Diagnostic Accuracy of Blind Bronchial Sample Testing by BioFire Pneumonia plus Panel in Pediatric Intensive Care Unit Patients and Its Impact in Early Adaptation of Antimicrobial Therapy: A Prospective Observational Study.

BACKGROUND: Community-acquired and nosocomial lower-respiratory-tract infections in critically ill pediatric patients require early appropriate antibiotic therapy to optimize outcomes. Using blind bronchial samples, we assessed the diagnostic performance of the rapid-multiplex polymerase chain reaction (PCR) assay BioFire Pneumonia plus Panel vs. reference standard culturing with antimicrobial susceptibility testing. METHODS: For this prospective observational study in a single pediatric intensive care unit, we included consecutive patients younger than 18 years admitted for suspected community-, hospital- or ventilator-associated pneumonia in 2021-2022. Sensitivity, specificity, positive predictive value and negative predictive value of the multiplex PCR assay were determined. The kappa coefficient was computed to assess agreement, and univariate analyses were done to identify factors associated with discrepancies between the 2 diagnostic methods. RESULTS: Of the 36 included patients (median age, 1.4 years; interquartile range, 0.2-9.2), 41.7%, 27.8%, and 30.5% had community-, hospital- and ventilator-associated pneumonia, respectively. The overall κ was 0.74, indicating good agreement. Overall, the sensitivity of the multiplex PCR assay was 92% (95% CI: 77%-98%) and specificity 95% (95% CI: 92%-97%), with variations across microorganisms. The median time from sample collection to antimicrobial susceptibility test results was 3.9 (2.5-15) hours with the multiplex PCR assay and 60.5 (47.6-72.2) hours with the reference technique. CONCLUSION: The BioFire Pneumonia plus Panel used to test blind bronchial samples had satisfactory diagnostic performance in critically ill pediatric patients. The rapid results provided by this test may improve the appropriateness of antimicrobial therapy and help minimize the use of antibiotics.

Sporadic Shiga Toxin-Producing Escherichia coli-Associated Pediatric Hemolytic Uremic Syndrome, France, 2012-2021.

Shiga toxin-producing Escherichia coli-associated pediatric hemolytic uremic syndrome (STEC-HUS) remains an important public health risk in France. Cases are primarily sporadic, and geographic heterogeneity has been observed in crude incidence rates. We conducted a retrospective study of 1,255 sporadic pediatric STEC-HUS cases reported during 2012-2021 to describe spatiotemporal dynamics and geographic patterns of higher STEC-HUS risk. Annual case notifications ranged from 109 to 163. Most cases (n = 780 [62%]) were in children <3 years of age. STEC serogroups O26, O80, and O157 accounted for 78% (559/717) of cases with serogroup data. We identified 13 significant space-time clusters and 3 major geographic zones of interest; areas of southeastern France were included in >5 annual space-time clusters. The results of this study have numerous implications for outbreak detection and investigation and research perspectives to improve knowledge of environmental risk factors associated with geographic disparities in STEC-HUS in France.

Fatal Meningitis from Shiga Toxin-Producing Escherichia coli in 2 Full-Term Neonates, France.

We report fatal meningitis in 2 neonates in France caused by Shiga toxin 1-producing Escherichia coli. Virulence factors capsular K1 antigen and salmochelin were present in both strains, potentially representing a new hybrid pathotype. Clinicians should remain aware of emerging pathotypes and design therapeutic strategies for neonatal E. coli infections.

Case Report: Hyperammonemic Encephalopathy Linked to Ureaplasma spp. and/or Mycoplasma hominis Systemic Infection in Patients Treated for Leukemia, an Emergency Not to Be Missed.

