A one-step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis

Stellrecht, Kathleen A.; Harding, Ian; Hussain, F.M.; Mishrik, Nada; Czap, R.T.; Lepow, Martha L.; Venezia, Richard A.

doi:10.1016/s1386-6532(00)00082-2

Cited by 33 publications

(24 citation statements)

References 14 publications

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“…PCR product detection. Ureaplasma and M. genitalium PCR products were detected by enzyme-linked oligosorbent assay (ELOSA) as previously described (38). Briefly, 5 l of heat-denatured biotinylated-PCR product was added to 100 l of hybridization solution containing 1 pmol of horseradish peroxidase-labeled probe specific for the target region (Table 1) (Synthetic Genetics), 7.5ϫ Denhardt solution, 3.5ϫ SSPE, and 0.2 mg of herring sperm DNA (Invitrogen Corp., Carlsbad, Calif.)/ml in streptavidin-coated microtiter plates (Roche Molecular, Indianapolis, Ind.).…”

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Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

et al. 2004

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Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.

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Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

et al. 2004

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“…A cytopathic effect (CPE) is not recognizable within a few days, and some EVs do not grow in cell culture and therefore do not cause a CPE at all (15,25). In recent years, several EV RT-PCR assays which are sensitive and rapid have been developed (8,9,14,25,26). In those previous studies, EV RNA was purified without an internal control (IC) RNA that monitored both RNA extraction efficiency and RT-PCR efficiency.…”

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Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

et al. 2004

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The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics ( The human enteroviruses (EVs) are members of the family Picornaviridae, are ubiquitous, and are mainly enterically transmitted. EVs have traditionally been identified by serotype-specific antisera in a virus-neutralizing test, and 66 EV types are known to infect humans (19). The 66 EV serotypes were initially recognized and divided into five major groups: polioviruses (PV; types 1 to 3), coxsackieviruses A (CVAs; types 1 to 22 and 24), coxsackieviruses B (CVBs; types 1 to 6), echoviruses (types 1 to 7, 9, 11 to 27, and 29 to 33), and EV types 68 to 71 (17). Recent molecular analyses have proved that echovirus types 22 and 23 are genetically distinct from the members of the genus Enterovirus and have been reclassified in a separate genus, Parechovirus, in the family Picornaviridae (13,20,23).Infections with EVs cause a wide range of clinical outcomes, such as asymptomatic infections, aseptic meningitis (meningeal inflammation in the absence of a bacterial pathogen), encephalitis, paralytic poliomyelitis, and myocarditis. Although the majority of EV infections do not cause significant disease, infection can cause serious illness, especially in infants and immune-compromised patients. EV infections are the most common cause of aseptic meningitis and account for 80 to 90% of all cases of central nervous system infections for which a possible causative agent is identified (24). In the neonate, aseptic meningitis-induced complications and poor outcomes of EV infections generally occur within the first 2 days of life (1, 2). Aseptic meningitis in immune-competent adults is characterized by sudden onset of fever, but neurological abnormalities are rare, and both short-term and long-term outcomes are generally good. Encephalitis caused by EV infections is a less common but a more severe disease than aseptic meningitis (18,29,30). Immune-compromised children and adults who are infected with EV may develop chronic meningitis and encephalitis, which may last for years before becoming fatal (16).The early clinical symptoms of meningitis caused by viruses, bacteria, and fungi are quite similar and are difficult to distinguish, but the diagnosis, therapy, and outcome of disease caused by these pathogens vary considerably. A reli...

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“…Whether CSF pleocytosis plays an important role in defining aseptic meningitis or in prioritizing the differential diagnosis has been investigated but is still not completely clear (7,14,17). In this era of rapidly rising health care costs, it is important, if possible, to limit laboratory-based testing by encouraging syndrome-directed testing and actively discouraging testing that is not consistent with the clinical picture or with other laboratory results.…”

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Correlation of Cerebrospinal Fluid (CSF) Cell Counts and Elevated CSF Protein Levels with Enterovirus Reverse Transcription-PCR Results in Pediatric and Adult Patients

Mulford

Buller²,

Arens³

et al. 2004

J Clin Microbiol

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We found that in the group of patients aged >2 months, the absence of pleocytosis was highly predictive of a negative RT-PCR result. Elevated CSF protein level was not a good predictor of RT-PCR positivity for enterovirus and did not add to the diagnostic sensitivity or specificity of pleocytosis.Enteroviruses (EVs) cause annual seasonal epidemics of aseptic meningitis in temperate climates and are the most common cause of this clinical syndrome in the United States. The infections are most clinically noticeable in children, but they are also clearly present in adults (9). The viruses are maintained within the human population in tropical climates year-round and at low but detectable levels in both children and adults during the cold months in temperate climates (3).Reverse transcription (RT)-PCR has become the method of choice for the rapid and efficient diagnosis of EV infections from cerebrospinal fluid (CSF). The use of commercially available (6,11,12,20) and in-house assay methods (6) is currently common in molecular diagnostic virology laboratories and often results in fewer tests being performed, less medication being used, and earlier release from the hospital than with previous diagnostic methods for patients with CSF samples with positive RT-PCR results for EV (EV RT-PCR) and negative Gram stains (4,8,10,14,15).Whether CSF pleocytosis plays an important role in defining aseptic meningitis or in prioritizing the differential diagnosis has been investigated but is still not completely clear (7,14,17). In this era of rapidly rising health care costs, it is important, if possible, to limit laboratory-based testing by encouraging syndrome-directed testing and actively discouraging testing that is not consistent with the clinical picture or with other laboratory results. Additionally, quality control within the laboratory can be enhanced by monitoring laboratory-based data to look for important correlations that will provide insight for clinicians and laboratory workers with regard to future testing requests.During the 2001, 2002, and 2003 EV seasons, we collected all available CSF cell count data and CSF protein data (available for 2001 and 2002 only) for patients for whom EV RT-PCR of CSF samples had been requested. Our suspicion that CSF with a nucleated cell count of Յ5 per mm 3 from patients more than 2 months old was not likely to be RT-PCR positive proved to be true, although a large number of CSF samples with high cell counts did not test positive for EV. MATERIALS AND METHODSCSF specimens (n ϭ 1,706) were submitted to the St. Louis Children's Hospital Virology Laboratory during the 2001, 2002, and 2003 EV seasons (roughly May to November of each year) for EV RT-PCR testing. Relevant CSF cell count data was available from the laboratory information system for 728 of the patients who were seen at Washington University Medical Center and tested by EV RT-PCR. Of the patients included in the study, 549 were pediatric (Յ18 years of age) and 179 were adults. The data analyzed included patient age, EV RT-PC...

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A one-step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis

Cited by 33 publications

References 14 publications

Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

Correlation of Cerebrospinal Fluid (CSF) Cell Counts and Elevated CSF Protein Levels with Enterovirus Reverse Transcription-PCR Results in Pediatric and Adult Patients

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