Comparative analysis of membrane filter diameters for detection of selected viruses in wastewater samples.

Wastewater serves as a valuable source of information as it contains biological markers that have been shed by infected individuals and from other biological organisms such as plants and animals. Wastewater has been proven to indicate the presence of emerging pathogens in a community before the manifestation of clinical symptoms. Several methods of concentration and nucleic acid extraction have been employed all around the world without a unified method. One such method involves the use of the adsorption extraction method (AE-method), which involves the use of electronegative membrane filters of different pore sizes. The membrane filters also differ by diameter, but no study has been reported on the effect of diameter on capture efficiency. This study was aimed at evaluating the comparative capture efficiency of two different membrane filter diameters of 45 and 90 mm with pore sizes of 0.45 µm for the detection of indicative and pathogenic viruses. Primary influent samples were obtained from two wastewater treatment plants in Baltimore, Maryland, between April 27 and June 29, 2023. A total of twenty samples were processed using 45- and 90-mm membrane filters. Nucleic acids were extracted from the filters using the QiAmp Viral RNA Mini Kit and assayed for four different targets: PMMoV, Norovirus (GI and GII), and CrAssphage by RT-qPCR. The result showed that 45 mm membrane filters had a higher combined mean capture efficiency in log10 gene copies per liter (gc/l) for crAssphage (7.40) than 90 mm membrane filters (7.10). Similarly, the 45-mm filter had higher mean capture efficiency for norovirus GI (4.67) than the 90-mm filter (1.84) and likewise for Norovirus GII (2.14, 1.04). On the contrary, 90-mm membrane filters were observed to have better capture of PMMoV (6.84) compared to 45-mm membrane filters (6.69). This result therefore implies that 45-mm membrane filters could be more efficient for wastewater surveillance studies through the AE method for indicator viruses like CrAssphage and human disease-causing viruses like Norovirus.

Application of Skimmed-Milk Flocculation Method for Wastewater Surveillance of COVID-19 in Kathmandu, Nepal.

Wastewater surveillance (WS) has been used globally as a complementary tool to monitor the spread of coronavirus disease 2019 (COVID-19) throughout the pandemic. However, a concern about the appropriateness of WS in low- and middle-income countries (LMICs) exists due to low sewer coverage and expensive viral concentration methods. In this study, influent wastewater samples (n = 63) collected from two wastewater treatment plants (WWTPs) of the Kathmandu Valley between March 2021 and February 2022 were concentrated using the economical skimmed-milk flocculation method (SMFM). The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested by qPCR using assays that target the nucleocapsid (N) and envelope (E) genes. Overall, 84% (53/63) of the total samples were positive for SARS-CoV-2 according to at least one of the tested assays, with concentrations ranging from 3.5 to 8.3 log10 gene copies/L, indicating the effectiveness of the SMFM. No correlation was observed between the total number of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater collected from the two WWTPs (p > 0.05). This finding cautions the prediction of future COVID-19 waves and the estimation of the number of COVID-19 cases based on wastewater concentration in settings with low sewer coverage by WWTPs. Future studies on WS in LMICs are recommended to be conducted by downscaling to sewer drainage, targeting a limited number of houses. Overall, this study supports the notion that SMFM can be an excellent economical virus-concentrating method for WS of COVID-19 in LMICs.

Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.