TITLE PAGE Rapid and accurate Diagnosis of Human Intestinal Spirochaetosis by Fluorescence in situ Hybridization 5 Running title: Intestinal spirochaetosis diagnosed by FISH

Human intestinal spirochaetosis (HIS) is associated with overgrowth of the large intestine by 50 spirochaetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is a histopathological diagnosis and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows visualization and identification of single bacteria within tissue sections. 55 In this study, we analyzed intestinal biopsies from five patients with possible HIS. All specimens yielded positive results with histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene was performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to B. pilosicoli. 60 Three phylotypes showed mismatches at distinct nucleotide positions as compared with Brachyspira spp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus specific 16S rRNA directed FISH probe that detects all of the so far published Brachyspira spp. FISH on biopsy samples resulted unequivocally in strong signals of brush-like formations at 65 the crypt surfaces. It allowed simultaneous visualization of single spirochaetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique to visualize and identify intestinal spirochaetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS. 70 on N ovem er 3, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom INTRODUCTION Human intestinal spirochaetosis (HIS) is a histologically defined condition of the human distal intestinal tract characterized by helical-shaped microorganisms attached at one end to the surface 75 epithelium of the colonic mucosa. This association forms a so-called “false brush border” (13). While certain Brachyspira spp. are recognized as the causative agents of swine dysentery and porcine intestinal spirochaetosis (11, 3), their clinical significance and pathogenic potential in humans remains unclear. Various studies have reported on the association of these bacteria with intestinal disorders such as chronic watery diarrhoea (8, 12) and on clinical improvement 80 following antimicrobial therapy (29). In contrast, others have suggested that intestinal spirochaetes are harmless commensals in humans (7). The prevalence of HIS varies from 1.2% (23) to > 40% (17, 34), depending upon presumable patient risk factors, such as origin from developing countries, immunodeficiency or homosexuality. Recently, Peruzzi et al. (30) discovered a prevalence of 12% in a selected population, indicating that HIS is an important 85 differential diagnosis for patients with chronic gastrointestinal disorders and risk factors. Two intestinal spirochaetes have been identified in humans so far, i.e. Brachyspira aalborgi (15) and B. pilosicoli (36). Both species require selective media, and B. aalborgi is an extremely slowgrowing, fastidious microorganism that requires anaerobic incubation for up to 4 weeks (34, 4). 90 For this reason, HIS is primarily a histopathological diagnosis. The fuzzy basophilic fringe, 4 – 7 μm thick, on the epithelial layer of the colonic mucosa is visible in hematoxylin-eosin (HE) stains of histological sections and considered pathognomonic for HIS. Tissue morphology usually remains unaltered, and an inflammatory reaction is not observed (20). However, diagnosis of HIS on the basis of HE staining requires experienced laboratory personnel 95 and accurate interpretation, and still often silver staining is additionally needed to confirm the on N ovem er 3, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom diagnosis (10). Therefore, a significant portion of cases may be missed, especially as B. pilosicoli might also colonize the epithelium without the characteristic end-on attachment, resulting in an impeded identification by light microscopy at low magnification (24). Furthermore, histopathology does not provide information about the identity of the microorganisms, thereby 100 precluding epidemiological studies. More importantly, it also hampers accurate therapeutics, as the intestinal spirochaetes are suspected to differ in virulence and therefore some cases of HIS may require more urgent antibiotic therapy than others (29, 5). The genus Brachyspira currently comprises 7 established and several proposed species. Among 105 some Brachyspira species, highly conserved 16S rDNA precludes inter-species differentiation by 16S rDNA methods and necessitates further molecular analyses. However, all known species isolated from humans can be identified and differentiated via 16S rDNA. Corresponding to the genetic variation discovered in Brachyspira species, e. g. B. hyodysenteriae (2) and B. innocens (9), recent molecular studies identified human Brachyspira strains genetically distinct from B. 110 aalborgi and B. pilosicoli. This heterogeneity was confirmed by sequencing of 16S rRNA (14, 21) or NADH oxidase genes (25), fluorescence in situ hybridization (FISH) (16, 17), as well as multilocus enzyme electrophoresis (33). Pettersson et al. (31) analyzed biopsies from two adults by 16S rDNA sequencing and consecutively proposed to divide the B. aalborgi lineage in three phylogenetic clusters, type strain B. aalborgi 513A T being included in the first cluster. 115 The extent of intraspecies genetic variation in human intestinal spirochaetes is unclear and difficult to estimate as only few complete 16S rDNA sequences are available. Further epidemiologic and phylogenetic investigations are needed to elucidate spirochaete genetic diversity and to facilitate the evaluation of presently available molecular diagnostic tools. on N ovem er 3, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom FISH is a microscopic method that allows simultaneous visualization and identification of 120 microorganisms. Jensen et al. (3, 16, 17) designed several genusor species-specific oligonucleotide probes targeting the 16S or 23S rRNA of Brachyspira spp. and applied them successfully on porcine and human intestinal biopsy specimens. However, a 16S rRNA directed genus-specific probe for diagnostic use targeting all so far known Brachyspira spp. has not been developed. 125 In the present study, intestinal biopsy specimens from 5 patients with possible HIS were analyzed histopathologically and by culture, FISH, PCR amplification and 16S rDNA sequencing. Biopsy specimens from a healthy control group were analyzed retrospectively by histopathology and FISH. The purpose was to (i) acquire further information about the phylogenetic structure within 130 the Brachyspira spp. associated with HIS, (ii) design a FISH probe covering all Brachyspira spp. with respect to the currently available sequence data, and (iii) evaluate FISH as a fast and robust diagnostic screening tool for HIS.