Background: Hyperammonemic encephalopathy caused by Ureaplasma spp. and Mycoplasma hominis infection has been reported in immunocompromised patients undergoing lung transplant, but data are scarce in patients with hematological malignancies. Case Presentation: We describe the cases of 3 female patients aged 11-16 years old, developing initially mild neurologic symptoms, rapidly evolving to coma and associated with very high ammonia levels, while undergoing intensive treatment for acute leukemia (chemotherapy: 2 and hematopoietic stem cell transplant: 1). Brain imaging displayed cerebral edema and/or microbleeding. Electroencephalograms showed diffuse slowing patterns. One patient had moderate renal failure. Extensive liver and metabolic functions were all normal. Ureaplasma spp. and M. hominis were detected by PCR and specific culture in two patients, resulting in prompt initiation of combined antibiotics therapy by fluoroquinolones and macrolides. For these 2 patients, the improvement of the neurological status and ammonia levels were observed within 96 h, without any long-term sequelae. M. hominis was detected post-mortem in vagina, using 16S rRNA PCR for the third patient who died of cerebral edema. Conclusion: Hyperammonemic encephalopathy linked to Ureaplasma spp. and M. hominis is a rare complication encountered in immunocompromised patients treated for acute leukemia, which can lead to death if unrecognized. Combining our experience with the few published cases (n=4), we observed a strong trend among female patients and very high levels of ammonia, consistently uncontrolled by classical measures (ammonia-scavenging agents and/or continuous kidney replacement therapy). The reversibility of the encephalopathy without sequelae is possible with prompt diagnosis and adequate combined specific antibiotherapy. Any neurological symptoms in an immunocompromised host should lead to the measurement of ammonia levels. If increased, and in the absence of an obvious cause, it should prompt to perform a search for Ureaplasma spp. and M. hominis by PCR as well as an immediate empirical initiation of combined specific antibiotherapy.

Improved Molecular Diagnosis and Culture of the Emerging Heteropathotype Enterohemorrhagic Escherichia coli O80:H2 Using Its Non-Melibiose-Fermenting and Antibiotic-Resistance Properties.

Enterohemorrhagic Escherichia coli (EHEC) O80:H2, belonging to sequence type ST301, is among the main causes of hemolytic and uremic syndrome in Europe, a major concern in young children. Aside from the usual intimin and Shiga toxin virulence factors (VFs), this emerging serotype possesses a mosaic plasmid combining extra-intestinal VF- and antibiotic resistance-encoding genes. This hybrid pathotype can be involved in invasive infections, a rare occurrence in EHEC infections. Here, we aimed to optimize its detection, improve its clinical diagnosis, and identify its currently unknown reservoir. O80:H2 EHEC strains isolated in France between 2010 and 2018 were phenotypically and genetically analyzed and compared with non-O80 strains. The specificity and sensitivity of a PCR test and a culture medium designed, based on the molecular and phenotypic signatures of O80:H2 EHEC, were assessed on a collection of strains and stool samples. O80:H2 biotype analysis showed that none of the strains (n = 137) fermented melibiose versus 5% of non-O80 EHEC (n = 19/352). This loss of metabolic function is due to deletion of the entire melibiose operon associated with the insertion of a 70-pb sequence (70mel), a genetic scar shared by all ST301 strains. This metabolic hallmark was used to develop a real-time PCR test (100% sensitivity, 98.3% specificity) and a melibiose-based culture medium including antibiotics, characterized by 85% specificity and sensitivity for clinical specimens. These new tools may facilitate the diagnosis of this atypical clone, help the food industry to identify the reservoir and improve our epidemiological knowledge of this threatening and emerging clone.

Azithromycin Resistance in Shiga Toxin-Producing Escherichia coli in France between 2004 and 2020 and Detection of mef(C)-mph(G) Genes.

We described and characterized Shiga-toxin-producing Escherichia coli (STEC) strains with high levels of resistance to azithromycin isolated in France between 2004 and 2020. Nine of 1,715 (0.52%) STEC strains were resistant to azithromycin, with an increase since 2017. One isolate carried a plasmid-borne mef(C)-mph(G) gene combination, described here for the first time for E. coli. Azithromycin resistance, although rare, needs consideration, as this treatment may be useful in cases of STEC infection.

