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Enteric protozoa usually cause severe morbidity and mortality in humans. Protozoal infections contribute to the high burden of infectious diseases. Despite recent advances in the epidemiology, diagnostic tool, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist; hence, protozoal infections require further research. We are describing here some important enteric protozoal infections along with the immune dysfunction produced by them. Genus- 1. Entamoeba; 2. Giardia; 3. Cryptosporidium; 4. Cyclospora; 5. Cystoisospora; 6. Dientamoeba; 7. Blastocystis; 8. Balantidium.
chapter and author info
Renu Kumari Yadav
ABVIMS and Dr. RML Hospital, New Delhi, India
ABVIMS and Dr. RML Hospital, New Delhi, India
ABVIMS and Dr. RML Hospital, New Delhi, India
*Address all correspondence to: firstname.lastname@example.org
From the Edited Volume
Innate Immunity in Health and Disease [Working Title]
Parasites are living organisms, which live in or upon another organism and derive nutrients directly from it, without giving any benefit to host. There are three main classes of parasites: protozoa, helminthes, and ectoparasites.
In the current chapter, we are going to discuss about pathogenic protozoan parasites responsible for intestinal infections and the immune system disturbance produced by them. Protozoa are unicellular organisms classified as eukaryotes. Protozoa responsible for intestinal infections are Entamoeba, Giardia, Cryptosporidium, Cyclospora, Cystoisospora, Dientamoeba, Blastocystis, and Balantidium. These enteric protozoa are associated with diarrheal illnesses in humans, with some causing severe debilitating illnesses, especially in immunosuppressed populations .
Enteric protozoa have been given much attention in developing countries, because of their poor sanitary conditions and the unavailability of effective water treatment, which provide a suitable environment for their transmission [2, 3, 4]. In more industrialized settings, less focus has been placed on the impact of protozoal infection, presumably because of better health standards. As a result of underdiagnosis and lack of monitoring programs, reliable data are not available for the estimation of the protozoal disease burden in developed settings . The major focus in developed countries is on bacterial and viral infections; hence, in operational surveillance systems, only a few or no parasitic protozoa are included [6, 7]. However, evidence suggests that some protozoa, like Entamoebaspp., Cryptosporidium, and Giardia, are isolated frequently from diarrheal patients in developing countries (8). Others, such as Blastocystisspp. and Dientamoeba fragilis, are isolated mainly in developed countries [8, 9]. Despite the lower prevalence of parasitic diseases in developed countries, they may result in a greater economic burden on the country.
Many protozoal diseases appearing and reappearing in developed countries presented as a public health problem in developing countries long before. The factors that influence the emergence and reemergence of the protozoal disease are similar to other diseases like change in the parasite or host that favor increased transmission, environment, and demographic change that favor increased human-parasite contact and increased the recognition of previously existing problem due to availability of more advanced diagnostic techniques.
There is lack of sensitive diagnostic techniques to detect parasite in clinical specimens, and hence, the estimation of parasite prevalence is difficult, and the carrier stages and subclinical infections of parasites are often not diagnosed . So the development of technologies that can simultaneously detect several protozoa in stool is the current need of the hour [11, 12]. For the sensitive, accurate, and simultaneous detection of protozoan parasites, the molecular techniques are the most promising methods in comparison to conventional staining and microscopy methods . Unfortunately, the molecular methods are not used routinely for the detection of parasitic protozoa because these are costly and labor-intensive . Much effort must now be placed in the development of inexpensive molecular tools for routine laboratory applications.
The enteric protozoa that are considered as a major public health problem are Entamoeba histolytica, Giardia intestinalis, Cryptosporidiumspp., Dientamoeba fragilis, Cyclospora cayetanensis, Blastocystisspp., Cystoisospora belli, and Balantidium coli.
Genus Entamoebais divided into six species that have been described in humans, including Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni. Among these, E. histolyticais the only pathogenic species [10, 14, 15]. Infection of E. histolyticais mainly seen in immigrants from or travelers to areas of endemicity, male having sex with male (MSM), HIV-infected patients, and institutionalized populations . Millions of people are infected with E. histolytica,and more than 55,000 people die each year globally due to amoebic colitis, a leading cause of diarrhea .
Globally, the highest burden of amebiasis is seen in tropical and subtropical areas of developing countries because of their improper hygiene and water sanitation. It was estimated that E. histolyticaaffect millions of people and responsible for the death of more than 50,000 people each year globally . Amebiasis is also seen in the developed world, mostly in returning travelers or immigrants from endemic countries .
It has been noticed that the prevalence of E. histolytica/disparhas increased to 36.6% in developed world from 2000 to 2015  in comparison to 1986 in which it was 20% as per Boston report .
