1. Introduction

Toxoplasmosis is a zoonotic parasitic disease caused by the protozoan Toxoplasma gondii (T. gondii; ^[1]). The clinical manifestations of toxoplasmosis in humans, including congenital, cerebral and ocular toxoplasmosis, cause a substantial disease burden worldwide ^[2][3] Moreover, T. gondii can also cause clinical disease in its animal hosts, resulting in major losses in livestock industry and lower welfare for the affected animals ^[1].

It has been estimated that 42–61% of acquired toxoplasmosis cases are foodborne ^[4]. The food- and waterborne transmission routes of T. gondii are numerous, including ingestion of infective tissue-dwelling stages of the parasite in raw or undercooked meat of infected animals and ingestion of oocysts, shed by infected felines and sporulated in the environment, in contaminated water or food, such as fresh produce (fruits, vegetables, and juice) ^[5].

Although T. gondii is a highly prioritized zoonotic foodborne pathogen in Europe and worldwide ^[6][7], it is not systematically controlled. Evaluation of T. gondii oocyst contamination of fresh produce, such as ready-to-eat (RTE) salad leaves, is an unfilled need of both the public health sector and food industry, especially with an increasing consumer preference for these food items ^[8]. Although scientific literature has reported an association between the consumption of unwashed fresh produce and T. gondii infection, the relative importance of this infection source remains unknown ^[5] and outbreak investigations are scarce. The typically low numbers of T. gondii parasites in food matrices makes detection challenging, and at present, no specific regulations or ISO standards are available for detection of T. gondii in any food matrix ^[5]. Thus far, ISO standards have been developed to detect few other foodborne protozoan parasites in fresh produce. The ISO standard method (ISO 18744:2016) for the detection of Cryptosporidium spp. and Giardia spp. on leafy greens and berry fruits is based on visual detection by immunofluorescence microscopy and is not amenable to high-throughput testing. In order to address food safety risk assessment challenges typical for foodborne protozoan parasites, it is essential that the testing moves to standardised molecular assays, similar to e.g. US FDA—BAM 19b for “Molecular Detection of Cyclospora cayetanensis in Fresh Produce Using Real-Time PCR” ^[9].

2. Discussion

Detection of T. gondii in vegetables is challenging due to the low sensitivity of existing detection methods. This also holds true for other foodborne parasites (e.g. Cryptosporidium spp. and Giardia duodenalis) ^[10]. As oocysts of T. gondii are highly resistant to environmental conditions and do not multiply in the environment, oocyst recovery from fresh produce is the first and key step to enable successful detection. Molecular detection must then rely on efficient DNA extraction from the robust oocysts, together with a reduction of possible contaminants that could inhibit the DNA amplification. Finally, amplification must be specific and sensitive to detect DNA from low numbers of oocysts, ideally a single oocyst, avoiding any cross-amplification with closely related species.

