Marie Senant

Lucile Musset

Georges Chyderiotis

Laurence Guis-Cabanne

Jan Damoiseaux

Nicole Fabien

MD Marie-Agnès Dragon-Durey
email: marie-agnes.durey@aphp.fr

Precision of autoantibody assays in clinical diagnostic laboratories: What is the reality?

Keywords: Autoantibody, laboratory accreditation, coefficient of variation, analytic performances, Internal Quality Control. ABREVIATIONS ALBIA: addressable laser bead immunoassay B2GP1: β2 glycoprotein 1 CCP: cyclic citrullinated peptides CDL: clinical diagnostic laboratories CL: cardiolipin CLIA: chemiluminescent immunoassay coefficient of variation dsDNA: double stranded-DNA ELISA: enzyme-linked immunosorbent assay FEIA: fluorescent-enzyme immunoassay GBM: glomerular basement membrane

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Specialized laboratories have to take up the challenge of applying for and maintaining ISO 15189 accreditation, with specific difficulties relative to the laboratory equipment, the preexamination phase and examination procedures [START_REF] Bizzaro | Accreditation in autoimmune diagnostic laboratories. A position paper of the European Autoimmunity Standardisation Initiative (EASI)[END_REF]. In the field of autoimmunity, the laboratories have to face the crucial issue of the absence of assay standardization. This is linked to the nature of the antigens, the immunoglobulin conjugate, the methods of detection and even the units of the results [START_REF] Mulder | Validation conform ISO-15189 of assays in the field of autoimmunity: Joint efforts in The Netherlands[END_REF]. Consequently, laboratories have to manage the analytical performance of the large panel of assays available on the market [START_REF] Damoiseaux | Autoantibodies 2015: From diagnostic biomarkers toward prediction, prognosis and prevention[END_REF]. The evaluation of such performances involves not only the clinical relevance of the assays in terms of sensitivity and specificity, but also their measurement precision. Precision of measurement is the extent to which repeated measurements of the same sample give similar results. It is expressed as standard deviation, variance or, more often, coefficient of variation (CV). Precision is measured for repeatability conditions of measurement (also called intra-serial, within-run or intra-run precision) that includes the same measurement procedure, operator, measuring system, operating conditions, time, and location. Precision is also measured for intermediate conditions of measurement (or inter-serial, between run, within-lab or inter-run precision) that includes the same measurement procedure and location, and replicate measurements on the same or similar objects over an extended period of time, and, as such, may include other conditions involving changes that include other lots of reagents, calibrations, calibrators, operators, and measuring systems. Reproducibility conditions of measurement also include different locations, operators, measuring systems or procedures [START_REF]Joint Committe for guides in Metrology. International vocabulary of metrology -Basic and general concepts and associated terms (VIM) 3rd edition[END_REF]. While external quality control is used to determine reproducibility, the use of internal quality controls (IQC) allow the calculation of precision, both intra-and inter-run precision which are essential for detecting errors during the analytical phase. This helps to ensure day-to-day consistency of the process and determine whether patient results are reliable enough to be communicated.

The interpretation of precision data obtained in clinical diagnostic laboratories (CDL), i.e., the decision to consider them as acceptable or not, remains challenging in the field of autoimmunity. To date very few reference values have been defined for autoantibody assays and no reference is available on-line or published. Such reference books exist for different specialties, mainly for clinical chemistry [START_REF] Ricos | Current databases on biological variation: pros, cons and progress[END_REF][START_REF] Vassault | Quality specifications and allowable standards for validation of methods used in clinical biochemistry[END_REF] or hematology [START_REF] Vis | Verification and quality control of routine hematology analyzers[END_REF]. Only some reference CVs may be found for anti-thyroperoxidase (TPO), anti-thyroglobulin (TG) antibodies and rheumatoid factor (RF) assays [8,[START_REF]Clinical Laboratory Improvement Amendments of 1988 (CLIA) Proficiency Testing Regulations Related to Analytes and Acceptable Performance[END_REF]. The mere absence of reference values for autoantibody diagnostics is partly due to the absence of reference materials in autoimmunity [START_REF] Sheldon | Strategies for building reference standards for autoantibodies[END_REF]. The Federal Drug Administration (FDA) published guidelines for companies developing diagnostics assays [START_REF]Bioanalytical Method Validation Guidance for Industry[END_REF], giving global rules and analytical objectives to reach. However, these recommendations are not specific to the autoantibody measured neither to the method of detection used by each assay.

Considering the complexity of the field, the European Autoimmunity Standardization Initiative (EASI) has been formed to harmonize autoantibody diagnostics [START_REF] Damoiseaux | EASI -European Autoimmunity Standardisation Initiative: facing the challenges of diagnostics in autoimmunity[END_REF]. The objective of this study was to elaborate a reference support in autoimmunity that could be used in routine practice and for the accreditation of CDL. As an initiative of the French EASI group, a large number of French CDL were requested to participate in a survey aiming to analyze the CVs of intra-run and inter-run precision obtained for assays quantifying 14 different autoantibodies. The results, obtained from 32 specialist laboratories, allow the proposal of acceptability limits for the performances of the autoantibody assays, allowing accurate clinical interpretation of the test and being compatible with the diagnostic laboratories' reality.

MATERIAL AND METHODS

Selection criteria of autoantibody assays

The assays included in this study were selected according to the following criteria: quantitative autoantibody assays for which the quantitation is of clinical relevance and performed in a large number of laboratories. So the assays studied were: IgG and IgM isotypes for anti-beta 2 glycoprotein 1 (B2GP1) and anti-cardiolipin (CL) autoantibodies, IgG isotype for anti-myeloperoxidase (MPO-ANCA), anti-proteinase 3 (PR3-ANCA), antiglomerular basement membrane (GBM), anti-double stranded-DNA (dsDNA), antithyroglobulin (TG), anti-thyroperoxidase (TPO), anti-TSH-receptor (TSH-R), and anti-cyclic citrullinated peptides (CCP), IgA isotype for anti-tissue transglutaminase (TTG) autoantibodies and IgM isotype for rheumatoid factor (RF); the latter, however, is also often measured by nephelometry/turbidimetry and these assays are not specific for IgM isotype only [START_REF] Sieghart | Determination of Autoantibody Isotypes Increases the Sensitivity of Serodiagnostics in Rheumatoid Arthritis[END_REF].

Data collection

A participation invitation for this study was sent by email in March 2018 to 88 French CDL, 68 being public hospital laboratories and 20 private CDL. To ensure uniformity of the responses, this e-mail was accompanied by an Excel sheet, ready to be completed for predefined items by fixed drop down menus. Figure 1A depicts the data collected. In addition to the analyte, for both intra-and inter-run CVs, information was collected on the type of immunoassay, the type of automation (manual, semi-or full automation), the number of values used for the CV calculation, the level of IQC according to the positive threshold (low: <2 fold, medium: between 2 and 5 fold, high: superior to 5 fold) and the nature of the sample used as IQC (patient sample prepared in the CDL, kit-related commercial control, or kitindependent commercial control). Each line was to be completed for each analyte and level of IQC. It was possible to complete as many lines as there were controls for the respective analyte. A 5 weeks deadline was given to complete the file and a reminder email was sent 10 days before the end of the study. The questionnaire did not ask for information about the time frame when measurements for intra-and inter-run CV's were done.

Data analysis

Prior to analysis, CV calculated from less than 5 measurements were excluded. In addition, reported CVs below 0.1% or above 80% were excluded.

