Thus far, nitazoxanide is the only drug approved by the United States Food and Drug Administration (FDA) for the treatment of cryptosporidiosis in immunocompetent human patients (Checkley et al., 2015). Nitazoxanide is a member of the thiazole class of drugs that was initially developed as a veterinary anthelmintic but was later reported to have broad-spectrum activity against parasites, viruses, and bacteria. This drug acts by inhibiting the pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR), an enzyme essential for the anaerobic energy metabolism of various microorganisms (Hoffman et al., 2007). However, the exact mechanism of action against Cryptosporidium remains questionable since these parasites encode a unique PFOR with a fused C-terminal cytochrome P450 domain (Rotte et al., 2001). Interestingly, nitazoxanide was shown to inhibit the growth of C. parvum by more than 90% at a concentration of 10 µg/ml (32 µM) in cell culture but was ineffective in the anti-IFN-γ-conditioned SCID mouse model of cryptosporidiosis even at high doses (Theodos et al., 1998). Furthermore, nitazoxanide has also been found to be ineffective in other immunodeficient or immunocompromised animal models of cryptosporidiosis, questioning the true efficacy of the drug (Lee et al., 2017; Jumani et al., 2018).

Various randomized placebo-controlled studies have found nitazoxanide to be helpful in treating cryptosporidiosis in adults and children without HIV resulting in reduced duration of both diarrhea and oocyst shedding (Rossignol et al., 2001; Amadi et al., 2002; Rossignol et al., 2006; Hussien et al., 2013; Abaza et al., 2016). Studies conducted in Egyptian immunocompetent adults and children demonstrated significantly higher clinical and parasitological cure rates compared with the placebo-treated groups (Rossignol et al., 2001; Rossignol et al., 2006). In a randomized controlled trial involving malnourished children in Zambia, nitazoxanide treatment for 3 days yielded a partial but significantly better cure than placebo (Amadi et al., 2002). Recent controlled trials have also reported complete clinical and parasitological recovery in most immunocompetent children with cryptosporidiosis (Hussien et al., 2013; Abaza et al., 2016).

However, a meta-analysis of seven randomized controlled trials involving 169 participants with cryptosporidiosis confirmed the absence of obvious evidence of efficacy of nitazoxanide in HIV-seropositive patients (Abubakar et al., 2007). A course of nitazoxanide does not appear to improve the resolution of diarrhea and parasitological outcome in HIV-infected and immunocompromised patients (Doumbo et al., 1997; Rossignol et al., 1998; Amadi et al., 2002; Zulu et al., 2005; Amadi et al., 2009; Abaza et al., 2016). In the first randomized controlled trial of this drug in adult HIV patients with cryptosporidiosis, better overall parasite clearance rates were seen in the treated group compared with the placebo one, but significant differences were only seen in those with CD4+ T-cell counts above 50/mm³ (Rossignol et al., 1998). Other randomized placebo-controlled trials conducted by Amadi and others in HIV-positive children in Zambia have also documented no beneficial effect of nitazoxanide over placebo in terms of clinical and parasitological cure rates or mortality (Amadi et al., 2002; Amadi et al., 2009). Moreover, only moderate efficacy was achieved in a study of cryptosporidiosis in immunocompromised children even after prolonged nitazoxanide treatment of up to 28 days (Abaza et al., 2016). While prolonged therapy with higher doses of the drug is somewhat effective in treating cryptosporidiosis in patients with compromised immunity, normal prescribed doses and short-term duration of therapy are inadequate for preventing recurrence of disease symptoms after treatment discontinuation (Abraham et al., 2008; Krause et al., 2012; Ali et al., 2014; Bhadauria et al., 2015; Demonchy et al., 2021). Therefore, it is evident that nitazoxanide therapy is clearly futile in treating cryptosporidiosis in advanced AIDS patients and other severely immunocompromised patients.

Lack of efficacy in immunocompromised animal models and humans suggests that a healthy host immune system is essential to the effectiveness of nitazoxanide. Nitazoxanide treatment has been recently shown to result in broad amplification of the host cell innate immune response to viral infections, including an increase in interferon activities (Jasenosky et al., 2019). If an immune defect renders the host incapable of generating an interferon-γ-dependent response, nitazoxanide would be expected to be ineffective in such immunocompromised hosts (Theodos et al., 1998; Jumani et al., 2018), given the importance of these innate responses in controlling Cryptosporidium at its initial stages of infection (McDonald et al., 2013). Similarly, lack of curative effect of nitazoxanide in advanced AIDS patients (with low CD4+ T-cell counts) suffering from chronic cryptosporidiosis can be explained by the fact that adaptive immunity plays a crucial role in clearing the parasites completely from the infected host (Mead, 2014). Thus, the efficacy of nitazoxanide seems to be closely related to both the innate and adaptive immune status of the host.

