Molecular typing of methicillin-resistant Staphylococcus aureus by polymerase chain reaction: distribution of the typed strains in hospitals

Management of the neuropathic bowel is one of the major issues in the treatment of patients with severe spinal cord injury (SCI). Pulsed irrigation evacuation (PIE) has been evaluated in several small studies for the clearing of fecal impactions in patients with a neuropathic bowel. We evaluated our experience with 398 PIE procedures performed on inpatients and outpatients at our facility. It has proven to be both safe and effective in a wide variety of patients with this disorder, and is a useful addition to traditional methods in the management of the neuropathic bowel.     

High-resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.     

Fetal RhD typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization

OBJECTIVE: To review the specificity and sensitivity of diagnostic techniques using the polymerase chain reaction (PCR) on amniotic fluid (AF) samples for the determination of fetal RhD status. DATA SOURCES: A MEDLINE computerized search was conducted for January 1991 through March 1996 using the key terms "polymerase chain reaction," "rhesus," and "RhD typing." METHODS OF STUDY SELECTION: All articles describing the use of PCR in AF for RhD typing were reviewed. Only cases in which the results of PCR testing were confirmed by fetal or neonatal serology were included in the final analysis. TABULATION, INTEGRATION, AND RESULTS: The results of PCR typing were compared with serology to determine the sensitivity, specificity, and positive and negative predictive values of DNA-based techniques. A total of 500 cases were reviewed, in which four different sets of oligonucleotide primers were used. The sensitivity and specificity of PCR typing were 98.7% and 100%, respectively, and the positive and negative predictive values were 100% and 96.9%, respectively. In five cases, an RhD-positive fetus was incorrectly diagnosed: Two fetuses died, one neonate needed exchange transfusions, and another neonate needed phototherapy in conjunction with a simple transfusion. The remaining infant was lost to follow-up. A theoretical model indicated that amniocentesis with PCR-based techniques for fetal RhD typing would be associated with a fourfold reduction in perinatal loss compared with funipuncture and serology for fetal typing. CONCLUSIONS: This lower rate of procedure-related loss makes RhD typing using AF the preferred method for assessing the fetal Rh status in cases of a heterozygous paternal genotype.     

Application of interspersed repetitive sequence polymerase chain reaction for construction of yeast artificial chromosome contigs

This work is devoted to the comprehension of the sorption mechanism of uranyl ions on chitosan particle dispersions. The uranyl concentration measurements were obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES) and we considered the role of various physicochemical parameters (pH; nature and concentration of added salts; degree of acetylation, DA). The use of appropriate calculation software allowed us to determine the chemical nature of uranyl species in solution in relation to these different parameters. The optimal pH of fixation has been found to be within 6.5-7.5 and can be related to the necessity of having both deprotonated amino groups and no carbonate ions, which are a strong complexant of uranyl ions, thus inhibiting their interaction with chitosan. The decrease of metal uptake with an increase of DA and the lack of influence of ionic strength, confirm the results obtained with pH and allowed us to suppose the formation of a complex with chitosan amino groups rather than interactions of an electrostatic nature.     

Rapid detection and typing of human papillomaviruses by consensus polymerase chain reaction and enzyme-linked immunosorbent assay

