Advances in serological, imaging techniques and

Infection https://doi.org/10.1007/s15010-017-1111-3

REVIEW

Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii infection Ali Rostami1 · Panagiotis Karanis2 · Shirzad Fallahi3,4 Received: 23 June 2017 / Accepted: 22 December 2017 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Background Toxoplasmosis is worldwide distributed zoonotic infection disease with medical importance in immunocompromised patients, pregnant women and congenitally infected newborns. Having basic information on the traditional and new developed methods is essential for general physicians and infectious disease specialists for choosing a suitable diagnostic approach for rapid and accurate diagnosis of the disease and, consequently, timely and effective treatment. Methods We conducted English literature searches in PubMed from 1989 to 2016 using relevant keywords and summarized the recent advances in diagnosis of toxoplasmosis. Results Enzyme-linked immunosorbent assay (ELISA) was most used method in past century. Recently advanced ELISAbased methods including chemiluminescence assays (CLIA), enzyme-linked fluorescence assay (ELFA), immunochromatographic test (ICT), serum IgG avidity test and immunosorbent agglutination assays (ISAGA) have shown high sensitivity and specificity. Recent studies using recombinant or chimeric antigens and multiepitope peptides method demonstrated very promising results to development of new strategies capable of discriminating recently acquired infections from chronic infection. Real-time PCR and loop-mediated isothermal amplification (LAMP) are two recently developed PCR-based methods with high sensitivity and specificity and could be useful to early diagnosis of infection. Computed tomography, magnetic resonance imaging, nuclear imaging and ultrasonography could be useful, although their results might be not specific alone. Conclusion This review provides a summary of recent developed methods and also attempts to improve their sensitivity for diagnosis of toxoplasmosis. Serology, molecular and imaging technologies each has their own advantages and limitations which can certainly achieve definitive diagnosis of toxoplasmosis by combining these diagnostic techniques. Keywords Advances · Diagnosis · Toxoplasma gondii infection · Toxoplasmosis

Introduction

* Shirzad Fallahi [email protected]; [email protected] 1

Infectious Diseases and Tropical Medicine Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran

2

Qinghai Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining, China

3

Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran

4

Department of Medical Parasitology and Mycology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran

Infection with the protozoan parasite Toxoplasma gondii has a worldwide distribution. Humans as well as virtually all warm-blooded animals, including mammals and birds, can be infected by this obligate intracellular parasite [1–3]. The main sources of infection are undercooked meat, unwashed vegetables and contaminated soils [1, 4]. Primary infection with T. gondii in hosts with healthy immune system usually appears as mild flu-like symptoms whereas in patients with a weakened or impaired immune system can cause life-threatening infections. In addition, transplacental transmission of T. gondii during pregnancy may cause serious problems that may lead to abortion, stillbirth, or neonatal abnormalities [5–9]. Also recently, toxoplasmosis is known as a risk factor for neurological and psychiatric disorders [10]. Accordingly, the diagnosis of toxoplasmosis is most critical in four groups

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of individuals; immunocompromised patients such as HIVinfected or organ-transplanted patients, pregnant women who acquire the infection during pregnancy, congenitally infected fetuses and newborns, and those with retinochoroiditis [1, 11]. General physicians (GPs), as well as infectious disease specialists (IDSs) should not ignore the risks of toxoplasmosis in health care settings. They may encounter a number of toxoplasmosis issues, including clinical manifestation, accurate diagnosis and effective treatment. Clinical manifestations of T. gondii infection are non-specific and unreliable; therefore, diagnosis of toxoplasmosis is traditionally based on laboratory tests, particularly bioassay and serological methods. Variation in sensitivity and specificity, being time-consuming and inability of these methods to differentiating parasite strains and also high sensitivity and specificity of molecular methods in the detection of T. gondii infection led to the widespread use of these techniques to detect and differentiate of various strains of T. gondii [12, 13]. Having basic information on the traditional and new developed methods for the detection of toxoplasmosis is essential for GPs and IDSs for choosing a suitable diagnostic approach for rapid and accurate diagnosis of the disease and, consequently, timely and effective treatment. This review described recent advances in serological and molecular diagnosis of toxoplasmosis as well as briefly reviewed imaging techniques to diagnosis of infection. In this regard, we conducted English literature searches in PubMed from 1989 to 2016 using the keywords Toxoplasma gondii, toxoplasmosis, serological, molecular, diagnosis, imaging techniques and summarize the advances in diagnosis of toxoplasmosis.

