Evolution of Toxoplasma gondii Detection and Genotyping Methods

TL;DR

This paper reviews the development of methods to detect and genotype Toxoplasma gondii, a parasite affecting humans and other hosts.

Contribution

The study compiles and evaluates both traditional and modern molecular methods for T. gondii detection and genotyping.

Findings

Molecular methods like PCR and LAMP are widely used for T. gondii detection.

Genotyping techniques such as MLST and microsatellite analysis help assess parasite diversity.

Newer methods like ddPCR offer improved sensitivity and specificity.

Abstract

Toxoplasma gondii is a parasitic protozoan that was first described over 100 years ago. Since then, its presence has been confirmed in a wide range of hosts, including humans. T. gondii infections are estimated to affect approximately 30% of the human population, representing a particular hazard to pregnant women, the elderly and immunocompromised individuals. In recent decades, diagnostic methods have been developed to detect the parasite, not only in clinical specimens but also in food or environmental samples. Nowadays, in addition to the mere confirmation of the presence of the parasite, the determination of its genotype is becoming increasingly important. The assessment of T. gondii diversity enables the investigation of the parasite′s circulation in host populations, the identification of atypical isolates and the inference of virulence. The study is aimed at gathering and presenting information on methods for detecting and genotyping T. gondii, including their range of applications, advantages and limitations. In view of the rapid development of molecular methods in recent years, particular emphasis was placed on this group of techniques. Therefore, both popular methods, such as one‐step PCR and nested PCR and relatively new techniques, such as LAMP or ddPCR, are included in the discussion. The review is complemented by the presentation of the most common techniques used in T. gondii genotyping, including PCR‐RFLP, MLST and microsatellite sequence polymorphism studies.

Tables

Table 1. Comparison of methods used in diagnostics of Toxoplasma gondii.

Method	Sensitivity	Specificity	Cost	Scalability	Labour intensity	Typical application/comments
Bioassay (in mice or cats)	High	High	Moderate–high	Low	High	Gold standard for detecting viable parasites, ethically and logistically demanding and rarely used in routine diagnostics.
Sabin–Feldman dye test (SFT)	High	High	Moderate	Low	High	Classical serological reference test, requires live tachyzoites and limited to reference laboratories.
ELISA	Moderate–high	Moderate–high	Low–moderate	High	Low	Routine serological screening (IgM/IgG), easily automated and dependent on antigen quality.
Western blot	High	Very high	High	Moderate	High	Confirmatory test after ELISA, provides banding pattern and more time‐consuming and expensive.
End‐point PCR	Moderate–high (depends on the target genes)	Moderate	Moderate	Moderate	Moderate	Detects parasite DNA, prone to contamination, requires good‐quality nucleic acids and sensitivity depends on the target sequence
Nested PCR	High–very high (depends on the target genes)	High	Moderate	Moderate	High	Two‐step amplification increases sensitivity and specificity but raises contamination risk, mostly used in research.
qPCR (real‐time PCR)	Very high	High–very high (especially in case of probe‐based assays)	High	High	Moderate	Quantitative detection, probe‐based assays provide higher specificity than dye‐based (e.g., SYBR Green) and widely used in clinical labs.
LAMP (Loop‐mediated isothermal amplification)	High	High	Low–moderate	High	Low	Rapid and cost‐effective alternative to PCR, suitable for field diagnostics and limited quantitative potential.
Digital PCR (dPCR)	Very high	Very high	Very high	Moderate–high	Moderate–high	Provides absolute quantification and ultrahigh sensitivity, ideal for low–parasite‐load samples and has costly instrumentation.

Table 2. Comparison of methods used in genotyping of Toxoplasma gondii.

Method	Sensitivity	Specificity	Cost	Scalability	Labour intensity	Informativity	Typical application/comments
Single‐locus RFLP	Moderate	Moderate	Low	Low	Moderate	Low	Classical PCR–restriction enzyme assay, differentiates mainly major lineages, limited for atypical strains, insufficient for full genotyping and requires adequate DNA quantity.
Multilocus PCR‐RFLP	Moderate–high	High	Moderate	Moderate	High	High	Uses several polymorphic loci, is standard in many epidemiological studies, enables lineage‐level classification and full profiles often fail when DNA is limited or degraded.
Single‐locus SNP analysis	Moderate	High (followed by the sequencing)	Moderate	Moderate	Moderate	Low–moderate (depends on amplified marker)	Simple and rapid and is suitable for confirming known polymorphisms but limited application in genotyping as single locus provide limited amount of data. Performance depends on the used marker
MLST (multilocus sequence typing)	Moderate–high	Very high	High	Moderate	High	High–very high	Sequence‐based typing approach with high discriminatory power. Complete profiles often unattainable from low‐DNA clinical samples without prior parasite culturing (e.g., bioassay).
Probe‐based qPCR typing	High	Very high	Moderate–high	High	Moderate	Low–moderate	Quantitative and specific (e.g., TaqMan assays), useful for detecting particular alleles but not a comprehensive genotyping tool.
HRM (High‐Resolution Melting) typing	Moderate–high	High	Low	High	Low	Low–moderate	Closed‐tube method, fast and cost‐effective, discriminates between particular SNPs but not a comprehensive genotyping tool.
Microsatellite typing	Moderate–high	High	Moderate	High (possibility of multiplexing)	Moderate	Very high	Highly discriminatory, provides high informativity due to analysis of many (usually 10–15) polymorphic loci and detects clonal and atypical strains. Low‐DNA samples (Ct > 35) frequently yield incomplete profiles.

Full text

1. Introduction

Toxoplasmosis is a disease caused by a parasitic protozoan, Toxoplasma gondii, which was first detected in the early 20th century (1908) in the tissues of a rodent, Ctenodactylus gundi, by Nicolle and Manceaux. Since then, the presence of T. gondii has been confirmed in many warm‐blooded animal species, including humans [1].

The life cycle of the parasite comprises the asexual phase, which can occur both in the cells of intermediate hosts (e.g., birds or mammals) and of the definitive host (representatives of the felid family) and the sexual phase occurring only in the intestinal epithelial cells of the definitive host [2]. In the case of co‐infection with two (or more) T. gondii strains, recombination of the genetic material of the parasites may occur [3]. The oocysts, which are excreted by cats, provide a source of infection for intermediate hosts and the spread of the parasite is promoted by its relatively long deposition time in the environment, which is estimated to be approximately 18 months [2, 4]. A meta‐analysis conducted by Montazeri et al. [5], including papers published in the years 1967–2017, indicated that the prevalence of anti‐T. gondii antibodies in the global domestic cat population accounted for approximately 35%, whereas for wild felids, the presence of antibodies was confirmed in approximately 59% of the individuals under study.

