WHO/BS/2015.2269 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 12 to 16 October 2015 Collaborative Study to Enlarge the First WHO Repository of Platelet Transfusion-Relevant Bacterial Reference Strains Eva Spindler-Raffel, Kay-Martin Hanschmann, Thomas Montag-Lessing†* and the Collaborative Study Group** *Division of EU cooperation / Microbiology, Head Dr Isabelle Bekeredjian-Ding Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany **ISBT WP Transfusion Transmitted Infectious Diseases Subgroup on Bacteria Chairs: Carl P McDonald and Richard J Benjamin See Appendix 1
NOTE: This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 14 September 2015 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr M. Nübling at email: nueblingc@who.int © World Health Organization 2015 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site: (http://www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.
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Summary Bacterial contamination of platelet concentrates (PCs) remains a persistent problem in transfusion [1, 2, 3, 4 and 8]. For method validation and for assessment of blood safety measures it is crucial to use bacterial strains that are able to proliferate in blood components, e.g. in PCs under usual storage conditions [5, 6]. Four bacterial strains were adopted by ECBS in 2010 as the 1st WHO International Reference Repository of Platelet Transfusion Relevant Bacterial Reference Strains: Staphylococcus epidermidis PEI-B-P-06, Streptococcus pyogenes PEI-B-P-20, Escherichia coli PEI-B-P-19 and Klebsiella pneumoniae PEI-B-P-08. [7] The committee requested detailed instructions for use, which were provided by PEI in February 2011. The four bacterial strains have been cultivated and distributed by the Paul-Ehrlich-Institut since 2014 (PEI code number 8483/13). A proposal for future expansion of the repository was endorsed by WHO ECBS in 2010. To characterize new candidate strains, a second international collaborative study was performed. This study was coordinated by the Paul Ehrlich Institut (PEI) in cooperation with the ISBT Working Party Transfusion Transmitted Infectious Diseases (WP-TTID), Subgroup on Bacteria. Eleven further bacterial candidate strains were evaluated together with the already established strains in an international study under routine conditions, which means the simulation of bacterial contamination during blood donation by low bacterial cell count spiking (< 1 Colony Forming Unit per milliliter) directly into PC-bags and determination of their ability to proliferate in PC from multiple donors. Bacterial counts were performed at days 2, 4 and 7 after inoculation to assess the bacterial growth kinetics. The candidate strains included the 2 spore forming bacterial strains (Bacillus cereus PEI-B-P-07S and Bacillus thuringiensis PEI-B-P-57-S), Gram-negative species (Enterobacter cloacae PEIB-P-43, Morganella morganii PEI-B-P-74, Proteus mirabilis PEI-B-P-55, Pseudomonas fluorescens PEI-B-P-77, Salmonella choleraesuis PEI-B-P-78, Serratia marcescens PEI-B-P56), and Gram-positive species (Staphylococcus aureus PEI-B-P-63, Streptococcus dysgalactiae PEI-B-P-71, and Streptococcus bovis PEI-B-P-61). The study was performed in fourteen centres (ten different countries) that tested each bacterial strain in triplicate. With the exception of the Morganella morganii strain, all bacterial strains showed moderate to excellent growth at day 7 after inoculation. The individual growth curves showed variation from slow to fast growth. Bacillus cereus, Bacillus thuringiensis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas fluorescens, Serratia marescens, Staphylococcus aureus and Streptococcus dysgalactiae showed growth of significantly more than 2 log10 CFU/mL up to 8 log10 CFU/mL by day 2 of storage. For Enterobacter cloacae, Proteus mirabilis, Staphylococcus epidermidis, Streptococcus bovis and Streptococcus pyogenes, this growth level was reached at day 4. Growth for Salmonella choleraesuis was lower than for the other strains and showed a high variability among the results of the different participants. In addition, the study provided information regarding the growth behaviour and kinetics of different bacterial species in PC. Stability testing was performed at PEI to ensure the growth ability at a defined content of living cells in deep frozen suspensions.
WHO/BS/2015.2269 Page 3 Platelet Transfusion Relevant Bacteria Reference Strains which are provided as ready to use deep frozen suspensions in a defined cell count are a feasible tool for validation and assessment of various microbiological methods for improving blood safety. Neither resuspension nor cultivation is needed prior to use. In the collaborative study referenced, 11 candidate strains were characterized. Those that demonstrate growth independent of donor effects under “real life” conditions, will be recommended for inclusion in the WHO International Reference Repository of Platelet Transfusion Relevant Bacterial Reference Strains.
Introduction Essential instruments suitable for preventing bacterial contamination of blood components include careful donor selection, selection of the punction site, effective skin disinfection, separation of the initial volume from the blood donation (pre-donation sampling, also called diversion). Nevertheless bacterial contamination is considered as one of the most common transfusion-associated causes of death [1, 2, 3, 4, 11]. The fundamental difference between contamination by viruses in comparison to bacteria is that the latter can replicate strongly in a PC during its shelf life [5, 6]. Under the usual storage conditions at 22.5°C with agitation, microorganisms contaminating a PC can grow up to 10 log10 Colony Forming Units (CFU) per bag. In addition to bacterial cells themselves, pyrogenic substances (i.e. endotoxins and/or exotoxins) may accumulate in the PC bags, depending on the bacterial species and strain. Even relatively apathogenic bacterial strains can cause lifethreatening infections in the recipient after transfusion [10]. The International Society of Blood Transfusion (ISBT) Working Party Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria (former chair: Dr Thomas Montag-Lessing) had organized an international validation study on Platelet Transfusion-Relevant Bacteria Reference Strains (PTRBRS) to be used as a tool for development, validation and comparison of the respective methods. Four blinded bacterial strains had been sent in replicates to participating laboratories worldwide for bacterial count calculation, strain identification and evaluation of growth properties in PCs. The results were submitted to the Expert Committee on Biological Standardization (ECBS) and were established as the WHO Repository of Platelet TransfusionRelevant Bacteria Reference Strains [7]. They are available at PEI as the First WHO Platelet Transfusion Relevant Bacterial Reference Strain Repository. In the first collaborative study (WHO BS/10.2154, 2010) the four strains were sent to the participating laboratories. The study partners had to identify, enumerate and spike the bacterial strains into the PCs in two different concentrations (10 CFU and 100 CFU per bag). Sampling and enumeration was performed on day 4. In contrast, in the enlargement study, the candidate bacterial strains were not blinded with respect to the strain identity and only one concentration was spiked into PCs (10 to 25 CFU per bag). To get more information of the growth kinetics of the candidate strains sampling and enumeration was carried out on day 2, 4, and 7 after inoculation. To ensure the stability of the bacterial strains (number of living cells in the deep
WHO/BS/2015.2269 Page 4 frozen suspension) during the study period and even beyond, cell counting had been performed routinely at PEI. Those 9 strains that demonstrated donor independent growth properties under “real life” conditions and showed stability during the storage time will be recommended for inclusion in the WHO International Reference Repository of Platelet Transfusion Relevant Bacterial Reference Strains. As the tested Morganella morganii PEI-B-P-74 strain showed no growth, a second strain was tested in 8 laboratories in accordance with the study protocol. The new strain showed growth in all tested PCs. The data are very consistent as the statistical evaluation shows. The growth potential as well as the match of inoculum is comparable to the already existing WHO bacteria repository. It is recommended to add this strain to the bacteria extension list.
