**2. Clinical perspective, diagnosis, and treatment**

Despite *Cryptosporidium* species infecting the epithelial cells of the gastrointestinal and respiratory epithelium tracts, human cryptosporidiosis is a usually self-limiting infection in immunocompetent individuals with a low fatality rate [39–41]. In general, onset of the symptoms occurs 5–7 days following exposure and resolves in 2–3 weeks [42]. Clinical manifestations vary from subclinical infection to watery diarrhoea, sometimes profuse. Other common symptoms include abdominal cramps, fever, flatulence, nausea, vomiting, and low-grade fever [43–45]. Clinical presentation of cryptosporidiosis in individuals with impairment of T-cell functions, mainly those with HIV infection, varies according to the level of immunosuppression, from asymptomatic disease, to transient disease, to relapsing chronic diarrhoea or even cholera-like diarrhoea that is debilitating and potentially life-threatening [46]. Spreading of infection beyond the extra-intestinal site (in the biliary or respiratory tract) has been documented in children and immunocompromised people, resulting in a potentially life-threatening disease [47, 48]. Sclerosing cholangitis and other biliary involvements are common in AIDS patients with cryptosporidiosis. Both innate and adaptive immunity of the host have major impacts on the severity of cryptosporidiosis and its prognosis.

*Cryptosporidium* has been diagnosed using a variety of approaches, such as microscopy, immunofluorescent antibody (IFA), enzyme-linked immunosorbent assay (ELISA), and DNA-based detection methods [18]. However, identification of the parasite's morphologic features through examination of stool smears is widely employed in diagnostic laboratories, particularly in resource-limited health systems. The oocysts are shed intermittently [49]; therefore, three faecal samples collected on alternate days are recommended. To maximise the recovery of oocysts, Sheather's sucrose flotation, saturated salt flotation, and Allen and Ridley's formol-ether method are the stool concentration techniques most frequently used prior to the use of the microscopy staining technique [50, 51]. Stain differential is required due to the small size of the specimen (ranging from 4 to 6 μm), similar in shape to yeasts and faecal debris [52]. Safranin-methylene blue, Kinyoun Ziehl-Neelsen, and dimethyl sulfoxide-carbol fuchsin are the most commonly used stain methods [11, 53–55]. However, in the absence of staining solution, phase contrast microscopy has proven to be highly specific for the detection of *Cryptosporidium* oocysts in human stool

**335**

*State of the Art and Future Directions of* Cryptosporidium *spp.*

samples [56]. In general, conventional microscopy lacks sensitivity, is time-consum-

