egg count

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I’m researching incubation methods, and hopefully in a few weeks I will embark on my first McMaster egg count. There seem to be 3 choices for incubating the hookworm larvae; the Baermann, Harada-Mori, and the AP method (agar plate.) Here’s a nice visual and thorough list of requirements for the first two methods:

http://books.google.com/books?id=8AWz0cS6e9kC&pg=PA115&lpg=PA115&dq#PPA115,M1

This article goes into great detail on the advantages of each method:

http://www.ajtmh.org/cgi/content/full/77/6/1087

Incubation temperature is important, and 30C (86 degrees farenheit) is optimal:

http://www.ncbi.nlm.nih.gov/pubmed/3569472

I’ll provide an equipment list and write about the process once I’ve ordered the supplies, and tried it out. Oh why didn’t I take a science class in college? An art major in oil painting is not helping me out here…perhaps I’ll start a larval triptych!

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There are very few studies on the effect of helminths on humans. As a patient trying this therapy, there are few immune markers we have on hand to check immune response. We have measures of inflammation, like CRP and SED rates, but only in the research setting can one measure IL-10, the T 1 and T2 arms, etc. All we have is eosinophilia and standard stool tests to assess worm burden.

Eosinophils rise in response to hookworm infection, seeming to peak between weeks 3-10. This study describes that eosiniphils peak between days 38-64 :

http://www.ajtmh.org/cgi/content/abstract/37/1/126

Peaks between weeks 3-9:

http://www.ajtmh.org/cgi/content/full/75/5/914#F5

Starts to be elevated at days 14-21, peaked on day 42 and declined to
a persistently elevated level:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1809522

Peaked week 5, declined by week 20:
“In the CD cohort, blood eosinophilia developed from week 5 (mean
2.60×109/l (1.89) v week 1 0.18×109/l (0.10) v week 20 0.59 (0.20)). ”

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1856386

But my favorite study, the MS study in Argentina, where they tracked 12 MS patients already infected with helminths and compared them to 12 other MS patients over a 4.6 year period, only recruited the helminth infected patients if their eosinophelia was high, (800-1800 mm3) and it stayed that way for the duration of the study. Their quantitative egg counts were also high: between 1,180 and 9,340 eggs/gram.

Eosinophils reflect parasitic infection, and the higher the number, usually the larger the worm burden. One indication that one has lost their worm infection would be having an elevated EOS for an extended length of time, then having it fall to baseline. Obviously, stool tests would confirm this, as well as symptom regression. In the dose-ranging trial, the higher doses resulted in higher EOS counts, though they did not test longer than 12 weeks.

I only tested my EOS at baseline and 18 weeks, so I never tracked a rise and fall. Baseline values were 74 cells/mcL and only rose to 192 post infection. (Normal is 15-550). Remember, I added worms from weeks 10-18, which may have provoked an immune response that curtailed the new worms from attaching, and possibly displaced some of the first 10, like this capsule endoscopy study shows. So by week 18, perhaps I had very few worms…

I will be testing EOS at weeks 3, 6, 9, and 12 to see how they respond to 10 larvae. I don’t think 10 hookworms are going to be enough to cause persistent eosinophilia. And like the MS study, it seems important to get and maintain a large enough worm burden to stimulate eosinophilia, and maintain a higher egg count. I’m very curious what my results will be this time…

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Just found the on-line protocol for the Crohn’s study at Nottingham University. For those of you with Crohn’s experimenting with hookworms, though most of the immune blood tests will be unavailable to us, it might be nice to follow some of the other protocol. The CRP and SED rate are taken at baseline, then at weeks 3, 6, 9, and 12. (They eradicate the hookworms at week 12, but follow up for another month.) Fecal egg counts are done at week 12. Of course, there’s also the CDAI, the Harvey Bradshaw Index (HBI), and quality of life IBD questionairre (IBDQ).

One of the frustrations I have in experimenting with this therapy, and of AIT, is the lack of diagnostic pursuit of patient’s effects. Most patients are not doing before and after screening, and often aren’t even communicating about their long-term effects from hookworms. Patient’s doses, frequency of doses, and clinical response are only known by AIT and none of the rest of us are able to access that data to learn from. I wish there was an anonymous database accessible online that would list the diseases, patient’s blood scores at baseline, side effects within the first 4 months, and when efficacy began, if it did. Although each person’s reaction to hookworm is different, the more we quantify and correlate our response, the more patterns might emerge that would help future patients. But alas, it’s not my business, so I am correlating what I find here, and when I redose in a few weeks, will try to take my own advice and do all I can to track my response to help future people wishing to experiment outside the trials.

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Light, long-lasting Necator infection in a volunteer.

School of Public Health, Tulane University, New Orleans, Louisiana 70112.

