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02. Environmental Monitoring
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Charting and Evaluation of Environmental Microbial Monitoring Data- more
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Risk Profiling and Proactive Response (RPPR) to Bio-contamination in GMP classified and controlled areas- more
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Comparison of different incubation conditions for microbiological environmental monitoring.- more
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How long do nosocomial pathogens persist on inanimate surfaces? A systematic review- more
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A review of cleanroom microflora: types, trends, and patterns.- more
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Comparison of Different Calculation Approaches for Defining Microbiological Control Levels Based on Historical Data - more
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Structure, function and diversity of the healthy human microbiome - more
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Environmental monitoring: myths and misapplications- more
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! NEW ! Evaluation of an Environmental Monitoring Program for the Microbial Safety of Air and Surfaces in a Dairy Plant Environment- more
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! NEW ! Environmental Monitoring: A Correlation Study Between Viable and Nonviable Particles in Clean Rooms - more
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! NEW ! Optimal conditions for the recovery of bioburden from pharmaceutical processes: A case study- more
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! NEW ! Multicenter Study on Incubation Conditions for Environmental Monitoring and Aseptic Process Simulation- more
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! NEW ! Facility-based case study: A comparison of the recovery of naturally occurring species of bacteria and fungi on semi-solid media when incubated under standard and dual temperature conditions and its impact on microbial environmental monitoring approach- more
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! NEW ! Effect of Impact Stress on Microbial Recovery on an Agar Surface - more
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! NEW ! Air sampling procedures to evaluate microbial contamination: a comparison between active and passive methods in operating theatres - more
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! NEW ! Validierung der Inkubationsbedingungen In-situ-Studie zu Abklatschplatten aus dem mikrobiologischen Umgebungsmonitoring - more
Bar R
PDA J Pharm Sci Technol.
Abstract
Statistical tools are required to organize and present microbial environmental monitoring data for the purpose of evaluating it against regulatory action limits and of determining if the microbial monitoring process is in a state of control. This paper applies a known methodology of a simple and straightforward construction of control XmR (X data and moving range) charts of individual microbial counts as they are or of contamination rates derived from them, irrespective of the type of the parent data distribution and without the need to transform the data into a normal distribution. Plotting of monthly and cumulative sample contamination rates, as newly suggested by USP <1116>, is also shown. Both types of the control charts and plots allow an evaluation of the behavior of the microbial monitoring process. After addressing the magnitude of microbial counts expected in environmental monitoring samples, this paper presents the rationale behind the use of XmR charts. Employing data taken from environmental monitoring programs of pharmaceuticals manufacturing facilities, this paper analyzes examples of (1) microbial counts from passive or active air sampling in area Grade D or B or Class 100,000 in XmR charts, (2) contamination recovery rates as suggested by USP <1116> from active air samples in area Grade B and contact plates in area Grade C, and (3) instantaneous contamination rates with calculations illustrated on microbial counts of contact plates in area Grade D.
LAY ABSTRACT
Pharmaceutical companies conduct environmental monitoring programs, and samples of air (active and passive sampling) and of surfaces (contact plates) are routinely tested for microbiological quality. Thus, hundreds of microbial counts of tested environmental monitoring samples are routinely generated and recorded. Statistical tools are required to organize and present this abundant data for the purpose of evaluating it against regulatory action limits and determining if the microbial monitoring process is a state of control. This paper has a two-fold purpose. The first purpose is to provide microbiologists and quality assurance personnel simple and straightforward tools of statistical process control for evaluating the behavior of the microbial monitoring process: individual XmR (X data and moving range) control charts of microbial counts as they are or of rates derived from them are constructed irrespective of the type of the parent data distribution and without the need to transform the data into a normal distribution. Plotting of monthly and cumulative sample contamination rates, as newly suggested by USP <1116>, is also shown. The second purpose is to present examples of the charting of (1) microbial counts, (2) contamination recovery rates as suggested by USP <1116>, and (3) instantaneous contamination rates using data taken from environmental monitoring programs of pharmaceuticals manufacturing facilities.
