Human Cryptosporidiosis: An insight into Epidemiology, Modern Diagnostic Tools and Recent Drug Discoveries

Review Article

Human Cryptosporidiosis: An insight into Epidemiology, Modern Diagnostic Tools and Recent Drug Discoveries

Arsalan Zafar1, Muhammad Kasib Khan1*, Zaheer Abbas1, Rao Zahid Abbas1, Zia ud Din Sindhu1, Zafar Iqbal1, Muhammad Shafi Hasni1, Hasnain Javed2, Muhammad Nadeem1, Hammad Ur Rehman Bajwa1, Muhammad Adnan Sabir Mughal1, Fatima Yasin1

Adv. life sci., vol. 6, no. 2, pp. 60-70, February 2019
*- Corresponding Author: Muhammad Kasib Khan (Email: mkkhan@uaf.edu.pk)
Authors' Affiliations

 1- Department of Parasitology, University of Agriculture, Faisalabad, Pakistan
2- Punjab AIDS Control Program, Health Department, Government of Punjab, Pakistan
 [Date Received: 12/12/2018; Date Revised: 11/02/2019; Date Published Online: 25/02/2019]

Abstractaa download_button
Introduction
Methods
Discussion
Conclusion
References 

Abstract

Cryptosporidiosis is an emerging food and water borne zoonotic disease, which is caused by genus Cryptosporidium. The first Cryptosporidium spp. was isolated from mice in 1907 and gained importance when it was found in an HIV positive patient. It usually causes self-limiting diarrhea in young children and immunocompetent patients. However, it may lead to chronic diarrhea with life threatening condition in immunocompromised patients. Other complications related to this transmittable infection may include respiratory problems, skin rashes and headache. HIV/AIDS patients are highly susceptible host for this parasite. Cryptosporidium parvum and Cryptosporidium hominis are the known pathogenic species, prevalent among humans and they are being transmitted through contaminated food and water. Usually, the diagnosis of Cryptosporidium spp. is dependent on microscopic technique in many countries, which has a low sensitivity and specificity leading to false positive results. However, for a step forward to successful epidemiological studies, advanced techniques (Serological and DNA-based) provide us the better ways of diagnosis with more sensitivity and specificity. Furthermore, no antiparasitic drug has found to be effective against Cryptosporidium spp. except Nitazoxanide which is FDA-approved and effective only when administered along with antiretroviral therapy. In this regard, present review summarizes the various epidemiological studies conducted around the globe along with modern diagnostic tools and the suitable treatment available now a days. This systemized review will help the scientists to better understand all the aspects of cryptosporidiosis at one platform which may help in designing surveillance studies through selection of sensitive diagnostic techniques. The new drugs mentioned in this review may also help to better control this parasite in humans, especially immunocompromised individuals.

Keywords: Cryptosporidium; Opportunistic protozoa; Zoonosis; Recent advancements

Introduction6th button-01

Cryptosporidium spp., an opportunistic intracellular protozoan parasite, belong to Phylum Apicomplexa and causes high economic losses in case of morbidity and mortality by affecting the gastrointestinal tract of sheep, goat, pig, cattle and human [1]. The first specie was isolated from gastric juice of mice, named as Cryptosporidium muris, in 1907 by Tyzzer [2]. Later, Cryptosporidium was also detected, from human stool sample in 1976 and became most emergent protozoa due to its zoonotic importance. Despite, Cryptosporidium spp. are host specific, there are 26 recognized species causing chronic diarrhea, dysentery and malabsorption in humans as well as in farm animals. Most prevalent species in humans are C. parvum and C. hominis. However, new species or new strains are going to be discovered due to rapid genetic recombination as i) two or more species are affecting the same host at the same time ii) it undergoes both sexual and asexual production leading to genetic variation [3].

Chances of infection; chronic diarrhea/dysentery, are more in immunocompromised patients like HIV patients as compared to Immunocompetent patients; who suffer with a short-term and self-limiting diarrhea [4,5]. In the past, Cryptosporidium was considered to be linked with childhood diarrhea, malnutrition. Situation became worse in 1993 when 4 million people got Cryptosporidium infection in USA and Milwaukee. Despite of the evidence, cryptosporidiosis was under-diagnosed, and the treatment was only symptomatic [6].

