Advancement in the Identification of Parasites and Obstacles in the Treatment of Intestinal Parasitic Infections: A Brief Overview

Abstract

Nowadays, intestinal parasite infections (IPIs) continue to be a serious public health issue worldwide. Helminth and protozoa are common examples of infections caused by poverty and inadequate sanitation, which act as two variables linked to IPIs. In response to the growing impact of IPIs, more advanced detection techniques have been researched and developed. To identify these parasites, the diagnostic method’s efficacy is paramount. In view of the above, microscopy as a traditional method is now assisted by serology and molecular biological tools. The modern technological tools will help to assess the efficacy of eliminating these parasitic illnesses and future control programs.

1. Introduction

Infectious illnesses have long posed a hazard to the global population throughout history. Nowadays, intestinal parasites account for a significant portion of the world’s illness burden. A variety of parasite species are known to be common worldwide, particularly in sub-Saharan Africa, the United States, and Asia [1, 2, 3]. The intestinal parasitic infections (IPIs) are widely documented in underdeveloped nations and nowadays also continue to impair human well-being in affluent countries. Ascaris lumbricoides, Trichuris trichiura, and hookworm are examples of helminths that commonly cause infections, while protozoa such as Blastocystis hominis, Cryptosporidium sp., Entamoeba histolytica, and Giardia duodenalis causes infections that are typically due to significant morbidity and mortality, especially in endemic countries [4]. An estimated 2 billion people suffer intestinal parasite infections, and the number is estimated to have increased significantly every year, with an additional 4 billion people at risk of contracting infections [5, 6]. Among the reasons causing these diseases are extreme poverty, inadequate sanitation, social stigma, and a lack of knowledge about the prevention and treatment of these conditions [7]. Moreover, in a study it was reported that the host immune system, the afflicted organ, and the type of parasite were all factors that affect IPIs [8]. IPIs seldom result in death, but they can stunt a person’s development, especially in young children [9].

As the parasite infections increase, new advanced techniques and methods for the identification of parasite have been used [10]. Modern methods should also take less time to complete without sacrificing the caliber of the output. Many researchers prompt diagnosis is so essential and has remained a top goal to accurately identify the parasites, provide the proper treatment, and ultimately prevent patient deaths [11]. This brief review article will go over the various methodologies that were frequently employed in previous laboratory diagnostics, although each method has its own pros and cons. Here provides an additional summary of the techniques utilized in the diagnosis of many parasites that cause IPIs, the data was tabulated (Table 1) [40, 41, 42].

	Diagnostic approaches
	Microscopy-based	Serology-based	Molecular-based	Proteomics	References
Helminth
Schistosoma species	Using Kato-Katz technique	IHA, ELISA, dipstick	PCR, real-time PCR, multiplex PCR	LC-MS/MS	[12, 13, 14, 15, 16, 17, 18]
Soil-transmitted helminths	Using sedimentation or concentration techniques	ELISA	Multiplex real-time PCR	—	[19, 20]
Taenia solium	Using sedimentation or concentration techniques	ELISA, Immunoblo	Nested PCR	LC-MS/MS	[21, 22, 23, 24]
Protozoa
C. parvum, C. hominis	Using modified acid-fast staining	DFA, ICT assay kit	PCR, real-time PCR, multiplex real-time PCR, LAMP, Luminex	LC-MS/MS	[21, 25, 26, 27, 28, 29]
Giardia lamblia	Using trichrome or ion hematoxylin staining; Using concentration or sedimentation techniques	DFA, ICT assay kit	Multiplex real-time PCR	—	[21, 30, 31, 32, 33]
Entamoeba histolytica	Using staining methods	IHA, IIF, ELISA, ICT assay	Multiplex real-time PCR, LAMP	LC-MS/MS	[34, 35, 36, 37, 38, 39]

Table 1.

Diagnostic approach for the detection of intestinal parasites.

2. Advancement in parasite diagnosis

Several assays have been developed during the past few decades to improve the sensitivity and specificity of the test done for parasite identification. The diagnosis of parasitic illnesses has reached a new height because of advances in knowledge and technology. These methods have been used in many research projects around the globe, which has made it possible to combat with disease [41].

