Cryptosporidium: An Emerging Zoonosis in Southern Khyber Pakhtunkhwa (KPK), Pakistan

Naimat Ullah Khan1,2, Muhammad Hassan Saleem1, Aneela Zameer Durrani1, Nisar Ahmad3, Ayesha Hassan1, Sultan Ayaz2, Sajid Umar4, Muhammad Luqman Sohail5,*, Muhammad Shafee6, Ikramullah Khan7, Mumtaz Ali Khan1, Azmat Ullah Khan8, Naimat Ullah Malik1 and Abdul Razzaq9

1Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan

2College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University Mardan, Pakistan

3Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan

4Department of Pathobiology, Faculty of Veterinary Sciences, Pir Meher Ali Shah Arid Agriculture University, Rawalpindi, Pakistan

5University College of Veterinary and Animal Sciences, The Islamia University of Bahwalpur, Pakistan

6Centre for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan

7Department of Livestock Management, Animal Breeding and Genetics, The University of Agriculture, Peshawar, Pakistan

8Department of Livestock and Dairy Development, The University of Agriculture, Peshawar, Pakistan

9Department of Veterinary Anatomy and Histology, Sindh Agriculture University, Tandojam, Pakistan

ABSTRACT

Zoonotic Cryptosporidium is chief cause of diarrhea in both developing and developed countries, causing millions of deaths each year. This study was designed to estimate the zoonotic potential of Cryptosporidium in three districts of Khyber Pakhtunkhwa (KPK), i.e. Bannu, Lakki Marwat and Kohat. For this purpose 360 stool samples were collected from the District Headquarter Hospitals of all the three districts and were screened through microscopy. Results yielded an overall prevalence of 11.11%, with significant difference (P<0.05) among different study areas. Prevalence was highest in district Bannu (11.66%), followed by district Lakki Marwat and Kohat with 10% in each district. Statistical analysis revealed significant difference (P<0.05) among children of different age groups with highest disease prevalence in children of less than 5 years of age (21.40%) and lowest in children of 11-15 years of age (15.99%). Cryptosporidium was significantly higher (P<0.05) in study subjects having livestock animal contact and persistent diarrhea, highlighting its zoonotic potential from livestock animals to human beings in the study area. This is first study addressing the one of the main cause of neonatal mortality in Southern KPK and its zoonotic potential.

Article Information

Received 21 March 2017

Revised 11 April 2017

Accepted 13 May 2017

Available online 25 July 2017

Authors’ Contribution

HS, AZD, NA, AH and SA designed the study plan. NUK, MS, IUK, MAK, NUM and AK collected samples and executed the experimental work. AR, MLS and SU recorded and analyzed the data and wrote the article.

Key words

Children, Cryptosporidium, epidemiology, Livestock.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.4.1455.1461

* Corresponding author: vet.luqman@gmail.com

0030-9923/2017/0004-1455 $ 9.00/0

Introduction

Diarrheal diseases are second leading cause of mortality among young children around the globe. It causes 9% of child deaths worldwide (Bhutta and Black, 2013). Cryptosporidium is among the top four causes of moderate to severe diarrheal disease in young children in developing countries and is a major opportunistic co-infection with HIV (Bodager et al., 2015). It is ranked 5th among the 24 most important food borne parasites globally (Aniesona and Bamaiyi, 2014). It is mostly prevalent in hot and humid weather during the year, with worldwide distribution. (Jafari et al., 2013). Cryptosporidium infection has been reported in humans from 3 days to 95 years of age, but data suggest that young children are most susceptible to infection (Fayer, 2004), along with immunocompromised hosts (Sow et al., 2016). Epidemiologic studies have demonstrated that Cryptosporidium is more prevalent in developing countries (5% to 10%) than in developed countries (1% to 3%) (Iqbal et al., 1999).

