سورة الرعد ايه ( 8 )

1. {اللَّهُ يَعْلَمُ مَا تَحْمِلُ كُلُّ أُنثَى وَمَا تَغِيضُ الأَرْحَامُوَمَا تَزْدَادُ وَكُلُّ شَيْءٍ عِندَهُ بِمِقْدَارٍ} سورة الرعد ايه ( 8 )

2. Serological and Molecular Comparative Diagnosis of Toxoplasmosis and infection with Rubella virus in aborted women in Al-Najaf province

3. Introduction Maternal infections play a critical role in pregnancy wastage and their occurrence in patients with bad obstetric history (BOH) is a significant factor. . The rate of spontaneous abortion from fetal infection by the infectious agents like TORCH group (Toxoplasma gondii , Rubella virus, Cytomegalovirus and Herpes Simplex virus) and others is ranged from 10 -15 % . Congenital intrauterine infections have been associated with congenital abnormalities, intrauterine growth retardation and intrauterine death of the fetus, as well as late sequelae such as developmental delay, blindness and deafness of the infected child.

4. Rubella is a common childhood infection usually with minimal systemic upset although transient arthropathy may occur in adults. Serious . Acquired ( not congenital) Rubella is transmitted via airborne droplet emission from the upper respiratory tract of active cases (can be passed along by the breath of people sick from Rubella .

5. Rubella virus specific IgM antibodies are present in people recently infected by Rubella virus but these antibodies can persist for over a year and a positive test result needs to be interpreted with caution. This review focuses on the application of Real-Time PCR in the clinical microbiology laboratory.

6. Conventional methods for the detection of antibodies to Toxoplasma and rubella include immunofluorescence assay (IFA) , enzyme immunoassay (EIA), and enzyme-linked fluorescent assay (ELFA) , these techniques have been used for years in both diagnostic and screening protocols for (TORCH) infection and have demonstrated reliable performance

7. Recently, Real Time- PCR technology emerged as a novel approach to assess the molecular response to various infectious diseases. This technology is similar to traditional PCR but allows for the simultaneous detection and identification of multiple samples

8. Objectives of study

9. 1.Detection of relationship between toxoplasmosis and certain types of viral infection (cross reaction or false positive for pregnant women ). 2.Diagnosis of acute TORCH infection in pregnant women by demonstration of specific IgM. 3. Screening for IgM and IgG antibodies against TORCH complex in a group of patients with bad obstetric history . 4. Real Time- PCR technique to confirm of positive cases of TORCH.

10. Materials and Method

11. 57 blood sample ( cases) divided into 2ml blood in EDTA tube and 3ml in plane tube to separate serum Negative Postive Serological diagnosis by ELISA test of serum sample to each TORCH infection IgM and IgG Molecular diagnosis to each sample TORCH infection by extraction DNA Extraction Each sample( TO ,CMV and herpes ) RT-PCRfor each sample DNA or RNA RNA Extraction Each sample (Rubella ) Excluded

12. Results Results

13. Table (1): Frequency of women with positive Toxoplasma antibodies detected by ELISA in the study sample (n=57).

14. Table (2): Frequency of women with positive Rubella antibodies detected by ELISA in the study sample (n=57) .

15. Serological and Molecularresult of TORCH Table (1): Proportion of women (N=57) with positive IgM, IgG antibodies in comparison with RT-PCR positivity . X² = 5.375 df= 6 P= 0.496

16. Table (2) :frequency of mixed infection of Toxoplasmosis and Rubella by ELISA X² = 3.67 df=3 P= 0.299

17. Table (7): Seropositive IgG for Toxoplasma and Rubella virus IgG Cross tabulation. X² = 0.026 df=1 P= 0.585

18. Table (10) : PCR Result for Toxoplasmosis and Rubella virus X² = 1.427 df=1 P= 0.296

19. Toxoplasma IgM positive Toxoplasma IgM negative RT-PCR Toxo. positive Fig.( 1):Comparative seropositive IgM from Toxoplasma with RT-PCR Result of Toxoplasma

20. JOE positive control FAM positive samples Threshold Cycle Fig.(2): Detection of Toxoplasma gondii by Real Time PCR instrument (Stratagene for Agilent 3005 ) (amplification plots positive )

21. Fig. (3): Detection of Rubella virus by Real Time PCR instrument (Bioneer ) • (amplification Curve) Positive control Positive samples Threshold cycle Samples with control negative

23. Conclusions 1- The accurate diagnosis of TORCH infection should be done by molecular method . 2- No role of treatment for mixed TORCH infection in pregnant ladies unless confirmed by RT-PCR . 3- Low prevalence of Rubella IgG in Iraq is mainly due to default of vaccination program that concentration on secondary school girls only . 4. ELISA test should be used as screening test . 5- There is no mixed RT-PCR results of TORCH in contrary to ELISA test which reflect the specificity and sensitivity of RT- PCR and in same time reflect the cross reaction and false result of ELISA technique .

24. Recommendations 1.Advice the gynecologist to take care before give management for pregnant women that present with more than one infection at same time unless confirm the diagnosis by more than one technique . 2- Rubella virus IgG should be included in the listed tests for pre married girls in order to give vaccine to negative one . 3. Pre pregnant screening for antibodies to T. gondii, rubella, CMV and herpes simplex virus type 1 (HSV-1) and HSV-2 are recommended to be done as a routine practice that prevent fetus from infection by TORCH in uterus. 4- The type of sample detection should be according to shedding of microorganisms to enhance more perfect test .

25. مرحبا بالضيوف الكرام Thank You 29/8/2014Thikra