BackgroundBrachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials.MethodsThe study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced.ResultsSensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named “Brachyspira hominis”, in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p = 0.028).ConclusionsThis newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species.

e Brachyspira species have been implicated as a potential cause of gastroenteritis in humans; this is, however, controversial. In 733 gastroenteritis cases and 464 controls, we found 29 samples positive for Brachyspira species (2.3% of cases and 2.6% of controls; P ‫؍‬ 0.77). Brachyspira species were not associated with gastroenteritis in humans.H uman intestinal spirochetosis (HIS) is caused by spirochetes from the genus Brachyspira (1) and is histologically characterized by the apical attachment of these bacteria to the mucosa of the colon (2). Three Brachyspira species have been reported to colonize humans: Brachyspira aalborgi, Brachyspira pilosicoli, and the provisionally named "Brachyspira hominis" (3-7). Although it is unclear whether HIS is associated with clinical symptoms, there is evidence suggesting an association between B. pilosicoli and diarrhea in humans (3,(8)(9)(10)(11). However, due to the obligate anaerobic culture requirements for these bacteria, most studies on HIS are based on histopathology without species differentiation, as histopathology requires additional techniques to differentiate between species (3, 12). As a result, the clinical significance of HIS has remained controversial for decades (1,(13)(14)(15)(16). Several studies have investigated the prevalence of Brachyspira species in patients with diarrhea, and various incidences have been reported, depending on the investigated population (high-versus low-income countries) and techniques used (culture versus histopathology). In this study, we investigated the prevalence of Brachyspira species in Dutch primary care patients with and those without symptoms of gastroenteritis. For this purpose, we used fecal samples stored from a case-control study reported in 2001 (17). DNA was extracted as previously described (18), directly from the fecal samples without selenite enrichment. Amplification was performed by using a previously described real-time PCR amplifying a 142-bp region of the 16S rRNA of all Brachyspira species (3), with some minor modifications. The modifications consisted of three species-specific probes targeting B. pilosicoli, B. aalborgi, and "B. hominis" and another reverse primer. These probes were validated against a known set of 66 Brachyspira-positive samples (cultured isolates and histopathologically positive samples) and 26 Brachyspira-negative samples (3). The PCR protocol and primer and probe sequences are supplied in Table 1. PCR inhibition was defined as a PhHV cycle threshold value greater than the average of all PCRs plus 2 times the standard deviation. Samples exceeding this value and all Brachyspira-positive samples were reextracted from the original fecal samples. Samples were excluded from analysis if the internal control exceeded the cutoff again. Statistical analyses were performed by using IBM SPSS Statistics version 20 (release 20.0.0) for Macintosh OS X.Samples from 736 cases and 465 controls were available. Four samples (three cases and one control) had to be excluded due to inhibition of the PCR. T...

BackgroundAcute 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.MethodsArchival 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.ResultsFive 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.ConclusionThe results obtained do not provide evidence for a role of Brachyspira species infection in the aetiology of acute appendicitis in children.

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