|
 
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| REVIEW ARTICLE |
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| Year : 2023 | Volume
: 3
| Issue : 1 | Page : 13 |
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Cryptosporidium infections in Nepal: A narrative review
Pitambar Dhakal1, Junqiang Li2, Longxian Zhang2
1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Central Department of Zoology, Tribhuvan University, Kathmandu 44600, Nepal
2 College of Veterinary Medicine, Henan Agricultural University; International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou 450046; Key Laboratory of Quality and Safety Control of Poultry Products, Ministry of Agriculture and Rural Affairs, China
| Date of Submission | 02-Apr-2023 |
| Date of Decision | 21-Apr-2023 |
| Date of Acceptance | 16-May-2023 |
| Date of Web Publication | 17-Jul-2023 |
Correspondence Address:
Longxian Zhang
College of Veterinary Medicine, Henan Agricultural University; International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou 450046; Key Laboratory of Quality and Safety Control of Poultry Products, Ministry of Agriculture and Rural Affairs
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2773-0344.380553
Cryptosporidium, a ubiquitous intracellular protozoan parasite, is capable of causing zoonotic infections in humans and domestic and wild animals. The primary aim of this review is to summarize the infection prevalence of Cryptosporidium spp. in Nepal. Based on all the available papers published until November 2022, we have also summarized the pattern of diagnostic methods, epidemiological factors, prevalence, and its genotypes distributed in humans, animals and environmental samples (mainly water). Among humans, the overall prevalence of cryptosporidiosis was 7.6% (1328/17527), of which the highest prevalence was identified in children (9.0%; 1225/13545), followed by HIV patients (4.6%; 51/1107) and adults (1.8%; 52/2875). The domestic animals were more infected (23.2%; 252/1088) than the wild animals (18.2%; 63/347) by Cryptosporidium, and water (32.6%; 29/89) remained the major source of environmental contamination by Cryptosporidium oocysts. Molecular epidemiological studies regarding the zoonotic transmission of cryptosporidiosis in Nepal are very few. However, molecular techniques have confirmed Cryptosporidium (C.) parvum isolates from humans as genotype Ia2, C. ryanae in zebu cattle and water buffalo, and C. ubiquitum in swamp deer by PCR-RFLP and sequence analysis of SSU rRNA gene. Extensive studies of Cryptosporidium at molecular level are warranted to find out its genomic variants in human and other vertebrates, both domestic and wild, in Nepal to elucidate its zoonotic potential in the future.
Keywords: Cryptosporidium; Humans; Animals; Water contamination
How to cite this article:
Dhakal P, Li J, Zhang L. Cryptosporidium infections in Nepal: A narrative review. One Health Bull 2023;3:13 |
| 1. Introduction | |  |
Cryptosporidiosis, a food- and water-borne disease, is one of the predominant causes of enteric infection in humans and other vertebrates. Cryptosporidium was firstly described by Tyzzer[1] as an intestinal protozoan parasite from the gastric mucosa of mice (Mus musculus), and classified as Cryptosporidium(C.) muris. It has high risk of infection through faecal-oral route through both anthroponotic and zoonotic transmission cycles owing to its low infectious dose (<10 oocysts) and resistance to various disinfectants including chlorine[2]. Studies have identified several risk factors like poor hygiene, exposure to animals, overcrowding, and unsafe drinking water in relation to Cryptosporidium infection in low- and middle-income countries[3],[4].
Globally, Cryptosporidium is the most common pathogen causing diarrhoea after rota virus in young children[5],[6]. Cryptosporidium infection leads to a consequence of diarrhea and nutritional deficiencies, making it a significant health problem in immunocompromised persons with HIV infection[4],[7],[8], but it may result in an asymptomatic course in persons with normal immune systems[9]. Extensive genetic variation within the genus Cryptosporidium has been reported. Of the 44 valid Cryptosporidium species and more than 120 genotypes, until now 19 species and four genotypes have been reported in humans in whom C. hominis, C. parvum, C. meleagridis, C. canis and C. felis are the most prevalent[10].
