Can environmental wastewater be used as a sentinel to improve public health surveillance in low-resource settings?

Methods

Summary

Sample collection and concentration:

Wastewater samples will be collected from drainage canals in different areas of Thailand, each with a volume of 40 mL, using the grab sampling technique with plastic bottles. The collected samples will be transferred into 50 mL centrifuge tubes. The samples will then be transported to a Biosafety Level 2 laboratory at Naresuan University.

Upon arrival at the laboratory, the samples will be heat-inactivated at 56°C for 30 minutes in a water bath to neutralize potential viruses without compromising sample quality. They will then be centrifuged at 5,000 × g at 4°C for 30 minutes to remove large debris, and the supernatant will be retained. To concentrate viral particles, 15% (w/v) PEG6000 and 2% (w/v) NaCl will be added, and the mixture will be incubated overnight at 4°C with shaking. Following incubation, the samples will be centrifuged at 12,000 × g for 90 minutes, the supernatant will be discarded, and the pellet will be washed by an additional centrifugation at 5,000 × g for 5 minutes. The final pellet will be resuspended in 100 μL of phosphate-buffered saline (PBS) and treated with DNase and RNase to remove free nucleic acids. The reaction will then be terminated under controlled conditions, and the samples will be stored at -80°C until nucleic acid extraction.

Nucleic Acid Extraction:

The resuspended pellet treated with DNase and RNase will be pooled into 1.5 mL microcentrifuge tubes (250 μL total) according to the collection sites. Nucleic acids will be extracted using the PureDireX Virus Nucleic Acid Isolation Kit. A 200 μL sample will be mixed with 400 μL Buffer V1, followed by a 10-minute incubation. Next, 450 μL of Buffer V2 (pre-mixed with ethanol) will be added and mixed. The mixture will be loaded onto a VN column and centrifuged at 16,000 × g, followed by sequential washes with Buffers W1 and W2. After a final centrifugation, the nucleic acids will be eluted with 50 μL of RNase-free water and stored at -80°C.

cDNA Synthesis:

For complementary DNA (cDNA) synthesis, 6 μL of extracted nucleic acids will be combined with dNTPs, random hexamer primers, and nuclease-free water. The mixture will be briefly centrifuged, incubated at 65°C for 5 minutes, and then chilled on ice. The reverse transcription reaction will be prepared with a reaction buffer, RNase inhibitor, additional dNTPs, and RevertAid M-MuLV Reverse Transcriptase. The mixture will undergo incubation at 25°C for 5 minutes, followed by 42°C for 60 minutes, and will conclude with an inactivation step at 70°C for 5 minutes. The resulting cDNA will be stored at -20°C for short-term use or -70°C for long-term storage. Double-stranded cDNA will be synthesized using a Klenow reaction, followed by quality assessments using 2% agarose gel electrophoresis and NanoDrop spectrophotometry.

Random Hexamer PCR and Sequencing:

PCR amplification will be conducted using 7 μL of the double-stranded cDNA template combined with Platinum™ II Hot-Start PCR Master Mix, random hexamer primers with linkers, and GC Enhancer. Thermal cycling will begin with an initial denaturation at 94°C for 2 minutes, followed by 35 cycles of denaturation, annealing (40°C–55°C), and extension, with a final extension at 68°C. Amplified PCR products will be verified through 2% agarose gel electrophoresis and quantified using a NanoDrop Spectrophotometer. Purified PCR products will then be used for library construction and shotgun metagenomic sequencing.

Bioinformatics Analysis:

Raw sequencing data will be assessed using FastQC to evaluate quality, using a Phred quality score threshold of 20. Low-quality reads and adaptors will be trimmed using Cutadapt. The cleaned reads will then undergo de novo assembly using MEGAHIT, and viral contigs will be identified by aligning them with the GenBank database using BLASTx, with an E-value cut-off of <10⁻⁵.

Phylogenetic Analysis:

Phylogenetic analysis will be performed using nucleotide or amino acid sequences of the closest related viral strains identified through BLASTx. Reference sequences will be aligned using MEGA 11, and phylogenetic trees will be constructed using the Maximum Likelihood method with 1,000 bootstrap replicates to validate the reliability of the results.