Recent advances in serological diagnosis Serological methods Immunological methods were the preferred and most common diagnostic method for diagnosis of toxoplasmosis in clinical laboratories in past century. These methods are based on recognition of T. gondii surface antigens by host T. gondii-specific immunoglobulins [1]. Different immunological methods often measure different antibodies (IgA, IgM, IgG, and IgE) that possess unique patterns of rise and fall with time after infection [1, 14]. During the past several years, many immunological testing methods, including Sabin Feldman dye test (SFDT), indirect fluorescent antibody test (IFAT), latex agglutination test (LAT), indirect hemagglutination test (IHA), enzyme-linked immunosorbent assays (ELISA), modified agglutination test (MAT), western blotting (WB) and IgG avidity test were used for detection of T. gondii infection in different patients and these diagnostic methods have been

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reviewed by previous publications [1, 13–15]. Although in two last decades, other immunological methods, including chemiluminescence assays (CLIA), enzyme-linked fluorescence assay (ELFA), immunochromatographic test (ICT), serum IgG avidity test and immunosorbent agglutination assays (ISAGA) have been developed to improve the ability of diagnosis of T. gondii infection [12, 15]. CLIA and ELFA are fully automated variations of the standard ELISA that have several advantages including reproducibility, cost effectiveness and fast and precise measurement of IgG and IgM antibody levels. Furthermore, CLIA is useful in measurement of the IgG avidity index even at low levels of Toxoplasma-specific IgG antibodies [16, 17]. ICT is a suitable method for field application because it has low cost, simple procedure and no requirements for skilled technicians. In ICT, a cellulose membrane is used as the carrier and a colloidal gold-labeled antigen or antibody is used as the tracer [18]. Recently, Wang et al. developed a rapid ICT strip for detection of excretory/secretory antigens (ESA) in acute infection of T. gondii as early as 2–4 days after infection [19]. In ISAGA, anti-human IgM antibodies are used for diagnosis of acute or congenital toxoplasmosis. Disadvantage for this method is requirement of large numbers of T. gondii tachyzoites. Although, this problem was resolved by replacing T. gondii tachyzoites with latex beads coated with soluble antigens [12, 20, 21]. The most of the above-mentioned serological methods used whole Toxoplasma lysate antigen (TLA) or native antigens from tachyzoites grown in mice and/or tissue culture. In DT, live tachyzoites are used that are severely hazardous. Soluble antigens are used in LAT and IHA. Formalinfixed and killed tachyzoites are used in MAT and IFA tests, respectively. The procedures for producing these antigens are very different between laboratories and also are associated with high costs and lengthy preparation and the possibility of staff infection [22]. In addition, the prominent limitations of these methods are their inability to estimate the accurate time of T. gondii infection and difficult in standardization of tests [15, 23]. A recently developed approach to improve the diagnosis of T. gondii infection and also discriminating between different phases of infection is to use recombinant antigens in place of the TLA.

