Trypanosoma cruzi

Judging by the number of blog entries I have dedicated to what I am doing in the lab one would think I really never do anything there. This would be what we call, “incorrect.” It is true that recently much of my time previously devoted to pure technical work has been taken over by helping plan and coordinate our upcoming project on Chagas/leishmaniasis in the oriente but we have still been progressing in the lab. One of the main reasons I haven’t posted as much is that in the monthly Fulbright updates I write to my program coordinators I discuss in-depth all things concerning the lab – however, I’ve been writing them in Spanish since month 3 and this wouldn’t help out the majority of people who actually read this thing. So, this entry is an attempt balance things out.

We recently procured a live Trypanosoma cruzi culture; this is the parasite responsible for Chagas disease for those of you who didn’t know. We have been growing it for several weeks know and boy have all those little parasites been asexually reproducing like mad!




(Parasite video, 40X power with zoom from my camera added in. Watch ‘em squirm! This is a good sign, growing well.)


Every couple of days we withdraw 80% of the medium (and parasites), which we spin out with a centrifuge and store the resultant pellet. The remaining sample has more medium added (medium is basically a super parasite food) and is placed back in the incubator at 28-30°C to continue growing. Now, why are we building up a huge stock of parasite bodies? So we can later use (after a series of more complicated steps that I won’t go into here) key T. cruzi surface proteins in an in-house ELISA procedure to test for the antibodies against the parasite (thus determining if a person is infected – although confirmatory tests are always carried out). This in-house ELISA, when dialed in, can be more effective and cost waaaay less than buying commercial kits.




(Manuel withdrawing parasites for examination under the microscope. Based on their shape and motility we can determine which phase of growth they are in and if it is the right time to harvest them. Note the small alcohol burner; everything must be passed through the flame to avoid contamination.)


Another in-house test we are preparing (once again using the parasites) is involves a process known as immunofluorescence. The test is actually quite straightforward and involves putting a known quantity (I will explain in a bit how we determined this) of parasites in a small well of a modified microscope slide. They are allowed to dry on the slide overnight and stored until use at -20°C. To run the test a small quantity of an individual’s serum is placed on the well and left for a time. If they have been infected with T. cruzi they will have antibodies to the parasite, which will bind to specific proteins on the parasites previously bound to the slide. A specific conjugate is then added which binds to the other end of the antibody and the slides are then washed, removing excess conjugate. The conjugate contains a compound that fluoresces under UV light and this can be observed with a fluorescence microscope. Thus, if fluorescence is observed it indicates T. cruzi infection. At this point we have only reached the prepared slide step; I will explain how we determined the parasite concentration.




(This tiny device, known as a Chamber of Neubauer, lets us count parasites. A drop of medium filled with parasites is placed in the middle of one of the metal looking middle sections and a cover slip is applied. The contraption is then placed under the microscope.)




(This is what you see at 20X, we used 40X to count. Zoom in to see the parasites. Each square surrounded by 3 lines is counted, there are 25 in total (a 5x5 square), which are all screened and then totaled. This box of 25 squares corresponds to a volume of 1mm x 1mm x 0.1mm and using this volume and the number of parasites we can calculate our parasite concentration per milliliter. One wants around 1-2 million/milliliter in order to have a sufficient number to make a useful slide for the immunofluorescence test.)




(Me counting parasites, oh yeah!)


During free time Theo (a lab compatriot) and I like practicing various techniques useful for working with infectious diseases. We’ve done a fair amount of blood draws after which we’ve centrifuged our own blood to get the serum, which we have used as negative controls in several Chagas tests in the lab! We’ve also learned how to do a finger prick and use the drop of blood to make a blood smear on a glass slide and then stain it. This may sound easy, and it is pretty easy, but there is a great trick Manuel showed us for getting just the right thickness. Too thin and you don’t have enough to examine, too thick and the red blood cells (RBCs) pile up making it easy to miss anything abnormal. This basic test can detect acute stage Chagas disease and malaria among others.




(Some of our blood (stained). The numerous cells are RBCs and the big purple guy is a white blood cell or WBC. This one happens to be an eosinophil, which if detected in elevated levels can indicate a parasitic worm infection, such as gnathostomiasis. The really purple part is a multi-lobed nucleus while the little reddish granules contain powerful enzymes.)