Table 3.

Features of methods used to detect and quantify drinking water-associated pathogens that can cause infections in immunocompromised individuals (DWPI). Molecular methods are abbreviated as follows: polymerase chain reaction (PCR), quantitative-PCR (qPCR) and digital PCR (dPCR).

Method		What reaction/substance does it measure?	Live/dead?	Sample type (typical)	Description of method	Specificity of target	Enable type matching? §	Primary question it answers	Questions it cannot answer	Sector used by	ISO approved?	Time required
Urinary antigen test		Antigen	Y	Urine, bodily fluids	Confirms presence/absence of antigen in sample	Serogroup (only for L. pneumophila SG1)	N	Is or was the human body fighting this organism recently?	Was the body fighting it before, or a different strain of it?	Health	N *	Minutes
Culture		Growth	Y	Sputum, Water (sometimes filtered), tissue	Quantifies growth in specific nutrient rich conditions.	Specificity varies, typically species.	Y – must do prompt-ly	Can the organism grow right now under specific conditions?	Could the organism grow before, later, or under more realistic conditions? Is the organisms in a VBNC state?	Health, Ecology, Engineering	Y (N for NTM)	7+ days
PCR, qPCR, ddPCR	DNA-based	DNA (in all cells)	N	Water (filtered), swabs	PCR – confirms presence/absence of a sequence. qPCR and dPCR – quantify a sequence.	Specificity varies typically genus, species or indicator genes.	Y ***	Is or was a gene present in the sample, and how much, absolutely?	Is it capable of infection/the thing that caused infection? (for DNA – is it alive?)	Ecology, Engineering, [Health as secondary method]	Y‡	8–24 hrs
	RNA-based	RNA (only in live cells)	Y								N
	PMA/EMA based	DNA [live/dead stained]	Y**								N	8–24 hrs**
‘omics methods (e.g., amplicon sequencing, metagenomics, shotgun sequencing)		DNA (in all cells)	N	Water (filtered), swabs	Identifies relative abundance of sequences in communities. For taxonomic profiling or genome assembly.	Varies: Amplicon sequencing – resolution to genus level, metagenomics to species and in some cases strain level.	Y***	Is or was a gene present in the sample, and how much, relatively? How does occurrence of several DWPI compare?	Is it capable of infection/the thing that caused infection? (for DNA – is it alive?)	Ecology	N	1–2 wks
		RNA (only in live cells)	Y
		DNA [live/de ad stained]	Y**
Pseudalert		Growth	Y	Water	Most probable number based on growth in chambered trays	Species – P. aeruginosa only	Y – must do prompt-ly	Can the organism grow right now under specific conditions?	Could the organism grow before, later, or under more realistic conditions? Is the organisms VBNC?	Engineering	Y	1 day
Legiolert		Growth	Y			Species – L. pneumophila only					N	7 days
Adenosine tri-phosphate (ATP)		Chemical cycled by living cells	Y	Drinking water	Quantifies ‘total activity’ of microbes	Non-specific – total bacterial measures****	N	Are cells generally active? More so than usual?	Is there a specific organism of concern present? Total growth not consistently linked with any DWPI, but can indicate general temporal/spatial conditions conducive to microbial growth.	Engineering, Ecology (primarily in research now)	N	Seconds
Flow cytometry		Nucleic acids within cells	Y	Drinking water	Quantifies number of cells (total or intact)		N	How many cells are there? (How many are intact?)			N	Mins.
Heterotrophic Plate Counts		Growth	Y	Drinking water (sometimes filtered)	Quantifies growth of heterotrophs in relatively nutrient rich conditions.		N	Can heterotro phs grow right now under specific conditions?		Engineering (regulated drinking water method)	Y	2–3 days

^§As detection methods – none of these can alone be used for definitively determining the exact source of a disease. Typically, a more in-depth method must be applied to determine sequence, type, or serogroup matching.

^*ISO does not typically approve diagnostic tests. This is CDC approved, however.

^**Success of PMA and EMA based methods for differentiating between live and dead cells is debated.

^***Extracted nucleic acids (RNA, DNA, or EMA/PMA differentiated DNA) used w/metagenome sequencing can identify more in depth characteristics. This is typically expensive.

^****Flow cytometry measures have been developed specifically for Legionella, involving a immunomagnetic separation pre-processing step – but have not been widely validated.

^‡In 2019, ISO approved a molecular method for Legionella and Legionella pneumophila.