Clinical impact of a gastrointestinal PCR panel in children with infectious diarrhoea.

OBJECTIVES: Multiplex gastrointestinal PCR (GI-PCR) allows fast and simultaneous detection of 22 enteric pathogens (including Campylobacter, Salmonella, Shigella/enteroinvasive Escherichia coli (EIEC), among other bacteria, parasites and viruses). However, its impact on the management of children with infectious diarrhoea remains unknown. PATIENTS/DESIGN: All children eligible for stool culture from May to October 2018 were prospectively included in a monocentric study at Robert-Debré University-Hospital. INTERVENTION: A GI-PCR (BioFire FilmArray) was performed on each stool sample. MAIN MEASURES: Data on the children's healthcare management before and after GI-PCR results were collected. Stool culture results were also reported. RESULTS: 172 children were included. The main criteria for performing stool analysis were mucous/bloody diarrhoea and/or traveller's diarrhoea (n=130). GI-PCR's were positive for 120 patients (70%). The main pathogens were enteroaggregative E. coli (n=39; 23%), enteropathogenic E. coli (n=34; 20%), Shigella/EIEC (n=27; 16%) and Campylobacter (n=21; 12%). Compared with stool cultures, GI-PCR enabled the detection of 21 vs 19 Campylobacter, 12 vs 10 Salmonella, 27 Shigella/EIEC vs 13 Shigella, 2 vs 2 Yersinia enterocolitica, 1 vs 1 Plesiomonas shigelloides, respectively. Considering the GI-PCR results and before stool culture results, the medical management was revised for 40 patients (23%): 28 initiations, 2 changes and 1 discontinuation of antibiotics, 1 hospitalisation, 2 specific room isolations related to Clostridioides difficile infections, 4 additional test prescriptions and 2 test cancellations. CONCLUSION: The GI-PCR's results impacted the medical management of gastroenteritis for almostone-fourth of the children, and especially the prescription of appropriate antibiotic treatment before stool culture results.

Emergence of New ST301 Shiga Toxin-Producing Escherichia coli Clones Harboring Extra-Intestinal Virulence Traits in Europe.

O80:H2 enterohemorrhagic Escherichia coli (EHEC) of sequence type ST301 is one of the main serotypes causing European hemolytic and uremic syndrome, but also invasive infections, due to extra-intestinal virulence factors (VFs). Here, we determined whether other such heteropathotypes exist among ST301. EnteroBase was screened for ST301 strains that were included in a general SNP-phylogeny. French strains belonging to a new heteropathotype clone were sequenced. ST, hierarchical clusters (HC), serotype, resistome, and virulome were determined using EnteroBase, the CGE website, and local BLAST. The ST301 general phylogeny shows two groups. Group A (n = 25) is mainly composed of enteropathogenic E. coli, whereas group B (n = 55) includes mostly EHEC. Three serotypes, O186:H2, O45:H2 and O55:H9, share the same virulome as one of the O80:H2 sub-clones from which they derive subsequent O-antigen switches. The O55:H9 clone, mainly present in France (n = 29), as well as in the UK (n = 5) and Germany (n = 1), has a low background of genetic diversity (four HC20), although it has three Stx subtypes, an H-antigen switch, and genes encoding the major extra-intestinal VF yersiniabactin, and extended-spectrum beta-lactamases. Diverse heteropathotype clones genetically close to the O80:H2 clone are present among the ST301, requiring close European monitoring, especially the virulent O55:H9 clone.

Clavulanate combinations with mecillinam, cefixime or cefpodoxime against ESBL-producing Enterobacterales frequently associated with blaOXA-1 in a paediatric population with febrile urinary tract infections.

