{'fraction_non_alphanumeric': 0.03146106736657918, 'fraction_numerical': 0.022537182852143484, 'mean_word_length': 5.341766533510874, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 3, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 1, 'zh_pornsignals.json': 0}}
Cryptosporidium infection in solid organ transplantation

Diarrhea is a common complication in solid organ transplant (SOT) recipients and may be attributed to immunosuppressive drugs or infectious organisms such as bacteria, viruses or parasites. Cryptosporidium usually causes self-limited diarrhea in immunocompetent hosts. Although it is estimated that cryptosporidium is involved in about 12% of cases of infectious diarrhea in developing countries and causes approximately 748000 cases each year in the United States, it is still an under recognized and important cause of infectious diarrhea in SOT recipients. It may run a protracted course with severe diarrhea, fluid and electrolyte depletion and potential for organ failure. Although diagnostic methodologies have improved significantly, allowing for fast and accurate identification of the parasite, treatment of the disease is difficult because antiparasitic drugs have modest activity at best. Current management includes fluid and electrolyte replacement, reduction of immunosuppression and single therapy with Nitazoxanide or combination therapy with Nitazoxanide and other drugs. Future drug and vaccine development may add to the currently poor armamentarium to manage the disease. The current review highlights key epidemiological, diagnostic and management issues in the SOT population.


INTRODUCTION
Cryptosporidium is a parasitic protozoan causing a gastroenteritis syndrome [1] . It is a common intestinal pathogen, not detected by routine ova and parasite evaluation. Because testing for Cryptosporidium is not routinely sought, the infection is often underdiagnosed, posing important epidemiological problems. In immu nocompetent persons, cryptosporidiosis is usually a selflimited disease lasting between just a few days up to 1014 d [1,2] . In immunocompromised patients, clinical presentation can vary from asymptomatic to acute gastroenteritis, chronic diarrhea or even extra intestinal manifestations [1,324] . The parasite binds on the apical surface of the intestinal epithelium fostering its own reproduction and causing direct injury of the epithelial cells and a local inflammatory response, leading to impairment of the absorption and secretory function of the intestine [1,25] . Several Cryptosporidium spp. have been associated with human disease, of which Cryptosporidium parvum (C. parvum) and Cryptosporidium hominis (C. hominis) account for > 90% of the cases [2628] . In this review, we examine the current epidemiology of Cryptosporidium in solid organ transplant (SOT) recipients, review its pathogenesis and clinical manifestations, diagnostic approach, discus sionavailable treatment options and possible future approaches.

EPIDEMIOLOGY
The incidence and prevalence of cryptosporidiosis varies according to socioeconomic status in both developed and developing countries. In the United States, it is estimated that 748000 cases occur every year [29] , but prevalence in patients with diarrhea can be as high as 12% in developing countries. In SOT recipients are largely unknown (Table 1). Cryptosporidiosis is most likely underreported in SOT, with most of the data being confined to case reports and case series, many of them from endemic areas such as Brazil, India and Middle East [3,10,30,31] . In a study from Brazil, Cryptosporidium infections were more common in renal transplant reci pients (35%) and hemodialysis patients (25%) com pared to the control group (17.4%) [30] . Similarly, in a study from Turkey, the prevalence of cryptosporidiosis in kidney transplant recipients was found to be signi ficantly higher than in healthy immunocompetent patients (21.2% vs 3.0%, P = 0.01) [10] . A recent study from India, shows that cryptosporidiosis accounts for the majority of infectious diarrhea (28.5%) in adult transplant recipients [3] . Children and immunocom promised patients are disproportionately affected, especially in developing countries [32] . Between 1.8% and 3.8% of immunocompetent children in childcare settings in the United States, United Kingdom, Spain, and France have been found to be asymptomatic carriers for C. hominis [31,33,34] . This proportion may be underestimated as up to 70% seroprevalence was found in children living in the United StatesMexican border [35] . Bandin et al [8] reported that Cryptosporidium infections were diagnosed in 3.5% of the new pediatric kidney recipients, and was responsible for 18% of the cases of infectious diarrhea over a period of 3 years. This marked heterogeneity in the prevalence of cryptosporidiosis in SOT from different studies (Table  1) is probably the result of different inclusion criteria used in each study, the geographical distribution, the sensitivity and specificity of the diagnostic tests used, type of induction and maintenance immunosuppression regimen [3,11] . Epidemiological studies, animal models and human case reports show that Cryptosporidium is trans mitted from person to person spread via fecaloral route, including sexual transmission and possibly via respiratory secretions [28,3540] . Infectivity depends on the number of oocysts and Cryptosporidium species and subtypes [41,42] . Outbreaks of cryptosporidiosis in developed countries have been described in daycare centers [43,44] in association with animal petting farms [45,46] and recreational water use [47,48] . During the last few decades, several waterborne outbreaks have been reported after ingestion of contaminated recreational water or drinking water, one of these was thought to affect more than 400000 people [4958] . Risk factors in SOT recipients reported in the literature are described in Table 2. Cryptosporidium oocysts are resistant to chlorine disinfection and can survive for days in treated recreational water despite adequate chlorination [36,59] . Cryptosporidium can be eliminated by boiling the water or just heating it to 62 ℃ for few seconds and by filtration through < 1 µm filters [40] . Transmission of cryptosporidiosis via respiratory secretions is less common; isolation of Cryptosporidium DNA in the sputum of children with intestinal cryptosporidiosis and cough supports the respiratory route of transmission of this organisms [60] . Even more, all of the life stages of Cryptosporidium have been described in the microvillus border of epithelial cells and within the bronchial mucus glands [61] . Cryptosporidiosis has also been reported as a donorderived infection after intestinal trans plantation [14] .

