I'm doing prevalence study of Toxoplasmosis using both serology (ELISA, Platelia Toxo IgG & Platelia Toxo IgM kit, BioRad USA) and molecular method (PCR). During ELISA assay, my samples were found positive for anti-T. gondii IgG and anti-T. gondii IgM antibodies. But when proceed with PCR, the samples were negative. Is it acceptable or maybe there is something wrong with the technique/kit?
Dear Aisha,
It is acceptable for IgM positive samples to be PCR negative at some rate (~5%). Since PCR is a more specific test, this usually suggests a false positive IgM result. However, if the number of IgM +, PCR - is too high there should be some problem with the second. In this case, it might be related to the DNA isolation method more than to the PCR kit. I recommend you use positive and negative controls in every process (DNA isolation and PCR) in order to assess they are working correctly.
Best regards
I am not an expert in diagnostics but aren't you measuring two different things with the two techniques? From what I understand, you are measuring anti-toxo antibodies (host immune response) by ELISA and the presence of toxoplasma (pathogen) by PCR. So the results don't always have to be concordant.
I understand your concern that the presence of antibodies in the absence of the pathogen does not make sense. However, your inability to detect the pathogen does not mean that it was never there. It could be present at levels lower than the limit of detection (LOD) of PCR (sensitivity of the assay) or you are not using the right sample type (the pathogen might be hiding in some other tissue/sample type)or it could be that the pathogen has been cleared from the body and can't be detected anymore.
If you are unsure your PCR is optimized, try adding appropriate +ve and negative controls to your setup like Luis Antonio Ochoa-Ramírez suggested.
Luis Antonio Ochoa-Ramírez I used positive and negative control in my pcr and both works well. But the samples which positive for IgG totally negative in pcr.
Only samples positive for IgM are candidates for a positive PCR result. A little reminder: IgM is associated with an active response against the pathogen so it means the immune system is currently "fighting" against the pathogen, thus it is present and you should be able to detect by direct means such as PCR. On the other side, IgG is related to memory against the pathogen meaning that a positive IgG result does not imply it is currently infecting the patient. In summary:
IgM+ IgG- = Prime, active infection --> PCR + (most of the time)
IgM- IgG+ = Past, no current infection --> PCR -
IgM+ IgG+ = Secondary, active infection --> PCR + (as in the first case)
Hope this helps,
Cheers
Serology detects antibodies in the sample, it doesn't indicate the current presence of the antigen it self, you should try to use kit for antigen detection. then compare the results to PCR
In samples with IgG positivity the PCR negativity is the most plausible result. In the samples whit IgM positivity the possibility to have a PCR positivity depends on the IgM titre: the lower it is, the greater the chances of having a PCR negative and vice versa. The better situation to have a PCR positivity should be that whit an isolated IgM positivity or associated with IgG having a high avidity
Luis Antonio Ochoa-Ramírez Julie Joseph Oded Babai Antonio La Gioia Can I have any article or paper regarding this? I've found 3 articles that compared between PCR and ELISA method but still not enough. Thanks in advance and your replies are so much appreciated :)
http://egyptianjournal.xyz/33_12.pdf
Article Molecular and Serological Detection of Acute and Latent Toxo...
http://archrazi.areeo.ac.ir/article_109841_9d3eeb9077900c408d7be88c64972034.pdf
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