Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens® Basic Kit

Fox, Julie D.; Han, Song; Samuelson, Agneta; Zhang, Yadan; Neale, M L; Westmoreland, D.

doi:10.1016/s1386-6532(01)00241-4

Cited by 78 publications

(38 citation statements)

References 23 publications

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“…It is possible that use of three enzymes in the EV NASBA, instead of one or two in RT-PCR, may make NASBA more susceptible to inhibitors. A recent publication on EV NASBA did not report invalid results, but an IC was not used, and thus inhibition of amplification may not have been recognized (3). It is also possible that the invalid rate could be reduced with an automatic extraction procedure.…”

Section: Discussionmentioning

confidence: 99%

Rapid Enterovirus RNA Detection in Clinical Specimens by Using Nucleic Acid Sequence-Based Amplification

2003

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Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.Enteroviruses (EV) are a diverse group of small, nonenveloped RNA viruses that are transmitted largely by the fecal-oral route, that replicate in high titer in the enteric tract, and that are carried by the blood to target organs. EV meningitis is a common infection in the United States and usually has a benign outcome. Nevertheless, it leads to a large number of hospitalizations of both children and adults. In addition to meningitis, other EV neurological manifestations include encephalitis, poliomyelitis, Guillain-Barré syndrome, transverse myelitis, cerebellar ataxia, peripheral neuritis, and Reye's syndrome. Other serious EV infections that can lead to hospitalization include those causing neonatal sepsis, myocarditis, and pericarditis and those in immunocompromised hosts. The ability to rapidly differentiate EV infections from bacterial illness can reduce hospitalization time, antimicrobial usage, and diagnostic tests (9), as well as reduce the anxiety of patients and families. Furthermore, specific antiviral agents are becoming available (11).Currently over 60 distinct EV serotypes, which differ antigenically and in their abilities to grow in various cell cultures and suckling mice, are recognized. The mainstay of EV diagnosis in diagnostic laboratories has been isolation of virus in cell culture. However, many coxsackievirus group A serotypes grow poorly or not at all in cell culture and require suckling mouse inoculation (4). Successful isolation of multiple EV serotypes increases with the number of cell lines inoculated (2, 4, 5, 7). Identification of cytopathic effect (CPE) due to an EV necessitates subpassage in culture, staining of infected cultures with pools of EV monoclonal antibodies, and/or neutralization of infectivity with type-specific antiserum. Time to detection of CPE is usually 2 to 7 days.In contrast, nucleic acid amplification techniques can provide results within 1 day, can detect serotypes that grow poorly in cell culture, and can significantly alter the medical care offered to patients (9, 10). The target for amplification is within the highly conserved 5Ј untranslated region in order to detect the broadest spectrum of EV serotypes. Most publications have focused on reverse transcription PCR (RT-PCR), including a commercial Amplicor kit from Roche Molecular Systems (8-10, 12, 14). However, the Amplicor kit is no longer available, and clinical laboratories must find other alternatives.Recently, nucleic acid sequence-based amplification (NASBA) has been used for rapid diagnosis of EV (3; F. Zhang, C. C. Ginocchio, A. Malhotra, and C. Chakrabarti, Abstr. 18th Annu. Clin. Virol. Symp., abstr. T5, 2002). In this study, we have compared NASBA with isolati...

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Section: Discussionmentioning

confidence: 99%

Rapid Enterovirus RNA Detection in Clinical Specimens by Using Nucleic Acid Sequence-Based Amplification

2003

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“…Rapid diagnostic tests have been developed in recent years and are usually based on nucleic acid amplification technology, such as reverse transcription-PCR and nucleic acid sequencebased amplification (3,7,9,12,20,24,31). These assays have proven to be sensitive and specific but, unfortunately, are often in a (semi-)nested format or require specific detection methods, which poses serious hazards for amplification product carryover.…”

