This was an analytical, descriptive, cross-sectional, and etiological diagnostic study conducted on resistant, multidrug-resistant, and extensively resistant bacteria from 07/01/2012 to 17/12/2025, which was conducted and executed at or at:

- Bacteriology Unit and Laboratory Mycobacteria Unit of the National Reference Hospital Center (CHURN) of N’Djamena;

- Bacteriology Unit of the Research, Diagnostic, and Scientific Expertise Laboratory (Labo-ReDES) of the Faculty of Human Health Sciences (FSSH) of the University of N’Djamena;

- Pasteur Institute of Paris and the laboratory of the University Hospital of Montpellier (France);

- University Center for Clinical Research (CURC), University of Sciences, Techniques, and Technologies of Bamako (USTTB), Bamako, Mali.

Inclusion Criteria

Any consenting individual in community or hospital settings who presents with symptoms of infectious diseases (acute carriers) or is asymptomatic (chronic carriers) and has benefited from diagnostic tests related to the objective of the study.

Exclusion Criteria

The following items are not included in this study:

- Any individual who has not undergone diagnostic testing for the main biological markers used to assess infectious disease control;

- Patients/individuals who do not consent to participate in this study.

Convenience sampling was used in relation to the duration of the study.

Samples of pathological biological products were collected from patients in community and hospital settings in different localities of Chad throughout the study period.

Antibiotics were chosen based on their prescription for the treatment of various infectious diseases (Table 1) [14]. Antituberculosis drugs (1st line: rifampicin, isoniazid; and second line: levofloxacin, amikacin, kanamycin, and capreomycin) and antileprosy drugs (1st line: dapsone, rifampicin, and clofazimine; and second line: ofloxacin, minocycline, and clarithromycin) were administered according to the management guidelines recommended to national tuberculosis and leprosy control programs.

Table 1.Antibiotics chosen for the susceptibility test.

Quality control was performed using the reference strain E. coli ATCC 25922. Legend: Nalidixic acid (NAL); trimethoprim-sulfamethoxazole (SXT). Susceptible = Treatment success; Resistant = Treatment failure; Intermediate = Uncertain.

The isolation of bacteria was carried out in different agar media: Hektoen (all enterobacteria), Chapman (staphylococci), chocolate agar with cooked blood or fresh blood (streptococci), TCBS agar (vibrios), Sabouraud chloramphenicol agar (molds and yeasts), etc.

The galleries: API20E, STREP, STAPH, etc., were used for the biochemical identification of the isolated bacteria. The MYCOPLASMA IST 3 gallery (BioMérieux, Marcy l’Etoile, France) enabled the biochemical identification and antibiogram testing of Mycoplasma hominis and Ureaplasma spp. Six (6) antibiotics are incorporated into the gallery; five (levofloxacin, moxifloxacin, tetracycline, erythromycin, and telithromycin) were tested against Ureaplasma, and four (clindamycin, levofloxacin, moxifloxacin, and tetracycline) were tested against Mycoplasma hominis.

The sensitivity test method used is the diffusion method in MH agar (disc method or Kirby-Bauer technique) following the recommendations of the Antibiogram Committee of the French Society of Microbiology and the European Committee on Antimicrobial Susceptibility Testing, the Clinical Laboratory Standards Institute (CLSI), and the Food and Drug Administration (FDA) of the United States of America [15][16].

ESBL production was detected on Muller-Hinton agar using the double-disc synergy assay. Discs of cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), and aztreonam (30 µg) were placed 30 mm (center to center) from an amoxicillin/clavulanic acid (20/10 µg) disc and then incubated at 35˚C - 37˚C (Figure 1). After 18 - 24 hours of incubation, ESBL production by the test organism was determined based on the partial inhibition of ESBL by clavulanic acid. The existence of even a weak synergy between cefotaxime, ceftazidime, cefepime, aztreonam, and clavulanic acid is characterized by a champagne cork-shaped image [17].

