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Environmental Pollution 259 (2020) 113903

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Environmental Pollution

journal homepage: www.elsevier .com/locate/envpol

Waterborne protozoan pathogens in environmental aquatic biofilms:Implications for water quality assessment strategies*

Frederick R. Masangkay, RMT MSMT a, b, *, Giovanni D. Milanez, RMT MSMT a, b,Amalia Tsiami, PhD c, Freida Z. Hapan, PhD d, Voravuth Somsak, PhD a,Manas Kotepui, PhD a, Jitbanjong Tangpong, PhD a, **, Panagiotis Karanis, PhD e, f

a Biomedical Sciences Program, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, 80160, Thailandb Department of Medical Technology, Institute of Arts and Sciences, Far Eastern University Manila, 1015, Philippinesc London Geller College of Hospitality and Tourism, University of West London, St Mary’s Road, Ealing, London, W5 5RF, United Kingdomd Department of Medical Technology, College of Pharmacy, The Pontifical and Royal University of Santo Tomas, Espa~na Blvd, Sampaloc, Manila, 1008,Philippinese Medical Faculty and University Hospital of Cologne, Cologne, 50923, Germanyf University of Nicosia Medical School, Nicosia, 2408, Cyprus

a r t i c l e i n f o

Article history:Received 19 November 2019Received in revised form29 December 2019Accepted 29 December 2019Available online 7 January 2020

Keywords:AsiaBiofilmsCryptosporidiumGiardiaLakes

* This paper has been recommended for acceptanc* Corresponding author. Department of Medical Tec

Sciences, Far Eastern University Manila, 1015, Philipp** Corresponding author. Biomedical Sciences ProgSciences, Walailak University, Nakhon Si Thammarat,

E-mail addresses: frederick_masangkay2002@yagmilanez@feu.edu.ph (G.D. Milanez), amalia.tsiahapanmfz@gmail.com (F.Z. Hapan), voravuth.smanaskote@gmail.com (M. Kotepui), rjitbanj@wu.acunic.ac.cy (P. Karanis).

https://doi.org/10.1016/j.envpol.2019.1139030269-7491/© 2020 Elsevier Ltd. All rights reserved.

a b s t r a c t

Biofilms containing pathogenic organisms from the water supply are a potential source of protozoanparasite outbreaks and a significant public health concern. The aim of the present study was todemonstrate the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens (WBPP)in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottomwater (BW) counterparts using manual filtration and elution from low-volume samples. For scenariopurposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collectedfrom the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques forCryptosporidium, and Immunofluorescence staining for the simultaneous detection of Cryptosporidiumand Giardia. WBPP were found in 43% SAB, 39% SW, and 39% BW of the samples tested in the presentstudy with SAB results reflecting SW and BW results. Further highlights were demonstrated in the po-tential of using low-volume samples for the detection of parasites in source water. Scanning electronmicroscopy of OM-SAB samples revealed a naturally-associated testate amoeba shell, while Cryptospo-ridium oocysts spiked samples provided a visual profile of what can be expected from naturallycontaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatialoccurrence of waterborne protozoan pathogens in low-volume aquatic matrices and further warrantsSAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS).

© 2020 Elsevier Ltd. All rights reserved.

e by Christian Sonne.hnology, Institute of Arts andines.ram, School of Allied Health80160, Thailand.hoo.com (F.R. Masangkay),mi@uwl.ac.uk (A. Tsiami),o@wu.ac.th (V. Somsak),.th (J. Tangpong), karanis.p@

1. Introduction

Cryptosporidium and Giardia are protozoan agents of diarrhealoutbreaks worldwide (Baldursson and Karanis, 2011; Efstratiouet al., 2017a; Karanis et al., 2007; Mac Kenzie et al., 1994). Crypto-sporidiosis is the second leading global cause of infantile mortalitynext to rotavirus infection (Checkley et al., 2015), while giardialinfections are reported to be at more than 200 million casesannually worldwide. Both Cryptosporidium and Giardia are listed inthe ‘Neglected Diseases Initiative’ by the World Health Organiza-tion (Savioli et al., 2006). The immunocompromised population is

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 1139032

at highest risk in contracting these waterborne protozoan patho-gens (WBBP) with low infectious dose; ingestion of as few as 10(oo)cysts have been reported to cause morbidity even in theimmunocompetent population (Dupont et al., 1995; Okhuysenet al., 1999; Ortega and Adam, 1997; Steiner et al., 1997).

Biofilms are formed from the transition of planktonic cells tosessile cells, which leads to the accumulation and establishment ofstable interactions between pure and multi-species communities(Matos et al., 2017; Suba and Masangkay, 2013). The complexity ofthe biofilm community is brought about by the introduction ofother unicellular organisms like protozoans (Murphy et al., 2018;Waiser et al., 2016; Xu et al., 2014), which can facilitate the inter-species co-evolutionary processes as recently reported and dis-cussed in the biofilm inhabitation of Acanthamoeba spp. infectedwith bacteria containing bacteriophages with the possibility ofharboring virophages as well (Bekliz et al., 2016; Masangkay et al.,2018).

