Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study

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.

Distribution of Enterotoxin Gene-positive <i>Clostridium perfringens</i> Spores among Human and Livestock Samples and its Potential as a Human Fecal Source Tracking Indicator

“…Previous studies of potential human fecal source-tracking indicators have suggested that viruses, such as polyomaviruses, somatic coliphage, adenoviruses, and pepper mild mottle virus, may all be suitable indicators [30][31][32][33]. Human fecal pollution-source tracking is extremely important for investigations of pathogens such as noroviruses or Cryptosporidium hominis, which cause illnesses via person-to-person transmission.…”

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

“…For example, Ahmed et al detected human adenoviruses in 73% of sewage samples in Australia (45), while Wolf et al detected adenovirus F in 100% of sewage samples in New Zealand (46) but found adenovirus C in only 36% of sewage samples. A multilaboratory study found that viruses were highly specific indicators of sewage in water, but the sensitivity of nine different qPCR methods for human virus detection was very poor, ranging from 0% to 13.2% (47). The sensitivity of the microarray was estimated using only those probes for which we have sufficient evidence in the literature to assign consistent "host association."…”

Human-Associated Bacteroides spp. and Human Polyomaviruses as Microbial Source Tracking Markers in Hawaii

“…Although several evaluation studies of molecular PCR markers have been conducted on the U.S. mainland (19)(20)(21) and elsewhere (22)(23)(24), no marker validation studies have been conducted in Hawaii or on other Pacific Islands. These validation studies have demonstrated that bacterial MST markers can be found in species of which they are not indicative, although at less frequently and at lower concentrations.…”