Introduction

Prostate cancer is the second most frequent cause of mortality due to cancer in males in the United States [1]. Transuretral resection, radical prostatectomy, radiation therapy and hormone therapy are the usual prostate cancer treatments [2]. Prostate removal leads to the observation of prostatic calculi. Because almost 99% of surgically removed prostates contain stones, these stones are generally considered clinically insignificant [3]. Therefore, only a few papers have investigated these calcifications.

We noted the large chemical diversity of these prostatic stones. Carbonated calcium phosphate apatite (carbapatite; CA) seems to be the major component, but several investigations show the presence of other mineral phases such as calcium oxalate monohydrate and dihydrate [4], brushite [5], and whitlockite [6]. More recently, several other mineral phases previously not reported in prostatic calculi were octacalcium phosphate pentahydrate and amorphous carbonated calcium phosphate [7]. Such chemical diversity indicates significant variations in the local biochemistry, which may be linked to different conditions.

A previous investigation involving stone culture revealed that infected calculi in the prostate were implicated in relapsing urinary tract infection [6]. This work aimed to assess a possible relationship between infection and prostatic calculi, taking into account the chemical and structural characteristics of such calculi. We combined chemical analysis with Fourier transform infra-red (FTIR) spectroscopy and structural investigation at the mesoscopic scale by scanning electron microscopy (SEM). FTIR spectroscopy has helped us to examine the presence of chemical phases involved in infection of other organs [8], [9]. Moreover, SEM observations allowed for assessing the presence of bacterial imprints on prostatic calculi [10]. FTIR spectroscopy and SEM have been used in several studies of pathological calcifications [11], [12], [13], [14], [15].

Materials and Methods

SEM

Each prostatic stone was observed by Field-effect SEM (Zeiss SUPRA55-VP with an Everhart-Thornley secondary electron detector). To maintain sample integrity, each measurement was taken at low voltage (≤2 keV). Stones were imaged at similar magnification for comparison.

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Table 1. Major phases in inner and peripheral layers (minor phases in italics) in 23 samples of prostate stones, along with presence of urinary infection and bacterial imprints.

https://doi.org/10.1371/journal.pone.0051691.t001

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Table 2. Nature and frequency of main chemical phases in 23 prostatic stones.

https://doi.org/10.1371/journal.pone.0051691.t002

Results

Table 1 summarizes the age of patients, the chemical components of the 23 samples in the core and peripheral layers (main phases [>5%], minor phase [<5%]), the clinical data regarding urinary tract and prostate infection, and the presence of bacterial imprints seen by SEM. Several chemical phases (9 phases) were identified by FTIR spectroscopy. All stones mainly consisted of calcium phosphate, with CA the most common. Interestingly, the frequency of 2 phases, namely whitlockite and amorphous carbonated calcium phosphate (ACCP), as the main components was higher but not significantly in the core than in peripheral layers (48% vs 21%, p = 0.07). SEM revealed bacterial imprints in 18 stones (78%, p = 0.001), but clinical signs of urinary tract infection were reported for only 6 patients.

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Figure 1. Fixed-effects SEM of prostatic stones.

Red arrows show bacterial imprints. a) prostatic carbapatite-amorphous carbonated calcium phosphate (CA-ACCP) stone without any visible imprint; b) spherical prostatic CA stones without any visible imprint; c) prostatic CA-ACCP stone; d) prostatic ACCP stone, e) prostatic CA stone; f) prostatic CA-whitlockite stone. Bar, 4 µm.

https://doi.org/10.1371/journal.pone.0051691.g001

Table 2 shows the proportion and frequencies of the main chemical phases in the 23 stones. Whitlockite was detected in the core of 11 stones (48%) and as the main component in 8 (35%) but was the main component of the stone surface in only 4 stones (17%). ACCP was identified in the core of 6 stones (26%) and was the main phase in 3 (13%). ACCP was the main component of the stone surface in only 1 stone (4%).

Representative SEM images of normal stones and those with bacterial imprints (size about 500 nm) on the surface and in the core of stones are in Figure 1.

Discussion

Our SEM study revealed a high occurrence of bacterial imprints (78%) in 23 prostatic stones, which reveals a past or present infection of the prostate tissue; however, urinary tract infection was detected in only 6 (26%) cases. The large difference between number of reported infections and markers of infection within stones implies that aging may not be the only cause of prostatic calcifications. Infection and a lithogenic process induced by infection may play a role in most of the 99% of surgically removed prostate-containing stones.

The bacterial imprints had a specific spherical shape, which suggests infection by cocci germs. More precisely, the grape-like clustering, shape and size are common with staphylococci infection [18]. In 2 cases of proven infection, the species was Staphylococcus aureus. Nevertheless, these imprints were not seen on all stone surfaces. Similarly to kidney stones, the bacteria can imprint on a particle surface such as CA or ACCP but not other crystal types such as whitlockite, octacalcium phosphate or brushite. Indeed, the size of CA and ACCP crystals is smaller than that of other phases, such as struvite [19]. As well, SEM examination was restricted to some parts of small and partial samples collected during prostate removal. However, all stones contained at least 15% CA or ACCP, so a careful observation by SEM allows for detection of bacterial imprints on the surface of these minerals.

We discuss only the major phases. As previously reported, the main compounds of prostatic stones are calcium phosphate [20]. The most common and abundant phase is CA, well known as a common form of ectopic calcification in the kidney [21], vascular system [22], [23] or breast [24]. Particular crystalline phases, namely whitlockite and ACCP, a marker of infection stones in the urinary tract, were identified in the core of 17 (74%) prostatic stones. Whitlockite is an infrequent component of kidney stones and has been associated with chronic urinary tract infection in most calculi from women [8]. This phase has been also found in infections such as tuberculosis [9]. These different markers (imprints, specific phases, etc.) led to the conclusion that 83% of calculi (19 stones, p<0.0001) could be linked to an infection process.

The deleterious consequences of chronic infection and inflammation have been well described in the cellular model [25] and chemical model [26]. A number of papers have highlighted the relation between infection-related inflammation and cancer in various organs such as stomach, liver, lung, colon or bladder [27], [28]. The same reasoning may be applied to prostatic stones. Moreover, recent studies suggest epidemiological and pathologic links between benign prostate hypertrophy and prostate cancer [29]. Chronic infection, as well as the resulting stones, may induce persistent inflammation and could contribute to prostatic hypertrophy. In fact, the inflammatory process, associated with tumor phenomena, seems to influence the formation and evolution of these concretions [30]. We previously reported a high content of proteins in prostate stones [7]. Lactoferrin was found among proteins identified in both corpora amylacea and stones. This protein is considered a marker of inflammation and infiltration by neutrophil polynuclear factors and is implicated in the cancerization process [30].

The clinical interest of this paper is to draw attention to the high occurrence of asymptomatic infection of the prostate.

History of urinary tract infection and risk of renal cell carcinoma have been found to be related. As reported by Parker et al. [31], analysis of epidemiological data suggests a positive association of history of urinary tract infection and renal cell carcinoma development. Similar results were reported by MacLaughin et al. [32] and by Meares for prostate tissue [6]. Clearly more research is needed to establish a relationship because intratissular infection necessitates antibiotic treatment during several weeks. Our data suggest that the presence of stones or calcifications within the prostate could indicate chronic, often asymptomatic infection, the consequence of which remains to be assessed for medical management.