… a day in the life of Crohn’s disease …
You are currently browsing the archive for the egg count category.
So I did another egg count just now, my second since a few months ago. I followed these instructions:
http://www.docstoc.com/docs/599020/Macmaster-counting-Technique-ppt
except rather than making a large volume of flotation fluid, I mixed 40 g of salt with 100 ml. of water. I wrote about the technique and where I got my supplies here. Read the rest of this entry »
Eureka! I finally managed to see some hookworm larvae under the microscope. For a full visual description of the McMaster egg count, see: http://www.docstoc.com/docs/599020/Macmaster-counting-Technique-ppt
Results from 10 larvae? 800 epg
I’ll walk through my steps one by one. First, I bought the materials:
digital scale: myweigh ibalance 500 (more expensive then the cheaper models, but comes with a 30 year guarantee)
microscope: Wesco binocular, with lenses from 4X to 100X
measuring devices: one glass cylinder 50 ml., 1 plastic graduated flask, 500 ml, and 1 200 ml. glass beaker
cheesecloth, spoon, salt, water
pipettes
1 McMaster grid slide, bought from this site: http://www.vetslides.com/
First, I went to the bathroom in a plastic container. I weighed a piece of paper on my scale, then added 40 grams of salt. I then filled a plastic beaker with 100 ml. of water. Mixed well, until the salt dissolved. Then I measured a small plastic container and added 4 g. of stool to it. (If the plastic weighs 1.5 grams, for example, you would add stool until you get to 5.5 g.) I put the stool into a glass flask and added 56 ml. of the salt water mixture. I made sure to rinse the plastic cup with stool in the salt water, so as to capture all of the contents of the stool. Then I stirred the contents with a plastic knife until it was well mixed. I took the contents and strained it through a single layer of cheesecloth several times (it helps to have two glass open flasks for this.) Then I pipetted the mixture into both chambers of the McMaster slide and let it sit for 5 minutes. (Well, really 10 as I paused to have breakfast.)
I put the slide into the microscope at 10X 10 magnification. It takes a while to get used to the binocular lenses; I’m sure the technique improves with experience. The challenge I had is one focus allowed me to see the grid, the other focus I could see the eggs. A faint blurry line sometimes could be seen, but not always. I suggest shelling out extra for the green lined grid slide.
The eggs should look like this: 
(photo courtesy of www.flickr.com/photos/prep4md/2772121447/)
Because I couldn’t see the grid lines well, every time I identified an egg, I’d notice something around it, like little food blobs that had a particular shape or color to them, so I wouldn’t count an egg twice. I carefully went up and down each grid slide, and counted eggs. On the left side, I found 7 eggs, on the right, 9.
I added these up and multiplied by 50, resulting in 800 eggs per gram.
I should do multiple stool samples for accuracy. Right now my eyes hurt. I’m just happy I actually found and identified something.
So yes, 10 little hookworms result in 800 epg 4 months later. Happy egg counting!
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!
Tags: egg count, hookworm, necator americanus
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…
Tags: dose ranging study, egg count, egg count studies, helminth immunology worm burden, hookworm dosing, Nottingham trial
I’ve been wondering why there are so many false negatives when using standard ova tests that are offered through such labs as Quest, etc. This study shows that even waiting 3 hours between stool sample and test allows for a 50% loss of eggs, so I wonder if the sample is over 24 hours old, and a light infection, if any eggs could be detected at all. Which really argues for those of us doing this to learn the techniques ourselves, since I don’t think we can rely on a standard lab. When I dropped off a stool test at Quest in November, and asked how many hours it was until processed, they claimed it didn’t matter, but obviously the understanding of parasite biology is not exactly a requirement for phlebotomists.
http://www.ncbi.nlm.nih.gov/pubmed/16824566
“Time delays between patient and laboratory selectively affect accuracy of helminth diagnosis.
London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
Studies of intestinal helminth infections are influenced by the constraints of sample collection, as identification of helminth ova in stools is affected by the time since evacuation from the host. Different methods may be required to optimise diagnostic sensitivity under different study conditions. In the context of studies in rural Malawi, we collected stool samples with different time delays from production by subjects to sample collection by field staff, to examination in the laboratory. Stools were processed by Kato-Katz (KK) or formol-ether concentration (FEC) methods. Hookworm and Schistosoma mansoni were the most common helminths identified. The prevalence of hookworm was higher with KK (270/988, 27%) than with FEC (191/988, 19%). Comparison was made between the results from the two methods according to the timing of the processing steps. Delays in processing did not affect retrieval of S. mansoni. A decrease in sensitivity of almost 50% for detection of hookworm was observed with either method when preservation/refrigeration was delayed by more than 3h. A delay of 1 day from refrigeration or preservation to laboratory processing also reduced the sensitivity for hookworm by 50% for both methods. Care must be taken in studies of multiple helminth infections owing to the selective reduction of hookworm ova during transport. This is particularly critical when samples are not preserved, even over short periods of time, and even with formalin preservation.”
Tags: egg count studies
Recent Comments