# Final Inter-Laboratory Proficiency Testing Program Report (FQPCRMV24): PCR detection and/or quantification of Coxiella burnetii for the diagnosis of abortion in ruminants.

Elodie Rousset, Myriam Prigent

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Elodie Rousset, Myriam Prigent. Final Inter-Laboratory Proficiency Testing Program Report (FQPCRMV24): PCR detection and/or quantification of Coxiella burnetii for the diagnosis of abortion in ruminants.. Rapport Final - Version 01, ANSES - Laboratoire de Sophia Antipolis, Unité fièvre Q animale (UFQa). 2024, 26 p. anses-05112766

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FINAL INTER-LABORATORY PROFICIENCY TESTING PROGRAMME REPORT
RF – FQPCRMV24 – V1 – ENG

PCR detection and/or quantification of
*Coxiella burnetii* for the diagnosis of abortion in
ruminants

Session : 2024

Final Report - Version 1

IPTP CODE: FQPCRMV24

Written by Elodie Rousset

<table><thead><tr><th></th><th>Last name, First name</th><th>Position</th><th>Date</th><th>Signature</th></tr></thead><tbody><tr><td>APPROVAL</td><td>ROUSSET Elodie</td><td>Coordinator</td><td>2024-12-23</td><td></td></tr><tr><td>AUTHORISATION FOR DISTRIBUTION</td><td>THIERY Richard</td><td>Technical Manager</td><td>2024-12-24</td><td></td></tr></tbody></table>

LAB.EIL.ENG.01 V01

LABORATOIRE DE SOPHIA ANTIPOLIS
105 Route des Chappes - Les Templiers - BP 111 – 06902 Sophia-Antipolis – Tél : +33 (0)4.92.94.37.00
## CONTENT

1. INTRODUCTION .................................................................................................................... 3

2. GENERAL INFORMATION ........................................................................................................ 3

2.1 P STAFF INVOLVED IN THE DESIGN AND MANAGEMENT OF THE CAMPAIGN .................................................... 3

2.2 INFORMATION AND INSTRUCTIONS FOR THE PARTICIPANTS ........................................................................ 3

3. COMPARISON MATERIALS ........................................................................................................ 5

3.1 PREPARATION OF COMPARISON MATERIALS.................................................................................... 5

3.2 HOMOGENEITY AND STABILITY ........................................................................................................ 5

3.3 IDENTIFICATION OF THE COMPARISON MATERIALS (CODING) ........................................................................ 6

4. ASSESSMENT METHODS ................................................................................................................ 6

4.1 DEFINITION OF ASSIGNED VALUES ........................................................................................................ 6

4.2 CRITERIA AND ASSESSMENT METHODS.................................................................................... 7

4.3 ASSIGNED VALUES (EXPECTED RESULTS) .................................................................................... 7

5. PARTICIPATING LABORATORIES AND USED METHODS ........................................................................ 8

6. QUALITATIVE RESULTS : SPECIFICITY AND SENSITIVITY ........................................................................ 9

6.1 QUALITATIVE RESULTS (TABLE) ........................................................................................................ 9

6.2 ANALYSIS OF SENSITIVITY (SAMPLES SD, S3, S4 ET S5) ........................................................................ 10

6.3 ANALYSIS OF SPECIFICITY (SAMPLES SN ET SN/I) ........................................................................ 10

6.4 RESULTS OF CQ VALUES FOR THE COXIELLA BURNETII TARGET (TABLE) .................................................... 11

7. QUANTITATIVE RESULTS .................................................................................................................. 12

7.1 QUANTITATIVE RESULTS (TABLE) ........................................................................................................ 12

7.1 SPECIFICITY AND SENSITIVITY ........................................................................................................ 13

7.2 REPEATABILITY (PRECISION)........................................................................................................ 13

7.3 TRUENESS (Z-SCORES).................................................................................................................... 14

8. RELATIVE RESULTS TO THE RMIT ........................................................................................................ 15

8.1 ORDERED QUALITATIVE RESULTS OR 'RELATIVE TO THE RMIT (TABLE).................................................... 15

8.2 SPECIFICITY AND SENSITIVITY ........................................................................................................ 16

8.3 TRUENESS .................................................................................................................................................... 16

9. CONCLUSION AND DISCUSSION ........................................................................................................ 17

9.1 SUMMARY OF RESULTS BY PCR TYPE ........................................................................................ 17

9.2 LIMITATIONS AND OBSERVED DEVIATION WITH INVESTIGATIONS PATHWAYS ........................................ 18

9.3 OVERALL LABORATORY PERFORMANCES .................................................................................... 19

10. OTHER INFORMATION .................................................................................................................... 20

11. REFERENCES ........................................................................................................................................ 20

12. CLAIMS.................................................................................................................................................... 21

13. APPENDICES ........................................................................................................................................ 22

13.1 DÉTAILED RESULTS OF IPC CQ VALUES (TABLE) .................................................................................... 22

13.2 LIST OF RECOMMENDED METHODS ........................................................................................................ 23

13.3 CODING OF THE SAMPLES IN THE PANEL .................................................................................... 24
Skimming through? We've got you covered! Head straight to the
summary table in section 9.1 for a quick overview of the results

# 1. INTRODUCTION

An Inter-Laboratory Proficiency Test (ILPT) on **PCR methods** was organized in **2024** by the Anses laboratory in Sophia Antipolis, Animal Q fever Unit, as part of its national and international mandates. The objective was to provide veterinary diagnostic laboratories with an external assessment of the quality of their tests on vaginal swab suspensions for the detection of *Coxiella burnetii*, the agent of Q fever. These analyses are carried out as part of the **differential diagnosis of abortions in sheep, goats, and cattle**, as well as to identify **vaginal shedders during investigations**.

This ILPT involved **50 participants**, including the 7 laboratories officially approved in France for this type of analysis. The **methods used were preferably those validated** according to the NF U47-600 standard (AFNOR) and the requirements of the French Reference National Laboratory for Q Fever (RNL-QF), or those whose performances had been verified (by comparison with a validated method), in compliance with RNL-QF recommendations. Participants could report **qualitative, quantitative, or results relative to a reference material at interpretation threshold (RMIT)**.

# 2. GENERAL INFORMATION

## 2.1 P STAFF INVOLVED IN THE DESIGN AND MANAGEMENT OF THE CAMPAIGN

<table><caption>**Table 1.** Contributors to the ILPT.</caption><thead><tr><td>NOM, Prénom</td><td>Fonction</td></tr></thead><tbody><tr><td>ROUSSET Elodie</td><td>Coordinator, Data Analyst</td></tr><tr><td>PRIGENT Myriam</td><td>Technical Manager</td></tr><tr><td>GRONCIN Stéphane</td><td>Information Systems Manager (coding, database constitution)</td></tr><tr><td>TORRES Christine</td><td>Logistics and Administrative and Financial Services</td></tr><tr><td>THIERY Richard</td><td>Technical Manager</td></tr></tbody></table>

## 2.2 INFORMATION AND INSTRUCTIONS FOR THE PARTICIPANTS

### 2.2.1 Timeline

* 05/07/2024 : Announcement of the ILPT, with the publication of the ILPT presentation sheet

* 16/09/2024 : Publication of instructions and result submission form.

* 24/09/2024 : Shipment of sample panels.

* 16/10/2024 : Deadline for result submission.

<figure><img src="image_1.png" /></figure>
### 2.2.2 Variables analysed in the ILPT

Participants were required to use validated or verified methods and report their results according to their routine practices. Performances were assessed based on the following variables:

* **qualitative results**, specifying whether the result was:

- "Detected", in the presence of a Cq value derived from a characteristic amplification curve,

- "Not detected", in the absence of a Cq value or a characteristic amplification curve.

* **quantitative results:**

- "Not detected" in the absence of a PCR signal (Cq value),

- "Detected not quantifiable", when the result is below the limit of quantification of the method (LOQ),

- the number of bacteria per mL in $log_{10}$ when the result was within the quantification range (with re-dilution and re-analysis if necessary).

* **relative to the RMIT:**

- "Not detected",

- "Detected", specifying the comparison with the target quantity of the RMIT (conversely to the Cq value)

- "with load lower than the RMIT",

- "close to the RMIT",

- "with load higher than the RMIT".

The term "close to the RMIT" applies to any result within the range of ± 1.0 Cq compared to the Cq value obtained for the RMIT included in the analysis series (corresponding to ± 0.30 $log_{10}$).

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Overview of the PCR result reporting workflow in the ILPT.</figcaption></figure>

In this ILPT, the variables analyzed were qualitative (binary or ordinal) for qualitative PCR and relative against a RMIT (rPCR), respectively, or quantitative for quantitative (qPCR).
# 3. COMPARISON MATERIALS

## 3.1 PREPARATION OF COMPARISON MATERIALS

### 3.1.1 Origin and composition

**a) Raw materials specifications**

Negative suspensions were prepared from vaginal swabs collected from ewes raised in salt marsh pastures in France. The suspensions, organized into batches of 25 swabs and verified to be free of Coxiella burnetii by digital PCR, served as a matrix for negative samples and as a base for samples spiked with inactivated bacteria (reference strain Nine Mile, phase 2).

**b) Description of the sample panel**

The panel provided to participants consisted of 10 blind samples, distributed as follows:

* **2 dummy suspensions** to reduce the risk of collusion between participating laboratories. These samples were randomly selected from the "FQPCRMV21" panels.

* **8 suspensions for evaluation**, containing bacteria at defined concentrations or none at all:

* **SN**: 1 negative level from a pool of suspensions with no detectable bacteria.

* **SN/I**: 1 negative level with a delayed or undetectable internal control Cq, from a different pool of suspensions.

* **4 independent positive levels**:

* **SD**: A load at the detection limit.

* **S3**: A low load, also near the detection limit.

* **S4**: A medium load (in 3 replicates).

* **S5**: A high load.

Each positive level was prepared using a ratio of 100 µL of the bacterial suspension to 7.6 mL of the base suspension to ensure a homogeneous matrix effect.

### 3.1.2 Packaging

Each suspension sample was aliquoted into tubes containing 200 µL per tube. The panels were stored at a temperature of -16°C or lower until shipment. A total of 800 samples was produced to meet the needs of the 50 participating laboratories.

## 3.2 HOMOGENEITY AND STABILITY

### a) Homogeneity tests

All packaged samples were verified to ensure their homogeneity prior to distribution. Negative samples were excluded from the tests as they were not relevant.

