Laboratory diagnosis of cryptosporidiosis.

Cryptosporidiosis is a major etiology of persistent and chronic diarrhea in children and immunocompromised patients. In addition, it is also one of the important pathogens causing zoonotic or waterborne outbreaks. A number of conventional and molecular tests are available, but each test is riddled with few limitations. Further, there is a need to develop point of care tests and multiplexed tests for simultaneous detection of multiple pathogens.

INTRODUCTION

Cryptosporidiosis, i.e., infection due to Cryptosporidium spp. is recognized globally as a major etiology of persistent and chronic diarrhea in immunocompromised patients leading to significant morbidity and mortality. In addition, it is also one of the important pathogens causing zoonotic or waterborne outbreaks mostly associated with the contamination of recreational water sites and drinking water.[1] In children, cryptosporidiosis is recognized as an important cause of diarrhea that is associated with chronic malnutrition and premature mortality, especially in developing countries. A large epidemiological survey in African and Asian countries involving about 22,500 children <5 years old has documented Cryptosporidium to be one of the four most common pathogens causing severe diarrhea and increased risk of death in toddlers aged 12–23 months.[23] Due to increasing importance of the parasite, The WHO has included this in the Neglected Diseases Initiative recently.[4] Currently, 30 species are recognized as valid based on mainly molecular data.[5] Human infections are mainly attributed to Cryptosporidium hominis and Cryptosporidium parvum, the former being anthroponotic and latter zoonotic.[567] In addition, few other species, namely, Cryptosporidium muris, Cryptosporidium meleagridis, Cryptosporidium canis, and Cryptosporidium felis have been less commonly associated with human infections.[567] The infection is acquired mainly by ingestion of oocysts and rarely by inhalation. The incubation period in humans can range from 2 days to 2 weeks, and symptoms can last anywhere from few days to months. In spite of this knowledge, it is underdiagnosed and underreported mainly due to lack of suspicion and unavailability of diagnostic modalities available. Even in resource-rich countries like the USA, it is estimated that only 1% of cases are diagnosed and reported.[8]

WHY DETECT CRYPTOSPORIDIA?

Laboratory diagnosis of cryptosporidiosis is required for appropriate management of patients while in the environmental samples, it is usually required for identification of outbreaks, source tracking, assessment of risk factors, and interventions. Since most of the laboratories may not look for Cryptosporidium unless specifically asked for, some selection criteria can be used to look for Cryptosporidium even in the absence of specific request. These include immunocompromised individuals; children <5 years; travelers from nonendemic to endemic countries; farm visits; and local outbreaks.

Feces are the most commonly examined sample while sometimes small bowel aspirates, biopsies or tissue samples may be available.

Presently, Cryptosporidium can be diagnosed by a number of techniques including microscopic examination either by the wet mount preparation or staining the smears with modified acid-fast stain or by fluorescent stains. Immunological methods detecting both antigen and antibody are available. Histological examination of the biopsy and various molecular methods for detection of DNA are also available.

Since the oocysts are shed intermittently, therefore three samples collected on alternate days are considered ideal. Cryptosporidium is classified as a biosafety level II organism, and therefore, the sample should be processed accordingly in a safety cabinet.[9]

