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Collaborative study to establish the 1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique (NAT)-based assays

3 декабря 2013 г. Всемирная организация здравоохранения

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WHO/BS/2013.2222 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 21 to 25 October 2013 Collaborative Study to Establish the 1st World Health Organization International Standard for Mycoplasma DNA for Nucleic Acid Amplification Technique (NAT)-Based Assays K-M O Hanschmann1, T Montag-Lessing1 , S A Baylis1, M Chudy1, J Kreß1, U Ulrych2, S Czurda2, R Rosengarten2, C M Nübling1 2

Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, D 63225 Langen, Germany Mycosafe Diagnostics GmbH, BioTech Center, Muthgasse 11/2, 1190 Vienna, Austria

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 04 October 2013 and should be submitted electronically to the Responsible Officer: Dr Ana Padilla at email: padillaa@who.int, with a copy to Dr David Wood at email: woodd@who.int. © World Health Organization 2013 All rights reserved. Publications of the World Health Organization can be obtained 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, at the above address (fax: +41 22 791 4806; email: permissions@who.int). 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 [or editors as appropriate] alone are responsible for the views expressed in this publication.

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Contents Summary Introduction Materials and Methods Mycoplasma preparations Lyophilization Post-lyophilization investigations Design of feasibility study Design of comparability study Statistical methods Results (a) Feasibility Study Statistical evaluation of feasibility study results Harmonization of mycoplasma NATs Comments from feasibility study participants (b) Comparability Study Proposed unitage for the WHO IS Discussion Comments from participants Acknowledgements References Tables Figures Attachments (1) Feasibility study protocols (a) (Semi)Quantitative NAT assays (b) Qualitative NAT assays (2) Instructions for Use (draft) Page 3 4 6 6 7 7 8 10 10 11 11 12 13 14 15 15 16 18 18 18 19 30 43 43 51 61

WHO/BS/2013.2222 Page 3 Summary This report describes the World Health Organization (WHO) project to develop an International Standard (IS) for mycoplasma DNA for the use with nucleic acid amplification technology (NAT) based test kits. The international collaborative study consisted of two parts: (a) an initial study to investigate the feasibility of standardization or harmonization in the field of mycoplasma NAT-based assays and, if successful, for selection of the candidate preparation (feasibility study), and (b) a comparability study for value assignment to the candidate preparation. The feasibility study revealed that a mycoplasma species may be representative for other, even distantly related mycoplasma species facilitating the harmonization of NAT assays designed for generic mycoplasma detection. As a result of these studies, the WHO IS candidate chosen is a culture-based preparation of Mycoplasma fermentans which has been freeze-dried for long-term stability. The freeze-dried candidate standard was characterized in parallel with the original liquid bulk material to evaluate potential effects on DNA detectability introduced by the lyophilization process; no detrimental effects were observed. Stability studies have been initiated, and interim data obtained under accelerated conditions already indicate long-term stability of the analyte. It is proposed to establish the 1st World Health Organization International Standard for mycoplasma DNA for nucleic acid amplification technique (NAT)-based assays with a unitage of 2 x 105 IU/ml.

WHO/BS/2013.2222 Page 4 Introduction The detection of different species of mycoplasmas by nucleic acid amplification technology (NAT)-based assays (NAT assays) is playing an increasing role both in the testing of materials used for the manufacture of biopharmaceutical products and for the differential diagnosis of bacterial infections in humans or animals. Throughout this report we use the term “mycoplasmas” or “mycoplasma species” as the trivial name for the bacteria in class Mollicutes, as it is in common use in many publications. Regulations in different parts of the world (e.g. Europe, USA, and Japan) increasingly accept NAT assays for testing biological materials for mycoplasma contamination, either with NAT replacing culture tests or with NAT being performed as a supplement to culture assays.1 These NAT assays are usually designed for generic detection by equivalent amplification of distantly related mycoplasma species. It has been shown that NAT assays of different design may result in quite different qualitative or quantitative results2, and these differences may be due to different assay features and/or to lack of standardization of assay. The World Health Organization (WHO) standardization program for biologicals aims to harmonize diagnostic methods by the use of a common reference preparation, e.g. a WHO International Standard (IS), which may be assigned an arbitrary unitage, the International Unit (IU). If possible, WHO ISs are produced as freeze-dried preparations to facilitate distribution worldwide without requiring a cold chain. The approach of establishing freeze-dried preparations containing the target analyte of biological origin has been proven successful in many fields, e.g. for the harmonization of diagnostic and screening NAT assays for blood borne viruses like hepatitis viruses (including HBV, HCV, HEV) or HIV. The Paul-Ehrlich-Institut (PEI, Langen, Germany) is a “WHO Collaborating Centre for Quality Assurance of Blood Products and in vitro Diagnostic Devices” and initiated the project to develop a WHO IS for mycoplasma NAT assays, endorsed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2010. The collaborative study summarized in this report consisted of two parts: 1) a feasibility study to investigate potential harmonization of assays and to select a candidate material; 2) a follow-up comparability study to characterize the candidate WHO IS and to assign an International Unitage. Both parts of the collaborative study were presented and discussed in different scientific forums. The idea behind the project was first discussed at the Parenteral Drug Association (PDA) Mycoplasma Task Force meeting in 2009 in Berlin and in several subsequent Task Force meetings since. The project was also reviewed at the 3rd Clinical Diagnostics meeting of the Scientific Working Group on the Standardization of Genome

WHO/BS/2013.2222 Page 5 Amplification Techniques (SoGAT) in London (UK) in January 2011 and at the joint 24th Blood Virology and 4th Clinical Diagnostics meeting of SoGAT in Ljubiljana (Slovenia) in May 2013. Feasibility Study. The feasibility study was designed to investigate if a preparation containing a mycoplasma strain would improve harmonization of NAT assays designed for generic mycoplasma DNA detection. The study was also intended to select a suitable mycoplasma species for further development as the candidate WHO IS. In 2012, the feasibility study was performed with worldwide representation of different mycoplasma NAT assays. Volunteer laboratories representing regulatory authorities, manufacturers of biological medicinal products, IVD manufacturers and contract research organizations received coded panels of lyophilized specimens representing four mycoplasma species: Acholeplasma laidlawii, Mycoplasma (M.) fermentans, M. orale and M. pneumoniae. These four species represent distant branches of the phylogenetic tree of the bacteria class Mollicutes (Figure 1). For the study, quantitative NAT assays were defined as assays reporting analyte concentrations in a unitage (e.g. “copies/ml”, “genome copies/ml”, “genomes/ml”), after respective calibration of the assays. Semi-quantitative NAT assays were defined as reporting signal intensities which correlate with the concentration of the analyte, e.g. threshold cycle (CT) values of realtime PCR. Semi-quantitative or quantitative assays were differentiated from qualitative NAT assays reporting test results as either “positive” or “negative”. Results were evaluated by the PEI after all participating laboratories had completed the study. The feasibility study report, proposing a M. fermentans preparation as the most suitable WHO IS candidate material, was distributed to participants. There were no objections to the proposal, and where comments were received from the participants these were incorporated into the final report, as appropriate. Comparability study. After completion and evaluation of the feasibility study, the selected WHO IS candidate material (Panel Member 8; M. fermentans) was prepared under identical conditions as those used for the production of the feasibility study panel members. The candidate material was characterized in terms of residual moisture and for potential effects introduced by the lyophilization process. A stability study under accelerated conditions was started. The lyophilized WHO IS candidate preparation was characterized by preselected NAT assays of different design, in parallel with Panel Member 8 from the feasibility study. Based on results of the comparability study and on potency estimates for Panel Member 8, the candidate material was assigned a value in IU/ml close to the mean “copies/ml” values determined for the quantitative assays and the mean “NAT detectable units” determined for qualitative assays after limiting dilution.