Transmission—Amebiasis is acquired by the ingestion of cyst via the fecal-oral route, but now it is found that fecal contact can occur during sexual contact also (MSM–male having sex with men).
Life cycle—Following ingestion cyst converts into trophozoites known as invasive form of Entamoeba histolytica.In asymptomatic individuals, cysts and trophozoites pass into the stool without invading intestinal mucosa, whereas in a few cases, it invade intestinal mucosa and lead to invasive manifestations of Entamoeba histolyticalike dysentery, amoebic colitis, and ameboma. After invading intestinal mucosa, trophozoites can travel to other organ via bloodstream and manifest as brain abscess and liver abscess [21, 22]. Males are more commonly affected from this invasive disease than females (Figure 1).
Pathophysiology—The virulence factors of E. histolyticaare its enzymes and proteases that help in the invasion of the epithelial cells, penetrating the intestinal mucosa and degradation of the extracellular matrix proteins [23, 24]. More recently it is thought that some individuals are genetically resistant to infection, while malnourished children are more susceptible, and a polymorphism in the leptin receptoris found to be associated with increased susceptibility to amebiasis [24, 25].
2.1.4 Immune dysfunction
In some cases, E. histolyticacolonizes the colon by high-affinity binding to MUC2 mucin without producing any symptoms, whereas in some it causes an aggressive inflammatory response upon invasion of the colonic mucosa. The parasite has cysteine protease that cleaves C-terminus of MUC2 that dissolves the mucus layer followed by binding of E. histolyticato the mucosal epithelium. As a result, the host mounts a pro-inflammatory response that causes tissue damage and participates in disease pathogenesis. E. histolyticaescapes the host immune system by mechanisms that are not completely understood. The parasite can destroy effector immune cells by inducing neutrophil apoptosis and suppressing respiratory burst. It also suppresses the production of nitric oxide (NO) from macrophages. Following the adherence of E. histolyticato the host cells, multiple cytotoxic effects occur that can promote cell death through phagocytosis and apoptosis, which might play critical roles in immune evasion .
For the early diagnosis of invasive and extraintestinal amebiasis, a high level of clinical suspicion is necessary .
Morphological similarities of the pathogenic species E. histolyticato the nonpathogenic species E. disparand E. moshkovskiimake the diagnosis difficult. Hence, microscopy is usually considered insufficient for the differentiation of these species . In microscopic techniques, trichrome-stained smears and wet mount preparations of stool specimens are used in routine for the identification of Entamoebaspp. . Other methods for Entamoebaidentification include culture, antigen-based, and molecular tests.
In culture methods, polyxenic culture media like Balamuth’s medium and axenic culture media like diamond’s medium are used. A new axenic culture medium CLUPSfor the growth of E. histolyticawas also there which is superior to previously used PEHPSmedia . However, cultivation is more sensitive than microscopy, but these methods are time-consuming, not cost-effective, and hence not routinely utilized by most diagnostic laboratories. Cultivation can effectively distinguish between E. disparand E. histolytica.
Enzyme immunoassay (EI) kits are commercially available that detect E. histolyticaor differentiate between E. histolyticaand E. disparby detecting 170 kDa of lectin antigen.
A rapid immunochromatographic testing (ICT) assay is available that detects antigens of E. histolyticaand E. dispar(29 kDa surface antigen) in stool; however, this assay cannot differentiate between E. histolyticaand E. dispar. This assay also detects antigens of Giardia(alpha 1 Giardia antigen) and Cryptosporidium(protein disulfide isomerase antigen).
For the detection of E. histolyticaantibodies in human serum serological methods such as latex agglutination, indirect hemagglutination (IHA), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA) are highly sensitive [10, 32]. However, in endemic areas serology is of limited use because of the difficulty in distinguishing between past and present infections .
Molecular assays (like PCR) for the detection and differentiation of Entamoebaspecies are now considered as the gold standard for diagnosis of Entamoeba[28, 33]. When species differentiation is difficult or not possible than E. histolytica/E. disparcomplex should be reported.
BioFire FilmArray system is the fully automated PCR system which can detect bacterial, viral, parasitic (E. histolytica, Giardia, Cyclospora, Cryptosporidium)diarrheal pathogen by using gastrointestinal panel .
For the diagnosis of invasive disease, a combination of microscopy, culture, and serology should be used with a PCR assay or with abdominal imaging (when PCR is unavailable) .
