Background:Vancomycin-resistant Enterococcus faecium is classified among World Health Organization priority pathogens for Research and development of new antibiotics. This study presents the results of 13-year surveillance of antibiotic resistance in E. faecium, with a specific focus on vancomycin and linezolid resistance in thirteen university hospitals across various regions of Tunisia. Methods:All participating laboratories followed a standardized methodology for clinical sample culture, bacterial identification, antibiotic susceptibility testing according to CA-SFM guidelines, result interpretation, epidemiological data collection. Additionally, they implemented quality controls. Findings:During the surveillance period, 2,931 E. faecium isolates were collected, predominantly from urine (49.1%) and blood cultures (24%), with high prevalence in Surgery Departments (30.3%) and Intensive Care Units (23.6%). Resistance rates were high for ampicillin (87.1%), erythromycin (93.6%), gentamicin (62.8%) and vancomycin (26%). Since 2019, six linezolid-resistant isolates have been isolated and only one isolate resistant to tigecycline was identified in 2021. Vancomycin resistance had significant upward trend since 2012, peaking at 38.1% in 2017. It was significantly associated to gentamicin (84.9% vs. 53.8%) (p<0.0001) and erythromycin95.8% vs. 89.6%) (p=0.0001) in a five year period (2018 - 2022). Conclusion:This worrying situation requires deeper investigations to develop adapted strategies with targeted measures in high-risk departments.

BACKGROUND:Healthcare-associated infections (HAI) remain a major challenge in neonatal intensive care units (NICU), particularly in resource-limited settings. The combination of high neonatal vulnerability and the rise of multidrug-resistant organisms substantially increases morbidity, mortality, and healthcare costs. AIM:This study aimed to describe the epidemiological, clinical, and bacteriological characteristics of HAI in a neonatal intensive care unit of a developing country, to assess associated morbidity and mortality. METHODS:A retrospective analytical study was conducted over three years (November 2020-November 2023) in the Neonatology Department of Hédi Chaker University Hospital, a level III referral center in Tunisia. All neonates hospitalized for ≥48 h with clinically or biologically suspected HAIs confirmed by positive blood culture were included. RESULTS:Among 4382 hospitalized neonates, 82 cases of HAI were identified. Preterm (85.4%) and low-birth-weight (82.9%) infants were the most affected. The main clinical signs were altered skin color (68.3%) and hemodynamic instability (65.8%). Gram-negative bacilli accounted for 93.9% of all isolates, with Klebsiella pneumoniae being the predominant pathogen (57 cases, 69.5%), showing multidrug resistance in 91.2% of isolates. Empirical therapy most often combined imipenem and amikacin (46.3%). The overall mortality rate was 58.5%, significantly associated with hemodynamic instability and birth weight <1400 g. CONCLUSION:HAI remains a serious issue in resource-limited settings, driven by high antimicrobial resistance and specific vulnerability factors. Strengthening infection prevention protocols and multidisciplinary surveillance is crucial to reduce morbidity and mortality.

Purpose. Invasive pneumococcal disease (IPD) remains a major global public health concern due to its high morbidity and mortality rates, particularly among children and the elderly. This study aimed to apply whole-genome sequencing (WGS) to characterize Streptococcus pneumoniae strains responsible for IPD in south Tunisia, including serotype distribution, clonal relationship and antimicrobial resistance (AMR) profiles. Methods. A total of 148 IPD S. pneumoniae isolates were collected from the microbiology laboratory at Habib Bourguiba University Hospital in Sfax, Tunisia, between 2012 and 2022. These isolates underwent WGS using Illumina technology. Bioinformatic analyses were performed to determine serotype distribution, sequence types (STs), Global Pneumococcal Sequence Clusters (GPSCs), phylogenetic relationships and AMR determinants. Results. Twenty-six different serotypes were identified, with the most prevalent being 14 (18%), 3 (13%), 19A (12%) and 19F (11%). The isolates showed high genomic diversity, as they belonged to 32 GPSCs and 59 STs. The most common GPSCs were GPSC-6, GPSC-10 and GPSC-44, associated with serotypes 14, 19A and 19F, respectively. The most frequent STs were ST2918, ST179 and ST3772. The most common resistance genes were erm B (53%) and tet M (55%), which were linked to resistance against erythromycin and tetracycline, respectively. There was a considerable concordance between WGS-based and phenotypic resistance profiles for most tested antibiotics, with few major and very major errors for most antibiotics. Temporal analysis showed a decline in serotypes 19F and 9V throughout the study period, which was associated with slight decreases in GPSC-6 and GPSC-44, while serotype 19A and GPSC-10 sharply increased. Conclusion. This study highlights the substantial genomic diversity, serotype distribution and high prevalence of AMR among IPD S. pneumoniae isolates in south Tunisia, underscoring the need for continued surveillance and effective vaccination strategies to combat this persistent public health threat.

