Appendiceal microbiome in uncomplicated and complicated acute appendicitis: A prospective cohort study.

BACKGROUND: Uncomplicated and complicated acute appendicitis seem to be two different forms of this common abdominal emergency. The contribution of appendiceal microbiota to appendicitis pathogenesis has been suggested, but differences between uncomplicated and complicated appendicitis are largely unknown. We compared the appendiceal microbiota in uncomplicated and complicated acute appendicitis.
METHODS: This prospective single-center clinical cohort study was conducted as part of larger multicenter MAPPAC trial enrolling adult patients with computed tomography or clinically confirmed uncomplicated or complicated acute appendicitis. The microbial composition of the appendiceal lumen was determined using 16S rRNA gene amplicon sequencing.
RESULTS: Between April 11, 2017, and March 29, 2019, 118 samples (41 uncomplicated and 77 complicated appendicitis) were available. After adjusting for age, sex, and BMI, alpha diversity in complicated appendicitis was higher (Shannon p = 0.011, Chao1 p = 0.006) compared to uncomplicated appendicitis. Microbial compositions were different between uncomplicated and complicated appendicitis (Bray-Curtis distance, P = 0.002). Species poor appendiceal microbiota composition with specific predominant bacteria was present in some patients regardless of appendicitis severity.
CONCLUSION: Uncomplicated and complicated acute appendicitis have different appendiceal microbiome profiles further supporting the disconnection between these two different forms of acute appendicitis. STUDY REGISTRATION: ClinicalTrials.gov NCT03257423.

Introduction

With current knowledge, acute appendicitis can be both epidemiologically and clinically classified by severity in two different entities of uncomplicated and complicated acute appendicitis [1-3] allowing a stratified approach to management [2]. The dogma of acute appendicitis inevitably always progressing to perforation without early appendicectomy seems to apply only to complicated acute appendicitis. As non-operative management of uncomplicated appendicitis has been identified as feasible and safe [4-9], it has become very relevant to clinically differentiate between uncomplicated and complicated acute appendicitis. This differential diagnosis still remains challenging and evident findings of complicated acute appendicitis include perforation, abscess or a suspicion of a tumor. The presence of an appendicolith has also been shown to be associated with a more complicated course of the disease [8,9] and should be considered a finding of complicated acute appendicitis not eligible for non-operative treatment [6,7,10]. Despite appendicitis being one of the most common general surgical emergencies worldwide, the ethiology still remains poorly understood and bridging the knowledge gaps in the pathogenesis of these different forms of appendicitis severity is necessary [2].

The understanding of both microbiological etiology of acute appendicitis and the potential differences in etiology and pathogenesis between uncomplicated and complicated acute appendicitis is very limited, but the involvement of bacteria or the appendicular microbiome has been suggested [2]. As majority of bacteria in the appendix are anaerobes, the role of culturing methods is limited and next generation sequencing (NGS) methods are somewhat a necessity in the detection of the microbiological factors in the appendicitis etiology. All studies that have characterised the appendiceal microbiome by NGS show that appendiceal microbiome is highly diverse harboring large interindividual variation in both healthy and inflamed appendixes [11-15]. Members of gram negative Fusobacteria were considered to be infectious, associating with more severe appendicitis in a study using fluorescence in situ hybridization (FISH) [16]. Studies using 16S rRNA gene amplicon sequencing with both adult and pediatric patients have indeed confirmed Fusobacteria as an important microbial factor [11,12,17-19]. In addition, increased levels of Parvimonas and Porphyromonas in pediatric appendicitis samples have been reported [17,18].

To our knowledge, only one previous study utilizing NGS has specifically compared the microbial composition of the appendix in adult patients with uncomplicated and complicated appendicitis showing no significant difference between perforated and unperforated acute appendicitis samples [14]. However, this study was limited by both a very small sample size and lack of standardized clinical definitions of appendicitis severity focusing on the methodological comparison of bacterial culture and NGS. In light of the limited available data, we performed this prospective study consisting of patients with CT-confirmed uncomplicated or complicated acute appendicitis with thorough prospective clinical data and standardized appendix samples aiming to assess the potential differences in appendiceal microbiota composition between uncomplicated and complicated appendicitis using 16S rRNA gene amplicon sequencing.

Methods

Study design and patients

This prospective cohort study was a single-center arm of a multicenter MAPPAC (Microbiology Appendicitis Acuta) trial (ClinicalTrials.gov NCT03257423) and was conducted at Turku University Hospital in Finland. Study design and key methods have been reported previously [20]. The study enrolled adult patients with CT or clinically confirmed either uncomplicated or complicated acute appendicitis undergoing appendicectomy in order to have the appendix both as the reference standard for the clinical diagnosis and the availability of appendiceal samples. MAPPAC trial was conducted concurrently with two randomized controlled trials (RCTs) APPAC II [7] and APPACIII [10] enrolling patients with a CT-confirmed uncomplicated acute appendicitis. APPAC II was an open-label, noninferiority trial comparing oral moxifloxacin with intravenous ertapenem followed by oral levofloxacin and metronidazole. APPAC III was a double-blind, placebo-controlled, superiority study comparing antibiotic therapy (intravenous ertapenem followed by oral levofloxacin and metronidazole) with placebo in the treatment of uncomplicated appendicitis.

Intramural neutrophil invasion in the histopathological examination of the removed appendix was required for the diagnosis of acute appendicitis. Acute appendicitis was defined uncomplicated if no features of complicated acute appenditis was present. Complicated appendicitis was defined as the presence of an appendicolith, perforation, abscess, gangrene, or suspicion of tumor or the combination of these. To validate the accuracy of the differential diagnosis between uncomplicated and complicated acute appendicitis, all patients were assessed using clinical data, CT, surgical, and histopathological findings by two investigators (S.S. and J.H.) unaware of the other’s evaluation. In cases of disagreement, the clinical diagnosis was reviewed by a third investigator (P.S.).