Bovine cryptosporidiosis is considered as one of the major causes of diarrhoea and diarrhoea-related deaths in neonatal calves[11]. Four specie of Cryptosoporidium such as C. parvum, C. bovis, C. andersoni and C. ryanae have been reported to be common species infecting cattle resulting in bovine cryptosporidiosis[12],[13]. Cryptosporidiosis cases have also been detected in various species of captive wild animals and wild birds, indicating its wide host range and zoonotic potential[14],[15],[16].
Cryptosporidium has high potential for zoonotic dissemination in low- and middle-income countries[4], however, comprehensive epidemiological studies are required to track its transmission dynamics and host specificity[10]. Compared with other countries, very few data are available on the prevalence of Cryptosporidium, its genotypes and subtypes in humans and animals in Nepal. Taylor et al.[17], first time in Nepal, have reported Cryptosporidium in 5% of the residents and travellers to Nepal by acid-fast staining method. Some pioneering studies on prevalence of cryptosporidiosis in Nepal were conducted by Sherchand et al.[18], Shrestha et al.[19] and Sherchand et al.[20]. The aim of this review is to summarize the findings of cryptosporidiosis from communities, fields and hospitals-based studies. Similarly, emphasis was given to find the diagnostic methods practiced mainly in hospitals, epidemiological factors and burden in various groups of people and animals, and recent trend of Cryptosporidium related research in context to Nepal. This review has pointed out the research gaps and opportunities for further studies on cryptosporidiosis.
| 2. Search strategy and selection criteria | | |
We searched PubMed, Web of Science, ResearchGate and Google Scholar with the search terms “cryptosporidium”, “cryptosporidiosis”, “epidemiology”, “diagnosis”, “Nepal” and collected all the papers available until November 30, 2022. We included 33 relevant papers with citations in English providing detail information regarding diagnostic tools associated to Cryptosporidium, disease prevalence either in humans, domestic farm animals, wild animals and/or environmental samples in context to Nepal.
| 3. Diagnostic methods | | |
3.1. Morphological and immunological tools
Direct microscopy, wet mount, concentration techniques (filtration, flocculation, Sheather’s sucrose floatation, immunomagnetic separation, saturated sodium chloride floatation, centrifugation), detection techniques (immunofluorescence microscopy, enzymatic immunoassay, enzyme linked immunosorbent assay), staining techniques, differential interference contrast microscopy, immunochromatographic dip strip test, and cell culture have been practiced by several researchers throughout the globe. However, modified Ziehl-Neelson staining technique followed by microscopy is most widely used by most of the researchers, and for clinical diagnosis of Cryptosporidium infections in Nepal. The specificity of staining techniques and antigen detection has remarkable variation while the PCR for Cryptosporidium has been found to be highly sensitive[21],[22],[23],[24],[25],[26].
3.2. Molecular approach
Various genotyping tools have been developed for exact identification of various species of Cryptosporodium. PCR assay targeting the small subunit (SSU) rRNA gene has been used as a common Cryptosporidium-genotyping tool[27],[28]. Similarly, qPCR-based genotyping assays[29],[30], SSU rRNA-based PCR assays using fluorescence resonance energy transfer probes and melt curve analysis can be used for rapid genotyping of Cryptosporidium spp. in humans[31], and MboII in RFLP analysis of PCR products from Cryptosporidium spp. in ruminants have been applied[32]. Subtyping based on analysis of DNA sequence of the 60 kDa glycoprotein (gp60) has been common in recent days[11],[33]. There were merely two reports regarding the molecular surveillance data of Cryptosporidium spp. in context to Nepal[34],[35].
| 4. Reported infections of Cryptosporidium | | |
4.1. Cryptosporidium in humans
Several incidences had been reported for potential transmission of the Cryptosporidium oocysts among humans mainly through contaminated water sources (tap, well and river water) and animals in Nepal. The study sites as mentioned in the selected papers have been depicted in [Figure 1] and the overall studies conducted in human subjects are summarized in [Table 1]. | Figure 1: Study sites of Cryptosporidium infections in humans, domestic animals, wild animals and water samples in Nepal.