Criteria for acceptable performance were defined for each autoantibody assay from data of CVs sent by participating laboratories. According to the model used by the French Society of Biochemistry [START_REF] Vassault | Quality specifications and allowable standards for validation of methods used in clinical biochemistry[END_REF] (SFBC), we fixed two performance goals corresponding to the 90 th percentile and the 50 th percentile (median) from CVs distribution. The first target is the lowest CV value reached by 90% of laboratories and the second by 50% of laboratories. These performance goals were defined for the 3 levels of autoantibodies.

In addition, we analyzed the impact of the number of measurements, the nature of the IQC used, as well as the method (type of immunoassay and automation), on the assay performance.

Statistical analysis

Prism-GraphPad™ software was used to preform different statistical analysis. Unpaired ttest, Mann-Whitney test and one-way ANOVA with multiple comparisons (Turkey's multiple comparison test) were used. .

RESULTS

The overall response rate of the invited laboratories was 36% (32/88). 20% of private laboratories (4/20) and 41% of hospital laboratories (28/68) participated, allowing collection of 440 values of intra-run CV (25 values were to be excluded, leaving 414 for analysis) and 654 values of inter-run CV (38 values were excluded leaving 616 for analysis). A mean of 34 values of CVs was provided by the participating laboratories. 41% of the assays were declared accredited by laboratories at the time of the data collection, according to the ISO 15189 guidelines.

The number of CVs obtained for each analyte is depicted in figure 1B. It varied from 7 to 53 and from 14 to 68 values per analyte for the CVs of intra-run and inter-run precision, respectively. The highest numbers of collected data were for anti-CCP and RF. The lowest numbers of collected CVs were for anti-TSH-R autoantibody assays.

Impact of the number of measurements on CV results

We first analyzed the results of CVs according to the number of measurements used for their determination (5-10, 11-20, 21-30 or >30 values) (Figure 2). No significant difference was observed between the 4 groups (one-way ANOVA) when considering all data (intra-and inter-run CVs).

Impact of the source of the quality controls on CV results

Different types of samples may be used as IQC to perform the measurements allowing the determination of the intra-and inter-run CVs. They may be provided by the manufacturer of the respective immunoassay. Reference values are usually provided in the technical data sheet and are to be used for acceptance of the data obtained. These reagents are frequently ready to use without dilution and may also be lyophilized. Alternatively, IQCs may be obtained from providers independent of the immunoassay manufacturer, possibly requiring adapted dilutions. Lastly, the CDL may use single or pooled patient samples. In the latter two situations the CDL has to determine the target value of the respective IQC. We analyzed if the CVs of the IQCs are different depending on the source of the IQC sample (Figure 3). We observed that lower CVs were obtained with commercial controls when compared to IQCs derived from in-house samples, especially for the intra-run CVs (p<0.0001).

Determination of acceptable precision performance

Acceptable precision performance was calculated for each autoantibody according to the levels of the IQC. The highest CV values were obtained with low level IQCs (<2 fold the positive threshold). CV values also depend on the type of autoantibody measured. In particular, assays for anti-CL or anti-B2GP1 autoantibodies have higher CVs. Irrespective of the analyte, the method and the level of the IQC, the 90 th percentile precision performance goals (lowest CV values reached by 90 % of laboratories) were between 3.3% and 16.2% for the intra-run CVs, and between 6.7% and 23.2 for the inter-run CVs. At the 50 th percentile precision performance goal values (lowest CV values reached by 50 % of laboratories) were between 2.9% and 10.7% for the intra-run CVs, and between 2.8% and 13.2% for the interrun CVs (Table 1).

Impact of the type of immunoassay on CV results

The main types of immunoassays used by participants were fluorescent-enzyme immunoassays (FEIA: 39%), chemiluminescence (CLIA; 26%), enzyme linked immunosorbent assay (ELISA; 23%), addressable laser bead immunoassay (ALBIA; 7%), and nephelometry/turbidimetry (5%). Other methods (radio-immunoassays or immunocompetition) represented less than 1% of the methods reported. In total twenty suppliers of in vitro diagnostic reagents were represented in the study (supplemental Figure 1).

We analyzed the results of intra-run and inter-run CVs and calculated the performances at 90 th and 50 th percentiles according to the type of immunoassay used (Figures 4 and5).

This analysis revealed larger distribution of the intra-run CVs (Figure 4A) and higher CV values with ELISA compared to the other immunoassays (Figure 4B, range of CVs at 90 th percentile: 11.4-13.5% for ELISA vs 3.2-12.2% for the other immunoassays), and especially for the low level IQCs. We then analyzed the impact of the type of immunoassay for specified autoantibodies on precision performance. We observed significant differences in inter-run CVs for IgG anti-CL assays, with higher values observed with ELISA as compared to FEIA, CLIA and ALBIA.

For RF, significantly lower values were observed with nephelometry/turbidimetry as compared to FEIA and ELISA. For the other autoantibodies low or no differences were observed (Mann and Whitney, supplemental data, Figure 2).

DISCUSSION

This article presents the results of a study proposed to French CDL, aiming to collect and analyze the CVs of intra-run and inter-run variations of assays used for the detection of 14 autoantibodies, obtained in real life conditions. Despite the low rate of responses (36% of the invited laboratories), the data collected allowed the analysis of 414 values of intra-run CV and 616 values of inter-run CV. The difference in the number of data for intra-and inter-run variations is linked to the fact that inter-run variation is a parameter followed by all laboratories but the determination of intra-run variation is usually performed only for the initial verification of the methods.

The number of measurements used for the CV calculations had no significant influence on the intra-and inter-run CVs of autoantibody assays. This information is very important because the accreditation rules were defined for biochemistry analytes, such as blood electrolyte concentration, for which low levels of variation may have important clinical significance.

Most of the accreditation guidelines have applied these rules to all laboratory specialties without any adaptation according to the clinical significance, the cost-effectiveness, or even feasibility. Indeed the cost for measuring an autoantibody is considerably higher than the measurement of blood electrolytes [START_REF]Bioanalytical Method Validation Guidance for Industry[END_REF]. In France, the accreditation technical guidance for the use of IQC, recommends calculating the inter-run variability during at least 15 days of measurements, or at least 30 values [START_REF] Gta | Guide technique d'accréditation : contrôle de qualité en biologie médicale[END_REF]. Based on our results we conclude that more than 10 measurements for either intra-or inter-run CVs will not improve the estimation of the assay precision, whatever the type of immunoassay used. These findings could substantially reduce cost of achieving accreditation if the criteria was reduced from a minimum of 30 observations to 10, if our findings are validated by other studies.

