All updates to make this article pertinent for today (April 3, 2002) appear in italics within the text.

Bacteria is a ubiquitous problem familiar to most dialysis processionals, and monitoring for it is not as easy as it may appear. There are many ways that staff and laboratory personnel can cause unintentional miscued results.

During a single dialysis treatment, the patient's blood may be exposed to as much as 150 liters of product (reverse osmosis {RO}, deionization {DI}) water. Therefore, maintaining bacterial counts at a minimum level in the product water is of utmost importance for two reasons:

1. The blood and dialysate are separated only by a thin, semi-permeable membrane--the dialyzer; and

2. The expansive use of dialyzer reprocessing systems.

Standards set by the Association for the Advancement of Medical Instrumentation (AAMI) and the US Food and Drug Administration (FDA) state that 200 cfu (colony forming units)/ml is the maximum allowable bacterial concentration in product water used for dialysis purposes. AAMI RD62: 2001 has also added an action level of 50 cfu/ml.  If the test is above 50, an action must be taken (such as disinfection of the loop and RO system and reculturing). The AAMI standard expounds that the total microbial count in proportioned dialysate should not exceed 2,000 cfu/ml. It further directs that voluntary endotoxin (lipopolysaccharide) levels in water used for reuse shall not exceed 1 ng/ml as demonstrated by the Limulus amebocyte lysate (LAL) assay. In the AAMI RD62: 2001 LAL testing is no longer voluntary, it is suggested for all water used for dialysis purposes and should not exceed 2 EU/ml. with an action level of 1 EU/ml.

If the water used for preparing dialysate for reused purposes or the dialysate itself exceed the allowable limit for bacteria and endotoxins, the dialysis patient can exhibit clinical signs such as shaking chills, fever, hypotension, myalgia, nausea and vomiting (pyrogenic reactions) and possible sepsis. The onset of these symptoms usually occur one to two hours after initiation of the treatment but may happen as early as hour.

BACTERIA GENERAL

First, let's review water borne bacteria. Gram negative bacteria (gram stain negative rods) and the nontuberculous mycobacteria genera (gram stain positive rods) are the primary microbial contaminants present in hemodialysis (HD) systems. Both organisms grow in water and have the ability to thrive on the few nutrients or organics that the water may contain (eg., water treated by RO).

The gram negative bacteria can proliferate rapidly in the water; therefore, bacterial counts can rise extremely high in a short period of time. They harbor endotoxins in their cell walls that can cause pyrogenic reactions in patients. Endotoxins are not destroyed by germicides, but the bacteria is easily killed during the germicides, but the bacteria is easily killed during the disinfection process unless it forms a glycocalyx (biofilm) that protects the bacteria. This protective film allows the microorganism to colonize in the HD systems and makes it more difficult to remove. Bacteria can start forming biofilm within 15 minutes after attaching to the side of piping and tubing.

Unlike the gram negative bacteria, the nontuberculous mycobacteria species grow slower in water. They don't form endotoxins and, therefore, do not cause pyrogenic reactions, but they are more resistant to chemical sterilants, which can pose a heath risk. While septicemia due to contaminated dialysate is rare, occurrences have been attributed to this bacterial due to either higher than acceptable levels of bacteria in the water used for dialyzer reprocessing or reuse germicide concentration inadequacies.

The goal is to prevent and control these microorganisms. A well designed water purification system and delivery loop is the first step in prevention. The best strategy is to properly disinfect the entire HD system, from the water treatment to the dialysis machines, on a routine basis according to the manufacturers' guidelines, and based on routine bacterial monitoring. Keep in mind that the testing for bacteria underestimates the bacterial bioburden of the system, and to disinfect on a routine basis, and not just when bacteria counts or endotoxin levels dictate.

AAMI RECOMMENDATIONS

AAMI recommends bacterial monitoring be performed monthly on the RO/DI product water and dialysate. The newest AAMI standard does not specify how often the bacteria and endotoxin samples should be taken but does state it must be often enough to determine that the system is within AAMI standards at all times.  Currently, the acceptable frequency for bacteria and endotoxin testing in non-problematic systems is once a month. The product water samples "shall be collected at a point where water enters the proportioner, or where it is placed in mixing tanks, depending on the type of system used for preparing dialysate." Bacteria testing on the water should be performed according to the newest AAMI standards, at all points of use.  These include dialysis machines, bicarbonate filling stations, reprocessing equipment, repair stations, anywhere water is dispensed for use in a dialysis facility. The dialysate specimens "shall be collected during or at the termination of dialysis at or beyond that point where the dialysate leaves the hemodialyzer." To test for worst case scenario, collect the dialysate sample exiting the dialyzer at the end of the day.

More frequent bacteria monitoring should be performed if the system is opened for any reason, a new piece of equipment is incorporated, or the water or dialysate culture result exhibits higher than allowable counts.

WORST CASE SCENARIO

The RO/DI water samples need to be drawn in relation to your worst case scenario. How do you know you only need to disinfect the RO system once a month if your unit is culturing after the monthly disinfection? This procedure only proves efficacy of the sanitation process, but it doesn't show if the frequency of disinfection is adequate. Draw your culture samples before you disinfect the water purification system and HD machines, and this will demonstrate if your procedures are sufficient.

