A prospective study of microorganisms in urine and bladder biopsies from interstitial cystitis patients and controls

A PROSPECTIVE STUDY OF MICROORGANISMS IN URINE AND BLADDER BIOPSIES FROM INTERSTITIAL CYSTITIS PATIENTS AND CONTROLS* S. KEAY, M.D., PH.D. R.S. SCHWALBE, PH.D. A.L. TRIFILLIS, PH.D.

J.C. LOVCHIK, PH.D. S. JACOBS, M.D. J.W. WARREN, M.D.

From the Division of Infectious Diseases, Department of Medicine, and the Departments of Pathology, Pediatrics, and Surgery, University of Maryland School of Medicine, and the Research Service, and Pathology and Laboratory Medicine Services, Department of Veterans Affairs Medical Center, Baltimore, Maryland

ABST~CT~bj~~. Interstitial cystitis [IC) is a chronic inflammato~ condition of the bladder of unknown etiology. We tested the hypothesis ti--‘at ainlcorganism would be found at higher prevalence in urine or bladder tissue from wome :n with lthan from control women. Methods. Urine and bladder tissue were obtained i at cystoopy from 1 1 IC patients and 7 control subjects. These specimens were cultured for a variety of fastidious and nonfastidious bacteria, mycobacteria, fungi, and viruses. In addition, special staining techniques were used to examine biopsy specimens and c~ospun urine, and tissue sections and outgrowths of explanted bladder cells were examined by electron microscopy. Results. Cultures of urine from 6 of 11 IC patients grew five different bacteria (Corynebacterkm sp, ~e~s~e~~a~~e~~oniae, iacto~acji/~s sp,, Streptococcus coffste/~u~s, and Streptococcus morbillorum), human cytomegalovirus, or Torulopsis glabrata; one of these organisms ~~acto~aci//ussp) was found in urine from 2 patients. Although contamination by urethral organisms is possible, the prevalence of microorganisms in urine of IC patients (6 of 1 1) was significantly greater than in urine of control subjects (0 of 7) (P
by funding from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes oJHealth (R5f DK44818). Submitted (Rapid Comman~cation~; September 14, 1994, accepted (with revisions): September 28, 1994

*This

work

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was supported

/ FEBRUARY1995 / VOLI/ME 45, NUMBER 2

Interstitial cystitis (IC) is a chronic inflammatory condition of the bladder that affects 20,000 to 90,000 women in the United States at any given time’; approximately one tenth as many men

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also have this condition.* Typical cystoscopic findings include petechial hemorrhages (glomerulations); ulcers that extend into the lamina propria (Hunner’s ulcers) are present in 10% of cases3 Inflammatory cell infiltrates may be seen in the lamina propria and often consist of T lymphocytes with or without mast cells.3,4 The etiology of IC is unknown, but several features suggest that it may be an infectious disease. Patients with IC are twice as likely to have a history of urinary tract infection as do control subjects, and 10 to 12 times as likely to have a history of childhood urinary tract disorders.1,5 In addition, the predominance of women with the syndrome, the usually acute onset of symptoms, the inflammatory changes seen on histopathologic examination, and the presence of immunoglobulin A deposits in bladder epithelium suggest that IC may be caused by an infectious organism.6-g However, no microorganism has been incriminated as a cause of IC. Nevertheless, a critical review of the literature suggests that an infectious etiology has not been properly sought or excluded.lO Indeed, most investigators have used only routine cultures of urine to rule out infection. We therefore undertook a prospective, controlled study to look for an infectious cause of IC in women by obtaining urine and bladder tissue at cystoscopy, and culturing these specimens for microorganisms, including bacteria, viruses, and fungi. Because some microorganisms might be culturable yet fastidious, we also used special culture techniques. Finally, because some organisms in urine or tissue might be viable but nonculturable, we used specific staining techniques and electron microscopy for discovery and identification. MATERIAL AND METHODS