VIRULENCE IMMUNOPATHOGENICITY
The severity and duration of illness (from asymptomatic shedding of oocyts to severe lifethreatening disease) depends on the infecting species, virulence of the parasite and the host immune response (the degree of the immunodeficiency that impacts mainly T cell function), and the incubation period can range from 2 d up to 2 wk [1,2] . Cryptosporidium significantly affects intestinal cells with consequent alterations in absorptive and secretory functions. This may be either caused by direct cell injury or alternatively by activation of the immune system with release of proinflammatory cytokines [1] . Tolllike receptors (TLR2 and TLR4) play an important part in initiating immune activation following mucosal injury by the parasite [6264] and inducing cytokine release (IL12, IL15, IL18, TNFα and IFNα/β) followed by activation of the NFkB cells with IFNγ production, mononuclear cell infiltration in the lamina propria, crypt cell hyperplasia, villous atrophy and blunting [6567] . Toll like receptors also have a role in establishing immunity to infection [62] . Innate immunity controls infection, but elimination of the parasite seems to require adaptive immunity [62] . IFNγ is an important cytokine determining CD4 + T cell response to infection, including memory response against Cryptosporidium infection in the intestine [62,68,69] [17] 1160 Yes (1) 1 Hong et al [9] 1 NA 7 (NA) Kidney TAC + MMF + S N, V, D Yes No Krause et al [4] 6 NA 3.7 (1.1-6.6) Kidney (4)   . Those on tacrolimus who developed cryptosporidium also had graft dysfunction, likely due to dehydration and increased tacrolimus levels [3] .

CLINICAL PRESENTATION
Most of the Cryptosporidium infections in the SOT population have been reported in renal transplant recipients (Table 1). Cryptosporidium can cause asymp tomatic infection in transplant recipients and because of that, many cases may be missed [30,71] . A relatively high prevalence of oocyst excretion in asymptomatic transplant population might be detected in the stool with random stool screening [71] . When clinically evident, patients with AIDS and CD4 count < 50 cells/mm 3 , and improvement of the symptoms after introduction of highly active antiretroviral therapy [70] (Change reference for more recent one) or after decreasing immunosuppression in transplant recipients that allows recovery of the immune system. Antibodies have a minor role in elimination of the infection, being more an indirect marker of the cellular immune response [68] . All these changes at the level of the epithelium lead to malabsorption and secretory diarrhea [12,65] .
In SOT the type of immunosuppression might play an important role in development of cryptosoridiosis. A recent study showed that patients on a tacrolimus based immunosuppressive regimen had a significantly higher risk of Cryptosporidium infection compared to the patients on a cyclosporinebased regimen. Being on