Another well studied drug, a poorly absorbed aminoglycoside paromomycin, has been investigated against cryptosporidiosis in three published controlled trials (White et al., 1994; Hewitt et al., 2000; Hussien et al., 2013), but results have been highly divergent and mostly discouraging. Paromomycin, like other aminoglycoside antibiotics, inhibits protein synthesis by binding to the 30S ribosomal subunit and shows broad spectrum activity against bacteria and some protozoa (Lin et al., 2018). Several uncontrolled trials and case studies in AIDS patients suffering from cryptosporidiosis have reported favorable clinical outcomes after paromomycin treatment (Gathe et al., 1990; Clezy et al., 1991; Armitage et al., 1992; Danziger et al., 1993; Fichtenbaum et al., 1993; Wallace et al., 1993; Bissuel et al., 1994; Scaglia et al., 1994; Hashmey et al., 1997). However, the patient responses in most cases were short-lived and continuous maintenance therapy was required to prevent frequent relapses after treatment discontinuation suggesting that complete parasitological cure was not achieved in these cases. Children suffering from cryptosporidiosis have been reported to respond favorably after treatment with paromomycin, although the overall clinical and parasitological response is reduced as compared to nitazoxanide or azithromycin (Trad et al., 2003; Vandenberg et al., 2012; Hussien et al., 2013).

Macrolides are a class of antibiotics that disrupt bacterial protein synthesis by binding to the 50S subunit of the ribosome (Lin et al., 2018). Among the macrolides tested for efficacy against human cryptosporidiosis, azithromycin remains the most studied. In a multi-center, placebo-controlled, double-blind study, preliminary data analysis revealed no significant improvement in clinical symptoms and oocyst numbers in azithromycin-treated AIDS patients with cryptosporidiosis (Soave et al., 1993). Interestingly, however, a statistically significant decrease in cryptosporidial oocyst shedding was reported in patients with appropriate azithromycin serum concentrations (Soave et al., 1993). By contrast, azithromycin was found to have no therapeutic or prophylactic efficacy in the management of cryptosporidial diarrhea in AIDS patients (Blanshard et al., 1997; Holmberg et al., 1998). While short-term azithromycin treatment for cryptosporidiosis was unable to achieve total parasitological clearance and prevent relapses in AIDS patients, long-term and low dose maintenance therapy was associated with noticeable clinical and parasitological benefits (Dionisio et al., 1998; Kadappu et al., 2002). Nevertheless, azithromycin seems to be more effective in treating children with cryptosporidiosis. Prompt clinical improvement and high parasite clearance rates have been reported after azithromycin therapy in both immunocompetent and immunocompromised children (Vargas et al., 1993; Hicks et al., 1996; Allam and Shehab, 2002; Trad et al., 2003).

Clarithromycin has been tested in humans with AIDS for prophylactic effectiveness against cryptosporidiosis. But studies have reported conflicting results with some indicating a highly protective effect against the development of cryptosporidiosis (Jordan, 1996; Holmberg et al., 1998) while others concluding that the drug is not useful in preventing cryptosporidiosis in this patient population (Fichtenbaum et al., 2000). Similarly, spiramycin has also shown inconsistent results in treating cryptosporidiosis in controlled and uncontrolled trials involving infants (Saez-Llorens et al., 1989; Wittenberg et al., 1989) and adult patients (Woolf et al., 1987; Moskovitz et al., 1988) with reports of acute intestinal injury in some patients receiving high doses of the drug (Weikel et al., 1991). Another macrolide, roxithromycin has proven effective in uncontrolled studies of AIDS patients with cryptosporidial enteritis. However, complete parasite clearance was only achieved in half of the treated patients and the results were not compared with a placebo-treated control group (Sprinz et al., 1998; Uip et al., 1998).