The life cycle of the parasite comprises three developmental forms: tachyzoites, tissue cyst with slow‐dividing bradyzoites and oocysts filled with sporozoites, which are spread in the environment by the definitive hosts [6]. T. gondii usually enters the host′s body either as oocysts or tissue cysts, depending on the source of infection. Subsequently, invasive forms are released from the tissue cysts—bradyzoites and from the oocysts—sporozoites [2, 7]. In the small intestine, the parasite differentiates into motile, rapidly dividing tachyzoites, which penetrate through the intestinal epithelium, first colonise enterocytes and cells of the reticuloendothelial system and then reach subsequent organs via the cardiovascular and lymphatic systems [8]. T. gondii has a particular affinity for cells of the muscular and nervous systems, with the subsequent course of infection being dependent on the host′s immune status [9].

In most cases, T. gondii infection occurs as a subclinical or asymptomatic form. In immunocompetent individuals, tachyzoite divisions are inhibited, the parasite transforms into slow‐dividing bradyzoites and the acute invasion phase turns into a chronic phase associated with the formation of tissue cysts [10]. It is noteworthy that, due to immunosuppression, the infection may shift into the acute phase, and the bradyzoites present in the cysts may transform back into tachyzoites [11]. As for primary infections in immunocompetent hosts, flu‐like symptoms, such as fever, fatigue, muscle aches and enlarged lymph nodes, occur in about 10%–20% of cases [12, 13].

Groups of people particularly vulnerable to a severe course of toxoplasmosis include immunocompromised patients (individuals undergoing immunosuppressive treatment and those infected with HIV), the elderly and pregnant women [14, 15]. These groups are the most likely to develop inflammatory foci and show signs of disease [2].

In Europe, human infection usually occurs as a result of the consumption of meat or meat products contaminated by cysts that have not been properly heat‐treated, water‐containing oocysts excreted in the faeces of felids, vegetables or fruit contaminated by T. gondii oocysts [16–18]. The important routes of infection are the vertical transfer of T. gondii from mother to foetus via the placenta [19]. Alternative routes of infection are the transmission of the parasite via organ transplantation [20] or blood transfusion from infected donors [21]. Toxoplasmosis, one of the most serious parasitological hazards transmitted with contaminated food and water, represents a significant public health problem in many countries.

2. Epidemiology and Molecular Characterisation of T. gondii

In European countries, the average seroprevalence of T. gondii is estimated at approximately 32% (data from 2000–2020) [22], whereas globally, T. gondii infection affects between 30% and 50% of the population [23]. However, the prevalence of anti‐T. gondii antibodies varies from region to region and ranges from less than 10% in the United Kingdom and Iceland to over 20% in Greece, approximately 50% in Germany and up to 60% in Côte d′Ivoire and Brazil [24–27].

It should be noted that the results for individual countries, depending on the year of the survey, the region or the target study group, can vary significantly. For example, in Portugal, studies conducted in the 1980s on a group of pregnant women showed the prevalence of antibodies at more than 60%, whereas studies conducted in the years 2010–2011 confirmed the presence of antibodies in less than 25% of women of childbearing age [28, 29]. On the other hand, studies conducted on the German population indicate that the prevalence of antibodies against T. gondii varies depending on the age of the subjects and ranges from 20% in younger age groups to more than 70% in those over 70 years of age [25, 30].

Irrespective of the above‐mentioned differences, a downward trend in the incidence of toxoplasmosis has been noticeable in many countries over the past decades. This is confirmed by studies conducted, inter alia, in Slovakia (38.6% in 1961 vs. 21% in 2020), France (64.5% in 1997 vs. 54.7% in 2013), Portugal (47% in the years 1979/1980 vs. 22% in 2013), the United States (16% in the years 1988–1994 vs. 10.1% in the years 2009–2010), Germany (59% in the years 1994–1996 vs. 49% in the years 2008–2011) and the Netherlands (40.5% in the years 1995–1996 vs. 26% in the years 2006–2007) [25, 31–35]. The trend may be a result of higher public awareness and the maintenance of better hygiene standards, which reduces infections resulting from the consumption of contaminated food or water.

In view of the zoonotic potential of the parasite and the fact that the parasite circulates among many host species, monitoring for the presence of T. gondii in a wide range of hosts, including populations of free‐living animals that are reservoirs of the parasite, is necessary to obtain a complete epidemiological picture. The presence of T. gondii has been studied in populations of marine mammals [36], primates [37], rodents [38], marsupials [39], canids [40], wild birds [41], wild boars [42] and felids [43]. Felids are particularly important for the genetic diversity of the parasite, as sexual reproduction of the parasite, its recombination and the formation of new genetic variants of T. gondii, which may differ in virulence, can occur in their bodies. Therefore, in addition to determining the prevalence of the parasite, it is essential to study its genetic variability.

One level of molecular characterisation of the parasite is to determine the clonal line or archetype (type) from which it originates. Most isolates, especially from Europe and North America, belong to one of the three main Types: I, II (with clonal Type 2 and Type 2 variant distinguished within the second type) and III [44–46]. Khan et al. [47] confirmed the existence of a fourth clonal lineage, referred to as 12 (XII), whose representatives have been detected in free‐living animals and, occasionally, in humans in North America. Furthermore, the picture of the global genetic structure of T. gondii is completed by the existence of genotypes that cannot be attributed to any line and are referred to as exotic or atypical [44, 48].

In the northern hemisphere, the parasite population appears to be quite genetically homogeneous, with the dominance of Types II and III, which is due to the clonal structure of the population [49, 50]. The situation is different in the southern hemisphere, for example, in Central and South American countries, where the pathogen exhibits a significantly higher genetic variability, which translates into the isolation of atypical genotypes, including those endemic in nature [51].

In the mouse model, the individual types differ in virulence. The isolates assigned to Type I are characterised by high host mortality even with low (< 10 tachyzoites) inoculum, whereas Type II exhibits intermediate virulence and Type III shows the lowest mortality and is referred to as avirulent [44, 52]. Strains described as atypical are often associated with high mortality in a mouse model, as well as a severe course of toxoplasmosis in humans, including the development of ocular toxoplasmosis [53–56].

In addition to assigning the isolate to one of the three clonal groups or indicating it as atypical, it is possible to characterise the parasite more precisely in molecular terms. In view of the use of different methods for assessing the genetic variability of the parasite, a comparison of genotypes obtained by different methods is difficult. A particular isolate can be assigned to a genotype using the restriction fragment length polymorphism (RFLP) method and the ToxoDB database integrated with the method (the database contains 231 genotypes determined on the basis of 11 markers), assigned to one of 16 clades on the basis of a profile obtained using the multilocus sequence typing (MLST) method or classified into a specific group on the basis of a microsatellite profile [46, 57–59]. Su et al. [46], while integrating data from the three main genotyping methods, analysed over 950 isolates, among which they distinguished 138 genotypes belonging to 15 haplogroups grouped into six main clades.

