Evaluation of serotypes of Streptococcus pneumoniae isolated from otitis media patients by multiplex polymerase chain reaction

The increasing difficulty in the management of pneumococcal acute otitis media in parallel with increases in antimicrobial-resistant strains has led to much interest in pneumococcal capsular types for the adoption of effective prevention by vaccines. This study shows that multiplex polymerase chain reaction is a valuable and expeditious method for the capsular typing of pneumococci. The multiplex polymerase chain reaction method accurately detects the majority of serotypes and serogroups frequently isolated from pediatric patients with acute otitis media, allowing the characterization of the colonization patterns for further implications of pneumococcal vaccines.     

Bacteriology of chronic maxillary sinusitis and normal maxillary sinuses: using culture and multiplex polymerase chain reaction

BACKGROUND: Although many investigations have been performed on bacteriology of chronic sinusitis and normal sinuses, there still is much discussion. Also a new bacterial agent, Alloiococcus otitidis determined in the nasopharynx and middle ear specimens can be thought as a causative agent of sinusitis. METHODS: The bacteriology of chronic maxillary sinusitis and maxillary sinuses with normal radiogram and endoscopic findings were studied by culture methods for aerobic and anaerobic bacteria. Multiplex polymerase chain reaction (PCR) was used to investigate four bacteria in study and control groups. There were 27 specimens in the study group and 28 specimens in the control group. RESULTS: In the study group, the bacteria commonly isolated were Staphylococcus aureus (11.1%), alpha-hemolytic streptococci (11.1%), Streptococcus pneumoniae (11.1%), Haemophilus influenzae (7.4%), coagulase-negative staphylococci (7.4%), and anaerobes (33.3%). Coagulase-negative staphylococci (14.3%), alpha-hemolytic streptococci (10.7%), and anaerobes (35.7%) were isolated also in the control group. PCR was used to investigate S. pneumoniae, H. influenzae, Moraxella catarrhalis, and A. otitidis in the study and control groups. None of these bacteria was determined in the control group whereas detection rates of these bacteria in the study group were 11.1, 11.1, 3.7, and 7.4%, respectively. It should be considered that PCR yielded faint amplification band for A. otitidis. CONCLUSION: Using multiplex PCR can help to increase detection rates of bacterial etiology. Healthy sinuses are not sterile. A. otitidis may be one of the pathogens causing sinusitis.     

The quantitative analysis of the human papillomavirus DNA load in submandibular gland lesions with droplet digital polymerase chain reaction

Background: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial.

Objectives: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16^INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated.

Materials and Methods: Tissue samples from 31 submandibular gland lesions were evaluated. p16^INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed.

Results: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16^INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16^INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers.

Conclusions: HPV-DNA positivity and p16^INK4A-immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.     

Fungal speciation using quantitative polymerase chain reaction (QPCR) in patients with and without chronic rhinosinusitis

OBJECTIVES/HYPOTHESIS: The objectives of this study were to determine the mycology of the middle meatus using an endoscopically guided brush sampling technique and polymerase chain reaction laboratory processing of nasal mucous; to compare the mycology of the middle meatus in patients with sinus disease with subjects without sinus disease; to compare the responses on two standardized quality-of-life survey forms between patients with and without sinusitis; and to determine whether the presence of fungi in the middle meatus correlates with responses on these data sets. STUDY DESIGN: The authors conducted a single-blind, prospective, cross-sectional study. METHODS: Patients with sinus disease and a control group without sinus disease were enrolled in the study. A disease-specific, validated Sinonasal Outcomes Test survey (SNOT-20) was completed by the subjects and a generalized validated Medical Outcomes Short Form 36 Survey (SF-36) was also completed. An endoscopically guided brush sampling of nasal mucous was obtained from the middle meatus. Fungal specific quantitative polymerase chain reaction (QPCR) was performed on the obtained sample to identify one of 82 different species of fungus in the laboratory. Statistical analysis was used to categorize the recovered fungal DNA and to crossreference this information with the outcomes surveys. RESULTS: The fungal recovery rate in the study was 45.9% in patients with sinus disease and 45.9% in control subjects. Patients with chronic rhinosinusitis had a mean SNOT-20 score of 1.80 versus the control group mean score of 0.77 (P < .0001). SF-36 data similarly showed a statistically significant difference between diseased and control populations with controls scoring a mean of 80.37 and patients with chronic rhinosinusitis scoring a mean of 69.35 for a P value of .02. However, no statistical significance could be ascribed to the presence or absence of fungi recovered, the type of fungi recovered, or the possible impact of fungi on the quality-of-life survey results. CONCLUSION: The recovery rate of fungi from the middle meatus of patients with chronic rhinosinusitis and a control population without chronic rhinosinusitis is 45.9% using QPCR techniques. No direct causation with regard to fungal species or presence was proven; however, a species grouping for future studies is proposed based on trends in this data and other reports. Disease-specific outcomes surveys revealed a statistically significant difference between the two groups.     

