الفهرس 
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Abstract
Toxoplasmosis is a zoonosis of worldwide distribution. It is caused by Toxoplasma gondii (T. gondii); an obligate intracellular coccidian parasite that infects most warm-blooded animals including birds, humans, domestic and wild animals. It affects up to 50% of the world’s population and approximately three billion humans have antibodies against T. gondii all over the world. The rate of human infection varies from 10% to 90% in different countries, depending on several factors such as environmental conditions, animal species, prevalence in animals and dietary habits. The global incidence of congenital toxoplasmosis (CT) was found to be 190,100 annual cases with an incidence rate of approximately 1.5 cases per 1000 live births. In Egypt, the prevalence of toxoplasmosis among pregnant women was about 42% in Alexandria, 46.5%- 57.6% in Qualyobia, 59.6% in Dakahlia, 45.8% in El Fayoum and was 67.5% in Menoufia.
About one-third of women who acquire infection during pregnancy transmit the infection to fetuses. Early maternal infection (first and second trimester) leads to less frequent transmission but results in severe symptomatology and also spontaneous abortion or intrauterine fetal death. However, late maternal infection (third trimester) leads to more frequent transmission but usually results in more subclinical disease and few cases of congenital infection. About 85% of congenitally infected children with subclinical disease develop signs and symptoms of toxoplasmosis. Features of CT are including chorioretinitis, cataract, blindness, epilepsy, mental retardation, anaemia, thrombocytopenia, encephalitis and pneumonitis. About 10% of congenitally infected infants suffer from severe CT which presents with Sabin’s tetrad; hydrocephalus, convulsions, cerebral calcification and retinochoroiditis.
Immunocompromised patients (AIDS, receiving corticosteroids, malignancies and organ transplant recipients) are at risk of reactivation of latent infection. Toxoplasmic encephalitis is the most common presentation of the reactivation of latent infection in AIDS patients. Pneumonia was reported in up to 5% of advanced cases of AIDS with a mortality rate of 35%. Reactivation of the disease eventually causes death of the patient if left untreated. In bone marrow transplant, toxoplasmosis frequently involves the lung and is associated with a mortality rate of more than 90%. Primary T. gondii infection in immune-compromised patients leads to multi-organ lesions including the CNS and frequently results in death of the patients.
There are four groups of individuals in whom the diagnosis of toxoplasmosis is most critical: pregnant women who acquire the infection during gestation, fetuses and newborns who are congenitally infected, immunocompromised patients, and those with chorioretinitis. The diagnosis of toxoplasmosis constitutes an important measure for the treatment of the disease. It can be achieved by a number of different indirect methods; serological tests and molecular techniques such as polymerase chain reaction (PCR) with its varieties. Serological testing whether for detection of parasite-specific antibodies or antigens are the most widely used method for the diagnosis of toxoplasmosis. The most commonly used serological tests are those detecting T. gondii IgG and IgM, such as indirect haem-agglutination test (IHAT), differential agglutination test, indirect fluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), IgG avidity test and Western blots. IFAT and ELISA are the most widely used assays for the diagnosis of toxoplasmosis.
The tests that measures IgG is used to determine if a person has been infected or not, however it cannot distinguish between current and past infection. Acute infection can be diagnosed by sero-conversion of T. gondii IgG, or its rising titre at three week intervals, and finally by the presence of IgM or IgA antibodies. However, diagnosis of acute infection on the basis of IgM detection is not usually reliable as it persists in many cases for more than one year post infection. The diagnosis of toxoplasmosis by conventional antibody- detecting serological methods may be particularly difficult in patients who are severely immunocompromised. In these patients, the antibody production is decreased due to depletion of Th lymphocytes and deficient B cell maturation and differentiation. So, detection of T. gondii antigen can provide specific diagnosis of acute infection in those patients. Moreover, it can provide an early diagnosis of acute stage during pregnancy, and in high risk neonates.
The major surface antigen (SAG1 or P30) with a molecular weight of 30 KDa is one of the most immunogenic T. gondii antigens. It is considered as an important candidate for the development of diagnostic techniques. This antigen is suitable for use in diagnostic systems for detecting anti SAG1 specific IgG and IgM antibodies. Moreover, it has no cross reactivity with proteins from other microorganisms and is highly conserved in T. gondii strains. One of the most important bio-chemical techniques for detection of T. gondii antigens is ELISA. The simplicity, low cost, easy reading, acceptability and safety of ELISA make it widely used for detection of cancer protein markers, pathogen antigens and/ or antibodies, and other proteins relative to various diseases. There are multiple ELISA formats for antigen detection: direct, indirect, sandwich and competitive ELISA. ELISA assays vary considerably due to different substrate and final signal detection system.
