Dr Markov CBIS Theory of ME/CFS - General Discussion

[Guwop2]

Dr Markov tells all patients to wash the genital area around urethra to prevent bacteria on the genital skin from contaminating the urine. Hip also warned of this when he made a post on how to do this test at home.

I did remember to clean the area, but I only used soap and water whereas others used anit-bacterial/chemical solutions that i guess would yield more acurate results. Im going to grow some more I think, but use anti-bacterial just to be 100%.

 

[Hipsman]

I did remember to clean the area, but I only used soap and water whereas others used anit-bacterial/chemical solutions that i guess would yield more acurate results. Im going to grow some more I think, but use anti-bacterial just to be 100%.

I just use soap and water too, this is how clinic recommends to do it. Most of the time nothing grows on my slides. But you can go one step further to be extra sure of course.

 

[BrightCandle]

If people keep coming back positive its got a shot at being a pretty good diagnostic test, better than anything seen so far, nothing has been this accurate so far. I am sure it will fail for someone but the chance its random is vanishingly small already, its a significant result statistically speaking.

 

[Breagjam]

If people keep coming back positive its got a shot at being a pretty good diagnostic test, better than anything seen so far, nothing has been this accurate so far. I am sure it will fail for someone but the chance its random is vanishingly small already, its a significant result statistically speaking.

I wish we had a few more healthy controls to test negative to reinforce the significance of positive tests.

 

[anne_likes_red]

Are the slides I need here? I could do one patient and one or two controls if so.
From: https://www.nzms.co.nz/379/dipslides-dimanco/

 

[Breagjam]

Are the slides I need here? I could do one patient and one or two controls if so.
From: https://www.nzms.co.nz/379/dipslides-dimanco/
View attachment 46030

Great 👍🏻

the SCT may be what you need, but here are the ones I ordered (I’m in US)

https://mfimedical.com/products/lifesign-uricult-cled-polymyxin-macconkey

Maybe @Hipsman can confirm

 

[Hip]

Are the slides I need here? I could do one patient and one or two controls if so.
From: https://www.nzms.co.nz/379/dipslides-dimanco/

Yes, this product is good:

TT2 - Standard urine dipslide with CLED/MacConkey.

Any dipslide which has CLED agar and MacConkey agar is good for urine bacteria testing.

If you cannot get hold of that product, there are other urine dipslide products listed in the pdf in this post. Several suppliers in the list will ship internationally. Bluecare may be a good option for you, as they ship from Hong Kong (shipping is $8), and this product is good, it has the necessary CLED & MacConkey.

 

[GlassCannonLife]

You are the 8th ME/CFS patient to test positive. No ME/CFS patients have so far tested negative, but the two healthy controls who were tested (by Nuno) were both negative. Dr Markov found that only 7.1% of healthy controls were positive.

Markov high sensitivity urine culture results of Phoenix Rising ME/CFS patients:

1. Hipsman — positive
2. Hip — positive
3. Martin aka paused||M.E. — positive
4. Cipher — positive
5. Nuno — positive
6. BrightCandle — positive
7. Guwop2 — positive
8. Breagjam — positive

Really? @Nuno shared images of control dipslides with me on discord (that's you right?) and they were very similar to his. The CLED media was still green (mixed positive, just didn't have white dots that his had) and the enterococcus/MacConkey looked pretty much identical (~10^3 enterococcus). He didn't have the second healthy person's one on hand.

I think we should aim to very much expand the healthy patient pool before drawing conclusions.

It might also be worthwhile to incubate dipslides that have just been left alone, or irrigated with sterile saline instead of urine as a negative control, to check both that this looks different to the ME/CFS samples, and that the test is indeed able to provide a completely negative result.

 

[Hip]

@Nuno shared images of control dipslides with me on discord (that's you right?) and they were very similar to his.

I don't recall seeing the dipslide images of @Nuno healthy controls, are you saying that there was some bacterial growth (dots, spots or a matt haze) on the healthy controls?

It might also be worthwhile to incubate dipslides that have just been left alone, or irrigated with sterile saline instead of urine as a negative control, to check both that this looks different to the ME/CFS samples, and that the test is indeed able to provide a completely negative result.

It might be, but generally you will not get growth on agar unless the agar has been touched by your fingers or any object, which will then transfer some bacteria. This is why you have to be careful not to touch the agar on the dipslides, and careful not to let the agar touch any object or surface, and only hold the dipslide by its plastic handle at the end.

There is nothing in my urine dipslide instructions (uploaded to this post) which says to compare to a control agar when performing a urine test.

Some more urine dipslide instruction manuals here (bottom of page).



If we do compare with healthy controls, it would be important to compare the same number of tests. In ME/CFS patients, if bacteria cannot be detected after 3 days of urine testing on a dipslide, then often the patients will take some additional dipslide tests to see if they can capture some bacteria. So in this case, it would be important to compare the results of the first 3 days of testing only.

 

[GlassCannonLife]

I don't recall seeing the dipslide images of @Nuno healthy controls, are you saying that there was some bacterial growth (dots, spots or a matt haze) on the healthy controls?

