Cryptosporidium spp.: Challenges in Control and Potential Therapeutic Strategies

Abstract

Cryptosporidium parasites (Cryptosporidium hominis and Cryptosporidium parvum) are prominent for playing a crucial role in the high prevalence of diarrheal infection across the globe, with immunocompromised individual at risk. The parasites’ remarkable resilience in the environment due to several adaptive strategies is responsible for persistent challenge in control especially in regions with inadequate sanitation. In tackling these challenges, exploring promising potential therapeutic strategies to combat Cryptosporidium infections is of critical importance. This encompasses investigations into experimental drugs, immunotherapies, and vaccine development efforts, all aimed at reducing the burden these parasites impose. This review aims to present the current state of research and development to shed light on the future prospects for managing Cryptosporidium infections and their profound impact on public health.

1. Introduction

Cryptosporidium is a well-established obligate gastrointestinal protozoan parasite [1]. It is widely distributed both environmentally and geographically, encompassing numerous species with a broad host range. Over 40 species of Cryptosporidium have been documented, with at least 20 implicated in human infections [2, 3]. However, the predominant agents of human infection are C. hominis and C. parvum, with C. hominis being a predominantly human-related transmission and C. parvum both human- and animal-borne transmission [3].

Cryptosporidium infection occurs through the ingestion of oocysts, which are released by a host already infected with the parasite and are present in contaminated environments through oral-fecal exposure. Transmission primarily occurs via water and, to a lesser extent, through food. Upon ingestion of oocysts, sporozoites are liberated and subsequently adhere to intestinal epithelial cells, undergoing internalization. A thick, actin-rich layer known as the parasitophorous vacuole membrane forms, separating them from the host cytoplasm within the microvillus layer of the host cell membrane [4]. Following maturation, the organism undergoes a bifurcating life cycle. Asexual reproduction yields merozoites, which can subsequently invade further intestinal epithelial cells, perpetuating the infection within a single host. Conversely, sexual multiplication culminates in the release of oocysts, which are shed from the host and serve to establish infection in new hosts.

Cryptosporidium has become a major worldwide contributor to diarrheal illness, posing a significant health risk, especially for young children and individuals with weakened immune systems. Research indicates its substantial impact in developing countries, where it is recognized as a leading cause of moderate-to-severe gastrointestinal illness in children under 5 years old [5]. A recent in-depth investigation into gastrointestinal diseases has underscored the significant public health burden posed by Cryptosporidium. This study estimates that Cryptosporidium species caused over 1 million deaths, with nearly half occurring in children under 5 years of age. Furthermore, it resulted in more than 71 million disability-adjusted life years (DALYs) between 2005 and 2015 [6]. The data reveals that developing countries, particularly those in sub-Saharan Africa, suffer the highest mortality rates. While Cryptosporidium is endemic in most developing nations, it also has the potential to cause waterborne outbreaks on a large scale, impacting both developing and developed countries [7]. Especially worrisome are the long-term consequences observed in children, such as stunted growth and cognitive impairments that persist beyond the initial recovery from cryptosporidiosis [5].

Cryptosporidiosis is a noteworthy health concern not only in wild animals but also in domesticated ones. Within farm animal management, it has been linked to the development of a severe and frequently fatal diarrheal syndrome in newborn calves and other young ruminant animals. This parasite infestation leads to substantial economic losses, both directly and indirectly [8]. Furthermore, cryptosporidiosis can have lasting detrimental effects on infected animals. Studies have shown diminished weight gain and reduced production performance in both cattle and sheep [9, 10]. For example, research by [9] found that beef calves exposed to cryptosporidiosis as newborns can experience an average weight deficit of 34 kg at 6 months of age compared to their counterparts without signs of infection.

In recent times, research has been dedicated to identifying compounds with novel mechanisms of action that are effective against Cryptosporidium spp. Presently, only one drug, nitazoxanide, holds approval from the US Food and Drug Administration (FDA) for treating cryptosporidiosis in immune-competent individuals in the United States, excluding immunocompromised individuals like AIDS patients. However, nitazoxanide’s efficacy is limited, and its mechanism of action remains unclear [11]. Compounding the challenge, there is a lack of FDA-approved medications to treat cryptosporidiosis in animals. Halofuginone lactate (Halocur), although approved for veterinary use in calves and lambs in some countries, demonstrates only partial effectiveness against Cryptosporidium and fails to entirely eliminate oocyst shedding [12].

