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Biomedical subjects

A Telenti

Publications and source records attributed to A Telenti.

At least 109 records · Page 6Linked to original sources
PubMed1993

Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis.

Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis. Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance. We set out to determine the molecular basis of resistance to rifampicin, a major component of multidrug regimens used for treating tuberculosis. Resistance to rifampicin involves alterations of RNA polymerase. The gene that encodes the RNA polymerase subunit beta (rpoB) was cloned. Sequence information from this gene was used to design primers for direct amplification and sequencing of a 411 bp rpoB fragment from 122 isolates of M tuberculosis. Mutations involving 8 conserved aminoacids were identified in 64 of 66 rifampicin-resistant isolates of diverse geographical origin, but in none of 56 sensitive isolates. All mutations were clustered within a region of 23 aminoacids. Thus, substitution of a limited number of highly conserved aminoacids encoded by the rpoB gene appears to be the molecular mechanism responsible for "single step" high-level resistance to rifampicin in M tuberculosis. This information was used to develop a strategy (polymerase chain reaction-single-strand conformation polymorphism) that allowed efficient detection of all known rifampicin-resistant mutants. These findings provide the basis for rapid detection of rifampicin resistance, a marker of multidrug-resistant tuberculosis.

Base Sequence↗
PubMed1993

Epstein-Barr virus infection in HIV-positive patients.

The relationship between Epstein-Barr virus (EBV) viral load in peripheral blood and HIV infection was determined in 103 HIV-infected patients. Epstein-Barr virus was detected by polymerase chain reaction in 75% of the patients, 21% of whom had the more uncommon EBV subtype 2. The highest levels of EBV were found in patients with 100-400 CD4+ cells/mm3 and not in those with more profound immunosuppression. An association was identified between EBV load and HIV proviral levels (p < 0.001), an IgM response to EBV early antigens (p < or = 0.01) and p24 antigenemia (p < 0.01 in patients with > 100 CD4+ cells), but not with other clinical or laboratory parameters. Combinations of different EBV and HIV parameters identified a subgroup of patients with a 2.2- to 4.8-fold risk of > or = 35% decline in CD4+ counts over six months. The association between EBV and HIV markers may reflect a significant pathogenic interaction between the two viruses.

Adult↗
PubMed1993

Candidemia from a urinary tract source: microbiological aspects and clinical significance.

Twenty-six cases of candidemia associated with a well-defined urinary tract source were retrospectively identified and reviewed. Urinary tract abnormalities were present in 23 of 26 patients (88%), 19 (73%) of whom had urinary tract obstruction. Nineteen patients had undergone urinary tract procedures before the onset of candidemia. Episodes of candidemia were brief and low-grade in intensity (median duration, 1 day; median colony count, 1.5 cfu/10 mL of blood). Only eight patients (31%) received > or = 500 mg of amphotericin B. There were five in-hospital deaths (19%); two of these deaths were attributed to candidiasis. No late complications of candidemia were documented for the surviving patients. Patients with urologic pathology and candiduria who undergo surgery or manipulation of the urinary tract are at significant risk for candidemia, and further studies should examine the issue of administration of prophylaxis to this group.

Adolescent↗
PubMed1993

Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis.

A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.

Base Sequence↗
PubMed1993

Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis.

A method for the rapid identification of mycobacteria to the species level was developed on the basis of evaluation by the polymerase chain reaction (PCR) of the gene encoding for the 65-kDa protein. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria. Using two restriction enzymes, BstEII and HaeIII, medically relevant and other frequent laboratory isolates were differentiated to the species or subspecies level by PCR-restriction enzyme pattern analysis. PCR-restriction enzyme pattern analysis was performed on isolates (n = 330) from solid and fluid culture media, including BACTEC, or from frozen and lyophilized stocks. The procedure does not involve hybridization steps or the use of radioactivity and can be completed within 1 working day.

Bacterial Proteins↗
PubMed1993

A simple and rapid technique for the detection of rifampin resistance in Mycobacterium leprae.

