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Biomedical subjects

P E Klapper

Publications and source records attributed to P E Klapper.

At least 55 records · Page 3Linked to original sources
PubMed1995

Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.

Acute Disease↗
PubMed1995

New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay.

A new, rapid, and simple method for the isolation of either RNA or DNA from cerebrospinal fluid samples for subsequent amplification by specific polymerase chain reaction (PCR) assays is described. The technique involves a single extraction with a guanidinium thiocyanate acid (GuSCN) buffer, and does not require the use of organic solvents. Applied to the recovery of enteroviral RNA, herpes simplex virus (HSV) and Varicella-zoster virus (VZV) DNAs the method has proved to be of equivalent or better efficiency than established methods of nucleic acid separation but is less laborious and time consuming. The simplicity of the procedure permits the processing of large numbers of samples and the use of a single preparative method for either RNA or DNA PCR makes it an attractive method for the routine laboratory.

Cells, Cultured↗
PubMed1995

Adenovirus cross-infection: a continuing problem.

Nosocomial outbreaks of epidemics of adenovirus keratoconjunctivitis are frequently reported even though the simple measures to prevent or limit such occurrences are well documented. There have been two such outbreaks associated with the accident and emergency department (A&E) of a large, urban eye hospital in recent years. In the first--involving at least 200 cases--there was a delay of two months in initiation of control measures, with consequent potentiation and prolongation of the outbreak. The delay resulted because of the time taken for isolation of the virus responsible (adenovirus type 8). In the second outbreak--23 cases--the use of a rapid diagnostic test--adenovirus immune dot-blot (IDBT)--allowed prompt identification of adenovirus and enabled early introduction of control measures. Important to this strategy was the routine surveillance of eye infections in patients attending the A&E. Many clinicians are reluctant to investigate possible ocular adenovirus disease because of the cost and the delay involved in isolation of the virus in cell culture. Adenovirus IDBT provides a rapid and economic alternative (mean time for IDBT reporting five days compared to 33 days for cell culture isolation). Concerns over sensitivity of IDBT (67-84%) vs. culture are now being addressed with molecular biological approaches to diagnosis offering sensitivity close to that achieved by culture (91% vs. culture) but with the added advantage of same day reporting.

Adenoviridae↗
PubMed1995

Prospective study of adenovirus antigen detection in eye swabs by radioimmune dot-blot.

Rapid laboratory diagnosis of ocular adenovirus infection is crucial in the containment of nosocomial transmission of the virus. In a large prospective study of adenovirus assay in eye swabs, antigen detection by radioimmune dot-blot (turnaround time 72 hours) achieved a sensitivity of 67% (239/355) and a specificity of 93% (3065/3285) in comparison with virus culture (median turnaround time 14 days). When specimens weakly reactive for adenovirus antigen, or equally reactive for both adenovirus antigen and Chlamydia trachomatis antigen, were considered falsely reactive in the adenovirus test, the sensitivity of the latter was reduced and false positive reactions were only marginally less frequent. The radioimmune dot-blot provides a more rapid diagnosis of ocular adenovirus infection than virus culture, but the high risk of false negative and in particular false positive results limits its clinical utility.

Adenovirus Infections, Human↗
PubMed1994

Corneal donor infection by herpes simplex virus: herpes simplex virus DNA in donor corneas.

Three corneoscleral discs (from two donors) underwent subtotal endothelial loss during routine "long-term" organ culture storage. Laboratory studies of these corneas revealed evidence of herpes simplex virus (HSV) infection. The fellow cornea from one of the donors had been issued for transplant to a patient with keratoconus. Deterioration of the graft was noted 5 days after surgery; the disc was removed at 2 months and was shown to be infected with HSV. In an experiment designed to simulate initial "cleansing" of donor globes, 0.1% polyvinylpyrolidone-iodine protected cells from infection with HSV. It was concluded that the detection of HSV in these corneas could not be explained by external contamination of the ocular surface. Furthermore, culture of conjunctival and pharangeal swabs taken from 47 consecutive donors confirmed that HSV is rarely isolated at or around the time of death. Five pairs of donor corneas destined for use in transplantation were selected at random and investigated for the presence of HSV. HSV DNA was detected by polymerase chain reaction (PCR) in tissue from two of the corneal donors. Sequential stepwise sectioning suggested that HSV DNA when present was distributed in discrete foci within the cornea. These observations suggest that HSV infection may be a cause of severe endothelial loss during corneal organ culture and possibly provide an explanation for some "failures" of corneal grafting.

Adult↗
PubMed1993

Computer network for a diagnostic virology laboratory.

A data base for a large diagnostic virology laboratory is described. The system uses a network of personal computers. It allows the entry, long-term storage, and subsequent retrieval of specimen and patient records (comprising personal identifiers and specimen and result information), and hard-copy results reporting. Sited entirely within the laboratory, the network is not connected to a modem. Within the laboratory there is restricted access to human immunodeficiency virus test results to guarantee patient confidentiality. Retention of a hard-copy of specimen request cards ensures the availability of the original clinical information. The data base is copied on a second file server to facilitate searches, and daily streaming onto magnetic tape provides system protection in the event of hard disc failure. Matching of old and new patient records is done by surname, date of birth, and sex, and therefore duplicate records accumulate when patient names are misspelt on specimen request forms. The system requires further development to speed searches of the data base and to achieve automatic generation of laboratory worksheets. Future goals are the replacement of hard-copy records of clinical information and hard-copy reporting with on-line access to hospital data bases and on-line requesting by and reporting to the clinician.

Clinical Laboratory Information Systems↗
PubMed1993

Herpes simplex type 1 encephalitis in acquired immunodeficiency syndrome.

