calcified plaques composed of calcium salts including phosphate , carbonate , and ammonium - magnesium salts in the bladder mucosa accompanied with inflammation and ulcerations characterize pathologically encrusted cystitis . despite its increasing incidence in nineties , especially in immune - depressed patients and renal transplant recipients ( meria et al 1998 ) , according to a pubmed search , by spring 2008 , only 60 patients have been reported in 60 published articles , mainly single case reports .
etiology of this rare entity , which is characterized by calcifications in the urinary bladder wall , is presently controversial .
the most popular of them proposes combined action of mucosal alterations ( lesion of any type ) and microorganisms that split urea forming ammonia and thus creating an alkaline environment leading to the disease by precipitation of calcium salts ( russell 1901 ) .
corynebacterium group d2 ( fernandez natal et al 1992 ) , escherichia coli ( romero perez et al 1992 ) , streptoccocus hemolyticus , s. viridians , and proteus ( jameson 1966 ) species were denoted as the most frequent culprits .
calcifying nanoparticles ( previously called nanobacteria ) are self - propagating , cultivable macromolecular complexes ( kajander 2006 ) generally regarded as bacteria but with a lack of definitive genomic proof . the defining characteristic of calcifying nanoparticles ( nanobacteria ) is their outer envelope composed of calcium phosphate that presents on transmission electron microscope images as an electron - dense shell surrounding a translucent central core .
the name is derived from the very small size of these nanoparticles , which varies from 50 nanometers to 500 nanometers ( nm ) .
calcifying nanoparticles ( nanobacteria ) are the smallest ( 50500 nm ) cultivable replicating agents on earth , which were isolated from bovine and human blood for the first time by kajander and colleagues ( 1997 ) in the last decade of the 20th century .
the most important characteristic of nanobacteria is that they produce carbonate apatite on their cell envelope ( kajander and iftioglu 1998 ) from soluble calcium and phosphorus at physiologic concentrations and conditions .
kajander s discovery initiated further studies and nanobacteria have been implicated in the wide array of human diseases associated with calcifications including : kidney stone formation ( iftioglu et al 1999 ) , valvular calcifications ( jelic et al 2004 ; miller et al 2004 ) , psammoma bodies seen in ovarian cancer ( hudelist et al 2004 ) , calcified atherosclerotic vascular plaques ( miller et al 2004 ; puskas et al 2005 ) , cholelithiasis ( wen et al 2005 ) , microcalcification in breast cancer ( altundag et al 2006 ) , calcific aortic valve stenosis ( jelic et al 2007 ; bratos - perez et al 2008 ) , calcifications of placental villi ( agababov et al 2007 ) , and randall s plaque ( iftioglu et al 2008 ) . the etiologic role of nanobacteria in kidney stone formation was confirmed in a small study ( garcia - cuerpo et al 2000 ) when nephrolithiais in rats resulted from intrarenal injection of nanobacteria that were previously isolated from kidney stones .
bratos - perez and colleagues ( 2008 ) successfully cultured self - replicating calcifying nanoparticles from the aortic valves of patients with calcific aortic stenosis .
some authors interpret nanosize calcifications as precipitation of calcium salts on macromolecules ( cisar et al 1999 ) , or on protein fetuin ( raoult et al 2008 ) and these and some other authors doubt the mere existence of nanobacteria as living microorganisms ( abott 1999 ; urbano and urbano 2007 ) .
this is partially due to the fact that genomic structure of nanobacteria has not yet been elucidated .
for this reason kajander ( 2006 ) suggested changing the name of nanobacteria to calcifying nanoparticles , while emphasizing that they are self - propagating , cultivable , and infectious agents .
recently mathew and colleagues ( 2008 ) documented that calcifying nanoparticles were self - replicators in physiological conditions and not simple crystals of precipitated inorganic apatite .
the patient is a 43-year - old man , a heavy smoker ( 34 packs a day for over 10 years ) who presented with complaints of hematuria and low back pain .
he was found to have multiple recurring papillary noninvasive urothelial ( transitional ) cell carcinomas at multiple bladder sites grade 1 and grade 2 ( in a scale 14 ) that were completely removed during a 6-month period .
after the second resection ( july 2006 ) a single instillation of 40 milligrams of mitomycin c was administered .
four months later he received six courses of bacillus calmette - guerin ( bcg ) over a six - week therapy to prevent bladder tumor recurrence per current recommended guidelines .
calcium and creatinine blood concentrations were normal 9.2 mg / dl and 0.7 mg / dl , respectively .
the patient was not treated with antibiotics or uroantiseptics except during cystoscopy procedure to prevent iatrogenic infection .
cystoscopy performed 3 months after the second surgery and before bcg application identified the recurrent papillary tumor .
these plaques were associated with at least two ulcerations that were hard on palpation due to these calcifications .
the small papillary tumor was extirpated and the calcifications were removed from the dome piece by piece .
repeated bladder biopsy 6 months later showed no evidence of malignancy but encrusted cystitis was still present .
biopsies of the tumor and of the bladder dome were fixed in 10% buffered formaldehyde neutral solution and embedded into paraffin blocks .
paraffin blocks were cut on standard rotary microtomes with a routine setting of 4 microns .
one ribbon from each paraffin block was placed on glass slide in order to be stained with hematoxylin and eosin .
the staining procedure was performed by an automated robotic stainer with a line up of solutions as follows : biocleer - f , absolute alcohol , 95% and 80% alcohol , gill s iii hematoxylin ( 7 minutes ) , running tap water , 1% acid alcohol , and eosin ( 1 min ) , row of alcohol solutions , and finally biocleer - f . after staining the glass slide
was placed in a xylene rinse to activate the cover slip adhesion on the automated cover - slipping machine .
