Accepted Manuscript Title: Comparison of current methods used to detect Cryptosporidium oocysts in stools Authors: Shahira A. Ahmed, Panagiotis Karanis PII: DOI: Reference:
S1438-4639(17)30469-8 https://doi.org/10.1016/j.ijheh.2018.04.006 IJHEH 13213
To appear in: Received date: Revised date: Accepted date:
17-7-2017 17-4-2018 17-4-2018
Please cite this article as: Ahmed SA, Karanis P, Comparison of current methods used to detect Cryptosporidium oocysts in stools, International Journal of Hygiene and Environmental Health (2010), https://doi.org/10.1016/j.ijheh.2018.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
REVIEW Comparison of current methods used to detect Cryptosporidium oocysts in stools
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Shahira A. Ahmeda,*, Panagiotis Karanisb,*
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt.
b
State Key Laboratory of Plateau Ecology and Agriculture, Center for Biomedicine and Infectious
Diseases, Qinghai University, Xining, Qinghai 810016, P.R. China & Medical School, University
authors:
tel:
+20-10-96238140
and
E-mail
address
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*Corresponding
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of Cologne, Cologne, Germany
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[email protected] (Dr. Shahira); tel: +86-971-152-37040. E-mail address:
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[email protected] (Prof. Karanis).
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Stool Material
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1. Introduction 2. Methods for preservation, concentration and purification of Cryptosporidium oocysts 2.1. Preservatives 2.2. Concentration 2.3. Purification 3. Detection 3.1. Non-molecular 3.1.1. Staining techniques 3.1.2. Immune assay techniques 3.1.2.1. Fluorescence techniques (IFA, FC) 3.1.2.2. Copro-antigen detection techniques (ELISA, EIA, ICT) 3.1.3. Microscopy 1
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3.1.3.1. Light microscopy (LM) 3.1.3.2. Phase contrast microscopy (PCM) 3.1.3.3. Electron microscopy (EM) 3.1.3.4. Laser scanning confocal microscopy (LSCM) 3.2. Molecular 3.2.1. Polymerase chain reaction (PCR) 3.2.2. Fluorescence in situ hybridization (FISH) 3.2.3. Loop-mediated isothermal amplification (LAMP) 3.2.4. Fingerprinting 3.2.5. DNA sequencing 3.2.6. Electrophoretic mutation scanning (EMS) 4. Conclusions
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Abstract
In this review all of the methods that are currently in use for the investigation
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of Cryptosporidium in stool material are highlighted and critically discussed. It appears that
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more qualifications and background knowledge in this field regarding the diagnosis of
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the Cryptosporidium parasite is required. Furthermore, there is no standardization for the protocols that are commonly used to either detect oocysts in faeces or to diagnose
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the Cryptosporidium infection. It is therefore necessary to initiate further education and research that will assist in improving the accuracy of the diagnosis of Cryptosporidium oocysts in the
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faecal micro-cosmos. Where ambient concentrations of oocysts are low in stool material, detection becomes a formidable task. Procedures for ring tests and the standardization of multilaboratory testing are recommended. It is also necessary to enhance the routine surveillance capacity of cryptosporidiosis and to improve the safety against it, considering the fact that this disease is under diagnosed and under reported. This review is intended to stimulate research that 2
could lead to future improvements and further developments in monitoring the diagnostic methodologies that will assist in harmonizing Cryptosporidium oocysts in stool diagnosis.
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Abbreviations ACMV: Analine carbol methyl violet AF: Acid flocculation AOAR: Acridine orange, auramine-rhodamine C. hominis: Cryptosporidium hominis
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C. parvum: Cryptosporidium parvum
ddPCR: Droplet digital PCR
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DFA: Direct immunofluorescence
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CsCl: Cesium chloride
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COWP: Cryptosporidium oocyst wall protein
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DGGE: Denaturing gradient gel electrophoresis DMSO-MAF: Dimethyl sulfoxide-modified acid-fast
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DNA: Deoxyribonucleic acid
DSIP: Discontinuous sucrose and isopycnic percoll gradients
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EIA: Enzyme immunoassay ELISA: Enzyme linked immunosorbent assay
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EM: Electron microscope EMS: Electrophoretic mutation scanning FA: Fluorescent antibody FC: Flow cytometry
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FCCS: Flow cytometry coupled with cell sorting FEA: Formalin-ethyl acetate FG: Ficoll gradient
FITC-C-mAb:
Fluorescein
isothiocyanate-conjugated
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FISH: Fluorescence in situ hybridization anti-Cryptosporidium
antibodies GBC: Glass bead column gp60: 60- kilodalton glycoprotein
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HDA: Heteroduplex analysis
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hsp: Heat shock protein
IFA: Indirect immunofluorescence
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ICT: Immunochromatographic dipstick assay
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hsp70: 70-kilodalton heat shock protein
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IMS: Immunomagnetic separation
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ITS: Internal transcribed spacer
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KAF: Kinyoun acid-fast
K-dichromate: Potassium dichromate
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LAMP: Loop-mediated isothermal amplification LM: Light microscope
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LSCM: Laser scanning confocal microscope MAF: Modified acid-fast
MgSo4: Magnesium sulphate
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monoclonal
Opg: Oocysts per gram stool PAS: Periodic acid-Schiff PBDG: Potassium bromide discontinuous gradient
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PCM: Phase contrast microscope PCR: Polymerase chain reaction PVA: Poly-venyle alcohol qPCR: Real-time or quantitative PCR RFLP: Restriction fragment length polymorphism
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RIHS: Regressive iron hematoxylin-stain
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RNA: Ribonucleic acid
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SAF: Acid-formalin fixatives
SGS: Simple gravity sedimentation
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SEM: Scanning electron microscope
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SMA: Silver methenamine acridine
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SSCP: Single-strand conformation polymorphism
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SSF: Sheather sucrose flotation
SSSF: Saturated sodium chloride salt flotation
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SSU: Small-subunit
TEM: Transmission electron microscope
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ZNAF: Ziehl-Neelsen acid-fast ZSF: Zinc sulphate flotation
Keywords: Cryptosporidium, preservation, detection, microscopy, antigenic, molecular.
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1. Introduction
Cryptosporidium is an Apicomplexan parasite that targets the gastrointestinal tract of most
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vertebrates and humans. From the current 34 species that have been recognised (Koehler et al., 2017; Kváč et al., 2016; Ryan et al., 2016), C. hominis and C. parvum are the most commonly reported pathogenic species in humans. C. hominis can be transmitted directly or indirectly from person to person, while C. parvum is considered to be a zoonotic parasite, with bovines being the main reservoir (Cacciò and Chalmers, 2016; Plutzer and Karanis, 2009; Ryan et al., 2016). The
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species C. hominis and C. parvum are responsible for nearly a million deaths every year (Kotloff
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et al. 2012; Villanueva, 2017). Most infections are self-limited, however, recurrence is frequent
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in endemic areas. Acute cryptosporidiosis can elicit abdominal pain, diarrhoea, vomiting and
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fever. Chronic infection can lead to malabsorption, weight loss and stunted growth in children.
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Cryptosporidium spp. causes numerous outbreaks worldwide compared to any other parasite
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(Baldursson and Karanis, 2011; Efstratiou et al., 2017a; Karanis et al., 2007). To date, at least 905 outbreaks are associated with the waterborne transmission of protozoan parasites. The most
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common aetiological agent was Cryptosporidium spp. (reported in 60-63% of these outbreaks) (Baldursson & Karanis 2011; Efstratiou et al., 2017a; Karanis et al., 2007).
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Cryptosporidium oocysts are shed in the stools of both humans and animals, once shed the oocysts lead to the contamination of water, soil and plants (Leitch and He, 2012). To control
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cryptosporidiosis it is essential that precise diagnosis is achieved through accurate identification and characterization. The examination of fresh faecal samples is essential for the diagnosis of Cryptosporidium oocysts. Oocysts are small in size (4-6 µm) and they can be mistaken for yeasts because of their shape. Many researchers therefore face challenges in the accurate identification
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and diagnosis of Cryptosporidium which impacts the accuracy of the reporting on the infection during a simple faecal examination (native or flotation procedure, see below). In developed countries, the diagnosis of Cryptosporidium oocysts relies on using several
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diagnostic tools, aimed to either observe the morphology of the parasite or to characterize it molecularly to the species/genotypes level. In developing countries, the scenario is quite different; accessibility to molecular equipment is limited in some labs and totally absent in others. Morphological identification of Cryptosporidium oocysts by microscopy is the most widely used method for the diagnosis due to its relatively low cost. Different species of Cryptosporidium
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have different sizes of oocysts but they also overlap and morphological identification is therefore
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difficult. Researchers are obligated to use staining techniques for the diagnosis, with
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considerable limitation to further analysis.
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Even though cryptosporidiosis is one of the most common communicable diseases Cryptosporidium infections are still under-diagnosed and under-reported (Cacciò and Chalmers,
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2016; Karanis, 2017). The fact that some laboratories do not include Cryptosporidium in their
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routine examination of stools contributes to this problem.
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The quantity and quality of the faecal sample and the amount of oocysts/antigen are essential factors that will determine the success of the detection method. For an accurate diagnosis, several
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sample investigations are necessary, particularly in the case of subclinical infections. Diagnosis via biopsy is not the method of choice for humans and it would only be ethically accepted if
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symptoms are persistent and stool analysis still remains negative. The diagnostic methods (Fig. 1) have their limitations, particularly those based on stains and rapid immuno-chromatography assays as they might not be sufficiently sensitive for diagnosing vulnerable patients. Increased laboratory testing would improve the epidemiological knowledge,
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speciation and subtyping of the Cryptosporidium parasite (Cacciò and Chalmers, 2016; Painter et al., 2015; Eftsratiou et al., 2017b). The final step of the diagnostic procedure is the detection and identification of the species. Methods used over the years for the detection of Cryptosporidium
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vary, with the most notable ones being microscopic and molecular based. Therefore the purpose of this review is to investigate the methods used in the diagnosis (directly or indirectly, routinely or not routinely used), of Cryptosporidium oocysts in stools, with a focus on their favourable and unfavourable characteristics and to summarize all of the techniques in the bibliography that have been used.
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2. Methods for stool preservation, concentration and purification of Cryptosporidium oocysts 2.1. Preservatives
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Stool material is the most diagnostic material available for the diagnosis of pretty much all
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intestinal pathogens. It can provide information on enteric pathogens in the gastro-intestinal-
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tract . Identification of Cryptosporidium oocysts in faecal samples is the most widely used
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method for the diagnosis. For both conventional and/or molecular investigations of Cryptosporidium spp. a stool sample is required. To keep the morphological integrity of such
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parasites in faecal material, preservation is necessary. The most commonly used stool preservatives (Table 1) are formalin 10%, sodium acetate-acetic, acid-formalin fixatives (SAF),
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poly-venile alcohol (PVA), potassium dichromate (K-dichromate) and absolute ethanol (Garcia
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et al., 1983; Greene, 2013; Jongwutiwes et al., 2002). To eliminate the oocyst infectivity, formalin, SAF and PVA are acceptable reagents (Greene, 2013). These reagents help to reduce the risk of infections amongst the hospital staff and the staff in the clinical laboratories (Garcia et al., 1983), however, a strong inhibitory effect of formalin drastically decreases PCR detection ability, resulting in non-reproducible outcomes (Johnson et 8
al., 1995; Jongwutiwes et al., 2002). Many oocyst staining methods are not compatible with PVA preserved specimens (Fayer and Xiao, 2007; Greene, 2013). To overcome such disadvantages, the addition of D' Antoni's iodine to the specimen will facilitate the removal of mercuric chloride;
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the smear could then be stained with Giemsa or acid-fast stains (Garcia et al., 1983). Mercury is one of the PVA ingredients, however, the high cost of its disposal becomes a problem in most laboratories (Garcia et al., 1993; Pietrzak-Johnston et al., 2000). Such issues guide numerous laboratories to use other preservatives.
