Table 2.

Examples of available analytical methods for OPPP detection in environmental samples

Targeting genus	Species(if any)	Method	Sample characteristics	Detection or quantification limit	Notes	Ref.
Legionella		Culturing	Pool water and shower water	LOD: 5 cfu/l or pool water and 10 cfu/l for shower water	Agglutination tests were used following cultivation to distinguish L. pneumophila serogroup 1–6, L.bozemanii, L. dumoffii, L.gormanii, and L. micdedei	(Leoni et al., 2001)
		Culturing (ISO 11731)	Hot water from hotels	LOD:25 cfu/l	Agglutination tests were used to separate L. pneumophila isolates serogroup 1 and 2-14, as well as seven species of non- L. pneumophila legionellae	(Bargellini et al., 2011; Borella et al., 2005)
	Legionella spp. & L. pneumophila	Culturing, PCR, &q-PCR	Drinking water from treatment plants	LOD (Legionella spp., direct culturing, without concentration): 1000 cfu/l LOD (L. pneumophila, q-PCR): 1000 gene copies/L	Semiquantitative PCR was used for concentration assessment. PCR products were cloned and sequenced for genitive diversity exploration	(Wullings and van der Kooij, 2006)
	Legionella spp. & L. pneumophila	Culturing, PCR, &q-PCR	Hospital water	LOD (Legionella spp. culturing): 1 cfu/100 ml LOD (Legionella spp. q-PCR): 2.3 cfu/100 ml; LOQ (Legionella spp. q-PCR): 23 cfu/100 ml LOD and LOQ for L. pneumophila: <2.3 and 23 cfu/100 ml, respectively	Correlated q-PCR and culturing results (P<0.001) with higher q-PCR enumeration number in relative to culturing; Correlated results between genus-specific and species-specific assays	(Wellinghausen et al., 2001)
	Legionella spp. & L. pneumophila	Culturing & q-PCR	Cooling towers Hot and cold water	LOD for q-PCR and culturing: 750 GU/l for water samples from cooling tower and 190 GU/l for samples from hot and cold water systems.	Greater discrepancy between q-PCR and culturing results for cooling tower samples compared to hot and cold water samples.	(Lee et al., 2011b)
		q-PCR	Spa water	LOD:40 GU/l LOQ: 1000 GU/l	Results revealed weak correlation between culturing and q-PCR	(Guillemet et al., 2010)
		Culturing, q-PCR, EMA-q-PCR	Hot water samples	LOD for EMA-q-PCR: 200 GU/ml for 1 ml of sample treated with EMA; 250GU/l for 1L of sample water	v-PCR counts were equal to or higher than those obtained by culture, and lower than or equal to conventional qPCR counts	(Delgado-Viscogliosi et al., 2009)
		Immunofluorescent labeling combined with solid-phase flow cytometry		LOD: 10-100 bacteria/l	Obtained numbers are higher than CFU counting
Mycobacterium		Culturing	Drinking water samples	LOD: 10 cfu/l	After culturing, PCR amplification of the hsp-65 gene followed by enzyme restriction of the PCR product was used for identification	(Falkinham et al., 2001)
		q-PCR	Cooling tower water	LOQ: 500 cells/l		(Adrados et al., 2011)
		q-PCR and culturing	Drinking water and other environmental samples	LOD for q-PCR: 6 genome equivalents for M. chelonae LOQ for q-PCR:100 genome equivalents	Higher concentration level but lower detection rates with q-PCR in relative to culturing method	(Radomski et al., 2013)
	Mycobacterium avium subsp. paratuberculosis	q-PCR	Drinking water and biofilm	Assay LOD: 1.8 gene copy		(Beumer et al., 2010)
P. aeruginosa		Pseudalert®/Quanti-Tray® MPN Test, Culturing method	pool water samples, artificially contaminated samples		Comparable results between Pseudalert®/Quanti-Tray® MPN Test and ISO 16266 and MoDW Part 8 methods	(Sartory et al., 2015)
		q-PCR	Clinical and environmental isolates		Duplex q-PCR assay with two targeted genes (ecfX and gyrB), requires simultaneous confirmation of P. aeruginosa by two genes	(Anuj et al., 2009)
		q-PCR and culturing	Hospital faucets (water, aerator and drain swabs)		qPCR revealed 50% positivity for P. aeruginosa remaining in the water compared with 7% by culture	(Bedard et al., 2015)
		q-PCR, PMA-q-PCR, standard cultivation-based technique	Drinking water and process water	LOD of q-PCR and PMA-q-PCR :102~103 cells/l	>80% samples yield accordant results by q-PCR, PMA-q-PCR, and cultivation based method. PMA-q-PCR reduced 4% false positive rates when compared to q-PCR.	(Gensberger et al., 2014)
Acanthamoeba		Culturing followed by morphological identification	Household water		Fungal overgrowth of the samples occurred more often with biofilm sample than water samples.	(Stockman et al., 2011)
		Culturing followed by morphological identification and PCR/sequencing	Tap water			(Winck et al., 2011)
		q-PCR	Anthropogenic water and biofilms	Assay LOD (trophozoites): 3 cells for water samples, 10 cells for biofilm samples	Qvarnstrom assays outperforms Riviere assay for Acanthamoeba detection and quantification	(Chang et al., 2010)
		PMA-q-PCR	Culture suspension, water samples from eyewash station, cooling tower, wastewater treatment plant	Detection range: 5~1.5×105 cells		(Chang et al., 2013)
Naegleria fowleri		q-PCR and MPN methods	Cooling water samples	LOQ for q-PCR: 320 cells/l	Samples with concentration <200 cells require culture method analysis; high q-PCR estimated numbers compared to MPN method	(Behets et al., 2007a)
		q-PCR and NNA-E. coli culturing method	N. fowleri cells spiked into water and biofilm samples	LOD in water samples: 5 cells in 250 ml water for a 66% detection rate and 10 cells for a 100% detection rate; DOL in biofilm samples: 1 cell for a 66% detection rate and 5cells for a 100% detection rate	Culturing method is less sensitive compared to q-PCR method	(Puzon et al., 2009)
		IMS-q-PCR	N. fowleri seeded lake water	LOD: 14 cells/l;	The methods has an average recover rate of 46%	(Mull et al., 2013)
		ELISA	Environmental water samples	LOD: 2000 cells/l	Can detect N. fowleri at three morphological stages, with 97.4% sensitivity and 97% specificity	(Reveiller et al., 2003)