Monday, December 17, 2018

Case of the Week 523

I have a bit of a puzzle for you this week. The following are transmission electron microscopy (TEM) images of a liquid-based stock culture that we used to keep refrigerated for use as a positive control. This positive control organism was plated at the same time as patient specimens to ensure that our culture media was capable of sustaining the organism's growth. Cultures were accomplished by plating the control or the patient specimen onto a non-nutrient agar overlain with a lawn of bacteria as a food source. We recently did away with this culture in favor of a faster real-time PCR assay that is performed daily (cultures used to take up to 5 days to become positive). As someone who loves microbe morphology, I decided to preserve this organism forever by submitting our stock culture for TEM analysis. Here are some representative images that we took:

What is this organism?


Bernardino Rocha said...

It appears to be trophozoites and cyst of Acanthamoeba sp.

Anonymous said...

The clue is the liquid culture seeded with bacteria. This clue points to an amoebic culture where the bacteria serve as food for the trophozoite stage of the amoeba. The polygonal appearance of the organism give us another clue compatible with Acanthamoeba sp. as observed through various stains.
Florida Fan

Sam said...

Trophozoites and cysts of Acanthamoeba species.

Luis said...

Trofozoitos, no son quistes, porque crecen en medios con bacterias

Old One said...

It never ceases to amaze me how quick and accurate everyone is with their identifications. I found this case to be very challenging.

The specimens appear to be protozoan in both cyst and trophozoite forms. Having a single nuclei with a large centrally located karyosome. Many inclusion bodies present in cytoplasm, possibly food vacuoles (probably bacteria, consistent for a facultative parasite). Cross sections of cyst-forms show a bilaminar cyst wall (photos 1,3,4,5). The ectocyst wall is wrinkled, the endocyst wall is smoother by comparison. Endocyst and ectocyst come together and meet at ostioles (photos 4 and 5). These ostioles (pores) have an operculum inside the endocyst. These characteristics appear to be those of Acanthamoeba sp..

I'd like to say "the eyes have it", but I may be wrong.

Sir Galahad said...

Cisti e trofozoiti di Acanthamoeba spp.

Old One said...

I would like to wish everyone a happy holiday season and extend a blessing we often see in our Christmas cards but I think it applies to most everyone.

"Peace on earth and good will to all"

Also to pass on a little seasonal parasite story.

Many years ago about this time of year, I was asked to collect some Ascaris suum for class. The school was willing to pay milage for my car (a wonderful cash infusion for a needy technician). So I loaded the car and headed for the Hormel packing plant (creator and maker of Spam canned meat).

Upon arrival I was met by a plant Supervisor who had me suit up with protective (for the meat not me) coverings. This included covering my face with head and beard nets. I looked like a real Dork (a total fool).

The Supervisor walked me to the gutting floor where I met the Forman. I couldn't believe my eyes. Leaning back in his chair was a rotund man in white shirt and suspenders. His face round, his cheeks rosy, his full luxuriant white beard and hair- this could only be one person. In my mind I yelled, "Santa, it really is you.

Santa looked at me and with a jolly Ho Ho Ho said " take that sh_t off your face".

Needless to say the rest of the collection went well. Santa's elves did pull some gutting line humor, but it was all done in good spirits.

I came away that day with three realizations.

1. Minnesota hogs are loaded with ascarids.
2. My car made more money than I did.
3. Santa is alive and well in Austin, Minnesota.

Blaine A. Mathison said...

Well, if not for the narrative I may not have immediately recognized this as Acanthamoeba, since my experience with electron microscopy is limited to microsporidia. But the cyst wall morphology is c/w Acanthamoeba.

Anonymous said...

Bilaminar cyst wall = Acanthamoeba.
Trilaminar cyst wall = Balamuthia.

-HLCM fan

Idzi P. said...

Definitely Acanthamoeba sp.
Cysts and trophozoites.
I’ve seen it twice in the lab. Each time in lens recipients.
Too bad we can’t see the typical “exploding” pseudopoda here...
It is not unusual for this parasite to form cysts when growing in a culture medium.

Old One said...

Would it be possible for someone to review the morphological features used to differentiate the cysts of Naegleria, Acanthamoeba, and Balamuthia ?

Anonymous said...

Old One,
There is an excellent lecture on line given by Dr. Visvesvara of CDC on the free living amoebas. If my memory is still correct, he named the amoeba Balamuthia in honor of his teacher Dr. Balamuth (just the same way Alexander Yersin named the Pateurella pestis, BTW Dr. Alexander Yersin was burried at the Suoi Dau facility of the Pasteur Institute in Vietnam during the Japanese occupation in World War II.)
Hope this help,
Florida Fan

Bernardino Rocha said...

Old one:

Naegleria fowleri:
Trophozoite Biphasic (amebic) and flagellate forms; 8–15 (7–35) μm; lobate pseudopodia pseudopodia
(amebic form);
Cysts not present in tissue; small, smooth, rounded; 7–15 μm;
Require living cells (bacteria or cell culture); do not grow with >0.4% NaCl by 0.85% NaCl
Appearance in tissue: Smaller than Acanthamoeba spp.; dense endoplasm; less distinct nuclear staining

Acanthamoeba spp.:
Trophozoite large (15–25 μm): no flagella; filiform pseudopodia (acanthopodia);
Cysts present in tissue; large with wrinkled double wall;
May grow without bacteria; not affected by 0.85% NaCl
Appearance in tissue: Large; rounded; less endoplasm; nucleus more distinct

Balamuthia mandrillaris:
Trophozoites are usually irregular in shape, and actively feeding amebae may measure from 12 to 60 μm in length (normal 30 μm). Possesses a nucleus but may possess more than one nucleolus. In tissue culture, broad pseudopodia are usually seen; however, as the monolayer cells are destroyed, the trophozoites develop fingerlike pseudopodia:
Cysts are somewhat smaller than the trophozoites, are usually spherical, and measure from
13 to 30 μm in diameter, in EM are characterized by having three layers in the cyst wall: an outer wrinkled ectocyst, a middle structureless mesocyst, and an inner thin endocyst;
Shown not to grow well on E. coli-seeded nonnutrient agar plates, can be cultured in mammalian cell cultures;
Both trophozoites and cysts of B. mandrillaris are found in many of the same CNS tissues as are Acanthamoeba spp. B. mandrillaris may have more than one nucleolus in the nucleus in some tissue sections.

Old One said...

Thank you one and all

Blaine A. Mathison said...

Well you can rule-out Balamuthia from the case narrative, because you cannot culture Balamuthia on bacteria. In nature, they feed on other free-living protozoa. In the lab you would need to culture them on cell lines.

Idzi P. said...

Indeed Blaine!
We tended to use the supernatant resulting from boiling bird seeds in water, called “eau de blé”. A perfect medium for growing acanthamoeba after it had been spiked with bacteria.