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PubMed2003

Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus.

Automated fluorogenic (5' nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or clotted blood, milk and oesophageal-pharyngeal fluid ('probang') samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak serotype O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12 h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probang and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture. The combined tests of ELISA, virus isolation in cell culture and RT-PCR might, therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then either ELISA plus RT-PCR or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory diagnostic test results.

Animals↗
PubMed2004

Laboratory diagnosis of SARS.

The virologic test results of 415 patients with severe acute respiratory syndrome (SARS) were examined. The peak detection rate for SARS-associated coronavirus occurred at week 2 after illness onset for respiratory specimens, at weeks 2 to 3 for stool or rectal swab specimens, and at week 4 for urine specimens. The latest stool sample that was positive by reverse transcription-polymerase chain reaction (RT-PCR) was collected on day 75 while the patient was receiving intensive care. Tracheal aspirate and stool samples had a higher diagnostic yield (RT-PCR average positive rate for first 2 weeks: 66.7% and 56.5%, respectively). Pooled throat and nasal swabs, rectal swab, nasal swab, throat swab, and nasopharyngeal aspirate specimens provided a moderate yield (29.7%-40.0%), whereas throat washing and urine specimens showed a lower yield (17.3% and 4.5%). The collection procedures for stool and pooled nasal and throat swab specimens were the least likely to transmit infection, and the combination gave the highest yield for coronavirus detection by RT-PCR. Positive virologic test results in patient groups were associated with mechanical ventilation or death (p < 0.001), suggesting a correlation between viral load and disease severity.

Adolescent↗
PubMed2005

Laboratory diagnosis in ulcerative keratitis.

AIMS: To identify the common bacterial and fungal isolates from corneal ulcers and to determine the antimicrobial susceptibility patterns of bacterial isolates to commonly used antibiotics at B.P. Koirala Institute of Health Sciences (BPKIHS), eastern Nepal. Culture and direct microscopic correlation and reliability were also compared. METHODS: All patients with suspected corneal ulceration presenting to the Ophthalmology Department of BPKIHS from 1st August 1998 to 31st July 2001 were evaluated. Corneal scraping was performed and processed for direct microscopy and culture for bacterial and fungal isolates. Bacterial isolates were subjected to antimicrobial susceptibility testing. RESULTS: Of 447 specimens examined direct microscopy was positive in 216 (48%) specimens. Culture positivity could be correlated with direct microscopy in 179 (83%) of specimens. Growth of etiologic agents was found in 303 (67.8%) samples. Of these 145 (47.8%) had pure fungal growth, 103 (34%) had pure bacterial growth and 55 (18.2%) had mixed fungal and bacterial infection. The commonest fungal pathogen was Aspergillus spp.in 78 (38.4%) followed by Fusarium spp. in 45 (22%). Aureobasidium sp. was isolated in 25 (12.3%) samples. Staphylococcus aureus (93, 56.7%) dominated the scene as the commonest bacterial agent. Streptococcus pneumoniae (33, 20%) was second in the list. Most of the bacterial isolates were sensitive to commonly used antibiotics. CONCLUSION: This study emphasizes the importance and need of the continued surveillance of the agents and their antimicrobial susceptibility for the prevention and management of corneal ulcers and their complications.

Adolescent↗
PubMed2002

Routine use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections.

Real-time quantitative polymerase chain reaction (PCR) on the LightCycler instrument (LC-PCR) was developed to measure the Epstein-Barr virus (EBV) load in clinical samples. LC-PCR detected two copies of the EBV genome per 500 ng of DNA. Its specificity was confirmed by assays in EBV-negative cell lines, other human herpesviruses and EBV-seronegative individuals. Excellent inter-assay reproducibility of LC-PCR was obtained in 43 samples (r = 0.983). LC-PCR results were compared with a routinely used ELISA-PCR of 150 samples and a good correlation was found (r = 0.956). A total of 88 individuals were studied, including healthy EBV-seropositive adults (n = 32), patients with EBV-associated disease (n = 34), and HIV-infected patients (n = 22); 37.5% of PBMC samples from healthy individuals contained EBV DNA, while no serum sample was positive. The viral load was significantly higher in PBMCS and saliva specimens in patients recently infected with HIV (19 and 39,400 copies/microg DNA, respectively), as well as in AIDS patients (122 and 331,130 copies/microg DNA) than in the control population (0 and 35 copies/microg DNA). This study confirmed that EBV load measurement with LC-PCR is helpful in the management of EBV-related post-transplantation lymphoproliferative disorders and probably of EBV-associated primary central nervous system B-cell lymphoma.

