Answer to Case 558

Answer to Parasite Case of the Week 558: Plasmodium falciparum malaria, >10% parasitemia. NEGATIVE rapid antigen.

So why is the rapid antigen test negative???

As noted by our readers, there are many possible reasons for a positive blood smear and negative rapid malaria antigen test (RDT). Here are our options, along with the reasons why each is or isn't a likely explanation in this case:

1. This is babesiosis, and not malaria. This is a very important consideration given the morphologic similarities between Babesia spp. and Plasmodium falciparum. However, the moprhologic features in this case are highly consistent with P. falciparum, including the presence of relatively-homogenous rings, without the size and shape pleomorphism usually seen with Babesia spp. There are also applique forms and headphone forms (arrows, below) which are characteristic, but not definitive, for P. falciparum infection. There may also be a hint of hemazoin (malaria pigment), but it is not obvious. Overall, we can rule out babesiosis based on the microscopic morphology.
   
2. The negative RDT is due to deletion of the P. falciparum histidine rich protein II repeat region in the parasite infecting this patient. This deletion has been reported in some African and South American countries, including Kenya where this patient had recently traveled. While this is a good thought, it would not explain why the pan-malaria antigen band (in this case, aldolase) is also absent, resulting in a completely negative RDT result. Thus, we can also exclude this as the reason for the negative RDT. 
3. This is the well-described prozone (or 'hook') phenomenon, where antigen excess (seen in cases like this with high parasitemia) binds to both the capture and detection antibodies and interferes with the formation of an antibody-antigen-antibody 'sandwich'. (You can refresh your memory on how a lateral flow immunoassay works by reading this fairly well-written Wikipedia article on malaria RDTs HERE). Prozone effect is well-described for malaria RDTs. You can read a nice study on how often this might occur HERE. In my mind, this is the most likely cause of the false negative result seen in this case.
4. The kit is faulty, or the test was not performed correctly. We unfortunately can't rule this out.

What additional testing would help us sort out the cause of the false negative RDT?

As some of you mentioned, confirmatory testing by another method would be useful to prove this was indeed malaria. Fortunately, we were able to perform PCR and it was strongly positive for P. falciparum, thus confirming our microscopic impression. To evaluate for possible RDT prozone phenomenon, we could also test serial dilutions of the fresh blood specimen to see if specimens with diluted antigen become positive. Repeat RDT testing would confirm that the test was performed correctly to begin with. Finally, we could easily confirm that the test kit isn't expired by checking the box that was used, and checking the storage conditions to ensure that they were appropriate for the test kits.

While we don't have a definitive answer for this case, I thought it was an excellent opportunity to discuss how the prozone phenomenon is a real risk in cases of high malaria parasitemia and may result in false negative results. This is one reason why malaria RDT results should always be followed gold standard blood film examination (or PCR). Also, we always need to remember that no lab test is perfect, and must always be interpreted in the context of the individual patient. 

Thank you all for the excellent comments and discussion on this case!