Advances in serological methods based on recombinant antigen of T. gondii As mentioned above, although several methods are applied for diagnosis of toxoplasmosis but ELISA remains the most common method in clinical laboratories. Crude tachyzoite antigen and/or excreted–secreted antigen were the primary testing antigens in conventional indirect ELISAs in which showed satisfactory sensitivity and specificity. However, above-mentioned limitations for serological

Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii…

methods such as difficult to standardize, contamination with extra-parasitic materials and discrepancy in results there are also for ELISAs that used of these native antigens [14, 24]. Replacing of the TLA with recombinant antigens is an alternative approach of improving these tests. Use of more than one defined antigen, standardized purified antigens, standard preparation methods, simple standardization of method and ability of discriminating between the acute and chronic stages of T. gondii infection are the major advantages of recombinant antigens [14, 23]. In recent years, several recombinant antigens have been expressed in Escherichia coli or yeast by gene cloning and expression techniques, and their potential diagnostic value for detection of specific IgG and IgM antibodies against T. gondii parasite was assessed in different infected hosts. These antigens are including surface antigens SAG1 (P30), SAG2 (P22), SAG3 (P43), and P35 [23, 25–30]; dense granule antigens GRA1 (P24), GRA2 (P28), GRA4, GRA5, GRA6 (P32), and GRA7 (P29) [30–39]; microneme antigens MIC2, MIC3, MIC4, and MIC5 [12, 40, 41]; matrix antigens MAG1 [42]; and rhoptry antigens ROP1 (P66) and ROP2 (P54) [30, 43, 44]. The prominent advantage of these recombinant antigens is the identification of specific antibodies from acute or chronic phases and the discriminating between these two stages of T. gondii infection using single sample of serum [23]. In addition to conventional ELISA, the application of T. gondii recombinant antigens in other serological methods such as IgG avidity test, CLIA and ICT have demonstrated very promising results for detection of acute T. gondii infection [45–54]. It is demonstrated that combining of complementary recombinant antigens significantly improves the sensitivity and specificity of the tests when it is compared with single antigen [55–57]. It reported that mixture of GRA7, GRA8, ROP1 and SAG2A, GRA2, GRA4, ROP2, GRA8 and GRA7 recombinant antigens are potentially useful to detect IgM and IgG, respectively [55, 58]. Further combined recombinant antigens and their diagnostic characteristics are reviewed in recently published review articles [12, 23, 41]. Moreover in 2014, Drapała et al. [59] evaluated the utility of two mixtures of recombinant antigens (rROP1 + rSAG2 and rROP1 + rGRA6) in IgG avidity test. Their results have shown that these two mixtures have high sensitivity (100 and 95.4%, respectively) for detection of acute toxoplasmosis, suggesting that new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. Variations in sensitivity and specificity and contamination with proteins of the organisms that are used for the production of recombinant antigens (e.g., Escherichia coli) are the major limitations of recombinant antigens [60]. Use

of chimeric and/or synthetic peptide antigens is alternative approach to resolve of these limitations.

Advances in serological methods based on chimeric antigens and multiepitope peptides of T. gondii A growing number of recently conducted studies have demonstrated the potential advantages of chimeric or synthetic peptide antigens for the serological diagnosis of T. gondii infection, such as simple standardization, high sensitivity, low contamination with proteins of the organisms, reducing the costs of production, and increasing the probability of discriminating different stage of toxoplasmosis [61–66]. These chimeric and/or synthetic peptide antigens are including various immunoreactive epitopes from different T. gondii antigens which have been properly selected. These immunoreactive epitopes are hydrophobic and generally are well exposed on the antigen surface. During the recent years, a variety of innovative tools including peptide microarray analysis by bioinformatic methods, epitope mapping, phage display of cDNA libraries and reactivity with monoclonal antibodies have been developed for the prediction of specific epitopes and their localization on T. gondii antigens [23, 60, 67–70]. Beghetto et al. [61] for first time evaluated the diagnostic utility of two chimeric antigens, EC2 (containing MIC1 and MIC2 antigenic regions) and EC3 (containing M2AP, GRA3 and GRA7 antigenic regions) for serological diagnosis of acute toxoplasmosis. Their results have shown that chimeric antigens significantly improved the ability of RecELISAs (IgM ELISAs with chimeric recombinant antigens) compared with standard assays, especially in the diagnosis of congenital toxoplasmosis. Holec-Gąsior et al. [64] have demonstrated that sensitivity of MIC1–MAG1 recombinant chimeric antigen for the serodiagnosis of human toxoplasmosis is comparable with the TLA (90.9 and 91.8%, respectively). In subsequent study, the same authors have found that the chimeric antigen from three antigenic regions MIC1–MAG1–SAG1 have yielded better results than the chimeric antigen containing only two fragments from the MIC1 and MAG1 proteins [65]. In 2012, Dai et al. developed a recombinant multiepitope fusion peptide (rMEP) that was composed of antigenic determinants from SAG1, SAG2, and SAG3 antigens. In this study, IgG and IgM ELISAs with this rMEP-based assay were performed on human sera and results have shown that rMEP has sensitivity as high as commercially available ELISA kits and also has a significant potential to distinguish between acute and chronic toxoplasmosis [62]. Ferra et al. [22] evaluated diagnostic utility of five chimeric antigens in compression with mixtures of three recombinant antigens. Their study demonstrated that chimeric antigens were generally more reactive than mixtures of recombinant antigens. In addition, they showed that