Our study also revealed an impact of the source of the IQC on intra-and inter-run CVs, with lower values obtained when using commercial IQC as compared to IQCs derived from in-house patient samples. This effect doesn't seem to be explained by a biased use of the type of controls according to the method (Supplemental data, Figure 3). Commercially produced IQCs have the advantage of long term stability and low vial to vial variability. However, the matrix effect on the measurement of analytes may be different from a patient serum and could give erroneously low CVs, which could be detrimental for the interpretation of some patient results, mainly for quantitative assays. For autoantibody assays, this is of high importance considering that the serum to be tested contains high concentrations of IgG, IgM and IgA being non-specific for the autoantigen but potentially influencing the interaction of the antigen-specific autoantibodies to their target. In addition, natural autoantibodies of IgM or IgG isotype may interfere in these immunoassays and their effect on precision should be taken into account [START_REF] Maddur | Natural Antibodies: from First-Line Defense Against Pathogens to Perpetual Immune Homeostasis[END_REF]. In our analysis we also differentiated data from IQC included in the assay versus IQC independent of the assay manufacturer. Indeed commercial IQC should be bought from a manufacturer different from the assay's provider to ensure an independent assessment of performance [START_REF] Gta | Guide technique d'accréditation : contrôle de qualité en biologie médicale[END_REF][START_REF] Kinns | Internal quality control: best practice[END_REF][START_REF]Statistical Quality Control for Quantitative Measurement Procedures : Principles and definitions[END_REF]. Moreover, positive and negative controls included in commercial assays are frequently ready to use and thus are not treated in the same manner as patient samples. This type of IQC does not reflect the exact uncertainty of measurement and potentially provides a false acceptance limit. For all of these reasons, the use of patientderived IQC samples has been recently recommended for antinuclear antibody testing by indirect immunofluorescence [START_REF] Bogaert | Harmonizing by reducing inter-run variability: performance evaluation of a quality assurance program for antinuclear antibody detection by indirect immunofluorescence[END_REF].

Regarding the type of immunoassays, we noticed an over-representation of the fully automated methods, mainly FEIA and CLIA which represented more than half of the immunoassays. This has a significant impact on the proposed acceptable performance, mainly on the 50 th percentile performance (inter-run CV lower than 9% at low levels vs 14.6% for ELISA).

To investigate the differences observed between the 50 th and the 90 th percentile performances, we performed sub-analyses of the results of autoantibody assays according to the immunoassay method. No major difference in performance according to the type of immuneassay was observed except for RF and anti-CL autoantibodies for which higher CVs were observed when using ELISA, as compared to the other types of immunoassay. This difference is not explained by the use of different kinds of automatization or of manual techniques for the same parameter because the data didn't demonstrate lower CVs obtained by manual techniques (data not shown). In addition, this phenomenon seems particularly important when the IQC are at low concentration levels. Indeed, our results show clearly the higher imprecision when the autoantibody levels of the IQC are below 2-fold the positive threshold of the assay. When studying the 90 th percentile values, the highest CVs of intra-and inter-run CVs were also observed with low level IQCs. The imprecision observed with low level IQCs is part of the so-called "grey zone" of the autoantibody assays. This zone corresponds to borderline positivity and may have a significant effect on the patient diagnosis and care.

That's why it is very important to be aware of the assay performances at these levels. For the diagnosis or classification of several autoimmune diseases, guidelines propose using serological criteria of relatively high autoantibody levels. This applies for the antiphospholipid syndrome [START_REF] Miyakis | International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)[END_REF], rheumatoid arthritis [START_REF] Aletaha | Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative[END_REF] and coeliac disease [START_REF] Lebwohl | Coeliac disease[END_REF].

The determination of the positive cut-off, and consequently of the grey-zone, remains debated for autoantibody detection. Indeed, their presence and levels in the normal population don't fit a Gaussian distribution and their prevalence may vary from one autoantibody to another [START_REF] Sepe | Definition of cut-off points for autoantibody assays in cohorts of healthy individuals. The Sardinian School Children-IDDM (SSI) & Newborn-IDDM (SNI) Study Groups[END_REF].

Due to this imprecision a commentary to the physicians should be added to the results within the "grey zone", in which measurement uncertainty is very high. Indeed, the laboratory should give information about the precision allowing appropriate interpretation of each patient result, at least at the clinically relevant levels.

No reference values for intra-and inter-run CVs are available for autoantibody detection assays in biorepositories. Some data are present on the Westgard website for anti-TPO and anti-TG autoantibodies and for RF, but they are based on only two studies with very few data (33 and 34 samples, respectively). These studies didn't really measured the intra-and inter-run CVs of these parameters but rather the biological variation observed in different samples collected from the same individuals during a short period of time. These values of withinsubject variations (anti-TG: 8.5%, anti-TPO: 11.3%, RF: 8.5%) don't inform specifically about the analytical performances of the assays per se [START_REF] Jensen | Biological variation of thyroid autoantibodies and thyroglobulin[END_REF], [START_REF] Jimenez | Usefulness of reference limits and evaluation of significant differences. An example of the biological variation of serum rheumatoid factors[END_REF]. In addition, they are of limited interest in the field of autoimmunity in which for some autoantibody, the levels may vary quickly in patients with active disease

In conclusion, our study revealed that autoantibody measurements exhibit higher imprecision than those observed in clinical chemistry and hematology. However, the imprecision of the autoantibody assays that we report here, is not prejudicial for patient care according to the different clinical guidelines. Though variable according to the different autoimmune diseases, such as in coeliac disease, lupus or vasculitis, [START_REF] Petroff | Antibody Concentrations Decrease 14-Fold in Children With Celiac Disease on a Gluten-Free Diet but Remain High at 3 Months[END_REF][START_REF] Babaoglu | Predictors of predominant Lupus Low Disease Activity State (LLDAS-50)[END_REF][START_REF] Damoiseaux | Evaluation of a new fluorescent-enzyme immuno-assay for diagnosis and follow-up of ANCA-associated vasculitis[END_REF][START_REF] Osman | Anti-neutrophil Cytoplasmic Antibodies (ANCA) as Disease Activity Biomarkers in a "Personalized Medicine Approach" in ANCA-Associated Vasculitis[END_REF] the significant clinical variations, i.e levels variation that have an impact on the patients diagnostic, prognosis or management, are usually higher than the imprecision of the assays. However, it would be useful for the physicians to be informed about the measurement uncertainty inherent to each autoantibody assay.

This study allows us to propose that an acceptable performance for intra-and inter-run CVs should be below the 90 th percentile. Robustness of an assay should thus be re-evaluated if

CVs are above the 90 th percentile value.

Similar larger studies should be performed by international societies in the field of autoimmunity in order to validate our findings. Nevertheless, regarding the absence of any available data, our work represents a first step in the analysis of proficiency testing of the CDL implicated in autoantibody diagnostics.

Recommendations:

• IQC must be defined in terms of source and levels of autoantibody according to the positive threshold of the assay.

• IQC should comprise at least one positive sample obtained from a source different from the manufacturer of the assay.

• IQC should comprise at least one positive sample that is to be treated in an identical manner as patient samples.

• Intra-and inter-run CVs should be comprised between the 50 th and 90 th percentile provided by this study; robustness must be re-evaluated if they are >90 th .

• Intra-and inter-run CVs should be calculated from at least 10 values

• The precision of the autoantibody assays must be adapted to their clinical significance and their results must be accompanied by appropriate biological comments. FEIA: fluorescent-enzyme immunoassay, CLIA: chemiluminescent immunoassay, ELISA: enzyme-linked immunosorbent assay, ALBIA:

addressable laser bead immunoassay, IQC: internal quality control; IQR: Inter-quartile ranges.

Legends of the figures. A.

