Amboceptor

Microbiological, virological, bacteriological, immunological, medical, epidemiological, historical, anecdotal

Medical privacy, 1950 style

Without really working in a medical field, I still hear a lot about medical privacy and HIPAA. And how any public presentation of patient information needs to be anonymized, any identifying details need to be removed, any irrelevant personal details need to be removed, populations need to be profiled rather than individuals, and generally all publications about human patients must run through a battery of administrators to make sure everything is in compliance with federal rules, state rules, institutional rules, the institutional rules of other institutions that collaborated with us, and heaven knows what else.

Not so long ago, the standards were different. Today we’ll look at a 1950 article on “Primary Cutaneous Cryptococcosis” from Archives of Dermatology and Syphilology (later renamed Archives of Dermatology, and then JAMA Dermatology). An actual doctor could explain what parts of this article would be scrubbed now, and what parts are still OK. I don’t really know. But what struck me was that nearly all the personal details seem unrelated to the actual topic of the article. Meanwhile, author William M. Gandy, M.D. points out that “family history was irrelevant”. Indeed, it would be even more irrelevant to point out the patient’s mother’s alcoholism, or grandfather’s yaws and phossy jaw.

gandy-title

Information we can glean from the piece:

- “The patient had been under continuous observation and treatment at the Charity Hospital of New Orleans since July 2, 1945. Successive complaints brought her at one time or another to the attention of the medical, surgical, gynecologic and dermatologic services.”

- “She stated that she had been ‘anemic’ and afflicted with ‘enlarged glands’ since childhood. She had measles, mumps, chickenpox and scarlet fever in early life.”

- She was “first seen by us” (the dermatology clinic) on August 14, 1947, at age 29.

- She was white and a lifetime resident of New Orleans, Louisiana.

- At this time she was divorced with one child.

- That child was conceived in 1942 in Cleveland, Ohio, but born in New Orleans.

- For months after the birth of the child, she was given weekly X-ray treatments for “extensive” genital warts. The “Roentgen rays” did no good and she was then given podophyllin ointment, which led to “a severe burn followed by a slough of much of the vulva”.

- During 1942 she was diagnosed with Boeck‘s sarcoid.

- During 1947 she had stones removed from her right kidney.

- During 1947 she underwent a vulvectomy and a ureterotomy.

- The whole point of this paper, a cutaneous Cryptococcus infection on her face, was probably the least of her problems and had little to do with all this other stuff anyway.

- Very unusual for cryptococcosis, this infection was limited to a patch of skin and not found in the blood, cerebrospinal fluid, urine, sputum or mucosal swabs.

- 12 mice were injected with half a milliliter of a broth culture of her strain of Cryptococcus. 6 of the mice died within 12 days, and the remaining 6 died within 21 days.

- Between the end of 1947 and April 1949 she left Charity Hospital, married her second husband, and returned to Charity Hospital, this time the urology clinic, after developing a renal abscess and uremia. This was not a Cryptococcus infection.

- “Further physical and laboratory investigations were not possible at the time because of the patient’s uncooperative attitude and the severity of her illness.”

- Finally… here’s a picture of her face.

gandy-cryptococcus-1950

If you know this woman, you may be glad to know that she received a large amount of medical care free from top clinicians, as Charity Hospital was adjacent to the LSU Health Sciences Center. Was this care better than what would have been provided in 1736, when the hospital was founded? Of course! What kind of question is that? Or anyway, I think so, but I’m no historian.

Gallery: The amazing ink-proof yeast capsule

Observed doctors and medical students as they learn about the workings of the clinical microbiology lab, I’m impressed by their love of the India ink test for cryptococcus. The way this test works is: Cryptococcus is a type of infectious yeast that looks a lot like Candida if you just do a gram stain. But it has a polysaccharide capsule around each cell (unless for some odd reason it isn’t producing a capsule), wider than the cell itself. So if you put Cryptococcus in a colored liquid, most famously a solution of India ink, the polysaccharide capsule shows up as a huge empty white area around the cell. Whereas with Candida, only the cell itself is white.