Nevertheless, new diagnostic tools and literature has improved the estimate of global burden. However, there are still gaps which need to be filled, particularly in the fields of epidemiological studies, diagnosis and proper cure for cryptosporidiosis. Moreover, there is a deficiency of well-organized and comprehensive reviews covering all the aspects of cryptosporidiosis i.e. disease outbreaks, modern diagnostic tools and therapeutic controls especially in HIV patients. Such detailed reviews are very important for the young parasitologists and researchers for better understanding the current scenarios regarding the disease epidemics, modern diagnostic tools and the latest drug discoveries.  Considering all the above-mentioned prospects, following review was planned to summarize a detailed information about the disease burden of Cryptosporidium infection in many countries, modern diagnostic tools and the new drugs available against Cryptosporidiosis.   

Methods6th button-01

Literature survey and selection criteria 

The main objective of writing this review was to summarize recent advancements in some aspects of cryptosporidiosis important for controlling the disease, including epidemiology and recent disease outbreaks in various countries, virulent factors, latest diagnostic techniques and recently available drugs to cure Cryptosporidium infection, particularly in HIV/AIDS patients. Doing so, a massive search was carried out on google scholar by providing different terms like “Cryptosporidiosis”, “Disease outbreaks of Cryptosporidium parvum”, “Epidemiological studies on Cryptosporidium parvum”, “Cryptosporidium infection is HIV/AIDS patients”, “Cryptosporidium infection in children”, “diagnostic techniques in Cryptosporidium infection”, “modern techniques for the identification of Cryptosporidium parvum”, “DNA based techniques for the identification of Cryptosporidium in humans”, “Mortality rate in immunocompromised patients due to Cryptosporidium spp.”, “how to treat Cryptosporidium infection in humans”, “latest drug discoveries against Cryptosporidium infection”, “FDA approved drugs for cryptosporidiosis”. A total of 65 citations were reviewed after screening according to the contents of this review. 

Discussion6th button-01

Epidemiology

Early epidemiological studies have revealed the prevalence of Cryptosporidium to be 1% in developed countries while 5-10% in poor countries [6]. However, results of recent studies, based on serological and molecular techniques, revealed that the previous studies have underestimated the frequency of C. parvum infection indicating the prevalence rate of 15-25% in diarrheal patients [6].

Disease burden

Numerous researches have been conducted on Cryptosporidiosis throughout the world due to its zoonotic and opportunistic behavior. Regarding the global disease burden published in 2010, highest prevalence rate of 3.5-35.8% has been documented in America, followed in order by Africa (2.6-21.3%), Europe (0.1-14.4%) and Asia (1.3-13.1%) [7]. However, a brief history of current occurrence of cryptosporidiosis documented in various hosts along with various diagnostic techniques is listed in Table 1.

 

 

 

 

 

 

Transmission and Pathogenesis

C. parvum mostly transmit either by direct or indirect ways. Direct transmission occurs when humans drink contaminated water or food with infected fecal material or by direct touching the infected person. Chances of disease spread are higher in day care centers and schools, where children get exposed to the contaminated surface and mud while playing. Meanwhile, a person can also get infection with sexual practices. Moreover, indirect transmission may also occur by drinking or eating contaminated water and food, respectively. It is clear with evidence that C. parvum can easily be passed through water filtrations plants due to its very small size of one micron. Furthermore, there is no effect of chlorination on these parasites during water treatment. So, drinking water is a main source of its transmission [13,41,42].

Pathologically, Cryptosporidium cause inflammatory damage to the epithelium of small intestine, which leads to the malabsorption and increased intestinal secretions resulting in diarrhea, dysentery, vomiting and nausea which may last for more than seven days. Situation may lead to death due to sever and persistent diarrhea in very young children and immunocompromised/HIV patients. By entering the host, they attach with intestinal epithelium with the help of gp900 and gp60 (glycoproteins) and start proliferating with the help of micronemes and rhoptries, resulting in the formation of parasitophorous vacuoles where they undergo sexual and asexual reproduction. The whole situation is called cryptosporidiosis [42]. Some studies have shown that Cryptosporidium may also affect the respiratory tract and cause some respiratory disorders along with skin rashes and headache [4,7,43]. Several virulenec factors cause damage to the host cells are mentioned in Table 2 [1].

