Unveiling the emergence of SARS-CoV-2 JN.1 sub-variant: Insights from the first cases at Charles Nicolle Hospital, Tunisia.

The JN.1 sub-variant is a new variant of the SARS-CoV-2 Omicron strain, derived from the BA.2.86 sub-variant. It was first detected in late 2023 and has quickly spread to many countries, becoming the most prevalent variant in some regions. JN.1 exhibits a unique mutation (L455S) in the spike protein compared to the BA.2.86 lineage, which may affect its transmissibility and immune evasion capabilities. JN.1 has been designated as a "variant of interest" by the World Health Organization due to its rapidly increasing spread and is being closely monitored for its impact on the COVID-19 pandemic. This study describes the emergence of SARS-CoV-2 JN.1 sub-variant in Tunisia, and reports its mutation profiles.Nasopharyngeal samples collected over a four-month period (October 2023 to January 2024) were subjected to RNA extraction and real-time RT-PCR confirmation of SARS-CoV-2 infection. The whole-genome sequencing was performed by an iSeq 100 sequencer and COVIDSeq kit reagents (Illumina, USA).Mutation analysis, using the NextClade platform and GISAID database, revealed the presence of JN.1 in 15 out of 80 positive cases (18.75%) during the study period.The emergence of JN.1 highlights the ongoing evolution of SARS-CoV-2 and the need for continued surveillance and research to better understand the characteristics and impact of emerging variants.

Genomic surveillance of SARS-CoV-2 in North Africa: 4 years of GISAID data sharing.

Objectives: This study aimed to construct geographically, temporally, and epidemiologically representative data sets for SARS-CoV-2 in North Africa, focusing on Variants of Concern (VOCs), Variants of Interest (VOIs), and Variants Under Monitoring (VUMs). Methods: SARS-CoV-2 genomic sequences and metadata from the EpiCoV database via the Global Initiative on Sharing All Influenza Data platform were analyzed. Data analysis included cases, deaths, demographics, patient status, sequencing technologies, and variant analysis. Results: A comprehensive analysis of 10,783 viral genomic sequences from six North African countries revealed notable insights. SARS-CoV-2 sampling methods lack standardization, with a majority of countries lacking clear strategies. Over 59% of analyzed genomes lack essential clinical and demographic metadata, including patient age, sex, underlying health conditions, and clinical outcomes, which are essential for comprehensive genomic analysis and epidemiological studies, as submitted to the Global Initiative on Sharing All Influenza Data. Morocco reported the highest number of confirmed COVID-19 cases (1,272,490), whereas Tunisia leads in reported deaths (29,341), emphasizing regional variations in the pandemic's impact. The GRA clade emerged as predominant in North African countries. The lineage analysis showcased a diversity of 190 lineages in Egypt, 26 in Libya, 121 in Tunisia, 90 in Algeria, 146 in Morocco, and 10 in Mauritania. The temporal dynamics of SARS-CoV-2 variants revealed distinct waves driven by different variants. Conclusions: This study contributes valuable insights into the genomic landscape of SARS-CoV-2 in North Africa, highlighting the importance of genomic surveillance in understanding viral dynamics and informing public health strategies.

HBHA-IGRA and cytotoxic mediators release assays for the diagnosis of cervical tuberculous lymphadenitis.

IMPORTANCE: Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is currently a major health problem in Tunisia and in several regions around the world. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis. This study demonstrates the added value of the heparin-binding hemagglutinin-interferon-gamma release assay as an immunoassay in the context of CTL.

Tunisian Multicenter Study on the Prevalence of Colistin Resistance in Clinical Isolates of Gram Negative Bacilli: Emergence of Escherichia coli Harbouring the mcr-1 Gene.

