Method For Diagnosis Of Toxoplasmosis Research Articles

Development of a LAMP method with lateral flow DNA chromatography to diagnose toxoplasmosis in immunocompromised patients

BackgroundRapid and accurate diagnosis of toxoplasmosis is critical, particularly for immunocompromised patients. Several molecular methods could have value for toxoplasmosis diagnosis, but often require sophisticated and expensive equipment, and as such are impractical for use in resource-limited countries. Our study aimed to develop a new rapid diagnostic test for toxoplasmosis that can be used in developed countries as well as low- or middle-income countries.MethodsCommon primers for conventional loop-mediated isothermal amplification (LAMP) and the new LAMP DNA chromatography method were designed based on a 529-bp repeat present in Toxoplasma gondii genomic DNA. A total of 91 clinical samples from 44 patients suspected of having toxoplasmosis who were treated at several hospitals across Japan were tested using the new LAMP DNA chromatography method, conventional LAMP, and nested PCR and the sensitivity and specificity of the methods was compared.ResultsThe LAMP DNA chromatography method showed better sensitivity and specificity (68.2% and 100%, respectively) compared with the nested PCR (45.4% and 100%, respectively) and conventional LAMP (63.6% and 100%, respectively) methods for diagnosis of toxoplasmosis in immunocompromised patients. LAMP DNA chromatography also has better sensitivity and specificity (75% and 100%, respectively) than nested PCR (50.0% and 93.5%, respectively) and conventional LAMP (62.5% and 100%, respectively) to diagnose toxoplasma encephalitis using CSF samples.ConclusionWe developed a LAMP DNA chromatography method to detect T. gondii DNA in clinical samples. This method also successfully detected T. gondii DNA in CSF from patients with toxoplasma encephalitis. This newly developed method can be a valuable rapid diagnostic test for toxoplasmosis in a range of settings, including resource-limited areas like those in low- or middle-income countries.

Immune blotting as a method for diagnosis of toxoplasmosis

Test kit for detection of IgG-antibodies to individual antigens of Toxoplasma gondii by immune blotting («Western blot» format) has been developed. Laboratory testing with first international WHO standard «Anti-toxoplasma serum (IgG), human, Lyophilized, 20 IU / ampoule» (NIBSC, Great Britain) demonstrated the analytical sensitivity of the new kit equal to 10 IU / ml. Study of the diagnostic efficiency of the new kit showed its high sensitivity, equal to 98.51 - 100%, and high specificity, equal to 99.5 - 100%. New test kit is intended for confirmatory testing in laboratory diagnostics of toxoplasmosis.

Detection of Toxoplasma gondii by Loop-mediated Isothermal Amplification in Blood and Urine Samples from Women in Saudi Arabia

Introduction: Toxoplasmosis is the disease which is caused by the protozoan parasite Toxoplasma gondii (T. gondii), which has the ability to infect all warm-blooded animals. Several molecular techniques for the diagnosis of Toxoplasma gondii includes normal and Real Time Polymerase Chain Reaction (RT-PCR), and gene sequencing. Aim: To apply Loop-Mediated Isothermal Amplification (LAMP) for the diagnosis of T. gondii infections in urine and blood samples from female population in Saudi Arabia. Materials and Methods: In this cross-sectional study, genomic DNA was extracted from 22 blood and urine samples (11 each), two LAMP assays based on B1 and Surface Antigen 2 (SAG2) genes of T. gondii was performed. Conventional PCR was done for the LAMP product followed by sequencing to confirm the specificity of LAMP method. Statistical Package for the Social Sciences (SPSS) version 20.0 was used to summarise continuous and categorical variables. Results: From 22 samples, 17 samples were LAMP positive in both urine and blood samples, four were negative in blood and positive in urine samples, and one was positive in blood and negative in urine. Sequencing of PCR product confirm the specificity of the method used. Conclusion: LAMP detection of T. gondii DNA is an appropriate, sensitive and specific method for diagnosis of toxoplasmosis from urine and blood specimens in humans.

