Abstract
The diagnosis of toxoplasmosis is of great importance due to the damage caused by this parasite in immunosuppressed people or in pregnant women, the diagnosis of an active toxoplasmosis represents a sign to initiate a pharmacological treatment immediately. The diagnosis of Toxoplasma can be performed with direct methods through intraperitoneal inoculation of serum or cerebrospinal fluid, in susceptible mice evaluating the survival and detection of tachyzoites of biological samples. Indirect methods detecting the IgM and IgG isotypes against Toxoplasma have been the tools mostly used and had leaded to discriminate between an active and acute, from a chronic toxoplasmosis. Molecular methods actually Toxoplasma-DNA identification by molecular biology tests like the polymerase chain reaction (PCR) allow the direct detection of the parasite. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) have been used to identify three strain linages (type I, II and III). Recently, a high-resolution melting method was described to determine the genotype of the infection by Toxoplasma gondii directly from biological samples.
Keywords
- Toxoplasma infection
- diagnosis
- toxoplasmosis
- methods
1. Introduction
The diagnosis of toxoplasmosis represents a very important decision to whether to initiate or not an anti-parasitic treatment. The immunological methods that detect the IgM and IgG isotypes against
Several methodologies have been applied historically, including the isolation of the parasite in biological specimens and the xenodiagnostic evaluation through intraperitoneal inoculation of serum or cerebrospinal fluid, in susceptible mice evaluating the survival and detection of tachyzoites in cerebrospinal fluid. Serologic diagnosis is frequently performed for the detection in serum of IgM and IgG anti-
2. Direct methods
2.1. Isolation of the parasite or bioassay
The diagnosis of toxoplasmosis could be performed by the inoculation of suspicious blood samples, cerebrospinal fluid, lymph node or other corporal fluids or tissues, in the peritoneal cavity of the immunosuppressed mice or in a susceptible strain like BALB/c. The presence of tachyzoites in the peritoneal fluid of the mice is analysed by phase contrast microscopy from 6 to 10 days after inoculation. The parasite is observed in its characteristic half-moon fixing the specimen with methanol and stain with Wright, Giemsa or May-Grunwald and observed by a microscope. The parasites present sometimes a granular membrane, dyed blue cytoplasm and a red nucleus at the center of the cytoplasm.
In cases of new-borns with toxoplasmosis, inoculation of mice with blood of the child obtained during the first week of birth reaches 75% of positive results; however, if the sampling is delayed by 1 month, the positive results are reduced to 52%. A drawback of this method is the need of specialized personal because of the risk of contamination by the handling of
2.2. Cellular culture
Cellular culture is more utilized nowadays for the diagnosis and isolation of parasite
The cell culture to identify tachyzoites from biopsy or corporal fluid of infected patients, leads to the destruction of the cellular monolayer and is related with the initial amount of tachyzoites in the biological sample. The sensitivity of parasite detection by cell culture from intraocular liquid (aqueous and vitreous humor) is 91%, compared with antibodies detection by ELISA with 67%.
2.3. Histology
The detection of tachyzoites in histological sections from suspicious biopsies indicates an acute infection; on the contrary, the detection of tissue cysts that contain bradyzoites in histological samples only confirms a chronic toxoplasmosis infection. The presence of cysts in the placenta, foetus or new-born indicates a congenital infection, according to the number of cysts in histological sections, can be suggested as an active infection and needs immediate treatment. In biopsy or autopsy material, the encysted tachyzoites and bradyzoites are usually demonstrable with wright, hematoxylin and eosin dyes, where the bradyzoites contain glycogen vacuoles. The size of the cyst is important in histological sections of the brain (Figure 1). There are other stain techniques like Schiff-periodic acid, specific immunohistochemistry stained with inmunoperoxidase that are equally efficient. These procedures have great diagnosis value but are limited due to the difficulty to grow parasites
3. Immunodiagnostics’
3.1. Sabin and Feldman test
The Sabin and Feldman reaction, also known as the dye test [12], is considered as golden standard by detection of
3.2. Hemagglutination
The hemagglutination assay was first described by Jacobs L. in 1957. This test is based on the capacity of anti-
3.3. Modified agglutination test (MAT)
For this assay, the tachyzoites are fixed with formaldehyde in microtiter plates in the form of U plates with diluted serum added. In positive serum samples, a thin layer of agglutination is produced, while in negative samples compact sediment is formed due to precipitated tachyzoites at the bottom of the well. This assay was first described by Fulton and Turk [17], with low specificity and sensitivity, due to the union of normal IgM at the surface of the parasite and improved by the preparation of the antigen using a buffer with 2-mercaptoethanol, that eliminates the non-specific IgM. This assay detects IgG antibodies, without limitation of host species, but false negative results could occur during early stages of acute infection [17]. The specificity and sensitivity of MAT are similar to the Sabin and Feldman assay in most species. Also, MAT can be used in corporal fluids, being of much used in field work.
