Occurrence of Cryptosporidium spp . , Giardia spp . and other pathogenic intestinal parasites in the Beberibe River in the State of Pernambuco , Brazil

Introduction: Transmission of pathogenic protozoa and helminths by water is a serious public health problem. In this study, we analyzed the presence of these organisms in the Beberibe River in Pernambuco, Brazil. Methods: Parasite analysis was performed using the Hoffman, Pons, & Janer method followed by centrifugation and preparation of slides by staining with acetic acid and Lugol’s solution. Protozoan oocysts were isolated by the modifi ed Ziehl Neelsen method. Results: Cryptosporidium spp., Giardia spp. and other parasites were found in the Beberibe River. Conclusions: Sanitation companies must assess pathogenic intestinal parasites in water basins providing public water and subsequently develop improved treatment systems for removal of such parasites.

Transmission of pathogenic protozoa by water is a major public health concern (1) and therefore a challenge for producers and distributors of water systems (2) .For example, the pathogenic intestinal protozoa Giardia spp.and Cryptosporidium spp.exhibit resistance to chemical disinfectants and are longlived in the environment; thus, these pathogenic parasites are highly problematic for sanitation companies and public health offi cials (3) .Although conventional water treatment stations are able to remove 99% of Cryptosporidium spp.oocysts from water sources, there is still a risk of transmission (4) .Indeed, because oocysts exhibit high compressibility during fi ltration, these oocysts can behave as small particles (2-4μm), allowing them to pass through commonly used fi lters and enter treated water provided to consumers (5) .Therefore, improved methods for detection and removal of such pathogenic parasites is necessary.
Cryptosporidium spp.and Giardia spp.have been found in surface water in several countries, such as the United States on Lake Texoma (on the border of Texas and Oklahoma) (6) , in France on Seine River and its tributary Marne River (7) , in Hungary on the Danube River (8) , and in China in the reservoir of the Three Gorges Dam (9) .In Brazil, in accordance with decree number 2914 of the Health Ministry (year 2011), which describes water quality procedures for human consumption and potability patterns, sanitation companies are expected to survey water intended for human consumption for the presence of Giardia spp.and Cryptosporidium spp. (10).However, methods for detection of Cryptosporidium spp.are complex, and proper survey of water supplies would require specialized human resources, which are scarce.Additionally, there is little information about these pathogens in national surface water bodies (1) .Therefore, while many researchers are studying Cryptosporidium spp.and Giardia spp., more studies are needed to further characterize these pathogens (6) .Indeed, in the northeast region of Brazil, few studies have characterized or identifi ed pathogenic intestinal parasites, such as some species of helminths and protozoa.An initial report describing water supply sources showed contamination by Cryptosporidium spp. in the Capibaribe River and the Tapacura reservoir (11) in State of Pernambuco, Northeast Brazil.
In this study, we characterized the presence of intestinal parasites in the Beberibe River in Pernambuco.The human population at the Beberibe River basin is approximately 590,000 inhabitants, with an average population density of 7,300 inhabitants/km 2 .Moreover, this area has many low-income settlements in the Recife metropolitan region (RMR); these settlements are scattered in risk areas, hills, and fl ooded areas and are characterized by poor urban infrastructure.
The Beberibe River is one of the most polluted rivers in Pernambuco due to the insuffi cient sanitary conditions of Olinda  and Recife.Despite this, the Beberibe River is one of the water sources for the public water system, supplying about 10% of the volume of water distributed throughout the RMR.In particular, the Beberibe River contributes enough water for approximately 100,000 people in the RMR (12) .Between May 2012 and May 2013, water samples were collected in duplicate from the Beberibe River at two locations where sampling was easy (i.e., convenience sampling).Samples were collected from the Beberibe River in sterile 1-L plastic bottles and transported to the Laboratory of Parasitology, Department of Tropical Medicine of the Federal University of Pernambuco [University Federal of Pernambuco (UFPE)] for analysis of intestinal parasites.Protozoa and helminths were analyzed using the Hoffman, Pons, & Janer method or spontaneous sedimentation.The time to sedimentation was between 24 and 48h for 250-mL samples.Subsequently, aliquots of the sediment were centrifuged at 900rpm for 5-10 min.During the water sampling, turbidity (portable turbidity meter, Hanna Instruments model HI93703), water temperature, and depth of the river water were also analyzed.The pH of water samples was measured using the standard method (SM 4,500-H+) and electrometric method (13) .

Hymenolepis diminuta eggs A
For each sample in duplicate, two slides were made with approximately 1mL of the centrifuged sample.Because collections were made at two points of the river, a total of eight slides were made for each collection day; samples were collected on 16 days, yielding a total of 128 slides.The slides were stained with Lugol iodine and analyzed under an optical microscope at 10× and 40× magnifi cation for qualitative evaluation of protozoa and helminth parasite contamination.
A modifi ed Ziehl Neelsen method was used for identifi cation of Cryptosporidium spp.oocysts, with fuchsin-phenolated solution followed by spontaneous sedimentation.For the identification of Cryptosporidium spp.oocysts by optical microscopy, an oil-immersion 100× objective lens was used to visualize the whole smear, observing the shape, color, and size of oocysts.A micrometer was coupled to the microscope for confi rmation of the morphometric identifi cation of the parasite.
Bacteriological parameters were evaluated in water samples by determination of total coliforms and Escherichia coli between June 2012 and May 2013.Samples (250mL) were collected in sterilized polyethylene containers.Fermentation in multiple tubes was performed with chromogenic substrate (13) , with results presented as the most probable number (MPN) in 100-mL samples (MPN/100mL).
Due to the detection of parasite contamination in the Beberibe river, we suggest that water production and distribution companies should assess the presence of intestinal parasites (particularly protozoa and helminths) in public water supplies and should consider improving their treatment systems to ensure removal of such pathogens, which are known to be highly resistant to chemical disinfectants and to be particularly long-lived in the environment.
We hope that the information presented in this study, i.e., the presence of pathogenic intestinal parasites in the Beberibe River in Pernambuco, Brazil, may contribute to improvements in public health.In addition, public authorities need to create documentation for informing the population of the occurrence of parasites in watersheds providing public water supplies.

Freitas DA et al. -
Intestinal parasites in the Beberibe River