Outcomes

The objective of this study was to compare the microbial composition of an inflamed appendix between uncomplicated and complicated acute appendicitis. Microbial composition was assessed by the relative levels of bacterial phyla, genera, and species. Species richness and diversity, as well as dissimilarity of microbiomes, i.e. beta diversity, were measured and differential abundance analysis was used to identify differentiating phyla, genera, and species associated with appendicitis severity.

Sample collection and processing

Microbiological swabs from the appendiceal lumen were collected right after the appendicectomy. Sample collection and bacterial DNA extraction have been previously described in detail [20]. Briefly, extraction of DNA from appendiceal swabs was performed by semiautomated GXT Stool Extraction kit (Hain Lifescience GmbH, Nehren, Germany). DNA concentration was quantified with Qubit fluorometer (Life Technologies, Carlsbad, California, USA).

16S rRNA amplicon sequencing

16S rRNA amplicon sequencing was performed targeting the V3-V4 hypervariable region. Negative and positive control samples were included in the sequencing: negative DNA extraction control, negative PCR control, and a mock community (ZymoBiomics microbial community DNA standard, Zymo Research, Irvine, California, USA) as a positive control. Amplicon libraries were generated following the Illumina protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) with the exception that increased amount of 75 ng of DNA template was used in the amplicon PCR reaction. Amplicon libraries were quantified using Qubit fluorometer and 10% of Phix (Illumina, San Diego, California, USA) was added to each equimolar pool. Sequencing was performed using MiSeq Reagent Kit v3 and paired-end 2×300 bp protocol on a MiSeq System (Illumina).

Statistical and bioinformatical analysis

Group differences in numerical (age, body mass index (BMI)) and categorical (sex) baseline characteristics were tested with Wilcoxon rank sum test and Pearson´s chi-squared test, respectively. All bioinformatical analyses were performed in R 4.0.3. Amplicon sequence variants (ASVs) of the 16S rRNA gene sequences were formed using the dada2 package, version 1.18.0 [21]. Raw reads were quality filtered and trimmed using the function filterAndTrim. The ASVs were assigned to taxa using the SILVA rRNA (v. 138) database as reference [22-24]. Contaminants, eukaryotes and mitochondrial sequences were removed from bacterial data. Archaeal sequences remained in the data. Decontamination was performed using R package decontam with the method “Prevalence” and a threshold of 0.225 [25]. This method compares prevalence of each ASV in true samples to the prevalence in negative controls to identify contaminants. To ensure that decontamination worked well, results were inspected and manually corrected. In normalization and all further analyses, the samples with more than 1800 sequences were included. Shannon’s index did not strongly correlate with library sizes of samples even in the in non-normalized raw data (Pearson correlation = 0.17). Thus, raw sequence counts of ASVs were transformed to relative abundances that were used in downstream analyses and for alpha diversity calculations [26]. Principal coordinate analysis (PCoA) was calculated using function cmdscale in R package vegan [27] and permutational multivariate analysis of variance (PERMANOVA), with control variables age, sex and BMI, was performed using function multiconstrained in R package BiodiversityR. Both analyses were based on Bray-Curtis dissimilarities. Significantly differentiating bacterial species, genus, and phyla between groups were detected using the DESeq2 method [28]. The p-values were adjusted using Benjamini-Hochberg method and adjusted p-values <0.01 were considered as statistically significant. All the reported p-values related to differential abundance analyses are adjusted p-values. Alpha diversity analyses were performed using standard linear regression models with alpha diversity measure (Chao1, Shannon entropy or number of observed species) as the response variable and appendicitis severity, age, sex, and BMI as the predictors.

Study approval

This study has been accepted by the Ethical Committee of the Hospital district of Southwest Finland (Turku University Hospital) and Finnish Medicines Agency (Fimea). Trial was performed in accordance with the Declaration of Helsinki and all patients and in the case of minors their legal guardians gave written informed consent to participate in the study.

Results

Altogether 308 patients were enrolled in the MAPPAC study between April 11, 2017, and March 29, 2019; Fig 1 shows the study flow. Out of the 169 patients with uncomplicated or complicated acute appendicitis undergoing appendicectomy, 118 (70%) samples were included in the final NGS analysis (41 uncomplicated, 77 complicated appendicitis) with 51 patients excluded due to failed library preparation (n = 26), incomplete sample collection (n = 21), or too low 16S amplicon library size (n = 4). Patient baseline characteristics and the subtypes of complicated appendicitis are presented in Table 1. The groups were similar regarding age and BMI, but the proportion of women in the uncomplicated appendicitis group was higher compared to the complicated appendicitis group (66% vs. 45%, respectively, p = 0.0346). Out of the 77 patients with complicated acute appendicitis, an appendicolith was present in 57 (74%) patients.

Fig 1

Flow chart of the study.

*Appendectomy for primary acute appendicitis. Only patients treated with appendectomy and histopathologically confirmed acute appendicitis study were included in the analysis to have the microbiological sample and to have confirmation for the differential diagnosis. a) Randomized, multicenter, open-label, noninferiority clinical trial comparing oral moxifloxacin with intravenous ertapenem followed by oral levofloxacin and metronidazole. b) Randomized, multicentre, placebo-controlled, double-blind trial comparing antibiotic therapy with placebo in the treatment of uncomplicated acute appendicitis.

Table 1

Patient baseline characteristics and subtypes of complicated appendicitis.