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chimeric antigens composed of SAG2–GRA1–ROP1L are most sensitive to detection of toxoplasmosis. In subsequent study, the same team demonstrated that this chimeric antigens (SAG2–GRA1–ROP1L) has also high sensitivity and specificity in IgG CLIA [45]. In summarizing the information presented in this section, although nowadays the majority of serological tests and also commercial kits used of TLA, but due their inability to distinguish between acute and chronic toxoplasmosis and also other limitations, it is necessary to develop of new strategies for resolving of this problem. Use of that recombinant antigens and mixtures of recombinant antigens is a good alternative approach in the diagnosis and also discriminate different stages of T. gondii infection. In addition, more recent studies using chimeric antigens and multiepitope peptides demonstrated very promising results for development of new strategies capable of discriminating recently acquired infections from chronic infection.

Molecular techniques for the detection of T. gondii Despite the significant improvements in serological methods, there are still unresolvable limitations such as inability of these methods to confirm the presence of parasite in fetus and brain in congenital transmission and/or immunocompromised patients. Development of the molecular methods during the recent three decades induced a great revolution in the diagnosis of infectious diseases in general. To overcome the limitations of the serological tests, different molecular methods including PCR, nested PCR, real-time PCR and also loop-mediated isothermal amplification (LAMP) techniques have been developed to detect T. gondii DNA in biological samples (Table 1) [71–73].

PCR‑based techniques In PCR-based methods, a specific fragment of the genome can be amplified resulting in many millions of copies of the target DNA molecule. The first PCR technique for T. gondii detection was established by Burg and colleagues in which the 35-repeat B1 gene of T. gondii genome was amplified [74]. Following it, several multi-copy targeting genes including 18S rRNA-, P30-, B1-genes, 529-bp repeat fragment or the AF146527 element have been used for the detection of T. gondii in different biological samples [39, 73–79]. It has been reported that PCR with the 529-bp RE gene is 10–100 times more sensitive than the B1 gene [76, 77]. To diagnose congenital and ocular toxoplasmosis, the PCR has been successfully used with amniotic fluid, placental and brain tissues, aqueous humor and vitreous fluid. Moreover, whole blood, urine, cerebrospinal fluid (CSF),

