Direct fecal smear

Grassi and Parona first employed a direct fecal smear in 1878 as a diagnostic tool for the detection hookworm infection in humans (Rockefeller 1922). For many years, this method was used for the identification of different parasites with different modifications. For the simple direct fecal smear examination, a small sample of feces is placed on a glass slide, mixed with a drop or two of saline, spread thinly over the slide, and then covered with a glass coverslip. The smear needs to be thin enough to read newsprint through them. Later, Faust (Reference Faust, Sawitz, Tobie, Odom, Peres and Lincicome1929) compared the sensitivity of the fecal smear technique without staining and with staining with hematoxylin. He observed that the staining of the fecal smear did not significantly improve diagnostic sensitivity compared to a direct fecal smear without staining. Thick smear technique was introduced by Kato and Miura in 1954 for fecal research (Kato and Miura Reference Kato and Miura1954). They placed 60–70 mg fecal sample on the glass slide, covered it with a cellophane strip soaked with glycerol-malachite green, pressed it with a rubber stopper or finger, and kept it at a room temperature for about 30–60 min. Although the thick smear technique proved more sensitive than a direct fecal smear with a cover glass, it exhibited a low sensitivity for hookworm eggs, which are present in low density in fecal samples and often are destroyed during the long preparation period because hookworm eggs have a delicate consistency, quickly clear with glycerin, and after 30–60 minutes, they cannot be seen (Santos et al. Reference Santos, Cerqueira and Soares2005; https://iris.who.int/bitstream/handle/10665/324883/9789240014497-rus.pdf).

Katz adopted and modified this technique for use in routine epidemiological studies, resulting in its recognition by WHO for the quantitative and qualitative diagnosis of intestinal helminth infections such as Ascaris lumbricoides, hookworms, and Schistosoma mansoni (Katz et al. Reference Katz, Chaves and Pellegrino1974; Santos et al. Reference Santos, Cerqueira and Soares2005). For the diagnosis of S. mansoni, this method may have low sensitivity. One of the main parameters affecting the sensitivity of the analysis is the minimum infection intensity, which depends on the egg output of one worm. One pair of S. mansoni produces about 100 eggs per day, so feces contain many eggs only in the case of intense invasion. Given the small amount of feces analyzed, detection of mild S. mansoni infections with only a few female worms is almost impossible (Barenbold et al. Reference Barenbold, Raso, Coulibaly, N’Goran, Utzinger and Vountasou2017).

However, fecal smear examination is the simplest and most economical procedure used in laboratories worldwide. The technique has been adopted for the detection of a wide range of parasites, including roundworms, whipworms, hookworms, flukes, and tapeworms. Among the numerous modifications of the direct fecal smear technique, Kato-Katz is recognized as a ‘gold standard’ and is widely used by both human and veterinary healthcare practitioners (Hong et al. Reference Hong, Choi, Kim, Chung and Ji2003).

However, due to the small amount of stool being analyzed (41.7–70 mg), the sensitivity of the method suffers and can give false negative results due to low concentrations of helminth eggs in the stool sample or may appear highly clustered. Sensitivity can be increased by examining multiple Kato-Katz smears prepared from the same stool sample or from multiple samples from the same host or employing a combination of the Kato-Katz smear with a direct fecal smear (Glinz et al. Reference Glinz, Silué, Knopp, Lohourignon, Yao, Steinmann, Rinaldi, Crindoli, N’Goran and Utzinger2010; Komiya and Kobayashi Reference Komiya and Kobayashi1966). In addition, this method requires rapid sample processing – optimally within 20–30 minutes after slide preparation. The acceptable processing time for samples is within 24 hours. When stool samples are stored overnight, regardless of temperature, the number of parasite eggs decreases, which makes their detection difficult and does not allow reliably determining the intensity of infection (Bosh et al. Reference Bosh, Palmeirim, Ali, Ame, Hattendorf and Keiser2021).

Concentration method

Microscopic examination of the sedimented fecal matter was one of the first modifications of the fecal smear examination technique (Koutz Reference Koutz1941). Sedimentation relied on the concentration of helminth eggs, larvae, and protozoan cysts at the bottom of a tube to detect parasites or eggs occurring at low densities in feces.

