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OBJECTIVES: From September 2022 an increase in Corynebacterium diphtheriae (C. diphtheriae) infections was reported in Europe. Our study focuses on 31 adolescent and young adult refugees with cutaneous C. diphtheriae infections detected in Germany. We examined treatment regimens and outcomes to provide targeted insights into the management of this infection. METHODS: We distributed a standardized survey, focused on children and adolescents presenting to paediatric clinics through the German Paediatric Infectious Diseases Society (DGPI) and additional professional contacts in Germany. Data were extracted from routine medical documentation and reported anonymously. RESULTS: A total of 31 individuals with cutaneous C. diphtheriae infection were reported by 9 centres. Two of these showed diphtheria toxin (DT) related systemic symptoms and four exhibited systemic inflammation requiring complex management. The remaining 25 cases, with exclusively cutaneous manifestations, were afebrile. Treatment with topical antiseptics and systemic antibiotics, mainly aminopenicillin/beta-lactamase inhibitors (BLI) (35%) or clindamycin (25%), achieved eradication in all but two cases treated with aminopenicillin/BLI. Treatment duration varied between 5 and 17 days. CONCLUSIONS: In refugees presenting with chronic skin wounds, C. diphtheriae should be included into the differential diagnosis. Fever seems to be a valuable marker to differentiate severe cases with potentially DT-mediated sequelae from exclusively cutaneous diphtheria (CD). For afebrile CD, topical antiseptics and oral antibiotic therapy with clindamycin for 7 days, followed by clinical surveillance appears to be a safe treatment regimen. Patients with CD who present with fever or pharyngitis should be thoroughly investigated including blood and pharyngeal swab cultures.
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BACKGROUND: Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. OBJECTIVES: We aimed to evaluate broth microdilution and disc diffusion methods and create a dataset of MIC values and inhibition zone diameters (ZDs) from which breakpoints could be determined. METHODS: We included 400 recent clinical isolates equally distributed by species (Corynebacterium diphtheriae and Corynebacterium ulcerans) and by national surveillance programmes (France and Germany). Non-duplicate toxigenic and non-toxigenic isolates were chosen to enable the inclusion of a diversity of susceptibility levels for the 13 agents tested. Broth microdilution and disc diffusion, using EUCAST methodology for fastidious organisms, were used. RESULTS: The distributions of MIC and ZD values were largely in agreement among methods and countries. Breakpoints to allow categorization of WT isolates as susceptible, i.e. susceptible (S) or susceptible, increased exposure (I) were determined for 12 agents. The data supported a breakpoint for benzylpenicillin and amoxicillin of resistant (R)â>â1 mg/L since WT isolates were inhibited by 1 mg/L or less. WT isolates were categorized as I (Sâ≤â0.001 mg/L) for benzylpenicillin, emphasizing the need for increased exposure, and S (Sâ≤â1 mg/L) for amoxicillin. Erythromycin breakpoints were set at Sâ≤â0.06 mg/L and Râ>â0.06 mg/L. The corresponding ZD breakpoints were determined for all agents except amoxicillin, for which categorization was based on benzylpenicillin results. CONCLUSIONS: This work provided a large set of antimicrobial susceptibility data for C. diphtheriae and C. ulcerans, using a harmonized methodology. The dataset allowed EUCAST and experts in the diphtheria field to develop evidence-based breakpoints in January 2023.