AIDS-Related Opportunistic Infections↗
PubMed2002

Laboratory diagnosis of lupus anticoagulants for patients on oral anticoagulant treatment. Performance of dilute Russell viper venom test and silica clotting time in comparison with Staclot LA.

The detection of lupus anticoagulant (LA) in plasmas from patients on oral anticoagulants is problematic because of their prolonged clotting times. Mixing of patients and normal plasmas prior to testing for LA is employed to overcome this problem. We investigated the diagnostic efficacy of silica clotting time (SCT) and dilute Russell viper venom test (dRVVT) performed at low and high phospholipid concentrations, to diagnose LA in patients on oral anticoagulants, in comparison with Staclot LA (Stago) performed with and without hexagonal phospholipids and normal plasma. Case materials were 114 filtered plasmas from patients on oral anticoagulants with (n = 62) and without (n = 52) the antiphospholipid syndrome. Plasmas were considered LA-positive when Staclot LA (taken as the "gold standard") was diagnostic for LA. Forty-four plasmas were positive with Staclot LA. Forty and 39 of these were also positive with SCT and dRVVT (sensitivity relative to Staclot LA was 91% and 89%, respectively). Seventy plasmas were negative with Staclot LA. Three of these were positive with both SCT and dRVVT (specificity relative to Staclot LA was 96%). Kappa values for measure of agreement were 0.87 and 0.85 (p <0.001), respectively. In conclusion, SCT and dRVVT performed at low and high phospholipid concentrations without normal plasma can be considered as reliable as Staclot LA performed with hexagonal phospholipids and normal plasma to diagnose LA in patients on oral anticoagulants. Advantages of SCT and Drvvt over Staclot LA are easy automation, no need for normal plasma and relatively low cost.

Administration, Oral↗
PubMed1994

Laboratory diagnosis of APC-resistance: a critical evaluation of the test and the development of diagnostic criteria.

The APC-resistance test consists of two APTT's, one in the presence and one in the absence of a fixed amount of Activated Protein C (APC), and is a simple and reliable method to detect a reduced sensitivity to the anticoagulant action of APC (APC-resistance). At a fixed concentration of APC the prolongation of the APTT is dependent on the activator, the CaCl2 concentration, the citrate concentration in the sample, and on sample handling. The effect of sample handling can be reduced by calculating the APC-Sensitivity Ratio (APC-SR). The actual prolongation of the APTT is also influenced by low protein S levels (reduction of APC-SR) and by reduced levels of factors V, VIII and IX (increase of APC-SR). The APC-SR is most dramatically effected by reduced levels of factors II and X, which result often in "unmeasurable" APC-SR's in plasmas of patients on oral anticoagulant treatment. So at present no reliable APC-SR's can be measured in these patients. Patients treated with heparin can be tested after treatment of their plasma with Hepzym. The inter- and intra-assay variation in the APC-SR is 4% and 2%, respectively, when using the same batches of activator and APC. The variation which is introduced in the APC-SR by use of different batches of activator or APC, or by the use of different APC or CaCl2 concentrations, can effectively be avoided by expressing the result of the test in normalized-APC-SR (n-APC-SR).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2001

[In vitro interferon-gamma release--a laboratory diagnosis of cutaneous adverse drug reactions].

Diagnosis of cutaneous adverse drug reactions is an accepted terminology. Is a challenge. Drug-specific T-cell clones (CD4+ or CD8+), with a Th1- or a Th2-type cytokine-release pattern, may be generated from the peripheral blood in CADRs. In vitro drug-induced cytokine-release suggests a drug-specific immune response, and may implicate the drug as a possible inducer of the skin reaction. We evaluated the diagnostic role of in vitro drug-induced interferon-gamma (IFN-gamma) release from peripheral blood lymphocytes in patients with CADRs. We studied 22 patients with CADRs following intake of 45 drugs (1-4 drugs per patient). Drugs were classified into 3 categories of suspicion. 17 patients who took 39 drugs of the same type (1-4 drugs per patient) without developing adverse reactions, served as controls. In vitro drug-induced release of IFN-à from peripheral blood lymphocytes, following in vitro challenge with the unmodified drugs, was evaluated. The mean IFN-gamma increase following 45 drug tests (60.8 +/- 85.2%) was higher (p < 0.05) than in controls after 39 drug tests (30.1 +/- 27.7%). Significance was greater (p < 0.005) when the mean IFN-gamma increase for the 24 highly suspected drugs (75.1 +/- 93.4%) and that for the controls were compared. This study suggests that the in vitro drug-induced IFN-gamma release test may serve as a diagnostic tool in CADRs.

Cells, Cultured↗
PubMed1999

Laboratory diagnosis of heparin-induced thrombocytopenia.