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Materials and Methods Mycoplasma preparations (a) Feasibility Study Panel Strains of four mycoplasma species representing different phylogenetic branches within the bacterial class Mollicutes were selected for inclusion into the feasibility study panels: Mycoplasma fermentans (PG18T, NCTC 10117) Mycoplasma orale (CH19299T, NCTC 10112) Mycoplasma pneumoniae (FHT, NCTC 10119) Acholeplasma laidlawii (PG8T, NCTC 10116) For cultivation of the mycoplasma strains Mycosafe Friis medium (Mycosafe Diagnostics GmbH, Vienna, Austria) was used which had been tested negative for mycoplasma DNA. For the feasibility study panel, the different strains were grown to an approximate target concentration of 105 colony forming units (CFU) per ml (M. fermentans, M. orale and M. pneumoniae) or 107 CFU/ml (A. laidlawii) and harvested during the exponential growth phase. An aliquot of each preparation was additionally diluted 1:100 using Mycosafe Friis medium as diluent. The actual number of CFU was determined for both the neat and the 1:100 diluted preparations (Table 1a). Species identity was confirmed by 16S rDNA sequence analysis and strain identity verified by Randomly Amplified Polymorphic DNA (RAPD) PCR analysis. The number of CFU was determined only for the characterization of the source materials. However, the correlation between CFU and nucleic acid concentration may depend on various factors, including mycoplasma species, strain, growth conditions, proportion of dead cells and the assays for CFU and nucleic acid determination. (b) WHO IS candidate For manufacture of the WHO IS candidate preparation Mycoplasma fermentans (PG18T, NCTC 10117) was selected and grown to an approximate target concentration of 105 colony forming units (CFU) per ml and harvested during the exponential growth phase. Species identity was confirmed by 16S rDNA sequence analysis and strain identity verified by RAPD PCR analysis. The number of actual CFU/ml in the preparation was determined (Table 1b).

WHO/BS/2013.2222 Page 7 Lyophilization For the feasibility study panel members, both the neat and the 1:100 diluted preparations were kept frozen at -60°C until freeze-drying (lyophilization) which was performed by a EN ISO 13485 certified company in Switzerland. Materials were thawed and, with gentle stirring, aliquoted as 0.5 ml volumes into 3 ml screw cap glass vials and immediately frozen. The coefficient of variation (CV) of filling was 1.2 %, as calculated by weight determination. Lyophilization was performed for 62 hours using the Christ Epsilon 2-25 instrument together with LPC-16/NT process documentation. Before sealing of the vials, the system was flooded with dry nitrogen to replace oxygen and moisture. The freezedried preparations were kept at -20°C until delivery to the feasibility study participants. Approximately 200 complete panels were produced in January 2012 for the feasibility study, with the exception of the A. laidlawii preparation with a target concentration of 107 CFU/ ml (Panel Member 9) where >3,000 vials were prepared. The lyophilization protocol, used for the manufacture of feasibility study panels, was used to prepare the candidate WHO IS (M. fermentans). The coefficient of variation (CV) of filling was 1.0 %. The lyophilized candidate WHO IS preparation (PEI code number 8293/13) was prepared in January 2013 in a quantity of 5,126 vials. All vials of lyophilized materials are stored at PEI at -20 °C with regular temperature monitoring. All manufacturing and monitoring records are held at PEI and are available on request by the ECBS. Post-lyophilization investigations A number of investigations were performed to characterize the lyophilized materials. DNA detection after lyophilization The potential impact of lyophilization on the detectability of mycoplasma DNA was addressed by comparative evaluation of lyophilized specimens (feasibility study panel members, candidate WHO IS) and their corresponding liquid source materials which had been kept frozen at -80°C. By using three quantitative NAT assays of different design (Venor GeM qEP, Intego Mycoplasma (both Minerva Biolabs GmbH, Berlin, Germany) and MycoSEQ Mycoplasma Real-Time PCR (Applied Biosystems GmbH, Weiterstadt, Germany)) there was no significant difference in DNA detection determined between non-lyophilized (liquid) and lyophilized mycoplasma materials. Table 2 shows the results obtained with Intego Mycoplasma as a representative NAT assay. Sterility tests Sterility tests performed under aerobic and anaerobic conditions and using both liquid and solid media indicated no presence of viable bacteria in the reconstituted preparations

WHO/BS/2013.2222 Page 8 other than the chosen mycoplasma species, despite some manufacture steps (e.g. aliquoting) having been performed under non-aseptic conditions. CFU after lyophilization Post-lyophilization viability of the different mycoplasma preparations was determined for the lyophilized specimens and their direct source materials (including the additional freeze/thaw cycle). Viability of mycoplasma was reduced by freeze drying by a factor of approximately 0.2 log10 (M. orale), 1 log10 (M. fermentans, M. pneumoniae) and 2 log10 (A. laidlawii) (Table 3). Residual moisture The residual moisture in the freeze-dried samples was determined for a representative number of vials using an accredited method according to the European Pharmacopoeia. Residual moisture never exceeded 2% (0.6% - 1.9%) which is compliant with the “WHO recommendations for the preparation, characterization and establishment of international and other biological reference standards”.3 Stability testing An accelerated stability test programme was initiated simulating storage / transport temperatures of -20°C, 4°C, 23°C, 37°C and 45°C. The potential degradation of mycoplasma DNA under elevated temperature was determined at regular intervals using two quantitative NAT assays (Venor GeM qEP, Intego Mycoplasma). Over a period of 52 weeks no degradation of mycoplasma DNA for the A. laidlawii (code no. 9) preparation was observed, even at elevated temperatures (Figure 2); this is also the case for the M. fermentans candidate WHO IS preparation after 24 weeks of incubation. The stability studies are being continued.

