Quantification of multiple respiratory viruses in wastewater in the Kathmandu Valley, Nepal: Potential implications of wastewater-based epidemiology for community disease surveillance in developing countries.

Despite being the major cause of death, clinical surveillance of respiratory viruses at the community level is very passive, especially in developing countries. This study focused on the surveillance of three respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IFV-A), and respiratory syncytial virus (RSV)] in the Kathmandu Valley, Nepal, by implication of wastewater-based epidemiology (WBE). Fifty-one untreated wastewater samples were from two wastewater treatment plants (WWTPs) between April and October 2022. Among eight combinations of the pre-evaluated methods, the combination of concentration by simple centrifugation, pretreatment by DNA/RNA Shield (Zymo Research), and extraction by the QIAamp Viral RNA Mini Kit (QIAGEN) showed the best performance for detecting respiratory viruses. Using this method with a one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR), SARS-CoV-2 RNA was successfully detected from both WWTPs (positive ratio, 100 % and 81 %) at concentrations of 5.6 ± 0.6 log10 copies/L from each WWTP. Forty-six SARS-CoV-2 RNA-positive samples were further tested for three mutation site-specific one-step RT-qPCR (L452R, T478K, and E484A/G339D), where G339D/E484A mutations were frequently detected in both WWTPs (96 %). IFV-A RNA was more frequently detected in WWTP A (84 %) compared to WWTP B (38 %). RSV RNA was also detected in both WWTPs (28 % and 8 %, respectively). This is the first study on detecting IFV-A and RSV in wastewater in Nepal, showing the applicability and importance of WBE for respiratory viruses in developing countries where clinical data are lacking.

Detection of SARS-CoV-2 and Omicron variant RNA in wastewater samples from Manila, Philippines.

Manila, a highly urbanized city, is listed as one of the top cities with the highest recorded number of coronavirus disease 2019 (COVID-19) cases in the Philippines. This study aimed to detect and quantify the RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Omicron variant in 51 wastewater samples collected from three locations in Manila, namely Estero de Santa Clara, Estero de Pandacan, which are open drainages, and a sewage treatment plant (STP) at De La Salle University-Manila, between July 2022 and February 2023. Using one-step reverse transcription-quantitative polymerase chain reaction, SARS-CoV-2 and Omicron variant RNA were detected in 78 % (40/51; 4.9 ± 0.5 log10 copies/L) and 60 % (24/40; 4.4 ± 0.3 log10 copies/L) of wastewater samples collected from all sampling sites, respectively. SARS-CoV-2 RNA was detected frequently at Estero de Santa Clara (88 %, 15/17); its highest concentration was at the STP (6.3 log10 copies/L). The Omicron variant RNA was present in the samples collected (4.4 ± 0.3 log10 copies/L) from all sampling sites, with the highest concentration at the STP (4.9 log10 copies/L). Regardless of normalization, using concentrations of pepper mild mottle virus RNA, SARS-CoV-2 RNA concentrations exhibited the highest positive correlation with COVID-19 reported cases in Manila 5 days after the clinical report. These findings revealed that wastewater-based epidemiology may aid in identifying and monitoring of the presence of pathogens in open drainages and STPs in the Philippines. This paper provides the first documentation on SARS-CoV-2 and the Omicron variant in wastewater from Manila.

Long-term SARS-CoV-2 surveillance in wastewater and estimation of COVID-19 cases: An application of wastewater-based epidemiology.

The role of wastewater-based epidemiology (WBE), a powerful tool to complement clinical surveillance, has increased as many grassroots-level facilities, such as municipalities and cities, are actively involved in wastewater monitoring, and the clinical testing of coronavirus disease 2019 (COVID-19) is downscaled widely. This study aimed to conduct long-term wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Yamanashi Prefecture, Japan, using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay and estimate COVID-19 cases using a cubic regression model that is simple to implement. Influent wastewater samples (n = 132) from a wastewater treatment plant were collected normally once weekly between September 2020 and January 2022 and twice weekly between February and August 2022. Viruses in wastewater samples (40 mL) were concentrated by the polyethylene glycol precipitation method, followed by RNA extraction and RT-qPCR. The K-6-fold cross-validation method was used to select the appropriate data type (SARS-CoV-2 RNA concentration and COVID-19 cases) suitable for the final model run. SARS-CoV-2 RNA was successfully detected in 67 % (88 of 132) of the samples tested during the whole surveillance period, 37 % (24 of 65) and 96 % (64 of 67) of the samples collected before and during 2022, respectively, with concentrations ranging from 3.5 to 6.3 log10 copies/L. This study applied a nonnormalized SARS-CoV-2 RNA concentration and nonstandardized data for running the final 14-day (1 to 14 days) offset models to estimate weekly average COVID-19 cases. Comparing the parameters used for a model evaluation, the best model showed that COVID-19 cases lagged 3 days behind the SARS-CoV-2 RNA concentration in wastewater samples during the Omicron variant phase (year 2022). Finally, 3- and 7-day offset models successfully predicted the trend of COVID-19 cases from September 2022 until February 2023, indicating the applicability of WBE as an early warning tool.