BACKGROUND: Actually, no data on the prevalence of plasmid colistin resistance in Tunisia are available among clinical bacteria. OBJECTIVES: This study aimed to investigate the current epidemiology of colistin resistance and the spread of the mcr gene in clinical Gram-negative bacteria (GNB) isolated from six Tunisian university hospitals. METHODS: A total of 836 GNB strains were inoculated on COL-R agar plates with selective screening agar for the isolation of GNB resistant to colistin. For the selected isolates, mcr genes, beta-lactamases associated-resistance genes and molecular characterisation were screened by PCRs and sequencing. RESULTS: Colistin-resistance was detected in 5.02% (42/836) of the isolates and colistin-resistant isolates harboured an ESBL (blaCTX-M-15) and/or a carbapenemase (blaOXA-48, blaVIM) encoding gene in 45.2% of the cases. The mcr-1 gene was detected in four E. coli isolates (0.59%) causing urinary tract infections and all these isolates also contained the blaTEM-1 gene. The blaCTX-M-15 gene was detected in three isolates that also carried the IncY and IncFIB replicons. The genetic environment surrounding the mcr-carrying plasmid indicated the presence of pap-2 gene upstream mcr-1 resistance marker with unusual missing of ISApl1 insertion sequence. THE CONCLUSIONS: This study reports the first description of the mcr-1 gene among clinical E. coli isolates in Tunisia and provides an incentive to conduct routine colistin susceptibility testing in GNB clinical isolates.

Extensively drug-resistant Acinetobacter baumannii co-producing VIM-2 and OXA-23 in intensive care units: Results of a one-day point prevalence in a Tunisian hospital.

OBJECTIVE: The main objective of this study was to identify carbapenem resistance mechanisms among clinical, commensal and environmental carbapenem-resistant Acinetobacter baumannii (CRAB) strains isolated in 5 Tunisian intensive care units (ICUs). MATERIALS AND METHODS: CRAB isolates were recovered from different sources: clinical specimens, rectal and environmental swabs. Bacterial identification was carried out using conventional methods and susceptibility testing according to EUCAST recommendations. Evaluation of phenotypic carbapenemase production of was performed using seven different methods, and molecular detection of carbapenemase-coding genes (blaOXA23, blaOXA24, blaOXA58, blaNDM, blaGES, blaOXA48, blaIMP, blaVIM and blaKPC) was done by PCR. The genetic relationships between CRAB isolates were analyzed by pulsed-field gel electrophoresis. RESULTS: All in all, 46 CRAB isolates were identified in clinical specimens (n = 26/26), rectal swabs (n = 17/36) and environmental swabs (n = 3/63). Most of them (n = 41/46) were clonally related and found in the different ICUs. All of the CARB isolates harbored blaOXA-51-like and blaOXA-23 genes, while blaVIM-2 gene was detected in 42 isolates (91.3 %). Phenotypic carbapenemase activity was found in all 46 strains, using the CIM-Tris test with 100 % sensitivity for OXA-23 and VIM-carbapenemase detection, thereby justifying its routine use. CONCLUSION: The emergence and diffusion of clonal CRAB strains inducing high mortality rates in ICUs is a major public health concern. Enhanced infection control practices and mandatory staff education are needed to control the spread of these multidrug-resistant bacteria.

Evaluation of Three Carbapenemase-Phenotypic Detection Methods and Emergence of Diverse VIM and GES Variants among Pseudomonas aeruginosa Isolates in Tunisia.

BACKGROUND: Since 2012, few reports on the molecular epidemiology of Pseudomonas aeruginosa were reported in Tunisia. OBJECTIVES: This study aimed to evaluate carbapenem-resistance determinants and molecular epidemiology and to compare the carbapenemase-phenotypic detection methods of multidrug-resistant P. aeruginosa isolates. METHODS: During a period of four years (2014 to 2017), all imipenem-ceftazidime-resistant P. aeruginosa isolates were retrospectively selected at the microbial laboratory of Charles Nicolle hospital of Tunis. These isolates were examined by the modified Hodge test, modified carbapenem inactivation method (mCIM), and another mCIM, called CIMTris, and their performance was evaluated using PCR analysis as the gold standard. RESULTS: A total of 35 isolates were recovered among patients hospitalized in different units. All strains were colistin-susceptible.All carbapenem-resistant isolates showed a high-level resistance to carbapenems. CIMTris and mCIM showed 96.15% and 46.15% sensitivity and 44.44% and 100% specificity, respectively, for detecting carbapenemase production. CONCLUSIONS: CIMTris is a promising approach for detecting carbapenemase activity in P. aeruginosa and merits further testing. Moreover, this study described the first detection of GES-5- and GES-9-producing P. aeruginosa in Tunisia as well as the co-occurrence of the blaGES-5 and blaVIM-11 carbapenemase genes in one isolate. These findings are of great concern because the rapid dissemination of MDR strains represents a major therapeutic and epidemiological threat.