We apparently don’t use this test regularly anymore, but we still show it to people in case they need to know what it is.

Something about the India ink test just makes people happy. A lot of diagnostic microbiology uses techniques that were developed several generations ago, but this one is just so simple, requiring not “acid alcohol” or various toxic red and purple substances, but merely the simplest form of ink, developed millennia ago. And to use the phrase “India ink”, instead of “colloidal carbon” or something, is such an anachronism in the 21st century. Most of us last saw that phrase when reading some classic of literature like The Secret Garden or A Bear Called Paddington. And aside from the name, there’s something magical about seeing this invisible capsule appear around what seemed to be a normal yeast cell. Like lemon-juice ink made visible.

* * *

So here are some depictions of India-ink-stained Cryptococcus in the literature. First, camera lucida drawings from a 1935 JID paper by Rhoda W. Benham (Cryptococci — their identification by morphology and serology) that must have been a handy field guide to Cryptococcus species. The top right corners of the dishes are shaded to show how they look under India ink.

crypto-benham

* * *

Now, some photos of patient tissues directly stained with India ink.

From Wilson HM, Duryea AW (1951), Cryptococcus meningitus (Torulosis) treated with a new antibiotic, actidione®. Archives of Neurology & Psychiatry 66(4):470-480.

crypto-duryea

* * *

From Carnecchia BM, Kurtzke JM (1960), Fatal toxic reaction to amphotericin B in cryptococcal meningo-encephalitis. Annals of Internal Medicine 53(5):1027-1036.

crypto-carnecchia

* * *

From Schupbach CJ, Wheeler CE Jr, Briggaman RA, Warner NA, Kanof EP (1976), Cutaneous manifestations of disseminated Cryptococcosis. Archives of Dermatology 112(12):1734-1740. Note “Tzanck preparation”, looking for multinucleated giant cells.

crypto-schupbach

* * *

From Love GL, Boyd GD, Greer DL (1985), Large Cryptococcus neoformans isolated from brain abscess. Journal of Clinical Microbiology 22(6):1068-1070.

crypto-love-1985

* * *

From Bottone EJ, Kirschner PA, Salkin IF (1986), Isolation of highly encapsulated Cryptococcus neoformans serotype B from a patient in New York City. Journal of Clinical Microbiology 23(1):186-188.

crypto-bottone

* * *

And some images of cells grown in culture. Ending with one in color!

From Neill JM, Abrahams I, Kapros CE (1950), A comparison of the immunogenicity of weakly encapsulated and of strongly encapsulated strains of Cryptococcus neoformans (Torula histolytica). Journal of Bacteriology 59(2):263-275.

crypto-neill

* * *

From Littman ML, Tsubura E (1959), Effect of degree of encapsulation upon virulence of Cryptococcus neoformans. Proceedings of the Society for Experimental Biology & Medicine 101:773-777.

crypto-littman

* * *

From Bulmer GS, Sans MD, Gunn DM (1967), Cryptococcus neoformans I: Nonencapsulated mutants. Journal of Bacteriology 94(5):1475-1479.

crypto-bulmer

* * *

From Dykstra MA, Friedman L, Murphy JW (1977), Capsule size of Cryptococcus neoformans: Control and relationship to virulence. Infection & Immunity 16(1):129-135.

crypto-dykstra

* * *

From Chang YC, Kwon-Chung KJ (1994), Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Molecular & Cellular Biology 14(7):4912-4919.

crypto-chang

* * *

From Doering TL (2000), How does Cryptococcus get its coat? Trends in Microbiology 8(12):547-553.

crypto-doering

* * *

From Zaragoza O, Casadevall A (2004), Experimental modulation of capsule size in Cryptococcus neoformans. Biological Procedures Online 6(10):10-15.

crypto-zaragoza-2d

* * *

From Zerpa R, Huicho L, Guillén A (1996), Modified India ink preparation for Cryptococcus neoformans in cerebrospinal fluid specimens. Journal of Clinical Microbiology 34(9):2290-2291.

crypto-zerpa

* * *

And a bonus: High-tech 3-dimensional visualization! These are 40 focal “slices” of a single cell. From Zaragoza O, McClelland EE, Telzak A, Casadevall A (2006), Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule.

crypto-zaragoza-3d

Wikipedia: Zinaida Ermolieva

Zinaida Vissarionovna Ermolieva (Russian: Зинаида Виссарионовна Ермольева; 15 October 1898 [O.S. 27 October] – 2 December 1974) was a Russian microbiologist and epidemiologist who led the Soviet effort to generate penicillin during the Second World War.