In asymptomatic carriers of E. histolytica, luminal agents should be used to minimize the spread of disease. The treatment of choice is different for intestinal and invasive disease; hence, diagnosis is important before treatment. Metronidazole, a tissue amoebicide, is highly effective and used for invasive amoebic disease along with a luminal agent for the elimination of intestinal colonization . Other agents like tinidazole and ornidazole can also be used for the treatment of invasive disease. For the treatment of intestinal and asymptomatic infections, the luminal agents paromomycin, iodoquinol, and diloxanide furoate are strictly recommended, as they are effective in eliminating cysts from the intestinal tract .
Regimen: asymptomatic carriage—luminal agent, Idoquinolone 650 mg tid for 20 days or Paromomycine 500 mg tid for 10 days.
Acute colitis—Metronidazole 750 PO or IV for 5–10 days or tinidazole 2 g/d PO for 3 days Plus luminal agent as above.
Amebic liver abscess—Metronidazole 750 PO or IV for 5–10 days or tinidazole 2 g/d PO once or ornidazole 2 g/d PO once Plus luminal agent as above.
2.1.7 Prevention and control
By avoiding the consumption of contaminated food and water and by treating the asymptomatic person, the infection can be prevented.
Certain vaccinesagainst E. histolyticaare under trial like colonization blocking vaccine targeting SREHP, 170 kDa subunit of lectin antigen, and 29 kDa cysteine-rich protein. Till now no vaccines against E. histolyticaare licensed for human use [37, 38].
2.2 Giardia intestinalis
Giardia intestinalisalso known as Giardia duodenalisand Giardia lambliais a common cause of parasitic diarrhea. It belongs to the flagellate group and first discovered by Leeuwenhoek in 1681 in his own stool.
Giardia lambliais distributed worldwide and reported from both temperate and tropical countries of the world. The prevalence of Giardiain low- and high-income countries is 4–43% and 1–7%, respectively [39, 40]. It was reported that Giardiaaffect approximately 2% of adults and 8% of children in developed countries .
Because of the increasing burden of illness from Cryptosporidiumspp. and Giardiaand their ability to impair development and socioeconomic improvements, they were included in the WHO Neglected Diseases initiative in 2004 .
Transmission—The most important mode of infection is fecal-oral route, but various studies have also found evidence of zoonotic transmission . Giardiasis presents mainly as acute or chronic diarrhea associated with abdominal pain, nausea, malabsorption, and weight loss. Giardiainfection can lead to growth retardation in malnourished children  and zinc deficiency in school-aged children . Giardiainfection is also common in dogs and cats . They are also a potential source of human infection that may be acquired through handling, sleeping together, licking, and kissing, as the zoonotic genotypes of Giardiawere isolated from cats and dogs . Giardiasis is also associated with waterborne disease outbreaks and is related to travel-associated diarrhea .
Life cycle—Cyst is the infective form of Giardiaand ingestion of as low as 10 cysts can initiate infection. After ingestion excystation occurs in the small intestine, and its cyst converts into trophozoites. These trophozoites do not disseminate hematogenously and remain in the small bowel. It can either attach to the intestinal epithelium via suckling disc or remain free in the intestinal lumen. Trophozoites convert into cyst form, and both trophozoite and cysts are excreted in feces, but only the cysts are capable of surviving in the outside environment and disease transmission (Figure 2).
Pathophysiology—After attachment to the intestinal epithelium, it feed on the mucus secretions and lead to apoptosis of enterocyte; epithelial detection tests are used for screening and are more sensitive than routine microscopic examination. Loss of brush border is also seen.
All of these consequences manifest as lactose intolerance and malabsorption syndrome.
2.2.4 Immune dysfunction
Both B cell-mediated antibody production and T cell-mediated immune responses play an in important role in protection against Giardiasis. Humans with immunodeficiency disorders like common variable immunodeficiency (CVID) and impaired IgA function have an increased risk of developing chronic Giardiainfections. People living in endemic areas are less prone to infection or reinfection, indicating that acquired immunity exists.
According to a study, cellular immune response to Giardiacan be understood by murine models. The mice model shows that in the absence of CD4+ T cells, poor control or chronic infection to giardia occurs, indicating that these cells are crucial for the murine defense against G. lamblia. Cytokines like tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) has been shown to be important for determining the parasite load and the duration of an infection.
Cytokines that are secreted by CD4+ T cells were found in the mice infected with Giardia, indicating that a range of TH responses may contribute to the protection against Giardia.
In another study, it was found that following infection of G. lamblia, there is an upregulation of peroxisome proliferator-activated receptor alpha and interleukin-17A (IL-17A) . IL-17A was also found to be upregulated in another mouse study also, where IL-17A and its receptor were important for defense against the parasite . Hence, on the basis of these studies, IL-17A may be linked to protection against Giardia.