### b) Stability tests

Stability tests were conducted to simulate the various transport and storage conditions.
<table><caption>**Table 2.** Stability tests performed on all samples used for evaluation.</caption><thead><tr><td>Condition</td><td>Description</td></tr></thead><tbody><tr><td>« Day 0 »</td><td>No modification of storage conditions (≤ -16°C), corresponding to the test performed during the homogeneity study.</td></tr><tr><td>« Transport »</td><td>Samples initially frozen and placed with a cold pack, left at room temperature to simulate the maximum transport duration (4 days). This maximum duration was estimated based on transport times recorded during the previous ILPT.</td></tr><tr><td>« Pre-session »</td><td>Samples subjected to "transport" conditions and laboratory storage (4 days of "transport" and 11 days at ≤ -16°C) to test the maximum session duration from shipment to analysis. Instructions required storage at ≤ -16°C upon receipt.</td></tr><tr><td>« Parallel session »</td><td>Samples tested simultaneously with the participants' session under identical conditions, except with a transport duration of 6 days</td></tr></tbody></table>

The objective was to ensure that the participants' performance evaluation was not affected by sample quality.

### 3.3 IDENTIFICATION OF THE COMPARISON MATERIALS (CODING)

Each sample was assigned a randomly generated number between 1 and 10. Additionally, a random code consisting of 1 to 3 digits was assigned to each participating laboratory to ensure the anonymity of the results. Each participating laboratory received the panel with its specific laboratory code, which was indicated on each sample. The coding of each sample in the **FQPCRMV24** panel is presented in the **Appendix** for each participant laboratory's code.

## 4. ASSESSMENT METHODS

### 4.1 DEFINITION OF ASSIGNED VALUES

The determination of assigned values (or expected results) for the various samples was conducted using two approaches depending on the type of variables:

* **Consensus values:** For quantitative variables, the assigned value (X*) and the standard deviation for proficiency testing evaluation (σpt or S*) were calculated based on robust statistics (as described in chapters 7.7 and 8.6 of the NF EN ISO 13528 standard) using the results reported in log10 (Table in 7.1)

* **Organizer-determined values:** For qualitative variables (binary or ordinal), the expected results were based on the outcomes of homogeneity and stability tests conducted by the organizing laboratory. The expected results were established based on these observations, and their alignment with the consistency of participants' results was observed.
## 4.2 CRITERIA AND ASSESSMENT METHODS

All participants received the same samples (except dummy samples). Depending on the three types of PCR, the evaluation criteria were as follows:

* ✓ sensitivity and specificity for qualitative PCR,

* ✓ sensitivity, specificity and trueness for PCR relative to the RMIT,

* ✓ sensitivity, specificity, trueness and precision for quantitative PCR.

<table><caption>**Table 5.** Definitions of evaluation criteria based on the types of PCR methods.</caption><tr><td>Specificity</td><td>The laboratory's ability to report "not detected" for samples expected to yield "not detected."</td></tr><tr><td>Sensitivity</td><td>The laboratory's ability to report "detected" for samples expected to be positive.</td></tr><tr><td>Trueness</td><td>The laboratory's ability to achieve a satisfactory z-score (quantitative variables).<br>The laboratory's ability to provide a correct (concordant) interpretation for qualitative "relative to the RMIT" variables.</td></tr><tr><td>Precision (repeatability)</td><td>The laboratory's ability to achieve a satisfactory Mandel k-ratio (quantitative variables).</td></tr></table>

## 4.3 ASSIGNED VALUES (EXPECTED RESULTS)

The expected results for the 8 comparison materials in the ILPT are presented in the table below for each type of PCR evaluated (Table 4).

<table><caption>**Table 4.** Expected results for ILPT comparison materials and evaluation criteria. The analytical performance of participants was evaluated based on 2, 3, or 4 criteria depending on the type of PCR used.</caption><thead><tr><th rowspan="2">Type of PCR</th><th rowspan="2">Sample Id</th><th colspan="4">Evaluation criteria</th><th rowspan="2">Expected result</th></tr><tr><th>Specificity</th><th>Sensitivity (Detectability)</th><th>Precision</th><th>Trueness</th></tr></thead><tbody><tr><td rowspan="5">Qualitative</td><td>S3</td><td></td><td>(X)</td><td></td><td></td><td>Detected (or Not detected)</td></tr><tr><td>S4 (x3)</td><td></td><td>X</td><td></td><td></td><td>Detected</td></tr><tr><td>S5</td><td></td><td>X</td><td></td><td></td><td>Detected</td></tr><tr><td>SD</td><td></td><td>(X)</td><td></td><td></td><td>Detected or Not detected</td></tr><tr><td>SN/I</td><td>X</td><td></td><td></td><td></td><td>Not detected or Uninterpretable</td></tr><tr><td>SN</td><td>X</td><td></td><td></td><td></td><td>Not detected</td></tr><tr><td rowspan="6">Quantitative</td><td>S3</td><td></td><td>(X)</td><td></td><td>X</td><td>2,8 log<sub>10</sub> ± 0,7 bact. / mL</td></tr><tr><td>S4 (x3)</td><td></td><td>X</td><td>X</td><td>X</td><td>3,7 log<sub>10</sub> ± 0,7 bact. / mL</td></tr><tr><td>S5</td><td></td><td>X</td><td></td><td>X</td><td>4,8 log<sub>10</sub> ± 0,7 bact. / mL</td></tr><tr><td>SD</td><td></td><td>(X)</td><td></td><td></td><td>Not detected, Not quantifiable or Quantifiable</td></tr><tr><td>SN/I</td><td>X</td><td></td><td></td><td></td><td>Not detected or Uninterpretable</td></tr><tr><td>SN</td><td>X</td><td></td><td></td><td></td><td>Not detected</td></tr><tr><td rowspan="5">Semi-quantitative (Relative to the RMIT)</td><td>S3</td><td></td><td>(X)</td><td></td><td></td><td>Detected (or Not detected)</td></tr><tr><td>S4 (x3)</td><td></td><td>X</td><td></td><td></td><td>Detected</td></tr><tr><td>S5</td><td></td><td>X</td><td></td><td>X</td><td>Détecté (en charge > au MRSI)</td></tr><tr><td>SD</td><td></td><td>(X)</td><td></td><td></td><td>Detected (or Not detected)</td></tr><tr><td>SN/I</td><td>X</td><td></td><td></td><td></td><td>Not detected or Uninterpretable</td></tr><tr><td>SN</td><td>X</td><td></td><td></td><td></td><td>Not detected</td></tr></tbody></table>

Note: Multiple results may be possible for the same sample.
The S3 and SD samples are at the detection limit. The SN/I sample is negative and, depending on the
IPC Cq value, could be reported as "Uninterpretable" if justified with clear explanations.

When the ILPT instructions were not followed (particularly regarding the result format, thresholds, or formulations), or when a participant's results failed to meet at least one of the criteria established for this ILPT, the laboratory was required to either immediately correct the issue or investigate the root causes and implement corrective actions, if necessary with support from the RNL as the coordinating body. A second panel may be offered to verify improvement or the restoration of performance.

**5. PARTICIPATING LABORATORIES AND USED METHODS**

Au total, 50 laboratoires ont participé à l'essai inter-laboratoire :

- 35 laboratories in France:

o 33 departmental veterinary diagnostic laboratories within the French network (including **all 7 approved laboratories**),

o 2 kit suppliers based in France,

- 15 international laboratories from Germany (1 participant), Austria (1), Belgium (1), Croatia (1), Denmark (1), Finland (2), Ireland (1), Poland (1), Romania (1), the United Kingdom (1), Serbia (3), Switzerland (1), and the UK (1).

The 7 French laboratories officially approved for PCR analyses met the ILPT requirements: They used one of the methods recommended by the RNL-QF and reported results in the quantitative and/or relative format compared to a reference material at the interpretation threshold (RMIT) of 10⁴ bacteria/mL.

In total, 28 methods were used, including 17 of the 24 recommended methods and 11 "other" methods (see Appendix). Results were reported in the following formats (Figure 2).

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Distribution of result formats.</figcaption></figure>
# 6. QUALITATIVE RESULTS : SPECIFICITY AND SENSITIVITY

## 6.1 QUALITATIVE RESULTS (TABLE)

<table><thead><tr><th>code lab</th><th>Used method</th><th>S3</th><th>S4</th><th>S4</th><th>S4</th><th>S5</th><th>SD</th><th>SN/I</th><th>SN</th></tr></thead><tbody><tr><td>1</td><td>Autre/Other</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>2(A)</td><td>Autre/Other</td><td>13</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>3</td><td>Autre/Other</td><td>23</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>4</td><td>Autre/Other</td><td>13</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>5</td><td>Autre/Other</td><td>13</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>6 (A)</td><td>Autre/Other</td><td>1</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>interprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>7</td><td>Autre/Other</td><td>13</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>8</td><td>Autre/Other</td><td>non détecté / not detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>9</td><td>Autre/Other</td><td>5</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>10</td><td>Autre/Other</td><td>5</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>11</td><td>Autre/Other</td><td>19</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>12</td><td>Autre/Other</td><td>19</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>13</td><td>Autre/Other</td><td>5</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>14</td><td>Autre/Other</td><td>7</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>15</td><td>Autre/Other</td><td>17</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>16</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>17</td><td>Autre/Other</td><td>11</td><td>non détecté / not detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>18</td><td>Autre/Other</td><td>non détecté / not detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>19</td><td>Autre/Other</td><td>7</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>20</td><td>Autre/Other</td><td>4</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>interprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>21</td><td>Autre/Other</td><td>5</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>22 (A)</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>interprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>23</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>24 (A)</td><td>Autre/Other</td><td>7</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>25</td><td>Autre/Other</td><td>3</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>26</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>27</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>28</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>29</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>30</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>31</td><td>Autre/Other</td><td>6</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>32 (A)</td><td>Autre/Other</td><td>1</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>33</td><td>Autre/Other</td><td>2</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>34</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>35</td><td>Autre/Other</td><td>7</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>36</td><td>Autre/Other</td><td>12</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>37</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>38</td><td>Autre/Other</td><td>19</td><td>non détecté / not detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>39</td><td>Autre/Other</td><td>5</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>40</td><td>Autre/Other</td><td>13</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>3</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>42</td><td>Autre/Other</td><td>14</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>43 (A)</td><td>Autre/Other</td><td>16</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>44</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>45</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>46</td><td>Autre/Other</td><td>15</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>47</td><td>Autre/Other</td><td>24</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>48</td><td>Autre/Other</td><td>17</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>49</td><td>Autre/Other</td><td>17</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>50</td><td>Autre/Other</td><td>non détecté / not detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>détecté / detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr></tbody></table>

French approved laboratories are marked with an (A) in the lab code column.
The results were unsatisfactory for two laboratories (codes 17 and 33) in terms of specificity (Tables in 6.1 and 6.4). No discrepancies identified regarding sensitivity.