Materials and Methods Participants and Study Design Sixteen laboratories world-wide were asked, fourteen which participated in the enlargement study and received the samples. All fourteen study partners finished the tests and sent the results. The participants were from Germany (3), Austria (1), The Netherlands (1), England (1), Canada (1), USA (3), Mexico (1), Pakistan (1), Japan (1), and South Africa (1). Details on participants and laboratories are given in Appendix 1: Design of the collaborative study The study protocol (scheme shown in Fig.1) was discussed and confirmed by the TTID WP subgroup on bacteria and presented at the meeting in Amsterdam 2013. The results were presented to the TTID WP in several meetings (i.e. ISBT Congress Seoul 2014, extraordinary meeting TTID WP subgroup on bacteria Philadelphia 2014, ISBT Regional Congress London 2015).
WHO/BS/2015.2269 Page 5 Fig. 1: Scheme of international enlargement study
Fifteen selected bacterial strains (Table 1) were sent to the 14 study-partners frozen on dry ice primarily for the evaluation of bacterial strain growth properties in PCs. As shown in Fig. 1 enumeration and bacterial strain identification was performed for inoculums control and bacterial strain identity confirmation after growth in PCs. For each bacterial strain, three PCs (buffy coat derived pooled and/or apheresis PCs) were inoculated with 10-25 CFU/bag. Before inoculation the baseline sterility of the PCs was confirmed by microbiological control in accordance with the routine standard operating procedure used in the participating laboratory. For inoculation, the bacterial reference strain solution was serially diluted in sterile NaCl to achieve a final concentration of ~10-25 CFU/mL in accordance with a standardized provided protocol (Fig. 2). To enumerate the inocula, 100 µl of the last three dilution steps were plated onto agar plates and colonies were counted the day after incubation. After contamination, the PCs were stored under blood bank routine storage conditions. Aseptic sampling was performed in accordance with the protocol on day 2, 4 and 7 from all 3 PC bags. A dilution series up to dilution 6 was performed from each sample (in total 9 dilution series per strain) and 100 µl of each dilution is plated in triplicate onto agar plates in accordance with the provided protocol. Colonies were counted after they became visible. Strain identification was performed from day 7 isolates of PC bag 1, 2 and 3 to guarantee that the inoculated bacterial strain grew in the PC bag. The study protocol is attached in Appendix 2.
WHO/BS/2015.2269 Page 6 Fig.2: Inoculation and sampling procedure
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Selection and characterization of bacterial candidate strains In total, 15 bacteria strains (4 established repository strains, 11 new candidate strains, 4 vials each strain) were sent to the participants in purpose-built containers on dry ice. Each vial was labelled with the name of the bacterial strain and PEI-identification/lot number (PEI-B-P-XX). The candidate strains are bacterial strains which were selected for their ability to replicate in PCs under routine storage conditions used in transfusion medicine. The strains are prepared using a specifically developed procedure which guarantees defined bacterial suspensions (deep frozen, ready to use, stable, shippable, defined in count of living cells) [7]. The enlargement strains are either isolates from blood products (blood bag and/or recipient) or tested regarding their growth ability in PCs at PEI. The bacterial strains were characterized for their ability to grow in PCs under current routine blood bank conditions (original bag volume, storage under agitation, temperature controlled). Enumeration and stability testing was performed at PEI before and after production/deep freezing and, additionally, during the operating time of the study. For the stability testing six vials of each bacterial strain solution were defrosted and two dilution series of each vial were produced. Samples were transferred directly from deep freezer to a dry incubator at 37ºC for 10 minutes. If ice crystals were still evident, the vial was warmed in the hand until the content had melted. The stock suspensions were used immediately after thawing. Plating assays were carried out (n = 6) for one defined dilution of both dilution series. Thereafter, mean values were calculated. The identity of the bacteria strains was tested by a combination of classical and molecular microbiological procedures. Classical characteristics used are growth properties, colony morphology, Gram-staining, and biochemical parameters like metabolism of certain sugars (APISystem). Additionally, part of the 16s ribosomal RNA gene was sequenced.
WHO/BS/2015.2269 Page 8 Tab. 1: List of tested bacteria strains
Sterility control for baseline sterility of platelet concentrates All PCs were sampled before bacterial inoculation to assure baseline sterility of the original platelet bags. Sterility testing was performed based on the commonly used methods in the laboratories (e.g. aerobic and anaerobic cultivation in automated systems).
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Dilution procedure and artificial contamination For low count spiking the bacteria reference strain solutions were diluted. The test strains were defrosted and vortexed for 15 seconds. Serial dilution of each vial was performed in sterile saline, as described in the dilution procedure in the protocol. The first dilution was termed the D1 (10-1) dilution, the following was termed D2, D3 etc. to the final dilution step containing ~10-25 CFU/mL. Inoculation of each PC bag was perfomed in accordance with the study protocol.
Sampling, enumeration and documentation Sampling was performed on day 2, 4 and 7 (48, 96, 168 hours) after inoculation during PC storage (storage at 20 - 24°C under agitation). Sample withdrawal was performed following the study protocol (Fig.2).
Stability testing of the bacterial strains The stability of all strains was tested at PEI continuously during the storage time and the results were statistically evaluated. For this purpose, 6 vials per lot (start, mid, end of production) were diluted and the colony forming units (CFU) were determined by plate counting method on 6 agar-plates in parallel (Fig.3). Fig. 3: Inoculation and sampling procedure performed for stability testing
Vial with bacterial suspension (Test of 6 vials per lot)
2 Dilution series per vial
6 Agarplates: plate counting method
Statistical methods Statistical analysis was performed at PEI based on the raw data sent by the participants. Evaluation was based on log10 CFU/mL; zero CFU/mL concentrations were set to 0.1 before logtransformation. Taking into account an inoculation of 10-25 CFU/bag and bag sizes of 100 to 300 mL, inoculation per milliliter ranged from 0.033 to 0.250 CFU/mL (i.e. -1.48 to -0.60 log10 CFU/mL).
