A targeted LC-MRM3 proteomic approach for the diagnosis of SARS-CoV-2 infection in nasopharyngeal swabs.

Since its first appearance, SARS-CoV-2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein (NCAP). A highly selective multiple reaction monitoring MRM3 strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral NCAP peptides from nasopharyngeal swabs samples presenting a cycle threshold (Ct) value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immuno-purification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with Ct-values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted mass spectrometry.

Taxonomic and metabolic diversity of Actinomycetota isolated from faeces of a 28,000-year-old mammoth.

Ancient environmental samples, including permafrost soils and frozen animal remains, represent an archive with microbial communities that have barely been explored. This yet unexplored microbial world is a genetic resource that may provide us with new evolutionary insights into recent genomic changes, as well as novel metabolic pathways and chemistry. Here, we describe Actinomycetota Micromonospora, Oerskovia, Saccharopolyspora, Sanguibacter and Streptomyces species were successfully revived and their genome sequences resolved. Surprisingly, the genomes of these bacteria from an ancient source show a large phylogenetic distance to known strains and harbour many novel biosynthetic gene clusters that may well represent uncharacterised biosynthetic potential. Metabolic profiles of the strains display the production of known molecules like antimycin, conglobatin and macrotetrolides, but the majority of the mass features could not be dereplicated. Our work provides insights into Actinomycetota isolated from an ancient source, yielding unexplored genomic information that is not yet present in current databases.

Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.

Prediction of Bloodstream Infection in Pediatric Acute Leukemia by Microbiota and Volatile Organic Compounds Analysis.

INTRODUCTION: Bloodstream infections (BSIs) cause treatment-related mortality in pediatric acute leukemia. We explored the potential of intestinal microbiota and fecal volatile organic compounds (VOCs) analyses to predict BSI. METHODS: In this case-control study, fecal samples of pediatric acute leukemia patients were collected. Microbiota composition and fecal VOC profiles of BSI cases and matched non-BSI controls were compared. RESULTS: In total, 6 patients were included, of which 1 developed BSI and 1 neutropenic fever. Both showed reduced microbial diversity and stability of Bacteroidetes. In the BSI case, Pantoea was identified 15 days before BSI. Significant differences in fecal VOC profiles were measured between the case and controls. CONCLUSION: Microbiota and fecal VOC could serve as biomarkers to predict BSI in pediatric leukemia.

The potential of rectal swabs to differentiate simple and complex appendicitis in children with a microbiota-based test.

Currently, accurate biomarkers differentiating simple (phlegmonous) from complex (gangrenous and/or perforated) appendicitis in children are lacking. However, both types may potentially require different treatment strategies, and the search for diagnostic modalities remains warranted. Previously, we demonstrated a distinct microbiota (both an increased bacterial diversity and abundance) in the appendix of children with complex compared to simple appendicitis. From the same cohort of patients we have collected 35 rectal swabs under general anesthesia prior to appendectomy and microbiota analysis was performed by IS-pro, a 16S-23S rDNA-based clinical microbiota profiling technique. Using the obtained IS-profiles, we performed cluster analyses (UPGMA), comparison of diversity (Shannon Diversity Index) and intensity (abundance in relative fluorescence units) on phylum level, and comparison on species level of bacteria between simple and complex appendicitis. Regarding these analyses, we observed no clear differences between simple and complex appendicitis. However, increased similarity of the microbial composition of the appendix and rectal swab was found within children with complex compared to simple appendicitis. Furthermore, PLS-DA regression analysis provided clear visual differentiation between simple and complex appendicitis, but the diagnostic power was low (highest AUC 0.65).   Conclusion: Microbiota analysis of rectal swabs may be viable to differentiate between simple and complex appendicitis prior to surgery as a supervised classification model allowed for discrimination of both types. However, the current diagnostic power was low and further validation studies are needed to assess the value of this method. What is Known: • Simple and complex appendicitis in children may require different treatment strategies, but accurate preoperative biomarkers are lacking. • Clear differentiation can be made between both types in children based upon the microbial composition in the appendix. What is New: • Increased similarity was found between the microbial composition of the appendix and rectal swab within children with complex compared to simple appendicitis. • Using a supervised classification model rectal swabs may be viable to discriminate between simple and complex appendicitis, but the diagnostic power was low.

Esophageal microbiota composition and outcome of esophageal cancer treatment: a systematic review.