Design of feasibility study For the feasibility study two different panels of mycoplasma species were designed for either (semi)quantitative or qualitative NAT assays. Both panels contained the four selected species represented by panel members common between the two approaches. All panel members were coded. Proposed study protocols differed between the two approaches. Users of (semi)quantitative assay were asked to perform one dilution step (1:10) for the high concentration panel members and to report numerical results, e.g. “copies/ml” or CTvalues. Users of qualitative assays were asked to perform end point dilutions and to report back positive or negative results obtained for the respective dilutions. Reconstitution of panel members was performed with 0.5 ml H2O; all dilutions were to be performed with the diluent representing the usual negative test matrix of the

WHO/BS/2013.2222 Page 9 laboratory. Therefore a variety of different diluents was used including isotonic buffers, saline, culture medium, cultured cells, cell culture supernatant, virus bulk harvest or just H2O. All participants received detailed and specific study protocols and tables for result reporting attached to this report (Attachment 1a: Study protocol for (semi)quantitative NAT assays; Attachment 1b: Study protocol for qualitative NAT assays). See Figure 3 for graphical presentation of the panels. a) Panel for (semi)quantitative NAT assays For (semi)quantitative NAT assays, the feasibility study panel consisted of 9 coded members representing lyophilized preparations of mycoplasma species of different concentrations and a medium control. The concentrations are indicated as the target CFU/ml before lyophilization. Code no. 1: A. laidlawii, 105 CFU/ml Code no. 2: M. fermentans, 103 CFU /ml Code no. 3: M. orale, 103 CFU /ml Code no. 4: M. pneumoniae, 103 CFU /ml Code no. 5: negative (Friis medium) Code no. 6: M. pneumoniae, 105 CFU /ml Code no. 7: M. orale, 105 CFU /ml Code no. 8: M. fermentans, 105 CFU /ml Code no. 9: A. laidlawii, 107 CFU /ml Each participant received three identical panels for three separate test runs. Participants were asked to test all preparations (1-9) neat and the preparations coded 6-9 additionally as 1:10 dilution, using the usual laboratory diluent as the matrix. Following this protocol, each participant performed 3 separate runs, resulting in at least 3 results per panel member and per 1:10 dilution, where proposed. b) Panel for qualitative NAT assays For qualitative NAT assays a feasibility study panel was designed which consisted of lyophilized preparations, coded as follows: Code no. 1: A. laidlawii, 105 CFU/ml Code no. 2: M. fermentans, 103 CFU/ml Code no. 3: M. orale, 103 CFU/ml Code no. 4: M. pneumoniae, 103 CFU/ml Code no. 9: A. laidlawii, 107 CFU/ml

WHO/BS/2013.2222 Page 10 Each participant received three identical panels for three test runs. In the first test run, participants were asked to test the coded preparations neat and in log10-dilution series until test results became negative in order to determine the preliminary end point dilution for each panel member (lowest concentration tested as positive). For the subsequent two test runs, five half log10 dilutions around the pre-determined end point were proposed. Design of comparability study For comparison of the candidate WHO IS preparation with the M. fermentans preparation included in the feasibility study panel (Panel member 8) users of selected (semi)quantitative NAT assays were asked to determine the relative concentrations by testing replicates of both preparations neat and as 1:10 dilution and to report back either copies/ml or CT-values; selected users of qualitative NAT assays were asked to test in replicates end-point dilution series of the two preparations, following a protocol analogous to the feasibility study. Statistical methods The statistical analysis was performed with SAS®/STAT software, version 9.3, SAS System for Windows. Estimation of end-point dilution and relative potency were determined using CombiStats Software, version 5.0, Release 2013, EDQM / Council of Europe. Relative potencies Evaluation of quantitative assays was performed without removing any outlying data. Assays giving CT values and those reporting copies were evaluated separately. Potencies of samples were estimated relative to the Panel Member 8 (M. fermentans; neat material) or Panel Member 2 (M. fermentans; 1:100 dilution) with an assigned arbitrary value of 5.00 log10 International Units/ml (IU/ml) or 3.00 log10 International Units/ml (IU/ml), respectively, by parallel line assay on log transformed data (quantitative protocol) or probit transformed data (end-point dilution protocol). Absolute potencies Quantitative assays Evaluation of the results reported by quantitative assays was restricted to dilutions in the range where the assays produce comparable data (linear range). For comparison of laboratories, the replicate results of each laboratory, corrected for the dilution factor, were combined as arithmetic mean of log10 copies/ml. Furthermore these estimates were combined to obtain an overall estimation for each sample.

WHO/BS/2013.2222 Page 11 Qualitative assays: End-point dilution procedure The results from the independent runs were pooled to give a series of number positive out of number tested at each dilution. The pooled results of the single assays were evaluated by probit analysis to estimate the concentration at which 63% of the samples tested were positive (i.e. the dilution at which on average one single copy per sample tested could be expected under the assumption of an underlying Poisson distribution). The calculated end-point was used to give estimates expressed in NAT detectable units/ml after correcting for an equivalent volume of the test sample. The correction included volume extracted, volume eluted and fraction used for amplification.

Results (a) Feasibility study The feasibility study was announced in 2011 on the homepage of the “International Organization for Mycoplasmology” (IOM), in the “PDA Letter” (September 2011), by communication at various meetings and within the WHO Collaborating Centers network. Interest in voluntary participation was received from numerous organizations representing governmental authorities, biopharmaceutical manufacturers, IVD manufacturers and contract test services. There was no pre-selection of laboratories or NAT assays; all laboratories that expressed a willingness to participate were included. After resolution of various issues with customs and safety regulations in some countries, panels were dispatched at ambient temperature between May and August 2012 to the participating organizations. Feasibility study panels, for evaluation in a total 25 (semi-)quantitative or qualitative mycoplasma NAT assays, were provided to 20 participating organizations. The participants’ names and organizations are provided in alphabetical order in Table 4. A set of panels for qualitative mycoplasma NAT was sent to one applicant who has not yet provided feedback (not included in Table 4). Most participants only performed one assay. The amplification/detection systems and extraction procedures used by participants are listed in Table 5. There were few assays performed by more than one participant. The numbers representing commercially available tests are indicated, with the agreement of the manufacturers. All assays of this evaluation targeted mycoplasma DNA, with the exception of two assays targeting mycoplasma RNA. However, results reported back for one of the RNA detection assays were incomplete and did not allow statistical evaluation, therefore this assay is not included further in this study report. The remaining qualitative assay for mycoplasma RNA detection was included in the feasibility study at a later stage (MilliPROBE; assay 26). The data obtained with this RNA assay were not included in

WHO/BS/2013.2222 Page 12 relative and absolute potency calculations which were restricted exclusively to assays detecting mycoplasma DNA. NAT assays for mycoplasma DNA detection were numbered 1-25 (1–16 (semi-) quantitative; 17-25 qualitative), not reflecting the order in Tables 4 or 5. Two of these assays were able to differentiate mycoplasma species (PLEX-ID Mycoplasma Detection assay, Abbott; CytoInspect PCR/Microarray, Greiner Bio-One). Both assays correctly identified the mycoplasma species of the different panel members. There were no false-positive results obtained with the negative specimen (Panel Member 5) included in the panel for (semi-)quantitative assays, with exception of one in-house RT-PCR (assay 13) reporting high CT-values for this panel member. All assays were able to detect the four mycoplasma species included in the panels, with the exception of a (semi-)quantitative assay designed for detection of M. pneumoniae exclusively which did not cross react with the other species (assay 6) and two qualitative assays which failed to amplify either M. orale (assay 20) or A. laidlawii and M. pneumoniae (assay 22). These assays were excluded from statistical evaluation for the respective species. The results reported by quantitative assays for the same panel member (e.g. in “copies/ml”) differed between assays by a factor of up to 5 log10. The distribution of concentrations reported for neat panel members of the four mycoplasma species are shown in Figure 5. The results reported for qualitative assays show a difference in sensitivity of the diluted samples between assays of up to 2 log10 for most assays and strains, with exceptions already mentioned. Figure 6 illustrates reactivity of individual qualitative assays (assay numbers 17 – 26) with replicate testing of the respective dilution series, providing for each assay either consistently positive or negative results (Figure 6, black or white boxes) or inconsistent results (Figure 6, grey boxes).

