The cross-sectional study was conducted from April to December 2017 in an indigenous community inhabited by the Tuwalis, an ethnolinguistic group in Boliwong, Lagawe, Philippines (16.8221°N, 121.1424°E). This area is covered by high mountains with a total land area of 956.4726 ha and an elevation of 563.2 m above mean sea level. The community was reported to have poor sanitation, unimproved water sources, and low socio-demographic status. Figure 1 shows the location of Boliwong in the northeast part of the Philippines.

Stratified random sampling was performed by forming 3 strata in the study location: (1) lowland, (2) midland, and (3) upland. From the lowland region, 30 consenting participants were drawn from 560 population members (5.3%), and the corresponding participation rates from the midland and upland regions were 35 of 550 (6.4%) and 72 of 570 (12.6%), respectively. The total sample was 137 (8.1%) of the total population of 1,680 in Boliwong. Participants were randomly chosen with substitution for data gathering and faecal sample collection.

A standardized questionnaire, probing general information, household income, water sources, sanitation, and hygiene practices, was distributed among participating heads of households or adults over 18 years old. The participants were asked to sign the consent form after indicating that they understood the main objective of the study.

To complete the participation, each participant was asked to submit a faecal sample. A spatula that came with the container was used to place the human faecal sample in a clean screw-top container. The participants were advised to fill the container with a pea-sized sample, without placing it into contact with urine and water from the toilet. Faecal samples were also collected from domestic dogs (n=12), domestic cats (n=7), domestic swine (n=4), goats (n=6), carabaos (n=2), and chicken (n=6). If defecation was not directly observed, the presence of high-moisture sheen on the surface of the faeces was observed to ensure faecal freshness. All samples were collected from surfaces that had not been in contact with the substratum to avoid contamination.

To detect Cryptosporidium oocysts from the animal and human faecal samples, modified Ziehl-Neelsen staining was done. This method has already been established for the detection of Cryptosporidium oocysts. Briefly, faecal smears were made from the concentrated deposit. It was air-dried, fixed in methanol for 3 minutes, and stained with strong carbol fuchsin for 15-20 minutes. Tap water was used for thorough rinsing, followed by decolorizing in acid alcohol (1.0% hydrochloric acid [HCl] in methanol) for 15-20 seconds. The sample was rinsed thoroughly again in tap water and was counterstained with 0.4% malachite green for 30-60 seconds. After final rinsing and air drying, the sample was examined with cedarwood oil under the oil immersion objective [18]. A zigzag method was used to view the fields of the cover slip. Pink or red stains corresponded to Cryptosporidium oocysts. The oocysts were measured, and ranged from 4 to 6 μm in diameter. Morphological details, including non-homogeneous colour and the appearance of a hollow portion of the oocyst, were also observed. Any particle that did not meet these criteria was excluded from the Cryptosporidium-positive samples [19].

A total of 24 surface-grab water samples was collected from the river (n=9), creek (n=9), and water pumps (n=6). The water samples were filtered using a nitrocellulose membrane (47 mm in diameter, 0.2 μm pore size) by mild suction using a vacuum pump. The obtained sediment was carefully washed using 0.1% Tween-80. The washed sediment was centrifuged at 3,000×g for 15 minutes and was aspirated into a 10-mL syringe. To isolate the oocysts, an anti-Cryptosporidium bead conjugate was added to the sample, allowing the oocysts to bind with the beads. The tube was then placed in a magnetic particle concentrator (MPC-1) to magnetize the sample and separate the oocyst-magnetic complex from the contaminating debris. The supernatant was then removed and discarded. The beads were separated again with 50 μL of 0.1 N HCl using a MPC-M. Each sample concentrate was placed in the well of a microscope slide. When dried, it was fixed with methanol and was labelled with anti-Cryptosporidium fluorescein isothiocyanate (FITC) monoclonal antibody. The assay was carried out using a commercial kit Dynabeads GC-Combo (Thermo Fisher Scientific, Waltham, MA, USA). Cryptosporidium oocysts were recognized based on fluorescence. Round or oval shapes with bright apple-green fluorescence that measured 4 to 6 μm were confirmed as Cryptosporidium. To confirm the presence of nuclei in the oocysts, light or intense blue internal staining was observed upon examination using 4′, 6-diamidino-2-phenylindole of the samples identified as positive based on the FITC examination.

The prevalence rate was computed by dividing the number of parasite-positive human/animal faecal samples by the total number of samples collected within each risk-factor category/animal group. SPSS version 23.0 (IBM Corp., Armonk, NY, USA) was used to analyse the data. Univariate logistic regression analysis evaluated the association between each risk factor (e.g., location, age, sex, water source, and sanitation- and hygiene-related factors) and Cryptosporidium infection. The results were calculated as crude odds ratios (cORs). Risk factors at p-value <0.05 were considered significant and were subjected to a multivariate regression analysis to further clarify their associations. The intensity of oocyst shedding among the animal groups was determined by computing the mean count of oocysts per animal group and their respective 95% confidence intervals (CIs).