Numerous obstacles in the discovery of drugs against cryptosporidiosis include the scarcity of parasite materials, lack of a robust and standardized in vitro cultivation system, lack of conventional drug targets, the need for the development of in vitro phenotypic screening platforms, the complexity of genetic manipulation techniques despite their availability, and the availability and constraints of animal models, among other challenges [13].

The complexity of combating Cryptosporidium spp. infections presents a formidable challenge to public health efforts worldwide. Despite significant advancements in understanding its biology and pathogenesis, effective control measures remain elusive. However, the exploration of potential therapeutic strategies offers a glimmer of hope in the quest to mitigate the impact of this ubiquitous pathogen. In this review, thelandscape of Cryptosporidium control is examined, with a focus on both the hurdles faced and the promising avenues for future intervention.

2. Cryptosporidiosis: transmission strategies, clinical manifestations, and public health impact

Cryptosporidium infection exhibits diverse transmission pathways. Humans can contract the parasite through direct contact with infected individuals (anthroponotic transmission) or animals (zoonotic transmission). Indirect transmission also occurs via contaminated food (foodborne transmission) or water (waterborne transmission). Person-to-person and waterborne transmission appear to be the most common routes, evidenced by the higher frequency of reported cases and outbreaks associated with these modes [14]. Outbreaks associated with contaminated recreational water, particularly swimming in polluted rivers, lakes, or pools, underscore the diverse modes of Cryptosporidium transmission [15]. Several other factors include migration, interaction with animals or young children experiencing diarrhea, and engaging in certain sexual practices (multiple sexual partner and anal intercourse) that carry a higher risk of transmission [13].

A worldwide study revealed a troubling number of waterborne protozoan illness outbreaks, totaling 936 between 1946 and 2016 [16]. Cryptosporidium was responsible for over half (58%) of these outbreaks. Even more concerning is its ability to bypass filtration systems, contaminating both filtered and unfiltered drinking water supplies in communities [16]. The resilience of these Cryptosporidium spp. Oocytes to conventional water treatment, environmental resilience, zoonotic strains and wide range of reservoir host complicate control. Oocysts can also seep from polluted surface water into groundwater [17, 18].

The most substantial outbreak of cryptosporidiosis linked to drinking water occurred in Milwaukee, Wisconsin, in 1993, impacting an estimated 403,000 individuals out of a population of around 800,000. It also resulted in high hospitalization (4400) and a hundred mortality rates [19]. The number of documented foodborne outbreaks is lower compared to waterborne incidents, potentially attributed to lack of standard foodborne oocyst detection tool and the less frequent occurrence of food contamination [20]. However, Cryptosporidium oocysts, the infectious stage of the parasite, have been found in a concerning variety of food products, including raw vegetables, meat, salads, fermented milk products, cider, raw milk, and apple [21]. This highlights the diverse points of contamination throughout the food chain, from production and harvest to processing, transportation, and even preparation in the home [21].

The clinical presentation of enteric cryptosporidiosis lacks distinct signs or symptoms, resembling various other forms of diarrheal diseases. Initial infections typically manifest with symptoms such as nausea, vomiting, abdominal cramps, watery diarrhea, and fatigue. Stool specimens rarely show the presence of blood or leukocytes. Unlike typical enteric infections, Cryptosporidiosis in developing countries often presents with a low-grade fever and cough less frequently [22]. Repeated exposure can lead to asymptomatic infections in children, and symptomatic cases tend to be shorter, lasting a median of just 2 days. However, weight reduction and dehydration remain a concern, especially for malnourished children or those with prolonged diarrhea [22]. In developed nations, immunocompetent individuals with Cryptosporidiosis experience severe symptoms. Diarrhea can last 9–11 days, with some requiring hospitalization [23]. Joint pain, fatigue, and other issues may also occur, especially with C. hominis. Cryptosporidiosis may even be linked to chronic bowel problems [22].