The rifampin resistance of Mycobacterium leprae is due to missense mutations in the rpoB gene encoding the beta-subunit of the essential enzyme RNA polymerase. A rapid and very simple method has been developed to detect rifampin resistance in small numbers of M. leprae present in biopsies. It involves polymerase chain reaction amplification of a defined region of the rpoB gene followed by single-strand conformational polymorphism analysis (PCR-SSCP). The reliability of the method has been tested on a sample of known drug-resistant and susceptible isolates of M. leprae.

Alleles↗
PubMed1992

Detection of JC virus by polymerase chain reaction in cerebrospinal fluid from two patients with progressive multifocal leukoencephalopathy.

The polymerase chain reaction (PCR) was used to identify JC virus (JCV) in the cerebrospinal fluid of two patients with progressive multifocal leukoencephalopathy confirmed by brain biopsy. In addition, JCV viremia was demonstrated by PCR in one case. JCV detection in spinal fluid by PCR may be the first non-invasive technique available for the diagnostic confirmation of progressive multifocal leukoencephalopathy.

DNA, Viral↗
PubMed1992

Competitive polymerase chain reaction using an internal standard: application to the quantitation of viral DNA.

A general strategy for the construction of an internal standard for the polymerase chain reaction (PCR) is described together with its application in the evaluation of clinical samples. This internal standard is a plasmid containing a modified target sequence that is co-amplified with the native target using the same set of primers. The co-amplification reaction will generate two fragments of different size that are readily separated without the need for restriction enzyme digestion. Thereafter, they are detected and quantitated by hybridization to the same probe. Detection of HIV proviral DNA was chosen as a model for this competitive PCR. The assay proved to be a sensitive tool for the detection of PCR inhibitors and allowed quantitation of HIV with a 20-30% variation coefficient. Despite limitations that appear inherent to the amplification process, internal standards appear to be useful tools for quantitative analysis by PCR.

Base Sequence↗
PubMed1992

[The EBNA2 coding region in the viral genome of Epstein-Barr virus (EBV)-positive cases of Hodgkin's disease].

Epstein-Barr virus (EBV)-genome containing cases of Hodgkin's disease (HD) are known to express latent membrane protein (LMP) of EBV but no EBV nuclear antigen 2 (EBNA2). We investigated 35 cases of HD for the presence of EBV genome sequences to know whether EBNA2 coding region is deleted in HD. 27 cases had general EBV sequences, none had evidence of a deletion of EBNA2. 26 cases turned out to harbour EBV type 1, one case had EBV type 2. The absence of EBNA2 protein expression in HD can not be explained by a deletion of EBNA2 coding region. EBV type 1 is the prevalent subtype of EBV in our series of HD.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Survey of urine from transplant recipients for polyomaviruses JC and BK using the polymerase chain reaction.

Using polymerase chain reaction (PCR), we examined 108 urine specimens from 39 post transplant patients for polyomaviruses JC (JCV) and BK (BKV). Urine sediments were collected and subjected to 30 cycles of amplification. PCR products were resolved by agarose gel electrophoresis, transferred to nylon membranes by Southern blot, and hybridized with radiolabelled probes. Polyomavirus DNA was found in urine specimens from 17 out of 39 patients (44%). Both viruses were detected in specimens from nine patients, JCV alone in five, and BKV alone in three. In comparison, polyomavirus was detected in only five of 22 PCR positive specimens by shell vial cell culture assay. Our results show a high prevalence of polyomavirus shedding after transplantation and suggest a higher rate of JC viruria than previously reported.

DNA, Viral↗
PubMed1991

Quantitative blood cultures in candidemia.