Optimization and performance evaluation of an automated filtration method for the recovery of SARS-CoV-2 and other viruses in wastewater.

A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.

Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates.

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

Development of a magnetic nanoparticle-based method for concentrating SARS-CoV-2 in wastewater.

Several virus concentration methods have been developed to increase the detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater, as part of applying wastewater-based epidemiology. Polyethylene glycol (PEG) precipitation method, a method widely used for concentrating viruses in wastewater, has some limitations, such as long processing time. In this study, Pegcision, a PEG-based method using magnetic nanoparticles (MNPs), was applied to detect SARS-CoV-2 in wastewater, with several modifications to increase its sensitivity and throughput. An enveloped virus surrogate, Pseudomonas phage φ6, and a non-enveloped virus surrogate, coliphage MS2, were seeded into wastewater samples and quantified using reverse transcription-quantitative polymerase chain reaction to assess the recovery performance of the Pegcision. Neither increasing MNP concentration nor reducing the reaction time to 10 min affected the recovery, while adding polyacrylic acid as a polyanion improved the detection sensitivity. The performance of the Pegcision was further compared to that of the PEG precipitation method based on the detection of SARS-CoV-2 and surrogate viruses, including indigenous pepper mild mottle virus (PMMoV), in wastewater samples (n = 27). The Pegcision showed recovery of 14.1 ± 6.3 % and 1.4 ± 1.0 % for φ6 and MS2, respectively, while the PEG precipitation method showed recovery of 20.4 ± 20.2 % and 18.4 ± 21.9 % (n = 27 each). Additionally, comparable PMMoV concentrations were observed between the Pegcision (7.9 ± 0.3 log copies/L) and PEG precipitation methods (8.0 ± 0.2 log copies/L) (P > 0.05) (n = 27). SARS-CoV-2 RNA was successfully detected in 11 (41 %) each of 27 wastewater samples using the Pegcision and PEG precipitation methods. The Pegcision showed comparable performance with the PEG precipitation method for SARS-CoV-2 RNA concentration, suggesting its applicability as a virus concentration method.

Circulating Genotypes of Rotavirus Prior to Rotarix?vaccine Introduction in Kathmandu, Nepal.

BACKGROUND: In Nepal, it is estimated that about 3 million children under 5 years of age are prone to diarrhea and previous studies have shown rotavirus as the major etiological agent. Given the high burden of rotavirus, Rotarix® vaccine was introduced in the national immunization schedule in July 2020. This study was carried out in a tertiary health center from January- September 2018 to determine the burden of rotavirus diarrhea as well as genotypic variations in the circulating virus prior to vaccine introduction in Kathmandu, Nepal. METHODS: Hospital based cross sectional study was conducted among children less than 5 years of age attending Kanti Children's Hospital. Rotavirus antigen detection was performed by enzyme immunoassay using ProSpecT Rotavirus Microplate Assay. Rotavirus A positive samples were further confirmed by genotyping using Reverse-Transcription Polymerase Chain Reaction. RESULTS: A total of 530 children that included 184 males and 346 females were enrolled in this study. Rotavirus antigen was detected in 112 (21.1%) stool samples. Of the total 112 positive EIA stool samples that were genotyped, G12P[6] (30.3%) was found to be the most common type, followed by G3P[8] (26.8%), mixed type (14.3%), and G1P[6] (13.4%). CONCLUSIONS: Continued surveillance should be carried out nationwide in Nepal to understand the effectiveness of the vaccination program and to report any new trends in the circulating genotypes.

Multidrug-resistant and extended-spectrum beta-lactamase-producing uropathogens in children in Bhaktapur, Nepal.

BACKGROUND: The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing uropathogens has complicated the treatment of urinary tract infections (UTI). Paediatric UTI is a common illness, which if not treated properly, may lead to acute and long-term complications, such as renal abscess, septicaemia, and renal scarring. This study aimed to determine the prevalence of MDR and ESBL-producing uropathogens among children. METHODS: During the study period (April 2017-April 2018), midstream urine samples were collected following aseptic procedures from children < 16 years in Siddhi Memorial Hospital. Standard culture and biochemical tests were performed to identify uropathogens and antimicrobial susceptibility test was done by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL-producing uropathogens were screened by ceftazidime (30 µg) and cefotaxime (30 µg) discs, and confirmed by the combination disc tests: ceftazidime + clavulanic acid (30/10 µg) or cefotaxime + clavulanic acid (30/10 µg) as recommended by CLSI. RESULTS: We processed 5545 non-repeated urine samples from the children with symptoms of UTI. A significant growth of uropathogens was observed in 203 samples (3.7%). The median age of the children was 24 months (interquartile range (IQR), 12-53 months). Escherichia coli (n = 158, 77.8%) and Klebsiella pneumoniae (n = 30, 14.8%) were common among the uropathogens. Among them, 80.3% were resistant to amoxycillin and 51.2% were resistant to cotrimoxazole. Most of them were susceptible to amikacin, nitrofurantoin, and ofloxacin. MDR was detected in 34.5% (n = 70/203) and ESBL producers in 24.6% (n = 50/203) of them. The proportion of MDR isolates was higher in children < 5 years (n = 59/153, 38.6%) than children ≥ 5 years (n = 11/50, 22%) (P = 0.03). CONCLUSIONS: Nitrofurantoin, ofloxacin, and amikacin can be used for the empirical treatment for UTI in children in Bhaktapur, Nepal. MDR and ESBL-producing uropathogens are prevalent; this warrants a continuous surveillance of antimicrobial resistance.

