Development of Magnetic Particle Concentrator for Cryptosporidium and Giardia
Pathogens Capture in Shellfish
Palsiri Srirungruang1*, Pimchanok Witayaudom1, Narongsak Boonkhao2, Pranee Inprakhon3,
Warawoot Thowladda 4, Kittipong Harncharoen1, Mayuna Srisuphanunt1#
1
Programe of Studies, Faculty of Public Health, Mahidol University, Bangkok 10400,
Thailand
2
Institute of Medical and Public Health Technology, Nonthaburi 11150, Thailand
3
Faculty of Science, Mahidol University, Bangkok 10400, Thailand
4
Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520,
Thailand
*Palsiri.pal@hotmail.com, #Mayunaph@yahoo.com
Abstract
Cryptospordium and Giardia pathogens are the significant protozoan parasites cause
diarrhea in humans worldwide via drinking water and food. The detection of both parasites
requires special methods and laboratory equipments, due to the small size and frequently low
number of their oo(cysts) in water and food. Thailand posts recently a trade deficit as such
equipments imports. The aim of the study is to develop domestic Magnetic Particle
Concentrator (MPC) for Cryptospordium and Giardia oo(cysts) detection in shellfish. The
developed thai MPC was validated by the recovery efficiency of the oo(cysts) detection in
seeding water samples to compare with the imported MPC. The efficiency comparison of
thai MPC and imported MPC for oo(cysts) recovery detection was no significant difference
(p < 0.05). Total of 100 oysters, economical shellfish, were collected from local markets in
Bangkok and its surroundings. All samples were homogenized and Cryptosporidium and
Giardia (oo)cysts were enumerated using a combined Immunomagnetic Separation and
Immunofluorescence antibody method. Cryptosporidium oocysts and Giardia cysts were
found in 9 % and 8 %, respectively. The results indicated that thai MPC was developed with
low cost and instead use of imported products with no difference quality. The presence of
Cryptosporidium and Giardia in the oysters provide in epidemiological study and a situation
of sanitary risk with determining the safety of shellfish for human consumption.
Keywords: Cryptosporidium, Giardia, Magnetic Particle Concentrator (MPC), shellfish
Introduction
Cryptosporidium and Giardia are the important protozoan parasites found commonly in
water and food and cause diarrhea in humans worldwide. Infections by either
Cryptosporidium oocysts or Giardia cysts are upon transmission by drinking water and food
with a low infectious dose (1 and 2) and fatalities can occur in widespread outbreaks where
the children and elderly or immunosuppressed are infected ,due to no effective drug
treatment or vaccine available at present (3). Moreover, numerous waterborne outbreaks of
cryptosporidiosis and giardiasis have been documented (4 - 11). Oocysts and cysts are often
excreted in large quantities with the feces of infected animals and humans and remain
infective for months in environmental waters and are highly resistant to chemical
disinfectants (12). There has been several disease outbreak evidences from these waterborne
protozoa in different from other countries in the United States and European regions (13). In
Thailand, cryptosporidiosis has been reported in HIV infected patients with the prevalence
rate between 2.5% and 25% (14 - 19) and few reported in water and food contamination (18,
20 and 21). Although parasitic water and food-borne disease is a significant public health
problem, water and food are rarely tested for parasite contamination. This may be due to the
small size and frequently low number of Cryptosporidium oocysts and Giardia cysts in
naturally contaminated water and food, and lack of the promising method to determine the
enteric parasites in water and foods. The detection of these parasites is difficult and requires
special methods and also efficient laboratory equipments. Most of the equipments in the
country are imported and it is also lopsided for Thailand. No previous data is available
describing the detection equipments of these parasitic enteropathogens in water and food in
Thailand. The aims of the study is the development of domestic Magnetic Particle
Concentrator (MPC), one of the laboratory equipments, by using local materials for the
detection of Cryptospordium and Giardia oo(cysts) in shellfish.
Methodology
Magnetic Particle Concentrator Development
The description and specification data of imported MPC were studied and used as the
prototype model for the development of the domestic MPC by using local materials in
Thailand.
Efficiency Comparison between thai-MPC and imported MPC
The comparison of the efficacy between imported MPC and thai MPC was evaluated
and validated for the recovery levels of Cryptosporidium and Giardia oo(cysts) detection in 6
different concentrations of seeding water samples. For the seeding experiment,
Cryptosporidium and Giardia oo(cysts) were diluted with PBS buffer for the initial
concentration as 400,300,200,100,50,25 oocysts/l and 300, 200, 100,50,25,10 cysts/l ,
respectively and spiked 1 ml each of these concentration into the seeding water samples with
brought up to 10 ml with sterile water and subjected to IMS, using the Dynal Dynabeads®
anti-Cryptosporidium kit and the Aureon anti-Giardia IMS kit (ImmTech, Inc. New Windsor,
MD), according to the manufacturer's instructions. The oo(cysts) in seeded water samples
were evaluated for the efficiency of thai MPC compared to the imported MPC.
