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Updated: May 9, 2026
Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses
Published on: December 3, 2011
Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study
Valerie J Harwood1, Alexandria B Boehm, Lauren M Sassoubre
1Department of Integrative Biology, University of South Florida, Tampa, FL 33620, USA.
Abstract:
An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.
Insights
This study evaluated microbial source tracking (MST) methods for detecting human fecal contamination. Virus methods showed high specificity but low sensitivity, while bacteriophage methods were less specific, highlighting the need for improved concentration techniques.
Area of Science:
- Environmental microbiology
- Water quality assessment
- Molecular biology techniques
Background:
- Microbial source tracking (MST) is crucial for identifying fecal contamination in water bodies.
- Existing MST methods vary in accuracy and reliability, necessitating inter-laboratory validation.
- Human viruses and bacteriophages are commonly used indicators for fecal pollution.
Purpose of the Study:
- To assess the accuracy of various virus and bacteriophage-based MST methods for detecting human fecal or sewage contamination.
- To evaluate the performance characteristics (sensitivity, specificity, predictive values) of these methods in a blind inter-laboratory study.
- To identify limitations and areas for improvement in current MST techniques.
Main Methods:
- An inter-laboratory study using blind fecal and sewage spiked samples in artificial freshwater.
- Testing of human viruses: adenoviruses (HAdV), enteroviruses (EV), noroviruses (NoV), and polyomaviruses (HPyVs).
- Testing of bacteriophages: somatic coliphages and F-specific RNA bacteriophages (FRNAPH) using plaque assays and genotyping.
Main Results:
- Human virus methods demonstrated high specificity (near 100%) but low sensitivity and negative predictive values due to small sample volumes and low target concentrations.
- Bacteriophage methods generally showed lower specificity for human sewage compared to virus methods.
- F-specific RNA bacteriophage (FRNAPH) II genotyping achieved 18% sensitivity and 85% specificity.
Conclusions:
- High specificity of human virus methods provides confidence in positive identification of contamination.
- Low sensitivity and negative predictive values indicate a need for enhanced sample concentration methods and larger sample volumes.
- Further optimization of MST techniques is required for accurate assessment of human fecal pollution and absence thereof.