Biographyzinaida


Born on a farm in the Frolovo region, Ermolieva attended school in Novocherkassk and studied medicine at Don University in Rostov-on-Don (now part of Southern Federal University), graduating in 1921. Continuing to work at Don University’s bacteriological institute, she collaborated with Nina Kliueva on a study on encephalitis lethargica [1], before moving to Moscow in 1925. There she worked at the People’s Commissariat of Health, as head of microbiology at a biochemical institute [2] that would later be named for its founder Aleksey Nikolayevich Bakh [3]. Early in her career she was known for her work on characterizing lysozyme and employing it as an antimicrobial agent [4].

During the Second World War Ermolieva became famous for her role in the independent Soviet effort to extract penicillin from mold, using the species Penicillium crustosum [4] (rather than P. notatum, the species employed by Alexander Fleming and other British scientists). To test this penicillin treatment, she was one of many scientists to travel to Abkhazia and make use of the monkey colonies at Sukhumi’s Institute of Experimental Pathology and Therapy [5].

Ermolieva also led the efforts to control a cholera outbreak in Stalingrad, as part of which she spent six months in the besieged city, and was credited with creating a bacteriophage-based vaccine against Vibrio cholerae in addition to developing the new Soviet source for penicillin.

Now an eminent scientist and patriotic hero, she was awarded the State Stalin Prize and spent the rest of her career in Moscow, being named director of the All-Union Research Institute for Antibiotics in 1947, and chair of the department of microbiology at the Central Postgraduate Medical Institute in 1952. She was also a founder and editor of the Moscow-based journal Antibiotiki [4]. According to Soviet propaganda, Ermolieva chose to redirect the proceeds from her Stalin Prize into building fighter jets, one of which was inscribed with her name. She was also publicly recognized as a self-experimenter, reportedly swallowing 1.5 billion cells of a glowing blue Vibrio strain in order to show that it caused a cholera-like illness [7].

Ermolieva was named an Academician of the USSR Academy of Medical Sciences in 1965, and was named an Honored Scientist of the RSFSR in 1970 [8]. She received other state honors including the Order of the Red Banner of Labour, the Order of the Badge of Honour, and the Order of Lenin. Credited with over 500 scientific papers and as adviser for 34 doctoral theses in her career, Zinaida Vissarionovna Ermolieva died in Moscow in 1974.


Personal Life


Ermolieva was married twice, both times to fellow microbiologists. She was important in the efforts to free her ex-husband Lev Alexandrovich Zilber, who had been imprisoned in labor camps on suspicions of spying for Germany and misusing his research on tick-borne encephalitis virus and Japanese encephalitis virus [9]. Zilber was freed permanently in 1944 and later rehabilitated in the eyes of the Kremlin, receiving several of the same state honors as Ermolieva [10]. Her second husband, Aleksey Aleksandrovich Zakharov, was also a microbiologist who was denounced during the Second World War, and died in a prison hospital in 1940 [11].

She became a model for aspiring Soviet female scientists as the basis for protagonist Tatiana Vlasenkova in The Open Book, a trilogy of novels written between 1949 and 1956 by Veniamin Alexandrovich Kaverin, the brother of Lev Zilber [12]. The Open Book was adapted in feature film form in 1973 [13], and as a television series in 1977 [14]. She is also the basis for the character Anna Valerievna Dyachenko in the Russian TV series “Black Cats” (Чёрные кошки), set in postwar Rostov-on-Don [15].


References


1. Krementsov, Nikolai (2007). The Cure: A Story of Cancer and Politics from the Annals of the Cold War. Chicago: University of Chicago Press. p. 40. ISBN 9780226452845.