In view of the prevalence of T. gondii, the presence of atypical strains resulting in a disease with a varied clinical picture and a diversity of infection routes, a special role is played by diagnostics, genotyping of the pathogen, research into potential reservoirs of the parasite and environmental monitoring [18].

3. Diagnosis of T. gondii

3.1. Classical Methods

The bioassays used in animal models (usually mice, less frequently cats) or on cell lines [60] are considered the gold standard for the diagnosis of T. gondii. Biological tests conducted on experimental animals enable unambiguous confirmation of the presence of a live parasite in the test material, which is not provided by molecular methods when detecting the parasite DNA [61, 62]. Bioassays involve administering the test material to the animal in the form of inoculum or via the oral route (especially in the case of cats), followed by the observation of symptoms and intravital examination of faeces and blood serum and the collection of material post‐mortem in order to apply other confirmatory techniques (such as tissue histopathological examination, polymerase chain reaction [PCR] or serological methods) [63, 64]. Bioassays have several significant disadvantages, including ethical aspects, the long waiting time for results and high culturing costs [63].

Due to these limitations, alternative culturing methods are carried out on cell lines in which the parasite propagates are applied. Depending on the aim and type of the study, various cell lines are used [65], including i.a. dendritic cells derived from mouse bone marrow to investigate the course of the invasion and the regulation of cytokine production [66], the Vero cell line (monkey kidney cells) used for tachyzoite propagation [67] and the HCT‐8 (human ileocecal adenocarcinoma cells) line to investigate the immune response to invasion [68].

In vitro studies on cell lines also have certain limitations. One of them is the loss of natural properties of the original strain during subsequent passages, manifested, for example, in changes in virulence, the capacity to form bradyzoites or the loss of the capacity to form oocysts in the final host′s body [65]. In addition, in most cellular models, investigating the entire developmental cycle of the parasite and obtaining oocysts are difficult. This problem is partly solved by using cell lines from the feline epithelium, which exhibit high susceptibility to infection and enable the investigation of the sexual cycle [69].

In addition to classical methods for detecting T. gondii in faeces, water or tissue samples, the microscopic observation of the test material is carried out in order to confirm the presence of oocysts. To this end, observation under a light microscope is used after staining the preparation using the Giemsa method or with haematoxylin and eosin [70–72]. It is also possible to use an electron microscope to investigate the detailed structure of parasites [73, 74]. In order to improve the efficacy of parasite isolation from environmental matrices (water, faeces and soil), concentration‐and‐purification methods, for example, filtration and centrifugation (e.g., continuous flow centrifugation), are employed. Due to the morphological similarity of T. gondii to other protozoa belonging to the Apicomplexa, it is possible to obtain false positive results from a simple microscopic examination [43, 60]. Therefore, microscopic methods should be complemented by serological or molecular techniques in order to confirm the diagnosis.

3.2. Serological Methods

Serological methods are most commonly used in diagnosing toxoplasmosis and enable monitoring of the invasion phase. In response to T. gondii infection, the host′s body starts producing specific antibodies acting against the parasite. During the initial period of infection, antibodies of IgA and IgM classes are produced, starting about 1 week after the onset of invasion, reaching the highest concentration after approximately 1–3 months, after which their concentration decreases to undetectable levels after about 4 months for IgA and 6–9 months for IgM [75]. The class of antibodies that enable confirmation of not current but past invasion is the IgG class antibodies that appear approximately 2 weeks after infection. The maximum IgG titre (concentration) is reached a few (usually 2–3) months after infection, remains stable for up to approximately 6 months and then gradually decreases in the following years. However, it can be detectable for the rest of the host′s life [76].

Numerous serological methods are currently available, and they differ in the course of determination, sensitivity, specificity and informativeness. One of the first methods based on the reaction between an antigen and an antibody was the Sabin–Feldman method (dye test), developed in the late 1940s [77], which, due to its high sensitivity and specificity, has remained the gold standard among serological methods to this day. The technique relies on complement‐dependent cytolysis of antibody‐coated tachyzoites, which are unable to attach the dye (originally, methylene blue). In the absence of antibodies in the serum, when the result is negative, tachyzoites remain intact and become stained [78, 79]. Although the method is considered to be the gold standard for serological methods, its application is limited, and it is not routinely used in most laboratories. This is due to the major disadvantage of this technique, as for the determination, live tachyzoites, derived, for example, after the inoculation of a mouse with the parasite, are required [79, 80]. Therefore, in many laboratories, alternative serological techniques are predominant, including the indirect fluorescent antibody test (IFAT), the indirect hemagglutination test (IHA), the immunosorbent agglutination assays (ISAGA), the latex agglutination test (LAT), the Western blot method, the enzyme‐linked immunosorbent assay (ELISA) test, chemiluminescence assay (CLIA) and enzyme‐linked fluorescence assay (ELFA) measurement‐based techniques [81].

Villardi et al. [75] divided the available serological methods into two main classes: the first class comprises screening methods characterised by a relatively low cost, simplicity of determination, a small amount of material required for analysis performance or the possibility of automating the determination. This group includes techniques such as the haemagglutination test, ELISA and CLIA, among others. The second class of methods, referred to as confirmatory tests, comprises techniques that are more complex or have a higher cost of analysis, such as the Sabin–Feldman method, IFAT and ISAGA. Confirmation or exclusion of the presence of antibodies of particular classes and monitoring their titres over time allow an inference to be made about the phase of invasion. Consequently, a number of guidelines and treatment algorithms, determined by serological results (mainly for antibodies in the IgG and IgM classes) and the patient category (e.g., immunocompromised patients, pregnant women and newborns) have been developed [75, 76, 82].

In addition, in the case of T. gondii, as well as many other parasitic organisms, serological methods are used to assess the prevalence of specific antibodies in the population in terms of epidemiological data. Seroprevalence of T. gondii in European countries varies depending on the region, study group, age and the techniques applied; for example, in Germany, it accounted for 55% [25], in the Netherlands for 31% [14] and in the United Kingdom for 28% [83]. In contrast, seroprevalence noted, for example, in Mexico accounted for 62% [84] and 11% in the United States [85]. Analysis of the distribution of anti‐T. gondii antibodies in a particular population also enables the identification of risk factors and groups particularly vulnerable to parasite infection, for example, a serological study conducted by Feckova et al. [31] in Slovakia showed 21% seropositive results, with the prevalence of antibodies varying between groups to reach the highest values among farmers (42.5%), hunters (28.5%) and shelter workers (22.6%). The high proportion of antibodies in the farmer group (66.9%) was also confirmed by a study conducted in Poland [86], whereas the risk of the occurrence of an invasion among hunters is indicated, for example, by a study by Machado et al. [87], who found 32.7% positive results. Information on the differences in the epidemiology of toxoplasmosis among the particular groups, obtained using serological tests, enables targeted and more effective implementation of prevention programmes and educational campaigns. Importantly, serological methods can be used not only to detect antibodies in blood serum but also, for example, in meat juice. This is important in that it enables testing of samples where serum is not available, thus allowing the parasite to be detected in samples of commercially available meat [6, 88–90].