URINESPECIMENS

At the time of cystoscopy, IC patients and control subjects received a 7.5% povidone-iodine scrub (Betadine, Perdue Frederick Co., Norwalk, Corm.) of the lower abdomen, vaginal vestibule, and urethral meatus, followed by swab application of 10% povidone-iodine to these areas. Urine was then obtained by suprapubic aspiration; if not gotten by aspiration, urine was obtained by urethral catheterization. Urine specimens were placed on ice for transport to the laboratory. BLADDERTISSUE

Cystoscopy was performed under general anesthesia using nonbacteriostatic normal saline as a bladder irrigant. The diagnosis of IC was confirmed by the presence of glomerulations on the second filling of the bladder with normal saline or the presence of Hunner’s ulcers. Rigid cold cup biopsy forceps (Olympus Corp., Lake Success, NY) were used to acquire six 4 mm* pieces of transitional epithelium with submucosa. Biopsies were transported from the operating room on saline-soaked Telfa (Kendall Co., Boston, Mass.) in a sealed sterile container at room temperature. One specimen was placed into cold viral transport medium (RPMI1640 [GIBCO] containing 3% fetal calf serum and 1% antibiotic and antimycotic solution) for viral cultures; another was placed into 2SP (sucrose phosphate)-based Chlamydia transport medium for Chlamydia cultures; two specimens were placed into minimum essential medium (MEM) for growth of bladder epithelial cells (as previously describedll) and for electron microscopy and histologic examination; a fifth specimen was placed into nonbacteriostatic saline for bacterial, fungal, mycobacterial, and Mycoplasma/Ureuplasma cultures; and a sixth was frozen dry at -80°C.

PATIENTS

Women with IC were referred by physicians, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and the Interstitial Cystitis Association. Control patients were women who were scheduled to undergo cystoscopy for other urologic diseases. IC patients underwent cystoscopy at least 3 months following the most recent known bacterial urinary tract infection and 1 month following the.last antibiotic use; control patients were required to have no symptoms of urinary tract infection or antibiotic use for at least 1 month. Participants were at least 18 years old and were enrolled in accordance with guidelines of the Institutional Review Board at the University of Maryland School of Medicine. 224

MICROSCOPY

Sediment from 1 mL of centrifuged urine and a sterile portion of bladder homogenate (in sterile saline) was added to sterile glass slides and stained for bacteria (Gram’s stain) and acid-fast bacilli (Kinyoun’s cold carbolfuchsin stain.)l* In addition, cells from 1 mL of urine were cytospun by centrifugation onto glass microscopy slides at 400 r-pm, fixed with 100% methanol, and stained with Bacto Acridine Orange Stain (DIFCO Laboratories, Detroit, Mich.) to detect microorganisms, including viable but nonculturable bacteria.13 The investigator was blinded to the results of urine culture at the time slides were examined. One specimen from each bladder biopsy was fixed in 10% phosphate-buffered formalin and

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embedded in paraffin. Sections (0.5 km> were stained routinely with hematoxylin-eosin, periodic acid-Schiff stain (to detect fungi), and Dieterle stain (to detect bacteria and fungi). Biopsy specimens and monolayer outgrowths of biopsy explants were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and routinely processed for electron microscopy, as previously described.14 Ultrathin sections were cut, mounted on carbon-coated grids, and double-stained with uranyl acetate and lead citrate prior to examination with a Joel 1200 EX electron microscope. Entire grids were scanned at a magnification of 8000 to detect bacteria and 20,000 to detect viral particles. BACTERIAL CULTURES AND IDENTIFICATION