This long-term prospective study evaluates the clinical results of subsequent laminectomy in 103 consecutive patients who initially underwent chemonucleolysis (CNL) or laminectomy for lumbar disc herniation. Between 1981 and 1994, 53 patients who had received CNL initially and then underwent laminectomy and 50 patients treated initially with laminectomy underwent a repeat laminectomy. Clinical assessment at 6 weeks showed a success rate of 80.8% for post-CNL laminectomy and 78% for repeat laminectomy. At 6 months, the success rate for patients treated with CNL was 86% versus 78.7% for laminectomy. At 12 months, the overall success rate for the CNL group was 80.4% versus 83.3% for the laminectomy group, but in patients who had not obtained relief from the first procedure the success rate for the second procedure was higher for the post-CNL patients. A questionnaire was sent to all patients for 1- to 13-year follow-up review. The average follow-up period was 6.6 years for post-CNL laminectomy and 5.2 years for repeat laminectomy. The long-term success rate (81.8%) was higher in the post-CNL group compared to 64.4% in the repeat laminectomy group. Seven patients in the post-CNL group and nine in the repeat laminectomy group had undergone a third operation. When these originally successfully treated patients were reassigned after unsuccessful outcomes, the success rate for the CNL groups was 72.7%, versus 51.1% in the laminectomy group (p = 0.049). Employment rates were 80% for patients with CNL (21.8% changed jobs) and 76.3% for patients undergoing laminectomy (48.3% changed jobs) (p = 0.036). In conclusion, patients who underwent laminectomies after receiving CNL had significantly better long-term results than those who had repeat laminectomies.     

The polymerase chain reaction in pathology

Membrane-fixed CD14 acts as a receptor for the protein-bound endotoxin (LPS) complex and mediates the cellular effects of endotoxin. Soluble CD14 (sCD14) is suggested to neutralize circulating LPS, i.e., acting as an endotoxin antagonist. The aim of this study was to elucidate the release of both sCD14 and endotoxin in traumatized patients, starting from the earliest phase after trauma. A total of 15 patients (O ISS = 19, 9-75) suffering major trauma were enrolled in this prospective study. Blood samples were collected as early as immediately at the site of accident, on hospital admission, and thereafter hourly, then daily. For patients (O ISS = 47) died within 24 h because of their severe injuries. Immediately after the accident as well as during the first 2 h after hospital admission, the mean sCD14 levels of surviving patients did not differ from those of healthy volunteers (n = 53). Thereafter, however, sCD14 increased continuously in the trauma group. The concentrations remained elevated throughout the entire observation period. There was, however, no relation between the sCD14 release and the pattern or the severity of injury. In contrast, endotoxin levels revealed a pattern-specific release. The highest plasma concentrations of LPS were observed in patients suffering from (additional) thoracic injury. On the basis of these results we conclude that the release of sCD14 after trauma does not reflect a strict principle such as action/reaction caused by the appearance of endotoxin immediately after the injury. Soluble CD14 is more likely release by an endotoxin-independent mechanism.     

Bordetella pertussis diagnosed by polymerase chain reaction

The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of PCR for the diagnosis of BP, we used known concentrations of BP, Bordetella parapertussis and Bordetella bronchiseptica in aqueous solutions. PCR was furthermore carried out on species of bacteria that might be isolated from the nasopharynx. The applicability of PCR to patient specimens was tested in 25 patients in whose nasopharyngeal secretions BP had been demonstrated after 4-7 days of culture. The detection limit of PCR in aqueous solution was 1-2 BP bacteria per reaction tube. PCR was 100% specific for BP, showing no response with other Bordetella species or other bacteria known to colonize the nasopharynx. Of 25 patient specimens, 16 were PCR-positive 4-7 days after the positive primary culture had been established; only 5 out of 13 patient specimens were positive by repeated conventional nasopharyngeal culture at that time. We conclude that PCR is a possible alternative to culture for the demonstration of BP, as PCR is considerably faster than culture and might be more sensitive.     

Detection of West Nile virus by the polymerase chain reaction and analysis of nucleotide sequence variation

A polymerase chain reaction (PCR) assay was developed to rapidly detect and identify West Nile (WN) virus. The RNA from seven isolates of WN virus from six countries and four other flaviviruses (Kunjin, Japanese encephalitis, St. Louis encephalitis, and yellow fever viruses) was reverse-transcribed (RT) and amplified by PCR. The nucleotide sequences of the amplified products were determined by a rapid, automated DNA sequencing method. The WN virus RT/PCR assay detected the target gene segment of sequencing method. The WN virus RT/PCR assay detected the target gene segment of isolates from both the African-Middle Eastern group and the Indian group with a sensitivity of approximately 0.05 pg of viral RNA. Kunjin virus was the only other flavivirus tested that produced a band of the appropriate size. Five of seven WN virus isolates showed 92-98% homology in the nucleotide sequence of their PCR products. The sequence of one isolate was virtually identical to the published sequence of the Nigerian isolate (99.5% homology). No correlation was established between the degree of nucleotide homology, geographic location, time of isolation, or source of the isolates.     