Biofilms are potential reservoirs of human and zoonotic path-ogens in environmental aquatic systems and contribute to thepersistence and transmission of waterborne protozoan pathogens(WBPP) and other microorganisms (Ryan et al., 2016). The biofilmroughness contributes to the attachment of protozoan (oo)cysts(DiCesare et al., 2012a; Wolyniak et al., 2009; Wolyniak et al., 2010)and the biofilm mass as a whole provides UV protection for (oo)cysts trapped within its matrix, further contributing to environ-mental persistence of potentially-pathogenic protozoans (DiCesareet al., 2012b).

The aim of this study was to demonstrate the presence ofCryptosporidium and Giardia (oo)cysts in environmental aquaticsubstrate-associated biofilms (SAB) and compare its results againstsurface water (SW) and bottom water with sediment (BW) coun-terparts through manual filtration and elution of low-volumesample and to introduce the significance of biofilms as biologicalreservoirs for WBPP.

2. Materials and methods

2.1. Study sites

A University Lake in Nakhon Si Thammarat, Thailand (Fig. 1A)was chosen as one of the study sites due to its popularity withstudents and tourists and its importance as a habitat for localamphibians, reptiles, small mammals, livestock, and aquatic birds.Fig. 1B illustrates the other study sites in the Philippines (Laguna deBay, Taal Lake, Pantabangan watershed and The Seven Lakes ofLaguna, and Ipo watershed), four of which were tested for thepresence of WBPP. The Ipo watershed site was selected for the one-month-old SAB (OM-SAB) in-situ culture experiment. Laguna deBay is the largest lake in the Philippines and is the major site forfreshwater aquaculture in Luzon (Guzman, 2006; Israel, 2007). TaalLake is the third largest lake in the country and the primary sourceof freshwater fish in the surrounding provinces (Martinez, 2011).The Pantabangan watershed was constructed by flooding a townand erecting a dam structure for hydroelectric power. The SevenLakes of San Pablo Laguna is a system of crater lakes, where Sam-paloc and Bunot Lake are used for aquaculture. Palakpakin Lakeconnects to a river system and Mohicap Lake is enclosed by naturalsurroundings that offer tourists and local residents a breath-takingview of the area. The remaining Yambo, Pandin, and Kalibato Lakesare mainly used for picnic and water activities. The Ipo watershedhas been created by a river system that drains the larger Angat dam,which provides the majority of the water supply to Manila and itsnearby metropolis.

2.2. Sample collection

Surface water samples (SW) were collected in 50-mL sterilepolyethylene containers by collecting no more than 30 cm belowthe water surface from each sampling area (Table 1). Bottomwaterwith sediments (BW) were collected from a water depth of at least60 cm and no more than 90 cm along the shoreline that werecomposed of one-part sediments and four-parts bottom water.Substrate-associated biofilms (SAB) were harvested from aquaticplants whenever present. Short segments of small aquatic plantswere collected and washed with sterile distilled water to removenon-adherent cells, cut into smaller portions in order to fit looselyinside 50-mL sterile polyethylene containers with a final volume of50-mL sterile distilled water. In the absence of aquatic plants,adherent biofilms (approximately one bottle cap full) were scrapedfrom rocks no more than 30 cm below the water surface. Sampleswere transported to the laboratory and processed within 48 h aftercollection. The 50-mL samples in this study aimed to provide initialdata on the capability and feasibility of detecting pathogenic (oo)cysts from environmental aquatic matrices using a low-volumesampling technique. The low sample volume of 50-mL was elec-ted to represent the hypothetical volume of water sample thatcould be accidently ingested or inhaled by an individual duringwater activities, which could lead to the infection of Cryptospo-ridium and/or Giardia if present in the water source.

2.3. Sample processing

Each sample was vortexed for 1 min to dislodge adherent cellsfrom any larger organic substances and debris for even distributionthroughout the sample matrix and left to stand for 5 min to settleheavier solids. The SW, BW and SAB sample suspensions were eachmanually filtered through a 1.2-mm glass microfiber filter fittedinside a 50-mL sterile disposable syringe (Masangkay et al., 2016).Glass microfiber filters were recovered and placed in steriledisposable polyethylene plates, where the filtered sediments werescraped using a sterile inoculating loop and 5-mL sterile distilledwater as eluent. The 5-mL eluates were transferred to sterile testtubes and centrifuged at 1500 g for 15 min (US EPA Method 1623,2005). The 3-mL of the supernatants were discarded and theremaining 2-mL and pellet were mixed to form a suspension,where they were subsequently transferred to microcentrifugetubes and smeared for microscopy within 24 h.

