Does anyone know when endotoxins reach their highest levels in dialysis equipment? Do endotoxins increase immediately after disinfection, due to the destruction of the cell wall? Bacterial levels reach their maximum levels the longer the duration between disnfection. However, are endotoxins elevated immediately after disnfection and reflect the effect of the disinfecton and the breakdown of the cell wall, and the subsequent release of endotoxins?

I have never really tested this but my assumption would be that they would be at their highest level sometime within 48 hours after a sanitization if you have bacteria levels in your loop. Our water provider waits for 48 hours after a sani to test for LALs to give any dead bacteria time to flush through the system or preferreably get caught in the ultrafilter. If your system is free of bacteria then it really wouldn't be possible for endotoxins to be there. How many colony forming units does it take to create an endotoxin level above the limits, I don't know. What you could do if you have bacteria counts present before you sani your system. After the sani run LAL test every 12 hours for the next 48 hours just to see. This definitely would be much easier if you are doing your LALs in house instead of sending them out and much cheaper. I do not know of any studies that have looked into this but if I really wanted to know that is how I would test it. You don't have to do them every 12 hours, you could start by doing them every 24. But over all my assumption would be that the LALs would be at their highest levels immediately after the sani then as your system rinses and runs through your ultrafilter the endotoxins would be caught there and in turn lower the exdotoxin count as time goes on.

jdbiomed,
My assumption would be the same as joebiomed, except I would think the UFs would take out the endos much sooner. Great question.

To play the role of Devil's Advocate:
Isn't the intent of the water quality standard to protect the patient and wouldn't this goal best be served by sampling at worst case?

Olddog,

There isn't a "WORST" case. The longer your wait the more bacteria less endotoxins, the less you wait the more endotoxins less bacteria

The worst case would be to test immediantly before and immediantly after disinfection would it?

I have not tested this out either.

Keep in mind that as you flush out your disinfectant, you are also flushing out your endotoxins. Therefore, I would think that the endotoxins are the highest just before disinfection when the most bacteria is present. You may have more endotoxin if you have a lot of biofilm. With biofilm, you may not see the bacteria, but you may see the endotoxin.

Thanks for all the excellent responses. The question came to mind recently when we were discussing "worst case" scenarios for sampling water. Of course, we need to test during our "worst case" scenario but we weren't sure when that would be for endotoxins. As far as I know, the source of endotoxins, is dead bacteria and live bacteria. When the cell lyses it sheds endotoxins. I guess we need a thorough testing of water throughout the entire "cycle" leading up to each disinfection in order to determine when it is best to test for endoxins.

I think it might go like this. You can have bacteria and no endotoxins but if you have endotoxins you also have bacteria. Since it takes bacteria to cause endotoxins (the broken particles of dead bacteria). Is there always endotoxins present if there is bacteria, I'm not sure. What in a system would cause the bacteria to die or rupture? If nothing is destroying the bacteria wouldn't they just continue to multiply or do they have a short life span? From what I understand is that they continue to multiply. If bacteria is present and you do your sani ideally you are killing off the bacteria and the endotoxin levels would probably be at the highest when the disinfectant is in the system, but you probably can't get a good test in that situation and that also is not a normal operating condition for patient care. I would have to say that highest level of endotoxins would be after the disinfectant is cleared out and system is ready to treat patients. I would be very interested to hear what you find if you do this testing. I think I would do the test immediately after the disinfectant is cleared and maybe repeat every 24 hours for the next 48 or more and I would check the levels at the pre uf and the return. You definitely got my curiousity going on this one.

I think a good working definition for "worst case" would be: immediately prior to disinfection. Test for both cultures and LALs, but look a the big picture. If you test immediately prior to disinfect and one of these tests comes back high and you repeat it and it is acceptable, you really don't know much because you just disinfected. Test again, cultures and LALs always, a few days or a week later. If the results are good, test again in a week, etc. to determine if you are maintaining good counts until the next "worst case" which, per the regs, should be one month. If you have established this pattern, re-check periodically. If you are not maintaining good results you either have a point of "infection" that you are not getting at and/or you have a biofilm problem.

Fellow technicians:
This has been one of the most simulating and thoughtful discussions I have seen on the site in quite a while.
Thank-you

There seems to be some confusion as to the source of bacteria and endotoxins in the dialysis water distribution system. The bacteria that are detected in a grab sample primarily result from the biofilm on the piping surfaces. If the majority of the bacteria and/or endotoxins were to originate upstream by passing through leaks or defects in the RO membrane, then disinfection of the distribution loop would do nothing to reduce their number.

Endotoxins are continuously released from the biofilm – not just when the bacteria colonies are destroyed by a chemical disinfectant.

If the disinfection successfully removes the biofilm, then after rinse out, the endotoxins should be at a minimal level. If the biofilm persists after the disinfection and rinse out then it is not clear what would happen. Yes, testing of such a situation would be revealing.

Assuming that the biofilm is removed by each disinfection, then the highest pyrogen level would be expected just prior to disinfection.

Are you saying that if you are getting counts even below the action level that that is and indication that there is a biofilm present? I thought it took quite and accumulation of bacteria to form a biofilm. If you are seeing low levels of bacteria does it always mean that the bacteria has grabbed onto something in the pipe and has started to grow a biofilm?

Neither membrane nor seals are perfect so some bacteria and pyrogens can be expected in RO product water, especially if the feed levels are high or if biofilm has established itself on the feed surface or downstream of the RO membrane on the p[roduct side. This is the primary reason for also disinfecting the RO itself. (Sometimes an RO may need disinfection because growth on the feed side of the membrane is causing reduced flux or rejection).

Still, the presence of contamination in the distribution system immediately after disinfection may be the result of biofilm that was not removed completely from some portion of the piping or tank walls. Comparison of the contamination level at the RO versus the loop should confirm the origin.

This message has been edited. Last edited by: StephenCCarr,

I know this is off topic but I've always wondered why we don't use better and more effective chemicals or processes to disinfect our systems. I'm no microbiologist, but I've read that bleach in any concentration is a very poor killer if biofilm. It seems like biofilm is our biggest enemy. There are other things like heat, chlorine dioxide, and ozone that do a much better job at removing biofilm, or so I have read. Anyone know if there is any truth to this? It is apparent that 1:100, 1:10, and even straight bleach is just not getting the job done in the water loop.