Streptococcal erythrogenic toxin spe A gene detection by polymerase chain reaction in strains isolated in Albania

A considerable proportion of cases of myeloproliferative and lymphoproliferative disorders exhibit renal involvement. However, it is unclear whether the cytologic features, immunophenotype or grade of malignancy of the cells infiltrating the kidney differ from those of the primary tumor. This study was performed on 120 autopsy cases with the following diagnoses: acute myelogenous leukemia (AML, n = 22; subtypes M1 + M2, n = 12, subtype M4, n = 10), chronic myelogenous leukemia (CML, n = 7), agnogenic myeloid metaplasia/myelofibrosis (AMM/MF, n = 6), acute lymphocytic leukemia (ALL, n = 6), chronic lymphocytic leukemia (CLL, n = 9), other low-grade non-Hodgkin's lymphomas (low-grade NHL, n = 24), high-grade NHL (n = 21) and multiple myeloma (MM, n = 25). Renal involvement was investigated by light microscopy and immunohistochemistry. It was found in 34% of the cases, and was most common in ALL (83%) and low-grade NHL (50%) and least common in high-grade NHL (10%) and MM (12%). Dense infiltration of almost the entire kidney was most commonly seen in AML, low-grade NHL and ALL. Infiltration was bilateral and involved both the cortex and medulla in the majority of cases. When involvement of other organs was compared with that of the kidney, the lung was found to be involved in approximately the same number of cases, but liver involvement was more common and heart involvement less common. Reactive lymphocytic infiltration of the kidney was found in 18 of the 120 cases (15%), and was distinguished from scanty tumorous infiltration by immunohistochemical staining. No major phenotypical differences were found between the tumor cells infiltrating the kidney and those of the primary tumors in the bone marrow or lymph nodes. However, in one case of CML, the cells infiltrating the kidney were negative for KP1 and chloroacetate esterase, but could be identified by reactivity for CD34. The grade of malignancy in NHL was similar in both the nodal and renal manifestations.     

Multiplex polymerase chain reaction of Alu polymorphic insertions

Alu sequences are present in humans in excess of 500,000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. Polymorphic Alu insertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)-based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to the Alu polymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five different Alu polymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimize Alu multiplex PCR.     

Fluorescence cross-correlation: a new concept for polymerase chain reaction

Infusion of sodium selenite to the occipital cortex of the rat was used for the specific tracing of zinc-rich pathways. Large numbers of labeled somata were found ipsilaterally in the visual, orbital and frontal cortices, and contralaterally in homotopic and heterotopic visual areas. Labeled neurons were also found ipsilaterally in the retrosplenial, parietal, sensory-motor, temporal and perirhinal cortex. In contrast to the cortico-cortical connections, ascending afferents to the visual cortex were not zinc-rich except for a few labeled neurons in the claustrum. Additional injections showed reciprocal zinc-rich connections between the visual cortex and the orbital and frontal cortices. The latter cortices also received ascending zinc-rich afferents from the claustrum. Selenite injections revealed the layered distribution and the morphology of these labeled neurons in the neocortex. Zinc-rich neurons were found in layers II-III, V and VI. However, none was found in layer IV. Zinc-rich somata appeared as pyramidal and inverted neurons. The contrasting chemical properties of cortical and subcortical visual afferents may account for the functional differences between these systems.     

The in situ polymerase chain reaction for detection of chlamydia trachomatis

The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features.     