Although both serological techniques and culture methods are important research and diagnostic tools, recent decades have seen many rapid, specific techniques that enable not only the detection of the pathogen from a variety of matrices (including environmental samples) but also the comparison of T. gondii isolates from different outbreaks of disease. Further on, the paper reviews the molecular methods most commonly used in diagnosis and in studies on the molecular epidemiology of T. gondii.

3.3. Molecular Methods

Because most serological techniques involve the detection of specific antibodies produced by the host organism in response to contact with a pathogen, the aforementioned group of methods does not always give complete information on the actual course of the invasion and the actual presence of the parasite [6]. One of the limitations of serological methods is that they can yield false‐negative results arising from the serological window, as antibodies can only be detected approximately 1–3 weeks after infection. In addition, for immunocompromised individuals, the immune response may be insufficient for the antibody level to reach the limit value for the methods applied [91, 92]. Although techniques such as the IgG antibody avidity test enable the determination as to whether the infection is acute or chronic, based on differences in the affinity of IgG antibodies to the T. gondii antigen, the presence of the genetic material of the parasite in the test sample can be an important indicator of active invasion [93]. Molecular methods enable the detection of parasite DNA across a wide range of diverse matrices, from clinical samples through food samples to environmental samples [94–100].

3.3.1. PCR

The PCR method, which enables the amplification of specific, selected gene fragments, is considered the primary molecular method for detecting the DNA of pathogens, including T. gondii. This technique, along with its modifications (e.g., nested PCR, multiplex PCR or real‐time PCR), is currently a valuable diagnostic tool.

In the case of T. gondii, primers flanking fragments of different genes are used, but the most common are B1 gene sequences with 35 repetitions in the genome, as well as the RE 529‐bp‐sequence (a 529‐bp long, repeatable fragment found in approximately 200–300 repetitions) and ITS-1 (110 copies) [46]. There are numerous articles comparing the effectiveness of the B1 and RE 529‐bp‐sequence primers. Homan et al. [101], who identified a fragment of the RE 529‐bp‐sequence and designed primers for it, indicate the higher sensitivity of the reaction amplifying this fragment in relation to the primers amplifying the B1 gene. Similar conclusions were reached by Cassaing et al. [94], who compared the efficiency of amplification of the two target fragments in clinical samples (amniotic fluid, the placenta, cerebrospinal fluid and bronchoalveolar fluid) and by Fallahi et al. [102], who tested samples derived from patients with a confirmed neoplastic disease. Sağlam et al. [103] indicated the feasibility of using the PCR method in the detection of the genetic material of the parasite in water samples, also indicating, in this case, the higher efficacy and sensitivity of the reaction based on the RE 529‐bp‐sequence, both in irrigation and drinking water samples. However, subsequent studies demonstrated that certain Toxoplasma genotypes have lost some or all of the RE gene repeats, which may yield false‐negative results. Amplification of a B1 gene fragment is equally frequently used in the detection of T. gondii, not only in clinical samples [104, 105] but also in environmental samples [106].

3.3.2. Nested PCR

The real‐time PCR and nested PCR variants enhance diagnostic capabilities due to the higher sensitivity and specificity of reactions. The nested PCR method involves the elongation of the diagnostic protocol through the application of two amplification stages for two pairs of primers. The technique relies on the use of external primers flanking the target region in the first amplification step and then on the use of the amplification products as a matrix in the second step with internal primers. The initial ‘amplification’ of the matrix in the first reaction and the use of two pairs of primers significantly improve the specificity and sensitivity of the method. On the other hand, the nested PCR method, due to amplification being carried out in two rounds, is also a more time‐consuming method that is more difficult to optimise and more prone to potential contamination [107, 108].

Similar to conventional PCR, in the case of nested PCR, the most commonly used target fragments are the B1 gene and the RE 529‐bp‐sequence repeatable fragment [109, 110]. Nevertheless, attempts are also being made to design primers for other regions of the genome, which is particularly important in view of the occurrence of mutations and polymorphisms that can affect the efficiency of amplification processes. Because the emergence of polymorphisms in the primer attachment regions increases the risk of false‐negative results, it is necessary to periodically review the effectiveness of the primers used, especially for new T. gondii variants. Costa et al. [111] indicate the high conservativity of the GRA7 gene between different T. gondii variants. These authors indicated the sensitivity and specificity of the nested PCR method, with primers for GRA7 being comparable or higher than those obtained for primers for the B1 and RE 529‐bp‐sequence fragments. The observations were confirmed, inter alia, by Liu et al. [112], who observed a higher reaction efficacy with primers for the GRA7 gene than for the amplification of the B1 gene.

3.3.3. Real‐Time PCR

One of the molecular methods used for the detection of a wide variety of pathogens, including T. gondii, is the real‐time PCR (quantitative polymerase chain reaction [qPCR]) method, in which shorter amplicons (less than 200 bp) are usually obtained, allowing a more efficient analysis of the material with a higher degree of degradation. In addition, the application of molecular probes improves the specificity of reactions. Real‐time PCR enables real‐time monitoring of the reaction course, including the quantitative determination of copies of the studied gene in the sample. The real‐time PCR method also has the advantage of not requiring electrophoretic separation of the products, which speeds up the analysis time and reduces the risk of contamination [93].

Similar to the classical PCR and nested PCR reactions, in the case of the real‐time PCR technique, the target fragments most commonly used in diagnosing T. gondii are the B1 gene and the RE 529‐bp‐sequence fragment [93, 94, 113]. The qPCR method has been applied, inter alia, to confirm the presence of T. gondii genetic material in clinical samples [94, 114, 115], food [95, 96], soil [97], faeces [98] and water samples [99, 100]. Other study results indicate that, compared with nested PCR, real‐time amplification is characterised by a higher sensitivity and lower risk of contamination, making real‐time PCR one of the most commonly used molecular diagnostic methods [96, 100, 115, 116].