The Vitek Compact 60 was also used to detect resistance phenotypes in isolated bacteria. The system includes the Vitek^® 2 Compact instrument, a computer (workstation), and a printer. The software provided with the Vitek^® 2 Compact system includes data analysis and management programs. A bidirectional computer interface automatically transfers results to the user’s Laboratory Information System (LIS) and to various product and patient reports. A quality control system is available to validate a Vitek^® 2 Compact system test kit. An Advanced Expert System^TM (AES) (for clinical use) is available to allow systematic online validation of results and interpretation of resistance phenotypes identified by antibiograms. The Vitek 2 range allows for same-day access to the information needed to initiate targeted antibiotic therapy, thus ensuring better patient care and cost savings in healthcare. Vitek has also been used to identify the genes (mecA, ACC (6') and vanA).

Figure 1. Evolution of multidrug-resistant and extensively drug-resistant bacteria associated with healthcare in Chad from 2012-2025.

Molecular characterization of the isolated V. cholerae O 1 strains was performed at the National Reference Center for Vibrio and Cholera (CNRVC) of the Pasteur Institute in Paris, France (IPP/F). The procedures were carried out according to the CNRVC’s instructions at the IPP/F. PCR testing for pathogenicity factors was systematically applied to all V. choleraeO 1 strains sent to the CNRVC at the IPP/F. PCR was also used to confirm the V. choleraeO 1 strains from Chad. After detection of the PCR product on a 2% agarose gel, the electrophoretic migration program was as follows: initial pulse time 60 s, final pulse time 120 s, migration time 72 hours at 6 V/cm², angle 120˚, temperature 14˚C. At the end of the migration, the gel was stained with ethidium bromide (ET, 1.5%) for 30 min. Visualization was performed under a UV lamp with an imager, incorporating IPP/F software.

Rapid immunochromatographic tests were used to detect hepatitis A, B, C, and E viruses, as well as dengue viruses and non-culturable bacteria. The tests were performed according to the manufacturer’s instructions.

The CDC MAC-ELISA 72 test at the Laboratory of the National Reference University Hospital Center (LCHU-RN) in N’Djamena was used for the detection of the yellow fever virus. Samples collected from the various provinces were received, sorted, prepared (aliquoted), and stored in a cold room (−80˚C) at the Virology Unit of the National Reference University Hospital Center in N’Djamena for analysis.

Thick blood film and thin blood film, as well as rapid detection tests, were used to detect Plasmodium falciparum.

The following molecular techniques have been used to detect resistance genes in order to map them [18]:

1)TuberculosisDetection Usingthe TB-LAMPAutomated System

TB-LAMP is a molecular diagnostic technique that has enabled sensitive and accurate detection of tuberculosis. It is easy to use and provides reliable results within one hour. The process consists of four steps:

- Sputum preparation: 60 µL of sputum is transferred to the heating tube, stirred to mix well, and incubated in the HumaLoop T heating unit at 90˚C for 5 minutes;

- DNA extraction: The pretreated (lysed) sample in the heating tube was transferred to the absorbent tube containing absorbent powder, which eliminates any potential inhibitors of the LAMP reaction. The purified DNA was extracted and directly transferred to the LAMP reaction tube.

- Amplification: The DNA transferred to the LAMP reaction tube was incubated for 2 minutes at room temperature to reconstitute the reagents in the cap. It was then homogenized several times and tapped until the reaction mixture settled at the bottom of the tube. The reaction tube was then transferred to the HumaLoop T reaction unit at 67˚C for 45 minutes.

Reading the results

The reaction tube was finally inserted into the UV lamp detection unit, and the lamp was switched on.

- A positive result produces a green light;

- A negative result produces no fluorescence.

Real-time turbidity readings and automatic reporting of results.

2)Detection of rifampicin sensitivity and resistance using the GeneXpert analyzer

Principle: It is based on amplification of a fragment of the rpoB gene containing the central region of 81 base pairs and fragments of the target sequences of the IS1081 and IS6110 insertion elements with multiple copies by primers.

Using a Pasteur pipette, 2 mL of sputum and 4 mL of the reagent were collected and mixed in another sterile jar. The mixture was vortexed and incubated at room temperature for 10 minutes. The jar was vortexed again and incubated at room temperature for 5 minutes. Then, 2 mL of the liquefied mixture was aspirated and transferred to the Xpert^® MTB/RIF ULTRA cartridge, and the test was launched for a duration of 1 hour and 30 minutes.