K-dichromate preservative maintains oocyst viability (Johnson et al., 1995; Simjee, 2007);
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however, it causes PCR inhibition problems, washing will therefore be an essential step prior to
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DNA amplification (Johnson et al., 1995; Jongwutiwes et al., 2002). K-dichromate preserved
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specimens need to be stored in a cold environment (4 °C). The most challenging part of requiring
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a refrigerator is the amount of space needed to store a large number and/or volume of samples. To reduce bio-hazardous risk for laboratory workers, ethanol is used as an alternative
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preservative. Oocysts morphology and its DNA property can be preserved for more than 2 years.
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Cold storage of ethanol preserved specimens is not required and even though there are benefits to
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ethanol, it will not maintain oocyst viability (Jongwutiwes et al., 2002; Lalonde and Gajadhar, 2009). Freezing (-20 °C) is another method to preserve DNA property. It was the best method for
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excellent PCR yield when compared with formalin 10%, K dichromate and ethanol (Abdelsalam et al., 2017).
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Preservation of specimens allows for later processing. As noted above, the choice of preservative mainly depends on what further investigations are required (stain, maintain oocyst viability and/or proceed for molecular testing) (Table 1).
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2.2.
Concentration techniques
The excretion of oocysts in stools is characterized by daily irregularity and heterogeneous distribution, hence, the concentration technique being highly recommended (Table 2).
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Concentration techniques eliminate faecal debris which may cofound the correct diagnosis and increase the yield of Cryptosporidium oocysts, especially in asymptomatic individuals with a low discharge of the parasite (Casemore, 1991; Pacheco et al., 2013). Concentration techniques are presented by either flotation or sedimentation methods. Flotation methods use a liquid suspending medium that is denser than the oocysts being concentrated. This can be used for the
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purpose of both concentration and purification. The most commonly used solution or media are:
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sheather sucrose flotation (SSF), zinc sulphate flotation (ZSF) and saturated sodium chloride salt
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flotation (SSSF).
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SSF and ZSF are easy techniques to perform (Ma and Soave, 1983), they selectively concentrate viable oocysts (Bukhari and Smith, 1995). In SSF preparations the oocysts appear as clean, pink,
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refractile objects (Ma and Soave, 1983). Although oocysts were easy recognizable, preparation
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of SSF solutions requires exact specific gravity and it is quite difficult to handle its high viscosity.
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The oocysts tend to collapse and disappear within 15 minutes of SSF wet mount preparation; this is an issue that necessitates immediate examination. SSF elicits inhibition of the process when it
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is used with staining techniques. This therefore explains its inferiority to some of the other concentration techniques f.e. Formalin-ethyl acetate (FEA) (MacPherson and McQueen, 1993;
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McNabb et al., 1985; Shaista et al., 2016; Sheather, 1923; Weber et al., 1992, 1991). Acid flocculation concentration method (AF) separates oocysts from fibrous faecal material. When SSSF pursues AF, the detection level of Cryptosporidium oocysts increased to five oocysts / gram of faeces. Using a large starting sample (50 gram) with this combination could
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explain its efficiency (Wells et al., 2016). Even considering the advantages of the flotation methods (SSF, ZSF and SSSF), none of them were able to reach a 100% positive detection rate (Rezende et al., 2015). This reinforces the benefit of combining diagnostic techniques.
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The most commonly used sedimentation techniques for the concentration of Cryptosporidium oocysts are simple gravity sedimentation (SGS), water ether and formalin-ether (FE) or (FEA) (Truant et al., 1981). SGS is a cost effective technique but it uses minimal materials with less financial resources. Although SGS produces large amount of faecal debrissome researchers prefered it to FEA (Pacheco et al., 2013; Rezende et al., 2015). FEA produces a cleaner
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preparation compared to SGS as it removes fats and fibers from the stool material. FEA is
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recommended for the diagnosis of human coccidian and intestinal protozoa (Garcia and Shimizu,
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1981; Pacheco et al., 2013). Through the FEA procedure, significant loss of Cryptosporidium
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oocysts was noted in the fat layer, this requires slide preparation from both the faecal pellet and fatty plug to avoid potential false-negative tests (Pacheco et al., 2013).
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During fuchsin staining of the FEA prepared specimen, ghost oocysts (weak colour or unstained)
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were formed. This might be due to ethyl acetate residues. In which, it could verify why FEA
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produced inconsistant results with some researchers (Casemore et al., 1985; Clavel et al., 1996, 1994; McNabb et al., 1985; Pacheco et al., 2013; Weber et al., 1992, 1991). In this respect,
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Weber et al. (1992) modified the FEA sedimentation to be followed by layering and flotation over a hypertonic sodium chloride solution to separate the oocysts from stool debris. This
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modification enhanced the detection of Cryptosporidium oocysts in diarrhoeic and regular stool samples. Although this technique requires a longer centrifugation time and additional pipetting it separated oocysts from stool debris very well (Weber et al., 1992). Higher recovery of oocysts was also obtained when the weight of the stool was increased with 10 min centrifugation (Clavel
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et al., 1996). Arguments among researchers raised concerns about the superiority of the concentration techniques (SSF, ZSF, FEA) used for Cryptosporidium oocysts (Bukhari and Smith, 1995; McNabb et al., 1985; Shaista et al., 2016; Weber et al., 1992, 1991).
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Even though previous concentration techniques work well with Cryptosporidium oocysts, they may still not be sufficient with the asymptomatic infected hosts (Coklin et al., 2011; Davies et al., 2003). Immunomagnetic separation (IMS) is nominated as an additional or alternative concentration step to separate Cryptosporidium oocysts but this assay is not commonly used in diagnostic laboratories for the detection of oocysts in stools because it is expensive and
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cumbersome. IMS is mainly used for the detection of oocysts in water samples but not in stools
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and it was used with environmental samples to concentrate/separate oocysts from food, water
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and soil (Bukhari et al., 1998; Di Giovanni et al., 1999; Hohweyer et al., 2016; Orlofsky et al.,
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2013; Perreira et al., 1999; Rochelle et al., 1999; Sturbaum et al., 2002). While in faecal samples, it is used to optimize the detection by PCR, IFA or FC (Atwill et al., 2003; Coklin et al., 2011;
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Davies et al., 2003; Gao et al., 2014; Pereira et al., 1999; Power et al., 2003). IMS removes PCR
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inhibitors (bilirubin, bile salts and complex polysaccharides) from faecal debris, enabling high
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quality target DNA to be extracted (Coklin et al., 2011; Gao et al., 2014; Hadfield et al., 2015). Despite IMS improving the detection rate to less than five oocysts / gram in stool
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material(Robinson et al., 2008), it is affected by the pH during oocyst capture (Davies et al., 2003), it application has been limited and only done for specific studies such as evaluating the
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technique used for the detection of oocysts in stool in human feaces (Robinson et al. 2008) or for the generation of whole genome sequences of C. hominis and C. parvum isolates directly from stool samples (Hadfield et al. 2015) In symptomatic hosts (animals and humans), higher concentrations of oocysts are being shed and the diagnosis in asymptomatic hosts is therefore
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expected to be more challenging. In such situations with low or intermittent shedding the IMS step would be valuable for providing a higher sensitivity detection of oocysts. When IMS is used in diagnoses or surveillance studies, the high costs of beads, the increased time of processing and
2.3.
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the potential for human error should also be taken into consideration (Coklin et al., 2011).
Purification techniques
Purification techniques are used to purify contaminated samples for further sensitive detection
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methods (Table 2).
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The simple or multistep gradient purification strategies to isolate oocysts from contaminants are:
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SSF, SSSF, ZSF, percoll, glass bead column (GBC), dialysis, cesium chloride (CsCl), ficoll
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gradient (FG), discontinuous sucrose isopycnic percoll gradients (DSIP), potassium bromide discontinuous gradient (PBDG) and magnesium sulphate (MgSo4) (Entrala et al., 2000;
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Kuczynska and Shelton, 1999; Suresh and Rehg, 1996; Truong and Ferrari, 2006).
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Percoll® is an expensive reagent and its gradient procedure is time consuming. Although its use
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is labour intensive, it yields a pure oocyst fraction that is free of bacterial contaminants (Entrala et al., 2000); a feature that is lacking with glass beads and dialysis techniques (Suresh and Rehg,
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1996). The low cost PBDG technique can keep viability of purified oocysts and a pure product can only be obtained after multiple washing due to the effect of the faecal fat (Entrala et al.,
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2000).
Eight concentration and purification solutions (MgSo4, ZSF, FEA, CsCl, NaCl, cold sucrose, SSU, and percoll) were evaluated to determine the recovery rate of C. parvum from calves stools. NaCl flotation gave the highst recovery rate among all methods. Over a wide range of oocysts
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concentrations (25 to 105opg); NaCl provided constant recovery rate. NaCl flotation used relatively short time and saving compared to other methods. With other animal’s stools (Cow, horse, pig, sheep, chicken); NaCl produced different recovery percentages. The highest was with
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cow faeces (17%) and the lowest was with chicken faeces (3.2%). Researchers should bear in mind that separate correction factors must be determined for different feacal types (Kuczynska and Shelton, 1999).
It seems that flotation methods are simple, cost efficient and time saving rather than the gradient
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concentration methods.
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3. Detection
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Detection confirmation depends on finding Cryptosporidium oocysts, antigen, and/or DNA in the
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cases of cryptosporidiosis.
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3.1 Non-molecular 3.1.1 Staining techniques
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The small size of Cryptosporidium oocysts and lack of distinctive visibility of their internal/external structures can cause confusion in its diagnosis. Numerous staining techniques
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are used to stain faecal smears (Table 3). Detection and differential diagnosis of other faecal
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parasites such as Cyclospora cayetanensis and Isospora belli is an added benefit (Shimelis and Tadesse, 2014).
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D’antoni iodine is a simple and easy wet mount stain. This impermanent stain can differentiate an unstained and colourless structure of Cryptosporidium oocysts from the brown colour of the other yeasts and faecal contents (Ma and Soave, 1983). The negative stain technique of periodic acid -Schiff (PAS) is a good stain for bacteria and fungi. In contrast, PAS stains protozoa poorly. Such stain identifies Cryptosporidium oocysts with its 14
refractive, spherical and unstained appearance, whereas, other artefacts stain reddish purple, allowing proper differentiation to oocysts and therefore eliminating the confusion (Horen, 1983). The negative staining technique of Heine (undiluted carbol fuchsin) is another simple and
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efficient non-permanent stain of Cryptosporidium oocysts. Oocysts appear as light red due to the carbol fuchsin used in this technique (Heine, 1982; Ignatius et al., 2016; Potters and Esbroeck, 2010). The Heine negative stain is modified by using other reagents such as “malachite green, methylene blue and crystal violet”, in place of carbol fuchsin. Malachite green provided to be the most sensitive stain to detect oocysts in faecal specimens (Khanna et al., 2014).
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Previous techniques (D’antoni iodine, negative stains) could be examined at x400, however, the
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recognition of the colourless oocysts remains tricky to researchers. Thickness of the smear
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should be considered as presence of debris in the stool and it adversely affects the visibility of
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the oocysts. FEA concentration or purification of the faecal sample can overcome such situation. Immediate examination (15-30 min) of the stained slides is also necessary otherwise the oocysts
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will dry out and become less visible.
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Kinyoun acid-fast (KAF), Ziehl-Neelsen acid-fast (ZNAF), modified acid-fast (MAF), dimethyl
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sulfoxide-modified acid-fast (DMSO-MAF), acridine orange auramine-rhodamine (AOAR), silver methenamine acridine (SMA) safranin and leishman are appropriate stains for formalin
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and K-dichromate preserved oocysts, however, Giemsa, Gomori's and Trichrome are appropriate to stain PVA preserved oocysts (Babxy and Blundell, 1983; Bronsdon, 1984; Garcia et al., 1983).
