A study to detect Helicobacter pylori in fresh and archival specimens from patients with interstitial cystitis, using amplification methods




To detect Helicobacter pylori DNA in fresh and paraffin-embedded bladder biopsy specimens, and thus determine any possible role in interstitial cystitis (IC).


Thirty-three bladder biopsy samples were examined from patients with IC (29 paraffin-embedded and four freshly frozen) diagnosed according to National Institute of Diabetes, Digestive and Kidney Disease criteria. The positive control was a gastric biopsy sample from a patient with a known gastric ulcer caused by H. pylori infection.


The anticipated polymerase chain reaction product size of 109 base pairs was obtained with the positive control, whereas none of the other biopsy samples (paraffin embedded or fresh) showed positive amplification specific for H. pylori.


As there was no H. pylori DNA in any of the samples from patients with IC, it is an unlikely candidate in the pathogenesis of IC.


Helicobacter pylori has been firmly established as a cause of gastritis and peptic ulcer in recent years, and investigators are now searching for its role as a likely factor in other diseases of unknown cause. Interstitial cystitis (IC) is one such chronic debilitating and painful bladder condition. Its sudden onset in a healthy person, leading to chronic pain and nonspecific LUTS, infiltration of the bladder wall with chronic inflammatory cells, ulceration and eventually contraction of the bladder, have similarities to chronic peptic ulcers [1]. The diagnosis and detection of H. pylori infection is not easy and is the explanation for its late discovery after years of research into peptic ulcer disease.

Molecular methods for detecting microbial pathogens avoid difficulties with the culture and recovery of viable organisms, and the problems associated with there being few culturable organisms. A PCR-based method for successfully identifying all forms of H. pylori was reported by Ho et al.[2] and can be used for amplifying H. pylori DNA from paraffin-embedded archival gastric biopsy specimens.

In the present study we used the same molecular approach to investigate the presence of H. pylori nucleic acid in fresh and paraffin-embedded bladder biopsy specimens, and hence its potential causal role in IC.


Thirty-three samples (29 paraffin-embedded and four fresh frozen) were collected from patients with an established diagnosis of IC conforming to the criteria of the National Institute of Diabetes, Digestive and Kidney diseases [3], with some modifications [4]. Biopsy samples were used because centrifuged urine samples may be contaminated by normal vaginal flora. The positive control was a paraffin-embedded gastric biopsy specimen from a patient with a known peptic ulcer caused by H. pylori infection.

Using a microtome, 5 µm thick tissue sections were cut from the paraffin-embedded biopsy specimen blocks. The first five sections from the blocks were discarded to ensure that the outer surface remained free of exterior contamination. Five thin sections were then collected in an Eppendorf tube. The cutting knife was changed frequently and all surfaces coming into the contact with the specimen were wiped clean with alcohol between each specimen and between the first five discarded sections and the five used for DNA extraction. Pre-treatment of all specimens with lysozyme and lysostaphin preceded DNA extraction. DNA was extracted from paraffin-embedded biopsies by the following procedure; specimens were first dewaxed and then crushed in centrifuge tubes using a pipette tip, frozen in liquid nitrogen and crushed further. The specimens were suspended in xylene for 1 h, centrifuged at 13000 g for 15 min and the xylene removed. This procedure was repeated and the specimens then suspended in 100% ethanol for 1 h, centrifuged at 13000 g for 15 min and the ethanol removed. The same procedure was repeated. Samples were then air-dried to remove traces of ethanol and used for DNA extraction. Frozen biopsy specimens were defrosted in Eppendorf tubes and 200 µL of sterile normal saline added. The tissue was crushed with a needle and homogenized. The samples were then centrifuged at 10 000 g for 15 min and the supernatant discarded. DNA (from the pellet) was extracted with Easy-DNATM genomic DNA isolation kit version 1 (Invitrogen plc, Paisley, UK) using the manufacturers’ instructions. All paraffin-embedded specimens were amplified for the presence of a 97 bp product of the human mitochondrial genome as evidence of human DNA presence, with human placental DNA as a positive control [5]. DNA from all the specimens was used to attempt to amplify a 109 bp product in PCR reactions specific for H. pylori. The primers are specific for H. pylori, and DNA from the closest relatives of H. pylori is not amplified [2]. The sequences were: Hp1, CTG GAG AGA CTA AGC CCT CC (bp 834–853); and Hp2, ATT ACT GAC GCT GAT TGT GC (bp 744–763) [2]. PCR conditions and analysis of amplified products from PCR reactions were as described previously [4].


