Nested PCR for detection of mutans streptococci in dental plaque

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.     

Development of 16S rDNA-based PCR assay for detecting centipeda periodontii and selenomonas sputigena

To detect oral motile bacteria directly from dental plaque, specific PCR primers for Centipeda periodontii and Selenomonas sputigena were designed based on the sequence analysis of their 16S rDNA. The primers were specific and sensitive enough to amplify DNA fragments from the available oral bacteria. The detection limit was fewer than 10 bacterial cells per sample. It was also possible to detect these bacteria in dental plaque. The prevalence of these bacteria varied in each sample. The specific primers designed in this study may clarify the epidemiology of periodontal disease.     

Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections

The role of Trichomonas tenax as a pathogen had been clearly implicated in various pathological processes that arise outside the boundaries of the mouth. Although a relationship between the increased occurrence of this protozoan and progression of periodontal disease has been demonstrated, the ability of T. tenax in causing oral infections and the precise mechanism of tissue damage is not well known. The present study aimed to investigate different isolates of T.tenax from individuals having oral infections. Plaques and/or calculi samples were collected from 70 individuals who were diagnosed as having periodontitis and/or gingivitis, then subjected to parasitological examination and culture on modified trypticase, yeast and iron medium (TYI-S-33). Isolates successfully maintained in culture were further subjected to analysis of protein profile of lysates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and analysis of proteinases by non-denaturing gelatin-SDS-PAGE. Comparison of growth kinetics of seven T. tenax isolates showed a wide variability in the growth characteristics. Protein profiles of the seven isolates revealed a total 53 bands ranged in molecular weight (MW) from 5 to 95kDa using 12% resolution gel. Also, T. tenax isolates were found to possess 19 proteinase bands ranged in MW from 14 to 66kDa. The proteolytic bands were intensified by a cysteine proteinase activator and totally disappeared by treatment with a cysteine proteinase inhibitor suggesting that the proteinases were of cysteine proteinases type. The high frequency of T. tenax detected (28.6%) along with the variability in protein profiling and proteolytic activity of the isolates supports the possible pathogenicity of T. tenax and clarifies a conclusion that different strains with possibility of variable pathogenic potential may exist.     

Trichomonosis of the oral cavity complicated by mycosis.

Protozoa and fungi of the oral cavity, although frequently occurring and connected to considerable clinical adverse effects, are as yet insufficiently known. The aims of the study were (i) to estimate the prevalence of common invasions of Trichomonas tenax and fungi, (ii) to associate the symptoms with the diagnosis of trichomonosis complicated by mycosis, and (iii) to determine trichomonacidal properties of ornidazole, tinidazole and metronidazole. A sample of 936 dentist patients with different diagnoses were included in the study. The collected material consisted of rinsings, with simultaneous application of selective media, different for protozoa or fungi cultures. Using ornidazole, tinidazole and metronidazole, we examined in vitro their influence on 30 strains of T. tenax. Among the examined patients T. tenax was found in 90 cases including 85 cases where it occurred together with fungi, on the basis of which diagnosis of trichomonosis complicated by mycosis of the oral cavity was established. It was the most frequent in patients with leukoplakia and lichen Wilsoni. We recorded a statistically significant association for T. tenax with fungi and xerostomia, burning sensation, periodontal pockets and denuded teeth. T. tenax was never found in patients with caries and with aphtha recidivans. The curves of ornidazole activity were obtained within the solution range of 130-4350 micrograms/ml; the curves of tinidazole activity within the concentration range of 500-16870 micrograms/ml; metronidazole in the highest concentration killed from 5 to 100% of the population of all strains.     

Trichomonads in pleural effusion: case report, literature review and utility of PCR for species identification

Trichomonas tenax is a flagellated protozoan commonly found in the human oral cavity but of unusual occurrence in pulmonary infections. We describe a case of a 67-year-old patient with glioblastoma who presented with severe pleurisy in the post-operative period while she was receiving high-dose corticotherapy. Several motile flagellated protozoa were identified in the pleural fluid. Trichomonas tenax was identified by molecular methods. Pulmonary infections with Trichomonads might be underestimated because of diagnostic difficulties. The utility of molecular biology for species identification is underlined and the pathogenicity of Trichomonad parasites in human lungs is discussed in light of previously reported cases.     