OBJECTIVES: Oral treatment of febrile urinary tract infections (FUTIs) can be impaired by MDR Enterobacterales often combining ESBL and inhibitor-resistant genes. We studied the impact of ß-lactamases and Enterobacterales' genotypes on the cefixime, cefpodoxime and mecillinam ± amoxicillin/clavulanate MICs. MATERIALS AND METHODS: In this multicentric study, we included 251 previously whole-genome-sequenced ESBL-producing Enterobacterales, isolated in French children with FUTIs. The MICs of cefixime, cefpodoxime, mecillinam alone and combined with amoxicillin/clavulanate were determined and analysed with respect to genomic data. We focused especially on the isolates' ST and their type of ß-lactamases. Clinical outcomes of patients who received cefixime + amoxicillin/clavulanate were also analysed. RESULTS: All isolates were cefixime and cefpodoxime resistant. Disparities depending on blaCTX-M variants were observed for cefixime. The addition of amoxicillin/clavulanate restored susceptibility for cefixime and cefpodoxime in 97.2% (MIC50/90 of 0.38/0.75 mg/L) and 55.4% (MIC50/90 of 1/2 mg/L) of isolates, respectively, whatever the ST, the blaCTX-M variants or the association with inhibitor-resistant ß-lactamases (34.2%). All isolates were susceptible to mecillinam + amoxicillin/clavulanate with MIC50/90 of 0.19/0.25 mg/L, respectively. Neither therapeutic failure nor any subsequent positive control urine culture were reported for patients who received cefixime + amoxicillin/clavulanate as an oral relay therapy (n = 54). CONCLUSIONS: Despite the frequent association of ESBL genes with inhibitor-resistant ß-lactamases, the cefixime + amoxicillin/clavulanate MICs remain low. The in vivo efficacy of this combination was satisfying even when first-line treatment was ineffective. Considering the MIC distributions and pharmacokinetic parameters, mecillinam + amoxicillin/clavulanate should also be an alternative to consider when treating FUTIs in children.

Shiga Toxin-Associated Hemolytic Uremic Syndrome in Adults, France, 2009-2017.

We conducted a retrospective study on hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC) in 96 adults enrolled in the cohort of the National Reference Center for Thrombotic Microangiopathies network in France during 2009-2017. Most infections were caused by STEC strains not belonging to the O157 or O104 serogroups. Thirty (31.3%) patients had multiple risk factors for thrombotic microangiopathy. In total, 61 (63.5%) patients required dialysis, 50 (52.1%) had a serious neurologic complication, 34 (35.4%) required mechanical ventilation, and 19 (19.8%) died during hospitalization. We used multivariate analysis to determine that the greatest risk factors for death were underlying immunodeficiency (hazard ratio 3.54) and severe neurologic events (hazard ratio 3.40). According to multivariate analysis and propensity score-matching, eculizumab treatment was not associated with survival. We found that underlying conditions, especially immunodeficiency, are strongly associated with decreased survival in adults who have hemolytic uremic syndrome caused by STEC.

Contribution of Serological Rapid Diagnostic Tests to the Strategy of Contact Tracing in Households Following SARS-CoV-2 Infection Diagnosis in Children.

Background: The contact tracing and isolation of contagious individuals are cornerstones in the control of the COVID-19 pandemic. Strategies to identify household contacts who should be isolated around index children that tested positive for SARS-CoV-2 remain to be clarified. We aimed to compare contact tracing strategies around an index child positive for SARS-CoV-2 using serological rapid diagnostic testing (RDT, chromatography immunoassay). Methods: We conducted a contact tracing study in households of index cases children in the Paris region, France, between May 8 and July 27, 2020. We compared two strategies, one using SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) and one combining RT-PCR and serological RDT, initiated once RDT was available. The contacts RT-PCR-/RDT+ were considered to have been previously infected and not requiring quarantine. The primary outcome was the proportion of contacts that could avoid quarantine with the two screening strategies. Results: We included 34 children as index cases. Median age was 7 years. They generated 184 contacts (111 adults, 73 children) tested by RT-PCR: 24/184 (13%) were positive. The strategy combining RDT and RT-PCR was performed in 120/184 contacts (77 adults, 43 children) of 26 index children: 16/120 (13%) were RT-PCR+ and 47/120 (39%) were RDT+. Among the 16 individuals who were RT-PCR+, 14 (87%) were also RDT+. Among the 104 individuals who were RT-PCR-, 33 were RDT+. Hence 33/120 (27%) individuals were not isolated. Conclusions: Following the diagnosis of SARS-CoV-2 infection in children, a strategy combining serological RDT and nasopharyngeal RT-PCR enabled us to identify around one fourth of contacts with past infection and avoid unnecessary quarantine of these individuals.