Evaluation of polymerase chain reaction amplification of Mycobacterium leprae-specific repetitive sequence in biopsy specimens from leprosy patients

Biopsy specimens were obtained from 102 leprosy patients before chemotherapy and examined by polymerase chain reaction (PCR) using the primers amplifying the 372-bp DNA of a repetitive sequence of Mycobacterium leprae. The PCR results were then compared with bacterial indices (BI) of slit-skin smears and biopsy specimens. The intensities of DNA bands were in general correlated with the numbers of acid-fast bacilli, and even a sample with only one organism gave a PCR positive result. Ten 5-micron sections from each frozen tissue sample were pooled and processed for DNA preparation. PCR was positive for 11 (73.3%) of 15 biopsy specimens with BI of 0 determined for the paraffin sections from the same biopsy samples. PCR also gave positive results for 84 (96.6%) of 87 BI positive biopsy samples. Although the difference in overall results between the two methods was not statistically significant, PCR seemed to have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli. Further evaluation of PCR using more specimens from leprosy patients who are bacteriologically negative is warranted to ensure PCR's advantage over the conventional microscopic examination for the diagnosis of leprosy.     

Detecting undetected HIV-1 variants in African children using degenerate polymerase chain reaction and sequence analyses

CONTEXT: Scientific journals issue press releases to disseminate scientific news about articles they publish. OBJECTIVE: To assess whether press releases about journal articles were associated with publication of subsequent newspaper stories. DESIGN: Retrospective content analysis of newspaper stories, journal press releases, and journal tables of contents. From December 1, 1996, to February 28, 1997, press releases and tables of contents were collected from BMJ, Nature, Science, and The Lancet, along with newspaper stories on scientific research published in The New York Times (United States), Le Figaro and Le Monde (France), El País and La Vanguardia (Spain), La Repubblica (Italy), and the International Herald Tribune. MAIN OUTCOME MEASUREMENTS: Number of newspaper stories that contained reference to articles appearing in the 4 scientific journals, number of newspaper stories that referred to journal articles described in press releases, and the order in which journal articles were mentioned in press releases. RESULTS: Of the 1060 newspaper stories analyzed, 142 referred to journal articles; of these, 119 (84%) referred to articles mentioned in press releases and 23 (16%) referred to journal articles not mentioned in press releases (comparison of proportions, P=.03). Articles described first or second were referenced in more newspapers than articles described later in the press release (P=.01 by chi2 analysis). CONCLUSIONS: Journal articles described in press releases, in particular those described first or second in the press release, are associated with the subsequent publication of newspaper stories on the same topic.     

Single-fiber polymerase chain reaction for detection of mutant mitochondrial DNA]

Single-fiber PCR amplifies mitochondrial DNA (mtDNA) in single muscle fiber isolated from cross frozen section. The PCR products are digested with a restriction enzyme to distinguish mutant mtDNA from wild-type mtDNA. The proportion of mutant mtDNA is higher in ragged-red fiber (RRF) than in non-RRF in mitochondrial encephalomyopathies with mutations of mtDNA. This method may be applied to evaluate amount of mtDNA and mRNA in single muscle fiber, and become a powerful tool to elucidate the pathogenetic mechanism in mitochondrial encephalomyopathies.     

On-line polymerase chain reaction (PCR) monitoring

Outcomes management has received increased attention in the current health care environment, but nursing participation has been limited due to the lack of standardized data about the effects of nursing practice. The Nursing Outcomes Classification (NOC) provides a standardized language that can be used to measure the effects of nursing practice on patient outcomes. An overview of the classification and implementation methods is provided.     

Detection of Francisella tularensis by the polymerase chain reaction

A 27-year-old woman and a 13-year-old girl diagnosed with juvenile dermatomyositis in childhood developed clinical findings of partial lipodystrophy 10 years after diagnosis. Exhaustive clinical and laboratory examinations showed an association with other abnormalities: hypertrichosis, steatohepatitis, and an abnormal insulin response to the glucose loading test in the first patient. Hypertrichosis, steatohepatitis, insulin-resistant diabetes mellitus, and acanthosis nigricans were observed in the second patient. Renal function was normal in both patients. Although a localized form of lipodystrophy has been reported associated with connective tissue disease (connective tissue lipoatrophy), the partial form has been infrequently described in association with juvenile dermatomyositis.