After identification of Cryptosporidium in 1907, its zoonotic potential was revealed in late 1970s (Shoultz et al., 2016). The most noteworthy symptom in immunologically healthy persons is diarrhea, usually voluminous and watery. Abdominal discomfort, anorexia, nausea, vomiting, weight loss, fever, fatigue and respiratory problems may accompany diarrhea (Masood et al., 2013). Prolonged diarrhea (7-14 days) and persistent diarrhea (≥14 days) is also reported during Cryptosporidiosis (Moore et al., 2010). Which may lead to malnutrition, hospitalizations and even death (Kotloff et al., 2013). Fecal shedding of Cryptosporidium oocysts can continue for weeks after clinical illness resolves. Since Cryptosporidium oocysts tolerate chlorination, waterborne outbreaks also occur in industrialized countries (Shirley et al., 2012).

For epidemiological studies, serological tests might also be used but microscopy is an important diagnostic method and because of the low cost of reagents, it is well practiced in lower or middle income countries (Checkly et al., 2015).

To the best of our knowledge, no data is available regarding the prevalence of cryptosporidium in children of Southern KPK, exposed to livestock animals and still is a neglected area of study. This paper will provide the information about the epidemiological study and related risk factors responsible for occurrence of the Cryptosporidium in children. Limited access of human population to health care facilities will further aggravate the issue if left, unreported.

Materials and Methods

Study area

The present study was conducted in three selected districts of Southern Khyber Pakhtunkhwa, district Bannu, Lakki Marwat and Kohat. Coordinates of the study areas were determined by global positioning system (GPS). Bannu is located at 32.99° North latitude, 70.61° East longitude and have an elevation of 371 meters from the sea level. Lakki Marwat, the neighboring district of Bannu, have coordinates 32° 36’ 27” North, 70° 54’ 45” East. Kohat district lies between North latitude 32° 47’ and 33° 53’ and East longitude 70° 34’ and 72° 17.

Experimental site

All the samples were processed at Diagnostic Laboratory, Department of Clinical Medicine and Surgery and in the Parasitology Laboratory, Department of Parasitology, University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan.

Inclusion criteria

Individuals visiting District Headquarter Hospitals of study area with abdominal anomalies i.e., acute diarrhea (three loose stools within last 24 h prior to hospital visit) or persistent diarrhea, vomiting (once in last 24 h before hospital visit) and abdominal cramps were included in the study. All the participants had exposure to livestock and other animals.

All the participants were informed about the study’s purpose and procedures. Written informed consent was obtained from the parents or the guardian on behalf of the study children prior to study enrolment.

Sample size and collection

Fecal samples from 360 individuals, satisfying the inclusion criteria were collected through convenient sampling with the assistance of Registered Medical Practitioners (RMP) of Pakistan Medical and Dental Council (PMDC) from Outdoor Patients Department (OPD) of three District Headquarter Hospitals (DHQ’s) of district Bannu, Lakki Marwat and Kohat from January to June, 2016.

All the collected samples were preserved in 10% formalin (2:3). The samples were collected into sterile wide mouthed plastic bottles, properly labeled and transported to the experimental site till further analysis. Collected samples were examined microscopically and screened out for the presence of Cryptosporidium oocysts.

The purpose of this study was to determine the prevalence of Cryptosporidium infection in children and adults due to ignorance, poor literacy rate and lack of information about the zoonotic aspect of the cryptosporidiosis in South KPK, Pakistan.

Laboratory analysis of fecal samples

The samples were concentrated by formalin-ethyl-acetate sedimentation method and stained using the modified Ziehl-Neelsen technique (MZN) for the detection of cryptosporidiosis (Shafieyan et al., 2016). Briefly, Smears were fixed using absolute methanol and were stained by carbol fuchsin solution for 15 min. Non-Cryptosporidium materials were decolorized by acid alcohol solution for 2 min. Then, the slides were treated with malachite green solution for one minute, washed, dried and examined for presence of the oocysts under light microscopy.

Identification of Cryptosporidium oocysts

Cryptosporidium oocysts appeared as bright red granules on a blue-green background in MZN stained fecal smears and were identified on the basis of morphology, size and other key features as described by Wantanabe et al. (2005).

A fecal sample was considered positive if at least one, clearly identifiable oocyst was recognized. The total number of oocysts per gram (OPG) of feces was calculated by multiplying the total number of oocysts on slide by 50.

