The protozoan parasites Entamoeba histolytica, Giardia lamblia, and Cryptosporidium spp. are the causative agents of amebiasis, giardiasis, and cryptosporidiosis, respectively. From 2005 to 2019, a total of 28,229 cases of amoebic dysentery and seven resulting deaths were reported in China (Huang et al., 2020). Annually, 280 million people worldwide are estimated to have clinically diagnosable giardiasis (Feng and Xiao, 2011; Ryan and Cacciò, 2013; Einarsson et al., 2016; Squire and Ryan, 2017). And approximately 28.5 million giardiasis cases are estimated to occur in humans per year in China (Feng and Xiao, 2011; Li et al., 2017). The weighted infection rate of Entamoeba histolytica and Giardia lamblia was 0.06 and 0.60%, respectively (Zhou, 2018). In China, an average prevalence of 2.97% involving at least 200,054 people from 27 provinces has been revealed in a retrospective epidemiological analysis of human Cryptosporidium infections (1987–2018); the burden of disease caused by Cryptosporidium varies between and within areas (Liu et al., 2020). The three intestinal protozoa discussed in this article have very simple biological cycles without intermediate hosts. Infection occurs via the fecal-oral route and these agents can infect the human intestinal tract, causing serious disease (Ghenghesh et al., 2016; Li et al., 2017; Xu et al., 2018).

E. histolytica is an internationally recognized pathogenic entamoeba that is invasive in animals and humans and is found in the human colon. It can cause intestinal amoebiasis (e.g., amoebic dysentery) and extraintestinal amoebiasis (e.g., amoebic liver abscess). E. histolytica is one of the main pathogens causing diarrhea in patients. Amebiasis is still a major cause of morbidity and mortality in developing countries, and it remains an important public health problem (Smith, 1997; Li et al., 2021).

G. lamblia is one of the most common intestinal parasites in the world, affecting approximately 200 million people annually. Giardia is transmitted via the fecal to oral route, most often by ingestion of contaminated food or water (Einarsson et al., 2016). Symptoms of Giardia infection include foul-smelling diarrhea, abdominal cramping, bloating, gas, and nausea. Immunocompromised individuals and undernourished children from developing countries are more susceptible to serious manifestations of untreated Giardia infection (Júlio et al., 2012; Bartelt and Sartor, 2015).

C. parvum is a specialized intracellular protozoan parasite that has a monoxenous life cycle (Zhu and Su, 2018). Infection takes place via oral ingestion of oocysts containing invasive sporozoites. These sporozoites enter intestinal epithelial cells and form a parasitophorous vacuole that is located at the apical part of the host cell, just underneath the brush border (Hemphill et al., 2019). This infection may cause diarrhea and abdominal pain in the host; the course of the disease is mostly self-limiting. Patients with immunodeficiency are more prone to developing severe symptoms and are more often complicated by extraintestinal cryptosporidiosis. Cryptosporidiosis is considered one of the original AIDS-defining illnesses and a major risk factor for mortality, especially compared with other AIDS-defining illnesses (Ahmadpour et al., 2020).

Currently, microscopic examination of stool specimens remains the primary method for diagnosing intestinal protozoan parasites, especially in developing countries. Light microscopy is its most commonly used detection method. However, microscopy can lead to incorrect results, with harmless parasites being interpreted as disease-causing, or life-threatening parasites being missed (Halligan et al., 2014). Many protists are only present in small quantities in fecal samples. Additionally, the quality of the microscopic examination is highly dependent on the skills of the laboratory technician (Khare et al., 2014). There are several PCR-based methods that are used in the diagnosis of E. histolytica, G. lamblia, and C. parvum (Friesen et al., 2018; Tsui et al., 2018; Jerez Puebla et al., 2020; Robinson et al., 2020; Shahbazi et al., 2020; Cai et al., 2021; Calle-Pacheco et al., 2022). Among the PCR methods developed for better diagnostics, real-time quantitative PCR (qPCR) methods are considered the leading ones as they are highly sensitive and specific. To the best of the authors’ knowledge, a few studies refer to use of multiplex qPCR commercial kits for these three protozoan parasites (Laude et al., 2016; Argy et al., 2022). However, these kits have national or regional limitations and are not available in some countries, including China. However, some key parameters, such as sequences of the specific primers and probes or reaction conditions, cannot be disclosed due to commercial confidentiality. Thus, the purpose of this study was to establish a TaqMan-based triplex qPCR assay for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum.

Amebiasis, giardiasis, and cryptosporidiosis are difficult to prevent and control because of their zoonotic characteristic and large number of reservoir hosts (Ghenghesh et al., 2016; Li et al., 2017, 2021; Xu et al., 2018). The ability to rapidly detect E. histolytica, G. lamblia, and C. parvum is important in laboratory diagnosis because these pathogens cause serious diseases (Helmy et al., 2014; Hemphill et al., 2019; Li et al., 2020). PCR-based molecular diagnostic techniques have obvious advantages in the accurate identification of intestinal protozoan species and detection of mixed infections of multiple intestinal protozoa. These techniques can compensate for the lack of sensitivity and high skill requirements of microscopic examination (Friesen et al., 2018; Tsui et al., 2018; Jerez Puebla et al., 2020; Robinson et al., 2020; Shahbazi et al., 2020; Cai et al., 2021; Calle-Pacheco et al., 2022). In particular, the qPCR method does not require electrophoresis and the results can be analyzed directly using images and data such as amplification curves and Ct values, making it ideal for use in a variety of medical settings where specialist parasitological microscopy staff are scarce. With the current global COVID-19 pandemic and the need for nucleic acid detection, the coverage of qPCR instruments is increasing at all levels in medical institutions throughout China and worldwide. Therefore, the acceptance of qPCR assays at these institutions is likely to be high.