Statistical evaluation of feasibility study results (a) (semi)quantitative NAT assays The design of panel members combined with the proposed test protocol facilitated results for three different (by log10) concentrations (“high”, ”medium”, ”low”) for each of the four mycoplasma species included in the panel: The panel itself included two preparations with 100-fold difference (“high”, “low”), with the “high” concentration also being 1:10 diluted by the participant to the “medium” concentration.

WHO/BS/2013.2222 Page 13 The relative potency (relative to a “reference preparation” assigned with an arbitrary potency) determined for the residual panel members by the individual NAT assay was calculated. For this calculation either the “copies/ml” (reported by quantitative assays) or the CT values reported by semi-quantitative real-time NATs were taken. For the statistical evaluation geometric mean values obtained for three individual panels in three test runs were used. Overall, there was a high level of reproducibility between different test runs of an assay (intra-laboratory variability) with only a few outlier results excluded. Estimation of relative potencies was done by means of a parallel line model with the validity preconditions of: (1) linearity within a dilution series for a mycoplasma species and (2) parallelism between the results obtained for different mycoplasma species (Table 6a). The linearity is necessary for potency calculation covering at least three concentration levels per species, and the parallelism (similar slopes) confirms that amplification efficiency of a NAT system is equivalent between the different mycoplasma species. If both preconditions are fulfilled for two different mycoplasma species, the relative potency between these species may be calculated, as reported by the specific NAT assay (Table 6a). For the vast majority of assays, and mycoplasma preparations, these preconditions were fulfilled. Figure 4 shows a graphical representation of quantitative (“copies/ml”) and/or semiquantitative (“CT”) results for the three log10 dilutions (1:1, 1:10, 1:100) for each assay. As an example, it becomes obvious that assays 3, 4 and 8 are non-linear within the A. laidlawii dilutions, but linear with the three other mycoplasma species. (b) qualitative NAT assays The data obtained by a NAT assay in three different dilution series were pooled to give a number positive out of number tested, at each dilution. As for (semi)quantitative assays, relative potencies of individual panel members were estimated relative to preparations included in the panel used as reference, e.g. M. fermentans (Table 6b). This estimation was performed by parallel line assay on probit transformed data using CombiStats Software, version 4.0, Release 2008, EDQM / Council of Europe.

Harmonization of mycoplasma NATs In the relative potency calculation, a mycoplasma preparation may be defined as a “common calibrator”, assigned an arbitrary unitage (e.g. candidate IU/ml), and the relative potency may be calculated for the other mycoplasma preparations in candidate IU/ml, too. The overall distribution of results expressed in candidate IU/ml by the different assays will reveal if the use of a “common calibrator” helps to harmonize results compared to analysis where no common calibrator is used, e.g. “copies/ml”.

WHO/BS/2013.2222 Page 14 Harmonization is reflected by reduction of inter-assay or inter-laboratory variation. Respective calculations were performed using A. laidlawii, M. orale or M. fermentans as candidate calibrators. With A. laidlawii (preparation 9) a harmonization of NAT results was obtained, when compared to the distribution of “copies/ml” reported by the assays. However, the A. laidlawii preparations were not consistently picked up by all assays and different concentration levels were non-linear for some assays. Furthermore, compared to M. fermentans, the results appeared less consistent for A. laidlawii between runs of some assays. M. orale was missed or underestimated by some assays, and M. pneumoniae, as an agent with higher biological hazard classification, would cause logistical shipment problems. In contrast, if the M. fermentans preparation as common calibrator (Panel Member 8, assigned arbitrary concentration of 105 candidate IU mycoplasma DNA/ml) the harmonization in candidate units reported for the other panel members was striking, especially for M. orale or M. pneumoniae, slightly less for A. laidlawii (Figure 7). The results reconfirm the lower consistency of relative results reported for the A. laidlawii preparations. A potential reason might be the higher concentration of mycoplasma DNA in Panel Member 9 compared to Panel Member 8, requiring more dilution steps for the qualitative assay study compounded further by higher variation between assays, and, additionally, being closer to the upper end (less linear) of the “linear range” of (semi)quantitative NATs, again contributing to higher variation between assays.

Comments from feasibility study participants Few specific comments were received on the feasibility study report: After completion of the feasibility study Laboratory 13 recognized an underestimation by 0.5 – 1.0 log10 of the reported results due to suboptimal reference DNA preparation; Laboratory 15 identified a consistent overestimation of the reported results by 1.0 log10 due to use of an incorrect calculation formula. These comments are not reflected in the feasibility study results where all results were included as reported; however, the absolute potency estimates were performed without and with the respective correction (see below).

WHO/BS/2013.2222 Page 15 (b) Comparability Study The outcome of the feasibility study was the selection of M. fermentans as the most suitable candidate for a WHO IS. This decision was confirmed by discussions within the PDA Mycoplasma Task Force and at different meetings (e.g. SoGAT Ljubljana) and presented to the ECBS in October 2012 in an update report for the whole project. A new lyophilized M. fermentans preparation was manufactured in January 2013 using the identical protocol as for preparation of the feasibility study panels. The M. fermentans concentration of the candidate WHO IS was targeted in the range of Panel Member 8. The mycoplasma DNA concentration and the CFU/ml were slightly lower both in the source material used for the candidate WHO IS and in the lyophilized materials when compared to the respective materials of Panel Member 8 (Tables 2b, 3b). Several NAT assays, shown to be proficient in the feasibility study, were used to compare the two lyophilized preparations. In total, the two materials were compared by four different quantitative assays reporting results in copies/ml, by two semi-quantitative assays generating CT values and by five qualitative assays in regard to potency differences. In all assays, the mycoplasma DNA content in the candidate WHO IS was determined to be slightly lower compared to Panel Member 8. The weighted mean difference factor was 0.316 (Table 7). Proposed unitage for the WHO IS Analytes in biological materials are often not traceable to SI units, e.g. mol or gram. In such cases WHO ISs are assigned an arbitrary unitage, the International Unit (IU). For NAT ISs developed for infectious diseases, generally the IU has been as closely aligned to the nucleic acid copy (e.g. genome copy) or to the NAT-detectable unit as possible. Therefore the mean “copies” reported by different quantitative assays for the feasibility study panel members were combined with the corresponding mean of “NAT detectable units” calculated from different qualitative assays’ end point dilution results. For this calculation, the results of the feasibility study were used. The absolute potencies for the neat materials of the feasibility study are shown in Table 8 and the mean values in Table 9. The distribution of absolute potencies, calculated for qualitative and quantitative assays, is illustrated in Figure 8. Calculations were performed without and with using the corrected values for assays 13 and 15; however, there is only a slight difference in the mean potency values of the different materials after correction for laboratories 13 and 15. The corrected mean value of the M. fermentans concentration in Panel Member 8 is 5.76 log copies/NAT detectable units/ml (575,439 copies/NAT detectable units/ml). The weighted mean difference between the candidate WHO IS and Panel Member 8 is factor 0.316, as determined in the comparability study (Table 7), resulting in 181,838 copies/NAT detectable units/ml for the candidate WHO IS. It is proposed to assign a unitage of 2 x 105 IU/ml to the candidate WHO IS.