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James L. Drinkwater
http://www.europeanpharmaceuticalreview.com/advent-calendar/bio-contamination-in-gmp-classified-areas/
This paper introduces an initiative based on microbiological risk profiling and a proactive response to bio-contamination risk escalation in classified and controlled environments. The concept is included in the PHSS Bio-contamination monograph 201. The monograph considers four principle requirements of a systematic approach to bio-contamination risk management and a microbial control strategy with guidance on best practice (see Figure 1); risk profiling is one key area where a systematic approach could yield significant benefits.
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Gordon O, Berchtold M, Staerk A, Roesti D
PDA J Pharm Sci Technol.
Abstract
Environmental monitoring represents an integral part of the microbiological quality control system of a pharmaceutical manufacturing operation. However, guidance documents differ regarding recommendation of a procedure, particularly regarding incubation time, incubation temperature, or nutrient media. Because of these discrepancies, many manufacturers decide for a particular environmental monitoring sample incubation strategy and support this decision with validation data. Such validations are typically laboratory-based in vitro studies, meaning that these are based on comparing incubation conditions and nutrient media through use of cultured microorganisms. An informal survey of the results of these in vitro studies performed at Novartis or European manufacturing sites of different pharmaceutical companies highlighted that no consensus regarding the optimal incubation conditions for microbial recovery existed. To address this question differently, we collected a significant amount of samples directly from air, inanimate surfaces, and personnel in pharmaceutical production and packaging rooms during manufacturing operation (in situ study). Samples were incubated under different conditions suggested in regulatory guidelines, and recovery of total aerobic microorganisms as well as moulds was assessed. We found the highest recovery of total aerobic count from areas with personnel flow using a general microbiological growth medium incubated at 30-35 °C.
The highest recovery of moulds was obtained with mycological medium incubated at 20-25 °C. Single-plate strategies (two-temperature incubation or an intermediate incubation temperature of 25-30 °C) also yielded reasonable recovery of total aerobic count and moulds.
However, recovery of moulds was found to be highly inefficient at 30-35 °C compared to lower incubation temperatures. This deficiency could not be rectified by subsequent incubation at 20-25 °C. A laboratory-based in vitro study performed in parallel was inconclusive. We consider our results potentially conferrable to other pharmaceutical manufacturing sites in moderate climate zones and believe that these should represent a valuable reference for definition of the incubation strategy of microbiological environmental monitoring samples.
LAY ABSTRACT:
Microbiological environmental monitoring confirms that pharmaceutical cleanrooms are in an appropriate hygienic condition for manufacturing of drug products. Guidance documents from different health authorities or expert groups differ regarding recommendation of the applied incubation time, incubation temperature, or nutrient media. Therefore, many pharmaceutical manufacturers perform studies that aim to identify the optimal incubation setup for environmental monitoring samples. An informal survey of the results of such studies, which had been performed at Novartis or European manufacturing sites of different pharmaceutical companies, highlighted no consensus regarding the optimal incubation conditions for microbial recovery. All these studies had been conducted in the laboratory using selections of cultured microbial strains. We tried to solve this disagreement by collecting a significant amount of real environmental monitoring samples directly from the environment in pharmaceutical production and packaging rooms during manufacturing operation. These samples were then incubated under different conditions suggested in the regulatory guidelines. We believe that the results of our study are more meaningful than laboratory-based experiments because we used environmental samples with microorganisms directly isolated from the manufacturing area. Therefore, we believe that our results should represent a valuable reference for definition of the incubation strategy of microbiological environmental monitoring samples.
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Axel Kramer, Ingeborg Schwebke and Günter Kampf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1564025/
Background
Inanimate surfaces have often been described as the source for outbreaks of nosocomial infections. The aim of this review is to summarize data on the persistence of different nosocomial pathogens on inanimate surfaces.
