Prevalence and Management of Invalid GeneXpert Enterovirus Results Obtained with Cerebrospinal Fluid Samples: a 2-Year Study

Abstract

A total of 525 cerebrospinal fluid (CSF) samples submitted during the 2007 and 2008 enteroviral seasons were included in a study to determine the prevalence of and potential risk factors for invalid Cepheid GeneXpert enterovirus assay (GXEA) results, as well as possible solutions for the problem. The invalid GXEA results were reported for 43 (8.2%) specimens and correlated with increased visibility of red blood cells (P < 0.0001) but not with CSF xanthochromia and clotting. Invalid GXEA result rates were markedly diminished by 82.1% and 96.0% and test sensitivities were minimally decreased by 1.7% and 3.6% when these specimens were tested at a 1:5 dilution and after a freeze-thaw cycle, respectively.Enterovirus (EnV) meningitis can be difficult to distinguish from disease caused by other etiologic agents when patients present with nonspecific pathogenic symptoms and signs, such as fever, headache and stiff neck, and pleocytosis in cerebrospinal fluid (CSF) (4,8,9). Nucleic acid amplification-based procedures for the detection of EnV RNA in CSF have replaced cell culture as the test of choice (10,11,14). The GeneXpert enterovirus assay (GXEA; Cepheid, Sunnyvale, CA) is designed as an integrated system combining specimen processing, EnV amplification, and detection in a disposable cartridge which takes 2.5 h to detect EnV from CSF (6,7,12). It is designed for on-demand testing, such that "stat" PCR results can be returned to the emergency room physicians in time for patient management decisions to be made in real time. The system includes an internal control that provides a means to detect amplification inhibitors. When the internal control does not amplify, the presence of an amplification inhibitor is assumed and the result is reported as "invalid." Invalid results, if and when they occur, could delay patient management in the emergency setting.We performed a 2-year prospective study to determine the prevalence of invalid GXEA results and explore the potential risk factors related to the occurrence of invalid results. We also validated two alternative procedures to minimize the occurrence of these invalid results for EnV detection in CSF.(This study was presented in part at the 25th Annual Meeting of the Pan American Society for Clinical Virology, Daytona Beach, FL, 19 to 22 April, 2009.) Clinical samples. CSF specimens submitted to Vanderbilt University Medical Center between 1 April 2007 and 20 September 2008 for detection of EnV by PCR were collected consecutively. Specimens with enough leftover volume and still unfrozen after the completion of diagnostic testing were included in the study.Determination of CSF characteristics. Each CSF sample was visually examined for visible red blood cells (RBCs), clotting, and the presence of xanthochromia. RBC visibility was graded semiquantatively on a scale using rankings of clear, 1ϩ, 2ϩ, 3ϩ, and 4ϩ. These grades were based on specimen colors and turbidities which roughly correspond to CSF RBC counts of Ͻ200/l (clear), 200 to 5,000/l (1ϩ), 5,000 to 75,000/l (2ϩ), 75...

Comparative detection of enterovirus RNA in cerebrospinal fluid: GeneXpert system vs. real-time RT-PCR assay

“…The results used for comparative analysis with those obtained with the reference assay used in the core laboratory on the molecular virology diagnostics platform [5] were those recorded in the biological validation network system of the hospital. There are major points concerning these results: (i) the PPV of the POC-operated-Xpert EV kit was excellent (97.6%); (ii) the 16% 'indeterminate' results were of the same order of magnitude as results reported previously [8]; and (iii) these 16% indeterminate results were not included in the calculation of Sensibility, Specificity, PPV, and negative predictive value, because our goal was not to evaluate the intrinsic technical value of the Xpert EV kit, but rather to produce an estimate of its value in a real-life POC strategy for EV RNA detection. In these conditions, POC-'indeterminate' results (not validated) were not considered to be useful for clinicians, and were therefore not transmitted as results.…”

Comparison of the Cepheid GeneXpert CT/NG assay to the Hologic Aptima Combo2 assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in self-collected rectal swabs

“…(Goldenberg 2012) Sample adequacy control failures could have also been caused by potential interference of the TE buffer with the GeneXpert assay or potential inhibition of fecal/rectal material, which seemed to be resolved with the freeze-thaw cycle of the primary aliquot. Two prior studies also showed resolution of INVALID results in cerebral spinal fluid (CSF) samples on GeneXpert Assays by utilizing a free-thaw method (Seme 2008; Seters 2009). As seen in table 2 had only genital samples been analyzed and rectal sites not been screened 13 cases of CT infection and 6 cases of GC infection would have gone undiagnosed and subsequently untreated increasing transmission of these infections.…”

Evaluation of a Rapid and Completely Automated Real-Time Reverse Transcriptase PCR Assay for Diagnosis of Enteroviral Meningitis

“…The actual rate of invalid results may be higher in routine practice. Sefers et al (17) reported an 8.2% invalid rate over a 2-year period of routine use. These authors found that either a 1:5 sample dilution of CSF in saline, or freezing and thawing the undiluted CSF markedly reduced the invalid rate with minimal impact on the sensitivity of the assay.…”