Direct fluorescent antibody tests (DFAs), enzyme-immunoassays (EIAs), and

rapid immunochromatographic assays (dipsticks) are commercially available [60–63]. The EIA kits have been evaluated with human stool specimens only, presumably from patients infected with *C. hominis* or *C. parvum*. The direct fluorescent antibody tests have been widely used for the detection of *Cryptosporidium* in faecal smears, water, and food [60, 62–66]. However, the antigenic variability of oocyst wall epitopes contributes to reducing specificity, and the sensibility of all immunological-based methods is low. High specificity (99–100%) has been generally reported for EIA kits. Sensitivities, however, have been reported to range from 70 to 100% [62–65]. Dipsticks and EIAs are available for individual and for all-in-one tests for *Giardia*, *Cryptosporidium*, and *Entamoeba histolytica* [66–69]. The tests are fast and easy to perform. However, EIA kits and rapid format assays present a potential problem with false positives, so results need to be interpreted and evaluated with caution [70]. To overcome these barriers, one of the most notable advances in public health in recent decades has been the development of tools based on molecular biology for the diagnosis of infectious diseases. These polymerase chain reaction (PCR) techniques have enabled specific sensitive detection of oocysts (a single oocyst) in clinical and environmental samples [71–77]. Examples of such techniques include conventional PCR, quantitative PCR real time, and high-resolution melt. A wide variety of PCR methods targeting different genes have been developed for the detection of *Cryptosporidium* at the species/genotype/subtype levels. However, no targeted tests have been patterned for the detection of *Cryptosporidium* in clinical laboratories. Recently, the simultaneous qualitative detection and identification of multiple viral, parasitic (including *C. parvum* and *C. hominis*), and bacterial nucleic acids in human stool specimens were approved by the Food and Drug Administration (FDA) [78]. In general, PCR tools solely amplify the DNA of *C. parvum*, *C. hominis*, *C. meleagridis*, and species/genotypes closely related to *C. parvum* [18]. For genotyping, nested PCR-RFLP was the most commonly used method in the past. Nowadays, DNA sequencing of 18S has been required to reliably detect all *Cryptosporidium* spp. The HSP70 and COWP targets fail to detect the DNA of *C. felis*, *C. canis*, and *C. muris* [79]. Subtyping tools are indispensable from the epidemiological point of view and are helpful in knowing the possible transmission routes of *Cryptosporidium* species and zoonotic potential of the parasite. Several subtyping tools have been developed to evaluate the diversity within *C. parvum* or *C. hominis*, including analysis of the microsatellite, GP-60 gene, HSP70 gene, 47-kDa protein, small double-stranded (ds) RNA virus, serine repeat antigen, and T-rich gene fragment [73, 80–85]. The 18S ribosomal RNA (rRNA) gene and the hypervariable 60-kDa glycoprotein (gp60) gene have been widely used as targets to identify species and track transmission [18, 86, 87]. The 60-kDa glycoprotein (gp60, also known as gp40/gp15) gene presents a wide genetic heterogeneity in the number of trinucleotide repeats (TCA, TCG, or TCT). This gene encodes a precursor protein that is cleaved to produce mature cell surface glycoproteins (gp45/gp40 and gp15) implicated in the attachment to, and invasion of, enterocytes [18, 87]. Identification of subtypes using GP60 subtype families has revealed the subtype families (Ia-Ik) in *C. hominis* [87–91] and two zoonotic subtypes (IIa, IId), subtypes (IIb, IIc, IIe, IIf, IIi, IIj-IIt) in *C. parvum* [4, 87, 92–94], and subtype families (IIIa to IIIg) in *C. meleagridis* have been acknowledged [87, 95, 96]. Subtyping tools targeting the gp60 gene have been developed recently for several other human-pathogenic *Cryptosporidium* species [87]. Species and subtype identification are not necessary for clinical care and therapeutic options but are important for epidemiological surveillance and for drug investigations and clinical trials. Novel diagnostic tools and biomarkers for

ing, and requires a skilled and well-experienced microscopist [57–59].

*DOI: http://dx.doi.org/10.5772/intechopen.88387*

#### *State of the Art and Future Directions of* Cryptosporidium *spp. DOI: http://dx.doi.org/10.5772/intechopen.88387*

*Parasitology and Microbiology Research*

perspective.

to inactivation by chlorine; and without the use of filtration, it is challenging to remove it from drinking water [19–21]. *Cryptosporidium* prevalence is higher in areas lacking a sanitation infrastructure, mainly drinking water and sewage, which led the World Health Organization (WHO) to include it in the water sanitation and health programme [22]. The scarcity of sustained access to safe water creates tremendous burdens of *Cryptosporidium* diarrhoea in developing countries [23]. Treatment and diagnosis options are still not totally effective [2, 24–26]. No fully effective drug therapy or vaccine is available for *Cryptosporidium*, and the diagnosis of cryptosporidiosis has been based on the demonstration of oocysts in faeces, which present low sensibility [25]. However, the ability to culture relevant *Cryptosporidium* isolates in vitro, the development of novel gene-editing tools (knockout genes, CRISPR/ Cas9, and RNAi) [26–30], and 'omic' research (genomics, transcriptomics, and proteomics) represent essential paths towards significant advancements in the control of cryptosporidiosis [30–38]. In the future, those approaches will show a holistic view of the biology of *Cryptosporidium*. In this chapter, we present recent advances and remaining challenges regarding human cryptosporidiosis under a public health