Performances of single tube nested polymerase chain reaction and GeneXpert ultra on Formalin fixed paraffin embedded tissues in the diagnosis of tuberculous spondylodiscitis.

INTRODUCTION: Tuberculous Spondylodiscitis is the most common form of musculoskeletal tuberculosis. Molecular techniques on fresh tissues are proved to improve the diagnosis of tuberculous spondylodiscitis and to allow a rapid diagnosis to initiate the treatment and prevent neurological complications. OBJECTIVES: The objective of the present study was to assess the diagnostic performances of single tube nested PCR and GeneXpert ultra in the diagnosis of tuberculous spondylodiscitis on formalin fixed paraffin embedded tissues. METHODS: This study included 63 tuberculous spondylodiscitis cases collected from June 2014 to January 2020 and corresponding to 27 definite tuberculous spondylodiscitis with positive microbiology, and 36 probable tuberculous spondylodiscitis, with histopathological, clinical and radiological findings consistent with tuberculous spondylodiscitis but with negative microbiology. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR and GeneXpert ultra were determined with reference to microbiology. RESULTS: Nested PCR was positive in 47 (75%) cases: 26/27 definite tuberculous spondylodiscitis and 21/36 probable tuberculous spondylodiscitis. GeneXpert ultra was positive in only 6 (10%) cases corresponding to definite tuberculous spondylodiscitis. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR on formalin fixed paraffin embedded tissues were 96%, 100%, 100%, 83% respectively. For GeneXpert ultra, these rates were 22%, 100%, 100% and 25% respectively. CONCLUSION: Nested PCR and GeneXpert ultra on formalin fixed paraffin embedded tissues are useful tools for the diagnosis of tuberculous spondylodiscitis, especially for cases where microbiological investigations were not carried out. Both techniques have excellent specificity but single tube nested PCR is more sensitive. Key Points • Molecular techniques are routinely performed on fresh tissues • GeneXpert and nested PCR on formalin fixed paraffin embedded tissues are reliable for the diagnosis of tuberculous spondylodiscitis • Nested PCR is more sensitive than Genexpert for diagnosing tuberculous spondylodiscitis.

Performance of GeneXpert ultra in the diagnosis of Tuberculous Cervical lymphadenitis in formalin fixed paraffin embedded tissues.

The diagnosis of Tuberculous Cervical lymphadenitis (TCL) is challenging. The present study aimed to assess the performance of GeneXpert ultra (GXu) in the diagnosis of TCL on Formalin Fixed, Paraffin Embedded Tissues (FFPET). This study included 35 TCL cases confirmed by positive microbiology and/or positive GXu on Fresh Tissues (FT). The diagnostic performance parameters of GXu on FFPET were determined with reference to microbiology (positive Ziehl Neelsen and/or positive culture) and with reference to positive microbiology and/or positive GXu on FT. The GXu on FFPET was positive in 26/35 (74%) cases. With reference to positive ZN and or culture, the sensitivity, specificity, positive predictive value, and negative predictive value of GXu on FFPET were 63%, 100%, 100% and 71% respectively. With reference to positive microbiology and/or positive GXu on FT, these rates were 74%, 100%, 100% and 40% respectively. GXu on FFPET is a reliable tool for the detection of Mycobacterium tuberculosis complex particularly for cases where microbiological investigations have not been performed.

Assessment of sample pooling for SARS-CoV-2 molecular testing for screening of asymptomatic persons in Tunisia.

The aim of this study is to test a pooling approach for the RT-PCR test to detect low viral loads of SARS-CoV-2. We found that a single positive specimen can still be detected in pools of up to 10. Each laboratory should conduct its own evaluation and validation of pooling protocols according to its specific context.