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KAF, ZNAF and MAF are simple, fast and cost effective techniques for the diagnosis of Cryptosporidium oocysts. Oocysts stain bright red due to the staining effect of undiluted carbolfuchsin. Such stains remain the basic diagnostic tool for Cryptosporidium in many laboratories worldwide. Large numbers of samples can be processed at the same time with KAF,
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ZNAF and MAF; however, many oocysts appear as unstained clear oval holes. Poorly stained oocysts against a background of similar size and shape yeast like cells will cause a matter of confusion. A disadvantage that might occur due to the interference of various factors (sugar from
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flotation, KOH digestion of mucus, formalin used to preserve the faeces, PVA and Schaudinn's fixative) and might explain the lack of sensitivity and specificity of such techniques. To avoid false positive results by these stains the faecal sample will require concentration and trained technical staff (Anderson, 1987; Babxy and Blundell, 1983; Bronsdon, 1984; Casemore, 1991; Casemore et al., 1985; Chartier et al., 2002; Garza, 1983; Ghoshal et al., 2018; Jex et al., 2008a;
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McNabb et al., 1985; Pacheco et al., 2013; Potters and Esbroeck, 2010; Shaista et al., 2016;
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Stibbs and Ongerth, 1986).
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DMSO is nominated as a modification of the acid fast technique (Bronsdon, 1984). It simply
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stains oocysts as brilliant pink structures against a pale green background. The slide handling requires less time and oocysts can be easily identified with a low power field (x10). A superior
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penetrating quality of DMSO outperforms the conventional acid fast stain. With such a property,
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the oocysts internal morphology could be preserved. In MAF, disintegration of the acid fast
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particles can cause formation of ghost oocysts. DMSO-MAF shares the disadvantage which requires careful decolourisation (Bronsdon, 1984).
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Auramine/carbol-fuchsin is a fluorescence staining method in combination with MAF. In this technique, the fixation step of MAF is not required anymore and when compared to DMSO-
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MAF, less than 10 oocysts could be identified. Such superiority makes it a rapid and simple screening method. Even though expensive fluorescence microscope is mandatory, yeasts do not fluoresce in this stain. Auramine-stained Cryptosporidium oocysts appear as yellow discs with a pale halo against a dark background, such an advantage efficiently identifies oocysts, even in
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unconcentrated direct smears of diarrheal stools. While, the fluorescence oocysts characteristics are easily observed by an experienced researcher, frequent quality control is still required. Attention moreover should be paid to the potential carcinogenic property of the staining fluid
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(Casemore et al., 1985; Lenga, 1985; MacPherson and McQueen, 1993; Nichols, 1984; Stibbs and Ongerth, 1986; Weikel et al., 1985).
Giemsa is an easy stain for the diagnosis of Cryptosporidium oocysts. Oocysts can be differentiated from yeasts and other artefacts. Oocysts stain as faintly blue with reddish to purple corpuscle even though weak colour contrast must be considered. They can be well differentiated
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regardless of the formation of reddish to dark granules that might cause difficultly in reading.
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Although, this technique might be time consuming, oocysts can be identified well with low
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magnification in heavy infections (Anderson, 1983, 1981; Babxy and Blundell, 1983; Casemore
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et al., 1985; Horen, 1983; MacPherson and McQueen, 1993; Mata et al., 1984; Perez-Schael et al., 1985).
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Analine carbol methyl violet (ACMV) when followed by tartrazine, works as a differential
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staining of Cryptosporidium oocysts in faecal smears. In this stain, oocysts splatter blue to blue-
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violet in colour on a yellow to yellow-green background. With ACMV the smear preparation is transparent regardless of its thickness and yeasts don’t take to the stain. Such merits prove the
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reliability of ACMV (Milacek and Vftovec, 1985). Regressive iron hematoxylin-stain (RIHS) is a simple stain of Cryptosporidium oocysts. The
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faecal smears are overstained and then destained (regressive). Before RIHS, there is no need to fix the wet mount smear; air drying would be enough. In its manoeuvre, hematoxylin solutions need to be ripened quickly, otherwise, a non stain substance will be formed and destroy the staining properties. To preserve the image quality with good differentiation the smears should be
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over stained and the duration of the final washing step must be increased, therefore maintaining the reliability of the stain (Ferreira et al., 2001). Safranin 1% can likewise stain Cryptosporidium oocysts with sufficient colour contrast.
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Crescent-shaped oocysts appear bright orange with a lighter stained centre (clear halo). In the safranin procedure, it is necessary to heat the slide until steam appears, this will require careful individual handling to avoid overheating the slide. Even though the stain manipulation consumes time and might appear inconvenient to some researchers, safranin worked well with five month old samples (Babxy and Blundell, 1983; Casemore et al., 1985).
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Leishman stain (Romanowsky staining) is a known stain for blood samples. The stain was
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effective to provide presumptive diagnosis of Cryptosporidium oocysts (Brar et al., 2017);
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however, still remains for trial with faecal samples from other hosts. From the aforementioned,
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each stain worked well in identifying Cryptosporidium oocysts, however, it allowed for some error. The number of samples, available reagents and experience of the investigator are important
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factors to be considered. Low levels of infection and sporadic shedding is another important
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issue as it could possibly go unnoticed with staining techniques that mainly depend on
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conventional microscopy with low detection limit of oocysts (Weber et al., 1991) (Ghoshal et al., 2018). This matter could possibly be overcome with screening multiple faecal samples on
CC
separate days or combining the staining techniques with another more sensitive and more specific approach to declare a true negative result. It is important to consider that oocysts from
A
different Cryptosporidium species can not be unequivocally differentiated or identified by these staining techniques.
3.1.2. Immunoassay techniques
18
When compared with the conventional staining methods, immunoassay procedures offer both increased sensitivity and specificity. They prove to be helpful when large numbers of patients are screened. The advantage appears particularly when screening patients with minimal symptoms
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and outbreaks situations (Garcia and Shimizu, 1997).
3.1.2.1. Fluorescence techniques
The immunofluorescent antibody (IFA) staining of oocyst walls with fluorescein isothiocyanate-
U
conjugated anti-Cryptosporidium monoclonal antibody (FITC-C-mAb) is a sensitive and specific
N
method of diagnosis in faecal samples from both human and animal sources (Chalmers et al.,
A
2011; Rossle and Latif, 2013; Ryan et al., 2016; Stibbs and Ongerth, 1986). The high sensitivity
M
of this procedure returns to the stability of the parasite antigens in the stool preparations and the
D
assumption that cross-reaction with other organisms does not occur (Jex et al., 2008a; Sterling et
TE
al., 1986; Sterling and Arrowood, 1986). IFA allow visualization of the intact parasites, providing a definitive diagnosis.
EP
IFA has achieved high sensitivity (96-100%), specificity (98.5-100%) and reliability in the diagnosis of cryptosporidiosis (Alles et al., 1995; Garcia and Shimizu, 1997; Jex et al, 2008a
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Mirhashemi et al., 2015). Its high sensitivity in the detection of oocysts appeared when compared
A
with other conventional staining methods; DMSO-MAF, MAF and auramine (Rossle and Latif, 2013; Stibbs and Ongerth, 1986). Researchers therefore use it as the gold standard to measure the sensitivity and specificity of other rapid methods for the detection of oocysts (Chartier et al., 2013; Mirhashemi et al., 2015; Roellig et al., 2017). 19
The type of mammal (human, cattle, sheep, horse and monkey) (Table 4) plays a significant role in the selection of the IFA being used as a solo method for oocysts detection or if it needs to be combined with another one. IFA, in combination with PCR was used for screening subclinical
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horse samples and IFA in combination with Kinyoun’s was deemed as the suitable method for cattle and sheep (Mirhashemi et al., 2015). In comparison with auramine, IFA was equally sensitive in the detection of oocysts from human faeces; however, with monkey faecal specimens (containing sparse oocysts), IFA sensitivity doubled auramine. The sensitivity of IFA and auramine exceeds 13 times MAF in the detection of oocysts in human specimens; and doubled
U
sensitivity with monkey specimens (Stibbs and Ongerth, 1986). It seems that IFA has greater
N
sensitivity particularly with low oocysts concentration, an advantage that makes it the method of
A
choice in studies on the prevalence of cryptosporidiosis in human or animal populations.
M
Although IFA allows rapid scanning of oocysts under low magnification, relatively lengthy processing and cross reactivity with certain faecal yeasts should be considered (Johnston et al.,
D
2003; Simjee, 2007; Sterling et al., 1986; Sterling and Arrowood, 1986). The requirement of a
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fluorescence microscope is another important concern for this technique. In resource poor areas,
EP
it would be problematic to find the instrument and the expertise to work on it (Chalmers et al., 2011; Ryan et al., 2016; Stibbs and Ongerth, 1986).
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Fluorescence assay when linked with monoclonal antibodies proved to be valuable in detecting oocysts in faecal samples. Such combination provides excellent screening techniques and offers
A
useful data for epidemiological studies. In comparison to ELISA, a monoclonal based fluorescence assay increased the sensitivity and specificity of oocysts detection (Adeyemo et al., 2018).
20
The commercial monoclonal antibodies are used for the detection and enumeration of Cryptosporidium oocysts in faecal samples. These monoclonal antibodies can differ substantially in their diagnostic specificity and sensitivity. (Jex et al., 2008a; Johnston et al., 2003; Weitzel et
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al., 2006). The rapid screening of infections and epidemiological studies in comparison to the direct microscopy or microscopy after staining makes IFA a favorable tool.
Flow cytometry coupled with cell sorting (FCCS) has been used to detect oocysts in human stools with a 4 to 34 times increase in sensitivity over IFA and MAF (Cole et al., 1999; Simjee, 2007; Valdez et al., 1997). This procedure has the ability to discriminate C. parvum oocysts from
U
debris or other microorganisms with no cross-reactions between the C. parvum antibody and the
N
most common microorganisms present in the stool (Barbosa et al., 2008). With flow cytometry,
A
Valdez and colleagues have found that 5 x 104 oocysts/ml is the threshold of oocysts detection in
M
human faecal samples (Valdez et al., 1997). The same threshold was reached with equine faecal samples (Cole et al., 1999). Before the use of FCCS, it is necessary to optimize the ideal
D
concentration of the fluorescent stain and establish the analytical protocol of cytometry.
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Optimization of these factors decreased the threshold of oocyst detection to 2 x 103 oocysts / ml
EP
(Barbosa et al., 2008). Clearing faecal samples from large particles (high fibre content, debris) would be beneficial with this technique, otherwise the apparatus might be clogged and sensitivity
CC
might be decreased. This therefore suggests that the use of zink sulphate solution flotation for 24 h, combined with increased centrifugation time would recover good concentration of oocysts
A
without background fluorescence (Barbosa et al., 2008; Cole et al., 1999). The dependence of this technique on the use of FITC-C-mAb makes it vulnerable to the antigenic variability in oocyst epitopes (Barbosa et al., 2008; Simjee, 2007; Valdez et al., 1997). Few studies have addressed this technique, possibly due to infrequent availability in diagnostic
21
parasitological labs due to the cost and the need of technical expertise. Moreover, its sensitivity with an asymptomatic carrier has not yet been estimated.
