Detection of avian leukosis virus subgroup J using the polymerase chain reaction

PURPOSE: To compare neutral, external rotation, and abduction external rotation positions of the glenohumeral joint during magnetic resonance (MR) arthrography in the assessment of the joint capsule, biceps-labral complex, and glenohumeral ligaments. MATERIALS AND METHODS: MR imaging with intraarticular administration of gadopentetate dimeglumine was performed in 10 adult cadaveric glenohumeral joints. Fat-suppressed oblique coronal, oblique sagittal, and axial. T1-weighted spin-echo imaging and axial three-dimensional spoiled gradient-recalled imaging were performed with each shoulder in the neutral, external rotation, and abduction external rotation positions. Shoulders were sectioned in the planes that yielded optimal MR images. Anatomic and MR imaging findings were correlated. RESULTS: The biceps-labral complex was best visualized on oblique coronal and axial images obtained in external rotation. Oblique axial abduction external rotation imaging best delineated the inferior glenohumeral ligament but did not improve assessment of the superior and middle glenohumeral ligaments in comparison with findings in neutral and external rotation. CONCLUSION: Although MR arthrography of the glenohumeral joint clearly delineates the biceps-labral complex and glenohumeral ligaments, external rotation of the shoulder optimizes visualization of the former structures. Abduction external rotation is the best position for evaluation of the inferior glenohumeral ligament and anterior capsular attachment.     

Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR)

Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.     

Detection of Mycoplasma pneumoniae by polymerase chain reaction in lung aspirates from patients with community-acquired pneumonia

STUDY OBJECTIVE: This study was designed to evaluate the usefulness of polymerase chain reaction (PCR) to detect Mycoplasma pneumoniae DNA in samples obtained by transthoracic needle aspiration (TNA). DESIGN: Prospective study of cases. SETTING: A university hospital in Lleida, Spain. PATIENTS: A total of 101 unselected patients, admitted between January 1993 and March 1994 in the emergency department, with a clinical and radiologic picture of community-acquired pneumonia, and without contraindications for TNA application. INTERVENTIONS: Patients were studied with conventional diagnostic techniques for community-acquired pneumonia. In addition, a sample obtained by TNA was processed by the following methods: culture in standard media, culture in selective media for Legionella, detection of capsular antigens for Streptococcus pneumoniae and Haemophilus influenzae, and detection of M pneumoniae specific genome by PCR. RESULTS: Serologic data were not available in eight patients and were excluded from this analysis. M pneumoniae PCR amplification was possible in eight cases, well correlated with serologic responses indicating current infection. Samples from ten additional patients, negative by PCR, were found to be demonstrative of recent M pneumoniae infection by serologic study. Finally, in all the remaining 75 cases, including the 59 patients for whom a different microbial diagnosis was established, M pneumoniae PCR test gave negative results. CONCLUSION: This study indicates that PCR, applied to samples obtained by TNA, appears to be a moderately sensitive and highly specific method for rapid detection of M pneumoniae lung infection.     

Mycoplasma pneumoniae detection from throat swab by two-step polymerase chain reaction

Two-step polymerase chain reaction (PCR) with primers designated against 16S rRNA gene of Mycoplasma pneumoniae for diagnosis of infection was evaluated in comparison with the conventional single-step PCR and culture methods. The two-step PCR method showed specific amplification of M. pneumoniae DNA and higher sensitivity (1.5 fg/assay) than the single-step PCR method. With the two-step PCR method, 76 of 322 throat swabs (23.6%) from patients with acute respiratory complaints gave positive results whereas 20.2% were positive in the culture method. Seven of 13 samples which were negative in the single-step PCR method but positive in either serological or the culture method showed positive results by the two-step PCR method. In addition, 5 samples which were weakly positive in the single-step PCR method showed distinctly positive results in the two-step PCR. These results indicate that the two-step PCR method is a useful tool for detection of M. pneumoniae in clinical specimens, although it requires a relatively sophisticated in technique.     

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.     