In collecting samples, wear long sleeves and gloves and a mask. This will help prevent skin and respiratory bacteria contamination of the samples. Also, don't draw the specimens from a silastic or silicone type hose since many bacteria can colonize in this stagnate portion of tubing. Allow the sample port to flush for at least 30 seconds before collecting. Cleaning with betadine is optional. The CDC recommends not using betadine, bleach or other disinfectants to clean the sampling port, only flush with water.  The disinfectants will get into the sample and kill the bacteria, rendering a false negative result.

The "samples must be assayed with 30 minutes of collection, or immediately stored at 4-6 degrees C and assayed within 24 hours of collection."(1) If the samples are allowed to stand, any bacteria present will proliferate, resulting in a false positive result.

STANDARD PLATE COUNT

The laboratory should be instructed to do a standard plate count of viable microorganisms via a spread plate or membrane filter technique. The membrane filter technique is a more accurate way of testing for planktonic bacteria in the system.  Instead of one milliliter of fluid being tested, 1 liter is ran through a screen.  The screen is then placed on the agar to grow.  Think of this as a short video versus a snapshot of what is growing in the system. Commercial diptest devices (e.g. red colored Millipore samplers) are also an acceptable medium.  For accuracy, the laboratory may use a pipette to one-tenth of a milliliter.  The final bacteria count must be times ten to equal one milliliter. The calibrated loop method used for urine culturing cannot be used for water or dialysate sampling. This is not a sensitive enough test, and the minimum count is 1,000. If you frequently see results that have many zeroes, be suspicious that the calibrated loop method is being used. If you often have"no growth" test returns, investigate! Many times, the lab considers a low bacteria count as just contamination and doesn't report it. Also, since we are testing for planktonic (free-floating) bacteria, many no growth results may indicate a massive biofilm in the system, especially with a lot of down time and a dismal disinfection regime. Don't accept "negative" or "positive" as a result. Negative simply means less than 200 cfu/ml. Most dialysis units take aggressive action at much lower counts because of the tenacious nature of bacteria (an action level of >50 cfu/ml. is recommended by AAMI). A positive report means you are at or above 200 cfu/ml and beyond the AAMI maximum limit.

"Culture media should be trypticase soy agar or equivalent." Blood or chocolate agar should be avoided because it is too nutrient rich for the waterborne bacteria and actually kills these microorganisms. Dialysate samples should only be grown on trypticase soy agar (or equivalent) because the salt loving bacteria do well with this media. There is evidence that other agars may be better suited for growing bacteria such as R2A and TGEA types.  However these may be more expensive and more difficult to acquire.

The incubation temperature should be between 35-38 degrees C, and the colonies should be counted after a 48 hour period using a magnifying device.  However, there are some slower growing forms of water-borne bacteria that may just begin showing at this hour, so incubating to 72 hours has some merit. Many laboratories incubate at the lower 35 degree C temperature instead of 37 degree C, so the AAMI standard was recently changed to accommodate that fact.

Lastly, when you receive your valuable lab slips, do not tape them away in a notebook. This makes it difficult to track the trends in your dialysis equipment. Rather, make a spreadsheet that contains all the stations and numbered sample ports where the microbial specimens were drawn, including on it all the months of the year and the results in written or graph format. In other words, perform trend analysis (see example).

Keep open the lines of communication with staff and laboratory personnel. Let them know your special dialysis requirements and your concerns.

By following the above recommendations, you will not be "bugged with bugs" nearly as much as you might otherwise be. And, if you do see a high count, you'll know it's true--but check anyway.

REFERENCES

1. Association for the Advancement of Medical Instrumentation (AAMI): American National Standard: Hemodialysis Systems (ANSI/AAMI RD5-1982, 1993 revision). Arlington, VA: AAMI, 1982

2. Food and Drug Administration (FDA): A manual on Water Treatment for Hemodialysis (section 2.2.2). Rockville, MD; FDA, 1989

3. Association for the Advancement of Medical Instrumentation (AAMI): Reuse of Hemodialyzers (AAMI ROH-1986). Arlington, VA: AAMI, 1986

4. Favero, MS, et al. Factors that Influence Microbial Contamination of Fluids Associated with Hemodialysis Machines. Appl Microbial 28:822-830, 1974

5. Favero, MS, et al. Gram Negative Water Bacteria in Hemodialysis Systems. Helath Laboratory Science 12:321-334, 1975

6. Bland, LA, et al. Microbial Contamination Control Strategies for Hemodialysis Systems. PTSM Series/No.3:30-36, 1989

7. Carson, LA, et al. Growth Characteristics of Atypical Mycobacteria in Water and Their Comparative Resistance to Disinfectants. Appl Environ Microbial 36:839-846, 1978

8. Gordon, SM, et al. Pyrogenic Reactions Associated With the Reuse of Disposable Hollow-fiber Hemodialyzers. JAMA 260:2077-2081, 1988