 

 

 

 

 

 

Associated risk factors

It is evident from various studies that many associated risk factors may likely to be linked with occurrence of cryptosporidiosis in humans. Some of them found associated with varied prevalence of cryptosporidiosis have been listed below:

Seasonal variations

Cryptosporidiosis has been found endemic throughout the year in various parts of the world, but infection is most likely to be happened in rainy season due to water contamination [44]. In a critical study, another aspect was highlighted when Cryptosporidium along with Giardia and Entamoeba showed clear seasonal variation. The prevalence was stable before rainy season but during the rainy season, an increased rate of infection was noticed. However, this analysis does not support the pattern in case of cattle host.

Age

As a result of cryptosporidiosis, different immune responses can be observed which are dependent on age variation of host. Cryptosporidiosis is found to be more prevalent in children of age less than 24 months as they have less developed immune system as compared to adults [11]. Some supporting results were obtained in a research conducted at a goat farm in Brazil. This study showed more prevalence of Cryptosporidium in juvenile (age less than 12 months) as compared to adults (age more than 12 months) as all 4.8% prevalence was observed in juvenile animals [45]. To observe the prevalence of cryptosporidiosis, a farm level study was performed which revealed the risk to be high in calves of age 8-21 weeks in comparison with 0-7 days aged calves. During the same year, similar results were found in buffalo calves which showed high risk of prevalence of infection in calves of age 1-15 days [46]. In another farm level study, PCR study disclosed cryptosporidiosis to be more prevalent in pre-weaned calves as compared to post weaned calves. In human, Cryptosporidium was comparatively more prevalent in children of age 7-15 days than children of 3-12 years.

Gender

Occurrence of cryptosporidiosis is higher in male population as compared to female population. However, it may vary due to the exposure level of males and females. In china, It has been observed that the prevalence of Cryptosporidium infection was 3 times higher in males (12.6%) as compared to those of females (4.4%) [47].

Socio-economics behavior

Socio-economic behavior of host directly affects the transmission of Cryptosporidium. Cryptosporidium is transmitted through ingestion of parasites which could be by means of waterborne, person to person and food borne whereas the middle one is occasional. The cryptosporidium mostly affects children with immunosuppression while suppression is more prominent in malnourished children as compared to children aged less than 6 months which are breastfed [48]. In a study, it was observed that cryptosporidium spp., were more prevalent in rural areas especially in the persons exposed to public toilets and infected with HIV [49]. In another investigation, samples from 1731 drug abuser rehabilitation patients were collected to record prevalence of Cryptosporidium spp., infection and the results showed 19.6% persons to be positive with infection.

Diagnostic Tools

  • Conventional Method

Traditionally, Cryptosporidium oocysts detection in environment, food, water, tissue and fecal sample has greatly relied on microscopic examination. But when it comes to the identification of Cryptosporidium, morphological characters are few. Therefore, identification based on light microscopy is time consuming and unreliable. Several staining techniques are used to identify Cryptosporidium oocysts. The least simple stains include modified Ziehl–Neelsen, Kinyoun, dimethyl sulphoxide carbol fuchsin and safranin–methylene blue. However, samples containing less oocysts number can suffer low sensitivity and/or specificity. Moreover, negative staining methods are also available. For example, by using chemicals like malachite green and green perbromide, background of slide is stained leaving the oocysts unstained. These latter staining techniques are time consuming, laborious and require perfection. Moreover, none of the techniques is helpful to identify Cryptosporidium species. For the identification of Cryptosporidium oocysts, modified Ziehl-Neelsen staining and wet mount preparation methods are also commonly used [36]. Another method for the identification and detection of Cryptosporidium oocysts is flow cytometry. To identify Cryptosporidium in sewage samples and water, Vesey used a skytron Argos 100-5 instrument spiked with oocysts stained with an FITC-C-mAb. The outcome of this study is encouraging by means of flow cytometry because it detects as few as 1000 oocysts/L.

