Cryptosporidium and waterborne outbreaks – A mini review

Abstract

Cryptosporidium spp. is one of the prime agents of infectious diarrhea. Cryptosporidium spp. has been gaining awareness as a pathogen of public health importance in India and other developing countries. Owing to the nature of multiple transmission routes such as person-to-person, animal-to-person, waterborne and foodborne, the epidemiology of cryptosporidiosis in humans is not well known. A deeper understanding of the pathogenesis may lead to better diagnosis and better treatment of the condition. Asymptomatic human and animal transmission illustrates that the spread of infection through the environment is a more plausible explanation, waterborne transmission in particular. The disease burden is underestimated and its global impact is yet to be quantified due to the lack of country-specific estimates. Assessment of the disease itself has been crucial since the morphological indistinguishability, differences in distribution and transmission, and variations in the genotypes.

INTRODUCTION

Cryptosporidium is an intracellular protozoan parasite belonging to the phylum Apicomplexa. It has emerged as one of the important causes of diarrheal diseases worldwide. It causes disease in humans and other vertebrates also. Immunocompromised individuals, pediatric age group, and preweaning animals are particularly vulnerable. Water is an important transmission medium for Cryptosporidium which is attributed to the capacity of Cryptosporidium to crack through several barriers to water treatment due to its small size and survival in the environment for long durations. Treatment options are limited and also there is a lack of vaccines against this organism. Since it is a highly infectious and transmissible protozoan, it is classified under category B bioterrorism agent by Centre for Disease Control and Prevention.[1]

This waterborne infectious agent is a major public health concern among both developed and developing nations. The incidence and the burden of this parasite have always been underestimated. Protozoa has been identified as the cause of numerous waterborne epidemics in the last three decades.[2] Cryptosporidium has been ranked among the top food and waterborne parasites by the World Health Organization. There are currently over 40 recognized species of Cryptosporidium.[1] However, not all the species and subtypes of the organism pose a risk to humans, and detection of these parasites may not prove to cause infection.

The infection is spread by oocysts and is primarily acquired through the fecal–oral route. Infection is often caused by oocyst ingestion, either by direct contact with human or animal waste or by indirect exposure to polluted water or food.[1] The spherical thick-walled, environmentally hardy oocysts, shed in the fecal material of the infected host, are immediately infectious, unlike other coccidian parasites, and have a low infectious dose.[3] Incubation period normally ranges from 3 to 12 days and the symptoms vary in severity. Severity varies in part with the patient's general health, and those with moderate, self-limiting diarrhea are unlikely to seek medical attention. It is distinct from other coccidial parasites in such a way that it does not go deep into the host cells but is limited to an extracytoplasmic area intracellular. All the stages of development take place in a vacuole along the brush border epithelium of the small intestine of the host.[3]

LIFE CYCLE

Cryptosporidium sp undergoes a complete life cycle in a single host. The parasites form three developmental stages: meronts, gamonts, and oocysts. Its life cycle comprises multiple rounds of the asexual division followed by subsequent sexual division and fertilization leading to the formation of oocysts. The oocysts contain four sporozoites which on the action of pancreatic enzymes and bile get excysted and the sporozoites are released in the intestine.[4] The sporozoites are then taken up or ingested into an extracytoplasmic vacuole which is called the parasitophorous vacuole in the brush border epithelium of the intestine.[5] The sporozoite undergoes schizogony within the vacuole, which results in the formation of eight merozoites called type one meronts. These merozoites are capable of invading the adjacent epithelial cells and spread the infection to other intestinal sites. These merozoites undergo repeated schizogony to form type two meronts. Four merozoites are released by this type two meront. Such merozoites undergo gametogony and differentiate further into microgamete and macrogamete, which fertilize further to form a zygote. The diploid zygote will form four sporozoites inside thick- or thin-walled oocysts through a process called sporogony. The thick-walled oocyst, protected by a resistant wall, after releasing in the feces is shed into the environment, ready to infect a new individual.[4]

