Hospitals and wastewater treatment plants (WWTPs) are high-risk point sources of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria. This study investigates the occurrence of clinically relevant ARGs (sul1, tet(B), bla_CTX-M, bla_NDM-1, qnrS) and a class one integron (intI1) gene in urban rivers, hospitals, and municipal wastewater in the Kathmandu Valley, Nepal. Twenty-five water samples were collected from three rivers, six hospitals, and a wastewater treatment plant to determine the concentrations of ARGs and intI1 using quantitative polymerase chain reactions. From the results, all tested ARGs were detected in the river water; also, concentrations of ARGs in WWTP and hospital effluents varied from 6.2 to 12.5 log₁₀ copies/L, highlighting the role of a WWTP and hospitals in the dissemination of ARGs. Except for bla_NDM-1, significant positive correlations were found between intI1 and other individual ARGs (r = 0.71–0.96, p < 0.05), indicating the probable implications of intI1 in the transfer of ARGs. Furthermore, this study supports the statement that the bla_NDM-1 gene is most likely to be spread in the environment through untreated hospital wastewater. Due to the interaction of surface water and groundwater, future research should focus on ARGs and factors associated with the increase/decrease in their concentration levels in drinking water sources of the Kathmandu Valley.

Digital polymerase chain reaction (dPCR) is a significant advancement in health-related water microbiology, enabling absolute quantification without standard curves. By partitioning samples into thousands of individual reactions, dPCR allows for precise quantification even in the presence of inhibitory substances common in environmental samples. This study evaluated the applicability of dPCR to detect gastroenteritis-causing enteropathogens (Salmonella spp., Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, JC and BK polyomaviruses, and human adenovirus), crAssphage, and four antibiotic resistance genes (ARGs) (sul1, bla_NDM-1, bla_CTX-M, and intI1) in wastewater and drinking water source samples. Wastewater samples were collected in 2018 from two municipal wastewater treatment plants (WWTPs) that used an oxidation ditch system (n = 12) and a stabilization pond system (n = 10) in the Kathmandu Valley, Nepal, whereas the drinking water source samples were collected from shallow wells between 2015 and 2016 (n = 22). The enteropathogens and ARGs were analyzed using the QIAcuity dPCR System. The highest detection ratio was observed by crAssphage in wastewater (100 %, 22/22) and by sul1 and intl1 in drinking water sources (91 %, 20/22). The log₁₀ reduction values evaluated in both WWTPs were <1 using dPCR, consistent with those obtained in previous quantitative PCR studies, and may be used to cross-validate between methods. However, this study also observed low detection ratios and concentrations of enteropathogens, likely due to factors such as low sample volumes, dead volume, thermal conditions, and the dPCR platform used. Thus, optimizing these variables is imperative to enhance the applicability of dPCR in environmental assessments.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in Wuhan, China. The clinical manifestation of COVID-19 varies from asymptomatic to severe infection. The World Health Organization has reported over 248 million cases and more than 5 million deaths worldwide due to COVID-19. Additionally, developing countries are still struggling to achieve the rapid diagnosis of suspected cases to break the chain of transmission of COVID-19. There may be undiagnosed or missing cases in these countries. Various studies have reported that the SARS-CoV-2 can be discharged in the stool and other bodily fluids. Moreover, it is possible that SARS-CoV-2 can be transmitted in the environment via municipal wastewater. In developing countries, such as Nepal, the molecular-based diagnosis of SARS-CoV-2 for the entire population is not practical owing to insufficient diagnostic material and budgetary constraints. Wastewater-based epidemiology (WBE) is a promising public health surveillance tool for the early warning of disease outbreaks and the understanding the prevalence of viruses for the development of intervention measures. This study highlights the importance of the utilization of WBE as an alternative tool for the mass detection of SARS-CoV-2 RNA in wastewater.

The analysis of host-associated microorganisms by quantitative polymerase chain reaction (qPCR) has long been widely applied as a microbial source tracking (MST) tool for identifying the sources of fecal contamination. Although numerous studies have been conducted across Asia, relevant research on Indonesia is scarce. In this study, we evaluated the performance of various human- and animal-specific MST markers and applied the results to the assessment of the water quality of a river in West Java, Indonesia. Fecal samples from seven animal species (n = 43), sewage samples (n = 7), and river water samples (n = 48) were collected from eight locations along a river in West Java from August to September 2023 (dry season) and February to March 2024 (wet season). Among the 16 host-specific MST markers tested using qPCR, the detection ratio of human-specific markers in the target samples showed considerable variation (43 %-100 %). Ruminant-specific markers, including bacterial (BacCow and BacR) and mitochondrial DNA (Bovine-mtDNA) markers, demonstrated a 100 % detection ratio in their target samples. In the nontarget samples, the positive ratio of MST markers varied widely from 0 % (AiV-1) to 81 % (BacCow). crAssphage and Bovine-mtDNA were identified as the most effective MST markers specific to humans and ruminants, respectively. pBI143, as a newly established human-specific marker, demonstrated commendable performance with 100 % sensitivity and 77 % specificity. This study confirmed the presence of MST markers in the river water, with a positive ratio of 38 % for Bovine-mtDNA and 67 % for crAssphage during the dry season; the values increased during the wet season to 54 % and 88 %, respectively. The results emphasize the importance of using several types of MST markers to obtain comprehensive results and insight into contamination patterns, as well as the necessity for further research across diverse sub-watersheds and other water sources.

Inadequately treated effluents discharged from wastewater treatment plants (WWTPs) severely affect the environment and the surrounding population. This study analyzed the presence of the Shiga toxin-producing Escherichia coli (STEC) genes, stx1, and stx2, and the E. coli gene, sfmD, in municipal WWTP A (n = 11) and B (n = 11) where the reductions were also evaluated; hospitals (n = 17), sewage treatment plants (STPs) (n = 4) and non-functional WWTPs (not-working WWTPs) (n = 5) in the Kathmandu Valley, Nepal. The sfmD gene was detected in 100% of the samples in WWTPs, hospitals, and not-working WWTPs and 50% of STP samples. The highest detection of stx1 and stx2 was shown in the WWTP influents, followed by WWTP effluents, not-working WWTP wastewater, hospital wastewater, and STP wastewater. Log₁₀ reduction values of sfmD, stx1, and stx2 in WWTP A were 1.7 log₁₀, 1.7 log₁₀, 1.4 log₁₀, whereas those in WWTP B were 0.5 log₁₀, 0.6 log₁₀, 0.5 log₁₀, respectively, suggesting the ineffective treatment of STEC in the wastewater in the Kathmandu Valley. The high concentrations of the stx genes in the wastewaters suggest the increasing presence of aggressive STEC in the Kathmandu Valley, which should be a major public health concern.