2. http://www.inbi.ras.ru/english/history.html

3. Kretovich, W.L. (1983), “A.N. Bach, Founder of Soviet School of Biochemistry”. in Semenza, G. Selected Topics in the History of Biochemistry: Personal Recollections (Comprehensive Biochemistry Vol. 35). Amsterdam: Elsevier Science Publishers. p. 346.

4.(pdf) S. Navashin (1975), Obituary of Prof. Zinaida Vissarionovna Ermolieva, The Journal of Antibiotics vol. XXVIII, no. 5, p. 399.

5. http://www.redorbit.com/news/science/1503065/renowned_science_facility_suffers_in_postsoviet_era/

7. Fiks, Arsen P (2003). Self-Experimenters: Sources for Study. Westport, CT: Praeger Publishers. p. 70.

8. “Zinaida Ermol’eva”. The Great Soviet Encyclopedia, 3rd edition (1970-1979).

9. Zlobin, V.I. et al. (2005). “Tick-Borne Encephalitis”. in Ebert, Ryan A. Progress in Encephalitis Research. New York: Nova Science Publishers, 2005. p.32. ISBN 1-59454-345-3.

10. “Lev Zil’ber”. The Great Soviet Encyclopedia, 3rd edition (1970-1979).

11. http://panov-a-w.narod.ru/stati/zilber.html

12. Eremeeva, Anna (2006). “The Woman Scientist in Soviet Artistic Discourse”. in Saurer, Edith; Lanzinger, Margareth; Frysak, Elisabeth. Women’s Movements: Networks and Debates in Post-Communist Countries in the 19th and 20th Centuries. Köln: Böhlau Verlag GmbH & Cie. p. 347. ISBN 9783412322052.

13. http://www.imdb.com/title/tt0170345/

14. http://www.imdb.com/title/tt0075554/

15. http://www.kino-teatr.ru/kino/movie/ros/104901/annot/

All facts not otherwise cited are from the Russian Wikipedia page on Zinaida Ermolieva, accessed via Google Translate on 24 August 2014.

Data update: Destroyed by freezing does not equal alive

In the experiments reported in the present paper a number of active agents, some undoubtedly living, others equally unquestionably not living, and still others of a doubtful nature, were subjected to repeated freezing (-185°C.) and thawing. By these tests it has been possible to determine that mere destruction or inactivation of a substance cannot be accepted as proof that it existed in a living state.

In 1926, legendary virologist / bacteriologist Thomas M. Rivers addressed the still-extant question “Are viruses alive?” through the simple experimental method of freezing them over and over. The paper in question came out in the Rockefeller Institute house publication Journal of Experimental Medicine (vol.45[1]: pp. 11-21). Not exactly a gripping title.

repeated-freezing-rivers

What kind of data is in here? Well, for most of the graphs they take an “active agent” (either bacteria, virus, or enzyme), freeze-thaw it “as often as desired”, and then see if it’s still active. The data is pretty straightforward, with a separate section for colon bacilli, a section for Virus III, a section for “a bacteriophage lytic in colon bacilli”, and so on. But it’s never summarized in a way that compares the different “agents” to each other. Let’s try to do that.

The low temperature (-185°C.) used in the experiments to be reported was obtained by means of commercial liquid air which was transported from the plant to the laboratory in Dewar flasks. Desired amounts of the air were transferred to deep Dewar beakers where small amounts of the substances to be frozen, enclosed in Noguchi tubes, were completely immersed for several minutes. After the substances had been completely frozen they were quickly thawed in tap water (16-18°C.).

What does “active” mean for these various substances? How did they determine that freeze-thawing had destroyed the substance’s activity? This was different for each substance.

rivers-activity

Bacteria can be measured the same way we measure them now, by making serial dilutions and plating each dilution on agar, then seeing how many colonies grow. Bacteriophage can be measured the same way, by first making a “lawn” (agar plate fully covered with bacteria) and applying different dilutions of bacteriophage, then seeing how many “plaques” (holes in the lawn) are formed by the phage killing the bacteria.