Brucella melitensis is a zoonotic pathogen that poses a worldwide public health challenge. In recent years, whole-genome sequencing has become a widely accepted molecular typing method for the genomic epidemiology of brucellosis. This study reports the genomic characteristics of 24 B. melitensis strains isolated from human infections in southern Tunisia over 35 years (1988–2022). We utilized WGS to analyze the clonal relationships of these strains, their relatedness to international sequences, their antimicrobial resistance determinants, and their virulence factors. Our findings revealed a high genetic stability over three decades. All isolates were identified as B. melitensis biovar 3 and were assigned to the same sequence type, ST11, using the MLST-9 scheme. Using the MLST-21, Tunisian sequences shared 20 out of 21 alleles and were assigned to 2 closely related STs (ST89 and ST114). Phylogenetic analysis indicated that all Tunisian sequences were grouped into a single subcluster within lineage I, the West Mediterranean clade, and were highly related to other strains from the Maghreb region (Morocco and Algeria). Antimicrobial resistance analysis revealed no classical resistance determinants. However, mprF, bepCDEFG genes, and missense mutations in rpoB, gyrA, gyrB, and parC genes were identified. Virulence analysis identified 67 genes, predominantly involved in lipopolysaccharide biosynthesis and the type IV secretion system. To our knowledge, this study represents the first genomic investigation of B. melitensis strains circulating in Tunisia. Our findings underscore the importance of genomic surveillance in understanding the epidemiology and evolution of brucellosis in North Africa.

West Nile virus (WNV) is a mosquito-borne pathogen of the Flaviviridae family that poses recurring public health threats in Tunisia, where Culex pipiens is recognized as the primary vector. Identification of circulating strains in different mosquito species is essential for targeted prevention and control. Between November 2021 and October 2022, mosquitoes were collected at four high-risk sites, and human samples were obtained through the national meningitis surveillance program. Human serum, cerebrospinal fluid (CSF), and urine samples were tested for WNV-specific IgM and IgG antibodies using ELISA, and molecular diagnosis was performed using Real-time RT-PCR (RRT-PCR). Positive samples underwent sequencing for phylogenetic characterization. Serological investigation on human serum revealed the presence of IgM and/or IgG antibodies reactive to WNV antigens, which may indicate exposure to WNV or related flaviviruses. RNA of WNV was detected in 21 mosquito pools (10.19%) belonging to Culex pipiens, Cx. perexiguus, Aedes caspius, and Ae. detritus, as well as in three human cases. Phylogenetic analysis of positive human and mosquito samples showed that all detected WNV strains belonged to sublineage 1a. The concurrent detection of WNV in vectors and humans confirms active circulation in Tunisia and underscores the role of Culex spp. Mosquitoes in transmission. Sustained multidisciplinary surveillance integrating entomological and clinical data is critical for early detection, guiding control measures, and preventing future outbreaks in humans and animals.