In-Vitro Biofilm Formation and Antimicrobial Resistance of Escherichia coli in Diabetic and Nondiabetic Patients.

BACKGROUND: Diabetic patients are more susceptible to urinary tract infection compared to nondiabetic patients, Escherichia coli being the most common uropathogen causing UTI. Unreasonable and incorrect antibiotic prescription for UTI in these patients may induce the development of antibiotic-resistant urinary pathogens resulting in delayed recovery and longer hospitalization. In addition to these, biofilm forming capacity of the pathogen may worsen the problem. The main aim of this cross-sectional study (conducted from March to September 2015) is to detect the biofilm forming capacity of UTI causing micro-organisms and compare the antibiotic resistance pattern of Escherichia coli, the most common cause of UTI, which will help the physician in choosing the best antibiotic. METHOD: Total of 1,099 clean-catch mid stream urine (CCMSU) was processed by standard microbiological technique; 182 were from the diabetic group and 917 nondiabetic. Following identification, all isolates were subjected to antibiotic susceptibility testing using modified Kirby-Bauer disc diffusion method. In-vitro biofilm forming capacity of the isolates were detected by Microtitre plate method. The data were analyzed using SPSS software 16. RESULT: Urinary tract infection was found to be significantly higher in diabetic patients (42.9%) compared to nondiabetic patients (17.4%) with Escherichia coli as the most common uropathogen in both diabetic and nondiabetic groups. Similarly, UTI was more common in elderly population (29.5%). Imipenem, nitrofurantoin and amikacin were found to be the most effective drug for uropathogenic E. coli in both diabetic and nondiabetic patients, whereas amoxicillin, ciprofloxacin, and cotrimoxazole were least effective. Of the total bacterial isolates, 43.3% showed positive results for in-vitro biofilm production by the Microtitre plate method. A significantly higher resistance rate was observed among biofilm producing E. coli for quinolones, cotrimoxazole, and third generation cephalosporin ceftriaxone. Most of the biofilm producers (79.5%) were found to be MDR (p-value 0.015). CONCLUSION: Elderly populations with diabetes are at a higher risk of UTI. Higher biofilm production and resistance to in-use antimicrobial agents in this study render its inefficacy for empirical treatment and point out the importance of biofilm screening to ensure the effective management of infection.

Acute gastroenteritis associated with Rotavirus A among children less than 5 years of age in Nepal.

BACKGROUND: Rotavirus gastroenteritis is a major public health problem in Nepal. This study was conducted to obtain information associated with Rotavirus gastroenteritis and to perform genotyping of Rotavirus A. METHODS: Hospital based cross sectional study was conducted from January to December 2017 among children less than 5 years of age attending Kanti Children's Hospital and Tribhuvan University Teaching Hospital. Rotavirus A antigen detection was performed by Enzyme Linked Immunosorbent Assay (ELISA) using ProSpecT Rotavirus Microplate Assay. Rotavirus A positive strains were further confirmed by genotyping using Reverse-Transcription Polymerase Chain Reaction (RT-PCR). RESULTS: A total of 1074 stool samples were collected, of them 770 were hospitalized, and 304 were non-hospitalized cases. Rotavirus A infection was found in 28% of children with infection rate higher in hospitalized (34%) than in non-hospitalized (14%) children. Rotavirus A detection was higher in male (31%) than in female (24%), but this was statistically not significant (p > 0.05). Rotavirus A positivity was higher in children of age group 0-23 months, this result was statistically not significant (p > 0.05) with higher frequency found in the months of November, December, January, February and March (p < 0.05). On the basis of molecular analysis of Rotavirus A genotyping, G12P[6] (46.39%) was found to be the predominant followed by G1P[8] (35.05%), G3P[8] (7.21%) and G1P[6] (5.15%) while 4.12% was mixed infection and 1.03% was partially typed (p < 0.05). CONCLUSION: Rotavirus A infection occurred throughout the year, but the infection was significantly higher during the month of March. The higher frequency of rotavirus infection was observed among children of age group 0-23 months; however this was not found to be statistically significant. In this study, G12P[6] is predominant genotype observed. The results of genotyping are essential for the introduction of Rotavirus vaccine in Nepal.