BACKGROUND: The role of esophageal microbiota in esophageal cancer treatment is gaining renewed interest, largely driven by novel DNA-based microbiota analysis techniques. The aim of this systematic review is to provide an overview of current literature on the possible association between esophageal microbiota and outcome of esophageal cancer treatment, including tumor response to (neo)adjuvant chemo(radio)therapy, short-term surgery-related complications, and long-term oncological outcome. METHODS: A systematic review of literature was performed, bibliographic databases were searched and relevant articles were selected by two independent researchers. The Newcastle-Ottawa scale was used to estimate the quality of included studies. RESULTS: The search yielded 1303 articles, after selection and cross-referencing, five articles were included for qualitative synthesis and four studies were considered of good quality. Two articles addressed tumor response to neoadjuvant chemotherapy and described a correlation between high intratumoral Fusobacterium nucleatum levels and a poor response. One study assessed surgery-related complications, in which no direct association between esophageal microbiota and occurrence of complications was observed. Three studies described a correlation between shortened survival and high levels of intratumoral F. nucleatum, a low abundance of Proteobacteria and high abundances of Prevotella and Streptococcus species. CONCLUSIONS: Current evidence points towards an association between esophageal microbiota and outcome of esophageal cancer treatment and justifies further research. Whether screening of the individual esophageal microbiota can be used to identify and select patients with a predisposition for adverse outcome needs to be further investigated. This could lead to the development of microbiota-based interventions to optimize esophageal microbiota composition, thereby improving outcome of patients with esophageal cancer.

Profound Pathogen-Specific Alterations in Intestinal Microbiota Composition Precede Late-Onset Sepsis in Preterm Infants: A Longitudinal, Multicenter, Case-Control Study.

BACKGROUND: The role of intestinal microbiota in the pathogenesis of late-onset sepsis (LOS) in preterm infants is largely unexplored but could provide opportunities for microbiota-targeted preventive and therapeutic strategies. We hypothesized that microbiota composition changes before the onset of sepsis, with causative bacteria that are isolated later in blood culture. METHODS: This multicenter case-control study included preterm infants born under 30 weeks of gestation. Fecal samples collected from the 5 days preceding LOS diagnosis were analyzed using a molecular microbiota detection technique. LOS cases were subdivided into 3 groups: gram-negative, gram-positive, and coagulase-negative Staphylococci (CoNS). RESULTS: Forty LOS cases and 40 matched controls were included. In gram-negative LOS, the causative pathogen could be identified in at least 1 of the fecal samples collected 3 days prior to LOS onset in all cases, whereas in all matched controls, this pathogen was absent (P = .015). The abundance of these pathogens increased from 3 days before clinical onset. In gram-negative and gram-positive LOS (except CoNS) combined, the causative pathogen could be identified in at least 1 fecal sample collected 3 days prior to LOS onset in 92% of the fecal samples, whereas these pathogens were present in 33% of the control samples (P = .004). Overall, LOS (expect CoNS) could be predicted 1 day prior to clinical onset with an area under the curve of 0.78. CONCLUSIONS: Profound preclinical microbial alterations underline that gut microbiota is involved in the pathogenesis of LOS and has the potential as an early noninvasive biomarker.

Composition of the vaginal microbiota during pregnancy in women living in sub-Saharan Africa: a PRISMA-compliant review.

BACKGROUND: The vaginal microbiota (VMB) are the set of microorganisms residing in the human vagina. During pregnancy, their composition is Lactobacillus-dominant in most Caucasian women. Previous studies suggest that the VMB of women with African ancestry is more likely to be non-Lactobacillus dominant (dysbiotic) compared to other populations, and possibly relate to the high incidence of pregnancy complications, such as preterm birth. This work reviewed the literature on VMB composition in pregnant women from sub-Saharan Africa. METHODS: A search was conducted in PubMed and Embase databases following PRISMA guidelines. Observational and intervention studies analysing VMB communities from sub-Saharan African pregnant women using molecular techniques were included. RESULTS: Ten studies performed in seven sub-Saharan African countries were identified. They independently showed that Lactobacillus-dominant VMB (particularly L. iners or L. crispatus) or VMB containing Lactobacilli are the most prevalent, followed by a more diverse anaerobe-dominant VMB, in the studied populations. The majority of pregnant women with a sexually-transmitted infection had a Lactobacillus-dominant VMB, but with a significantly higher presence of anaerobic species. CONCLUSION: In agreement with studies performed in other populations, Lactobacillus species are the most prevalent VMB species during pregnancy in sub-Saharan African women. The frequency of diverse anaerobe-dominant VMB is high in these populations. In Africa, studies on VMB in pregnancy are scant, heterogeneous in methodology, and knowledge remains limited. More insights on VMB composition and their possible sequalae among these populations is needed.