There is a need to improve the sensitivity of the current simple ELISA method for highly sensitive detection of protein biomarkers, which are important for early diagnosis of cancer, neurodegenerative and infectious diseases. The use of nanoparticles (typically in the size range of 1-100 nm) promises to promote in vitro diagnostics to the next level of performance. Quantum dots, super-paramagnetic nanoparticles and gold nanoparticles (AuNPs) are the most promising nanostructures for in vitro diagnostic applications. These nanoparticles can be conjugated to recognition moieties such as antibodies or oligonucleotides for detection of target biomolecules. Nanoparticles have been utilized in immunoassays, immunohistochemistry, DNA diagnostics, bio-separation of specific cell populations, and cellular imaging. Nanoparticle-based diagnostics may open new frontiers for diagnosis of tumors, infectious diseases, bio-terrorism agents, and neurological disorders.
AuNPs have unusual optical properties and high absorption coefficients. A phenomenon called plasmon–plasmon interaction allows the use of AuNPs as labels for colorimetric detection of biomolecules. Furthermore, AuNPs have high atomic number, thus they are readily detectable by electron microscopes or by X-rays. The multivalent surface structure of AuNPs offers the diversity to incorporate multiple therapeutic drugs or biomolecules by covalent or noncovalent conjugation on their surface. AuNPs have found their way among therapeutics in today’s medicine based on the functional moieties and their capabilities in the delivery of nucleic acids, proteins and genes in vivo. Therapeutic research of AuNPs had extended to involve parasitic infectious diseases such as toxoplasmosis. Gold nanorods were conjugated with anti-T. gondii P30 antibody to selectively target the extracellular tachyzoites in infected cell cultures. AuNPs can be conjugated with DNAs, antibodies, enzymes and other bio-molecules, which can afford them promising applications in signal enhancement of bio-chemical detection. They were conjugated to specific antibodies for the development of a microchannel immunoassay that detected Eschericia coli and Helicobacter pylori antigens. In addition, an assay was developed for simultaneous detection of HBV and HCV antibodies in human sera using AuNPs-labelled staphylococcal protein A to amplify the detection signals.
Regarding the diagnostic applications of AuNPs in parasitic infections, mRNA derived from viable purified Cryptosporidium parvum oocysts was detected using oligonucleotide-functionalized AuNPs. Moreover, a study was carried out for detection of Plasmodium falciparum HSP 70 in malaria-infected human blood cultures using AuNPs- based fluorescence immunoassay with promising results. Furthermore, electrodeposited AuNPs were used in a microfluidic system for quantitative detection of IgG antibodies in Chagas’ disease showing good yields. Moreover, a novel electrochemical immuno-sensor based on gold-magnetic nanoparticles and graphene sheets was developed for T. gondii IgM detection resulting in high test sensitivity. Recently, a novel dynamic flow immunochromatographic strip test was constructed using T. gondii antigens and AuNPs functionalized with staphylococcal protein A and showed good results.
Recently, a highly sensitive protein detection method based on nanoparticles and ELISA “nano-ELISA” was developed, in which the antibody-conjugated AuNPs are used to detect a cancer protein marker “P53” with very promising results. This method combines the nanotechnology with the classic sandwich ELISA. The detector antibody mixed with HRP at a certain ratio was immobilized on the AuNPs to form Au nano probes for signal amplification. The promising yields due to application of nano assays in the diagnosis of cancer and parasitic infectious diseases, gave a challenge to use AuNPs in the diagnosis of experimental toxoplasmosis. In this context, the current work was designed to apply nano-ELISA for early diagnosis of acute Toxoplasma infection, particularly in high risk groups; immunocompromised animals, pregnant mice and their pups through detection of T. gondii SAG1antigen in their sera. Nano-ELISA was further compared to a modified form of sandwich ELISA “capture ELISA”.
In fulfillment of this aim, 216 laboratory-bred Swiss Albino mice were used. 18 mice served as control group (group I), this group was equally subdivided into three subgroups; subgroup I-a (non-infected, non-pregnant, immunocompetent mice), subgroup I-b (non-infected, non-pregnant, immunosuppressed mice) in which animals were intraperitoneally (IP) injected twice with cyclophosphamide seven days apart, and subgroup I-c (non-infected, pregnant, immunocompetent mice and their offsprings). 198 mice served as Experimental group (Group II), this group was subdivided into three main subgroups; subgroup II-a (Infected, non-pregnant, immunocompetent subgroup): It involved 66 mice and it was further subdivided into two subgroups: subgroup II-a1 in which 30 mice were infected IP with the virulent T. gondii RH HXGPRT (-) strain in a dose of 2 x 102 tachyzoites/ mouse and subgroup II-a2 in which 36 mice were orally infected with the avirulent T. gondii Me49 strain in a dose of ten cysts / mouse. Subgroup II-b (Infected, non-pregnant, immunosuppressed subgroup) involved 66 mice that were immunosuppressed as in control subgroup I-b, these mice were further subdivided into two subgroups: Subgroup II-b1 in which 30 mice were infected with T. gondii virulent strain as in subgroup II-a1, but two days after the second cyclophosphamide dose and subgroup II-b2 in which 36 mice were infected with T. gondii avirulent strain as in subgroup II-a2, but two days after the second cyclophosphamide dose. Six mice from each subgroup were anesthetized then sacrificed on the following dates: day zero (the day of infection), the first, second, seventh and 14th day post infection (PI) and a blood sample was collected from each mouse. The remainder six mice of subgroups II-a2 and II-b2 were kept up to 60 days PI and then they were sacrificed for detection of T. gondii brain cysts as a proof of infection. Subgroup II-c (Infected, pregnant, immunocompetent subgroup) included 66 mice which were subdivided into two subgroups; subgroup II-c1 in which 30 pregnant mice were IP infected with T. gondii virulent strain on 10-14th day of gestation by the same dose as in subgroup II-a1. Subgroup II-c2 included 36 pregnant mice that were infected with T. gondii avirulent strain by the same dose as in subgroup II-a2 on the 10-14th day of gestation. Six mice from each subgroup were anesthetized and sacrificed on day zero, the first, the second, the seventh day PI (on delivery) and on the 14th day PI (the seventh day post-delivery). A blood sample was collected from each mother mouse and from their living pups (on delivery). The remainder six mothers of subgroup II-c2 were kept after delivery up to 60 days PI, and then they were sacrificed for parasitological confirmation of the infection. The control animals were sacrificed simultaneously with their corresponding experimental groups. Sera were separated to be tested for T. gondii SAG1 antigen by both capture and nano-ELISA techniques.