WHO/BS/2015.2269 Page 10 Growth data were analyzed per strain and day. Overall mean for each strain was estimated by means of a mixed linear model with log10 CFU as dependent variable and random factors bacterial strain and participant. Analysis of stability of inoculum data at PEI was performed for up to five determinations per test strain by means of a linear regression model with dependent variable log10 CFU and date of determination as explanatory variable. The statistical analysis was performed with SAS®/STAT software, version 9.3, SAS System for Windows. Results for bacterial growth were presented in Box- and-Whisker plots (Fig. 4.)
Fig. 4: Box-and-Whisker plots for growth
Results Recovery of inoculum The bacteria reference strain solutions were diluted for low count spiking. The cell count of each bacterial strain was provided in the study protocol and displayed in colony forming units per mL (CFU/mL). The match of inoculum by the participating labs was statistically evaluated and results are shown in Fig. 5 and 6.
WHO/BS/2015.2269 Page 11 Fig. 5: Box-and-Whisker-Plot for the recovery of inoculum
Box-and-Whisker-Plot of recovery of inoculum values by test strains (rhombus: PEI inoculum; A=Bacillus cereus spores, B=Bacillus thuringiensis spores, C=Enterobacter cloacae, D=Escherichia coli, E=Klebsiella pneumoniae, F=Morganella morganii, G=Proteus mirabilis, H=Pseudomonas fluorescens, I=Salmonella choleraesuis, J=Serratia marcescens, K=Staphylococcus aureus, L=Staphylococcus epidermidis, M=Streptococcus bovis, N=Streptococcus dysgalactiae, O=Streptococcus pyogenes)
With the exception of Enterobacter cloacae (reflected as rhombus ◊ in Figure 5) the participating labs confirmed the inoculum value calculated by PEI. Nevertheless, Enterobacter cloacae was also inoculated successfully.
WHO/BS/2015.2269 Page 12 Fig. 6: Recovery of PEI inoculum values by participants, mean log10 CFU/mL plotted as stacked boxes (PEI results dark grey shaded).
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Bacterial Growth in PCs To gain more information about the growth behaviour of each bacterial strain, bacterial cell counting was performed at day 2, 4 and 7 after spiking. Cell counts are summarized in Table 2 and results are presented as Box- and Whisker plots in Fig. 7 and 8. Tab. 2: Statistical evaluation of growth ability for each bacterial strain and day post inoculation
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WHO/BS/2015.2269 Page 22 Fig. 7: Box- and Whisker plots of growth kinetics - WHO-Repository strains
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Fig. 8: Box- and Whisker plots of growth kinetics - Candidate strains
Bacillus cereus spores Bacillus cereus, spores, PEI-B-P-57 12 11 10 9 8 7
Bacillus thuringiensis spores Bacillus thuringiensis, spores, PEI-B-P-07 12 11 10 9 8 7
log10 cfu/ml
6 5 4 3 2 1 0 -1 -2 2 4 Day 7
log10 cfu/ml
6 5 4 3 2 1 0 -1 -2 2 4 Day 7
Enterobacter cloacae, PEI-B-P-43 Enterobacter cloacae 12 11 10 9 8 7 12 11 10 9 8 7
Morganella morganii, Morganella morganiiPEI-B-P-74
log10 cfu/ml
6 5 4 3 2 1 0 -1 -2 2 4 Day 7
log10 cfu/ml
6 5 4 3 2 1 0 -1 -2 2 4 Day 7
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Stability testing Tab. 3: Estimation of stability parameters by means of a Linear Regression (Intercept and Slope=Time) for each test strain
WHO/BS/2015.2269 Page 27 Fig. 9: Stability WHO-Repository Strains (mean values) performed by PEI
Fig. 10: Stability Candidate Strains (mean values) performed by PEI
CFU/mL
CFU/mL
WHO/BS/2015.2269 Page 28 Stability testing – Plots Specification was set by data from first determination at PEI as mean (bold base line) ± 0.5 log10 CFU/mL (dotted lines). A strain can be regarded as stable until the lower 95% confidence interval for an individual prediction (thin line; including variance of the error as well as variance of the parameter estimates) for the linear trend (thick line) intersects the lower specification limit (dotted line).
WHO/BS/2015.2269 Page 29 Fig 11: Stability WHO-Repository Strains performed by PEI
WHO/BS/2015.2269 Page 30 Fig 12: Stability Candidate strains
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WHO/BS/2015.2269 Page 33 Bacillus thuringiensis spores, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus bovis showed stability with good precision. Another six strains, Bacillus cereus spores, Escherichia coli, Klebsiella pneumoniae, Pseudomonas fluorescens, Streptococcus dysgalactiae, and Streptococcus pyogenes, are stable over the observed period with variability of data. For Pseudomonas fluorescens and Streptococcus pyogenes this could be due to outliers at the second (third) timepoint. Enterobacter cloacae was stable until April 2014 (upper prediction limit intersects the dotted specification line). Here a sampling error at the first stability testing might be responsible for the “increase” in bacteria cells during storage. Serratia marcescens showed stability until July 2014 (for 1.5 years). This could be due to outliers at the third timepoint. The two remaining strains (Morganella morganii and Salmonella choleraesuis) are less stable than the other strains.
Bacterial Identification After the testing of the growth kinetics of the bacterial strains the participants identified the strains following their routine protocols as used in the respective microbiological lab (Tab. 4). The results of identification corresponded with the results provided by PEI. According to classification issues Enterobacter cloacae is a gene complex containing six genovars. One of them is Enterobacter hormaechei steigerwaltii. Salmonella choleraesuis (hemolytic synonym and serotype of S.enterica) identified as Salmonella enterica. Streptococcus bovis (biotype I, taxonomy of the group) is currently named Streptococcus gallolyticus pasteurianus due to phylogenetic results.