All specimens were processed within 2 hours of collection. Urine was swirled, and inocula of 0.001 mL and 0.1 mL were plated onto the appropriate agar media to detect organisms present ab 1000 colony-forming units (CFU)/mL or 10 CFU/mL, respectively. Bladder tissue was homogenized in sterile saline and 0.1 mL of the homogenate was plated onto appropriate agar media. - For nonfastidious aerobic bacteria, specimens were plated on nonselective tryptic soy agar containing 5% defibrinated sheep blood agar, incubated in a 5% carbon dioxide (CO,) incubator at 35’C, and examined at 24 and 48 hours for bacterial growth. Bacteria were identified using a Sceptor identification system (Becton-Dickinson, Mountain View, Calif), and, when necessary, identification was confirmed by API (Analytab Products, Inc., Hicksville, NY). Lactobacillus sp and Corynebacterium sp were identified using conventional biochemical tests.15 For anaerobic bacteria, samples were plated on prereduced blood agar plates (CDC anaerobic blood agar; BBL, Cockeysville, Md), incubated in anaerobic jars (Gas-Pak, BBL) at 35”C, and examined every 2 days for 10 days. A portion of each specimen was also added to prereduced thioglycolate broth to ensure recovery of organisms present in low numbers. For Huemophilus sp and Neisseria gonorrhoeae, chocolate agar plates were inoculated, incubated for 96 hours in a 5% CO, incubator at 35’C and examined daily for bacterial growth. For Helicobacter pylori and Campylobacter sp, samples were plated on additional chocolate agar plates (for H pylori) and Campylobacter blood agar plates and incubated in a microaerophilic environment (Campy-Pak, BBL) at either 35°C (H pylori) or 42°C (Campylobacter sp). Cultures were examined daily for 5 days.

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For Mycoplasma/Ureaplasma, 1 mL of bladder biopsy homogenate was mixed with an equal amount of 2SP medium and urine was diluted 1:l with 2SP medium. Two tenths mL of each diluted specimen was inoculated into 2 mL of U9/10B medium and into 2 mL of SP4/HSI medium. Serial lo-fold dilutions were made to 10”. Tubes were incubated at 36°C and examined for color change daily for 2 weeks. At the end of 2 weeks or when color change was observed, 0.3 mL of culture was plated onto A7 agar (Northeast Laboratory, Waterville, Me), incubated at 36°C in 5% CO,, and examined daily for 1 week, then again on day 14 of culture. For Chlamydia, 0.2 mL of each ZSP-diluted urine and biopsy specimen, prepared as for Mycoplasma and Ureaplasma isolation, was inoculated by centrifugation onto buffalo green monkey kidney cell monolayers in 48-well plates. After incubation at 37°C in 5% CO, for 2 days, monolayers were fixed with methanol, stained with fluorescein isothiocyanate (FITC)-conjugated antibody to Chlamydia antigen (Bartels, Bellevue, Wash), and examined by fluorescence microscopy. For Mycobacteria, Lowenstein-Jensen agar slants (BBL) were inoculated with urine or tissue homogenate, then incubated at 35°C in a 5% CO, incubator for 6 weeks. VIRALISOLATION AND IDENTIFICATION

Cultures of confluent human embryonic lung cells (HEL-299) (ATCC, Rockville, Md), primary human foreskin fibroblasts, human lung carcinoma cells (A549) (ATCC), and rhesus monkey kidney cells (Viromed, Minnetonka, Minn) were inoculated with 2 mL of pH-adjusted urine specimen or 1 mL of homogenized tissue suspension (in cell culture medium). These cultures were incubated at 37°C with 5% CO, and examined daily for cytopathic effect for 1 week, then biweekly for at least 12 weeks. Attempts were made to passage possible viruses from cultures exhibiting cytopathic effect using supematant and sonicated cell suspensions. The cytomegalovirus isolate was identified by immunofluorescence using monoclonal antibodies specific for a late viral antigen (Chemicon Intemational, Segundo, Calif) followed by FITC-conjugated goat anti-mouse immunoglobulin (Caltag Laboratories, South San Francisco, Calif). CULTURE AND IDENTIFICATION OFYEASTS AND FILAMENTOUS FUNGI

Specimens were added to Sabouraud’s dextrose agar, incubated at 3O”C, and examined every 2 days for 2 weeks. Yeasts were speciated based on

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TABLE Method for Obtaining Urine

I.