Statistical analysis

The data collected was fed in the Microsoft Excel version 2013 and was exported to the Statistical Product and Service Solutions (SPSS) version 20.0 to analyze associations between prevalence of Cryptosporidium and the risk factors at 95% level of confidence. The prevalence was the proportion of positive individuals out of the total number of individuals analyzed and was presented in percentage (%). Statistical differences in prevalence and other variables were determined using Chi-square test (X2). All values at statistical difference P < 0.05 were considered significant.

Table I.- Prevalence of Cryptosporidium in adults and children in three districts of Southern KPK.

Factor	Infected / Total (Prevalence)
Factor	Bannu	Lakki Marwat	Kohat	Overall
Adult Humans	3/60 (5%)	3/60 (5%)	2/60 (3.33%)	10/180 (5.55%)
Children	11/60 (18.33%)	9/60 (15%)	10/60 (16.66%)	30/180 (16.66%)

Statistically mean values that are carrying same superscript are differ from each other non-significantly (P>0.05) while those statistical mean values having different superscripts are differ significantly (P< 0.05).

Results

Results showed significantly higher prevalence (P < 0.05) of Cryptosporidium in children (30/180) 16.66% as compared to adults (10/180) 5.55%. Similar findings were recorded in all the three study areas. In Bannu, children (18.33%) had higher prevalence as compared to adults (5%). Similarly, in Lakki Marwat prevalence in children was 15% and in adults it was 5%. In Kohat 16.66% of children had Cryptosporidium whereas adults had 3.33%.

Table II shows age wise prevalence of Cryptosporidium in children having abdominal disturbances. Highest prevalence (5%) was recorded in children in less than 5 years of age (21.42%), followed by children of age 6-10 years (17.30%) and the lowest prevalence was recorded in children from 11-15 years of age (10.34%). Statistical analysis revealed significant difference (P < 0.05) among the study subjects of different ages.

Higher prevalence was recorded in children having close contact with domestic animals (20%) as compared to those having no contact with domestic animals (12.94%). Statistical analysis revealed significant difference between the two study groups (P < 0.05) (Table III).

Table II.- Prevalence of Cryptosporidium according to age in young children in three districts of Southern KPK.

Age

Infected / Total (Prevalence)

Bannu

Lakki Marwat

Kohat

Overall

0 -5 years

5/23

(21.73%)

5/21

(23.80%)

5/26

(19.23%)

15/70

(21.40 ͣ%)

6-10 years

3/17

(17.64%)

3/18

(16.66%)

3/17

(17.64%)

9/52

(17.29ᵇ%)

11-15 years

3/20

(15%)

1/21

(4.76%)

2/17

(11.76%)

6/58

(10.33ᶜ%)

Statistically mean values that are carrying same superscript are differ from each other non-significantly (P>0.05) while those statistical mean values having different superscripts are differ significantly (P< 0.05).

Table III.- Prevalence of Cryptosporidium according to animal contact in three districts of South KPK.

Contact with animals	Infected / Total (Prevalence)
Contact with animals	Bannu	Lakki Marwat	Kohat	Overall
Domestic animals	7/34 (20.58%)	6/29 (20.68%)	6/32 (18.75%)	19/95 (20 ͣ%)
Others	4/26 (15.38%)	3/31 (9.67%)	4/28 (14.28%)	11/85 (12.94ᵇ%)

Statistically mean values that are carrying same superscript are differ from each other non-significantly (P>0.05) while those statistical mean values having different superscripts are differ significantly (P< 0.05).

In Bannu, the higher prevalence (20.58%) was recorded in those children having direct contact with domestic animals as compared to children with no contact with domestic animals (15.58%). Similar results were observed in other two study areas (Lakki Marwat and Kohat).

Results showed an overall higher prevalence (25.84%) in those children having persistent diarrhea as compared to the children suffering from acute diarrhea (7.69%). On statistical analysis significant difference (P < 0.05) was found on the basis of nature of diarrhea (acute or persistent) among study subjects (Table IV).

Table IV.- Prevalence of Cryptosporidium according to nature of diarrhea in three districts of South KPK.