2.4. Microscopy of Cryptosporidium and Giardia (oo)cysts

25-mL of each suspensionwas made into a 1-cm diameter smearon a clean glass slide in duplicate. Screening for Cryptosporidiumoocysts was performed by staining with modified Kinyoun’s (MK),modified Safranin Methylene Blue (SMB), and Auramine (Aura).Microscopic confirmation of Cryptosporidium spp. and Giardia spp.(oo)cysts were performed by Direct Antibody fluorescent testing(IFT) using the Aqua-GloTM G/C Direct Comprehensive Kit(Waterborne Inc. USA) according to the manufacturer’s in-structions. Light microscopy of MK and SMB smears was performedby examining 200 oil immersion fields using a Nikon Model EclipseE100LED light microscope. An upright epifluorescence incidentlight excitation trinocular UB microscope with a three-megapixelcamera was used to examine 200 high power fields of the Auraand IFT smears. Suspected (oo)cysts were compared to stainedpositive controls (A100FLR-1X Aqua-Glo G/C Direct positive con-trol), where MK and SMB stained Cryptosporidium (oo)cysts stoodout as round bodies measuring 4 to 6-mm in diameter with occa-sionally visible sporozoites that are bright red against a bluebackground. Aura and IFT stained oocysts fluoresced bright apple-

Fig. 1. Geographical representation of study sites and sampling areas of substrate-associated biofilms in a University lake in Tha Sala district, Nakhon Si Thammarat, Thailand (A)and in the Island of Luzon, Philippines (B).

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 113903 3

Table 1Simultaneous and multi-spatial occurrence of WBPP in study sites and sampling areas.

Sampling areas (n ¼ 23) Coordinates WBPP status in sample matrices Contributory sources of contamination

SW BW SAB

ThailandTS1A1 Rest house 8.639596, 99.883423 e C C Z4, E2TS1A2 Cattle farm 8.641208, 99.883122 C C C Z4, E2TS1A3 Fish nets 8.640635, 99.875751 C, G C C A1, A4, Z1, Z2, Z3, Z11, E2PhilippinesPS1 Laguna de BayPS1A1 14.510793, 121.144123 C C e A1, A3, A4, Z2, Z8, E1PS1A2 14.505526, 121.072599 C C e A1, E1, E2PS1A4 14.184165, 121.201272 C C e A1, E1, E2PS2 Taal LakePS2A1 14.069887, 120.951694 e e C A1, A4, Z2PS2A2 14.006203, 121.091681 e e C A2, A4, Z2, E2PS2A3 13.938634, 121.035967 C e e A2, A4, Z2, E2PS3 Pantabangan watershedPS3A1 15.835721, 121.191528 C C e A2, A4, Z4, E2PS4 7 Crater Lakes of LagunaPS4L1 Sampaloc LakePS4L1A1 14.074873, 121.326266 C e C A1, A2, A4, Z2, Z5, Z7, Z9PS4L1A2 14.081366, 121.327980 e e e A1, A2, Z2, Z5, Z7, Z9PS4L1A4 14.073950, 121.333047 e e e A1, A2, Z2, Z5, Z7, Z9PS4L2 Bunot LakePS4L2A1 14.078870, 121.341471 e e C A1, A2, Z2, Z5, Z7, Z6, Z8, Z9, E1PS4L2A2 14.082432, 121.346738 e e C A1, A2, Z2, Z5, Z7, Z6, Z8, Z9, E1PS4L3 Palakpakin lakePS4L3A1 14.112446, 121.336483 C e e A1, A2, A3, E2PS4L4 Mojicap LakePS4L4A1 14.123512, 121.335790 e C e A2, A4, Z2, E2PS4L4A2 14.121030, 121.335897 e e e A2, A4, Z2, E2PS4L5 Yambo LakePS4L5A1 14.121134, 121.369148 e e G A4, A5, E2PS4L5A2 14.117229, 121.368207 e G e A4, A5, E2PS4L6 Pandin LakePS4L6A1 14.116827, 121.368650 e e G A4, A5, E2PS4L6A2 14.112670, 121.364796 e e e A4, A5, E2PS4L7 Kalibato LakePS4L7A2Positivity rate for sampling area ¼ 78%

14.105084, 121.366484 e

39%e

39%e

43%A4, Z2, E2

PS5 Ipo watershed 14.889154, 121.162871 Planting site of OM-SAB A2, A4, Z6, Z8, Z9, Z10, E2 chicken

Legend: (WBPP) waterborne protozoan pathogen; (SW) surface water; (BW) bottomwater with sediment; (SAB) substrate-associated biofilm; (C) Cryptosporidium spp. oocyst;(G) Giardia spp. oocyst; (�) none seen.Sampaloc lake sampling area 3 was not included in the study as this sampling area had no SAB sample.Positivity for Cryptosporidium was detected by any one of the microscopy methods of MK, SMB, Aura, and IFT.Positivity for Giardia was detected only through IFT.(A) Anthropogenic: (A1) Domestic wastewater; (A2) Fishing; (A3) Industrial wastewater; (A4) Swimming; (A5) Tourism activities.(Z) Zoogenic: (Z1) Amphibians; (Z2) Aquaculture; (Z3) Birds; (Z4) Cattle; (Z5) Cats; (Z6) Chicken; (Z8) Ducks; (Z9) Goats; (Z10) Pigs; (Z11) Reptiles.(E) Environmental: (E1): Aquatic plants; (E2) Soil run-off.

