Appendiceal spirochaetosis in children
© Westerman et al.; licensee BioMed Central Ltd. 2013
Received: 19 November 2013
Accepted: 2 December 2013
Published: 13 December 2013
Acute appendicitis is a surgical emergency in which the appendix is surgically removed to prevent peritonitis due to perforation of the appendix. Depending on age and gender, up to 17% of removed appendices do not show the histopathological changes pathognomonic for acute appendicitis and are called ‘pseudo-appendicitis’. Intestinal spirochaetes have been reported in up to 12.3% of these non-inflamed appendices obtained from adults. Although children carry the highest risk for acute appendicitis, not much is known on the prevalence of intestinal spirochaetes in children. The aim of this study was to determine whether there is an association between pseudo-appendicitis and appendiceal spirochaetosis in children.
Archival appendix specimens from paediatric patients (less than 18 years old) were obtained from two Dutch hospitals (acute appendicitis, n = 63; pseudo-appendicitis, n = 55; control appendices, n = 33) and microscopically analysed by H&E staining and spirochaete-specific immunohistochemistry and Brachyspira species specific real-time PCR.
Five out of 142 appendices were found to be positive, all in male patients: one in the acute appendicitis group, two in the pseudo-appendicitis group and two in the control group.
The results obtained do not provide evidence for a role of Brachyspira species infection in the aetiology of acute appendicitis in children.
KeywordsAppendicitis Spirochaetes Brachyspira species Human intestinal spirochaetosis Appendiceal spirochaetosis Real-time PCR
Acute appendicitis is a clinical syndrome characterized by peri-umbilical pain and/or pain in the right lower abdominal quadrant, anorexia, fever, vomiting, and signs of generalized or localized peritoneal irritation (guarding or rebound tenderness). Definitive proof of acute appendicitis is the histopathological evaluation of the removed appendix: acute appendicitis is characterized by a massive invasion of neutrophils in the entire appendiceal wall, usually combined with focal or extensive ulceration and/or obliteration of the mucosa. The pathogenic mechanisms for this inflammatory process have remained unclear, despite numerous research-efforts on this subject. The term pseudo-appendicitis is used to describe clinical conditions mimicking acute appendicitis, where the removed appendix fails to show the characteristic histopathological changes defining ‘true acute appendicitis’.
Numerous infectious agents have been implied in the aetiology of acute appendicitis, such as common intestinal pathogens (Salmonella species, Campylobacter species, Clostridium species, various intestinal parasites) and intestinal spirochaetes[1–7]. The latter are anaerobic gram-negative bacteria and are occasionally found in the colon and appendix of humans with abdominal complaints where they cause a condition called human intestinal spirochaetosis (HIS). Human intestinal spirochaetosis was first described by Harland and Lee in 1967 and is characterized by the attachment of spirochaetes to the epithelium of the colonic mucosa (Additional file1: Figure S1). In 1982, Hovind-Hougen et al. reported the first culture of a spirochaete isolated from a colon biopsy-sample, and named it Brachyspira aalborgi. Our knowledge regarding both the prevalence and pathogenic potential of these putative human pathogens is scarce, as they can only be cultured under strict anaerobic conditions. Therefore, microscopic examination of biopsy samples is the gold standard for diagnosing intestinal spirochaetes in humans, but sensitivity may decrease in acute appendicitis where the mucosal structure is disrupted. PCR detection on formalin-fixed paraffin-embedded (FFPE) samples does not depend on intact mucosal structure and also allows species identification, as was demonstrated recently.
In veterinary medicine, intestinal spirochaetes are recognized as important pathogens in pigs (B. hyodysenteriae and B. pilosicoli)[12, 13] and poultry (B. intermedia and B. pilosicoli)[14–16]. Only two species have ever been isolated from humans: Brachyspira aalborgi and Brachyspira pilosicoli[11, 17–19], and there are reports of a third species tentatively named “B. hominis”[11, 20–22]. However, this species has recently been shown to be a 16S-rDNA variant of B. aalborgi. While B. pilosicoli has been isolated on numerous occasions, B. aalborgi is notoriously difficult to culture, with only a few successful descriptions of its isolation in the literature[10, 21, 24–26].
Results from two recent studies provided evidence for an association between B. pilosicoli and inflammatory changes in colon biopsy specimens and B. pilosicoli-induced pathological changes in cultured Caco-2 cells in vitro[11, 27]. Yet, the pathogenicity of this bacterium for humans has not been unequivocally demonstrated.
The first observation of spirochaetes and appendicitis stems from 1911. Thiroloix and Durand described a 35 year-old woman with acute appendicitis with a spirochaete isolated from her blood. Twenty years later, in 1930, Mazza examined 394 appendices using light-microscopy and identified spirochaetes in 9.6%. Unfortunately, he did not differentiate between acute appendicitis and pseudo-appendicitis. Since then the role of spirochaetes in appendices removed for either histopathological acute appendicitis, pseudo-appendicitis and/or other reasons has been investigated and a prevalence of up to 12.3% has been observed in pseudo-appendicitis[2–7]. However, all evidence stems from samples obtained from adults or is not corrected for age, whereas children are at risk for acute appendicitis. The aim of this paper was to investigate whether Brachyspira species are associated with pseudo-appendicitis. This was tested by investigating the presence of Brachyspira species in appendices of patients between 2 and 18 years of age with acute appendicitis, pseudo-appendicitis and without clinical symptoms of appendicitis obtained from the histopathological archives of two Dutch hospitals.