Correlation between superficial and intra-operative specimens in diabetic foot infections: results of a cross-sectional Tunisian study.

OBJECTIVE: To determine the correlation between superficial, and intra-operative specimens in diabetic foot infections (DFIs). METHODS: We conducted a cross-sectional study in patients with DFIs hospitalized in a Tunisian teaching hospital. Superficial specimens were collected for all patients, and intra-operative specimens were collected in operated patients. The specimens were processed using standard microbiology techniques. Antimicrobial susceptibility testing was carried out according to the protocol established by the European Committee on Anti-microbial Susceptibility Testing. Intra-operative and superficial specimens were considered correlated if they isolated the same microorganism(s), or if they were both negative. RESULTS: One hundred twelve patients, 81 males and 31 females, mean age 56 years, were included. Superficial samples were positive in 77% of cases, and isolated 126 microorganisms. Among the positive samples, 71% were monomicrobial. The most frequently isolated microorganisms were Enterobacteriaceae (53%), followed by streptococci (21%) and Staphylococcus aureus (17%). Nine microorganisms (7%) were multi-drug resistant. Intra-operative samples were positive in 93% of cases. Superficial specimens were correlated to intra-operative specimens in 67% of cases. Initial antibiotic therapy was appropriate in 70% of cases. The lower-extremity amputation and the mortality rates were 41% and 1%, respectively. CONCLUSION: In our study, DFIs were most frequently caused by Enterobacteriaceae and superficial specimens were correlated to intra-operative specimens in only two thirds of cases. Clinicians should emphasize on the systematic practice of intraoperative specimens in all patients with DFIs treated surgically, while well-performed superficial specimens could be useful for prescribing appropriate antibiotic therapy in other patients.

First report of extensively-drug-resistant Proteus mirabilis isolate carrying plasmid-mediated blaNDM-1 in a Tunisian intensive care unit.

Emergence of the New Delhi metallo-ß-lactamase (NDM-1), an Ambler class B metallo-ß-lactamase able to hydrolyse all ß-lactams except monobactams, constitutes a critical and increasingly important medical issue. The acquisition of blaNDM-1 is of particular concern for Proteus mirabilis, which is intrinsically resistant to tetracycline, tigecycline and colistin, as this will make clinical treatment extremely difficult. To the authors' knowledge, this is the first report of the blaNDM-1 gene in an extensively-drug-resistant P. mirabilis clinical isolate carrying plasmid-mediated resistance to carbapenems (blaNDM-1), cephalosporins (blaCMY-4), aminoglycosides (aph3 VIa and aph3 Ia) and fluoroquinolones (qnrA6).

Carriage of multidrug-resistant bacteria among pediatric patients before and during their hospitalization in a tertiary pediatric unit in Tunisia.

The pandemic spread of multidrug-resistant (MDR) bacteria (i.e., methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum b-lactamase-producing Enterobacteriaceae (ESBLPE), vancomycin-resistant enterococci, carbapenemase-producing Enterobacteriaceae (CPE), multiresistant Pseudomonas aeruginosa and multiresistant Acinetobacter baumannii) pose a threat to healthcare Worldwide. We found limited data of MDR bacteria in pediatric patients hospitalized in Tunisian tertiary healthcare.The aim of the study is to evaluate the acquisition rate of MDR acquisition during hospitalization and to explore some of the associated risk factors for both carriage and acquisition at the pediatric department, Sahloul University Hospital. During September and October 2016, newly admitted patients were screened, at admission, during care and at discharge. Risk factors for colonization were explored by multivariate analysis. Of 112 newly admitted patients, 8.92% were colonized with at least one MDR. No risk factor was identified at admission. During hospitalization, five newly acquisition MDR (4.9%) were detected and eight (7.84%) at discharge. The specie most frequently detected on admission was Escherichia coli (50%), whereas, on discharge, Escherichia coli and K. pneumoniae were the species most frequently detected (52.7%). The pediatric intensive care unit (PICU) hospitalization, the length of hospital stay (more than 3days) and age under 2 years were identified as risk factor for acquisition of MDR during hospitalization. We identified several independent risk factors for contracting MDR bacteria during hospitalization in a tertiary pediatric department. The incidence of symptomatic MDR Infection among those colonized should be under close surveillance and long-term screening for those children is required. An institutional screening program for MDR especially in PICU might be discussed in regards to cost effectiveness.

