Treatment of Cryptosporidium: What We Know, Gaps, and the Way Forward (2024)

Abstract

Cryptosporidiosis is increasingly recognized as an important globalhealth concern. While initially reported in immunocompromised such as AIDSpatients, cryptosporidiosis has now been documented as a major cause ofchildhood diarrhea and an important factor in childhood malnutrition. Currently,nitazoxanide is the only proven anti-parasitic treatment forCryptosporidium infections. However, it is not effective inseverely immunocompromised patients and there is limited data in infants. Immunereconstitution or decreased immunosuppression is critical to therapy in AIDS andtransplant patients. This limitation of treatment options presents a majorpublic health challenge given the important burden of disease. Repurposing ofdrugs developed for other indications and development of inhibitors for noveltargets offer hope for improved therapies, but none have advanced to clinicalstudies.

Keywords: Cryptosporidium, Cryptosporidium parvum, Cryptosporidium hominis, cryptosporidiosis, nitazoxanide, paromomycin

Introduction

Cryptosporidium species are increasingly recognized asimportant enteric pathogens [1-3]. Cryptosporidiosis was initiallyrecognized as a cause of diarrhea in compromised hosts. Shortly thereafter, zoonoticand waterborne transmission of the parasite was identified. Cryptosporidium is nowconsidered one of the major causes of childhood diarrhea. In addition,Cryptosporidium has been documented as a key component of the viciouscycle of infection and malnutrition that are major contributors to childhoodmorbidity and mortality worldwide. The majority of humanCryptosporidium infections are attributed to two species:C. hominis and C. parvum [1, 2]. However, at least 13 other species may infect humans,[3-5]. Clinically, cryptosporidiosis causes watery diarrhea inhealthy patients. In contrast to other causes, diarrhea caused by cryptosporidiosistends to be more prolonged and can be chronic in compromised hosts, such as childrenwith malnutrition.

Cryptosporidium parasites develop within the microvillus layer of intestinalepithelial cells, mainly found in the small intestines in immunocompetent hosts, butmay be found throughout the GI tract and even the respiratory tract. Persistentinfection is associated with villus atrophy, crypt hyperplasia, and variableincreases in leucocytes in the lamina propria. The symptoms of watery diarrhea andmalabsorption are thought to be related to sodium malabsorption, electrogenicchloride secretion, and increased intestinal permeability, and severity of diseasecorrelates with altered intestinal permeability [6, 7].These effects are likely mediated by the host response and neuropeptides such assubstance P may be key contributors [8, 9]

The burden of disease caused by Cryptosporidium worldwidehas been significantly underestimated. For example, only about 1% of theestimated 750,000 cases that occur annually in the US are reported [10, 11]. Historically, Cryptosporidium was thought ofprimarily as a cause of chronic diarrhea in AIDS and other immunocompromisedpatients. More recent data has shed light on the parasite's effect onchildren in resource poor areas. Older studies, using acid fast staining to identifythe organisms, found Cryptosporidium in <5% ofcases of childhood diarrhea. However, more recent studies using antigen andmolecular assay have detected Cryptosporidium infection in15-20% of all childhood diarrhea [1, 12]. In a multicenterstudy of childhood diarrhea in Sub-Saharan Africa and South Asia,Cryptosporidium was second to rotavirus as a cause ofmoderate-to-severe diarrhea in children two years of age and was a major cause ofmorbidity in the second year of life [13]. Subsequent studies using molecular methods demonstratedthat even that study had under diagnosed cryptosporidiosis [14].

There are also chronic sequellae of Cryptosporidiuminfection. Lima et al showed that infection in children less than one year old wasfollowed by recurrent diarrhea and growth stunting that continued for two yearsafter the initial episode [15]. Follow-up studies demonstrated deficits in cognitivedevelopment and physical fitness when patients were examined 5 years later[16].