The relationship between quantitative data on peripheral blood cultures and source of infection was studied in 172 episodes of candidemia that occurred in 169 patients. Clinically, the source of candidemia was an intravascular device in 67 episodes, an extravascular source in 73 episodes, and endocarditis in 2 patients; no source was identified for the other 30 episodes. Colony counts were determined in peripheral blood specimens on the first day of candidemia by the lysis-centrifugation system. High-grade and low-grade candidemia were defined as 25 colony-forming units or more per 10 ml and 10 colony-forming units or fewer per 10 ml of blood, respectively. Of 48 episodes of high-grade candidemia, 43 (90%) were associated with an infected intravascular device; therefore, the presence of high-grade candidemia should prompt the removal of intravascular devices. In contrast, 92 of the 112 episodes of low-grade candidemia (82%) had an extravascular or an unidentified source of candidemia. In patients with infections associated with an intravascular device, colony counts declined significantly within 72 hours after removal of the device in the absence of antifungal therapy; failure to decline suggests an alternative source of persistent infection. Quantitative data from peripheral blood cultures may help distinguish intravascular from extravascular sources of candidemia and aid in assessing the response to the removal of infected intravascular devices.

Academic Medical Centers↗
PubMed1991

Epstein-Barr virus and persistent graft dysfunction after liver transplantation.

Epstein-Barr virus infection has been associated with a broad spectrum of clinical manifestations, depending on the immune status of the host. In this report, we describe two liver transplant patients who received hepatic allografts from donors serologically positive for Epstein-Barr virus and who experienced primary infection with Epstein-Barr virus associated with prolonged liver graft dysfunction. In both patients, Epstein-Barr serologies converted within 3 mo of liver transplantation, and hepatic histological study revealed mononuclear infiltration of the sinusoids evolving to pronounced immunoblastic features suggestive of evolving lymphoma. In both cases, in situ hybridization studies confirmed the presence of Epstein-Barr virus genome in the liver. Furthermore, polymerase chain reaction analysis suggested that high levels of Epstein-Barr virus DNA were present in biopsy specimens obtained during the episode of acute hepatitis that followed Epstein-Barr virus seroconversion. The degree of Epstein-Barr virus DNA estimated by polymerase chain reaction appeared to increase in parallel with the progression of parenchymal lymphocytic infiltrates. In one patient, a biopsy sample from a cervical node also revealed high levels of Epstein-Barr virus DNA estimated using the polymerase chain reaction technique. Furthermore, in these patients, Epstein-Barr virus DNA levels appeared to decrease dramatically after discontinuing azathioprine administration and beginning treatment with acyclovir. These two cases illustrate the dynamics of Epstein-Barr virus immune regulation and confirm chronic hepatic allograft dysfunction related to Epstein-Barr viral infection.

Adult↗
PubMed1990

Hepatic cyst infection in autosomal dominant polycystic kidney disease.

To characterize the syndrome of hepatic cyst infection in autosomal dominant polycystic kidney disease (ADPKD) and to review its diagnosis and management, we retrospectively studied five such cases in patients from our institution and nine detailed case reports from the literature. The clinical manifestations were an acute (58%) or subacute (42%) febrile illness, typically associated with tenderness in the right upper quadrant, leukocytosis, a very high erythrocyte sedimentation rate, but minor abnormalities of liver function tests. Bacteremia was present in 7 of 11 patients. Enterobacteriaceae grew in pure culture from the cyst fluid in 9 of 12 patients. Complex cysts were observed by ultrasonography (in four of eight patients), computed tomography (in six of nine), and magnetic resonance imaging (in two of two). 111In leukocyte scans were positive in all four patients in whom they were done, and 67Ga scans were positive in only one of three patients. An unfavorable outcome was observed in six of seven patients treated with only antibiotics, in contrast with one of seven patients who received antibiotics and early drainage. In two patients, ciprofloxacin cyst levels were 2.3 and 4.8 times higher than the level in serum; in a third patient, cyst levels remained in therapeutic range 30 hours after the last dose of ciprofloxacin, at which time serum levels were undetectable. Clinical and laboratory features and the use of modern scanning techniques facilitate a prompt diagnosis of infection in hepatic cysts in ADPKD. The treatment of choice is a combination of percutaneous drainage and antimicrobial therapy.

Aged↗
PubMed1990

Detection of JC virus DNA by polymerase chain reaction in patients with progressive multifocal leukoencephalopathy.