Purpose. The prevalence of multidrug-resistant (MDR) Shigella sonnei is increasing globally, raising concerns for public health. In 2022, an outbreak of MDR S. sonnei was observed in Tunisia. We aimed to evaluate the genetic profile of S. sonnei isolates during the outbreak, including their clonal relationship, antimicrobial determinants and connection to international strains.Methods. In this study, we sequenced the whole genome of 24 S. sonnei strains collected from South Tunisia between July 2022 and November 2023. Bioinformatic analysis was conducted to confirm species identification, assign sequence types, determine core genome sequence types, analyse phylogenetic relationships and identify antimicrobial resistance determinants. Phylodynamic and phylogeographic analyses were performed to trace the spatiotemporal spread of the outbreak genotype.Results. Our investigation revealed that 23 out of 24 isolates were grouped into the HC10-20662 genotype within the 3.6.3 subclade. All isolates carried the blaCTX-M-15 gene associated with extended-spectrum beta-lactamase production, as well as the dfrA1 and qnrS1 genes, along with the D87G mutation in gyrA. Additionally, the sul2, tet(A) and mph(A) resistance genes were present in most isolates (96%, 96 and 83, respectively). Phylogeographic analysis suggested that the outbreak genotype likely spread in Europe before being introduced into Tunisia.Conclusion. To the best of our knowledge, this is the first MDR S. sonnei outbreak in the country. The HC10-20662 genotype appears to be responsible for a multi-country outbreak, affecting both Tunisia and Europe. Continued genomic surveillance efforts, both nationally and internationally, are essential for monitoring the dynamic evolution and global spread of MDR S. sonnei.

Background: Changing patterns in community respiratory virus activity were reported in different geographical locations during the COVID-19 pandemic. In this study, we aimed to assess the prevalence of circulating respiratory viruses, including SARS-CoV-2, during the season 2021-2022 in Tunisia. Methods: We retrospectively enrolled 328 nasopharyngeal samples received at the Triage Center of Habib Bourguiba Hospital from patients with acute respiratory symptoms during September 2021-May 2022. All samples were screened for both SARS-CoV-2 and common respiratory viruses. This latter detection was performed using end-point multiplex RT-PCRs, Real-Time PCR, and AllplexTM Respiratory Panel 1 kit (Seegene) for Influenza Virus A (IFVA) and Respiratory Syncytial Virus (RSV) subtyping. Results: Among included patients, at least one viral pathogen was identified in 118 (35.9 %) patients. The detection rate of SARS-CoV-2 was 21.6 %. A low viral coinfection rate was observed (3.3 %). The most prevalent pathogen among non-SARS-CoV-2 viruses was Enterovirus/Rhinovirus (HEV/HRV) (59.6 %) followed by IFVA (15.3 %) and Adenoviruses (ADV) (11.5 %). Only IFVA H3N2 was found to circulate during the study period. A negative virus interaction was eventually induced by SARS-CoV-2, as it was shown by lower levels of activity of non-SARS-CoV-2 viruses (not exceeding 17.7 %) while infections due to pandemic Omicron variants of concern became widespread. Conclusions: This study highlights the relative return of community IFVA circulation during the 2021-2022 season in Tunisia. A negative viral interaction between SARS-CoV-2 and other respiratory viruses is highly suggested, which explains, in addition to the easing of COVID-19 restriction measures, the epidemiological changes in non-SARS-CoV-2 viruses circulation.

Background: Early and accurate diagnosis is crucial for preventing the spread of SARS-CoV-2 infection. The rapid antigen test was developed for testing infection, and it was necessary to assess its performance before widespread use in Tunisia. Aim: To evaluate the effectiveness of a rapid antigen test for the detection of SARS-CoV-2 in nasopharyngeal swabs in Tunisia. Methods: Nasopharyngeal samples were taken from COVID-19 suspected cases between October and December 2020 and tested using the Standard Q COVID-19 Ag test (SD-Biosensor, Republic of Korea) and real-time reverse transcription polymerase chain reaction (RT-PCR). Results: Overall, 4539 patients were tested. Of the total study population (N = 4539), 82.5% of positive samples remained positive with the rapid antigen test, while 20.2% (470/2321) of samples that were negative with rapid antigen test were confirmed positive with RT-PCR, giving a negative predictive value of 79.8% for the rapid antigen test. The sensitivity and negative predictive value of the rapid antigen test were 70.2% and 65.8%, respectively. These results improved to 96.4% and 92.8%, respectively, when considering the cycle threshold value by RT-PCR below 25. Conclusion: Although the rapid antigen test was less sensitive than RT-PCR, its ability to rapidly detect individuals with high viral loads makes it suitable for use during an epidemic.