Rifamycins are a group of drugs that are highly active against mycobacterial infections. Members of this antibiotic class inhibit RNA synthesis by selective binding of the bacterial DNA-dependent RNA polymerase (Hartmann et al., 1967). Several uncontrolled studies have evaluated rifamycin derivatives, namely rifabutin and rifaximin, for prophylactic and therapeutic efficacy respectively, against cryptosporidiosis in HIV-infected humans. Rifabutin has been found to be highly effective in preventing the development of cryptosporidiosis in AIDS patients receiving chemoprophylaxis for Mycobacterium avium complex infection (Holmberg et al., 1998; Fichtenbaum et al., 2000). Similarly, some studies have demonstrated a significant clinical and parasitological benefit of rifaximin, a poorly absorbed rifamycin, in the treatment of cryptosporidiosis in solid organ transplant recipient patients (Burdese et al., 2005) and a small number of HIV-infected adults and children with CD4+ T-cell counts ranging from <50 cells/mm³ to >200 cells/mm³ (Amenta et al., 1999; Gathe et al., 2008). Thus, these results warrant the testing of these drugs in larger randomized controlled clinical trials for confirmation of anti-Cryptosporidium efficacy.

Diclazuril and letrazuril have been both shown to be active against Eimeria species, parasites closely related to Cryptosporidium, although the exact mode of action of these drugs is currently unknown. Yet, diclazuril failed to show any obvious effect in severe cryptosporidiosis in adults with HIV infection (Connolly et al., 1990). In another study, a single patient showed both clinical and parasitological response to diclazuril treatment but the infection was less severe and the patient also received antiretroviral therapy during and after the treatment course (Menichetti et al., 1991). Additionally, letrazuril, the p-fluor analog of diclazuril, shows only partial efficacy against advanced AIDS-related cryptosporidial diarrhea (Harris et al., 1994; Loeb et al., 1995; Blanshard et al., 1997) and treated patients develop temporary drug-related side-effects including abnormal liver function tests and skin rashes that tend to resolve after treatment discontinuation (Murdoch et al., 1993; Harris et al., 1994; Loeb et al., 1995).

Although most studies on the treatment of cryptosporidiosis have been carried out by repurposing the use of antibacterial drugs, some studies have tested other treatments with alternate modes of action such as immune cell extracts, immunoglobulins, hyperimmune colostrum, somatostatin analogs, and highly active antiretroviral therapy (HAART) ( Table 2 ).

Several combination therapies involving the use of either nitazoxanide or paromomycin in conjunction with macrolides, rifamycin derivatives, or HAART have shown promising efficacy for cryptosporidiosis in small uncontrolled trials and case studies with both clinical improvement and parasite elimination in a range of affected immunocompromised individuals ( Table 3 ). However, these results need to be replicated in large, controlled trials before any definite conclusions can be drawn regarding the efficacy of such combinations. Huang and colleagues conducted a randomized placebo-controlled trial to investigate the therapeutic effects of acetylated spiramycin and garlicin on Cryptosporidium infection in institutionalized drug users. Although the combination treatment achieved high parasitological cure rates, this study was carried out in asymptomatic Cryptosporidium carriers without ascertaining the HIV/immune status of the enrolled individuals, and therefore has limited clinical significance (Huang et al., 2015).

Certain clinical case reports have documented favorable clinical and parasitological outcomes in HIV-infected and organ transplant recipient patients diagnosed with extra-intestinal and intestinal cryptosporidiosis, after antimicrobial combination therapy with azithromycin and paromomycin (Palmieri et al., 2005; Meamar et al., 2006; Denkinger et al., 2008). In a small open-label, uncontrolled study, patients with AIDS (<100 CD4+ T-cells/mm³) and chronic cryptosporidiosis, showed a marked improvement in stool frequency and a significant decrease in fecal excretion of Cryptosporidium oocysts in response to azithromycin/paromomycin combination therapy (Smith et al., 1998). However, follow-up study after the completion of treatment revealed the persistence of chronic, mild diarrhea in some patients.

Complete resolution of diarrhea as well as elimination of the parasite has been reported in immunosuppressed children and adults suffering from cryptosporidiosis after dual therapy with azithromycin and nitazoxanide (Legrand et al., 2011; Bakliwal et al., 2021; Dupuy et al., 2021). Additionally, triple therapy involving azithromycin, nitazoxanide, and paromomycin or rifaximin led to complete clinical and parasitological cure with no relapse in renal transplant patients (Hong et al., 2007; Tomczak et al., 2022). Another study successfully treated cryptosporidiosis in a pediatric renal transplant patient using a triple therapy consisting of spiramycin, nitazoxanide, and paromomycin (Acikgoz et al., 2012).

