Why does the dialysis community still culture bacteria?

A lot of money is spent taking, shipping, culturing, counting, and reporting bacteria in water and dialysate.

I write as a taxpayer concerned about our out of control health costs – the biggest issue of our day. I have no financial interest in the LAL reagent or testing business.

Years ago a correlation was noted between high levels of bacteria in the dialysate and dialysis patient health. The mechanism of this correlation was not understood. The analytical tool available for managing the threat was to take samples and culture them on a specific growth medium at a specified temperature and for a specified time.

Today this bacteriological test mandates that water samples be assayed within 30 minutes, or stored at 4-6 degrees Celsius and assayed within 24 hours. The bacteria are to be cultured on tryptic soy agar medium and cultured for 48 hours at 35-37 degrees Celsius.

This test does not measure all the bacteria that are in the water but is only a rough indicator of sterility. Different growth media and longer incubation at a different temperature can detect other strains of bacteria that do not show up with the standard procedure. The sample size of 1 milliliter is also limiting; if one really wants to find all the bacteria, he/she will filter many liters on a submicron filter and then incubate or examine the filter microscopically.

Fortunately science advances and it has been discovered and acknowledged that the threat never was from planktonic bacteria in the dialysate per se, but rather the metabolic byproducts of bacteria we call endotoxins or pyrogens. A solution can be completely sterile (no viable bacteria) and still be full of endotoxins.

The old test for pyrogenicity was to inject a rabbit with a sample of the fluid and then monitor its body temperature. Then the LAL test tool was discovered. The pyrogenicity could be determined in a two hour (pass or fail) test and the gross level measured by serial dilutions. Later the test was improved by measuring the rate of floc formation which gives an exact level of pyrogenicity.

It was also realized that, typically, the pyrogens seen in water or dialysate did not come from the potable water supply passing though all the filtration, but indeed came from biofilms growing on the wetted surfaces of the purified water system.

So other than tradition, why de we continue to sample and incubate dialysis fluids? Plate counts are not a very good indicator, they don’t give results for 2-3 days, and it is common to contaminate the sample and receive a false high count.

Stephen,

The only answer that I can think of is because AAMI in their infinite (actually finite) wisdom says we have to.

I do personally know of at least one instance recently (last month) where the RO water did have a high colony count and endotoxin level despite multiple disinfections. We determined the problem to be either a minute pinhole in a membrane or bad o-rings. The RO continued to produce AAMI quality water (chemically). What pointed us to the membranes and/or o-rings is, we noted that just a few days prior to the cultures the TDS rose from consistently being 3 up to 5 and the % rejection had dropped from 99% down to 97.5%.

I'm no microbiologist but as far as I know, waterborne bacteria are too large to pass through a dialyzer membrane. Though, I do think I recall reading a paper that said that bacteria just being in proximity to the membrane can cause an inflammitary responce in a patient. If this is true or not I have no idea.

The only part of your post that I really don't agree with is...

quote:

It was also realized that, typically, the pyrogens seen in water or dialysate did not come from the potable water supply passing though all the filtration, but indeed came from biofilms growing on the wetted surfaces of the purified water system.

A number of years ago we had an endotoxin issue at one of our facilities. I sent a TAP water sample to the lab and it was reported as being >10.0 eu/ml. I sent another sample with a special request for an actual count, it was reported as 80 eu/ml if I remember correctly.

My point being, endotoxin can be/is present in potable water.

I did not mean to imply that there are not pyrogens in domestic water or that they would not pass through an imperfect filter. Despite chlorine and chloramine, there are still biofilms in water distribution systems. Certainly pyrogens would be expected downstream of a carbon filter. Rather, I meant that even presuming a perfect filtration of incoming endotoxin, biofilm will develop in purified distribution piping and must be controlled by periodic disinfection.

Stephen,

Do you feel it is not necessary to perform colony counts at all? I have had some test points where there will be some bacterial growth; but the LAL will be <0.1, which is as low a Spectra goes. I would say contamination could be a cause, but it is always the same test points in the distribution system.

Don't you think performing both tests gives you a better indication of what is going on with your water treatment system?