2.1 Microscopy-based approach

The only method available at the time for identifying parasites from cerebrospinal fluid, feces, blood smears, and tissue specimens was routine laboratory diagnosis, which includes the conventional microscopy technique. For the morphological identification of parasites, this technique has been widely used as this approach just needs a microscope and cheap reagents or dyes [11]. Nevertheless, over the time, even though microscopy examination has been regarded as the gold standard, it has become increasingly challenging to identify or differentiate the species with the unaided eye. This is because high-quality results require the expertise of a skilled microscopist, and the entire process—from sample collection to the concentration of the parasite’s identification—takes time. This condition was further demonstrated by the fact that morphological inspection alone was insufficient to differentiate between the species. Dual procedures such formalin-ether sedimentation, trichrome, and Ziehl-Neelsen staining are typically applied jointly despite the drawback.

Furthermore, other methods like Kato-katz and McMaster counting methods are also widely used nowadays and have been considered as standard techniques for the detection and quantification of IPIs for nearly forty years. Since then, the WHO has recommended them. In the meanwhile, the McMaster counting method is widely used to evaluate STHs or soil-transmitted helminths. The sensitivity of both approaches was shown to vary significantly across trials. The McMaster technique is based on the floating of eggs, whereas the Kato-Katz method covers a larger quantity of feces but has a downside when the infection intensity is relatively low. Additionally, both methods can be used to diagnose IPIs, though, because of its robust factor, the latter is better suited for additional standardization.

2.2 Serology-based approach

If the parasite density is low, for example, as in the case of Toxoplasma gondii, it cannot be directly identified because of its life cycle in the host. Therefore, an indirect method of parasite identification employed a serology-based technique needs to be used. Together with the ability to view the parasites under a microscope, the advent of serology-based approaches has made it possible to diagnose IPIs more quickly and effectively. The two subcategories of serology-based diagnosis are antigen detection assays and antibody detection assays. It includes hemagglutination (HA) test, complement fixation (CF) test, immunoblotting, enzyme-linked immunosorbent assay (ELISA), indirect or direct immunofluorescent antibody (IFA or DFA), and rapid diagnostic tests (RDTs) [41]. In the laboratory diagnosis techniques, ELISA test is the most widely used antibody detection test. Although, dipstick assays that are simpler and have a higher sensitivity than microscopy when it comes to identifying intestinal schistosomiasis, have also been thought to be a more sensible option [16].

Because of their sensitivity, two more serology-based tests are also frequently used in laboratories, i.e., indirect hemagglutination (IHA) and indirect immunofluorescence (IIF). However, few researches have been done to examine their repeatability [43]. Moreover, immunoassays have emerged as a primary diagnostic method for parasites [44, 45]. Several commercial kits in the market use an immunoassay-based method to test the parasites using FITC-monoclonal antibodies that target cell wall antigens to detect Giardia and Cryptosporidium [41]. It took less time to conduct the test and is easier to comprehend the assay’s results. But one drawback of the serology-based approach is that, because antibodies are present at different times after infection, the diagnosis is made retrospectively (Figure 1) [40, 41].

Figure 1.

Representing serology-based approach (figure taken from De Vriese et al. [46], see reference [47]).

2.3 Molecular-based approach

Polymerase chain reaction (PCR) technology has grown in importance as a tool for quantifying parasites and assessing the effectiveness of treatment regimens. This method provides higher specificity and sensitivity than the existing diagnostic tests. As technologies have developed, nested, multiplexed, and real-time PCR have replaced classical PCR. By focusing on the 18S rRNA, the PCR technique has effectively identified Cryptosporidium from ambient samples in cases of protozoan illnesses [26]. Furthermore, as noted, the multiplex real-time PCR assay, which was utilized to identify E. histolytica, G. lamblia, and C. parvum/C. hominis, was shown to be similar to microscopy and permit the simultaneous detection of several sequences within a single reaction tube [21]. By using a pentaplex real-time PCR approach, in a previous study four species of soil-transmitted helminths: Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis was effectively detected [20]. For the detection of DNA of Taenia solium based on the TSO31 gene, nested PCR has demonstrated 100% sensitivity and specificity [48]. Other earlier research has shown the sensitivity of real-time PCR in identifying Giardia and Cryptosporidium (oo) cysts [49, 50]. The traditional PCR-based approach takes a long time and yields non-quantitative results [51]. Though cost is an issue for both real-time and multiplex PCR, both have produced quick results in comparison to 64 Advances in Parasite Diagnosis and Challenges using the traditional approach [11]. Additionally, one of the most used methods for identifying parasites like Toxoplasma gondii is restriction fragment length polymorphism (RFLP) [11, 52]. This method can identify several genotypes from a single sample, based on the digestion of PCR products by restriction enzymes, is appropriate for environmental samples [53].