Despite *Cryptosporidium* species infecting the epithelial cells of the gastrointestinal and respiratory epithelium tracts, human cryptosporidiosis is a usually self-limiting infection in immunocompetent individuals with a low fatality rate [39–41]. In general, onset of the symptoms occurs 5–7 days following exposure and resolves in 2–3 weeks [42]. Clinical manifestations vary from subclinical infection to watery diarrhoea, sometimes profuse. Other common symptoms include abdominal cramps, fever, flatulence, nausea, vomiting, and low-grade fever [43–45]. Clinical presentation of cryptosporidiosis in individuals with impairment of T-cell functions, mainly those with HIV infection, varies according to the level of immunosuppression, from asymptomatic disease, to transient disease, to relapsing chronic diarrhoea or even cholera-like diarrhoea that is debilitating and potentially life-threatening [46]. Spreading of infection beyond the extra-intestinal site (in the biliary or respiratory tract) has been documented in children and immunocompromised people, resulting in a potentially life-threatening disease [47, 48]. Sclerosing cholangitis and other biliary involvements are common in AIDS patients with cryptosporidiosis. Both innate and adaptive immunity of the host have major impacts on

*Cryptosporidium* has been diagnosed using a variety of approaches, such as microscopy, immunofluorescent antibody (IFA), enzyme-linked immunosorbent assay (ELISA), and DNA-based detection methods [18]. However, identification of the parasite's morphologic features through examination of stool smears is widely employed in diagnostic laboratories, particularly in resource-limited health systems. The oocysts are shed intermittently [49]; therefore, three faecal samples collected on alternate days are recommended. To maximise the recovery of oocysts, Sheather's sucrose flotation, saturated salt flotation, and Allen and Ridley's formol-ether method are the stool concentration techniques most frequently used prior to the use of the microscopy staining technique [50, 51]. Stain differential is required due to the small size of the specimen (ranging from 4 to 6 μm), similar in shape to yeasts and faecal debris [52]. Safranin-methylene blue, Kinyoun Ziehl-Neelsen, and dimethyl sulfoxide-carbol fuchsin are the most commonly used stain methods [11, 53–55]. However, in the absence of staining solution, phase contrast microscopy has proven to be highly specific for the detection of *Cryptosporidium* oocysts in human stool

**2. Clinical perspective, diagnosis, and treatment**

the severity of cryptosporidiosis and its prognosis.

**334**

samples [56]. In general, conventional microscopy lacks sensitivity, is time-consuming, and requires a skilled and well-experienced microscopist [57–59].

