Study on Distribution of Four Pseudomonas Species in Living Environment Using Multiplex PCR

Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Investigation of Bacteria Species Most Involved in Peri-Implantitis

Purpose: Currently, bacteriological examinations of implant treatments target periodontopathic bacteria such as red complex bacteria, including Porphyromonas gingivalis, and detect them qualitatively or quantitatively. However, it seems that those examinations do not reflect the peri-implant tissue conditions precisely, because periodontopathic bacteria are also frequently detected from healthy peri-implant sites. The purpose of the present study was to investigate bacteria species most involved in peri-implantitis using a PCR method. Methods: Polymerase chain reaction (PCR) primers in this study were designed based on partial sequences of 16S rDNA of bacteria species involved in peri-implantitis that were described in numerous previous studies. Peri-implant sulcus fluid (PISF) samples were collected from thirty periodontally healthy patients with implants (HI) and thirty patients with peri-implantitis (PI). Each detection frequency of bacteria species in PISFs of both groups was investigated using a PCR method, and was compared using Fisher’s exact test. Results: In PI group, detection frequencies of Corynebacterium durum, Fretibacterium fastidiosum and Slackia exigua were significantly higher than those of HI group (p P. gingivalis and Tannerella forsythia belonging to red complex were frequently detected in the PISF samples of HI group (p > 0.05). Conclusion: It was suggested that monitoring C. durum and F. fastidiosum levels in PISF samples was useful as a clinical indicator for the evaluation of peri-implant tissue conditions.

Study on the Distribution at Species Level of Genus Candida in Human Oral Cavities, Using Culture and Multiplex PCR Methods

Purpose: Although the genus Candida is frequently isolated from human oral cavities, the distribution at the species level of these organisms has been little reported. The purpose of the present study was to assess the distribution at the species level of the genus Candida in human oral cavities. Methods: This study was performed using culture and Multiplex PCR methods. Moreover, the genotyping classification of C. albicans was analyzed with a PCR. Results: Of all subjects (n = 90), detection frequency of genus Candida was 42.2%. Genus Candida was not detected in the subjects between 0 to 9 years old, and there was no difference in the detection frequencies of this organism among each generation from 10s to 80s. C. albicans was the most dominant species, followed by C. parapsilosis, C. glabrata, and C. dubliniensis. Plural Candida species tended not to be detected in the individual sample. Genotype A was dominant in the C. albicans isolates. Conclusion: These results indicated that C. albicans of genotype A was dominant and that the genus Candida rarely coexists with other Candida species, in each individual oral cavity.

Isolation and Identification Methods for Oral Klebsiella pneumoniae Involved in Onset of Inflammatory Bowel Disease

Purpose: Recently, it was reported that Klebsiella pneumoniae is related to the onset of inflammatory bowel disease including the Crohn disease. It was frequently reported that K. pneumoniae was detected in human oral cavities. Regrettably, it currently remains unclear whether K. pneumoniae is part of the normal oral flora. The aim of this study was to establish the isolation and identification methods for K. pneumoniae from human oral cavities, and investigate its transmission pattern. Methods: A selective medium, OKPSM, for the isolation of K. pneumoniae from oral cavities was developed in this study. Also, PCR primer for the identification and detection at subspecies level of K. pneumoniae was designed. Results: OKPSM and PCR method using the primers designed in this study were useful for the isolation and identification of K. pneumoniae from human oral cavities. K. pneumoniae subsp. pneumoniae was detected at 10.0% in 30 saliva samples. On the other hand, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis were detected from no sample. Moreover, K. pneumoniae subsp. pneumoniae isolates from same subject at 0 month and after 3 months showed same genotypes on AP-PCR using OPA-07 primer. Conclusion: These results indicated that human oral cavities were not suitable for the habitat of K. pneumoniae.

Origin of Candida albicans in Human Oral Cavity

Purpose: Candida albicans is regarded as a part of normal flora in the human oral cavity. However, it remains unclear whether the genus Candida, especially C. albicans, is an oral resident microorganism and causes marital infection or not. The purpose of the present study was to elucidate the origin of oral C. albicans by investigating the colonization and infection route to oral cavities of this organism with arbitrarily primed polymerase chain reaction (AP-PCR). Methods: After C. albicans was isolated from four subjects (average age: 42.2, range: 33 - 56), the isolations of this organism from them were performed six months later again. To investigate whether C. albicans is an oral resident microorganism, the genotype homology of each C. albicans isolates that were isolated twice from the same subjects was compared. Moreover, C. albicans was isolated from five pairs of married couples (average period of cohabitation: 12.4 years, range: 5 - 31). To investigate whether C. albicans causes marital infection, the genotype homology of C. albicans isolates that were isolated from each pair of married couples was compared. Results: AP-PCR patterns of C. albicans that were isolated from each subject at o month and after 6 months showed the identical genotypes among each individual. C. albicans isolates from five pairs of married couples showed the identical genotypes between a husband and wife of each pair on AP-PCR. Conclusion: These results indicated that C. albicans was an oral resident microorganism and caused the marital infection.