Role of Toll-Like Receptor 5 (TLR5) in Experimental Melioidosis.

The Gram-negative intracellular pathogen Burkholderia pseudomallei is the causative agent of melioidosis, an important cause of sepsis in Southeast Asia. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is essential for an appropriate immune response during pathogen invasion. In patients with melioidosis, TLR5 is the most abundantly expressed TLR, and a hypofunctional TLR5 variant has been associated with improved survival. Here, we studied the functional role of TLR5 and its ligand flagellin in experimental melioidosis. First, we observed differential TLR5 expression in the pulmonary and hepatic compartments upon infection with B. pseudomallei Next, we found that B. pseudomallei-challenged TLR5-deficient (Tlr5-/- ) mice were more susceptible to infection than wild-type (WT) mice, as demonstrated by higher systemic bacterial loads, increased organ injury, and impaired survival. Lung bacterial loads were not different between the two groups. The phenotype was flagellin independent; no difference in in vivo virulence was observed for the flagellin-lacking mutant MM36 compared to the wild-type B. pseudomallei strain 1026b. Tlr5-/- mice showed a similar impaired antibacterial defense when infected with MM36 or 1026b. Ex vivo experiments showed that TLR5-deficient macrophages display markedly impaired phagocytosis of B. pseudomallei In conclusion, these data suggest that TLR5 deficiency has a detrimental flagellin-independent effect on the host response against pulmonary B. pseudomallei infection.

The Skin Microbiota in Patients Hospitalized for Cellulitis and Association With Outcome.

BACKGROUND: The skin microbiota plays a key role in the pathogenesis of several skin diseases, but its role in cellulitis remains unknown. We investigated the skin microbiota in patients with cellulitis, studied whether its analysis could help determine the causative pathogen, and explored whether skin microbiota composition was associated with clinical outcomes. METHODS: We prospectively included 58 patients hospitalized for cellulitis. Skin swabs obtained from the lesion sites were compared with swabs from identical sites on the contralateral unaffected limbs and with swabs obtained from 19 age- and sex-matched control subjects without cellulitis. Bacterial profiling of the skin microbiota was performed by interspacer profiling (IS-pro). RESULTS: A large interpersonal variation in the skin microbiota composition of patients hospitalized with cellulitis was observed. Firmicutes were the dominant phylum, and Staphylococcus and Streptococcus the dominant genera. In most patients, a strong correlation between the microbiota of the affected lesion and the microbiota of the unaffected, contralateral limb was seen. Overall, the composition of the cellulitis microbiota could not be distinguished from the skin microbiota of controls. No consistent association could be found between traditional culture results and skin microbiota signatures in patients with cellulitis. Lastly, we found that neither microbiota composition nor diversity were associated with clinical parameters and outcomes in patients with cellulitis. CONCLUSIONS: In this exploratory study on the skin microbiota in patients hospitalized with cellulitis, we were unable to identify a typical cellulitis microbiota. The diagnostic and prognostic information that could be derived from skin microbiota profiling in this patient cohort was limited. CLINICAL TRIALS REGISTRATION: NCT02032654.

Effect of Daily Intake of Lactobacillus casei on Microbial Diversity and Dynamics in a Healthy Pediatric Population.

Emerging evidence exists that an altered gut microbiota is a key factor in the pathophysiology of a variety of diseases. Consequently, microbiota-targeted interventions, including administration of probiotics, have increasingly been evaluated. Mechanisms on how probiotics contribute to homeostasis or reverse (effects of) dysbiosis remain yet to be elucidated. In the current study, we assessed the effects of daily Lactobacillus casei strain Shirota (LcS) ingestion in healthy children aged from 12-18 years on gut microbiota compositional diversity and stability. Results were compared to healthy children without LcS exposure. For a period of 6 weeks, fecal samples were collected weekly by both groups. In total, 18 children were included (6 probiotics; 12 non-probiotics). At 1-week intervals, no differences in diversity and stability were observed in children exposed to LcS versus controls. LcS ingestion by healthy children does not result in a more diverse and stable gut microbiota composition. Large double-blind placebo-controlled randomized clinical trials in children should be performed to gain more insight on potential beneficial health consequences.