Cross-resistance to cefiderocol and ceftazidime-avibactam in KPC ß-lactamase mutants and the inoculum effect.

OBJECTIVES: Ceftazidime-avibactam (CZA) and cefiderocol are recently commercialized molecules active against highly drug-resistant bacteria, including carbapenem-resistant members of the Enterobacteriaceae. Mutants resistant to CZA have been described, notably in Klebsiella pneumoniae carbapenemase (KPC) producers. Considering the structural similarities between ceftazidime and cefiderocol, we hypothesized that resistance to CZA in KPC-producing members of the Enterobacterales may lead to cross-resistance to cefiderocol. METHODS: CZA-resistant mutants from three clinical isolates of the Enterobacterales carrying either blaKPC-2 or blaKPC-3 were selected in vitro. Mutants with increased MIC to CZA compared to the ancestral allele were cloned in a pBR322 plasmid and expressed in Escherichia coli TOP10. We evaluated the impact of these mutations on cefiderocol MICs and minimal bactericidal concentrations (MBCs), and we assessed the impact of bacterial inoculum size on cefiderocol MICs. RESULTS: We used 37 KPC mutants with increased CZA MICs. Of these, six have been described previously in clinical isolates. Compared to the wild-type alleles, increases in the cefiderocol MICs of 4- to 32-fold were observed for 75.6% of tested mutants (28/37), MICs reaching up to 4 mg/L in E. coli TOP10 for KPC-31 (D179Y-H274Y mutations). MBCs and MICs of cefiderocol were similar, confirming the bactericidal activity of this drug. Finally, when using higher inocula (107 CFU/mL), a large increase in cefiderocol MIC was observed, and all isolates were categorized as resistant. CONCLUSION: We observed that most of the CZA-resistant KPC variants have a possible impact on cefiderocol by increasing the cefiderocol MICs. In addition, cefiderocol is greatly impacted by the inoculum effect, suggesting that precautions should be taken when treating infections with a suspected high inoculum.

Shiga Toxin-Associated Hemolytic Uremic Syndrome: Specificities of Adult Patients and Implications for Critical Care Management.

Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is a form of thrombotic microangiopathy secondary to an infection by an enterohemorrhagic E. coli. Historically considered a pediatric disease, its presentation has been described as typical, with bloody diarrhea at the forefront. However, in adults, the clinical presentation is more diverse and makes the early diagnosis hazardous. In this review, we review the epidemiology, most important outbreaks, physiopathology, clinical presentation and prognosis of STEC-HUS, focusing on the differential features between pediatric and adult disease. We show that the clinical presentation of STEC-HUS in adults is far from typical and marked by the prevalence of neurological symptoms and a poorer prognosis. Of note, we highlight knowledge gaps and the need for studies dedicated to adult patients. The differences between pediatric and adult patients have implications for the treatment of this disease, which remains a public health threat and lack a specific treatment.

Tuberculosis in Children Hospitalized in a Low-burden Country: Description and Risk Factors of Severe Disease.