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bronchoalveolar lavage, pleural and peritoneal fluids have been effectively used to diagnose toxoplasmosis in immunocompromised patients. Nested PCR is used to increase the specificity of DNA amplification as well as to detect the pathogens that occur with very few amounts. In a study reported by Alonso et al., nested PCR was found to be a rapid, sensitive and effective molecular method in the early detection of toxoplasmosis in patients with HIV [80]. In another study, it was demonstrated that nested PCR has high sensitivity for detection of T. gondii oocysts in river, well and sea waters [81]. Mousavi et al. compared RE and B1 genes for diagnosis of toxoplasmosis in diabetic patients using the nested PCR assay. Their findings showed that in comparison with the RE gene, the B1 gene is able to detect more positive samples and can be utilized for the detection of toxoplasmosis [82]. Vitale et al. developed a high-sensitive nested PCR assay targeting a region between the 28S and 18S rDNA for the detection of toxoplasmosis in animal and food samples [83]. It has been shown that additional development nested PCR assay can be a valuable, accurate, and specific method for diagnosis of different types of toxoplasmosis [84, 85]. Martınez et al. developed a nested PCR–ELISA technique to detect low quantity of T. gondii DNA using PCR coupled to a colourimetric detection step, by hybridization with a polystyrene sphere-bound probe [86]. This study reported that nested PCR–ELISA–beads assay is a valuable tool for the detection of T. gondii DNA [86]. Multiplex PCR is another type of PCR methods to detect multiple pathogens using multiple primer sets that each one targets a particular pathogen. This method permits the simultaneous analysis of multiple targets in a single sample. Nowakowska et al. used the amniotic and cerebrospinal fluids samples from congenital toxoplasmosis cases to determine the parasite genotype by nested multiplex PCR. They reported a sensitivity of > 25 parasites/ml in amniotic and cerebral spinal fluids samples by multiplex-nested PCR [87]. Rahumatullah et al. developed a triplex PCR assay targeting the B1 gene, 529 repeat region and ITS1 region of T. gondii. According to their report, this assay was capable of detecting as little as 10 pg T. gondii DNA, 1 04 tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice [88].

Real‑time PCR (RT‑PCR) RT-PCR-based methods are recently developed assays that made quantification of parasite burden possible and have a high degree of complex-specific diagnostic accuracy for diagnosis of pathogens in clinical samples. RT-PCR is the most sensitive molecular assay for the detection of pathogens, especially when target DNA is in low concentrations. Moreover, these assays are more rapid, sensitive

Not known

Difficult to setup

0.02–1 genome Not suitable for equivalent typing, expensive equipment are needed 0.1–1 genome False positive equivalent results, relative difficult primer designing, distinct partitions for each stage of test

~ 10 genome equivalent

Less sensitive, false positive results

Less sensitive, false positive results

90–100

85–100

75–100

90–100

90–100

80–100

NPV

100

85–100

98–100

95–100 10 fg of genomic DNA/1–10 tachyzoites/ml

85–100

100

Not known Not known Not known

100

95–100%

85–100%

Specificity PPV

1–50 fg of genomic DNA/10–103 tachyzoites/ml

Not known

2–5 tachyzoites DNA per PCR

100 genome equiva- 10–100 pg lent of genomic DNA/105–109 tachyzoites/ml 1–10 genome 5–200 fg of equivalent genomic DNA/103–105 tachyzoites/ml

Insensitive, isolation of parasite needed

Sensitivity

Results

Disadvantages

CSF cerebrospinal fluid, AF amniotic fluid, ASF ascitic fluid, BAF bronchoalveolar fluid, FT fetal tissues, SM skeletal muscle, CNS central nervous system, PPV positive predictive value, NPV negative predictive value, Mn-PCR multiplex-nested PCR, MS microsatellite

LAMP

RT-PCR & Realtime PCR

Multiplex PCR

Mn-PCR-based sequencing

Nested PCR

Species detection, B1 gene, 529 bp RE, CSF, AF, ASF, blood, urine, BAF high resolution in 18S rDNA, ITS-1, typing SAG1, SAG2, BAG1, GRA1 B1 gene, 529 bp RE, Blood, urine, FT Species detection, 18S rDNA high resolution in typing, isolation of parasite may not be needed Blood, urine Species detection, SAG1, SAG2, high resolution in SAG3, BTUB, typing, isolation GRA6, c22-8, of parasite may c29-2, L358, PK1, not be needed Apico AF, CSF, FT, BF High resolution in SAG2, SAG3, typing, suitable GRA6, BTUB, for epidemiology MS markers, B1, studies RE, ITS1 B1 gene, 529 bp RE, Blood, FT, AF, CSF Highly sensitive in SAG1, BAG1 detection, isolation of parasite not needed Water, brain, heart, Highly sensitive, B1 gene, TgOWP, specific and rapid liver, spleen, SAG1, SAG2, in detection, isolakidney, blood, 529 bp RE, GRA1 tion of parasite not SM, CNS needed, easy to perform,