## 6.2 ANALYSIS OF SENSITIVITY (SAMPLES SD, S3, S4 ET S5)

**Sensitivity for S5 and S4** : All laboratories reported the expected result, "Detected".

No deficiencies were observed for these samples, which is consistent with the expectation for a high load, such as **S5**, and a medium load, such as **S4** (approximately 10 times the detection limit of the validated recommended methods).

**Detection limit (LOD) for S3 and SD:**

The **S3** sample (approximately $10^3$ bacteria/mL) was detected by the majority of laboratories.

Exceptions: Five participants (**codes 8, 17, 18, 38, and 50**) did not detect S3.

This suggests a slightly higher detection limit or variability in their methods.

These cases warrant further analysis to improve their detection limit:

- Examine the methods used (kits, protocols).

- Enhance test repeatability and robustness for low loads.

The **SD** sample is at the detection limit (with a load lower than S3).

SD was reported as "Not detected" by 24 participants.

Conversely, **26 laboratories demonstrated good detectability performance** (Table 6 in Conclusion).

## 6.3 ANALYSIS OF SPECIFICTY (SAMPLES SN ET SN/I)

For **SN/I**, several laboratories reported "Uninterpretable" due to undetected IPC Cq and undetected *Coxiella burnetii* Cq values. In each case, the "Uninterpretable" result was justified, explaining that insufficient volume for a second analysis (pure, 1/10 dilution) prevented confirmation of potential inhibition.

The majority of laboratories reported "Not detected" (or "Uninterpretable" for SN/I).

Exceptions: -Laboratoire 33 reported "Detected" for both **SN** and **SN/I**.

-Laboratoire 17 reported "Not detected" for **SN** and **SN/I**, despite the presence of Cq values for the *Coxiella burnetii* target. The interpretation should have been "Detected.".

These results indicate a specificity issue for laboratories 33 and 17.

* These errors could be attributed to cross-contamination or excessive sensitivity to non-specific signals.

Cq values exceeding 35 (very late signals) indicate weak signals. This issue is considered minor if qualitative PCR is used solely as a screening method for positives, to be retested with PCRr or PCRq for confirmation.
## 6.4 RESULTS OF CQ VALUES FOR THE COXIELLA BURNETII TARGET (TABLE)

<table><thead><tr><th rowspan="2">code lab</th><th rowspan="2">Used method</th><th rowspan="2">S3</th><th rowspan="2">S4</th><th rowspan="2">S4</th><th rowspan="2">S4</th><th rowspan="2">S5</th><th rowspan="2">SD</th><th rowspan="2">SN/I</th><th rowspan="2">SN</th><th colspan="3">S4 Cq</th><th rowspan="2">Cq Différence RMIT - Mean S4</th></tr><tr><th>RMIT Cq</th><th>Mean</th><th>CVr (%)</th></tr></thead><tbody><tr><td>1</td><td>Autre/Other</td><td>34,2</td><td>31,0</td><td>30,5</td><td>31,0</td><td>27,1</td><td>35,2</td><td>nd</td><td>nd</td><td>0,0</td><td>30,8</td><td>0,8%</td><td></td></tr><tr><td>2 (A)</td><td>13</td><td>34,7</td><td>29,9</td><td>29,7</td><td>30,2</td><td>26,8</td><td>nd</td><td>nd</td><td>nd</td><td>30,3</td><td>29,9</td><td>0,7%</td><td>0,4</td></tr><tr><td>3</td><td>23</td><td>36,0</td><td>32,8</td><td>33,1</td><td>33,1</td><td>29,2</td><td>nd</td><td>nd</td><td>nd</td><td>27,8</td><td>33,0</td><td>0,4%</td><td>-5,2</td></tr><tr><td>4</td><td>Autre/Other</td><td>36,3</td><td>32,5</td><td>32,6</td><td>32,3</td><td>29,3</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>32,5</td><td>0,4%</td><td></td></tr><tr><td>5</td><td>Autre/Other</td><td>32,4</td><td>30,6</td><td>30,5</td><td>30,1</td><td>27,0</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>30,4</td><td>0,7%</td><td></td></tr><tr><td>6 (A)</td><td>1</td><td>35,4</td><td>31,3</td><td>31,2</td><td>31,2</td><td>27,9</td><td>38,4</td><td>nd</td><td>nd</td><td>0,0</td><td>31,2</td><td>0,2%</td><td></td></tr><tr><td>7</td><td>13</td><td>33,5</td><td>30,0</td><td>29,6</td><td>29,7</td><td>26,2</td><td>nd</td><td>nd</td><td>nd</td><td>29,9</td><td>29,7</td><td>0,5%</td><td>0,2</td></tr><tr><td>8</td><td>Autre/Other</td><td>nd</td><td>34,1</td><td>31,9</td><td>33,9</td><td>29,7</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>33,3</td><td>3,0%</td><td></td></tr><tr><td>9</td><td>5</td><td>33,7</td><td>29,5</td><td>29,5</td><td>29,3</td><td>25,8</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>29,4</td><td>0,3%</td><td></td></tr><tr><td>10</td><td>5</td><td>32,4</td><td>29,6</td><td>30,1</td><td>29,3</td><td>26,1</td><td>38,5</td><td>nd</td><td>nd</td><td>0,0</td><td>29,7</td><td>1,1%</td><td></td></tr><tr><td>11</td><td>19</td><td>35,3</td><td>33,0</td><td>33,8</td><td>33,0</td><td>29,8</td><td>nd</td><td>nd</td><td>nd</td><td>33,3</td><td>33,3</td><td>1,1%</td><td>0,0</td></tr><tr><td>12</td><td>Autre/Other</td><td>33,4</td><td>30,7</td><td>30,9</td><td>30,5</td><td>27,5</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>30,7</td><td>0,6%</td><td></td></tr><tr><td>13</td><td>5</td><td>33,9</td><td>31,0</td><td>31,1</td><td>31,1</td><td>28,0</td><td>35,8</td><td>nd</td><td>nd</td><td>30,8</td><td>31,1</td><td>0,2%</td><td>-0,3</td></tr><tr><td>14</td><td>7</td><td>33,3</td><td>31,0</td><td>31,2</td><td>32,1</td><td>28,2</td><td>37,3</td><td>nd</td><td>nd</td><td>30,6</td><td>31,4</td><td>1,6%</td><td>-0,8</td></tr><tr><td>15</td><td>17</td><td>33,6</td><td>31,1</td><td>30,6</td><td>31,0</td><td>27,4</td><td>35,1</td><td>nd</td><td>nd</td><td>31,0</td><td>30,9</td><td>0,6%</td><td>0,1</td></tr><tr><td>16</td><td>15</td><td>34,6</td><td>29,3</td><td>29,5</td><td>29,4</td><td>26,0</td><td>33,6</td><td>nd</td><td>nd</td><td>30,0</td><td>29,4</td><td>0,3%</td><td>0,6</td></tr><tr><td>17</td><td>11</td><td>35,3</td><td>28,8</td><td>29,4</td><td>28,8</td><td>25,9</td><td>35,5</td><td>35,7</td><td>nd</td><td>28,7</td><td>29,0</td><td>1,1%</td><td>-0,3</td></tr><tr><td>18</td><td>Autre/Other</td><td>nd</td><td>38,5</td><td>36,7</td><td>36,4</td><td>32,8</td><td>nd</td><td>nd</td><td>nd</td><td>42,5</td><td>37,2</td><td>2,5%</td><td>5,3</td></tr><tr><td>19</td><td>7</td><td>35,5</td><td>31,1</td><td>31,1</td><td>31,3</td><td>27,3</td><td>34,9</td><td>nd</td><td>nd</td><td>0,0</td><td>31,2</td><td>0,4%</td><td></td></tr><tr><td>20</td><td>4</td><td>35,1</td><td>31,0</td><td>30,6</td><td>31,6</td><td>27,2</td><td>38,2</td><td>nd</td><td>nd</td><td>31,8</td><td>31,0</td><td>1,3%</td><td>0,8</td></tr><tr><td>21</td><td>5</td><td>33,6</td><td>30,4</td><td>30,6</td><td>30,2</td><td>26,8</td><td>nd</td><td>nd</td><td>nd</td><td>30,5</td><td>30,4</td><td>0,5%</td><td>0,1</td></tr><tr><td>22 (A)</td><td>2</td><td>34,7</td><td>31,0</td><td>31,4</td><td>30,9</td><td>27,8</td><td>36,6</td><td>nd</td><td>nd</td><td>30,4</td><td>31,1</td><td>0,7%</td><td>-0,7</td></tr><tr><td>23</td><td>2</td><td>34,0</td><td>30,7</td><td>30,8</td><td>30,4</td><td>27,1</td><td>37,3</td><td>nd</td><td>nd</td><td>33,0</td><td>30,6</td><td>0,6%</td><td>2,4</td></tr><tr><td>24 (A)</td><td>7</td><td>35,2</td><td>31,9</td><td>31,6</td><td>32,4</td><td>29,0</td><td>nd</td><td>nd</td><td>nd</td><td>33,7</td><td>32,0</td><td>1,0%</td><td>1,7</td></tr><tr><td>25</td><td>3</td><td>36,1</td><td>32,3</td><td>32,9</td><td>32,6</td><td>28,9</td><td>nd</td><td>nd</td><td>nd</td><td>32,3</td><td>32,6</td><td>0,8%</td><td>-0,3</td></tr><tr><td>26</td><td>2</td><td>36,1</td><td>31,0</td><td>30,9</td><td>32,3</td><td>28,0</td><td>37,4</td><td>nd</td><td>nd</td><td>32,8</td><td>31,4</td><td>2,0%</td><td>1,4</td></tr><tr><td>27</td><td>7</td><td>33,4</td><td>29,7</td><td>30,1</td><td>29,1</td><td>26,4</td><td>34,5</td><td>nd</td><td>nd</td><td>30,7</td><td>29,6</td><td>1,4%</td><td>1,1</td></tr><tr><td>28</td><td>2</td><td>35,3</td><td>32,6</td><td>32,7</td><td>32,5</td><td>29,0</td><td>36,4</td><td>nd</td><td>nd</td><td>30,2</td><td>32,6</td><td>0,2%</td><td>-2,4</td></tr><tr><td>29</td><td>2</td><td>37,1</td><td>31,3</td><td>31,6</td><td>31,2</td><td>28,0</td><td>35,6</td><td>nd</td><td>nd</td><td>0,0</td><td>31,4</td><td>0,5%</td><td></td></tr><tr><td>30</td><td>15</td><td>33,3</td><td>30,0</td><td>29,4</td><td>29,7</td><td>26,0</td><td>36,5</td><td>nd</td><td>nd</td><td>30,1</td><td>29,7</td><td>0,8%</td><td>0,4</td></tr><tr><td>31</td><td>6</td><td>35,4</td><td>31,3</td><td>31,6</td><td>31,6</td><td>28,2</td><td>nd</td><td>nd</td><td>nd</td><td>30,8</td><td>31,5</td><td>0,5%</td><td>-0,7</td></tr><tr><td>32 (A)</td><td>1</td><td>35,8</td><td>33,1</td><td>32,7</td><td>32,8</td><td>28,9</td><td>nd</td><td>nd</td><td>nd</td><td>31,1</td><td>32,9</td><td>0,5%</td><td>-1,8</td></tr><tr><td>33</td><td>7</td><td>34,3</td><td>30,3</td><td>30,8</td><td>30,6</td><td>27,4</td><td>36,0</td><td>36,6</td><td>nd</td><td>32,3</td><td>30,6</td><td>0,7%</td><td>1,7</td></tr><tr><td>34</td><td>15</td><td>32,8</td><td>30,0</td><td>30,0</td><td>29,4</td><td>26,7</td><td>37,5</td><td>nd</td><td>nd</td><td>28,5</td><td>29,8</td><td>0,9%</td><td>-1,3</td></tr><tr><td>35</td><td>7</td><td>32,9</td><td>28,5</td><td>28,9</td><td>29,4</td><td>25,5</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>28,9</td><td>1,2%</td><td></td></tr><tr><td>36</td><td>12</td><td>30,5</td><td>28,2</td><td>28,6</td><td>28,6</td><td>25,5</td><td>36,7</td><td>nd</td><td>nd</td><td>29,6</td><td>28,5</td><td>0,7%</td><td>1,1</td></tr><tr><td>37</td><td>15</td><td>34,0</td><td>30,1</td><td>30,0</td><td>30,1</td><td>26,7</td><td>nd</td><td>nd</td><td>nd</td><td>29,9</td><td>30,1</td><td>0,2%</td><td>-0,2</td></tr><tr><td>38</td><td>19</td><td>nd</td><td>31,4</td><td>32,4</td><td>32,7</td><td>27,4</td><td>nd</td><td>nd</td><td>nd</td><td>32,6</td><td>32,2</td><td>1,7%</td><td>0,4</td></tr><tr><td>39</td><td>5</td><td>35,4</td><td>32,1</td><td>32,0</td><td>32,3</td><td>28,9</td><td>36,6</td><td>nd</td><td>nd</td><td>0,0</td><td>32,1</td><td>0,4%</td><td></td></tr><tr><td>40</td><td>Autre/Other</td><td>35,9</td><td>33,0</td><td>32,8</td><td>32,7</td><td>29,5</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>32,8</td><td>0,4%</td><td></td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>35,7</td><td>32,7</td><td>32,6</td><td>32,6</td><td>29,3</td><td>38,2</td><td>nd</td><td>nd</td><td>30,8</td><td>32,6</td><td>0,1%</td><td>-1,8</td></tr><tr><td>42</td><td>3</td><td>38,1</td><td>33,6</td><td>34,4</td><td>35,0</td><td>31,5</td><td>40,1</td><td>nd</td><td>nd</td><td>0,0</td><td>34,3</td><td>1,7%</td><td></td></tr><tr><td>43 (A)</td><td>14</td><td>34,5</td><td>31,3</td><td>31,4</td><td>30,5</td><td>27,3</td><td>nd</td><td>nd</td><td>nd</td><td>30,0</td><td>31,1</td><td>1,3%</td><td>-1,1</td></tr><tr><td>44</td><td>16</td><td>33,5</td><td>29,3</td><td>30,4</td><td>30,4</td><td>27,2</td><td>34,9</td><td>nd</td><td>nd</td><td>29,5</td><td>30,0</td><td>1,7%</td><td>-0,5</td></tr><tr><td>45</td><td>Autre/Other</td><td>34,4</td><td>30,8</td><td>31,6</td><td>30,7</td><td>27,5</td><td>nd</td><td>nd</td><td>nd</td><td>30,2</td><td>31,0</td><td>1,3%</td><td>-0,8</td></tr><tr><td>46</td><td>15</td><td>35,1</td><td>30,6</td><td>30,8</td><td>31,1</td><td>27,6</td><td>nd</td><td>nd</td><td>nd</td><td>0,0</td><td>30,8</td><td>0,7%</td><td></td></tr><tr><td>47</td><td>Autre/Other</td><td>34,5</td><td>31,3</td><td>30,8</td><td>31,2</td><td>28,0</td><td>35,9</td><td>nd</td><td>nd</td><td>0,0</td><td>31,1</td><td>0,7%</td><td></td></tr><tr><td>48</td><td>24</td><td>35,2</td><td>33,1</td><td>32,8</td><td>32,2</td><td>29,4</td><td>36,9</td><td>nd</td><td>nd</td><td>0,0</td><td>32,7</td><td>1,1%</td><td></td></tr><tr><td>49</td><td>17</td><td>36,8</td><td>33,0</td><td>33,0</td><td>33,3</td><td>29,5</td><td>nd</td><td>nd</td><td>nd</td><td>34,7</td><td>33,1</td><td>0,4%</td><td>1,6</td></tr><tr><td>50</td><td>Autre/Other</td><td>nd</td><td>34,8</td><td>35,1</td><td>33,8</td><td>30,9</td><td>nd</td><td>nd</td><td>nd</td><td>30,5</td><td>34,6</td><td>1,6%</td><td>-4,1</td></tr></tbody></table>
# 7. QUANTITATIVE RESULTS