Diagnosis Co-infection Tacrolimus levels (early on admission)
Abdo et al [15] N/A C. parvum N/A No No Acikgoz et al [23] Petting animals N/A ELISA Modified acid fast staining

No Increased
Arslan et al [10] N/A N/A Modified acid fast staining N/A N/A Bandin et al [8] Swimming pool (3) Traveler diarrhea (1) [3] N/A N/A Modified acid fast staining CMV (8) Increased Bonatti et al [5] Travel (water exposure) (4) 1 Camping (1) [18] N/A N/A N/A No N/A Chieffi et al [30] N/A C. parvum Carbol-fuchsin staining N/A N/A Clifford et al [21] Public water supply N/A N/A No No Delis et al [16] N/A N/A Microscopy Biopsy

No Increased
Franco et al [100] N/A N/A Gastric and small bowel biopsies and hematoxillin staining [17] N Hong et al [9] Swimming pool N/A Modified acid-fast staining DFA

N/A Increased
Krause et al [4] None N/A Immunochromatographic test No Increased (5/6) Ok et al [19] N/A N/A N/A Blastomycsis hominis, Giardia intestinalis, Dientamoeba fragilis, Entamoeba coli Pozio et al [14] Allograft C. hominis Microscopy No N/A N/A C. parvum Biopsy Rodríguez Ferrero et al [7] N/A N/A Modified Kinyoun stain No No Tran et al [12] N/A N/A Modified acid fast staining Microscopy Biopsy

No No
Udgiri et al [13] N/A N/A Modified acid fast stain Giardia spp. (7) 1 Entamoeba butschili (1) 1 N/A Vajro et al [24] N/A N/A Monoclonal antibody fluoresceinconjugated stain No NA Ziring et al [11] Nosocomial (1)   SOT recipients typically present with profuse and prolonged watery diarrhea, sometimes associated with nausea, vomiting, abdominal pain and fever [1,410,1224] . Other nonspecific symptoms have been described in immunocompetent and immunocompromised pa tients such as malaise, generalized weakness, myalgia, anorexia and headache [1,5,17] . Persistent vomiting and diarrhea can lead to dehydration and wasting and have been associated with increased morbidity [4,7,8,17] . Several study described acute renal failure, most likely secondary to dehydration, hypotension and sometimes tacrolimus toxicity [35,79,16,23] . Atypical manifestations such as respiratory tract disease, pancreatitis, cholan gitis and urinary tract infection, have been reported in patients with immune deficiencies, mainly AIDS [7275] . Biliary involvement with Cryptosporidium inducing scl erosing cholangitis has been reported in few SOT reci pients [12,15,18] . However, elevated liver enzymes should not be equivalent to the diagnosis of sclerosing cholangitis as they can be abnormal in the settings of hypotension or high tacrolimus levels [11] . Radiologic findings in support of the diagnosis of sclerosing cholangitis: Abdominal ultrasound can show dilation of the biliary duct; Techne tium 99m iminodiacetic scan might show biliary stasis, irregularity of the biliary ducts, focal strictures [18] ; endos copic retrograde cholangiography or magnetic resonance cholangiopancreatography could demonstrate dilation and/or irregularity of the biliary ducts [15,76] . Infection of the biliary tree in immunocompromised patients could represent an extraintestinal reservoir that would allow the organism to avoid certain antiparasitic agents (paromomycin) and would lead to relapses. Drugs with biliary excretion such as nitazoxanide should be preferred in these patients [2,77] . Relapse rates in cryptosporidiosis are high (up to 40%60%) due to incomplete eradication of the oocysts, especially from the biliary tree and possibly due to inadequate intestinal drug levels in patients with severe diarrhea [12,14] . Respira tory cryptosporidiosis can present as an upper or lower respiratory tract infection manifested by nasal discharge, voice change, cough, dyspnea and hypoxemia [7881] .

