Polymerase chain reaction-based strain characterization of noncapsulate Haemophilus influenzae

A polymerase chain reaction-based typing method for noncapsulate Haemophilus influenzae was developed. Randomly amplified polymorphic DNA fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during putative outbreaks of chest infection and validated by comparison with sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of outer membrane protein-enriched preparations and rRNA gene restriction analysis. There was complete concordance between the three techniques. The results show that randomly amplified polymorphic DNA analysis provides a highly discriminatory method of characterizing strains of noncapsulate H. influenzae which is eminently suitable as an epidemiological tool for the rapid investigation of outbreaks of infection.     

Polymerase chain reaction-based assays of vitreous samples for the diagnosis of viral retinitis. Use in diagnostic dilemmas

OBJECTIVE: This study aimed to review the authors' results using polymerase chain reaction (PCR)-based assays for the diagnosis of viral retinitis. DESIGN: The study design was a retrospective case series. PARTICIPANTS: Thirty-seven patients (38 eyes) with active retinitis from whom vitreous biopsy specimens were received in the authors' laboratory for diagnostic evaluation. INTERVENTION: Vitreous biopsy specimens were evaluated with previously described PCR-based assays for cytomegalovirus (CMV), varicella zoster virus (VZV), and herpes simplex virus (HSV) DNA; clinical histories were reviewed. MAIN OUTCOME MEASURES: Laboratory findings and clinical course were measured. RESULTS: The results of the authors' assays were consistent with the long-term clinical course of each patient. Cytomegalovirus, VZV, or HSV DNA was detected in the vitreous from 24 patients. Cytomegalovirus DNA was detected in vitreous biopsy specimens from 10 patients (11 eyes). Nine patients (ten eyes) with acquired immune deficiency syndrome ultimately were diagnosed with CMV retinitis as they were followed clinically over time. Varicella zoster virus DNA was detected in vitreous biopsy specimens from eight patients; seven adult patients were ultimately diagnosed with acute retinal necrosis or progressive outer retinal necrosis. Herpes simplex virus DNA was detected in vitreous biopsy specimens from six patients; five patients had previous or subsequent herpes encephalitis. No viral DNA was detected in the vitreous from 13 patients; all were ultimately diagnosed with toxoplasmosis, syphilis, Behcet disease, fungal endophthalmitis, or idiopathic inflammation. CONCLUSIONS: These data further support the use of PCR-based assays of vitreous specimens in the diagnostic evaluation of patients with infectious retinitis.     

Polymerase chain reaction-based diagnosis of Onchocerca volvulus infection: improved detection of patients with onchocerciasis

Definitive diagnosis of Onchocerca volvulus (Ov) infection requires the identification of the parasite in either the skin or subcutaneous nodules. These parasitologic approaches suffer from poor sensitivity. To assess the efficacy and utility of a polymerase chain reaction (PCR)-based diagnosis for Ov infection, skin snips were examined from 94 persons in an Ov-endemic region of Ecuador, and results were compared in a blinded fashion with those of a PCR assay based on the Onchocerca-specific repetitive DNA sequence, O-150. All 60 patients microfilaria-positive on skin snip examination were positive in the PCR-based assay. In addition, 13 of 34 who were microfilaria-negative by skin snips were positive in the PCR assay. This suggests that the PCR-based assay is significantly more sensitive than current methods and overcomes many deficiencies of parasitologic and serologic methodologies in diagnosing active onchocerciasis.     

Polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion

Eight of 38 patients (21%) with familial and 5 of 175 patients (3%) with sporadic amyotrophic lateral sclerosis (ALS) had missense mutations in the SOD-1 gene. Two novel mutations were identified. One in exon 4 substituting leucine with phenylalanine (L84F) in a familial patient and the second in exon 3 at substituting glycine with serine (G72S) in an "apparently" sporadic patient. Over 60 point mutations have now been described in all five exons of SOD-1, involving 43 of the 153 residues. Hypotheses about the toxic role of mutant SOD-1 in the pathogenesis of ALS must account for this molecular diversity.     

Polymerase chain reaction-based microsatellite polymorphism analysis of follicular and Hürthle cell neoplasms of the thyroid

Follicular and Hürthle cell carcinomas of the thyroid cannot be differentiated from adenomas by either preoperative fine needle aspiration or intraoperative frozen section examination, and yet there exist potentially significant differences in the recommended surgical management. We examined, by PCR-based microsatellite polymorphism analysis, DNA obtained from 83 thyroid neoplasms [22 follicular adenomas, 29 follicular carcinomas, 20 Hürthle cell adenomas (HA), and 12 Hürthle cell carcinomas (HC)] to determine whether a pattern of allelic alteration exists that could help distinguish benign from malignant lesions. Alterations were found in only 7.5% of informative PCR reactions from follicular neoplasms, whereas they were found in 23.3% of reactions from Hürthle cell neoplasms. Although there were no significant differences between follicular adenoma and follicular carcinoma, HC demonstrated a significantly greater percentage of allelic alteration than HA on chromosomal arms 1q (P < 0.001) and 2p (P < 0.05) by Fisher's exact test. The documentation of an alteration on either 1q or 2p was 100% sensitive and 65% specific in the detection of HC (P < 0.0005, by McNemar's test). In conclusion, PCR-based microsatellite polymorphism analysis may be a useful technique in distinguishing HC from HA. Potentially, the application of this technique to aspirated material may allow this distinction preoperatively and thus facilitate more optimal surgical management. Consistent regions of allelic alteration may also indicate the locations of critical genes, such as tumor suppressor genes or oncogenes, that are important in the progression from adenoma to carcinoma. Finally, this study demonstrates that Hürthle cell neoplasms, now considered variants of follicular neoplasms, differ significantly from follicular neoplasms on a molecular level.     

