Detection Of Escherichia Coli DNA From Interstitial Cystitis Bladder Biopsies Provides Little Evidence Of A Causal Microbe

摘要

The aetiology of interstitial cystitis in patients remains unknown and despite many investigations no single micro-organism has been implicated. We have studied the possible role of bacteria in interstitial cystitis by investigating PCR amplified DNA sequences from 33 individually well-documented interstitial cystitis patients. The frozen or archived bladder biopsy specimens have been examined for the presence of bacterial DNA by the amplification of 16S rRNA and subsequent RFLP of cloned DNA fragments. Bacterial DNA was detected from 21/33 (64%) of frozen or archived bladder biopsy samples. RFLP analysis of cloned DNA strongly resembled the fragment profile of Escherichia coli DNA in 10/33 (30%) of the IC biopsy samples overall or 10/21 (48%) of the samples that had yielded any DNA. Bacterial DNA was however identified in 4/5 (80%) of samples from non-interstitial cystitis bladder biopsy patients. The significance of the presence of E.coli DNA in 48% of interstitial cystitis bladder biopsies is clearly tempered by the finding of a high proportion of E.coli DNA in non-interstitial cystitis patients. The issues of appropriate control groups and adequate methods to ensure authentic DNA are discussed. Introduction The aetiology of interstitial cystitis (IC) in patients continues to elude us and despite intensive research, the possible causative agent of the disease and its ideal treatment remains unknown. Acute onset with exacerbations and remissions, a previous history of urinary tract infections, virtual confinement of the disease to female patients in which the short urethra allows easy access to bacteria, all strongly suggest an infective aetiology [1]. However, over the past three decades, numerous studies using traditional isolation techniques have failed to convincingly identify any bacterial, viral or protozoal agent(s) responsible for the disease. The requirement to recover organisms from patients by growing them on artificial media or identify extremely fastidious or ‘non-culturable’ organisms has limited progress in the investigation of IC. Advances in molecular-based diagnosis, however, have allowed a new strategic approach by which workers use nucleic acid-based methodology to exclude the presence of specific exogenous microbes as the aetiological cause. PCR amplification of bacterial DNA for the potential identification of specific causal pathogens in frozen IC biopsy specimens has been investigated previously. Using this approach, Hampson et al [2] excluded the specific presence of mycobacterial DNA in IC biopsy specimens and Hukkanen et al [3] excluded the presence in IC specimens of the adenovirus or BK virus genome. Agarwal and Dixon [4,5] specifically excluded the bacterial pathogens Gardnerella vaginalis and Helicobacter pylori respectively and various other pathogens have also been excluded [6]. Escherichia coli remains a possible candidate in the pathogenesis of IC especially as data from Anderson et al [7] suggests that E coli in mice is internalized into bladder epithelial cells and persists for months. In some patients, onset of IC can correlate with evidence of UTI or urinary inflammation originating from episodes of E.coli infection. (J.W. Warren - personal communication). Investigations into the aetiology of IC using patient groups has remained limited, often due to insufficient frozen biopsy specimens and to date, studies have been small and conclusions difficult to make. In the present study, we are attempting to detect DNA from any bacteria present not only from frozen biopsies but a larger number of material preserved in paraffin wax. Materials and Methods Thirty-three bladder biopsy specimens (including 29 paraffin-embedded and 4 fresh frozen specimens) were collected from patients with a clinical diagnosis of IC fulfilling (National Institute of Diabetes and Digestive and Kidney diseases - NIDDK) criteria [8] with some modifications (criteria based on cystometrogram were not strictly adhered to,