真菌特异性引物内转录片段PCR检测和培养的比较研究

目的:比较真菌特异性引物内转录片段(ITS)PCR检测方法与培养的敏感性,探讨真菌ITS PCR检测方法的敏感性与特异性及其在真菌性鼻窦炎诊断中的意义.方法:取18例临床诊断为真菌性鼻窦炎患者的鼻窦内分泌物,分别做ITS PCR及培养,比较2种方法阳性率的差异及检出真菌的相符率;同时以20例临床诊断为非真菌性慢性鼻窦炎患者鼻窦内分泌物为对照,进一步探讨ITS PCR对真菌性鼻窦炎临床诊断的意义.结果:18例真菌性鼻窦炎的标本中,ITS PCR方法检测真菌阳性的例数为14例(78%);培养法的阳性例数为8例(44%).20例慢性鼻窦炎ITS PCR方法检测阳性例数为1例(5%).结论:ITS PCR方法对于真菌性鼻窦炎的快速诊断很有价值,并能通过测序明确感染菌种,是一种高敏感性和高特异性的方法.     

Human papillomavirus (HPV) in sinonasal papillomas: A study of 78 cases using in situ hybridization and polymerase chain reaction

To determine the role of human papillomavirus (HPV) in the etiology of sinonasal papillomas, 57 inverted papillomas including 5 cases associated with carcinomas, 16 exophytic papillomas, and 5 cases of columnar cell papillomas were examined for the presence of HPV DNA by in situ hybridization and polymerase chain reaction (PCR). The genetic studies were performed on the formalin-fixed, paraffin-embedded material. In only 6% of the 52 benign inverted papillomas was HPV DNA identified, whereas 69% of the exophytic papillomas were infected by HPV DNA. In none of the 5 cases with columnar cell papillomas could HPV be demonstrated. HPV 6/11 was identified in all of these HPV-positive cases. In the carcinoma area, HPV was detected in 2 (1 HPV 6/11 and 1 HPV 18) of the 5 inverted papillomas associated with carcinomas. The findings confirm the presence of HPV DNA in sinonasal papillomas. The results also indicate that HPV 6/11 may be involved in the pathogenesis of, solely, exophytic papillomas. We found that in situ hybridization and PCR seem equally sensitive in detecting HPV in sinonasal papillomas.     

Detection of fungi in the nasal mucosa using polymerase chain reaction

HYPOTHESIS: Fungi have been increasingly recognized as important pathogens in sinusitis. However, detection of fungus with conventional culture techniques is insensitive and unreliable. Polymerase chain reaction (PCR) is an exquisitely sensitive assay that can detect the DNA of 10 or less fungal elements. The aim of this study was to compare the sensitivity of conventional culture techniques using PCR analysis. METHODS: Nasal swabs and DNA samples were collected from the nasal cavities of control subjects and patients with chronic sinusitis. Fungal-specific PCR analysis and standard cultures were performed on every sample. chi2 analysis was used to test for statistical differences between groups. RESULTS: PCR analysis detected fungal DNA in 42% and 40% of control subjects and patients with chronic sinusitis while standard cultures were positive in 7% and 0%, respectively. There was no statistically significant difference in the prevalence of fungi in the normal volunteers and patients with chronic rhinosinusitis. CONCLUSION: PCR is significantly more sensitive than nasal swab cultures in detecting the presence of fungi in nasal mucosa. In addition, our study suggests that the presence of fungi alone is insufficient to implicate it as the pathogen in chronic sinusitis.     

Characterization of fungi in chronic rhinosinusitis using polymerase chain reaction and sequencing

The role of fungi in chronic rhinosinusitis (CRS) remains unknown. Fungi were also determined as one of the responsible agents in the etio-pathogenesis, while several studies found fungi in 6–93% of the cases. The aim of this study is to test the presence of fungi in samples taken from the middle meatus of patients with CRS, using traditional culture methods and polymerase chain reaction (PCR), and to compare the efficacy of these methods. Thirty patients diagnosed with CRS, with or without nasal polyposis, undergoing an operation in the Otorhinolaryngology Clinic, were prospectively included in the study. Nasal mucosa samples from ten patients, who were operated for pathologic evaluation, and without CRS, were used as controls. Nasal samples were taken from each patient by swabbing with a cytology brush. Middle meatus culture samples were taken by using nasal cotton swab, and the polyp and/or sinus mucosa samples were taken during endoscopic sinus surgery. Fungal specific PCR, using 18S rRNA primers and standard cultures, were performed on every sample. All amplicons were sequenced. There was no fungal growth in the Sabouraud dextrose agar (SDA) medium from middle meatus samples and tissue parts. Of 30 tissue and brush samples, 3 and 2 were positive for fungal DNA, respectively. Sequence analysis showed that four amplicons were homologus to Cladosporium herbarum and one to Aspergillus amstelodami. We concluded that fungal etiology is overestimated and fungi rarely play a role in patients with CRS. Large-scale studies should be done using molecular methods.     