“A Necator infection produced by percutaneous exposure of a volunteer to three larvae was followed with periodic egg counts for 4 months beyond 18 years when passage of eggs in the feces ceased permanently. During the 2nd year of infection, there was unintentional exposure to two additional larvae. Egg counts per gram of feces (EPG) were approximately 1,000 during the 1st year, 1,500 over the next 5 years, and less than or equal to 200 over the final 3 years, during which time there were 6 periods of approximately 1 month each when Kato thick-smear examinations revealed no eggs. During 6 of the 1st 14 years, when egg counts were done by the standard direct smear method, up to 37% of the eggs were infertile. Based on the assumption that two female worms were present during the 1st year, three during the next 3 years or more, and only one during the final 3 years, the estimated output per female in this light infection was 500 EPG in the worm’s prime of life, and less than 200 EPG in the final year of the 17 or 18 year life span.”

From: http://www.ncbi.nlm.nih.gov/pubmed/3189697

This study is quite amazing in that such a small infection could last so long. (I’d like to know how he was accidentally exposed to 2 larvae, and how did he know they were two? Was he fooling around under the microscope and his hand slipped and they splashed onto his arm? I wish they provided details!) Also, that anyone would do fecal egg counts for 18 years, and monthly! If such a high EPG is found from only a few females, then why are we messing around with doses of 25-50? I’m still probably going to dose with 10, I just want to do my egg counts to see what happens. And if they all die, I’ll think of this man, who hosted 5 for two decades. Clearly, the modern lifestyle didn’t affect his hookworm status. Amazing creatures!

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Dosing

I was thinking about worm dose and efficacy. (Brace yourself; this is
long and convoluted.) If one were to be dosed once with 10 larvae,
then accounting for attrition, there would be perhaps 7 left in the
gut. (And this number may be a high estimate.) Assuming a 50% gender
split, there would be 3 or 4 males. (And there could be 2, 1.) This
Cornell transcript claims the males die off after copulation.
(http://www.cumc.columbia.edu/dept/ps/2007/para/old/transcript_02_pd02.pdf)
Even if they don’t, this paper claims the lifespan, though averaging
3-5 years, is really much shorter than that.
(http://www.jimmunol.org/cgi/content/full/173/4/2699) Would 3 or 4
worms be enough to maintain a steady immunological state? How many
months/years would they live and would there be an average drop off
around the same time? (meaning; do they all die at once?) As
Nottingham is only studying their Crohn’s patients for 6 months, they
will not collect this data, which is unfortunate. (length of
efficacy, worm lifespan, wormcount after x number of months/years,
timing of necessary reinfection, etc.) So we’re on our own…

This study says that adding a population of 50 in two healthy people
who hosted existing hookworms resulted in some old worms dying and new
ones replacing them, with no netted increase in number.
(http://www.ncbi.nlm.nih.gov/pubmed/17035088) So for these two
people, adding worms at a high, one time dose, didn’t result in a
greater population. What the paper doesn’t address is age of the
worm. How many new worms replaced the old? Would one end up with a
mixed age population? That should extend efficacy, assuming the new
ones live longer than the first cohorts. (Some worms would be old
crones…surrounded by fresh whippersnappers.)

It seems like the only choice to maintain a healthy, thriving
population is to redose periodically, with a small enough number not
to illicit enough of an allergic response to expel the new or old
population. What is the number before adding becomes displacing? If
one started with 10, was left with 5, added 15, they merely displaced,
then one is left with 5 again. Would adding 2 be more effective?

And then I wonder how much of the acquisition of new larvae is part of
the immunological puzzle. If there is an additional, temporary
benefit from each subsequent infection, ( the bounce, or the “hookworm
high”) then perhaps maintaining both the immunological and
psychological health of the human and an effective worm burden is
based on the constant periodic addition of small numbers of worms.

Which means the patients who get one dose only are incomparable to the
ones who get 3 doses, who are incomparable to Jasper, for example,
who’s had multiple tiny doses. Perhaps his efficacy would have
waned if a small, steady population wasn’t supplied?

If there is a psychological component to small reinfections, along
with a temporary immunological benefit, perhaps man evolved to have
this periodically. Sort of a natural antidepressant to go with the
immune regulation. This study finds antidepressant qualities in the
soil bacteria itself
(http://www.medicalnewstoday.com/articles/66840.php), showing even
greater depth to the whole hygiene hypothesis. Depression as a
consequence of our sterility. We may need not only the worms, but the
soil they came from.

Which means trying to recreate the natural state by supplying one
aspect of it; the worms, in an unnatural dose (one time, a great
number) and removing everything else may not work. What is the
natural number and frequency of infection in the wild for a healthy
adult anyway?

This really argues against a one time inoculation, at least based on
the theory that the worms’ benefit will last as long as they are
alive. Yet it also argues against multiple inoculation therapy, as
that might cause one to end up with the same amount as one began with,
or less. It seems the only way to recreate the natural state is to
add 1 or 2 worms at a time.

There are no answers to any of these questions yet…Will we die
before we learn how to properly live?

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