Population Medicine considers the following types of articles:• Research Papers -reports of data from original research or secondary dataset analyses.• Review Papers -comprehensive, authoritative, reviews within the journal's scope.These include both systematic reviews and narrative reviews.• Short Reports -brief reports of data from original research.• Policy Case Studies -brief articles on policy development at a regional or national level.• Study Protocols -articles describing a research protocol of a study.• Methodology Papers -papers that present different methodological approaches that can be used to investigate problems in a relevant scientific field and to encourage innovation.• Methodology Papers -papers that present different methodological approaches that can be used to investigate problems in a relevant scientific field and to encourage innovation.

BACKGROUND AND STUDY AIM:During the natural course of HBeAg-negative chronic hepatitis B (CHB), fluctuations in hepatitis B virus (HBV) DNA and alanine aminotransferase (ALT) levels are often observed, making the classification of patients difficult. We aimed to describe spontaneous short-term HBV DNA level fluctuations and to assess the usefulness of qHBsAg in Tunisian patients with HBeAg-negative chronic HBV infection. PATIENTS AND METHODS:We included 174 treatment-naive Tunisian patients with HBeAg-negative chronic HBeAg-negative HBV infection. A prospective 1-year follow-up was conducted with serial determinations of HBV DNA, ALT levels, and qHBsAg. The patients were classified into three groups: inactive carriers (G1), patients with negative HBeAg CHB (G2), and patients with an "indeterminate state" (G3). For the latter group, a liver biopsy was indicated. RESULTS:Only genotype D was detected. During follow-up, 21.6% and 19.5% of patients with a low initial (<2,000 IU/ml) and intermediate viral load (2,000-20,000 IU/ml) experienced a subsequent increase in their HBV DNA levels above 2,000 and 20,000 IU/ml, respectively. Significant variations in viral load were observed in 61.1% of patients at 6-month intervals. Among the 174 patients, 89 (51.1%) belonged to G1, 33 (19%) to G2, and 52 (29.9%) to G3. Fourteen patients have undergone a liver biopsy, of whom seven showed moderate to severe liver disease. Combination of HBV DNA < 2,000 IU/ml and qHBsAg < 832 IU/ml excluded CHB in 98.4% of cases. A cutoff point for qHBsAg < 100 IU/ml associated with an annual decline of > 0.5 log 10 IU/ml is a good predictor marker of functional cure for hepatitis B. CONCLUSIONS:This study highlights the large short-term fluctuations in HBV DNA in patients with HBeAg-negative chronic HBeAg-negative HBV infection with genotype D. Thus, using the cutoff value of 832 for qHBsAg combined with that of 2,000 for HBV DNA makes it possible to exclude CHB for most patients.

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

Human respiratory infections caused by a large variety of microbial pathogens are the most common diseases responsible for hospitalization, morbidity and mortality. Parachlamydia acanthamoebae , a Chlamydia -related bacterium, has been found to be potentially associated with these diseases. An early and accurate diagnosis of this pathogen could be useful to avoid the potential respiratory complications linked especially to COVID-19 patients and to set suitable outbreak control measures. A TaqMan-PCR assay was developed to detect and quantify Parachlamydia acanthamoebae in environmental and clinical samples from patients of all ages with COVID-19. The selected hydrolysis probe displayed no cross-reaction with the closely related Chlamydia or the other tested pathogens. This q-PCR achieved good reproducibility and repeatability with a detection limit of about 5 DNA copies per reaction. Using this q-PCR assay, Parachlamydia acanthamoebae was detected in 2/78 respiratory specimens and 9/47 water samples. Only one case (1.3%) of Parachlamydia acanthamoebae and SARS-COV-2 co-infection was noticed. To our knowledge, the combination of these two respiratory pathogens has not been described yet. This new TaqMan-PCR assay represents an efficient diagnostic tool to survey Parachlamydia acanthamoebae on a large-scale screening programs and also during outbreaks.