Laryngeal verrucous carcinoma: A clinicopathologic study and detection of human papillomavirus using polymerase chain reaction

Laryngeal verrucous carcinoma (LVC) is a rare, well-differentiated variant of squamous carcinoma with a low malignant potential. Human papillomavirus (HPV)–16 DNA has been identified in a small number of LVC and an etiologic relationship has been suggested. A correlative clinical and molecular pathological study was performed in order to determine the prevalence and typing of HPV DNA in LVC. Possible associations between patient and tumor subsets, and the presence of HPV DNA were also investigated. Formalin-fixed, paraffin-embedded tissue samples from 29 patients with LVC were examined by polymerase chain reaction (PCR) using DNA primers specific for HPV types 6b/11, 16, and 18. Overall, HPVDNA was detected in 13 (45%) of the cases. Of these, HPV-16 DNA, HPV-18 DNA, and both HPV-16 DNA and HPV-18 DNA were detected in 4 (14% overall; 31% of positive cases), 4, and 5 (17% overall; 38% of positive cases), respectively. HPV-6b/11 DNA was not detected in any LVCs. In 16 cases, no HPV DNA was detected. There was a trend toward HPV DNA detection in higher stage tumors. HPV DNA detection was unrelated to patient age, tumor site, or radiotherapeutic responsiveness. The detection of HPV DNA in 45% of LVCs suggests an association between the presence of HPV-16 DNA and HPV-18 DNA, and some LVCs.     

General primer-mediated polymerase chain reaction for simultaneous detection and typing of human papillomavirus DNA in laryngeal squamous cell carcinomas

The aim of this study was to investigate the prevalence of different human papillomavirus (HPV) types in laryngeal squamous cell carcinomas using general primer-mediated polymerase chain reaction (PCR). Tumour sections from 42 patients with laryngeal carcinomas were investigated. For HPV DNA amplification, consensus primers were used which were directed to the LI coding region of the HPV genome. Analysis of the PCR products was done using 2% agarose gel electrophoresis followed by restriction enzyme analysis to identify different HPV types. Amplification of the human TGF-β DNA was successfully performed in 36/42 (85.7%) of samples confirming the presence of sufficient DNA for viral amplification. HPV DNA was detected in 8/36 (22.2%) of the tumours examined (three HPV-6, two HPV-16, one HPV-11, two unknown HPV types). HPV DNA was not detected in any of the non-neoplastic laryngeal mucosa which was used as control (n = 15). Fifty per cent of women had HPV-positive tumours compared with 8% of men (x²= 5.8, P<0.05). Our data indicate that while the overall prevalence of HPV in laryngeal carcinomas is fairly high (22.2%), the frequency of high-risk types (HPV-16 & HPV-18) is low (5.5%). HPV probably acts as a promoter in the multistep process of carcinogenesis in squamous mucosal cells of the larynx.     

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection

Conclusions. In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. Objective. To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. Material and methods. This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. Results. mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.     

Polymerase chain reaction detection of Mycobacterium tuberculosis from fine-needle aspirate for the diagnosis of cervical tuberculous lymphadenitis

BACKGROUND: Despite its well-established usefulness in the diagnosis of cervical tuberculous lymphadenitis, fine-needle aspiration cytology (FNAC) has several limitations in its clinical applications, especially when the presence of acid-fast bacilli is not proven. Furthermore, fine-needle aspirate is sometimes inadequate for diagnosis, and the sensitivity and specificity of this technique for cervical tuberculous lymphadenitis has not been firmly established. OBJECTIVE: The authors performed Mycobacterium tuberculosis polymerase chain reaction (PCR) for mycobacterial DNA sequences from the remainder of fine-needle aspirate after cytological examination and evaluated its diagnostic efficacy in clinical situations. METHODS: Conventional diagnostic procedures including FNAC and M tuberculosis PCR were performed simultaneously in 29 cases that had been suspected to be cervical tuberculous lymphadenitis on patients' first visit. The results of FNAC and M tuberculosis PCR were compared with the clinical outcomes after several months of follow-up and pathological results from open biopsy of some cases. RESULTS: Among the 17 cases of cervical tuberculous lymphadenitis diagnosed in clinical situations, M tuberculosis DNA was found by PCR in 13 cases (76.4%). Negative findings on PCR were achieved in 12 cases, which revealed non-granulomatous lymphadenopathy. CONCLUSION: From these results, we conclude that M tuberculosis PCR using the remainder of aspirate for cytological examination is a very useful tool for the diagnosis of cervical tuberculous lymphadenitis, and its clinical application with FNAC could reduce the necessity for open biopsy.     