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 1139034

green against a black background (CDC DPDx Cryptosporidiosis).Giardia cysts stained with IFT were ovoid, measuring 10 to 20-mm,and fluoresced bright apple-green against a black background (CDCDPDx Giardiasis).

2.5. OM-SAB grown in-situ

Naturally grown in situ one-month-old substrate-associatedbiofilms (OM-SAB) were produced from the Ipo Watershed usingglass coverslip substrates that were secured to 20 X 3-cm plasticpanels held together by a 20 X 5-cm Styrofoam body in the hori-zontal and vertical orientation on either side (Supplemental ma-terial 1). The constructed substrates were immersed 30 to 120 cmbelow the surface of the water from October 1, 2018 to November 1,2018.

2.6. OM-SAB microscopy panel

To be able to determine the presence of naturally-associatedCryptosporidium and Giardia (oo)cysts in OM-SAB, coverslip

panels with OM-SAB were collected and washed with steriledistilled water to remove non-adherent cells and individuallyplaced into 50-mL sterile polyethylene containers, transported tothe laboratory, and harvested by scraping the coverslip substrate-side (in contact with the water column) with sterile scalpelblades, and prepared into 2-mL microcentrifuge tube suspensionsusing sterile distilled water. OM-SAB suspensions were vortexed for1 min and left to stand for 5 min to settle the heavier particles. 25-mL of the OM-SAB suspension was aspirated and prepared induplicate into 1-cm diameter smears on clean glass slides andstained with MK, SMB, Aura, and IFT (Aqua-GloTM G/C DirectComprehensive Kit) according to the manufacturer’s directions.

2.7. Scanning electron microscopy (SEM) of OM-SAB

To be able to provide SEM visualization of the natural archi-tecture and composition of an environmental aquatic substrate-associated biofilm, horizontal and vertical OM-SAB grown onglass coverslips substrates were fixed with absolute methanol andallowed to dry for 24 h. The coverslip substrates were gently broken

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 113903 5

into shards of approximately 3 X 3-mmwith care not to disrupt theOM-SAB, then attached to a carbon tape secured on the SEM(TM3000 Hitachi Tabletop SEM) metal platform for examination atthe University of Santo Tomas, Thomas Aquinas Research Centre.

2.8. Spiking of OM-SAB for Aura, IFT, and SEM

In order to simulate environmental contamination of OM-SABwith Cryptosporidium oocysts, 25-mL of OM-SAB suspensions werespiked with 10-mL of C. parvum oocysts in 1 X 106 cells/mL con-centration (P102C @ 1x10/6, Cryptosporidium parvum oocysts, 1million, in 4 mL, Waterborne Inc. USA) and prepared in duplicateinto 1-cm diameter smears on clean glass slides and stained withAura and IFT. For SEM analysis of spiked OM-SAB, one vertical andone horizontal OM-SAB were fixed with absolute methanol,allowed to dry for 24 h, then manually broken into shards ofapproximately 3 X 3-mmwith care not to disrupt the OM-SAB, thenspiked with 5-mL of C. parvum oocysts, and allowed to dry for 24 hfor visualization through SEM.

Table 2SAB results in comparison to SW and BW counterparts.

Study sites and sampling areas Sample matrix, method, and WBPP

SAB

Cryptosporidium spp. oocysts

Method: Screening and confirmatory

MK SMB Aura

Thailand samplingTS1A1 e e þTS1A2 þ þ þTS1A3 e þ þPhilippine samplingPS1 Laguna de BayPS1A1 e e e

PS1A2 e e e

PS1A4 e e e

PS2 Taal LakePS2A1 e e þPS2A2 e e þPS2A3 e e e

PS3 Pantabangan watershedPS3A1 e e e

PS4 7 Crater Lakes of LagunaPS4L1 Sampaloc LakePS4L1A1 e e þPS4L1A2 e e e

PS4L1A4 e e e

PS4L2 Bunot LakePS4L2A1 e e þPS4L2A2 e e þPS4L3 Palakpakin LakePS4L3A1 e e e

PS4L4 Mojicap LakePS4L4A1 e e e

PS4L4A2 e e e

PS4L5 Yambo LakePS4L5A1 e e e

PS4L5A2 e e e

PS4L6 Pandin LakePS4L6A1 e e þPS4L6A2 e e e

PS4L7Kalibato LakePS4L7A2 e e e

Total sampling areas (n ¼ 23)Positive for WBPP 1/23 2/23 9/23Positivity rate 4% 7% 39%

Legend: (WBPP) waterborne protozoan pathogen; (þ) presence of WBPP; (�) no WBPPassociated biofilm; (MK) Modified Kinyoun’s; (SMB) Modified Safranin Methylene Blue; ((C) Cryptosporidium spp. oocyst; (G) Giardia spp. cyst.