Microbiological

results Acridine Orange Stain of Cytospun Urine

Urine Culture

Bladder Tissue Culture

IC Patients

1

Urethral catheter

2

Suprapublc

aspiration

3 4 5 6 7 8 9 10 1I

Suprapublc aspiration Urethral catheter Suprapubic aspiration Urethral catheter Urethral catheter Suprapubic aspiration Urethral catheter Urethral catheter Urethral catheter

Controls 1 2 3 4

Suprapubic aspiration Urethral catheter Urethral catheter Urethral catheter

5 6 7

Urethral catheter Urethral catheter Suprapubic aspiration

10,000 CFUimL Corynebacten’um human cytomegalovirus > 100,000 CFUimL Kiebsieiia pneumoniae; Lactobacillus sp * 4000 CFUimL Lactobacihs sp Toruiopsis g/abrata+

sp,

Long rods Medium

rods

Long rods Long rods Yeast; medium Medium rods

60 CFU Lactobacilius rods

sp

Toruiopsis giabrata’

Streptococcus constellatus * Streptococcus morbillorum’ 10 CFU Pseudomonas 15 CFU Pseudomonas

sp sp

5 CFU Staphylococcus epidermidis

Cocci

‘Grew in thioglyc-olotubmh only ‘Grew m viral cullur.rr only

morphologic appearance in corn meal Tween 80 agar plus biochemical reactions using the API Yeast-Ident System. STATISTICAL ANALYSIS

Data were analyzed using unpaired Student t test and Fisher’s exact test (one-tailed and twotailed analysis). Statistical significance was determined at the 5% level. RESULTS PATIENTS Eleven IC patients, all previously diagnosed by cystoscopy, and 7 control patients were enrolled in our study over a 2-year period (February 1992 to March 1994). The average age of the IC patients was 49.2 years versus 61.3 years for the controls (P >O.l). Race was distributed evenly, with 1 black woman and the remainder white women in each group. The IC patients had variable durations of symptoms, with a median of 4 years and a mean of 5.6 years. Ten patients fulfilled the criteria for IC established by the NIDDK,16 but 1 patient had experienced symptoms for only 6 months at the time of enrollment. The control patients were diagnosed with papillary adenocarcinoma of the bladder, renal cell carcinoma, ureteral carcinoma, renal vascular hypertension, nephrolithiasis, ureteral endometriosis, or urethrocele.

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Ten of 11 IC patients had previously taken antibiotics for suspected or reportedly cultureproven urinary tract infections compared with 3 of 7 control patients. The total number of previous antibiotic courses for urinary tract infections averaged at least 6.5 for the IC patients and 0.6 for the control subjects. The most recent antibiotic therapy was at least 1 month prior to enrollment for IC patients and at least 4 months prior to enrollment for control patients. PATHOLOGY All 11 of the IC patients but none of the control patients had glomerulations or Hunner’s ulcers, part of the NIDDK criteria for IC.lh Histopathologic studies were available on biopsy specimens from 10 IC patients, and revealed nonspecific inflammation (3), chronic inflammation with mast cell infiltrate (2), hemorrhage (1)) mild to moderate edema with or without associated hemorrhage (3), or normal bladder mucosa (1). MICROBIAL CULTURES Urine cultures from all 7 controls were negative. In comparison, urine cultures from 6 of 11 (55%) IC patients were positive for microorganisms (P co.02 for one-tailed analysis, P co.05 for twotailed analysis). Four were positive for bacteria alone (Klebsiella pneumoniae (l), Lactobacillus sp (2) or

Streptococcus constellatus and Streptococcus movbillorum (l)), one was positive for bacterial and viral isolates (Corynebacterium sp and human cytomegalovirus), and one was positive for fungus (Torulopsis glabrata) (Table I). The Klebsiella sp and one Lactobacillus sp isolated from both urine and biopsy specimens were cultured on a variety of media and could be quantified; the other Lactobacillus sp and the two Streptococcus sp grew only in the thioglycolate broth. The Corynebacterium sp grew only on BHVhorseblood medium. The Torulopsis sp was subcultured from the viral cell culture medium; original cultures on Sabouraud’s medium were negative. Urine from 2 additional IC patients and from 2 control subjects had evidence for cytopathic effect in HEL or HFF cells; in none of these 4 cases could this effect be passaged to secondary cultures, suggesting a toxic rather than an infectious cause. Bladder biopsy specimens from 3 of 7 control subjects yielded small numbers of bacteria. Biopsy specimens were culture positive for 2 of the 11 IC patients, each for the same species grown from the patient’s urine. Again, the Torulopsis sp grew only from subculture of viral culture medium. MICROSCOPY