Cryptosporidiosis is more frequent and severe disease in children withmalnutrition, including higher incidence of deaths [2, 17, 18]. Malnutrition predisposes patientsto infection and creates a vicious infection-malnutrition cycle with long termconsequences including cognitive impairment and stunting [19]. Using an animal model, that closelyresembles the complex interaction between nutritional status and infection, Costaet al. showed 20% additional weight loss whenmalnourished mice were infected with C. parvum, higher fecalshedding and failure to prevent weight loss or parasite stool shedding despitetreatment with nitazoxanide [20]. In addition, children under the age of one year withCryptosporidium infections fail to have catch-up growth that istypically observed with children infected at a later age. The diarrheal morbidityfor this young age group is significantly increased as well [18, 21].

Symptomatic therapy is vital in cases of cryptosporidiosis. Replacement offluids and electrolytes in cryptosporidiosis is critically important as in othercauses of diarrhea. Anti-motility drugs are also a key element of therapy. Mostpublished patient studies utilize narcotic agents such as loperamide anddiphenoxylate/atropine. Other reports suggest that tincture of opium (paregoric) maybe a more effective agent in AIDS patients. Nutritional support is also imperativefor successful treatment, which includes continued breast-feeding of infantpatients.

Because cryptosporidiosis is typically self-limited in immunocompetent hosts,restoration of immune function is a key component of management. Immunereconstitution in response to effective combination antiretroviral therapy has beenlinked to parasite clearance, as well as reduced long term morbidity and mortalityassociated with cryptosporidial infection of AIDS patients [2, 22, 23]. Nevertheless, even with effectiveantiretroviral therapy, chronic diarrhea is associated with early mortality[24]. There are anecdotesof better responses when anti-motility and anti-parasitic drugs are used as part ofthe initial therapy [23].However there is no conclusive evidence that this is the case. Interestingly, someHIV protease inhibitors have activity against Cryptosporidium bothin vitro and in vivo [25].

Current Therapeutics

Despite the fact that cryptosporidiosis has been recognized as an importantcause of diarrheal disease for over 3 decades, anti-parasitic treatments has beenlimited. Initial screening of available compounds failed to identify effectivetreatments for cryptosporidiosis. A number of drugs previously reported to beeffective have failed in clinical trials [22].

The only drug that has FDA approval for treatment ofCryptosporidium is nitazoxanide [2]. Nitazoxanide was synthesized in the 1980s bycombining a thiazole ring (similar structurally to metronidazole) with a benzamidinering (similar to the tapeworm drug niclosamide). Nitazoxanide is a broad spectrumanti-parasitic with reports of use as a deworming agent as well as in controlledtrials in giardiasis and cryptosporidiosis [22]. Three placebo-controlled trials of treatment ofcryptosporidiosis with nitazoxanide in non-AIDS patients have been reported[26-28]. Studies reported up to 93% oftreated patients experienced parasite clearance as opposed to 37% of placebotreated patients [27]. Thedrug also has been shown to improve diarrhea and mortality rates among infected,malnourished children [26].However, the response rate in malnourished children was only 56%[26]. Unfortunately,nitazoxanide has not been found to be effective in AIDS patients [29].

Nitazoxanide is only FDA approved for patients one year of age or older. Arecent study among hospitalized children in Egypt aged 6 months to 10 yearspresenting with persistent diarrhea compared paromomycin and nitazoxanide with noantiparasitic treatment. [30]. Overall, 86.6% of children treated with 100 or 200 mgof nitazoxanide every twelve hours for three days demonstrated complete clearance ofoocysts and cessation of clinical symptoms. Among children treated with 25 mg/kg/dayof paromomycin for two weeks, 68.8% experienced stool clearance and werecompletely cured. Both treatments were better than no anti-parasitic treatment.