Methods
The literature was systematically reviewed in MedLine without language restrictions. In addition, cited articles in a report were assessed and standard textbooks on the topic were reviewed. All reports with experimental evidence on the duration of persistence of a nosocomial pathogen on any type of surface were included.
Results
Most gram-positive bacteria, such as Enterococcus spp. (including VRE), Staphylococcus aureus (including MRSA), or Streptococcus pyogenes, survive for months on dry surfaces. Many gram-negative species, such as Acinetobacter spp.,
Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Serratia marcescens, or Shigella spp., can also survive for months. A few others, such as
Bordetella pertussis, Haemophilus influenzae, Proteus vulgaris, or
Vibrio cholerae, however, persist only for days. Mycobacteria, including
Mycobacterium tuberculosis, and spore-forming bacteria, including Clostridium difficile, can also survive for months on surfaces.
Candida albicans as the most important nosocomial fungal pathogen can survive up to 4 months on surfaces. Persistence of other yeasts, such as
Torulopsis glabrata, was described to be similar (5 months) or shorter (Candida parapsilosis, 14 days). Most viruses from the respiratory tract, such as
corona, coxsackie, influenza, SARS or rhino virus, can persist on surfaces for a few days. Viruses from the gastrointestinal tract, such as
astrovirus, HAV, polio- or rota virus, persist for approximately 2 months. Blood-borne viruses, such as HBV or HIV, can persist for more than one week. Herpes viruses, such as CMV or HSV type 1 and 2, have been shown to persist from only a few hours up to 7 days.
Conclusion
The most common nosocomial pathogens may well survive or persist on surfaces for months and can thereby be a continuous source of transmission if no regular preventive surface disinfection is performed.
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Sandle T
PDA J Pharm Sci Technol.
Abstract
Cleanroom microflora are of importance for microbiologists and quality control personnel in order to assess changes in trends. Shifts in the types of microflora may indicate deviations from the "norm" such as resistant strains or problems with cleaning practices. Given the few published studies of the typical microflora, this paper uniquely reviews over 9000 microbial isolates from a range of different grades of cleanroom. The paper concludes that the typical flora are primarily those associated with human skin (Gram-positive cocci), although microorganisms from other sources such as the environment (Gram-positive rods) and water (Gram-negative rods) are also detected, although in lower numbers.
LAY ABSTRACT:
It is of importance that pharmaceutical manufacturers and healthcare pharmacies review the types and numbers of microorganisms found within their clean areas. Such examination should be carried out over a long period of time so that the complete picture can be revealed. This is important in order to understand if certain species are being recovered pose a product or environmental risk and to check if the cleaning and sanitization practices are effective.
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Gordon O, Goverde M, Pazdan J, Staerk A, Roesti D.
http://www.ncbi.nlm.nih.gov/pubmed/26048745
Abstract
In the present work we compared different calculation approaches for their ability to accurately define microbiological control levels based on historical data. To that end, real microbiological data were used for simulation experiments. The results of our study confirmed that assuming a normal distribution is not appropriate for that purpose. In addition, assumption of a Poisson distribution generally underestimated the control level, and the predictive power for future values was highly insufficient. The non-parametric Excel percentile strongly predicted future values in our simulation experiments (although not as good as some of the parametric models). With the limited amount of data used in the simulations, the calculated control levels for the upper percentiles were on average higher and more variable compared to the parametric models. This was due to the fact that the largest observed value was generally defined as the control level. Accordingly, the Excel percentile is less robust towards outliers and requires more data to accurately define control levels as compared to parametric models. The negative binomial as well as the zero-inflated negative binomial distribution, both parametric models, had good predictive power for future values. Nonetheless, on basis of our simulation experiments, we saw no evidence to generally prefer the zero-inflated model over the non-inflated one. Finally, with our data, the gamma distribution on average had at least as good predictive power as the negative binomial distribution and zero-inflated negative binomial distribution for percentiles ≥98%, indicating that it may represent a viable option for calculating microbiological control levels at high percentiles. Presumably, this was based on the fact that the gamma distribution fitted the upper end of the distribution better than other models. Since in general microbiological control levels would be based on the upper percentiles, microbiologists may exclusively rely on the gamma distribution for calculation of their control levels. As the gamma distribution can conveniently be calculated in standard office calculation software, it may represent a superior alternative to the widely used percentile functions or other distribution models.