Acridine orange stains of cytospun urine specimens were positive for cocci in urine from 1 control patient. In comparison, rodlike organisms were seen in specimens from 6 IC patients. These included the four IC specimens that were culture positive for bacteria, and in each the morphology of the rods was compatible with the bacterium cultured. Other microscopic techniques did not reveal additional microorganisms. Acridine orange stains of bladder tissue smears were negative except for the specimen culture positive for Torulopsis sp, which revealed extracellular yeastlike organisms. Gram’s stains and Kinyoun stains of urine sediment and tissue, and Dieterle stains of tissue were all negative. Electron microscopy was performed on tissue biopsy specimens from 10 IC patients and on monolayer outgrowths of biopsy explants from 7 IC patients; no intracellular or extracellular microorganisms were seen. COMMENT This prospective, controlled study provides no evidence that IC is the result of an infection or colonization by a single microorganism. The methods used did not reveal the presence of a uniform bacterial species or of any Mycoplasma, Ureaplasma, Chlamydia, mycobacteria, anaerobic bacteria, or fastidious bacteria (including Gardnerella sp, Helicobacter sp, Campylobacter sp, Haemophilus sp, or

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Neisseria sp). Only one of our patients had evidence of a virus (cytomegalovirus) infection and 1 had a fungus (Torulopsis sp) infection. No patient had evidence of active infection with other viruses culturable with our methods: herpesviruses (herpes simplex, cytomegalovirus, and varicella zoster), enteroviruses (poliovirus, coxsackievirus groups A and B, echovirus, and others), measles, mumps, rubella, or respiratory viruses (adenovirus, influenza A and B, respiratory syncytial, parainfluenza, and rhinovirus). The lack of evidence for mycobacterial infection in IC patients concurs with the findings of Hampson et al.,” in which mycobacteria were not detected in bladder tissue from IC patients using the polymerase chain reaction technique. Furthermore, the lack of widespread evidence for herpesvirus or other viral replication agrees with the findings of Fall and colleagues,18 who were unable to culture any viruses from IC patients’ urine or to find a difference in serologic titers for herpes simplex virus, cytomegalovirus, and varicella zoster virus between IC patients and control subjects. Our viral culture data also confirm the findings of Hanash and Pool,” who were unable to find evidence of viral infection in 30 urine specimens or three biopsy specimens from IC patients. However, our findings generate the hypothesis that IC patients have a higher prevalence than controls of various microorganisms in their urine. In this type of study, contamination of urine specimens by urethral organisms is a main concern. Therefore we obtained urine by suprapubic aspiration, or urethral catheterization following perineal disinfection. The fact that none of seven control urine specimens had any growth, even with methods that could detect as few as 10 CFU/mL, suggests that our methods of urine collection adequately avoided contamination. This conclusion is strengthened by the fact that a similar proportion of specimens from IC patients, whether obtained by suprapubic aspiration or by urethral catheterization, yielded organisms (two of four versus four of seven, respectively). Finally, five of six IC patient urine specimens that grew bacteria or yeast were positive for organisms of appropriate morphology by acridine orange staining, and two of the microorganisms present in small numbers in the urine of IC patients (Torulopsis sp and Lactobacillus sp) were also grown from bladder tissue. Although our sensitive methodology allowed the growth of bacteria in urine from 5 of 11 patients and should theoretically exclude the diagnosis of IC according to NIDDK criteria,16 we believe these patients did have IC. All had a prolonged history of urinary symptoms compatible with IC. All had

