Answer to Case 630

 Answer to the Parasite Case of the Week 630:  nematode larvae; primary differential is Strongyloides sp., hookworm, Trichostrongylus, and free-living nematodes (e.g., Rhabditis sp.). 

The agar plate culture (a.k.a., Koga plate) is a relatively safe and straight-forward method to increase detection of S. stercoralis in feces, and can also be used to culture other nematodes by allowing the eggs in feces to hatch and mature. The procedure is performed by placing a small amount of stool (as shown here) in the center of a nutrient agar. Any agar that supports the growth of enteric bacteria will do, including sheep blood agar and Mueller Hinton agar. We make our own in-house agar using beef broth. If larvae are present in the specimen, they will move over the agar and carry bacteria from the feces with them. The bacteria grow in their tracks, leaving a visible trail of their journeys. Our technologists examine the plates daily for these serpiginous tracks of bacteria. If they are seen, they examine the plate under a light microscope (4x or 10x objective) to look for larvae. HERE is a link to the publication by Dr. Koga detailing the modified agar culture method that we use in our lab.

This case didn't have very pronounced bacterial tracks. However, you can see great examples in some of my previous cases:

Case of the Week 110 and Answer (S. stercoralis in sputum; microscopic track on our in-house agar)

Case of the Week 112 and Answer (Hookworm in a duodenal aspirate; very clear track on our in-house agar)

Case of the Week 137 and Answer (S. stercoralis tracks on sheep blood agar; this was an incidental finding on bacterial stool culture and shell vial viral culture)

Case of the Week 151 and Answer (video of S. stercoralis larvae and tracks on our in-house agar)

Case of the Week 218 and Answer (video of S. stercoralis larvae in agar)

Case of the Week 337 and Answer (S. stercoralis; incidental finding on bacterial stool culture: sheep blood agar and McConkey agar)

Wow, I didn't realize how many cases I've posted of this over the years!

As Marc mentions, the next step is to examine the larvae to identify the genus present, since other nematode eggs in the stool can hatch over a period of days and produce similar-appearing larvae. Free-living larvae may also be present if the fecal specimen was contaminated with soil (which would be unlikely in this case). 

To safely examine the larvae, we flood the plate with formalin and let it sit for several minutes. We then carefully remove some of the peripheral material on the plate and place it on a slide to examine microscopically. 

This case was particularly remarkable because many of the larvae had matured to be free-living Strongyloides adults. We saw both males and gravid females! You can actually see them on the agar itself, which is really impressive. We suspect that the stool was not submitted immediately after passage by the patient, thus explaining the presence of these advanced forms. 

Gravid free-living females:

Male

Adults seen on the plate:

The last question to answer is whether these are S. stercoralis or S. fuelleborni - but that is a subject for a future post!