Since 1948, various modifications and simplified improvements have been employed by a number of researchers in clinical laboratories (Blagg et al. Reference Blagg, Mansour, Schloegel and Khalaf1955; Knight et al. Reference Knight, Hiatt, Cline and Ritchie1976; Manser et al. Reference Manser, Saez and Chiodini2016). The method uses several pieces of apparatus, which must be washed after use with each sample and includes several safety precautions since it involves the use of formalin (an irritant) and ether, which is flammable. These kits are tightly sealed to reduce the hazards of formalin. The major modification of sedimentation method is using the ethyl acetate, a more stable and less flammable alternative, rather than ether (Truant et al. Reference Truant, Elliott, Kelly and Smith1981; Young et al. Reference Young, Bullock, Melvin and Spruill1979).

The TF-Test® is another modification of the concentration method for use in routine parasitological surveys. This complex method combines multiple sampling (on three consecutive days), a fixative (SAF-sodium acetate-acetic acid formalin), a concentration method, and a permanent stain (Chlorazol Black dye) (Van Gool et al. Reference van Gool, Weijts, Lommerse and Mank2003).

A highly competitive method for field studies is MIF (merthiolate–iodine–formalin). It is a concentration-based method that requires a centrifuge. This technique uses the MIF solution (50 mL formaldehyde at 37%, 10 mL glycerin at 87%, filled to 1 L with distilled water as stock solution I) as a preservative and staining (with 2 g potassium iodide in 10 mL distilled water as stock solution II). Ether is added to dissolve the fecal fats. The MIF method showed higher sensitivity for hookworms, while for T. trichiura and A. lumbricoides, Kato-Katz performed better. However, MIF detected Strongyloides stercoralis for which Kato-Katz method is not specific. Another advantage of this technique is that it allows for the preservation of fecal samples for a long time. Overall, the MIF method is simple and inexpensive, and is suitable for diagnosis and assessment of infection intensity in the field (Incani et al. Reference Incani, Homan, Pinelli, Mughini-Gras, Guevara and Jesus2016).

Flotation techniques

The principle of fecal flotation is based on the ability of a solution to allow less dense material (including parasites) to rise to the top (Ballweber et al. Reference Ballweber, Beugne, Marchiondo and Payne2014; Dryden et al. Reference Dryden, Payne, Ridley and Smith2010).

The specific gravity (SG) of the flotation solution must be higher than the SG of helminth eggs, so commonly used flotation solutions have an SG of around 1.18–1.33 g/L. Common flotation solutions are made by adding a measured amount of sugar (sucrose or dextrose) or salts such as sodium chloride, sodium nitrate, magnesium sulfate, or zinc sulfate to a specific amount of water to achieve the desired SG. Ideally, all helminth eggs would float without loss of morphological structures, while fecal debris would sink. Despite their low cost and simplicity, the flotation methods are highly influenced by several factors, such as floatation time, tube filling, and precise removal of the coverslip. Flotation methods have many modifications, ranging from simple passive flotation in solution with high SG, the addition of a centrifugation step, and using a chambered slide or special devices that utilize flat-bottomed vials in which the feces/flotation fluid mixture is placed (Ovassay Plus Fecalyzer) (Ballweber et al. Reference Ballweber, Beugne, Marchiondo and Payne2014).

The presence of a large amount of fecal debris is a major obstacle for the fecal flotation technique. One modification to alleviate this problem was the addition of a centrifugation step to remove large floating debris. This technique was first introduced by Lane (1924) and later modified by several others (Dryden et al. Reference Dryden, Payne, Ridley and Smith2010; Zajac et al. Reference Zajac, Johnson and King2002). The centrifugation time varies from 5 to 20 min depending on the characteristics of the centrifuge used such as the size of the rotor and the relative centrifugation force. For example, Egwang and Slocombe (Reference Egwang, Slocombe and Can1982) showed that centrifuging for 4 or 5 min at 264 ×g provided statistically significantly better egg recoveries than shorter (1 min) or longer (20 min) durations. Dryden et al. (Reference Dryden, Payne, Ridley and Smith2010) also observed that a 5-min centrifugation time at 280 ×g showed a significantly higher fecal egg count when compared with the passive flotation method. Another modification of flotation techniques is a fecal egg count method based on microscopy of an aliquot of fecal suspension from a known volume of a fecal sample. This method allows the investigator to express the number of parasitic elements (eggs, larvae) in the fecal sample in terms of eggs per gram of feces and can be used to measure the distribution of infections for epidemiological surveys to detect the presence or build-up of anthelmintic resistance, and to quantify the efficacy of anthelmintic treatment (Dryden et al. Reference Dryden, Payne, Ridley and Smith2010).