## 7.1 QUANTITATIVE RESULTS (TABLE)

<table><thead><tr><th>code lab</th><th>Used method</th><th>S3</th><th>S4</th><th>S4</th><th>S4</th><th>S5</th><th>SD</th><th>SN/I</th><th>SN</th></tr></thead><tbody><tr><td>6 (A)</td><td>1</td><td>2,8</td><td>3,9</td><td>3,9</td><td>3,9</td><td>4,8</td><td>détecté non quantifiable / detected not quantifiable</td><td>Ininterprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>7</td><td>13</td><td>2,6</td><td>3,7</td><td>3,8</td><td>3,8</td><td>4,9</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>12</td><td>Autre/Other</td><td>3,3</td><td>4,0</td><td>4,1</td><td>4,1</td><td>4,8</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>14</td><td>7</td><td>détecté non quantifiable / detected not quantifiable</td><td>3,1</td><td>3,0</td><td>détecté non quantifiable / detected not quantifiable</td><td>3,9</td><td>détecté non quantifiable / detected not quantifiable</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>15</td><td>17</td><td>3,2</td><td>4,0</td><td>4,1</td><td>4,0</td><td>5,1</td><td>2,8</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>17</td><td>11</td><td>détecté non quantifiable / detected not quantifiable</td><td>4,0</td><td>3,8</td><td>4,0</td><td>4,9</td><td>détecté non quantifiable / detected not quantifiable</td><td>détecté non quantifiable / detected not quantifiable</td><td>détecté non quantifiable / detected not quantifiable</td></tr><tr><td>19</td><td>7</td><td>détecté non quantifiable / detected not quantifiable</td><td>3,3</td><td>3,3</td><td>3,2</td><td>4,3</td><td>détecté non quantifiable / detected not quantifiable</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>20</td><td>4</td><td>2,4</td><td>3,7</td><td>3,8</td><td>3,5</td><td>4,9</td><td>détecté non quantifiable / detected not quantifiable</td><td>Ininterprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>21</td><td>5</td><td>3,2</td><td>4,1</td><td>4,0</td><td>4,1</td><td>5,0</td><td>non détecté / not detected</td><td>Ininterprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>22 (A)</td><td>2</td><td>2,6</td><td>3,8</td><td>3,7</td><td>3,8</td><td>4,9</td><td>détecté non quantifiable / detected not quantifiable</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>26</td><td>2</td><td>2,8</td><td>4,3</td><td>4,3</td><td>3,9</td><td>5,1</td><td>2,4</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>31</td><td>6</td><td>détecté non quantifiable / detected not quantifiable</td><td>3,8</td><td>3,7</td><td>3,6</td><td>4,7</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>33</td><td>2</td><td>3,0</td><td>4,2</td><td>4,0</td><td>4,0</td><td>5,0</td><td>2,4</td><td>2,3</td><td>2,2</td></tr><tr><td>38</td><td>19</td><td>non détecté / not detected</td><td>3,4</td><td>2,9</td><td>3,1</td><td>4,5</td><td>non détecté / not detected</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>39</td><td>5</td><td>2,9</td><td>3,8</td><td>3,9</td><td>3,8</td><td>4,8</td><td>2,5</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>2,3</td><td>3,2</td><td>3,2</td><td>3,2</td><td>4,2</td><td>1,6</td><td>non détecté / not detected</td><td>non détecté / not detected</td></tr><tr><td>43 (A)</td><td>14</td><td>2,4</td><td>3,4</td><td>3,4</td><td>3,7</td><td>4,7</td><td>non détecté / not detected</td><td>Ininterprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>45</td><td>Autre/Other</td><td>2,8</td><td>3,8</td><td>3,6</td><td>3,8</td><td>4,8</td><td>non détecté / not detected</td><td>Ininterprétable / Uninterpretable</td><td>non détecté / not detected</td></tr><tr><td>Robust mean X*</td><td></td><td>2,8</td><td>3,7</td><td>3,7</td><td>3,7</td><td>4,8</td><td>Not detected or Not quantifiable or Quantifiable</td><td>Not detected or Uninterpretable</td><td>Not detected</td></tr><tr><td>Robust standard deviation S*</td><td></td><td>0,4</td><td>0,4</td><td>0,4</td><td>0,4</td><td>0,3</td><td>NA</td><td>NA</td><td>NA</td></tr></tbody></table>

Out of the 50 participants, 18 laboratories reported results in the quantitative format (36 %).
Overall, the quantitative results from the 18 participants are satisfactory: A slight issue with repeatability was observed for one participant (no. 38), and an issue with trueness for another (no. 14).