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Discussion The collaborative study for the establishment of a WHO IS for mycoplasma DNA consisted of two parts. In the first part, the feasibility study, it could be shown that most of the assays designed for generic detection are able to consistently detect the different mycoplasma species provided in the panel. In the study there was only one assay which was designed for the specific detection of M. pneumoniae (Assay 6) which did not crossreact with the other mycoplasma species. The majority of assays of generic design detected the four mycoplasma species contained in the panel. Few in-house developed qualitative assays missed the one or other mycoplasma species, or showed quite different sensitivity limits between different species. In this study the need for an international reference preparation was confirmed by the high variation of quantitative assay results for the same preparations, best explained by lack of standardization of assays. We performed different calculations assuming that individual panel members would be used as common calibrator. Harmonization of assays would have been achieved with any panel member, however, the M. fermentans preparation appeared to be the most suitable candidate. The effect of harmonization of assays by the M. fermentans preparation as common calibrator is obvious. In this study each participant used the routine diluent matrix; spiking of a common mycoplasma preparation (harvested in Friis medium) into different commonly used diluents appeared to have no detrimental effect. Due to the complex composition of biological materials the target analyte e.g. the nucleic acid of a virus or an organism, has to be extracted and purified prior to analysis. This kind of analyte is often not traceable to SI units, e.g. gram or mol. WHO ISs are often assigned instead with IU/ml to have a common value for the content of the analyte. In the field of NAT assays it has been sensible that the “IU/ml” of the WHO ISs are in the similar range to the “copies/ml” reported by quantitative assays and the “PCR-detectable units/ml” calculated from qualitative assay results obtained by replicate limiting dilutions. In the comparability study the weighted mean difference factor between Panel Member 8 and the corresponding candidate WHO IS was calculated using a number of assays of different type (quantitative, semiquantitative, qualitative) and design. Based on this differential factor and the absolute mean potency of Panel Member 8 in the feasibility study a concentration of 2 x 105 IU/ml is proposed to be assigned to the WHO IS for mycoplasma DNA. This value is in the same range as the copies/ml both reported by different quantitative NAT assays (Table 7) and obtained by an NAT independent method for measuring genomic mycoplasma DNA in Panel Member 8 after staining with PicoGreen, a fluorochrome selectively binding to dsDNA (S. Czurda, data not shown). The candidate WHO IS is proposed to be associated with an unitage for mycoplasma DNA, not RNA. First, there was only one assay in the feasibility study delivering

WHO/BS/2013.2222 Page 17 complete results based on RNA detection. Results obtained for the assay 26 (MilliPROBE) are reported for information and, of course, were not included in the statistical evaluation of the DNA detecting assays. Second, mycoplasma RNA is a measurand potentially very different from mycoplasma genomic DNA. The composition and levels of different mycoplasma RNAs depend on the metabolic status of the mycoplasma cell (e.g. cultivation conditions) and test results may strongly differ between different target RNAs selected by the assay(s). Furthermore, there are additional results from the stability study that mycoplasma RNA in lyophilized specimens appears to be clearly less stable under accelerated and stress conditions when compared to mycoplasma DNA (T. Hämmerle, personal communication). An analogous observation had been made in the collaborative study for establishment of a WHO IS for Plasmodium falciparum NAT assays.4 In conclusion, assays targeting (potentially different) mycoplasma RNAs may be much more difficult to standardize by a reference preparation of the chosen design.

WHO/BS/2013.2222 Page 18 Comments from participants In July 2013 the collaborative study draft report was circulated among all participants of the feasibility and comparability study for comments and feedback. Minor changes were proposed which are now included in this version of the report. The feedback was unanimously supportive for the establishment of the 1st WHO IS for mycoplasma DNA and the proposed unitage. Several participants proposed as next step the establishment of a WHO International Reference Panel containing different mycoplasma species for NAT assays.

Acknowledgements We thank all the participants in the feasibility and the comparability study who invested much time and efforts in this exercise. We thank the members of the PDA Mycoplasma NAT Task Force (B. Potts, K. Brorson, S. Deutschmann, T. Hämmerle, L. Mallet) for their continued support and advice. Last not least, we are grateful to the manufacturers of mycoplasma assays who provided kits free of charge for this study.

References 1. Volokhov D, Graham LJ, Brorson KA, Chizhikov VE. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. Molecular and Cellular Probes 2011, 25:69-77. 2. Milne C, Daas A. Establishment of European Pharmacopeia Mycoplasma Reference Strains. Pharmeuropa Bio 2006, 1:57-71. 3. Recommendations for the preparation, characterization and establishment of international and other biological reference standards. WHO Expert Committee on Biological Standardization. Fifty-fifth report, 2004. (WHO Technical Report Series, No. 932). 4. Padley DJ, Heath AB, Baylis SA, Chiodini PL and the Collaborative Study Group. An International Collaborative Study to Establish A WHO International Standard for Plasmadium falciparum DNA Nucleic Acid Amplification Technology Assays. WHO Expert Committee on Biological Standardization, 2006. WHO/BS/06.235.

WHO/BS/2013.2222 Page 19 Tables Table 1: Colony forming units (CFU) in mycoplasma source materials prior to lyophilization (a) Feasibility study panel members Mollicutes species/strain T

Titer Total Cells (CFU/ml) Targeted Titer Actual Titer 0.98 x 106 8.59 x 104 1.67 x 105 1.75 x 103 0.84 x 105 0.87 x 103 1.47 x 105 1.47 x 103 0 7

Acholeplasma laidlawii PG8 , NCTC 10116 Mycoplasma fermentans PG18T, NCTC 10117 Mycoplasma orale CH19299 T, NCTC 10112 Mycoplasma pneumoniae FHT, NCTC 10119 Friis culture medium

105 105 103 105 103 105 103 0

(b) Candidate WHO IS Mollicutes species/strain Mycoplasma fermentans PG18T, NCTC 10117 Titer Total Cells (CFU/ml) Targeted Titer 105 Actual Titer 0.79 x 105

WHO/BS/2013.2222 Page 20 Table 2: Mycoplasma DNA quantification before/after lyophilization by NAT (Intego Mycoplasma, Minerva) (a) Feasibility study panel members Mollicutes species/strain Code Acholeplasma laidlawii PG8T, NCTC 10116 Mycoplasma fermentans PG18T, NCTC 10117 Mycoplasma orale CH19299 T, NCTC 10112 Mycoplasma pneumoniae FHT, NCTC 10119 Friis culture medium 1 9 2 8 3 7 4 6 5 “Copies/ml” Liquid 4.77E+05 7.02E+07 1.44E+04 2.62E+06 1.02E+03 1.44E+05 2.53E+03 1.99E+06 0 Lyophilized 4.08E+05 4.03E+07 1.71E+04 2.23E+06 1.56E+03 2.37E+05 5.90E+03 1.07E+06 0