Cryptosporidiosis poses a greater risk for immunocompromised people. As their immune system weakens, asymptomatic infections can worsen, leading to the development of symptoms [24]. Furthermore, the parasite can invade the bile ducts, causing inflammation (cholangitis), and potentially impact the respiratory system, leading to mild oxygen deficiency and shortness of breath [24]. Previously thought uncommon, respiratory infections with Cryptosporidium have been identified in a surprising number of children with intestinal infections [25, 26, 27, 28]. This suggests potential transmission through coughed-up sputum. For individuals with HIV/AIDS, Cryptosporidiosis can be life-threatening, significantly increasing mortality and shortening lifespan [28]. The severity varies greatly, with a particularly aggressive form causing massive fluid loss, severe pain, and rapid weight loss, leading to death within days or weeks. Variations in immune function, infection location, and parasite species all contribute to these differences in disease severity and patient outcomes [28].

Cryptosporidiosis poses a significant global public health concern, ranking as the sixth most frequent foodborne parasitic infection in humans and animals [29]. In developing countries, the parasite disproportionately impacts malnourished children, where it is a leading cause of death. The full scope of human Cryptosporidium epidemics, particularly in these less developed regions, remains unclear [30]. For children suffering from acute, chronic, or persistent diarrhea caused by Cryptosporidium, the consequences are severe, including stunted growth, reduced physical development, cognitive impairment, and even death [13].

Cryptosporidium species are prominent diarrheal pathogens worldwide, affecting a staggering 20% of children experiencing diarrhea in developing countries, compared to a much lower range of 1 to 5% in North America and Europe [26]. Landmark studies conducted in West Africa during the 1990s underscored the severity of this disparity. Children with Cryptosporidiosis displayed significantly higher mortality rates, with a nine-fold increase in death risk even after accounting for nutrition-related factors. Infection typically occurs earlier in life: before age 2 in developing regions and before age 5 in developed ones. Additionally, the prevalence is higher in rural areas and during rainy seasons. Exposure to the parasite appears to be continuous, as evidenced by increasing seropositivity with age [13]. However, malnutrition, HIV/AIDS, and other conditions that weaken the immune system dramatically elevate the risk, severity, and duration of Cryptosporidiosis. This makes it a defining opportunistic infection in AIDS patients and a frequent complication for malnourished children suffering from persistent diarrhea [31].

3. Current preventive and control measures and their constraints

Several control and preventive measures have been initiated toward curtailing the transmission of Cryptosporidium spp. most of which focus on environment-related factors and reservoir host. This is because reservoir characteristics and environmental factors significantly influence the epidemiology and transmissibility of Cryptosporidium spp. [32]. Oocysts, shed by the host, exhibit sensitivity to various unfavorable environmental conditions before finding a suitable new host [32]. Environmental factors like higher temperatures, age, and dryness significantly impact the survival and infectiousness of Cryptosporidium oocysts. While prolonged sunlight exposure completely inactivates them, traditional water treatment methods like coagulation and filtration often prove inadequate [33]. This highlights the resilience of the oocyst and the need for more robust treatment strategies. Advancements in UV disinfection research have shown promise, with UV light effectively killing Cryptosporidium oocysts. In addition, solar photocatalytic disinfection (SPCDIS) using titanium oxide has demonstrated effectiveness, even in less-than-ideal light conditions [33]. Chlorination has proven inadequate in removing Cryptosporidium spp., prompting ongoing efforts to develop vaccines for animals [34].

Similarly, [35] reported modern microbial reduction process designs, such as the integrated disinfection design framework (IDDF) which ensure the provision of low-risk drinking water by addressing the shortcomings of traditional treatment methods. Also, the prevention of reservoir hosts from contact with water supplies and the construction of wetlands for wastewater treatment have emerged as effective and cost-efficient strategies for removing parasite like Cryptosporidium spp. [36]. Practical measures, such as educational campaign, handwashing initiatives, and point-of-use water treatment (especially in developing nation) are key ways to curb Cryptosporidium transmission. Boiling water, using reliable bottled water, and home filtration units offer further protection [34]. However, in many developing countries, several challenges hinder efforts to prevent the transmission of diseases. These include a lack of access to clean water, open grazing and extensive rearing of animals, poor sanitation infrastructure leading to water source contamination, limited healthcare resources, porous borders, and high population density. These factors collectively serve as significant setbacks to various preventive measures aimed at curbing the spread of diseases.

From pharmaceutical perspective, anti-cryptosporidial agents, including paromomycin, nitazoxanide, and azithromycin, have shown limitations in treating Cryptosporidium enteritis. Nitazoxanide, the only FDA-endorsed medication, exhibits limitations, especially in Individuals with HIV/AIDS [37]. Studies have indicated that current chemotherapy has a limited role in managing cryptosporidiosis in immunocompromised individuals, emphasizing the importance of restoring immune status using antiretroviral drugs [37].