3.3.4. Loop‐Mediated Isothermal Amplification (LAMP)

There are two relatively new methods that enable the detection of nucleic acids of the parasite, that is, the LAMP method and the digital droplet polymerase chain reaction (ddPCR) method. The LAMP technique was developed in 2000 by a Japanese team of Notomi et al. [117]. The method is characterised by such advantages as the short analysis time and the possibility of carrying out the reaction under isothermal conditions, which means that the reaction does not need to be carried out in a thermocycler, and its adaptation for field studies is much easier than for other molecular methods [92, 118]. Another feature that differentiates LAMP from classical amplification is the stage of primer design and selection. As for LAMP, four to six primers are used, among which a few outer primers, internal primers and the so‐called loop primers are distinguished, with the latter increasing the specificity and rate of amplification [119]. The difference between PCR and LAMP also relates to the polymerase used. In the case of classical amplification, the Taq polymerase is used, whereas for LAMP, the Bst polymerase isolated from Bacillus stearothermophilus, which exhibits both replication and displacement activity, is used. The LAMP method enables the use of various detection systems, from electrophoretic separation of the reaction products, through real‐time evaluation of the increase in fluorescence levels to the visual assessment of colorimetric or turbidimetric changes taking place in the sample [118, 120].

Attempts have been made to apply LAMP in diagnosing T. gondii, using primers both for the genes that occur in the genome in single copies and for sequences such as the B1 gene and RE 529‐bp‐sequence [121]. Research results indicate that the LAMP method is characterised by a shorter reaction time and a higher sensitivity than that of conventional PCR [122] or nested PCR [123]. A comparison of the technique with qPCR is inconclusive, as some researchers indicate that LAMP has a sensitivity comparable with that of qPCR [121, 124], whereas other teams indicate a slightly lower sensitivity of isothermal amplification [125, 126]. The differences in the results may be due to the specificity of test matrices and the different fragments for which the primers were developed. The LAMP method has been applied, inter alia, for the detection of the genetic material of T. gondii in soil samples from parks [127] or swine‐breeding farms [128]. In both cases, isothermal amplification yielded a higher percentage of positive results than classical PCR. Other types of challenging matrices, for which the LAMP method has been successfully applied to confirm the presence of the parasite, include water [129, 130] and faecal samples [118, 120].

3.3.5. ddPCR

Another method based on nucleic acid amplification is ddPCR, which enables the quantitative assessment of the target test sequence without the use of external standards. Although the technique stems from the research conducted as far back as the early 1990s by Sykes et al. [131], its development and adaptation to diagnostic purposes is a relatively new solution. The ddPCR technique uses solutions that transform the sample into an emulsion, which is subsequently divided (partitioned) in a generator for 15,000–20,000 droplets, each representing a single, separate reaction environment. The use of dyes or fluorescent probes allows the signal to be measured, and the droplets in which amplification has occurred to be calculated [132]. This, along with the implementation of appropriate statistical methods (Poisson statistics), allows for the quantification of amplicons [133].

In view of the quantitative nature of the method, it seems natural to compare it with qPCR. One of the major differences is the fact that ddPCR, unlike qPCR, is an end‐point method and thus does not enable real‐time monitoring of product gain. A comparison of the sensitivity of these two methods yields inconclusive results, with some studies indicating a similar sensitivity and specificity of ddPCR and qPCR while emphasising the higher reproducibility of ddPCR [132, 134, 135]. In contrast, other teams have noted the superiority of ddPCR over qPCR for samples containing very low amounts of the target genetic material of the pathogen [136], thus confirming the higher sensitivity of the ddPCR method [137, 138]. The undoubted advantage of ddPCR is the very small sample amount used for amplification, which translates into two benefits. Firstly, it is the minimal consumption of the material, which, especially in the case of difficult matrices, is concentrated and suspended in low volumes of buffers; in addition, potential inhibitors are also diluted along with the sample, resulting in a reduced risk of false‐negative results and improved reproducibility of the reaction [134].

Nevertheless, the ddPCR method also has disadvantages that do not currently allow it to be implemented into routine diagnostics. One of them is the cost of analysis, which, for ddPCR, is twice as high as the qPCR method [139, 140]. The disadvantages of ddPCR also include the narrower dynamic range of determination, which translates into a decrease in the reliability of the result for samples in which the target sequence is found at high concentrations [139, 141].

There are several reports on the use of ddPCR for the detection of T. gondii. Nabet et al. (2023), when applying qPCR and ddPCR in a study on retrospective clinical trials, obtained a similar efficacy for both methods, with the ddPCR method enabling more precise quantitative assessment. Mancusi et al. [134] confirmed the feasibility of using ddPCR for the detection of T. gondii in meat samples after obtaining a method sensitivity of 97.5%, specificity of 100% and the detection limit of 8 genomic copies/μL of T. gondii. Mancusi et al. [136] applied ddPCR to confirm the presence of T. gondii DNA in mussels. Of the 100 pooled samples (comprising 10 individual samples), the presence of genetic material of the parasite was confirmed in 16 samples. Quantitative analysis by the ddPCR method showed that the concentration of T. gondii DNA ranged from 0.1 to 1.9 genomic copies/μL. Testing the same samples using the qPCR method yielded positive results for none of them. This indicates a higher sensitivity and efficacy of ddPCR, especially for samples with a low number of target sequence copies. The results confirming the higher ddPCR sensitivity compared with that of qPCR were obtained, inter alia, from the detection of Bartonella species [137] as well as Plasmodium knowlesi and Plasmodium vivax [138].

Molecular techniques offer numerous solutions that can be applied to detect the genetic material of parasites from a wide range of matrices (Table 1). Of the molecular methods discussed above, the nested PCR and qPCR methods remain the most commonly used for diagnosing T. gondii. Nevertheless, methods based on nucleic acid amplification, despite their broad applicability, also have a number of limitations. One of the major ones is the lack of standardisation of methodologies, as a result of which the use of different pairs of primers, different reagents (ready‐to‐use mixes, ‘in‐house’ PCR assays) or different thermal‐time programmes can lead to results that are inconsistent between individual laboratories and the need to confirm the result by other methods. In addition, the variability found between individual T. gondii variants necessitates periodic evaluation of the effectiveness of the primers used. However, despite their limitations, these methods have been increasingly used in diagnostic practice. Their use goes beyond merely detecting the pathogen′s genetic material; it also allows for the assessment of genetic variability, an analysis of the functional basis for changes in individual gene sequences and the investigation of T. gondii circulation in the environment.

4. Genotyping of T. gondii

Although the classical PCR method and the modifications discussed above enable the detection of the genetic material of T. gondii, techniques that are based on nucleic acid amplification also exist, which allow the variability between individual isolates to be assessed. Therefore, their application is of particular importance when genotyping T. gondii, studying the circulation and evolution of the parasite or in epidemiological investigations [142, 143]. Originally, the assignment of T. gondii to particular clonal groups, and thus the inference of virulence, was possible thanks to the use of the MLEE (multilocus enzyme electrophoresis) technique and the analysis of different enzyme isoforms [144–147]. The method enabled the assignment of the isolates obtained to one of three clades, originally formed by four zymodemes. Major drawbacks of MLEE included its low resolution and the large amount of material required to carry out the determination [44].