Gordon and Whitlock (Reference Gordon and Whitlock1939) proposed the first egg count method using a chambered slide while working with sheep feces, which later came to be known as the McMaster method (Gordon and Whitlock Reference Gordon and Whitlock1939). This procedure used 2 g of feces mixed with 30 mL of flotation solution (Sheater’s sugar or saturated sodium chloride), which was then shaken by hand to make a slurry. An aliquot of 1 mL was drawn from the center of the tube and added to three areas of the McMaster counting chamber. Since then, several modifications, including variations in the ratio of feces to fluid, centrifugation time, number of chambers counted, and area of slide counted, have been described. The sensitivity of this technique depends on the weight of the feces examined and the dilution ratio (g of feces/mL of water). In a study by Vadlejch et al. (Reference Vadlejch, Petrtýl, Zaichenko, Čadková, Jankovská, Langrová and Moravec2011), three modifications of the McMaster technique, like Wetzel, Zajíček, and concentration modification according to Roepstorff and Nansen, were compared; the concentration modification according to Roepstorff and Nansen used 4 g of the feces examined and determined that a low dilution ratio (1:14) was more sensitive than when a lower weight of feces (1–2 g) or a higher dilution ratio (1:30) was used (Blagg et al. Reference Blagg, Mansour, Schloegel and Khalaf1955).

A modern modification of the McMaster Technique is FLOTAC®, introduced by Cringoli (Reference Cringoli2006), which incorporates a centrifugation-enhanced flotation method in a chambered device with a detection limit of 1 or 2 EPG (eggs per gramm) (Cringoli Reference Cringoli2006; Utzinger et al. Reference Utzinger, Rinaldi, Lohourignon, Rohner, Zimmermann, Tschannen, N’Goran and Cringoli2008). A further modification of FLOTAC^® was proposed by Barda et al. (2013) called Mini-FLOTAC. In this method, a simple device Mini-FLOTAC apparatus comprises two physical components – namely, the base reading disc and two accessories, the key and the microscope adaptor (Barda et al. Reference Barda, Cajal, Villagran, Cimino, Juarez, Krolewiecki, Rinaldi, Crindoli, Burioni and Albonico2014). There are two 1-mL flotation chambers, designed for optimal examination of fecal sample suspensions in a total volume of 2 mL. A major advantage of this method is the lack of a centrifugation step.

Coproantigen detection

Highly sensitive and specific immunological assays employing antibodies have been developed to identify helminth parasite antigens released in the host feces. The helminth coproantigens are detected by enzyme-linked immunosorbent assays (ELISAs). One major advantage of such coproantigen ELISAs over serum antibody assays is that this method indicates only the current infection and avoids handling of serum that may contain parasitic products from previous infestations. The first such assay using agar gel diffusion was developed for the detection of Echinococcus granulosus (Babos and Nemeth Reference Babos and Nemeth1962). Other coproantigen assays have been developed for many human and animal cestode parasites to identify of major flatworm infections such as Opisthorchis viverrini, Fasciola hepatica, F. gigantica, and Echinostoma capronii) (Abdel-Rahman et al. Reference Abdel-Rahman, O’Reilly and Malon1998; Estuningsih et al. Reference Estuningsih, Widjayanti, Adiwinata and Piedrafita2004; Fraser and Craig Reference Fraser and Craig1997; Mezo et al. Reference Mezo, González-Warleta, Carro and Ubeira2004; Watwiengkam et al. Reference Watwiengkam, Sithithaworn, Duenngai, Sripa, Laha, Johansen and Sithithaworn2013).