## 7.1 SPECIFICITY AND SENSITIVITY

The performances remain consistent with the results obtained for qualitative PCR.

The SD sample was used to assess analytical sensitivity, and the results demonstrate good detectability for the majority of laboratories.

## 7.2 REPEATABILITY (PRECISION)

The repeatability performance, based on the results of three repetitions of S4, was analyzed using a Mandel's h-statistic histogram, highlighting intra- and inter-laboratory variations (Figure 3).

<figure><img src="image_4.png" /><figcaption>Figure 3. Mandel's histogram for the three repetitions of S4 showing k-ratios.</figcaption></figure>

All laboratories demonstrated acceptable repeatability, except for laboratory no. 38. Mandel's precision ratio for laboratory no. 38 (k = 2.2) exceeded the critical value calculated at 2.06, indicating a repeatability issue for this sample.
## 7.3 TRUENESS (Z-SCORES)

An analysis of z-scores for samples S3, S4, and S5 was conducted to evaluate the trueness of the numerical values obtained (Table 5).

Out of the 18 participants, 17 achieved accurate results for samples S4 and S5 (94 %).

<table><caption>Table 5. Z-scores obtained for the 5 quantifiable samples.</caption><thead><tr><th>code lab</th><th>Used method</th><th>Score Z: S3</th><th>Score Z: S4</th><th>Score Z: S4</th><th>Score Z: S4</th><th>Score Z: S5</th></tr></thead><tbody><tr><td>6 (A)</td><td>1</td><td>-0,1</td><td>0,4</td><td>0,5</td><td>0,5</td><td>0,0</td></tr><tr><td>7</td><td>13</td><td>-0,5</td><td>0,0</td><td>0,3</td><td>0,2</td><td>0,5</td></tr><tr><td>12</td><td>Autre/Other</td><td>1,4</td><td>0,7</td><td>0,9</td><td>1,1</td><td>0,2</td></tr><tr><td>14</td><td>7</td><td>NA</td><td>-1,7</td><td>-1,8</td><td></td><td>-3,2</td></tr><tr><td>15</td><td>17</td><td>1,2</td><td>0,7</td><td>1,1</td><td>0,8</td><td>1,1</td></tr><tr><td>17</td><td>11</td><td>NA</td><td>0,7</td><td>0,3</td><td>0,7</td><td>0,6</td></tr><tr><td>19</td><td>7</td><td>NA</td><td>-1,2</td><td>-1,2</td><td>-1,4</td><td>-1,7</td></tr><tr><td>20</td><td>4</td><td>-0,9</td><td>0,0</td><td>0,3</td><td>-0,5</td><td>0,5</td></tr><tr><td>21</td><td>5</td><td>1,1</td><td>0,9</td><td>0,8</td><td>1,1</td><td>0,9</td></tr><tr><td>22 (A)</td><td>2</td><td>-0,5</td><td>0,2</td><td>-0,1</td><td>0,3</td><td>0,3</td></tr><tr><td>26</td><td>2</td><td>0,0</td><td>1,4</td><td>1,5</td><td>0,5</td><td>1,2</td></tr><tr><td>31</td><td>6</td><td>NA</td><td>0,1</td><td>-0,1</td><td>-0,2</td><td>-0,4</td></tr><tr><td>33</td><td>2</td><td>0,5</td><td>1,1</td><td>0,8</td><td>0,8</td><td>0,8</td></tr><tr><td>38</td><td>19</td><td>NA</td><td>-0,8</td><td>-2,0</td><td>-1,6</td><td>-1,0</td></tr><tr><td>39</td><td>5</td><td>0,3</td><td>0,3</td><td>0,4</td><td>0,1</td><td>0,0</td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>-1,3</td><td>-1,3</td><td>-1,3</td><td>-1,3</td><td>-2,0</td></tr><tr><td>43 (A)</td><td>14</td><td>-1,1</td><td>-0,8</td><td>-0,9</td><td>-0,2</td><td>-0,2</td></tr><tr><td>45</td><td>Autre/Other</td><td>-0,1</td><td>0,3</td><td>-0,3</td><td>0,3</td><td>0,1</td></tr></tbody></table>

Interpretation of the z-score (according to the NF EN ISO 17043 standard):

If $|z| \le 2$ performance is "acceptable"

If $2,0 < |z| < 3,0$ performance is "questionable" (to be monitored)

If $|z| > 3,0$ performance is "unacceptable" (need to investigate)

### Laboratory no. 14 showed two trueness issues:

- One for one of the three replicates of S4,

- Another for sample S5.
# 8. RELATIVE RESULTS TO THE RMIT

## 8.1 ORDERED QUALITATIVE RESULTS OR 'RELATIVE TO THE RMIT (TABLE)

<table><thead><tr><th>code lab</th><th>Used method</th><th>S3</th><th>S4</th><th>S4</th><th>S4</th><th>S5</th><th>SD</th><th>SN/I</th><th>SN</th></tr></thead><tbody><tr><td>2 (A)</td><td>13</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>3</td><td>23</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>7</td><td>13</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>11</td><td>19</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>13</td><td>5</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load lower than the RMIT</td><td>not detected</td><td>not detected</td></tr><tr><td>14</td><td>7</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load lower than the RMIT</td><td>not detected</td><td>not detected</td></tr><tr><td>15</td><td>17</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load lower than the RMIT</td><td>not detected</td><td>not detected</td></tr><tr><td>16</td><td>15</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load lower than the RMIT</td><td>not detected</td><td>not detected</td></tr><tr><td>17</td><td>11</td><td>not detected</td><td>detected close to the RMIT</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>18</td><td>Autre/Other</td><td>not detected</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>20</td><td>4</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load lower than the RMIT</td><td>Ininterprétable / Uninterpretable</td><td>not detected</td></tr><tr><td>21</td><td>5</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>Ininterprétable / Uninterpretable</td><td>not detected</td></tr><tr><td>22 (A)</td><td>2</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load lower than the RMIT</td><td>not detected</td><td>not detected</td></tr><tr><td>23</td><td>2</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>24 (A)</td><td>7</td><td>detected with load lower than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>25</td><td>3</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>Ininterprétable / Uninterpretable</td><td>not detected</td></tr><tr><td>26</td><td>2</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>27</td><td>7</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>28</td><td>2</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>30</td><td>15</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>31</td><td>6</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>32 (A)</td><td>1</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>33</td><td>2</td><td>detected with load lower than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>34</td><td>15</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>36</td><td>12</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>37</td><td>15</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>38</td><td>19</td><td>not detected</td><td>detected with load higher than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>43 (A)</td><td>14</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>Ininterprétable / Uninterpretable</td><td>not detected</td></tr><tr><td>44</td><td>16</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>45</td><td>Autre/Other</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>Ininterprétable / Uninterpretable</td><td>not detected</td></tr><tr><td>49</td><td>17</td><td>not detected</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>detected with load higher than the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr><tr><td>50</td><td>Autre/Other</td><td>not detected</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected with load lower than the RMIT</td><td>detected close to the RMIT</td><td>not detected</td><td>not detected</td><td>not detected</td></tr></tbody><tfoot><tr><td>Expected results</td><td></td><td>Detected &lt; or close to RMIT (or not detected)</td><td></td><td>Detected &lt;, close or &gt; to RMIT</td><td></td><td>Detected close or &gt; to RMIT</td><td>Detected or not detected</td><td>Not detected or uninterpretable</td><td>Not detected</td></tr></tfoot></table>

The indication "close to the RMIT" was to be used for any result falling within the measurement uncertainty range of ±1 Cq at the interpretation threshold (deviation of ±0.30 log₁₀). NB: a deviation of ±0.70 log₁₀ corresponds to 2.33 Cq.
Overall, the relative results to RMIT from the 33 participants are satisfactory: 31 out of 33 participants (94%) reported accurate results for S5.

## 8.2 SPECIFICITY AND SENSITIVITY

The results relative to the RMIT confirm the performances observed for the qualitative results.

## 8.3 TRUENESS

No misinterpretation of loads relative to the RMIT was observed.

Trueness was assessed by comparing the reported results to the expected result for sample S5. Overall, the performance was satisfactory :

* 31 out of 33 participants (94%) reported accurate results for S5 (Table in 8.1).

* A bias was identified for participants **no. 3 and 50**.
# 9. CONCLUSION AND DISCUSSION

This ILPT aimed to evaluate the ability to achieve correct results using real-time PCR analysis as part of a differential diagnosis of abortion in ruminants<fnref n="1" />. To achieve this, 10 samples prepared from vaginal swab suspensions as biological matrices were analyzed blindly.

## 9.1 SUMMARY OF RESULTS BY PCR TYPE

<table><thead><tr><th>Category</th><th>Aspect</th><th>Summary of observations</th></tr></thead><tbody><tr><td rowspan="3">Qualitative results</td><td>Specificity</td><td>- All 50 participants reported qualitative results.</td></tr><tr><td>Identified deviations</td><td>- Two laboratories (no. 17 and 33) reported incorrect results for samples SN and SN/I (late Cq values).</td></tr><tr><td>Sensitivity and limit of detection (LOD)</td><td>- All laboratories correctly detected samples S5 (high load) and S4 (medium load).<br/>- Sample S3 was detected by most laboratories, except for no. 8, 17, 18, 38, et 50 (analytical sensitivity limits).<br/>- Sample SD revealed inter-laboratory variability:<ul><li>26 laboratories detected SD, with Cq values between 33 and 35 for labs no. 16, 19, 27 et 44.</li><li>24 laboratories reported "Not detected".</li></ul></td></tr><tr><td rowspan="3">Quantitative results</td><td>Performance générale</td><td>- Satisfactory performances for samples S5 (high load, X* = 4,8 log<sub>10</sub>) and S4 (medium load, X* = 3,7 log<sub>10</sub>).</td></tr><tr><td>Limite de quantification</td><td>- Sample S3 (X* = 2,8 log<sub>10</sub>) allowed evaluation of the initially validated quantification limit.</td></tr><tr><td>Identified deviations</td><td>- Laboratory no. 38 showed a repeatability issue (Mandel's k-ratio > 2.06).<br/>- Laboratory no. 14 emonstrated a trueness issue for S4 and S5 (deviations assessed with z-scores).</td></tr><tr><td rowspan="2">Results relative to RMIT au MRSI</td><td>General performance</td><td>- Performances aligned with expectations for 31 out of 33 participants.</td></tr><tr><td>Identified deviations</td><td>- Laboratories no. 3 and 50 exhibited biases for S5, highlighting difficulties in classification relative to the RMIT.</td></tr></tbody></table>

**Overall result:** 44 out of 50 laboratories produced compliant results, including the 7 accredited French laboratories.