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 | Table 1: Cryptosporidium identification in various groups of humans in Nepal.
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We have included 33 studies for analysis. Of the 20 studies conducted among humans, 13 were associated to hospital-based investigations among children, four in HIV seropositive cases from various hospitals and only three studies were conducted in adult humans visiting hospital. Overall 16 studies were based on hospital in-and out-patients, four community-based studies and only one study had combination of both hospital patients and community people. Based on the available studies, a total of 17527 stool samples from human subjects were analyzed by using concentration techniques and modified Ziehl-Neelsen stain followed by microscopy. Cryptosporidium infection prevalence was highest in children (9.0%; 1225/13545) followed by HIV cases (4.6%; 51/1107) and adults (1.8%; 52/2875), however the overall prevalence was 7.6% (1328/17527).
Cryptosporidiosis is considered to be a major threat to AIDS patients who are deprived of highly active antiretroviral therapy especially in developing countries[53]. In a hospital-based study in western Nepal, Cryptosporidium was found to be associated with persistent diarrhoea in HIV infected immunocompromised children, and malnutrition[47]. In Nepal, the prevalence of opportunistic cryptosporidiosis has been reported in HIV seropositive patients[39],[54],[55] as well as in HIV positive cases seeking for CD4 T-cell count[38],[56]. The discrepancy of cryptosporidiosis in rural and urban settings could not be categorized because most of the studies as mentioned above were hospital-based (in-patients and out-patients), and demographic characteristics like rural and urban data were lacking. In the studies conducted in hospital settings in Kathmandu, it can be assumed that most of the samples examined belong to the people from rural setting as many people from rural areas visit hospitals in Kathmandu on their own or on a referral basis. Most of the cases have been reported from urban areas as hospitals in urban settings are provided with laboratory facilities for epidemiological studies. However, studies have not segregated the results based on rural and urban areas. A wider surveillance on cryptosporidiosis in various human communities of different ethnic origin is worthwhile in order to estimate the disease burden.
4.2. Cryptosporidium in domestic animals
Of the total 1 088 faecal samples examined from various domestic animals, 23.2% (252/1 088) were found shedding Cryptosporidium oocysts. The highest prevalence was recorded in buffalo (47.3%; 98/207), followed by cattle (42.3%; 116/274), cats (11.2%; 12/107), pigs (10%; 10/100) and goats (4.0%; 16/400) [Table 2]. Domestic ungulates contribute as potential sources for environmental contamination of Cryptosporidium oocysts[57]. Paudyal et al. reported overall 32.0% (16/50) calves positive for Cryptosporidium oocysts in the periphery of Manohara and Bagmati river basin of Kathmandu[37]. A cross sectional study was conducted among cattle and buffalo calves near the river basins of Mahakali and Karnali, Nepal[58]. Of the total 350 faecal samples tested, 48.6% were positive for Cryptosporidium with higher prevalence in cattle calves (50.3%; 72/143) than the buffalo calves (47.3%; 98/207). For both the groups the highest prevalence of infection was recorded in the age group 3-6 months (53.7%; 101/188) and males had a higher prevalence (53.1%; 78/147) than females (45.3%; 92/203). Similarly, in the same study, the exotic breeds had a higher prevalence of infection (50.6%; 82/162) compared to indigenous breeds (46.8%; 88/188), the highest prevalence was observed in Bardiya (57.7%; 75/130) followed by Kanchanpur (46.4%; 64/138) and Kailalil (37.8%; 31/82), and the Karnali river basin with higher incidence (50.0%; 106/212) than the Mahakali river basin (46.4%; 64/138)[58].
Based on questionnaire survey, thatched sheds, more grazing hours and use of surface water were considered to be the probable risk factors of its prevalence[58]. Goats have been incriminated as a probable source for dissemination of cryptosporidial oocysts to the urban areas[59]. As the livestock survey in Nepal comprises a large number of cattle (2280542 holdings; 6430397 heads), buffaloes (1668820 holdings; 3174389 heads), yaks/chauri (6235 holdings; 48865 heads), sheep (98464 holdings; 612884 heads), goats (2463253 holdings; 11225130 heads) and pigs (477984 holdings; 870197 heads)[62], a nationwide surveillance on Cryptosporidium sp. is necessary in order to determine the prevalence, genotypes and their zoonotic potential.
