Unusual Oropharyngeal Asymptomatic Manifestations Caused by Atypical Pathogens Detected by PCR into Altered Ecosystems of an Infertile Couple

study. statistical GN clinical evaluations. GDBOC laboratory ABSTRACT The young couple casually arrived at our observation presented two different and painless altered ecosystems. The female partner exhibited inflamed lingual surface, while the man manifested several genital ulceration areas. Microbiological analysis of several sites belonging to each ecosystem revealed a latent dismicrobic ecosystem, as also confirmed by altered pH values. Interestingly, microbiological analysis for Mycoplasma hominis , Ureaplasma urealyticum , and Chlamydia trachomatis gave different results in both ecosystems, highlighting that each partner was differently infected by the same “silent infections”. The frequency of Ear Nose and Throat (ENT) chronic morbidity for these atypical infections increases, in the couple, the probability that mycoplasmosis and chlamydiasis serve as a reservoir of infection and/or reinfection for the urogenital tract. Although it is well known that several common bacteria and “atypical pathogens” like mycoplasmas and C. trachomatis - can colonize both oral and urogenital ecosystems, rarely happens that they can clinically manifest their presence by unusual clinical manifestations we observed in different ecosystems investigated, hiding the unique true problem of infertility in the young couple. In this regard, our results showed that only starting from an accurate anamnesis, using an adequate sampling modality, adopting a new procedure to quantify microbial populations into asymptomatic ecosystems, and introducing the constant use of PCR analysis, it will be possible to disclose similar precocious problems of couple infertility, in genetically predisposed subjects.


INTRODUCTION
A young caucasian couple (woman: 26 years old; man: 30 years old) manifested different inflamed ecosystems together with a referred infertility problem. At the urological visit, the male partner declared the presence of inflamed areas on the glans that periodically peeled without any particular pain (Fig. 1A). Previous local treatment with gentamicin-betamethasone had not given any significant stable improvement. Furthermore, after sexual activity the patient referred for a sudden burning into meatus with acute pain that spontaneously regressed in 2-3 days (Fig. 1B). This clinical manifestation resulted to be ever more persistent during the first emiction of the morning. Urine culture carried out during these clinical manifestations resulted negative, and urine analysis showed no erythrocytes and leucocytes in the sediment. He presented ulcerations on both sides of glans and on scrotum since about 3 months (Figs. 1A, 1C). No other symptoms were referred for other ecosystems.
The female partner showed a furry tongue with intense viscous salivation highly suggestive for a clinically altered lingual ecosystem (Fig. 2). No particular sign or pharyngeal alteration could be observed in both partners, although the male partner referred for infrequent pharyngitis episodes during the year. On demand, the couple confirmed a frequent oral intercourse in their sexual ménage.
From clinical history -collected by an Ear Nose and Throat (ENT) specialist team together with an expert of "atypical infections" -emerged that the infertility of the young couple represented, at that moment, the sole early and important reversible problem.

MATERIALS AND METHODS
Patients were studied according to the declaration of Helsinki, obtaining written informed consent from each partner. No personal hygiene was carried out by both partners before microbial investigations.

Prostatic and Urological Evaluation
According to National Institutes of Health guidelines (NIHCPPSI: Chronic Pelvic Pain Symptom Index) [1], the patient was classified as belonging to category IV of asymptomatic inflammatory prostatitis.  Urethra was also sampled for cellular material by introducing asharp Dacron swab into urinary meatus for 1 cm and turning it once. Sample was dispersed by stirring in 1.0 mL of concentrated urine, and this suspension was used for culture and molecular analyses.

Genital Ulcer Evaluation
The cutaneous ulcer observed on scrotum (genital ulcer, GU) was sampled by scraping. Sample was dispersed into 1.0 mL of sterile saline and assayed for the presence of three major agents of genital ulcer disease (T. pallidum, Haemophilus ducreyi, C. trachomatis), and N. gonorrhoeae by molecular analyses, as previously reported [2][3][4].