Samples collection and preparation
A total of 100 raw oyster samples were collected from different local markets in
Bangkok and its surroundings, including Tai market, Marketing Organization for farmers,
Yin Charoen market, Prannok market and Huai Khwang market. All samples were kept cool
at 4°C in an insulated container with ice for immediate transportation to the laboratory and
processed within 24 hours. Each sample consisted of the whole meat from 10 oysters was
pooled and kept separately. An aliquot of each individual sample was stored before the
constitution of the pool for further Cryptosporidium and Giardia species identification. The
pooled samples were homogenized in PBS (0.04 M, pH 7.2) using a stomacher or a Moulinex
blender machine. The resulting homogenates of each pool were then passed through a sieve
(mesh size 45 and 150 μm), subjected to lipid extraction with PBS/diethyl ether (2:1, v/v),
and concentrated by centrifugation at 1,250 × g, for 5 min, at 4 °C. The sediments from each
pool were examined for the presence of Cryptosporidium and Giardia (oo)cysts.
Isolation, Concentration and Purification of (oo)cysts by IMS
To recover Cryptospordium and Giardia oo(cysts) from the homogenate pellets, each
10-ml sample was processed by IMS according to the manufacturer's instructions (Dynabeads
anti-Cryptosporidium ;Dynal and anti-Giardia kit; Aureon). Briefly, 100 μl of antiCryptosporidium and anti-Giardia beads were added to a 10-ml homogenate sample obtained
after shellfish preparation. The sample was then processed according to the recommendations
of the manufacturer, using thai MPC equipment for IMS processing. After dissociation from
magnetic beads, (oo) cysts were transferred to a new microcentrifuge tube and treated with 5
μl of 1 N NaOH to neutralize the pH. The resultant suspension for screening was clean and
approximately 50 μl total volume. This resulted final sample was divided into two portions,
and one portion was examined by the immunofluorescent assay (IFA) to detect
Cryptosporidium oocysts and Giardia cysts.
Detection and identification of Cryptosporidium and Giardia (oo)cysts by IFA
A direct immunofluorescence antibodies (IFA) assay was applied to detect
Cryptosporidium and Giardia oo(cysts) in the sediments obtained. Samples were placed on
glass microscope slides. Slides were fixed with methanol and air dried, oo(cysts) were
enumerated by
IFA using a commercially available test kit (MeriFluor
Cryptosporidium/Giardia; Sanofi Diagnostics Pasteur, France) per the manufacturer's
instructions. The oo(cysts) were identified under an FITC filter using fluorescence
microscopy (OLYMPUS, Model BX 51), according to the criteria proposed by Graczyk et al.
(22), i.e. brilliant green fluorescence, correct shape and size of the FITC-labelled objects with
clearly visible oocyst wall.
Statistical data analysis
Statistical analyses were performed on logarithmically transformed data of
Cryptosporidium oocysts and Giardia cysts numbers and then process using Pearson
correlation analysis. Values of P < 0.05 were considered statistically significant. All the
analyses were performed using SPSS for Windows software (version 11.5) software.
Results
The thai MPC was successfully developed by using local materials for
Cryptosporidium and Giardia detection in shellfish samples. The detection limit in seeding
water samples for Cryptosporidium and Giardia at the initial inoculum were 50 oocysts/l and
25 cysts/l, respectively. The efficacy of imported MPC and thai MPC for Cryptosporidium
and Giardia oo(cysts) detection in oysters was no significant difference (p < 0.05),as shown
in Figure 1. The detection of Cryptosporidium and Giardia oo(cysts) in total of 100 raw
oysters were found in 9 % and 8 %, respectively. (Table 1).
Figure 1. Efficiency comparison of imported MPC and thai MPC for Cryptosporidium
and Giardia oo(cysts) detection.
Table 1. Prevalence of Cryptosporidium and Giardia oo(cysts) in raw oyster samples.