Characteristic	Uncomplicated appendicitis (N = 41)	Complicated appendicitis (N = 77)	P value
Sex, n (%)
Female	27 (66)	35 (45)	0.0346
Male	15 (34)	42 (55)
Age (years), median (range)	37 (16–75)	43 (18–69)	0.7090
Body mass index (kg/m2), median (range)	25.8 (20.4–39.5)	26·6 (18·4–42.7)	0.3395
Complicated subtypes, n (%)
Appendicolith		30 (39.0)
Gangrenous/perforation		14 (18.2)
Gangrenous/perforation and appendicolith		21 (27.3)
Periappendicular abcess (circumscribed closed perforation)		6 (7.8)
Gangrenous/perforation, abscess and appendicolith		3 (3.9)
Abscess and appendicolith		1 (1.3)
Tumour and appendicolith		1 (1.3)
Gangrenous/perforation, tumour and appendicolith		1 (1.3)

Differing microbial signatures in uncomplicated and complicated acute appendicitis

Appendiceal microbiome composition in uncomplicated acute appendicitis was significantly different compared with complicated acute appendicitis regarding both alpha and beta diversities. Alpha diversity indices Shannon and Chao1 describing diversity and richness were lower in uncomplicated acute appendicitis compared to complicated appendicitis samples (Fig 2A, p = 0.011 and p = 0.006, respectively). Similar to alpha diversity, Principal coordinate analysis based on Bray-Curtis dissimilarity (Fig 2B) also indicated different beta diversities in appendiceal microbiota between the two forms of appendicitis severity. Pairwise comparisons revealed significantly different Bray-Curtis distance measure between the two groups (p = 0.002).

Fig 2

The difference in appendiceal microbiota between uncomplicated and complicated acute appendicitis.

A) Violin plot representing alpha diversity measures Shannon index and Chao1 in uncomplicated and complicated acute appendicitis. * p < 0.05 and ** p < 0.01). B) Principal Coordinates Analysis (PCoA), i.e. beta diversity, based on Bray-Curtis distances. The percentage of variation explained by the two first PCoA dimensions is indicated on the respective axes. C) Barplot showing significantly (adj p < 0.01) differentiating species in uncomplicated compared to complicated that were present in meaningful levels (average relative abundance >0.1%). Species are listed along the y-axis and x-axis indicates the log2 fold change. UNK = unknown species.

To investigate which bacteria are responsible for the observed beta diversity difference, the relative abundances of bacteria between groups were compared in phylum, genus, and species level. When a complete taxonomical name was not possible to assign to species level, they are referred as unknown (UNK) species. Bacterial species in which abundance differed statistically significantly (p<0.01) between uncomplicated and complicated acute appendicitis (S1 Table) and that were present at average relative abundance >0.1%, are illustrated in Fig 2C. Seven species that were more abundant in uncomplicated acute appendicitis were Aggregatibacter aphrophilus, B. fragilis, Enterobacteriaceae UNK, Haemophilus UNK, Streptococcus UNK, Veillonella parvula, and Veillonella UNK. Seven species were more abundant in complicated acute appendicitis: Bacteroides faecis, Christensenellaceae UNK, Clostridia UCG-014, Dialister UNK, Oscillospiraceae UCG-005 UNK, Phocaeicola abscessus, and Porphyromonas endodontalis. Genus Aggregatibacter was more abundant in uncomplicated acute appendicitis (adj. p = 0.00016), where the mean relative abundance was 6.4% compared with 1.0% in complicated acute appendicitis. Nine other, significantly different genera were Veillonella and an unknown genus from the family Enterobacteriaceae, which were enriched in uncomplicated acute appendicitis and an unknown genus from Christensenellaceae, Defluviitaleaceae UCG-011 UNK, Family XIII UCG-001, Oscillibacter, Paludicola, Phocaeicola, and Subdoligranulum were more abundant in complicated acute appendicitis (S1 Table). The abundances of bacterial phyla were not significantly different between uncomplicated and complicated acute appendicitis.

Species poor appendiceal microbiomes are predominated by specific bacterial species

The overall microbial profiles of the appendiceal lumen are illustrated at species level in Fig 3 and in phylum and genus level in S1 Fig. Interindividual variation in both the composition and diversity was high in both forms of appendicitis severity. Further, in some samples only few predominant species were present at significant levels reflected in the low values of alpha diversity (Fig 3), while in other samples, hundreds of species were present and comprised an even microbial profile. The appendiceal microbiome was dominated by one bacterial species with a relative abundance of more than 50% in 17% (7/41) and 12% (9/77) of patients with uncomplicated and complicated acute appendicitis, respectively. B. fragilis and Escherichia UNK appeared as predominating species regardless of appendicitis severity. Aggregatibacter aphrophilus or segnis, and Streptococcus UNK (Fig 3) were found to dominate only in uncomplicated appendicitis and Fusobacterium UNK, Haemophilus parainfluenzae, B. faecis, B. dorei or Bacteroides UNK only in complicated appendicitis. Altogether 419 different genera and 599 different species were identified. Mean relative abundances of bacterial species composing more than 0.1% in each group are presented in S2 Table.

Fig 3

Stacked barplots of appendiceal microbiome (n = 118) in species level in uncomplicated and complicated acute appendicitis.

Microbiome profiles of individual appendix samples with the 40 most abundant species plotted and lower abundance species grouped to “other”. On top of barchart is overlaid the number of observed species in each sample as a line chart. UNK = unknown species.

Discussion

In this prospective clinical study on 118 patients with confirmed acute appendicitis, multiple differences between uncomplicated (n = 41) and complicated acute appendicitis (n = 77) patient samples were detected in the microbial composition of the appendiceal lumen supporting the theory of dividing acute appendicitis into two separate forms of acute inflammation processes with different clinical presentation and fates. There were differences in overall alpha and beta diversities between uncomplicated and complicated acute appendicitis. In addition, several bacterial taxa were identified to be significantly different between these two forms of appendicitis severity.