Polymerase chain reaction (PCR) was used in the detection of JC virus (JCV) DNA in formalin-fixed, paraffin-embedded brain tissue sections from 24 patients with progressive multifocal leukoencephalopathy. Brain sections from normal autopsies (n = 17) and other neurologic conditions (n = 4) were used as controls. Specific amplified products were detected in 20 (83%) of 24 patients with progressive multifocal leukoencephalopathy. PCR did not amplify JCV or human beta-globin gene sequences in four patients with characteristic demyelinating lesions of progressive multifocal leukoencephalopathy that were positive for JCV by in situ hybridization or immunocytochemistry. PCR from biopsy sections resulted in more intense amplification signals than PCR from autopsy tissue. Normal brain sections from 17 autopsies were negative by PCR. Low-grade amplification of JCV was observed in one patient with herpes simplex virus encephalitis. PCR served as a more rapid test for confirmation of progressive multifocal leukoencephalopathy than in situ hybridization. However, PCR performance was altered by prolonged formalin fixation of tissue and undetermined inhibitors of the amplification reaction. Laboratories and clinicians should be aware of the difficulties encountered when using paraffin-embedded tissue for PCR.

Base Sequence↗
PubMed1990

Detection of Epstein-Barr virus by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to study DNA extracted from the blood of 25 transplant patients, 5 patients with infectious mononucleosis, and 13 healthy subjects and autopsy or biopsy tissue from 29 patients with lymphoproliferative disorders. Primers were directed to conserved regions of the Epstein-Barr virus (EBV) genome encoding capsid protein gp220 and Epstein-Barr nuclear antigen 1. Specific EBV amplification was found in the blood of 11 of 25 transplant patients and all patients with infectious mononucleosis. All patients with lymphoproliferative disorders occurring in the presence of immunosuppression (eight organ transplant patients and two patients with acquired immunodeficiency syndrome) had biopsies positive for EBV by PCR. Only 1 of 19 samples from lymphomas or leukemias unrelated to immunosuppression contained EBV. PCR confirmed the very close association of EBV and lymphoproliferative disorders occurring in the presence of immunosuppression. The significance of detecting EBV sequences in the blood of transplant patients, particularly in relationship to lymphoproliferation, requires further study.

Adult↗
PubMed1990

Rapid detection of polyomavirus BK by a shell vial cell culture assay.

Polyomavirus BK (BKV) causes asymptomatic latent infection in the human host that is reactivated during periods of immune suppression. Detection by conventional tube cell culture is difficult and time consuming because BKV exhibits slow growth with late (14 to 28 days) and subtle cytopathic effects. We developed a shell vial cell culture assay (SVA) using a cross-reactive monoclonal antibody to the T antigen of simian virus 40 to detect BKV rapidly by indirect immunofluorescence. Nuclear fluorescence was seen in BKV-infected cells as early as 16 h postinoculation; 6 to 28 times more foci were present at 36 h postinoculation. Human embryonic kidney cells infected with BKV produced 7 to 42 times more fluorescent foci than MRC-5 or rhabdomyosarcoma cells did. Centrifugation enhanced the infectivity of BKV in the SVA. To define the clinical utility of SVA, urine specimens from organ transplant patients were tested. Of 27 patients, 4 (15%) were found to be positive by SVA. SVA offers a simple and rapid method for detection of BKV that can be of use in clinical studies of this virus.

Animals↗
PubMed1989

Fungal blood cultures.

It is generally accepted that fungemia is an important and often life-threatening event. Unfortunately, it often remains undetected due to the lack of physician and/or laboratory awareness of the usefulness of fungal blood cultures. Many laboratories do not offer the possibility of performing fungal blood cultures and, if offered, some use methods that are not optimal for recovery or require extended incubation for recovery. Of the methods considered satisfactory, the lysis-centrifugation method (Isolator) has the highest recovery rate and the shortest recovery time. The Bactec and Septi-Chek systems are alternative methods, but have limitations.

Blood↗