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been previously diagnosed by cystoscopy. All were confirmed to have glomerulations or Hunner’s ulcer during the current study Finally, only 1 of the patients would have had bacteriuria reportable using routine laboratory procedures in place at the time the NIDDK criteria were established. Examination of bladder tissue by these methods did not provide additional information regarding a possible infectious etiology of IC, however. In patients with a history of recurrent urinary tract infection, Elliott and colleaguesZo found organisms in the bladder tissue of patients whose urine cultures were sterile. In this study, we did not find evidence for organisms in bladder tissue of IC patients with sterile urine. Furthermore, our negative findings by electron microscopy of bladder tissue concur with an earlier study in which no microorganisms could be found in bladder biopsy specimens from IC patients.21 In the biopsy specimens from control subjects, none of the three organisms grown from tissue was also cultured from the patient’s urine or seen by acridine orange staining; Staphylococcus epidermidis is a frequent skin colonizing organism, and the two Pseudomonas sp were not identifiable using the API system and therefore may represent environmental organisms. Although several studies of microorganisms in IC have been reported, we are aware of only two that have used a spectrum of microbiologic techniques and catheterized urine specimens. Although details of the methodologies used by Wilkins et al.,22 are not well described, cultures of urine and bladder biopsies were performed. Their findings were similar to ours, in that 10 of 13 patients with history and cystoscopy findings consistent with IC had microorganisms present in urine: three were known uropathogens (eg, Escherichia cob, Proteus mirubilis, and group B Streptococcus) and the remaining seven organisms were Gardnerella vaginalis or Lactobucillus sP. 22 Because specimens from control subjects were not obtained, the interpretation of these data was uncertain. However, the similarity of our findings in IC patients, contrasted with the low prevalence of microorganisms in the urine of our control patients, makes the report of Wilkins et al.22 more intriguing. In the second study, by Hanash and Pool,lg Candida albicans was grown from the urine of 1 of 30 IC patients; all bacterial cultures were negative. However, these investigators used a calibrated loop-streak technique that detects organisms only in relatively high concentrations. The methods used in the current study, including culturing 0.1 mL of urine and inoculating thioglycolate broth, resulted in greater sensitivity for detecting low numbers of organisms.

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If the association of IC with positive urine culture for various microorganisms is confirmed by other controlled studies, two possibilities exist: that the organisms are either a result or a cause of IC. That these organisms may be the result of IC implies that colonization of the bladder, or possibly the urethra, is secondary to epithelial cell damage, inflammation, or other features of IC pathology. Increased bacterial colonization of epithelium in other organ systems has been reported to result from underlying disease states, such as viral infection23 or cystic fibrosis.24,25 Vaginal organisms such as corynebacteria, lactobacilli, and streptococci26 that often gain entry to the female bladder,27 therefore may be more prevalent in the urinary tracts of women with IC than of control subjects because of an underlying difference in lower urinary tract epithelium. The other possibility, that these organisms are involved in the etiology of IC, in turn generates two hypotheses. The first is that IC is a consequence of the direct effect of virulence factor or factors of these organisms. This is unlikely, however, because several different species of microorganisms were isolated, intimating that no common factor or cluster of factors characterizes IC-associated organisms. Furthermore, the organisms are lacking in known virulence factors and are nonpathogenic in normal hosts. A recent study by Kunin et aL2* demonstrated that low concentrations of corynebacteria, lactobacilli, and streptococci are significantly more likely to be found in the urine of asymptomatic young women than of those with urinary tract symptoms. If these organisms, which are vaginal colonizers that often gain entry to the female bladder, were capable of causing a sustained inflammatory response in the normal host, one would expect a high incidence of associated symptomatic disease in many women. The second hypothesis is that these organisms encounter an abnormal host response. The presence of organisms that cause disease primarily in immunocompromised patients or that do not often cause urinary tract infections in the normal host is consistent with the possibility that the epithelial, inflammatory, or immune response to these microorganisms may not be normal in IC patients. An example of an abnormal host defense has been described for malacoplakia, a urinary tract inflammatory disorder in which a defect exists in the intracellular killing of ingested microorganisms by macrophages. 29 Although it is unlikely that IC and malacoplakia share the same host defect, additional studies of the host response of IC patients to organisms cultured from the urine may be warranted.

