Applications of Microbiolgical Data Tim Sandle Microbiology information resource: http://www.pharmamicroresources.com/

Introduction Distribution of microbiological data Use of trend charts Calculation of warning and action levels

Introduction Examples from environmental monitoring and water testing Broad and illustrative overview Written paper with more detail

Distribution of microbiological data Why study distribution? Impact on sampling Impact on trending Impact upon calculation of warning and action levels

Distribution Most statistical methods are based on normal distribution, and yet…. Most microbiological data does NOT follow normal distribution

Distribution Micro-organisms, such as those in a typical, free-flowing water system, follow Poisson distribution For example…

Distribution S1 S2 S3 S4 S5 Where S = sample = micro-organism

Distribution And microbial counts tend to be skewed (or positive or negative exponential distribution) For example, a Water-for-Injection system…

Distribution

Distribution So, what can we do about it? ? Skewed question mark

Distribution Well: a) Use complex calculations and Poisson distribution tables, or b) Attempt to transform then data We’ll go for the second option

Distribution A general rule is: For low count data e.g. Grade A monitoring and WFI systems, take the square root For higher count data, e.g. Grade C and D environmental monitoring or a purified water system, convert the data into logarithms

Distribution For example, some counts from a WFI system:

Distribution When the data is examined for its distribution, using a simple ’blob’ chart:

Distribution Whereas if the square root is taken:

Distribution We move closer to normal distribution:

Distribution Logarithms work in a similar way for higher counts Remember to add ‘+1’ to zero counts (and therefore, +1 to all counts)

Trend Analysis There is no right or wrong approach There are competing systems This presentation focuses on two approaches, both described as ‘control charts’: The cumulative sum chart The Shewhart chart

Trend Analysis Control charts form part of the quality system They can be used to show: Excessive variations in the data How variations change with time Variations that are ‘normally’ expected Variations that are unexpected, i.e. something unusual has happened

Trend Analysis Control charts need: A target value, e.g. last year’s average Monitoring limits: Upper limit Lower limit Control line / mean So the data can be monitored over time and in relation to these limits

Trend Analysis Of these, The warning limit is calculated to represent a 2.5% chance The action level is calculated to represent a 0.1% chance So, if set properly, most data should remain below these limits These assumptions are based on NORMAL DISTRIBUTION Various formula can be used to set these or validated software

Trend Analysis Cumulative sum chart (cusum) Shewhart chart Suitable for large quantities of low count data. It is very sensitive to small shifts Shows shifts in the process mean Shewhart chart Suitable for higher count data. It shows large changes more quickly.

Trend Analysis Cusums Harder to interpret Displays the cumulative sum of a rolling average of three values and plots these in comparison with the target value The direction and steepness of the slope are important Significant changes are called ‘steps’ V-masks can be used as a prediction to the future direction

Trend Analysis For example, a Grade B cleanroom Contact (RODAC) plates are examined A target of 0.2 cfu has been used, based on data from the previous year

Trend Analysis

Trend Analysis Shewhart charts Powerful for distinguishing between special causes and common causes Common causes are inherent to the process and are long-term Special causes are where something has changed and maybe of a long or short term

Trend Analysis Examples of special causes: a) A certain process b) A certain outlet c) A certain method of sanitisation, etc. d) Sampling technique e) Equipment malfunction e.g. pumps, UV lamps f) Cross contamination in laboratory g) Engineering work h) Sanitisation frequencies

Trend Analysis For example, a Grade C cleanroom Active air-samples are examined A target of 1.5, based on historical data

Trend Analysis

Trend Analysis The previous charts were prepared using a statistical software package However, MS Excel can also be used The next example is of a WFI system Notice the data has been converted by taking the square root of each value

Trend Analysis

Trend Analysis Alternatives: Individual Value / Moving Range charts Exponentially Weighted Moving Average charts (EWMA) These are useful where counts are NOT expected, e.g. Grade A environments They look at the frequency of intervals between counts

Trend Analysis Summary

Limits Alert and action levels Based on PDA Tech. Report 13 (2001): Alert level: a level, when exceeded, indicates that the process may have drifted from its normal operating condition. This does not necessarily warrant corrective action but should be noted by the user. Action level: a level, when exceeded, indicates that the process has drifted from its normal operating range. This requires a documented investigation and corrective action.

Limits Why use them? • Assess any risk (which can be defined as low, medium or high) • To propose any corrective action • To propose any preventative action

Limits “Level” is preferable to “Limit” Limits apply to specifications e.g. sterility test Levels are used for environmental monitoring

Limits Regulators set ‘guidance’ values e.g. EU GMP; USP <1116>; FDA (2004) These apply to new facilities User is expected to set their own based on historical data Not to exceed the published values Many references stating this Views of MHRA and FDA

Limits Things to consider: The length of time that the facility has been in use for How often the user intends to use the limits for (i.e. when the user intends to re-assess or re-calculate the limits. Is this yearly? Two yearly? And so on). Custom and practice in the user’s organisation (e.g. is there a preferred statistical technique?) They be calculated from an historical analysis of data. Uses a statistical technique.

Limits Historical data Aim for a minimum of 100 results Ideally one year, to account for seasonal variations

Limits Statistical methods: Percentile cut-off Normal distribution Exponential distribution Non-parametric tolerance limits Weibull distribution Recommended by PDA Technical Report, No. 13

Limits Assumptions: a) The previous period was ‘normal’ and that future excursions above the limits are deviations from the norm b) Outliers have been accounted for

Limits Percentile cut-off Good for low count data May need to use frequency tables May need to round up or down to nearest whole zero or five Warning level = 90th or 95th Action level = 95th or 99th

Limits Percentile cut-off Data is collected, sorted and ranked 90th percentile means that any future result that exceeds this is 90% higher than all of the results obtained over the previous year. Refer to PharMIG News Number 3 (2000) for excellent examples.

Limits Normal distribution Can only be used on data that is normally distributed! Could transform data but inaccuracies can creep in Most data will be one-tailed, therefore need to adjust 2nd and 3rd standard deviation Warning level = 1.645 + the mean Action level = 2.326 + the mean

Limits Negative exponential distribution Suitable for higher count data Warning level: 3.0 x mean Action level: 4.6 x mean

Limits For all, do a ‘sore thumb’ activity by comparing to a histogram of the data Does it feel right?

Conclusion We have looked at: Distribution of microbiological data Trending Cusum charts Shewhart charts Setting warning and action levels Percentile cut-off Normal distribution approach Negative exponential approach

Conclusion Key points: Most micro-organisms and microbial counts do not follow normal distribution Data can be transformed Inspectors expect some trending and user defined monitoring levels Don’t forget to be professional microbiologists – it isn’t all numbers!

Just a thought… This has been a broad over-view If there is merit in a more ‘hands on’ training course, please indicate on your post-conference questionnaires.

Thank you Any questions?