Complement can be measured by seeing how long it takes to destroy “given amounts of red blood cells in the presence of a great deal of amboceptor“.

Trypsin can be measured somehow, Dr. Rivers doesn’t specify except by saying that his colleague Dr. Northrop took care of that part of the study. Nowadays Dr. Northrop would be a co-author. But the paper would then have to list Rivers as both the first author and the last author somehow, since Northrop didn’t do enough to merit either status.

Anyway, to find “details of the technic” we are directed to a paper by Northrop and Hussey (1923), showing a very clever method by which a solution of gelatin is exposed to trypsin, and at different timepoints the gelatin’s viscosity is measured.

northrop-graph

And how do you measure viscosity? With a viscosimeter.

northrop-viscosimeter

I’m having trouble understanding sentences like “The gelatin-water time ratio was approximately 3″, but the point is that you can measure the amount of trypsin by measuring how fast it turns gelatin into runny gelatin. Nowadays you would use a colorimetric assay, in which trypsin cuts the protein that has some sort of colored label attached to it, and you would measure how much colored label gets released into solution.

* * *

Finally, the three mammal viruses.

“Vaccine virus” and “Virus III” are both introduced to the skin of rabbits, probably by scarifying and then rubbing the virus into the scratches. They look for a “virus reaction” in the skin surrounding where the virus was inoculated, and measure this semi-quantitatively based on how bad of a sore forms. I hope they aren’t simply measuring the immune response to the inoculation, because even killed virus should produce some immune response.

“Vaccine virus” is basically what we now call vaccinia virus. “Virus III” is a more interesting question.

All stocks of Virus III were lost some time before the invention of electron microscopy. Nobody can now be sure what exactly this rabbit-specific virus was, but it was probably Leporid herpesvirus 2, as described by Nesburn in 1969. For an objective summary of the Virus III story, read the page on LHV-2 (p. 380) in The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents (Academic Press, 2012).

“Herpes virus” (Herpes simplex) activity is measured differently. They inject HSV into rabbit brains, and they look for dead rabbits. It seems like this assay should actually be quantitative, based on looking at how long it takes the rabbits to die, but all their rabbits either died within a week or lived longer than a month, so the results were clear without measuring time to death.

 * * *

The results are pretty simple, which is why I would like a summary figure instead of a series of tiny individual tables for each thing they studied.

rivers-summary

Some comments on the data:

  • “Locke’s solution” is like what we call Ringer’s solution, the intravenous fluid given to people who have suffered blood loss. This page indicates that it’s the same as Ringer’s solution, but buffered by bicarbonate instead of lactate. But most other sources indicate that it also includes glucose, making it closer to Tyrode’s solution.
  • Locke’s solution is basically a physiological salt solution, so what’s the difference between that and the “physiological salt solution” used in the phage experiments? The latter doesn’t contain glucose, and isn’t buffered; instead of a pH of roughly 7.6, the pH is “between 6 and 7″.
  • Vaccinia virus was the most stable, retaining the ability to create a skin lesion even after 34 freeze-thaws. Virus III was destroyed after 12, and the other herpesvirus was destroyed after 24.
  • Both bacteria and bacteriophage had more than 99% of their activity destroyed by freeze-thawing in Locke’s solution. All three big viruses were more hardy than that.
  • I don’t know what a “1:10 dilution of trypsin” is. How many USP Trypsin Units is that? What’s the molarity? We have to take Dr. Northrop’s word for it, that it’s the same system for trypsin dilution he always uses.
  • At a mere 1:10 dilution, complement and trypsin were not damaged by 12 freeze-thaws. But when diluted further, they were very susceptible to freeze-thaw.
  • In fact, bacteria and bacteriophage were also more susceptible to freeze-thaw at low dilutions. Even vaccinia virus lost all its effect after 34 dilutions at 1:100,000 dilution, although it didn’t have much activity at that dilution to begin with.
  • Of further interest: the difference between high and low dilutions was only observed in Locke’s solution or salt solution, not broth. Remember these cutting-edge findings when you make your own aliquots.
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