The microbial signatures in uncomplicated and complicated acute appendicitis were different, but in both forms of appendicitis severity, we found several opportunistic pathogens. Patients with uncomplicated acute appendicitis presented with a higher proportion of gram negative Aggregatibacter species (spp) consisting mainly of A. segnis and A. aphrophilus (former Haemophilus aphrophilus). Aggregatibacter belongs to the HACEK group (gram negative bacteria Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella) that are most notable for causing infective endocarditis, but they are also significant causes of periodontitis, abscesses, and septic arthritis [29]. In addition Veillonella parvula and an unknown species from the genus Streptococcus both linked to gastrointestinal tract and oral microbiota [30,31], were found to be enriched in uncomplicated acute appendicitis. In complicated acute appendicitis, a group of gram negative bacteria Porphyromonas endodontalis, an unknown species from the genus Dialister, and Phocaeicola abscessus were more abundant and all of these are considered to be oral pathogens [32,33]. Genera Porphyromonas and Dialister have been previously associated with complicated appendicitis in pediatric patients [18,34].

Microbial diversity was lower in uncomplicated acute appendicitis compared to complicated acute appendicitis. In both forms of appendicitis severity, we observed individual samples with very low diversity. These species poor appendiceal microbiomes were often dominated by one bacteria representing at least 50% of the appendiceal microbiota. The observed predominance may play a role as an etiological factor on an individual level as it could be an indication of infection by these specific predominant species. Both uncomplicated and complicated acute appendicitis had microbial profiles where either B. fragilis or Escherichia UNK were the predominant bacteria, which are well reported in appendicitis [13,35]. A. aphrophilus or A. segnis and Streptococcus UNK were predominant only in patients with uncomplicated acute appendicitis. The most abundant Fusobacteria species F. UNK and F. nucleatum were present in both disease forms. However F. UNK as the predomint species was discovered only in the complicated appendicitis samples. Fusobacteria is the only group prominently associated with appendicitis [12,16] and our results corroborate the previous studies suggesting it to be specifically associated with complicated acute appendicitis [16].

Based on the results of this study, both infection and a disturbance of normal appendiceal microbiota could be etiological factors for some patients in both uncomplicated and complicated acute appendicitis. However, the observed difference in the microbiota diversity suggests that an infection may be more common in uncomplicated acute appendicitis partially associated with different bacterial species. This is further supported by the presence of an appendicolith in the majority (74%) of patients presenting with complicated acute appendicitis as appendicoliths often lead to obstruction of the appendiceal lumen [36]. In large RCTs, the presence of an appendicolith has been shown to be associated with a more complicated course of the disease [8,9].

This study has several limitations. First, a major limitation of the study is the lack of a healthy non-appendicitis control group. The perception of appendicitis as an opportunistic infection, where the abruption of the healthy appendiceal microbiota would drive the inflammation, is gaining ground [12,13]. In the search for etiological agents of a disease where the infection site contains normal microbiota, a healthy control group would be crucial. However, the acquisition of healthy appendix and appendiceal microbiota for this purpose is naturally both clinically and ethically impossible as the negative appendicectomy rate with the current CT diagnostic accuracy is very low and appendicectomy for healthy patients would be unethical. Second, further limitation is that patients received standard prophylactic antibiotics preoperatively, which could have affected the microbial composition of the appendiceal microbiota. However, all patients received antibiotics and the time between antibiotics administration and appendicectomy was relatively short. Third weakness of the study is the utilization of 16S rRNA gene amplicon sequencing, which is partly insufficient in species level analysis. Strain level identification is required in some bacteria to fully estimate the pathogenicity of bacteria present in the sample. For example, it has been suggested that specific virulent strains of E. coli [37] or the enterotoxic strains of B. fragilis [38] might be involved in the infection of the appendix and the resolution of taxonomical information gained in this study characterising appendiceal microbiota can be considered too low to drawn conclusions about the role of these bacteria in different forms of appendicitis severity. Future studies should harness methods, such as shotgun metagenomic sequencing combined with culturing methods, enabling the assessment of appendiceal microbiome function and virulence.

To our knowledge, this is the first prospective study with a large patient cohort and standardized clinical definitions of appendicitis severity comparing the microbial composition of the appendix in adult patients between uncomplicated and complicated acute appendicitis. The thorough prospective clinical data is a major strength in our study in addition to the so far largest number of patients.

In conclusion, uncomplicated and complicated acute appendicitis have different appendiceal microbiome profiles further supporting the disconnection between these two different forms of appendicitis severity.

Appendiceal microbiome in phylum and genus level in uncomplicated and complicated acute appendicitis.

(PDF)

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Differentially abundant bacterial species and genera between uncomplicated and complicated appendicitis.

(PDF)

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Relative abundance of species in uncomplicated and complicated appendicitis.

(PDF)

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Reviewer #1: In this manuscript, the authors describe the examination of the appendiceal microbiome comparing patients with complicated and uncomplicated appendicitis. They appear to show differences in the microbiome as their main finding.

While the idea is great and important, there are significant issues that create confounders. One of the most important issues is that there is no control group. I know that the authors recognize this in their discussion as a limitation. However, this is a huge limitation for a number of reasons outlined below.

In table 1, the authors outline the types of complications. Many of these are concerning in that they might create irregularities in data. For example, an appendicolith may simply be a stool fragment imbedded in the appendix. During sequencing would the microbiome of the appendicolith be what is determined rather than the appendicitis?

Another concern is the perforation. In essences the perforation means the appendix is "draining"...albeit into the peritoneal cavity. Nonetheless it is decompressing the appendix. There are a number of events that might happen in this state. First the pathogenic microbes may diminish. Secondly, the perforated appendix patient may be more symptomatic with systemic symptoms such as fever. As a result is more likely to be on antibiotics. What antibiotics may or may not have been given to subjects? I cannot find a list of antibiotics or the use of antibiotics. This will most certainly influence the microbiome results.

To correct these, at a minimum, the authors should disclose all antibiotic use and to which group these were used. Secondly, the number of days of antibiotics. Also, the number of days that the patients had symptoms and whether fever or other factors were present. Also, to analyzed perforation with out abscesss, appendicolith or tumor, would be helpful.

