PCR for Detection of Herpes Simplex Virus in Cerebrospinal Fluid: Alternative Acceptance Criteria for Diagnostic Workup Paula López Roa, a Roberto Alonso, a Viviana de Egea, a Rafael Usubillaga, a Patricia Muñoz, a,b,c Emilio Bouza a,b,c Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain a ; Red Española de Investigación en Patología Infecciosa (REIPI), Sevilla, Spain b ; Department of Medicine, Universidad Complutense de Madrid, Madrid, Spain c The determination of herpes simplex virus (HSV) infection using a PCR assay is one of the most commonly requested tests for analysis of cerebrospinal fluid (CSF), although only a very low proportion of results are positive. A previously reported study showed that selecting only those CSF samples with >5 leukocytes/mm 3 or a protein level of >50 mg/dl was adequate for the di- agnostic workup. The aim of the present study was to assess the reliability of alternative acceptance criteria based on elevated CSF white blood cell counts (>10 cells/mm 3 ). We analyzed all requests for HSV PCR received between January 2008 and Decem- ber 2011. CSF samples were accepted for analysis if they had >10 cells/mm 3 or if the sample was from an immunocompromised patient or a child aged <2 years. In order to evaluate our selection criteria, we identified those CSF samples with a leukocyte count of 5 to 10 cells/mm 3 or protein levels of >50 mg/dl in order to test them for HSV type 1 and 2 (HSV-1 and HSV-2) DNA. During the study period, 466 CSF samples were submitted to the microbiology laboratory for HSV PCR. Of these, 268 (57.5%) were rejected, and 198 (42.5%) were tested according to our routine criteria. Of the tested samples, 11 (5.5%) were positive for HSV DNA (7 for HSV-1 and 4 for HSV-2). Of the 268 rejected specimens, 74 met the criteria of >5 cells/mm 3 and/or protein lev- els of >50 mg/dl. Of these, 70 (94.6%) were available for analysis. None of the samples yielded a positive HSV PCR result. Accep- tance criteria based on CSF leukocyte counts, host immune status, and age can help to streamline the application of HSV PCR without reducing sensitivity. D etection of herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) using PCR assay has been validated for the diag- nosis of central nervous system (CNS) herpes infections. HSV PCR is currently recognized as the reference method (1, 2). This sensitive but expensive test is included in the routine evaluation of many patients with suspected CNS infection, although most of the tests performed yield negative results (3). Therefore, screening for suitable diagnostic samples is necessary. Acceptance criteria based on elevated CSF leukocyte counts (5 cells/mm 3 ) and protein levels (50 mg/dl) have been pro- posed as a way to save health care costs without reducing sensitiv- ity (4, 5); however, other acceptance criteria have not been evalu- ated. Most patients with viral CNS infection have an abnormal CSF leukocyte count, which usually ranges from 10 cells/mm 3 to 200 cells/mm 3 (6). The CSF leukocyte counts reported in previous studies (1, 2, 5–13) are summarized in Table 1. In our institution, we accepted CSF specimens for HSV PCR testing if they had 10 cells/mm 3 . This cutoff was based both on our experience before the study and on data from the studies cited above. We also ac- cepted specimens collected from immunocompromised patients and children younger than 2 years of age. Clinical laboratories attempt to decrease costs while maintain- ing the quality of the diagnostic approaches used. As our criteria are stricter than those reported by Hanson et al. (5), we achieved a greater reduction in workload with the consequent laboratory savings. In order to evaluate the quality of our approach, we com- pared both criteria by performing HSV PCR in rejected specimens with leukocyte counts of 5 to 10 cells/mm 3 or protein levels of 50 mg/dl. We also analyzed the correlation between PCR results and leukocyte counts and protein levels in all CSF specimens submit- ted to the microbiology laboratory. MATERIALS AND METHODS Acceptance criteria. From January 2008 to December 2011, we accepted CSF specimens for HSV PCR analysis if they had an elevated CSF leuko- cyte count (10 cells/mm 3 ) or if the sample was collected from an immu- nocompromised patient (HIV-positive patient or transplant recipient) or a child younger than 2 years of age. Specimens which did not meet the acceptance criteria were rejected and frozen immediately at -80°C if an adequate volume was available. In the case of an urgent request by the attending physician for analysis of a specimen, HSV PCR was carried out even if the patient had normal CSF parameters. Laboratory record review. We reviewed laboratory records (protein level and leukocyte counts) for all CSF specimens submitted for HSV PCR during the study period. We identified frozen specimens with a leukocyte count of 5 to 10 cells/mm 3 or protein levels of 50 mg/dl in order to test them for HSV types 1 and 2 (HSV-1 and HSV-2) DNA. HSV PCR analysis. DNA was extracted using a NucliSENS EasyMAG system (bioMérieux, Boxtel, The Netherlands) according to the manufac- turer’s instructions. HSV-1 and -2 were detected using real-time reverse transcriptase PCR (affigene HSV 1/2 tracer, Cepheid AB, Sweden) in a Stratagene MX3000 thermocycler (Stratagene, La Jolla, CA). Five positive samples were randomly thawed and tested retrospec- tively for HSV PCR in order to analyze the viability of HSV DNA after 1 freeze-thaw cycle. Received 9 April 2013 Returned for modification 29 April 2013 Accepted 18 June 2013 Published ahead of print 26 June 2013 Address correspondence to Paula López Roa, paulalopezroa@gmail.com. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.00950-13 2880 jcm.asm.org Journal of Clinical Microbiology p. 2880 –2883 September 2013 Volume 51 Number 9 on May 26, 2020 by guest http://jcm.asm.org/ Downloaded from on May 26, 2020 by guest http://jcm.asm.org/ Downloaded from on May 26, 2020 by guest http://jcm.asm.org/ Downloaded from