Some human studies for Giardia-specific immune responses were also there. In one study, IFN-γ was secreted, and cells were proliferating after the stimulation of CD4+ T cells with Giardiatrophozoites, suggesting that Giardia-specific proliferation of CD4+ T cells exists in humans as well .
The relative importance of the cytokines IL-17A, IFN-γ, TNF-α, IL-4, and IL-10 in the T cell response to Giardiainfection in humans has not been determined .
In outline the diagnosis of Giardiais based on the microscopic detection of Giardiacysts or trophozoites in a stool specimen by using wet mount, iodine mount, and trichrome stain method.
The string test (entro-test) may be useful for revealing Giardiatrophozoites from duodenal sample .
Antigen detection assays that are available for G. intestinalisinclude EIAs, ELISAs, and direct fluorescent-antibody tests [53, 54]. For the cultivation of Giardiaaxenic media like diamond media can be used .
Molecular methods have higher sensitivity than conventional methods; however, many of them are still not commercially available [10, 52]. The use of real-time PCR is increasing for the detection of Giardia. Conventional single, nested, and multiplex PCRs have also been developed [10, 52].
Metronidazole 250 mg TDS for 5 days or tinidazole 2 g once PO has been used as the therapy of choice. Due to the emergence of resistant isolates of Giardia, treatment failures and clinical relapses have been known to occur . Alternatively, nitazoxanide 500 mg BD for 3 days can be used. Other drugs like furazolidone, albendazole, and paromomycin are also effective against giardiasis.
2.2.7 Prevention and control
By improving food and personal hygiene and by treating asymptomatic person, Giardiainfection can be prevented.
One vaccine (GiardiaVax)has been licensed for dogs and cats in the United States [56, 57]. No human vaccines are currently available [57, 58].
It is the coccidian parasite which causes self-limiting illness in immunocompetent person but can lead to severe disease in immunocompromised (AIDS) person. Two species of Cryptosporidium—C. parvumand C. hominis—found to be associated with human disease.
Epidemiology—The distribution of Cryptosporidiumspp. is seen worldwide, and it mainly affects or causes severe illness in children and immunocompromised population. The prevalence of cryptosporidiosis in children of developing and developed nations was found to be 20 and 9%, respectively . In adult population the change in prevalence of Cryptosporidiumspp. was seen in the year 2000; before its prevalence was 3.8% in Los Angeles, and after 2000 it increased to 13% in the developed world .
Different clinical manifestations are produced by species of Cryptosporidiumand subtype families of Cryptosporidium hominis.
Transmission—It is a waterborne disease transmitted via the fecal-oral route, by ingestion of contaminated salads, contaminated water supply, or recreational water, swimming in public pools, and it is observed that it can be transmitted from person to person, men who have sex with men, as well as through zoonotic infections . Cryptosporidiumspp. have a wide host range including humans, domestic pets, and wildlife . Zoonotic transmission occurs by direct contact with infected animals or their feces or indirectly through the consumption of contaminated water [61, 62].
Life cycle—The environmentally resistant oocysts are produced by Cryptosporidium, and they sporulate when excreted in feces, and therefore, these oocysts are immediately infectious . After ingestion of oocyst, excystation occurs in the intestine to liberate sporozoite which further develops into meront (with four merozoite). Meront form converts into macrogamete and microgametes that lead to zygote and finally unsporulated oocyst excreted into the feces.
Pathophysiology—In the small intestine, Cryptosporidiumpresent inside the intracellular vacuoles and no characteristic changes were found on biopsy. Infection of Cryptosporidiumis characterized by self-limiting diarrhea along with abdominal pain, dehydration, and malabsorption. It is an important infection in both immunosuppressed (especially HIV-infected) and in transplant recipients  Figure 3 (Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Disease; 8th ed. Philadelphia. Elsevier saunder’s 2015 Fig. 284–1. Life cycle of Cryptosporidium, p. 3174).
2.3.3 Immune dysfunction
The occurrence of cryptosporidiosis is closely related to the immune status of its host. It primarily affects the infants and immunocompromised individuals. In recent years, several studies have highlighted the importance of innate immunity in cryptosporidiosis. Intestinal epithelial cells play a key role in cryptosporidiosis as these are the exclusive host cell for the replication of the parasite and also participate in the protective immune response. Epithelial cells produce chemokines and attract immune cells to the infected area. They also release antimicrobial peptides with parasiticidal activity and induce apoptosis. Intestinal dendritic cells induce adaptive immunity and control Cryptosporidium parvuminfection in the early stage .
Both innate and adaptive immune responses are important for controlling cryptosporidiosis. Innate immune responses are mediated by Toll-like receptor pathways, antimicrobial peptides, prostaglandins, mannose-binding lectin, cytokines, and chemokines. Cell-mediated responses, particularly those involving CD4+ T cells and IFN-γ, play an important role. The parasite has developed several escape mechanisms to slow down these protective mechanisms .
