Point-Counterpoint: Meningitis/Encephalitis Syndromic Testing in the Clinical Laboratory.

INTRODUCTIONSyndromic panels were first FDA cleared for detection of respiratory pathogens in 2008. Since then, other panels have been approved by the FDA, and most recently, the FilmArray meningitis/encephalitis panel (BioFire, Salt Lake City, UT) has become available. This assay detects 14 targets within 1 h and includes pathogens that typically cause different manifestations of infection, although they infect the same organ system. Several studies have reported both false-positive and false-negative results with this test, and all agree that the cost is significant. As with other panels, health care systems have adopted different strategies for offering this assay. Some have implemented strategies to limit the use of the test to certain patient populations, others have elected not to offer the test, and others have elected not to offer the test and instead request that providers order specific PCRs for the pathogens that best fit the patient's symptoms. In this Point-Counterpoint, Jennifer Dien Bard of the Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, and of the Keck School of Medicine at the University of Southern California explains why laboratories should offer these assays without restriction. Kevin Alby of the University of Pennsylvania explains the concerns about the use of these assays as first-line tests and why some limitations on their use might be appropriate.

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.

Homogenization with heat treatment: A cost effective alternative to nucleic acid extraction for herpes simplex virus real-time PCR from viral swabs.

Most laboratories use expensive commercial kits to purify nucleic acids and remove PCR inhibitors that may be present in clinical specimens. In this study a simple homogenization with heat treatment of herpes simplex virus types 1 and 2 (HSV-1/2) was shown to be equivalent to commercial kit-based nucleic acid extraction methods. With a cost of less than $1 USD per extraction, this method provides an economical, rapid, and effective method to recover HSV-1/2 DNA from swabs suitable for real-time HSV PCR.

Comparison between turnaround time and cost of herpes simplex virus testing by cell culture and polymerase chain reaction from genital swabs.

Clinical diagnosis of genital herpes simplex virus (HSV) (GH) infection is insensitive and non-specific and requires laboratory confirmation. In this study we compared viral culture and the amplification of HSV DNA by polymerase chain reaction (PCR) with respect to turnaround time and cost. We compared 182 swabs submitted to our laboratory between March and May 2005, which were tested using MRC-5 cell culture, with 168 genital swabs submitted during the same months in 2006, and were tested by PCR. We concluded that PCR testing in our laboratory has significantly improved the turnaround time, with 68.4% of tests having been reported in less than 24 hours. This in turn significantly improved the service provided for the diagnosis of genital herpes without additional costs.

A preclinical assessment of the safety and biodistribution of an adenoviral vector containing the herpes simplex virus thymidine kinase gene (Cerepro) after intracerebral administration.

BACKGROUND: Cerepro (sitimagene ceradenovec) is an adenoviral vector containing herpes simplex virus thymidine kinase gene (HSV-tk), which is being developed for the treatment of high-grade glioma with oral ganciclovir (GCV). The nonclinical safety and biodistribution of Cerepro were assessed following intravenous (i.v.) or intracerebral (i.c.) injection. METHODS: Crl : WI(GLX/BRL/Han) rats (n = 198) were injected i.c. or i.v. with Cerepro or vehicle control, with GCV by intraperitoneal (i.p.) injection to selected groups. Safety was assessed by observation of animal behaviour and post mortem histology. Antibody response was assessed, and biodistribution measured using the quantitative polymerase chain reaction (PCR) and reverse transcriptase-PCR in blood and tissues. RESULTS: Following i.v. or i.c. injection, there was no antibody response and no effect on behaviour, body weight, food consumption or haematological and clinical chemistry parameters. Minor needle track changes were observed in control and Cerepro-i.c. injection groups. Transient myeloid hyperplasia was observed in five of the 24 animals in the i.v. injection group and spleen weight increased in both the i.c. and i.v. groups. Cerepro was detected in the brain and at low levels in blood and spleen following i.c. injection, decreasing with time. Following i.v. injection, Cerepro was detected in viscera and blood, decreasing with time. Transcription of Cerepro was detected in the brain following i.c. injection, with lower levels in spleen; following i.v. injection, transcription was seen in viscera. Germline integration was not seen. CONCLUSIONS: Intracerebral injection of Cerepro is safe and produces a high level of transgene expression in the brain, with limited biodistribution.

A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods.

BACKGROUND: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. OBJECTIVES: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. STUDY DESIGN: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol ) gene of HSV isolates. RESULTS: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). CONCLUSION: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.

In vivo experimentation with simian herpesviruses: assessment of biosafety and molecular contamination.