3.4. Indirect immunofluorescence (IFI)
This assay was described by Garin in 1967 [18], consists of fixation of tachyzoites on microscope slides. Upon contact with the antibodies present in the patient’s serum, the antibody antigen reaction is carried out, which is visible with a second antibody labelled with fluorescein. The antigen-antibody reaction is evidenced by fluorescence microscope. In positive cases, a bright yellow greenish fluorescence is evident and a red colour without fluorescence is observed in negative cases. The colour of the fluorescence depends on the fluorochrome conjugated to the antibodies. The immunofluorescence is a test that shows high precision when it is adapted for specific antibodies. It is known that when the human is infected, it responds first with the production of IgM that disappears later. The detection of specific IgM in the new-born shows difference in the antibodies transmitted from the mother to the product (IgG goes through placental barrier and IgM is not capable to do it). The detection of specific IgG contributes to the diagnosis most of the times; however, considerable titles of antibodies may persist for months or years [18].
3.5. Enzyme linked immunosorbent assay (ELISA)
The enzyme linked immunosorbent assay (ELISA) was described initially in 1971 by Engvall and Perlman, uses antibodies conjugated to an enzyme [19]. The antibody conserves its capacity of specific union to the antigen, while the enzyme is capable of a redox reaction, in which the substrate precipitates in a coloured product. In this system, the antigen or the antibody is absorbed to an insoluble solid phase (micro polystyrene plates). There are several variants of ELISA: direct, indirect, capture and competitive. All of them allow the determination of antigens in biological fluids except the indirect method that only detects antibodies.
The direct ELISA methods involve attachment of the antigen to the solid phase, followed by an enzyme-labelled antibody. This type of assay generally makes measurement of crude samples difficult, since contaminating antigens compete for plastic binding sites. Whereas, indirect ELISAs also involve attachment of the antigen to a solid phase, but in this case, the primary antibody is not marked. An enzyme-conjugated secondary antibody, directed at the first antibody, is then added. This format is used most often to detect specific antibodies in serum.
The captured ELISA assay involves attachment of a captured antibody to a solid phase support. Samples containing known or unknown antigens are then added in a matrix or buffer that will minimize attachment to the solid phase. An enzyme-labelled antibody is then added for detection.
The most used enzymes are horse radish peroxidase (HRP), alkaline phosphatase (FA) and the β-d-galactosidase (BG), the substrates for the HRP enzyme are hydrogen or urea peroxide, which on reducing make certain chromogens oxidized in the enzymatic reaction, developing a coloured product. The oxidized ortho-phenylene diamine remains soluble after the oxidation reaction and is the most used. The FA substrate p-nitrophenyl phosphate that produces colour when degraded by the enzyme at temperatures over 30°C, the 5 bromo-4cloro-3-indoilphosfate can be employed in ELISA in tube or in dot-ELISA. The ELISA method detects from 70 to 80% of cases in congenital infections, a rise in the titles of IgM that extends more over the first week is indicative of acute infection, since the average of duration of maternal IgM is from 3 to 5 days. Also, antibodies can be determined in serum and aqueous humour.
Interpretation of the test: If ELISA is qualitative, a development of colour is observed in seropositive samples. If the test is quantitative, the intensity of the developed colour is measured at a determined wavelength (nanometres) and the lecture of absorbance is proportional to the magnitude of the reaction antigen-antibody [20–22].
