Impact of cerebrospinal fluid syndromic testing in the management of children with suspected central nervous system infection.

The aim of the study was to evaluate the impact of the use of BioFire® FilmArray® meningitis/encephalitis(FA-ME) panel which enables rapid automated CSF testing for 14 common viral, bacterial, and yeast pathogens that cause CNS infections, in the management of children with suspected CNS infection. A prospective cohort study was performed on children admitted to a tertiary pediatric hospital, over a period of 1 year, with possible CNS infection and CSF pleocytosis (> 15 cells/mm3). Children were randomized 1:1, either to use FA-ME or separate molecular CSF microbiological tests according to usual pediatric practice in the hospital. Length of hospital stay, duration of antimicrobials, and total cost of hospitalization were compared between groups. A total of 142 children were included in the study (71 cases). A pathogen was detected in 37/71(52.1%) children with the use of FA-ME and in 16/71(22.5%) in the control group (P value < 0.001). In aseptic meningitis cases a virus was detected in 27/61(44.2%) and in 11/66(16.7%) controls (P value < 0.001). Median (IQR) length of stay in cases and controls with aseptic meningitis was 5(4-8) and 8(6-10) days, respectively (P value < 0. 001). The median (IQR) duration of antimicrobials in cases and controls was 4(2-5.7) and 7(5-10) days, respectively (P value < 0.001). The hospitalization cost was calculated in cases and controls 1042€ (932-1372) and 1522€ (1302-1742), respectively (P value < 0.001). The use of FA-ME was able to reduce significantly the use of antimicrobials, the hospitalization days, and the total cost comparing to the control group in children with suspected CNS infection.

[Clinical utility of HIV viral load assessment in cerebral spinal fluid, a case report]. / Utilidad de la cuantificación de carga viral de VIH en líquido cefalorraquídeo, a propósito de un caso.

Detection of virus in cerebrospinal fluid (CSF) in HIV-infected patients with HIV viral load (VL) undetectable in plasma has been termed viral escape. These leaks may be asymptomatic from a neurological point of view, similar to plasma blips, or associated with neurological disease, with discordant VL between plasma and CSF, and may be evidence of a compartmentalization of the virus and the possibility of identifying quasispecies with mutations that confer resistance to ART. We present the case of a man with AIDS and disseminated tuberculosis who presented neurological symptomatology evidenced by headache and convulsive syndrome, who presented a discordance between plasma and CSF HIV VL; the genotypic test of the virus, obtained by lumbar puncture, identified new mutations that determined a change in ART with subsequent satisfactory evolution.

Utilidad de la cuantificación de carga viral de VIH en líquido cefalorraquídeo, a propósito de un caso / Clinical utility of HIV viral load assessment in cerebral spinal fluid, a case report

Resumen La detección de virus en el líquido cefalorraquídeo (LCR) en pacientes infectados por VIH con carga viral (CV) indetectable en el plasma se ha denominado escape viral. Estas fugas pueden ser asintomáticas o asociadas con enfermedad neurológica. La discordancia de la carga viral de VIH entre plasma y LCR evidenciaría la presencia de distintos compartimentos del virus, con la posibilidad de identificar quasiespecies con mutaciones específicas que confieran resistencia a la TARV. Presentamos el caso clínico de un paciente con infección por VIH en etapa SIDA y una tuberculosis diseminada que presentó un cuadro neurológico manifestado por cefalea y un síndrome convulsivo, en que se encontró una discordancia entre la CV para VIH en plasma y LCR. El estudio genotípico del virus obtenido del LCR identificó nuevas mutaciones que determinaron un cambio de la TARV, con evolución posterior satisfactoria.

Detection of virus in cerebrospinal fluid (CSF) in HIV-infected patients with HIV viral load (VL) undetectable in plasma has been termed viral escape. These leaks may be asymptomatic from a neurological point of view, similar to plasma blips, or associated with neurological disease, with discordant VL between plasma and CSF, and may be evidence of a compartmentalization of the virus and the possibility of identifying quasispecies with mutations that confer resistance to ART. We present the case of a man with AIDS and disseminated tuberculosis who presented neurological symptomatology evidenced by headache and convulsive syndrome, who presented a discordance between plasma and CSF HIV VL; the genotypic test of the virus, obtained by lumbar puncture, identified new mutations that determined a change in ART with subsequent satisfactory evolution.

Cost-Effectiveness Study of Criteria for Screening Cerebrospinal Fluid To Determine the Need for Herpes Simplex Virus PCR Testing.