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The Human Microbiome Project Consortium
http://www.nature.com/nature/journal/v486/n7402/full/nature11234.html
Abstract
Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet,
environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort
and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat's signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and
among individuals. The project encountered an estimated 81−99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among
individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of
structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.
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Akers J, Agallaco J.
http://www.ncbi.nlm.nih.gov/pubmed/11417108
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Zacharski KA, Southern M, Ryan A, Adley CC
Journal of Food Protection, Vol. 81, No. 7, 2018, Pages 1108−1116
https://www.ncbi.nlm.nih.gov/pubmed/29916731
Abstract:
Microbiological hazards can occur when foodstuffs come into contact with contaminated surfaces or infectious agents dispersed by air currents in the manufacturing environment. An environmental monitoring program (EMP) is a critical
aspect of sustainable and safe food manufacturing used to evaluate the effectiveness of the microbial controls in place. An effective EMP should be based on risk analysis, taking into account previous sampling history to determine
the selection of the sampling points, the scope of the test, and the frequency of analysis. This study involved evaluation of the environmental monitoring regime and microbiological status of a medium-sized dairy plant manufacturing
food ingredients, e.g., proteins, milk powders, and dairy fats. The data specific to microbial tests (n¼3,468), recorded across 124 fixed sampling locations over a 2-year period (2014 to 2015) from air (n¼1,787) and
surfaces (n¼1,681)
were analyzed. The aim of this study was to highlight the strengths and weaknesses of the EMP in a select dairy processing plant. The results of this study outline the selection of sampling locations, the scope of the test, and
the frequency of analysis. An analysis of variance revealed subsections of the manufacturing areas with high risk factors, especially the packaging subsection specified for bulk packaging, the atomizer, and the fluidized bed.
The temporal and spatial analysis showed the potential to reduce or relocate the monitoring effort, most notably related to total coliforms and Staphylococcus aureus, across the dairy plant due to homogeneity across the sampling
subsections with little or no deviations. The results suggest a need to reevaluate the current EMP and the corrective action plan, especially with regard to detection of pathogens. Recommendations for optimization of the EMP are
presented to assist the dairy industry with reviewing and revising the control measures and hazard assessment with regard to existing contamination issues.
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De Abreu CS, Pinto Tde JA, De Oliveira DC
PDA J Pharm Sci and Tech 2004, 58 45-53
https://www.ncbi.nlm.nih.gov/pubmed/15053054
Abstract:
This article describes an environmental monitoring data study for clean rooms. Two parameters were compared in order to investigate the possibility of correlation between them: (1) total airborne nonviable (0.5 and 5_m) and
viable (CFU) particulates, and (2) surface contamination (Rodac_). The investigation took into account A, B, and C classified areas, in operational moment or status, which involved "at rest" (just equipment working) and "dynamic"
(equipment and operators working) conditions. Available data in the literature indicates that the parameters are not particularly dependent upon the layout or classification of areas, but rather on the use of the areas and operator
behaviour. Our results showed a correlation around 0.6 between different sampling places for nonviable 0.5- and 5-_m particles. There was no correlation between nonviable 0.5- and 5-_m particles in places with laminar flow.
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Sandle T, Skinner K, Yeandle E
European Journal of Parenteral & Pharmaceutical Sciences 2013; 18(3): 84-90
https://www.researchgate.net/profile/Tim_Sandle/publication/...pdf
Abstract:
Bioburden testing is an important part of pharmaceutical microbiology and provides data in relation to the quality of pharmaceutical products during manufacture. Little guidance is provided in relation to test methodology, culture media and incubation parameters. The quality control laboratory, therefore, needs to establish the most appropriate method. This paper outlines a case study for the selection of incubation parameters for the bioburden assessment of in-process samples using the Total Viable Count technique and pour plate method. While the outcome of the experiment contained within the paper relates to a specific set of processes, the approach taken can be used by other laboratories to compare or to develop their test methods and techniques for bioburden determinations.