3)Detection of rifampicin and isoniazid resistance genes by the automated Line Probe Assay (Hain)

Principle: It is based on DNA-STRIP technology and includes DNA extraction, master mix preparation, multiplex amplification with biotinylated primers, and detection by reverse hybridization.

Sputum samples were decontaminated using the BD BBL^TM MycoPrep kit containing N-Acetyl L-Cysteine-sodium hydroxide and phosphate buffer. DNA was extracted from the samples using the GenoLyse kit (Hain Life Science). 500 µL of the decontaminated sample was taken and transferred to Eppendorf tubes and centrifuged at 10,000 rpm for 15 minutes. After centrifugation, only the pellet was kept, to which 100 µL of lysis buffer solution (A-LYS) was added, mixed by vortexing, and incubated for 5 minutes at 95˚C in a water bath. Afterwards, we added 100 µL of neutralization buffer (A-NB), then vortexed the mixture for 5 seconds and centrifuged quickly for 5 minutes in order to bring the DNA from the pellet to the supernatant. Our interest was focused on the supernatant. The extracted DNA was then amplified by PCR and to do this, we prepared 45 µL of mixture (10 µL of MixA + 35 µL of MixB) for each sample, then introduced 45 µL of mixture (MixA + MixB) into each tube. Then, 5 µL of the DNA from each sample was added. After amplification, the PCR products (amplicons) were hybridized with the probes fixed on the strip. To do this, 20 µL of denaturation solution (DEN, blue) was distributed in the corner of each of the wells used, to which 20 µL of amplified DNA was added. The mixture was then homogenized and incubated at room temperature for 5 minutes. 1 mL of preheated hybridization buffer was added to each well and then the tray was gently shaken until the solution had a homogeneous color. Subsequently, a strip was placed in each well. We placed the tray in the shaking water bath/TwinCubator and incubated it for 30 minutes at 45˚C. We then aspirated the hybridization buffer completely and added 1 mL of stringent wash solution to each strip, incubating for 15 minutes at 45˚C in a shaking water bath/TwinCubator. The stringent wash solution was then completely vacuumed up. Each strip was washed once with 1 mL of rinse solution for 1 minute on the shaking platform/TwinCubator (pour out the RIN after incubation), then 1 mL of diluted conjugate was added to each strip and incubated for 30 minutes on the shaking platform/TwinCubator. The solution was removed, then each strip was washed twice for 1 minute with 1 mL of rinsing solution and once for 1 minute with 1 mL of distilled water on a shaking platform/TwinCubator (pour the solution off each time), then 1 mL of diluted substrate was added to each well, incubated away from light for 5 minutes, rinsed briefly twice with distilled water, and the strips were dried.

Line Probe Assay (LPA) or reverse hybridization strip tests are DNA strip tests which make it possible to determine the drug resistance profile of a strain of the Mycobacterium tuberculosis Complex through the pattern of amplicon binding (amplification products DNA) to probes targeting the mutations most often associated with resistance of the Mycobacterium tuberculosisComplex to first- and second-line anti-tuberculosis drugs, and to probes targeting the corresponding wild-type DNA sequence.

4)Detection of mycobacteria by thespoligotypingtechnique[19]

Culture and spoligotyping were performed at the University Clinical Research Center (CURC), University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako, Mali. All culture-positive samples were used for spoligotyping. Spoligotyping was performed on boiled bacterial lysates using a commercially available kit (Ocimum) or an internally prepared membrane. In short, this technique amplifies the polymorphic region called DR (Direct Repeat) using two external primers (DRa and DRb). We performed real-time PCR for amplification. Hybridization was then carried out on an Isogen membrane. Development was performed using a streptavidin conjugate and the ECL (Enhanced Chemi-Luminescence) detection kit. A film strip was used on the Isogen membrane in a dark chamber for development and detection. The results were recorded in octal code on the film strip, and strain comparisons were performed with SPOTCLUST (based on SpolDB3) and SITVIT2 (available at http://www.pasteur-guadeloupe.fr:8081/SITVIT2). This database is currently a major genotypic marker database with over 111,635 genotypes isolated from 163 countries. Approximately 174 isolates were used for spoligotyping, the results of which are also recorded in octal code.

