Reflection About The Hemodialysis Water Microbiological Quality in Brazil Ⅲ

Current legislation on dialysis water in Brazil 

The criteria used for the evaluation of dialysis water arose through the awareness of competent authorities regarding the potential risk to which patients undergoing treatment were exposed (Faria, 2011).

HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK?

The Resolution of the Collegiate Board of Directors (RDC) nº 33/2008 provides for the technical regulation for planning, programming, elaboration, evaluation, and approval of Water Treatment and Distribution Systems for Hemodialysis in the Brazilian health regulatory agency (Anvisa, 2008). 

RDC nº 11/2014 provides for the Good Practice requirements for Dialysis Services, it applies to all dialysis services, whether public, private, philanthropic, civilian, or military, including those performing teaching and researching activities. It also determines that the samples for microbiological analyses are collected at least monthly at the point of return of the distribution loop and in one of the points in the processing room. In addition, it determines that the water microbiological quality has to be verified whenever there are pyrogenic manifestations, bacteremia, or suspicion of sepsis in patients on dialysis. This RDC nº 11/2014 establishes the water quality standard for dialysis, with the biological and microbiological attributes being highlighted in Table 1 (Anvisa, 2014). 

TABLE I - Biological and microbiological quality standard for dialysis water

The International Organization for Standardization (ISO) in its standard nº 13959:2014 provides for hemodialysis water and related therapies, specifying as a quality standard a total of microbiological count less than 100 CFU / mL or less if so, provided in local legislation. It also specified levels of endotoxins below of 0.25UE / mL or equally minor if so, provided by local legislation (International Organization for Standardization, 2014).

CONVENTIONAL AND ALTERNATIVE MICROBIOLOGICAL METHODS

Heterotrophic bacteria enumeration

It can be considered as an initial mark of the microbiological tests when in the middle of 1610 and, in 1665 the microscopes were invented by Galileo Galilei and Van Leeuwenhoek respectively. Although Leeuwenhoek was probably not the first to observe bacteria and protozoa, he was the first to make reports with drawings and descriptions of his observations. Among these, in 1675, he described living things present in water (Dias, 2018; Pelczar, Reid, Chan, 1980). 

However, German scientists had observed the growth of colonies in boiled potatoes, which characterized the practice of microbial cultivation and the development of culture media. It was Koch, who initiated the cultivation of microorganisms in a solid medium. He named agar the substance extracted from algae that could solidify at room temperature. Richard Petri developed a glass plate for depositing the culture medium (Jay, 2001; Pelczar, Reid, Chan, 1996). 

After 200 years of the discovery of living beings in water by Leeuwenhoek, Louis Pasteur, Robert Koch, Theobald Smith and a few other scientists associated microscopic beings with diseases. Joseph Lister, in 1878, obtained the first pure cultures of bacteria employing serial dilutions in a liquid medium (Pelczar, Reid, Chan, 1980). 

At the end of the 19th century, cultivation techniques were adopted to analyze the quality of potable water. For E. coli analysis and other coliforms, culture broth, through the most probable number (MPN), became the main method, as well as Koch's agar medium or solid medium for the total count, both of which have gone through a few modifications. In 1950 the use of membrane filters for bacterial enumeration was introduced (Pelczar, Reid, Chan, 1996; Sartory, Watkins, 1999). 

MNP is simple but requires a longer time of analysis (up to 5 days), whereas in membrane filtration presumptive results are available after 3-5 days of incubation (Anvisa, 2019). As for the solid medium, one of the options consists of plaque counting agar (PCA), media poor in nutrients and unsuitable for bacteria recovery that is already stressed in water. Nutritionally weaker culture media may also be used, for example, because they can recover a larger number of bacteria, but not the entire viable population (Sartory, Watkins, 1999). Reasoner's 2A Agar (R2A), is an example of nutritionally weaker culture media and is the most recommended for water microbiological analysis (USP, 2017).

Currently, there are many conventional microbiological methods, such as plate method, membrane filtration, and multiple tubes by the MNP process (Anvisa, 2019). Although they are simple, efficient, and economical, they have some limitations: low selectivity of the culture medium, variability of the biological response, and late results in the detection of microorganisms compromising the time to determine preventive measures to reduce patients' injuries (Anvisa, 2019). 