Isolated non-conformities related to specificity and accuracy were observed, requiring follow-up or even corrective actions.

<footnote n="1">¹ https://www.plateforme-esa.fr/page/thematique-diagnostic-differentiel-des-avortements</footnote>
<table><caption>**Table 6. Participants with the best detectability (Sample SD).**</caption><thead><tr><td>Lab Code</td><td>Used method</td><td>Cq SD</td></tr></thead><tbody><tr><td>16</td><td>15</td><td>33,6</td></tr><tr><td>27</td><td>7</td><td>34,5</td></tr><tr><td>44</td><td>16</td><td>34,9</td></tr><tr><td>19</td><td>7</td><td>34,9</td></tr><tr><td>15</td><td>17</td><td>35,1</td></tr><tr><td>1</td><td>Autre/Other</td><td>35,2</td></tr><tr><td>17</td><td>11</td><td>35,5</td></tr><tr><td>29</td><td>2</td><td>35,6</td></tr><tr><td>13</td><td>5</td><td>35,8</td></tr><tr><td>47</td><td>Autre/Other</td><td>35,9</td></tr><tr><td>33</td><td>2</td><td>36,0</td></tr><tr><td>28</td><td>2</td><td>36,4</td></tr><tr><td>30</td><td>15</td><td>36,5</td></tr><tr><td>39</td><td>5</td><td>36,6</td></tr><tr><td>22 (A)</td><td>2</td><td>36,6</td></tr><tr><td>36</td><td>12</td><td>36,7</td></tr><tr><td>48</td><td>24</td><td>36,9</td></tr><tr><td>23</td><td>2</td><td>37,3</td></tr><tr><td>14</td><td>7</td><td>37,3</td></tr><tr><td>26</td><td>2</td><td>37,4</td></tr><tr><td>34</td><td>15</td><td>37,5</td></tr><tr><td>20</td><td>4</td><td>38,2</td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>38,2</td></tr><tr><td>6 (A)</td><td>1</td><td>38,4</td></tr><tr><td>10</td><td>5</td><td>38,5</td></tr><tr><td>42</td><td>3</td><td>40,1</td></tr></tbody></table>

The detection limit (LOD) is a critical aspect of this analysis and must be closely monitored, especially considering the coexistence of over 20 equivalent performance methods used across the inter-laboratory network.

For sample SD, which corresponds to a bacterial load lower than S3:

* 26 laboratories reported a "Detected" result, with Cq values ranging from 33 to 35 for laboratories no. 16, 19, 27 and 44.

* 24 laboratories reported "Not detected," confirming the challenge of detecting very low bacterial loads.

No clear trend emerges regarding the methods associated with these results.

## 9.2 LIMITATIONS AND OBSERVED DEVIATION WITH INVESTIGATIONS PATHWAYS

### Specificity

* Verify the absence of contamination during the process (sample surrounded by positive samples).

* Re-test DNA extracts with additional negative controls to detect potential weak contamination.

* Examine the characteristics of amplification curves, which can be challenging for late Ct signals.

### Limit of detection

* These cases require an in-depth analysis to improve their detection limit.

### Repeatability issues:

* Hypotheses: Operational error, calibration issue, or instability in experimental conditions.

### Trueness issue:

* The observed deviations could be due to a calibration problem or an error in preparing critical dilutions (standard range, RMIT).

* Investigation Pathway: Verify the calibration and robustness of RMIT thresholds or the control chart to reduce variability for loads near the RMIT threshold.
## 9.3 OVERALL LABORATORY PERFORMANCES

* **Performance of accredited laboratories:** The 7 laboratories accredited in France for this official analysis **achieved results that met expectations** (quantitative format or relative to a reference material at interpretation threshold (RMIT)), with special recognition for laboratories 22(A), 41(A), and 6(A) for their exceptional detectability.

* This ILPT highlighted the high quality of results produced by a large number of laboratories (44 out of 50, 88%).

* The vast majority of laboratories demonstrated the ability to meet diagnostic requirements.

* The overall analysis confirms the strong capability of participating laboratories for high (S5) and medium (S4) bacterial loads.

* The diversity of methods (more than 20 equivalent approaches) underscores the importance of **maintaining continuous performance monitoring** and promoting the harmonization of practices within the network.

* Critical points include the **detection limit** (tested with samples SD and S3) and **accuracy at the interpretation threshold** (tested through repeatability and z-scores on sample S4 and concordance on sample S5).

<figure><img src="image_5.png" /></figure>
# 10. OTHER INFORMATION

This PCR ILPT is organised every three years, alternating with the ELISA ILPT.

Accredited laboratories are encouraged to participate in these ILPTs, as are the volunteer departmental laboratories of the OSCAR scheme (ObServatory for Causes of Abortions in Ruminants).

# 11. REFERENCES

AFNOR NF EN ISO/CEI 17043 "Conformity assessment – General requirements for proficiency testing".

COFRAC LAB CIL REF 02 "Requirements for the accreditation of organisers of inter-laboratory comparisons according to the NF ISO/CEI 17043 standard".

AFNOR NF U47-400 "Animal health analysis methods - Guide for the organisation of interlaboratory proficiency testing in animal health".

NF EN ISO 13528 "Statistical methods for use in proficiency testing by interlaboratory comparison".

NF EN ISO 5725-2 "Accuracy (trueness and precision) of measurement methods and results – Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method".

NF EN ISO/CEI 17025 "General requirements for the competence of testing and calibration laboratories".

AFNOR NF U47-600 "Animal health analysis methods – PCR – Part 1: requirements and recommendations for the implementation of veterinary PCR, Part 2: requirements and recommendations for the development and the validation of veterinary PCR".

Euroreference. Elodie Rousset, Elise Yang, Philippe Dufour, Myriam Prigent, Karim Sidi-Boumedine, Richard Thiéry. A National Reference Laboratory's interactions with veterinary diagnostic laboratories: example of Q fever, an abortive disease in ruminants and a zoonosis. 2018. DOI: http://doi.org/10.5281/zenodo.1171994.

17_12_ED_ER 03-1 ROUSSET.PDF (anses.fr)
## 12. CLAIMS

The participants were given the opportunity to submit any comments or expectations to the organiser of this ILPT via the satisfaction survey sent to each participant.
# 13. APPENDICES