PEI Method of Identification AccuGENX-ID MALDI-TOF ID AccuPRO-ID Gram stain/API Gram stain/API Maldi-TOF MS /Vitek MS BD Phoenix Identification Mass spectrometry Vite ID / BBL chystal gram positive 4.0 Gram stain/Colony morphology
Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9 Gram stain / Microscan/ API
Lab 10 MALDI mass spectrometry system / Biolog ELx808
Lab 11
Lab 12
Lab 13
Lab 14
Maldi-TOF
MALDI-TOF
16rRNA gene sequence
16S rRNA
Gram stain/ Colony morphology
WHO Repository Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus pyogenes PEI-BP-06
a a a a Bacillus cereus group
a a a a a a a a a a a a a a a a a a yes (Streptococcus type A)
a
a
a
a
Gram positive coccus Gram positive coccus Gram negative rods Gram negative rods
a a a a Gram positive rods
a a a a a a
a a Escherichia coli / (Stenotropomona s maltophilia in bag 1)
a
100%
98%
Gram positive coccus
Streptococcus pyogenes PEI-BP-19 PEI-BP-08 Klebsiella pneumoniae Escherichia coli Serotyping: Escherichia coli, Serovar O6:H16
PEI-BP-20
a
99%
98%
Gram positive coccus
Escherichia coli Klebsiella pneumoniae Candidates for Enlargement study
a a Bacillus cereus/ thuringiensis
99%
98%
Gram negative rods
a
99%
99%
Gram negative rods
Tab. 4: Identification of the strains by the participating labs
Bacillus cereus spores Bacillus cereus Bacillus spp Bacillus genus
PEI-BP-57
a
Bacillus cereus / Bacillus thuringiensis (2 species had equal scores ) 100 '%
99%
Gram positive rods
Bacillus thuringiensis spores PEI-BP-07 Bacillus thuringiensis
a
Possibility of Bacillus Bacillus cereus thuringingiensis group low discrimination
a
Bacillus cereus
a
Gram positive rods
a
a
Bacillus cereus/ thuringiensis
a
Bacillus cereus , Bacillus thuringiensis , Bacillus toyonensis (3 species had equal scores) 99 %
98%
Gram positive rods
Enterobacter cloacae
PEI-BP-43
Enterobacter hormaechei steigerwaltii
Enterobacter hormaechei Serotyping: Enterobacter cloacae
a no growth
a no growth
a no growth
a no growth
a a
a no growth (identifiation of control)
Gram negative rods
a Gram negative rods
a a a
a no growth
a
99%
99%
Gram negative rods
Morganella morganii Proteus mirabilis Pseudomonas fluorescens Salmonella cholerasuis (heterotypic synonym) Serratia marcescens Serratia PEI-Bmarcescens P-56 marcescens / nematodiphila Staphylococcus aureus / PEI-BStaphylococcus P-63 aureus ATCC 6538 Control Streptococcus dysgalactiae equisimilis Staphylococcus aureus Streptococcus dysgalactiae PEI-BP-71 Streptococcus bovis (old name reclassified) Streptococcus gallolyticus pasteurianus PEI-BP-61 Salmonella PEI-B- enterica enterica P-78 NCTC 6017 Control PEI-BP-55 Proteus mirabilis
Morganella PEI-Bmorganii subsp. P-74 morganii Morganella morganii
Morganella morganii morganii
no growth
99%
99%
Gram negative rods
a a Salmonella spp/Salmonella cholerae
a no growth
a a Salmonella spp Salmonella spp (Serotyp: heidelberg)
a a Salmonella spp.
a a yes
a a Salmonella spp.
Gram negative rods Gram negative rods
a a Gram negative rods
PEI-BPseudomonas sp Pseudomonas fluorescens P-77 Salmonella enterica Serotyping: Salmonella Heidelberg
Yes - Gram negative rods /API no / Pseudomonas putida
a a a Salomenella Antisera typing: species (unable Salmonella to speciate choleraesuis organism)
a
100%
99%
Gram negative rods
a
99%
98%
Gram negative rods
Maldi-TOF/ Kaufman-white schema: Salmonella choleraesuis Serratia marcescens
no growth
98%
Gram negative rods
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a Staphylococcus aureus
a a Streptococcus dysgalactiae
a a a Streptococcus gallolyticus no growth
a a a a a a a a a
a a a a
a a Streptococcus dysgalactiae ssp. equisimilis
Gram negative rods
a Gram positive coccus
a a Gram positive coccus
Serratia spp.
a a a a Gram positive coccus Staphylococcus aureus ss aureus Streptococcus dysgalactiae ssp. equisimilis
99%
100%
Gram negative rods
a a a a
100%
99%
Gram positive coccus
a Streptococcus gallolyticus
100%
99%
Gram positive coccus
Streptococcus gallolyticus
Streptococcus bovis / Streptococcus pasteurianis (equal scores) 100 %
99%
no growth
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Discussion All participants received the deep frozen bacteria strains in good condition without complaint. As in the first study, deep frozen, pathogenic bacteria strains could be shipped worldwide without any difficulties. The tested inocula proliferated well and were successfully used for spiking. The bacterial identification performed by the study partners complied with the ID of PEI. The results of bacteria counting of all participants are homogenous since the measured divergence factors represent an acceptable value in the estimation of high bacteria cell counts. The results of the four strains of the existing WHO Repository are equivalent to the first study. With an initial bacterial count of approx. 0.03 CFU/mL (spiking of 10 to 25 CFU per PC unit) Escherichia coli and Staphylococcus epidermidis grew up until day 7 in 100%, Klebsiella pneumoniae in 95% of the tested PC units. These three strains showed consistent growth at all test sites. Streptococcus pyogenes proliferated in 73% of the tested PC units. This strain showed no growth at all at only one site. In the previous collaborative study, this strain did not grow at 3 sites at an inoculum of 10 CFU per bag. The most likely interpretation of these failures are specific or unspecific inhibitory mechanisms directed at the bacterial strain in the donor population [7]. The actual results confirmed the suitability of the strains as 1st WHO Repository. Nine of the eleven candidate strains showed good (70%) to excellent growth (100%). Enterobacter cloacae and Streptococcus bovis failed to proliferate at two sites. The tested Morganella morganii strain grew only at two sites. Salmonella choleraesuis showed no growth at five sites. These results might be explained by antibodies against these strains in the blood donor population. These two strains also exhibited lower stability [Fig.12]. The statistical evaluation of the growth ability showed for Bacillus cereus spores, Bacillus thuringiensis spores, Escherichia coli, Klebsiella pneumoniae, Pseudomonas fluorescens, Serratia marcescens, Staphylococcus aureus and Streptococcus dysgalactiae a growth to significantly more than 2 log10 CFU/mL by day 2. For Enterobacter cloacae, Proteus mirabilis, Staphylococcus epidermidis, Streptococcus bovis and Streptococcus pyogenes this growth milestone was reached at day 4. Growth for Salmonella choleraesuis was lower than for other strains and showed a high variability among participants which might be explained by the low storage temperature or preexisting antibodies. Morganella morganii failed to grow beyond that amount of bacteria in the initial inoculation. As Morganella morganii caused transfusion incidents in several countries it was decided to qualify another strain of this species. In an additional study 8 partners tested a second strain in accordance with the study protocol as a proposal for replacing the Morganella morganii strain from the main study. The results are documented in Appendix 3 in Fig. 12 and 13.