3. Results

Table 1 outlines the coordinates (geographical location), WBPP,and contributory contamination sources for each sampling area.The majority of the samples tested positive for waterborne proto-zoan pathogens in Thailand (3/3) and the Philippines (15/20)sampling areas. Overall, 78% (18/23) of the aquatic sample matricestested in this study contained at least one WBPP in at least onesample matrix. Table 2 highlights the natural association of Cryp-tosporidium and Giardia (oo)cysts in SAB relative to SW and BWmatrices tested in this study. IFT confirmed 22% (5/23) Cryptospo-ridium and 4% (1/23) Giardia (oo)cysts in SAB which collates with39% positivity from both SW and BW counterparts. Table 3 shows a33% (3/9) agreement between SW and SAB samples positive forWBPP and the increased detection capacity of WBPP by 50% (7/14)as compared to SW negative for WBPP. The comparison of BW andSAB demonstrated similar results. Table 4 provides a panel of mi-croscopy results for OM-SAB, where horizontal and vertical sam-ples were negative for naturally-associated WBPP. The absence of

SW BW

Giardia spp. cyst

Method: Confirmatory only

IFT IFT

þ e e C: 1,3e e C: 1 C: 1,3,4þ e C: 1; G: 4 C: 1,3

e e C: 1 C: 2e e C: 1 C: 2e e C: 3 C: 3

e e e e

e e e e

e e C: 3 e

e e C: 3 C: 1

e e C: 1 e

e e e e

e e e e

þ e e e

þ e e e

e e C: 3 e

e e e C: 3e e e e

e þ e e

e e e G: 4

þ e e e

e e e e

e e e e

5/23 1/23 9/23 9/2322% 4% 39% 39%

observed; (SW) surface water; (BW) bottom water with sediment; (SAB) substrate-Aura) Auramine; (IFT) immunofluorescence test; (1) MK; (2) SMB; (3) Aura; (4) IFT;

Table 3Percent agreement and increased detection in the analysis of SAB along with SW and BW.

(þ) for WBPP (þ) SAB and (þ) SW % agreement (�) for WBPP (þ) SAB and (�) SW % higher detection

SAB n ¼ 23

SW9 3 33% (3/9) 14 7 50% (7/14)BW9 3 33% (3/9) 14 7 50% (7/14)

Positivity for WBPP (Cryptosporidium) was detected by any one of the microscopy methods of MK, SMB, Aura, and IFT.Positivity for WBPP (Giardia) was detected only through IFT.

Table 4Microscopy results of OM-SAB grown in situ.

Substrate orientation Microscopy panel Spiked OM-SAB

Natural OM-SAB

30 cm vertical Negative1,2,3,4 C. parvum3,4

30 cm horizontal Negative1,2,3,4 C. parvum3,4

Legend.1 ¼ MK; 2 ¼ SMB; 3Aura; 4 ¼ IFT Cryptosporidium spp. and Giardia spp. (oo)cysts.

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 1139036

Giardia cysts was consistent in both natural and spiked OM-SABafter staining with Aura and IFT, which confirms its absence fromthe samples tested in the study. Fig. 2 demonstrates positive con-trols of Cryptosporidium and Giardia (oo)cysts (Waterborne Inc.)that were used as reference for microscopic identification. Fig. 3shows the microscopic detection of Cryptosporidium and Giardia(oo)cysts in SW, BW, and SAB using the elected staining techniques.Fig. 3a-c demonstrate typical Cryptosporidium oocysts stained withMK and SMB as round (oo)cysts with internal structures thatstained red by Carbol fuchsin, while Fig. 3d-f demonstrate apple-green fluorescence attributed by Auramine and IFT stains. AllCryptosporidium oocysts (Fig. 3a-e) fit within the 4e6 mm diameterrange and had morphologic characteristics similar to the positivecontrol of C. parvum (as shown in Fig. 2a-d). Fig. 3 f demonstratesthe oval to ellipsoid morphology and size range of 10e14 mm ofGiardia cysts, similar to the G. lamblia positive control found inFig. 2e. Fig. 4 shows pictures of OM-SAB grown in situ in Ipowatershed on glass coverslip substrates with microscopic archi-tecture and associated structures visualized through SEM. Thicknetworks of exopolysaccharide matrix are observed in Fig. 4 d,which is responsible for binding the contents of the biofilmmicrocosm. A naturally-associated testate amoeba shell (Fig. 4e)was incidentally identified in a vertical OM-SAB. Fig. 4 g demon-strates simulated environmental contamination with spikedC. parvum (oo)cysts for scenario purposes and the relative com-parison of the size of C. parvum oocysts against contents of the OM-SAB. Evidence of the temporal accumulation and the diversity oforganic materials in the biofilm matrix, mainly, freshwater diatoms(Fig. 4f), contributes to the variable surface roughness and porositywhich can potentially facilitate attachment and trapping of path-ogenic (oo)cysts and other organic materials. Horizontal substrates

Fig. 2. Photomicrographs of Positive controls of C. parvum and G. lamblia (oo)cysts (WaterboG. lamblia cyst after IFT staining; a and b 1000X magnification; c to e 400X magnification;

and substrates immersed at 120 cmdepth had denser biofilmgrowth compared to vertical and 30 cm depth counterparts (Sup-plemental material 2 and 3). All staining methods detected 0e1(oo)cyst per 25-mL smeared suspension thereby providing anapproximate (oo)cyst load of 2000 (oo)cysts per 50-mL or 40,000(oo)cysts per 1-L of sample matrix. On very rare occasions, 0e2 (oo)cysts per 25-mL were observed.