## 13.1 DÉTAILED RESULTS OF IPC CQ VALUES (TABLE)

<table><thead><tr><th>code lab</th><th>Used method</th><th>S3</th><th>S4</th><th>S4</th><th>S4</th><th>S5</th><th>SD</th><th>SN/I</th><th>SN</th></tr></thead><tbody><tr><td>1</td><td>Autre/Other</td><td>26,1</td><td>26,4</td><td>26,6</td><td>25,8</td><td>26,4</td><td>28,4</td><td>32,2</td><td>24,7</td></tr><tr><td>2 (A)</td><td>13</td><td>25,1</td><td>25,3</td><td>25,1</td><td>25,6</td><td>25,7</td><td>26,8</td><td>34,2</td><td>23,6</td></tr><tr><td>3</td><td>23</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4</td><td>Autre/Other</td><td>33,2</td><td>33,5</td><td>32,1</td><td>33,0</td><td>32,6</td><td>31,5</td><td>28,6</td><td>31,1</td></tr><tr><td>5</td><td>Autre/Other</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>6 (A)</td><td>1</td><td>29,2</td><td>29,6</td><td>29,5</td><td>29,4</td><td>28,6</td><td>30,4</td><td>0,0</td><td>27,3</td></tr><tr><td>7</td><td>13</td><td>23,1</td><td>23,0</td><td>22,9</td><td>23,5</td><td>23,2</td><td>25,4</td><td>28,2</td><td>22,6</td></tr><tr><td>8</td><td>Autre/Other</td><td>33,8</td><td>33,4</td><td>32,5</td><td>33,2</td><td>32,7</td><td>32,9</td><td>33,2</td><td>33,0</td></tr><tr><td>9</td><td>5</td><td>34,2</td><td>34,5</td><td>34,7</td><td>34,5</td><td>34,4</td><td>34,5</td><td>0,0</td><td>31,3</td></tr><tr><td>10</td><td>5</td><td>33,1</td><td>32,6</td><td>34,0</td><td>34,0</td><td>33,1</td><td>37,3</td><td>40,2</td><td>32,0</td></tr><tr><td>11</td><td>19</td><td>34,9</td><td>36,1</td><td>34,9</td><td>34,5</td><td>34,8</td><td>32,7</td><td>0,0</td><td>29,0</td></tr><tr><td>12</td><td>Autre/Other</td><td>27,5</td><td>27,4</td><td>27,6</td><td>27,4</td><td>27,3</td><td>27,4</td><td>27,5</td><td>27,7</td></tr><tr><td>13</td><td>5</td><td>44,5</td><td>0,0</td><td>42,3</td><td>0,0</td><td>0,0</td><td>37,8</td><td>0,0</td><td>35,7</td></tr><tr><td>14</td><td>7</td><td>39,2</td><td>39,1</td><td>39,6</td><td>38,6</td><td>40,1</td><td>34,3</td><td>36,3</td><td>31,7</td></tr><tr><td>15</td><td>17</td><td>29,2</td><td>29,3</td><td>29,2</td><td>29,2</td><td>29,3</td><td>28,8</td><td>37,7</td><td>27,8</td></tr><tr><td>16</td><td>15</td><td>26,9</td><td>27,1</td><td>27,6</td><td>26,4</td><td>26,3</td><td>27,3</td><td>0,0</td><td>24,3</td></tr><tr><td>17</td><td>11</td><td>25,5</td><td>25,6</td><td>26,0</td><td>25,9</td><td>26,0</td><td>27,2</td><td>30,0</td><td>25,2</td></tr><tr><td>18</td><td>Autre/Other</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>19</td><td>7</td><td>33,8</td><td>33,7</td><td>33,1</td><td>34,3</td><td>32,6</td><td>33,3</td><td>0,0</td><td>31,1</td></tr><tr><td>20</td><td>4</td><td>28,2</td><td>28,8</td><td>27,6</td><td>28,6</td><td>27,6</td><td>29,0</td><td>0,0</td><td>25,4</td></tr><tr><td>21</td><td>5</td><td>32,5</td><td>33,4</td><td>34,2</td><td>33,5</td><td>32,5</td><td>34,0</td><td>0,0</td><td>30,7</td></tr><tr><td>22 (A)</td><td>2</td><td>27,4</td><td>27,6</td><td>27,6</td><td>27,6</td><td>27,1</td><td>28,6</td><td>37,0</td><td>25,4</td></tr><tr><td>23</td><td>2</td><td>25,7</td><td>25,7</td><td>26,1</td><td>25,8</td><td>25,5</td><td>27,7</td><td>32,8</td><td>24,3</td></tr><tr><td>24 (A)</td><td>7</td><td>34,8</td><td>35,9</td><td>35,6</td><td>35,3</td><td>35,7</td><td>35,9</td><td>44,1</td><td>34,5</td></tr><tr><td>25</td><td>3</td><td>33,1</td><td>33,8</td><td>33,6</td><td>33,9</td><td>44,9</td><td>32,7</td><td>0,0</td><td>28,9</td></tr><tr><td>26</td><td>2</td><td>27,8</td><td>27,1</td><td>27,4</td><td>27,3</td><td>27,7</td><td>28,7</td><td>36,9</td><td>25,7</td></tr><tr><td>27</td><td>7</td><td>31,4</td><td>30,8</td><td>31,3</td><td>31,0</td><td>31,7</td><td>31,7</td><td>41,4</td><td>29,8</td></tr><tr><td>28</td><td>2</td><td>29,2</td><td>26,6</td><td>29,2</td><td>28,5</td><td>28,4</td><td>30,1</td><td>38,9</td><td>26,6</td></tr><tr><td>29</td><td>2</td><td>25,4</td><td>25,3</td><td>25,2</td><td>25,5</td><td>25,3</td><td>27,5</td><td>31,2</td><td>24,1</td></tr><tr><td>30</td><td>15</td><td>26,1</td><td>25,7</td><td>25,3</td><td>25,5</td><td>25,5</td><td>27,6</td><td>34,1</td><td>24,2</td></tr><tr><td>31</td><td>6</td><td>0,0</td><td>41,0</td><td>0,0</td><td>0,0</td><td>0,0</td><td>40,9</td><td>0,0</td><td>38,1</td></tr><tr><td>32 (A)</td><td>1</td><td>35,3</td><td>36,8</td><td>34,5</td><td>35,2</td><td>41,2</td><td>36,0</td><td>38,3</td><td>33,4</td></tr><tr><td>33</td><td>2</td><td>27,0</td><td>26,8</td><td>26,7</td><td>26,9</td><td>27,0</td><td>28,3</td><td>34,6</td><td>25,1</td></tr><tr><td>34</td><td>15</td><td>24,9</td><td>24,6</td><td>24,7</td><td>24,7</td><td>24,9</td><td>27,4</td><td>30,5</td><td>23,8</td></tr><tr><td>35</td><td>7</td><td>32,4</td><td>31,3</td><td>31,7</td><td>31,5</td><td>30,3</td><td>32,7</td><td>42,8</td><td>30,8</td></tr><tr><td>36</td><td>12</td><td>23,1</td><td>23,2</td><td>23,2</td><td>23,1</td><td>23,5</td><td>24,9</td><td>31,5</td><td>21,9</td></tr><tr><td>37</td><td>15</td><td>29,0</td><td>29,0</td><td>28,4</td><td>28,5</td><td>27,7</td><td>29,5</td><td>38,1</td><td>26,1</td></tr><tr><td>38</td><td>19</td><td>26,0</td><td>27,6</td><td>27,0</td><td>26,0</td><td>26,3</td><td>26,0</td><td>25,9</td><td>26,5</td></tr><tr><td>39</td><td>5</td><td>33,4</td><td>32,4</td><td>32,4</td><td>32,7</td><td>33,6</td><td>34,7</td><td>38,0</td><td>31,8</td></tr><tr><td>40</td><td>Autre/Other</td><td>33,9</td><td>33,6</td><td>34,8</td><td>32,9</td><td>33,7</td><td>35,3</td><td>0,0</td><td>32,3</td></tr><tr><td>41 (A)</td><td>Autre/Other</td><td>30,6</td><td>30,6</td><td>30,5</td><td>30,4</td><td>29,8</td><td>30,1</td><td>0,0</td><td>27,1</td></tr><tr><td>42</td><td>3</td><td>30,6</td><td>30,4</td><td>30,3</td><td>31,1</td><td>30,3</td><td>32,9</td><td>36,8</td><td>29,4</td></tr><tr><td>43 (A)</td><td>14</td><td>29,6</td><td>26,6</td><td>27,4</td><td>26,8</td><td>26,9</td><td>28,5</td><td>33,7</td><td>25,4</td></tr><tr><td>44</td><td>16</td><td>24,6</td><td>24,6</td><td>24,6</td><td>24,6</td><td>24,5</td><td>26,7</td><td>30,3</td><td>23,6</td></tr><tr><td>45</td><td>Autre/Other</td><td>30,0</td><td>29,6</td><td>29,4</td><td>29,9</td><td>29,6</td><td>30,3</td><td>0,0</td><td>27,2</td></tr><tr><td>46</td><td>15</td><td>31,9</td><td>33,2</td><td>34,0</td><td>32,1</td><td>32,1</td><td>30,8</td><td>0,0</td><td>31,3</td></tr><tr><td>47</td><td>Autre/Other</td><td>28,8</td><td>28,6</td><td>28,6</td><td>28,6</td><td>28,9</td><td>30,8</td><td>35,2</td><td>27,2</td></tr><tr><td>48</td><td>24</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>49</td><td>17</td><td>32,8</td><td>31,2</td><td>31,0</td><td>32,1</td><td>30,2</td><td>35,8</td><td>37,5</td><td>29,4</td></tr><tr><td>50</td><td>Autre/Other</td><td>28,1</td><td>28,3</td><td>28,2</td><td>27,9</td><td>28,0</td><td>27,8</td><td>27,9</td><td>28,5</td></tr><tr><td></td><td>Robust mean</td><td>29,7</td><td>29,3</td><td>29,3</td><td>29,3</td><td>29,1</td><td>30,6</td><td>24,4</td><td>28,0</td></tr><tr><td></td><td>Robust SD</td><td>4,3</td><td>4,4</td><td>4,4</td><td>4,4</td><td>4,3</td><td>4,0</td><td>18,7</td><td>3,9</td></tr></tbody></table>

(0,0 corresponds to Undet.)

Four participants did not report IPC results (shaded rows). Two of the accredited laboratories recorded late Cq values (codes 24 A and 32 A).

The obtained Cq values are fairly consistent across different methods and laboratories, as shown by the robust standard deviations of the calculated means. The higher robust standard deviation for SN/I confirms a broader range of Cq values for this sample.
13.2 LIST OF RECOMMENDED METHODS

<table><thead><tr><th>Supplier</th><th>N°</th><th>DNA Extraction Kit</th><th>PCR Kit</th><th>Standard Operating Procedure</th></tr></thead><tbody><tr><td rowspan="4">AB™ by Thermo Fisher Scientific (LSI S.A.S)</td><td>1</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td><td rowspan="4">VetMAX™ C. burnetii Absolute Quant Kit (FQPAQ)</td><td>Notice MAN0007807<br/>Notice MAN0008765<br/>QF-RNL Recommandations</td></tr><tr><td>2</td><td>x robot: MagMAX CORE</td><td>MAN0008871<br/>MAN0008873</td></tr><tr><td>3</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td><td rowspan="2">VetMAX™ Ruminant Abortion Screening Kit (SARP)</td></tr><tr><td>4</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td><td>MAN0008882<br/>MAN0008884</td></tr><tr><td rowspan="10">Adiagene - Bio-X-Diagnostics</td><td>5</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td><td rowspan="2">ADI143<br/>ADIAVET COXIELLA REAL TIME</td><td rowspan="2">Notice NF143</td></tr><tr><td>6</td><td>x manuel: Nucleospin tissue (Macherey Nagel)</td></tr><tr><td>7</td><td>x robot: Billes magnétiques ARN/ADN (Bio-X-Diagnostics)b</td><td rowspan="4">418025<br/>ADIAVET CHLAMCOX REAL TIME (C. burnetii and Chlamydia abortus)</td><td rowspan="4">Notice NF313</td></tr><tr><td>8</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td></tr><tr><td>9</td><td>x manuel: Nucleospin tissue (Macherey Nagel)</td></tr><tr><td>10</td><td>x robot: Billes magnétiques ARN/ADN (Bio-X-Diagnostics)</td></tr><tr><td rowspan="6">BioSellal</td><td>11</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td><td rowspan="3">Bio-T kit Coxiella burnetii BIOTK016</td><td rowspan="3">MU/qFQ/002/FR</td></tr><tr><td>12</td><td>x robot: BioExtract SuperBall (BioSellal)</td></tr><tr><td>13</td><td>x manuel: BioExtract Column (BioSellal)</td></tr><tr><td>14</td><td>x manuel: Qiaamp DNA mini kit (Qiagen)</td><td rowspan="3">Bio-T kit Coxiella burnetii &amp; Chlamydia abortus BIOTK039</td><td rowspan="3">MU/qFQChab/002/FR</td></tr><tr><td>15</td><td>x robot: BioExtract SuperBall (BioSellal)</td></tr><tr><td>16</td><td>x manuel: BioExtract Column (BioSellal)</td></tr><tr><td rowspan="8">IdVET</td><td>17</td><td>x manuel: Spin Universal SPIN50/250 (ID Gene™)</td><td rowspan="2">Kit IDQF (ID Gene™ Q Fever Triplex)</td><td rowspan="2">IDQFCH</td></tr><tr><td>18</td><td>x robot: Mag Universal Extraction Kit MAG192/384 (ID Gene™)</td></tr><tr><td>19</td><td>x robot: Mag Fast Extraction Kit MAGFAST384 (ID Gene™)</td><td rowspan="4">Kit IDQFCH (ID Gene™) Q Fever-Chlamydophila spp</td><td rowspan="4">IDQFCH</td></tr><tr><td>20</td><td>x manuel: Spin Universal SPIN50/250 (ID Gene™)</td></tr><tr><td>21</td><td>x robot: Mag Universal Extraction Kit MAG192/384 (ID Gene™)</td></tr><tr><td>22</td><td>x robot: Mag Fast Extraction Kit MAGFAST384 (ID Gene™)</td></tr><tr><td>23</td><td>x DNeasy® Blood &amp; Tissue kit (Qiagen)</td><td rowspan="2">DV IDABORT10 (ID Gene™)<br/>Ruminant Abortion Multiplex HRM</td><td rowspan="2">/<br/>Insert_ABORT10-MAGFAST (lyse LMAP)</td></tr><tr><td>24</td><td>x robot: Mag Fast Extraction Kit MAGFAST384/768 (ID Gene™)</td></tr></tbody></table>
13.3 CODING OF THE SAMPLES IN THE PANEL