Direct fluorescent antibody tests (DFAs), enzyme-immunoassays (EIAs), and rapid immunochromatographic assays (dipsticks) are commercially available [60–63]. The EIA kits have been evaluated with human stool specimens only, presumably from patients infected with *C. hominis* or *C. parvum*. The direct fluorescent antibody tests have been widely used for the detection of *Cryptosporidium* in faecal smears, water, and food [60, 62–66]. However, the antigenic variability of oocyst wall epitopes contributes to reducing specificity, and the sensibility of all immunological-based methods is low. High specificity (99–100%) has been generally reported for EIA kits. Sensitivities, however, have been reported to range from 70 to 100% [62–65]. Dipsticks and EIAs are available for individual and for all-in-one tests for *Giardia*, *Cryptosporidium*, and *Entamoeba histolytica* [66–69]. The tests are fast and easy to perform. However, EIA kits and rapid format assays present a potential problem with false positives, so results need to be interpreted and evaluated with caution [70]. To overcome these barriers, one of the most notable advances in public health in recent decades has been the development of tools based on molecular biology for the diagnosis of infectious diseases. These polymerase chain reaction (PCR) techniques have enabled specific sensitive detection of oocysts (a single oocyst) in clinical and environmental samples [71–77]. Examples of such techniques include conventional PCR, quantitative PCR real time, and high-resolution melt. A wide variety of PCR methods targeting different genes have been developed for the detection of *Cryptosporidium* at the species/genotype/subtype levels. However, no targeted tests have been patterned for the detection of *Cryptosporidium* in clinical laboratories. Recently, the simultaneous qualitative detection and identification of multiple viral, parasitic (including *C. parvum* and *C. hominis*), and bacterial nucleic acids in human stool specimens were approved by the Food and Drug Administration (FDA) [78]. In general, PCR tools solely amplify the DNA of *C. parvum*, *C. hominis*, *C. meleagridis*, and species/genotypes closely related to *C. parvum* [18]. For genotyping, nested PCR-RFLP was the most commonly used method in the past. Nowadays, DNA sequencing of 18S has been required to reliably detect all *Cryptosporidium* spp. The HSP70 and COWP targets fail to detect the DNA of *C. felis*, *C. canis*, and *C. muris* [79]. Subtyping tools are indispensable from the epidemiological point of view and are helpful in knowing the possible transmission routes of *Cryptosporidium* species and zoonotic potential of the parasite. Several subtyping tools have been developed to evaluate the diversity within *C. parvum* or *C. hominis*, including analysis of the microsatellite, GP-60 gene, HSP70 gene, 47-kDa protein, small double-stranded (ds) RNA virus, serine repeat antigen, and T-rich gene fragment [73, 80–85]. The 18S ribosomal RNA (rRNA) gene and the hypervariable 60-kDa glycoprotein (gp60) gene have been widely used as targets to identify species and track transmission [18, 86, 87]. The 60-kDa glycoprotein (gp60, also known as gp40/gp15) gene presents a wide genetic heterogeneity in the number of trinucleotide repeats (TCA, TCG, or TCT). This gene encodes a precursor protein that is cleaved to produce mature cell surface glycoproteins (gp45/gp40 and gp15) implicated in the attachment to, and invasion of, enterocytes [18, 87]. Identification of subtypes using GP60 subtype families has revealed the subtype families (Ia-Ik) in *C. hominis* [87–91] and two zoonotic subtypes (IIa, IId), subtypes (IIb, IIc, IIe, IIf, IIi, IIj-IIt) in *C. parvum* [4, 87, 92–94], and subtype families (IIIa to IIIg) in *C. meleagridis* have been acknowledged [87, 95, 96]. Subtyping tools targeting the gp60 gene have been developed recently for several other human-pathogenic *Cryptosporidium* species [87]. Species and subtype identification are not necessary for clinical care and therapeutic options but are important for epidemiological surveillance and for drug investigations and clinical trials. Novel diagnostic tools and biomarkers for

cryptosporidiosis, which could also be used for therapeutic or vaccine trials, are necessary for accurate identification.

Current treatment options for cryptosporidiosis are limited. So far, there is no vaccine against *Cryptosporidium* [97]*,* and nitazoxanide (NTZ) is the only drug approved by the FDA for treatment of cryptosporidiosis in children and immunocompetent adults [98]. However, it is not effective without an appropriate immune status and, consequently, is ineffectual for the treatment of immune-compromised patients, particularly those with AIDS [25, 99]. NTZ is a nitrothiazole benzamide compound with a broad spectrum of activity against a wide range of parasites, bacteria, and viruses. In protozoa, NTZ inhibits the enzyme pyruvate ferredoxin oxidoreductase, which is essential to anaerobic energy metabolism [100]. Due to the prevalence of cryptosporidiosis, the development of novel therapeutic targets and vaccines against *Cryptosporidium* spp. is a public health priority. The ongoing need to develop new anti-cryptosporidial drugs has spurred the process of finding new uses for existing drugs. Repurposing drug provides an attractive alternative to drug development [101]. Two compounds, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, pitavastatin and auranofin (approved for the treatment of rheumatoid arthritis), have been shown to be effective against *Cryptosporidium* in vitro [102]. Auranofin has been shown to be 10 times more potent than metronidazole against *Entamoeba histolytica*, the protozoan agent of human amoebiasis [103]. HMG-CoA and auranofin have particular promise in fasttracking for further in vivo testing in animals and humans.