4. Discussion

4.1. Microscopy panel and low-volume sampling

The results of this study show that traditional staining methodsof MK and SMB are cost-effective but provided lower detectioncompared to fluorescence techniques. The low-cost MK stainingmethod has been reported to exhibit 66% sensitivity and up to 88%specificity (Elsafi et al., 2014; Johnston et al., 2003), and only re-quires a light microscope (Current and Garcia, 1991). In our study,only 4% (1/23) SAB tested positive for Cryptosporidium (oo)cystsusing MK, which collates with SW and BW results (Tables 1 and 2).In similar studies, MK detected only 2% (3/135) Cryptosporidium(oo)cysts from water samples in Turkey, which were identified asC. parvum after polymerase chain reaction (Aslan et al., 2012) Inaddition, MK presented with 71% (53/75) positivity for Cryptospo-ridium oocysts in a metropolitan watershed in the Philippines,which suggests heavy parasite contamination during the three-daysampling period (Masangkay et al., 2016). In the present study, thestaining characteristics of Cryptosporidium were identical for bothMK and SMB, where 7% (2/23) positivity for SMB was relativelyclose to the results obtained from MK but lower than Aura with a39% (9/23) screening positivity. These results suggest variability inthe power of (oo)cysts detection depending on the microscopicmethod and the utility of Aura as a convenient screening methodfor detection of Cryptosporidium oocysts in water samples (Ahmedand Karanis, 2018; Hanscheid et al., 2008; Smith et al., 1989). In thepresent study, IFT findings were positive for Cryptosporidium andGiardia (oo)cysts at 22% and 4%, respectively. IFT kits are expensiveand require a fluorescence microscope. However, the cost may bejustified from the high degree of sensitivity (99%) and specificity(100%) for Cryptosporidium and 96%e100%, respectively, for Giardia(Adeyemo et al., 2018; Pacheco et al., 2013). Researchers in different

rne Inc. USA). a to d C. parvum oocysts after staining with a MK; b SMB; c Aura; d IFT; eScale bar for a and b ¼ 5-mm; Scale bar for c, d, and e ¼ 5-mm.

Fig. 3. Photomicrographs of aquatic sample matrices positive for Cryptosporidium spp. and Giardia spp. (oo)cysts. a to e Cryptosporidium spp. oocysts; f Giardia spp. cyst; a TS1A1 BW(MK); b TS1A3 SAB (SMB); c PS1A4 BW (MK); d TS1A3 SW (AURA); e PS4L2A2 SAB (IFT); f PS4L5A1 SAB (IFT); a to c 1000X magnification. d to f 400X magnification; Scale bar for ato c ¼ 5-mm; Scale bar for d to f ¼ 5-mm.

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 113903 7

parts of the globe have extensively contributed to establishing theimportance of water analysis methodologies in the effectivedetection of WBPP (Estratiou et al., 2017a; Estratiou et al., 2017b;Plutzer and Karanis, 2016). Likewise, the establishment of cost-effective concentration methods for WBPP in water samples likeflotation and flocculation being applied to large and lower volumewater samples have gained traction over the past two decades(Gallas-Lindemann et al., 2013; Gallas-Lindemann et al., 2016;Karanis et al., 2006; Karanis and Kimura, 2002; Koloren et al., 2016;Koloren et al., 2018; Kourenti et al., 2003; Kourenti and Karanis,2004; Kourenti and Karanis, 2006; Ma et al., 2019; Tsushimaet al., 2001; Tsushima et al, 2003a; Tsushima et al., 2003b). Low-volume water sampling for the detection of Cryptosporidium andGiardia is not routinely performed but a number of studies havealready confirmed its benefits as reported in the PCR positivity ofCryptosporidium in 50 mL raw river water samples in China (Xiaoet al., 2012), and studies in the Philippines that reported both MKand PCR confirmation and sequence identification of C. hominisdirectly from 50 mL samples (Masangkay et al., 2016; Masangkayet al., 2019a; Masangkay et al., 2019b). These methods are cost-effective alternatives to Method 1623 or other methodologies,providing effective detection of Cryptosporidium and Giardia,particularly in high-turbidity aquatic samples (Efstratiou et al.,2017). In the present study, low-volume water sampling offeredthe advantages of ease of collection and transport, multiple matrixsampling (SW, BW, and SAB), multiple area sampling per study site,reproducibility, and significantly lower test cost. The (oo)cyst loadof approximately 2,000 (oo)cysts per 50 mL or 40,000 (oo)cysts per1 L sample matrix indicated high contamination of the samplingareas withWBPP. These estimates, however, should not be taken asabsolute counts as (oo)cysts can be unevenly dispersed and asso-ciated with biocolloids, thereby further complicating its’ non-homogeneous distribution in environmental aquatic matrices.The variation of positive and negative results per staining methodacross each 50 mL sample may have been influenced by the non-homogeneous distribution of (oo)cysts in the aquatic samplematrices.