Enzyme
linked
immunosorbent
assay
(ELISA),
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3.1.2.2. Coproantigen detection techniques enzyme
immunoassay
(EIA)
and
immunochromatographic dipstick assay (ICT) are antigen detection formats that depend on the detection of Cryptosporidium antigen in faecal samples. Copro-antigen techniques provide quick and easy diagnostic tools (Aghamolaie et al., 2016); Clark, 1999; Simjee, 2007; Shimelis and
U
Tadesse, 2014). Shedding of intact oocysts usually stops when a patient is on treatment, while
N
stool antigens continue. A condition will be more likely to be missed by microscopy and will be
A
better diagnosed by antigen techniques; however, variable diagnostic sensitivities (70-100%) of
M
these techniques should be carefully considered (Ryan et al., 2016). The variability of sensitivity
D
and specificity of coproantigen assays are affected by the quantity of Cryptosporidium oocysts,
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per gram of stool.
ICT assays provide results within 10-15 min. These advantage lead to its expanded use for the
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routine detection and reporting of cryptosporidiosis in the United States (Roellig et al., 2017). Despite their convenience of use, poor sensitivities were noticed (75–88%) with high false
CC
positives (Garcia et al., 2000; Ghoshal et al., 2018; Johnston et al., 2003; Weitzel et al., 2006).
A
False negatives were also expected with a few numbers of oocysts, leading to misdiagnosis in many cases (Ghallab et al., 2016; Garcia et al., 2003; Magi et al., 2006). Such factors explained why half of ICT positive results from different community laboratories in US couldn’t be confirmed by IFA (Roellig et al., 2017). The US Council of State and Territorial Epidemiologists (CSTE) excluded cases from cryptosporidiosis surveillance and specifies that cases diagnosed 22
with these laboratory tests (ICT) be considered probable rather than confirmed (Ryan et al., 2017). Enzyme immunoassays (ELISA and EIA) offer higher sensitivity and specificity than
detection limit of 103-104 oocysts / ml (Ghoshal et al., 2018).
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conventional microscopy as a diagnostic procedure for Cryptosporidium copro-antigen with a
The procedure does not need concentration of faecal samples before processing. The spectrophotometric reading will eliminate subjectivity of microscopist interpretations. A large numbers of stool samples can be easily and quickly examined with increased reliability of results (Clark,
U
1999; Ghoshal et al., 2018; Nair et al., 2008; Simjee, 2007). Longer processing (1-2 h) should be
N
considered with enzyme assay. Attention should be drawn to false positives have been reported
A
in previous studies with human and animal (cattle, horse, and sheep) stools (Doing et al., 1999);
M
Fathy et al., 2014; Ghallab et al., 2016; Ignatius et al., 1997; 2016; Johnston et al., 2003; Mirhashemi et al., 2015; Simjee, 2007). Variable sensitivity (59-100%) and specificity (93-100%)
D
with this technique are also reported (Chalmers et al., 2011; Ghoshal et al., 2018; Ignatius et al.,
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1997; Rossle and Latif, 2013; Ryan et al., 2016; Youn et al., 2009). Such variability can arise
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from the use of different commercial kits, the work on different populations (human/animal) and because of the use of different references for evaluation. Even though combining enzyme assay
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and MAF provided better results in the detection of cryptosporidiosis; optimization of this approach to meet maximum cost effectivity and patient benefit is still required (Ghoshal et al.,
A
2018).
3.1.3. Microscopy 3.1.3.1. Light Microscopy (LM)
23
The detection of Cryptosporidium oocysts in faecal samples is traditionally dependant on examination with microscopy. The LM is used as a tool to read carbol fuchsin stained Cryptosporidium and other permanent slides and it remains to be the most commonly used
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technique for detecting the presence of active infections (Destura et al., 2015; Khanna et al., 2014; Potters and Esbroeck, 2010). LM is a cost effective method and offers detection of unexpected parasites (Khanna et al., 2014; van Lieshout and Roestenberg, 2015). Morphological characters for Cryptosporidium spp. are not adequately available. Therefore microscopic detection can be difficult, time consuming and does not include species identification (Rossle
U
and Latif, 2013). When there is low oocyst shedding, infection might pass unnoticed with the
N
low sensitivity and specificity of this technique (Destura et al., 2015; Jex et al., 2008a; Ryan et
A
al., 2016). The experience of microscopists is also essential for accurate diagnosis (Ryan et al.,
M
2016; van Lieshout and Roestenberg, 2015). Although, microscopic visualization is in fact a limitation in such studies, it was the chosen method for an unlimited number of previous studies
D
and yielded invaluable outcomes with various microorganisms, including Cryptosporidium and
EP
TE
its first discovery by Tyzzer at the beginning of the previous century (Karanis, 2017).
3.1.3.2. Phase contrast microscopy (PCM)
CC
PCM (in the absence of any staining solution) proved to be an inexpensive for the detection of Cryptosporidium oocysts in human faecal samples (Ignatius et al., 2016; Khanna et al., 2014;
A
Potters and Esbroeck, 2010). There is a kind of “misconception that PCM is indispensible with inferior results caused by Köhler-illumination” (Potters and Esbroeck, 2010). The sensitivity of this approach is affected by the number of oocysts excreted (Ignatius et al., 2016). Similar to LM, species differentiation is not provided by PCM, however, it is a good tool for epidemiological
24
and training/educational studies where various Cryptosporidium spp. are prevalent (Ignatius et al., 2016).
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3.1.3.3. Electron microscopy (EM) Electron microscopy has been used by researchers to describe the ultra-structure of Cryptosporidium. EM can provide useful information by focusing on the 3D structure of the specimen with assessing its electrical properties and chemical composition (Pomeranz, 1976). Both types of EM, scanning (SEM) and transmission (TEM) were used to introduce different
U
views and details to Cryptosporidium developmental stages (Baxby et al., 1984; Connolly et al.,
N
1991; Ghazy et al., 2015; Koh et al., 2014; Pohlenz et al., 1978). SEM also achieved high
A
resolution imaging of the Cryptosporidium extracellular stages in biofilms (Koh et al., 2014).
M
Although it is not common to use TEM with faecal samples and detect Cryptosporidium, it has been proven as a very useful procedure to visualize developmental stages of Cryptosporidium in
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2016b; 2016c).
D
in vitro axenic cultures with a high ultra-structural resolution (Aldeyarbi and Karanis, 2016a;
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While EM demonstrated the presence of Cryptosporidium for different and mainly ultra-structure research purposes, it is considered to be time-consuming, labour-intensive, expensive and it
CC
requires a high level of expertise (Fayer et al., 2013; Aldeyarbi and Karanis, 2016a; 2016b; 2016c). Routine electron microscopic examination of stool samples is not necessary and does not
A
increase the detection rate, when compared to microbiological/parasitological techniques and/or histological investigations (Connolly et al., 1991). This could explain its limited use in faecal samples examination.
3.1.3.4. Laser scanning confocal microscopy (LSCM) 25
LSCM is a technique that provides confocal epifluorescence imaging and optical sectioning of semi-opaque samples. It eliminates any interference of an auto-fluorescence background and haze in the image plane (Campbell et al., 1992). This technique has the ability to identify,
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enumerate and assess the viability of C. parvum oocysts in different matrices like faecal samples and soils (Anguish and Ghiorse, 1997). LSCM offers accurate analysis of oocysts even if they were buried in the sample, however, lengthy processing and experience should be considered. While this approach provides useful automated characteristics the depths of the penetration of the laser beam is a limiting factor (Anguish and Ghiorse, 1997; Fayer et al., 2013). When
U
samples contain a small numbers of oocysts, the detection sensitivity of LSCM is lowered as
N
well as its viability assessments (Anguish and Ghiorse, 1997). Based on its availability in
M
can be generated (Karanis et al., 2008).
A
laboratory facilities its application is restricted to specific studies, although impressive images
D
3.2. Molecular diagnostic tools 3.2.1. Polymerase chain reaction (PCR)
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Identification at the species level is important to determine the source of the infection and tounderstand the transmission dynamics and outbreak events, especially for zoonotic species
EP
(Algabban, 2008; Ryan et al., 2016). The PCR technique is an automated, repeatable technique
CC
and it includes large quantity processing (Fayer et al., 2000; Rossle and Latif, 2013; Ryan et al., 2016; Adeyemo et al., 2018). The ability for quantification (q-PCR) and molecular typing, the
A
possibility to combine multiple targets with one assay (multiplex PCR) and rapid processing time when automated DNA extraction is used makes this approach a powerful tool for genotyping of Cryptosporidium (Ryan et al., 2017). This approach offers advantageous features over the previously mentioned methods (Table 5). The increased sensitivity and specificity of PCR over traditional methods is a significant 26
advantage reported in various studies that amplified oocyst DNA from human and animal faecal samples (Checkley et al., 2015; Clark, 1999; da Silva et al., 1999; Gibbons et al., 2001; Ignatius et al., 2016; Morgan et al., 1998; Plutzer and Karanis, 2009; Simjee, 2007). However, the
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sensitivity and reliability of PCR detection of many protozoan parasites from faecal material including Cryptosporidium is critically affected by the purity and quality of DNA (Checkley et al., 2015; Elwin et al., 2012; Ghallab et al., 2016; Stroup et al., 2012). Five DNA kits were assessed for their efficacy in obtaining DNA from enteric protozoa. For efficient release of cryptosporidial oocyst DNA, the procedures combining chemical, enzymatic, mechanical lysis at
U
temperatures of at least 56 °C should be used (Paulos et al., 2016). Le Govic et al. (2013)
N
stressed the importance of including mechanical or thermal pre-treatment of oocysts as a
A
supplementary step (if not included in the used DNA extraction method provided by supplier).
M
The reliability of the primer sets and the validation of the protocol are added factors to be considered with the PCR system (Leetz et al. 2007).
D
Nested PCR protocols have been used to determine Cryptosporidium spp. in both human and
TE
animal faecal samples (Koehler et al., 2017; Mirhashemi et al., 2015; Ryan et al., 2003; Xiao et
EP
al., 1999). Nested PCR has proven more beneficial than primary PCR, producing more positive results and enhancing the amplification sensitivity of Cryptosporidium DNA with faecal samples
CC
(Amar et al., 2001, 2005; Bairami Kuzehkanan et al., 2011; Sadek, 2014). Two primer sets are utilized, so if there is nonspecific binding of a DNA template with the 1st primer sets, it is
A
unlikely happen with the 2nd primer sets. Previously established nested PCR protocols (Nichols et al., 2010, 2003; Ryan et al., 2003; Xiao et al., 1999) were compared for their reliability to detect cryptosporidial 18S rRNA in animal faecal samples (cattle, sheep and horses) (Mirhashemi et al., 2015). Reliability of primer sets
27
explained (Ryan et al. 2003) why it was the best protocol for screening oocysts in faecal samples from different animals. However, the combination of three nested PCR assays is favourable to provide a better understanding of diversity in species, in livestock and to detect mixed infection.
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With human faecal samples some PCR protocols (within SSU) produced relatively specific amplification from Cryptosporidium DNA; however, when DNA originates from an animal faecal sample, PCR assays do not achieve high specificity. In animal stools, cross-amplification’ of SSU from DNA of some Cryptosporidium related apicomplexans, selected alveolates and/or dinoflagellates has been observed (Koehler et al., 2017).
U
PCR-restriction fragment length polymorphism (PCR-RFLP) improved diagnostics of
N
Cryptosporidum spp.. PCR-RFLP detects and identifies species and subtypes of Cryptosporidium
A
in a sensitive way when the appropriate primers and restriction enzymes are used. This approach
M
is an important tool for investigating the relationship between the genotypes and phenotypes, as well as the molecular basis of the pathogenesis, virulence and genetic population structure of
D
Cryptosporidium (Wu et al., 2003).