Screening Survey of Pain Intensity in Patients with Temporomandibular Disorders

Objective: Pain tends to be the chief complaint in patients suffering temporomandibular disorders (TMD). Previous studies on pain and psychosocial factors have reported on the relationship between presence of pain and mental disorders. To date, however, few studies have addressed the relationship between intensity of pain and psychosocial factors. In this study, we investigated the relationship between intensity of pain and age, gender, palpation scores (PPS), tendencies toward depression, anxiety, and somatization, and oral parafunctional habits. Methods: This screening survey encompassed 104 patients (70 women and 34 men;mean age of 46.1 ± 19.3) who visited our clinic. We gathered the following data: age;gender;PPS included in Axis I diagnosis;and characteristic pain intensity (CPI), depression, anxiety, somatization, and oral parafunctional habits (assessed by the Oral Behavior Checklist) included in Axis II diagnosis. Based on the results of CPI, we divided patients into two groups: those experiencing low pain intensity (LP group) and those experiencing high pain intensity (HP group). The statistically significant level was set to below 5%. IBM SPSS Statistics V25 was used to perform all statistical analyses. Results: We observed no gender differences between LP and HP groups. The HP group included significantly more patients with higher scores for depression, anxiety, somatization, and oral parafunctional habits than the LP group. While no gender differences were observed in CPI, depression, anxiety, somatization, and oral parafunctional habits were significantly more common in women than in men. We observed no differences in age or PPS between the LP and HP groups. However, scores for depression, anxiety, somatization, and oral parafunctional habits were significantly higher in the HP group than in the LP group. We performed multiple regression analysis using the CPI score as the dependent variable and scores for depression, anxiety, somatization, and oral parafunctional habits as independent variables in both the LP and the HP groups. We identified no significant predictors for the LP group, but extracted depression as a significant predictor in the HP group. On evaluating the correlation of PPS with depression, anxiety, somatization, and oral parafunctional habits in both the LP and the HP groups, we found no correlation between the PPS and the seven-item generalized anxiety disorder (GAD-7) scale in the LP group but identified a significant correlation between the PPS and GAD-7 scores in the HP group. Moreover, the correlation coefficient between the patient health questionnaire (PHQ)-9 and GAD-7 scores was higher in the HP group than in the LP group. Conclusion: In those reporting more intense pain, we found a stronger correlation among psychological factors in patients diagnosed with TMD. Greater tendency toward depression was directly associated with pain intensity. The results point to the need to consider differences in psychosocial factors associated with pain intensity when treating TMD.

Study on Distribution of Acinetobacter baumannii Complex in Dental Hospital Using Multiplex PCR

Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

One Step Multiplex PCR for Identifications at Subspecies Level of Fusobacterium nucleatum and Fusobacterium necrophorum

Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nucleatum and F. necrophorum are classified into five and two subspecies, respectively. Conventional identification methods were technically hard to distinguish each subspecies of two Fusobacterium species accurately. The purpose of the present study was to design primers to identify two medically important Fusobacterium species at the subspecies level, using one-step multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene, RNA polymerase B (rpoB) gene, and DNA gyrase subunit B (gyrB) of each subspecies of F. nucleatum and F. necrophorum. Results: These primers were able to distinguish each subspecies of F. nucleatum and F. necrophorum and did not display cross-reactivity with representative Fusobacterium species other than F. nucleatum and F. necrophorum. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

One Step Multiplex PCR for Identification of Candida haemulonii complex and Candia auris

Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emerged as an opportunistic fungal pathogen associated with various infectious diseases of humans, and most of those isolates have displayed antifungal resistance. Because it is difficult to differentiate these microorganisms by a current technique, unfortunately, it is important to establish a method for identifying them accurately. The purpose of the present study was to design species-specific primers in order to identify and detect C. auris, C. pseudohaemulonii, and C. haemulonii complex, i.e. , C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 26S rRNA, 18S rRNA, and RPB1 genes and ITS region of five Candida species. Results: The multiplex PCR method developed in this study was able to distinguish five Candida species clearly. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction.