Recently, an ELISA developed with monoclonal mouse IgG antibodies against Clonorchis sinensis was used for its detection in experimentally infected rats (Rahman et al. Reference Rahman, Choi, Bae and Hong2012). However, only a handful of coproantigen assays have been reported for nematodes, including assays for the detection of a number of species like Haemonchus contortus (Ellis et al. Reference Ellis, Gregory, Turnor, Kalkhoven and Wroth1993), Ascaris suum (Schniering Reference Schniering1995), Strongyloides ratti (Nageswaran et al. Reference Nageswaran, Craig and Devaney1994), and Heligmosomoides polygyrus (Johnson et al. Reference Johnson, Behnke and Coles1996). These tests have been shown to be relatively sensitive and species-specific.

PCR based detection methods

The first polymerase chain reaction (PCR) based assay for the identification of parasite DNA from eggs in feces was demonstrated by Flisser et al. (1988, 1990) and then by Bretagne et al. (1993). Common techniques such as conventional PCR, multiplex PCR, and real-time PCR are used for detecting parasite DNA in host feces (Bergquist et al. Reference Bergquist, Johansen and Utzinger2009). Conventional PCR involves the amplification of a sequence of target DNA using a primer pair to detect foreign DNA belonging to any parasite stage in either tissue, blood, or urine. The conventional PCR technique is slightly modified to perform a multiplex PCR that allows amplification of multiple target DNA sequences simultaneously (Gordon et al. Reference Gordon, Gray, Gobert and McManus2011; Toze Reference Toze1999). Multiplex PCR can also be used for the detection of multiple parasites species from the same individual’s fecal sample or blood.

The development of the real-time PCR introduced researchers to a powerful tool to study gene expression profiles as a function of the relative abundance of the target gene in the sample. Real-time or quantitative PCR (qPCR) does this by measuring the fluorescence released as a by-product of the PCR reaction and presenting it as a graph of fluorescence intensity relative to time. Thus, samples with a higher concentration of the target DNA show a peak in fluorescence at an earlier time point during the PCR (Frickmann et al. Reference Frickmann, Schwarz, Rakotozandrindrainy, May and Hagen2015; Heid et al. 1996). PCR, however, has a number of limitations, such as the risk of contamination, false positive results due to the presence of naked nucleic acids and non-viable microorganism, and difficulty in quantification in water and wastewater (Toze Reference Toze1999).

Modern diagnostic molecular-based techniques includes DNA sequencing, DNA barcoding and Loop-Mediated Isothermal Amplification (LAMP).

DNA barcoding was established as a rapid, powerful method for taxonomic research. DNA barcoding uses molecular data for identification and differentiation of species. PCR uses for amplification of short DNA fragments which compared to a pubilc DNA databases for the possible sequences matches. Nuclear and mitochondrial genes, such as 18S rRNA, 16S rRNA, ITS regions, and cox1, are used for DNA barcoding of eukaryotic organisms.