3.6. Recombinant antigens
At the final of the nineteenth century, several recombinant antigens of different sites of the parasites were analysed [23, 24]. The dense granule antigen—GRA1, GRA2, GRA4, GRA6, GRA7 and GRA8, rhoptries protein—ROP1 and ROP2, the matrix protein—MAG1, protein micronemes—MIC2, MIC3, MIC4 and MIC5 or the surface antigen—SAG1 and SAG226, were evaluated as diagnostic markers in human beings or animals by ELISA to detect IgG and IgM specific antibodies [25]. The combinations of recombinant antigens have showed more sensibility and specificity for the use of only one antigen. For example, the combinations of SAG2A, GRA2, GRA4, ROP2, GRA8 and GRA7 are potentially useful to detect antibodies IgG in human beings with the infection recently acquired [26], ROP1, SAG1, GRA7, GRA8 and GRA6 are promising to detect specific IgM antibodies while GRA7 and GRA8 are used to detect IgA specific antibodies. A specific protein of sporozoites related with embryogenesis (TgERP) was identified in a study, it can react with specific antibodies of oocysts and can be used to detect an early infection by sporozoites implying the oocysts as infection source [27, 28].
3.7. Avidity test
The IgG avidity test was described by Hedman et al. [24]. This assay measures the avidity of the binding of specific antibodies to
3.8. Western blot
Western blot is an immunoenzymatic technique, based on the binding of
Western blot has the advantage to identify specific antigens recognized by IgG, IgM and IgA antibodies from serum and cerebrospinal fluid of patients with chronic and acute toxoplasmosis, specially in immunocompromised patients with low titers of antibodies. In immunocompetent patients this method has been used to identify parasite proteins as markers for toxoplasmosis diagnosis (Figure 2) [33]. Also, diagnosis of congenital toxoplasmosis from colostrum samples by Western blot is very useful, since IgA antibodies recognize a greater number of antigens [34]. This method has also been used with other corporal fluids such as saliva [35].
3.9. Immunocytochemistry and immunohistochemistry
The immunocytochemistry involves detection of epitopes expressed by the antigens of
4. Molecular techniques
4.1. Generalities of the genome of T. gondii
Molecular biology has permitted the advance in molecular knowledge of
4.2. Molecular diagnosis by the polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) consists in making a repetitive replication
In patients with AIDS, the detection of
The polymerase chain reaction (PCR) can be in the future, undoubtedly a successful tool in the diagnosis of toxoplasmosis, when a satisfactory correlation exists between the patient’s clinical profile and the presence of
4.3. PCR in real time
Unlike the conventional PCR, the real-time PCR using fluorescence probes measures the product of amplification in each cycle and can be quantified with the use of known concentration standards. Real-time PCR has been successfully used to detect the
4.4. Loop mediated isothermal amplification (LAMP)
Loop mediated isothermal amplification (LAMP) is a technique of DNA amplification under isothermal conditions, which combines speed, efficiency and high specificity. The amplification of DNA proceeds from the repetition of two reactions of elongation in a structure that has loop form. It uses DNA polymerase with strand displacement activity and two inner primers and two outer primers. The inner primers contain sense and anti-sense sequences of DNA and initiate the LAMP reaction. The outer primers hybridize a few bases of the inner and initiate the DNA synthesis with strand displacement, forming a loop structure, which initiates a DNA synthesis by auto recognition. In this technique, 10 copies of DNA can be produced in less than one hour [52]. The final products are multiple loop DNA formed by alignment between inverted repeated sequences present in the same chain [53]. It only requires a water bath or thermo block to realize the amplification of DNA and the amplified products can be analysed by real time PCR equipment or by electrophoresis [54]. This technique is more sensitive than conventional PCR but low sensitive than real time PCR [53]. The LAMP assay has been evaluated identifying REP 529-bp, SAG1, SAG2 and B1
4.5. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs)
The PCR-RFLP is based on the capacity of restriction endonucleases that recognize polymorphisms of only one nucleotide (SNPs). Polymerase chain reaction (PCR) products treated with restriction enzymes to generate polymorphic DNA fragments of variable length, that are visualized after electrophoresis in agarose or polyacrylamide gels with a band pattern [55]. In 1995, Howe and Sibley identified three main linages (type I, II and III) from 106 strains of
Since then, in different laboratories, several groups of different multi-locus markers have been selected, to characterize by PCR-RFLP
The microsatellites (MS) are short tandem repeats (STR) sequences of DNA (2 to 6 pb), that are widely distributed throughout the nuclear genomes of eukaryotes. They are highly polymorphic, the MS length varies depending the numbers of STR. The tandem repetitions in
4.6. High-resolution melting (HRM)
In homogeny sample, the HRM method can analyse genetic variations, which allows to characterize polymorphisms based on the melting temperature generated by the DNA length and GC content [62]. On the base of only one SNP from gene B1 which is found in multiple copies, the HRM can classify correctly three different types of strains of
References
- 1.