Simultaneous Amplification of Two Bacterial Genes: More Reliable Method of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Detection in Microbial Rich Dental Plaque Samples

Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.     

Study on the relationship between Helicobacter pylori in the dental plaque and the occurrence of dental caries or oral hygiene index

Background: The aims of our study were to determine the presence of Helicobacter pylori DNA in the dental plaque of Chinese children aged 3–6 years by nested polymerase chain reaction (PCR) and to investigate the relationship between this infection and the occurrence of dental caries or oral hygiene index.
Methods: Two hundred and fourteen children from a kindergarten in Guangzhou City of China were evaluated. The children's plaques were assessed by plaque indices of Quigley–Hein. Dental plaque was analyzed using nested PCR for two sets of primers directed to the 860-bp fragment of H. pylori genomic DNA, which have been reported to be highly sensitive and specific by other researchers.
Results: H. pylori was detected in dental plaque samples from 126 children, and 70 children with dental caries carried H. pylori in dental plaque. Of these children without infection, only 36 of 88 suffered dental caries. Besides, the average dental plaque index of 126 H. pylori -positive children was higher than that of 88 children without infection. In the present study, there was a significant correlation between H. pylori infection and dental caries or dental hygiene.
Conclusion: The oral cavity may be a reservoir for H. pylori infection in children. H. pylori in dental plaque may play a role in the occurrence of dental caries, and poor oral hygiene may represent a risk factor for H. pylori in the oral cavity.     

Axenic Cultivation of Trichomonas tenax, the Oral Flagellate of Man I. Establishment of Cultures

SYNOPSIS. A technique for the axenic cultivation of Trichomonas tenax , the oral flagellate of man, is presented. The medium employed consists of a nutrient broth supplemented with horse serum and a cell-free extract of chick embryo. Three strains established in this medium have been maintained for 16, 23 and 48 months respectively.
All the cultures were initiated with trichomonads grown in association with Trypanosoma cruzi. Attempts to establish axenic cultures with trichomonads obtained from xenic cultures containing a bacterial flora of unknown composition met with failure. This suggests that the successful cutcome of the process of axenization is to a certain extent dependent upon the type of organism(s) with which T. tenax is associated in culture. Furthermore, these findings may serve to explain earlier failures since not only were all of these attempts made with media lacking tissue extract supplements but all were made using bacteria-trichomonad cultures as a source of trichomonads.     

Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.     

Trichomonas vaginalis and trichomoniasis in the Republic of Korea

Vaginal trichomoniasis, caused by Trichomonas vaginalis, is the most common sexually transmitted disease. More than 170 million people worldwide are annually infected by this protozoan. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vaginalis. However, despite its high prevalence, the pathogenesis of T. vaginalis infection has not been clearly characterized although neutrophil infiltration is considered to be primarily responsible for the cytologic changes associated with this infection. We hypothesized that trichomonads in the vagina sometime after an acute infection secrete proteins like excretory-secretory product that have a chemotactic effect on neutrophils, and that these neutrophils are further stimulated by T. vaginalis to produce chemokines like IL-8 and GRO-alpha, which further promote neutrophil recruitment and chemotaxis. Thus, neutrophil accumulation is believed to maintain or aggravate inflammation. However, enhanced neutrophil apoptosis induced by live T. vaginalis could contribute to resolution of inflammation. Macrophages may constitute an important component of host defense against T. vaginalis infection. For example, mouse macrophages alone and those activated by lymphokines or nitric oxide are known to be involved in the extracellular killing of T. vaginalis. In the host, T. vaginalis uses a capping phenomenon to cleave host immunoglobulins with proteinases and thus escape from host immune responses. Recently, we developed a highly sensitive and specific diagnostic polymerase chain reaction (PCR) technique using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650), and found that the method enables the detection of T. vaginalis at concentrations as low as 1 cell per PCR mixture.     