The cytochrome c oxidase (cox1) is the most slowly evolving gene, acknowledged as the ‘gold standard’ for DNA barcoding of eukaryotic organisms. For the determination of partial cox1 sequences of Platyhelminthes, the primer set of JB3 (5’-TTT TTT GGG CAT CCT GAG GTT TAT-3’) and JB4.5 (5’-TAA AGA AAG AAC ATA ATG AAA ATG-3’) (Bowles et al. Reference Bowles, Hope, Tiu, Liu and McManus1993) was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. A PCR, followed by sequencing, on the mitochondrial genes cox1 and nad1 with primers published by Bowles and co-authors was the standard test at our laboratory for addressing both the Taenia sp. determination as the detection of Echinococcus sp. with subsequent species determination (Bowles et al. Reference Bowles, Blair and McManus1992; Bowles and McManus Reference Bowles and McManus1993; Gasser et al. Reference Gasser, Woods, Huffman, Blotkamp and Polderman1999). The polymorphisms in these genes, which are so useful for typing, also interfere with the PCR. The annealing site for these primers is polymorphic as well. With several specimens of cestodes, the PCRs – most often the nad1 PCR – failed to amplify one of the two targets. Many different PCR based approaches to detect and type various Cestodes have been described (Abbasi et al. Reference Abbasi, Branzburg, Campos-Ponce, Abdel Hafez, Raoul, Craig and Hamburger2003; Al-Sabi & Kapel Reference Al-Sabi and Kapel2011; Bart et al. Reference Bart, Bardonnet, Benchikh-Elfegoun, Dumon, Dia, Vuitton and Piarroux2004; Boubaker et al. Reference Boubaker, Macchiaroli, Prada, Cucher, Rosenzvit, Ziadinov, Deplazes, Saarma, Babba, Gottstein and Spiliotis2013; Dinkel et al. Reference Dinkel, Njorogeb, Zimmermanna, Walza, Zeyhleb, Elmahdic, Mackenstedta and Romiga2004; Gonzalez et al. Reference Gonzalez, Monteroa, Puenteb, Lopez-Velezc, Hernandez, Sciutto, Harrisone, Parkhouse and Garate2002; Jeon et al. Reference Jeon, Chai, Kong, Waikagul, Insisiengmay, Rim and Eom2009; Knapp et al. Reference Knapp, Millon, Mouzon, Umhang, Raoul, Ali, Combes, Comte, Gbaguidi-Haore, Grenouillet and Giraudoux2014; Maurelli et al. Reference Maurelli, Rinaldi, Capuano, Perugini and Cringoli2009; Mayta et al. Reference Mayta, Gilman, Prendergast, Castillo, Tinoco, Garcia, Gonzalez and Sterling2008; Schneider et al. Reference Schneider, Gollackner, Edel, Schmid, Wrba, Tucek, Walochnik and Auer2008; Siles-Lucas and Gottstein Reference Siles-Lucas and Gottstein2001; Stefanic et al. Reference Štefanić, Shaikenov, Deplazes, Dinkel, Torgerson and Mathis2004; Trachsel et al. Reference Trachsel, Deplazes and Mathis2007; van der Giessen et al. Reference van der Giessen, Rombout, Franchimont, Limper and Homan1999; von Nickisch-Rosenegk et al. Reference von Nickisch-Rosenegk1999; Yamasaki et al. Reference Yamasaki, Allan, Sato, Nakao, Sako, Nakaya, Qiu, Mamuti, Craig and Ito2004). Most of these studies, however, use different PCRs for the detection of different cestodes. The PCRs are not specifically designed for human diagnostics. For routine diagnostics, a single test that is able to detect and type a range of cestodes is preferable. A widely published PCR target is the 12SrRNA gene, hereafter referred to as 12S. A number of different primers have been designed for the amplification of this gene because different research groups had different purposes (Boufana et al. Reference Boufana, Campos-Ponce, Naidich, Buishi, Lahmar, Zeyhle, Jenkins, Combes, Wen, Xiao, Nakao, Ito, Qiu and Craig2008; Dinkel et al. Reference Dinkel, Njorogeb, Zimmermanna, Walza, Zeyhleb, Elmahdic, Mackenstedta and Romiga2004; Stefanic et al. Reference Štefanić, Shaikenov, Deplazes, Dinkel, Torgerson and Mathis2004; Trachsel et al. Reference Trachsel, Deplazes and Mathis2007; van der Giessen et al. Reference van der Giessen, Rombout, Franchimont, Limper and Homan1999). The primers on 12S were designed to amplify a broad range of cestodes. The reverse primer is essentially the same as one of the primers published by Trachsel and co-authors for Echinococcus sp. and Taenia sp. (Trachsel et al. Reference Trachsel, Deplazes and Mathis2007). In addition to the cestode 12S primers, the Taenia sp. specific primers designed by Trachsel and co-authors were also used.

Additionally, the primers for the nad5 gene specifically for amplification of Echinococcus sp. were designed. Both the 12S and nad5 genes are mitochondrial, which ensures high sensitivity. The genes are also genetically highly variable, which is especially useful for species determination within the genus Echinococcus sp. We investigated the sensitivity and specificity of the PCRs on these genes. Furthermore, material from suspected echinococcosis patients was tested with cox1, nad1, 12S, and nad5 PCRs to compare sensitivity in clinical samples. To compare the targets for their use in species determination, we tested a variety of different Echinococcus sp. samples.

