2002/2 EDTNA/ERCA Journal Club Discussion Summary 

The February 2002 EDTNA/ERCA Renal Care Journal Club discussed the paper entitled 'Dialysis Fluid Contamination of Pathways and Life of Microbes ' by Dr. Rolf NYSTRAND (Sweden) – EDTNA/ERCA Journal XXVII, n° 3 July-September 2001, ISSN 1019-083x.

Without doubt this paper is of great importance in order to obtain more knowledge and recommendations about what has to be observed and performed to get and keep the dialysis fluid at an acceptable level. It is important, not only the number of micro organisms, but mainly what these micro organisms do in the fluid systems. Inlet lines and couplings ( dialysis fluid + concentrates ) are seldom sufficiently disinfected and consequently microbial growth is seen in these “ weak “ parts. The only way to ensure good microbiological quality of the water system is to disinfect, and disinfect preventively! This must be carried out frequently so that micro organisms do not get the necessary time to multiply. Very little is known about micro organisms in dialysis fluid systems as they have never been of economic interest. Disadvantages of microbiological tests are that it takes a long time before results are available and not all micro organisms will grow in the lab! Disinfection performed less frequent than twice a week cannot prevent growth in a system.

This paper was discussed and circulated in a group of 475 JC Members within the world. Anyone interested in Renal Care can simply become a JC Member by subscribing for free at the EDTNA/ERCA website ) at the Journal Club section; or by sending me your email-address and full name at devosjy@skynet.be

The paper has been actively discussed between Lee FISCHBACH (USA), Roelf SMIT ( the Netherlands ), Stu (Canada ), Ian Morgan ( UK ), André STRAGIER ( Belgium ), Matthew ARDUINO (USA), Frans VOGELS ( the Netherlands ),Hans TRAEGER (Germany), Dave Weatherill (Canada), Jean- Yves DE VOS (Belgium ) and Dr. Rolf NYSTRAND ( Sweden ).

DISCUSSION SUMMARY

What about microbial growth in acid concentrates?

Microbial growth in a central acid distribution system is an hygienic problem. In principle the systems used for medical purposes must not be contaminated. In principle they should be constructed in such a way that it is possible for the user to disinfect. Microbial growth in acid concentrate lines takes a long time to be seen in samples. Numbers are low in the fluid but significant in the system. For all three groups of organisms ( yeast, fungi, gram negative bacteria ) the counts are from 0.1 – 100 CFU/ml. All bacterial species have been gram negative. Yeast and fungi show gram positive gramstaining reactions. Special measures to prevent this growth are not more than what should be general practice for all fluid systems: preventive disinfection. Glucose in connection with acid concentrate becomes more and more interest and than conditions may change. The acid concentrate without glucose is an environment that is more challenging for the micro organisms compared to water, but the amount of nutrient will be higher. It takes longer time until a micro organism that finds the conditions excellent comes, but when it is there, it grows good.

What is CFU?

CFU means Colony Forming Unit. Such a unit can be everything from 1 to 10,000 living cells which keeps together. The CFU is in reality very seldom 1 but most often 10 to 1,000 cells.

What about Ultra filters and on-line substitution fluid producing systems?

There are different systems on the market. Some have two ultra filters in series, some have three ultra filters in series of which the last normally is a single use ultra filter. More complex molecules will be adsorbed when more filters are used, especially if new filters are used. Adsorption will be the mechanism for low molecular weight molecules. Large molecules will be filtered off.

What about Ultra filters to cope with poor quality feed water?

Ultra filters used for this purpose are efficient in removing microbial cells. For endotoxins however it is little more tricky. An area of some concern is microbial metabolites. For small molecules there is not much of reduction, especially not in a single step filtration ( = one filter only ). With respect to the efficiency the kind of the membrane and the standing time are factors of importance.

How long can canisters with concentrates be used once opened?

Canisters of today are of good quality microbiologically seen. The standing time in open state is a parameter controlled by the user. For acid concentrates 24 hours is advised to be the maximum. For sodium bicarbonate concentrates 12 hours is advised to be the maximum. These are acceptable limits of time from a microbiological standpoint.

Which temperature must be reached and for how long in heat disinfection?

The temperature which should be achieved is 85°C. Micro organisms, depending on type, are starting to be damaged already at about 60°C. This does not mean that they will be killed, but that they will have to spend some time to repair damages in order to have the most sophisticated mechanism, the one of reproduction, in order again. And by that time the next disinfection should not be far away! Time of exposure is decided by several factors: surfaces of the system which must be heated up as growth generally occurs there; the sensitivity of the micro organisms to heat; the amount of organisms to be inactivated. This means that a system has to be heated up for up to a couple of hours depending on the temperature. But as for all methods of disinfection it is the frequency which is most important. It is better to disturb a system often with maybe not the best method compared to seldom ( too little ) with a better method. Reverse Osmosis systems as such should be disinfected at least twice a week. Acid concentrate systems need a disinfection frequency of once a month. However when glucose is used once a week may be necessary. Do not forget it is not only the water but also the water system that must be in a good hygienic shape! Heat disinfection is to be used in a proactive way from the first minute. A clean system is in favour as less food for organisms is there. The whole idea is to integrate maintenance in the systems so that cleaning and disinfection is done automatically with a low degree of work from the staff. When heat is used, insulation of piping is necessary to keep up the temperature along the whole system and to protect staff against burning. Warning signs are necessary at taps saying that there might be hot water!

What about Hansen couplings?