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

口腔中幽门螺杆菌PCR检测方法的建立

全世界大约有50%的人口感染幽门螺杆菌(Helicobacter pylori, Hp),Hp感染容易造成慢性胃炎、消化性溃疡、低度恶性胃MALT淋巴瘤及胃癌,对人类造成严重的危害。为寻求一种简便、准确、非创伤性的方法用于口腔中Hp的检测,本研究建立了口腔中幽门螺杆菌的PCR检测方法,并对建立的PCR方法的敏感性、特异性进行分析,并应用于检测唾液样品和牙菌斑共50份。结果显示,50份样品中,阳性率为82%。结果表明,本研究建立的口腔中Hp的PCR检测方法特异性强、敏感性高,可以用于临床上Hp诊断的重要参考。     

Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1

Abstract Pulse labelling experiments and enzyme studies show that Thermoproteus tenax is able to degrade glucose via the Embden-Meyerhof-Parnas (EMP) pathway. T. tenax is the first archeum for which the glycolytic EMP pathway could be established. One of the key enzymes, 6-phosphofructokinase of T. tenax depends on (pyrophosphate-fructose-6-phosphate-1-phosphotransferase) instead of ATP as found in some bacterial and eucaryal species. In addition to the intermediates of the EMP pathway the intermediary products of the non-phosphorylated Entner-Doudoroff pathway were also detected by pulse labelling experiments indicating that under chemoorganotrophic conditions at least 2 glycolytic pathways are operative in T. tenax .     

PCR for diagnosis of male Trichomonas vaginalis infection with chronic prostatitis and urethritis

The aim of this study was to assess the usefulness of PCR for diagnosis of Trichomonas vaginalis infection among male patients with chronic recurrent prostatitis and urethritis. Between June 2001 and December 2003, a total of 33 patients visited the Department of Urology, Hanyang University Guri Hospital and were examined for T. vaginalis infection by PCR and culture in TYM medium. For the PCR, we used primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Voided bladder urine (VB1 and VB3) was sampled from 33 men with symptoms of lower urinary tract infection (urethral charge, residual urine sensation, and frequency). Culture failed to detect any T. vaginalis infection whereas PCR identified 7 cases of trichomoniasis (21.2%). Five of the 7 cases had been diagnosed with prostatitis and 2 with urethritis. PCR for the 5 prostatitis cases yielded a positive 330 bp band from bothVB1 and VB3, whereas positive results were only obtained from VB1 for the 2 urethritis patients. We showed that the PCR method could detect T. vaginalis when there was only 1 T. vaginalis cell per PCR mixture. Our results strongly support the usefulness of PCR on urine samples for detecting T. vaginalis in chronic prostatitis and urethritis patients.     

Structure and function of a regulated archaeal triosephosphate isomerase adapted to high temperature

Triosephophate isomerase (TIM) is a dimeric enzyme in eucarya, bacteria and mesophilic archaea. In hyperthermophilic archaea, however, TIM exists as a tetramer composed of monomers that are about 10% shorter than other eucaryal and bacterial TIM monomers. We report here the crystal structure of TIM from Thermoproteus tenax, a hyperthermophilic archaeon that has an optimum growth temperature of 86 degrees C. The structure was determined from both a hexagonal and an orthorhombic crystal form to resolutions of 2.5A and 2.3A, and refined to R-factors of 19.7% and 21.5%, respectively. In both crystal forms, T.tenax TIM exists as a tetramer of the familiar (betaalpha)(8)-barrel. In solution, however, and unlike other hyperthermophilic TIMs, the T.tenax enzyme exhibits an equilibrium between inactive dimers and active tetramers, which is shifted to the tetramer state through a specific interaction with glycerol-1-phosphate dehydrogenase of T.tenax. This observation is interpreted in physiological terms as a need to reduce the build-up of thermolabile metabolic intermediates that would be susceptible to destruction by heat. A detailed structural comparison with TIMs from organisms with growth optima ranging from 15 degrees C to 100 degrees C emphasizes the importance in hyperthermophilic proteins of the specific location of ionic interactions for thermal stability rather than their numbers, and shows a clear correlation between the reduction of heat-labile, surface-exposed Asn and Gln residues with thermoadaptation. The comparison confirms the increase in charged surface-exposed residues at the expense of polar residues.     