Sabin AV, Elizky PK. Toxoplasma and obligate intracellular parasitism. Science. 1937;85: (336–8):17815128. DOI: 10.1126/science.85.2205.336. - 2.
Chai JY, Lin A, Shin EH, Oh MD, Han ET, Nan HW, Lee SH, Chai JY, Lin A, Shin EH, Oh MD, Han ET, Nan HW, Lee SH. Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea. J. Parasitol. 2003;41: (147–154):PMID: 12972728. DOI: 10.334/kjp.200341.3.147. - 3.
Stajner T, Vasiljević Z, Vujić D, Marković M, Ristić G, Mićić D, Pasić S, Ivović V, Ajzenberg D, Djurković-Djaković O. Atypical strain of Toxoplasma gondii causing fatal reactivation after hematopoietic stem cell transplantion in a patient with an underlying immunological deficiency. J. Clin. Microbiol. 2013;51: (2686–2690):PMID: 23761151. DOI: 10.1128/JCM.01077-13. - 4.
Ibebuike K1, Mantanga L, Emereole O, Ndolo P, Kajee A, Gopal R, Pather S. Ibebuike K, Mantanga L, Emereole O, Ndolo P, Kajee A, Gopal R, Pather S. Cerebellar toxoplasmosis in HIV/AIDS infant. Cerebellar toxoplasmosis in HIV/AIDS infant: case report and review of the literature. Neurol. Sci. 2012; 33: (1423–1428):PMID: 22286317. DOI: 10.1007/s10072-012-0960-x. - 5.
Monaco SE, Monaghan SA, Stamm JA, Khalbuss WE, Nichols L, Pantanowitz. Toxoplasmosis in a post-transplant bronchoalveolar lavage: a case report. Diagn. Cytopathol. 2012; 40: (629–634):PMID: 21591274. DOI: 10.1002/dc.21646. - 6.
Değirmenci A1, Döşkaya M, Caner A, Ciçek C, Korkmaz M, Gürüz Y, et al. Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method. Exp. Parasitol. 2011;128: (1–8):PMID: 21296077. DOI: 10.1016/j.exppara.2011.01.019. - 7.
Mahadevan M, Ramalingaiah AH, Parthasarathy S, Nath A, Ranga U, Krishna SS. Neuropathological correlate of the “concentric target sign” in MRI of HIV associated cerebral toxoplasmosis. J. Mag. Res. Imag. 2013; 38: (488–495):PMID: 23440973. DOI: 10.1002/jmri.24036. - 8.
Garweg JG. Determinants of immunodiagnostic success in human ocular toxoplasmosis. Parasite Immunol. 2005; 27: (61–68):PMID: 15882232. DOI: 10.1111/j.1365-3024.2005.00747.x. - 9.
Miller D, Davis J, Rosa R, Diaz M, Perez, E. Utility of tissue culture for detection of Toxoplasma gondii in vitreous humor of patients diagnosed with toxoplasmic retinochoroiditis. J. Clin. Microbiol. 2000;38: (3840–3842):PMID: 11015415. - 10.
Peyron F, Lobry JR, Musset K, Ferrandiz J, Gomez-Marin JE, Petersen E,et al. Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America). Microbes Infect. 2006;8: (2333–40):PMID: 16938480. DOI: 10.1016/j.micinf.2006.03.023. - 11.
Fuentes I, Rubio JM, Ramírez C, Alvar J. Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples. J. Clin. Microbiol. 2001;39 (4):(1566–1570). DOI: 10.1128/JCM.39.4.1566-1570.2001 - 12.
Sabin A, Feldman HA. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoan parasite ( Toxoplasma ). Science. 1948;108: (660–3). DOI: 10.1126/science.108.2815.660 - 13.
Jacobs L, Lunde MN. Hemoaglutination test for toxoplasmosis. J. Parasitol. 1957; 43: (308–314):PMID: 13439469. - 14.
Desmonts G, Remington JS. Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity and specificity. J. Clin. Microbiol. 1980;11: (562–568):PMID: 7000807. - 15.