Background:The COVID-19 pandemic changed the typical patterns of respiratory infections globally. While SARS-CoV-2 illness exhibited explosive growth since 2020, the activity of other respiratory viruses fell below historical seasonal norms. The objective of this study was to assess the prevalence of seasonal respiratory viruses during the COVID-19 pandemic in Tunisia.Methods:This is a retrospective cross-sectional study including 284 nasopharyngeal samples tested negative for SARS-CoV-2 during the period October 2020-May 2021. All samples were screened for fifteen common respiratory viruses. Either a fast syndromic approach using Biofire FILM ARRAY respiratory 2.1 (RP2.1) Panel, or end-point multiplex RT-PCRs detecting RNA viruses and Real-Time PCR detecting Adenoviruses were used.Results:Overall, 30.6% (87/284) of samples were positive for at least one virus. Mixed infections were detected in 3.4% of positive cases. Enterovirus/Rhinovirus (HEV/HRV) was the most detected virus throughout the study period, especially during December 2020 (33.3% of all HEV/HRV being detected). During the 2020-2021 winter season, neither Respiratory Syncytial Virus nor Influenza Viruses circulation was observed. Metapneumovirus and Parainfluenza Viruses infections were detected during the spring season. The highest rate of respiratory viruses detection was observed in children and adults aged [0-10] years (50%) and [31-40] years (40%). HEV/HRV was the most detected virus regardless of age group.Conclusions:Public health measures used to prevent SARS-CoV-2 spread in Tunisia were also effective to reduce transmission of the other respiratory viruses, especially Influenza. The higher resistance of HEV/HRV in the environment could explain their predominance and continuous circulation during this period.

Introduction:The Delta variant posed an increased risk to global public health and rapidly replaced the pre-existent variants worldwide. In this study, the genetic diversity and the spatio-temporal dynamics of 662 SARS-CoV2 genomes obtained during the Delta wave across Tunisia were investigated.Methods:Viral whole genome and partial S-segment sequencing was performed using Illumina and Sanger platforms, respectively and lineage assignemnt was assessed using Pangolin version 1.2.4 and scorpio version 3.4.X. Phylogenetic and phylogeographic analyses were achieved using IQ-Tree and Beast programs.Results:The age distribution of the infected cases showed a large peak between 25 to 50 years. Twelve Delta sub-lineages were detected nation-wide with AY.122 being the predominant variant representing 94.6% of sequences. AY.122 sequences were highly related and shared the amino-acid change ORF1a:A498V, the synonymous mutations 2746T>C, 3037C>T, 8986C>T, 11332A>G in ORF1a and 23683C>T in the S gene with respect to the Wuhan reference genome (NC_045512.2). Spatio-temporal analysis indicates that the larger cities of Nabeul, Tunis and Kairouan constituted epicenters for the AY.122 sub-lineage and subsequent dispersion to the rest of the country.Discussion:This study adds more knowledge about the Delta variant and sub-variants distribution worldwide by documenting genomic and epidemiological data from Tunisia, a North African region. Such results may be helpful to the understanding of future COVID-19 waves and variants.