Routinely the diagnosis of cryptosporidiosis is done by the identification of oocysts in stool by microscopic examination. The staining and preservation technique enhance the sensitivity of the test. The staining technique for Cryptosporidiumspp. includes modified Ziehl-Neelsen technique, Kinyoun’s acid-fast staining technique, modified Sheather’s flotation technique, and the iron-hematoxylin staining technique. Out of these staining techniques, modified Ziehl-Neelsen technique is widely used [67, 68].
Antigen detection assay is also used widely in the diagnosis of cryptosporidiosis; it is more effective in cases where oocyst numbers are low. These assays are thought to be more sensitive than conventional staining [69, 70]. Other tests like fluorescence microscopy and direct fluorescent-antibody (DFA) assay have been used for the detection of Cryptosporidiumoocysts with relatively high specificities (96–100%) and sensitivities (98.5–100%) [70, 71]. Molecular methods for the detection and differentiation of Cryptosporidiumspp. include nested PCR, real-time PCR, multiplex real-time PCR, reverse transcription-quantitative real-time PCR, and multiplex tandem real-time PCR [12, 72]. More recently, an automated multiplex tandem PCR is used; it is based on a robotic platformand used to detect Cryptosporidiumspp. and coinfecting diarrheal pathogens from the human fecal genomic sample . Some new methods such as reverse line blot (RLB) hybridization, loop-mediated isothermal amplification method, and nucleic acid sequence-based amplification (NASBA) methods that amplify RNA from either RNA or DNA templates are also used for the diagnosis of Cryptosporidiumspp. [72, 74]. Another new technique for the detection of Cryptosporidiumspp. includes next-generation sequencing, DNA microarray,etc. which are also available  (Figure 4).
In immunocompetent persons, cryptosporidiosis is usually self-limiting, requiring little or no treatment, but in immunosuppressed person, treatment is required . Nitazoxanide 500 mg BD for 3 days has proven to be effective against cryptosporidiosis. In patients with dehydration, rehydration fluids and nutritional management may be required.
Nitazoxanide is the only licensed drug for the treatment of cryptosporidiosis, and it is given only in persons after the first year of life and with healthy immune systems. Recently trials are going on clofazimine, a drug used for leprosy, to know its effectiveness against Cryptosporidiumspp. .
2.3.6 Prevention and control
Prevention can be done by avoiding exposure to infectious oocyst in human or animal feces, proper hand washing, and improved personal hygiene. No vaccines are available for use against Cryptosporidiumspp. .
This is a coccidian parasite, and the only species of this genus found in humans is Cyclospora cayetanensis. Similar to Cryptosporidium, Cyclosporais also associated with severe illness in immunocompromised patients in comparison to immunocompetent patients.
Epidemiology—Human cyclosporiasis is distributed worldwide, and cases are reported both from tropical and subtropical areas of the world. According to a study from China, the prevalence of Cyclosporaspp. from 2007 to 2009 was 0.70%, whereas in the United Kingdom at the same time, the prevalence was 0.07% .
Transmission—An important feature of the C. cayetanensisis that the direct fecal-oral transmission from fresh stool is not seen because the oocysts require days to weeks outside the host to sporulate and become infectious.
Life cycle and pathophysiology—Similar to cryptosporidiumspp.
Clinical feature—Infection due to Cyclosporamanifest as persistent diarrhea, fever, bloating, flatulence, abdominal cramps, constipation, and fatigue, weight loss, etc.
Immune dysfunction—similar to Cryptosporidiumspp.
Routinely the diagnosis of Cyclosporais done by using microscopic methods. Cyclosporaoocyst produces autofluorescence (white-blue) under an epifluorescence microscope [78, 79]. By demonstration of Cyclosporaoocysts, autofluorescence and staining characteristics diagnosis can be made. Oocysts appear as acid-fast round or ovoid very small (8–10 μm) structures. The diameter measurement of oocyst is important to differentiate it from smaller Cryptosporidiumoocysts, which measure 4–6 μm in size. The procedures used to diagnose Cyclosporaincludes concentration by the formalin-ethyl acetate technique followed by either (i) UV epifluorescence and bright-field microscopy, (ii) examination of a modified acid-fast-stained stool slide, or (iii) examination using a modified safranin-based technique .
Molecular techniques have been developed like various PCR tools that target the internal transcribed spacer region, which use primers for the 18S rRNA gene . These tools include conventional PCR, reverse transcriptase PCR in combination with agarose gel electrophoresis, and nested PCR . Cyclosporais included in the BioFire FilmArray systems GIT panel; hence, syndromic diagnosis can be made.