Research on alternative treatments involves traditional medicinal plants such as Allium sativum (garlic) and various plant extracts. Garlic possesses potential as a preventative measure (prophylactic) and treatment (therapeutic) for Cryptosporidiosis, with studies indicating its potential in reducing the shedding of oocysts. Furthermore, studies on plant extracts like Peganum harmala and Artemesia herba-alba have shown promising anti-parasitic activity, providing potential alternatives to conventional pharmaceuticals [38, 39]. Research into alternative treatments for Cryptosporidium, particularly utilizing traditional medicinal plants, faces several challenges. The lack of comprehensive scientific validation, standardization of effective dosage, bioavailability issues of active compounds, cultural variability, interactions with conventional medications, long-term effects, and many more are several issues linked with the utilization of traditional medicinal plant.

4. Existing diagnostic methods and limitation

Given that Cryptosporidium poses a significant threat to people with weakened immune systems and is associated with serious illness and death, it is important for clinical laboratories to come up with effective screening criteria and reliable testing methods. However there is still no agreed-upon international standard for diagnosing cryptosporidiosis [40]. In labs and field research, there are multiple ways to detect and study Cryptosporidium spp. Each method has its strengths and weaknesses, like sensitivity, cost, processing time, specificity, detection limits, difficulty, and equipment needs [41].

Microscopy-based methods are commonly used in identifying Cryptosporidium oocysts in various samples, such as water, food products, and stool. However, their reliability to identify different Cryptosporidium species based on Conventional microscopy alone is questioned due to the similarities in morphological characteristics among many species [42]. Also, these methods cannot determine oocyst infectivity [42]. While a range of staining techniques, such as the Ziehl–Neelsen stain (acid-fast stain) and dimethyl sulfoxide, have been utilized to distinguish Cryptosporidium oocysts from other particles on glass slides [41]. However, staining methods have limitations such as variation in stain uptake, low sensitivity, lack of specificity for samples with small oocyst numbers, and the need for a skilled microscopist. Furthermore, staining method is limited by its slow speed, time-consuming nature, and potential for subjectivity compared to other options [43].

Immunology-based methods, particularly immunofluorescence assays using monoclonal antibodies (mAbs), provide higher specificity and sensitivity compared to microscopy [43]. The direct fluorescent antibody (DFA) assay, which utilizes the fluorescein isothiocyanate-conjugated anti-Cryptosporidium mAb, is commonly employed for this purpose [43]. Commercially available kits have demonstrated high specificity and improved sensitivity [41]. However, indirect immunofluorescence assays involve additional steps compared to DFA and utilize a second fluorophore-conjugated antibody. The process is longer and potentially more prone to nonspecific binding [44].

Immunomagnetic separation (IMS) offers a valuable technique for isolating Cryptosporidium oocysts, particularly from samples containing low numbers of oocysts [44]. This method utilizes magnetic beads coated with specific antibodies to target and capture Cryptosporidium oocysts from environmental samples. Studies report high sensitivity and specificity for C. parvum oocysts, demonstrating its effectiveness in detecting this particular species [45]. However, a key limitation of IMS lies in its inability to differentiate between Cryptosporidium species or genotypes. Additionally, the time-consuming nature of the process and the high cost of commercially available IMS kits pose challenges for widespread implementation [46]. Immunochromatographic lateral-flow assays (ICLFAs) are popular rapid tests that use antibody strips to detect Cryptosporidium antigens in stool and water samples. Their advantages include rapid detection, cost-effectiveness, and no need for specialized equipment or trained personnel. However, despite high accuracy of ICLFA, cases of false positive and negative results have been reported. This has led to cautious consideration of positive results until further improvement of these tests [47].