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Guinet R, Berthoumieu N, Dutot P, et al.
PDA J Pharm Sci and Tech 2017, 71 43-49
https://www.ncbi.nlm.nih.gov/pubmed/27593691
Abstract:
Environmental monitoring and aseptic process simulations represent an integral part of the microbiological quality control system of sterile pharmaceutical products manufacturing operations. However, guidance documents and
manufacturers practices differ regarding recommendations for incubation time and incubation temperature, and, consequently, the environmental monitoring and aseptic process simulation incubation strategy should be supported by
validation data. To avoid any bias coming from in vitro studies or from single-site manufacturing in situ studies, we performed a collaborative study at four manufacturing sites with four samples at each location. The environmental
monitoring study was performed with tryptic soy agar settle plates and contact plates, and the aseptic process simulation study was performed with tryptic soy broth and thioglycolate broth. The highest recovery rate was obtained with
settle plates (97.7%) followed by contact plates (65.4%) and was less than 20% for liquid media (tryptic soy broth 19% and thioglycolate broth 17%). Gram-positive cocci and non-spore-forming Gram-positive rods were largely predominant
with more than 95% of growth and recovered best at 32.5 °C. The highest recovery of molds was obtained at 22.5 °C alone or as the first incubation temperature. Strict anaerobes were not recovered. At the end of the five days of
incubation no significant statistical difference was obtained between the four conditions. Based on these data a single incubation temperature at 32.5 °C could be recommended for these four manufacturing sites for both environmental
monitoring and aseptic process simulation, and a second plate could be used, periodically incubated at 22.5 °C. Similar studies should be considered for all manufacturing facilities in order to determine the optimal incubation
temperature regime for both viable environmental monitoring and aseptic process simulation.
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Symonds ID, Martin DL, Davies MC
European Journal of Parenteral & Pharmaceutical Sciences 2016; 21(1): 7-15
https://cdn.ymaws.com/phss.co.uk/resource/resmgr/files/Facility-based_case_study_A_.pdf
Abstract:
This study compared the recovery of naturally occurring micro-organisms on tryptone soya agar (TSA) and Sabouraud dextrose agar (SDA), when incubated under three different temperature conditions. The micro-organisms were sourced
directly from a factory environment. The incubation conditions employed were 20−25°C, 30−35°C and a combination of each, termed dual temperature incubation, for a period of 5 days. The results demonstrated that TSA was the most
effective medium for the primary isolation of Environmental Monitoring both bacterial and fungal/yeast micro-organisms. Bacteria were recovered best at 30−35°C with human commensals providing the largest numbers, while fungi and
yeasts showed best recovery at 20−25°C. The use of dual temperature incubation at 20−25°C for 3 days followed by 2 days at 30−35°C gave reduced recovery for both types of micro-organisms. The authors
recommend that similar studies
should be considered for all manufacturing facilities in order to determine the optimal incubation temperature regime for the recovery of local, naturally occurring species of bacteria and fungi which may present a threat to aseptic
manufacturing areas. This process should be undertaken as part of an overall risk assessment for the establishment and maintenance of a viable environmental monitoring programme and may also be relevant to the incubation conditions
employed in process simulation studies.
