the RNase P internal control. Additionally, regional COVID‑19 case counts were at their nadir during these weeks, supporting the interpretation that lower community prevalence led to undetectable viral loads in wastewater. This variability underscores the value of frequent sampling and highlights why composite or increased volume sampling could further reduce non‑detect frequency in future studies. The persistence of viral RNA in wastewater is influenced by environmental conditions, particularly temperature, pH, and rainfall20,21. All samples in this study were collected during dry weather conditions, with no rainfall recorded on any of the sampling dates (based on Behbahan meteorological station data). Daily air mean temperature during sampling was 18 °C in January and 22 °C in February, conditions that favor moderate RNA stability22. Lower temperatures can reduce RNA degradation rates, potentially contributing to the successful detection of viral RNA in 75% of the samples. In contrast, samples with non-detect results (samples 2, 6, 8, 13, 19, 22) coincided with relatively lower community case numbers, but not with unusual weather conditions. Thus, the non-detects are more likely due to low viral shedding or sampling-time variability, rather than environmental degradation of RNA. In future studies, incorporating flow-adjusted composite sampling and parallel viral decay modeling would further refine the understanding of these effects. It is important to note that this study focused exclusively on the liquid phase of untreated wastewater. Previous studies have shown that SARS-CoV-2 viral particles may adsorb to solid or sludge components, often resulting in higher RNA concentrations in the solid fraction compared to the supernatant23,24,25. Therefore, inclusion of both fractions could potentially enhance detection sensitivity.

Wastewater-based epidemiology (WBE): a tool for disease surveillance

The ability of WBE to reflect community infection trends has been validated across numerous countries. In our study, the viral RNA concentrations in untreated wastewater with the number of weekly clinically confirmed COVID-19 cases reported by Jundishapur University of Medical Sciences were compared. These data are summarized in Table 2. Also, the Correlation between SARS-CoV-2 RNA concentrations in municipal wastewater samples and weekly confirmed COVID-19 cases is presented in Fig. 1.

Table 2 Weekly average SARS-CoV-2 RNA concentrations and confirmed COVID-19 cases.
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Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Correlation between SARS-CoV-2 RNA concentrations and weekly confirmed COVID-19 cases.

A Pearson correlation analysis between mean weekly RNA concentrations and case numbers yielded r = 0.76 (p < 0.05), indicating a strong positive correlation. Also, the coefficient of determination (R² = 0.9683) obtained from the linear regression analysis indicates a very strong positive correlation between SARS-CoV-2 RNA concentrations in wastewater and the number of clinically confirmed COVID-19 cases. This high R² value suggests that approximately 96.83% of the variation in reported COVID-19 cases can be explained by fluctuations in viral RNA levels in the wastewater samples. This supports the utility of WBE as a non-invasive early warning system, especially in regions with limited access to clinical testing. The increasing viral RNA trends in weeks 4 to 6 coincided with a local spike in case reports, while the decline in viral load in week 8 suggests a tapering of community transmission. These findings are consistent with international reports and reinforce WBE as a practical, scalable, and cost-effective strategy for real-time disease surveillance, especially during pandemics. The approach is particularly valuable in resource-limited settings like Behbahan, where rapid clinical testing capacity may be restricted. The success of WBE in tracking SARS-CoV-2 shows its potential to monitor other infectious diseases such as influenza, norovirus, and antimicrobial resistance genes in the future. By utilizing existing wastewater infrastructure, WBE can provide real-time insights into community health, enabling early detection of outbreaks and informed decision-making for public health interventions. This approach can be used in areas with limited access to clinical trials, which provides a scalable and sustainable solution for disease surveillance. Due to the increasing threat of emerging infectious diseases in the world, WBE plays a critical role in enhancing global health security and pandemic preparedness. While WBE offers valuable insights, it has limitations such as variability in individual viral shedding, environmental factors affecting RNA stability, and lack of standardized methods. Additionally, it provides community-level data only and cannot pinpoint specific infection sources. These factors should be considered when interpreting results.

Conclusion

This study provides evidence of SARS-CoV-2 RNA detection in untreated municipal wastewater in southwestern Iran, with a focus on Behbahan County. Based on the results, SARS-CoV-2 RNA was detected to be in the concentration range of 0 to 2100 (gene copies/L) in most of the studied samples. Our findings revealed a positive correlation between SARS-CoV-2 RNA concentrations in wastewater and weekly confirmed COVID-19 cases in the region, supporting the utility of WBE as a complementary surveillance tool. However, due to sample size limitations and variability in clinical reporting, these results should be interpreted with caution. These results highlight the important role of WBE as a simple, cost-effective, and non-invasive approach for monitoring the spread of SARS-CoV-2 within communities. Further studies are required to investigate seasonal variations in virus shedding, optimize detection methods to improve sensitivity, and further investigate the relationship between wastewater viral loads and disease transmission dynamics to enhance pandemic preparedness and response.