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Conclusion and Proposals Platelet Transfusion-Relevant Bacteria Reference Strains are a practical tool for the objective validation and assessment of methods for screening and pathogen reduction in blood components. The collaborative study confirmed the growth ability, stability and consistency of the already approved four WHO strains. Nine of the candidate strains fulfilled the predefined requirements for addition to the repository. After spiking with low bacteria counts they grew up to high counts in platelet concentrates under routine conditions independently from individual donors properties. As the candidates cover very fast growing bacteria (high bacterial counts already after day 2) and intermediate growers (to high bacterial counts after day 4 to day 7) the bacterial panel well reflects the different needs both of validation of pathogen inactivation technologies and of bacterial detection systems. It is proposed to add these bacterial strains to the existing bacteria repository as WHO Platelet Transfusion-Relevant Bacteria Reference Strains. As the tested Morganella morganii PEI-B-P-74 strain showed no growth, a second strain was tested in 8 laboratories in accordance with the study protocol. The new strain showed growth in all tested PCs. The data are very consistent. It is recommended to add this strain to the WHO bacteria list as well.
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Acknowledgements Christian Gabriel, Susanne Süssner, Claudia Renke, Ingrid Lindlbauer, Austrian Red Cross, Blutzentrale Linz Dana Devine, Sandra Ramirez-Arcos, Heather Perkins, Yuntong Kou, Adriana Zapata, Canadian Blood Service, Ottawa Blood Service, Ottawa Axel Seltsam, Bernd Lambrecht, Birgit Jarck, German Red Cross Blood Service NSTOB, Springe, Germany Birgit Gathof, Melanie Störmer, Transfusion Medicine, University Hospital Cologne, Germany Erhard Seifried, Kai Hourfar, Simone Schwientek, German Red Cross, Frankfurt, Germany Carl McDonald, Kate Aplin, Anjana Roy, NHS Blood and Transplant, London, England Dirk de Korte, Willy Karssing, Herbert Korsten, Sanquin Blood Supply Foundation, Jan Marcelis, Jaap van Meeteren, Eveline Thijssen, Elisabeth Hospital, Tilburg, The Netherlands Richard J. Benjamin, Stephen J. Wagner, Anne Hapip, American Red Cross, Blood Component Dep, Rockville, USA Roslyn Yomtovian†, Michael R. Jacobs, Caryn Good, Case Western Reserve University, Cleveland, USA Truscha Niekerk, Katlego Moagi, Xoliswa Mpumlwana, Nokuthula Chilwane, Nolwazi Nkambule, South African National Blood Service, Weltevreden Park, South Africa Susanne Marschner, Shawn D. Keil, Denise Gilmour, Meghan Hudziec, Therumo, BCT, Lakewood, USA Julieta Rojo, Gabriela Ibañez- Cervantes, Juan Manuel Bello-López, Centro Nacional de la Transfusión Sanguínea, Mexico Masahiro Satake, Hideto Nagumo, Mami Matsumoto, Kumiko Shinozaki, Kumi Kinno, Moe Kaneko, Japanese Red Cross, Tokyo, Japan Zainab Mukhtar, Shaheen Sharafat, Institute of Biological, Biochemical and Pharmaceutical Sciences, Dow Medical College, DUHS Karachi, Pakistan Julia Brachert, Rekia Beshir, Anna-Maria Scheder, Annemarie Mück, Anja Schneider, Sigrid Hanitsch, Utta Schurig, Ute Sicker, Jan-Oliver Karo, Ingo Spreitzer, Paul-Ehrlich-Institut, Langen, Germany
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References 1 Brecher ME, Blajchman MA, Yomotovian R, et al: Adressing the risk of bacterial contamination of platelets within the United States: A history to help illuminate the futuere. Transfusion 2013; 53:221-231 2 Lohmann A, Halbauer j, Witzenhausen C, et al: Hemovigilance Report oft he Paul-EhrlichInstitut, 2012 3 Funk M, Günay S, Lohmann A: Hämovigilanzbericht des Paul-Ehrlich-Instituts, 2014 4 Fatalities Reported to FDA Following Blood Collection and Transfusion – Annual Summary for Fiscal Year 2013 5 Montag T: Perspectives and limitations in the bacterial screening of platelet concentrate. J Lab Med 2006; 30: 60-65 6 Palaveino EL, Yomotovian RA, Jacobs MR: Bacterial contamination of platelets. Transfusion Apheres Sci 2010; 42:71-82 7 Störmer M, Arroyo A, Brachert J, Montag T. et al: Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion transmitted infectious diseases (WP-TTID), subgroup on bacteria. Vox Sang. 2012; 102:22-31. 8 Müller B, Walter-Wenke G, Kalus M, Alt T, Bux j, Zeiler T, Schottstedt V: Routine bacterial screening of platelet concentrates by flow cytometry and is impact on product safety and supply. Vox Sang. 2015, 108 209-218. 9 Schrezemeier H, Walter-Wenke G, Müller TH. Et al: Bacterial contamination of platelet concentrates: results of a prospective multicenter study comparing pooled whole blood-derived platelets and apheresis platelets. Transfusion 2007; 47:644-652 10 Montag T: Strategies of bacteria screening in cellular blood components. Clin Chem Lab Med. 2008; 46:926-32. 11 Störmer M, Vollmer T. Diagnostic Methods for Platelet Bacteria Screening: Current Status and Developments. Transfus Med Hemother 2014; 41:19–27.
WHO/BS/2015.2269 Page 39
Appendix 1 Study partners
WHO/BS/2015.2269 Page 40
Appendix 2
WHO/BS/2015.2269 Page 41 Project Team:
Co-Chair ISBT WP-TTID, Subgroup on Bacteria Dr. Carl P. McDonald Head of Bacteriology National Bacteriology Laboratory NHS Blood and Transplant Charcot Avenue Colindale London NW9 5BG ENGLAND Co-Chair ISBT WP-TTID, Subgroup on Bacteria Dr. Richard J. Benjamin MD PhD FRCPath Chief Medical Officer American Red Cross Holland Laboratories 15601 Crabbs Branch Way Rockville, MD 20855-2743 USA Dr. Melanie Störmer Transfusion Medicine University Hospital Cologne Kerpener Str. 62 50937 Cologne Germany Dr. Eva Spindler-Raffel (Corresponding address) FG Bakteriologische Sicherheit / Microbial Safety Paul-Ehrlich-Institut Paul-Ehrlich-Str. 51-59 63225 Langen Germany
WHO/BS/2015.2269 Page 42 CONTENTS
1. 2. 3.
Background ..........................................................................................................43 Study Design ........................................................................................................45 Shipping and Storage ..........................................................................................46
3.1. 3.2. 4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 4.7. 5.1. 5.1.1. 5.1.2. 5.1.3. 5.2. 6. 7.