Nature of diarrhea	Infected/Total (Prevalence)
Nature of diarrhea	Bannu	Lakki Marwat	Kohat	Overall
Acute	2/24 (8.33%)	3/35 (8.57%)	2/32 (6.25%)	7/91 (7.69 ͣ%)
Persistent	9/36 (25%)	6/25 (24%)	8/28 (28.57%)	23/89 (25.84ᵇ%)

Statistically mean values that are carrying same superscript are differ from each other non-significantly (P>0.05) while those statistical mean values having different superscripts are differ significantly (P< 0.05).

Discussion

Prevention is the only way out to protect masses from getting infection as no vaccine is currently available against Cryptosporidium infection and limited understanding of the its specific biology and challenges encountered in vaccine development, masks the availability of its vaccine in near future (Ryan and Hijjawi, 2015). Hence, to design effective disease control and prevention strategies a comprehensive understanding of various risk factors contributing to the spread of disease among human and animal population is unavoidable (Collinet-Adle et al., 2015).

Cryptosporidium infects a wide range of livestock animals and humans, causing substantial economic losses and serious public health concern (Li et al., 2016). Various molecular techniques are used for oocysts detection, which are costly, time consuming, laborious and require a range of equipment, rendering them not the best candidate for screening studies, so for large scale investigations, microscopy of stained fecal smears is more useful. Taking these aspect of diagnosis in consideration modified Ziehl–Neelsen method is arguably a useful tool for Cryptosporidium diagnosis (Shafieyan et al., 2016). Despite of the improved sensitivity of molecular technique, microscopy remains the gold standard in different labs (Helmy et al., 2013). To the best of our knowledge, this communication is the first documented study on zoonotic aspect of Cryptosporidium in population of Southern KPK, Pakistan.

Exposure to animals, poor sanitation and hygienic conditions are the factors responsible for transmission of the parasitic diseases (Zambrano et al., 2014) and these factors are highly prevalent in the study area, where poor sanitation, open toiletries, shared water sources of animals and humans fuel the fire of its spread among the human population.

This debut study on human beings of Southern KPK yielded higher prevalence (%) of Cryptosporidium in children (16.66%). Similar findings were reported from the other regions of the country, reporting 20.8% prevalence in Sakurdu (Khushdil et al., 2016). A hospital based study in an urbanized city of similar province yielded comparatively lower prevalence (9%), which can be attributed to better health care facilities in urban areas as compared to our study area (Mumtaz et al., 2010).

An extensive range of prevalence of the disease have also been described in children from other countries, 1.9% in Philippine, 25.3% in Uganda (Natividad et al., 2008; Tumwine et al., 2003). Dissemination of the parasite in each community and country depends on extent of contamination of water and food, animal contact and health measurements (Sharbatkhori et al., 2015).

Age of children was found to be significant risk factor for the occurrence of Cryptosporidium in Southern KPK. Similar principle age pattern of Cryptosporidium prevalence (less than 5 years of age) was reported by literature from developing countries (Helmy et al., 2013). Similar higher prevalence in pre-school children were reported by Eibach et al. (2015), supporting studies from various other tropical countries. This high incidence of the disease in children may be related to the lack of pre-existing immunity, as older people may get exposed to Cryptosporidium infection in their lifetime. Moreover, children were more exposed to water during playing, increasing the chance of getting infected, and there was a more frequent attendance of diarrheic children than adults at DHQ’s (El-Badry et al., 2015). Cryptosporidium in early age in the study area can be related to malnutrition which has been described as potential risk factor for the diarrhea due to Cryptosporidium (Checkly et al., 2015). According to Mehmood et al. (2016) more than 50% of children in this study area are victim of malnutrition, which further aggravate the situation.

Diarrheal diseases are one of the most leading cause of morbidity and mortality in children under 5 years of age throughout of the world (Walker et al., 2013). Present study reports significantly higher prevalence in study subjects having persistent diarrhea, when compared to people having acute diarrhea. One of the pathological hallmarks of intestinal cryptosporidiosis is villous atrophy (villi became stunted and shortened), with a diffuse shortening or loss of brush border microvilli (Huang and White, 2006). The imbalance of absorption and secretion is likely a major contributor to disease manifestation, specially diarrhea (Zhang et al., 2016).