Materials and methods
Patients, and/or their legal representatives, visiting a Dutch hospital are actively informed of the ‘opt-out’ system regarding research on archival patient material. Dutch law requires all studies using such materials to obtain an official approval by the local ethics committee. This study was approved by the Medical Ethics Committee of the University Medical Centre Utrecht, The Netherlands, under protocol number 11-198/C. Only material from patients that did not opt-out has been included in this study.
Selection of archival appendix-specimens
Formalin-fixed paraffin-embedded (FFPE) appendix resection specimens collected between May 1988 and February 2011 from paediatric patients (between 2 and 18 years old) were selected retrospectively from stored collections in two Dutch hospitals (University Medical Centre Utrecht, Utrecht and Tergooiziekenhuizen, Hilversum). Three clinico-pathological groups were created: histopathologically proven acute appendicitis, pseudo-appendicitis and a control group (surgically removed for non-acute, non-inflammatory pathology). Per group the aim was to select 60 samples, equally distributed over three age groups (2 < 6, 6 < 12 and 12 < 18 years) and with equal gender distribution within the group. Samples were selected consecutively in time (starting from 2010 and going back to 1988) from the automated archive, based on the following criteria: clinical suspicion of acute appendicitis (both true appendicitis and pseudo-appendicitis) or appendices removed for other surgical reasons (non-acute, non-inflammatory pathology). Samples were excluded if the pathology report mentioned total fibrous obliteration of the appendiceal mucosa, obstruction due to tumours or other architectural disturbances, extensive mucosal ulceration and when not enough material remained to process the FFPE-sample as required.
Of each FFPE-sample, three 4 μm sections were cut and mounted on glass-slides for histopathology, and two subsequent 20 μm sections were cut and placed in two separate 1.5 ml Eppendorf tubes (Eppendorf, Hamburg, Germany), then three additional 4 μm controls were cut and mounted on glass-slides. The knife was then discarded and the microtome cleaned thoroughly with 96% ethanol to prevent contamination of the next FFPE-block with DNA from the previous sample. The tubes were frozen at -80°C until DNA isolation.
Samples were processed by a semi-automatic deparaffinization protocol on a VERSANT® kPCR Sample Prep Machine according to the protocol supplied by the manufacturer (Siemens Healthcare Diagnostics, Breda, The Netherlands). Each DNA-isolation run included a negative control (HPLC-grade water). Each sample was centrifuged at 15,000 g for one minute, then 700 μl buffer consisting of 10 mmol/L tris(hydroxymethyl)aminomethane (TRIS), 0.1 mmol/l ethylenediaminetetraacetic acid (EDTA), 50 g/l sodium dodecyl sulphate (SDS), pH 8.0) was added to each sample. Samples were then incubated at 80°C for 30 minutes in a shaking heat-block (1,400 rpm). Subsequently, samples were centrifuged at 15,000 g for 30 seconds and 100 μl proteinase K and 40 μl Phocine Herpes Virus (PhHV, internal control) were added. Samples were then incubated at 65°C for 30 minutes in a shaking heat-block (750 rpm). Subsequently, all samples were centrifuged at 15,000 g for five minutes and 500 μl of the supernatant was transferred to 5 ml sterile polypropylene round bottom tubes (BD Biosciences, Breda, The Netherlands) and 100 μl of HPLC-grade water was added to the sample. One hundred μl DNA-eluate was extracted from 500 μl of the sample according to the manufacturers instructions.
If the internal controls exceeded a Ct-value of 36 for either PhHV (suggesting inhibition of PCR) or β-globulin (suggesting insufficient quality of DNA), samples were manually deparaffinised from the second tube. Samples were excluded from further analyses if the Ct-values for the internal controls exceed the cut-off values again. The manual DNA isolation protocol for purification was described previously. Briefly, several wash-steps consisting of xylene, ethanol, and acetone remove the paraffin, followed by digestion with proteinase K and heat-inactivation of proteinase K. DNA was extracted using the Siemens Healthcare Diagnostics VERSANT® kPCR Sample Prep Machine as described above.