Advantages

Conventional PCR

Sample(s)

DNA target regions

Technique(s)

Table 1 Comparison of the molecular methods for the diagnosis of toxoplasmosis


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and reproducible than conventional PCR, and are useful in investigations such as the kinetics of infections and monitoring therapeutic response [89]. The main advantages of RT-PCR over conventional PCR are including no need to open the reaction tubes and, therefore, minimized risk of environmental contaminations of amplicons, reduction of false positive results, measuring of amplification products during the each cycle using probes or intercalating dyes and providing precious diagnosis as choice of specific treatments for patients harboring several pathogens [79, 89–91]. In the last decade, several studies applied RT-PCR for diagnosis of toxoplasmosis in various biological, environmental and food samples [79, 92–98]. The majority of these studies reported that RT-PCR is more sensitive method compared with other molecular and biological methods [79, 92–98]. Lin et al. developed a RT-PCR-based assay for the detection of T. gondii using specific primers and a fluorescence-labeled TaqMan probe targeting the B1 gene of T. gondii. The assay was capable of detecting as little as 0.05 T. gondii tachyzoites in a reaction. Using this assay, they were able to detect Toxoplasma infection in 10 sections (33%) of 30 paraffin-embedded fetal tissue sections [99]. In another study, Costal et al. developed a RT-PCR assay using fluorescence resonance energy transfer (FRET) hybridization probes to detect and quantify T. gondii DNA in serum sample. Their results have demonstrated that the RT-PCR is a valuable method to identify and follow-up of Toxoplasma reactivation in immunocompromised patients [91]. In subsequent studies, it has been shown that sensitivity RT-PCR was improved about 10–100 fold when the 529-bp targeting was used instead to B1 [77, 79, 100]. In addition, it demonstrated that RT-PCR using the 529-bp target was found better than B1 gene for the diagnosis of congenital toxoplasmosis in both prenatal (81.3 versus 64.6%) and birth (36.0 versus 20.0%) periods [100]. The combination of a sequence-specific magnetic-capture method and RT-PCR has demonstrated very promising results for detection of T. gondii cysts and predilection-infected site in animal meat [94, 101].

Loop‑mediated isothermal amplification (LAMP) LAMP is a one-step modified PCR method, with high sensitivity, specificity, efficiency and rapidity. This method amplifies DNA under isothermal conditions (60–65 °C) using a DNA polymerase (Bst) with strand settlement activity and 4–6 specific primers, which are able to recognize a total of 6–8 distinct regions within the target DNA [102–104].