The absence of markers of inflammation in the cerebrospinal fluid (CSF) commonly predicts the absence of herpes simplex virus (HSV) central nervous system (CNS) infection. Consequently, multiple authors have proposed and validated criteria for deferring HSV PCR testing of CSF in immunocompetent hosts with normal CSF white blood cell and protein levels (≤5 cells/mm3 and ≤50 mg/dl, respectively). Hosts are considered immunocompetent if they are ≥2 years old and have not had HIV or an organ transplant. Adoption of the criteria may erroneously exclude HSV-infected persons from a necessary diagnostic test or, alternatively, reduce the costs associated with HSV tests with minimal to no effect on patient care. Little is known about the cost-effectiveness of this approach. A decision analysis model was developed to evaluate the adoption of criteria for screening HSV tests of CSF. Estimates of input parameter values combined available literature with a multiyear multisite review at two of the largest health care systems in the United States. Adoption of criteria to screen for HSV test need proved cost-effective when less than 1 in 200 patients deferred from testing truly had an HSV CNS infection. Similar to prior studies, none of the deferred cases had HSV encephalitis (n = 3120). Adoption of these criteria in the United States would save an estimated $127 million ($95 million to $158 million [±25%]) annually. The model calculations remained robust to variation in test cost, prevalence of HSV infection, and random variation to study assumptions. The adoption of criteria to screen HSV PCR tests in CSF represents a cost-effective approach.

Criteria for reducing unnecessary testing for herpes simplex virus, varicella-zoster virus, cytomegalovirus, and enterovirus in cerebrospinal fluid samples from adults.

Excessive utilization of laboratory diagnostic testing leads to increased health care costs. We evaluated criteria to reduce unnecessary nucleic acid amplification testing (NAAT) for viral pathogens in cerebrospinal fluid (CSF) samples from adults. This is a single-center split retrospective observational study with a screening cohort from 2008 to 2012 and a validation cohort from 2013. Adults with available results for herpes simplex virus 1/2 (HSV-1/2), varicella-zoster virus (VZV), cytomegalovirus (CMV), or enterovirus (EV) NAAT with CSF samples between 2008 and 2013 were included (n = 10,917). During this study, 1.3% (n = 140) of viral NAAT studies yielded positive results. The acceptance criteria of >10 nucleated cells/µl in the CSF of immunocompetent subjects would have reduced HSV-1/2, VZV, CMV, and EV testing by 63%, 50%, 44%, and 51%, respectively, from 2008 to 2012. When these criteria were applied to the 2013 validation data set, 54% of HSV-1/2, 57% of VZV, 35% of CMV, and 56% of EV tests would have been cancelled. No clinically significant positive tests would have been cancelled in 2013 with this approach. The introduction of a computerized order entry set was associated with increased test requests, suggesting that computerized order sets may contribute to unnecessary testing. Acceptance criteria of >10 nucleated cells/µl in the CSF of immunocompetent adults for viral CSF NAAT assays would increase clinical specificity and preserve sensitivity, resulting in significant cost savings. Implementation of these acceptance criteria led to a 46% reduction in testing during a limited follow-up period.

Laser capture microdissection assessment of virus compartmentalization in the central nervous systems of macaques infected with neurovirulent simian immunodeficiency virus.

Nonhuman primate-simian immunodeficiency virus (SIV) models are powerful tools for studying the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in the brain. Our laboratory recently isolated a neuropathogenic viral swarm, SIVsmH804E, a derivative of SIVsmE543-3, which was the result of sequential intravenous passages of viruses isolated from the brains of rhesus macaques with SIV encephalitis. Animals infected with SIVsmH804E or its precursor (SIVsmH783Br) developed SIV meningitis and/or encephalitis at high frequencies. Since we observed macaques with a combination of meningitis and encephalitis, as well as animals in which meningitis or encephalitis was the dominant component, we hypothesized that distinct mechanisms could be driving the two pathological states. Therefore, we assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection. Viral RNAs were isolated from representative areas of the meninges, brain parenchyma, terminal plasma, and cerebrospinal fluid (CSF) and from the inoculum, and the SIV envelope fragment was amplified by PCR. Phylogenetic analysis of envelope sequences from the conventional progressors revealed compartmentalization of viral populations between the meninges and the parenchyma. In one of these animals, viral populations in meninges were closely related to those from CSF and shared signature truncations in the cytoplasmic domain of gp41, consistent with a common origin. Apart from magnetic resonance imaging (MRI) and positron-emission tomography (PET) imaging, CSF is the most accessible assess to the central nervous system for HIV-1-infected patients. However, our results suggest that the virus in the CSF may not always be representative of viral populations in the brain and that caution should be applied in extrapolating between the properties of viruses in these two compartments.

Herpes simplex virus testing and hospital length of stay in neonates and young infants.