His CLED medium was green with some white looking spots (not on the picture in the legend for interpretation), and the control was also green but with very few amber spots (also not on that picture). The manual just has a textured green as "mixed growth". I'm not sure what to make of it.

The other medium strips were exactly the same between the two, yes, both low density enterococcus.

It might be, but generally you will not get growth on agar unless the agar has been touched by your fingers or any object, which will then transfer some bacteria. This is why you have to be careful not to touch the agar on the dipslides, and careful not to let the agar touch any object or surface, and only hold the dipslide by its plastic handle at the end.

There is nothing in my urine dipslide instructions (uploaded to this post) which says to compare to a control agar when performing a urine test.

If we do compare with healthy controls, it would be important to compare the same number of tests. In ME/CFS patients, if bacteria cannot be detected after 3 days of urine testing on a dipslide, then often the patients will take some additional dipslide tests to see if they can capture some bacteria. So in this case, it would be important to compare the results of the first 3 days of testing only.

Yeah sure, I'm not saying we have to do negative controls, and I realise you should be careful, but that is somewhat the point - that it might be useful to see if people are potentially contaminating them in some way by accident.

If the strips are cheap then it seems worthwhile to do it at least once to make sure that we aren't getting results inadvertently.

Unless you've already had a few trials where you had negatives even with your urine? It would be great if you could share photos of the results.

 

[Hip]

His CLED medium was green with some white looking spots (not on the picture in the legend for interpretation), and the control was also green but with very few amber spots (also not on that picture). The manual just has a textured green as "mixed growth". I'm not sure what to make of it.

If there are any spots or dots at all, then I believe it is a positive result, no matter how few.

These instructions for the Uricult Plus urine dipslide show what bacterial growth on the agars can look like.

 

[Hip]

Yeah sure, I'm not saying we have to do negative controls, and I realise you should be careful, but that is somewhat the point - that it might be useful to see if people are potentially contaminating them in some way by accident.

Yes true, that's good point.

I guess it will be hard to conduct a test with forum patients in any rigorous way. People may make errors which contaminate the agar, or may not understand what a positive result is.


In any case, even if it were demonstrated by proper published study that bacteria are found in ME/CFS patients urine much more frequently than in healthy controls, it would be interesting, but it would not prove that these bacteria are the cause of ME/CFS.

 

[GlassCannonLife]

If there are any spots or dots at all, then I believe it is a positive result, no matter how few.

These instructions for the Uricult Plus urine dipslide show what bacterial growth on the agars can look like.

Yes, those are the instructions that I had. Am I correct in interpreting that indeed any "green" result in the CLED medium is a "mixed result" ie both gram negative and gram positive? And a negative result would not be green. What colour is the CLED medium prior to testing? I thought I saw images of the medium online and it was green already so I wasn't sure..

In either case they both had a small amount of spotting (maybe 5-10x more on the ME sample, of white dots, and very few but still 3 or 4 amber dots on the control, as I said previously). The interpretation panel doesn't give these as options though, it just has a single green result and not green with various spots..

 

[GlassCannonLife]

Yes true, that's good point.

I guess it will be hard to conduct a test with forum patients in any rigorous way. People may make errors which contaminate the agar, or may not understand what a positive result is.


In any case, even if it were demonstrated by proper published study that bacteria are found in ME/CFS patients urine much more frequently than in healthy controls, it would be interesting, but it would not prove that these bacteria are the cause of ME/CFS.

Speaking of rigorous, I think we could definitely conduct it in a slightly more scientific way. If we're worried about people contaminating the strips and they're relatively cheap (no idea sorry), then including a negative control with some water for injection (cheap plastic single use tube for example) could be an option.

People could share their photos, and we could use a coded system eg everyone is assigned a number, and your two strips are just 1a and 1b. We then take a number of people who are interested, and they each go through all of the images and independently grade them based on the given information sheet. We keep it all blinded, and then reveal the results at the end of the scoring, taking either the most common classification per image or whatever seems most appropriate as a "mean" or "median" measurement (in this case "mode" made sense off the top of my head).

 

[Hip]

Am I correct in interpreting that indeed any "green" result in the CLED medium is a "mixed result" ie both gram negative and gram positive? And a negative result would not be green.

I have two types of urine dipslide, Uricult Plus and Dimanco. CLED is green on both of these; the MacConkey agar is red on the Uricult, and orange on the Dimanco.

Growth of bacteria on these agars is not detected by color change, but by the appearance of tiny spots.

Color changes can in certain circumstances can help identify the species of bacteria growing though.

CLED is designed to turn yellow when lactose positive bacteria are growing on it, and remains green when lactose negative bacteria are growing.

On MacConkey agar, lactose positive bacteria colonies turn red or pink on MacConkey agar, whereas lactose negative bacteria colonies do not change color, forming white colonies.

MacConkey agar will grow both gram positive and gram negative bacteria. Whereas MacConkey No.3 agar is a selective medium which will only grow gram negative bacteria. So if you have a urine dipslide with MacConkey No.3 (rather than regular MacConkey), that can help with bacterial identification.