<table><thead><tr><th>code</th><th>S3</th><th>S4</th><th>S4</th><th>S4</th><th>S5</th><th>SD</th><th>SN/I</th><th>SN</th><th>L1</th><th>L2</th></tr></thead><tbody><tr><td>1</td><td>9</td><td>10</td><td>7</td><td>8</td><td>4</td><td>6</td><td>2</td><td>1</td><td>3</td><td>5</td></tr><tr><td>2</td><td>10</td><td>2</td><td>1</td><td>7</td><td>6</td><td>8</td><td>5</td><td>4</td><td>3</td><td>9</td></tr><tr><td>3</td><td>1</td><td>5</td><td>9</td><td>8</td><td>10</td><td>3</td><td>7</td><td>6</td><td>4</td><td>2</td></tr><tr><td>4</td><td>2</td><td>4</td><td>1</td><td>8</td><td>3</td><td>5</td><td>6</td><td>7</td><td>9</td><td>10</td></tr><tr><td>5</td><td>10</td><td>9</td><td>2</td><td>3</td><td>1</td><td>8</td><td>6</td><td>7</td><td>5</td><td>4</td></tr><tr><td>6</td><td>7</td><td>1</td><td>10</td><td>5</td><td>3</td><td>6</td><td>9</td><td>4</td><td>2</td><td>8</td></tr><tr><td>7</td><td>6</td><td>5</td><td>7</td><td>9</td><td>4</td><td>2</td><td>10</td><td>1</td><td>3</td><td>8</td></tr><tr><td>8</td><td>9</td><td>3</td><td>7</td><td>2</td><td>8</td><td>6</td><td>4</td><td>10</td><td>1</td><td>5</td></tr><tr><td>9</td><td>4</td><td>7</td><td>9</td><td>10</td><td>2</td><td>6</td><td>8</td><td>1</td><td>5</td><td>3</td></tr><tr><td>10</td><td>1</td><td>6</td><td>8</td><td>3</td><td>10</td><td>7</td><td>4</td><td>5</td><td>2</td><td>9</td></tr><tr><td>11</td><td>10</td><td>5</td><td>2</td><td>8</td><td>6</td><td>4</td><td>3</td><td>9</td><td>1</td><td>7</td></tr><tr><td>12</td><td>5</td><td>9</td><td>4</td><td>3</td><td>10</td><td>6</td><td>2</td><td>1</td><td>7</td><td>8</td></tr><tr><td>13</td><td>9</td><td>6</td><td>2</td><td>7</td><td>3</td><td>8</td><td>5</td><td>1</td><td>4</td><td>10</td></tr><tr><td>14</td><td>2</td><td>8</td><td>6</td><td>7</td><td>1</td><td>5</td><td>3</td><td>9</td><td>4</td><td>10</td></tr><tr><td>15</td><td>10</td><td>7</td><td>4</td><td>5</td><td>9</td><td>6</td><td>2</td><td>3</td><td>1</td><td>8</td></tr><tr><td>16</td><td>9</td><td>3</td><td>1</td><td>6</td><td>2</td><td>5</td><td>10</td><td>8</td><td>4</td><td>7</td></tr><tr><td>17</td><td>2</td><td>3</td><td>1</td><td>5</td><td>9</td><td>7</td><td>10</td><td>8</td><td>4</td><td>6</td></tr><tr><td>18</td><td>10</td><td>4</td><td>8</td><td>2</td><td>6</td><td>1</td><td>3</td><td>7</td><td>9</td><td>5</td></tr><tr><td>19</td><td>7</td><td>2</td><td>10</td><td>9</td><td>3</td><td>4</td><td>6</td><td>5</td><td>1</td><td>8</td></tr><tr><td>20</td><td>7</td><td>9</td><td>6</td><td>8</td><td>5</td><td>2</td><td>1</td><td>4</td><td>3</td><td>10</td></tr><tr><td>21</td><td>8</td><td>3</td><td>2</td><td>7</td><td>5</td><td>9</td><td>4</td><td>6</td><td>10</td><td>1</td></tr><tr><td>22</td><td>1</td><td>5</td><td>6</td><td>10</td><td>8</td><td>9</td><td>3</td><td>4</td><td>7</td><td>2</td></tr><tr><td>23</td><td>9</td><td>1</td><td>7</td><td>2</td><td>8</td><td>3</td><td>5</td><td>4</td><td>6</td><td>10</td></tr><tr><td>24</td><td>1</td><td>7</td><td>3</td><td>5</td><td>8</td><td>2</td><td>10</td><td>6</td><td>9</td><td>4</td></tr><tr><td>25</td><td>10</td><td>2</td><td>1</td><td>7</td><td>9</td><td>4</td><td>3</td><td>6</td><td>5</td><td>8</td></tr><tr><td>26</td><td>7</td><td>6</td><td>2</td><td>10</td><td>8</td><td>1</td><td>5</td><td>9</td><td>4</td><td>3</td></tr><tr><td>27</td><td>1</td><td>8</td><td>9</td><td>10</td><td>6</td><td>3</td><td>7</td><td>5</td><td>4</td><td>2</td></tr><tr><td>28</td><td>1</td><td>8</td><td>9</td><td>7</td><td>10</td><td>6</td><td>2</td><td>4</td><td>5</td><td>3</td></tr><tr><td>29</td><td>4</td><td>3</td><td>9</td><td>7</td><td>5</td><td>2</td><td>10</td><td>6</td><td>1</td><td>8</td></tr><tr><td>30</td><td>1</td><td>7</td><td>8</td><td>9</td><td>10</td><td>2</td><td>4</td><td>6</td><td>5</td><td>3</td></tr><tr><td>31</td><td>8</td><td>1</td><td>9</td><td>4</td><td>5</td><td>2</td><td>6</td><td>7</td><td>3</td><td>10</td></tr><tr><td>32</td><td>1</td><td>4</td><td>7</td><td>10</td><td>3</td><td>8</td><td>5</td><td>6</td><td>2</td><td>9</td></tr><tr><td>33</td><td>3</td><td>10</td><td>2</td><td>7</td><td>9</td><td>6</td><td>8</td><td>4</td><td>5</td><td>1</td></tr></tbody></table>
<table><thead><tr><td>code</td><td>S3</td><td>S4</td><td>S4</td><td>S4</td><td>S5</td><td>SD</td><td>SN/I</td><td>SN</td><td>L1</td><td>L2</td></tr></thead><tbody><tr><td>34</td><td>1</td><td>4</td><td>8</td><td>5</td><td>2</td><td>3</td><td>6</td><td>9</td><td>10</td><td>7</td></tr><tr><td>35</td><td>6</td><td>10</td><td>3</td><td>7</td><td>5</td><td>9</td><td>4</td><td>8</td><td>2</td><td>1</td></tr><tr><td>36</td><td>10</td><td>8</td><td>3</td><td>9</td><td>7</td><td>4</td><td>2</td><td>5</td><td>6</td><td>1</td></tr><tr><td>37</td><td>4</td><td>9</td><td>1</td><td>10</td><td>5</td><td>8</td><td>3</td><td>2</td><td>7</td><td>6</td></tr><tr><td>38</td><td>9</td><td>4</td><td>10</td><td>8</td><td>3</td><td>2</td><td>5</td><td>6</td><td>1</td><td>7</td></tr><tr><td>39</td><td>10</td><td>3</td><td>8</td><td>5</td><td>7</td><td>2</td><td>6</td><td>4</td><td>1</td><td>9</td></tr><tr><td>40</td><td>2</td><td>4</td><td>3</td><td>1</td><td>8</td><td>5</td><td>9</td><td>10</td><td>7</td><td>6</td></tr><tr><td>41</td><td>7</td><td>8</td><td>5</td><td>9</td><td>1</td><td>2</td><td>10</td><td>3</td><td>4</td><td>6</td></tr><tr><td>42</td><td>10</td><td>4</td><td>5</td><td>2</td><td>6</td><td>3</td><td>7</td><td>8</td><td>9</td><td>1</td></tr><tr><td>43</td><td>5</td><td>3</td><td>6</td><td>1</td><td>10</td><td>9</td><td>4</td><td>2</td><td>8</td><td>7</td></tr><tr><td>44</td><td>7</td><td>10</td><td>1</td><td>5</td><td>6</td><td>8</td><td>3</td><td>4</td><td>9</td><td>2</td></tr><tr><td>45</td><td>2</td><td>3</td><td>8</td><td>9</td><td>4</td><td>5</td><td>6</td><td>10</td><td>1</td><td>7</td></tr><tr><td>46</td><td>10</td><td>2</td><td>4</td><td>7</td><td>5</td><td>8</td><td>3</td><td>1</td><td>9</td><td>6</td></tr><tr><td>47</td><td>10</td><td>8</td><td>6</td><td>1</td><td>9</td><td>7</td><td>5</td><td>3</td><td>4</td><td>2</td></tr><tr><td>48</td><td>1</td><td>10</td><td>5</td><td>7</td><td>8</td><td>2</td><td>6</td><td>4</td><td>3</td><td>9</td></tr><tr><td>49</td><td>8</td><td>4</td><td>3</td><td>1</td><td>10</td><td>6</td><td>5</td><td>9</td><td>7</td><td>2</td></tr><tr><td>50</td><td>7</td><td>2</td><td>3</td><td>9</td><td>8</td><td>4</td><td>10</td><td>6</td><td>1</td><td>5</td></tr></tbody></table>
<figure><img src="image_6.png" /></figure>

The organising laboratory gratefully acknowledges its collaborators who enabled it to obtain large volumes
of biological matrices combined with relevant diagnostic and epidemiological data.

END OF REPORT