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At least 19 recordsLinked to original sources
PubMed2006

Detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-PCR, in children with acute respiratory infections during a winter season.

Five hundred seventeen consecutive nasopharyngeal aspirates were collected between October 1998 and May 1999 for episodes of acute respiratory tract infections in children presenting at the University Hospital of Antwerp. Culture and nucleic acid amplification techniques--nucleic acid sequence-based amplification (NASBA) and reverse transcription-PCR (RT-PCR)--were applied to detect rhinoviruses (RVs). Other respiratory viruses were detected by immunofluorescence (IF) analysis of the specimens and IF analysis of shell vial cultures. Among the 517 specimens, 219 viral agents were identified. They were, in decreasing order, rhinoviruses (93 [18.0%]), respiratory syncytial virus (76 [14.7%]), adenoviruses (16 [3.1%]), influenza viruses (15 [2.9%]), enteroviruses (15 [2.9%]), and herpes simplex virus (4 [0.8%]). For the evaluation of rhinovirus detection, culture positivity and/or a positive reaction in the two independent amplification methods was used as an expanded "gold standard." Based on this standard, the sensitivity, specificity, positive predictive value, and negative predictive value of culture were 44.7, 100, 100, and 99.8%, and those of NASBA and RT-PCR were 85.1, 98.3, 83.3, and 98.5% and 82.9, 93.4, 55.7, and 98.2%, respectively. NASBA and RT-PCR produced comparable results and were significantly more sensitive than virus culture. RVs showed the highest incidence in acute respiratory tract infections in children.

Acute Disease↗
PubMed2003

Calibration of HIV-1 working reagents for nucleic acid amplification techniques against the 1st international standard for HIV-1 RNA.

Many laboratories use working reagents/run controls to monitor the performance of their nucleic acid amplification techniques (NAT) for the measurement of HIV-1 RNA. A collaborative study was carried out in order to calibrate seven internationally available working reagents, QC105 (National Serology Reference Laboratory [NRL], Australia), B5 and B10 (Center for Biological Evaluation and Research [CBER], USA), Pelispy (Central Laboratory of the Netherlands Blood Transfusion Service [CLB], The Netherlands), PWS-1 and PWS-3 (National Institute for Biological Standards and Control [NIBSC], UK) and IRC (Virology Networks [VN], The Netherlands) against the 1st International Standard for HIV-1 RNA (code 97/656). Twenty-one laboratories from 12 different countries participated in the collaborative study and from the results it was determined that QC105 contained 4.0 log(10) International Units (IU)/ml, B5 2.2 log(10) IU/ml, B10 3.8 log(10) IU/ml, Pelispy 4.4 log(10) IU/ml, PWS-1 3.6 log(10) IU/ml, PWS-3 2.7 log(10) IU/ml and IRC 4.3 log(10) IU/ml. The seven working reagents calibrated in this international study may be used to validate and standardise the large number of qualitative and quantitative, commercial and in-house NAT assays that are currently being applied in the fields of blood safety and patient management. They will also help laboratories to comply with the sensitivity requirements that may be brought in by the regulatory authorities and may contribute to further harmonisation of guidelines on NAT published by organisations such as the European Medicines Evaluation Agency (EMEA), Paul-Ehrlich Institute and CBER, FDA.

Calibration↗
PubMed1997

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.

Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for excluding false-positive and false-negative results.

Bacterial Infections↗
PubMed2005

Enhanced enzyme immunoassay with negative-gray-zone testing compared to a single nucleic Acid amplification technique for community-based chlamydial screening of men.

We evaluated a low-cost diagnostic strategy for detecting Chlamydia trachomatis in a low-prevalence population. We used an amplified enzyme immunoassay (EIA) with a reduced-cutoff "negative gray zone" to identify reactive specimens for confirmation by a nucleic acid amplification test. As part of the Chlamydia Screening Studies project, men provided a first-pass urine specimen, which they returned by post for testing. We tested 1,003 specimens by IDEIA PCE EIA (Dako) and Cobas PCR (Roche). There were 32 (3.2%) true positive specimens according to a combined standard using an algorithm requiring concordant results from at least two independent tests. All of these were positive by Cobas PCR and 24 were confirmed to be positive by PCE EIA, including 2 that gave results in the negative gray zone. There were 971 true negative specimens, 2 of which were positive by Cobas PCR and 19 of which were initially inhibitory for PCR. The relative sensitivity, specificity, positive predictive value, and negative predictive value of PCE EIA with PCR confirmation were 75.0% (95% confidence interval [CI], 56.6 to 88.5%), 100% (95% CI, 99.7 to 100%), 100% (95% CI, 88.3 to 100%), and 99.2% (95% CI, 98.4 to 99.6%), respectively. The corresponding values for Cobas PCR were 100% (95% CI, 89.1 to 100%), 99.8% (95% CI, 99.3 to 100%), 94.1% (95% CI, 76.9 to 98.2%), and 100% (95% CI, 99.6 to 100%), respectively, with 1.9% (19/1003) of the samples being initially indeterminate. When the prevalence of C. trachomatis is low, the use of an amplified EIA on urine specimens, with confirmation of results in the negative gray zone by use of a nucleic acid amplification technique, is not suitable for screening asymptomatic men. In addition, positive nucleic acid amplification test results should be confirmed and an inhibition control should be used.