TE
Nested PCR followed by RFLP-PCR were able to diagnose and identify Cryptosporidium spp. in
EP
various hosts (pigs and calves) (Danišová et al., 2016). Three PCR protocols (RFLP of SSU, nested of Gp60, semi nested of COWP) followed by sequencing were used to characterize
CC
bovine Cryptosporidium isolated from diarrheic calves. A difference of PCR sensitivity was allocated with the use of different genes, especially the low proportion of positive results for
A
COWP (Taha et al., 2017). COWP is heterogenous gene with a single copy that is used for Cryptosporidium spp. identification but suitable primers need to be designed for the described species (Homan et al., 1999; Leetz et al. 2007; Simjee, 2007; Spano et al., 1997). SSU rRNA
28
gene is expressed to a higher extent and thus allows a more sensitive PCR (Checkley et al., 2015; Simjee, 2007; Taha et al., 2017; Xiao, 2010; Zahedi et al., 2016). PCR-RFLP was able to identify eighteen isolates as C. hominis in different human populations
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with a high degree of polymorphism based on Gp60 (Essid et al., 2017). Owing to similarity on the DNA sequence level between C. hominis and C. parvum (Mazurie et al., 2013) the Gp60 gene has high heterogenous protein and its sequencing presents as a good tool for subtyping within species (Cai et al., 2017; Glaberman et al., 2002; Plutzer and Karanis, 2009; Simjee, 2007; Xiao and Ryan, 2004). Only PCR-RFLP of the ssrRNA identified Cryptosporidium in a human
U
sample, challenging all of the other tests used (Chalmers et al., 2005).
N
Quantitative PCR (qPCR) offers quantification of genetic targets over a wide dynamic range
A
when compared with conventional PCR. qPCR is generally accepted to be the most sensitive
M
laboratory assay for the detection and enumeration of Cryptosporidium oocysts in faecal samples from different hosts (Elwin et al., 2012; Hadfield et al., 2011; Yang et al., 2015). Its superior
D
sensitivity was reported when compared to MAF (Fathy et al., 2014; Ignatius et al., 2016;
TE
Kaushik et al., 2008; Khurana et al., 2012; Martín-Ampudia et al., 2012; Morgan et al., 1998;
EP
Yang et al., 2013, 2014; Zaidah et al., 2008). When compared with nested PCR, qPCR provides rapid, cost effective and accurate screening,
CC
identification and quantification of C. parvum and C. hominis. It has the ability to detect all the C. parvum and C. hominis isolates separately or in a mixture (1:100) with no cross reaction with
A
other genera (Yang et al., 2013). Hadfield et al. (2011) used qPCR assay to target the SSU rRNA gene to detect and differentiate C. hominis and C. parvum in human faecal samples. Through analytical sensitivity and specificity; the assay allows detecting as low as 200 opg equivalent to 2 oocysts per PCR with no
29
cross reaction with other genera; offering high sensitivity and specificity. When tested prospectively with COWP PCR-RFLP qPCR was more sensitive, detecting Cryptosporidium spp. in two more samples.
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LAXER qPCR, 18S qPCR, and nested PCR were evaluated prospectively to detect oocysts in human faecal samples. 18S qPCR detected all positive samples while LAXER qPCR and nested PCR induced false negative results (Le Govic et al., 2016).
The presence of inhibitors in stools has an impact on PCR protocol. It might not completely prevent DNA amplification with 18S qPCR but it led to underestimation of DNA quantities and
U
lowered sensitivity of LAXER qPCR (Le Govic et al., 2016). Affection to the Ct value should
N
also be considered (Yang et al., 2014). Additionally, PCR inhibitors [bilirubin, bile salts and
A
complex polysaccharides, stool fixative 10% buffered formalin] have adversely caused inhibition
M
of Taq polymerase and block the DNA extraction columns (Clark, 1999; Monteiro et al., 1997; Simjee, 2007; Wells et al., 2016). An internal amplification control (IAC) would be of great
D
value to overcome these difficulties.
TE
With qPCR, choice of primer probe affects the specificity of the assay through efficient
EP
differentiation between C. parvum and C. hominis. The specificity of qPCR was enhanced by using MGB probe in comparison to standard TaqmanTM probes. MGB probe lowered the
CC
background signal resulting in better precision in quantitation (Yao et al., 2006). Yang et al. (2013) used MGB probe in the qPCR assay resulting in high sensitivity and specificity of 100%
A
compared to the 18S nested assay, [96.9% and 98.4 %]. Multiplex real-time PCR assays have recently become available for the detection and identification of enteric protozoan parasites being highly valuable with mixed infection (Nurminen et al., 2015; Taniuchi et al., 2013; Van Lint et al., 2013). Many panels are used within
30
the system of multiplex real time PCR assays to detect Cryptosporidium with other protozoa and with/without viruses and bacteria. For Cryptosporidium these panels’ sensitivity ranged from (95-100%) and specificity from (99.6-100 %) (Ryan et al., 2017). BD MAX™ Enteric Parasite
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Panel “Giardia duodenalis, Entamoeba histolytica, and cryptosporidia (C. parvum and C. hominis)” was compared with previous results of multiplex in house PCR for the detection of the same parasites. With CryptosporidiumBD MAX™ assay detected all positive samples offering high sensitivity and specificity (100%) (Parčina et al., 2018). In earlier multicenter study BD MAX™ gave positive results with other Cryptosporidium spp. (Madison-Antenucci et al., 2016);
U
however, less prevalent zoonotic Cryptosporidium spp. remains to be evaluated (Parčina et al.,
N
2018). The BD MAX™ assay was compared prospectively to DFA, trichrome staining and
A
conventional PCR combined with bidirectional sequencing (APCR). The sensitivity of the BD
M
MAX™ for C. parvum and C. hominis ranged from 91.6% (compared to microscopy) to 98.9% (compared to APCR). Specificity with the assay was 98.9% for all methods (Madison-Antenucci
D
et al., 2016). When BD MAX™ compared with microscopy, multiplex PCR displayed more
TE
objectivity and sensitivity (Ögren et al., 2016).
EP
Droplet digital PCR (ddPCR) offers absolute quantitation of nucleic acid without the need for calibration curves as qPCR. The quantitation of Cryptosporidium DNA from faecal samples
CC
(cattle, sheep and humans) was compared by using ddPCR and qPCR based on two different loci (18S rRNA and actin). For most samples, ddPCR provided higher estimates with precision on
A
both loci compared to qPCR. PCR inhibitors affected negatively on qPCR but not on ddPCR. The ddPCR would be useful method for calibrating qPCR standards (Yang et al., 2014). For resource poor countries, the high cost of the PCR technique form a significant obstacle. Adding to the cost of PCR, chemicals are the cost of instruments required for the procedure.
31
There is also a shortage in the abundance of expertise working with this technique which will result in an additional cost for training.
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3.2.2. Fluorescence in situ hybridization (FISH) FISH technique is based on hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. It is one of the most widely adopted techniques in diagnostic, ecological, environmental and phylogenetic studies in microbiology (Adeyemo et al., 2018). This procedure has been used in the detection of Cryptosporidium oocysts in environmental and
U
faecal samples (Alagappan et al., 2008; Bednarska et al., 2006; Lemos et al., 2005; Smith et al.,
N
2004; Vesey et al., 1995). FISH allows species-specific visualization of the oocysts when used in
A
combination with a Cryptosporidium-specific antibody (Alagappan et al., 2008). As rRNA
M
presents in large copy numbers in viable organisms it moreover facilitates enumeration of viable pathogens. However, optimization of the FISH technique is a necessary step. Hybridization time,
D
temperature, permeabilization and fixation conditions as well as a specific probe should be
TE
considered before using this technique.
EP
Cryptosporidium RNA and DNA in preserved samples are stable for years (Amar et al., 2005), The storage of faecal samples up to 36 months at -20 oC or up to 7 months at 4 oC in 2.5%
CC
potassium dichromate did not affect C. parvum rRNA (Bednarska et al., 2006). Therefore FISH can be applied retrospectively to the stored faecal samples for identification of C. parvum
A
oocysts.
No FISH probes have been reported for the successful differentiation of C. hominis from C. parvum (Alagappan et al., 2008); however, a two-colour FISH assay, based on species-specific probes for C. parvum and C. hominis, was able to distinguish between these two species
32
(Alagappan et al., 2009). Yet, the sensitivity and specificity has not been attempted on faecal samples. The use of FISH to assess the viability of C. parvum oocysts is intricate. FISH was used to
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evaluate the survival of C. parvum oocysts over time in water. Even though it appeared to be a reliable indicator of viable oocysts, there was modest agreement between FISH and cell culture infectivity assays (Jenkins et al., 2003). False negative and false positive results with FISH and oocysts viability should also be considered. While rRNA from viable cells may be degraded during sample processing giving false negative result; extended preservation of rRNA in
U
nonviable oocysts might produce false-positive FISH results. In this state adaptation of FISH is
N
desirable (Smith et al., 2004). Even with such adaptation, gamma-irradiated oocysts of
A
Cryptosporidium have shown to provide positive FISH signals when dead (Davies et al., 2005).
M
A current issue still remains to be confirmed, doubting the reliability of this approach in the
D
assessment of oocyst viability.
TE
3.2.3. Loop-mediated isothermal amplification (LAMP)
EP
LAMP is a cost effective, accurate, simple and very sensitive method used for the detection of Cryptosporidium species with the possibility to differentiate between six Cryptosporidium
CC
human pathogenic species (Bakheit et al. 2008; Karanis et al. 2007; Karanis and Ongerth, 2009). It has high specificity, sensitivity, efficiency and rapidity that amplify DNA under isothermal
A
conditions (Nago et al., 2010; Notomi et al., 2000). It has been proposed that it has performed in economical heating blocks or in water baths and that it could be used to diagnose pathogens and diseases in the field and in clinical laboratories; it could also replace labour-intensive and experience dependent microscopic examination (Nago et al., 2010; Notomi et al. 2000). It can
33
amplify the DNA with high efficiency and it can detect a few copies in comparison to PCR without significant influence of the co-presence of non-target DNA (Notomi et al., 2000). In addition, the inhibitors that interfere with the PCR results have no effect on LAMP which makes
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it superior in efficiency and sensitivity, besides it also has a major advantage for detection of Cryptosporidium species at very low numbers (Bakheit et al., 2008; Karanis et al., 2007). The sensitivity of PCR methods to inhibitors could be overcome by use of LAMP, it has for the first time been developed for Cryptosporidium in water samples (Karanis et al., 2007). Advantages of this method include its cost-effectiveness compared to other molecular assays, its speed and high
U
sensitivity as well as its specificity. On the other hand, disadvantages include the requirement for
N
primer designs and protocol evaluation for specificity and sensitivity. Continuing technological
A
developments, including the loop-mediated isothermal assay, offer the possibility of significantly
M
simplifying the overall sample processing scheme with potential critical savings in time and
D
effort in order to produce meaningful results.
TE
3.2.4. Fingerprinting
EP
This method depends on the screening of genome(s) to check if there is any kind of variation in its organization or sequence (Jex et al., 2008a). The arbitrarily primed-polymerase chain reaction
CC
or random amplification of polymorphic DNA (RAPD) (Morgan et al., 1996, 1995), amplified fragment length polymorphism (AFLP) (Blears et al., 2000), satellite DNA (microsatellites and
A
minisatellites) and multilocus satellite analysis (MLST) (Grinberg et al., 2008; Mallon et al., 2003) are all various methods that are coupled with PCR to fingerprint the Cryptosporidium isolates. For characterizing the parasite through fingerprinting, no prior genome sequence is required, forming an advantage. This approach also provided valuable information concerning
34
the role of genetic exchange in C. parvum and C. hominis, population structure of Cryptosporidium and its diversity (Jex et al., 2008a). The genetic fingerprint of a protozoan species entitles a population of organisms rather than an individual profile. Therefore, all
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individuals in a population might not be represented. In addition, data sets from the analysis of mini/micro satellite fingerprinting could be phenotypic rather than genetic unless defined by sequence, limiting the interpretation (Jex et al., 2008a).