PCR Characterization of Microbiota on Contracted and Non-Contracted Breast Capsules.

BACKGROUND: The aetiology of capsular contracture around breast implants remains unclear. The leading theory is that a subclinical infection around the implant plays a role in the development of capsular contractions. Several studies found associations between the presence of bacteria and the occurrence of capsular contraction. However, it is unclear whether detected bacteria originate from the breast capsule, breast glandular tissue or skin contamination. Moreover, this has never been investigated with molecular techniques. The aim of this study was to assess the bacterial microbiota on breast capsules, glandular tissue and skin using a highly sensitive PCR assay. MATERIALS AND METHODS: Fifty breast capsules were collected during implant removal or replacement. Ten specimens of glandular breast tissue and breast skin were collected in females who were undergoing reduction mammoplasty. A sample specimen (4 mm) was sterilely obtained from all tissues. All specimens were analysed by IS-pro, a 16S-23S interspace region-based PCR assay. RESULTS: Low numbers of Staphylococcus spp. (four species in four capsules) were found on breast capsules. There was no difference in bacterial presence between normal and contracted capsules. The skin of the breast-harboured Streptococcus spp. and Staphylococcus spp. while the glandular tissue was sterile. CONCLUSION: The low numbers of bacteria found on the capsules are most likely caused by contamination during capsule removal. More and larger studies are needed to investigate the bacterial presence on breast capsules using a PCR assay. This is the first study in which breast capsules have been studied using a highly sensitive PCR assay. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Development of severe bronchopulmonary dysplasia is associated with alterations in fecal volatile organic compounds.

BackgroundThe aim of this study was to evaluate the potential of fecal volatile organic compounds (VOCs), obtained by means of an electronic nose device (Cyranose 320), as early non-invasive biomarker for BPD.MethodsIn this nested case-control study performed at three Neonatal Intensive Care Units, fecal samples obtained at postnatal age of 7, 14, 21, and 28 days from preterm infants with severe bronchopulmonary dysplasia (BPD) were compared with fecal VOC profiles from matched controls. Microbiota analysis was performed by means of IS-pro technique on fecal samples collected at 28 days postnatally.ResultsVOC profiles of infants developing severe BPD (n=15) could be discriminated from matched controls (n=15) at postnatal age of 14 days (area under the curve (±95% confidence interval), P-value, sensitivity, specificity; 0.72 (0.54-0.90), 0.040, 60.0%, 73.3%), 21 days (0.71 (0.52-0.90), 0.049, 66.7%, 73.3%) and 28 days (0.77 (0.59-0.96), 0.017, 69.2%, 69.2%) but not at 7 days. Intestinal microbiota did not differ between BPD subjects and controls.ConclusionFecal VOC profiles of infants developing BPD could be differentiated from controls at postnatal day 14, 21, and 28. VOC differences could not be directed to intestinal microbiota alterations but presumably reflect local and systemic metabolic and inflammatory pathways associated with BPD.

Composition and stability of intestinal microbiota of healthy children within a Dutch population.

Numerous diseases linked to microbial imbalance can be traced back to childhood, illustrating the impact of the juvenile microbiota development from infancy toward adulthood. However, knowledge on this subject is currently very limited. The primary aim of this study was to characterize composition and short- and long-term stability of the intestinal microbiota in healthy children. Between November 2011 and June 2014, 61 children 2 to 18 yr of age from different areas in The Netherlands were included and instructed to collect fecal samples weekly, for 6 wk, and a follow-up sample after 18 mo. The intergenic spacer profiling technique (IS-pro) was used to analyze all available fecal samples. Microbial diversity was calculated by the Shannon diversity index and individual compositional stability by comparing all collection time points. Microbial stability varied per phylum (P< 0.0005), declined rapidly in a short time period, and subsequently stabilized on the long run with very gradual variation, leading to an overall compositional stability of 70% on average over a period of 18 mo. Higher species diversity was correlated to a higher overall compositional stability (P< 0.001). We observed an age-independent bacterial shared core consisting of a limited number of species. In conclusion, in this study, we showed that microbial composition stability in children varied per phylum, at both short-term and long-term intervals. Healthy children seem to share a microbiome core consisting of a limited number of species.-De Meij, T. G. J., Budding, A. E., de Groot, E. F. J., Jansen, F. M., Kneepkens, C. M. F., Benninga, M. A., Penders, J., van Bodegraven, A. A., Savelkoul, P. H. M. Composition and stability of intestinal microbiota of healthy children within a Dutch population.

Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples.

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice.

Composition and diversity of the duodenal mucosa-associated microbiome in children with untreated coeliac disease.

BACKGROUND: Intestinal microbiome may play a role in the pathogenesis of coeliac disease (CD). Studies comparing intestinal microbiome in children with and without CD are contradictory. AIM: To compare the composition and diversity of the duodenal mucosa-associated microbiome in children with untreated CD and control children without CD and to identify specific gut bacteria associated with CD at diagnosis. METHODS: Total microbiome profile in small bowel biopsies of 42 children (21 with untreated CD and 21 age-matched controls) were analyzed by means of IS-pro, a 16S-23S interspacer (IS) region-based profiling method. RESULTS: Both groups showed a similar mucosa-associated microbiome pattern and diversity, with high concentrations of the genera Streptococcus, Lactobacillus, and Clostridium. CONCLUSION: Mucosa-associated duodenal microbiome composition and diversity did not differ between children with untreated CD and control children. Duodenal mucosa-associated bacteria do not seem to play an important role in the pathogenesis of CD.

Bacterial Contamination Is Involved in the Etiology of Soft-Tissue Filler, Late-Onset, Inflammatory Adverse Events.

BACKGROUND: The treatment algorithm in late-onset inflammatory adverse events with soft-tissue fillers depends primarily on the assumed causative factor: immunologic or bacterial. METHODS: The authors included 29 patients, 13 of whom experienced late-onset inflammatory adverse events to fillers (inflammatory group) and 16 who did not (reference group). Biopsies were acquired from both groups with an 18-G needle. Before taking the biopsy, the authors acquired skin swabs for 25 of the 29 patients. The IS-pro method-a new and very sensitive method to detect microbiota-was used. This is a novel broad-range polymerase chain reaction technique based on length and sequence variations of the 16S to 23S ribosomal interspacer region. IS-pro can detect bacteria at low abundances and identify them up to species level. To exclude contamination from skin microbiota, the authors compared the microbiota found on skin swabs with that found in the corresponding biopsies. RESULTS: A high level of Gram-positive bacteria was found in biopsies of soft-tissue fillers, predominantly in patients from the inflammation group. This suggests that these bacteria were introduced during the primary filler injection treatment. The composition of the microbiota on the skin differed markedly from that in the filler, indicating that contamination during the sampling process did not influence results. CONCLUSIONS: Bacteria adherent to soft-tissue fillers or bacteremia probably play a causative role in adverse events. Contamination of samples in the biopsies with skin microbiota was excluded. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Enhanced Th17 responses in the appendix of children with complex compared to simple appendicitis are associated with microbial dysbiosis.

Introduction: Appendicitis is one of the most common causes of acute abdominal surgery in children. The clinical course of appendicitis ranges from simple to complex appendicitis. The mechanisms underlying the heterogeneity of appendicitis in children remain largely unclear. Dysregulated T cell responses play an important role in several inflammatory diseases of the intestine, but the extend of T cell dysregulation in appendicitis in children is less well known. Methods: To characterize appendiceal T cells in simple and complex appendicitis we performed in-depth immunophenotyping of appendiceal-derived T cells by flow cytometry and correlated this to appendiceal-derived microbiota analyses of the same patient. Results: Appendix samples of twenty children with appendicitis (n = 8 simple, n = 12 complex) were collected. T cells in complex appendicitis displayed an increased differentiated phenotype compared to simple appendicitis, including a loss of both CD27 and CD28 by CD4+ T cells and to a lesser extent by CD8+ T cells. Frequencies of phenotypic tissue-resident memory CD69+CD4+ T cells and CD69+CD8+ T cells were decreased in children with complex compared to simple appendicitis, indicating disruption of local tissue-resident immune responses. In line with the increased differentiated phenotype, cytokine production of in particular IL-17A by CD4+ T cells was increased in children with complex compared to simple appendicitis. Furthermore, frequencies of IL-17A+ CD4+ T cells correlated with a dysregulation of the appendiceal microbiota in children with complex appendicitis. Conclusion: In conclusion, disruption of local T cell responses, and enhanced pro-inflammatory Th17 responses correlating to changes in the appendiceal microbiota were observed in children with complex compared to simple appendicitis. Further studies are needed to decipher the role of a dysregulated network of microbiota and Th17 cells in the development of complex appendicitis in children.