Reviewer #2: Important note: This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ [like medical importance, relevance of the study, ‘clinical significance and implication(s)’ of the whole study, etc.] are to be evaluated [should be assessed] separately/independently. Further please note that any ‘statistical review’ is generally done under the assumption that (such) study specific methodological [as well as execution] issues are perfectly taken care of by the investigator(s). This review is not an exception to that and so does not cover clinical aspects {however, seldom comments are made only if those issues are intimately / scientifically related & intermingle with ‘statistical aspects’ of the study}. Agreed that ‘statistical methods’ are used as just tools here, however, they are vital part of methodology [and so should be given due importance].

COMMENTS: In my opinion, since this study was a single-center arm of a multicenter MAPPAC (Microbiology Appendicitis Acuta) trial, the study should be treated as having different ‘Study design and methodology’. Therefore, quoting previously published protocol describing these [Study design and key methods have been reported previously (30)] is not correct. This quoted ‘study protocol is for the ‘MAPPAC trial’ and is definitely excellent, however, since this publication is not ‘one of the series’, giving sufficient details regarding ‘Study design and methodology’ are expected. That protocol is for two arms two clinical trials but the present one is single arm study. How can the ‘Study design and methodology’ be same? Therefore, is that really relevant quoting previously published protocol? In ‘Results’ section saying that “Altogether 308 patients were enrolled in the MAPPAC study between April 11, 2017, and March 29, 2019; Figure 1 shows the study flow” do you think has any meaning/relevance?

May I request you to make a small change in title of study [“Appendiceal microbiome in uncomplicated and complicated acute appendicitis: a prospective cohort study (MAPPAC)”] because ‘MAPPAC’ is another trial. This is only a suggestion (as inclusion of the term ‘MAPPAC’) may cause confusion and average reader like me, will take the present study as MAPPAC. You may not follow this as this one is my subjective opinion [please free to take appropriate decision].

Since this study is based on ‘single-arm design’, may I further request to read the following [which is pasted from one standard textbook on ‘Research Methodology’]:

For a pilot study it is alright to have ‘single-arm design’, or it is alright when that is the only possibility’, however, It is very essential to keep the limitations in mind while interpreting results. Further, note that a classical/ideal clinical trial/study needs/requires a concurrently {but similarly} handled/treated appropriately selected/chosen control/comparison parallel group/arm.

Note further that “Inferential statistics (i.e., hypothesis testing + estimation of CI) is built on the population model [which means the underlying assumption is that there is/are population(s) and we are dealing with random sample(s) drawn from that/those population(s)]. Although in clinical trial (involving at least two groups) we do not really deal with random samples (generally a non-probabilistic convenience sampling), ‘allocation’ to treatment groups is ‘randomly’ done which enable us to evoke the population model and we can use inferential statistics safely. But when there is only one group (so that there is no question of random allocation), with ‘non-random’ selection, it may be questionable to use inferential statistics even if you have two measurement sets as ‘pre-post’ or use ‘internal grouping for comparison” [like here uncomplicated acute appendicitis compared to complicated appendicitis but this is an ‘internal grouping’].

I am sure that these learned authors already know these things, however, it is very essential to keep the limitations in mind while interpreting results {note that I am not asking you to change the study design}.

What exactly do you mean by ‘excluded due to failed library preparation (n=26)’? Please note that any ‘regression models / regression techniques’ are not originally developed for head-to-head between group comparison(s), [Alpha diversity analyses were performed using standard linear regression models with alpha diversity measure (Chao1, Shannon entropy or number of observed species) as the response variable]. Nevertheless, limitations of the study highlighted on page 14 are highly appreciated. However, as pointed out in ‘important note’ above “This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ should be assessed separately/independently [one should carefully consider/look at the clinical implications of the study].

One last minor doubt, refer to page 15 where you said “To our knowledge, this is the first prospective study with a large patient cohort and standardized clinical definitions of appendicitis severity comparing the microbial composition of the appendix (may be true) in adult patients between uncomplicated and complicated acute appendicitis” and quoted reference number 14 for this. My doubt is ‘What evidence this reference provided?’ to say you so or ‘what in this reference is indicative of this fact?’.

Except these minor points, in my opinion, this article/manuscript is good and needs a small amount of re-vision (which is quite possible). After minor revision, this manuscript may be accepted [provided clinical implications of these findings/results are valuable].

Reviewer #3: I have read a very interesting manuscript and I provide the following minor queries for the authors to address.

1. Provide a succinct definition of how was a complicated versus a non complicated acute appendicitis defined.

2. Microbiome dysbiosis of the appendix has been recently advanced as causal re the pathogenesis of appendicits, was there scope from the data reported to further highlight appendix microbiome dysbiosis as a feature in this study?

3. The presence of oral bacterial pathogens in the large bowel and consequently the appendix elicits the query as to how do these pathogens travel to these sites. As such then were any patients on proton pump inhibitors that in reducing stomach acid reduces this barrier to orally ingested microbes? This perhaps explaining in part how a pathogen such as Porphyromonas endodontalis is in the appendix.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Luis Vitetta

**********

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24 Aug 2022

Response to Reviewers

PONE-D-22-06832

Appendiceal microbiome in uncomplicated and complicated acute appendicitis: a prospective cohort study (MAPPAC)

PLOS ONE

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Our response: This information has now been added: “Trial was performed in accordance with the Declaration of Helsinki and all patients or in the case of minors, their legal guardians gave written informed consent to participate in the study.”

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“The MAPPAC study was supported by research grants from the Mary and Georg C. Ehrnrooth Foundation, the Sigrid Jusélius Foundation, the Finnish Academy, Government research grant awarded to Turku University Hospital (EVO foundation), The Maud Kuistila Memorial Foundation, Paulo Foundation, Doctoral Programme in Clinical Research of University of Turku, and Turku University foundation.”

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“The MAPPAC study was supported by research grants from the Mary and Georg C. Ehrnrooth Foundation, the Sigrid Jusélius Foundation, the Finnish Academy, Government research grant awarded to Turku University Hospital (EVO foundation), The Maud Kuistila Memorial Foundation, Paulo Foundation, Doctoral Program in Clinical Research at the University of Turku, and Turku University foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”

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Our response: The current funding statement is correct; we have added that in the cover letter and omitted all funding information from the manuscript. Thank you for revising this for us in the online system.