PATHOGENESIS

Cryptosporidium biology is not well known due to the lack of in vitro culture systems, defined developmental stages, stage-specific protein markers, and genetic studies. The main mode of entry may be due to the ingestion of the infective stage which is the oocyst. The Cryptosporidium oocyst wall protein (COWP 1-9) encodes proteins involved in oocyst wall formation and helps as contributing factor for its survival in the environment for a long time.[6] Cryptosporidium causes infection by inflammatory damage to the intestinal epithelium. It alters the function of the intestinal barrier, increasing its permeability, absorption, and secretion of fluid and electrolytes. A sporozoite-specific lectin adhesion factor was found to be the attachment agent on the intestinal surface.[7] In Cryptosporidium parvum, gp 40, is the key surface protein being examined and analyzed for its function in the invasion, but the entire mechanism of the sporozoite or merozoite invasion into host cells has not yet been identified. It is known to be secreted by sporozoites during gliding motility. It has been hypothesized that after sporozoite attachment, epithelial mucosa cells release cytokines such as TNF-alpha, IL-8, and prostaglandins that activate macrophages. These factors increase intestinal secretion of chloride and water and decrease the sodium absorption coupled to glucose transport. Cell damage could occur through T-cell-mediated inflammation. Recent studies indicate that cryptosporidiosis may be transmitted by inhalation of aerosolized droplets via respiratory secretions or by coughing, in addition to the well-documented fecal–oral transmission.[8]

WATERBORNE TRANSMISSION

The emergence of large outbreaks of Cryptosporidium from drinking water sources in the 1990s underscored the need for close monitoring by health authorities and water quality requirements and regulations were developed thereafter. The actual burden of this disease is unknown due to the underreporting of infections and outbreaks. In 2019, of 71 potable water samples analyzed from in and around Chandigarh, 16% were contaminated with Cryptosporidium oocysts and/or Giardia cysts.[9] Cryptosporidium and Giardia concentrations in deep and shallow tube wells were measured in the period 2012–2013 in 206 tube wells in Odisha, out of which Cryptosporidium oocysts were detected in 14% of deep (n = 110) and 5% of shallow (n = 96) tube wells.[10] Cryptosporidium is the most common cause of outbreaks in recreational water sources like swimming pools due to its chlorine resistive capacity and challenges in removing it by filtration. Despite a high prevalence of human infection, their transmission is underrecognized.[9] Cryptosporidium infects a wide variety of humans and animals and the dose of infectious oocysts is sufficient to pollute the aquatic ecosystem. Based on a global review and meta-analysis, the rank order of prevalence of Cryptosporidium spp based on water type was wastewater > surface water > raw water > drinking water > reservoirs water > groundwater > swimming pool water > marine water.[11] Treatments such as disinfection and chlorination have also been shown to be ineffective against these pathogens if not done properly. According to the CDC, a concentration × time (CT) value of 15300 is required to inactivate Cryptosporidium sp. The CT inactivation value refers to the concentration of free chlorine in parts per million (ppm) multiplied by time in minutes at a specific pH and temperature.[12]

A very small load typically exists in water while they can cause the infection but may not be sufficient for detection.[13] Thus, it is important to include parasitological control in treatment plants and to establish regulations for acceptable concentrations of oocysts based on the subsequent use of water.

DETECTION AND DIAGNOSIS

Human samples

Cryptosporidium infects the mucosal epithelium which causes the release of numerous oocysts in the stool, sputum, or bile which can be used as specimens for detection of oocysts.[12] The oocyst which is shed in feces is the usual diagnostic target. It can be difficult to detect Cryptosporidium oocysts; three fecal samples collected during separate days should be examined microscopically to detect oocysts.14 Visualization of oocysts requires special staining, such as Kinyoun Modified Acid Fast, Ziehl–Neelsen acid-fast, or safranin.[15] Permanent staining provides contrasting colors between the background debris and the organism if present. It enables the examination and recognition of detailed organism morphology of acid-fast organism characteristic under oil immersion microscopy. Cryptosporidium oocysts are round 4–6 μm in diameter having a granular appearance. Concentration techniques such as flotation-zinc sulfate and Sheather's sucrose and sedimentation-formalin ether and formalin ethyl acetate sedimentation allow detection of organisms that may be missed on a direct wet mount.[14] Fluorescent staining like auramine phenol stain is used to stain Cryptosporidium species and they appear brilliant greenish yellow against a dark background when viewed under a fluorescent microscope.