The enzyme-linked immunosorbent assay (ELISA) is a widely employed method in detecting Cryptosporidium parvum in human and animal stool samples. ELISA offers high sensitivity for Cryptosporidium, exceeding acid-fast staining [48]. However, its effectiveness can vary depending on the kit and population [48, 49]. Longer processing times and potential false positives limit its use in water sample testing for C. parvum [50]. Flow cytometry excels in fast, large-scale analysis of cells with high accuracy, making it a valuable tool for Cryptosporidium detection. When combined with immunomagnetic separation (IMS), FC enhances oocyst recovery from water samples [51]. Although FC is more sensitive than traditional methods like DFA, its infrequent use in diagnostic laboratories is attributed to high costs and technical expertise requirements [43]. Cell culture immunofluorescence assays use lab-grown C. parvum oocysts to assess viability and infectivity. The challenges include low yields and difficulties in long-term propagation [52].

Nucleic acid methods excel in detecting parasites like Cryptosporidium, offering high sensitivity and improved accuracy. Conventional PCR, nested PCR, and quantitative PCR (qPCR) have been extensively used for Cryptosporidium detection in various samples [43, 45, 47, 50]. qPCR, in particular, allows real-time monitoring with high sensitivity and specificity [41]. Also, droplet digital PCR (ddPCR) improves nucleic acid quantification, but its use for Cryptosporidium detection is limited due to cost considerations [53, 54]. PCR-restriction fragment length polymorphism (PCR-RFLP) has been a useful tool for genotyping; its role is diminishing due to the affordability and accessibility of DNA sequencing technologies [55]. Furthermore, whole-genome sequencing using Sanger sequencing and next-generation sequencing platforms has also helped in understanding Cryptosporidium diversity [56].

DNA fingerprinting methods that include random amplified polymorphic DNA (RAPD) provide valuable information on genetic variation. However, Aptamer-based methods, which adopt synthesized molecular recognition probes, present a good approach for direct and sensitive Cryptosporidium detection. Aptamer’s advantages over antibodies include cost-effectiveness and stability [57].

Aptamer-based aptasensors have demonstrated success in identifying Cryptosporidium oocysts in various samples, such as water, food products, and stool [41]. Despite challenges in aptamer research, recent advancements have addressed issues like rapid degradation and lack of standardized protocols. Emerging research suggests aptamers have the potential to become widely used in diagnostics, therapy, and biosensing applications [41].

5. Present therapeutic options

The primary pharmaceutical intervention for Cryptosporidium infection in children over 1 year of age remains nitazoxanide, a thiazole compound approved by the FDA in 2003. Placebo-controlled studies have demonstrated its increased efficacy in non-HIV-infected cryptosporidiosis patient [58]. The treatment’s efficacy wanes in immunocompromised individuals, even when administered at higher doses for extended periods [59]. While nitazoxanide is currently the only FDA-approved treatment for Cryptosporidium, its effectiveness is reduced in children with malnutrition or weakened immune systems [60]. Studies have shown that nitazoxanide treatment only leads to a stop in diarrhea for 56% of malnourished children with chronic Cryptosporidium compared to 23% receiving a placebo [59]. Combining nitazoxanide with new anti-Cryptosporidium drugs may offer a more effective approach for treating malnourished children and immunocompromised patients with HIV/AIDS.

While paromomycin offers some promise and partial effectiveness in AIDS patients with Cryptosporidium, it lacks official treatment approval. Similarly, azithromycin shows minimal effectiveness and fails to outperform a placebo in AIDS patients with the infection [61].

Halofuginone shows promise as a treatment for coccidiosis in animals by targeting a specific parasite enzyme [62, 63]; its use in humans is limited. This compound is even licensed for cryptosporidiosis in calves in some countries [62, 63] However, unfavorable side effects on the gastrointestinal system and higher toxicity compared to other options prevent its use for human cryptosporidiosis treatment [63]. These limitations on effective drug treatments force many developing countries to rely on basic strategies like rehydration and electrolyte replacement for managing Cryptosporidiosis.

6. Challenges in the development of Cryptosporidium infections

Limited access to pure Cryptosporidium for research impedes progress. While C. parvum is commonly used, obtaining large quantities remains challenging [64]. Although in vitro culture techniques for C. parvum have advanced over decades, continuous cultivation faces persistent limitations. The parasite primarily replicates asexually, hindering the production of unlimited materials needed for drug discovery and a deeper understanding of the life cycle [65, 66].

Limited drug targets due to Cryptosporidium’s simple biology (minimal metabolism and nutrient synthesis) are a hurdle. However, recent sequencing of various Cryptosporidium genomes has opened exciting possibilities for identifying new drug targets [13]. While genetic manipulation tools like CRISPR/Cas9 have addressed issues in drug target validation, challenges persist in the adaptation of these tools for routine laboratory use. C. tyzzeri, a mouse-specific species, serves as a valuable genetic model, offering convenience for laboratory manipulation. Additionally, gene-silencing strategies have shown success in knockdown experiments [67, 68].