As shown in this review, many different methods have been described for each step of the molecular detection of T. gondii oocysts and different combinations of them have been used to analyse fresh produce as well as other matrices. This variability, which was also evident in the results of the questionnaire survey ^[11], prevents a direct comparison of the studies to identify the most promising method for a sensitive and reliable detection of T. gondii oocysts in fresh produce (as well as in other matrices). Although specific characteristics of different vegetable matrices can interfere with oocyst recovery due to e.g. trapping and adhesion force and, later on, with molecular detection (i.e. different concentrations of PCR inhibitors), the overall molecular detection procedure should be harmonised and standardised. The oocyst recovery step from fresh produce is particularly important but challenging to standardise due to a large variability in the reported methods (e.g. washing procedure, washing buffers and oocyst concentration). For instance, stomaching with an appropriate setting of homogenisation power and speed to account for brittleness of the vegetable samples, would be a fast procedure to apply for large scale analysis and easy to standardize. Due to the presence of high amounts of natural detergents in some types of fresh produce (e.g. saponins in spinach), the use of washing buffers with detergent (i.e. Tween-80) might not be recommended as they could exacerbate foaming and potentially trap oocysts in the foam, thus lowering the recovery rate. The 1 M glycine solution is potentially the buffer of choice, as it is inexpensive and did not generate an excess of debris during stomaching of lettuce as the sample matrix ^[12], which could eventually interfere with downstream oocyst concentration and DNA extraction. Although oocysts concentration by centrifugation might be time consuming and require a centrifuge, other procedures might be more complicated or less efficient. For instance, flocculation of water samples with Fe₂(SO₄)₃ resulted in PCR inhibition ^[10]. The risk of oocyst loss following NaNO₃ flotation was highlighted in one study on soil samples ^[13], suggesting that NaNO₃ flotation is suitable when oocyst contamination is ≥103/40 g soil. One paper discussed that while flocculation is simple and inexpensive, filtration is more robust for processing turbid wastewater (and possibly the washing suspensions of vegetables), and PCR inhibitors appeared to be eliminated by using 1-μm pore-sized polyethersulfonate membrane filters ^[14]. Additionally, filtration would be preferable when large volumes (litre) of a sample need to be processed.

The reported DNA extraction protocols substantially differ in their approach to break the robust oocyst wall (FT, US and BB), whereas further DNA purification and clean up from inhibitors are mostly performed using silica-column-based DNA extraction kits. Although FT does not require expensive equipment, in contrast to the use of a bead beater, the choice of the most promising and efficient FT procedure is difficult due to the large variability of settings applied in different studies (e.g. length and number of reported freeze and thaw cycles were quite different). Moreover, the requirement of several cycles of FT is time consuming especially when a large panel of samples is tested. According to most of the manufacturer’s protocols, kits using pre-packed silica spin columns allow the use of only a fraction of the supernatant obtained from the initial sample lysis per single extraction. This might lead to a considerable loss of material and reduction of the final assay sensitivity, as either only a portion of the original sample is used for the DNA extraction step, or might require multiple DNA extraction from the same sample with consequent increase in assay time and costs. Commercial kits including a mechanical disruption step (e.g. BB) have already been successful in detecting Hammondia spp. and T. gondii oocysts by PCR with a high sensitivity ^[15]. Furthermore, they have the advantage of using larger sample volumes without substantial adaptation of the kit that are loaded with a silica matrix onto empty columns and could, therefore, favour a higher assay sensitivity. Whether the performance of different commercial kits based on BB is comparable or not was not specifically assessed in any of the papers included in this study, but might be presumed by the comparability of two kits tested in Herrmann et al., 2011 (specifically NucleoSpin Soil from Marcherey-Nagel vs ZymoResearch fecal DNA Kit from Zymo) ^[15]. However, since available kit formulations and producers might change over time and in different countries, kit performance should always be evaluated prior to a study, in order to select the most suitable kit.

For the purpose of this entry, we did not further consider nested-PCR and LAMP (loop-mediated isothermal AMPlification) assays as suitable for routine testing of fresh produce. Despite their higher sensitivity and specificity compared to conventional PCR, both techniques suffer from a high risk of background and cross-contamination, and nested PCR requires two consecutive rounds of amplification. Concerning the reported molecular assays, qPCR targeting either the B1 gene and/or the 529RE both provide a very high sensitivity, due to multiple copies of both targets in the T. gondii genome. Although double-strand DNA-intercalating fluorescence dyes (e.g. SYBR Green) combined with melting curve analysis (MCA) are relatively cost beneficial and easy to use, dual-labeled TaqMan probes have the advantage of combining detection with confirmation of the amplification products without the need for further amplicon sequencing. It should be noted that the specificity of the amplification product can be of concern, especially when targeting 529RE, due to potential cross amplification with parasites closely related to T. gondii (i.e. Hammondia hammondi, Sarcocystis spp. Neospora caninum) ^[16][17].