The transmission of Cryptosporidium oocysts is usually waterborne. Contaminated water can be a direct source of human infection through drinking or accidental ingestion of contaminated water. Contaminated water can also infect humans if it is used in washing and preparing food, which is considered foodborne transmission [2].

There is one long stretch of river and another stretch of creek in this community. These water systems traverse the upstream, midstream, and downstream regions of Boliwong, serving a large portion of the community. These bodies of water, together with water pumps, are used by the human inhabitants for bathing, swimming, and laundry activities. Aside from domestic use, these also serve as the main water sources of the terraced ponds and rice paddies of the farmers. The creek, for example, is connected to the tubing system in the rice field that is used for watering purposes. A high prevalence of Cryptosporidium in the creek was identified in this study, which poses the risk that the oocysts may spread widely, not only to the food of the people in the community, but also to the regions that receive the harvested rice.

Furthermore, various animals have access to the water system—specifically to the river and creek—to drink, bathe, and defecate. Voided goat, carabao, and dog faecal samples were observed less than a meter away from the river. This close proximity between animals and the water system might cause the massive dispersal of oocysts in the environment.

Several animal groups were visibly present in the community. Carabaos and goats were usually tied in the backyard, while pigs were put inside a cage. Of note, most owners of these pets did not practice any manure management. Manure and other pet waste, and even human faeces, visibly pooled on the surrounding ground. The pooling of animal faeces on the ground, with subsequent drainage into waterways, was considered as a significant contributor to infections. Dogs and chickens had the highest intensity of oocyst infection. These animal groups, together with cats, are free roamers in the community, with access to houses, gardens, rice fields, and other places. Birds and dogs are among the non-human hosts of Cryptosporidium that are considered reservoirs for human infections because they harbour C. meleagridis and C. canis, which have been reported to infect humans as well [20]. Several studies have demonstrated contrasts in clinical manifestations among the different species of Cryptosporidium. C. hominis, for instance, is associated with diarrhea, nausea, and vomiting. C. parvum, C. meleagridis, and C. canis, in contrast, are only associated with diarrhoea. Furthermore, there are other species that can infect animal hosts, but not necessarily humans [21]. This study did not identify the species of the detected Cryptosporidium via molecular techniques. Despite this gap, the potential for Cryptosporidium infections in the community can still be considered high due to the important ecological characteristics of the community that favour the widespread transmission of oocysts.

The 28.5% prevalence of Cryptosporidium infection among human inhabitants in Boliwong should not be dismissed. Poor hygiene and sanitation, particularly regarding the widespread use of open pits as toilet facilities in the community, can worsen the present condition. People regularly drink water from the natural spring, which possibly harbours the parasite. They are also in direct contact with pets and farm animals that increase the risk of contracting Cryptosporidium infection. The habitual use of unsafe water sources (i.e., river, creek, and water pumps) in the community supports the conjecture that Cryptosporidium may be emerging as a zoonosis in the community.

Interventions should be made to address the factors found to be influential in the spread of parasitic infections. It is advisable that people in Boliwong should avoid accidental intake of water from the river during bathing and when engaging in other recreational activities. It is also not advisable to have contact with the creek, as it was contaminated with Cryptosporidium. The dispersal of animal groups throughout the environment should be minimized by placing chicken in cages and maintaining other pets at a distance. If a person cannot avoid contact with animals, proper handwashing should be a habit.

Furthermore, providing a developed water source and improved toilet facilities to the households in the community is strongly recommended and would make a major positive impact on the prevention of Cryptosporidium infections. The people in Boliwong should also understand the various means of transmission of parasites from animals to the environment and to humans. Proper hygiene and sanitation behaviour should be further encouraged in the community through community-wide health education. An One Health approach should be applied to the eradication of parasites, since several sources were identified as infected or contaminated.

An estimated 14-17 million indigenous people inhabit the Philippines [22]. The findings of this study should receive attention and lead to the implementation of similar epidemiological surveillance programs among other indigenous communities in the country. The impact of waterborne pathogens such as Cryptosporidium from the public health perspective has not been highlighted in the Philippines due to the paucity of domestic studies regarding this important protozoan parasite. Sufficient data on Cryptosporidium in various areas and from different sources could be used by experts to formulate possible solutions.

In conclusion, the people of Boliwong are at risk of contracting Cryptosporidium infections, as Cryptosporidium may be emerging as a zoonosis in the community. Unimproved water sources and poor sanitation practices support the wide dispersal of oocysts throughout large parts of the community. Cryptosporidium oocysts were detected from several key point sources (i.e., humans, animals, and the water system), which calls for consistent interventions before any waterborne outbreak involving Cryptosporidium or other relevant intestinal protozoan parasites occurs in the near future.