Montoya JG, Berry A, Rosso F, Remington JS. The differential agglutination test as a diagnostic aid in cases of toxoplasmic lymphadenitis. J. Clin. 2007; 45: (1463–1468). - 16.
Yamamoto YI, Hoshino-Shimizu S, Camargo ME. A novel IgM-indirect hemagglutination test for the serodiagnosis of acute toxoplasmosis. J. Clin. Lab. Anal. 1991; 5: (127–132):PMID: 2023058. - 17.
Fulton JD, Turk JL. Direct agglutination test for Toxoplasma gondii . Lancet. 1959;12: (1068–1069):PMID: 13825641. - 18.
Garin JP, Ambroise TP, Cornet AM, Dien-Truong T, Despeinges J. Interet de la fluoresence et de l’inmnofluoresence dans le diagnostic Parasitologique et serologique de la Toxoplasmosis. Revue de 1’ Institut Pasteur de Lyon. 1967; 68: (179–205). - 19.
Engvall E, Perlmann P. Quantitative assay of immunoglobulin G. Immunochemistry. 1971; 9: (871–4):PMID: 5135623. - 20.
Liu Q, Wang Z-D, Huang S-Y, Zhu X-Q. Diagnosis of toxoplasmosis and typing of Toxoplasma gondii . Parasites & Vectors. 2015;8: (292):PMID: 26017718. DOI: 10.1186/s13071-015-0902-6. - 21.
Kotresha D, Noordin R. Recombinant proteins in the diagnosis of toxoplasmosis. APMIS. 2010; 118: (529–542):PMID: 20666734. DOI: 10.1111/j.1600-0463.2010.02629.x. - 22.
Aubert D, Maine GT, Villena I, Hunt JC, Howard L, Sheu M, et al. Recombinant antigens to detect Toxoplasma gondii -specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J. Clin. Microbiol. 2000;38: (1144–1150):PMID: 10699010. - 23.
Hill D, Coss C, Dubey JP, Wroblewski K, Sautter M, Hosten T, et al. Identification of a sporozoite-specific antigen from Toxoplasma gondii . J Parasitol. 2011;97: (328–37):PMID: 21506817. DOI: 10.1645/GE-2782.1. - 24.
Hedman K, Lappalainen M, Seppaia I, Makela O. Recent primary Toxoplasma infection indicated by a low avidity of specific IgG. J. Infect. Dis. 1989;159: (736–740):PMID: 2926163. - 25.
Pfrepper KI, Enders G, Gohl M, Krczal D, Hlobil H, Wassenberg D, et al. Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clin. Diagn. Lab. Immun. 2005;12(8):(977–982):PMID: 16085916. DOI: 10.1128/CDLI.12.8.977-982.2005. - 26.
de Ory F, Casas I, Domingo CJ, Echevarria J. Application of fluoroimmunoassay to the identification of low avidity specific IgG against pathogenic human viruses and Toxoplasma gondii . Clin. Diagn. Virol. 1995;3: (323–332). - 27.
Deshpande PS, Kotresha D, Noordin R, Yunus MH, Saadatnia G, Golkar M, et al. IgG avidity Western blot using Toxoplasma gondii rGRA-7 cloned from nucleotides 39–711 for serodiagnosis of acute toxoplasmosis. Rev. Inst. Med. Trop. Sao Paulo. 2013;55: (79–83):PMID: 23563759. DOI: http://dx.doi.org/10.1590/S0036-46652013000200003. - 28.
Gutierrez J, Rodriguez M, Piedrola G, del Carmen MM. Detection of IgA and low-avidity IgG antibodies for the diagnosis of recent active toxoplasmosis. Clin. Microbiol. Infect. 1997; 3 (658-662):PMID: 11864209. DOI: http://dx.doi.org/10.1111/j.1469-0691.1997.tb00474.x - 29.
Ali-Heydari S, Keshavarz H, Shojaee S, Mohebali M. Diagnosis of antigenic markers of acute toxoplasmosis by IgG avidity immunoblotting. Parasite. 2013; 20: (18):PMID: 23688778. DOI: 10.1051/parasite/2013017. - 30.
Ashburn D, Joss AW, Pennington TH, Ho-Yen DO. IgA, IgE, and IgG avidity tests have any value in the diagnosis of Toxoplasma infection in pregnancy? J. Clin. Pathol. 1998;51: (312–315):PMID: 9659246. - 31.