4.3. Cryptosporidium in wild animals
Relatively very few studies are carried out on Cryptosporidium infection in wild animals in Nepal. A high prevalence was reported in swamp deer (62.5%; 20/32) followed by wild buffaloes (37.0%; 30/81), and the overall prevalence was found to be 18.15% (63/347) based on the studies available [Table 3]. Further investigations are essential to unveil the disease burden and the risk of zoonotic transmission.
4.4. Potential water-borne cryptosporidiosis
Water-borne gastrointestinal pathogens are found in high concentrations in the faeces of infected persons as primary source of infection and they are transmitted primarily through the faecal–oral route, mainly through consumption of contaminated food or water. Food- and water-borne diseases are considered to be the main reason for mortality and morbidity, and they are exacerbated by climate change effects globally[66]. Of the total deaths due to diarrhoea in low- and middle-income countries (60%), inadequate drinking water accounts for 35% followed by sanitation (31%) and hygiene (12%)[67]. About 83% of population in Nepal live in rural setting where more than 90% are devoid of toilet facilities[68], which indicates higher chances of fecal contamination to the water sources. Contamination of manure by Cryptosporidium oocysts is a potential source of further contamination of environment and water, and then to humans and animals[69],[70].
The Cryptosporidium oocysts positivity ranges from 20% to 50% (highest in river and sewage water) [Table 4]. Protozoa and helminth parasites are one of the major causes of diarrhoea in people of all age groups in the Kathmandu Valley, especially during the rainy season (June–September)[71],[72]. People residing in the Kathmandu valley especially rely on groundwater from shallow wells for their daily water requirement, but most of these wells do not meet the WHO microbiological guideline values for drinking water[73]. Tanker water seems to be one of the environmental determinants in transmission of cryptosporidiosis and other intestinal parasites in slum areas of Kathmandu[41] as sources of tanker water might be polluted[49]. The risk of acquiring water-borne diarrhoea from Cryptosporidium was reported to be higher than the acceptable limit in water resources for drinking and bathing[74]. Knowledge regarding the prevalence of water-borne pathogens is crucial to evaluate the risk of infection from pathogens transmitted via contaminated water. The prevalence study was mainly carried in ground water (wells) and river water in Kathmandu valley[74],[75],[76], however epidemic outbreak of food and water borne cryptosporidiosis at a large scale was not reported.
4.5. Molecular characterization of Cryptosporidium infections
C. parvum is mainly composed of two genotypes, I (human genotype or anthroponotic genotype) and II (bovine genotype or zoonotic genotype). The six isolates of C. parvum from humans in Nepal were identified as genotypes Ia2 by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP–single-strand conformational polymorphism (RFLP-SSCP) analyses of Cpgp40/15 encoding gene[34]. Feng et al. [35] carried out PCR-RFLP and sequence analysis of SSU rRNA gene in order to genotype Cryptosporidium specimens from calves of zebu cattle and water buffalo, and swamp deer (Cervus duvauceli) from the buffer zone of Chitwan National Park, Nepal [Table 5]. The C. ryanae variant reported in this study from zebu cattle indicates a unique cryptosporidiosis transmission in bovine animals and also deserves the possibility of cross-species transmission between allied host species sharing the same habitats. | Table 5: Genotypes of Cryptosporidium isolates from humans and other vertebrates in Nepal.
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4.6. Research gap, challenges and opportunities of Cryptosporidium research in Nepal
Research has confirmed that Cryptosporidium is one of the leading causes of food- and water-borne diarrhoea in children[5],[30], adults, immunocompetent and immunocompromised individuals[7], and domestic and wild animals[14],[35]. However, reports related to geriatrics, kidney patients, diabetics and organ transplant recipient with continuous use of immunosuppressants are lacking. In Nepal, most of the reports are related to hospital-based studies mainly for clinical diagnosis of diarrheal cases and very negligible number in community level. As more than 80% of Nepalese are relying on agriculture, the potential of zoonotic transmission of such parasites through animal excreta, soil, food and water are very common. Similarly, extensive research with epidemiological design are lacking in humans and wild life.