Serological Diagnosis of Toxoplasmosis in Household Cats in Egypt

im: the aim of this study is to use serological diagnosis for rapid detection of Toxoplasma gondii infection among household cats. Material and Methods: 212 serum samples collected from household cats of different sex, breeds and age groups in urban and rural area of Giza Province. Samples were checked by rapid chromatographic immune assay (IC) for detection of IgM and IgG antibodies against Toxoplasma gondii. Statistical analysis of the obtained results were carried out using Chi-square test to study the effect of sex, breed and age on the results. Results: the IgM antibodies against Toxoplasma gondii were detected in 19 cats (8.96%), and IgG antibodies were detected in 63 cats (29.71%), the overall positive were 82 cats (38.67), the sero prevalence against Toxoplasma gondii was high in Egyptian Mau breed and in cats of over one year age. Statistical evaluation of the results proved that there were significant differences between different sex, different cat breeds and different age groups. Conclusion: This study concluded that the serological diagnosis by IC for detecting IgM and IgG against Toxoplasma gondii, is a rapid, accurate method for diagnosis of Toxoplasmosis

Toxoplasmosis in immunocompromised patients in Iran: a systematic review and meta-analysis.

Although toxoplasmosis in immunocompetent individuals is generally asymptomatic, in immunocompromised patients (HIV/AIDS, cancer, and transplant patients), it can lead to serious pathological effects. This study included a systematic review and meta-analysis to comprehensively assess the seroprevalence rate of Toxoplasma infection in immunocompromised patients in Iran. Electronic English and Persian databases (PubMed, Google Scholar, ScienceDirect, Scopus, Magiran, Scientific Information Database [SID], IranMedex, and IranDoc), parasitology congresses, and projects and theses of Iranian medical universities, were systematically searched from 1997 to 2013 (published or unpublished data). In this paper, several studies that used serological methods for diagnosis of toxoplasmosis were selected. Analysis of seroprevalence estimates was pooled using a random-effects meta-analysis. Twenty-two studies, comprising 2,805 individuals, were included in the meta-analysis. Overall seroprevalence rate of Toxoplasma infection in Iranian immunocompromised patients was 50.01% (95% confidence interval, 43.85 to 56.17); however, there was significant heterogeneity among study groups. The results showed that seroprevalence rate of toxoplasmosis among transplant recipients, HIV/AIDS, and cancer patients in Iran was 55.1%, 50.05%, and 45.06%, respectively. In addition, IgM seroprevalence rate was estimated to be 4.85% (95% confidence interval, 2.22 to 8.41). This systematic review and meta-analysis identified a high seroprevalence rate of Toxoplasma infection among immunocompromised patients (50%). Consideration of management, design and provision of appropriate control measures of toxoplasmosis is highly recommended.

Comparative Study Between Serological and Molecular Methods for Diagnosis of Toxoplasmosis in Women and Small Ruminants in Egypt

Routine serological diagnosis of toxoplasmosis provides high sensitivity, but specificity varies depending on the test used; false-positive results (IgM) have been reported. Blood samples were collected from 88 women (59 pregnant and 29 nonpregnant) and 86 contact animals (62 sheep and 24 goats) at El Fayoum Governorate during the period from October 2005 to December 2006. All collected samples were tested for Toxoplasma gondii infection by serological tests (ELISA IgM & IgG and Sabin-Feldman dye test) and polymerase chain reaction (PCR). Results revealed specific IgG in 45.8% and 41.4%, IgM in 30.5% and 24.2%, and positive Sabin-Feldman dye test in 23.7% and 17.2% in pregnant and nonpregnant women, respectively. Positive PCR products were detected in 32.2% and 27.6% in pregnant and nonpregnant women, respectively. Regarding animals, positive ELISA IgG and PCR were detected in 98.4% and 67.7% of sheep and 41.7% and 25.0% of goats, respectively. It was concluded that serological tests can detect higher rate of toxoplasmosis than PCR, so ELISA combined with the PCR technique is a recommended tool for accurate diagnosis of toxoplasmosis.

Schizophrenia and Serological Methods for Diagnosis of Toxoplasmosis

Schizophrenia and Serological Methods for Diagnosis of Toxoplasmosis

Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.

We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.