3.2.5. DNA sequencing
U
The technology of DNA sequencing has been applied immensely in many fields of diagnostics. It
N
facilitates the detection of mutations and leads to the rapid discovery of single nucleotide
A
polymorphisms (SNPs) (Dwivedi et al., 2017).
M
Sequencing of a wide range of cryptosporidial PCR products has facilitated the identification of most of the Cryptosporidium species (Plutzer and Karanis, 2009; Ryan et al., 2016). Given the
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benefit of using partial SSU sequence data, to date more than 34 species and 40 genotypes of
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Cryptosporidium have been classified (Koehler et al., 2017).
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Genome sequencing of both C. parvum and C. hominis has been completed (Abrahamsen et al., 2004; Xu et al., 2004) and it has enabled researchers to accurately compare their genetic
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compositions and it provides the chance to re-asses their phenotypic differences (Mazurie et al., 2013). Such technology was able to assess the genomic variation within and among species of
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the Cryptosporidium parasite (Jex and Gasser, 2014). DNA sequencing based on the Gp60 gene of both cryptosporidial genotypes has been used to screen for genomic variation on the subtype level (Feng et al., 2017; Sikora et al., 2017).
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Even so, DNA sequencing provides a library of accurate reference genome sequences, its source is coming from cloning making it vulnerable to cloning artefacts. Additionally, during the investigation of the long-range order of the sequence data, gabs, the repeated conserved elements
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should be considered (Bankier et al., 2003). Multiple steps are required to directly sequence an amplicon, which is relatively time consuming and expensive, particularly, if a large number of samples will be analyzed. It also does not allow the various sequence types within an amplicon to be separated and / or characterized (Abs EL-Osta et al., 2003; Gasser, 2006; Jex et al., 2007b). Despite such limitations, DNA sequencing has been established as a common procedure in many
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commercial laboratories for the differentiation of the Cryptosporidium species and whole
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genome sequencing of Cryptosporidium isolates prepared directly from human stool samples has
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A
already been introduced (Hadfield et al., 2015).
3.2.6. Electrophoretic mutation scanning (EMS)
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PCR and DNA fingerprinting approaches provided a wide range of molecular data to
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characterize Cryptosporidium genetically and classify its members. Although useful, it doesn’t
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allow accurate analysis of sequence and length variation within an amplicon because they separate DNA molecules, depending on its size on agarose gel electrophoresis (phenotypic
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approaches) (Gasser et al., 2004, 2003, 2001). DNA sequencing represents a gold standard in many molecular labs, however, it does not provide separation and characterization of various
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sequence types within an amplicon and when used with large number of samples, time and cost should be considered (Abs EL-Osta et al., 2003; Gasser et al., 2007; Jex et al., 2007b; Power et al., 2011).
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To overcome such limitations, employing EMS methods will add a great value as it relies on separating the DNA molecules in a nucleotide sequence dependent manner. Therefore, EMS methods, heteroduplex analysis (HDA), denaturing gradient gel electrophoresis (DGGE) and
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single-strand conformation polymorphism (SSCP) can provide useful alternatives for the direct analysis of sequence variation (Gasser, 1997).
HDA technique is a simple approach that relies on determining whether one or more types of DNA sequence exist in a sample. It depends on the fact that under particular electrophoretic conditions heteroduplex molecules that contain one or more mismatches can be separated from
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identical molecules containing no mismatches (Gasser, 1997). HDA has been used with the
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Cryptosporidium parasite for the rapid characterization of sequence diversity in a 173-bp
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fragment of the small dsRNA element. It has been proven to be a useful subgenotyping tool for
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the analysis of outbreak samples. HDA has the ability to rapidly screen for distinguishing diversity in samples from cryptosporidiosis cases. Its reproducibility, simplicity and applicability
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to large volumes of samples made it a useful tool for typing Cryptosporidium in epidemiological
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investigations (Leoni et al., 2003a, 2003b). However, the HDA mutation detection rate for
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changes in a single base could be affected by the position of a mismatch, melting temperature of the fragment containing a mutation and gel matrix composition (Gasser, 1997).
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DGGE is dependent on the usage of high temperatures to drive electrophoresis of DNA fragments in an acrylamide gel containing a gradient of denaturant. It also has the ability to
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detect mutation at a high rate. Its capacity to display all sequence variants of a particular molecule in one step visually indicates its effectiveness in analyzing genomic DNA (Gasser, 1997). DGGE has been applied to detect polymorphism in the Cryptosporidium spp. (purified form) faecal samples from mice. The authors found it to be a useful tool for differentiating
37
species that PCR–RFLP failed to differentiate (Satoh and Nakai, 2007). Given its usefulness, it requires standardization before the beginning of any application, therefore DGGE will need an experienced technician, who recognizes that the length of DNA, electrophoresis time and DNA
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melting profile should be considerable (Gasser, 1997). SSCP is a method that is widely applied in biomedical research for the purpose of analysis and diagnosis and it has been applied more and more to Cryptosporidium genes (Abs EL-Osta et al., 2003; Gasser et al., 2003, 2004, 2007; Jex et al., 2007b). It also has a broader applicability compared to DGGE because it doesn’t need to be optimized for each type of sequence and
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organism (Gasser et al., 2007). SSCP is a simple, cost effective technique with capability of
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detecting mutation in high capacity. When it is combined with PCR, it proves to be a powerful
A
tool for the identification of Cryptosporidium species or genotypes. It can screen for unknown
M
mutations and screen genetic variabilities amongst and within a large number of samples, it can also detect mixed infection (Jex et al., 2007a).
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PCR-based isotopic SSCP (hot-SSCP) has been used for the display of sequence variation in
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different nuclear gene regions of Cryptosporidium (hsp70, SSU) (Abs EL-Osta et al., 2003;
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Gasser et al., 2001) and (SSU and ITS) (Gasser et al., 2003), using a small number of welldefined oocyst DNA samples. Even though this approach is useful, the use of a radio-isotope it is
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an environmentally problematic material (Gasser et al., 2004). Therefore, Gasser group modified this approach to non-isotopic SSCP (cold-SSCP) (Gasser et al., 2007, 2004) and likewise used it
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to display sequence variation in different cryptosporidial genes (SSU from different host species) (Jex et al., 2007a), (SSU and gp60) (Abeywardena et al., 2014; Chalmers et al., 2005; Gasser et al., 2007; Jex et al., 2007b; Jex and Gasser, 2009; Koehler et al., 2016) and (SSU and hsp70) (Jex et al., 2008b).
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A comparative study used six methods (DNA sequence analysis of HSP70 gene, DNA sequence analysis of ssrRNA, SSCP analysis of ssrRNA, SSCP analysis of ITS-2, gp60 gene sequencing after PCR-RFLP, MLG of 3 microsatellite markers) for typing C. parvum from human faecal
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samples. Of the six molecular methods compared the SSCP analysis of pITS-2, gp60 and MLG displayed superior discriminatory opportunities. The authors advocated that the SSCP is a rapid, cheap and high typability method over sequence analysis (Chalmers et al., 2005).
Mutation scanning tools provide good opportunity globally for the detailed genetic characterization of a wide range of parasite species (large sample sizes) and they permit
Conclusions
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4.
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comparisons amongst them within different host groups and geographical origins.
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This review is the first comparison of a wide range of methods that have been used for the detection of Cryptosporidium in stool material in the last 40 years of Cryptosporidium and
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cryptosporidiosis research development. There are still several challenges for parasitologists,
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public health professionals and other experts regarding the diagnosis of the Cryptosporidium
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parasite. From the simple techniques to the highly complicated ones, they are all being used to achieve the ultimate goal, the “detection and identification” of Cryptosporidium.
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The methods of the concentration of the oocysts from the investigated material that is selected for use by individual laboratories may depend on the type of examination for Cryptosporidium
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species oocysts and the number of specimens received for processing. Various limitations (performance, costs, time, availability, etc…) form a barrier in front of investigators to use diagnostic tests. Enumerating these limitations and favoured outcomes would be of great value to ‘open the eyes of researchers’ in guiding them on selecting the most suitable
39
method to hit this target. These critical insights should be taken into consideration to achieve a successful cryptosporidial diagnosis. The choice of the most suitable and accurate diagnostic method for detecting Cryptosporidium in
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stool material is not an easy decision, it also depends on the research goal that will be achieved and the facilities that the investigator has to assist him/her in reaching their target. Considering the advantages and disadvantages of the diagnostic methods used, knowledge transfer and interdisciplinary research will be significant for making decisions in the future.
Variability of the purification results using different techniques displayed that flotation methods
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were less complex procedures, inexpensive, cost efficient and less time consuming rather than
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the gradient centrifugation methods. After all, iodine, Giemsa staining, MZN, negative staining
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by PAS reagent and examination of unstained preparations floated on SSF, all seem to allow for
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some error or confusion on the part of inexperienced persons. Modified acid-fast (MAF) stain is the recommended stain for detecting Cryptosporidium.
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Because of its greater sensitivity, especially for smears with low oocyst concentrations, the IFA
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seems to be the detection method of choice in studies on the prevalence of Cryptosporidium
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infections in humans, animals and the environment. Coproantigen tests are easy to perform and are less time-consuming but they are less sensitive compared to the IFA and molecular tools and
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perhaps to the conventional detection microscopical and staining methods. An additional and major limitation in antigen detection methods is being unable to identify Cryptosporidium in
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species or subtype level, which is essential for understanding the transmission dynamics and outbreaks, especially for zoonotic species. Regarding microscopy, its diagnostic accuracy is largely dependent on the experience and the skills of the microscopist. PCM is species independent and it may therefore be well-suited for
40
field studies where various Cryptosporidium spp. are prevalent. Microscopy by MZN has also the limitation as is being able to identify Cryptosporidium only few species, however, the most common pathogenic human and animal species are included.
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LSCM has been claimed to be sophisticated microscopical methodology for specific laboratory studies, however, it is time consuming and requires high experience. Additionally, it was considered to be an expensive instrument and a labour intensive technique.
Electron microscopic examination of stool samples is suitable method for the investigation of the ultra-structure of various species.
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DNA-based methods have evolved remarkably over the last two decades, being an indispensable
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tool for the rapid detection and genetic differentiation of Cryptosporidium in stool material. A
A
DNA analysis can be carried out by the PCR. Thus, Cryptosporidium species can be identified
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and the exact species can even be differentiated after sequencing. In many countries, it is currently being used not only for research purposes, but it becomes more useful for routine
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examinations too, particularly in high equipped laboratories. Comparisons between published
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protocols are difficult because no standardized or optimized procedures for DNA extraction or a
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relieving inhibitory effects exist, there is also no unified gene that can be targeted,
standardized scheme is not currently available and no consolidated type of PCR that has been
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used.
LAMP is a promising DNA based assay that is a cost effective and accurate method in
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epidemiological surveillance studies for the detection of Cryptosporidium species. The inhibitors that interfere with the PCR results have no effect on LAMP, which makes it superior in efficiency and sensitivity, besides it has major advantage for detection of organisms at very low
41
concentrations. Currently assays that are available are only for a limited range but the most importantly the pathogenic species of Cryptosporidium. Direct immunofluorescence, and molecular diagnosis (PCR, real-time PCR, LAMP) are the most
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sensitive tests and have been shown to display comparable sensitivities in several reports. Stool is and will always remain the cheapest and easiest material in its processing for the detection of Cryptosporidium oocysts. It is the simplest substance to ever represent the infection of any patient with diarrhoea, besides it is available almost at any time. Visual confirmation of oocysts is strong evidence in front of experts and eliminates doubt. Although microscopical stool
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examination is of limited value to differentiate species of Cryptosporidium oocysts, it still
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remains the most important issue to evaluate the patient state. Simple processing with
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concentration and staining can answer the question of whether there are cryptosporidial oocysts
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present or not. Staining and its quality is many times an obstacle in front of finding the correct diagnosis. Many laboratories all over the world suffer mistaken detection or no detection at all
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cause of bad quality of staining powder. The presence of positive controls will always be of great
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value.