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“EM is currently working as full-time Medical Advisor for Biocodex Nordics. PS reports receiving personal fees for lectures from Merck and Orion Pharma. AJH reports receiving personal fees for lectures from BioCodex, Merck and Pfizer. All other authors declare no competing interests.”

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Our response: We apologize for missing this requirement in the original submission and we have now added the required statement in the competing interests section, which is added to the cover letter.

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Our response: We have revised the data availability statement accordingly.

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Reviewer comments

Reviewer #1

1. In this manuscript, the authors describe the examination of the appendiceal microbiome comparing patients with complicated and uncomplicated appendicitis. They appear to show differences in the microbiome as their main finding.

While the idea is great and important, there are significant issues that create confounders. One of the most important issues is that there is no control group. I know that the authors recognize this in their discussion as a limitation. However, this is a huge limitation for a number of reasons outlined below.

Our response: We thank the reviewer for constructive comments and fully agree that the lack of a healthy control group is a clear limitation as we have stated in the study limitations. However, as stated in the manuscript, it would be both clinically and ethically impossible to have a control group as there is no clinical indication of appendectomy for an uninflamed appendix. Removal of a healthy appendix is associated with similar complication rate as appendectomy for acute appendicitis and furthermore, the role of the appendix in general is still unclear underlining the impossibility of subjecting patients to appendectomy to achieve a healthy control group. Negative appendicectomy rate is currently very low due to high accuracy of the preintervention CT diagnostics, i.e. these few cases would not constitute such a healthy control group that would be needed. These issues have already been stated in the study limitations as follows and no further additions have been made: “However, the acquisition of healthy appendix and appendiceal microbiota for this purpose is naturally both clinically and ethically impossible as the negative appendicectomy rate with the current CT diagnostic accuracy is very low and appendicectomy for healthy patients would be unethical.”

2. In table 1, the authors outline the types of complications. Many of these are concerning in that they might create irregularities in data. For example, an appendicolith may simply be a stool fragment imbedded in the appendix. During sequencing would the microbiome of the appendicolith be what is determined rather than the appendicitis?

Our response: We thank you the reviewer for pointing out this important fact. Most recognized definition of the complicated appendicitis is the presence of gangrene, abscess, or perforation. Further, the presence of appendicolith has been shown to be associated with a more complicated form of the disease. However, in this patient cohort and in all concurrent trials, an appendicolith was a clearly visible concretion on CT and not a merely a stool fragment in the appendix. The appendicoliths can easily be avoided and have been avoided during the sample collection, i.e. our analysis actually implies the appendiceal microbiome and not the microbiome of an appendicolith. We do recognize that these cannot be fully isolated from each other and that the presence of and appendicolith may very well have an effect on the appendiceal microbiome.

3. Another concern is the perforation. In essences the perforation means the appendix is "draining"...albeit into the peritoneal cavity. Nonetheless it is decompressing the appendix. There are a number of events that might happen in this state. First the pathogenic microbes may diminish. Secondly, the perforated appendix patient may be more symptomatic with systemic symptoms such as fever. As a result is more likely to be on antibiotics. What antibiotics may or may not have been given to subjects? I cannot find a list of antibiotics or the use of antibiotics. This will most certainly influence the microbiome results.

Our response: We agree with the reviewer that perforated appendicitis does exactly decompress the appendix and as it is complicated acute appendicitis, it may be associated with more systemic symptoms as stated by the reviewer. Please see our response to comment #4 for the antibiotic use. In addition, the number of patients in the subgroups of complicated acute appendicitis are very small limiting the ability of any subgroup analysis in providing comparative outcomes data between these different groups. Thus these groups are only described in table 1 as no conclusions can be drawn in comparing these groups as pointed out by reviewer #2, comment #3.

4. To correct these, at a minimum, the authors should disclose all antibiotic use and to which group these were used. Secondly, the number of days of antibiotics. Also, the number of days that the patients had symptoms and whether fever or other factors were present. Also, to analyzed perforation with out abscesss, appendicolith or tumor, would be helpful.

Our response: We agree with the reviewer that the antibiotic usage is a very relevant point and thank the reviewer for pointing out this. For acute appendicitis, there is standard practice of antibiotic prophylaxis 30 minutes prior to appendectomy and this is valid for all appendicitis cases, i.e. none of the patients, not even the complicated cases, have a longer preoperative antibiotic duration. The effect of mere antibiotic prophylaxis given 30 minutes preoperatively presumably has either little or no effect to the appendiceal microbiota. As appendectomy is performed within the first 6-8 hours especially for the complicated acute appendicitis cases, even these patients did not have any major effect of antibiotics on the appendiceal microbiota. We have now clarified this information in the Methods section as follows:

“Study design and patients: The study enrolled adult patients having either uncomplicated or complicated acute appendicitis confirmed either CT or clinically. Patients are undergoing appendicectomy in order to have the appendix both as the reference standard for the clinical diagnosis and the availability of appendiceal samples. According to standard clinical practice, patients received antibiotic prophylaxis as a single dose of 1.5 g cefuroxime and 500 mg metronidazole approximately 30 minutes prior to appendectomy initiation. None of the patients had long antibiotic treatment durations preoperatively.”

In addition, the sequencing method does not actually differentiate between dead and living bacteria.

The duration of symptoms from a clinical point may not be that relevant as we have the information on appendicitis severity from a combination of CT, surgical finding, and histopathology. Fever is an important clinical parameter, but most likely with the differential diagnosis based on the previously mentioned findings, analyzing patient temperatures most likely may not add to the current analysis. In addition, the patients may have taken or received antipyretic medications further adding potential bias in adding this information. However, if this is evaluated necessary by the editors, patient temperature on admission can naturally be reported for both groups.