Despite the interest of researchers to use recent molecular diagnostic tools for the diagnosis and genomic identification of Cryptosporidium strains, most of the studies are relying on concentration method, wet mount and mostly modified Ziehl-Neelsen staining. The sensitivity and specificity of various diagnostic tools are different. The staining techniques (Ziehl-Neelsen and Safranine methylene blue) are less sensitive for detection of Cryptosporidium in HIV seropositive patients with CD4 count >200 cells/L. This finding suggests the chances of missing of cryptosporidiosis in patients who have CD4 count >200 cells/L, if only Ziehl-Neelsen and Safranine methylene staining technique is employed[21]. But these techniques are common practice in most of the teaching hospitals and universities in Nepal. The use of established molecular technique for species identification was very rare. The lack of awareness, education and fundamental health hygiene are predisposing factors for the seasonal outbreak of food- and water-borne diseases mainly in the rural areas. Most of the reports on cryptosporidiosis related diarrhoea are diagnosed only up to generic level. Therefore, diagnosis up to the species level and molecular characterization in relation to zoonotic potential are scarce. The chance of cross-species transmission between different species of domestic animals, wild animals to domestic animals or to the humans specially to the farmers or shepherds through pasture and water resource contamination cannot be ruled out. The existing drainage and pipe line system for distribution of drinking water in the urban regions are posing threat of fecal contamination. In addition to this, the trend of direct disposal of human excreta without treatment to the nearby streams and rivers also favour several water-borne diseases including cryptosporidiosis[78]. Presence of Cryptosporidium oocysts in water samples increases the chances of zoonotic transmission[79]. The presence of livestocks at home, use of untreated drinking water, habit of consuming raw fruits and vegetables, and poor sanitation are incriminated to the occurrence of Cryptosporidium in the low-income communities of Kathmandu[51]. Even if the drinking water is treated with chlorination at distribution centre or at home it is not certain to completely prevent the chance of infection of Cryptosporidium oocysts owing to its chlorine resistance. It is an utmost importance from the concerned authorities and research institutions to address the public health issues considering recent trend of urbanization, movement of the public, transportation of animal breeds within the country or cross border transport. At the same time, the use of integrated advanced molecular diagnostic tools along with well prepared and designed epidemiological field studies of cryptosporidiosis with participation of research scholars and scientists within the country as well as collaborative approach with foreign researchers are necessary so as to dig out the real scenario of its molecular diversity.
| 5. Conclusions | | |
The domestic animals are more infected (23.2%) than the wild animals (18.2%) followed by humans (7.6%), and water has been noticed as one of the major sources of environmental contamination by Cryptosporidium oocysts in urban areas. Most of the studies are based on microscopic observation in clinical specimens. Molecular epidemiological evidences regarding the zoonotic transmission of cryptosporidiosis in Nepal are negligible. Some studies carried out in domestic animals and wild animals in Nepal indicated that there could be more genotypes of Cryptosporidium with zoonotic potential. The role of domestic animals, wild animals and environmental determinants in transmission of cryptosporidiosis needs to be investigated in context to Nepal. Therefore, an integrated collaboration among parasitologists, molecular biologists, clinicians, veterinarians and epidemiologists is necessary to better understand the genetic variants of Cryptosporidium in humans, domestic animals and wild animals, and their zoonotic potential in Nepal.
Conflict of interest statement
The authors claim there is no conflict of interest.
Funding
This study receives no extramural funding.
Authors’ contributions
All the authors discussed and designed the study. Dhakal P collected, screened and analysed the literatures. Dhakal P drafted the first manuscript. Li J and Zhang L edited and finalized the manuscript. All the authors have read and approved the final version to publish.
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[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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