Laboratory diagnosis is important as the treatment of Cyclosporais different from the other protozoa of similar presentation. Trimethoprim-sulfamethoxazole (also known as co-trimoxazole) 160/800 mg BD for 7–10 days is the drug of choice for cyclosporiasis.
2.4.5 Prevention and control
No vaccine is available till now against Cyclospora; by avoiding contaminated food and water it can be prevented.
2.5 Cystoisospora belli(formerly Isospora belli)
Cystoisospora belliis responsible for intestinal disease in several mammalian hosts. It is also a coccidian parasite similar to Cryptosporidiumand Cyclospora. It is also found to be associated with more severe illness in immunocompromised host.
Very little is known about the epidemiology and prevalence of Cystoisospora belli. The worldwide reported prevalence of Cystoisospora belliwas 2.5–26.1%, and it is mostly seen in HIV-positive individuals .
Transmission—Infection occurs via the ingestion of mature sporulated oocysts present in contaminated food or water.
Life cycle and pathophysiology—Similar to Cryptosporidiumspp.
Clinical feature—Infection is manifested as watery diarrhea, abdominal cramps, anorexia, and weight loss, and it is almost indistinguishable from cryptosporidiosis. It is more common in AIDS patients and causes traveler’s diarrhea in travelers to developing countries. In HIV-infected patients, infection of Cystoisospora bellimay lead to chronic diarrhea, acalculous cholecystitis cholangiopathy, and extraintestinal infection . Other Cystoisosporaspecies causes of diarrhea in domestic animals, like Cystoisospora suis, lead to severe diarrheal illness in pigs .
Immune dysfunction—Similar to cryptosporidiumspp.
Diagnosisis made by demonstration of oocyst by direct microscopic examination of the feces, with acid-fast staining or fluorescent staining with auramine and rhodamine. Oocysts of Cystoisospora belliare large (20–23 μm × 10–19 μm) and ellipsoidal in shape in comparison to oocyst of Cryptosporidiumwhich is round and 4–6 μm in diameter.
Autofluorescence can be seen, but this property is not consistent in Cystoisospora.
In molecular techniques PCRs using SSU rRNA primer have shown excellent sensitivity and specificity for the detection of C. belliin fecal samples. But these tests are not commercially available [83, 84] (Figure 5).
Treatmentis done by giving oral co-trimoxazole 160/800 mg BD for 10 days and in case of HIV-infected patients three times daily for 3 weeks. Other drugs like ciprofloxacin, pyrimethamine, and folinic acid are good alternative.
2.5.4 Prevention and control
To date no vaccines are available for Cystoisospora belli; however, trials are going on to identify the suitable candidate of parasite for vaccine preparation . Hence, prevention can be done by avoiding contaminated food and water consumption.
2.6 Dientamoeba fragilis
It was initially considered as amoeba but recently classified under ameboflagellate as it has internal flagellum. It mostly involves the lumen of the cecum and upper colon of the human intestine.
Epidemiology—The reported prevalence of Dientamoeba fragilisin the year of 2015 was around 1–70% in developed settings . It varies globally as in United States and Australia; the reported prevalence is 5%, and in Netherland it was around 8% .
Many debates are there for the pathogenicity of D. fragilis[88, 89]. Previously it was thought that in D. fragilis, cyst stage has not been found , but recently both the cyst and precyst stagehave been identified . The nuclear chromatin of trophozoite of D. fragilisis fragmented into 3–5 granules; hence, it is known as fragilis. Although the mode of transmission remains unknown, previously it was thought that infection occurs via the pinworm vector because of its high rates of coinfection with Enterobius vermicularis. But according to recent studies, it is found that transmission occurs directly via the fecal-oral route .
Clinical symptoms of Dientamoeba fragilisinfection include abdominal pain, persistent diarrhea, loss of appetite, weight loss, and flatulence. Symptoms of D. fragilisare similar to those of irritable bowel syndrome (IBS); hence, it should be considered in the differential diagnosis of IBS .
2.6.3 Immune dysfunction
Dientamoeba fragilisis often described as “neglected parasite” . Recently, several studies have occurred on the life cycle and molecular biology of the organism, but the knowledge on immune response against the pathogen is lacking. Because of the close relativity of Dientamoeba fragilisto histomonads, the immunological research on H. meleagridiscan give an indication to the immunological responsiveness of host against D. fragilis.