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Stewart SL, Grinshpun SA, Willeke K, Terzieva S, Ulevicius V, Donnelly J
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1995, p. 1232−1239
https://aem.asm.org/content/61/4/1232
Abstract:
Microbial stress due to the impaction of microorganisms onto an agar collection surface was studied experimentally. The relative recovery rates of aerosolized Pseudomonas fluorescens and Micrococcus luteus were determined as a
function of the impaction velocity by using a moving agar slide impactor operating over a flow rate range from 3.8 to 40 liters/min yielding impaction velocities from 24 to 250 m/s. As a reference, the sixth stage of the Andersen
Six-Stage Viable Particle Sizing Sampler was used at its operating flow rate of 28.3 liters/min (24 m/s). At a collection efficiency of close to 100% for the agar slide impactor, an increase in sampling flow rate and, therefore,
in impaction velocity produced a significant decline in the percentage of microorganisms recovered. Conversely, when the collection efficiency was less than 100%, greater recovery and lower injury rates occurred. The highest
relative rate of recovery (approximately 51% for P. fluorescens and approximately 62% for M. luteus) was obtained on the complete (Trypticase soy agar) medium at 40 and 24 m/s (6.4 and 3.8 liters/min), respectively. M. luteus
demonstrated less damage than P. fluorescens, suggesting the hardy nature of the gram-positive strain versus that of the gram-negative microorganism. Comparison of results from the agar slide and Andersen impactors at the same
sampling velocity showed that recovery and injury due to collection depends not only on the magnitude of the impaction velocity but also on the degree to which the microorganisms may be embedded in the collection medium. Impaction
velocity, characterized by the sampler's operating flow rate and inlet design, is unique for each sampling device. The resulting impaction stress influences the recovery and injury of collected microorganisms and ultimately affects
the measurement data for colony enumeration. This can be one of the most important reasons for variations that occur when using different sampling devices to measure bioaerosols from the same environment.
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Napoli C, Marcotrigiano V, Montagna MT
BMC Public Health 2012, 12:594
https://www.ncbi.nlm.nih.gov/pubmed/22853006
Abstract:
Background: Since air can play a central role as a reservoir for microorganisms, in controlled environments such as operating theatres regular microbial monitoring is useful to measure air quality and identify critical situations. The aim of this study is to assess microbial contamination levels in operating theatres using both an active and a passive sampling method and then to assess if there is a correlation between the results of the two different sampling methods.
Methods: The study was performed in 32 turbulent air flow operating theatres of a University Hospital in Southern Italy. Active sampling was carried out using the Surface Air System and passive sampling with settle plates, in accordance with ISO 14698. The Total Viable Count (TVC) was evaluated at rest (in the morning before the beginning of surgical activity) and in operational (during surgery).
Results: The mean TVC at rest was 12.4 CFU/m3 and 722.5 CFU/m2/h for active and passive samplings respectively. The mean in operational TVC was 93.8 CFU/m3 (SD = 52.69; range = 22-256) and 10496.5 CFU/m2/h (SD = 7460.5; range = 1415.5-25479.7) for active and passive samplings respectively. Statistical analysis confirmed that the two methods correlate in a comparable way with the quality of air.
Conclusion: It is possible to conclude that both methods can be used for general monitoring of air contamination, such as routine surveillance programs. However, the choice must be made between one or the other to obtain specific information.
Keywords: Bioaerosol, Air sampling, Operating theatres, Surveillance
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Goverde M, Herzog S
PharmInd 2017, 79
http://ecv.de/suse_section.php?section=Z&suseOrder=hits&sp=736
Abstract:
In situ Study for Incubation Conditions for Contact Plates from Microbiological Environmental Monitoring The current study investigates different incubation conditions for agar plates from the microbiological environmental monitoring on the growth of aerobic mesophilic microorganisms and molds. The study was performed using naturally contaminated surfaces. Four different incubation conditions were tested. Additionally, the effect of a hold time step of the used agar plates for 3 days at room temperature before incubation was investigated. Neither the incubation condition nor the hold time at room temperature had a significant effect on the total aerobic microbial count. However, the hold time significantly increased the numbers of molds found, at least in 3 out of 4 incubations conditions. This effect probably relied on the increased incubation time due to the hold time step. In conclusion, the current study shows the minimal effect of the different investigated incubation conditions on the microbial counts of agar plates from the environmental monitoring.
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