In an attempt to minimize these limitations, alternative microbiological methods have been developed to provide a higher level of quality to the tests, greater sensitivity, and faster results, allowing corrective actions to be taken early (Anvisa, 2019)

Bacterial Endotoxins 

Theodor Billroth, in 1865, used the term pyrogen to refer to substances that caused fever (Kikkert, Groot, Aarden, 2008; Medical Staff Conference, 1978). Richard Pfeiffer's studies on cholera in 1892, which were encouraged by Robert Koch, consecrate him as the father of endotoxin for his discovery (Rietschel, Cavaillon, 2003). 

In 1942, the pyrogen test by in vivo method was added in the American Pharmacopoeia in its twelfth edition (Mc Closky et al., 1943). Since its inclusion, this test has been widely used to evaluate the contamination by pyrogens in parenteral drugs (Kikkert, Groot, Aarden, 2008). However, only in 1976, this test was included in the Brazilian Pharmacopoeia (Navega et al., 2015).

The test is based on the measurement of the rabbit's febrile response after intravenous injection of the test solution, and the interpretation of the results is used to characterize the biological control (Anvisa, 2019). There are, however, some limitations, such as animal management, biological variability, and ethical issues. These aspects encouraged researchers to develop alternative methods for the in vivo test of pyrogens (Kikkert, Groot, Aarden, 2008). In their research, Levin and Bang (1964), quoted by Kikkert, Groot, and Aarden (2008), observed that Escherichia coli endotoxin caused clotting in the hemolymph of the crab Limulus polyphemus.

The Limulus Amebocyte Lysate (LAL) test, was added in the American Pharmacopoeia in 1980, whereas it was included in the Brazilian Pharmacopoeia only in 1996 (Farmacopéia, 1996; USP, 1980). There are two types of LAL tests, the first is the semi-quantitative coagulation test, which is based on gel formation; the second one is the photometric, a quantitative test, which can be divided into a chromogenic method that is based on color development, and the turbidimetric method which is based on turbidity development (Anvisa, 2019).

However, LAL tests have some limitations such as the variability of the analyst technique, and the error inherent to the instruments used, compromising the analysis quality, in this context alternative methods that eliminate these limitations are desirable (Anvisa, 2019; Charles River, 2017; Lemgruber et al.,2011)

Alternative Microbiological Methods 

Alternative microbiological methods are desirable when it is sought to overcome the limitations of conventional methods. In different compendia, alternative methods are classified into qualitative, quantitative, or identification (Anvisa, 2019; PDA, 2013; USP, 2017). 

The Brazilian Pharmacopoeia highlights the main methods: viability-based, growth-based, and cellular component-based (Anvisa, 2019). The main types of alternative microbiological methods and their respective technologies are described in Table II. 

TABLE II - Main types of alternative microbiological methods and their respective technologies

Despite the importance of using rapid alternative methods to provide not only the possibility of corrective actions in real-time but also greater patient safety, few studies are being conducted to demonstrate their applicability in the field of dialysis-treated water analysis (Anvisa, 2019). Riepl et al. (2011) evaluated the applicability of solid-phase cytometry, revealed by epifluorescence microscopy, and observed a high correlation between the conventional and the alternative method.

The solid-phase cytometry application in dialysis water microbiological analysis is promising because, besides being reliable, it is a fast method, since the analysis time is around three hours, and its use is suggested for monitoring not only the water quality but also the dialysis fluid (Canaud, 2011; Riepl et al., 2011).

Cytometry, besides being a fast method, also allows the detection of viable non-cultivable microorganisms. These microorganisms are responsible for results divergences between the laboratory experiments and real water microbiota. They are unable to divide and form colonies, even though they have an active metabolic mechanism and remain alive. Many bacteria, especially gram-negative, have this ability. Mycobacterium species can be highlighted as potential VNC (Anvisa, 2019; Joux, Lebaron, 2000; Díaz et al., 2010; Sandle, 2015).

Cellular component-based techniques as 16S rDNA PCR analysis is also a promising technique because it is independent of culture and therefore able to detect non-cultivable viable microorganisms (Anvisa, 2019; Gomila et al., 2010; Gomila, Ramirez, Lalucat, 2007).