It is based on the microscopic detection of trophozoites in fresh or fixed stool specimens. The trophozoites degrade soon after passing in the stool; hence, the examination should be done immediately. Modified iron-hematoxylin or trichrome staining of fixed smears is considered the gold standard for the diagnosis of D. fragilisinfection . But in comparison to molecular methods, this method is time-consuming and relatively insensitive . The demonstration of the characteristic nuclear structure is needed for the definitive diagnosis of D. fragiliswhich cannot be seen in unstained fecal material. Hence, fixation and staining are necessary for definitive diagnosis of D. fragilis.
Antigen detection by rapid fecal immunoassays like enzyme immunoassays, fluorescent antibody, and rapid cartridge formats is previously under trial, but now enzyme immunoassay and immunofluorescence tests are available for antigen detection .
Antibody detection can be done by using an indirect immunofluorescence method .
For the cultivation of D. fragilis, no axenic cultures exist, and it will grow well in xenic cultures. For growth of D. fragilis, Loeffler’s slope medium and modified Earle’s balanced salt solution media are used .
Recently, the more advanced tests based on various conventional and real-time PCRs are used to detect the small subunit (SSU) rRNA gene of D. fragilis.These tests are more rapid, sensitive, and specific for the diagnosis in fresh stools . Molecular methods are more sensitive than microscopy and staining but still not commercially available. Dientamoeba fragilisis not included in the gastrointestinal panel of BioFire FilmArray system .
For the treatment of D. fragilisparomomycin, secnidazole, iodoquinol, tetracycline, ornidazole, and metronidazole have been used successfully. The treatment is recommended both in symptomatic patients and asymptomatic family members to prevent reinfection. There are few studies which show emergence of resistance or treatment failures of metronidazole therapy; it suggests that the combination therapy should be used for these protozoa . For the complete eradication of the parasite and in resolution of symptoms, combination therapy is effective .
2.6.6 Prevention and control
By improving personal hygiene, Dientamoebainfection can be prevented. No vaccines available yet for Dientamoeba fragilis.
Blastocystisspp. are commonly isolated from stools of humans and animals . There are numerous subtypes of Blastocystisspp., but it is unclear whether any of these subtypes are specific to humans disease. It was found that Blastocystissp. subtype 3 is most commonly associated with illness in human [13, 98]. It appears in various morphological forms like vacuolar, granular, amoeboid, and cyst forms, and less frequently avacuolar and multivacuolar cells and cells containing filament-like inclusions [99, 100].
Epidemiology—The prevalence of Blastocystisspp. in developed countries is 0.5–23% and in developing countries 22–100%. The prevalence varies in different geographical are according to the socioeconomic status of that area .
Transmission is usually taken place via the fecal-oral route through the consumption of cyst. These cysts may be waterborne or foodborne. There are many zoonotic subtypes of Blastocystisspp. which supports the increased potential for zoonotic transmission .
Life cycle—Infection is acquired through the consumption of cyst, and it converts to vacuolar, granular, and amoeboid form in human intestine. Cyst form is excreted in the feces and contaminates the food and water ingested by other animals.
Clinical features of Blastocystisspp. infection include mostly asymptomatic or nausea, anorexia, abdominal pain, flatulence, and acute or chronic diarrhea. Blastocystisspp. have been found to be the most common enteric organisms isolated from diarrheal patients but have been reported as noninfectious pathogens or a member of normal gut microbiota .
2.7.3 Immune dysfunction
According to a study, the incubation of Blastocystissp. ST1 cells or culture filtrates induces the production of proinflammatory cytokines interleukin (IL)-8 and granulocyte-macrophage colony-stimulating factor. It suggests that the parasite was able to modulate the host immune response. Blastocystissp. releases cysteine proteases, which induce human colonic epithelial cells (HT84) for the production of IL-8. Cysteine proteases of Blastocystissp. are involved in parasite survival in vivo and represent virulence factors. Blastocystissp. also secretes protease, which is able to cleave human secretory immunoglobulin A. Apart from proteases, hydrolases and protease inhibitors were predicted to be secreted by Blastocystissp. which participates in the blastocystosis physiopathology and its mechanisms for immune evasion .
The diagnosis of infection with Blastocystisspp. is done by using wet mount smears, iodine staining, trichrome staining, or iron-hematoxylin staining and detecting the vacuolar, granular, amoebic, or cystic form in stool samples [13, 100]. Trichrome staining of fecal smears or xenic in vitro culture systems has better sensitivity . Subtype 7 which is a slower-growing subtype of Blastocystisspp. may be missed with this procedure .
For antibody detection in serum ELISA and immunofluorescent assay are also available. ELISA for the antigen detection in the stool is also introduced recently .