Figure 1 :

 1 Figure 1: (A) Pre-defined Excel sheet sent to 88 French clinical diagnostic laboratories.

Figure 2 :

 2 Figure 2: Combined results of intra-and inter-run coefficients of variation according to

Figure 3 :

 3 Figure 3: Levels of CVs according to the source of IQC used (commercial control different

Figure 4 :

 4 Figure 4: (A) CVs for intra-run variation according to the levels of autoantibodies of the

Anti

Table 2

 2 depicts the 616 results of inter-run CVs collected according to the type of immunoassay and the levels of IQCs. Irrespective of the IQC levels, larger distribution was

observed for the inter-run CVs obtained with ELISA (median: 11.06%, IQRs

(7.15-15.82

) as compared to the other methods (FEIA: median: 8%, IQRs (6.07-10.71; CLIA: median: 6.78%, IQRs (4.1-9.67); ALBIA: median: 7.8%, IQRs (6.1-9.19); nephelometry/turbidimetry: median: 2.55%, IQRs (1.97-5.37) (Figure

5A

). Moreover, for the 90 th percentile values, higher CVs of intra-and inter-run CVs were observed for low level IQCs (mean 10.9% and 16.2%, respectively) as compared to high level IQCs (8.2% and 13.8%, respectively, p=0.008 and 0.09, unpaired t test), irrespective of immunoassay type.

Table 2 :

 2 Statistical description of the Inter-run CVs results, according to the type of immunoassay and the IQC levels.

	Method	Level of	Number of	Median	IQR	Ranges
		IQC	values
		Low	42	9.30	7.2-12.47	4.40-20.1
	FEIA	Medium	117	7.42	5.98-10.45	2.90-33.3
		High	75	8.00	6.00-10.30	3.36-32.95
		Low	57	7.73	6.15-10.82	2.39-10.82
	CLIA	Medium	69	6.68	4.78-9.91	1.40-14.56
		High	41	3.70	3.46-7.19	1.90-13.5
		Low	33	14.6	11.28-20.93	1.40-36.6
	ELISA	Medium	69	10.05	5.98-14.90	1.50-23.00
		High	53	10.00	6.77-15.00	3.58-26.40
		Low	5	5.10	4.1-12.71	4.10-14.61
	ALBIA	Medium	16	7.40	6.10-8.93	5.10-12.66
		High	9	7.95	7.42-10.53	4.90-13.50
		Low	16	2.38	2.16-4.11	1.80-14.9
	Nephelo/Turbidimetry	Medium	8	1.77	1.72-7.64	1.70-17.00
		High	6	4.07	3.49-7.59	1.97-8.60

Table ( B

 ( ) resumes the values corresponding to 90 th percentile (i.e the lowest intra-run CV value obtained by 90% of the laboratories). *: Not applicable, only one value reported.

	Name of Analyte	Immunoassay	Method of experiment	Number of values	IQC levels	Nature of the sample
	Anti-dsDNA	ELISA	Manual	<5	Low (<2x Cut off)	Serum/plasma
	Anti-CCP-IgG	CLIA	Semi-automatization	5-15	Medium (2-5x Cut off) Internal commercial control
	RF	FEIA	Full automatization	15-30	High (>5x Cut off)	External commercial control
	Anti-CL IgG	ALBIA		>30
	Anti-CL IgM	Nephelo/Turbidimetry
	Anti-B2GP1 IgG	Other
	Figure 5: (A) CVs for inter-run variation according to the levels of autoantibodies of the
	IQC and type of immunoassay. Dotted lines indicate acceptable limits corresponding to
	50th percentile and plain lines acceptable limits corresponding to 90th percentile. Table (B)

resumes the values corresponding to 90 th percentile (i.e the lowest inter-run CV value obtained by 90% of the laboratories). *: Acceptable limits calculated from less than 5 values.

A.

B.

CV90 (90th percentile) CV50 (50th percentile)

		Low	Medium	High	Low	Medium	High
	FEIA	13.5	12.7	12.5	9	7.4	7.8
	CLIA	12.4	10.8	9.3	7.6	6.7	3.7
	ELISA	30.8	18	18.9	14.6	9.9	10.0
	ALBIA	10.8	9.2	11.2	4.1	6.5	7.9
	Nephelometry/	7.5	10.0	7.3*	2.4	1.8	4.1*
	Turbidimetry

Acknowledgement

We thank all biologists from the participating French DBL for sharing their data (in alphabetical order) : Biomedical Laboratory of Hospital Ambroise Paré, Paris (Dr J Zhu), Biomedical laboratory of CH Alpes Leman, Contamine/Arve (Dr B Murienne), Laboratory of Immunology, Hospital Bichat, Paris (Dr P Nicaise), Private Biomedical Laboratory BIO7/Cerballiance, Lisses (Dr M Senant), Private Biomedical Laboratory Bioliance, Nantes (Dr S Le Quere), Private Biomedical Laboratory Biomnis-Eurofins, Ivry (Dr L Guis-Cabanne), Lyon, (Dr G Chyderiotis), Laboratory of Immunology of University hospital of Caen (Dr E Comby), Biomedical Laboratory of Hospital Metropole Savoie, Chambéry (Dr C Dumollard), Biomedical Laboratory of Hospital of Annecy (Dr S Boutruche), Biomedical

Funding None

Statement of author contributions

Marie Senant collected and analyzed the data. All authors participated in the design of the study, the drafting of the manuscript and provided approval of the final submitted version.


David Lupande-Mwenebitu

Larbi Sophie Alexandra Baron

Zakaria Nabti

Octavie Lunguya-Metila

Jean-Philippe Lavigne

Jean-Marc Rolain
email: jean-marc.rolain@univ-amu.fr

Seydina Mouhamadou Diene
email: seydina.diene@univ-amu.fr

Larbi Zakaria Nabti

Current status of resistance to antibiotics in the Democratic Republic of the Congo: A review

Keywords: Antimicrobial resistance genes, Democratic Republic of Congo, Enterobacteriaceae, Extended-spectrum β-lactamases, methicillin-resistant Staphylococcus aureus

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

There is a growing concern on antimicrobial resistance (AMR) worldwide [START_REF] Tadesse | Antimicrobial resistance in Africa: a systematic review[END_REF].

Resistance against antimicrobial agents s in clinically relevant bacteria is one of the most imminent threats to public health and especially to our most vulnerable patient populations [START_REF] Van Duin | The global epidemiology of carbapenemase-producing Enterobacteriaceae[END_REF][START_REF] Xian-Zhi | The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria[END_REF]. The treatment of bacterial infections in Africa is largely empirical and, in most instances, there are no laboratory results to guide therapy [START_REF] Mshana | Antimicrobial resistance in human and animal pathogens in Zambia, Democratic Republic of Congo, Mozambique and Tanzania: an urgent need of a sustainable surveillance system[END_REF][START_REF] Pitout | Transmission Surveillance for Antimicrobial-Resistant Organisms in the Health System[END_REF]. The spread of AMR bacteria is problematic for the medical community as a whole as it compromises empirical treatment regimens by delaying the administration of appropriate antibiotic therapy and reducing appropriate treatment options [START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Wong | Interventions to improve antimicrobial prescribing of doctors in training: the IMPACT (Improving Antimicrobial prescribing of doctors in Training) realist review[END_REF]. AMR remains a real challenge in resource-limited countries due to a lack of available antibiotics with multidrug-resistant isolates being labelled on the basis of a small number of antibiotics tested, with economic consequences and yet the use of the old molecules is very effective. The consequences of AMR are not limited to patients with infections, but to a whole system including the environmental and agro-pastoral sector that hemming enough resistance genes. Hence, there is a need for action to significantly reduce the expansion of the phenomenon, whose distribution varies considerably from one country to another [START_REF] Guardabassi | Antimicrobial resistance: a global threat with remarkable geographical differences[END_REF][START_REF] Doare | Systematic Review of Antibiotic Resistance Rates among Gram-Negative Bacteria in Children With Sepsis in Resource-Limited Countries[END_REF]. To combat the global threat of AMR, improved surveillance to detect emerging and long-term resistance trends is vital, several global initiatives, such as the Fleming Fund, have been recently established to improve laboratory capacity in low-and middle-income countries [START_REF] Huttner | Antimicrobial resistance: a global view from the 2013 World Healthcare-Associated Infections Forum[END_REF].

