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...

, and virus isolation. Eighty-two samples were tested, and 44 were positive, 34 by both NASBA and RT-PCR and 5 each by NASBA or RT-PCR only. Two nasopharyngeal samples positive only by RT-PCR were determined to be rhinovirus. Of 42 enterovirus-positive samples, NASBA detected 39 (92.9%) and RT-PCR detected 37 (88.1%). The NucliSens Basic kit and the Argene Biosoft RT-PCR had comparable sensitivities for detection of enterovirus RNA, and both molecular methods were more sensitive than culture, which detected only 60.5% of positive samples. NASBA could be completed in 6.5 h versus 9 h for the Argene Biosoft RT-PCR kit.Enterovirus (EV) infections are extremely common, with an estimated 10 to 30 million infections occurring annually in the United States alone (15). While most infections either are asymptomatic or result in minor illnesses, aseptic meningitis and neonatal sepsis syndrome bring many patients to the hospital (16-18).For the past 50 years, the mainstay of EV diagnosis has been virus isolation in cell culture (5). The optimal cell culture systems for the isolation of more than 60 recognized EV serotypes differ. To increase recovery, ideally five different cell systems should be employed (2, 5, 7). Use of E-mix cells has been reported to reduce the number of tubes inoculated and to increase recovery (1). However, the turnaround time for cell culture is usually 2 to 7 days for positive results and 10 to 14 days for negative reports. Lastly, many coxsackie group A serotypes require suckling mouse inoculation (5).Nucleic acid amplification techniques can detect most serotypes, including those that grow poorly or not at all in cell culture, can provide results within 24 h, and, consequently, can significantly alter patient management (13,14,20). Molecular methods also require a smaller volume of cerebrospinal fluid (CSF) than comprehensive culture, an important advantage. To inoculate five cell culture systems, 1 ml of CSF is needed, but this amount is often not available. In contrast, only 0.2 ml is needed for nucleic acid extraction. In addition, other pathogens that cause viral meningitis, such as herpes simplex virus (HSV) type 2, can also be detected by using the same nucleic acid extract.In the 1990s, several studies were published validating the AMPLICOR reverse transcription-PCR (RT-PCR) enterovirus kit from Roche (12,15,19,22). Subsequently, however, the AMPLICOR kit was withdrawn from the market. More recently, studies using real-time PCR for EV diagnosis have been reported (10, 21). However, many smaller laboratories may not have access to expensive real-time PCR equipment and may lack the expertise needed to develop molecular techniques in-house. Instead, they rely on commercial kits to bring molecular technology to the patients they serve.We and others have recently reported on nucleic acid sequence-based amplification (NASBA) using the NucliSens Basic kit for the diagnosis of EVs (3, 9; F. Zhang, C. C. Ginocchio, A. Malhotra, and C. Chakrabarti, presented at the 18th Annual Clinical Virology Sym...

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...

The Amplicor HIV-1 Monitor test was compared to the nucleic acid sequence-based amplification (Nasba) assay system for the quantitation of human immunodeficiency virus (HIV) RNA in three different types of clinical samples: plasma, serum, and plasma subjected to freeze-and-thaw cycles. Each assay detected HIV RNA in the same 73 (90%) of 81 samples tested, and the quantitative results obtained with the two assays were significantly correlated. Both assays detected higher RNA levels in patients with CD4 ؉ cell counts lower than 200 cells/mm 3 than in patients with CD4 ؉ cell counts higher than 200 cells/mm 3. In addition, RNA levels in plasma higher than 5 logs predicted higher numbers of clinical events than did RNA levels in plasma lower than 5 logs. Quantitation of HIV RNA in paired plasma and serum samples showed lower HIV RNA content in serum than in the paired plasma sample, with mean differences between HIV RNA contents of plasma and serum of 0.54 and 0.28 log RNA copy/ml by the Nasba assay and the Amplicor HIV-1 Monitor assay, respectively. No significant loss of HIV RNA was detected with either assay in plasma samples subjected to multiple freeze-and-thaw cycles. These studies demonstrate that the Nasba and Amplicor assays perform similarly with plasma and serum samples. Further, the results indicate that freeze-and-thaw cycles do not result in significant loss of detectable HIV RNA.

The antiviral effect of stavudine (2', 3'-didehydro-3'-deoxythymidine) against human immunodeficiency virus (HIV) type 1 was measured in 15 HIV-infected patients at baseline and at weeks 4, 10, 22, 34, and 52 of therapy. Patients received 0.1, 0.5, 1.0, or 2.0 mg/kg/day of stavudine. At all time points examined during the 52 weeks of therapy, the median virus titers in peripheral blood mononuclear cells were decreased 1-2 logs, and median immune complex-dissociated antigen levels were reduced 37%-67% compared with baseline values. Plasma RNA content measured by polymerase chain reaction was reduced approximately 0.5 log from baseline median values at both time points examined (weeks 10 and 52). These data demonstrate that stavudine has a substantial and durable antiviral effect.