von kossa s method for calcium staining is based on silver salt and was performed as follows : ribbon 6 microns thin was cut off paraffin block by standard rotary microtome and placed on glass slide .
after deparaffinization and hydration with distilled water the glass slide was placed in 5% silver nitrate solution for 60 minutes exposed to ultraviolet lamp .
after rinsing in distilled water glass slide was placed in 5% sodium thiosulfate ( hypo ) solution for 2 minutes . after rinsing in distilled water nuclear and cytoplsmic counterstaining
rinsing in distilled water , dehydration in alcohol , clearing in xylene and mounting with permount were final steps in von kossa s procedure for calcium staining .
standard procedure for obtaining electron microcope images was implemented as follows : the calcified area was cut out from the paraffin block , deparaffinized before embedding in a polymerized block of spur epoxy resin , and cut into 600 to 900 angstroms thin slices that were stained with uranyl acetate stain and lead citrate stain .
transmission electron microscopy was performed on a zeiss model cem 902 instrument manufactured in west germany .
light microscopic examination of the hematoxylin and eosin stained sections of the tumor showed noninvasive , low - grade papillary urothelial carcinoma .
biopsies from the bladder ulcers showed encrusted cystitis with an abundance of calcifications confirmed by von kossa stain ( figure 2a  c ) .
transmission electron microscopy of the encrusted cystitis calcifications revealed that the smallest constituents of calcifications were not amorphous substance or granules but formed particles ( elementary units of calcifications ) , composed of electron - dense shell rimming a central translucent core .
they vary in size ( figures 3 , 4 ) from165 nm to 440 nm and exhibit the size and spherical - to - ovoid shape diagnostic of calcifying nanoparticles / nanobacteria according to the relevant literature and our experience .
it is easy to notice in figure 3 that merging of the calcifying nanoparticles ( like stacking of snowballs ) forms micron - size calcium aggregates ( micron - size plaques ) measuring about 3 square microns that by further merging have ultimately become plaques of encrusted cystitis visible by light microscope ( figure 2 ) and naked eye ( figure 1 ) .
our electron microscopic study of the calcifications in the urinary bladder mucosa of the patient with encrusted cystitis revealed that the smallest formed structures , to wit elementary units of these calcifications are electron - dense rings with central lucent core diagnostic of calcifying nanoparticles .
as discussed in length in the introduction , controversy about nature of calcifying nanoparticles ( previously called nano - bacteria ) will continue in the future .
encrusted cystitis is a very rare form of cystitis still of controversial etiology and thus no standardized therapy exists , although the first of report for cure of an individual patient appeared 107 years ago ( russell 1901 ) .
papillary urothelial carcinomas in our patient were successfully eradicated but removal of calcified plaques was only partial and encrusted cystitis persisted . prevailing view is that encrusted cystitis is alkaline cystitis due to urea splitting bacteria ( mainly corynebacterium group d2 ) .
however , it was found later ( huget - perez et al 1999 ) that not all patients with encrusted cystitis have alkaline urine implying that other mechanisms for calcifications may play role in the genesis of encrusted cystitis .
our patient had acidic urine with a ph of 5 and standard urine culture ( not prolonged ) for common microorganisms showed no growth .
it has not been documented in the literature that nanobacteria or calcifying nanoparticles can split urea .
it has been demonstrated in vitro ( iftioglu et al 2008 ) that nanobacteria ( calcifying nanoparticles ) were inhibited by tetracycline , nitrofurantoin , trimethoprim , trimethoprim - sulfamethoxazole , and ampicillin at levels achievable in serum and urine .
encrusted cystitis has also been reported after the intravesical application of bcg ( spirnak et al 1993 ) or mitomycin c ( alter and malek 1987 ) for the treatment of bladder carcinoma .
our patient developed encrusted cystitis before bcg application . since the first report by alter and malek ( 1987 ) , only 6 additional cases of intravesical calcifications after local application of mitomycin c were reported , it is unlikely that intravesical instillation of this cytostatic drug in our patient caused encrusted cystitis but it can not be totally excluded . at this point in time ,
when nature of calcifying nanoparticles is unresolved and their bacterial structure uncertain , it is not warranted to speculate that mitomycin c facilitated infection with calcifying nanoparticles / nanobacteria .
we have revealed by electron microscopic study calcifying nanoparticles in the calcifications of the encrusted cystitis . from this finding
we have inferred that calcifying nanoparticles are associated with encrusted cystitis and are quite possibly the causative agents of this very rare disease . at this point in time with scant knowledge of pathogenesis and epidemiology of calcifying nanoparticles , a discussion about the mode of infection or their access to bladder mucosa is premature .
further studies are necessary in order to confirm whether calcifying nanoparticles are causative agents of encrusted cystitis or only its pathologic marker .
 encrusted cystitis is a subtype of chronic cystitis characterized by multiple calcifications in the form of plaques located in the interstitium of the urinary bladder mucosa and frequently associated with mucosal ulcers . 
 it is a very rare disease of controversial etiology . 
 our transmission electron microscopy of the calcified plaques of encrusted cystitis has revealed that the smallest formed particles ( elementary units ) of these calcifications are electron - dense shells surrounding an electron lucent core , diagnostic of calcifying nanoparticles ( previously called nanobacteria ) . 
 we pioneer the notion that calcifying nanoparticles are the causative agents of encrusted urinary bladder cystitis . 
Introduction
Case report
Material and methods
Results
Discussion