Lefevre-Pettazzoni M, Le Cam S, Wallon M, Peyron F. Delayed maturation of immunoglobulin G avidity: implication for the diagnosis of toxoplasmosis in pregnant women. Eur. J. Clin. Microbiol. Infect. Dis. 2006;25:(687–693):PMID: 17024503. DOI: 10.1007/s10096-006-0204-1. - 32.
Meek B, Diepersloot RJ, van Gool T, Speijer D, Peek R. IgM recognition of recombinant Toxoplasma gondii antigens by sera of acutely or latently infected humans. Diag. Microbiol. Infect. Dis. 2003;45: (45–52):PMID: 12573550. - 33.
Galván RML, Guillen VC, Saavedra DR, Islas RA. Analysis of Toxoplasma gondii antigens with sera from toxoplasmosis. Rev. Soc. Bra. Med. Trop. 1998;31: (271–277):PMID: 9612018. - 34.
de Morais Oliveira AC, Silva Borges DS, Reis Carvalho F, Gomes de Macêdo A, Mota CM, de Morais Oliveira AM. Evaluation of colostrum as an alternative biological sample for the diagnosis of human congenital toxoplasmosis. BMC Infect. Dis. 2015; 15 :(519):PMID: 26573138. DOI: 10.1186/s12879-015-1242-z. - 35.
Stroehle A, Schmid K, Heinzer I, Naguleswaran A, Hemphill A. Performance of a Western immunoblot assay to detect specific anti- Toxoplasma gondii IgG antibodies in human saliva. J. Parasitol. 2005;91: (561–563):PMID: 16108547. DOI: 10.1645/GE-423R. - 36.
Lang W, Miklossy J, Deruaz JP, Pizzolato GP, Probst A, Schaffner T, Gessaga E, Kleihues P. Neuropathology of the acquired immune deficiency syndrome (AIDS): a report of 135 consecutive autopsy cases from Switzerland. Acta Neuropathol. 1989; 77: (379–90):PMID: 2540610. - 37.
Budka H, Costanzi G, Cristina S, Lechi A, Parravicini C, Trabattoni R, Vago L. Brain pathology induced by infection with the human immunodeficiency virus (HIV). Neuropathology. 1987; 75: (185). DOI: 10.1007/BF00687080 - 38.
Miyamoto C, Mattos Neto RB, Cesare SD, Belfort Junior R, Burnier MN. Use of CD25 as an immunohistochemical marker for acquired ocular toxoplasmosis. Arq. Bras. Oftalmol. 2010; 73: (443–446):PMID: 21225130. DOI: dx.doi.org/10.1590/S0004-27492010000500011. - 39.
Anselmo LM, Vilar FC, Lima JE, Yamamoto AY, Bollela VR, Takayanagui OM. Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients. J. Neurol. Sci. 2014; 346: (231–234):PMID: 25240445. DOI: 10.1016/j.jns.2014.08.034. - 40.
http://protists.ensembl.org/Toxoplasma_gondii/Info/Annotation/) - 41.
http://www.toxodatabase.org/ - 42.
http://www.jcvi.org/cms/home/. - 43.
Yamagish J, Suzuki Y. The genome-wide identification of promoter regions in Toxoplasma gondii . Methods Mol. Biol. 2015;1201: (193–205):PMID: 25388115. DOI: 10.1007/978-1-4939-1438-8_11. - 44.
Jalal S, Nord CE, Lappalainen M, Evengard B, on behalf of the ESCMID, Study Group on toxoplasmosis. Rapid and sensitive diagnosis of Toxoplasma gondii infection by PCR. Clin. Microbio.l Infect. 2004;10 (937-939):PMID: 15373893. DOI: 10.1111/j.1469-0691.2004.00948.x - 45.
Kasper DC, Sadeghi K, Prusa AR, Reischer GH, Kratochwill K, Förster-Waldl E,et al. Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid. Diagn. Microbiol. Infect. Dis. 2009;63 :(10–15):PMID: 18990529. DOI: 10.1016/j.diagmicrobio.2008.09.009 - 46.