PCR inhibitors are important confounders that must be addressed in any PCR-based detection effort. Molecular detection of pathogens in food can be challenging due to a large variety of PCR inhibitors that can be co-extracted with DNA. Especially, DNA extracts from pelleted washing suspensions of plant-based food may contain diverse PCR-inhibiting substances from debris of the plants themselves (e.g. phenols, polyphenols, polysaccharides), but also from residual soil or irrigation water components (e.g. humic and fulminic acids) ^[18]. Depending on the food matrix and type and mechanism of inhibitory substances, different strategies can be evaluated to decrease their concentration in the sample or to reduce their inhibitory effect by e.g. using less-sensitive polymerases or specific PCR additives (e.g. BSA, DMSO) ^[18]. It should be noted that, for the detection of PCR inhibitors and to exclude false-negative results, the use of an internal amplification control (IAC) is mandatory for diagnostic PCR detection of foodborne pathogens according to CEN/ISO 22174 ^[19]. Competitive IACs are synthetic oligonucleotides that are amplified with the same set of primers as the target gene. Although they are amplified under the same conditions and thus mimic the amplification of the target gene, they also have a stronger potential to reduce the assay sensitivity and may require more optimization work ^[20]. As low sensitivity and inhibition is already an issue when analysing fresh produce for T. gondii, we rather propose the application of non-competitive IACs, ideally as a synthetic sequence with no homology with either the target parasitic DNA or with the matrix, as for example used in the US FDA—BAM 19b for “Molecular Detection of Cyclospora cayetanensis in Fresh Produce Using Real-Time PCR” ^[9]. As these non-competitive IACs are amplified with a different set of primers, they can universally be applied in different PCR detection systems and have the advantage of generally not competing with the target amplification, when used in low concentrations and with limiting primer concentrations.

We would like to stress that for any published qPCR assay, it is important to report the performance characteristics according to the MIQE guidelines ^[21][22][23]. This includes: i) use of an IAC to check for PCR inhibition; ii) preparation of a standard curve (10-fold serial dilution of at least five template concentrations) with background matrix (e.g., pelleted washing suspensions from uncontaminated food matrix); iii) evaluation of amplification efficiency and linearity with a R2 value (ideally ≥0.98); iv) determination of the LoD95%, supported by spiking studies.

A standardized procedure to be applied for the detection in fresh produce would not only be desirable for T. gondii but also for other foodborne protozoan parasites. Of course, implementation of slightly different methods might be necessary to reliably detect the target parasite. The problems associated with the availability of a large number of laboratory methods for pathogen detection are manifold. If prevalence data are not comparable from different regions or countries, they might result in inaccurate risk assessment conclusions. For instance, if quantification is required, it is necessary to define the quantified target (DNA amount, number of oocysts, target copy number ) as well as a standardized and harmonized procedure to convert this to equivalent numbers of oocysts (indeed oocysts load is the data that food stakeholders might expect). This is exacerbated by the fact that the large majority of published methods are not or insufficiently validated. Although ISO standards for the validation of parasitic methods are currently not available, method validations can be based on a number of available documents ^{[21][24][25][26][27][28][29][30]}.

Noteworthy, none of the articles reviewed reported on any attempt to evaluate the applied methodology through an inter-laboratory comparison. Results of ring trials are an important indicator for the inter-assay precision (reproducibility) of a method and an essential step for the better understanding of the method characteristics. As already described for the validation of microbiological methods in the food chain, inter-laboratory comparisons should also be performed as part of the validation of parasitological methods. In light of the increasing internationalisation of food supply chains, the need for conclusive data to better understand food-borne transmission or to provide a solid basis for risk assessments, has increased. For this, robust, validated and standardized laboratory methods for the detection of contamination of food sources with a high level of confidence are essential. This is especially important for T. gondii, a highly prioritized zoonotic foodborne pathogen, where laboratories are currently using a multitude of different diagnostic approaches.