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confidence: 99%

Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR

et al. 2002

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We developed a rapid and sensitive method for the routine detection of all members of the enterovirus genus in different clinical specimens by using real-time TaqMan quantitative PCR. Multiple primer and probe sets were selected in the highly conserved 5-untranslated region of the enterovirus genome. Our assay detected all 60 different enterovirus species tested, whereas no reactivity was observed with the viruses from the other genera of the picornaviridae family, e.g., hepatovirus and parechovirus. Weak cross-reactivity was observed with 7 of the 90 different high-titer rhinovirus stocks but not with rhinovirus-positive clinical isolates. Analysis of a well-characterized reference panel containing different enteroviruses at various concentrations demonstrated that the enterovirus real-time TaqMan PCR is as sensitive as most of the currently used molecular detection assays. Evaluation of clinical isolates demonstrated that the assay is more sensitive than the "gold standard" method, i.e., viral culture. Moreover, the PCR assay can be used on different clinical specimens, such as plasma, serum, nose and throat swabs, cerebrospinal fluid, and bronchoalveolar lavage, without apparent inhibition. Our data demonstrate that the real-time TaqMan PCR is a rapid and sensitive assay for the detection of enterovirus infection. The assay has a robust character and is easily standardized, which makes it an excellent alternative for the conventional time-consuming viral culture.

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“…NASBA with detection by ECL-labeled probes is a highly sensitive and specific amplification method for enteroviruses and other RNA targets (2,6,8,15). In this study, we compared NASBA-ECL with real-time NASBA using molecular beacons and found excellent agreement.…”

Section: Discussionmentioning

confidence: 74%

“…The impact on clinical decisionmaking as well as overall health care costs can be substantial (11,14). Although reverse transcription-PCR (RT-PCR) is the most commonly used test, nucleic acid sequence-based amplification (NASBA) using the Nuclisens Basic Kit has proved of equal or greater sensitivity for detection of enteroviruses (2,6,7).…”

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confidence: 99%

Real-Time Nucleic Acid Sequence-Based Amplification Using Molecular Beacons for Detection of Enterovirus RNA in Clinical Specimens

2005

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Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBAbeacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays were significantly more sensitive than culture. Real-time NASBA-beacon reagents and equipment rental were more expensive than those for NASBA-ECL; however, time to result was shortened by 1.5 h, hands-on time was reduced by 25 min, and the assay was much simpler for technologists to learn and perform.The detection of enteroviruses (EV) in clinical specimens, particularly spinal fluid, by molecular amplification techniques has important advantages over cell culture, namely, greater sensitivity and faster results. The impact on clinical decisionmaking as well as overall health care costs can be substantial (11,14). Although reverse transcription-PCR (RT-PCR) is the most commonly used test, nucleic acid sequence-based amplification (NASBA) using the Nuclisens Basic Kit has proved of equal or greater sensitivity for detection of enteroviruses (2, 6, 7).In the Nuclisens Basic Kit, amplified RNA products are detected by hybridization using electrochemiluminscently (ECL) labeled probes, a highly sensitive methodology. Recently, real-time RT-PCR using TaqMan chemistries has been applied to further shorten both technical hands-on time and time to result (3, 10). In this report, we compare the Nuclisens EasyQ Enterovirus Test, which utilizes real-time molecular beacons as probes (NASBA-beacon), to our standard NASBA with ECL probes (NASBA-ECL) for detection of EV in clinical specimens. MATERIALS AND METHODSSamples and patients. Samples submitted to the Clinical Virology Laboratory at Yale New Haven Hospital (YNHH) for EV diagnosis were used. For the retrospective study, RNA extracts from 53 samples stored at Ϫ70°C for 1 to 8 months were used. For the prospective study, 107 samples submitted for EV diagnosis from mid-May through August 2004 were tested within 0 to 3 days by NASBA with ECL detection as the standard clinical test. The RNA extracts were then retested by real-time NASBA-beacon (Nuclisens EasyQ Enterovirus Test) within 0 to 10 days. Extracts not tested the same day were stored at Ϫ70°C.The retrospective study included 53 cerebrospinal fluid (CSF) samples from 53 patients. The prospective study included 107 samples from 107 patients: 80 CSF and 27 stool samples. The stool samples were submitted as part of an epidemiologic investigation of an outbreak of meningitis, nausea, and vomiting in a church youth group returning from Mexico. Spinal fluid and stool samples were submitted in sterile containers. Patients' ages ranged from 5 days to 93 years, and 34 percent were over 18 years of age.Virus isolation. Viral cultures on CSF we...

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Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens® Basic Kit

Cited by 78 publications

References 23 publications

Rapid Enterovirus RNA Detection in Clinical Specimens by Using Nucleic Acid Sequence-Based Amplification

Rapid Enterovirus RNA Detection in Clinical Specimens by Using Nucleic Acid Sequence-Based Amplification

Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR

Real-Time Nucleic Acid Sequence-Based Amplification Using Molecular Beacons for Detection of Enterovirus RNA in Clinical Specimens

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