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Procedure steps of LAMP are shown in Fig. 1. The technical advantages of using LAMP instead of conventional PCR and RT-PCR include no need of highly sophisticated equipment, not expensive and time-consuming, no need for DNA extraction, and being more tolerant to PCR inhibitors such as blood, serum and food ingredients [104–109]. Although, sensitivity and amplification efficiency of LAMP are much higher than conventional PCR, but are slowly lower than RT-PCR [108, 110, 111]. In addition, due to primers binding to 6–8 distinct regions on the target DNA, the amplification specificity of LAMP is considered to be very high [106, 109, 112]. These attractive properties have motivated many research groups to use the LAMP for detecting various pathogens including T. gondii [39, 73, 78, 105, 113, 114]. Following widespread use of LAMP to identify the T. gondii, several targeting genes including 529-bp repetitive element, B1, 18S rRNA, SAG1, SAG2, GRA1 and oocyst wall protein (OWP) genes have been used for the detection of T. gondii in different biological and environmental samples [12, 73, 108, 110, 111, 113–116]. In a study on water samples and targeting the B1 and TgOWP genes, a sensitivity detection limit of 0.1 tachyzoites’ DNA was reported for both genes, suggesting the LAMP as a sensitive and specific tool for detection of T. gondii in water and other environmental samples [113]. Wang et al. reported that LAMP assay using SAG1 as target gene has a significant potential for early diagnosis of toxoplasmosis. In their study, LAMP was able to diagnose acute toxoplasmosis 2 days post-infection [110]. In another study, Lin et al. reported that LAMP assay using the SAG2 target was more sensitive than B1 and SAG1 genes for the diagnosis of active toxoplasmosis (87 versus 80 and 80%, respectively) [111]. The performance of LAMP and real-time PCR assays targeting a 529-bp repeated element of Toxoplasma genome was compared by Lin et al. The reported detection limits for LAMP and real-time PCR assays were 10 and 1 fg/μL of T. gondii DNA, respectively [111]. Kong et al. developed a LAMP assay targeting the RE gene to detect T. gondii DNA in blood samples of experimentally infected mice. They reported a detection limit as low as 0.6 fg of T. gondii DNA for the RE-LAMP assay. Moreover, the LAMP sensitivity was reported 100- and 1000-fold higher than that of the B1-LAMP and RE-nested PCR, respectively [117]. In 2013, Qu et al., using the conserved sequence of 18s rRNA, developed reverse transcription LAMP (RT-LAMP) method for the detection of T. gondii in meat with a detect limit of one tachyzoite in 1 g meat. Their results suggest that RT-LAMP technique provides a simple and reliable tool for inspecting


Fig. 1 Procedure steps and different product diagnostic methods of LAMP technique

meats which are T. gondii-contaminated [114]. In general, all advantages of RT-PCR assay including high sensitivity and specificity along with simple procedure for easy flexibility to field situations can be seen in the LAMP assay. Summarizing the information presented in this section, it must be mentioned that use of the high-sensitive molecular methods based on PCR has made a significant improvement in clinical diagnosis of different type of toxoplasmosis. Results of recent studies indicated that LAMP and RT-PCR using AF and CSF are more sensitive and specific methods in congenital infection and immunocompromised patients, respectively, and could be used for early diagnosis of infection and also in follow-up after treatment. Moreover, Improvement of bioinformatics technologies gives an ability to recognize potential targeting genes that could be very crucial to improve the sensitivity and specificity of these methods and provide prospects for the design of new complimentary diagnostic approaches for toxoplasmosis.

Imaging techniques Imaging techniques are useful for diagnosis of cerebral and ocular toxoplasmosis. In cerebral toxoplasmosis (encephalitis), a variety of imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), nuclear imaging and ultrasonography (US) could be useful, although their results might be not specific. A CT scan of the brain is often used as an initial screening test and may show single or multiple nodular lesions. The administration of intravenous (IV) contrast material with either modality improves the diagnostic yield and accuracy [118]. In CT scan studies, rounded isodense or hypodense lesions with thin- and smooth-wall were observed following the IV administration of contrast medium and calcified lesions with dot-like or thick-wall were observed following medical treatment. When double-dose delayed scan is used, central filling lesions are shown and detection rate