DIAGNOSIS
Stool microscopy is the main and cheapest method for diagnosis, however all microscopic methods are labor intensive and have low sensitivity unless a high concentration of oocysts are being released in stool. The size of the oocysts is also important (between 37 µm) as they can be confounded with yeast, so modified staining with ZiehlNeelsen or fluorescent techniques such as auraminerhodamine can be employed to improve detection. The sensitivity of these stains still remains low [82,83] , requiring about 500000 oocysts/mL in formed stools for detection [35] . The most commonly used test by microbiology laboratories is currently direct immunofluorescence which may be either a standalone test or a combined Cryptosporidium/Giardia diagnostic kit [35] . There are several enzyme linked immunosorbent assay (ELISA) kits available with sensitivities ranging from 66%-100% but excellent specificity and have the advantage of being more automated when compared to conventional staining methods [41,8489] . Immunochro matographic tests have lower sensitivity compared to other molecular or other antigen tests and are not as sensitive to detect species other than C. parvum or C. hominis but are easy to perform, correlate well with EIA/ELISA tests and provide results in a matter of minutes [89,90] . Molecular methods provide the highest diagnostic sensitivity and are the preferred methods for diagnosis given their superior sensitivity and specificity. There are several multiplex polymerase chain reaction (PCR) test that can detect different gastrointestinal pathogens including viruses, parasites and bacteria however, these may not available in all laboratories [91] . These tests usually have high sensitivity to detect Crypto sporidium, although speciation may require further testing and carry a higher cost [26,41,42,9294] . Tissue histopathology is a useful method for diag nosis, especially when intestinal biopsies are obtained. Parasites may appear lining epithelial surfaces or in the lumen. When hematoxylin is used to stain the tissue, intracellular parasites appear blue or purple [2,16,17,20] .
Intestinal transplant recipients may have negative stool examinations but the parasite may be readily seen on graft biopsies, highlighting the importance of endoscopic examination even in cases where diarrhea persists and routine stool examinations are negative [11,16,17] . Detection of Cryptosporidium in respiratory sample specimens is usually achieved with acidfast, modified acid-fast staining or and indirect immunofluorescence [28,74] although PCR testing may also be possible [28] . Histopa thology may show parasites lining the mucosal epithelium of trachea, bronchi or lung; tissue biopsies may also show intra or extracellular organisms [28] .