As recently reported, there is a link between antibiotic resistance genes found in human pathogens and those found in non-pathogenic, commensal and environmental organisms, prompting further studies of natural and human-associated reservoirs of resistance genes [START_REF] Baron | Human microbiomes and antibiotic resistance[END_REF]. Many hypotheses can be evoked, even though the relationship between antibiotic use and antibiotic resistance remains complex [START_REF] Peterson | Antibiotic Resistance Mechanisms in Bacteria: Relationships Between Resistance Determinants of Antibiotic Producers, Environmental Bacteria, and Clinical Pathogens[END_REF]. Resistance selection pressure continues as antimicrobials are released into the environment, largely while remaining in their active forms. These drugs frequently found in wastewater for example contributed greatly to the environmental selection of antibiotic-resistant bacteria [START_REF] Gbaguidi-Haore | Antibiotics involved in the occurrence of antibiotic-resistant bacteria: a nationwide multilevel study suggests differences within antibiotic classes[END_REF][START_REF] Drancourt | Antiquité de la résistance aux antibiotiques[END_REF][START_REF] Pehrsson | Novel resistance functions uncovered using functional metagenomic investigations of resistance reservoirs[END_REF][START_REF] Sommer | The human microbiome harbors a diverse reservoir of antibiotic resistance genes[END_REF][START_REF] Jarrell | Factors associated with in-hospital mortality among critically ill surgical patients with multidrug-resistant Gram-negative infections[END_REF]. As reported by many health care settings, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii have been identified as emerging organisms of concern for multidrug resistance, and have encouraged researches in this area [START_REF] Magiorakos | Multidrugresistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance[END_REF][START_REF] Corona | The analysis of the antibiotic resistome offers new opportunities for therapeutic intervention[END_REF]. Resistance to antibiotics is the result of either by reducing its affinity, lowering its concentration, or destroying it in order to effectively reduce or cancel the interaction with its target [START_REF] Durão | Evolutionary Mechanisms Shaping the Maintenance of Antibiotic Resistance[END_REF]. Therefore, depending on the involved mechanism, the resistance level can vary considerably [START_REF] Martinez | General principles of antibiotic resistance in bacteria[END_REF].

Over the past decade, the increase in multidrug resistance of Gram-negative rods and in particular the spread of resistance to carbapenems in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., has prompted the return of colistin (or polymyxin E) as a treatment of last resort. Extended-spectrum β-lactamase (ESBL)-producing pathogens and methicillin resistant Staphylococcus aureus (MRSA) are endemic in many hospitals worldwide and are also increasingly detected in the community [START_REF] Lee | Strategies to Minimize Antibiotic Resistance[END_REF][START_REF] Armand-Lefèvre | La résistance en ville, mythe ou réalité ? La menace des entérobactéries productrices de BLSE[END_REF][START_REF] Mitgang | Review and Mapping of Carbapenem-resistant Enterobacteriaceae in Africa: Using Diverse Data to Inform Surveillance Gaps[END_REF][START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. Carbapenem-resistant enterobacterial infections (CRE) are being increasingly observed, mainly in health care facilities, but also in the community; they are associated with management difficulties due to the lack of alternative active molecules and cause outbreaks difficult to control in health care facilities in low-income countries where hygiene is poor and the population is overcrowded, in addition to an epidemic context of ESBL that was still not effectively controlled [START_REF] Kariuki | Antibacterial resistance in sub-Saharan Africa: an underestimated emergency[END_REF].

Challenge of humanitarian disaster, infectious diseases and AMR spread in war context

In Democratic Republic of Congo (DRC), data on AMR are scarce; some bacteria are being included in surveillance cross sectional study, such as Salmonella spp. and S. aureus, but there are unknown prospects focused on other bacteria and in many parts of the country [START_REF] Kalonji | Invasive Salmonella Infections at Multiple Surveillance Sites in the Democratic Republic of the Congo, 2011-2014[END_REF][START_REF] Phaku | Unveiling the molecular basis of antimicrobial resistance in Staphylococcus aureus from the Democratic Republic of the Congo using whole genome sequencing[END_REF]. The DRC is a vast country (2.3 million km of area) divided into 26 provinces. Each province is divided into health districts, which in turn are divided into health zones (called "zones de santé"). A health zone in the DRC corresponds to what is internationally referred to as a health district [START_REF] Stasse | Improving financial access to health care in the Kisantu district in the Democratic Republic of Congo: acting upon complexity[END_REF]. The conflict in DRC has resulted in a health-system collapse and created a humanitarian disaster. An estimated 5.4 million excess deaths occurred from 1997 to 2004, with fewer than 10% attributable to violence and the rest to preventable and treatable medical conditions, such as malaria, diarrhea, pneumonia, and malnutrition [START_REF] Coghlan | Mortality in the Democratic Republic of Congo ; a nationalwide survey[END_REF]. Armed conflicts and other disastrous natural phenomena can lead to major population displacements.

An emergency unsustainable infrastructure is temporarily built, and often with overcrowding, insufficient if not insufficient drinking water and sanitation, which gives its populations a risk of exposure to communicable diseases, especially those with epidemic potential [START_REF] Gayer | Conflict and Emerging Infectious Diseases[END_REF]. The country had been the site of two wars involving multiple African nations and armed conflict between rebels and soldiers for the past 16 years, characterized by extreme violence, massive population displacements from east to west, and the collapse of all existing infrastructure [START_REF] Casey | Care-Seeking Behavior by Survivors of Sexual Assault in the Democratic Republic of the Congo[END_REF][START_REF] Kohli | A Congolese community-based health program for survivors of sexual violence[END_REF]. Massive population movements are at the root of most risk factors, people are displaced to regions and areas where resources and services are insufficient and where contact with other naïve populations with new vectors of infectious diseases is potentially more frequent. This leads to large numbers of people going to camps, often associated with overcrowding, inadequate housing and poor water conditions, resulting in the spread of pathogenic microorganisms, as reported by UNHCR in January 2018 in Eastern Congo (Fig. 1) [34,[START_REF] Hammer | Risk factors and risk factor cascades for communicable disease outbreaks in complex humanitarian emergencies: a qualitative systematic review[END_REF].

The deterioration of health services is probably one of the main risk factors for communicable diseases in this type of humanitarian disaster, both for individuals and populations. Access to health care in such conditions is impossible, which is a key risk factor for the serious progression of most communicable diseases in the individual. As the spread and detection of cases becomes a major challenge, emergency measures to be implemented remain unavailable, including public health services, vaccination, communicable disease prevention and control measures and surveillance, making epidemics more likely, more difficult to detect and more difficult to control [START_REF] Hammer | Risk factors and risk factor cascades for communicable disease outbreaks in complex humanitarian emergencies: a qualitative systematic review[END_REF][START_REF] Connolly | Communicable diseases in complex emergencies: impact and challenges[END_REF].

