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.     

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.     

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 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.     

Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10(0.2) colony forming units (cfu) of Mycoplasma synoviae and 10(0.7) cfu of Mycoplasma gallisepticum. When M. synoviae and M. gallisepticum were spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccines improved the sensitivity of PCR to detect both M. synoviae and M. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.     

Clonal analysis in acute myeloid leukemia by polymerase chain reaction

We have used PCR to amplify a polymorphic portion of the X-chromosome linked phosphoglycerate kinase gene (PGK) combined with a methylation-sensitive restriction enzyme digestion of the active X chromosome to examine the frequency of heterozygosity in Taiwanese females and analyze clonality in 18 female patients with acute myeloid leukemia (AML). We used hair follicles as normal tissue control. We found that the incidence of heterozygosity of the PGK gene in 102 hematological normal females and 18 patients tested was 35% (42/120). In five AML patients, a monoclonal X-inactivation pattern of leukemic blasts was found at presentation, which then returned to polyclonal at remission. In two of these five cases, a monoclonal pattern recurred at relapse. We also found that the hair follicles were readily accessible normal tissue for control, were easy to obtain, and were non-invasive.     

Detection of a mutator phenotype in cancer cells by inter-Alu polymerase chain reaction

A mutator phenotype due to a DNA mismatch repair deficiency is usually detected by typing a number of microsatellite markets. Here, eight hereditary nonpolyposis colon cancer patients with microsatellite instability were investigated by inter-Alu PCR, known to amplify DNA segments that may represent preferential targets of replication errors. Among 40-60 bands revealed in a single PCR experiment, more than 20% were found altered in tumoral DNA samples compared to matched normal samples from the same patient. Shifts and changes in signal intensity accounted for most of the alterations, whereas gains or losses of bands were rare. Certain bands were affected only in a single patient, whereas the instabilities in others were common. These results suggest that some genomic regions are more susceptible than others to the expression of a mutator phenotype. Four such bands altered in at least five patients were characterized further and shown to be unstable because of contractions of the Alu poly(A) tails. Interestingly, none of the bands representing loci shown previously to be polymorphic in the population displayed instability in the tumoral samples. Inter-Alu PCR appears to be a robust, cost-effective, and sensitive technique for revealing the mutator phenotype in cancer cells.     

Detection of ileal symbiont intracellularis in porcine faecal samples by polymerase chain reaction

Ileal Symbiont Intracellularis (ISI), the organism causing proliferative enteritis (PE) in pigs was detected in faeces by the application of polymerase chain reaction (PCR). The assay based on a 319 base pair DNA fragment was used on faecal and mucosal samples derived from pigs either affected or unaffected with PE. As few as 10(3) ISI could be detected in pig faeces spiked with ISI. No amplification product was detected in the faeces of unaffected pigs but faeces of confirmed clinical cases were positive. This method offers an accurate, sensitive, easy to perform alternative to monoclonal antibody tests or histological examination post-mortem for the presence of ISI in pig herds.     

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 Leishmania DNA by polymerase chain reaction in scars of treated human patients

Leishmaniasis is an endemic disease in developing countries. The efficacy of therapy is usually evaluated through clinical parameters. To define the parasitologic cure, 20 patients were biopsied before and 1 month to 8 years after treatment. Paraffin-embedded tissue was used for DNA isolation. All patients had a positive polymerase chain reaction (PCR) result before therapy, except 1, for whom no histopathologic material was available. The causative agent was identified as belonging to the Leishmania (Viannia) subgenus by hybridization. Despite clinical healing and absence of reactivation or development of mucosal lesions, PCR was positive in scars of 16 patients (80%). The results suggest that parasites persist in the skin for many years despite treatment. Depending on specific pathogenetic features of the parasite and the immune status of the host, this phenomenon might result in mucosal lesions. Alternatively, it could have a role in the maintenance of immunologic memory in patients living in areas in which leishmaniasis is endemic.