Occurrence of Corynebacterium striatum as an emerging antibiotic-resistant nosocomial pathogen in a Tunisian hospital.

Corynebacterium striatum is a nosocomial opportunistic pathogen increasingly associated with a wide range of human infections and is often resistant to several antibiotics. We investigated the susceptibility of 63 C. striatum isolated at the Farhat-Hached hospital, Sousse (Tunisia), during the period 2011-2014, to a panel of 16 compounds belonging to the main clinically relevant classes of antimicrobial agents. All strains were susceptible to vancomycin, linezolid, and daptomycin. Amikacin and gentamicin also showed good activity (MICs90 = 1 and 2 mg/L, respectively). High rates of resistance to penicillin (82.5%), clindamycin (79.4%), cefotaxime (60.3%), erythromycin (47.6%), ciprofloxacin (36.5%), moxifloxacin (34.9%), and rifampicin (25.4%) were observed. Fifty-nine (93.7%) out of the 63 isolates showed resistance to at least one compound and 31 (49.2%) were multidrug-resistant. Twenty-nine resistance profiles were distinguished among the 59 resistant C. striatum. Most of the strains resistant to fluoroquinolones showed a double mutation leading to an amino acid change in positions 87 and 91 in the quinolone resistance-determining region of the gyrA gene. The 52 strains resistant to penicillin were positive for the gene bla, encoding a class A ß-lactamase. Twenty-two PFGE patterns were identified among the 63 C. striatum, indicating that some clones have spread within the hospital.

Evaluation of the VITEK-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system for the identification of clinical Corynebacterium species / Evaluación del sistema VITEK-MS de espectrometría de masas MALDI-TOF para la identificación de corinebacterias de importancia clínica

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Serotype distribution and antimicrobial resistance of invasive and noninvasive pneumococcal isolates in Tunisia.

Pneumococcal conjugate vaccines have not yet been introduced into the national program for childhood vaccination in Tunisia. The aim of this 7-year study was to obtain local data about serotype distribution and antimicrobial resistance of Streptococcus pneumoniae. A total of 203 isolates of culture confirmed that S. pneumoniae was evaluated. Invasive (n=108) and noninvasive (n=95) pneumococcal isolates were obtained from patients aged from 1 month to 85 years old. Considering all age groups, vaccine coverage was 40%, 62%, and 68% for PCV7, PCV10, and PCV13 serotypes, respectively. Overall, 31% of these isolates were penicillin G nonsusceptible. The most prevalent serotypes identified were those found in currently available pneumococcal conjugate vaccines, emphasizing the importance of implementing the vaccine in the routine immunization schedule at the national level.

Antimicrobial and molecular analysis of Salmonella serovar Livingstone strains isolated from humans in Tunisia and Belgium.

INTRODUCTION: Salmonella Livingstone is one of the most common serotypes responsible for nosocomial outbreaks in Tunisia. In this study, 42 isolates of Salmonella Livingstone were analyzed. Most of these were isolated from humans (31 strains from Tunisia and 9 strains from Belgium) and 2 isolates came from food products (beef and pork). METHODOLOGY: All strains were characterized by antibiogram, multilocus sequence typing (MLST), and virulotyping. This last technique was carried out by simple PCR of five chromosomal genes (agfA, hin/H2, iroB, phoP/Q, and slyA) and two plasmid genes (spvA and spvC). RESULTS: All Tunisian strains were resistant to amoxicillin, amoxicillin-clavulanic acid, ticarcillin, cefalotin, gentamicin, and kanamycin. They were also resistant to third-generation cephalosporin antibiotics (cefotaxim and ceftazidim). Belgian isolates were susceptible to all antibiotics tested. Further to MLST analyses, Tunisian strains belonged to the same sequence type, ST543. For Belgian isolates, eight strains had a ST543 profile, two strains had a ST638 profile, and one strain had a ST457 profile. Analyses of the virulence gene contents showed that strains isolated in different years and from different origins had the same virulence profile. These carried all five chromosomal genes and lacked plasmid-located virulence genes spvA and spvC. CONCLUSIONS: A combination of different typing methods showed that the majority of Belgian strains and all Tunisian strains were closely related; they belonged to the same sequence type (ST543) and had the same virulence profile, but different antibiotic resistance profiles depended on the country of origin.