Algorithms↗
PubMed2004

Collaborative study for establishment of a European Pharmacopoeia Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150 per cent of the estimated potency was determined. For the quantitative assay the semi-weighted combination of the 5 potency estimates lead to a combined potency of 5.83 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.04 log( 10) which corresponds to 91 to 110 per cent of the estimated potency. The semi-weighted combination of the results from both types of assay lead to a final potency estimate of 5.80 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.05 log( 10) which corresponds to 86 to 117 per cent of the estimated potency. A threshold control suitable for quantitative testing of B19 DNA and reflecting the requirements of the European Pharmacopoeia monograph Human anti-D immunoglobulin should contain 104 IU/ml of B19 DNA. Taking into account the assigned titre of 5.80 log( 10) IU/ml (630.957 IU/ml), a dilution of 10(-1.8) (1/63) of the candidate BRP should yield a positive response. This concentration was easily detected by all participating laboratories in all tests. The candidate BRP thus appeared to be suitable for the intended purpose. The candidate BRP was adopted as the European Pharmacopoeia BRP by the European Pharmacopoeia Commission at its session in June 2003 and is available from the European Directorate for the Quality of Medicines (Catalogue Number: Y0000285).

DNA, Viral↗
PubMed2001

An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques.

BACKGROUND AND OBJECTIVES: Twenty-two laboratories from nine countries participated in an international collaborative study to establish a World Health Organization (WHO) international standard for hepatitis B virus (HBV) DNA nucleic acid amplification techniques (NAT). MATERIALS AND METHODS: Three samples, AA, BB (both of which were lyophilized) and CC (which was a liquid preparation), were analysed using several different NAT assays. The mean HBV DNA content of each sample was determined from the study. RESULTS: Despite the range of assays (commercial and in-house) used by participants, there was good agreement among the overall mean 'equivalents'/ml obtained by the different assays, except for one laboratory (laboratory 4). The variation in estimates of log10 'equivalents'/ml was 1.75-1.25 for the three samples if results from laboratory 4 were excluded. The mean log10 'equivalents'/ml for all laboratories were 6.42 for sample AA, 6.30 for sample BB and 5.03 for sample CC (exclusion of results from laboratory 4 made little difference). The difference in titres between the two lyophilized samples (AA and BB) was not statistically significant but the titre of the frozen sample (CC) was significantly lower. Material AA (code 97/746) was accepted as the first WHO international standard for HBV DNA NAT assays and assigned a potency of 10(6) international units (IU)/ml. CONCLUSIONS: The titres (genome equivalents/ml) of three HBV preparations were determined by several laboratories using different NAT assays. This study enabled the establishment of an international standard, 97/746, for HBV DNA NAT assays.

DNA, Viral↗
PubMed2002

Establishment of the first World Health Organization International Standard for human parvovirus B19 DNA nucleic acid amplification techniques.

BACKGROUND AND OBJECTIVES: A collaborative study, involving 26 laboratories from 14 countries, was carried out in order to establish a World Health Organization (WHO) International Standard for human parvovirus B19 (B19) DNA nucleic acid amplification techniques (NAT). MATERIALS AND METHODS: Four samples: AA, BB (which were lyophilized), CC and DD (which were liquid preparations) were analysed using several different NAT assays. The mean B19 DNA content of each sample was determined for each laboratory using an end-point dilution method. RESULTS: There was good agreement between the overall mean 'equivalents'/ml obtained by the different assays. The mean log(10) 'equivalents'/ml were 5.76 for sample AA, 5.73 for sample BB, 5.82 for sample CC and 7.70 for sample DD. The differences in titre among samples AA, BB and CC were not statistically significant, but the titre of DD was significantly higher. CONCLUSIONS: Despite the range of NAT assays used in the study, it was possible to calculate the mean B19 DNA concentrations in the four preparations. Lyophilized preparation AA was established as the first International Standard for B19 DNA NAT assays and was assigned a concentration of 10(6) international units (IU)/ml.

DNA, Viral↗
PubMed2004

Unexpected detection of DNA by nucleic acid sequence-based amplification technique.

Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective.

DNA, Bacterial↗
PubMed2001

A comparison of nucleic acid amplification techniques for the assessment of bacterial viability.

AIMS: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. METHODS AND RESULTS: In this study, we have assessed three amplification techniques, PCR, RT-PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat-killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.

Bacteria↗
PubMed2002

The use of nucleic acid amplification techniques to increase the viral safety of blood.

Despite recent significant improvements in the viral safety of blood and blood products there remains a small risk of contamination mainly due to the existence of a window period before the appearance of antibodies. Nucleic acid amplification technologies (NAT) permit a direct detection of the viral genome itself with an extreme sensitivity and specificity, without depending anymore on the delayed appearance of antibodies. These technologies can be applied to detect most blood-borne viruses. However, the usefulness and strategies will largely depend on different features specific to the type of the virus, such as pathogenicity, prevalence of the infection, viral load during preseroconversion, doubling time of the virus and infectious dose. Many studies have already been conducted in different parts of the world, and the results proving the feasibility of the NAT screening are more than encouraging. However, some problems still remain to be solved in theroutine application of these technologies.

