CrAssphage as a Novel Tool to Detect Human Fecal Contamination on Environmental Surfaces and Hands

Abstract

CrAssphage is a recently discovered human gut-associated bacteriophage. To validate the potential use of crAssphage for detecting human fecal contamination on environmental surfaces and hands, we tested stool samples (n = 60), hand samples (n = 30), and environmental swab samples (n = 201) from 17 norovirus outbreaks for crAssphage by real-time PCR. In addition, we tested stool samples from healthy persons (n = 173), respiratory samples (n = 113), and animal fecal specimens (n = 68) and further sequenced positive samples. Overall, we detected crAssphage in 71.4% of outbreak stool samples, 48%-68.5% of stool samples from healthy persons, 56.2% of environmental swabs, and 60% of hand rinse samples, but not in human respiratory samples or animal fecal samples. CrAssphage sequences could be grouped into 2 major genetic clusters. Our data suggest that crAssphage could be used to detect human fecal contamination on environmental surfaces and hands.

Keywords: crAssphage; cruise ship; enteric infections; environmental contamination; hand contamination; human fecal indicator; long-term care facility; norovirus outbreak; viruses.

Figure 1

Phylogenetic relationships and pairwise sequence comparison of crAssphage strains from swab samples collected during norovirus outbreaks on cruise ships. A) Phylogeny of crAssphage on cruise ships, showing ship and source for each strain. Inset shows position of cruise ship strains among reference strains; scale bar indicates number of nucleotide changes between sequences. B) Color-coded pairwise identity matrix for crAssphage strains. Each cell includes the percentage identity among 2 sequences (horizontally to the left and vertically at the bottom). Key indicates pairwise identity percentages.

Figure 2

Phylogenetic relationships and pairwise sequence comparison of crAssphage strains from hand rinse samples collected during norovirus outbreaks at long-term care facilities. A) Phylogeny of crAssphage strains. Strain identification includes facility (A–J), strain number, and human source (H, healthcare worker; R, resident) for each isolate. Sample source and genotypes are indicated. Red strain names indicate that both hand and stool sample are genetically related, blue strain names that paired hand and stool samples are genetically distinct. Black strain names indicate hand or stool sample pairs that tested negative for crAssphage. Inset shows position of long-term care strains among reference strains; scale bar indicates number of nucleotide changes between sequences. (B) Color-coded pairwise identity matrix for crAssphage strains. Each cell includes the percentage identity among 2 sequences (horizontally to the left and vertically at the bottom). Key indicates pairwise identity percentages