4.2. Spatial distribution of WBPP in aquatic matrices

Spatial distribution of Cryptosporidium and Giardia in samplematrices as shown in Tables 1 and 2 demonstrate that BW and SW,and in particular SAB are all suitable environmental aquaticmatrices for the detection of WBPP. Out of the 23 combined sam-pling areas (3 in Thailand and 20 in the Philippines), 78% (18/23)were positive for Cryptosporidium and/or Giardia in at least onesample matrix, where 39% (9/23) of both SW and BW, and 43% (10/23) SAB, tested positive. Results of this study on the presence ofCryptosporidium and/or Giardia in environmental drinking andrecreational waters can be supported by a study done in 2016 in theLa Mesa Watershed in the Philippines, where a high incidence ofCryptosporidium and Cyclospora was documented over a three-dayperiod of investigation (Masangkay et al., 2016). Similarly, a studyin Malaysia reported 100% (24/24) of SW to be positive for Cryp-tosporidium and Cyclospora (Bilung et al., 2017), with Giardia cystsreported to contaminate SW as well (Lass et al., 2017; Ramo et al.,2017). Studies investigating pathogenic protozoans from BW arerare; one exemplary study for BW was reported in 2017 from theYunlong Lake in China where 47% (28/60) BW tested positive forCryptosporidium (Kong et al., 2017). Although in vitro extracellularexcystation of Cryptosporidium has been elaborated in Pseudomonasaeruginosa aquatic biofilms (Koh et al., 2013; Koh et al., 2014), therehas been no documented case of the natural-association of Cryp-tosporidium in environmental aquatic biofilms until the first reportof C. hominis in SAB that was isolated from a freshwater sponge inthe Philippines in 2019 (Masangkay et al., 2019). In the presentstudy, the value of SAB as a supplemental sample matrix providedclose agreement with both SW and BW positivity and increasedreporting of WBPP as compared to analyzing SW alone (Tables 1and 3). The possible contributory factors for contamination of thesampling areas and the cycling of WBPP between humans and theenvironment can come frommany sources (Table 1) which includesanthropogenic activities, communities with poor sanitary andliving conditions, and improper domestic wastewater sanitizationprocedures (Adamska, 2014; Bhattachan et al., 2017; Masangkayet al., 2016). The presence of wildlife, domestic, and farm animals

Fig. 4. Photos of OM-SAB. a and b gross image; a horizontal coverslip substrate fixed with absolute methanol; b fragmented coverslip substrate spiked with C. parvum oocysts; c to gSEM photomicrographs; c OM-SAB vertical substrate; d OM-SAB horizontal substrate showing exopolysaccharide matrix created by resident microbes with trapped organic andinorganic materials (black circle); e OM-SAB vertical substrate with naturally-associated testate amoeba cyst (black circle); f predominance of diatoms in OM-SAB matrix (blackarrows); g OM-SAB spiked with C. parvum oocysts to simulate visualization of what can be expected from a high-density natural contamination (black circles).

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 1139038

in the surroundings of lakes and other water reservoirs plays asignificant role in parasite transmission to other animals and watersources thereby contributing to zoonotic transmission of Crypto-sporidium and Giardia (Gil et al., 2017; Wells et al., 2019; Zahediet al., 2016). In addition, birds, fish, amphibians, and small mam-mals have been tested and reported to be positive for Cryptospo-ridium and Giardia (Holubov�a et al., 2016; Karanis et al., 1996; Ryan,2010; Yang et al., 2015). Soil run-off, mixed with animal and humanexcreta can contribute to source water contamination with WBPP(Dai and Boll, 2003; Norman et al., 2013). Aquatic plants andimmersed substrates, as demonstrated in this study, can harborpathogenic (oo)cysts through the temporal accumulation of (oo)cysts in SAB. These contaminating factors are best exemplified inthe University Lake of Thailand, which simulates anthropogenic,zoogenic, and environmental cycling of WBPP in a small watercatchment (Fig. 3a-b and Table 1), where all sample matrices werepositive for Cryptosporidium oocysts and TS1A3 SW was

simultaneously positive for both Cryptosporidium and Giardia.

4.3. Aquatic biofilms: implications for water quality assessmentstrategies (WQAS)

Biofilms in relation to WQAS have been limited to tap watersystems (Wingender and Flemming, 2011; van der Kooij et al., 2017;Zhou et al., 2017), while biofilms in natural freshwater resources arenot screened for Cryptosporidium and Giardia contamination. Thenatural-association of a testate amoeba shell suspected of beingDifflugia (Qin et al., 2011) on the OM-SAB surface (Fig. 4e) dem-onstrates the potential for interactions between SAB and bio-colloids like Cryptosporidium and Giardia (oo)cysts (Luo et al., 2016;Searcy et al., 2006). Parasite load and temporal accumulationcontribute to the presence of WBPP in SAB as can be observed fromIpo watershed where OM-SAB grown in-situ were negative forCryptosporidium and Giardia (Table 4) seemingly because of low