A severe acute necrotizing herpes simplex virus type 1 encephalitis, apparently mediated by macrophages or cytokines or both, was seen in a patient with acquired immunodeficiency syndrome. The necrosis had a distribution similar to that seen in immunocompetent subjects. Viral inclusions were abundant and meningeal reaction was minimal, as previously described in one anergic patient.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Measles encephalitis during immunosuppressive treatment for acute lymphoblastic leukaemia.

Between 1971 and 1989 measles encephalitis was identified in five children receiving chemotherapy for acute lymphoblastic leukaemia. Review of these and previously reported cases of measles encephalitis in immunosuppressed patients failed to identify any pathognomonic features in the history, the clinical presentation, or the results of electroencephalography or computed tomography. Detection of measles virus antigen in nasopharyngeal secretions or intrathecal synthesis of specific antibody was not possible in all instances. Early diagnosis by direct detection of viral antigen in the brain was confounded by difficulties in identifying areas of the brain suitable for biopsy. Increasing herd immunity to measles in the general population by vaccination is the only effective intervention against measles encephalitis in immunosuppressed children. Measles encephalitis must be remembered as a possible explanation of encephalopathy in the immunocompromised child: the benefits of early use of antiviral agents need to be evaluated.

Brain↗
PubMed1993

The role of a rapid diagnostic test (adenovirus immune dot-blot) in the control of an outbreak of adenovirus type 8 keratoconjunctivitis.

Twenty-three patients were involved in an outbreak of adenovirus type 8 infection based at the Manchester Royal Eye Hospital in the spring of 1991. Confirmation of adenovirus infection was by means of the immune dot-blot test (IDBT) and virus culture, the latter, allowing serotype 8 to be identified as the cause. Epidemiological tracing and limited restriction endonuclease analysis of the virus isolated suggest that 15 patients contracted the infection within the Casualty Department following attendance for minor eye trauma, 10 being infected by the same junior doctor. Seven patients presented with an established adenovirus type 8 conjunctivitis at the first hospital visit and the source of their infection could not be identified. Laboratory confirmation of adenoviral involvement took an average of 5 days by IDBT, compared with 33 days by virus culture. Rapid identification of adenovirus by IDBT enabled early institution of control measures thereby limiting the size of the outbreak.

Adenovirus Infections, Human↗
PubMed1992

Detection of false seroconversions in cytomegalovirus seronegative platelet donors by serial complement fixation or IgG-specific radioimmunosorbent tests.

A panel of 421 cytomegalovirus (CMV) seronegative-platelet donors was followed using complement-fixation (CFT) and IgG-specific radioimmunosorbent tests (RIST) to detect seroconversion. During 4623 person-months of observation 2452 serum specimens were tested, and the annual rate of seroreactivity detected by RIST was 56%. However, most of the positive RIST results were followed by negative results with later serum samples. CFT seroreactivity was a poor predictor of RIST seroreactivity. The high rate of transient seroreactivity detected by RIST may have reflected the lack of absolute specificity of the test. Alternatively, the results could illustrate the difficulty of determining whether an individual harbours latent CMV infection using antibody tests when some seronegative individuals appear to be latently infected. In the light of our observations recommendations for CMV antibody testing of blood donors are proposed.

Antibodies, Viral↗
PubMed1991

CSF pretreatment and the diagnosis of herpes encephalitis using the polymerase chain reaction.

A number of techniques for extraction of DNA prior to polymerase chain reaction (PCR) amplification of herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) were compared to the use of "native" CSF in the PCR reaction. The results indicate that extraction of DNA (which allows efficient removal of inhibitors of Taq polymerase) is an essential pre-requisite of the PCR detection of CSF HSV DNA.

DNA, Viral↗
PubMed1990

Diagnosis of cytomegalovirus infection in cyclosporin-treated renal allograft recipients.

The relative merits of antibody response and virus shedding as markers of cytomegalovirus (CMV) infection among cyclosporin-treated renal allograft recipients were analysed. CMV-specific antibody was assayed by IgG-specific radioimmunosorbent test (RIST) and by complement fixation test (CFT). CMV shedding was assayed by virus isolation and by the rapid test immediate early nuclear antigen detection (IENAD). RIST and CFT detected seroconversion in similar numbers of patients, but the former test was the more sensitive when CMV antibody was sought in pretransplant sera to differentiate primary from recurrent infection. IENAD detected or excluded CMV shedding for more urine specimens than virus isolation (462/515 [90%] vs. 366/515 [71%]), but the reverse applied to saliva specimens (33/57 [58%] vs. 54/57 [95%]). The high specificity of IENAD allowed positive results by IENAD to be accepted when virus isolation failed to provide a result. IENAD was, however, less sensitive than virus isolation even when specimens yielding CMV by IENAD, but no result by virus isolation, were included in the analysis (27/44 [61%] vs. 38/44 [86%]). Assays of both antibody response and virus shedding were required to maximise the diagnosis of recurrent CMV infections, but the former assay detected all primary CMV infections. The diagnostic implications of these results are discussed.

Antibodies, Viral↗
PubMed1990

Diagnosis of herpes encephalitis via Southern blotting of cerebrospinal fluid DNA amplified by polymerase chain reaction.

Herpes simplex virus thymidine kinase gene specific polymerase chain reaction (PCR) amplification of DNA extracted from lumbar cerebrospinal fluid (CSF) and Southern blotting (SB) were evaluated as a method for the diagnosis of herpes simplex encephalitis (HSE). Positive PCR-SB results were obtained with CSF samples from 9 of 10 patients (11 of 12 CSF specimens) with proven herpes encephalitis as early as 2 days after onset of neurological illness. Our data support the suggestion that PCR techniques may provide a clinically relevant "non-invasive" method for the diagnosis of HSE.

Base Sequence↗