TREATMENT
The main treatment approach is oral rehydration when ever possible, however intravenous fluids that include sodium, potassium, glucose and bicarbonate may be required in severe cases. A lactose free diet is recom mended since Cryptosporidium destroys mature epithe lial cells that are located in the villi resulting in loss of enzymes such as lactase. The disease is associated with high intestinal transit that may interfere with fluid, electrolyte, and drug absorption. Antimotility agents may be used once other causes of diarrhea such as Clostridium difficile or dysentery are ruledout.
The first step in SOT patients is an attempt to restore immune function by adjusting or switching immu nosuppressive therapy, because severity of disease is likely related to the degree of immunosuppression and CD4 cell counts [3,10,13,19,37,74,82,95] . This example was illustrated in a renal transplant recipient with enteritis and sclerosing cholangitis, where an accidental redu ction of immunosuppression resulted in clearance of the disease [15] . Mycophenolate, a commonly used Florescu DF et al . Cryptosporidium in transplantation immunosuppressive agent may have some antiparasitic activity against Cryptosporidium by inhibiting folate metabolism [4] . Cryptosporidium induced diarrhea may also result in increased tacrolimus levels [37] as evidenced in two recently published case series [4,5] . The pathophysiology is not entirely clear but it is likely a combination of factors including reduced cytochrome 3A activity during inflammation [96] , interaction with other drugs, and reduced renal function due to fluid depletion [4] . Increased tacrolimus may in turn worsen renal function, and prolong immunosuppression [3] . Chole cystectomy may be indicated for cases with acalculous cholecystitis and sclerosing cholangitis usually needs endoscopic retrograde pancreatography with possible papillotomy and stenting [97] . To date, there has not been a highly effective agent to treat cryptosporidiosis in immunocompromised individuals [98] . A metaanalysis of seven trials including 130 patients with AIDS found no evidence for effective agents against cryptosporidiosis, although significant heterogeneity and flaws of individual trials may have influenced the negative results [95] . Moreover, whether any drug may have partial effect or the use of combination therapy were not investigated in this metaanalysis. To date, no randomized clinical trial with antiparasitic drugs has been conducted in SOT recipients with cryptosporidiosis and most experience is extrapolated either from data in immunocompetent hosts, patients with human immunodeficiency virus (HIV) infection [37] or case series and case reports (Table   3) [319, 21,23,24,30,99,100] . Several antiparasitic drugs such as paromomycin, nitazoxanide or azithromycin have been used with variable success. Nitazoxanide is the only FDA approved drug for treatment of cryptosporidiosis, it is available in tablets and suspension , it has no significant drugdrug interactions or dosing requirements in renal or hepatic failure [98] . Its activity, including the one of its metabolites has previously been shown in vitro [101] and it is believed to interfere with the pyruvate: Ferredoxin oxidoreductase enzymedependent electron transfer reaction, which is essential to anaerobic energy metabolism [102] . Nitazoxanide has been effective in 3 randomized clinical trials among immunocompetent adults and children, showing reduction in duration of diarrhea and eradication of cysts from stool [103,104] . Its effectiveness in immunocompromised patients has been variable with some clinical trials showing positive results whereas in other trials the drug was no better than placebo. In a randomized study of nitazoxanide in HIV infected patients with cryptosporidiosis treated with either 500 mg twice a day or 1 g twice a day vs placebo, good responses to nitazoxanide were seen in those with CD4 cell counts > 50/mm 3 although no difference to placebo was seen in the subgroup with CD4 < 50/ mm 3 [105] . Nitazoxanide effectiveness was also questioned in a randomized doubleblind trial in children with HIV infection who received the drug for 28 d and there was no difference with placebo for clinical and parasitological cure or mortality [106] . One difference with patients with HIV infection when compared to SOT recipients is in many cases the ability to more readily manage and adjust immunosuppression, whereas in HIV infection restoration of the immune system with antiretroviral therapy is key and may take longer time [98] . The recom mended nitazoxanide dose in SOT recipients is 500 mg twice daily for 14 d [37] , however data from randomized trials in SOT recipients is lacking and longer courses of therapy are sometimes employed [3,4,8] .
Paromomycin, a nonabsorbable aminoglycoside has limited activity against the parasite, probably the doses used in clinical practice are not enough to achieve the high concentrations needed to inhibit parasitic activity [97] . It was useful reducing oocyst excretion in a small clinical trial [107] . Because paromomycin has not been shown to be useful as a standalone agent, combination therapy with other antiparasitic agents such as azithromycin and Nitazoxanide may be an attractive option [5,7,9,11,14,16,23,108] . Macrolide antibiotics such as azithromycin, clarith romycin or spiramycin also have activity against crypto sporidium [98] , and were shown to reduce duration of symptoms and oocyst shedding in a clinical trial of treat ment of children with cryptosporidiosis [109] , but these findings were not replicated on a subsequent rando mized trial [110] . Several clinical trials and case series evaluating the use of azithromycin in immunocompetent and immunocompromised patients with cancer and also HIV infection have shown mixed results in clinical response including duration of symptoms, and oocyst shedding [110114] . Several case reports and case series have described successful use of spiramycin and azithromycin either alone, or in combination therapy with paromomycin or Nitazoxanide in SOT patients [5,7,9,11,13,14,1618,23] . Drug drug interactions between macrolides and immuno suppressive agents such as tacrolimus or cyclosporine should be considered before treatment is initiated and may further limit prolonged use of these antibiotics [99] . Rifamycins also have antiparasitic activity. Rifabutin was shown to decrease cell infection by Cryptospori dium [115] and rifaximin has also been shown to be active in vitro [98] . Interestingly, the incidence of cryptosporidiosis was dramatically decreased in patients with advanced HIV infection who used rifabutin as part of Mycobacterium avium chemoprophylaxis [116,117] . To date, there have been no randomized clinical trials to evaluate its efficacy in SOT recipients or other immunocompromised hosts. Drug drug interactions with rifabutin may also be an important issue in those who take tacrolimus or cyclosporine [15,99] . Tacrolimus levels are not affected by rifaximin, however an elevation of rifaximin levels may be seen as a result of Pglycoprotein inhibition.
Because individual drugs lack full activity against the parasite, use of combination therapy may be a more attractive option. Current guidelines recommend starting with nitazoxanide alone as preferred therapy, although combination therapy is listed as an alternative option [37] . Our review of the literature showed some authors have used nitazoxanide as standard therapy, while others have used this approach in relapsed or refractory cases, Florescu DF et al . Cryptosporidium in transplantation usually with long courses advocated [35,8,9,23] . Azithromycin has been combined with either nitazoxanide or paromo mycin also with reported success [5,82,115,118] . Caution should be exercised though, because large studies using combination therapy including nitazoxanide have not been carried out to date. Current data on combination therapy is derived from case reports and case series, which may only reflect positive outcomes, while negative results may not be necessarily reported.