DNA barcoding has emerged as a powerful, rapid, molecular-based method with significant contributions to both taxonomic and biodiversity research (Hajibabaei et al. Reference Hajibabaei, Singer, Hebert and Hickey2007; Hebert et al. Reference Hebert, Cywinska, Ball and deWaard2003; Pereira et al. Reference Pereira, Hanner, Foresti and Oliveira2013 ; Weigt et al. Reference Weigt, Driskell, Baldwin and Ormos2012). Organisms can be accurately identified at the species-level by this process, which uses polymerase chain reaction (PCR) to amplify short DNA fragments corresponding to standardized regions of the genome with associated discriminatory sequence variations that are then subject to DNA sequencing. These sequences are then compared to a public DNA database of all the possible sequence matches or to a defined reference library populated with DNA barcoding sequences of voucher specimens (e.g., BOLD Systems v3; http://www.boldsystems.org/).

Nuclear (e.g., 16S rRNA, 18S rRNA, ITS regions), mitochondrial (e.g., cytochrome b, mitochondrial control regions), and some chloroplastic genes have all been used in barcoding eukaryotic organisms (Patwardhan et al. Reference Patwardhan, Ray and Roy2014). The more slowly evolving gene of the mitochondrial genome, cytochrome c oxidase I (cox1), is acknowledged as the ‘gold standard’ for species identification and DNA barcoding of animals (Hebert et al. Reference Hebert, Cywinska, Ball and deWaard2003). However, for parasite identification, discovery, and diversity research, the nuclear 18S small subunit (SSU) rRNA gene has been more commonly used (e.g., Harris & Rogers Reference Harris and Rogers2011; Maia et al. Reference Maia, Gómez-Díaz and Harris2012; Netherlands et al. Reference Netherlands, Cook and Smith2014 ; Perkins & Keller Reference Perkins and Keller2001; Wozniak et al. Reference Wozniak, Telford and Mclaughlin1994).

Notomi and co-authors, in 2000, described a new method of DNA amplification called Loop-Mediated Isothermal Amplification (LAMP) that has since been used for identification of major human and animal parasites and demonstrated comparable sensitivity and specificity to that of qPCR (Notomi et al. Reference Notomi, Okayama, Masubuchi, Yonekawa, Watanabe, Amino and Hase2000).

LAMP employs a DNA polymerase with high strand displacement activity and a set of four primers that recognize six distinct sequences of the target DNA, giving it very high specificity. Although relatively new in the category of diagnostic techniques, the LAMP has distinct advantages over both conventional PCR and qPCR, such as high specificity of DNA amplification in the presence of non-target DNA, cost-effective amplification at isothermal conditions since no thermal cycler is necessary, and simple detection of amplification products using fluorescent dyes. However, the lack of data with this technique hinders the development of LAMP kits and their use in mass screening of helminth infections (Biswal Reference Biswal, Debnath, Kharumnuid, Thongnibah and Tandon2016).

Which method is the best?