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Great advances and the achievements have been reached after more than four decades in the Cryptosporidium field; however, it is so far unknown how well the described methods are
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harmonized amongst different laboratories. More highly monitoring approaches have been explored and some have the potential for recognizing Cryptosporidium.
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Further studies are required to simplify the diagnostic possibilities of Cryptosporidium and cryptosporidiosis, the types of the pathogens and its circulation in the hosts and the detection in the related material. We strongly recommend the validation of procedures for ring tests or multilaboratory test standardization and validation in different settings, e.g. laboratories with a high
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technical standard vs. field laboratories /laboratories in small hospitals to enhance routine surveillance capacity of cryptosporidiosis and to improve safety against cryptosporidiosis. Under these conditions it may be possible to better define the real epidemiological situation of
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cryptosporidiosis worldwide and to avoid differences in diagnosis among countries and continents. The prospect for a simple on-line and real-time diagnosis of Cryptosporidium in stool
A
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D
M
A
N
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samples within the next years appears to be difficult but it is achievable.
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A
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EP
TE
D
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72
Fig. 1. Flow chart describing methods of detection and identification of Cryptosporidium oocysts
A
CC
EP
TE
D
M
A
N
U
SC RI PT
in stool material.
73
Table 1. Features of common preservatives that have been used to preserve Cryptosporidium oocysts in faecal samples.
Formalin,
Disadvantage
SAF, Destroy oocysts Inhibit
PVA
infectivity
Comment
References
PCR Reduce the risk (Abdelsallam et
SC RI PT
Advantage
outcome
of high infectious al., 2017; Garcia
Require
cold samples
storage (4◦C)
et
al.,
1983;
(Tuberculosis
Greene,
2013;
and HIV)
Jongwutiwes
et
Keep
PCR Can’t
oocysts Affect outcome
with
used (Johnson et al., high 1995;
A
viability
be
N
K-dichromate
U
al., 2002)
samples
Require
cold (Tuberculosis
storage (4◦C)
al., 2002; Simjee, 2007)
term
storage may not retain oocysts for
CC A
et
and HIV) Long
EP
TE
D
proper washing
Ethanol
Jongwutiwes
M
therefore require infectious
molecular studies. Preserve morphology Keep
DNA Oocysts lose its Storage of faecal (Jongwutiwes et viability
samples
in al.,
DNA Require washing ethanol for ≥ 2 Lalonde
74
2002; and
property Doesn’t
before PCR
(22-38◦C) Gajadhar, 2009)
years
require
doesn’t
cold storage
change
oocysts
Freezing (-20◦C)
Preserve
DNA Require
property Excellent
SC RI PT
morphology. -20◦C Freezing
freezer
the
PCR
gave (Abdelsalam
best
PCR al., 2017)
outcome
product
followed by K
U
dichromate (4◦C) evaluated
with
formalin
M
A
N
when
10%
,
K
dichromate, and
D
ethanol
TE
Formalin 10 %
EP
should avoided
A
CC
PCR
75
be with
et
Table 2. Features of common concentration and purification techniques used to refine Cryptosporidium oocysts from different stools.
Pros
Cons
ion techniques
Comment
Reference
SC RI PT
Concentration/Purificat
(s)
Flotation techniques Easy
to Exacting specific
Selectively
gravity
concentrate
High
more
viscosity
A
viable
It
EP
TE
D
M
oocyst
CC
a and Smith, 1995; Ma
method rather and Soave, than
inhibits concentration
1983; MacPhers
staining
on
procedures
McQueen,
Collapse of
1993;
oocysts
McNabb et
within
A
as
purification
U
perform
Can be used (Bukhari
N
SSF
and
15
al., 1985;
min in wet
Shaista et
mount
al., 2016;
preparation
Sheather, 1923; Weber al.,
76
et
1992,
1991) Easy
to Low
Produce
the (Bukhari
perform
recovery
least recovery and Smith,
Selective
rate
rate of oocysts 1995; Ma
SC RI PT
ZSF
concentrati on of viable oocyst
when
and Soave,
compared
1983;
with SSF and Weber water ether
al.,
et
1992,
Reach
Long
rate 5 opg that
M
SSSF
procedure
A
followed by detection
N
AF
U
1991)
compared
suits with
only
D TE EP CC A
Large
Separate
starting
the oocysts sample from
needed
fibrous
(50g)
faecal material
Sedimentation
77
adult
for cattle samples
salt laboratories
flotation
(Wells
et
method to deal al., 2016)
to 100 opg research with
Effective
is
techniques Minimal
Large
For
proper (Rezende
materials
amount of oocysts
et
Minimal
fecal debris concentration
2015)
financial
interfere
this technique
resources
with
should
al.,
SC RI PT
SGS
be
microscope
combined
analysis
with
other
Ether
High
is
A
Water ether
N
U
concentration
M
recovery rate
when
D TE
to SSF and
can be used as and Smith,
reagent
an alternative 1995)
Procedure
to avoid ether
must done in
However,
flameproof
ZSF
EP CC
Ethyle acetate (Bukhari
flammable
compared
A
extremely
techniques
fume
ether is more
cupboards
effective
Doesn’t select
yield
to
cleaner
viable oocysts
faecal pellet FEA
Remove fats
Significant and loss
Layering FEA (Clavel et
of sediment and al.,
1996,
fibers from oocysts in flotation over 1994; stools
fat layer
78
hypertonic
Garcia and
Provide
a Presence of sodium
Shimizu,
cleaner
ghost
sediment
oocysts in solution
Pacheco et
stained
al., 2013;
1981;
improves
SC RI PT
slides
chloride
prepared
oocysts
Weber
detection
al.,
from FEA Diarrhea/form concentrate
ed
et
1992,
1991)
stools
N
U
affect
-iodine-
FEA
A
Remove
The
M
Merthiolate
and dye
fats
red Small amount (Garza of
fibers from particles
concentrati
stools
TE
D
formalin
might
EP
on
CC
(MIFC)
be be used with diarrhea
with
samples.
oocysts
Useful this concentration
technique
method when
combined
precede
with MAF
Auramine staining
79
et
ethyle al., 1985)
acetate should
confused
when
A
sensitivity of
method Increase
Affected by Valuable
sensitivity
the
of
pH technique
oocysts during
detection rate
oocysts
when capture
coupled
Need
with (PCR, longer
increase
U
IFA, DFA, processing time
sensetivity with
low
al., 2011;
or Gao et al., 2014;
shedding
Pereira et
A
al., 1999;
of cost
Power
DNA Increase
et
al., 2003;
product
potential
Robinson
Remove
for human
et
PCR
error
2008)
D TE EP
et
intermittent
M the
Coklin
detection with al., 2003;
Improve the Increase quality
to al., 2003;
oocysts Davies et
N
FC)
(Atwill et
SC RI PT
IMS
al.,
inhibitors
A
CC
Purification techniques
Percoll
The oocysts Expensive
This technique (Entrala et
yield
is reagent
should
pure
Labour
preceeded
Suresh and
(largely
intensive
with SSF
Rehg,
free
of Time
80
be al., 2000;
Percoll causes 1996)
bacterium)
consuming
high
oocysts
Recover
Doesn’t
loss
after
viable
work
purification
oocysts
efficiently
compared
SC RI PT
with large glass faecal
to
beads
and dialysis
volumes Glass beads Efficient
The
and dialysis
full
oocysts bacterial
N
of
rat contaminan
A
from
ts
M
faeces
can eliminated with
1.75%
hypochlorite
viable
Technique
oocysts but
allows oocysts
inferior
collection
to
Percoll
and Rehg,
be 1996)
Recover
D TE EP CC A
(Suresh
of contaminants
U
purification
yield Bacterial
from
large
faecal volumes
PBDG
Clear band Affected by High
(Entrala et
of
fat content
centrifugal
al., 2000)
separation
Require
force
Keeps
multiple
enhance purity
81
will
oocysts
washing
of
final
viability
before
suspension
Low cost
starting
Time
SC RI PT
procedure
A
CC
EP
TE
D
M
A
N
U
consuming
82
Table 3. Pros and cons of staining techniques used in the detection of Cryptosporidium oocysts in faecal samples. Type of
Name
stain
stains
of
Brief
description
of the method
Oocysts
Artefact
Pros
shape with
appearance
Cons
Referenc
pe
e (s)
can
read the
SC RI PT
stain
Microsco
stained slide
Non perman ent Mix
preparation
centrifuged
appear
formalinized stool
refractile
their
sediment one drop
structure
of 10% KOH on a
with
glass slide.
internal,
After
clear
EP
Artefacts
Slide must
PCM-
(Garcia et
Simple
be
LM
al., 1982)
Consume
examined
(x100)
natural
no time
within 10
condition
Almost
min.
no cost
Require
appears in
Fast
A
N
as
experience
vacuole
d
with some
microscop
dots
ist
between
Require
the
cell
concentrat
wall
and
ion
the vacuole
For mucoid
A
CC
Oocysts
D
clearing
TE
examine
of
M
drop
U
Wet
samples 10
drops
of
10%
KOH should be
83
added Sheather's
Put washed stool
Oocysts
Artefacts
Simple
Slide must
LM
(Ma and
sugar
in 15 ml tube
appeared
appears in
Fast
be
(x100)
Soave,
their
Consume
examined
natural
less time
within 10
slip flotation
Add
Sheather’s
Sugar
solution
(spg
as oval or spherical,
1.275)
condition
min.
structures, halfway then mix
Require
characteri Fill the tube with Sheather solution
experience
zed by a
d
pink hue
to the top
microscop
Place cover slip on
ist
min
Put
N
5
U
the top, centrifuge for
cover slip on slide
A
then examine
For
M
mucoid
D
samples
TE EP
ion
10
drops
of
10%
added Oocysts
iodine
formalinized
appear
CC A
concentrat
should be
Mix drop of faecal
with
Require
KOH
D’antoni
concentrate
as
unstained
Artefacts
Simple
Slide must
LM
(Ma and
stain
Fast
be
(x100)
Soave,
brown
Can
examined
drop of D’antoni
and
differenti
within 10
idodine
colourless
ate
min.
structures
oocysts
Low
from
number of
yeasts
oocysts
Cover
then
1983)
SC RI PT
cover
examine
could pass
84
1983)
unnoticed Sample requires concentrat ion
and
SC RI PT
purificatio n
before
examinati on
Oocysts
Artefacts
Effective
Slide must
LM
(Horen,
formalinized
appear
stain
negative
be
(x40-
1983)
refractive,
reddish
stain
examined
x100)
one drop of 0.5%
spherical
purple
Simple
within 15
periodic acid and
and
one
unstained
drop
of
Schiff's Reagent React 5-15 min
EP
TE
examine
then
D
Cover
structures
N
with
A
concentrate
U
Mix drop of faecal
M
PAS
min.
Can
Reaction
differenti
wasn’t
ate
obvious
oocysts
with
from
sucrose
yeasts
concentrat ed samples
Negative
Mix drop of faecal
Oocysts
Artefacts
Effective
Slide must
LM
(Heine,
stain of Hein
concentrate
appear
stain
negative
be
PCM
1982;
(Carbol
drop of undiluted
refractive,
(carbolfuch
stain
examined
(x10-
Ignatius
fuchsin)
carbol fuhsin
spherical
sin colour)
Simple
within 15
x40)
et
Left to dry
and
Cost
min.