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Antibacterianos , Corynebacterium diphtheriae , Corynebacterium , Pruebas de Sensibilidad Microbiana , Pruebas de Sensibilidad Microbiana/métodos , Humanos , Corynebacterium/efectos de los fármacos , Corynebacterium/aislamiento & purificación , Antibacterianos/farmacología , Corynebacterium diphtheriae/efectos de los fármacos , Corynebacterium diphtheriae/aislamiento & purificación , Corynebacterium diphtheriae/genética , Alemania , Infecciones por Corynebacterium/microbiología , Difteria/microbiología , FranciaAsunto(s)
Difteria , Refugiados , Humanos , Difteria/epidemiología , Análisis por Conglomerados , Alemania/epidemiologíaRESUMEN
Borrelia burgdorferi sensu lato is a species complex of spirochetal bacteria that occupy different ecological niches which is reflected in their reservoir host- and vector-associations. Borrelia genomes possess numerous linear and circular plasmids. Proteins encoded by plasmid genes play a major role in host- and vector-interaction and are important for Borrelia niche adaptation. However, the plasmid composition and therewith the gene repertoire may vary even in strains of a single species. Borrelia garinii, one of the six human pathogenic species, is common in Europe (vector Ixodes ricinus), Asia (vector Ixodes persulcatus) and in marine birds (vector Ixodes uriae). For the latter, only a single culture isolate (Far04) and its genome were previously available. The genome was rather small containing only one circular and six linear plasmids with a notable absence of cp32 plasmids. To further investigate B. garinii from marine transmission cycles and to explore i) whether the small number of plasmids found in isolate Far04 is a common feature in B. garinii from marine birds and presents an adaptation to this particular niche and ii) whether there may be a correlation between genome type and host species, we initiated in vitro cultures from live I. uriae collected in 2017 and 2018 from marine avian hosts and their nests. Hosts included common guillemots, Atlantic Puffin, razorbill, and kittiwake. We obtained 17 novel isolates of which 10 were sequenced using Illumina technology, one also with Pacific Bioscience technology. The 10 genomes segregated into five different genome types defined by plasmid types (based on PFam32 loci). We show that the genomes of seabird associated B. garinii contain fewer plasmids (6-9) than B. garinii from terrestrial avian species (generally ≥10), potentially suggesting niche adaptation. However, genome type did not match an association with the diverse avian seabird hosts investigated but matched the clonal complex they originated from, perhaps reflecting the isolates evolutionary history. Questions that should be addressed in future studies are (i) how is plasmid diversity related to host- and/or vector adaptation; (ii) do the different seabird species differ in reservoir host competence, and (iii) can the genome types found in seabirds use terrestrial birds as reservoir hosts.
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Grupo Borrelia Burgdorferi , Borrelia , Charadriiformes , Ixodes , Enfermedad de Lyme , Animales , Humanos , Grupo Borrelia Burgdorferi/genética , Enfermedad de Lyme/veterinaria , Enfermedad de Lyme/microbiología , Ixodes/microbiología , Evolución Biológica , Aves/microbiologíaRESUMEN
The Eurasian beaver (Castor fiber) has been reintroduced successfully in Germany since the 1990s. Since wildlife is an important source of zoonotic infectious diseases, monitoring of invasive and reintroduced species is crucial with respect to the One Health approach. Three Eurasian beavers were found dead in the German federal states of Bavaria, North Rhine-Westphalia and Baden-Wuerttemberg in 2015, 2021 and 2022, respectively. During post-mortem examinations, Corynebacterium (C.) ulcerans could be isolated from the abscesses of two beavers and from the lungs of one of the animals. Identification of the bacterial isolates at the species level was carried out by spectroscopic analysis using MALDI-TOF MS, FT-IR and biochemical profiles and were verified by molecular analysis based on 16-23S internal transcribed spacer (ITS) region sequencing. Molecular characterization of the C. ulcerans isolates using whole-genome sequencing (WGS) revealed a genome size of about 2.5 Mbp and a GC content of 53.4%. Multilocus sequence typing (MLST) analysis classified all three isolates as the sequence type ST-332. A minimum spanning tree (MST) based on cgMLST allelic profiles, including 1211 core genes of the sequenced C. ulcerans isolates, showed that the beaver-derived isolates clearly group on the branch of C. ulcerans with the closest relationship to each other, in close similarity to an isolate from a dog. Antibiotic susceptibility testing revealed resistance to clindamycin and, in one strain, to erythromycin according to EUCAST, while all isolates were susceptible to the other antimicrobials tested.
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Background: In industrialised countries diphtheria is a rare but still life-threatening disease with a recent increase in cases due to migration and zoonotic aspects. Due to the rarity of the disease, laboratory diagnosis of diphtheria is often carried out in central reference laboratories and involves the use of sophisticated equipment and specially trained personnel. The result of the diphtheria agent detection can usually be obtained after 5-6 days or more. Authors suggest a Lateral Flow Immunoassay (LFIA)-based laboratory algorithm for the diagnosis of diphtheria, which may render less time in issuing a result and could promote the testing be performed in laboratories closer to the patient. Methods: LFIA for diphtheria toxin (DT) detection was designed using a pair of monoclonal antibodies to receptor-binding subunit B of the DT, and validated with 322 corynebacterial cultures as well as 360 simulated diphtheria specimens. Simulated diphtheria specimens were obtained by spiking of human pharyngeal samples with test strains of corynebacteria. The simulated specimens were plated on selective tellurite agar and after 18-24 hours of incubation, grey/black colonies characteristic of the diphtheria corynebacteria were examined for the DT using LFIA. Results: The diagnostic sensitivity of the LFIA for DT detection on bacterial cultures was 99.35%, and the specificity was 100%. Also, the LFIA was positive for all pharyngeal samples with toxigenic strains and negative for all samples with non-toxigenic strains. For setting LFIA, a 6-hour culture on Elek broth was used; thus, under routine conditions, the causative agent of diphtheria could be detected within two working days after plating of the clinical specimen on the tellurite medium of primary inoculation. Conclusions: The availability of such a simple and reliable methodology will speed up and increase the accuracy of diphtheria diagnosis globally.