A number of single or duplex qPCR assays have also been established for E. histolytica, G. lamblia, or C. parvum (Helmy et al., 2014; Tsui et al., 2018; Li et al., 2020; Robinson et al., 2020; Cai et al., 2021; Calle-Pacheco et al., 2022). In this study, a triplex qPCR assay was developed that allows amplification of three target sequences in one reaction using three pairs of primers and probes to simultaneously identify these three target sequences. The sensitivity and specificity of the triplex qPCR assays are determined by the primers and probes. So, it is particularly important to find the optimal primers and probes. We designed the primers and probes in the conservative region of each three intestinal protozoa (Table 1), the specificity of the primers and probes were verified by BLAST and Primer-BLAST. We optimized the annealing temperature, primer and probe concentration to make sure that the developed assay had the greatest amplification efficiency and the lowest Ct value.

In this study, the specificity of each of the Emh, Gla, and CryP triplex qPCRs was evaluated individually against the DNAs from other two protozoa species. In addition, the specificity of each of the three triple qPCRs was evaluated in triplex formats against the DNAs from other intestinal parasites such as C. baileyi, T. saginata, T. solium, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, and Entamoeba coli (Clonorchis sinensis and Paragonimus westermani are not intestinal parasites, but pathogens can be detected in human fecal samples). The specific primers and probes for these three intestinal protozoa parasites did not exhibit cross-reactivity among them or among any of the seven human parasite species described above. That is to say, the developed assay has good specificity. The standard curves showed good linear correlation between the Ct values and logarithm of concentrations of the three standard plasmids. According to the results, the LOD of the triplex qPCR for the detection of Emh, Gla, and CryP was up to 500 copies/μL.

The results of the repeatability testing of the triplex qPCR revealed that the intra-assay CV and inter-assay CV were both below 2%. Consequently, this may indicate that the triplex assay has a high repeatability (intra-assay) and reproducibility (inter-assay) within the range of detection (Table 4). Noteworthy, in clinical practice, there exists the possibility of mixed infections of different parasites at different concentrations in fecal samples (Hemphill et al., 2019). Thus, the co-infection models detection was used for the determination of the detection efficiency of mixed infection. The result showed that these three intestinal protozoa at different concentrations with different combination could be determined, and the standard deviation of the co-infection detection was less than 1% (Table 5). Therefore, the developed triplex qPCR assay is highly reliable and accurate, and it can also increase throughput while reducing reaction costs by detecting these three intestinal protozoan parasites in just one amplification.

Clinical fecal samples were tested using the triplex qPCR assay developed in this study, and the results were positive for C. parvum in four samples and positive for Cryptosporidium in the copro-antigen assays. The PCR results of the remaining 159 fecal samples were negative. According to some scholars (Weitzel et al., 2006; Helmy et al., 2014; Uppal et al., 2014; Jerez Puebla et al., 2020), the results of immunological methods are not always accurate. It has also been pointed out that the reliability and reproducibility of results from only immunological assays are controversial (Danišová et al., 2018). Few commercial kits for the diagnosis of intestinal protozoan copro-antigens have been reported in China (Chen et al., 2012; Wang et al., 2021). Furthermore, the results of copro-antigen testing are susceptible to many factors, such as the length of time the samples are left after collection, storage, and transportation conditions. Thus, the role of the copro-antigen assays in this study was to initially screen fecal samples from individuals with suspected intestinal protozoan infections, to appropriately narrow the scope of nucleic acid detection, and to reasonably lessen the workload of laboratory staff. However, the triplex qPCR assay simultaneously amplified well one sample of E. histolytica, G. lamblia, and C. parvum (Figure 2), and the amplification curves of these samples showed significant exponential growth, indicating that the method can be initially applied for the laboratory detection of intestinal protozoa.

In summary, the triplex qPCR assay established in this study is highly sensitive, specific, stable, and repeatable, and it can be used for rapid, specific, and accurate quantitative detection of three important human intestinal protozoa, providing technical support for disease control and medical institutions. In future research, we will strengthen the combined application of molecular biologic, immunologic, and pathogenetic diagnostic methods for these three protozoa, which will help to improve the detection rate and epidemiological surveillance of intestinal protozoa to promote the prevention and control of intestinal protozoiasis.

The authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository, prior to publication. Frontiers cannot accept a manuscript that does not adhere to our open data policies.

The studies involving human participants were reviewed and approved by the Health Ethics Committee of Shanghai Municipal Center for Disease Control and Prevention (ref 2019-23). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

ZW and HW conceived the research project. YZ, ZW, and QZ performed the experiments. YZ and ZW performed the data analysis. JC, HP, XM, LJ, YZ, and ZW interpreted the data and discussed the results. YZ, ZW, and JC wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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