Labelling of Bacterial Reference Strains .................................................................. 46 Storage of Bacterial Reference Strains ................................................................. 47 Materials ................................................................................................................... 47 Sterility control for baseline sterility of platelet concentrates .................................. 47 Thawing / Defrosting Bacterial Reference Strains ................................................... 47 Dilution Procedure .................................................................................................. 48 Artificial contamination of platelets /platelet spiking ............................................... 49 Control (Enumeration) of inoculum...................................................................... 50 Sampling, Enumeration and Documentation ....................................................... 50 Enumeration .............................................................................................................. 53 Enumeration 1: after 2 days ...................................................................................... 53 Enumeration 2: after 4 days ...................................................................................... 54 Enumeration 3: after 7 days ...................................................................................... 55 Identification of grown microorganism .................................................................... 56 Lab Protocol ........................................................................................................57 Questionnaire .....................................................................................................61
WHO/BS/2015.2269 Page 43
1. Background Bacterial contamination of platelet concentrates (PCs) still remains a persistent problem in transfusion [1]. To mitigate the risk of bacterial contamination of blood components, blood centres have implemented donor screening along with bacterial detection systems or pathogen reduction technologies (PRT). In order to validate and to compare these methods, it is crucial to use bacterial strains which are able to proliferate in blood components, e.g. in PCs during storage [2]. The International Society of Blood Transfusion (ISBT) Working Party Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria (former chair: Dr Thomas Montag-Lessing) had organized an international validation study on Platelet Transfusion-Relevant Bacteria Reference Strains (PTRBRS) to be used as a tool for development, validation and comparison of the respective methods. Four blinded bacterial strains were sent in replicate to participating laboratories worldwide for bacterial count calculation, strain identification and evaluation of growth properties in PCs. The results were submitted to the Expert Committee on Biological Standardization (ECBS) and were established as the WHO Repository of Platelet TransfusionRelevant Bacteria Reference Strains [3]. Platelet Bacteria Reference strains (PBRS) are a feasible tool for objective validation and assessment of methods for screening and pathogen reduction in blood components. For this purpose, four PBRS were tested and approved. They are available as the WHO Platelet Transfusion Relevant Bacterial Reference Strains Repository. The next step will be to enlarge this bacterial panel to approve more strains as PBRS. In contrast to the previous validation study where both the bacterial count and the growth properties of several replicates in PCs was evaluated, only the ability to proliferate in PCs under “real life”/routine conditions after low spiking (< 1CFU/mL) requires evaluation in this WHO – ISBT enlargement study. For this reason 11 selected bacterial strains that were discussed during the meetings of ISBT WP-TTID Subgroup on Bacteria will be sent to the participating laboratories to evaluate their ability to proliferate in PCs in different regions of the world.
WHO/BS/2015.2269 Page 44 In addition to these 11 new strains, the 4 already approved repository strains will be tested again as a reference. For statistical reasons each bacteria strain has to be tested in 3 platelet bags for 7 days. Enumeration will be performed on day 2, 4 and 7. Whether tested in parallel or on 3 different days is up to the participating lab, but must be documented. References 1. Palavecino EL, Yomtovian RA, Jacobs MR. Bacterial contamination of platelets. Transfus
Apher Sci. 2010; 42:71-82. 2. Montag T, Strategies of bacteria screening in cellular blood components. Clin Chem Lab
Med. 2008; 46:926-32. 3. Störmer M, Arroyo A, Brachert J, Carrero H, Devine D, Epstein JS, Gabriel C, Gelber C,
Goodrich R, Hanschmann KM, Heath DG, Jacobs MR, Keil S, de Korte D, Lambrecht B, Lee CK, Marcelis J, Marschner S, McDonald C, McGuane S, McKee M, Müller TH, Muthivhi T, Pettersson A, Radziwon P, Ramirez-Arcos S, Reesink HW, Rojo J, Rood I, Schmidt M, Schneider CK, Seifried E, Sicker U, Wendel S, Wood EM, Yomtovian RA, Montag T. Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria. Vox Sang. 2012; 102:22-31.
WHO/BS/2015.2269 Page 45
2. Study Design 15 different bacterial reference strains will be sent to the partners. For each bacterial strain, three PCs (pooled and/or apheresis PCs) have to be artificially contaminated with 10-25 CFU/bag. Before contamination the baseline sterility of the PCs needs to be proven by microbiological control in accordance with the routine standard operating procedure used in the participating laboratory. For contamination, the reference strain solution needs to be diluted in sterile NaCl to achieve a final concentration of 10-25 CFU/mL in the tube. To enumerate the inocula, 100 µl of the last three dilution steps are plated onto agar plates and colonies are counted the following day after incubation. After contamination the PCs are stored under routine conditions. Sampling is performed according to the protocol on days 2, 4 and 7 from all 3 PC bags. A dilution series up to dilution 6 is performed from each sample (in total 9 dilution series per strain) and 100 µl of each dilution is plated in triplicate onto agar plates. Colonies are counted. Strain identification has to be performed from day 7 isolates of PC bag 1, 2 and 3 to guarantee that the inoculated bacterial strain grew in the PC bag.
WHO/BS/2015.2269 Page 46
3. Shipping and Storage The 15 bacterial strains will be sent in purpose-built containers with dry ice. Please check the containers immediately after receiving. To assure the stability of the bacterial load of the Bacteria Strain, the cold chain must not be interrupted and the strains must be used immediately after thawing.
Note: Check the vials immediately after arrival. If the samples show any signs of thawing, they must be discarded! In this case please inform the study coordinating team immediately.
3.1. Labelling of Bacterial Reference Strains 15 different bacteria strains are contained in vials in 4-replicates of 4. Each vial is labelled with the name of the bacterial strain and PEI-identification/lot number (PEIB-P-XX). List of Blood Bacteria Reference Strains WHO Repository
Staphylococcus epidermidis Streptococcus pyogenes Escherichia coli Klebsiella pneumoniae Candidates for Enlargement Study Bacillus cereus, spores Bacillus thuringiensis, spores
PEI-B-P-06 PEI-B-P-20 PEI-B-P-19 PEI-B-P-08
Enterobacter cloacae Morganella morganii Proteus mirabilis Pseudomonas fluorescens Salmonella cholerae-suis Serratia marcescens Staphylococcus aureus Streptococcus dysgalactiae Streptococcus bovis
PEI-B-P-57 PEI-B-P-07 PEI-B-P-43 PEI-B-P-74 PEI-B-P-55 PEI-B-P-77 PEI-B-P-78 PEI-B-P-56 PEI-B-P-63 PEI-B-P-71 PEI-B-P-61
WHO/BS/2015.2269 Page 47 3.2. Storage of Bacterial Reference Strains Store the vials immediately after arrival in a deep freezer at -80°C without secondary packaging. Test of growth of selected transfusion-relevant bacterial reference strains in platelet
concentrates (PCs)