A number of kits for antigen detection are commercially available and provide a more sensitive and specific detection than the routine microscopic examination. Various ELISA kits have been developed for the detection of Cryptosporidium antigen and antibody from stool sample.[16] Immunochromatographic tests are also available for simultaneous detection of Cryptosporidium, Giardia, and Entamoeba on which mouse monoclonal antibodies are precoated on the nitrocellulose membrane. Various ELISAs to test for the presence of IgM and IgG antibodies in patients' serum samples are also available.[16]

Cryptosporidium spp can be detected molecularly in fecal or other clinical samples, such as bile, small intestine aspiration, sputum, broncho alveolar lavage, antral washout, or liver biopsy. Polymerase chain reaction (PCR) amplification of sporozoite DNA in these samples is usually base of the molecular method.[17] Oocysts may first be partially purified by a flotation or sedimentation technique, or usually, DNA is extracted directly from feces and further characterized to confirm the presence of parasitic DNA. The common PCR targets for the detection of Cryptosporidium sp. are 435 bp SSU rRNA gene, COWP gene, and TRAP C2 gene.[2] The SSU rRNA gene is targeted since it is multi-copy and has been widely used in species-identification studies; COWP has broad specificity and is also commonly used, whereas the TRAP C2 gene is used for its small amplicon size.[2]

Environmental samples

Testing of nonclinical samples for Cryptosporidium supports risk assessment and monitoring and investigation of potential sources of contamination and transmission routes and provides microbiological evidence in outbreak investigations. The USA follows the US Environmental Protection Agency and CDC, which have collaborated to monitor the outbreaks, following which many countries have established their own surveillance systems.[18] However, in developing countries including India, there is no agency that organizes and reports such information. Epidemiological models can be developed and data from existing data that has been reported by these organized agencies. USEPA has approved Method 1623 for simultaneous detection of Cryptosporidium.[19] This method is considered the gold standard and requires filtration, immuno-magnetic separation of cysts and oocysts, immunofluorescence analysis to determine protozoan concentrations with confirmation via staining with live dyes (4.6-diamidinophenylindole), and microscopy of differential interference contrast.[20] In comparison with this method, molecular genotyping to identify the infective agents and their characterization by powerful molecular methods such as conventional and real-time PCR will help in a much better understanding of this parasite.

Concentration techniques have been applied for both the protozoan, but their efficiency of usage in environmental samples is questionable. Molecular methods of detection and genetic characterization of these pathogens are more relevant and preferred. The challenges involved in assessing environmental samples are the methods of sampling significant quantities of water, filtration duration, expensive methods of detection and in spite of employing all these a very low probability of identification of cysts and oocysts.[20]

DRUG RESISTANCE

Despite a gamut of studies on therapy for Cryptosporidium, nitazoxanide is the only medication for the treatment for Cryptosporidium infection. Drug and vaccine development against it is limited by a lack of continuous culturing, facile animal models, and less developed molecular tools. Nitroimidazoles like metronidazole is not effective against Cryptosporidium.[21] It is shown that paromomycin is also beneficial through placebocontrolled studies.[22] The parasite is known to form a feeder organelle which is a network of folded, convoluted structures. This feeder layer may sometimes obstruct the molecular transport. This junction might hinder the permeability of drugs and ends up as a major therapeutic obstacle in maintaining drug levels within the intestinal lumen. The inconsistent drug delivery is believed to account for the failure of the drug. Many protozoan parasites have described genes that could be responsible for membrane pump expression. These pumps, which belong to the ABC superfamily of transporters, translocate a wide range of substrates through a variety of cell membranes.[23] Only three ABC transporters have been characterized in C. parvum to date. These include CpABC1 and CpABC2, which are similar to the MRP subfamily, and CpABC3, which groups with the MDR subfamily.[24] These transporters play a major role in the efflux of drugs which lead to improper uptake of drugs.

CONCLUSION

There are currently few options for treating people with Cryptosporidium sp infection. The only FDA-approved drug is nitazoxanide and there are no vaccines against it yet. Understanding and looking deep into the pathogenesis and transmission of Cryptosporidium will further help in identifying new drug targets. With advanced molecular methods available, key intervention points can be established by studying the pathogenesis of the infection, through which further treatment options can be developed. To develop strategies for prevention and treatment, investigating the survival of oocysts inside the host is also crucial. Further research on what promotes immunity against this infection will aid the development of vaccines.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.