Location Sampling
(raw oysters)
No of positive
No. samples
Cryptosporidium
Giardia
local markets in Bangkok and surrounding
-Tai market
25
3
3
- Marketing Organization for farmers
25
1
1
-Yin Charoen market
20
1
1
- Prannok market
15
2
2
- Huai Khwang market
15
2
1
100
9
8
Total
Discussion and Conclusion
Cryptosporidium and Giardia present public health challenges due to their low
infective dose, ease of dispersal and persistence in the environment, difficulty to reliably
isolate and detect, small (oo)cyst size that may bypass common water treatments and relative
resistance to disinfection (23 and 24). Both parasites may be transmitted by direct fecal–oral
route (and thus by direct contact with infected animals or humans) or by ingestion of food or
water contaminated with (oo)cysts (25). Agricultural practices including manure storage and
manure application to arable land, discharge of contaminated water, pasturing of livestock
near water sources and disposal of fecally contaminated waste from abattoirs and wastewater
treatment facilities may result in contamination of the environment with large numbers of
(oo)cysts (26 and 27). With (oo)cysts circulating in environmental water supplies, and
waterborne outbreaks documented in developed countries throughout the world, attention has
been drawn to transmission potential of parasites in contaminated shellfish to consumers.
Through improved detection methods, studies continue to confirm the presence of these
parasites in livestock and contaminated water sources. Although studies examining shellfish
for these zoonotic protozoa are limited, literature available on bioaccumulation and retention
of (oo)cysts in shellfish highlights the need to examine the efficacy of current sanitation and
depuration protocols to prevent the public from acquiring cryptosporidiosis and/or giardiasis
from the consumption of contaminated shellfish.
The Thai market in 2010 is valued medical devices and laboratory equipment at
US$795 million. Moreover, the Ministry of Commerce showed the top ten imported goods in 2012,
scientific instruments was imported continuously and ranked number 6 at more than a billion baht
deficit (28). Even though the market is vibrant, it is also lopsided. Two-thirds of medical
devices in the country are imported, with products from the United States accounting for 29%
of inbound shipments, according to figures from the Thai Medical Device Technology
Industry Association (ThaiMed). In fact, Thailand needs to import about 10 billion baht in
medical devices annually to meet local demand. Although the country has a substantial
number of manufacturers, currently these tend to concentrate on a relatively small number of
product categories. Progress is being achieved, however, toward upgrading local production
of medical devices to reduce imports. Imports of diagnostic or laboratory reagents or
equipments hit 3.63 billion baht and exports were just 186.3 million baht in the 10 months.
Thus, this study was successfully developed the domestic Magnetic Particle Concentrator
(MPC) ,one of these laboratory equipments , which is promising use to replace the imported
product with no different quality, but lower cost and reduce the trade balance. Thailand, as a
leader in Asia, plays a vital role in many regional and international organizations dedicated to
promoting the advancement of medical devices in the area. Therefore, signifcant investment
opportunity exists in this medical sector, especially for artificial blood vessels, disposable
diagnostic test kits, respiratory systems, rehabilitation equipment, orthopedic and implant
devices, and neurosurgical devices and accessories. Thailand also takes part in groups such as
the Asian Harmonization Working Party and the ASEAN Consultative Committee on
Standards and Quality. Both of these strive to achieve conformity in medical device
regulations and upgrade professional training in the region.
Bivalve shellfish (mussels, oysters, clams, and cockles) are economically important sea
food that are harvested commercially and preferentially consumed raw. As shellfish filter
large volumes of water that trap suspended particles from water and many commercial
species thrive in coastal marine environments where nutrient levels are high, but these areas
may be contaminated with sewage and livestock effluent (29 and 30). Shellfish filter ~ 20–
100 L of seawater in 24 h (depending on species) and can accumulate very high (oo)cyst
loads in their digestive gland, intestinal tract and gills (30 and 31) and can concentrate
pathogenic organisms, which may act as potential vehicles of transmission to the consumer.
In contaminated water, the concentration of (oo)cysts significantly increases the potential risk
of infection to humans upon consumption of raw or undercooked shellfish (32 and 34).
However, few cases of cryptosporidiosis and giardiasis have been positively linked to eating
shellfish and this creates doubt regarding the significance of consuming protozoancontaminated shellfish as a public health concern (32).
As many species of shellfish are eaten raw or lightly cooked, there is potential for
consumers to ingest pathogens if they are not excreted or inactivated by the shellfish (30).
Oysters and clams may present a higher risk of seafood-borne disease as they are normally
eaten whole (including gills and intestinal tract) and raw or very lightly cooked (35).
Cryptosporidium oocysts and Giardia cysts have been identified in shellfish from different
regions of the world, many of which were marketable product destined for human
consumption (36 - 41).
Several studies record the presence of protozoa in water and shellfish, however these
reports are disconnected because the majority of research has been conducted in different
geographical locations or during different time periods and on different bivalve species (42).