4.1. Materials 1 vial of each bacteria test strain.
(The below mentioned materials are calculated for one strain and dilution series for 3 PC bags in parallel. If the spiking is done on different dates you will need more NaCl aqueous solution.) 3 platelet units for each strain (apheresis or whole blood derived PCs. Preferably use fresh PCs, if not available use outdated ones) Usual platelet storage device (agitation, temperature controlled, 22± 2°C) Dry incubator, 37°C and 30°C Sterile welding equipment (e.g. sterile connecting device) Sterile NaCl aqueous solution (0.85 %) in sterile tubes with caps Trypticase Soy Agar plates (alternatively Columbia Blood Agar) Sterile applicators (spattles / spreaders) Sterile syringes Luer Lock connection device/ sterile Coupler spike
Before starting the experiments please ensure sufficient supplies are available i.e. agar plates and NaCl.
4.2. Sterility control for baseline sterility of platelet concentrates All PCs have to be sampled before bacterial inoculation to assure baseline sterility of the original platelet bags. Perform sterility testing, commonly used in your laboratory (e.g. aerobic and anaerobic cultivation in automated systems) and describe the procedure in the result section.
4.3. Thawing / Defrosting Bacterial Reference Strains Transfer the vial directly from deep freezer to a dry incubator and defrost the vial at 37ºC for 10 minutes. If ice crystals are still evident, warm the vial in the hand until the content has melted completely.
WHO/BS/2015.2269 Page 48 Note: The bacterial strains (stock suspensions) must be used immediately after thawing. 4.4. Dilution Procedure For low count spiking the bacteria reference strain solutions need to be diluted. For this reason the cell count of each bacterial strain is provided by the organizing committee and displayed in colony forming units per mL (CFU/mL). For each strain the numbers of dilution steps are provided in the appendices. For all test strains, defrost the vials as described above (Section 4.3.) and vortex for 15 seconds at the highest speed. Perform a series of dilutions of each vial in sterile saline, as described in the dilution procedure (see Section 7 Appendices for each bacterial strain). The undiluted (stock) suspension is termed the D0 (100) dilution. Unless otherwise specified in the dilution procedure, prepare 1:10 serial dilutions using 9 mL of sterile saline (NaCl) each and 1 mL of the stock or 1 mL dilution from previous dilution step. Consequently, each dilution is 1/10th the concentration of the previous dilution. The first 10-fold dilution is termed the D1 (101
) dilution, the following is termed D2, D3 etc. to the final dilution step (containing 10-25
CfU/mL) that is needed.
1mL
1mL
1mL
1mL
1mL
1mL
D0
D1
D2
D3
D4
D5
D6
Dilution series
Dilute each bacterial strain down to approximately 10 to 25 CFU/mL in sterile NaCl.
Note: The final dilution step is dependent on the bacterial count stated in the strain dilution specification (see Section 7 Appendices).
WHO/BS/2015.2269 Page 49 Make sure… … that the dilution series of the stock tubes is prepared immediately after thawing the stock suspension. … that the stock suspension as well as each dilution is intensively vortexed (highest speed) for 15 seconds. … that tips are changed after each step!
4.5. Artificial contamination of platelets /platelet spiking If possible: All work mentioned below should be done in a Laminar Flow & Biosafety Cabinet to avoid contaminations. Connect each platelet bag with a luer-lock connection device (e.g. a short tube using Sterile Connecting Device), or insert a sterile Coupler spike (with luer-lock Safesite valve) through a port into the pack. Luer-Lock If using the luer-lock connection device - draw 5 mL out of the platelet bag using a sterile syringe but do not discard it (see below). Ensure aseptic technique is followed! Using a second sterile syringe, inoculate 1 mL of the final dilution through the same port into the platelet bag. Afterwards the final bacterial load will be 10 – 25 CFU per bag. Add the previously removed 5 mL PC sample back into the bag to flush the tube segment of the bag if using the luer-lock connection device. Close the luer-lock port. Coupler-Spike If using the sterile Coupler-Spike - inoculate 1 mL of the final dilution (~ 10 CFU/mL to 25 CFU/mL) through the port into the platelet bag. Rinse the syringe 3 times with PC. Incubate the contaminated PC units at 22-24°C under agitation for 7 days (168 hours after inoculation).
WHO/BS/2015.2269 Page 50 Note: Close the tube by clamp in case of any opening of the luer-lock device (e.g. before connection with a syringe, change of syringes etc.). The procedure described is used to overcome the “dead-volume” of the tube, i.e. to bring the inoculum directly into PCs main bag. Additionally, bacteria attached to the inner surface of the tube will be detached. Avoid any entry of air into the platelet unit during the inoculation process!
4.6. Control (Enumeration) of inoculum Plate 100 µl of the last three dilution steps from abstract 4.4 in triplicate onto agar plates and incubate strain specific at 30°C or 37°C for 24-48h. Count the colonies and document the results in the lab protocol (Section 6, “dilution of stock”).
4.7. Sampling, Enumeration and Documentation Following inoculation with approximately 10 to 25 CFU per bag, growth kinetics of the test strains during usual PC storage conditions (storage at 20 - 24°C), are monitored as described below:
If possible, all work mentioned below should be done in a Laminar Flow & Biosafety cabinet to avoid contaminations. Sampling will be performed on days 2, 4 and 7 (48, 96, 168 hours). Sample drawing shall be performed following the principles described in Section 4.5. (Artificial contamination of platelets /platelet spiking). If using the Luer-Lock connection device - remove the first 5 mL of the PC using a sterile syringe but do not discard it, use a second sterile syringe to take a sample (1 mL) of each platelet bag and then add back the previously removed 5 mL PC sample in order to enable a repetition if necessary.
WHO/BS/2015.2269 Page 51 If using the sterile Coupling-Spike device – rinse the syringe with PC from the pack 3 times to ensure the sample is from the bag, remove a 1 mL sample of each platelet bag and close the luer-lock port. Enumerate the bacterial count by diluting the 1 mL sample up to 10-6 (D6) and plating out (see Section 4.8). Complete documentation in Section 5.
WHO/BS/2015.2269 Page 52
WHO/BS/2015.2269 Page 53
Documentation 5.1. Enumeration Please copy for each bacterial strain!
5.1.1.
Enumeration 1: after 2 days
Bacterial strain: _____________________________________________ Pool or apheresis PC:_________________________________________ Inocula:____________________________________________________ PC Unit Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Plate 1 Plate 2 Plate 3 Mean value
Strain PC bag 1 date
PC Unit Strain PC bag 2 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
PC Unit
Strain PC bag 3 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
WHO/BS/2015.2269 Page 54 Please copy for each bacterial strain!