OBJECTIVE: To examine whether ordering testing of cerebrospinal fluid (CSF) for herpes simplex virus (HSV) by polymerase chain reaction (PCR) in neonates and young infants is associated with increased hospital length of stay (LOS) or increased hospital charges. STUDY DESIGN: This retrospective cohort study enrolled infants age

Prevalence and management of invalid GeneXpert enterovirus results obtained with cerebrospinal fluid samples: a 2-year study.

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.

Real-time polymerase chain reaction detection of herpes simplex virus in cerebrospinal fluid and cost savings from earlier hospital discharge.

Neonatal herpes simplex virus (HSV) can be a devastating illness and may be difficult to diagnose in those cases without a typical skin rash. As a result, physicians often rely on HSV polymerase chain reaction of cerebrospinal fluid to rule out HSV encephalitis. We developed a real-time polymerase chain reaction assay for HSV using the SmartCycler II (Cepheid, Sunnyvale, CA). End point dilution studies showed sensitivity comparable to that of two national reference laboratories that use LightCycler. In-house turnaround time was approximately 1.5 days versus approximately 5.2 days for sending the test to a reference laboratory. We hypothesized that the rapid availability of a negative test result would allow physicians to discharge appropriate patients earlier. Six months after implementation, clinical case analysis identified 12 pediatric patients who were discharged earlier based on more rapid test results, with a projected savings of approximately 55.2 hospital days throughout the first year. Actual length of stay for patients tested in-house was significantly less than that of historical controls and was projected to save approximately 70.2 hospital days in the first year. Including projected annual laboratory cost/test savings of approximately $11,000, a total savings of $38,000 to $43,000 was estimated for the first year of implementation, more than offsetting startup instrument and development cost.

[Serotype distribution of enteroviruses isolated from paediatric cases prediagnosed as aseptic meningitis between 2001-2004 period]. / 2001-2004 yillari arasinda aseptik menenjit ontanili pediatrik olgulardan izole edilen enterovirus serotiplerinin dagilimi.

Enteroviruses have major clinical and public health importance and are one of the leading causes of aseptic meningitis. There are many diseases with similar clinical symptoms and cerebrospinal fluid (CSF) findings of aseptic meningitis, thus virus isolation and identification is crucial for definitive diagnosis. Virological diagnosis is nonetheless important to distinguish between induced meningitis and other treatable causes of disease with a similar clinical picture. A total of 249 samples obtained from 246 cases (age range: 0-15 years), prediagnosed as aseptic meningitis, were sent to Virology Laboratory of Refik Saydam Hygiene Center. The patients were followed at Department of Pediatric Infectious Diseases in the Social Security Hospital, Ankara, Turkey, between 2001 and 2004. Stool (n: 180), CSF (n: 54) and throat swab (n: 15) samples have been inoculated to RD (rhabdomyosarcoma), Hep-2 (human epithelioma) and L20B (transgenic mice) cell lines, and followed up for the presence of cytopathic effects. A total of 95 enterovirus strains were isolated from 85 (34.6%) cases, and serotyped by using RIVM (National Institute of Public and the Environment, Nederlands) antisera with microneutralization method. As a result, the most frequently isolated types were found as echovirus type 30 (n: 24) and coxsackievirus type B (n: 19), which were most frequently isolated between July to October. This is the first report from Turkey for aseptic meningitis cases due to echovirus type 25 (n:3), 18 (n:2), 14 (n:1), 13 (n:4), 11 (n:6), 9 (n:1), 6 (n:9), 5 (n:1), 4 (n:1) and coxsackievirus type A9 (n:1).

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system.

The use of nucleic acid (NA) amplification techniques has transformed the diagnosis of viral infections of the central nervous system (CNS). Because of their enhanced sensitivity, these methods enable detection of even low amounts of viral genomes in cerebrospinal fluid. Following more than 10 years of experience, the polymerase chain reaction or other NA-based amplification techniques are nowadays performed in most diagnostic laboratories and have become the test of choice for the diagnosis of several viral CNS infections, such as herpes encephalitis, enterovirus meningitis and other viral infections occurring in human immunodeficiency virus-infected persons. Furthermore, they have been useful to establish a viral etiology in neurological syndromes of dubious origin and to recognise unusual or poorly characterised CNS diseases. Quantitative methods have provided a valuable additional tool for clinical management of these diseases, whereas post-amplification techniques have enabled precise genome characterisation. Current efforts are aiming at further improvement of the diagnostic efficiency of molecular techniques, their speed and standardisation, and to reduce the costs. The most relevant NA amplification strategies and clinical applications of to date will be the object of this review.

Impact of rapid polymerase chain reaction results on management of pediatric patients with enteroviral meningitis.