BACKGROUND: In high-income countries, few pediatric studies have described the clinical expression of tuberculosis (TB) according to age, and their results are discordant. Patients <2 years of age are usually considered to be at higher risk for severe disease than older children. Our aim was to better describe pediatric TB disease severity in a low-incidence country. METHODS: All children (<18 years of age) admitted with TB disease to the Robert Debré University Hospital, Paris, between 1992 and 2015 were included. Patients were classified by the severity of TB disease based on the original classification of Wiseman et al. Risk factors associated with severity were analyzed. RESULTS: We included 304 patients with a median age of 9.9 years (interquartile range 3.3-13.3) and a male to female ratio of 1.04. Overall, 280/304 (92%) were classified: 168/304 (55%) were classified as showing severe TB and 112/304 (37%) as showing non-severe TB. Central nervous system disease was more frequent among patients <2 years of age than patients 2-17 years of age (5/54; 9% vs. 5/229; 2% P = 0.024). An age of ≥10 years (P = 0.001) and being born abroad (P = 0.011) were both associated with disease severity in univariate analysis. In multivariate analysis, diagnosis through symptom-based screening was independently associated with severity (odds ratio 7.1, 95% confidence interval: 3.9-12.9, P < 0.0001). CONCLUSIONS: This description of the clinical spectrum of pediatric TB in a low-burden setting demonstrates that adolescents are the group most at risk of experiencing severe TB.

KPC Beta-Lactamases Are Permissive to Insertions and Deletions Conferring Substrate Spectrum Modifications and Resistance to Ceftazidime-Avibactam.

To explore the mutational possibilities of insertions and deletions (indels) in the Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase, we selected for ceftazidime-avibactam-resistant mutants. Of 96 screened mutants, we obtained 19 indels (2 to 15 amino acids), all located in the loops surrounding the active site. Three antibiotic susceptibility phenotypes emerged: an extended-spectrum-beta-lactamase-like phenotype, an activity restricted to ceftazidime, and a carbapenem-susceptible KPC-like phenotype. Tolerance for indels reflects the evolvability of KPC beta-lactamase, which could challenge the therapeutic management of patients.

Rapid Detection and Characterization of Carbapenemases in Enterobacterales with a New Modified Carbapenem Inactivation Method, mCIMplus.

The worldwide emergence and spread of antimicrobial resistance in Gram-negative bacteria are severely limiting therapeutic options and thus constitute a major public health threat. The timely accurate detection of carbapenemase producers and the determination of carbapenemase class according to the Ambler classification can guide antimicrobial therapy and facilitate infection control measures. A modified version of the carbapenemase inactivation method (CIM), mCIM, was described and approved by the CLSI in 2017. We evaluated the performance of a faster new mCIM-based assay, mCIMplus, which can detect carbapenemase activity within 8 h and characterize the carbapenemase according to the Ambler classification in 20 h. A panel of 137 isolates producing carbapenemases (GES, IMP, KPC, NDM, OXA-48, OXA-48-like, and VIM enzymes) and 22 non-carbapenemase-producing isolates was used to evaluate the performance of mCIMplus. We evaluated the detection of carbapenemase activity at 8 and 20 h. Carbapenemase class was determined, with specific inhibitors, at 20 h. The sensitivities of mCIMplus were 99.3% at 8 h and 98.5% at 20 h. Its specificity was 100% regardless of culture time. Based on a decision algorithm, this test successfully identified the carbapenemase class for 98.4% of the tested isolates (127/129). Characterization was correct for 100, 95, and 100% of Ambler class A, B, and D isolates, respectively. This test can, therefore, be used to detect carbapenemase activity within 8 h and to determine carbapenemase class within 20 h. It constitutes a very affordable (<€1 per isolate) and reliable technique requiring only basic laboratory equipment.

Be aware of Shiga-toxin 2f-producing Escherichia coli: case report and false-negative results with certain rapid molecular panels.

We report a hemolytic uremic syndrome (HUS) case due to Stx2f-producing E. coli illustrating the diagnostic difficulty of this Shiga-toxin subtype. Clinicians should be aware of limits of certain rapid molecular panels that are increasingly being used and may play a role in underestimating the global burden of such infections.