MICROSCOPY

Wet mount examination

Cryptosporidia can be demonstrated in the preserved as well as unpreserved stool samples. The samples suspected to be delayed should be preserved in either 10% formalin, sodium acetate formalin, or polyvinyl alcohol (PVA). However, some staining techniques may not be suitable with PVA preserved samples. Formalin-preserved samples may not be best suited for molecular diagnosis. Although oocysts can be detected by light microscopy or phase-contrast microscopy, however these are most often missed unstained. In light microscopy, they are seen as smooth, colorless, and spherical or slightly ovoid bodies, with size ranging from 3 to 8 μm.[9] The sensitivity of microscopy can further be increased by reducing the debris, which is achieved by oocyst concentration from feces. Common methods used for the concentration are centrifugation, Sheather's sucrose flotation method,[10] saturated salt flotation, and Allen and Ridley's formol-ether method.[11] Symptomatic cases generally contain large number of oocysts than asymptomatic ones. Various authors have different views regarding their superiority, but the modified formol-ether method is more widely used as it is more sensitive. Higher speed of centrifugation of 1200 ×g is recommended for better recovery of Cryptosporidium oocysts; however, if the same sample is also to be examined for other parasites including helminths, speed higher than 750 ×g is not advisable as helminthic ova can rupture. In case, there is high clinical and epidemiological suspicion with repeated negative results; a time-consuming and expensive option is immunomagnetic separation.[9] In this technique, the oocyst epitopes exposed on the surface adhere to the magnetizable beads which are coated with monoclonal antibodies (mAbs). The bead–oocyst complex is then concentrated in the presence of magnetic force as the complex gets attracted to the side of the test tube; subsequently, the suspending fluid is aspirated, and the acidic solution is added to liberate the concentrated and purified oocysts.

Staining methods

Cryptosporidium is very small which makes its detection in the presence of fecal debris, a challenge. To overcome this, multiple staining techniques and their modifications have been described in the literature. Romanowsky stains such as Giemsa and Jenner's stain were first to be used for the identification of the oocysts. Oocyst appears semi-translucent with a narrow clear halo around it and stains blue to azure with four to six red or purple eosinophilic granules appearing as dots. At times, “Ghost” forms may also be found with a frosted glass appearance without granules.[9] Although the technique was much easier and noninvasive, it lacked sensitivity and specificity. In 1981, Henriksen and Pohlenz used acid-fast Ziehl–Neelsen (ZN) stain to identify the oocyst, which was modified by Casemore et al. (modified ZN [mZN])[12] with better results, and subsequently, it became the widely used method for oocyst detection. Oocyst appears as red spherules against the pale green background on the slide. The degree of the stain taken by the individual oocyst varies and can be confused with various structures such as fecal debris, yeast cells, and bacterial spores which too stain red but are comparatively smaller; the parasites such as Cyclospora and Isospora oocysts are much larger. Moreover, only the internal structures of the oocyst such as sporozoites and residual body are red while the empty oocysts remain unstained reducing its sensitivity. Safranin-methylene blue stains the oocysts bright orange and can be used in the place of mZN stain.[1314] Various studies have recommended fluorogenic stain auramine-phenol as an alternative to the mZN stain.[141516] It is a rapid procedure and seems to be more sensitive than mZN method.[151617] It can be used as a simple rapid screening technique and may be confirmed later by mZN or Giemsa stain. Many laboratories consider it as a gold standard method as it provides the highest combination of sensitivity and specificity.[18] Smears stained with auramine-phenol or ZN stain have an advantage that the stained oocyst can be scraped off from the slide for subsequent DNA extraction for speciation.[19] The detection limit for unconcentrated stool sample by microscopy has been reported to be 1 × 104 to 5 × 104 while concentration increases the sensitivity by 10 folds.[9]

Electron microscopy

This was the method used for the confirmation of the infection in humans reported in the initial studies[2021] However, its major disadvantage lies in the equipment cost, installation cost, complex processing, and inability to analyze large number of samples.

IMMUNOLOGICAL METHODS

Immunological methods can be based on either antigen detection or antibody detection. These methods have reported to yield good sensitivity and specificity in the range of 93%–100%.[222324252627] While antigen detection tests are useful for diagnosis of acute infection, antibody detection tests are useful in seroepidemiological surveys.

ANTIGEN DETECTION METHODS

Using antibodies labeled with fluorescent reporters

Cryptosporidium oocysts can be using mAbs against oocyst wall antigen (C-mAbs). These mAbs basically recognize the epitopes on the surface of oocysts. Most of the commercially available mAbs are raised against the C. parvum, and no antibody preparation is as such available for the specific epitopes on human pathogenic or animal pathogenic Cryptosporidia. Therefore, different species and genotypes of genus Cryptosporidium which vary in the oocyst epitope expression will fluoresce less intensely. Therefore, the negative samples should always be confirmed by either conventional methods or polymerase chain reaction (PCR) methods.[9]