Belgium↗
PubMed2001

Utility of nucleic acid amplification techniques for the diagnosis of pulmonary tuberculosis in sub-Saharan Africa.

SETTING: Lusaka, Zambia. OBJECTIVES: To investigate the utility of nucleic amplification tests for the diagnosis of pulmonary tuberculosis in a resource-poor setting with a high incidence of human immunodeficiency virus (HIV). DESIGN: Sputum specimens from suspects attending a referral chest clinic were examined by low-cost 'in-house' one-tube nested polymerase chain reaction (PCR), the enhanced Gen-Probe Amplified Mycobacterium Direct Test (AMTD), auramine smear and Lowenstein-Jensen culture. RESULTS: PCR and AMTD detected respectively 80% and 92% of smear-positive specimens and 40% and 60% of smear-negative, culture-positive specimens. AMTD was positive for 18 culture-negative suspects; subsequent investigation indicated these to be six confirmed tuberculosis patients, nine judged from radiological data and clinical follow-up studies to have pulmonary tuberculosis, and three non-tuberculosis patients. Sensitivity for smear, culture, PCR and AMTD, when compared to a gold standard incorporating both microbiological and clinical data, was respectively 29%, 69%, 55% and 81%. CONCLUSION: In this setting, the sensitivity of the low-cost PCR proved insufficient for its effective use as a tool for diagnosing pulmonary tuberculosis, while AMTD performed considerably better than the current laboratory methods for diagnosis of pulmonary tuberculosis. However, the high cost of this technology may limit its application in the public sector of low-income countries.

AIDS-Related Opportunistic Infections↗
PubMed2006

Confirmation of electron microscopy results by direct testing of viruses adhered to grids using nucleic acid amplification techniques.

It is possible to visualize rapidly viral particles by electron microscopy (EM) in patient samples and in cell cultures, and characterize the particles on the basis of their size and morphology. In many instances, EM has contributed to the diagnosis of specific infectious agents. Four different types of viruses with different characteristics of particle size, capsid structure, the presence or absence of an envelope, genomic content and stability outside the host were screened and diagnosed by EM at the level of family/genus. The results were confirmed at the species level by elution of the sample material from the grids used for EM examination and nucleic acid amplification. This approach could be valuable in situations where the immediate diagnosis is unclear, or when new infectious agents appear.

Adenoviridae↗
PubMed1999

An international collaborative study on the detection of an HIV-1 genotype B field isolate by nucleic acid amplification techniques.

An international collaborative study to assess inter-laboratory variation in the sensitivity of gene amplification assays for the detection of HIV-1 RNA sequences was conducted using a panel of eight duplicate dilutions of an HIV-1 genotype B clinical isolate and negative control samples. Twenty-five laboratories participated in the study and used a variety of in-house assays and commercial assay systems. With few exceptions, the assays were more sensitive than a p24 antigen assay. Overall, the PCR-based Amplicor Monitor assay was the most sensitive and gave the highest mean copy number for any one sample. Some of the in-house assays gave results comparable with the Monitor assay whilst the NASBA and bDNA assays appeared to be less sensitive. As a result of this study, an HIV-1 Working Reagent for the standardisation of nucleic acid amplification assays was developed and assessed in a subsequent study. Similar differences in sensitivity between the different assay systems was observed. The discrepancies in viral copy number obtained using the Working Reagent highlights the need for an International Standard against which all Working Reagents may be calibrated.

Adult↗
PubMed1995

Identification of the major North American bluetongue viruses using nucleic acid amplification techniques.

A set of primers (BTV-pr1/2) were selected that hybridized to the VP3 gene of the major North American serotypes of bluetongue virus (BTV). Polymerase chain reaction (PCR) testing yielded positive results from specimens of major North American BTV isolates (serotypes 10, 11, 13 and 17) propagated in Vero cells. In addition, PCR assays were positive from samples of all other BTV serotypes, except BTV-16; however, an alternative primer pair (BTV-prN1/N2) was devised for amplification of this serotype and the major North American BTV serotypes. PCR products were not evident following amplification of related viruses, epizootic haemorrhagic disease virus (EHDV) serotypes 1 or 2, in either PCR test. In addition, slight modification of the nucleic acid extraction method allowed for the amplification of BTV template from ovine and cervine blood, but not from the respective control blood samples. Restriction endonuclease analysis (REA) using AluI and TaqI discriminated the PCR products of BTV serotypes 10, 13 and 11/17. Identification of BTV-11 and -17 was accomplished by PCR product nucleotide sequencing. Thus, using a single gene region (VP3), nucleic acid amplification methods were devised for expeditious serogroup-specific detection of all BTV serotypes and identification of individual North American BTV nucleotypes, which is expected to prove valuable for disease control strategies and retrospective epidemiological analyses.

Base Sequence↗