Currently, the dominant group of methods used for studying variability and genotyping the parasite are techniques based on DNA analysis. The greater interest in such markers is due, among other things, to the greater stability of nucleic acids and, frequently, to their higher polymorphism. Analyses at the DNA level enable the detection of synonymous mutations that cause changes in the nucleic acid sequence but do not result in changes in the amino acid sequence. In addition, the profiles obtained when applying such techniques as MLST or microsatellite sequence analyses are characterised by a higher possible number of alleles at polymorphic loci, which provides higher resolution of these methods [148, 149]. The most common genotyping methods used to assess T gondii diversity are outlined below.

4.1. Random Amplified Polymorphic DNA (RAPD)

One of the methods that enable studying the variability between individual isolates of the parasite is the RAPD‐PCR method. The method offers several key differences from classical PCR. Firstly, RAPD‐PCR usually uses primers with a length of 8–12 bp (in the case of T. gondii, mainly decamers), that is, significantly shorter than those used in PCR (20–30 bp), which can bind to many different sites in the genome [150, 151]. In addition, the primer binding (annealing) step is carried out at a much lower temperature than in PCR (under low stringency conditions), leading to primers hybridising even to nonfully complementary sequences. The products of RAPD‐PCR, after separation on agarose or polyacrylamide gels, allow the genetic profile of the isolate under study to be obtained, and, in this way, the relationships between closely related genetic variants can be studied. The technique was applied in the 1990s, for example, to compare Sarcosystis species isolated from sheep [152], Trichinella nativa isolated from polar foxes [153] and in the case of T. gondii, isolates derived from both humans and animals [154].

The RAPD‐PCR method can also be used to identify molecular markers, useful from an epidemiological perspective, including those responsible for virulence [150, 151, 155]. Although the technique requires no knowledge of the target sequence, is characterised by a relatively low cost and a short analysis time and requires small amounts of DNA for testing, it has several disadvantages that hinder its widespread use in genotyping T. gondii as well as many other pathogens. One of the main disadvantages of RAPD‐PCR arises from its nature, that is, relatively short primers with an arbitrary sequence, which, combined with low annealing temperatures, results in low reproducibility even within the same laboratory, with the results being susceptible to even small changes in conditions (amplification carried out on a different thermocycler, use of reagents from a different supplier or even a different batch from the same supplier) [156, 157]. In addition, in contrast to the PCR method, RAPD‐PCR cannot be used for direct analysis of clinical or environmental samples. The arbitrary nature of the primers used can result in the amplification products derived from the matrix (i.e., environmental DNA or host DNA). Therefore, the genetic material used for genotyping by the RAPD‐PCR method should be characterised by high purity [70].

4.2. High‐Resolution Melting (HRM) Analysis

In contrast to RAPD, some methods focus on the study of single nucleotide polymorphisms (SNPs), with examples of such techniques including the RFLP, allele‐specific amplification (ASA)‐PCR, Single‐strand conformation polymorphism (SSCP)‐PCR, HRM analysis or probe‐based qPCR. Some of the methods mentioned rely on the fact that the presence of polymorphic nucleotides determines the presence (or absence) of sites specific for restriction enzymes (RFLP), primers (ASA‐PCR) or molecular probes (qPCR). On the other hand, other techniques are based on differences in DNA strand conformation (SSCP) or different melting temperatures of the double‐stranded DNA structure (HRM), resulting from the presence of polymorphic nucleotides.

One method of genotyping T. gondii is the HRM analysis method, which is an extension of the qPCR based on intercalating dyes. HRM is a post‐PCR technique carried out after the actual amplification and involves the study of differences in melting temperatures and melting curves that result from, among other things, the presence of SNP‐type polymorphisms [158]. The method has found applications in parasitology, including genotyping of T. gondii [158–162]. Costa et al. [162] applied HRM to investigate polymorphism in the B1 gene of T. gondii, indicating that the method achieves a level of discrimination close to the amplification of five microsatellite markers. In addition to the analysis of the melting curve produced after the amplification of the B1 gene fragment [162, 163], the feasibility of applying a primer‐based reaction for the ROP8 gene, which enabled more precise genotyping while showing lower sensitivity compared with the B1 gene, was investigated [164]. The application of HRM for the analysis of multiply‐repeated sequences in the genome, such as the B1 gene or the RE 529‐bp‐sequence, is a kind of compromise between the discriminatory power and the sensitivity of the method. Analysis of even a few target fragments by the HRM method does not enable a comprehensive identification of the isolate as the amplification of 15 microsatellite loci. However, as these fragments occur in a higher number of repetitions, it is possible to obtain a result for the samples for which the number of parasites in the sample is lower, and STR (short tandem repeats) marker analysis does not yield complete profiles.

4.3. RFLP

The primary method for the initial analysis of T. gondii diversity is the RFLP method. The technique involves the digestion of PCR reaction products with restriction enzymes, which, under optimal reaction conditions, identify the target sequences and cleave them at sites specific to them.

Analysis of single RFLP markers is often insufficient to provide the resolution enabling reliable genotyping of the parasite, especially for recombinant T. gondii genotypes [165] or atypical strains [166]. Therefore, for the genotyping of isolates, the so‐called Mn‐PCR‐RFLP (multiplex multilocus nested polymerase chain reaction–restriction fragment length polymorphism) method is used, enabling the amplification and restriction analysis of multiple markers [167].

Howe and Sibley [168] demonstrated that using RFLP analysis based on six markers enables the assignment of a T. gondii isolate to one of three clonal lines. Attempts have been made to identify loci with particularly high informativeness. For instance, Howe et al. [169] identified fragments at the 3 ^′^ and 5 ^′^ ends of the SAG2 gene as sequences containing polymorphisms differentiating individual clonal lines. Restriction analysis of the SAG2–5   ^′^ fragment using the Sau3AI enzyme allowed Type III to be distinguished from Types I and II, whereas the digestion of the SAG2–3   ^′^ fragment with the HhaI enzyme yielded a restriction profile differentiating Type II from Types I and III. Grigg and Boothroyd [166] also attempted to use B1 gene digestion in order to assign the isolate to a clonal line. They proposed the application of two enzymes, XhoI and PmlI, which allowed Type I to be distinguished from Types II and III. Su et al. [170] proposed an RFLP assay based on the analysis of nine markers. Dubey et al. [171] extended the range of analysed markets to 10, additionally including a restriction profile of a SAG1 gene fragment. In subsequent years, further target sequences were included in the restriction analyses—for example, an analysis of the 5 ^′^ and 3 ^′^ fragments of the SAG2 gene, alternative SAG2 [167] or CS3 (virulence marker) was carried out separately [172–174].