(b) Candidate WHO IS “Copies/ml” Liquid Mycoplasma fermentans PG18T, NCTC 10117 1.24E+06 Lyophilized 7.33E+05

Mollicutes species/strain

WHO/BS/2013.2222 Page 21 Table 3: Mycoplasma CFU/ml determination before/after lyophilization

(a) Feasibility study panel members Mollicutes species/strain Code Acholeplasma laidlawii PG8T, NCTC 10116 Mycoplasma fermentans PG18T, NCTC 10117 Mycoplasma orale CH19299 T, NCTC 10112 Mycoplasma pneumoniae FHT, NCTC 10119 Friis culture medium 1 9 2 8 3 7 4 6 5 Liquid 6.99E+04 3.33E+06 5.78E+02 6.95E+04 4.57E+02 4.63E+04 1.10E+03 1.15E+05 0 CFU/ml Lyophilized 6.90E+02 5.45E+04 9.25E+01 1.00E+04 3.11E+02 2.44E+04 1.98E+02 2.44E+04 0

(b) WHO IS candidate preparation

Mollicutes species/strain Liquid Mycoplasma fermentans PG18T, NCTC 10117 7.54E+04

CFU/ml Lyophilized 2.63E+03

WHO/BS/2013.2222 Page 22 Table 4: Feasibility study participants (in alphabetical order) Name Eric Abachin, Laurent Mallet Freek Blanken, Nigel Stapleton Francesca Bonci Susan Brand-Hoefs Vicki Chalker Stefan Czurda, Ursula Ulrych, Renate Rosengarten Alena Dabrazhynetskaya, Vladimir Chizhikov Thomas Hämmerle Michael Hantman Matthias Hornschuh, Dirk Vollenbroich Holger Kavermann, Sven Deutschmann Claudia König, Oliver Karo Fabrizio Lecce Andreas Lindauer Cynthia Martino Dietmar Mayer Alexandra Priessner, Michael Molitor Brian Mondeja Rodriguez Walter Rudorfer, Joerg Stappert Rangarajan Sampath Yuko Sasaki Tropical Medicine Institute, Havana, Cuba Greiner bio-one, Frickenhausen, Germany IBIS Biosciences, Abbott, Carlsbad, USA National Institute of Infectious Diseases, Tokyo, Japan Paul Ehrlich Institut, Langen, Germany Merck Serono, Ivrea, Italy Synlab MVZ, Weiden, Germany Bionique Testing Laboratories, Saranac Lake, USA IDT Biologika, Dessau, Germany Novartis Vaccines and Diagnostics, Marburg, Germany Roche Diagnostics, Penzberg, Germany Baxter AG, Orth/Donau, Austria Charles River Biopharmac. Services, Malvern, USA Minerva Biolabs, Berlin, Germany FDA/CBER/LMD, Kensington, USA Organization Sanofi Pasteur, Paris, France Microsafe Laboratories, Leiden, NL Kedrion Biopharmaceuticals, Castelvecchio Pascoli, Italy Merck, Oss, Netherlands Health Protection Agency, London, UK Mycosafe Diagnostics GmbH, Vienna, Austria

WHO/BS/2013.2222 Page 23 Table 5: NAT assays included in the feasibility study Amplification / Detection Extraction Assay No. NAT assays used for (semi)quantitative evaluation In-house RT-PCR 1 In-house RT-PCR 2 In-house RT-PCR 3 In-house RT-PCR 4 Intego Mycoplasma Intego Mycoplasma Intego Mycoplasma Microsart AMP Mycoplasma MycoSEQ Mycoplasma Real-Time PCR QIAamp Viral RNA Mini Kit DNeasy Blood & Tissue Kit QiaSymphony Phenol / Chloroform InviMag Universal Kit/IG Chemagen RSMI QIAamp DNA Blood Mini Kit InviMag Universal Kit/IG PrepSEQ Sample Preparation Kit MycoSEQ Mycoplasma Real-Time PCR MycoTool Mycoplasma RealTime PCR PLEX-ID Mycoplasma Detection assay Venor GeM qEP NucliSENS easyMAG MagNA Pure Bead beating lysis QIAamp DNA Blood Mini Kit 4 11 15 3 5, 8, 9, 10 7 1 2 15

NAT assays used for qualitative evaluation CytoInspect PCR/Microarray CytoInspect DNA Extraction Kit In-house nested PCR In-house RT-PCR 5 In-house RT-PCR 6 MilliPROBE MycoTool Mycoplasma Amplif. and Det. Kits Venor GeM Advance Venor GeM Advance QIAamp DNA Mini Kit Phenol / Chloroform Silica columns Target capture (rRNA) Manual, 2-propanol MB DNA Extraction Kit QIAamp DNA Blood Mini Kit 26 19 20 25 18, 21, 24

WHO/BS/2013.2222 Page 24 Table 6: Potencies relative to Panel Member 8, Mycoplasma fermentans (a) (Semi)quantitative NAT assays A. laidlawii Relative 95%-Confidence potency Interval 7.59 7.31 7.92 5.87 5.45 6.35 M. orale 95%-Confidence Interval 4.88 5.28 4.45 5.45 3.74 4.36 M. pneumoniae Relative 95%-Confidence potency Interval 5.41 5.20 5.62 6.18 5.71 6.77 4.63 4.34 4.91 4.66 4.53 4.79 4.75 4.56 4.93 4.78 4.65 4.90 4.46 4.18 4.73

For the sample data including at least 3 dilutions (1:1, 1:10, and 1:100), potencies were calculated relative to the reference preparation, M. fermentans (Panel Member 8), using a parallel line model. For the reference preparation an arbitrary potency of 5.0 log10 candidate IU/ml was used for the calculations. Assay code Relative potency 5.08 4.94 4.07 Measured Remark Borderline linearity1 Borderline parallelism2 A. laidlawii non-linear A. laidl. non-linear; M. orale non-parallel A. laidlawii non-linear; borderline linearity1 A. laidl. non-linear; M. orale non-parallel Borderline linearity1, M. orale non-linear Only 2 dilutions Only 2 dilutions Borderline linearity1, M. pneum. non-lin. A. laidlawii non-linear Only 2 dilutions Borderline parallelism2 Only 2 dilutions Borderline parallelism2 A. laidlawii non-linear Only 2 dilutions