In addition to the PCR-based methods, numerous alternative amplification techniques also have been developed. Loop-mediated isothermal amplification (LAMP) is a unique gene amplification method that was first employed in several investigations [54]. The advantages of the LAMP approach are its excellent specificity for the target sequence and its capacity to amplify DNA with high efficiency under isothermal condition [54]. This procedure just needs four primers, DNA polymerase, and a standard laboratory water bath or heat block for the reaction; it is also thought to be straightforward and simple to carry out [11]. Additionally, LAMP may amplify RNA sequences very effectively when combined with reverse transcription. Furthermore, as stated reagents can be stored at room temperature without the need for any post-PCR procedures [41]. LAMP has been utilized in the identification of DNA and RNA viruses, including SARS and West Nile viruses, as per earlier research conducted [55, 56]. Additionally, a previous study compared LAMP with multiplex PCR using stool samples from taeniasis patients [57]. In the meantime, a recent work described a unique diagnostic strategy that combines the LAMP and MinION sequencing technology to identify human Plasmodium species [58].

Furthermore, in recent years, the analysis of proteins expressed by parasites has been a fast-expanding field of proteomics research [59]. The demand for sensitivity has since grown as a result of the present interest in proteomics, which helped researchers overcome the obstacles to early diagnosis and therapy [60]. Two methods were used to identify the proteins: top-down and bottom-up. In the top-down approach, two-dimensional polyacrylamide gel electrophoresis was used [40]. Proteomics has been applied to a number of different disorders, including taeniasis, malaria, and Chagas disease [61, 62, 63]. A different novel strategy known as microsatellites was based on simple sequence tandem repeats and was also reported in studies on parasites that could rapidly evolve and were taken from both people and animals [64, 65]. Due to the prominent polymorphism shown, microsatellites are regarded as valuable genetic markers [66]. Luminex-based assays, which integrate flow cytometry, fluorescent beads, lasers, and digital signal processing, have also surfaced as a potential method for identifying parasite infections [11, 67]. In a study, Luminex was used to distinguish between the C. hominis and C. parvum species using just one nucleotide. Other serological testing or antigen detection cannot differentiate between the two species [28]. The assay’s research enhances the other PCR procedures’ speed, accuracy, and dependability [11, 68, 69].

3. Challenges in the management of parasitic infections

A disease arises from the daily exposure of the human body to parasites from our surroundings. It has been demonstrated that intestinal parasites, which were formerly thought to be benign commensals, may potentially be pathogens [68]. More number of parasites can be discovered at the same time with further improvement of diagnostics procedures as the researchers increasingly try to concentrate on refining existing diagnostic techniques rather than developing new ones. Mass screening and quick diagnostics are helpful features that will increase our knowledge of parasites and lessen the spread of illness [69]. Moreover, to prevent significant delays, the development of field-based diagnosis is also required. Cost is still a problem, as are sensitivity and specificity. Particularly in endemic areas, renewed and sustainable intervention is required. The status of public health, particularly in the area of medical parasitology, can be improved globally in several ways, including by enacting appropriate regulations or policies and by tracking, assessing, and bolstering the surveillance of parasitic diseases [70, 71]. Monitoring the resistance to anti-parasite medications and other options is essential for creating improved patient treatments. Increased awareness is required, especially in targeted areas, and preventive interventions including routine deworming [72]. To improve and eradicate possible diseases, more financing for parasitological research and interventions is also required [73].

4. Conclusion

The utilization of microscopy-based techniques is still beneficial in the diagnosis of patients with parasitic illnesses, even with the rapid advancement of other methods. On the other hand, molecular and serology-based techniques are seen to be superior substitutes, particularly for infections with a narrow spectrum of parasites. The current state of research and methodologies for illness detection offers a better foundation for the development of more effective, dependable, and affordable approaches, thereby enhancing life quality and paving the way for future drops in the global disease burden. Higher authorities, including public health and healthcare organizations, medical experts, and funding suppliers, must fully commit to implementing these recommendations. The efforts to combat IPIs would be strengthened by the support of all stakeholders, and more work needs to be done to fulfill the largest gap in science.

Acknowledgments

Authors are grateful to the facilities provided by the head, department of Zoology, Chaudhary Charan Singh University, Meerut, India.

Conflict of interest

The authors declare no conflict of interest.