Objectives: Since the onset of the COVID-19 pandemic, cases of reinfection with SARS-CoV-2 have been reported, raising additional public health concerns. SARS-CoV-2 reinfection was assessed in healthcare workers (HCWs) in Tunisia because they are at the greatest exposure to infection by different variants. Methods: We conducted whole-genome sequencing of the viral RNA from clinical specimens collected during the initial infection and the suspected reinfection from 4 HCWs, who were working at the Habib Bourguiba University Hospital (Sfax, Tunisia) and retested positive for SARS-CoV-2 through reverse transcriptase-polymerase chain reaction (RT-PCR) after recovery from a first infection. A total of 8 viral RNAs from the patients' respiratory specimens were obtained, which allowed us to characterize the differences between viral genomes from initial infection and positive retest. The serology status for total Ig, IgG, and IgM against SARS-CoV-2 was also determined and followed after the first infection. Results: We confirmed through whole-genome sequencing of the viral samples that all 4 cases experienced a reinfection event. The interval between the 2 infection events ranged between 45 and 141 days, and symptoms were milder in the second infection for 2 patients and more severe for the remaining 2 patients. Reinfection occurred in all 4 patients despite the presence of antibodies in 3 of them. Conclusion: This study adds to the rapidly growing evidence of COVID-19 reinfection, where viral sequences were used to confirm infection by distinct isolates of SARS-CoV-2 in HCWs. These findings suggest that individuals who are exposed to different SARS-CoV-2 variants might not acquire sufficiently protective immunity through natural infection and emphasize the necessity of their vaccination and the regular follow-up of their immune status both in quantitative and qualitative terms. (c) 2022 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

We thank Lippi and Plebani for their comments on our article: "Evidence of SARS-CoV-2 Symptomatic Reinfection in Four Health Care Professionals from the Same Hospital Despite the Presence of Antibodies" (Gargouri et al., 2022Gargouri S Souissi A Abid N Chtourou A Feki-Berrajah L Karray R et al.Evidence of SARS-CoV-2 Symptomatic Reinfection in Four Health Care Professionals from the Same Hospital Despite the Presence of Antibodies.International Journal of Infectious Diseases. 2022; (0)https://doi.org/10.1016/j.ijid.2022.01.006Abstract Full Text Full Text PDF Scopus (4) Google Scholar). Our study confirmed SARS-CoV-2 reinfection in 4 health care workers (HCW). Although anti-S1 immunoglobulin G (IgG) was detectable before reinfection in 3 patients, all of them developed symptomatic secondary infection after a symptom-free interval ranging between 45 and 141 days, with a more severe clinical presentation in 2 cases. Lippi and Plebani pointed out that the presence of anti-SARS-CoV-2 antibodies does not necessarily reflect efficient neutralization. They suggested the following conclusion "reinfection occurred with the presence of low values of anti-S1 IgG antibodies" instead of "reinfection occurred despite the presence of antibodies". Indeed, we would like to clarify this point. Lippi and Plebani mentioned the work carried out by Montesinos et al. (Montesinos et al., 2021Montesinos I Dahma H Wolff F Dauby N Delaunoy S Wuyts M et al.Neutralizing antibody responses following natural SARS-CoV-2 infection: Dynamics and correlation with commercial serologic tests.J Clin Virol. 2021; 144104988https://doi.org/10.1016/j.jcv.2021.104988Crossref PubMed Scopus (10) Google Scholar), who compared different serological tests, including the one used in our study (VIDAS SARS-CoV-2 IgG serologic test), and who support the use of this test to monitor neutralizing antibody response following natural SARS-CoV-2 infection. However, contrary to Lippi et al., who mentioned negative predictive value (NPV) as a base to point out that several samples of patients with low values of these antibodies (and even samples of sero-reverted patients with a negative result) may retain significant neutralizing potential, in our study we chose to consider the specificity of the test and we will discuss the reason for that choice. It is worth noting that predictive values (both positive and negative predictive values; PPV and NPV) depend upon the prevalence of the disease in a population. As the prevalence of the disease increases (that is, true positives are more common), the likelihood of a false positive decreases. Therefore, predictive values can change over time or in different places, whereas sensitivity and specificity do not change, as these are characteristics of the test itself. Therefore, both PPV and NPV are dependent on the proportion of the test population that has the disease (otherwise known as prevalence) Montesinos et al., 2021Montesinos I Dahma H Wolff F Dauby N Delaunoy S Wuyts M et al.Neutralizing antibody responses following natural SARS-CoV-2 infection: Dynamics and correlation with commercial serologic tests.J Clin Virol. 2021; 144104988https://doi.org/10.1016/j.jcv.2021.104988Crossref PubMed Scopus (10) Google Scholar. carried out their work using samples collected between April 15, 2020, and December 7, 2020 (Belgium), whereas our samples were collected between August 2020 and October 2020. During this period, the prevalence of COVID-19 was very different between Tunisia and Belgium as well as within each country. For this reason, we rather considered the specificity of the test, which measures the proportion of negative test results out of all truly negative samples. The VIDAS test, used in our study, showed the best specificity (89% vs. 54.7%-79.7%). Thus, it is clear that these results support our conclusion "Reinfection despite the presence of antibodies", which means, based on the study of Montesinos et al., "despite the presence of potentially neutralizing antibodies". Second, according to Lumley et al. (Lumley et al., 2021Lumley SF O'Donnell D Stoesser NE Matthews PC Howarth A Hatch SB et al.Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers.N Engl J Med. 2021; 384: 533-540https://doi.org/10.1056/NEJMoa2034545Crossref PubMed Scopus (428) Google Scholar), the presence of anti-spike antibodies was associated with a substantially reduced risk of SARS-CoV-2 reinfection in the ensuing 6 months, and no symptomatic infections in HCWs with these antibodies were observed. Furthermore, Montesinos et al. (Montesinos et al., 2021Montesinos I Dahma H Wolff F Dauby N Delaunoy S Wuyts M et al.Neutralizing antibody responses following natural SARS-CoV-2 infection: Dynamics and correlation with commercial serologic tests.J Clin Virol. 2021; 144104988https://doi.org/10.1016/j.jcv.2021.104988Crossref PubMed Scopus (10) Google Scholar) showed that only a minority (16.9%) of the HCWs lost neutralizing antibodies within at least 6 months. Interestingly, in our study, all 4 reinfection cases occurred within a short period and all of them were symptomatic with a severe outcome in 2 patients. Finally, we appreciate this comment on our findings and hope that our work contributes to the growing body of knowledge about SARS-CoV-2 reinfection. All authors declare no competing interest. Not applicable