One major challenges of systemic drugs is inability to cross the epicellular delivery (ED) band. Significant knowledge gaps remain regarding the molecular makeup and function of the parasite–host interface, particularly the ED band. This results in unsatisfactory efficacy of some identified anti-cryptosporidial drugs in vivo. Systemic drugs must effectively cross the ED band; this emphasizes the importance of pharmacokinetic parameters in plasma for efficacy in mouse models. While animal experiments are informative, they are costly and time-consuming. This calls for better in vitro assays to evaluate how easily small molecule drugs can permeate the ED band [65]. The systemic drugs are also limited by severe watery diarrhea. This is because they can be easily flushed off from the gastrointestinal tract (GIT). Novel pharmacological modifications like exploration of the enterohepatic recycling pathway could improve drug absorption in individuals with severe diarrhea. Testing drug effectiveness in animals, especially mice that do not develop diarrhea, poses a challenge in evaluating drug absorption and efficacy [13].

An alternative strategy involves the development of drugs that target the parasite directly within the gut (nonsystemic drugs). Unlike systemic drugs, which can be flushed out by diarrhea, nonsystemic drugs offer a targeted approach. Nonsystemic drugs may serve as effective alternatives to systemic drugs. Further improvements could involve increasing its (nonsystemic drugs) adhesion to the gut lining (mucoadhesive properties) and potentially combining them with anti-diarrheal medications to maximize their effectiveness within the digestive tract [13].

7. Emerging therapeutic strategies

Significant progress has been achieved in identifying active compounds against Cryptosporidium, with aim of providing medication for childhood diarrhea in developing nations [69].

Benzoxaboroles, boron-heterocyclic compounds, have garnered attention globally. A compound called AN7973 shows promise as a treatment for cryptosporidiosis. This 6-carboxamide benzoxaborole inhibits the development of C. parvum within host cells and kills C. hominis parasites. AN7973 reduced fecal shedding of C. parvum by over 90% in both mouse models and neonatal dairy calves, suggesting its potential effectiveness. Furthermore, Pyrazolopyridines displayed over 60% inhibition of C. parvum. KDU731 (ayrazolopyridine) has shown efficacy against cryptosporidiosis in both immunocompromised mice and neonatal calves by reducing oocyst shedding [70].

Piperazine derivative MMV665917, initially identified in the Medicines for Malaria Venture “Malaria Box,” exhibited high efficacy against C. parvum and C. hominis in vitro. In newborn calves and gnotobiotic piglets, it was discovered the MMV665917 reduced oocysts, diarrhea severity, and intestinal mucosal damage. Further investigations are required for dosage optimization and understanding mechanisms of action [64, 71].

Bicyclic azetidines, known for their effectiveness against P. falciparum, have demonstrated efficacy against C. parvum and C. hominis both in vitro and in vivo. Targeting phenylalanyl-tRNA synthetase, these compounds showed potent inhibition of Cryptosporidium growth and protective effects in mice. Bicyclic azetidines present a promising series for cryptosporidiosis treatment [72].

Recent advancements in genetic modification, particularly CRISPR-guided relinking, have enabled a broad comprehension of Cryptosporidium biology. This approach, known for its simplicity, supports efficient reverse genetics for the parasite. The introduction of transgenic reporter strains, like the Nluc reporter strain, has speed of the process of drug development against Cryptosporidium. This is achieved through the provision of a rapid and scalable screening method. These strains allow lead compound testing in both immunocompromised mouse models and in vitro setting. Genetic modification promises to identify crucial drug targets and understand their mechanisms, expediting drug development against Cryptosporidium [72].

Natural products emerge as a rich source for potential therapeutic agents. A recent study that screened 800 natural products has identified 16 compounds with low to submicromolar anti-Cryptosporidium parvum activity in vitro [73]. Compounds like Ginsenoside-Rh2, Curcurbitacin-B, flavonoids, and isoflavones displayed Anticryptosporidia activity in mouse models. Chicory, curcumin, chitosan, and others have shown efficacy against C. parvum both in vivo and in vitro. The investigation of novel bioactive compound and natural products holds promise in the development of effective Anticryptosporidia compounds and potent candidates [73].