Lingual and Pharyngeal Evaluation
Samples obtained by lingual and pharyngeal scraping were suspended into 1.0 mL sterile saline, then immediately evaluated for the oropharyngeal ecosystems by culture analysis for saprophytic flora, common pathogens and atypical ones (C. trachomatis, M. hominis, and U. urealyticum). One microliter of this suspension was plated onto chromID CPS and Columbia CNA agar 5% sheep blood agar, then incubated under both aerobic and anaerobic atmosphere. Cultures were monitored daily up to 72 h for total colony counting. Total microbial charge (TMC), expressed as colony-forming unit per milliliter (CFU/mL), was calculated by adding the number of colonies developed onto chromID CPS agar to that anaerobically grown onto Columbia CNA agar. The relative abundance of each species was reported as percentage of TMC (considered as 100%). Since the oropharyngeal ecosystem is directly exposed to external environment, we could accept that this ecosystem present a TMC > 10 7 CFU/mL. By this way, the threshold of pathogenicity for a monoresident pathogen could be considered as ≥ 10 6 CFU/ml. The presence of mycoplasma was detected as above described. An aliquot of 0.1 mL of the initial suspension was used for pH measurement of each ecosystem investigated, while 0.5 mL was used for molecular assays.

Serologic Evaluations
VDRL test was negative for man, but positive for woman; both partners were negative at TPHA evaluation. Total PSA was 0.87 ng/mL (95%-ile of health population: 0.60 ± 0.35).

Oral and Pharyngeal Ecosystems
Culture and molecular analyses of different samples collected from the male partner showed that common microbial flora was differently distributed. Lingual scraping revealed the presence of C. ulcerans only (1.0 x 10 6 CFU/mL), while pharyngeal scraping revealed positivity for S. faecalis, C. ulcerans, E. coli, and K. pneumoniae (20%, 40%, 30%, and 10% of TMC, respectively; TMC: 0.5 x 10 6 CFU/mL), (Table 1). Mycoplasmas were present at low concentrations (10 3 CCU/mL), although differently distributed. Particularly, U. urealyticum was present in both lingual and pharyngeal scraping samples, while M. hominis was observed in lingual scraping samples only ( Table 1). The pH value measured in the lingual ecosystem of female partner resulted to be higher than the physiologic one measured in the male partner (pH: 8.9 vs 6.2, respectively).
Pharyngitis episode is one of the most common clinical evidence in patients at the consulting room of the ENT clinic [5,6]. Although female partner appeared in good health and without clinical pharyngeal history, the characteristic oral ecosystem observed led us to investigate the pharynx too. Ecosystems showed different microbial pattern for saprophytes and common opportunistic pathogens (Table 2). In particular, lingual scraping revealed the presence of C. ulcerans only (6.0 x 10 5 CFU/mL), contrarily to the polymicrobial growth observed onto pharyngeal mucosae, consisting of S. milleri, S. faecalis, C. ulcerans, K. pneumoniae (10%, 20%, 50%, and 20% of TMC, respectively; TMC: >10 7 CFU/mL). The two ecosystems differed as well when compared for atypical bacteria. In fact, only the lingual one showed the presence of M. hominis and U. urealyticum (both at >10 4 CCU/mL), and C. trachomatis. With regard to common microbial patterns, no significant differences were observed between different samples collected from the pharyngeal ecosystem. Pharyngeal swab resulted to be positive for S. milleri, C. ulcerans, E. coli, and K. pneumoniae (30%, 20%, 20%, and 30% of TMC, respectively; TMC: > 10 7 CFU/mL), but negative for sexual common atypical pathogens (C. trachomatis, M. hominis, and U. urealyticum). Pharyngeal scraping sample was positive for S. milleri, S. faecalis, C. ulcerans, and K. pneumoniae (10%, 20%, 50%, and 20% of TMC, respectively; TMC: > 10 7 CFU/mL). Pharyngeal scraping revealed the presence of M. hominis and U. urealyticum (both at 10 3 CCU/mL), although it was negative for C. trachomatis.