Using antibodies labeled with enzyme reporters

Kits for antigen detection based on the antibodies which are labeled with enzyme are commercially available for enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) or immunochromatographic (IC) formats. Coproantigen detection ELISA is reported to have variable detection limits of 3 × 105 to 106, which is similar to microscopy. However, depending on the antibody used in the kit, it can have low sensitivity in some cases. However, it has the advantage of good specificity of 98%–100%, and a large number of samples can be processed in a short time.[914152526272829] IC assay is another popular procedure practiced in various laboratories; it is known for its rapid results. Although the specificity is reported to be high (98%–100%), sensitivity is reported to be low.[9] EIA and IC kits are available for individual pathogens or in combination with Giardia and/or Entamoeba histolytica. These tests are suitable to be performed on fresh, frozen, or formalin-preserved samples. These offer the advantage of short test time and multiple results in one reaction device.

Antibody detection methods

The demonstration of the presence of antibodies to Cryptosporidium-specific antigens in serum, saliva, or fecal samples is an indirect diagnostic method for the evidence of infection or exposure. Detection of specific circulating antibody is of benefit only in case of seroconversion, demonstrating elevation in titer, or antibody isotype switching. In the absence of these, the demonstration of antibodies can merely reflect either the current infection or the past exposure to the antigen which is not useful. These tests are, therefore, useful mainly for the seroepidemiological surveys of the disease.

Serum antibodies are produced to the 27-kDa and 15/17-kDa antigens on the sporozoite surface following infection. There are many formats for antibody detection using native antigen or recombinant antigen of Cryptosporidium. Initially, the ELISA was done using crude C. parvum extracts with less specificity than Electro Immuno Transfer Blot, but now the availability of recombinant Cryptosporidium antigens has increased its specificity. ELISA showed promising results in studies with various recombinant proteins. A recombinant 23 kDa antigen (Cp23) has been reported to give comparable results to ELISA using native 27 KDa antigen. It has been reported that antibody to Cp23 correlates with past infection while that to Cp17 suggests recent infection.[30] There are some commercially available tests that employ finger prick blood or oral fluid for detection of these antibodies.[3132] Recombinant rCP41 (cloned and expressed in Escherichia coli) has been used for the determination of human seropositivity.[33] Cp23 (recombinant form of C. parvum 27-kDa antigen) has been used to determine longitudinal infection trends[34] and age-specific seroprevalence.[35] A native 17-kDa protein from C. parvum oocyst has also shown comparable results.[30] However, its reliability for routine use needs standardization of the antigens and further evaluation on diverse samples.

HISTOLOGY

Initial studies proving Cryptosporidiosis in humans were based on the histological findings of the various stages of the parasite in intestinal mucosal biopsy. In tissue sections, the diagnosis is made by demonstrating Cryptosporidium in brush border of intestinal mucosa. The parasites appear as small, basophilic, spherical structures measuring 3–5 μm, arranged in rows or clusters. Special stains are not necessary for tissue sections though these facilitate the screening of sections. PCR and immunohistochemistry may also be performed on paraffin-embedded tissue sections.[836] However, presently, it is rarely used due to multiple disadvantages such as the requirement of invasive procedures and careful processing of the sample. All regions of the intestine are not infected; therefore, sampling errors do occur. Moreover, it is an expensive, time-consuming technique not suitable for routine diagnosis.

MOLECULAR METHODS

For decades, conventional methods followed by immunological methods were the major diagnostics for cryptosporidiosis in most of the laboratories worldwide. These are time-consuming, need-skilled microscopist, are labor intensive, and are prone to false-positive and negative results, thereby reducing its sensitivity and specificity. Molecular methods have revolutionized the diagnostic laboratories with the advent of PCR which are more sensitive with the detection range from 1 to 106 oocysts,[9] are relatively rapid, and have the major advantage of speciation which is very important from epidemiological point and is also helpful in knowing the possible transmission routes.

Various nucleic acid detection methods used are PCR-restriction fragment length polymorphism (PCR-RFLP), multiplex allele-specific-PCR (MAS-PCR), and quantitative real-time PCR.