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is significantly improved. Delayed scan demonstrated more sensitive diagnostic features, as in several cases enhancing lesions were detected by delayed scan, whereas negative results were achieved in immediate CT scan [119]. In CNS toxoplasmosis, approximately 75% of lesions are multiple and basal ganglia are most prominent site to induce nodules. In the congenital toxoplasmosis, CT scans may demonstrate diffuse hydrocephalus and brain calcifications that are associated with multiple, irregular, nodular, cyst-like calcifications in the periventricular areas and the choroid plexus [120]. MRI is more suitable for the determination of the damage extent and improved the ability to distinguish between different CNS lesions [119]. The T1 and T2 MR spectroscopy patterns are useful to improve the ability for discrimination of different CNS lesions. On T1-weighted MRIs, the lesions are typically isointense or hypointense relative to brain tissue, whereas in T2-weighted MRIs, they are usually hyperintense, although they could sometimes be isointense to hypointense. Brightbill et al. reported that T2-weighted hyperintensity could be pathologically related with necrotizing encephalitis, whereas T2-weighted isointensity is linked with organizing abscesses. Moreover, they described change from hyperintensity to isointensity in T2-weighted MRI could be resulted from positive response to antibiotic treatment, a tool to evaluate the efficacy of medical therapy [121]. The differential diagnosis between CNS lymphoma and toxoplasmosis is an important issue among ADIS patients. In MR spectroscopy, lymphoma is characterized by increased lactate, lipid, choline peaks and decreased N-acetyl aspartate, creatine, and myoinositol signals, whereas in CNS toxoplasmosis only elevated lactate and lipid peaks are depicted. Moreover, lesions induced by toxoplasmosis have significantly greater values than lymphoma lesions. Moreover in recent years, nuclear imaging tools such as fluorodeoxyglucose positron-emission tomography (FDG-PET), thallous chloride (201Tl) and technetium-99 m ( 99mTc) sestamibi (MIBI) have shown promising features to distinguish CNS toxoplasmosis from CNS lymphoma in HIV-positive patients, although more prospective investigations are needed to confirm the ability of these imaging tools [122–125]. Prenatal US can be useful to detect brain abnormalities due to congenital toxoplasmosis [126]. In a comprehensive review [122], Khan states that “ultrasonographic findings in congenital toxoplasmosis included symmetrical ventriculomegaly, intracranial periventricular and hepatic/splenic

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densities, focal destruction of cerebral tissue that is related with enlargement of the neighboring cistern or ventricle, ocular calcification, microphthalmia, metaphyseal bands, irregularity of the metaphyseal border of the growth plate, fetal ascites, and increased placental thickness”. Postnatal US can be used to monitor ventricular size in infants up to 18 months of age. In adult patients, abdominal US can reveal hepatosplenomegaly and abdominal lymphadenopathy due to toxoplasmosis [126–128]. The ocular toxoplasmosis can be diagnosed by clinical findings such as visual acuity, intraocular pressure, biomicroscopy, and indirect ophthalmoscopy [129]. Although imaging techniques such as fundus autofluorescence (FAF), confocal scanning laser ophthalmoscopy (CSLO), optical coherent tomography (OCT), fluorescent angiography (FA), ultrasound, and indocyanine green angiography (ICG) may be useful for its diagnosis and management of complications that include macular edema, retinal detachments, retinal neovascularization, vascular occlusion, optic nerve atrophy, vitreoretinal lesions, glaucoma and cataracts [129].

Conclusion Early and accurate diagnosis of T. gondii infection is critical, especially in congenital transmission and immunocompromised patients. The choice of the appropriate diagnostic method is essential for this propose. In this review, we provide a summary of recent developed methods and also attempts to improve their sensitivity for diagnosis of toxoplasmosis. Serology, molecular and imaging technologies each has their own advantages and limitations which can certainly achieve accurate and effective results for diagnosis of toxoplasmosis by combining these diagnostic techniques. For this purpose, we suggest IgG/IgM ELISA in combination with RT-PCR/LAMP on AF and prenatal US for detection of acute infection in pregnant women and also to start preventive measures of congenital transmission. Serological finding (ELISA using TLA, recombinant or chimeric antigens) is gold standard in newborns, although RT-PCR/ LAMP on cord blood or whole blood can be useful for definitive diagnosis. Moreover, combining of ELISA (using TLA, recombinant or chimeric antigens), RT-PCR/LAMP on CSF and imaging findings by MRI or CT could result in definitive diagnosis in immunocompromised patients (Fig. 2).


Fig. 2 An algorithm for tests and test combinations for definitive diagnosis of toxoplasmosis in suspected individuals

Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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