Cryptococcus neoformans -- samples and results

Cryptococcus neoformans outbreak in a rural hsopital/clinic

We used a non-pathogenic Sacchromyces cereviseae to spike any positive samples. When this group requested to do an india ink stain for capsule, we provided them with fixed Cryptococcus neoformans as a sputum sample. Any encapsulated yeast would be useable.

For all student designed experiments, we tried to encourage students in the right direction. For example, groups were discouraged from testing four very similar organisms for the Western blot and we encouraged them to be as specific as possible (about antigens, samples, etc.) when designing the ELISA.

Cultured blood and sputum samples were positive; urine was negative. C. neoformans requires two days to culture. Therefore, plates were negative on day one, but were given back to the students a day later with yeast colonies present. They gram stained these colonies and saw large yeast cells. This provided an oppotunity to discuss possible explanations of this result with the entire class. Due to the time constraints of this course, we chose to give the students stool samples already streaked on LB agar plates, rather than simulate samples for them to plate from. This plate contained only E. coli in the interest of simplicity and time. However, for a longer course, it would be possible to give more normal flora species in this sample, allowing for practice in distinguishing mixed cultures.

For all of the students' ouchterlony experiments, we used the reagents provided in a Carolina Biologicals' ouchterlony kit. However, samples were relabeld as fungal, viral and bacterial extracts and a serum sample from one case patient. In this case, a positive reaction from the patient was seen only for the "fungal extract." This experiment was used more to get across the concept of antibody-antigen reactions and to prepare them for the Western blot and ELISA experiments than to help them distinguish their particular outbreak. However, some groups did use this experiment as further confirmation of their results.

This group did a viral plaque assay since only normal flora was seen on plates. The plaque assay was negative.

For the Western blot, students were asked to give us four candidate organisms against which they wished to test one patient serum. In the interest of time, we explained the concept of the Western blot to the students and then ran the gel and transferred it for them. Students were only involved in the probing of these blots. Blots can be done with any readily available samples and antibodies that will provide the appropriate pattern of positive and negative results. Additionally, groups that named two or more closely related organisms did get back false positives from cross-reacting antigens.

At the point in time in which we did the ELISA, the students understood basic immunology and their own cases well enough that we allowed them to design their own experiments. Samples, antibodies, etc. were obtained from a Carolina Biologicals ELISA kit and positive or negative samples were simply labelled appropriately for each group. Most groups used this to confirm organisms they were almost certain were causing their outbreak. Students were pushed to focus on including appropriate controls in their design of this experiment. THIS EXERCISE REQUIRES EXTENSIVE PRE-LAB PREPARATION FOR THE INSTRUCTOR.