The characteristics of the currently available platelet function assays (platelet aggregation, serotonin release, and flow cytometry) and enzyme-linked immunosorbent assays that quantitate antiheparin-platelet factor 4 antibody titers were studied using sera collected from clinically diagnosed heparin-induced thrombocytopenia patients, patients without heparin-induced thrombocytopenia, patients with platelet immune disorders other than heparin-induced thrombocytopenia, and normal individuals. The platelet aggregation assay was less sensitive than the serotonin release assay, which was less sensitive than the enzyme-linked immunosorbent assay (p < 0.001). Yet heparin-induced thrombocytopenia was identified by platelet aggregation assay in cases where the serotonin release assay and/or the enzyme-linked immunosorbent assay were negative. Patients with heparin-induced thrombocytopenia and thrombosis were more often positive than heparin-induced thrombocytopenia patients without thrombosis (p < 0.05). Positive platelet aggregation assay and serotonin release assay results were generally associated with a higher antibody titer; however, a minimum critical titer could not be identified. Over a 30-day period the percentage of positive responses did not change significantly even though clinical symptoms corrected in most heparin-induced thrombocytopenia patients. Multiple testing over several days enhanced the chance of detecting a positive, and combined results of the three assays further enhanced the positive response (p < 0.005). In patients without heparin-induced thrombocytopenia, false-positive results were obtained with the enzyme-linked immunosorbent assay. These data demonstrate that there is no direct correlation between the positive responses of these assays, that clinically positive patients can be missed by all assays, and the presence of antibody alone does not determine clinical heparin-induced thrombocytopenia. With these limitations, the combination of aggregation, serotonin release, and enzyme-linked immunosorbent assay testing with multiple samples offers the best chance of identifying a positive heparin-induced thrombocytopenia patient. Caution is advised for all assays as none is optimal.

Anticoagulants↗
PubMed1992

Laboratory diagnosis of parvovirus B19 infection.

The sensitivity and application of the polymerase chain reaction (PCR) for the diagnosis of parvovirus B19 (B19) infection was investigated by simultaneously assaying a collection of 279 consecutively received samples for presence of anti-B19 IgM and IgG antibodies by Western blot and for B19 DNA by PCR and dot-blot hybridization (dot-blot); samples were sera from patients with suspected B19 infection. PCR and dot-blot detected B19 DNA in 9% (16/179) and 1% (2/179), respectively of Ab-positive samples (IgM+/IgG-, IgM+IgG+, IgM-IgG+), and in 28% (15/54) and 2% (1/54), respectively, of IgM+ samples. PCR also detected B19 DNA in 2% (2/100) of IgM-/IgG- samples, both of which had normal total IgG and IgM levels. PCR is of unique value because it permits diagnosis of B19 infection even in the absence of specific acute phase (IgM) and in the presence or absence of convalescent-phase (IgG) Ab.

Antibodies, Viral↗
PubMed1989

Laboratory diagnosis of Japanese encephalitis using monoclonal antibodies and correlation of findings with the outcome.

Detection of virus, viral antigen, and class-specific antibody was carried out in cerebrospinal fluid (CSF) and sera of 27 children with Japanese encephalitis. The diagnosis could be confirmed in 78.57% of cases (22/27) by demonstration of virus-specific IgM in CSF (15/22), viral antigen in CSF (5/22), or by virus isolation (2/22). Absence of virus specific IgM in CSF was associated with a fatal outcome (P = 0.05).

Adolescent↗
PubMed2005

Laboratory diagnosis and surveillance of human respiratory viruses by PCR in Victoria, Australia, 2002-2003.

Respiratory viruses were identified by the polymerase chain reaction (PCR) in more than 4,200 specimens collected during 2002 and 2003 in Victoria, Australia from patients admitted to hospitals or participating in an influenza surveillance program. Influenza viruses and picornaviruses were important causes of morbidity in both years. Additional testing of picornavirus-positive samples suggested that rhinoviruses but not enteroviruses were more likely to be associated with respiratory symptoms, irrespective of the season in which they circulated. Detection of influenza viruses was strongly associated with the clinical symptoms of cough, fever, and fatigue; but each of the other respiratory viruses occasionally caused these symptoms or was responsible for symptoms severe enough to require hospitalization. Human coronaviruses HCoV-OC43 and HCoV-229E circulated at low levels throughout the study period with peak activity in winter, but overall did not circulate as widely as has often been reported for these agents. Evidence for the human metapneumovirus (hMPV) was only sought in the second year of the study and revealed low-level circulation of this virus, mainly in the cooler months among the very young and adult populations. The detection rate of all viruses declined with increasing age of the patient, particularly in hospital patients. Infection with more than one respiratory virus occurred in a small number of patients; picornaviruses were most commonly implicated in these dual infections.

Age Distribution↗