  • Immunological Assays

For detection and identification, Immunological methods have several advantages over microscopic techniques. For instance, direct fluorescent antibody (DFA) and fluorescence microscopy assay use fluorescence in isothiocyanate-conjugated anti-Cryptosporidium monoclonal antibody (FITC-C-mAb), which identify epitopes on the surface of Cryptosporidium oocysts with high sensitivity (98.5–100%) and specificity (96–100%) for Cryptosporidium oocysts detection in environmental samples and fecal smears. FITC-C, commercially available monoclonal antibody, is used for enumerating and detecting Cryptosporidium oocysts routinely in environmental and fecal samples. DFA assays which are based on monoclonal antibody can differ in their sensitivity and specificity of diagnosis which depends on many factors including ‘biophysics’ detection system, antibody conjugated reporter (enzyme fluorochrome), Cryptosporidium antigen used to develop mAb and the avidity and subclass/class of the antibody. IgG monoclonal antibody seems to have better avidity to Cryptosporidium oocyst surface antigens as compared to IgM resulting in better recoveries with immuno-magnetic separation (IMS) within water samples of high turbidity. Both molecular interaction and fluorochrome selection are the critical aspects and are responsible for false positive results due to autofluorescence in emission and excitation spectra. Though different monoclonal antibodies have been developed but commercially existing mAbs cannot identify Cryptosporidium specifically. Monoclonal antibodies have been developed with lesser number of Cryptosporidium parvum oocysts [50].

In the last few years, identification and diagnostic trends have been greatly changed towards EIA (Enzyme immunoassays), IC (Immuno-chromography) and ELISA (Enzyme Linked Immunosorbent Assay) for the detection of Cryptosporidium oocyst antigens. Specificity of these assays is reported to be high (98–100%). Advantage of these assays is that they detect infections when oocysts are not being excreted in feces [51]. while limitation of some kits may be seen because they mainly depend upon visual inspection which enable subjective interpretation of test results.

Furthermore, these assays like many other immunological approaches do not diagnose Cryptosporidium genotype or species. So far, no such immunological tool is present which makes a distinction among oocysts of dissimilar species. One effort has been made with the development of PCR-EIA which differentiates between C. hominis and C. parvum. In a recent study, results have shown the 100% specificity and 86.6% sensitivity of antigen ELISA for Cryptosporidium [52].

  • DNA Based Approaches

Molecular techniques are available to find out species, genotypes and sub-genotypes of Cryptosporidium to differentiate among human and animal pathogens. Nucleic acid-based methods have been evaluated for species identification in animals and human from environmental, water and fecal samples. Some methods are based on in-situ hybridization of probes within oocysts to specific genetic loci, while most methods are based on specific amplification of loci from genomic DNA by PCR. These applications have led to understanding Cryptosporidium species regarding biology, ecology, epidemiology, systematic and population genetics for control and prevention of cryptosporidiosis in animals and humans. DNA profiles from human isolates showed two different genotypes, C. hominis (anthroponotic) and C. parvum zoonotic genotype. Analysis of tri-nucleotide sequence has shown twelve subtypes of Cryptosporidium hominis and seven subtypes of Cryptosporidium parvum [53].

FISH (Fluorescent In-situ Hybridization) utilizes probes (fluorescently-labelled oligonucleotides) by targeting RNA or DNA sequences. For identification of Cryptosporidium, most of FISH assays are employed on RNA hybridization rather DNA by making target on a small subunit of nuclear rRNA of variable region [50,54]. Because within cell, small subunit (SSU) has a high copy number considered as rich target. Some limitations with the use of FISH probes depend upon the viability of oocysts as SSU-rRNA decay during cell death making useless the FISH technique. Degradation varies with environmental conditions including RNase contamination, pH, salinity and temperature etc.