1 CT values 2 copies 3 copies 3 CT values 4 4.12 3.91 4.31 copies 4 CT values 5 6.14 5.86 6.47 CT values 6 copies 6 CT values 7 6.30 6.17 6.43 4.40 4.29 4.51 copies 8 4.54 3.44 5.43 4.54 3.44 5.43 CT values 9 5.82 5.50 6.16 4.30 3.97 4.62 4.28 3.94 4.59 CT values 10 CT values 11 7.18 6.96 7.41 4.03 3.84 4.20 4.47 4.30 4.63 CT values 12 copies 13 6.59 6.43 6.76 4.25 4.10 4.39 4.74 4.60 4.88 copies 13 4.28 4.17 4.39 4.76 4.65 4.87 CT values 14 copies 15 6.45 6.16 6.77 3.89 3.60 4.16 4.76 4.49 5.01 copies 15 6.54 6.27 6.85 3.92 3.65 4.18 4.77 4.52 5.01 CT values 16 7.13 6.81 7.51 4.18 3.90 4.44 4.65 4.38 4.90 copies 16 7.23 7.06 7.42 4.24 4.10 4.38 4.55 4.42 4.68 CT values Mean3 6.23 – 7.02 4.10 – 4.50 4.54 – 5.01 6.62 4.30 4.77 1 – p-value for non-linearity between 0.01 and 0.05; 2 – p-value for non-parallelism between 0.01 and 0.05; 3 – Combined potency

Borderline parallelism2

WHO/BS/2013.2222 Page 25 (b) Qualitative NAT assays

For sample data including potencies were calculated relative to the reference preparation, Mycoplasma fermentans, using a parallel line assay on probit transformed data. For the M. fermentans reference preparation a potency of 3.0 log10 candidate IU/ml was used for the calculations.

Lab code

17 18 19 fixed slope 20 M. orale negative 21 5.02 4.19 5.88 22 4.25 3.73 4.77 A. laidlawii, M. pneumoniae neg. 23 6.00 5.34 6.66 4.40 3.74 5.06 4.80 4.14 5.47 6.20 5.54 6.87 fixed slope 24 5.82 5.13 6.51 3.80 3.10 4.47 4.23 3.53 4.90 5.42 4.72 6.08 25 7.53 7.10 8.02 4.16 3.66 4.56 4.85 4.41 5.28 7.49 7.06 7.90 Mean2 5.48 – 7.39 4.03 – 4.47 4.48 – 4.95 5.31 – 6.67 6.44 4.25 4.71 5.99 1 – Slope was not estimable and thus fixed to 0.869 (with 0.869 = 1/ln(3.16) and 3.16 as dilution step width) for potency estimation; 2 – Combined potency

A. laidlawii 1:100 Relative 95%potency Confidence Interval 6.80 6.14 7.47 5.56 4.78 6.27 5.00 4.29 5.71 8.52 7.13 10.61 6.26 5.41 7.17

M. orale 1:100 Relative 95%potency Confidence Interval 4.25 3.59 4.91 4.83 4.06 5.60 3.77 3.08 4.47

M. pneumoniae 1:100 Relative 95%potency Confidence Interval 4.59 3.93 5.25 4.57 3.80 5.29 4.36 3.67 5.04 5.00 3.57 6.43 5.58 4.76 6.42

A. laidlawii 1:1 Relative 95%potency Confidence Interval 6.20 5.55 6.87 4.97 4.21 5.69 5.59 4.89 6.29 6.65 5.30 8.51 5.41 4.59 6.23

Remark

WHO/BS/2013.2222 Page 26 Table 7 Relative potency of WHO IS versus Panel Member 8 (Reference) Assay Assay Type Potency of WHO IS candidate relative to Panel Member 8 0.288 0.272 0.282 0.309 0.270 0.392 0.150 0.750 0.603 0.747 0.383 95% Confidence Interval Copies/ml reported for WHO IS candidate 0.477 7.4 x 105 0.535 5.2 x 105 0.382 2.7 x 105 0.421 3.1 x 105 0.358 0.549 4.251 1.582 5.707 2.658 1.049

In-house 1 In-house 2 Microsart AMP Mycoplasma Intego Mycoplasma MycoTool Mycoplasma RealTime PCR MycoSEQ Mycoplasma Real-Time PCR In-house 3 In-house 4 CytoInspect PCR/Microarray Venor GeM Advance MycoTool Mycoplasma Amplification and Detection Kits Combined1

quantitative quantitative quantitative quantitative semiquantitative semiquantitative qualitative qualitative qualitative qualitative qualitative

0.163 0.110 0.204 0.223 0.202 0.274 0.000 0.356 0.062 0.204 0.140

0.316

0.277

0.360

1 – Weighted combination estimator Relative potencies were estimated by means of a parallel line model (quantitative data) and Probit analysis (qualitative data; Spearman-Kaerber method was used instead of Probit in cases, where the slope of the curves could not be estimated). The overall potency estimator is based on a weighted combination of results. No outliers were removed from combination due to relatively homogeneous results.

WHO/BS/2013.2222 Page 27 Table 8 Absolute Potency Estimates (Feasibility Study Panel Members) as log copies/ml (quantitative assays; 2-16) or log NAT detectable units/ml (qualitative assays; 17-25). For assays 13 and 15 both originally reported (not corrected) and corrected (* italic) values are shown

Assay 2 3 4 7 12 13 13* 14 15 15* 16 17 18 19 20 21 22 23 24 25

Acholeplasma laidlawii 95% Confidence Estimate Interval 5.57 3.66 7.48 6.82 5.30 8.33 6.65 5.21 8.09 7.55 7.20 7.90 7.66 4.78 10.54 6.30 5.77 6.83 6.80 6.27 7.33 6.91 4.67 9.15 9.11 8.72 9.50 8.11 7.72 8.50 9.66 9.28 10.04 7.14 6.60 7.69 5.84 5.48 6.20 6.58 6.23 6.92 7.03 5.43 8.63 6.58 5.84 7.32 6.24 6.97 7.30 5.91 6.64 7.09 6.57 7.31 7.51

Assay 2 3 4 7 12 13 13*

Mycoplasma fermentans 95% Confidence Estimate Interval 5.09 3.97 6.21 6.02 5.62 6.42 5.90 5.48 6.31 6.12 5.57 6.68 5.94 4.80 4.57 5.02 5.30 5.07 5.52

WHO/BS/2013.2222 Page 28 14 15 15* 16 17 18 19 20 21 22 23 24 25 5.35 7.70 6.70 7.51 5.64 5.58 6.27 4.48 5.76 5.80 5.14 6.36 4.79 3.73 7.47 6.47 7.11 5.14 5.03 5.77 3.32 5.14 5.32 4.67 5.85 4.47 6.98 7.92 6.92 7.91 6.08 6.11 6.78 5.39 6.33 6.14 5.61 6.84 5.10

Assay

Mycoplasma orale 95% Confidence Estimate Interval 2 4.44 3.77 5.12 3 5.09 4.74 5.44 4 4.99 4.73 5.25 7 5.43 4.86 5.99 12 5.64 13 4.09 4.00 4.18 13* 4.59 4.50 4.68 14 4.94 3.02 6.86 15 6.62 6.31 6.93 15* 5.62 5.31 5.93 16 6.68 6.35 7.01 17 4.89 4.39 5.34 18 5.40 4.85 5.96 19 5.05 4.57 5.53 20 21 5.78 5.17 6.38 22 5.05 4.57 5.39 23 4.54 4.07 5.00 24 5.16 4.66 5.60 25 3.95 3.57 4.22