Review methods

For this review, we searched PubMed/Medline, Google Scholar and African Journals for articles written in French and in English on bacterial resistance to antibiotics in DR Congo. We used the search terms "bacterial resistance", "antibiotic and resistance", "antimicrobial resistance", "microbial resistance", "susceptibility", "resistance" combined with the name of Democratic Republic of Congo. We screened the search results for relevant, methodologically rigorous studies and conducted a forward search of the references of many of the relevant results to identify additional studies. Articles were reviewed and publications using original data on antibiotics resistance in humans and environment samples were included. The studies site, study period, organism isolated, the resistance phenotype observed, the molecular methodology used as well as the antimicrobial resistance genes found were appraised and analysed. This review on antibacterial resistance in DRC focuses on available published literature on the subject dating from 1990 to December 31 st , 2019.

Resistance pattern among Gram-negative and Gram-positive bacteria

Enterobacteriaceae:

For Enterobacteriaceae, the emergence and spread of CTX-M type ESBLs are global, with the highest prevalence observed in low-income countries, particularly in the community with an estimated carrying prevalence of 70%, 35% and 15% respectively in Asia, the Eastern Mediterranean Basin and Africa [START_REF] Mitgang | Review and Mapping of Carbapenem-resistant Enterobacteriaceae in Africa: Using Diverse Data to Inform Surveillance Gaps[END_REF].

Salmonella

Salmonella spp. is the most studied bacterial genus in DRC as a result of the research set up and is still ongoing since 2008 on bacteremia. Indeed, since the first publications on bacteremia, the prevalence of resistance to ampicillin, chloramphenicol and cotrimoxazole are particularly high (90 -100%) [START_REF] Leekitcharoenphon | Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo[END_REF][START_REF] Ley | Invasive Salmonella enterica Serotype Typhimurium Infections, Democratic Republic of the Congo, 2007-2011[END_REF]. The most frequently isolated species are Salmonella enteritidis, Salmonella typhimurium and Salmonella Typhi. The level of resistance is as high for S. Typhi as it is for non-Typhi Salmonella (NTS). Ampicillin, chloramphenicol and cotrimoxazole are no longer susceptible in more than 70% of isolates [START_REF] Ley | Invasive Salmonella enterica Serotype Typhimurium Infections, Democratic Republic of the Congo, 2007-2011[END_REF][START_REF] Vandenberg | Microbiologic and Clinical Features of Salmonella Species Isolated From Bacteremic Children in Eastern Democratic Republic of Congo[END_REF][START_REF] Phoba | Epidemic increase in Salmonella bloodstream infection in children, Bwamanda, the Democratic Republic of Congo[END_REF]. For Muyembe et al. in 2009, all isolates (11 isolates) of S. Typhi were resistant to ampicillin and cotrimoxazole [START_REF] Veyi | An outbreak of peritonitis caused by multidrug-resistant Salmonella Typhi in Kinshasa, Democratic Republic of Congo[END_REF]. Finally, a decreased susceptibility to ciprofloxacin was also observed between 59 to 65% [START_REF] Bahwere | Community-Acquired Bacteremia among Hospitalized Children in Rural Central Africa[END_REF][START_REF] Phoba | Salmonella enterica serovar Typhi Producing CTX-M-15 Extended Spectrum β-Lactamase in the Democratic Republic of the Congo[END_REF][START_REF] Vandendriessche | Characterisation of Staphylococcus aureus isolates from bloodstream infections, Democratic Republic of the Congo[END_REF]. Several resistance genes were detected in Salmonella spp. strains isolated from DRC including, blaTEM-1b and blaSHV-2a genes [START_REF] Kalonji | Invasive Salmonella Infections at Multiple Surveillance Sites in the Democratic Republic of the Congo, 2011-2014[END_REF][START_REF] Leekitcharoenphon | Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo[END_REF][START_REF] Phoba | Salmonella enterica serovar Typhi Producing CTX-M-15 Extended Spectrum β-Lactamase in the Democratic Republic of the Congo[END_REF]. Moreover, S.

Typhi harboring blaCTX-M genes has been described in blood cultures in a bacteremic patient in 2018 [START_REF] Kakabadze | Selection of Potential Therapeutic Bacteriophages that Lyse a CTX-M-15 Extended Spectrum β-Lactamase Producing Salmonella enterica Serovar Typhi Strain from the Democratic Republic of the Congo[END_REF]. Resistance to chloramphenicol, aminoglycosides, quinolones and sulfonamides is correlated to the presence of catA1, strA/B, sul1, dfrA1 genes and the aac(6')-1aa gene [START_REF] Leekitcharoenphon | Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo[END_REF][START_REF] Phoba | Epidemic increase in Salmonella bloodstream infection in children, Bwamanda, the Democratic Republic of Congo[END_REF]. Some mutations on gyrA gene, as well as the presence of qnrB1 and aac(6')-Ib-cr genes were found in strains resistant to fluoroquinolones (Table 1 andTable 2). Puyvelde et al., in 2019, analysed the entire genome of 81 strains of S. typhimurium by high-throughput sequencing. They reported that 67% of the isolates (54/81) were ESBL-producers and 63% were multidrug resistant (MDR), according to the definition used for Salmonella spp., i.e. resistant to both ampicillin, chloramphenicol and cotrimoxazole. Several resistances genes were found, including catA, blaTEM-1, blaSHV-2A, dfrA, dfrA1, dfrA14, mphA and a mutation in the gyrA gene (S83Y) [START_REF] Van Puyvelde | An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation[END_REF]. Bieke et al., in 2019, analysed 295 S. Typhi, 93 of which were sequenced (WGS): 38.4% (114/295) were resistant to ampicillin, chloramphenicol and cotrimoxazole; 24.5% (73/295) had decreased sensitivity to ciprofloxacin, mainly due to mutations detected in the gyrA (S83E, S83Y, D87Y, D87G, A119E) and gyrB (E466D, S464Y) genes [START_REF] Tack | Salmonella Typhi From Blood Cultures in the Democratic Republic of the Congo: A 10-Year Surveillance[END_REF].