In vivo studies with highly pathogenic viruses prompt concerns regarding the persistence of infectious virus in pathology specimens. Although formalin fixation of tissues may inactivate infectious virus, fixation may also degrade viral nucleic acid and antigens, thereby limiting detection of virus in tissues by polymerase chain reaction (PCR) amplification or immunohistochemistry (IHC). We sought to: 1) assess the rate of inactivation of infectious virus in tissue specimens during formalin fixation, 2) assess IHC recognition of viral antigens and PCR detection of viral DNA after long-term (14 d) formalin fixation, and 3) investigate microtome contamination by DNA carry-over to subsequently sectioned tissues. Infectious baboon herpesvirus HVP2 could be recovered from fresh tissues of infected mice but not those fixed in formalin for >/=24 h. The intensity of IHC staining of viral antigen was unaffected by the duration of formalin fixation. PCR detection of viral DNA was negatively impacted by formalin fixation and/or heat inherent to paraffin processing; however, amplification of very short DNA sequences using real-time PCR was not affected. Lastly, microtome contamination by viral DNA was demonstrated by PCR screening of uninoculated control tissues that were sectioned after sectioning infected tissues. In summary, infectious virus is inactivated after only 24 h of formalin fixation whereas IHC staining remains sensitive in tissues fixed for up to 14 d. Formalin fixation does degrade DNA, but viral DNA can be detected by PCR amplification of very short DNA sequences. In addition, viral DNA can contaminate a microtome knife such that subsequently sectioned uninoculated control tissues exhibit false positive PCR amplification.

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.

[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.

Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods.

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

Genetic 'budget' of viruses and the cost to the infected host: a theory on the relationship between the genetic capacity of viruses, immune evasion, persistence and disease.

The nature of the pathogen-host relationship is recognized as being a dynamic coevolutionary process where the immune system has required ongoing adaptation and improvement to combat infection. Under survival pressure from sophisticated immune responses, adaptive processes for microbes, including viruses, have manifested as immune evasion strategies. This paper proposes a theory that virus immune evasion can be broadly classified into 'acquisition' or 'erroneous replication' strategies. Acquisition strategies are characteristic of large genome dsDNA viruses, which (i) replicate in the cell nucleus; (ii) have acquired host genes that can be used to directly manipulate responses to infection; (iii) are often latent for the lifetime of the host; and (iv) have little or no serious impact on health. Alternatively, erroneous replication strategies are characteristic of small genome RNA viruses, which are recognized as being the cause of many serious diseases in humans. It is proposed that this propensity for disease is due to the cytoplasmic site of replication and truncated temporal relationship with the host, which has limited or removed the evolutionary opportunity for RNA viruses to have acquired host genes. This has resulted in RNA viruses relying on error-prone replication strategies which, while allowing survival and persistence, are more likely to lead to disease due to the lack of direct viral control over potentially host-deleterious inflammatory and immune responses to infection.

Use of the polymerase chain reaction in the diagnosis of herpes simplex encephalitis: a decision analysis model.

PURPOSE: To evaluate the utility of an assay based on a polymerase chain reaction (PCR) of cerebrospinal fluid in the management of patients with suspected herpes simplex encephalitis. METHODS: A decision model was constructed and used to compare a PCR-based approach with empiric therapy. Inputs required by the model included the sensitivity (96%) and specificity (99%) of PCR (derived from review of the literature), the prevalence of herpes simplex encephalitis (5%, based on the actual prevalence at Barnes Hospital among patients treated empirically with acyclovir), the outcomes for patients with and without herpes simplex encephalitis (derived from clinical studies of the Collaborative Antiviral Study Group and the actual experience at Barnes Hospital), and the average duration of empiric acyclovir therapy for patients with possible herpes simplex encephalitis (5.3 days based on actual experience at Barnes Hospital). RESULTS: Using these input values, the decision model predicted better outcomes with empiric therapy. However, low rates of inappropriate discontinuation of empiric therapy in patients with herpes simplex encephalitis or improved diagnosis and outcome resulting from a negative PCR assay result in patients without herpes simplex encephalitis led to better outcomes with the PCR-based approach. The PCR-based approach was associated with 9.2 fewer doses of acyclovir per patient. CONCLUSION: Based on the decision model using conservative assumptions, a PCR-based approach can yield better outcomes and reduced acyclovir use compared with empiric therapy.

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.

Assessment of ganciclovir toxicity to experimental intracranial gliomas following recombinant adenoviral-mediated transfer of the herpes simplex virus thymidine kinase gene by magnetic resonance imaging and proton magnetic resonance spectroscopy.

Magnetic resonance imaging and in vivo localized H magnetic resonance spectroscopy were used to evaluate a gene therapy approach for treating experimental brain tumors. This approach involved the use of an adenoviral vector to transfer the herpes simplex virus thymidine kinase (HSVtk) gene into intracerebral 9L gliosarcomas in rats followed by systemic administration of the antiherpetic agent ganciclovir. Magnetic resonance imaging quantitation of changes in intracranial 9L tumor doubling times revealed a significant variation in therapeutic response. Localized H magnetic resonance spectra of 9L tumors treated with Ad.RSVtk/ganciclovir revealed a dramatic increase in the resonance intensity at 0.9-1.3 ppm, corresponding to mobile lipids and/or lactate. Changes in intracranial tumor doubling times correlated with changes in H tumor magnetic resonance spectra, suggesting that specific changes in tumor metabolite levels may be predictive of the effectiveness of this gene therapy approach.