None of the biopsy samples from patients with IC (either fresh-frozen or paraffin-embedded) showed positive amplification specific for H. pylori (data not shown). However, the sensitivity for detecting H. pylori DNA was ≈ 2 pg (Fig. 1). Amplification of region V of the human mitochondrial gene from DNA extracted from all the paraffin-embedded biopsy samples resulted in a specific band of 97 bp, indicating the presence of human mitochondrial marker DNA in the fresh-frozen or archival samples. The positive control sample (gastric biopsy specimen) showed the anticipated PCR product of 109 bp and confirmed the presence of H. pylori DNA.

Figure 1.

The PCR for the samples assessed: lane 1, 100 bp ladder; lane 2, negative control (no DNA added); lane 3–8, H. pylori DNA at 200, 160, 120, 80, 40, 4 and 2 pg. The 109 bp product is arrowed.


Since the discovery of the causal effect of Helicobacter in peptic ulcer disease, this organism has been isolated from other mucosal surfaces and from feces [6]. Therefore it is highly likely that the organisms may gain entry along the short female urethra from the perineal flora to the female genital tract and infect the bladder. H. pylori is a Gram-negative spiral or curved bacterium, which is strictly microaerophilic and requires CO2 for growth. For successful bacteriological culture of H. pylori, specimens must be < 2 h old. Agar plates are incubated at 37 °C in an atmosphere of high humidity and CO2, and left undisturbed for 3–7 days [7]. Biochemically, H. pylori produces an exceptionally powerful urease which, unusually, can be detected in biopsy material with no culture of the organism. Routinely, the tissue is placed in a urea solution with an indicator and if H. pylori is present, the pH changes from acid to alkaline because of the formation of ammonia. However, the organism undergoes coccal transformation under adverse conditions and unfortunately the culture techniques, urease test and other serological tests cannot identify this form of the organism [2]. The development of molecular methods of diagnosis in recent years has potentially overcome the problems in culturing the organism. Organisms that may be important clinically but are difficult or impossible to culture have been research targets for the application of PCR technology. Amplification of DNA from pathogens by PCR is rapidly becoming established as a method of locating very low quantities of an infective agent present in the clinical samples [8].

Only one study specifically addressed the issue of Helicobacter infection in IC. English et al.[9] assessed 23 patients with IC and 23 control subjects, and examined their serum for the presence of H. pylori IgG antibodies. They found positive antibodies in 22% of the patients and 35% of the controls. The controls were the relatives of patients attending various urology clinics and the presence of antibodies in them was explained by the fact that 11–24% of the USA population are seropositive for H. pylori, but few of them are symptomatic. The authors concluded that as the incidence of H. pylori infection was no greater in IC, it is unlikely to be the cause of IC [9]. However, the ELISA used can be problematic; false-positive and false-negative results are common [10], and it fails to recognize the coccoid forms of the organism [2].

In the present study, we attempted to detect H. pylori DNA by PCR; the negative results with a highly sensitive method makes H. pylori a very unlikely contender as a cause of IC.


We are grateful for the gift of paraffin-embedded bladder biopsies from Dr C.L. Parson, Veterans Administration Medical Centre, San Diego, USA and the kind cooperation of Messrs P.A. Hamilton Stewart and G.M. Flannigan, Bradford Royal Infirmary, Bradford. We also thank Mr P.H. O’Reilly (Stepping Hill Hospital, Stockport) for reviewing the manuscript.


interstitial cystitis.