Pemphigus vulgaris macroscopically and cytologically resembling oral squamous cell carcinoma

We describe the clinical, macroscopic, cytological, histopathological, immunohistochemical, serodiagnostic and aspects of pemphigus vulgaris (PV) in the oral gingiva that clinically mimicked oral squamous cell carcinoma (OSCC) in a 57-year-old Japanese man. He developed slight haphalgesia of the buccal gingiva around teeth numbers 18 and 19 2 years ago. A dentist diagnosed intractable ulcer, but the patient ignored the condition for about 2 years until a sharp pain in the gingiva worsened. He consulted an otolaryngologist, who referred the patient to our hospital under a cytological diagnosis of OSCC. An oral examination revealed several extensive painful erosions/ ulcers from the buccal and lingual gingiva around teeth numbers 18 to 21 to the distal alveolar mucosa of no. 18 and the buccal and lingual gingiva around tooth number 31. A presumptive diagnosis of PV with dysplastic changes was determined from cytological smears. The cytological Nikolsky test was positive. The diagnosis of PV was confirmed from clinical and histopathological findings of a biopsy specimen obtained from the perilesional site. Although the definitive diagnosis of PV required only 2 weeks after this patient presented at our hospital, 2 years had elapsed since the onset of oral lesions.

Morphometrical findings among dysplasias of oral, cervical and bronchial regions

The purpose of this study was to compare among dysplasia of oral, uterine cervix and bronchus. Using a computer cytomorphometry cell measurement program, the study was based on a retrospective review of smear cases diagnosed with dysplasia of oral, bronchial and uterine cervix, from 2002 to 2010. For 50 - 70 cells from each lesion, nuclear (N) and cytoplasm (C) variables were assessed: area (A), diameter (D), irregularity (I), stain brightness and granularity. NA and ND were highest in OSCC and higher according to dysplastic grading. By contrast, CA and CD were lowest in severe dysplasia. The significant difference of N/C ratio was observed among OSCC to inflammation, mild and moderate dysplasias (p < 0.05). The N/C ratios of mild and moderate dysplasias were equal. Brightness and granularity values of OSCC cases were significantly higher than those of another (p < 0.05). About the difference between mild to moderate dysplasias, it was the easiest to detect of the uterine cervix. All severe dysplasias among the 3 regions were easily identified morphometrically. The deficient in the difference between inflammation to mild dysplasia and mild to moderate dysplasia were obtained in the oral mucosal lesion. The results displayed a significant variation in cytomorphometrical values among the 3 regions. N/C values for uterine cervix and bronchus were well distinguished in comparison with oral dysplasias. Screening of mild and moderate dysplasias requires experience which carries out the comprehensive judgment of the color.

Palisaded encapsulated neuroma of the upper lip

Palisaded encapsulated neuroma (PEN;solitary circumscribed neuroma) is a benign and hyperplastic lesion consisting of Schwann cells. PEN of the lower lip was reported by Tomich and Moll [1] 35 years ago. However, the accumulation of the information about PEN which occurred in the oral mucosa was not enough. This article describes a case of a PEN on the upper lip of a 41-year-old woman. The lesion with 0.7 cm diameter was performed excisional biopsy. Histologically, the tumor was almost circumscribed by thin fibrous capsule, and consisted of diffusely and dense proliferation of the spindle shape cells arranged in interlacing fascicles. Focal suggestions of nuclear pal-isaded growth were indicated within the tumor. Immunohistochemicallly, the fascicles of tumor cells were positive for S-100 protein, and vimentin and negative for α-actin and GFAP. A few numbers of axons were demonstrated by anti-neurofilament antibody in this lesion. Therefore, the definitive diagnosis was PEN.

A clinico-pathological and cytological study of oral candidiasis

Candidiasis of the oral mucosa arises chiefly as a re- sult of infection with Candida albicans. Many clinico- pathological analyses of macroscopic findings have been described, although the clinical findings of oral candidiasis vary considerably and the conditions are complex. The present study analyzes the distribution, clinical, cytological and histological diagnoses of oral candidiasis, associated complex diseases and the di-agnostic value of cytology. The ratio of Candida in-fection was 28.9% among 1551 study participants. Females were infected significantly more often than men (p < 0.01) and the affected age range was 60 - 79 years (61.0%, p < 0.01). The predominantly affected areas were the tongue (48.3%, p < 0.01) and gingiva (20.0%, p < 0.01), and occurrence at multiple loci was seen in 43 (9.6%) patients. The typical clinical find- ings of oral candidiasis were ulcerative/erythematous lesions (33.2%, p < 0.01) and pseudomembranous candidiasis (31.6%, p < 0.01). A histopathological dia- gnosis of candidiasis based on biopsy specimens from 26 lesions in patients with Candida infection indicated by cytology was confirmed from cultures. The break- down of a cytological to a definite diagnosis was 6 positive (SCC 4, verrucous carcinoma 1, moderate to severe dysplasia 1), 6 suspected positive (mild dyspla- sia, 2;moderate to severe dysplasia, 2;papilloma, 1 and SCC, 1) and 14 negative (epulis, 3;papilloma, 3;granulation tissue, 2;fibrosis, 2 and others, 4). Exfo-liative cytology can easily judge the presence of Can-dida species, although experience is necessary for the presumptive diagnosis of an oral mucosal disease. The application of exfoliative cytology using the Pe- riodic acid-Schiff reaction is helpful for the earlier detection of oral candidiasis with various macrosco- pic findings.