Various gene targets for the species identification are 18S rRNA, TRAP C1, COWP, Hsp 70, and DHFR genes.[9] Further subtype determination can also be done using subtyping tools such as glycoprotein (GP) 60 gene, minisatellite, and microsatellite markers and also by analysis of extrachromosomal double-stranded RNA elements.

Nested assay detects most of the common pathogenic Cryptosporidium spp. with the small-subunit rRNA-based PCR-RFLP using external primers of 1325 bp and internal of about 826 bp. This is also preferred for detecting small number of oocysts (<100) in the sample and is, therefore, the most popular technique which has been validated in numerous laboratories globally.

MAS-PCR is based on dihydrofolate reductase gene sequence and basically differentiates between C. hominis (357 bp) and C. parvum (190 bp) in a single step.[37] Therefore, it can be a valuable tool in case of human outbreaks.

Milwaukee Health Department Laboratory developed and validated a 19-plex Gastrointestinal Pathogen Panel using Luminex xTAG analyte-specific reagents (ASRs).[38] This commercial test can simultaneously screen for diarrhea-causing pathogens, including 9 bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic E. coli, Shiga toxin-producing E. coli, E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Campylobacter difficile), 3 parasites (Giardia lamblia, Cryptosporidium spp., and E. histolytica), and 4 viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A) directly from fecal specimens.[38]

Last decade has witnessed number of publications of Cryptosporidium with real-time PCR; the parasite is identified by exploiting the genetic polymorphism of 18S rRNA gene. It promises increased sensitivity, increased speed of detection, and qualitative analysis. The closed system format of the assay also reduces the chances of contamination. Its major advantage of quantitation is invaluable for estimating the degree of contamination in the environment. Recently, a novel real-time PCR assay[39] has been reported to target, telomeric Chos-1 gene for the identification of subtelomeric regions of C. hominis and C. parvum with improved genomic analysis.

Loop-mediated isothermal amplification (LAMP) has become a practical diagnostic tool for many organisms due to its simplicity and specificity. Therefore, expecting the same in case of cryptosporidiosis, a number of environmental and fecal samples were evaluated with LAMP for the first time in 2007.[40] The primer set used in LAMP was from 60-kDa gp60 gene of C. parvum which amplifies a 189-bp product. The study suggested that the LAMP could be a useful diagnostic tool. In a study,[41] three LAMP assays (SAM-1, HSP, and gp60) were compared with nested PCR on fecal samples. In this study, the LAMP was proposed to be an effective and useful epidemiological surveillance tool.

Presently, there are a number of technical and practical challenges faced during the diagnosis of cryptosporidiosis. Moreover, the parasite cannot be studied in detail as its artificial culture does not last long in the laboratory, and there are no suitable experimental animal models. As a result very less research is ongoing. As of today, we have neither an effective treatment nor any vaccine for cryptosporidiosis. Only nitazoxanide is the Food and Drug Administration approved and has proved to be significantly effective in immunocompetent healthy participants only. Therefore, prevention or early diagnosis is the best way to fight the infection.

Detection of Cryptosporidia in environmental samples, water, food, etc

Standard method for detection of parasitic pathogens in water samples is large volume sampling of 10–1000 L, concentration by filtration, magnetic beads coated with chitin or specific antibodies, etc., Detection is made usually by indirect fluorescent microscopy or molecular techniques such as PCR.[42]

The food products are processed similarly by elution and detection.[43] However, all these are tedious and time-consuming, and some automated technologies are being developed to detect these parasites for their widespread applicability for environmental samples. However, presence of a low number of parasites in most of these samples presents a challenge for their detection. Another challenge of detection of these parasites in environmental samples is to differentiate main human pathogenic Cryptosporidia from other species commonly found in environment

CONCLUSION

It is thus emphasized that Cryptosporidium should ideally be looked for in all patients with diarrhea, but it must be investigated in children and immunocompromised patients. Further, there is a need to develop point of care tests and multiplexed tests for simultaneous detection of multiple pathogens.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.