Evaluation of a new HTLV-I/II polymerase chain reaction

AIM: Evaluation of a qualitative HTLV-I/II DNA polymerase chain reaction (PCR) test for the detection of HTLV-I/II DNA (Roche Diagnostic Systems, Branchburg, N.J., USA) in various panels. METHODS: The panels consisted of fresh EDTA blood samples from blood donors who were anti-HTLV-I/II ELISA repeatably reactive: 53 were Western blot (WB) positive, 228 were WB indeterminate and 15 were WB negative. Elevent ELISA-negative blood donors were used as negative controls. Furthermore, specimens from 1 HTLV-II-infected intravenous drug user and from 1 HTLV-II-infected blood donor were included in the panel. Peripheral blood lymphocytes were prepared by red blood cell lysis with the Roche washing solution and stored at < -23 degrees C until processing. Amplification products were analyzed with the HTLV-I/II detection kit. RESULTS: All 53 anti-HTLV-I/II ELISA- and WB-positive samples and both HTLV-II-positive samples tested positively by PCR. All 228 anti-HTLV-I/II ELISA-positive and WB-indeterminate, all 15 ELISA-positive and WB-negative and all II ELISA-negative control samples tested negative by PCR. CONCLUSION: The Roche Amplicor HTLV-I/II test is a simple test, suitable for the confirmation of HTLV-I and-II infection in individuals with indeterminate or positive WB patterns.     

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.     

Using polymerase chain reaction for rapid sex-determination of a case of sirenomelia

Developing endoluminal technology has permitted the management of selected aneurysms using stent-grafts, but the applicability and durability of these new devices has not yet been proven. Standard treatment of mycotic aneurysms generally requires arterial ligation, excision and debridement, and autologous or extraanatomic synthetic bypass. A saphenous vein-covered stent was used to exclude an expanding, mycotic, superficial femoral artery aneurysm in a critically ill patient. Although stent-graft exclusion was intended as a bridge to standard therapy, the mass resolved, the superficial femoral artery remains patent, and the patient is symptom-free at 1 year without further intervention. Additional experience is required to determine whether stent-grafts have a role in the management of mycotic aneurysms.     

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.     

Sex identification of turkey embryos using a multiplex polymerase chain reaction

A considerable portion of the W chromosome in Gallinaceous birds consists of tandem repetitive DNA. In the turkey, a 0.4-kb PstI element is repeated about 10,000 times in the female diploid genome but is undetectable as such a unit in males. In this study a multiplex polymerase chain reaction was developed to identify the sex of turkeys based upon the PstI repeat. The technique utilized two pairs of primers, the first pair was designed to amplify a region of the PstI repetitive element, resulting in the production of a 177-bp fragment in females. The other pair was designed to amplify a region of the adenosine triphosphate (ATP) synthase gene, present in both males and females. The simultaneous use of all four primers in the same reaction resulted in the coamplification of a 177-bp and a 250-bp fragment in females and a 250-bp fragment in males. This technique was used to verify the sex of 45 adults of known sex and to identify the sex of 74 embryos from Day 5 to hatch. This procedure is rapid and permits the sexing of many embryos in a short time. The ability to sex early embryos can facilitate studies on avian sex determination.     

Diagnosis of extrapulmonary tuberculosis by a commercial polymerase chain reaction kit

The Roche Amplicor Mycobacterium tuberculosis PCR test was compared with mycobacterial culture for direct detection of M. tuberculosis in extrapulmonary specimens. From January 1995 to October 1996, 124 clinical specimens from 112 patients were assessed, including 47 body fluids, 61 tissue specimens and 16 abscesses. The sensitivity and specificity of Amplicor PCR compared to culture were 63.6% and 93.1% respectively. Analysis of 7 PCR-positive, culture-negative specimens confirmed that all were from patients with recently diagnosed tuberculosis under treatment. Eight specimens were PCR negative-culture positive, including a pleural fluid containing inhibitory substances. On acid-fast bacilli (AFB) smear-negative specimens, sensitivity and specificity were 53 and 100% respectively. The best results for Amplicor PCR were obtained with abscesses and biopsies. It is concluded that this test, highly specific for the diagnosis of tuberculosis, is at least as sensitive on extrapulmonary specimens as on smear-negative respiratory specimens.     

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.     

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.     

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.     

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.     

Analyte detection with DNA-labeled antibodies and polymerase chain reaction

We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.     

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.     

Repetitive element sequence based polymerase chain reaction for typing of Brucella strains

A collection of 40 Brucella strains, including 19 clinical B.canis isolates, was analyzed using ERIC-PCR and REP-PCR. PCR amplification using primers REP 1R-I and REP 2-I provided distinct patterns for Brucella spp. All Brucella strains could be discriminated at least to the species level. ERIC-PCR, employing ERIC 1R and ERIC 2 as primers was less discriminative for Brucella, only allowing a discrimination to the genus level with occasional discrimination between individual strains. It can be concluded that rep PCR is a promising fingerprinting method for the evaluation of an outbreak.     

Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples

The nested polymerase chain reaction technique was compared with the conventional smear and culture methods for detection of Mycobacterium tuberculosis. The nested polymerase chain reaction used in this study showed excellent specificity, sensitivity, and agreement with the conventional methods for 417 clinical samples, indicating a contribution to the rapid diagnosis of mycobacterial infectious diseases.     

The tampon test for trichomoniasis: a comparison between conventional methods and a polymerase chain reaction for Trichomonas vaginalis in women

Vertebral artery tortuosity causing neural foraminal widening is a well described abnormality that should not be confused with other causes of neural foraminal enlargement, particularly on conventional roentgenograms. We, hereby, describe CT features of another cervical osseous change due to the vertebral artery tortuosity, the so called "tubular shaped vertebral artery canal", which is embedded in the vertebral body instead of causing neural foramen enlargement. Catheter and MR angiographic studies have also been performed to confirm the vertebral artery tortuosity causing the osseous changes.