Good question Stephen.
I see the advantages of the filtration vs incubation only, but are there disadvantages to the filtration method, or has it just been overlooked?

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

Dave. If the same test points consistently yield bacteria then it seems likely that disinfectant is not reaching these points adequately or that bacteria have colonized the test point downstream of the sampling valve. Are these test points the same where the LAL samples are taken?

B. I am not suggesting that more sensitive tests (larger sample, alternative growth media, alternate incubation time or temperature) be used. The bacteria themselves are not the problem. I would expect the more sensitive tests would also be much more costly.

Stephen,
I understand and share your concern about not wasting money and time, but I think testing for endos only is not the best marker for growth or disinfection. Correct me if I'm wrong because my understanding may be off base.
Live bacteria may not 'shed' many endos. So, where a culture would show growth, an LAL may not show endos. If not addressed, the bacteria may colonize and form biofilm, which would then be seen by the LAL, but would be too late.
IMO endos & cultures, together, serve as a much better marker.

August 19, 2009
The quotes that follow come from Theodore Meltzer’s authoritative book “High Purity Water Preparation.”
Planktonic (free floating) bacteria that are measured in a water sample either enter the water by passing through defects or seal leaks in the RO membrane, from atmospheric contact, or from biofilm on the piping surfaces. In high purity water systems “…the majority of bacteria are attached to surfaces. The ratio of sessile to planktonic organisms (i.e bacteria per square centimeter to bacteria per milliliter) may exceed 10,000.”
The bacteria colonize surfaces because that is where their ‘food’ attaches. “The situation, then, is that neither smoothness of surface, nor flow velocities long delay the advent of biofilm formation…Smoother surfaces delay the initial build-up of attached bacteria but do not reduce the total number attached. A similar observation has been made for the influence of flowrates on bacterial attachment.”
If the planktonic bacteria measured came by passing through the RO membrane, then even a 100% effective disinfection of the distribution loop would not reduce the concentration measured.
Besides being the actual health threat, the measurement of pyrogens by the LAL test is much more sensitive than a culture test of viable bacteria. There is also the situation that biofilm growth will increase dramatically in the typical 72 hours before getting results.
Don’t expect the laboratory industry to give up a good source of income without a fight and propaganda.

Good points Stephen. I guess in Brian's little world I like to think that my RO produces perfectly pure water and my pipes are perfectly clean....
OK, you got my vote.

Cultures can be processed in a way to get much more accurate results. The point of culturing is not exactness but consistency. It is quality control of your disinfection schedule. The usefulness comes in a comparison to the last month or two and other ports or machines. Cultures and endotoxin testing are just a small part of the picture-but an important part. You can not stick your results in a file and forget about them. It will eventually come back to bite you. One day you will have a change is water pressure and it will knock off that little piece of biofilm that was giving you problems at a different port each time. It was small and had not been shedding enough endotoxin to measure until it fell off in a chunk. Now it is Friday afternoon and you have several endotoxins that are >10. I have seen this happen on several occasions. Each little step has it's place in the process

I don’t dispute the importance of tracking test results over time. However 0 cfu/ml does not mean no bacteria are present. LAL testing is going to be more sensitive and a better indicator of biofilm than a 1 ml sample of planktonic viable bacteria. The problem is more not having a sufficiently sensitive measure of LAL. A 1.0 EU/ml action level is not very meaningful. Moreover, if the problem explodes on Friday afternoon, and you did sample to detect it, you wouldn’t know it for 2-3 days by culturing it.

Stephen you make very good points. I too have often wondered about the rational of bacteria and LAL testing. Does anyone know when AAMI plans to meet again to review standards? How often do they do this? I am currently working with CSA and we are reviewing dialysis standards with regards to water. Funny thing is though we are basically mirroring what AAMI and ISO do so I doubt we will come up with any major changes. Stephen you sound like a great candidate for an AAMI standards review committee.

Great post but seems like should be in technical. I measured for bacteria in my second carbon tank and found some but not a real lot. How am I going to know what's really in there?

quote:

Originally posted by North:
Stephen you sound like a great candidate for an AAMI standards review committee.

They wouldn't let him on the committee, he has too much common sense.

Chuck