Polymerase chain reaction in liposomes

BACKGROUND: Compartmentalization of biochemical reactions within a spherically closed bilayer is an important step in the molecular evolution of cells. Liposomes are the most suitable structures to model this kind of chemistry. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochemical reactions such as DNA replication can be carried out inside these compartments. RESULTS: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixture of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temperature conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amount of DNA is produced which can be observed by polyacrylamide gel electrophoresis. CONCLUSIONS: This work shows that it is possible to carry out complex biochemical reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are critical for carrying out this complex reaction within the liposome compartment.     

Polymerase chain reaction and other amplification techniques in mycobacteriology

Amplification methods for detection of Mycobacterium tuberculosis, such as polymerase chain reaction (PCR), have undergone much research and development in the last several years. The most common methods for extraction, amplification, and detection of mycobacterial nucleic acid sequences used in "in-house" PCR assays are discussed. A list of commercially prepared PCR and non-PCR amplification assays that should be available soon is included. The pros and cons of "in-house" versus commercial technology and issues of implementation of molecular technology in the clinical laboratory are reviewed.     

Normal and anomalous electrophoretic behavior of polymerase chain reaction-based DNA polymorphisms in polyacrylamide gels

The electrophoretic mobility of five single tandem repeats (STRs) and four amplified fragment length polymorphisms (AFLPs) in polyacrylamide gels was tested under denaturing and nondenaturing conditions. Relative anomalous mobility in nondenaturing conditions was found in one AT-rich AFLP (3'ApoB) and in two AT-rich STRs (HUMACTBP2 and HUMF13A1). In these cases, highly anomalous electrophoretic behavior was found even when changes were made in the %T value alone. In such cases typing results were affected by the gel composition. The mobility of these systems was nevertheless normal under denaturing conditions. As a consequence of this study, we recommended that the typing of these systems should only be performed under denaturing conditions and we also recommend the further study of the electrophoretic behavior of repetitive DNA polymorphisms, especially AT-rich systems, before they are used in forensic or genetic applications.     

Polymerase chain reaction for the detection of Bordetella pertussis in clinical nasopharyngeal aspirates

A PCR procedure for the detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) was developed with primers derived from the pertussis toxin promoter region. The amplification resulted in a 191-bp PCR product specific for B. pertussis. A total of 681 NPAs collected from children with cough lasting >7 days was evaluated by PCR and culture; 104 aspirates were positive by PCR and 93 by culture. Sixteen cases were positive only by PCR and five culture positive aspirates were negative by PCR. An internal control was included in the assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. The PCR method was more efficient than culture in detecting B. pertussis in samples collected late in the disease, in antibiotic-treated children and in patients with mild disease.     

Polymerase chain reaction to detect Mycobacterium tuberculosis in histologic specimens

BACKGROUND: Whether acute MS lesions are primarily inflammatory or demyelinative is unresolved. Our study examined acute MS lesions longitudinally by quantitative magnetization transfer (MT), an MRI technique that identifies tissue integrity and destruction. METHODS: Four MS patients were studied by serial MRI including MT, conventional T2-weighted images, and postgadolinium T1-weighted images for 9 to 12 months. In 15 new lesions, the MT ratio (MTR) was calculated retrospectively. RESULTS: In 13 lesions, a marked decrease in the MTR was present early during the first 2 months after the onset of the lesion and was followed by a variable increase. In two other lesions, the MTR progressively declined. CONCLUSIONS: These results suggest that major early structural changes compatible with demyelination and followed by remyelination and gliosis, or by continuous demyelination, occur in new MS lesions. The various MTR profiles provide in vivo confirmation of the current knowledge of the progression in MS lesions. Furthermore, MTR may be used to monitor in vivo drug efficacy in new MS lesions.     

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease

The structural and electronic properties of seventeen alkylxanthine derivatives were calculated using the MO program PM3 to elucidate the key features related to their inhibitory activity on phosphodiesterase (PDE) IV isoenzyme. Except for 7-alkylxanthine derivatives, a good correlation could be established between the distance between the tops of the two alkyl groups at the N1 and N3 positions of the xanthine skeleton (molecular length) and the PDE IV inhibitory activity (r=0.973, n=13). The same inhibitory activity could also be significantly correlated with the following electronic parameters of alkylxanthines: HOMO energy (r=0.850), absolute hardness (r=-0.806), and absolute electronegativity (r=-0.825). These results suggest that the electronic properties are partly responsible for PDE IV inhibition as far as the effects of structural properties associated with molecular length are concerned. Alkylxanthines may also act as electron donors in the charge-transfer interaction with the active sites on PDE IV isoenzyme.     

Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene

The aims of this study were to determine if there were any differences in the thickness of the lip levator musculature in men and women, and whether the height of the smile line in adults was related to the thickness of the lip levator musculature. Thirty Caucasian (13 males, 17 females), and 24 Asian (11 males, 13 females) undergraduate dental students participated in this study. The subjects were placed in high, medium, and low smile-line groups, according to the height of their upper lip while smiling. The thickness of the levator labii superioris and zygomaticus major muscles was measured on ultrasound scans of the relaxed muscles. Gender, right-left side, and ethnic differences in muscle thickness were determined, and the thicknesses of the lip levator musculature in subjects in the high, medium, and low smile-line groups were compared. There were significantly more women than men with high smile-lines and the zygomaticus major muscle was significantly thicker in the women, as compared with the men. There were, however, no statistically significant differences in muscle thickness in the three smile-line groups. While it appears that women have higher smile-lines and significantly thicker zygomaticus major muscles than men, the height of the smile line is not due to the thickness of either the levator labii superioris or zygomaticus major muscles.