DNA amplification by PCR targeting RLEP repeat sequences was performed as described by biologists at the Arnaud de Villeneuve University Hospital in Montpellier, France. After detection of the PCR product on a 2% agarose gel, the amplicons were processed according to the Sanger sequencing tube protocol [20].

Results, including control data for key biological markers and data from information forms, were entered into a spreadsheet using MS-Excel. Descriptive analysis consisted of presenting the collected data as counts, percentages, means, and graphics. GPS Maps was used to map and locate the MDRs and resistance genes on a map of Chad.

Healthcare-associated infections were dominated by the bacterium Escherichia coli (90%), followed by infections from blood-sucking parasites (Plasmodium falciparum; 67%) and fungal agents such as Candida albicans (55%).

The investigation of microorganisms associated with female and male infertility revealed 30.37% Chlamydia trachomatis detected by immunochromatographic tests, 18 (13.33%) Mycoplasma hominis, and 11 (8.14%) Ureaplasma spp. identified by biochemical tests (API-MYCOPLASMA IST 3 with antibiogram test) (Figure 2).

Figure 2. Frequency of healthcare-associated microorganisms in Chad: 2012-2025.

The multi-resistant bacteria detected were distributed according to the biochemical mechanisms of resistance, either to beta-lactams (β-lactams) by production of extended-spectrum beta-lactamases (ESBL), by production of acquired penicillinases, or by modification of penicillin-binding proteins (PBPs) induced by resistance genes. During 24 years (2012-2025) of study, 851 resistant bacterial phenotypes were identified, including 329 MDR (bacterial isolates that remain resistant to at least three or more classes of antibiotics), 407 ESBL (Extended Spectrum Beta-lactamases), 114 XDR (bacterial isolates that remain sensitive to antibiotics in only one or two classes), and 10 PDR (bacterial isolates that remain resistant to all classes of antibiotics tested locally). Among the 10 PDRs, there were 3 Pseudomonas aeruginosa, 3 Escherichia coli, 2 Proteus spp., and 2 Klebsiellapneumoniae. The main multidrug-resistant and extensively drug-resistant bacteria identified were named with their bacterial name and the name of the antibiotic to which they were resistant: methicillin-resistant Staphylococcus aureus (MRSA), VRE (Vancomycin-resistant Enterococcus), and Extended Spectrum Beta-Lactamases (ESBL) producing Enterobacteriaceae. The most frequently reported resistant bacteria (MRB) were Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae, followed by Salmonella spp. Among the identified MDR bacteria, 48 (11%) of the Mycobacterium leprae strains were detected positive by PCR-RLEP, the sequencing of which revealed two mutant strains resistant to rifampicin and ofloxacin, with a mutation rate of 1.2%. Molecular techniques (GeneXpert, TBLAMP, Line Probe Assay) detected 66 (15%) Mycobacterium tuberculosis-positive (MTB+) strains that were resistant to first-line antituberculosis drugs. Of these 66 MTB+ strains, 34 (13%) were resistant to rifampicin, 10 (4%) were resistant to isoniazid, and 22 (8%) were multidrug-resistant.

Conventional culture allowed the isolation of 329 bacteria commonly responsible for infections in hospital and community settings, the results of which revealed the following:

- From 1971 to 2025, Chad experienced 45 cholera epidemics, with 86,961 cases and 6567 deaths (7.55%). The spread of the disease was linked to sociocultural practices in Chad and neighboring countries, particularly those related to water systems. The first Vibrio cholerae O1 serotype Ogawa was isolated in 1971, and the last isolates, found during the 2025 epidemic in refugee camps in the east, were all susceptible to doxycycline. The susceptibility profiles of the strains (Vibrio cholerae O1, serotype Ogawa, biotype El Tor, and classical) to other classes of antibiotics varied. Resistance rates of 100% were observed with trimethoprim-sulfamethoxazole, nalidixic acid, and the vibriostatic compound O/129. The second most active antibiotics were tetracycline (99%), norfloxacin (78.14%), and ciprofloxacin (76%);