AuNPs were prepared and then conjugated with T. gondii SAG1 antibodies and horseradish peroxidase (HRP) molecules to form Au nano probes. Both AuNPs and Au nano probes were subjected to four characterization methods; the morphological study by TEM, the spectroscopic analysis using an UV-vis spectrophotometer, the particle size and size distribution analysis by particle size analyzer and the structural analysis using X ray diffractometer (XRD). A modified sandwich ELISA (capture ELISA) was done for detection of T. gondii SAG1 in the sera of both experimental and control animal subgroups after preparation Toxoplasma antigen, negative and positive control samples and performance of checkerboard titration. Nano-ELISA was performed the same as capture ELISA but using the Au nano probes instead of T. gondii SAG1 antibody-HRP conjugate.
In the present study, the following results were recoded; T. gondii tachyzoites were detected microscopically in the peritoneal exudate of subgroups II-a1, II-b1 and II-c1 (T. gondii virulent strain-infected animals) that were sacrificed on days seven and 14 PI. While Toxoplasma brain cysts were detected microscopically in subgroups II-a2, II-b2 and II-c2 (T. gondii avirulent strain-infected mice) which were sacrificed 60 days PI. TEM images revealed that both AuNPs and Au nano probes were spherical, distinct and regular in shape with smooth surface. Moreover, Au nano probes showed a contrast in the electron density between the dark spherical core of AuNPs and the surrounding lighter shell of the immobilized antibodies and HRP molecules. The optical spectra of AuNPs and Au nano probes showed a broadening and a red shift of the absorption peak from 512 nm up to 592 nm indicating the AuNPs- antibody- HRP conjugation. The average size recorded by the particle size analyzer was 24.7 nm for AuNPs and 42.3 nm for Au nano probes with quit narrow size distribution of both. XRD patterns of either AuNPs or Au nano probes were corresponding to the pure metallic gold with monophasic nature of nanoparticles. The cut off value (COV) of ELISA was estimated to be 0.075. There were no false positive results in all control subgroups neither by capture nor by nano-ELISA formats (100% specificity).
Both capture and nano-ELISA had the advantage of less reaction steps than that of conventional type, in turn both of them could be considered as time-saving techniques. Furthermore, the high sensitivity records (up to 100%) for nano-ELISA were similar to that of the capture assay on days seven and 14 PI in all experimental subgroups. Thus, both assays could be considered valuable in the diagnosis of toxoplasmosis in these durations, in comparison to other serological tests in which antibody detection is not reliable particularly in immunosuppressive conditions. Additionally, nano-ELISA had reported an early diagnosis with a high sensitivity (up to 100%) in all studied infected animal subgroups whether immunocompetent, immunosuppressed or pregnant. Moreover, it showed a significantly higher sensitivity than the capture format in the antigen detection among high risk pups of mothers either infected by virulent or avirulent strains. This finding foreshadows the value of such sensitive technique in the diagnosis of CT.
In conclusion, nano-ELISA is a promising sensitive method for an early and specific diagnosis of acute phase of toxoplasmosis in animal model especially when the immunity is suppressed. It is also of great value in early and accurate diagnosis of active toxoplasmosis in pregnant females. It is further valuable to exclude or verify foetal infection, as it could prompt an early decision for the proper treatment reducing the infection sequalae. Moreover, this assay would be reliable in the detection of T. gondii antigen in the amniotic fluid to diagnose intrauterine infection and in the aqueous humor for the diagnosis of toxoplasmic chorioretinitis. Thereby, nano-ELISA could be an addition to the available methods for the diagnosis of acute acquired toxoplasmosis and CT as well.