  • Advanced molecular assays

Different methods are available regarding PCR and many of them have been applied to Cryptosporidium in the past. A variety of techniques have been evaluated, but currently, only those methods are needed which are easy to perform, universally applicable and effective in approach.

  • Fingerprinting

Different techniques are available which permit genetic fingerprint to be developed for parasite sample. Such techniques based on genome(s) screening for distinction in organization and sequence. Positive approach of some methods is that no earlier genomic information is needed for a sample which is to be characterized. However, the disadvantage is that pooled organism’s genetic fingerprinting show organism’s population rather than individual, therefore, individual markers may not signify all population. However, these techniques have valuable applications. RAPD (Random Amplification of Polymorphic DNA) or AP-PCR (Arbitrarily Primed-Polymerase Chain Reaction) are developed, based on DNA fragments amplification usually using single primers (10-mer) following separation of amplicons by polyacrylamide or agarose gel electrophoresis. This technique has the benefit of being easy, fast and efficient. Band profiles reproducibility can be raised by using dissimilar thermal cyclers, primer sensitivity, DNA quality and template concentration.

  • AFLP (Amplified fragment length polymorphism)

AFLP has been used in parentage analysis, forensic science, for genetic investigation and disease diagnosis in humans and animals. This technique is based on (i) DNA digestion with two restriction enzymes (ii) ligation to 5′ and 3′- end with specific adapters of restriction fragments (iii) use of primers for amplifying restriction fragments (iv) analysis of subsequent restriction fragments through electrophoresis. AFLP has the advantage of being used to composite DNA of every origin. PCR can be applied with high stringency that can attained high discrimination level. Disadvantage of this technique is that it takes more time than other fingerprinting methods. Occasionally, in band profiles a high variability can limit the AFLP performance, but this technique has been utilized to find out the genotype of different organisms, including protozoa but has not been utilized to Cryptosporidium [55]. Some researchers have focused on satellite DNA utilization for genetic make-up analysis of parasite populations. Microsatellites and Minisatellites have been used as ubiquitous and profuse in all eukaryotic genomes. They are usually non-transcribed and sustain polymorphism because of alteration in repeat number. On gel electrophoresis, this change let the alleles to be scored by size and characterization is done by variability in alleles. Thus, it is utilized to investigate genetic mapping and genetic structure of organism’s populations. PCR is an excellent technique for the amplification of repeat region that can be examined by gel electrophoresis and visualized by auto-radiography or staining. Analysis with multi-locus satellite has been used to explore population structures and to identify genetic exchange role in Cryptosporidium [56]. Multi-locus satellite technique has also been utilized to investigate zoonotic threat from several protozoa including Cryptosporidium [57]. Fingerprinting method has also been utilized to explore differences between C. parvum and C. hominis. In electrophoretic band profiles, a similar method revealed the substantial heterogeneity in clinical isolates taken from sporadic diseases and homogeneity among clinical isolates from an outbreak. Based on these studies, it is now clear that these diagnostic tools have given useful information on population structure and diversity in C. hominis and C. parvum and similar methods are required for other Cryptosporidium species.

  • PCR-based sequencing and restriction fragment length

Different PCR-based methods, utilizing selective pairs of primers for the amplification of genetic loci followed by sequencing or enzymatic cleavage, have been employed to classify and characterize the Cryptosporidium species genotypes [4]. Several loci (key markers) comprise rRNA genes and spacers, COWP (Cryptosporidium oocyst wall protein), HSP70 (70 kDa heat shock protein), TRAP (Thrombospondin-related adhesive protein) genes and GP60 (genes and the 60 kDa glycoprotein). The SSU-rRNA gene gave a valuable genetic marker for Cryptosporidium identification having great interspecific and low intraspecific sequence differences [7,58]. Markers allow identification at species level include the hsp70 and actin gene which have been used together with SSU (small subunit) in phylogenetic (systematic) analysis of Cryptosporidium, giving basic structure for members classification in the genus [59]. The ITS (internal transcribed spacers) of ribosomal DNA are helpful for revealing variability because their sequences possess great intraspecific variation rather than rRNA gene regions. Extremely variable loci may have repetitive microsatellite regions including gp60, ML1 (microsatellite locus 1) and ML2 (microsatellite locus 2) have been used to explore population genetics of Cryptosporidium especially for C. parvum and C. hominis [60].

