Hi Hayder,

Overall, it is very difficult to obtain a positive result of pathogen amplification from the blood samples. Simply it is hard to find something small in four to six liters of blood. You have to have blood sample collected in parasitemia and moreover, blood samples contain many inhibitors. It is not clear if you extracted DNA from whole blood or from serum only. And also if you used any control of amplification and which genes were targeted. These information maybe could other researchers help you with you issue.

Regards,

marcela

Dear Marcela,

Thanks for reply. Actually, I was thinking the same concerning the low possibility of producing the parasite from 1 or 2 ml of whole blood but I found many papers following that. The genes I have used are SAG2, GR5 and GR6.

yet, we don't have a standard strain as a positive control

I think that you should start with searching someone who is willing to provide you a positive control (Toxoplasma DNA). Than you can be sure about the annealing temperature of primers. 

Although there can be a transient parasitaemia in a Toxoplasma infection, the organism's DNA will not necessarily be present in the peripheral blood. In fact, usually not, probably. Toxoplasma is primarily a tissue (non-blood) parasite, for all practical purposes. Furthermore, a positive IgG (assuming that the result is not falsely positive, which it can be) merely indicates that there has been "contact" with Toxoplasma (meaning, classically, ingestion of tissue cysts or oocysts). The infection is either chronic, or Toxoplasma is not present in the body anymore, despite the positive IgG. As for IgM, the test can sometimes, if not frequently, remain positive (in toxoplasmosis) for a long time, e.g. for more than a year. So it is not necessarily indicative of a recent infection. In pregnant women and immunocompromised individuals, interpretation of a positive toxoplasmic IgM result is not straightforward and additional tests can be done, depending on the laboratory facilities available [Markus, M.B. 2003. Toxoplasma gondii. In: International Handbook of Foodborne Pathogens (ed. M.D. Miliotis & J.W. Bier); Marcel Dekker Inc., New York]. But that is another story. Failure to detect Toxoplasma in your patients via PCR using blood samples is exactly the result that I would expect. It is more likely that Toxoplasma would be picked up in lymphatic tissue, for instance.

Dear Miles,

Thanks for reply. I agree with your opinion.

All samples we have are of recent Miscarriage and a history of toxoplasmosis. Given that other researchers succeeded to have a positive PCR with a pirepheral blood samples but only with IgM ELISA.

the puzzle here is many papers referring to simple "peripheral blood sampling" in EDTA tubes for DNA isolation & successful PCR products!

Thanks, Hayder. I didn’t know that the patients were women who had miscarried and not random toxoplasmic immunological positives (on statistical grounds, one would expect to normally get a negative PCR result in respect of the latter). Note that there can be false-positive toxoplasmic IgMs in pregnancy. But you are absolutely right. PCR using blood can indeed “work” in such IgG-positive or IgM-positive patients. Often it doesn’t, however. In one such study that comes to mind, PCR involving blood was successful 19% of the time. It varies. An example of a study concerning a different kind of patient, where PCR was not satisfactory, is the following: Bourdin, C. et al. 2014. PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis. Journal of Clinical Microbiology 52 (11): 3987-3991. It was concluded that “... PCR detection of Toxoplasma gondii in blood samples cannot currently be considered a sufficient tool for the diagnosis of OT [ocular toxoplasmosis], and ocular sampling remains necessary for the diagnosis of OT”. I think you are aware of these things.

Dear Hayder

I found that some investigators showed that SAG1-based PCR approach can be used for early diagnosis of toxoplasmosis using peripheral blood.

You can take a look at this review:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451882/pdf/13071_2015_Article_902.pdf

Good luck

The bottom line is that in suspected toxoplasmosis, a negative PCR result using blood is not evidence that Toxoplasma not present in the body. If the infection is acute, such as in toxoplasmic encephalitis, the chances can be greater that a positive PCR result will be obtained. If PCR is done in early toxoplasmosis at the time when there is a parasitaemia (I mentioned the parasitaemia previously), the result can be positive.

I agree with you Miles....

PCR of blood from patients with toxoplasmosis is likely to be valuable if the infection is associated with a disseminated spread of the parasite or severe encephalitis, which is not common since most cases result from local reactivation of latent brain cysts.

Dear Miles,

Thanks for your reply. Just to add here that our samples are limited to women who have spontaneous miscarriage.

Hence, I actually going to the same conclusion that the parasitaemia is the key, whenever the IgM or IgG is seropositive, it is still not a guarantee of actual existence parasites in the circulation (although IgM is much recommended for early infection). Some papers refer to peripheral blood PCR diagnosis but with low percentage of positive results comparing to serology- Gashout, et al. 2016, still not impossible tho. 

Dear Harith,

Thanks for reply and it is nice to hear from you here!

Primers we have chose wasn't arbitrary as they considered universal and conservative for Toxoplasma gondii.

Hi,

This could be a simple suggestion but i think better to look in to it.

Does your PCR reaction is working fine?

I know that you are not having the positive control to check. But can you do any other PCR (standardized and working well in your lab) along with your Toxoplasma PCR preferably using the same PCR reaction components.

Hi.

The bradizoite  of parasite found in the tissue , in acute stage can you found  the parasite in the blood .check your sample.

Hi

me too have this problem

I have tested 8 patient blood by pcr assay, using GrA6 gene which they had IgM psoitive eliza before but I have no amplification.