Development, Technical Performance, and Clinical Evaluation of a NucliSens Basic Kit Application for Detection of Enterovirus RNA in Cerebrospinal Fluid

Abstract

The combination of nucleic acid sequence-based amplification and electrochemiluminescence detection was used to develop an internally controlled, highly sensitive and specific assay for the detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF). The analytical performance of the assay was determined using both in vitro-transcribed EV RNAs and viral culture isolates. The sensitivity of the assay was 10 EV RNA copies per amplification reaction. The assay detected all enteroviral isolates tested with no cross-reactivity to 21 nonenteroviral species, including rhinovirus and parechovirus. The clinical performance of the assay was evaluated by testing 992 CSF specimens collected from adult and pediatric patients. NucliSens EV results from a subset of 327 CSF samples were compared to viral culture of nasopharyngeal specimens and rectal swabs (n ‫؍‬ 195) and/or CSF (n ‫؍‬ 212). Of the 212 CSF samples, 96 samples were positive by either the NucliSens EV assay (94/96; 97.9%) or culture (63/96; 65.6%), and 61/96 (63.5%) were positive by both methods. The inclusion of an EV-specific internal control monitored the entire process, including the efficiency of nucleic acid extraction, amplification, and detection. In total, only five blood-clotted CSF samples (0.5%) were inhibited. The NucliSens EV assay demonstrated superior sensitivity over viral culture (P < 0.001), excellent specificity, clear delineation of positive samples, and minimal amplification inhibition.Enteroviruses (EV) are RNA viruses that are members of the Picornaviridae family (6). The nonpolio enteroviruses, including the coxsackieviruses, echoviruses, and numbered enteroviruses, are responsible for approximately 5 to 10 million symptomatic infections per year in the United States (14,20). Enteroviruses cause a diverse array of illnesses, including respiratory, cardiac, and neurologic ones, in both adults and children. Children under 5 years of age appear to be the most susceptible to infection, due partly to a lack of acquired immunity and poor hygienic habits. In the neonate, enteroviruses can cause a sepsis-like picture (fever, irritability, failure to eat, poor responsiveness) or meningoencephalitis, which can be severe (14).Enteroviral infections of the central nervous system will often mimic bacterial infection, and therefore, children with meningitis or febrile infants are hospitalized until a bacterial etiology is either confirmed or ruled out. Many enteroviruses can be grown in primate cell lines; however, results of viral culture can take 3 to 8 days, and therefore, the results are generally not rapid enough to affect either treatment options or length of hospitalization (4,6,16,21). In addition, viral culture has a sensitivity of approximately 65 to 75%, in part because of the inability to culture all enteroviral serotypes, including several coxsackievirus group A strains that require mouse inoculation for detection (6). To increase the sensitivity of enteroviral detection and reduce the turnaround time for results, molecular methods, inc...

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