Though AIDS patients are the main immunocompromised population effected byCryptosporidium, the parasite is also problematic in organtransplant recipients as well [31-33]. Bhadauriaet al. conducted a retrospective review of living donor renaltransplant recipients admitted for evaluation of diarrhea [33]. Cryptosporidium was foundto be the most common cause of infectious diarrhea in these patients. Patientsreceiving combination immunosuppression including tacrolimus had higher rates ofinfection with Cryptosporidium compared to a combination ofcyclosporine. Bhadauria and colleagues reported a better response tonitazoxanide/flouroquinolone combination therapy than to nitazoxanide alone[33]. However, theauthors did not test the patients for E. coli enteropathogens.Thus, the benefit of the fluoroquinolone may have been on undiagnosed bacterialco-infection. Other groups have noted that transplant patients responded poorly tonitazoxanide alone. Still, there are anecdotes of better responses to combinationsof nitazoxanide, azithromycin, and/or paromomycin [34-36].

Another population effected by chronic Cryptosporidiuminfections includes patients with primary immunodeficiencies such as hyper-IgMsyndrome. This syndrome is a combined immune deficiency disorder caused by mutationsin CD40 ligand. Infections in these patients are similar to that of AIDS patients,with extra-intestinal manifestations including biliary involvement. Fan etal., showed that treatments with CD40 agonist antibody effectivelyreduced the number of oocytes shed by these patients. Although relapse occurredtreatment was stopped [37].

Physicians initially approached the devastating effects of cryptosporidiosisin AIDS patients by testing a wide range of available drugs. One such drug,paromomycin, was reported to ameliorate cryptosporidiosis in AIDS patients[38]. In neither of thetwo controlled trials in patients with AIDS were the effects more than modest (fewcures and mild decrease in diarrhea) [38, 39]. In onecontrolled trial, there was a statistically significant decrease in oocyst sheddingand diarrhea [40]. WhileHussien et al. demonstrated clearance of cryptosporidiosis amonghospitalized children with paromomycin treatment compared to no treatment. However,the response was significantly less active than with nitazoxanide [30].

Other drugs reported to have some effect in case series includeazithromycin, spiramycin, and bovine anti-cryptosporidium immunoglobulin[22]. However, all wereineffective in controlled trials in AIDS patients. Unfortunately, while all of thesestudies were presented at scientific meetings, none of these trials have beenpublished [22]. Azithromycinseemed to be better than two anthelminthic drugs for cryptosporidiosis in children[41]. It has also beenused in combination with nitazoxanide and/or paromomycin in compromised hosts withdecreased stool frequency and parasite clearance in some patients [34, 42].

Rifamycins have been studied for anti-Cryptosporidium activity. Rifabutin,was tested in vitro and showed a 25% decrease in C.parvum infection in vitro. When combined with nitazoxanide, infectiondecreased by 75% [43]. Holmberg et al. [44] reported an 85% decrease in theincidence of Cryptosporidium in the cohort of AIDS patientsreceiving rifabutin for M. avium complex prophylaxis, compared tothe groups receiving clarithromycin or azithromycin. Similar results were seen whencomparing AIDS patients taking rifabutin, clarithromycin or a combination[45]. A small group ofHIV patients were found to have resolution of diarrhea with rifaximin, anon-absorbed rifamycin [46, 47].

The Way Forward

One of the typical difficulties faced when developing anti-parasitictreatments is lack of drug targets that do not have human hom*ologues. Several keyenzymes have been identified as targets due to significant differences from humanenzymes. Many apicomplexan parasites have unique calcium-dependent protein kinases,including the Cryptosporidium CDPK1, an essential component of cellinvasion [52]. The parasiteslack amino acids blocking access to the active site by “bumped kinaseinhibitors”. Castellanos et al. has identified severalcompounds that bind to this enzyme, inhibiting enzyme function and ultimatelykilling the Cryptosporidium cell. A number of these inhibitors haveexhibited anti-cryptosporidium activity both in vitro as well as inSCID-beige immunocompromised mouse models [53].

