Microbiological Testing Equipment

Microbiological testing equipment and methods should be EPA-REGISTERED, conforming to the latest edition of Standard Methods for the Examination of Water and Wastewater,²⁴⁴ existing professional guidelines, or other recognized international guidelines or STANDARDS. At this time, routine microbiological testing for POOLS, SPAS, and other AQUATIC VENUES is not recommended in the MAHC. Routine MONITORING of chemical levels (e.g., pH, disinfectant concentration) and proper operation and maintenance of the AQUATIC VENUE have historically been considered to be sufficient to ensure that proper barriers are maintained to minimize potential infectious disease risks from CHLORINE sensitive pathogens. Currently, routine MONITORING for CHLORINE-tolerant microorganisms (e.g., Cryptosporidium spp.) is not a feasible or cost-effective disease prevention approach. Chemical tests such as FREE RESIDUAL CHLORINE, pH, CT VALUES, and others provide a good indication of operational control of an AQUATIC VENUE. However, while these tests provide an indication of DISINFECTION potential, they may not provide complete assurance of the microbial quality of AQUATIC VENUE water. While agencies such as the World Health Organization²⁴⁵, the South Australia Environmental and Public Health Service²⁴⁶, and the United Kingdom Health Protection Agency²⁴⁷ have established STANDARDS for routine MONITORING of public and semi-public AQUATIC VENUES for microbial parameters including enteric bacteria (fecal organisms or E. coli), Pseudomonas aeruginosa, and Legionella, there is insufficient scientific data for the purposes of the MAHC to indicate that these routine MONITORING STANDARDS provide an increased level of public health protection beyond adherence to current BEST PRACTICES. The routine MONITORING recommendations in the MAHC can be reconsidered to potentially include routine MONITORING for microbial parameters if compelling scientific data indicate that such testing provides additional, measurable public health protections beyond use of BEST PRACTICES for DISINFECTION in AQUATIC VENUE operation and maintenance. It should be noted that this section of the Annex is a minimum guideline for microbiological MONITORING. AQUATIC VENUE operators wishing to achieve additional microbial water quality characterization are encouraged to use the references in this Annex regarding water quality MONITORING techniques and STANDARDS established by the United States and in other countries. Microbial water quality STANDARDS established for AQUATIC VENUES by U.S. and international agencies include: * Alberta Public Health, Alberta Regulation 293/2006 (2006) Swimming Pool, Wading Pool and Water Spray Park Regulation; Alberta, Canada o Excerpt, Page 10, Bacterial Limits: Heterotrophic Plate Count less than 100/mL; Pseudomonas aeruginosa 0/100 mL, coliforms 0/100 mL * Code de la Santé Publique, France, (2007) Arrêté préfectoral en date du 15 juin 2007 fixant les upermens du contrôle sanitaire de la qualité des eaux des piscines (Prefectural order dated June 15, 2007 establishing standards for the control of swimming pool water quality) o Excerpt: Determination of the parameters to be analyzed in the field or laboratory: Standards for Bacteriological Analytical Parameters * Viable aerobic bacteria at 37°C <100/ml * Total coliforms <10/100ml * Fecal coliforms (E. coli) 0/100ml * Pathogenic staphylococci 0/100ml * Pseudomonas aeruginosa (in SPAS) 0/100ml * New Jersey Department of Health and Senior Services (2009) New Jersey State Sanitary Code, Chapter IX, Public Recreational Bathing N.J.A.C 8:26 o Excerpt: pages 20 – 21; Heterotrophic plate count do not exceed 200 colonies per one milliliter sample; Coliforms to be less than one colony per 100 milliliter sample, Pseudomonas aeruginosa not to exceed one colony per 100 milliliter sample. Although routine microbial testing is not recommended by the MAHC at this time, microbiological testing can be useful as supporting data for evaluating the need for (or effectiveness of) troubleshooting activities, remediation activities, and AQUATIC FACILITY upgrades. As indicated by WHO²⁴⁸ recommendations, microbiological testing of water samples from AQUATIC VENUES can be useful for the following reasons: * Before an AQUATIC VENUE is used for the first time, * Before it is put back into use after it has been shut down for repairs or cleaning, * If there are difficulties with the treatment system, or * As part of any investigation into possible adverse effects on BATHER or PATRON health. It is known that certain microorganisms, because of their ecology and/or structure, can be tolerant of chemical disinfectants (e.g., chlorine, bromine). Legionella pneumophila, Pseudomonas aeruginosa, Cryptosporidium parvum, Entamoeba