- Resistance frequencies were also noted in 120 isolated strains of Staphylococcus aureus. This study showed that the Staphylococcus strains were 100% resistant to methicillin, followed by 82% to vancomycin, 46% to erythromycin, 43% to gentamicin, and 42% to ciprofloxacin. Six (6) strains of Staphylococcus aureus were resistant to antibiotics from the following families: ampicillin/sulbactam (MIC ≥ 8), linezolid (MIC ≥ 8), and trimethoprim-sulfamethoxazole (MIC ≥ 320), respectively. The acquired penicillin resistance of these isolated strains was cross-resistant with the majority of beta-lactams, but at varying levels depending on the antibiotics used, allowing the use of the most active molecules. Another example of observed cross-resistance was methicillin resistance in Staphylococcus aureus strains (68.33%) due to a modification of a PBP that also confers resistance to other molecules in the beta-lactam family;

- Two strains of Klebsiella pneumoniae detected produced associated β-lactamases, which hydrolyzed carbapenems moderately and other β-lactams weakly by the biochemical mechanism allowing the reduction of the permeability of the outer membrane;

- Three strains of Salmonella identified were resistant to beta-lactams by production of ESBLs and to nalidixic acid (quinolones);

- Among the strains of bacteria producing extended-spectrum cephalosporinases (ESBLs) that inactivate 3rd generation cephalosporins (ceftriaxone, cefotaxime, ceftazidime, cefixime), 8 strains of Escherichia coli produced AmpC (cyclic adenosine monophosphate) type cephalosporinases, which is a resistance mechanism by enzymatic inactivation that increases the MIC of beta-lactam antibiotics: ceftriaxone (MIC ≥ 32), cefixime (MIC ≥ 64), cefotaxime (MIC ≥ 64), ceftazidime (MIC ≥ 6). Most of the enterococcal strains isolated in this study exhibited various multi-resistance phenotypes to antibiotics from different families;

- Twenty-one (21) BMRs have developed multi-resistance associated with the families (beta-lactams, glycopeptides, macrolides/lincosamides/ST/reptogramins), by modification of PBP induced by the mecA gene, or by inducible MLSb;

- Fourteen MDRs were resistant to the families (beta-lactams, aminoglycosides) either by production of ESBLs or by acquisition of plasmid resistance induced by the asparagine protein due to the presence of the AAC(6') nucleotide;

- Four strains of Neisseria meningitidis with reduced susceptibility to penicillins and two strains resistant to third-generation cephalosporins were detected in Goundi.

Ureaplasmaspp. developed an average resistance of 86.52% to fluoroquinolones, 32% to macrolides, and 36.36% to tetracycline. Mycoplasmahomonis developed an average resistance of 36.16% to fluoroquinolones, 39% to tetracycline, and 28% to lincosamides.

The main risk factors identified in the transmission of these multidrug-resistant bacteria in hospital settings were urinary tract infections and the presence of a urinary catheter in patients hospitalized in wards.

Finally, the study revealed the presence of 72 fungal agents (51 Candida albicans, 3 Cryptococcuslaurentii, 1 Candidakrusei, 8 Candidalipolytica, 4 Aspergillusniger, 5 Aspergillus fumigatus) resistant to varying degrees to antifungals (azoles (5-Fluorocytosine, Econazole, Clotrimazole, Fluconazole, Voriconazole) and polyenes (Amphotericin B, Nystatin)). The fungal strains were sensitive (83.63%) to azole derivatives and resistant (96%) to polyenes. Azole derivatives were more active than polyenes. The activity of fluconazole was limited.

Figure 3 illustrates the surveyed cities where resistance genes were distributed: Adré, Goz Beida, Farchana, Choukoyane, Abéché, Guera, Barh El Gazel, Hadjarlamis, Bol, N’Djamena, Bousso, Bongor, Kélo, Pala, Léré, Moundou, Bebedjia, Doba, Goundi, Sahr, and Goré.