Comparison of LED and conventional fluorescence microscopy for detection of acid-fast bacilli in an area with high tuberculosis incidence.

The objective of the study is to compare the performance of conventional fluorescence microscopy (CFM) and light-emitting diode (LED) fluorescence microscopy (FM) for detection of acid-fast bacilli (AFB) in clinical samples. We included AFB smears, stained using the auramine O method and blindly examined with both CFM and LED-FM. Culture results were used as reference for evaluating the reliability of the FM. We included 180 culture positive specimens and an equal number of culture negative specimens. Sensitivities for the CFM and LED-FM were 79.4% and 82.2%, respectively. Both microscopes had a high specificity (97.2%). The negative-positive (>1 cross) inter-reader agreement of LED-FM and CFM was excellent. Therefore, detection of scanty AFB was higher with LED-FM. Both microscopes were equivalent with respect to time required to read smears. Although it was not faster than CFM, the higher detection of scanty AFB smears combined with ease of use supports the consideration of LED microscopy by all tuberculosis diagnostic laboratories, as a replacement for conventional fluorescence microscopes.

Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection.

BACKGROUND: Escherichia coli is the predominant pathogen causing urinary tract infection (UTI), the most common bacterial infectious disease encountered in clinical practice, accounting for significant morbidity and high medical costs. The severity of UTI produced by E. coli is due to the expression of a wide spectrum of virulence factors. In this study we evaluated the role of E. coli virulence determinants in the pathogenesis of UTI. METHODS: A total of 90 uropathogenic E. coli strains were screened by PCR for the prevalence of seven virulence genes encoding type 1 fimbriae (fimH), pili associated with pyelonephritis (pap), S and F1C fimbriae (sfa and foc), afimbrial adhesins (afa), cytotoxic necrotizing factor (cnf), hemolysin (hly), and aerobactin (aer). RESULTS: The prevalence of genes coding for fimbrial adhesive systems was 68% for fimH, 41% for pap, and 34% for sfa/foc. The operons coding for afa afimbrial adhesins were identified in 20% of strains. The hly and cnf genes coding for toxins were amplified in 19% and 3% of strains, respectively. A prevalence of 52% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from hospitalized patients displayed a great diversity of gene associations compared to those isolated from ambulatory patients. CONCLUSIONS: Our study showed that investigation of the bacterial pathogenicity associated with UTI may contribute to a better medical intervention.

[Evaluation of chromogenic medium Uriselect4 in urine culture]. / Évaluation du milieu chromogène Uriselect4 au cours des urocultures.

We evaluated the performance and the cost of chromogenic medium Uriselect4 agar with regard to the standard medium for the detection and identification of urinary tract pathogens. A total of 503 clinical urine specimens containing leucocytes greater or equal to 104/mL were analysed prospectively, in parallel by two different persons on blood agar (GS) and Uriselect4 according to the manufacturers' instructions. Of the 503 urine specimens tested, 210 gave a positive culture on Uriselect4 versus 181 on GS. The majority of bacterial species grew on both media; enterobacteria grew on Uriselect4 better than GS. The identification of Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), KES group and Enterococcus faecalis (E. faecalis) did not require the use of galleries Api and has a gain of 24  h. Positive pure cultures on Uriselect4 corresponding to negative cultures of GS were noted in 17 ases. Conversely, in seven cases a positive pure culture on GS was noted while the corresponding Uriselect4 cultures were negative. The cost of identification on GS (including the cost of galleries Api), was about two times higher than Uriselect4. Uriselect4 medium isolates the most frequent urinary tract pathogens and identify them so almost immediately, with a lower cost.