histolytica cysts, and Mycobacterium avium complex are a few examples of pathogenic microbes that have been reported to show some tolerance to chemical disinfectants. In addition, sessile (in the biofilm) microorganisms in biofilm are likely to receive additional protection from oxidizers (such as chlorine) when the exposure concentration of these oxidizers is reduced at the interface with the biofilm due to reaction with biofilm material. Biofilm is a complex community of microorganisms which attach to the sides, piping, and filters of AQUATIC VENUES ²⁴⁹. Even at elevated concentrations, oxidizing and non-oxidizing chemicals have reduced effectiveness in controlling biofilm when their concentrations and contact times are not sufficient for penetrating the biofilm²⁵⁰. Biofilm formation in AQUATIC VENUES is also a concern because microorganisms in the biofilm or the biofilm itself can detach and multiply²⁵¹. Following BEST PRACTICE guidelines for AQUATIC VENUE cleaning and continuous DISINFECTION is critical to avoid biofilm growth and expansion problems²⁵²,²⁵³. If biofilm-related problems arise, it can be useful to incorporate biofilm sampling to develop a comprehensive evaluation of the risk factors for water quality impairment and potential solutions to identified problems²⁵⁴. MAHC Annex Table 4.7.3.6 (below) identifies microorganisms for which chlorination may have, or is known to have, reduced efficacy²⁵⁵,²⁵⁶,²⁵⁷. MAHC Annex Table 4.7.3.6 also identifies methods that may be used to detect these microbes in AQUATIC VENUE systems, but the methods identified are not necessarily rapid. Additional research is needed to evaluate the benefits of microbiological testing data for AQUATIC VENUES, especially for improving public health protection. This is particularly important for the protozoans, amoebas, and sessile bacterial pathogens that co-exist in biofilms. It should be noted that the use of fecal indicator organisms for AQUATIC VENUE water quality evaluation may not be sufficient for certain AQUATIC VENUE operation, maintenance, and public health investigations, especially in public health investigations related to inhalation, skin breaks, or ocular exposure routes. Since health risks in AQUATIC VENUES and similar environments may be fecal or non-fecal in origin, investigation of fecal indicators and non-fecally-transmitted microorganisms (e.g. P. aeruginosa, S. aureus and Legionella spp.) may be warranted. Table 4.7.3.6: Known Pathogenic Organisms of Concern in Chlorinated Aquatic Venues (continued on next page) Organism Illness Route of Infection Resistant To Chlorine Environmental Biofilm Amplification Test Method Pseudomonas aeruginosa1 * Hot tub * Folliculitis * Conjunctivitis * Pneumonia * Swimmer’s Ear * Skin * Eyes * Inhalation * Ears * Yes when planktonic2 * Yes when sessile in biofilms Yes * APHA Standard Method 9213 E-F * APHA Standard Method Rapid PCR test available Enteric Bacteria * Gastroenteritis * Hip and Knee joint replacement infections, replacement heart valve infections * Conjunctivitis * Pneumonia * Fecal/Oral * Skin breaks * Eyes * Inhalation * No when planktonic * Yes when sessile in biofilm Yes * APHA Standard Method for Coliforms 9221 A-F * APHA Standard Method 9260 A-L for specific pathogens * APHA pathogen specific PCR test * Bacteroides/Enterococci PCR tests under investigation by EPA to replace Coliforms Legionella * Legionnaires’ Disease, Pontiac Fever * Hip and Knee joint replacement infections, replacement heart valve infections * Inhalation * Skin breaks * Yes when planktonic3 * Yes when sessile in biofilm Yes * APHA Standard Method 9260 J * CDC/ISO Method is Gold Standard * APHA Standard Method * Rapid PCR test Mycobacterium avium complex (MAC) * Hypersensitivity pneumonitis * Dermatitis * Inhalation * Skin Breaks * Yes when planktonic * Yes when sessile in biofilm Yes * APHA Standard Method 9260 M * Rapid PCR Staphylococcus aureus & Methicillin resistant Staphylococcus aureus (MRSA) * Conjunctivitis * Antibiotic resistant skin infection possibly fatal * Eyes * Skin Breaks * No when planktonic * Yes when sessile in biofilm Yes * APHA Standard Method 9213 B 6 * and 7 * Rapid PCR test Organism Illness Route of Infection Resistant To Chlorine Environmental Biofilm Amplification Test Method Naegleria fowleri * Primary amoebic meningoencephalitis (uncommon but high mortality rate) * Water accidentally inhaled in nose or pharynx * Yes when planktonic Yes * APHA Standard * Method PCR test Acanthamoeba * Conjunctivitis and Keratoconjunc-tivitis (may cause blindness particularly in contact lens wearers) * Eye, skin, mucous membranes * Yes when sessile in biofilm Yes * APHA Standard Method 9711 C Cryptosporidium and Giardia * Gastroenteritis * Biliary Tract Infections * Reactive Arthritis * Fecal/Oral * Inhalation * Inhalation * Cysts are resistant in planktonic and sessile forms Yes * Standard Method 9711 B * APHA PCR test Adenoviruses * Conjunctivitis * Gastroenteritis * Eyes * Fecal/Oral * No Unknown * Cell culture * PCR Enteroviruses * Gastroenteritis * Viral meningitis * Fecal/Oral * No Unknown * APHA Standard Method 9510 * EPA Method 1615 Noroviruses * Gastrointestinal * Fecal/Oral * No Unknown * EPA Method 1615 * RT-PCR Methods Helminths and Roundworms * Ascariasis * Baylisascariasis * Fecal/Oral * Cysts are resistant in planktonic and sessile forms Yes * APHA Standard Method 10750 * PCR tests available for species identification Fungi * Ringworm * Skin * Spores are resistant Yes * Culture and PCR tests Table 4.7.3.6 Notes: 1. NOTE a. Many elderly and/or immuno- compromised people use SPAS making them more susceptible to disease; b. P. aeruginosa can be tolerant of CHLORINE and is found in biofilm; c. Hot tub folliculitis is the most common illness associated with hot tubs; and d. Coliform testing is not an indication of P. aeruginosa contamination; e. Since this is a non-reportable disease, we have no information on the incidence of this disease. 2. Grobe, Wingender, & Flemming, 2001; Price, 1988; Clements, 2000. 3. Muraca, Stout, & Yu, 1987; Clements, 2000. It is not feasible or cost effective to test for all infectious organisms. Therefore MAHC Annex Table 4.7.3.6 identifies those organisms which have readily available test methods and/or cause illnesses that are common, very serious, or fatal. It is important to note that these test methods may not allow for rapid remediation, decision making, or public health intervention on a timely basis. The Heterotrophic Plate Counts (HPC) method has not been included in the list of microbial water quality tests in MAHC Annex Table 4.7.3.6. While HPC data are generally a good indicator of microbial water quality and efficacy of POOL operations (e.g., water treatment), this parameter has been reported to show no correlation to the presence of Legionella²⁵⁸, planktonic pathogens²⁵⁹, or the presence of biofilm²⁶⁰. HPC tests (as do all culture tests) under-report the actual concentration of viable bacteria. Therefore, it is recommended that the use of this test be restricted for assessing the level of planktonic, non-pathogenic bacteria only. HPC data are not sufficient to assess the public health risk of POOLS, SPAS, and waterparks²⁶¹. Since the MAHC is intended to be a living document with changes anticipated as our knowledge increases, it is prudent to acknowledge that a paradigm shift is occurring in the world of microbiology that likely will impact how pathogen testing will be conducted and interpreted in the future. Culture tests are gradually being replaced with culture-independent test methods such as Polymerase Chain Reaction (PCR) testing and microarray testing. Years ago when PCR was first used commercially, the cost of the tests was prohibitively expensive. Now test costs have decreased and are competitive with culture dependent tests. A recent development is the commercialization of microarray testing which can screen for the presence of a wide variety of bacterial and viral pathogens without the need for an isolation step. However, the costs associated with microarray testing are prohibitively expensive as of this MAHC publication. EPA is re-evaluating the use of culture-based fecal indicator bacteria (FIB) tests in recreational water testing (i.e., total and fecal coliforms, E. coli and Enterococcus) and is researching the use of PCR for Bacteroides and Enterococcus testing as a possible replacement for these culture tests. Two of the most compelling reasons for this re-evaluation are: * Incubation times for culture tests prevent quick decision-making to minimize public exposure to water with a potentially elevated disease risk, and * Molecular tests are generally considered to have higher specificity (lower false positive rates) than traditional culture tests. PCR can be a good method for investigating whether pathogenic microbes were present in AQUATIC VENUES (e.g., sampling filter backwash) since the technique detects the DNA of pathogens regardless of whether they are live, dead, or viable-but-not-culturable. Another benefit is that PCR culture tests can be completed in hours versus days. However, while PCR can be effective for determining whether pathogens have been present in an AQUATIC VENUE, the technique is less effective as a measure of DISINFECTION effectiveness since it detects DNA from both viable and non-viable organisms. New techniques, such as the use of propidium monoazide (PMA) have been reported to enable PCR to characterize the viability status of microorganisms, so in the future PCR may be an effective option for DISINFECTION studies²⁶².