For many years, diagnosing cryptosporidiosis relied heavily on conventional techniques followed by immunological assays. While these methods served as the initial diagnostic tools in laboratories globally, they present significant drawbacks. Traditional approaches are often time-consuming, demand highly skilled microscopists, are labor-intensive, and are susceptible to both false-positive and false-negative results. These limitations inherently reduce their overall sensitivity and specificity in accurately detecting Cryptosporidium.
The advent of molecular methods, particularly Polymerase Chain Reaction (PCR), has revolutionized diagnostic laboratories. PCR-based techniques offer enhanced sensitivity, capable of detecting minute quantities of Cryptosporidium, ranging from just 1 to 106 oocysts.9 Furthermore, molecular methods are relatively rapid and provide a crucial advantage: the ability to differentiate Cryptosporidium species. This speciation capability is invaluable from an epidemiological perspective and is instrumental in understanding potential transmission routes of the parasite.
Several nucleic acid detection methods are now employed in Cryptosporidium diagnosis. These include PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), Multiplex Allele-Specific PCR (MAS-PCR), and quantitative Real-time PCR. Each technique offers unique benefits in terms of specificity, speed, and application.
Species identification in Cryptosporidium diagnosis using molecular methods often targets specific genes. Common gene targets include 18S rRNA, TRAP C1, COWP, Hsp 70, and DHFR genes.9 Beyond species identification, further subtyping is critical for detailed epidemiological studies and understanding transmission dynamics. Subtyping tools leverage genetic markers such as the glycoprotein (GP) 60 gene, minisatellite, and microsatellite markers, and can also involve the analysis of extrachromosomal double-stranded RNA elements. These advanced techniques provide a deeper understanding of Cryptosporidium diversity and strain variations.
Nested PCR assays have proven highly effective in detecting a broad range of pathogenic Cryptosporidium species. Utilizing small-subunit rRNA-based PCR-RFLP, nested PCR employs external primers of 1325 base pairs (bp) and internal primers of approximately 826 bp. This method is particularly advantageous for detecting low numbers of oocysts (<100) in a sample. Consequently, nested PCR has become a widely adopted and validated technique across numerous laboratories worldwide for sensitive cryptosporidium diagnosis.
MAS-PCR, based on the dihydrofolate reductase gene sequence, offers a streamlined approach to differentiate between Cryptosporidium hominis (producing a 357 bp amplicon) and Cryptosporidium parvum (yielding a 190 bp amplicon) in a single reaction step.37 This rapid differentiation capability makes MAS-PCR a valuable diagnostic tool, especially in human outbreak investigations where distinguishing between these two prevalent species is crucial for public health responses.
Commercial multiplex assays have also emerged, significantly enhancing the efficiency of pathogen detection. The Milwaukee Health Department Laboratory, for instance, developed and validated a 19-plex Gastrointestinal Pathogen Panel using Luminex xTAG analyte-specific reagents (ASRs).38 This comprehensive commercial test allows for the simultaneous screening of various diarrhea-causing pathogens directly from fecal specimens. The panel includes detection capabilities for 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 Clostridioides difficile), 3 parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and 4 viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A).38 Such multiplex assays represent a significant advancement in rapid and comprehensive cryptosporidium diagnosis and broader gastrointestinal pathogen screening.
Real-time PCR has gained prominence in Cryptosporidium diagnosis over the last decade. By exploiting the genetic polymorphism within the 18S rRNA gene, real-time PCR offers increased sensitivity, faster detection times, and the capability for quantitative analysis. The closed-system format of real-time PCR assays also minimizes the risk of contamination, enhancing assay reliability. A key advantage of real-time PCR is its quantitative nature, providing invaluable information for estimating the degree of environmental contamination. Recently, a novel real-time PCR assay targeting the telomeric Chos-1 gene has been reported.39 This innovative assay focuses on identifying subtelomeric regions of C. hominis and C. parvum, leading to improved genomic analysis and potentially more refined diagnostic capabilities.
Loop-mediated isothermal amplification (LAMP) has emerged as a user-friendly and specific diagnostic method for numerous organisms. Recognizing its potential in cryptosporidium diagnosis, researchers evaluated LAMP for the first time in 2007 using environmental and fecal samples.40 The primer set employed in this LAMP assay targeted the 60-kDa gp60 gene of C. parvum, amplifying a 189-bp product. This initial study suggested that LAMP holds promise as a valuable diagnostic tool for cryptosporidiosis. Further comparative studies have reinforced this potential. For example, one study compared three LAMP assays (SAM-1, HSP, and gp60) with nested PCR using fecal samples.41 The findings indicated that LAMP could serve as an effective and practical tool for epidemiological surveillance of Cryptosporidium, offering a simpler and potentially more accessible diagnostic option in various settings.
Despite the advancements in molecular methods, challenges persist in the diagnosis and study of cryptosporidiosis. The parasite’s limited ability to be cultured artificially in the laboratory and the lack of ideal experimental animal models hinder in-depth research. Consequently, research progress in this area remains relatively constrained. Currently, effective treatments and vaccines for cryptosporidiosis are still lacking. Nitazoxanide remains the only FDA-approved drug, and its effectiveness is primarily demonstrated in immunocompetent individuals. Therefore, emphasizing prevention and early cryptosporidium diagnosis are paramount strategies in managing and controlling this infection.
Detection of Cryptosporidia in Environmental Samples: Water and Food Safety
Detecting parasitic pathogens like Cryptosporidium in environmental samples, including water and food, presents unique challenges. Standard methods for water sample analysis involve large volume sampling (10–1000 L), followed by concentration techniques such as filtration and the use of magnetic beads coated with chitin or specific antibodies. Detection typically relies on indirect fluorescent microscopy or molecular techniques like PCR.42
Similar procedures are applied to food products, involving elution and subsequent detection methods.43 However, these processes are often laborious and time-consuming. Efforts are underway to develop automated technologies to streamline the detection of these parasites in environmental samples, aiming for broader applicability and faster results. A significant hurdle in environmental sample analysis is the typically low number of parasites present, which can challenge detection sensitivity. Another critical challenge is differentiating human-pathogenic Cryptosporidium species from other species commonly found in environmental matrices, which may not pose a significant threat to human health but can complicate diagnostic interpretation.
References
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