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BACKGROUND: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. RESULTS: Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. CONCLUSION: Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.
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Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Borrelia , Enfermedad de Lyme , Humanos , Borrelia/genética , Genoma Bacteriano , Filogenia , Borrelia burgdorferi/genética , Enfermedad de Lyme/microbiología , Grupo Borrelia Burgdorferi/genéticaRESUMEN
BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.
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Infecciones por Escherichia coli , Salmonella enterica , Humanos , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Salmonella enterica/genética , Alemania , Secuenciación Completa del Genoma/métodos , Epidemiología MolecularRESUMEN
PURPOSE: Omicron is rapidly spreading as a new SARS-CoV-2 variant of concern (VOC). The question whether this new variant has an impact on SARS-CoV-2 rapid antigen test (RAT) performance is of utmost importance. To obtain an initial estimate regarding differences of RATs in detecting omicron and delta, seven commonly used SARS-CoV-2 RATs from different manufacturers were analysed using cell culture supernatants and clinical specimens. METHODS: For this purpose, cell culture-expanded omicron and delta preparations were serially diluted in Dulbecco's modified Eagle's Medium (DMEM) and the Limit of Detection (LoD) for both VOCs was determined. Additionally, clinical specimens stored in viral transport media or saline (n = 51) were investigated to complement in vitro results with cell culture supernatants. Ct values and RNA concentrations were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: The in vitro determination of the LoD showed no obvious differences in detection of omicron and delta for the RATs examined. The LoD in this study was at a dilution level of 1:1,000 (corresponding to 3.0-5.6 × 106 RNA copies/mL) for tests I-V and at a dilution level of 1:100 (corresponding to 3.7-4.9 × 107 RNA copies/mL) for tests VI and VII. Based on clinical specimens, no obvious differences were observed between RAT positivity rates when comparing omicron to delta in this study setting. Overall positivity rates varied between manufacturers with 30-81% for omicron and 42-71% for delta. Test VII was only conducted in vitro with cell culture supernatants for feasibility reasons. In the range of Ct < 23, positivity rates were 50-100% for omicron and 67-93% for delta. CONCLUSION: In this study, RATs from various manufacturers were investigated, which displayed no obvious differences in terms of analytical LoD in vitro and RAT positivity rates based on clinical samples comparing the VOCs omicron and delta. However, differences between tests produced by various manufacturers were detected. In terms of clinical samples, a focus of this study was on specimens with high virus concentrations. Further systematic, clinical and laboratory studies utilizing large datasets are urgently needed to confirm reliable performance in terms of sensitivity and specificity for all individual RATs and SARS-CoV-2 variants.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Técnicas de Cultivo de Célula , ARNRESUMEN
From July 2022, cases of imported diphtheria with toxigenic Corynebacterium diphtheriae remarkably increased among migrants arriving in Germany. Up to 30 September 2022, 44 cases have been reported to the national public health institute, all laboratory-confirmed, male, and mainly coming from Syria (n = 21) and Afghanistan (n = 17). Phylogeny and available journey information indicate that most cases (n = 19) were infected along the Balkan route. Active case finding, increased laboratory preparedness and epicentre localisation in countries along this route are important.