Clan CA cysteine proteases have been found to be a potential target for thetreatment of cryptosporidiosis. Clan CA cysteine proteases are thought to be vitalfor host cell invasion and has been found to be structurally different thananalogous enzymes in humans [54]. This protease can be inhibited utilizingN-methyl-piperazine-Phe-hom*oPhe-vinylsulfone phenyl (K11777) inhibits growth ofCryptospordium in vitro and has been shown to rescueimmunocompromised mice from lethal infection [55].

The folate biosynthesis pathway, historically a target for cancer,bacterial, and malarial disease has also been identified as a potential target foranti-cryptosporidials. Cryptosporidium contains a bi-functionalthymidylate synthase/dihydrofolate reductase enzyme. Licensed anti-bacterial andanti-protozoan drugs do not inhibit the cryptosporidium enzyme but, research teamsare evaluating the activity of compounds designed to specifically block this keyenzyme in the folate synthesis pathway in vitro. Published data hasreported striking anti-cryptosporidial activity of these compounds within cellculture [56].

Another potential drug target is oxidoreductase inosine5′-monophosphate dehydrogenase (IMPDH) which is essential in guaninesynthesis in Cryptosporidium. Unlike human IMPDH,CpIMPDH seems to have originated from bacteria via lateral genetransfer thus the enzyme is structurally different than its human counterpart.Current research has found a series of inhibitors for this enzyme [57, 58]. Another study investigated the use ofPhylomer^® peptides to inhibit CpIMPDHfunctions. These studies were conducted in vitro and identified twoout of twelve peptides with anti-cryptosporidium functions [59].

The repurposing of already developed drugs or compounds is another promisingarea of anti-cryptosporidial research. A study done by Bessoff etal. utilized Human HMG-CoA Reductase and Isoprenoid Synthesisinhibitors from the NIH Clinical Collections drug library in a novel cell-basedassay [60]. Results showedthat there may be a surprising amount of overlap between previously FDA approveddrugs and potential anti-cryptosporidal activity [60]. The use of Malaria Box drug-like compoundsis also being investigated. These compounds have been created by the Medicines forMalaria Venture and are freely available to researchers [61]. A preliminary study identified severalpotential anti-cryptosporidiosis candidates however, further invitro and in vivo research has yet to be published[62]. Similarly, theanti-inflammatory drug auranofin demonstrates activity againstCryptosporidium in vitro however, no in vivoor clinical results have been published [63].

The re-evaluation of current oral rehydration therapies is also beingconducted to determine its role in the clearance of Cryptosporidiumin immunocompetent hosts. Castro et al. demonstrated thatsupplementation of L-arginine to infected, undernourished mice aided in weight gainand reduced parasite burden [64]. Similar findings were reported by Costa et al.and Argenzio et al. utilizing the supplementation of anoligodeoxynucleotide with unmethylated CpG motif, alanyl-glutamine, and tumornecrosis factor alpha in in vitro and in vivomodels [65, 66]. These findings indicate that currentrecommended oral rehydration therapies may need to be updated, however no clinicaltrials have been completed to asses these supplements in humans. Further research inthis area could potentially provide a cost effective treatment option formalnourished and immunocompromised patients in resource-limited areas.

Conclusions

Within the past few years, studies have increasingly focused on theimportance of Cryptosporidium as a cause of childhood diarrhea andassociated morbidity and mortality. However, there has been little clinicaladvancement in the treatment of cryptosporidiosis. The study conducted by Hussienet al. concluded that nitazoxanide treatment is superior toparomomycin in children with chronic diarrhea in endemic areas [30]. Successful development of noveldrugs could also aid in decreasing childhood malnutrition. Progress has been slowedby limitation in methods to propagate the organisms in vitro and geneticallymanipulate the organisms. Nevertheless, research focused on anti-cryptosporidialscontinues to make substantial advancements. Several enzymes have been identified aspotential drug targets, including calcium-dependent protein kinases [53], Clan CA cysteine proteases[55], IMPDH[57] and the folatebiosynthesis pathway [56].Other studies are testing compounds repurposed, which were developed for otherindications. However, none of these compounds have made it into clinical trials.

Footnotes

References