Escherichia coli

E. coli is one of the most isolated microorganisms in bacteriology laboratories. A single emerging clonal group of E. coli, designated sequence type 131 (ST131) by multilocus sequence typing, has been identified as a factor in the AMR epidemic in E. coli, especially for first-line agents such as fluoroquinolones and extended-spectrum cephalosporins. E. coli contains a wide array of genetic diversity, most notably among genes that confer virulence and resistance [START_REF] Hutton | Phylogroup and virulence gene association with clinical characteristics of Escherichia coli urinary tract infections from dogs and cats[END_REF][START_REF] Johnson | The Pandemic H30 Subclone of Sequence Type 131 (ST131) as the Leading Cause of Multidrug-Resistant Escherichia coli Infections in the United States (2011-2012)[END_REF]. In DRC, five studies carried out in 2001, 2014 and 2015 showed that the resistance rates of E. coli varied from 65 to 90% to penicillin [START_REF] Bahwere | Community-Acquired Bacteremia among Hospitalized Children in Rural Central Africa[END_REF][START_REF] Kadima | Profil bactériologique de l'infection urinaire gravidique (Cas de l'hôpital Bonzola à Mbujimayi en RD Congo)[END_REF][START_REF] Liesse | Antimicrobial susceptibility patterns of enterobacteriaceae isolated from HIV-infected patients in Kinshasa[END_REF], from 6 to 15% to ceftriaxone and 15.4% to 31.5% to ciprofloxacin [START_REF] Irenge | Antimicrobial resistance of bacteria isolated from patients with bloodstream infections at a tertiary care hospital in the Democratic Republic of the Congo[END_REF][START_REF] Irenge | Antimicrobial resistance in urinary isolates from inpatients and outpatients at a tertiary care hospital in South-Kivu Province (Democratic Republic of Congo)[END_REF]. For studies in which resistance genes were sought, the blaCTX-M-15 and blaSHV-18 genes were reported (Table 1) [START_REF] Irenge | Antimicrobial resistance in urinary isolates from inpatients and outpatients at a tertiary care hospital in South-Kivu Province (Democratic Republic of Congo)[END_REF][START_REF] Boeck | ESBL-Positive Enterobacteria Isolates in Drinking Water[END_REF]. In Bukavu, a recent study conducted in 2019 by Irenge et al. using whole genome sequencing of 21 E. coli strains showed that 33% of the strains (7 out of the 21 E. coli) belonged to ST131 and exhibited more virulence genes as compared to compared to the same clone in the NCBI database. The majority of ESBL genes included blaCTX-M-15 and blaSHV-12 in all the isolates. Several other AMR genes have been found including blaOXA-1, blaTEM-1, as well as genes encoding resistance to aminoglycosides, quinolones, chloramphenicol, rifampicin, tetracyclines and sulfonamides. As a matter of fact, blaCTX-M-15 gene is the most frequently found ESBL gene among African hospital strains [START_REF] Alonso | Antibiotic resistance in Escherichia coli in husbandry animals: the African perspective[END_REF][START_REF] Irenge | Wholegenome sequences of multidrug-resistant Escherichia coli in South-Kivu Province, Democratic Republic of Congo: characterization of phylogenomic changes, virulence and resistance genes[END_REF].

Klebsiella pneumoniae

K. pneumoniae is a frequent cause of nosocomial infections and has also emerged as an agent of severe community-acquired infections, including pyogenic liver abscess, pneumonia, and meningitis. K. pneumoniae clinical isolates are evolving toward increasing levels of antimicrobial drug resistance, placing this species among the infectious bacterial pathogens that are most challenging to control [START_REF] Bialek-Davenet | Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups[END_REF]. In a study on urinary tract infection aetiologies in Bukavu, Leonid et al. found that K. pneumoniae was the second most frequently identified microorganism after E. coli [START_REF] Irenge | Antimicrobial resistance in urinary isolates from inpatients and outpatients at a tertiary care hospital in South-Kivu Province (Democratic Republic of Congo)[END_REF]. Its resistance profile in studies published in the DRC in 2012 and 2015 showed a resistance rate of less than 35% for both amoxicillin/clavulanate acid (14%-19%), ceftriaxone (14% -19%) and ciprofloxacin (12%-33%). One out of the 21 strains of K. pneumoniae was phenotypically resistant to imipenem. No further studies were performed to screen for the carbapenemase genes. Studies have shown that the use of carbapenems promotes the emergence of carbapenemase-producing isolates. In Bukavu, the use of these molecules is increasingly becoming part of the therapeutic arsenal of clinicians [START_REF] Harris | Effect of Piperacillin-Tazobactam vs Meropenem on 30-Day Mortality for Patients With E coli or Klebsiella pneumoniae Bloodstream Infection and Ceftriaxone Resistance[END_REF]. The resistance genes described for K. pneumoniae in DRC are the blaCTX-M-15 and blaSHV-18 genes, as previously reported for E. coli [START_REF] Irenge | Antimicrobial resistance of bacteria isolated from patients with bloodstream infections at a tertiary care hospital in the Democratic Republic of the Congo[END_REF][START_REF] Boeck | ESBL-Positive Enterobacteria Isolates in Drinking Water[END_REF][START_REF] Boeck | Presence of extended-spectrum beta-lactamase-producing Enterobacteriaceae in waste waters, Kinshasa, the Democratic Republic of the Congo[END_REF].

Others Gram-negative bacteria (including non-fermenting rods)

The available data do not allow the resistance profile of these other bacteria to be established, given the small number of isolates tested (Table 2). Those microorganisms are Citrobacter freundii, Enterobacter spp., and Proteus spp. Enterobacter cloacae is an opportunistic pathogen that can cause several types of infections in the lower respiratory tract, surgical sites, urinary tract and central nervous system. Moreover, this species is frequently associated with nosocomial infections during outbreaks, emphasizing the need for rapid detection and typing of such pathogens. C. freundii is considered a commensal of the intestinal tract of humans and other animals. However, this species can also cause diarrhea and other infections in humans [START_REF] Yang | Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China[END_REF][START_REF] Liu | The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells[END_REF]. De Boeck et al. in Kinshasa reported the presence of blaCTX-M-15, blaSHV-18 and blaSHV-2-like genes in C. freundii and E. cloacae strains, demonstrating that the two ESBL genes are widely disseminated in Enterobacteriaceae [START_REF] Boeck | ESBL-Positive Enterobacteria Isolates in Drinking Water[END_REF][START_REF] Boeck | Presence of extended-spectrum beta-lactamase-producing Enterobacteriaceae in waste waters, Kinshasa, the Democratic Republic of the Congo[END_REF]. In a recent study conducted by Leonid et al., out of the 78 analysed strains of Vibrio cholerae, all were resistant to cotrimoxazole and nalidixic acid, 12% (9/78) had reduced susceptibility to ciprofloxacin and 14% (11/78) were resistant to ampicillin. They were still sensitive to chloramphenicol and tetracyclines. Many AMR genes were detected (Tab. 1) [START_REF] Irenge | Genomic analysis of pathogenic isolates of Vibrio cholerae from eastern Democratic Republic of the Congo (2014-2017) bioRxiv[END_REF].

Finally, there are no data available on the occurrence and resistance profile of non-fermenting Gram-negative bacilli in RDC.

Resistance pattern among Gram-positive bacteria

Staphylococcus aureus

S. aureus is one of major human pathogens, associated with wide spectrum of localized or systemic infections including wound infections, osteomyelitis, food poisoning, endocarditis, as well as more serious illnesses, such as pneumonia and bacteremia [START_REF] Ilczyszyn | Clonal Structure and Characterization of Staphylococcus aureus Strains from Invasive Infections in Paediatric Patients from South Poland: Association between Age, spa Types, Clonal Complexes, and Genetic Markers[END_REF][START_REF] Asadollahi | Distribution of the Most Prevalent Spa Types among Clinical Isolates of Methicillin-Resistant and -Susceptible Staphylococcus aureus around the World: A Review[END_REF]. In RDC, the prevalence of bacterial infections caused by methicillin-resistant S. aureus (MRSA) varied from 16 to 64% [START_REF] Phaku | Unveiling the molecular basis of antimicrobial resistance in Staphylococcus aureus from the Democratic Republic of the Congo using whole genome sequencing[END_REF][START_REF] Vandendriessche | Characterisation of Staphylococcus aureus isolates from bloodstream infections, Democratic Republic of the Congo[END_REF][START_REF] Boeck | Staphylococcus aureus nasal carriage among healthcare workers in Kisangani, the Democratic Republic of the Congo[END_REF][START_REF] Liesse | High prevalence of methicillin resistant staphylococci strains isolated from surgical site infections in Kinshasa[END_REF][START_REF] Lupande | Determinants of nasal carriage of MRSA in Bukavu city[END_REF], which is similar to the prevalence reported in other African countries [START_REF] Schaumburg | New epidemiology of Staphylococcus aureus infection in Africa[END_REF]. The reported prevalences of antibiotics resistance are as follows: 33 to 72 % against to tetracyclines, 5 to 54 % against cotrimoxazole, 31 % against gentamicin, 26 to 69% against erythromycin and 20 to 59% against ciprofloxacin [START_REF] Phaku | Unveiling the molecular basis of antimicrobial resistance in Staphylococcus aureus from the Democratic Republic of the Congo using whole genome sequencing[END_REF][START_REF] Vandendriessche | Characterisation of Staphylococcus aureus isolates from bloodstream infections, Democratic Republic of the Congo[END_REF][START_REF] Liesse | High prevalence of methicillin resistant staphylococci strains isolated from surgical site infections in Kinshasa[END_REF].