Probiotics, recognized as a natural alternative therapeutic approach, demonstrate results against Cryptosporidium infections. Probiotics can reduce the number of oocysts shed in feces (oocyst excretion) and lessen the severity of infection [74]. Certain probiotic strains, particularly Lactobacilli, have shown potential by the clearance of Cryptosporidium oocysts in mice and humans. Understanding probiotic mechanisms is pertinent in the development of effective probiotic treatments against Cryptosporidium infections [74].

Advances in next-generation sequencing technologies allow for detailed characterization of the gut microbiota’s role in various diseases, not exempting Cryptosporidium infection. Specific members of the microbiome, such as Megasphaera, may influence the severity of Cryptosporidium-induced diarrhea. Despite the complexity of the microbiota’s impact on Cryptosporidium development, it represents a promising target for interventions. Future research should focus on understanding specific genera or phyla within the microbiota that can positively influence immune responses to Cryptosporidium [75].

The development of a Cryptosporidium vaccine is driven by the necessity for effective preventive measures. Memory T cells, essential components of immunological memory, enhance the body’s ability to resist subsequent infections. This characteristic makes them a prime focus in vaccine development [76]. Identification of new vaccine candidates should prioritize antigens capable of eliciting T cell responses [77]. A molecule named gp40/15 polyprotein shows promise as a Cryptosporidium vaccine [78]. It triggers the production of IFN-γ, which activates memory T cells crucial for fighting off future infections. Also, Zoite surface proteins like Cpgp40 and Cpgp40/15 have demonstrated associations with the parasitophorous vacuole membrane, through the inhibition of C. parvum infection [79]. Antigens like gp40 elicit cellular immune responses in humans and animals, but their protective response against Cryptosporidium infection needs further examination. CpGP15 recombinant antigen eliminates C. parvum infection in cattle. It further addressed false-negative results on animal farms [80].

Furthermore, Peptides like CP15 and circumsporozoite-like antigen (CSL) stimulate antibody production and block parasite entry in vitro [81]. CpTSP8, a TRAP-like protein in C. parvum, is important for parasite movement and invasion or attachment to the host cell [82]. Despite the potential of TRAP proteins as vaccine candidates, further investigations are warranted.

Similarly, the discovery of Cp-P34, a sporozoite surface protein in Cryptosporidium, is crucial for developing a multiantigenic vaccine against cryptosporidiosis. This protein, primarily localized within the parasite, transiently appears on the surface of Cryptosporidium sporozoites, thereby stimulating host immune responses [83]. The mechanism by which Cp-P34 reaches the surface could be a valuable target for future vaccine development. Despite these advancements, further research is needed to fully understand the protective efficacy of these antigens and their potential as components of an effective vaccine against Cryptosporidium.

8. Conclusions

The review highlights the multifaceted challenges associated with Cryptosporidium spp. infections, focusing on the transmission strategies, clinical manifestations, public health impact, preventive measures, diagnostic methods, existing therapeutic options, and emerging strategies. The prevalence of waterborne and person-to-person transmission underscores the global significance of cryptosporidiosis, with substantial public health and environmental implications. The limitations of current preventive measures emphasize the need for innovative approaches to curb transmission. Diagnostic methods exhibit varied strengths and weaknesses. However, challenges like false positives and negatives persist, emphasizing the ongoing need for improvement and standardization in diagnostic techniques. Existing therapeutic options face limitations, particularly in immunocompromised patients, highlighting the necessity for novel anti-Cryptosporidium therapeutics. The challenges in drug development underscore the complexity of combating Cryptosporidium infections. Emerging therapeutic strategies, such as benzoxaboroles, pyrazolopyridines, and probiotics, show promise in preclinical studies, offering potential alternatives to current treatments. Genetic modification techniques, natural products, and the exploration of the gut microbiota’s role present exciting avenues for future research and drug development. The pursuit of an effective Cryptosporidium vaccine remains a critical goal, with promising candidates like gp40/15 polyprotein, CpGP15 recombinant antigen, and sporozoite surface proteins showing potential. Future research should focus on refining preventive measures, improving diagnostic accuracy, advancing drug development, and ultimately developing an effective vaccine to mitigate the global impact of cryptosporidiosis.