Urogenital Ecosystem
The male urogenital ecosystem, assayed by both culture and molecular analyses, showed a polymicrobic resident flora in each of different sites investigated ( FVU collected from the female partner resulted positive for a polymicrobic bacterial growth, consisting of S. faecalis, S. epidermidis, and C. ulcerans (10%, 40%, and 50% of TMC, respectively; TMC: 5.0 x 10 4 CFU/mL). Microscopic observation was negative for T. vaginalis. M. hominis was absent, while U. urealyticum was found at 10 3 CCU/mL. PCR was positive for C. trachomatisonly.

DISCUSSION AND CONCLUSION
In this study we investigated all the ecosystems involved in sexual activity of a young couple presenting a latent infertility problem only. In the male patient, lingual pH and PSA tot values, added to negative VDRL, were highly suggestive for apparent physiological conditions, despite of oropharyngeal and urogenital asymptomatic clinical pictures. However, culture analysis of lingual ecosystem highlighted the exclusive presence of C. ulcerans, while molecular analysis revealed the presence of M. hominis, U. urealyticum, and C. trachomatis. The bacterial load we observed in male pharyngeal ecosystem, lower than that observed in the lingual one, might reflect an apparent "physiologic status" at pharyngeal site, as supported by lack of clinical picture. Furthermore, this evidence of polymicrobial growth in pharyngeal ecosystem is in accordance with methodological preparation and standardization of the samples we carried out. Quantitative microbiological analyses allowed us to ascribe to the higher mycoplasmal lingual presences compared to pharyngeal site, the major clinical evidence of an asymptomatic condition of silent and/or low-grade inflammatory status. If lingual and pharyngeal weak evidence for C. trachomatis infection observed in male partner could explain asymptomatic conditions, on the other side it is possible to involve both the direct modality of lingual contagion and different cellular typology to colonize.In our previous studies [2,7], we have never found these situations because the patients were affected by chronic oropharyngeal and conjunctival inflammation. Alternatively, it is possible to evoke for both patients both different genetic individual susceptibility [8], and the always possible "time difference" with respect to contagion.
The high pH value observed in the female lingual ecosystem and its clinical manifestation are indicative of the altered microbial ecosystem observed in the oral cavity (Fig. 2). On the contrary, no demonstrated pharyngeal mucosae inflammation was noted. Considering microbiological findings obtained from lingual samples -that is the presence of C. ulcerans, the high mycoplasmas (U. urealyticum, and M. hominis) concentrations and the presence of C. trachomatis -it is plausible to hypothesize that this site would be the first one to be colonized during sex oral behaviour [8], while C. ulcerans could be considered as a microbiological marker of the altered lingual ecosystem following urogenital contamination.
With regard to the pharyngeal ecosystem, while swab samples suggested the absence of "atypical infections", M. hominis and U. urealyticum were observed, although at low concentrations, in scraping samples, thus demonstrating the latter as the best sampling modality. Additionally, microbiological evaluation of the female pharyngeal ecosystem by means of different sampling modalities showed a noteworthy overlapping of bacterial species, thus suggesting the marginal role of these bacteria in triggering inflammation of the pharyngeal ecosystem. They might, however, contribute in altering, together with mycoplasmas, the nutritional state of colonized cells, thus favouring the development of the critical condition of inflammatory status [9,10], triggering the unbalance of cellular potential redox state [11]. The absence of chlamydia at this uninflammed site could be explained by both an initial phase of contagion from tongue and a chlamydial condition not detectable by PCR [12][13][14]. In this regard, it is noteworthy to remember that during renewal of chlamydial infection in a patient with asymptomatic chronic infection, each reticular body produces much more progeny from a single inclusion, assuring a new potential infecting load for contiguous tissue or its diffusion in intestinal habitat.
It is known that higher pH value, increased number of leucocytes/hpf, and sperm agglutination are some of causes of asymptomatic inflammatory prostatitis, often incidentally diagnosed during infertility or prostate cancer evaluation [15][16][17][18]. Together with an accurate anamnesis and prostatic investigation, adequate evaluations concerning SF demonstrated an altered fertility due to low vitality test and high agglutination phenomenon in the presence of an adequate nemasperms concentration.