The couplings to the dialyser are often complex in structure and only the fluid path can be considered in principle to be disinfected. There are however areas not being part of the disinfection, including O-rings. A more simple design would be appreciated. Hansen couplings can be best submerged in hot water. Use a water boiler, used as for heating tea water, heat up the water to boil, submerge one coupling after another. Do not use the Hansen couplings for microbial sampling!

Which method – spread plate or membrane filtration – is best for cultivating micro organisms?

This is a question of how much is in the sample. If there is from 5 CFU/ml up to around 5,000 CFU/ml, spread plate with 0.2 ml on the plate is the method of choice. If lower counts are expected, membrane filtration will say the number. The volume to be filtered is depending upon the cleanliness of the fluid: 100 ml up to 1000 ml. It is cheaper to have a spread plate technique.

What is the difference between TGEA and R2A?

Tryptone Glucose Extract Agar (TGEA) is like home cooked food and Reasoner’s 2 Agar ( R2A) is like French cuisine. What is used and what is recommended? A cultivation technique consists basically of three main components: medium, temperature and time. Other components are pH, redox potential, oxygen, … As different countries and different branches of microbiology has different opinions based upon what they are looking for the result is many different cultivation techniques.

In the European Pharmacopoeia medium B ( Tryptone Soya Agar (TSA)) is recommended.

Time and temperature has a large influence.

Temperature should be low in order to allow as many different species as possible to grow. A wise philosophy is to see what are the temperatures in the system from where the samples come.

Time should be long: 7 days. For concentrates 14 days are recommended.

However, time of incubation 48 hrs, 5 days , 7 days, or … are arbitrary cut offs based on a particular standard. One must realize that there are slow growers and injured cells that require much greater time to grow. The longer one holds media the higher the number of organisms that will recover. As for injured cells we can speak about viable but not culturable (VBNC) organisms that are found in water samples. So with culture methods we are still only recovering a fraction of organisms that may be present. If we compare TSA, R2A and TGEA under the same time and temperature conditions, we find that TSA will often give about 10 to 100 times less colonies compared to the others. So TSA is not a good choice. TGEA and R2A are almost equal; TGEA is just a little better than R2A Regarding recommendations in Pharmacopoeias it is normally allowed to use other techniques as long as can be shown that they are better or equal compared to the recommended technique.

Which medium is to be used for bicarbonate concentrate cultivation?

Media with supplementation of 2% to 8% sodium bicarbonate were tested and 4% was found to be the optimum. It has not be found necessary to use this supplementation for bicarbonate dialysis fluid.

What about Fungi and Yeast?

Fungi is best on Malt Extract Agar (MEA). Yeast is best on TGEA.

Fungi and yeast do adhere very well to surfaces. They only occur in systems not disinfected with high frequency. In a well maintained system they do not occur and in others they are there but the full power never shows up. Fungi consists of a net of thin threads and spores can be formed. The threads are quite sensible but the spores are more resistant. Therefore fungi is hard to eradicate especially if the frequency of disinfection is low. When fungi is seen in a sample there is a lot of it in the system!

What about growth in a water system before Reverse Osmosis?

Here we always have microbes present in much larger numbers than we think. The important issue is that there are much more than we can see in our normal analyses or what we will be told by the water plant. The metabolites which are harmful will be removed by RO. Today we see the RO as the border and this is effective. If we try to do something before the RO we just move the problem to another point and we will have a larger system to maintain. As we still in most cases do not have full control over the real critical area, the one downstream the RO, it is far more important to solve that than putting resources in an area which is not that critical.

How to remove biofilm?

Due to the diversity of micro organisms there exists no number one method to remove biofilm. In principle, when a biofilm is formed, the frequency of disinfection must be increased as there is a lot of nutrients present which means that growth can occur. Hot water is the best in order to dissolve biofilm.

What about Ultra Violet lamps?

An UV-lamp does not harm but it is not doing much. Only organisms on the sunny side will be damaged and most bacteria have rather effective repair systems for UV damages. The UV light acts through damaging the DNA. If the damages are below a certain degree the cell has repair enzymes which fix the cell. The repair action will take several hours. The UV lamp is only effective were the light is. It reduces the number of living cells in the fluid phase passing by but does not influence surfaces beyond the lighted zone. Therefore it must always be accompanied by chemical disinfection. High Energy Ultra Violet sterilizers are used for the destruction of chloramines and chlorine prior to RO systems in Canada. UV lamps are replaced every 8000 hours. Quartz sleeves every 16000 hours. Installation of two UV systems in series are recommended.

Carbon Filters and Softeners: which one comes best first in the system?

A carbon filter offers good conditions for certain organisms and a softener, with different physical and chemical conditions, offer good conditions for other species. With respect to this it does not make any difference which one comes first. One point to make is that a carbon filter may adsorb some organic matter which in the case it is placed prior to the softener will not enter the softener.

Some general comments to the conception of sterility:

The word STERILE means that in 1 000 000 units of whatever kind processed with a certain process it is allowed to find 1 living micro organism. Sterility we do not have in dialysis fluid systems like water systems or central concentrate distribution systems. We better speak about disinfection. There is however one issue in dialysis where the item sterile applies: the production of substitution fluid for HDF/HF. That fluid when it leaves the machine to enter the patient must be sterile!

Closing words:

Today we have the European (EDTNA/ERCA ) recommendations for the control and monitoring of microbiological contamination in dialysis fluids on its way.

We have the new American (AAMI) recommendations.

One thing all new recommendations have in common is they are more stringent compared to the previous. But they do not give much in how to do in practice! This we will have to learn ourselves apparently.

Jean-Yves DE VOS (BELGIUM)
EDTNA/ERCA JC Manager