The utility of the RFLP polymorphism‐based genotyping system is considerably extended thanks to the ToxoDB database that collects, inter alia, restriction profiles for the individual genotypes. The database contains 231 genotypes (as of 24 June 2024) distinguished on the basis of 11 RFLP markers (SAG1, SAG2-5   ^′^, SAG2-3   ^′^, SAG2 alternative, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) [175]. Currently, the Mn‐PCR‐RFLP method is widely used in epidemiological research as part of the characterisation of the variants obtained, which is used in the genotyping of T. gondii isolates derived from human clinical trials [176, 177], as well as in samples isolated from birds [178], wild canids [40], pigs [172], cats [179] and sheep [180].

4.4. MLST

As mentioned above, the RFLP method is very often used to study the epidemiology of T. gondii. However, the profile obtained during RFLP analysis only enables inferences to be made about the presence or absence of a site recognised by a particular restriction enzyme without providing information on other possible polymorphic nucleotides that may be present in a particular sequence. Therefore, RFLP markers, although considered to be useful for distinguishing between individual clonal lines, do not provide sufficient information to infer variability within individual lines or genotypes (intragenotype variability) [148]. In addition, during the reaction with restriction enzymes, incomplete digestion can occur, which can lead to misinterpretation of the result [181].

These limitations are partly eliminated through the use of the MLST method, which involves the sequencing of amplicons. MLST is characterised by higher informativeness and discriminatory power than the RFLP method, and the genotyping itself can be conducted both when using Sanger method sequencing and the NGS (next‐generation sequencing) techniques [182]. The MLST method is primarily applied in the genotyping of bacteria and fungi, where polymorphisms in the so‐called ‘housekeeping genes’ are analysed [183, 184]. Nevertheless, the technique is also used for parasitological studies [184–186], including in the genotyping of T. gondii [61, 187].

Originally, in order to genotype and assess the variability of the parasite, attempts were made to analyse the encoding fragments or introns of single genes. Binas and Johnson [188], while comparing the sequence of one of the introns of the polα gene, identified 16 polymorphisms differentiating between virulent and avirulent strains. Fazaeli et al. [189], when carrying out an analysis of the GRA6 gene fragment (approximately 800 bp) in 30 T*. gondii* strains, detected 24 polymorphism sites, including 22 substitutions (of which 21 changed the amino acid sequence) and two deletions. The authors demonstrated that some of the polymorphisms obtained can be used to distinguish the three main genotypes of the parasite.

A simultaneous analysis of multiple loci significantly increases the resolution of the method. In practice, both sequence analysis of intron fragments (introns of the UPRT, MIC, BTUB, HP and EF genes, among others) and exon fragments (GRA6, GRA7 and SAG3) are used [46, 190, 191]. Frazão‐Teixeira et al. [187], when developing the genetic characterisation of T. gondii isolated from pig brains and hearts, applied the MLST method while taking into account the markers used in RFLP analyses (SAG1, SAG2, SAG3, BTUB, c22-8, c29-2, L358, PK1, Apico and GRA6) as well as a B1 gene fragment. The researchers confirmed that the MLST method enables the detection of polymorphisms and unique alleles that are impossible to distinguish using the restriction analysis, thus confirming the higher resolution of MLST as compared with RFLP. Therefore, MLST can be used to study differences between parasite populations and detect endemic strains. Bertranpetit et al. [190], when comparing the sequences of five genes (GRA6, GRA7, SAG3, UPRT1 and UPRT7) in T. gondii isolates derived from 13 populations, demonstrated the presence of 140 variable sites, based on which 60 haplotypes were distinguished. The application of the above‐mentioned markers enabled the confirmation of significantly higher variability of the parasite in South America (32 haplotypes, of which 29 are endemic) than in Europe (four haplotypes).

Joeres et al. [192] used MLST analysis, performed on the NGS platform (Illumina), to identify highly polymorphic regions in the T. gondii genome and to study genetic variability within European isolates belonging to clonal Type II. The authors demonstrated that the method enabled the differentiation between even closely related isolates and its resolution can be increased by including additional polymorphisms.

Despite the advantages of the MLST method, the Mn‐PCR‐RFLP remains the more common and popular method used to assess the variability of T. gondii. This is because, among other reasons, MLST is a more time‐consuming and costly method. Moreover, a comparison of the sequences obtained requires an additional step of preliminary bioinformatics analysis. Nevertheless, the increasing availability and the decreasing cost of sequencing (especially the NGS technology) suggest that in the near future, MLST will start to gain more importance in studying the epidemiology of T. gondii [192].

4.5. Microsatellite Sequences

Due to certain disadvantages of the RFLP (limited resolution) and MLST techniques (a persistent, relatively high cost and long analysis time), as mentioned above, methods that represent a kind of compromise between the discriminatory power and the cost and time of performing the analysis may become an alternative solution. Microsatellite sequence analysis appears to be such a method.

In view of the limited resolution of the RFLP method, Ajzenberg et al. [149] proposed a two‐step system for the analysis of genetic variability. The first step is to classify the isolate obtained into one of the three main genetic lines or to demonstrate that the strain obtained is an atypical one. The second level of the analysis, as proposed by the authors, is the so‐called ‘fingerprinting level’ characterised by a considerably higher discriminatory power. Whereas the first level of variability can be assessed using the RFLP technique, in the case of the second one, it is necessary to study markers that ensure higher resolution, such as microsatellite sequences.

In contrast to the RFLP technique, which allows the obtained T. gondii isolate to be assigned to individual types, analysing a sufficient number of microsatellite markers allows even closely related strains to be distinguished within types. The much higher discriminatory power of such markers is due to the much higher polymorphism of STR‐type markers, which have a higher number of alleles per loci than RFLP‐type markers. STR markers are currently used mainly in forensic genetics to determine genetic profiles, but they are also used in studies in the field of population genetics, or more specifically epidemiology, to conduct epidemiological investigations and to track the circulation of the parasite between disease foci [42, 193–195].

In practice, simultaneous amplification of multiple markers using the multiplex PCR method is used to determine the genetic profile of the material donor. Originally, the separation of the products of such a reaction was carried out by electrophoresis in agarose or polyacrylamide gels. Nowadays, capillary electrophoresis and fluorescently labelled primers are much more commonly used. Such a solution enables the separation and identification of alleles of a similar size.