The high need of rapid and flexible tools that facilitate the identification of circulating SARS-CoV-2 Variants of Concern (VOCs) remains crucial for public health system monitoring. Here, we develop allele-specific (AS)-qPCR assays targeting three recurrent indel mutations, AEF156-157, Ins214EPE and ALPP24-26, in spike (S) gene to identify the Delta VOC and the Omicron sublineages BA.1 and BA.2, respectively. After verification of the analytical specificity of each primer set, two duplex qPCR assays with melting curve analysis were performed to screen 129 COVID-19 cases confirmed between December 31, 2021 and February 01, 2022 in Sfax, Tunisia. The first duplex assay targeting AEF156-157 and Ins214EPE mutations successfully detected the Delta VOC in 39 cases and Omicron BA.1 in 83 cases. All the remaining cases (n = 7) were identified as Omicron BA.2, by the second duplex assay targeting Ins214EPE and ALPP24-26 mutations. The results of the screening method were in perfect concordance with those of S gene partial sequencing. In conclusion, our findings provide a simple and flexible screening method for more rapid and reliable monitoring of circulating VOCs. We highly recommend its implementation to guide public health policies.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century.

Introduction Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied by real time RT-PCR (rRT-PCR) in Cerebrospinal fluid (CSF), which often gives negative results due to too short virorachia and late sampling. rRT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples. Methods During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative rRT-PCR was performed on both type of samples. Results WNV RNA was detected in 50.5% of urine samples (48/95) and in 2.8% of CSF samples (1/35). WNV RNA was detectable from day 1 to day 41 from symptom onset, however, positive urine rate was 53.1% during the first 10 days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference (p > 0.005). Cycle threshold (Ct) values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ct value was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases (p < 0.001). Discussion/conclusion Our study reported the use of rRT-PCR on urine samples as a confirmatory diagnostic tool for WNV "probable cases" during an outbreak. Our findings underlined the reliability and the rapidity of this confirmatory tool, even late, and showed its superiority on CSF investigation.