F.R. Masangkay et al. / Environmental Pollution 259 (2020) 113903 9

parasite load where only one Cryptosporidium oocyst was detectedfrom all SW and BW from all four sampling sites (results notshown). Fig. 4c and 4 d demonstrate biofilm roughness and porosityand a network of exo-polysaccharide matrix, which can trapCryptosporidium and Giardia (oo)cysts (Wolyniak et al., 2010). Thisis in agreement with the observations presented in Fig. 4 g, whereC. parvum oocysts spiking experiment revealed the relative sizecomparison between Cryptosporidium oocysts and the size ofchannels and spaces on the OM-SAB surface, which can potentiallypermit the temporal accumulation and trapping ofWBPP, includinglarger testate amoeba shells among other biocolloids. Further, theabundance of organic debris in the OM-SAB permits UV radiationprotection, which further contributes to the maintenance of (oo)cyst viability (DiCesare et al., 2012b). As shown in Table 3, addi-tional analysis of SAB generated 33% agreement with positive SWresults and enhanced detection ofWBPP to 50% by not declaring thewater samples as not contaminated based on negative SW resultsalone with similar results observed from SAB and BW comparisons.As opposed to the real-time nature of contamination from SW, SABand BW are matrices that submits results based on the temporalaccumulation of WBPP similar to reports of temporal accumulationof Cryptosporidium oocysts in marine shellfish (Pagoso and Rivera,2017) and freshwater sponge in the Philippines (Masangkay et al.,2019). The results of this study support the hypothesis of thenatural-association of WBPP in SAB that can be exploited to detectCryptosporidium and Giardia (oo)cysts from environmental fresh-water resources and lead to improved water quality assessmentstrategies (iWQAS). The formulation of screening initiatives for thesimultaneous testing of SW, BW, and SAB in multiple samplingareas per study site can also provide important data for limiting theexposure of humans and animals to WBPP, stimulate governmentresponses, regulatory actions, and improved accessibility toscreening protocols.

5. Conclusions

Screening biofilms for waterborne protozoan pathogens hasbeen an underappreciated tool in the detection and mitigation ofwaterborne infections and outbreaks. The results of this studyprovide evidence that analysis of aquatic substrate-associatedbiofilms leads to improved water quality assessment strategies.Employing more than one microscopy method for the detection ofwaterborne protozoan pathogens in low-volume samples canimprove Cryptosporidium and Giardia detection from surface water,bottom water with sediments, and substrate-associated biofilms.Biofilms can act as biological reservoirs for waterborne protozoanpathogens by associating and protecting (oo)cysts from UV expo-sure within their matrices and surfaces.

Funding

This study was financially supported by The Walailak UniversityGraduate Studies Research Fund Contract No. 24/2562 awarded toFrederick R. Masangkay.

Declaration of competing interest

The authors declare no conflict of interest.

CRediT authorship contribution statement

Frederick R. Masangkay: Data curation, Formal analysis,Investigation, Methodology, Resources, Visualization, Writing -original draft, Writing - review & editing. Giovanni D. Milanez:Data curation, Formal analysis, Investigation, Writing - review &

editing. Amalia Tsiami: Validation, Writing - review & editing.Freida Z. Hapan: Funding acquisition, Investigation, Projectadministration, Resources, Supervision. Voravuth Somsak: Datacuration, Project administration, Supervision, Validation, Writing -review & editing. Manas Kotepui: Data curation, Project adminis-tration, Supervision, Visualization, Validation, Writing - review &editing. Jitbanjong Tangpong: Data curation, Formal analysis,Funding acquisition, Project administration, Resources, Supervi-sion, Visualization, Validation, Writing - review & editing. Pan-agiotis Karanis: Data curation, Formal analysis, Methodology,Project administration, Supervision, Visualization, Validation,Writing - review & editing.

Acknowledgments

We extend our deepest gratitude to Antoinette E. Dasig, MichaelChristopher P. Agustin, Rado Paul F. Cruz, Harrison Karl C. De Jesus,Jasmin Aliya L. Ellazar, Enrico B. Gozum, and Paula Marie Isabel F.Inocencio of the Department of Medical Technology, College ofPharmacy, The Pontifical and Royal University of Santo Tomas forbeing exemplary in their work and as undergraduate researchstudents whose roles played an integral part in the fruition of thisinternational collaboration. We thank Joseph Dionisio, Romel Sol-omon, Virgilio Bitagcul, Noel Cajuday, Maria Lourdes Policarpio, IvyTimogtimog, and Ernest Hidalgo of Far Eastern University-Manilafor laboratory logistics; Nina Caisido, Junee Linsangan of the USTAnalytical Services Laboratory Research Centre for the Natural andApplied Sciences, and Director of facility Dr. Christina Binag for SEMvisualizations, Harry Esmaya of Howell Medical supplies for thefluorescence microscopy facility, and Dr. Henry H. Stibbs, AngelaFaust, and Molly Rowe of Waterborne Inc. for the comprehensiveIFT kit. We thank Chad Schou for the editorial proofreading of themanuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.envpol.2019.113903.

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