According to another model, prostaglandins altered NaCl transport which primarily stimulates the enteric nervous system. Because of the profuse secretory diarrhea experienced by some patients, it has been hypothesized that Cryptosporidium produces an enterotoxin (Fayer, 2004).

Exposure to animals was concluded to be a significant risk factor for the occurrence of Cryptosporidium in humans because of its zoonotic transmission. Adamu et al. (2014) also reported livestock animals as a major reservoir for the zoonotic transmission of disease to both immunocompetent and immunocompromised individuals. Kinross et al. (2015) reported infected calves and other livestock animals as the potential source of transmission of infection to human beings. Another possible route of transmission can be the water bodies in the study area, shared by animals and humans, as Cryptosporidium has been found in water sources of different regions of KPK province in previous studies (Akbar et al., 2015).

Conclusion

This study warns about the zoonotic transmission of Cryptosporidium, one of four main causes of morbidity in pre-school children. It provides an evidence of Cryptosporidium transmission in young children from livestock animals. Furthermore, poor sanitation and contaminated water and environment fuel the fire of its spread. Due to limited treatment options available and poor health care facilities in study areas, situation can aggravate if remain unnoticed.

Statement of conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this article.

References

Adamu, H., Petros, B., Zhang, G., Kassa, H., Amer, S., Ye, J., Feng, Y. and Xiao, L., 2014. Distribution and clinical manifestations of Cryptosporidium species and subtypes in HIV/AIDS patients in Ethiopia. PLoS Negl. Trop. Dis., 8: e2831. https://doi.org/10.1371/journal.pntd.0002831

Akbar, N.U., Ayaz, S., Khan, M.A., Rahman, S., Khan, S., Khan, S.N., Waqar, M., Raza, F., Shagufta, B.I., Ali, S. and Saeed, S., 2015. Molecular detection of Cryptosporidium parvum in different water sources of District Peshawar, Pakistan. Adv. biol. Res., 9: 344-349.

Aniesona, A.T. and Bamaiyi, P.H., 2014. Retrospective study of cryptosporidiosis among diarrheic children in the arid region of North-Eastern Nigeria. Zoon. Publ. Hlth., 61: 420-426. https://doi.org/10.1111/zph.12088

Bhutta, Z.A. and Black, R.E., 2013. Global maternal, newborn, and child health—so near and yet so far. N. Engl. J. Med., 369: 2226–2235. https://doi.org/10.1056/NEJMra1111853

Bodager, J.R., Parsons, M.B., Wright, P.C., Rasambainarivo, F., Roellig, D., Xiao, L. and Gillespie, T.R., 2015. Complex epidemiology and zoonotic potential for Cryptosporidium suis in rural Madagascar. Vet Parasitol., 207: 140-143. https://doi.org/10.1016/j.vetpar.2014.11.013

Checkley, W., White, A.C., Jaganath, D., Arrowood, M.J., Chalmers, R.M., Chen, X.M., Fayer, R., Griffiths, J.K., Guerrant, R.L., Hedstrom, L. and Huston, C.D., 2015. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect. Dis., 15: 85-94. https://doi.org/10.1016/S1473-3099(14)70772-8

Collinet-Adler, S., Babji, S., Francis, M., Kattula, D., Premkumar, P.S. and Sarkar, R., 2015. Environmental factors associated with high fly densities and diarrhea in Vellore, India. Appl. environ. Microbiol., 81: 6053–6058. https://doi.org/10.1128/AEM.01236-15

Eibach, D., Krumkamp, R., Al-Emran, H.M., Sarpong, N., Hagen, R.M., Adu-Sarkodie, Y., Tannich, E. and May, J., 2015. Molecular characterization of Cryptosporidium spp. among children in rural Ghana. PLoS Negl. Trop. Dis., 9: e0003551. https://doi.org/10.1371/journal.pntd.0003551

El-Badry, A.A., Al-Antably, A.S., Hassan, M.A., Hanafy, N.A. and Abu-Sarea, E.Y., 2015. Molecular seasonal, age and gender distributions of Cryptosporidium in diarrhoeic Egyptians: distinct endemicity. Eur. J. clin. Microbiol. Infect. Dis., 34: 2447-2453. https://doi.org/10.1007/s10096-015-2502-y