RPC技术检测儿童龈炎中的卟啉单胞菌

根据牙龈卟啉单胞菌特异的纤毛亚单位蛋白结构基因,设计一对寡核苷酸引物,采用ＰＣＲ扩增了１３１ｂｐ特异片段,实验证明,ＰＣＲ直接检测临床标本中的牙龈卟啉单胞菌,不仅特异、敏感、而且快速,从而显示了较好的优越性。用该引物,分析牙龈卟啉单胞菌在儿童龈炎中的分布。经ＰＣＲ检测４６例龈下菌斑标本中的牙龈卟啉单胞菌,结果表明：２４例标本ＰＣＲ为阳性;对照组４６例标本中,牙龈卟啉单胞菌仅有５例为阳性,儿童龈炎中牙龈卟啉单胞菌的检出率明显高出正常组（ｐ＞０００５）。提示用ＰＣＲ检测儿童龈炎中的牙龈卟啉单胞菌具有重要意义。     

Trichomonas vaginalis: Investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.     

Detection of cariogenic bacterial genes by microchip electrophoresis

Allele-specific PCR primers were designed, based on the dextranase (dex) gene, to identify Streptococcus mutans and Streptococcus sobrinus in dental plaque; subsequently, PCR products were detected via microchip electrophoresis (ME). In order to amplify the dex gene fragment of S. mutans and S. sobrinus, the following two PCR methods were established. Duplex allele-specific PCR primers were designed on a region of low DNA homology; furthermore, 211 and 126-bp fragments were amplified for S. mutans and S. sobrinus, respectively. Common PCR primer for single allele-specific PCR was designed so as to sandwich a region exhibiting high homology and amplify PCR product of different DNA size due to deletion of small DNA fragment in two dex genes. S. mutans and S. sobrinus were amplified, leading to the generation of 202 and 226-bp products, respectively. Analysis of DNA base size by ME in order to achieve efficient separation employed a polymer mixture consisting of hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO). In the presence of a polymer mixture of 0.125% PEO/0.6% HPMC, two PCR products were obtained, displaying degree of separation of 226 bp/202 bp of 2.67 (Rs). Reproducibility (CV%, n = 7) was 0.3%; additionally, separation time was approximately 85 s. This method was applied to the detection of S. mutans and S. sobrinus in dental plaque. Detection of the dex genes of S. mutans and S. sobrinus characterized by quickness, precision and high sensitivity was possible.     

Quantitation of Bacteroides forsythus in subgingival plaque comparison of immunoassay and quantitative polymerase chain reaction

Our objective was to compare three methods (enzyme-linked immunosorbent assay [ELISA], endpoint and quantitative polymerase chain reaction [E-PCR and Q-PCR]) for detection and quantitation of Bacteroides forsythus in 56 plaque samples from seven subjects with progressive periodontal disease. Samples collected in buffer were pelleted and resuspended in 500 microl of water. Fifty microl aliquots were removed for an ELISA performed on bacteria or plaque immobilized on 96-well plates and probed with B. forsythus specific antibody. An occurrence of 3.7+/-0.6 x 10(4) or more bacteria were detected by ELISA in pure culture; 26 of 54 plaque samples were positive, two samples could not be analyzed. Samples for PCR were autoclaved for 10 min prior to use. The detection level of E-PCR using primers specific for B. forsythus 16S rRNA was 200 cells and 42 out of 56 samples were positive based on ethidium bromide stained agarose gels. Q-PCR using the same primers combined with a nested fluorescent oligonucleotide probe detected 10+/-0.32 bacteria in pure culture; 43 of 56 plaque samples were positive. The ELISA and Q-PCR obtained identical results with 36 of the 54 samples assayed; there were one false positive and 17 false negative ELISA results using Q-PCR as standard. The positive proportions of plaque samples were almost the same for E-PCR and Q-PCR. We conclude that the PCR methods are more appropriate for a multicenter study because of greater sensitivity and convenience of sample transportation from clinics to a central laboratory.