  • PCR-RFLP (PCR-based restriction fragment length polymorphism)

It has been used to classify Cryptosporidium spp. genetics in many researches. PCR-RFLP based genotypic analysis of gene fragment 18s rRNA has shown 82% isolates from C. hominis, while 18% from C. parvum in children [53]. Moreover, PCR-RFLP analysis for the gene (SSU rRNA) explains that native breeds of cattle do not transmit human Cryptosporidium in Nigeria (Kaduna State). In an advance study, four species of Cryptosporidium, C. bovis, C. andersoni, C. parvum and C. ryanae were explored by analyzing the SSU rRNA (18S) and COWP genes in polish breed cattle. This method does not identify all the sequence length variations in amplicons because endonuclease/s, which is utilized, identifies a limited number of variable sites. One of the gold standard methods remains the direct DNA sequencing to identify polymorphism or genetic variation and can be used for multi-copy (provided there is no sequence heterogeneity in copies) as well as single-copy genes. DNA sequence data can be employed for comparative genetic analysis and suitable for phylogenetic studies. But direct DNA sequencing have some restrictions, as small quantity of DNA from single oocyst of Cryptosporidium is not of practical approach for PCR amplification; amplicons are produced always from that isolate which represents oocysts population. If considerable polymorphism or heterogeneity present in that isolate as in first (ITS-1) and second (ITS-2), ITS regions may not be able to develop a precise sequence from an amplicon [61]. However, analysis of polymorphic amplicons can be done by mutation scanning methods.

  • Electrophoretic scanning

Some restrictions in the investigation of sequence variation can be minimized by high resolution electrophoretic methods. These methods are comprised of mutation scanning techniques, for instance, DGGE (denaturing gradient gel electrophoresis), TGGE (temperature gradient gel electrophoresis), SSCP (single strand conformation polymorphism) and hetero duplex analysis are being used. Particularly, SSCP is a valuable method that is based on the principle which states that electrophoretic movement of ssDNA molecule in non-denaturing gel is dependent mainly on two things, its structure and size, enabling the method to verify mutation at a single point in 500bp amplicons [18]. Thus, PCR-based SSCP has exhibited a useful diagnostic tool for identification and genotypes of Cryptosporidium species to verify genetic variation among and within large quantity of samples. In Cryptosporidium, this has also been utilized for exhibiting sequence variation in the regions of nuclear gene (SSU and hsp70) with lesser number of samples from proper oocyst DNA.

New drug discoveries

Several therapeutics have been tested against cryptosporidiosis such as macrolide Paromomycin aminoglycoside, ionophores and immunotherapy. However, Nitazoxanide was the only anti-protozoal drug against cryptosporidiosis, which was approved in 2006 by the U.S. food and drug administration. But this drug is only effective in immunocompetent patients. In immunocompromised patients, combination of medicine; paromomycin + protease inhibitors along with antiretroviral therapy is recommended [62-65]. The new drugs recently approved for the treatment of cryptosporidiosis along with their limitations have been mentioned in table 3.

 

 

 

 

 

 

Conclusion6th button-01

Cryptosporidium spp. is an emerging zoonotic protozoan parasite which is being transmitted through contaminated food, raw vegetables and drinking water which may be a leading cause of gastrointestinal illnesses in humans. Cryptosporidium parvum and Cryptosporidium hominis are most prevalent parasites among all kind of hosts. These species cause chronic diarrhea and dysentery in young children and particularly, in immunocompromised patients or HIV/AIDS patients. Recovery chances from this parasitic infection are very rare in HIV patients. Even, no antiparasitic drug against these virulent protozoa is efficacious in HIV infected/ AIDS patients until a suitable anti-retroviral medicine is given in combination. Nitazoxanide is a drug of choice against Cryptosporidium spp. and is recently approved by FDA. Moreover, the most appropriate option to avoid cryptosporidiosis is to adopt better hygienic and sanitation conditions.

Conflict of Interest Statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

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