Detection of Mycobacterium bovis in milk by polymerase chain reaction

A simple method was developed for DNA extraction of Mycobacterium bovis in milk and further detection of the bacterium by polymerase chain reaction (PCR). Milk previously seeded with M. bovis was used as the starting material. The procedure involved overnight digestion of a milk sample with proteinase K at 56 degrees C and phenol extraction, followed by ethanol precipitation and PCR. The amplification pattern obtained was analyzed with primers BW8-BW9 which amplify a 248 bp in strains of M. bovis. By using the BW8-BW9 primers, 10(3) CFU were detected on silver-stained PAGE gels. The procedure was validated by PCR analysis of milk in tuberculin-positive animals. It is anticipated that this method can be used for routine diagnosis of M. bovis in milk samples.     

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.     

Detection of Legionella pneumophila in wastewater by nested polymerase chain reaction

Behavioral economics defines unit price (UP) as the ratio of the response requirement to magnitude of reinforcer. When applied to drug self-administration, the UP model defines UP as the ratio of the response requirement to the unit dose of drug. This model makes two predictions about drug self-administration: increasing UP decreases consumption and consumption at a given UP will be constant regardless of the response requirement and dose that make up the UP. In previous experiments conducted in rhesus monkeys allowed to choose between an i.v. injection of cocaine and food, the UP model has failed to adequately predict drug consumption in that consumption varied (increased with dose) at a given UP. However, previous experiments have allowed a fixed number of choice trials/day, thereby imposing a procedural ceiling on consumption that may have influenced conformity to the UP model. In the present experiment, the number of choice trials available was varied in such a way that constant drug consumption was possible over the range of UPs tested. The response requirement for cocaine was varied between 15 and 1200 lever presses/injection and the dose of cocaine was varied between 0.05 and 0.2 mg/kg/inj, yielding UPs from 300 to 5600 responses/mg/kg. The response requirement for food was always 30. As predicted by the UP model, cocaine consumption decreased as UP increased. Moreover, in contrast to previous experiments, consumption did not vary significantly across the response requirement/dose combinations that made up a UP. A detailed analysis suggested that a decrease in magnitude of the alternative reinforcer (one rather than three food pellets), rather than the increase in trials, was responsible for the improved conformity to the UP model in the present experiment relative to previous experiments. Taken together with previous experiments, the present experiment suggests that conformity to the UP model of drug consumption in a choice situation is dependent upon the magnitude of alternative reinforcers that are available. Consumption was best predicted by the UP model when the magnitude of the alternative reinforcer was small.     

Detection of Pneumocystis carinii sequences in serum by polymerase chain reaction

The clinical significance of the detection of Pneumocystis carinii DNA was evaluated, as well as the detection of circulating P. carinii antigen from serum using previously collected samples. Fourteen serum samples from 13 patients were diagnosed positively for P. carinii DNA by polymerase chain reaction (PCR). Ten of 14 episodes (71.4%) of pulmonary complications were compatible with P. carinii pneumonia. Two patients were definitely diagnosed as having had P. carinii pneumonia at autopsy. All patients positive for circulating antigens were also positive for P. carinii DNA, suggesting that the detection of P. carinii DNA by PCR is more sensitive compared to the detection of circulating antigens by the Ouchterlony method. It is concluded that the detection of P. carinii DNA in serum by PCR provides useful information for identifying P. carinii pneumonia.     

Detection of hepatitis C virus with RNA polymerase chain reaction in fulminant hepatic failure

The role of hepatitis C virus (HCV) infection in fulminant hepatic failure is controversial. The frequency of serum HCV RNA positivity in previously reported patients with fulminant hepatic failure (FHF) of indeterminate cause ranged from 0 to 12% in the United States and Europe and from 43% to 59% in Asia. We assessed serum HCV RNA using polymerase chain reaction (PCR) and oligoprimers from the 5'UTR of the HCV genome in 26 consecutive patients with FHF. Another laboratory independently performed PCR on 21 of the serum samples using different oligoprimers from the 5'UTR and NS3 region of the HCV genome. Serum HCV RNA was detected in two of seven (28%) patients with hepatitis B, 9 of 15 (60%) with an indeterminate cause, and in none with hepatitis A (n = 2) or drug-induced hepatotoxicity (n = 2). HCV RNA PCR results were concordant between both laboratories in 17 of 21 (81%) of samples. In patients with an indeterminate cause, HCV RNA positivity was significantly associated with the transmission risk factor of low socioeconomic status and Hispanic ethnicity. Eighteen patients underwent liver transplantation (LT) and 15 (83%) survived. Among patients with FHF of indeterminate cause, recurrent or acquired HCV infection after transplantation occurred in three of five (60%) and one of four (25%) patients, respectively. Three of four (75%) patients with hepatitis C virus infection post-LT also developed histologic hepatitis. HCV appears to be the causative agent of a substantial number of cases of FHF classified as indeterminate in the Los Angeles area. Differences in patient populations or risk factors may explain the discordant incidences of HCV infection in FHF observed among different programs.     