Blay EA, Ghansah A, Otchere J, Koku R, Kwofie KD, Bimi L, et al. Congenital toxoplasmosis and pregnancy malaria detection post-partum: effective diagnosis and its implication for efficient management of congenital infection. Parasitol. Int. 2015; 64: (603–608). DOI: dx.doi.org/10.1016/j.parint.2015.08.004 - 47.
Belaz S, Gangneux JP, Dupretz P, Guiguen C, Robert-Gangneux F. A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR. J. Clin. Microbiol. 2015;53: (1294–1300):PMID: 25653416. DOI: 10.1128/JCM.02900-14. - 48.
Robert-Gangneux F, Sterkers Y, Yera H, Accoceberry I, Menotti J, Cassaing S, et al. Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study. J. Clin. Microbiol. 2015; 53: (1677–1684):PMID: 25762774. DOI: 10.1128/JCM.03282-14. - 49.
Switaj K, Master A, Skrzypczak M, Zaborowski P. Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clin. Microbiol. Infect. 2005;11: (170–176):PMID: 15715713. DOI: 10.1111/j.1469-0691.2004.01073.x. - 50.
Bretagne S. Molecular diagnostics in clinical parasitology and mycology: limits of the current polymerase chain reaction (PCR) assays and interest of the real-time PCR assays. Clin. Microbiol. Infect. 2003; 9: (6). DOI: 10.1046/j.1469-0691.2003.00677.x - 51.
Zainodini N, Zare-Bidaki M, Abdollahi SH, Afrooz M, Ziaali N, Ebrahimian M, et al. Molecular and serological detection of acute and latent toxoplasmosis using real-time PCR and ELISA techniques in blood donors of rafsanjan city. Iran. Iran J. Parasitol. 2014 ; 9: (336–341):PMC4316564. - 52.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000; 28: (e63):PMID: 10871386 PMCID. - 53.
Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y. Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated rsothermal amplification method using blood samples from patients. J. Clin. Microbiol. 2010; 48: (3698–3702). DOI: 10.1128/JCM.00462-10 - 54.
Zhang H, Thekisoe OM, Aboge GO, Kyan H, Yamagishi J, Inoue N, et al. Toxoplasma gondii : sensitive and rapid detection of infection by loop mediated isothermal amplification (LAMP) method. Exp. Parasitol. 2009;122: (47–50):PMID: 19545521. DOI: 10.1016/j.exppara.2009.01.012. - 55.
Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 1995;172: (1561–1566). DOI: doi.org/10.1093/infdis/172.6.1561 - 56.
Ajzenberg D, Cogne N, Paris L, Bessieres MH, Thulliez P, Filisetti D, et al. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J. Infect. Dis. 2002;186: (684–689). DOI: doi.org/10.1086/342663 - 57.
Khan A, Su C, German M, Storch GA, Clifford DB, Sibley LD. Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J. Clin. Microbiol. 2005;43: (5881–5887). DOI: 10.1128/JCM.43.12.5881-5887.2005J - 58.
Su C, Zhang X, Dubey JP. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Int. J. Parasitol. 2006;36: (841–848):PMID: 16643922. DOI: 10.1016/j.ijpara.2006.03.003. - 59.
Ferreira IM, Vidal JE, Costa-Silva TA, Meira CS, Hiramoto RM, de Oliveira AC P,et al. Toxoplasma gondii : genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers. Exp. Parasitol. 2008;118: (221–227):PMID: 17950282. DOI: 10.1016/j.exppara.2007.08.006. - 60.
Ajzenberg D, Collinet F, Mercier A, Vignoles P, Darde ML. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J. Clin. Microbiol. 2010;48: (4641–4645):PMID: 20881166. DOI: 10.1128/JCM.01152-10. - 61.
Ajzenberg D, Banuls AL, Tibayrenc M, Darde ML. Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. Int. J. Parasitol. 2002;32: (27–38):PMID: 11796120. - 62.
Ferreira Ade M, Vitor RW, Carneiro AC, Brandao GP, Melo MN. Genetic variability of Brazilian Toxoplasma gondii strains detected by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and simple sequence repeat anchored-PCR (SSR-PCR). Infect. Gen. Evol. 2004;4: (131–142). DOI: dx.doi.org/10.1016/j.meegid.2004.03.002 - 63.
Costa JM, Cabaret O, Moukoury S, Bretagne S. Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J. Microbiol. Meth. 2011;86: (357–363). DOI: http://dx.doi.org/10.1016/j.mimet.2011.06.017