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Corynebacterium diphtheriae , Difteria , Migrantes , Masculino , Humanos , Corynebacterium diphtheriae/genética , Difteria/diagnóstico , Difteria/epidemiología , Difteria/microbiología , Corynebacterium , Brotes de Enfermedades , Alemania/epidemiologíaRESUMEN
Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the virulence of a strain for the purpose of diphtheria agent monitoring. To determine the LTP of diphtheria agents, an immunoassay based on monoclonal antibodies (mAbs) has been developed. A pair of mAbs specific to the fragment B of DT was selected, which makes it possible to detect DT in a sandwich ELISA with a detection limit of DT less than 1 ng/mL. Sandwich ELISA was used to analyze 218 liquid culture supernatants of high-, low- and non-toxigenic strains of various corynebacteria. It was shown that the results of ELISA are in good agreement with the results of PCR and the Elek test for the tox gene and DT detection, respectively. The diagnostic sensitivity of the assay was approximately 99%, and specificity was 100%. It has been found that strains of C. ulcerans, on average, produce 10 times less DT than C. diphtheriae. The mAbs used in the ELISA proved to be quite discriminatory and could be further used for the design of the LFIA, a method that can reduce the labor and cost of laboratory diagnosis of diphtheria.
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Background: Despite a vaccination rate of 82.0% (n = 123/150), a SARS-CoV-2 (Alpha) outbreak with 64.7% (n = 97/150) confirmed infections occurred in a nursing home in Bavaria, Germany. Objective: the aim of this retrospective cohort study was to examine the effects of the Corminaty vaccine in a real-life outbreak situation and to obtain insights into the antibody response to both vaccination and breakthrough infection. Methods: the antibody status of 106 fully vaccinated individuals (54/106 breakthrough infections) and epidemiological data on all 150 residents and facility staff were evaluated. Results: SARS-CoV-2 infections (positive RT-qPCR) were detected in 56.9% (n = 70/123) of fully vaccinated, compared to 100% (n = 27/27) of incompletely or non-vaccinated individuals. The proportion of hospitalized and deceased was 4.1% (n = 5/123) among fully vaccinated and therewith lower compared to 18.5% (n = 5/27) hospitalized and 11.1% (n = 3/27) deceased among incompletely or non-vaccinated. Ct values were significantly lower in incompletely or non-vaccinated (p = 0.02). Neutralizing antibodies were detected in 99.1% (n = 105/106) of serum samples with significantly higher values (p < 0.001) being measured post-breakthrough infection. α-N-antibodies were detected in 37.7% of PCR positive but not in PCR negative individuals. Conclusion: Altogether, our data indicate that SARS-CoV-2 vaccination does provide protection against infection, severe disease progression and death with regards to the Alpha variant. Nonetheless, it also shows that infection and transmission are possible despite full vaccination. It further indicates that breakthrough infections can significantly enhance α-S- and neutralizing antibody responses, indicating a possible benefit from booster vaccinations.
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PURPOSE: Diphtheria, still present in many countries of the world, is caused by toxigenic strains of species of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and the emerging zoonotic pathogen C. ulcerans. The immunoprecipitation test according to Elek is the gold standard for detection of the major virulence factor diphtheria toxin (DT) in toxigenic corynebacteria. Due to its sophisticated methodological requirements, the classical Elek test is performed mainly by specialized reference laboratories. It was revealed that the current modification of the Elek test does not detect the toxin in weakly toxigenic isolates. Therefore, a more robust method for detecting free DT is urgently needed, especially for toxigenic C. ulcerans strains which are known to produce often much lower amounts of DT than C. diphtheriae. METHODS: Thirty-one tox-positive C. ulcerans isolates with a negative standard Elek test result previously determined as NTTB (non-toxigenic tox bearing) were re-analyzed in this study using a modified immunoprecipitation method optimized regarding different parameters including type and concentration of antitoxin, medium volume, inoculum distance from the antitoxin disk and position of controls. RESULTS: All 31 C. ulcerans strains tested positive in the optimized Elek test. CONCLUSION: Only with a reliable and easy-to-handle method for detecting the toxigenicity of C. ulcerans, it is possible to assess the etiological role of this emerging zoonotic bacterium in human pathology.