Polymerase chain reaction (PCR) identification of human papillomavirus (HPV) DNA in verrucous carcinoma of the larynx

The incidence of human papillomavirus (HPV) DNA in archival formalin-fixed, paraffin-embedded tissue sections from verrucous carcinoma of the larynx was determined using polymerase chain reaction (PCR) with consensus primers and by in situ hybridization designed to detect HPV types 6/11, 16/18, 31/33/35. HPV DNA was detected in 17 (85%) of 20 tissue samples by PCR; none of the 20 samples were positive for the seven genotype types tested by in situ hybridization. PCR is a valuable tool to detect HPV and therefore will significantly clarify the importance of HPV in squamous mucosal disorders.     

Comparison between polymerase chain reaction and fungal culture for the detection of fungi in patients with chronic sinusitis and normal controls

CONCLUSION: PCR using panfungal gene primers is a more sensitive method for fungus detection than fungus culture, both in patients with chronic sinusitis and in normal controls. The presence of fungi alone, however, was insufficient to implicate them as pathogens in chronic sinusitis. OBJECTIVE: Previous findings have suggested that polymerase chain reaction (PCR)-based methods are more sensitive and reliable than conventional culture methods for the detection of fungal DNA. We therefore compared these methods in 82 patients with chronic sinusitis and 40 normal controls. MATERIAL AND METHODS: The noses of the subjects were irrigated with sterile saline, and the samples collected. The sediment from each irrigation was used for fungus culture and PCR analysis. RESULTS: PCR analysis using panfungal gene primers showed that 76/82 (92.5%) patients with chronic sinusitis and 39/40 (97.5%) normal controls were positive. In contrast, fungus cultures were positive in 19/82 (23.2%) patients with chronic sinusitis and 12/40 (30.0%) normal controls. We observed no significant between-group differences in the prevalence of fungus or in the fungal species detected.     

Detection of herpesvirus DNAs in perilymph obtained from patients with sensorineural hearing loss by real-time polymerase chain reaction

OBJECTIVES/HYPOTHESIS: Perilymph and peripheral blood mononuclear cells (PBMCs) from patients with bilateral severe sensorineural hearing loss (SNHL) were evaluated for the presence of DNA from cytomegalovirus (CMV), herpes simplex virus (HSV), and human herpesvirus (HHV)6. STUDY DESIGN: A prospective clinical study. METHODS: The subjects were 14 patients who underwent cochlear implantation and 1 patient who underwent gentamicin injection in the inner ear. We attempted to detect viral DNA from perilymph and PBMCs by real-time polymerase chain reaction (rtPCR). RESULTS: CMV DNA was detected in two perilymph specimens obtained from patients who were diagnosed as congenitally symptomatic CMV infection, although no CMV DNA was detected in PBMCs. Neither HSV DNA nor HHV6 DNA was detected in any other perilymph specimens. CMV DNA was detected in three PBMC samples, HSV DNA was detected in two samples, and HHV6 DNA was detected in six samples. CONCLUSION: CMV may persistently infect the inner ear of patients with congenital CMV infection, and rtPCR analysis may prove to be a valuable tool for investigating the etiology of SNHL.     

Mutation detection of GJB2 using IsoCode and real-time quantitative polymerase chain reaction with SYBR green I dye for newborn hearing screening

OBJECTIVES/HYPOTHESIS: Recent developments in molecular genetics have opened a new era in genetic analysis accompanied by new concepts concerning genetic disorders. Although 30 genes responsible for nonsyndromic deafness have been discovered as of March 27, 2003, the connexin 26 gene (GJB2) is commonly found in cases of deafness of unknown origins. The GJB2 contains a predicted open reading frame of 785 base pairs, which makes it relatively easy to detect mutations. Accordingly, mutation analysis of GJB2 should be suitable for the screening of congenital deafness. STUDY DESIGN: Prospective study. METHODS: IsoCode Stix is a useful device to isolate DNA from small samples of blood, which can be delivered from remote areas. To apply the detection of common mutations of GJB2 to hearing screening, DNA was extracted from several droplets of blood applied to the IsoCode device, and an allele-specific amplification method with real-time quantitative polymerase chain reaction was performed using GeneAmp 7700 with SYBR Green I dye. RESULTS: DNA extracted from IsoCode was purified within 45 minutes, which was sufficient to detect the full sequence of GJB2. Four types of common GJB2 mutations were reliably detected within 2.5 hours. CONCLUSION: IsoCode and real-time quantitative polymerase chain reaction will be promising tools for newborn screening of deafness genes in the future in DNA-based deafness screening, allowing early diagnosis of deafness and prompt training for language development.