The number of loci analysed in studies on T. gondii has varied over the years. In 2005, Ajzenberg et al. [196] proposed a panel comprising five microsatellite sequences (loci—TUB2, TgM-A, W35, B17, B18), which allowed a parasite type to be identified (or atypical isolates to be indicated). The above‐mentioned set of microsatellite markers was applied by Sgroi et al. [42] for the genotyping of T. gondii in a free‐living wild boar population in southern Italy. The team showed the presence of the parasite in over 39% of the animals tested, and the genotyping of a proportion of positive samples (the full profile was obtained for n = 11) indicated a clear dominance of atypical genotypes (9 out of 11). The same set of microsatellite sequences was used, for example, by Dumètre et al. [197] for the genotyping of T. gondii isolated from sheep in France (market Type II dominance) or by de Sousa et al. [198] for the assessment of the variability of the parasite in a swine population in Portugal (also, Type II domination).

The resolution of the microsatellite analysis, based on the analysis of five loci, allows the isolate to be assigned to a specific type, thus achieving the first level of discrimination as mentioned by Ajzenberg et al. [149, 199] but is not fully sufficient to assess variability within individual types. Therefore, attempts have been made to include new polymorphic loci in the panel. Consequently, a panel comprising 15 microsatellite sequences was proposed in 2010 [149]. The panel comprises eight markers (in addition to five markers from the original set, the authors added three new markers (M33, IV.1 and XI.1), allowing the isolate under study to be assigned to a genetic line. The other seven markers (N60, N82, AA, N61, N83, M48 and M102) enable the examination of variability within individual genetic types.

In this form, the set was used, inter alia, in a study by Deiro et al. [179] to identify atypical genotypes of T. gondii in cats, to genotype a parasite in stray dogs by Valenzuela‐Moreno et al. [200] or in sheep by Pastiu et al. [201].

Despite the high resolution and the relatively rapid course of analysis, the use of microsatellite markers is not without disadvantages. Genotyping carried out using 15 polymorphic loci to obtain a complete and reliable profile requires a large amount of the genetic material of the parasite in the sample under study. Ajzenberg et al. [149] point out that the method is not recommended for samples with a small amount of genetic material of the parasite. Limited sensitivity often involves an additional step, that is, the propagation of the parasite through passages on experimental animals (mouse bioassay). This was the methodology employed by Lachkhem et al. [202] when they genotyped T. gondii from sheep and chicken brain and heart samples. In addition to samples isolated from mouse bioassay, the authors also selected tissue homogenates from sheep and chickens for genotyping for which the Ct value of the qPCR assay for the presence of genetic material of the parasite did not exceed 32 (< 32), which the authors indicated as the threshold to consider microsatellite genotyping.

Evidently, there are a number of methods for genotyping T. gondii, yet each has its own advantages and disadvantages, and it is difficult to clearly identify a technique that could be considered the gold standard (Table 2). In terms of universality, by far the most popular is the RFLP analysis, which is increasingly being supplemented with additional MLST or microsatellite markers.

5. Summary

This paper provides information on the prevalence, epidemiology and methods for the detection and genotyping of T. gondii. Due to the dynamic development taking place in these areas, it is reasonable to prepare studies that update and consolidate information that is often scattered in many sources. Therefore, the current review is an attempt to gather, organise and present the information contained in both older, well‐established studies and in studies published in recent years. In conclusion, several aspects should be particularly highlighted.

Firstly, the development and evolution of available methods, both genomic and proteomic, are undoubtedly expanding the possibilities for the detection and analysis of the circulation of pathogens, including T. gondii. There has been an increase in the sensitivity of the methods used (the implementation of modifications to classical PCR, such as qPCR, ddPCR), their specificity (multiplex PCR reactions amplifying multiple fragments of the genome of the same individual), informativeness (sequencing by the Sanger method and the increasingly widespread NGS) and a significant extension of the possibilities for data analysis and interpretation through the use of bioinformatic and biostatistical platforms. Undoubtedly, the development of new diagnostic solutions and an increase in throughput of new generation techniques can substantially not only improve knowledge of polymorphism in the parasite population but also provide new information on the molecular mechanisms regulating the parasite′s life cycle, virulence and adaptive capacity.

Secondly, in addition to developing new solutions, it is important to standardise and integrate solutions that have already been implemented and tested. Therefore, particularly important are studies that bring order to the existing state of knowledge, such as an interlaboratory study by Joeres et al. [148] concerning the standardisation and harmonisation of microsatellite sequence analyses or the integration of data obtained from different genotyping methods, as carried out by Su et al. [46]. What is also noteworthy is the number of studies summarising the state of the knowledge, for example, studies by Dardé et al. [44] or Liu et al. [70], which help in the dissemination and organisation of information.

Thirdly, it is important to integrate the information obtained from genotyping and molecular epidemiology studies into diagnostic practice. Examples of such activities include proposing new primers for PCR reactions that are adapted to the parasite′s current gene pool or identifying new polymorphic regions that can be used to genotype the parasite.

Many authors emphasise the relatively low variability of T. gondii in the northern hemisphere; however, a number of factors may lead to gradual changes in this regard. Due to climate change, the ecological niches of individual species, including potential hosts of T. gondii, are changing. In addition to climate change, a factor that expands the range of occurrence of pathogens, including T. gondii, is the intense global movement of animals and goods that are not routinely tested for toxoplasmosis. This can undoubtedly result in the emergence of new genotypes of the parasite in areas where they have not previously been detected and, consequently, hybridisation with the parasite population that is native to the area.

Finally, genotyping may also play an important role in shaping treatment strategies and preventive measures against toxoplasmosis. Currently, pyrimethamine and sulfadiazine remain the gold standard of therapy [203]. However, growing evidence suggests the emergence of drug‐resistant T. gondii strains. Characterising the molecular background of resistance, including the identification of markers associated with reduced drug susceptibility, may therefore be crucial for optimising therapeutic protocols and guiding individualised treatment approaches [204].

Genotypic information is also highly relevant for vaccine development. Although no effective vaccine is currently available for human use, numerous experimental platforms—particularly DNA vaccines—show promise due to their ability to induce both humoral and cellular immune responses. Vaccine candidates frequently target genes encoding key virulence factors such as ROP or SAG proteins [203]. Increasing attention is being directed toward multigene vaccine constructs, which tend to elicit broader and more robust protective immunity compared with single‐gene formulations [205]. Because molecular polymorphisms may involve nonsynonymous substitutions that alter protein structure and antigenic properties, integrating molecular data with bioinformatic analyses can facilitate the identification of immunogenic epitopes, which may significantly enhance the design of future vaccines [206].

Funding

This work was supported by the National Science Center, Poland, 2024/08/X/NZ6/01328.

Conflicts of Interest

The authors declare no conflicts of interest.