Regarding the subgroup analysis of the patients with perforation only, we agree with the reviewer that it would be interesting to assess the potential differences of the various forms of complicated acute appendicitis. For this, the number of patients is too low to draw any conclusions (n=14) and we have thus not performed this subgroup analysis

Reviewer #2

Important note: This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ [like medical importance, relevance of the study, ‘clinical significance and implication(s)’ of the whole study, etc.] are to be evaluated [should be assessed] separately/independently. Further please note that any ‘statistical review’ is generally done under the assumption that (such) study specific methodological [as well as execution] issues are perfectly taken care of by the investigator(s). This review is not an exception to that and so does not cover clinical aspects {however, seldom comments are made only if those issues are intimately / scientifically related & intermingle with ‘statistical aspects’ of the study}. Agreed that ‘statistical methods’ are used as just tools here, however, they are vital part of methodology [and so should be given due importance].

COMMENTS:

1. In my opinion, since this study was a single-center arm of a multicenter MAPPAC (Microbiology Appendicitis Acuta) trial, the study should be treated as having different ‘Study design and methodology’. Therefore, quoting previously published protocol describing these [Study design and key methods have been reported previously (30)] is not correct. This quoted ‘study protocol is for the ‘MAPPAC trial’ and is definitely excellent, however, since this publication is not ‘one of the series’, giving sufficient details regarding ‘Study design and methodology’ are expected. That protocol is for two arms two clinical trials but the present one is single arm study. How can the ‘Study design and methodology’ be same? Therefore, is that really relevant quoting previously published protocol? In ‘Results’ section saying that “Altogether 308 patients were enrolled in the MAPPAC study between April 11, 2017, and March 29, 2019; Figure 1 shows the study flow” do you think has any meaning/relevance?

Our response: We thank the reviewer for the thorough feedback and understand the issues pointed out by the reviewer. However, from a clinical perspective it is of vital importance to describe the recruitment of the patients in the larger MAPPAC trial in concurrence with the APPAC II and APPAC III trials as this is the basis for describing the whole patient population to the readers adding to the understanding of how representative this single-center study arm patient population is of the larger patient cohort. We have already stated the single-center arm nature of this study in the methods and we have further clarified the study rationale in the Methods section as follows.

”This prospective cohort study was a single-center arm of a multicenter MAPPAC (Microbiology Appendicitis Acuta) trial (ClinicalTrials.gov NCT03257423) and was conducted at Turku University Hospital in Finland. Study rationale and key methods have been reported previously (20).”

2. May I request you to make a small change in title of study [“Appendiceal microbiome in uncomplicated and complicated acute appendicitis: a prospective cohort study (MAPPAC)”] because ‘MAPPAC’ is another trial. This is only a suggestion (as inclusion of the term ‘MAPPAC’) may cause confusion and average reader like me, will take the present study as MAPPAC. You may not follow this as this one is my subjective opinion [please free to take appropriate decision].

Our response: Thank you for the suggestion. Please see our response to comment #1, this study is a key part of the MAPPAC trial, but we do understand the reviewer’s point of view and we have omitted the trial name from the title.

3. Since this study is based on ‘single-arm design’, may I further request to read the following [which is pasted from one standard textbook on ‘Research Methodology’]:

For a pilot study it is alright to have ‘single-arm design’, or it is alright when that is the only possibility’, however, It is very essential to keep the limitations in mind while interpreting results. Further, note that a classical/ideal clinical trial/study needs/requires a concurrently {but similarly} handled/treated appropriately selected/chosen control/comparison parallel group/arm.

Note further that “Inferential statistics (i.e., hypothesis testing + estimation of CI) is built on the population model [which means the underlying assumption is that there is/are population(s) and we are dealing with random sample(s) drawn from that/those population(s)]. Although in clinical trial (involving at least two groups) we do not really deal with random samples (generally a non-probabilistic convenience sampling), ‘allocation’ to treatment groups is ‘randomly’ done which enable us to evoke the population model and we can use inferential statistics safely. But when there is only one group (so that there is no question of random allocation), with ‘non-random’ selection, it may be questionable to use inferential statistics even if you have two measurement sets as ‘pre-post’ or use ‘internal grouping for comparison” [like here uncomplicated acute appendicitis compared to complicated appendicitis but this is an ‘internal grouping’].

I am sure that these learned authors already know these things, however, it is very essential to keep the limitations in mind while interpreting results {note that I am not asking you to change the study design}.

Our response: We thank the reviewer for the constructive criticism and the MAPPAC study in itself is not a randomized controlled trial, but the patients recruited to the MAPPAC trial were partially patients enrolled in two concurrent RCTs and partially patients excluded from these trials. This current single-center study is a prospective cohort study, as stated in the title, comparing the appendiceal microbiota in uncomplicated and complicated acute appendicitis in patients excluded from the RCTs as all of these patients in this single-center study underwent appendectomy. Based on the study design, we fully agree with the reviewer that this limitation has to be kept in mind when interpreting the results as the results are more descriptive and comparison between the two groups is more limited in this study design. We have thus aimed to focus on only describing the microbial findings i.e. bacterial profiles and signatures within the samples and further report potential associations between appendicitis severity and appendiceal microbiota. Since uncomplicated and complicated appendicitis are recognized as two different forms of acute appendicitis, the comparisons are essentially made between two populations with representative samples rather than internal grouping comparison.

We have now further underlined this limitation by revising the conclusion as follows. Abstract and Discussion conclusion: “Uncomplicated and complicated acute appendicitis seem to have different appendiceal microbiome profiles further supporting the disconnection between these two different forms of acute appendicitis.”

4. What exactly do you mean by ‘excluded due to failed library preparation (n=26)’? Please note that any ‘regression models / regression techniques’ are not originally developed for head-to-head between group comparison(s), [Alpha diversity analyses were performed using standard linear regression models with alpha diversity measure (Chao1, Shannon entropy or number of observed species) as the response variable]. Nevertheless, limitations of the study highlighted on page 14 are highly appreciated. However, as pointed out in ‘important note’ above “This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ should be assessed separately/independently [one should carefully consider/look at the clinical implications of the study].