I have been teaching myself a bit about bacterial identification tests. If you happen to have a microscope, then you can see certain identifying features using this: whether the bacteria are cocci (round shape) or bacilli (rod shape), and whether they can swim around (motility).

Another really easy bacterial identification test you can do at home is the catalase test: just place a few drops of hydrogen peroxide on the bacteria growing on agar, and if it fizzes up, then the bacterium is catalase positive (the catalase in the bacteria causes the hydrogen peroxide to break down releasing oxygen, hence the fizz).

I compiled a table of various easy-to-perform bacterial identification tests, and these can help identify the bacteria you have isolated on the agar. This table is attached as a pdf.

However, I think even for these simple tests, it may not be easy to interpret the results correctly unless you have some experience in bacteriology. But it's fun to perform these tests, as it helps learn about bacteria.

 

[Hip]

Speaking of rigorous, I think we could definitely conduct it in a slightly more scientific way. If we're worried about people contaminating the strips and they're relatively cheap (no idea sorry), then including a negative control with some water for injection (cheap plastic single use tube for example) could be an option.

People could share their photos, and we could use a coded system eg everyone is assigned a number, and your two strips are just 1a and 1b. We then take a number of people who are interested, and they each go through all of the images and independently grade them based on the given information sheet. We keep it all blinded, and then reveal the results at the end of the scoring, taking either the most common classification per image or whatever seems most appropriate as a "mean" or "median" measurement (in this case "mode" made sense off the top of my head).

That is a good plan, though maybe out energies should be directed at making ME/CFS researchers aware of this urine dysbiosis findings of Dr Markov, so that they can conduct and publish a replication study. That should have more impact that an informal study conducted on PR.

 

[BrightCandle]

Water should be boiled before introduction into the dipslide as water does usually contain bacteria and other live things in small quantities. Its one of the reason cpap users are recommended to use distilled water and the main substitute is a 0 TDS filtered water that is also boiled.

I shall set up a control today.

 

[Hip]

Water should be boiled before introduction into the dipslide as water does usually contain bacteria and other live things in small quantities. Its one of the reason cpap users are recommended to use distilled water and the main substitute is a 0 TDS filtered water that is also boiled.

Technically you need a pressure cooker temperatures of 121°C to fully sterilize water. Boiling water at 100°C for around 10 minutes will kill almost all organisms, but will not destroy bacterial spores in the water. To kill spores you need 121°C for about 20 minutes.

When hospitals sterilize medical equipment, they use autoclaves, which are in essence the same as a pressure cooker, and have an internal water temp of 121°C.


This incidentally is why even boiled rice is not safe to eat the next day, unless you store in a fridge. Rice contains bacterial spores, and boiling at 100°C does not kill them. So if the rice is left, these spores germinate into bacteria, and can cause food poisoning.

 

[Hufsamor]

I noticed last night that yandex.com, the Russian search engine, seem to be better than Google at finding products like urine dipslides.

I found lots of online stores selling CLED and MacConkey urine dipslides through Yandex, and I tabulated these into the pdf document attached below.

If you live in Continental Europe, you are in luck, as there are many places in Germany selling urine dipslides which can ship to you. Some of these German sellers will ship to the UK, others not.





Some of these online stores sell regular 2-agar CLED + MacConkey urine dipslides, as well as 3-agar urine dipslide with CLED + MacConkey + Enterococcus. The extra Enterococcus agar is designed to cultivate Enterococcus species bacteria (although Enterococcus bacteria will also grow on CLED). Enterococcus is the bacterium Dr Markov most commonly finds in CBIS.

I am going ask the Markov clinic whether there is any advantage of using these 3-agar urine dipslides.



Attachment: Online Stores Selling CLED + MacConkey Urine Dipslides:

@Hip I’ve downloaded your excellent overview, but I’m slightly confused.
For Europe, which one of the cheaper options would be the best by?
Did you ever ask dr markov?

Would the best option be CLED + MacConkey + Enterococcus. ?

Is enterococcal the same as enterococcus?:

• Uricult plus with CLED/MacConkey and enterococcal agar

For the moment I’m not going to do any treatment, I just want to test my healthy husband and myself.

 

[Hip]

For Europe, which one of the cheaper options would be the best by?
Did you ever ask dr markov?

Would the best option be CLED + MacConkey + Enterococcus. ?

As long as the dipslide contains CLED and MacConkey agars, you are fine. Those are the standard ones used in urine cultures in labs.

The Enterococcus agar is an additional one, on which only Enterococcus grows, so if you get bacteria growing on this Enterococcus agar, it's likely Enterococcus (although the Uricult Plus instructions say group B Streptococcus may also grow on the Enterococcus agar). So this is an aid to bacterial identification, but it is not a necessary requirement.


I don't know which would be the best buy for you, you would have to check prices and shipping costs, which are detailed in the pdf. All the dipslides linked to in that pdf are fine for urine testing, so you can buy any one which ships to your country.

Dr Markov does not use dipslides, he uses a similar product called a Diaslide, which is essentially the same thing, but not available online, as far as I could see.

Is enterococcal the same as enterococcus?:

Yes, adjective and noun.