Increase
2016;
Examine
unstained
effective
Thickness
the
Potters
structures
Can
of
sensitivit
and
detect
affect
y
Esbroeck,
other
negatively
A
CC A
Fast
with
85
red
slide
drop
2010)
al.,
protozoa
on
like
result
the
of oil can be added
cyclospor
and
a,
examine
isospora
d
Can
be
x100
used
as
SC RI PT
on
initial
screening
for slides to be later
U
confirme
if
N
d
necessary
Oocysts
Artefacts
The most
Slide must
LM
(Khanna
Negative
formalinized
appear
stain green
effective
be
PCM
et
stain of Hein
concentrate
refractive,
(malachite
and
examined
(x10-
2014)
(Malachite
drop of malachite
spherical
green
sensitive
within 15
x40)
green)
green
and
colour)
stain
min.
Increase
TE
D
with
A
Mix drop of faecal
M
Modified
unstained
among
Thickness
the
Examine
structures
other
of
sensitivit
negative
affect
y
stains
negatively
A
(carbul
on
of oil can
fuchsin,
result
A
CC
EP
Left to dry
the
drop
be added
methylen
and
e
examine
blue
and
d
crystal
x100
violet) Enhanced
86
slide
on
al.,
refractivit y
of
oocysts Practical and safe
SC RI PT
Perman
Fixation
Oocysts
Artefacts
Simple,
Presence
LM
(Anderso
(Methanol)
stains
take
fast
of
(x100)
n, 1987;
bright red
counter
Cost
oocyst
Babxy
fuchsin)
stain
efficient
Consume
and
Wash (H2O)
colour
Process
time
Blundell,
Destaining
Green
large
Require
1983;
(Sulphoric acid)
(Malachite
number
experience
Bronsdon
)
of
microscop
,
Blue
samples
ist
Casemore
(Methylene
Low
,
blue)
number of
Casemore
oocysts
et
Left to dry
could pass
1985;
A drop of oil &
unnoticed
Chartier
(Carbol
chite
M
Wash (H2O) Counterstain(Mala
TE
D
green/Methylene blue)
U
Staining
the
A
MAF
N
ent
1984;
1991;
et
EP
Examine
ghost
al.,
al.,
2002;
CC
Garza, 1983;
A
Ghoshal et
al.,
2018; Jex et
al.,
2008a; McNabb
87
et
al.,
1985; Pacheco et
al.,
2013;
SC RI PT
Potters and
Esbroeck, 2010;
Shaista et al., 2016;
U
Stibbs
N
and
A
Oocysts
Presence
LM
(Bronsdo
stains
stain
internal
of
(x10)
n, 1984)
brilliant
green
morpholo
oocyst
pink
gy can be
Unable to
structures
observed
detect
Destaining
Fast,
slides ≤ 10
(Sulphoric acid)
Simple,
oocysts
Wash (H2O)
Clean,
Counterstain
Versatile
(Malachite green)
Penetratin
Left to dry
g
A drop of oil &
qualities
Examine
of DMSO
Oocysts
(Methanol) (carbol
D
Staining
TE
fuchsin-DMSO)
CC
EP
Wash (H2O)
A
1986)
Artefacts
Fixation
M
DMSO-MAF
Ongerth,
pale
eliminate the need for
88
heat
ghost
or steam with carbol fuchsin Fixation
Oocysts
Artefacts
Efficient
Expensive
Fluoresc
Anderson
bol-fuchsin
(Methanol)
appear
don’t
Detect
Require
ent
,
Staining
yellow
fluoresce
oocysts ≤
technical
microsco
1981;
(Auramine)
discs with
10
expertise
pe (x40)
Babxy
Wash (H2O)
pale
oocysts
Destaining
against
(Sulphoric acid)
dark
Wash (H2O)
background
Counterstain
.
as
halo
N
U
a
(Potassium
and
used
Blundell,
is
carcinoge
1983;
nic
Casemore
Consume
et
time
1985;
Examine
al.,
Horen, 1983;
M
Left to dry
The fluid
A
permanganate)
1983,
SC RI PT
Auramine/car
MacPhers
D
on
TE
McQueen ,
1993;
Mata
et
EP
al., 1984; PerezSchael et
CC A
ACMV
and
al., 1985). Fixation
Oocysts
Artefacts
Reliable
(Methanol)
stains blue
appears
Yeast
Staining (ACMV)
to
yellow
Wash (H2O)
violet
Desataining
colour
bluein
yellowgreen
89
to
-
LM
(Milacek
(x100)
and
don’t take
Vftovec,
the stain
1985)
(Sulphoric acid)
backgroun
Wash (H2O)
d
Counterstain (Tartrazine)
A drop of oil & Examine Fixation
Oocysts
Artefacts
Inexpensi
Take long
LM
(Ferreira
(Methanol)
stain
appears
ve
time
(x100)
et
Immersion
with black
(mordant solution)
grey
Staining (Aqueous
granules
grey
light grey
N
solution)
A
(H2O)
Immersion
steps
Stain need to
be
ripened
helpful for the
D
(Mordant solution) (H2O)
diagnosis
TE
Wash
2001)
Not
M
several times
al.,
washing
hematoxylin
Wash
Multiple
U
RIHS
SC RI PT
Left to dry
several times
of oocysts
Left to dry
EP
A drop of oil & Examine
A
CC
Safranin
Fixation
(Acid-
Crescent
Artefacts
Work
Need
LM
(Babxy
shape
appears
well with
careful
(x100)
and
oocysts
blue
5 months
individual
Blundell,
appear
violet
old
handling
1983;
bright
backgroun
samples
Require
Casemore
steam appeared
orange with
d
Don’t
heating
et
Counterstain
a
require
Structures
1985).
(Methylene-blue)
stained
destining
are
methanol) Staining
(1%
safranin) Heating
until
lighter
90
to
not
al.,
Left to dry
centre
Simple
well
(clear halo) Dry drop of faecal
Oocysts
Artefacts
Effective
Less
LM
(Brar
concentrate
appear
as
appears
to
available
(x10-
al., 2017)
Staining
round
to
red
presumpti
Need
x40)
(Leishman stain)
oval,
ve
experience
work as fixative
hollow
diagnosis
too
unstained
of
Staining (Diluted
bodies
oocysts
leishman stain)
Left to dry Oocysts
(Methanol)
stain
Staining
faintly blue
grey
Giemsa stain 1:10
to
blue
from
with
stock
LM
(Anderso
to
careful
(x100)
n,
confirm
colourizati
1981;
oocysts
on
Babxy
identity,
formation
and
especially
of reddish
Blundell,
purple
where
to
1983;
corpuscle
fluoresce
granules
Casemore
nce
that might
et
microsco
cause
1985;
py
is
difficult
Garcia et
unavailab
reading.
al., 1982;
le
Consume
Horen,
time
1983;
Some
MacPhers
yeast cells
on
stain blue,
McQueen
their shape
,
is
Mata
as
azure
reddish
to
A
CC
EP
TE
Valuable
appears
D
Giemsa)
Require
A
(diluted
Artefacts
N
Fixation
M
Giemsa
U
Wash with water
make
et
SC RI PT
Leishman
defined
91
to
dark
more
1983,
al.,
and
1993; et
consistentl
al., 1984;
y oval and
Perez-
they tend
Schael et
to
al., 1985)
be
A
CC
EP
TE
D
M
A
N
U
SC RI PT
smaller
92
Table 4. Comparative studies of detection methods according to type of mammal in the detection of Cryptosporidium oocysts in stools. Type
State
of
of
mam
mamm
Type of Method*
Result
Comment
Refe
SC RI PT
renc e
mal as al a source
U
of
N
fecal
A
sampl
M
e Stain”Mi
Fluores Enzy
Nucleic
croscope
cence
acid
D
techniq assay
TE
”
me
assays
EP
ues
Sheep, NM
CC
Cattle,
A
Horse
MAF
IFA
ELISA
Three nested
Combined are
the
sensitive
PCRs most
protocol
IFA can be used as primary tool for screening
rRNA”
veterinary diagnostics
93
reliable tool hash for the detection
specific tool.
“18S
is (Mir
and
PCR
s
DFA
in
of
emi et
Cryptosporidiu m oocysts
in
cattle and sheep faecal samples.
al., 2015 )
Kinyoun's was
DFA
and
more PCR
are
specific
in recommend than ed
for
SC RI PT
sheep horses
and subclinical
cattle.
horse
ELISA
was
affected
by
samples
low
numbers
of
U
oocysts with false
test
N
positive
Asymp
Negative
n
tomati
stain
-
of
EIA
M
Huma
A
results.
RT-PCR
The sensitivity and specificity of PCM
PCM
with (Igna
diarrhea
tius
EIA were 89 %
samples
et
and 100 %.
work
well al.,
with
high 2016
and Hein
TE
c
D
compared with the
sympto “PCM”
PCM
identified
only
EP
matic
halve
positive
of
samples
CC
by RT-PCR “two of
them
after
specificity and sensitivity
A
reexamination”
very
close
to EIA With diarrhea
94
non
)
samples PCM
was
inferior
to
SC RI PT
PCR and is affected by number
of
excreated oocysts
MAF
-
-
Nested PCR
omatic
attempt
Low
TE
D
M
A
by RFLP
(Tah
to concentrati
N
followed
Unsuccessful
U
Calves Sympt
identify DNA on
of al.,
from halve of oocysts/
2017
positive
bad quality )
microscopic
of DNA can
samples
hamper
EP
PCR systems.
CC
False positive of
A
microscope based method must
95
a et
be
considered. Huma
Asymp
Glycerin
n
tomati
e, MAF,
-
qPCR
qPCR
was qPCR
is (Le
more sensitive able
and AR
than
to Govi
prevent the c et
SC RI PT
c
-
sympto
microscopy in misdiagnosi al.,
matic
patients risk
qPCR
at s
of 2016
cryptospori
)
was diosis
U
sensitive and qPCR
in quickly
N
accurate
M
A
estimating the identifys
shedding
D Trichrom
n
omatic
e
A
DFA
the sources in of infection
the course of in the case infection
TE EP
Sympt
CC
Huma
oocyst
of outbreaks.
-
“Multipl The ex
close
“PCR
APCR.
combine BD
diso
with can be well nsubstitute Max for
Ante nucc
with identified 12 microscopic i
bidirecti
96
BD (Ma
DB MAX™ was MAX™
max”-
d
BD The
positive
examinatio
al.,
et
onal
specimens
sequenc
that
ing
missed
n
or 2016
were immunoass
)
by ays. Cost
must
SC RI PT
DFA.
be
considered
IFA
-
Asymp
FC
was
10 High
s
tomati
with
times (5x104 content
c
Flow
opg)
more equine
N M
A
try
and affect
DFA
(5x105 detection
opg)
)
threshold of MAF while
D -
al.,
MAF
it
TE EP
CC A
-
of e et
sensitive than feces don’t 1999
cytome
Calves NM
fiber (Col
U
MAF
-
Horse
affects
IFA and FC -
Cryspov RT-PCR
The
irus-
amplification
Cryspovirus kins
specific
of
Semiqu
sequences
antitativ
specific
e
gene -specific
et
RT-PCR for assay
al., is 2016
RT- Cryspovirus is preferable
PCR
97
(Jen
a
sensitive over
SSU
)
(sqRT-
method
for rDNA PCR
PCR)
detecting
C. assays.
with
parvum
The
quantita
oocysts
PCR detect
SC RI PT
tive RT-
low
PCR
numbers
(qRTPCR) vs
N
A
PCR
M D omatic
moor
to
PCR Both qRTand
are functional
TE e
need
sqRT-PCR
by utilizing
EP A
Sympt
no
PCR
an internal standard to
CC Grous
with
secondary
U
SUU nested
RT-
avoid false negatives -
IMSIFA
-
Nested
PCR
18S
superior
rRNA
IMS-IFA
98
was PCR
has (Bai
to the advantage
nes et
PCR
detecting oocysts
of
being al.,
in more
2017
30% vs 20% sensitive in ) infected detecting
SC RI PT
of
grouse
oocysts
(