Evaluation of treated downstream water samples and neighbouring harvested shellfish
(Ruditapes decussates and Mytilus galloprovincialis) in a geographically closed environment
in Southern Italy found Giardia in 16/21 wastewater and 7/21 downstream water samples,
and Cryptosporidium in 5/21 and 8/21 wastewater and downstream water samples,
respectively (42). Surprisingly, IFA and molecular testing were negative for both protozoa in
shellfish and various physical, ecological, and climatic conditions were suggested to account
for the lack of contamination in shellfish. In Thailand, Cryptosporidium was found in 8.3%
and 15.6% of market shellfish from Bangkok and Samut Prakan, respectively. The variance
was attributed to differences in physical, ecological, and anthropogenic conditions including
different contamination levels of the water sources (41). In future studies water samples
should be collected at the same time as shellfish in order to correlate parasite levels in water
with shellfish contamination. Further, serial sampling of a location over time is needed to
understand the dynamics between shellfish contamination and their ability to naturally clear
the parasites. Nevertheless, this study was carried out to investigate the presence of
Cryptosporidium and Giardia protozoan parasites in oysters, the economically important
shellfish and the most destined for consumption in Thailand. Cryptosporidium oocysts and
Giardia cysts were detected in local markets of Thailand and its surroundings with 9 % and
8% of all oysters examined was positive. This is probably an underestimation of the true
fraction of oysters contaminated with these parasites, compared with the previous reports. In
addition, Fayer et al. (2002 and 2004) observed that only 50% of gill washings seeded with
500 Cryptosporidium oocysts tested positive with an immunofluorescence assay, indicating a
low sensitivity of this detection method. These differences in positive samples from the
different locations may be caused by physical, ecological and anthropogenic conditions. This
could relay to different contamination levels of marine water by Cryptosporidium and
Giardia (oo)cysts and consequently to contamination of harvested shellfish populations.
Isolation of Cryptosporidium oocysts from naturally contaminated shellfish is
technologically complex, and large variability exists regarding methods used to process
shellfish samples for the presence of protozoan parasites. Several methods are available (32
and 43 - 48) but few data are available on recovery efficiencies of the various methods, their
specificity or sensitivity, and no generally accepted gold standard method is currently
available (30 and 32). Variation in recovery efficiencies exists among similar methods (12–
70%) and depends on mollusc species as well as between tissues examined (33, 47, 49 and
50-51). A recently developed processing method utilizing whole shellfish tissues from which
protozoa (oo)cysts are isolated using immunomagnetic separation (IMS) and enumerated by
IFA has achieved the highest recovery efficiencies to date, 70–80% for both Cryptosporidium
and Giardia (30). Enumeration by IFA following IMS is the standard method of evaluation in
water described in the USEPA-1623 method and has been used for processing shellfish
samples, although underestimation in lightly contaminated oysters using this method has been
estimated to be 50% (52 and 53).
Purification of (oo)cysts by IMS also has contradictory results. IMS has been integrated
into the detection of Cryptosporidium and Giardia in shellfish similar to the USEPA test
descriptions for water sample concentrates (30, 39 and 54), but some researchers have found
that the mucoid quality of various shellfish tissues compromises IMS function and others
have discontinued use of the method (40 and 47). Furthermore, IMS is expensive (48), each
test costing at least US$ 90. This cost makes routine large-scale monitoring efforts cost
prohibitive. To counteract cost, most studies employing IMS either used a smaller sample
size or pooled samples to facilitate processing of larger quantities of shellfish, both of which
can compromise detection (39 and 47).
In future research should be studied by using molecular methods for the detection of
Cryptosporidium and Giardia in shellfish and drinking water supplies, which will be better
understanding the transmission of these water and food-borne diseases. However, with or
without IMS, shellfish samples are now commonly analyzed by both IFA and PCR. As there
are discrepancies in both methods (e.g. IFA can stain empty oocyst walls and depends on the
skill of the slide reader whereas PCR can appear negative due to a lack of sensitivity or
positive due to presence of DNA from degraded cells), using a combination of techniques
that rely on different substrates (e.g. proteins or DNA) can increase the likelihood of
detection (32, 50 and 53). Some studies have identified positive water, fecal, or homogenized
shellfish samples by IFA, and have then performed complementary PCR analysis for final
confirmation and genotyping (32, 49 and 55).
This study gave the efficient thai MPC equipment with low cost and instead use of
imported products with no difference quality. The presence of Cryptosporidium and Giardia
in the oysters provide in epidemiological study and a situation of sanitary risk with
determining the safety of shellfish for human consumption. Knowledge of food safety, good
personal hygiene and health education should be continually campaigned.
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Acknowledgements : This work was supported by the Thailand Research Fund under the
TRF MAG project and partial supported by E for L Co.Ltd. We would like to thank the local
network authorities for effort to facilitate data and sample collection and the Faculty of Public
Health, Mahidol University for institutional research support facilities.