The presence of asymptomatic recurrent genital ulcers, suggestive for chlamydial and/or mycoplasma infections, together with increasing trend of PSA value [19], were justified by microbiological evaluation of urogenital ecosystems. Considering culture and molecular analyses of FVU, GU, SF, and UAPM, it is highlighted that the positivity for mycoplasma infections are distributed differently on the several sources, indicating a major presence of these infections in parenchymal prostatic tissue compared to FVU, GU, and SF. In the same sources, chlamydial infection presented a different behaviour, being more positive in GU and UAPM, indicating in C. trachomatis the true aetiologic agent of ulcered areas, while the parenchymal prostatic tissue represents a major reservoir of chlamydial infection [9].
In our operative conditions, the polymicrobic colonization found in each ecosystem was highly suggestive for saprophytic flora. Lastly, yellowmucous secretion, high pH value, relevant presence of leucocytes/hpf, very low L. acidophilus concentration, replaced with an abundant presence of C. ulcerans, characterized an altered female genital environment due to asymptomatic chronic mycoplasma and chlamydial infections. Although C. ulcerans has been increasingly isolated as an emerging zoonotic agent of diphtheria and other infections from companion animals [20,21], we believe that in our case it has to be considered as an opportunist pathogen [5], a biomarker of altered oral, as well as ocular, ecosystems as already found in previous studies [6][7][8]22]. The relevant presence of C. ulcerans we observed in the female patient might in fact indicate that it concurred to the pathogenic determinism by increasing pH level and, therefore, favouring the decrease of cellular redox state [5]. Furthermore, toxigenic C. ulcerans could have contribute in developing the evidence of a "patinated whitish tongue" with mucoid salivation we observed.
There is currently no direct evidence of personto-person transmission of C. ulcerans. Although our findings rise the possibility of a sexual transmission, unfortunately strains from both partners were not subjected to genetic fingerprinting to demonstrate their clonal relatedness.
The female patient also showed VDRL positive test, indicating an early condition triggering of autoimmune diseases [23]. We have recently reported that these atypical infections -being "silent and/or at low grade inflammatory" -might developed, in patient genetically susceptible, a VDRL positivity after decades from contagion, indicating the first biochemical marker of silent mycoplasma infection, developed itself in any ecosystem of the host [2,7,9,24,25]. It is interesting to note that silent atypical chronic infections by M. hominis, U. urealyticum, and C. trachomatis determine several health problems on genital female reproductive system [23][24][25][26][27], but one of which, such as the precocious infertility of a susceptible genetically young couple [9], was solved following the holistic vision of "Human Being" and carrying out a double sampling modality. Gavazzi et al. [28] stated that "ageing may be the cause of infection but infection can also be the cause of ageing", considering the holistic vision "Human Being" as the best way to approach the human problems before these become irreversible decline. Franceschi et al. described the "inflammaging" phenomenon [29,30], caused by innate immune system chronically stressed. Furthermore, Chang et al. [31] reported that chronic mycoplasma infections are responsible of subsequent risk of cancer among elderly adults. In a recent study, Gallenga et al. [32] demonstrated that the standardization of sampling methodology by scraping of living cellsis crucial in establishing C. trachomatis presence in the ocular ecosystem during the early phase of conjunctivitis.
Results we obtained by adopting these two different sampling modalities, we also used in our previous studies [2,7,9,24,32], have borne out our beliefs regarding asymptomatic patients during initial chronic phase of infections. To correctly interpret this assertion it is necessary to highlight that this initial phase could last for decades. It is interesting to note as the two sampling modalities led to opposite results for C. trachomatis presence, nourishing once again the enigmatic behaviour of chlamydial infection. The failure of a laboratory diagnosis in clinically asymptomatic patients would underestimate these atypical and silent infections, keeping out control by any control program of public health.

ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.