WHO/BS/2013.2222 Page 29 Mycoplasma pneumoniae 95% Confidence Estimate Interval 5.35 4.43 6.28 5.65 5.06 6.24 5.64 4.97 6.30 5.28 3.57 6.98 5.39 4.60 6.18 5.67 4.55 4.33 4.78 5.05 4.83 5.28 5.07 2.99 7.15 7.46 7.04 7.88 6.46 6.04 6.88 7.15 6.93 7.37 5.23 4.73 5.67 5.14 4.60 5.63 5.63 5.17 6.10 4.48 3.32 5.39 6.34 5.74 6.91 4.94 5.59 4.64 4.47 5.08 4.33 5.42 6.04 4.94

Assay 2 3 4 6 7 12 13 13* 14 15 15* 16 17 18 19 20 21 22 23 24 25

Table 9 Absolute Potency Estimates (Feasibility Study Panel Members) Overall mean estimates with (bold) and without (in brackets) corrections for Lab 13 and 15 N Mean Estimate (assays) 18 7.02 (7.05) 19 5.76 (5.79) 18 5.13 (5.16) 19 5.48 (5.51) 95% Confidence Interval 6.55 (6.52) 7.50 (7.59) 5.41 (5.37) 6.12 (6.21) 4.81 (4.78) 5.45 (5.54) 5.16 (5.12) 5.81 (5.91)

Sample Acholeplasma laidlawii Mycoplasma fermentans Mycoplasma orale Mycoplasma pneumoniae

WHO/BS/2013.2222 Page 30

Figures Figure 1 Phylogenetic Tree of Mycoplasma species

Arrows indicate the different species included into the feasibility study panels

WHO/BS/2013.2222 Page 31

Figure 2 Accelerated stability testing of lyophilized mycoplasma (Intego, Minerva)

Acholeplasma laidlawii, Panel Member 9

WHO/BS/2013.2222 Page 32 Figure 3 Composition of coded feasibility study panels (a) Panel designed for (semi)quantitative mycoplasma NATs (the size of the bars reflects approximate content of CFU (log10 scale))

b) Panel designed for qualitative mycoplasma NATs (the size of the bars reflects approximate content of CFU (log10 scale))

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Figure 4 Result graphs for (semi)quantitative NAT assays (1-16) reported as “copies” or “CT” for three concentration levels of individual Mycoplasma species

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WHO/BS/2013.2222 Page 35 Figure 5 Distribution of “copies/ml” as reported by quantitative assays Assay numbers are indicated in boxes, each box represents a different assay. 8 7 6 NAT assays

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WHO/BS/2013.2222 Page 37 Figure 6 Result graphs for qualitative NAT assays (17-26) reported for replicates of serial dilutions (neat to -6.5 log10) of panel members Panel Member 1, A. laidlawii Assay No. neat 17 18 19 20 21 22 23 24 25 26 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 -6.0 -6.5

Panel Member 2, M. fermentans Assay No. neat 17 18 19 20 21 22 23 24 25 26 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 -6.0 -6.5

Panel Member 3, M. orale Assay No. neat 17 18 19 20 21 22 23 24 25 26 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 -6.0 -6.5

WHO/BS/2013.2222 Page 38 Panel Member 4, M. pneumoniae Assay No. neat 17 18 19 20 21 22 23 24 25 26 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 -6.0 -6.5

Panel Member 9, A. laidlawii Assay No. neat 17 18 19 20 21 22 23 24 25 26 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 -6.0 -6.5

“consistently positive” (black), “consistently negative” (white) or “pos/neg” (grey)

WHO/BS/2013.2222 Page 39 Figure 7 Combined relative potency evaluation for quantitative and qualitative Assays (Reference candidate: M. fermentans) Distribution of candidate IU/ml as calculated for (semi)quantitative assays (1-16; dark colour: calculation based on “copies”; light colour: calculation based on “CT-values”) and for qualitative assays (17-25) with using M. fermentans with an aribitrary concentration of 105 candidate IU/ml (Panel Member 8) or of 103 candidate IU/ml (Panel Member 2) as calibrator. The assay number is included in the box.

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9 5 24 24 21 19 18 5

7 21 20 15 15 13 17 16 16 11 7

23 23 2 17 6

25 25 1 8

19 18

20 9 10

Relative Potency [log10 Candidate IU/ml]

Qualitative assays: grey: for A.laidlawii 1:1 concentration (panel code 9); white: for A. laidlawii 1:100 concentration (panel code 1)

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13 12 11 10 9 Laboratories

Mycoplasma orale Calibrator: M. fermentans (10 5 Candidate IU / ml)

4 3 25 24 19 16 16 15 15 11 2 3 4

8 7 6 5 4 3 2 1

9 8 7 23 22 17 13 13 21 2 18 1 5 6 7 8 9 10

Relative Potency [log10 Candidate IU/ml]

13 12 11 10 9 Laboratories

Mycoplasma pneumoniae 9 8 5 3 3 19 18 17 16 16 13 24 2 3 4

Calibrator: M. fermentans (10 5 Candidate IU / ml)

8 7 6 5 4 3 2 1

4 4 25 23 20 15 15 13 5

21 1 2 6 7 8 9 10

Relative Potency [log10 Candidate IU/ml]

WHO/BS/2013.2222 Page 41 Figure 8 Combined absolute potency evaluation for quantitative and qualitative NAT assays

10 9 8 7

Acholeplasma laidlawii

Laboratories

6 5 4 3 2 1

24 20 17 21 14 25 23 19 13 12 2 18 4 3 7 15 16

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log10 copies/ml ---- log10 NAT detectable units/ml

10 9 8 7 Laboratories

Mycoplasma fermentans

6 5 4 3 2 1

22 21 18 12 25 17 7 24 23 14 4 19 20 2 13 3 15 16

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log10 copies/ml ---- log10 NAT detectable units/ml

WHO/BS/2013.2222 Page 42 10 9 8 7

Mycoplasma orale 24 22 19 17 18 23 14 15 13 4 12 25 2 3 7 21 16

Laboratories

6 5 4 3 2 1

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log10 copies/ml ---- log10 NAT detectable units/ml

10 9 8 7

Mycoplasma pneumoniae 24 19 12 23 7 18 6 17 4 25 14 3 20 13 2 21 15 16

Laboratories

6 5 4 3 2 1

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log10 copies/ml ---- log10 NAT detectable units/ml

Results from quantitative (copies/ml, shaded in grey) and qualitative assays (adjusted for dilution, not shaded).

WHO/BS/2013.2222 Page 43 Attachments Feasibility study protocols (a) (Semi)Quantitative NAT assays

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WHO/BS/2013.2222 Page 51 Attachments Feasibility study protocols (b) Qualitative NAT assays

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WHO/BS/2013.2222 Page 61 Instruction for Use (draft)

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Основные сведения

Организация Всемирная организация здравоохранения (ВОЗ / WHO)

Тип документа Technical Documents

Дата принятия 3 декабря 2013 г.

Источник Всемирная организация здравоохранения