PCR conditions and sequencing
Primer-sets, amplicon length, annealing temperature, amplification time and PCR-protocols
β glob FW (GH20)
β glob probe
β glob REV (PC04)
Step A FW
Step A REV
Step B FW
Step B REV
Step C FW
Step C REV
The first and last 4 μm slides were stained with standard haematoxylin and eosin (H&E). All H&E-slides from all appendices were revised by an experienced pathologist specialized in gastrointestinal pathology (MEIS). Mucosal remains were scored using five categories: totally absent; possibly some remains; some remains; relatively normal mucosal remains and normal mucosa. For Brachyspira detection all appendices were subjected to immunohistochemistry (Borrelia burgdorferi (Lyme) IgG antiserum, ILP 0301, ImmunoLogic, Duiven, The Netherlands) on the second 4 μm slide after the first H&E-slide. This staining is based on the immunological cross-reactivity of a universal spirochaete antigen and is used for routine diagnostic purposes in most Dutch Pathology Departments to confirm the presence of spirochaetes in histopathological samples. The specificity of this staining for Brachyspira species was demonstrated in a previous study. A five-point scale was used to score the result of this spirochaete specific colouring (negative (−); possibly positive (±); focally positive (+); positive (++) and strongly positive (+++)). The diagnosis of HIS was made if a slide scored at least focally positive (+).
Statistical analyses was performed using IBM® SPSS® Statistics Version 20 (release 20.0.0) for Macintosh OS X.
151 archival samples were obtained (63 of patients with acute appendicitis, 55 of patients with pseudo-appendicitis and 33 control patients). No statistical differences existed within the groups regarding age, sex and histopathological diagnosis. Inhibition of PCR (PhHV) or DNA quality (β-globulin) occurred in 83 samples. As per protocol, DNA was re-extracted from a second sample and this resolved the inhibition in 74 samples, leaving 142 samples for analysis (59 with acute appendicitis (27 females and 32 males), 50 with pseudo-appendicitis (21 females and 29 males) and 33 in the control group (18 females and 15 males)).
Immunohistochemistry and PCR result
Sequencing revealed the 16S-rDNA variant of B. aalborgi in two patients (one acute appendicitis and one pseudo-appendicitis) and B. aalborgi in three patients (one pseudo-appendicitis and two control patients). These sequences have been submitted to GenBank (accession numbers JX463020 through JX463024). There was no statistical correlation between the presence of Brachyspira species and histopathological outcome (p = 0.54, Chi-squared).
Only appendices of males were found to be positive for Brachyspira species (p = 0.041, Fisher’s Exact test).
To the best of our knowledge, this is the first study investigating the association between Brachyspira species and appendices removed for clinically acute appendicitis in children. In addition, we are the first to perform species identification in appendiceal spirochaetosis, using 16S-rDNA sequencing.
Prevalence of appendiceal spirochaetosis
Article (year of publishing)
Westerman (this study)
While PCR is, in general, considered to be more sensitive and specific than classical microscopy- or culture-based diagnoses, we found only one additional case using PCR versus immunohistochemistry specifically targeted at all spirochaetes. The use of two highly specific, independent techniques reduces the chance of false-positives, which is an important difference with previous studies on appendiceal spirochaetosis, since these were all H&E-stain based. This difference in used techniques might be an explanation as to why we did not confirm the previously reported association between Brachyspira species and pseudo-appendicitis. Alternatively, local differences in the prevalence of Brachyspira species or the fact that we included only children, whereas previous studies consisted predominantly of adults, might explain the differences in prevalence between our study and those previously performed.
A potential limitation of this study is that only appendices were available for analysis, as it might be hypothesised that the presence of Brachyspira pilosicoli in the colon, but not necessarily in the appendix, might be associated with the clinical symptoms of acute appendicitis. While this could be a limitation of microscopy-based studies as they can only observe the end-on attachment of Brachyspira species to the appendiceal mucosa, it is known from avian, canine, porcine and murine infections with B. pilosicoli that the presence of this bacterium is not limited to the end-on attachment to the mucosa, but is also present in faecal samples, indicating that it moves freely through the colon content[35, 36]. Thus, it can be expected that, as most appendix-specimens contain some faecal matter, the presence of B. pilosicoli would also have been detected by PCR. In fact, finding one additional positive sample by PCR versus microscopy supports this assumption.
Another potential limitation might be that sequencing of part of the 16S-rDNA gene does not fulfil all necessary requirements for definitive species identification. However, as was shown before, the obtained fragment is sufficient to differentiate the human Brachyspira species from each other.
Remarkably, only appendices from males were found positive for Brachyspira species (p = 0.041). This association has been reported before, but often more males than females were included in those studies, whereas this study aimed to include equal numbers of males and females. Thus far no clear biological explanation for this phenomenon exists.
Based on the results of this study there is no association between appendicitis and pseudo-appendicitis in Dutch children and the presence of Brachyspira species in the appendix.
Human intestinal spirochaetosis
Polymerase chain reaction
Ribosomal deoxyribonucleic acid
Phocine herpes virus
Siemens Healthcare Diagnostics (Breda, The Netherlands) supplied the use of the VERSANT® kPCR Sample Prep Machine and the necessary consumables. No other external funding or support was received.
The authors would like to thank prof. J.A. Wagenaar, DVM, PhD and prof. dr. S.G.M. Meuwissen, MD, PhD for their critical evaluation of the manuscript.
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