In molecular techniques, PCR using the SSU rRNA gene is being used increasingly for the detection of Blastocystisspp. with increased sensitivity, and it is more commonly used method nowadays . Based on SSU rRNA 17, subtypes of Blastocystisspp. were identified out of which ST3 is the commonest worldwide  (Figure 6).
Drugs used for the treatment of pathogenic Blastocystisspp. include metronidazole, co-trimoxazole, nitazoxanide, and a combination of paromomycin and metronidazole. Among these metronidazole is the drug of choice, although failures of this drug in eradicating the organism are common .
2.7.6 Prevention and control
Prevention can be done by avoiding the contaminated food and water consumption; no vaccines are available for Blastocystisspp. till now.
2.8 Balantidium coli
Balantidium coliis the largest protozoan that infects humans. It belongs to the ciliate group. The whole body of the organism is covered with a row of cilia which is responsible for the rotary motility of B. coli(Figure 7).
It mostly occurred in tropical and subtropical areas of developing countries. The estimated prevalence of B. coliworldwide is 1% as the human infection with this protozoa is rare .
Transmission—Pigs are the natural host for B. coli. Human acquires infection by ingestion of cysts present in water and undercooked food (fecal-oral route).
Life cycle—After ingestion of cyst, it converts into trophozoite in the large intestine of human. Both cyst and trophozoite are excreted into the feces, but in external environment, trophozoite disintegrates and cyst will survive. It contaminates the water and food, and the life cycle continues.
Clinical feature—Infections of B. coliare mostly asymptomatic, but in symptomatic patients, mild diarrhea and abdominal discomfort have been reported. In a few patients fulminating acute balantidiasis, with intestinal perforation, can develop which has a case fatality rate of about 30%. Fulminating dysentery, resembling amoebic dysentery, can also occur in B. coliinfection .
2.8.4 Immune dysfunction
Immune response in B. coliis poorly understood due to the lack of studies available. It was found that infection of B. coliis more severe in immunocompromised patients.
B. coliis diagnosed by its large size cysts (50–70 μm) and trophozoite (30–200 μm × 40–70 μm) and rotatory motility which is demonstrated by wet mount slide preparations. Trophozoites of B. coliare visible even with hand lens and, sometimes with the naked eye, in freshly collected diarrheic stools as well as in bronchoalveolar wash fluid . Collection of stool samples should be done for several days because the excretion of parasites can be erratic .
Balantidium colican be cultivated on Boeck and Drbohlav egg serum media and Balamuth media. It can also be cultivated on Pavlova xenic culture media, and the growth of trophozoites can be obtained within 72 h on this media .
B. coliis treated with tetracycline or metronidazole:
Tetracycline—500 mg qid for 10 days
Metronidazole 750 mg tds for 5–7 days
2.8.7 Prevention and control
By avoiding contamination of food and water with pig and human feces and by treating the carrier shedding the cyst, the infection can be prevented. No vaccines are available till now for Balantidium coliinfection.
Enteric protozoa are responsible for severe morbidity and mortality in humans. Protozoal infections contribute to the high burden of infectious diseases. Despite recent advances in the epidemiology, diagnostic tool, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist; hence, protozoal infections require further research. Both humoral and cellular immunities play an important role in the protection of host against enteric protozoal infection. Protozoa have some virulence factor or mechanism to evade the immune system of the host, for the production of disease. An already dysfunctional host immune system (immunocompromised conditions) helps the protozoa to cause easy and more serious disease. Among these enteric protozoal infection Entamoeba histolytica, Giardia intestinalis, Cryptosporidium, Cyclospora, Cystoisospora belli, Dientamoeba fragilis, Blastocystis, and Balantidium coliare common. In the recent years, increasing trends of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidiumis seen both in developing and developed countries. Blastocystis is an emerging infection to humans. Mostly enteric protozoal infections are acquired through fecal-oral route and present with abdominal pain and fever mainly. Some of them can lead to anemia and malabsorption syndrome. If not diagnosed or treated timely, enteric protozoal infection can be fatal. Diagnosis for most of them is based on microscopy,but recently many molecular methods are available to diagnose them. By using multiplex PCR, BioFire Gastrointestinal panel, common diarrheal illness-causing organisms can be identified in a shorter time span. ELISA and immunofluorescent assay are also available for some of them, e.g., Blastocystisspp. and Dientamoeba fragilis. For the treatment of protozoal infection, drugs like metronidazole, paromomycin, diloxanide furoate, nitazoxanide, cotrimoxazole, etc. are used. The infection can be prevented by good personal hygiene and by avoiding food and water contamination. Till date, no vaccines are available for human use against these protozoal infections.
I want to thank our technical staff for their support.
I also want to thank my juniors for their assistance and support.
Special thanks to the whole microbiology department, my family, and my teachers for their guidance.
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