BACKGROUND: Enterovirus (EV) infections can be rapidly detected by PCR. However, several studies suggest that results must be available early in the management of the patient to impact significantly on patient care. We evaluated this hypothesis directly during an outbreak of EV aseptic meningitis. METHODS: From June through November, 1998, EV PCR was performed 5 days a week on cerebrospinal fluid specimens from pediatric patients evaluated for meningitis. We compared antibiotic use, length of stay and hospital charges in a group of patients with EV meningitis whose positive EV PCR results were available within 24 h of specimen collection, to a group of similar patients whose results were available >24 h after collection. RESULTS: Cerebrospinal fluid specimens were submitted for EV PCR from 113 patients with suspected EV meningitis, and 50 of 113 (44%) were positive. Of these 50 EV-PCR-positive patients, 17 of 50 (34%) had EV PCR results available in < or = 24 h and 33 of 50 (66%) had results available in >24 h. Patients with EV-positive results reported < or = 24 h after specimen collection had 20 h less of antibiotic use (P = 0.006) and $2,798 less in hospital charges (P = 0.001) than patients with positive results available in >24 h. Hospitalized patients who received positive results rapidly did not have significantly less antibiotic therapy or shorter length of stay, but hospital charges were reduced by $2,331 (P = 0.009). CONCLUSION: Rapid reporting of PCR results can have a significant impact on several outcome measures for patients with EV meningitis.

Benefits of management strategy adjustments during an outbreak of enterovirus meningitis in adults.

Enteroviruses (EVs) are the most common identifiable cause of the aseptic meningitis syndrome. Widespread seasonal outbreaks of EV meningitis result in a high financial cost to the community, in part because of the difficulty of discriminating between viral and bacterial meningitis. During a nationwide outbreak of EV meningitis due to echovirus 30 in France we tested the hypothesis that a management strategy including early testing of cerebrospinal fluid (CSF) by means of EV PCR in all adult patients with acute aseptic meningitis on admission might reduce the duration of hospitalization and thus the expenditure on health resources. We compared the characteristics of adult patients with acute aseptic meningitis seen in our institution before (n = 21) and after (n = 27) implementation of this strategy. The strategy was cost-effective in that it significantly reduced (i) the mean duration of hospital stay (from 103 to 80 h; p = 0.04); and (ii) the mean duration of antibacterial treatment (from 115 to 69 h; p = 0.02). Systematic testing of CSF in adult patients with aseptic meningitis by means of EV PCR may be cost-effective during an outbreak of EV meningitis.

[Rapid diagnosis of herpetic meningoencephalitis by PCR]. / Diagnóstico rápido de la meningoencefalitis herpética mediante PCR.

OBJECTIVE: To evaluate the usefulness of a rapid and simple PCR method in the diagnosis of herpetic meningoencephalitis in a pediatric population. PATIENTS AND METHODS: One hundred twenty-three cerebrospinal fluid samples from 114 pediatric patients attending the Hospital Sant Joan de Déu in Barcelona for clinical suspicion of viral meningoencephalitis or to rule out a possible herpetic etiology were evaluated. In addition to classical methods, the diagnostic technique used was PCR amplification of a highly preserved region of the DNA polymerase gene common to herpes virus 1 and 2. All patients were administered acyclovir on admission and until the results of PCR were known. If the result was negative, withdrawal of acyclovir was considered after clinical reexamination. If the result was positive, the therapy was continued for 20 days. RESULTS: Herpes simplex DNA was detected in four patients. In all patients, clinical outcome confirmed the results of PCR, whether positive or negative. PCR results were available within 6.30 and 72 hours (mean: 18 hours). CONCLUSION: This simple and rapid PCR technique can be applied in the daily routine of the microbiology laboratory. It allows early diagnosis of herpetic meningocephalitis or, when lacking, exclusion of Herpes simplex etiology.

Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid.

In the past few years, application of the PCR to the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from patients with encephalitis and meningitis has become standard laboratory practice. However, from an operational perspective, the true diagnostic value of PCR in this setting is yet to be realized because most laboratories subject the amplification products to lengthy probe hybridization procedures by Southern blotting. As alternatives to Southern blotting, we evaluated colorimetric microtiter plate (MTP) systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc. (Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a system developed at the Mayo Clinic with the PCR ELISA system (Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern blotting, the sensitivity and specificity were 63.0 and 100.0%, respectively, for the PrimeCapture system, 98. 2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100. 0%, respectively, for the GEN-ETI-K system, and 100.0 and 96.9%, respectively, for the Mayo system. All four MTP systems had turnaround times 12 to 24 h less than that for Southern blotting. There were no significant differences in costs or technologist time between the Mayo system and Southern blotting. Other features of the Mayo system include type-specific genotypic identification of HSV and the potential for determination of drug resistance by DNA sequencing. Overall, we found that colorimetric MTP systems were likely to improve test turnaround times and patient care at no additional cost.