PREVENTION
Transplant recipients should be cautious about swimming in streams or lakes and if possible avoid untreated well or lake water. Drinking water should either be treated municipal, filtered by < 1 µm filters or bottled water. Contact with anyone who has diarrhea should be limited, (food and water may be contaminated by those infected) and handwashing for everyone, especially all household members is strongly encouraged. Moreover, all surfaces should be cleaned with running water and soap [37,119] . Safe sexual practice using condoms is also encouraged for anal intercourse, since it increases the risk of trans mission as well [119] .

PERSPECTIVE
Oral bovine immunoglobulin (hyperimmune colostrum) seemed an attractive alternative for treatment although it has not been effective at conventional doses and at higher doses oocyst excretion was decreased but diarrhea increased and clinical symptoms were not reduced [120] . More recently, monoclonal or polyclonal antibodies have shown to reduce oocyst shedding

MMF and TAC reduced Resolved No No
Tran et al [12] PAR (4 wk) Sirolimus discontinued Resolved No No Udgiri et al [13] Spiramycin (10 d) (2) 1 No Resolved No No Vajro et al [24] None No Resolved No No Ziring et al [11] PAR + AZM N/A Resolved No No and improve clinical symptoms [121] . Thus, although still controversial, using oral bovine immunoglobulin or monoclonal antibodies along with antiparasitic agents may be a strategy to consider in immunocompromised individuals with recurrent or recalcitrant disease [121] . The genome of both C. parvum and C. hominis has been decoded and this should also help develop antiparasitic drugs against specific targets such as calciumdependent protein kinases, microtubule forma tion inhibitors, hexokinase, lactate dehydrogenase, inosine5monophosphate dehydrogenase, and fatty acylCoA binding inhibitors among others [82,122] . Despite this, the full understanding of Crypto sporidium immunopathogenesis remains unclear [35,68] . Declines in infection rates with increasing age among children in developing countries points to possible acquisition of immunity against the parasite, although immune responses that may lead to protective immunity are not well understood [35,82] . A study conducted in healthy volunteers who were challenged with Crypto sporidium, showed that after second rechallenge episodes of diarrhea were similar but clinical severity was milder and fewer subjects were shedding oocysts [123] . Both IgG and IgA antibodies increased after exposure, however there was no correlation with infection [123] . Vaccination may be a viable alternative and vaccine has been evaluated in a mouse model [124] . The two most common species causing human disease, C. parvum and C. hominis share > 95% of their genome so it may be possible to have one vaccine for both species (Mead 2015). Several parasitic antigens such as gp15 and gp40 have been evaluated in vaccine development. Both elicit an immune response and production of interferon gamma by mononuclear cells in patients previously infected with cryptosporidium. A vaccine trial in Bangladesh using IgA against gp15 showed the antibody was not species specific and resulted in shorter duration of illness [82] . There are other targets being investigated including a recombinant DNA vaccine using Vaccinia, Salmonella or Lactobacillus as DNA vectors [82] . A successful vaccine would first have to be proven effective in immunocompetent hosts before moving on to immunocompromised patients, although the latter are the ones who would most likely benefit from vaccination.

CONCLUSION
Diarrhea caused by Cryptosporidium is a serious clinical syndrome in SOT recipients and diagnosis may be delayed if the infection is not routinely suspected or investigated. Advances in diagnostic methodologies has improved the sensitivity of detection, however, treatment remains problematic, especially in immunocompromised patients. Aggressive fluid and electrolyte replacement, reduction in immunosuppression along with antiparasitic therapy are the mainstay of therapy, but few partially active drugs are available and the infection may follow a protracted course with many relapses. Combination therapy with nitaxoxanide and paromomycin or macro lides may be the best approach, especially in SOT recipients. New therapies in the horizon such as hyperi mmune colostrum, monoclonal antibodies, and vacci nation may help increase the armamentarium to manage the disease.