Moreover, quite a few small uncontrolled studies suggest that a combination of antimicrobials and HAART (especially with protease inhibitors) dramatically accelerates the clinical response in AIDS patients suffering from cryptosporidiosis (Maggi et al., 2000; Schmidt et al., 2001; Moling et al., 2005). But data from randomized controlled trials is required to support these results given the self-limiting nature of the disease. Nevertheless, increasing evidence has demonstrated that combination therapy achieves better clinical and microbiological resolution rates than monotherapy for the treatment of cryptosporidiosis in immunocompromised patients (Maggi et al., 2000; Bhadauria et al., 2015; Lanternier et al., 2017; Bakliwal et al., 2021; Tomczak et al., 2022).

In addition to humans, paromomycin has also been extensively tested for anti-Cryptosporidium efficacy in various food animals. However, as has been the case in humans, results have been varied and the treatment failed to achieve complete parasitological cure in most studies. Prophylactic administration of paromomycin was found to decrease the duration and severity of diarrhea as well as the duration and intensity of oocyst shedding in calves experimentally infected with C. parvum (Fayer and Ellis, 1993). Similar positive results were reported in a controlled-blind field trial of natural infection in calves, but the treated group started shedding oocysts and developed diarrhea after the treatment withdrawal (Grinberg et al., 2002). However, paromomycin does seem to be more effective in small ruminants. Treatment has been shown to reduce both cryptosporidial oocyst output and severity of clinical signs, when used prophylactically in neonatal goat kids (Mancassola et al., 1995; Chartier et al., 1996; Johnson et al., 2000) and therapeutically in neonatal lambs (Viu et al., 2000). This agent was also proven to be therapeutically effective against moderate cryptosporidiosis but ineffective against severe cryptosporidiosis in infected gnotobiotic piglets (Tzipori et al., 1994; Theodos et al., 1998). However, paromomycin, like many aminoglycosides, is potentially nephrotoxic and detrimental effects on growth have been observed after treatment in young animals (Viu et al., 2000). In addition, the drug is expensive and therefore, its use in agricultural animals is impractical.

Nitazoxanide, the only licensed treatment available in humans, has also been tested for efficacy in animal cryptosporidiosis, although reports on treatment outcomes have been conflicting. While Ollivett and others found this medication to significantly reduce the duration of oocyst shedding and clinical severity in experimentally infected calves as compared to the placebo treated group (Ollivett et al., 2009), another controlled study found no prophylactic or therapeutic effect of nitazoxanide on clinical appearance or oocyst excretion in calves infected with C. parvum (Schnyder et al., 2009). Furthermore, while nitazoxanide reduced oocyst shedding in experimentally challenged newborn goat kids, no reduction in mortality rates or improvement in weight gains were recorded in the treated groups compared with the control group (Viel et al., 2007). Importantly, the authors of this study suggested that the mortalities seen in kid neonates in the nitazoxanide treated groups were caused by severe drug toxicity (Viel et al., 2007). In the gnotobiotic piglet diarrhea model, nitazoxanide demonstrated only partial efficacy at high doses in reducing C. parvum oocyst shedding, induced drug-related diarrhea, and was not as effective as paromomycin (Theodos et al., 1998). In another study performed in the same animal model but infected with C. hominis, nitazoxanide reduced diarrhea and oocyst shedding in only the initial phase of treatment and had no clinical or parasitological effect at the later stages of the disease (Lee et al., 2017).

Macrolides have been evaluated as anti-Cryptosporidium agents in a range of animals. Azithromycin significantly suppressed Cryptosporidium oocyst shedding and resulted in significant clinical improvement and weight gain in naturally infected dairy calves when used as a therapeutic at high doses, but high costs of treatment are a concern (Elitok et al., 2005). Similar reports of azithromycin efficacy against C. parvum infection in calves (Nasir et al., 2013) and a buffalo calf (Maurya et al., 2016) have also been published. Treatment of gnotobiotic neonatal piglets infected with C. hominis alleviated clinical disease only for the first few days and azithromycin treated piglets exhibited no reduction of oocyst excretion compared with untreated animals (Lee et al., 2017). In combination with nitazoxanide, azithromycin led to significant clinical improvement in infected piglets but did not eliminate oocyst excretion after producing a transient initial reduction in oocyst shedding in treated animals (Lee et al., 2017).

Experience with other macrolides has also been mixed. While tilmicosin failed to prevent severe cryptosporidiosis in newborn kids raised on a commercial dairy goat farm (Paraud et al., 2010), tylosin was found to be therapeutically effective in reducing fecal oocyst excretion and clinical signs of disease in naturally infected calves (Duru et al., 2013).