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It remains possible that organisms that are not easily demonstrated in urine or biopsy specimens, or are present only during early phases of this disease, could cause this inflammatory condition. Therefore different methods of microbial cultivation or microscopy, studies using polymerase chain reaction to look for evidence of microbial genomes, immunologic assays to seek evidence of specific antigens or antibodies, or investigation of recently symptomatic IC patients may be revealing. Susan Division

Keay, M.D.

ofInfectiousDiseases

University of Maryland School of Medicine Veterans Administration Medical Center 10 N. Greene Street Baltimore, MD 21201 ACKNOWLEDGMENT. To Edward W. Campbell, Jr., M.D., Michael J. Naslund, M.D., Linda Home, and Terry Wilson, R.N., for their assistance with patient enrollment and specimen acquisition; Cynthia Drachenberg, M.D., for assistance with light microscopy and histopathology; Xiao Ling Cui, M.D., for assistance with electron microscopy; Marcia Fullem for technical assistance; and J. R. Hebel, Ph.D., for helpful discussions.

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12. Ebersole L: Acid-fast stain procedures, in Isenberg, HD (Ed): Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992, pp 3.5.1-3.5.11. 13. Roszak DB, and Colwell RR: Survival strategies of bacteria in the natural environment. Microbial Rev 51: 365-379, 1987. 14. Trifillis AL, Cui X, Jacobs S, and Warren JW: Culture and characterization of normal epithelium from cystoscopic biopsies of human bladder. In Vitro Cell Dev Biol Anim 29A: 908-911, 1993. 15. Pratt-Rippin K, and Pezzlo M: Identification of commonly isolated aerobic gram-positive bacteria, in Isenberg, HD (Ed): Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC,. 1992, pp 1.20.1-1.20.44. 16. Division of Kidney, Urologic, and Hematologic Diseases (DKUHD) of the National Institute of diabetes and Digestive and Kidney Diseases (NIDDK). Diagnostic criteria for research studies (interstitial cystitis). Am J Kidney Dis 13: 353-354, 1989. 17. Hampson SJ, Christmas TJ, and Moss MT: Search for mycobacteria in interstitial cystitis using mycobacteria-specific DNA probes with signal amplification by polymerase chain reaction. Br J Urol 72: 303-306, 1993. 18. Fall M, Johansson SL, and Vahlne A: A clinicopathological and virological study of interstitial cystitis. J Ural 133: 771-773, 1985. 19. Hanash KA, and Pool TL: Interstitial and hemorrhagic cystitis: viral, bacterial and fungal studies. J Urol104: 705-706, 1970. 20. Elliott TSJ, Slack RCB, and Bishop MC: Scanning electron microscopy of human bladder mucosa in acute and chronic urinary tract infection. Br J Urol 56: 3843, 1984. 21. Collan Y, Alfthan 0, Kivilaakso E, and Oravisto KJ: Electron microscopic and histological findings on urinary bladder epithelium in interstitial cystitis. Eur Uro12: 242-247, 1976. 22. Wilkins EG, Payne SR, Pead PJ, Moss ST, and Maskell RM: Interstitial cystitis and the urethral syndrome: a possible answer. Br J Urol 64: 3944, 1989. 23. Pate1 J, Faden H, Sharma S, and Ogra PL: Effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable Haemophilus influenzae: implications for otitis media. Int J Pediatr Otorhinolaryngol 23: 15-23, 1992. 24. Saiman L, and Prince A: Pseudomonas aeruginosa pili bind to asialoGM1 which is increased on the surface of cystic fibrosis epithelial cells. J Clin Invest 92: 1875-1880, 1993. 25. Schwab UE, Wold AE, Carson JL, Leigh MW, Cheng PW, Gilligan PH, and Boat TF: Increased adherence of Staphylococcus aureus from cystic fibrosis lungs to airway epithelial cells. Am Rev Respir Dis 148: 365-369, 1993. 26. Raz R, and Stamm WE: A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infections. N Engl J Med 329: 753-756, 1993. 27. Buckley RM Jr, McGuckin M, and MacGregor RR: Urine bacterial counts after sexual intercourse. N Engl J Med 298: 321-324, 1978. 28. Kunin CM, White LV, and Hua TH: A reassessment of the importance of “low count” bacteriuria in young women with acute urinary symptoms. Ann Intern Med 119: 454460, 1993. 29. Mitchell MA, Markovitz DM, Killen PD, and Braun DK: Bilateral renal parenchymal malacoplakia presenting as fever of unknown origin: case report and review. Clin Infect Dis 18: 704-718, 1994.

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