Our response: In Results section, we report that 26 samples originally collected were not included in final statistical analyses. The PCR amplification step in 16S rRNA amplicon library preparation protocol of these 26 samples failed to produce enough DNA in order to proceed to sequencing. Criteria for a successful library preparation has now been added to methods section.

”16S rRNA amplicon sequencing

16S rRNA amplicon sequencing was performed targeting the V3-V4 hypervariable region. Negative and positive control samples were included in the sequencing: negative DNA extraction control, negative PCR control, and a mock community (ZymoBiomics microbial community DNA standard, Zymo Research, Irvine, California, USA) as a positive control. Amplicon libraries were generated following the Illumina protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) with the exception that increased amount of 75 ng of DNA template was used in the amplicon PCR reaction. Amplicon PCR products were verified (size, integrity) with DNA agarose gel and library preparation was considered successful if a clear and right sized (550 bp) DNA band was seen on gel. Amplicon libraries were quantified using Qubit fluorometer and 10% of Phix (Illumina, San Diego, California, USA) was added to each equimolar pool. Sequencing was performed using MiSeq Reagent Kit v3 and paired-end 2×300 bp protocol on a MiSeq System (Illumina).”

5. One last minor doubt, refer to page 15 where you said “To our knowledge, this is the first prospective study with a large patient cohort and standardized clinical definitions of appendicitis severity comparing the microbial composition of the appendix (may be true) in adult patients between uncomplicated and complicated acute appendicitis” and quoted reference number 14 for this. My doubt is ‘What evidence this reference provided?’ to say you so or ‘what in this reference is indicative of this fact?’.

Our response: Thank you for pointing out this mistake as the reference should not be there. We have now removed the reference from the sentence.

Except these minor points, in my opinion, this article/manuscript is good and needs a small amount of re-vision (which is quite possible). After minor revision, this manuscript may be accepted [provided clinical implications of these findings/results are valuable].

Reviewer #3

I have read a very interesting manuscript and I provide the following minor queries for the authors to address.

1. Provide a succinct definition of how was a complicated versus a non complicated acute appendicitis defined.

Our response: We thank the reviewer for the suggestions and have now added the more accurate definition to manuscript:

” The CT criteria for uncomplicated acute appendicitis included appendiceal diameter exceeding 6 mm with thickened and enhanced appendiceal wall and periappendiceal edema and /or minor fluid collection. Intramural neutrophil invasion in the histopathological examination of the removed appendix was required for the diagnosis of acute appendicitis. Acute appendicitis was defined uncomplicated if no features of complicated acute appenditis was present. Complicated appendicitis was defined as the presence of an appendicolith, perforation, abscess, gangrene, or suspicion of tumor or the combination of these.”

2. Microbiome dysbiosis of the appendix has been recently advanced as causal re the pathogenesis of appendicits, was there scope from the data reported to further highlight appendix microbiome dysbiosis as a feature in this study?

Our response: Thank you for disclosing the interesting concept of microbiome dysbiosis. However, the definition of the dysbiosis in this study field is still very much in progress, and this is the case especially related to appendicitis. Our primary aim was simply to describe the microbial ecosystems within the appendix and evaluate the possible differences in the signatures and profiles between the two forms of appendicitis severity. For the future trials, we agree with the reviewer that microbiome dysbiosis of the appendix needs to be addressed after bridging these existing knowledge gaps of the appendiceal microbiome.

3. The presence of oral bacterial pathogens in the large bowel and consequently the appendix elicits the query as to how do these pathogens travel to these sites. As such then were any patients on proton pump inhibitors that in reducing stomach acid reduces this barrier to orally ingested microbes? This perhaps explaining in part how a pathogen such as Porphyromonas endodontalis is in the appendix.

Our response: This is an interesting consideration pointed out by the reviewer. We do agree that the oral microbiome could have an effect on the appendiceal microbiome and this effect could be further enhanced by proton pump inhibitors. However, to investigate the role of specific oral pathogens such as Porphyromonas endodontalis in appendicitis etiology to our opinion it would require more profound analysis approach, such as shotgun metagenomic sequencing, in order to reach strain level identification of possible pathogens. The presence of oral bacterial pathogens and the medications potentially affecting the orally ingested microbes needs to be included in the scope of future research.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Sep 2022

Appendiceal microbiome in uncomplicated and complicated acute appendicitis: a prospective cohort study

PONE-D-22-06832R1

Dear Dr. Paulina Salminen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

PLOS ONE

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Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

Reviewer #3: Yes

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Reviewer #2: (No Response)

Reviewer #3: Yes

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Reviewer #2: (No Response)

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: (No Response)

Reviewer #3: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: COMMENTS: Since most of the comments made on earlier draft [though not all were/are attended/followed and I am not very much convinced for reasons given or arguments made (for example, in response to one query/comment you said “Since uncomplicated and complicated appendicitis are recognized as two different forms of acute appendicitis, the comparisons are essentially made between two populations with representative samples rather than internal grouping comparison”) with which I do not agree at all. I never said (and I can very-well understand it) that uncomplicated and complicated appendicitis are recognized as two different forms of acute appendicitis but how can the comparison was between two populations with representative samples when samples were not selected from these two populations?) and so definitely not happy], I recommend the acceptance, because the manuscript now has achieved acceptable level [I had earlier said that after minor revision (suggested), this manuscript may be accepted].

Reviewer #3: I have read the revised manuscript with all queries that have been addressed and as such I have no further remakrs to add.

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Reviewer #2: Yes: Dr. Sanjeev Sarmukaddam

Reviewer #3: Yes: Luis Vitetta

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6 Oct 2022

PONE-D-22-06832R1

Appendiceal microbiome in uncomplicated and complicated acute appendicitis: a prospective cohort study

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