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.     

Detection of pathogenic fungi in human blood by the polymerase chain reaction

The ability of the polymerase chain reaction (PCR) to detect pathogenic fungi in human blood was investigated. A DNA fragment of about 300 bp from the 18S rDNA, highly conserved in all fungi, was amplified with target DNA from 18 different species of fungi commonly isolated from clinical samples. The presence of PCR products was confirmed by hybridization with a fluorescein-labelled internal probe (21-mer). The PCR assay described is sensitive enough to detect 125 fg of purified Candida albicans DNA and 10 to 100 yeast cells per millilitre of blood.     

Detection of chlamydia trachomatis by polymerase chain reaction assay in nonbacterial prostatitis

BACKGROUND: Melasma is a common disorder of facial hyperpigmentation among Asian women. Many modalities of treatment are available but none is satisfactory. OBJECTIVE: This study was undertaken to see if glycolic acid peels are effective and safe in the treatment of melasma and fine facial wrinkling. METHODS: Ten Asian women with moderate to severe melasma were recruited into the study. The women had twice daily applications of a cream containing 10% glycolic acid and 2% hydroquinone (Neostrata AHA Age Spot and Skin Lightening Gel) to both sides of the face, and glycolic acid peels every 3 weeks (20-70%) to one-half of the face using Neostrata Skin Rejuvenation System. All patients had to use a sunblock (SPF 15%). At regular intervals and at the end of 26 weeks (or after eight peels) the degree of improvement of pigmentation and fine facial wrinkling on each side of the face were assessed. Any skin irritation or side effects were also noted. Assessment was by an independent dermatologist, the patients themselves, and the use of the Munsell color chart and photographs. The nonparametric Wilcoxon Rank-Sum test was used for statistical analysis. RESULTS: The melasma and fine facial wrinkling improved on both sides of the face. The side that received glycolic acid peels did better but the results were not statistically significant (P > 0.059). CONCLUSION: A cream containing 10% glycolic acid and 2% hydroquinone (Neostrata AHA Age Spot and Skin Lightening Gel) improved melasma and fine facial wrinkling in Asian women. In combination with glycolic acid peels at 3-week intervals the lightening of melasma is subjectively much better. This improvement does not reach statistical significance and the sample size is small (n = 10).     

Detection of Mycobacterium tuberculosis in transbronchial biopsy specimens by polymerase chain reaction

Polymerase chain reaction (PCR) for the rapid detection of Mycobacterium tuberculosis was used to test 30 clinical specimens (transbronchial biopsy specimens) from patients in whom tuberculosis was suspected on chest X-ray. In these patients, 15 cases were histologically diagnosed as tuberculosis. Among them, M. tuberculosis was detected in 8 specimens by PCR. Of 8 PCR positive specimens, 7 were also positive by smear and culture; the other was negative by smear and positive by culture. On the other hand, 15 cases were therapeutically diagnosed as the tuberculosis and these specimens showed negative results in neither PCR nor microbiological tests. We conclude that the PCR method is useful for rapid and direct detection of M. tuberculosis in transbronchial biopsy specimens.     

Diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients by polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) testing was performed on respiratory tract specimens obtained by throat swab in 21 children admitted to the hospital with suspected Mycoplasma pneumoniae pneumonia. Of 13 patients with a clinical condition compatible with mycoplasma infection and an immunological response to M. pneumoniae, 11 were positive by PCR. Eight patients were negative by serology and/or had a clinical condition not compatible with mycoplasma infection, and all were negative by PCR. The antibody response to M. pneumoniae was delayed for a week or more in 3 (23%) of the 13 patients with documented mycoplasma infection. These results suggest that PCR performed on a respiratory tract specimen obtained by a throat swab may be useful in the initial evaluation of children with suspected M. pneumoniae pneumonia, especially in patients in whom the serological response is delayed.