Streptococcus pneumoniae

S. pneumoniae is the leading cause of pneumonia and bacterial meningitis. It is commonly encountered in children and in immunocompromised and elderly populations and it has considerable implications for healthcare systems worldwide [START_REF] Hirvonen | Use of an automated PCR assay, the GenomEra S. pneumoniae, for rapid detection of Streptococcus pneumoniae in blood cultures[END_REF]. To date, multidrugresistant S. pneumoniae (MDR-SP) have been isolated from both adults and children around the world. These isolates are resistant to penicillin, clindamycin, cotrimoxazole and erythromycin [START_REF] Thummeepak | High prevalence of multi-drug resistant Streptococcus pneumoniae among healthy children in Thailand[END_REF]. In DRC, a study performed on 23 S. pneumoniae isolates, showed that the prevalence of penicillin resistance was 34%, 21% to cotrimoxazole and 13% to cefuroxime (Table 2) [START_REF] Kacou-Ndouba | Results from the Survey of Antibiotic Resistance (SOAR) 2011-14 in the Democratic Republic of Congo, Ivory Coast, Republic of Senegal and Kenya[END_REF]. Serotyping had not been completed, yet it would give us valuable information on circulating strains depending on the vaccine used. A recent study of 163 nasal carriage isolates of S. pneumoniae in children under 60 months of age reported 62% (n=101) of resistance to penicillin G, 42% (n=68) to ampicillin, and 37% (n=61) to ceftriaxone. Almost all strains were resistant to cotrimoxazole (94%) and 43% (n=70) were resistant to more than three classes of antibiotics [72].

Perspectives

Surrounded by nine neighboring countries, the DRC faces many security and health challenges, including population movement, high mortality rates and recurrent endemic diseases, which make it impossible for the health system to make antibiotic resistance a priority. With an annual budget of about US$6 billion, 1.2% of which is allocated to the health sector, for a population estimated at 85 million [START_REF] Innocent | The Access to health care in the Democratic Republic of Congo: Major challenge for the poor[END_REF]. Despite the existence of 39 officially recognized medical schools, most of which are located in urban areas, the training of clinical microbiologists remains a real problem because entirely self-financed, so that the number of microbiologists is very low in relation to actual needs [START_REF] Stasse | Improving financial access to health care in the Kisantu district in the Democratic Republic of Congo: acting upon complexity[END_REF]. The DRC is almost last with its human development index (168/169), public spending on health is among the lowest with two dollars per capita. In this context of advanced poverty, investment in laboratory materials and other diagnostic equipments for infectious diseases is therefore impossible. The WHO recommends a minimum of $35 per capita to safeguard a country's population [START_REF] Rajan | Health service planning contributes to policy dialogue around strengthening district health systems: an example from DR Congo 2008-2013[END_REF]. Out of the 424 hospitals in the country, very few have organized a microbiology service due to lack of electricity, infrastructure and especially qualified personnel, as the only university in the country that trains them markets 2 to 4 clinical microbiologists per year [START_REF] Ntembwa | The. Improving health systems efficiency: Democratic republic of Congo; Improving aid coordination in the health sector (world health report)[END_REF]. The serious threat of antibiotic resistance in the DRC is the ineffectiveness of the antibacterial molecules available on the market, which means that the use of old molecules which prove to be more active with multidrug resistant isolates elsewhere should quickly orient the regulatory authorities to change procurement policies.

In light of these challenges, there is an urgent need to create collaborative networks with all microbiology laboratories across the country, which will then be linked to external laboratories to effectively address the AMR problem.

Conclusion

This review summarizes the current state of our knowledge regarding antimicrobial susceptibility of the common organisms causing serious community and hospital-acquired infections in DRC. Many studies focused on Salmonella spp., S. aureus and V. cholerae.

Bacterial resistance to antibiotics concerns both

  Gram negative and Gram-positive bacteria. Multidrug resistance prevalence is almost common in half of S. pneumoniae isolates. More disturbing, we can note a worrying prevalence of MRSA with co-resistance to several other antibiotics, and of ESBL-producers in Enterobacteriaceae with the diffusion of blaCTX-M-15 and blaSHV-18 genes. Data on non-fermenting Gram negative bacilli, as well as carbapenem and colistin resistance, are not available.

Figure 2 :

 2 Figure 2: Localization of published data on antimicrobial resistance in Democratic Republic of the Congo.

Table 1 :

 1 Main antimicrobial resistance genes found in Democratic Republic of Congo 37.2%, respectively. MDR were 90.2% and 79.7%, respectively for S. typhimurium (386) and S. enteritidis (390). ESBL production was observed in 12.7% of S. typhimurium isolates Ib-cr, blaTEM-1 Salmonella Typhi (2015) Production of ESBL e was confirmed by PCR, the first time in DR Congo on S. Typhi. The isolate further showed decreased ciprofloxacin to pefloxacin and nalidixic acid with resistance

	539
	540
	Species	Methods	N° of strains	Antibiotic resistance genes	References
				blaCTX-M-15, blaSHV-2a, mph(A), gyrA mutation, qnrB1,
	Salmonella spp.	PCR, gene sequencing, whole genome sequencing	453	aac(6′)-Ib-cr, blaTEM-1, blaTEM-1b, catA1, strA/B, sul1, dfrA1, aac(6')-1aa	[27,37, 38, 40, 43,45,47,77]
				blaCTX-M-1, blaTEM, blaCMY, blaOXA, aac3, ant2'',
	E. coli	PCR, Whole genome sequencing	21	ant3'', aph3'', aph6, pbp2, aac6', qnrB, mphA, cat, catA, catB, arr, folp, sulI, sulII, tetA, tetB, tetD, dhfr	[53,56]
	Klebsiella spp.	PCR	3	blaCTX-M-15, blaSHV variants	[59]
	S. aureus	PCR, Whole genome sequencing	148	aac(6')-aph(2''), tetK, emrC, mecA gene found, mutation in femA gene found, dfrG, rpoB, emrC	[28,44]
				cardB, intSXT, sxt/R391, sulII, sulI, qnrVC, aph3-
	Vibrio cholerae	Whole genome sequencing	78	dprime, aph6, drfA, dhfr, flor, gyrA (S83I), parC	[46]
				(S85L)

and

Ethical issues

There are no ethical concerns with this paper and ethical review board approval was not required as no human subjects were involved.

Financial support

This work was supported by the French Government under the « Investissements

Transparency declaration:

The authors declare no conflict of interest. Although sensitivity to fluoroquinolones seemed to be preserved, strain resistance patterns continued to evolve with MLVA genotyping, no resistance genes searched [START_REF] Miwanda | Antimicrobial Drug Resistance of Vibrio cholerae, Democratic Republic of the Congo[END_REF]