5.1.2.
Enumeration 2: after 4 days
Bacterial strain: _____________________________________________ Pool or apheresis PC:_________________________________________ Inocula:____________________________________________________ PC Unit Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Plate 1 Plate 2 Plate 3 Mean value
Strain PC bag 1 Date
PC Unit
Strain PC bag 2 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
PC Unit
Strain PC bag 3 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
WHO/BS/2015.2269 Page 55 Please copy for each bacterial strain!
5.1.3.
Enumeration 3: after 7 days
Bacterial strain: _____________________________________________ Pool or apheresis PC:_________________________________________ Inocula:____________________________________________________
PC Unit
Strain PC bag 1 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
PC Unit
Strain PC bag 2 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
PC Unit
Strain xy PC bag 3 Date
Dilution 100µl of… Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6
Plate 1
Plate 2
Plate 3
Mean value
WHO/BS/2015.2269 Page 56 Please copy for each bacterial strain!
5.2. Identification of grown microorganism Strain: Identification (number) of sample: Growth after day: Macroscopic view Colony morphology:
Microscopic view: (shape: rod, coccus)
Result of Gram-staining: Gram-negative Gram-positive Description of identification method (down to species level, i.e. API) : (Identification panel)
WHO/BS/2015.2269 Page 57 6. Lab Protocol Please copy for each bacterial strain! Test strain_________________________________ Platelet Concentrates Volume PC: Shelf life data: : apheresis-PC pool-PC (donors n= )
Control inoculum (dilution of stock):__________________CFU/mL (mean value) (CFU plate 1: __________ CFU plate 2:____________ CFU plate 3: ______________ ) Result of enumeration of inoculum control: _________________CFU/ml Storage conditions: Bacterial proliferation:
Bacterial growth after storage
Sampling after 2 days (48 h) 4 days (96 h) 7 days (168 h)
Yes (growth)
no
Results of Identification: Method (please add details like reactions)
Microorganism identified: Match of inoculated strain (name) ________________ yes: no:
Notes Laboratory: Responsibility:
WHO/BS/2015.2269 Page 58
Appendices To obtain comparable results from the participants the strains are put together in groups. Please perform the working steps specified below and send the results of every group to the corresponding address after finishing the tests of each group. Group 1:
Staphylococcus epidermidis Bacillus cereus spores Enterobacter cloacae Morganella morganii Group 2:
PEI-B-P-06 PEI-B-P-57 PEI-B-P-43 PEI-B-P-74
Streptococcus pyogenes Bacillus thuringingiensis spores Proteus mirabilis Pseudomonas fluorescens Group 3:
PEI-B-P-20 PEI-B-P-57 PEI-B-P-55 PEI-B-P-77
Escherichia coli Serratia marcescens Staphylococcus aureus Streptococcus dysgalactiae Group 4:
PEI-B-P-19 PEI-B-P-56 PEI-B-P-63 PEI-B-P-71
Klebsiella pneumoniae Streptococcus bovis Salmonella cholerae-suis
PEI-B-P-08 PEI-B-P-61 PEI-B-P-78
WHO/BS/2015.2269 Page 59 Dilution steps WHO Repository
WHO/BS/2015.2269 Page 60 Dilution steps, enlargement strains
WHO/BS/2015.2269 Page 61 7. Questionnaire Please complete this questionnaire and return with the first set of completed results to allow accurate assessment. Study partner: Contact name: Contact details: (Postal address, fax, phone, email) Were you a participant of the first WHO-ISBT International Validation Study on Blood Bacteria Standards? Yes / No Lab equipment used: Microbiological Safety Cabinet (Class II) / Laminar flow hood: If yes, please give details: (Make, model) Yes / No
If no, please give other details (e.g. performed on bench, with bunsen burner): 37°C incubator: Yes / No If no, please give details of temperature used: 30°C incubator: Yes / No If no, please give details of temperature used: 22-24°C incubator: Yes / No If no, please give details of temperature used: Blood culture system (automated system for sterility testing): (Mark, model) Deep freezer (-80°C): Yes / No If no, please give details of alternative used:
WHO/BS/2015.2269 Page 62
Deviation to the protocol: If any deviations to the protocol have been used please describe: (e.g. different method of inoculation / sampling)
Microbiological procedures: Established identification system: (e.g. grams staining, biochemical methods, automated identification systems)
Established cultivation methods: (TSA / Columbia Blood Agar / other) Established enumeration methods: (Manual counting / automated plate counter)
Many thanks for taking the time to complete this questionnaire.
WHO/BS/2015.2269 Page 63
Appendix 3 Test of Morganella morganii PEI-A-91 Morganella morganii caused transfusion incidents in several countries. It would be very necessary to have this strain on the list for enlargement of the WHO repository. As the tested Morganella morganii PEI-B-P-74 strain showed no growth, a second strain was tested in 8 laboratories according the study protocol. The new strain showed growth in all tested PCs. The data are very consistent as the statistical evaluation shows. The growth potential as well as the match of inoculum is comparable to the already existing WHO bacteria repository. As the strain is very new the testing of stability is still under progress but very promising. It is recommended to add this strain to the WHO bacteria list. Fig. 12: Box and whisker plots of Morganella morganii PEI-A-91 Morganella morganii PEI-A-91 12 11 10 9 8 7 log10 cfu/ml
6 5 4 3 2 1 0 -1 -2 1 2 4 Day 7
WHO/BS/2015.2269 Page 64 Fig. 13: Match of inoculum Morganella morganii PEI-A-91 10 9 8 Laboratories
Morganella morganii PEI-A-91
7 6 5 4 3 2 1 2 12 8 15 7 4 1 3
Box-and-Whisker-Plot of recovery of inoculum values by test strains (rhombus: PEI inoculum; A=Bacillus cereus spores, B=Bacillus thuringiensis spores, C=Enterobacter cloacae, D=Escherichia coli, E=Klebsiella pneumoniae, F=Morganella morganii, G= Morganella morganii PEI-A-91, H=Proteus mirabilis, I=Pseudomonas fluorescens, J=Salmonella choleraesuis, K=Serratia marcescens, L=Staphylococcus aureus, M=Staphylococcus epidermidis, N=Streptococcus bovis, O=Streptococcus dysgalactiae, P=Streptococcus pyogenes).
2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 4. 0 4. 2 4. 4 4. 6 4. 8 5. 0 5. 2 5. 4 5. 6 5. 8 6. 0 6. 2 6. 4 6. 6 6. 8 7. 0 7. 2 7. 4 7. 6 7. 8 8. 0
log10 CFU/ml