The sensitivity and specificity of diagnostic tests significantly influence the results of epidemiological surveys for helminth infections and the particular efficacy of anthelmintic treatment or drug resistance. The sensitivity of the diagnostic tool used is also important in helminth control programs; a low sensitivity leads to false negative results that in turn may lead to premature treatment cessation. Among the different diagnostic techniques described above, the fecal smear examination methods are the most frequently used in epidemiological surveys. The World Health Organization recommends the Kato-Katz method of duplicate slides for the detection of soil-transmitted helminth infections (Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale). The Faust method with zinc sulfate solution is one of the most common methods used in veterinary studies (Faust et al. Reference Faust, Sawitz, Tobie, Odom, Peres and Lincicome1929). Other methods such as sedimentation, McMaster, and FLOTAC are also used in parasitological studies (Biswal Reference Biswal, Debnath, Kharumnuid, Thongnibah and Tandon2016). Comparative data on fecal examination methods are controversial since each procedure varies due to helminth species and study methodology. Moreover, the similar examination methods can have different sensitivities for the same parasite species in different studies. However, a summary of recently published data on the comparison of different fecal examination methods FLOTAC and mini-FLOTAC shows higher sensitivity for most parasites when compared with the gold-standard Kato-Katz method, McMaster egg count method, and sedimentation techniques. PCR-based methods have a higher sensitivity than traditional coproscopical examination techniques, ranging from 78.9% to 100% (Knopp et al. Reference Knopp, Glinz, Rinaldi, Mohammed, N’Goran, Stothard, Marti, Grindoli, Rollinson and Utzinger2009; Taniuchi et al. Reference Taniuchi, Verweij, Noor, Sobuz, van Lieshout, JrWA, Haque and Houpt2011) for different parasitic species. Coproantigen ELISA tests are also highly sensitive methods, especially for identification of tapeworm infections – for example, sensitivity of the coproantigen test for the detection of Taenia pisiformis is 87.5% (Schär et al. Reference Schär, Odermatt, Khieu, Panning, Duong, Muth, Marti and Kramme2013), and recently developed coproantigen assays for detection of fluke worm infections such as Opisthorchis and Clonorchis range in sensitivity from 93.3% to 100% (Craig et al. Reference Craig, Gasser, Parada, Cabrera, Parietti, Borgues, Acuttis, Agulla, Snowden and Paolillo1995; Teimoori et al. Reference Teimoori, Arimatsu, Laha, Kaewkes, Sereerak, Sripa, Tangkawattana, Brindley and Sripa2017).

In terms of specificity, coproantigen tests rank lower than PCR tests, ranging from 54.2% to 100% (Rahman Reference Rahman, Choi, Bae and Hong2012; Watwiengkam Reference Watwiengkam, Sithithaworn, Duenngai, Sripa, Laha, Johansen and Sithithaworn2013). In a study by Nageswaran et al. (Reference Nageswaran, Craig and Devaney1994), the coproantigen test for detection of S.ratti had cross-reactions with Necator americanus and S. muris (Nageswaran et al. Reference Nageswaran, Craig and Devaney1994).

One major advantage of PCR analyses over ELISAs is a significant decrease in the detection limits. For example, positive results have been obtained with a concentration 0.11–0.35 ng of parasite DNA (Ai et al. Reference Ai, Dong, Zhang, Elsheikha, Mahmmod, Lin, Yuan, Shi, Huang and Zhu2010), whereas ELISA coproantigen tests typically require around 0.3–0.6 ng of parasite antigens (Mezo et al. Reference Mezo, González-Warleta, Carro and Ubeira2004).

Coproscopical examination methods are still favored by diagnostic clinics owing to their simple and economical set-ups. Without the use of costly equipment like thermal cyclers or spectrophotometers, coproscopic examinations can be performed in small laboratories or field studies. In order to circumvent the possibility of false negative results due to low concentrations of helminth eggs, low detection limit techniques like the Wisconsin and FLOTAC methods (detection limit 1 EPG), the McMaster method (10 EPG), and Kato-Katz (24 EPG) are recommended (Glinz et al. Reference Glinz, Silué, Knopp, Lohourignon, Yao, Steinmann, Rinaldi, Crindoli, N’Goran and Utzinger2010; Knopp et al. Reference Knopp, Glinz, Rinaldi, Mohammed, N’Goran, Stothard, Marti, Grindoli, Rollinson and Utzinger2009; Levecke et al. Reference Levecke, Rinaldi, Charlier, Maurelli, Bosco, Vercruysse and Cringoli2012). The sensitivity can be augmented by multiple stool examinations (Bogoch et al. Reference Bogoch, Andrews, Speich, Utzinger, Ame, Ali and Keiser2013).

The choice of an appropriate examination method is also dependent on the study objectives, including host species, estimation of overall parasite prevalence or individual parasite infection, the intensity of parasite infection, and efficacy of anthelmintic treatment or drug resistance. Moreover, recently published reports claim an absence of the so-called ‘gold standard’ of helminth infections diagnostics (Nikolay et al. Reference Nikolay, Brooker and Pullan2014; Tarafder et al. 2010). Thus, different methods can be employed in parasitological studies.