Chad is located between 7˚ and 24˚ North latitude and 13˚ and 24˚ East longitude. It covers an area of 1,284,000 km² and is the fifth largest country in Africa after Sudan, Algeria, Zaire, and Libya. It stretches 1700 km from north to south and 1000 km from east to west. It shares borders with Libya to the north, Sudan to the east, the Central African Republic to the south, and Cameroon, Nigeria, and Niger to the west. The city of Abéché, located 900 km north of N’Djamena, shares borders with Libya to the north and Sudan to the east, while Goré, 600 km south of Chad, borders the Central African Republic and Cameroon to the west. Politically and economically, the country belongs to Central Africa, but due to similar climatic conditions, it is also considered part of the Sahel region. Chad’s geographical location has likely contributed to the distribution of multidrug-resistant (MDR) organisms, highly resistant (XDR), pan-resistant (PDR) bacteria, and resistance genes. Among the biopsies performed for Mycobacterium leprae testing, 60% were positive for PCR-RLEP. Sequencing revealed two mutant strains of Mycobacterium leprae associated with resistance to rifampicin and ofloxacin. A missense mutation at codon 456rpoB (Ser (TCG > Leu (TTG))) was observed in a secondary case of rifampicin resistance in a 46-year-old relapsing patient from N’Djamena. Additionally, a missense mutation at codon gyrA 91 (Ala (GCA > Val (GTA))) associated with ofloxacin resistance was found in a 37-year-old patient from Abéché who had not been sensitized to ofloxacin but had been exposed to other quinolones. No mutations were observed in the flop1 gene.

Of the positive cases of Mycobacterium tuberculosis (MTB+) detected by molecular techniques (TBLAMP and GeneXpert), 25% were resistant to first-line anti-tuberculosis drugs. Among the 25% of resistant MTB+ cases, the automated Line Probe Assay (Hain) detected 13% rifampicin-resistant genes (rpoB), 4% isoniazid-resistant genes (inhA), and 8% multidrug-resistant genes (mdr) (rpoB/KatG and/or KatG/inhA). Among the genes detected with antibiotic-associated mutations (Levofloxacin (Lfx), Amikacin (Am), Kanamycin (Km), and Capreomycin (Cm)) by the Line Probe Assay technique, 13.6% (gyrA, gyrB) were due to gyrase enzymes, 7.6% (eis), 6.1% (rrs and eis), and 10.6% were mutations affecting different alleles on the chromosomes of the Mycobacterium tuberculosis complex. The mechanism of resistance to Levofloxacin was manifested by point mutations occurring in specific regions of two enzymes: DNA gyrase A and DNA gyrase B.

Spoligotyping of Mycobacterium tuberculosis isolates resulted in 5 groups, ranging from 4 to 9 isolates per group. Of the twelve octal codes identified by the spoligotyping technique, three lineages exhibited the same genotypic characteristics as the major genotypic lineages (Euro-American, Indo-Oceanic, East-African-Indian).

The strains of Vibrio cholerae O 1 isolated during the successive epidemics of 2011-2012 in Chad were confirmed in 71/72 cases (98.61%) by molecular methods at the Pasteur Institute in Paris (France) as carrying the virulence genes encoding the synthesis of cholera toxin (ctxB and ctxA).

Eight Staphylococcus aureus strains (6.7%) producing acquired penicillinase through modification of PBP (penicillin-binding protein) induced by the mecA gene were also detected. Six (6) Staphylococcus aureus strains resistant to beta-lactams through penicillinase production and to the oxazolidone and sulfonamide families through acquisition of plasmids induced by the ACC (6') gene were identified. Seventeen (17) Staphylococcushaemolyticus strains were identified, of which 12/120 (10%) produced second-generation cephalosporinases through modification of PBP induced by the mecA gene.

Ten strains of enterococci (Enterococcus (9.1%), Enterococcus faecalis (67%), and Enterococcus faecium (33%)) isolated in this study exhibited multi-resistance phenotypes to antibiotics from different families by induction of the vanA gene (Figure 3).

Figure 3.distribution of resistance genes in the study sites.