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Antitoxinas , Corynebacterium diphtheriae , Difteria , Humanos , Difteria/diagnóstico , Difteria/microbiología , Toxina Diftérica , CorynebacteriumRESUMEN
Introduction: Here we report our results of a multi-center, open cohort study ("COVID-Kids-Bavaria") investigating the distribution of acute SARS-CoV-2 infections among children and staff in 99 daycare facilities and 48 elementary schools in Bavaria, Germany. Materials and Methods: Overall, 2,568 children (1,337 school children, 1,231 preschool children) and 1,288 adults (466 teachers, 822 daycare staff) consented to participate in the study and were randomly tested in three consecutive phases (September/October 2020, November/December 2020, March 2021). In total, 7,062 throat swabs were analyzed for SARS-CoV-2 by commercial RT-PCR kits. Results: In phase I, only one daycare worker tested positive. In phase II, SARS-CoV-2 was detected in three daycare workers, two preschool children, and seven school children. In phase III, no sample tested positive. This corresponds to a positive test rate of 0.05% in phase I, 0.4% in phase II and 0% in phase III. Correlation of a positive PCR test result with the local-7-day incidence values showed a strong association of a 7-day-incidence of more than 100/100,000 as compared to <100/100,000 (OR = 10.3 [1.5-438], p < 0.005). After phase III, antibody testing was offered to 713 study participants in elementary schools. A seroprevalence rate of 7.7% (students) and 4.5% (teachers) was determined. Discussion: During the initial waves of the SARS-CoV-2 pandemic, the risk of a positive SARS-CoV-2 result correlated positively with the local 7-day incidence. Hence, the occurrence of SARS-CoV-2 infections were reflected in schools and daycare facilities. An increased risk of SARS-CoV-2 transmission in the setting of daycare and elementary schooling was unlikely.
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The taxon names used in public databases are of critical importance in all areas of biology because they are needed for linking organisms to sequence data and other information. Since most users of taxonomic classifications may be unprepared for dealing with synonyms, the names that are preferred in such databases are of high impact. Using the genus Borrelia as an example, we here show how simplistic approaches for determining the preferred synonym may lead to biases regarding the preferences for taxonomic opinions. We highlight that in this and other cases where genera were split, for reverting to the previous "merged" genus it is neither possible nor necessary to generate validly published and legitimate names that are newer than those that were proposed as new combinations when the genus was split. The policy to always prefer the latest validly published name in a public database may thus render this database oblivious to reversals in taxonomic opinion. We emphasize that users of public databases should be aware of such potential shortcomings, and that curators of databases which provide nomenclatural information should be open-minded about taxonomic views expressed in the literature.
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Borrelia , SesgoRESUMEN
BACKGROUND: Five SARS-CoV-2 variants are currently considered as variants of concern (VOC). Omicron was declared a VOC at the end of November 2021. Based on different diagnostic methods, the occurrence of Omicron was reported by 52 countries worldwide on December 7 2021. First notified by South Africa with alarming reports on increasing infection rates, this new variant was soon suspected to replace the currently pre-dominating Delta variant leading to further infection waves worldwide. METHODS: Using VOC PCR screening and Next Generation Sequencing (NGS) analysis of selected samples, we investigated the circulation of Omicron in the German federal state Bavaria. For this, we analyzed SARS-CoV-2 surveillance data from our laboratory generated from calendar week (CW) 01 to 49/2021. RESULTS: So far, we have detected 69 Omicron cases in our laboratory from CW 47-49/2021 using RT-qPCR followed by melting curve analysis. The first 16 cases were analyzed by NGS and all were confirmed as Omicron. CONCLUSION: Our data strongly support no circulation of the new Omicron variant before CW 47/2021.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genéticaRESUMEN
Children have been disproportionately affected during the COVID-19 pandemic. We aimed to assess a saliva-based algorithm for SARS-CoV-2 testing to be used in schools and childcare institutions under pandemic conditions. A weekly SARS-CoV-2 sentinel study in primary schools, kindergartens, and childcare facilities was conducted over a 12-week-period. In a sub-study covering 7 weeks, 1895 paired oropharyngeal and saliva samples were processed for SARS-CoV-2 rRT-PCR testing in both asymptomatic children (n = 1243) and staff (n = 652). Forty-nine additional concurrent swab and saliva samples were collected from SARS-CoV-2 infected patients (patient cohort). The Salivette® system was used for saliva collection and assessed for feasibility and diagnostic performance. For children, a mean of 1.18 mL saliva could be obtained. Based on results from both cohorts, the Salivette® testing algorithm demonstrated the specificity of 100% (95% CI 99.7-100) and sensitivity of 94.9% (95% CI 81.4-99.1) with oropharyngeal swabs as reference. Agreement between sampling systems was 100% for moderate to high viral load situations (defined as Ct-values <33 from oropharyngeal swabs). Comparative analysis of Ct-values derived from saliva vs. oropharyngeal swabs demonstrated a significant difference (mean 4.23; 95% CI 2.48-6.00). In conclusion, the Salivette® system proved to be an easy-to-use, safe and feasible saliva collection method and a more pleasant alternative to oropharyngeal swabs for SARS-CoV-2 testing in children aged 3 years and above.