Lymphocytic Choriomeningitis Virus–associated Meningitis, Southern Spain

Lymphocytic choriomeningitis virus (LCMV) was detected in 2 patients with acute meningitis in southern Spain within a 3-year period. Although the prevalence of LCMV infection was low (2 [1.3%] of 159 meningitis patients), it represents 2.9% of all pathogens detected. LCMV is a noteworthy agent of neurologic illness in immunocompetent persons.

CSF and acute-phase serum samples were also tested for IgG and/or IgM against TOSV (EIA Enzywell Toscana virus IgG/IgM, Diesse, Siena, Italy), West Nile virus (ELISA IgG and IgM-capture ELISA; Focus Diagnostics, Cypress, CA, USA), and LCMV by indirect fl uorescent assay (IFA) with further confi rmation by Western blot (2).
Case-patient 1, a 21-year-old woman, came to the hospital's emergency unit in April 2008 exhibiting headache, chills, fever (38.9°C), confusion, nausea, vomiting, and slight nuchal rigidity. Aseptic meningitis was suspected, and samples of CSF and serum were obtained. Laboratory results were 415 leukocytes/mm 3 (100% mononuclear cells), 43 mg/100 dL glucose level (reference 35-65 mg/dL), and 128 mg/dL protein level (reference 15-45 mg/dL) in the CSF. Results of a computed tomographic scan of the brain were normal. IgG and IgM titers of 640 and 128 against LCMV were detected in the serum sample by IFA and titers of 400 and 200 by Western blot assay, respectively. Results of RT-PCR for arenavirus and LCMV were negative. Viral culture of the CSF in Vero cells revealed no cytopathic effect after 1 month of incubation. Cell culture supernatants from several passages were subjected to specifi c RT-PCR. LCMV PCR was positive at a dilution of 10 −4 at the third passage. Viral isolate (EEB-7) was used for genetic characterization. To sequence the genome, degenerate and specifi c primers were designed on the basis of an alignment of all complete LCMV large (L), glycoprotein complex and nucleocapsid protein sequences (Table). Terminal sequences were generated by using a universal arenavirus primer, targeting the conserved viral termini (5′-CGC ACM GDG GAT CCT AGG C-3′), combined with 4 specifi c primers positioned near the ends of each segment. Amplifi cation products were size-fractionated by electrophoresis in 1% agarose gels, purifi ed (MinElute, QIAGEN, Valencia, CA, USA), and sequenced in both directions on an ABI PRISM 3700 DNA analyzer (PE Applied Biosystems, Foster City, CA, USA). To determine the evolutionary history of isolate EEB-7, we performed Bayesian phylogenetic analysis of all available complete genome sequences of LCMV using BEAST, BEAUti, and Tracer analysis software packages (13). The analysis showed that EEB-7 (GenBank accession nos. JN872494-5) belonged to LCMV lineage I (Figures 1,  2). Clinical diagnosis was acute meningitis and the patient was discharged from hospital on day 9. Case-patient 2, a 39-year-old man, sought treatment in May 2010 with headache, nausea, vomiting, increased perspiration, and a temperature of 37.5°C. Aseptic meningitis was suspected, and CSF and serum samples were collected. CSF analysis demonstrated 1,715 leukocytes/mm 3 (95% mononuclear cells), normal glucose level (68 mg/dL), and elevated protein levels (240 mg/dL). Results of a cranial computed tomographic scan were normal. Further virologic investigation detected LCMV RNA in the CSF and an IgG titer of 640 by IFA and IgM antibodies against LCMV in the serum sample. Serum amount was insuffi cient to conduct IgM titration and Western blot assay. No CSF sample was available to attempt viral isolation. The sequence of a 194nt PCR product (nucleocapsid protein gene) obtained was most closely related to sequences of the lineage I. Sequence homology among the LCMV amplicon from case-patient 2 and lineage I strains was >87% versus 77%-79% sequence homology among case-patient 2 and strains from lineages II-IV. Clinical diagnosis was subacute meningitis, and the patient was discharged on day 16. PCR has become the reference standard for identifying common viruses involved in AME (6). However, no commercial tests are available for LCMV; and in-house PCRs have to be optimized according to the natural genetic diversity of the virus (14). Serologic testing has been found useful in detecting LCMV. Neurologic LCMV infection in Spain has been diagnosed primarily by serologic tests (5,6). The diagnosis in case-patients 1 and 2 in the current study was achieved by serologic testing as well. Nonetheless, PCR was useful because it allowed genetic characterization of the LCMV strain from case-patient 2. Furthermore, the viral isolate of case-patient 1 was evident only by detection of LCMV RNA in the cell culture supernatant because no cytopathic effect was observed.
Isolates from both LCMV case-patients belonged to the classical lineage I, which has been detected elsewhere in Europe. Lineage I is usually associated with human disease (as are lineages II and III) and is linked to the common house mouse as its reservoir (14). Lineage IV viruses were previously detected in Spanish wood mice (2) and have not been associated with human disease.

Conclusions
Human LCMV infections might be underdiagnosed because the clinical characteristics of LCMV meningitis are similar to those of other viral meningitis; no commercial tests are available for serologic or molecular diagnostic assays, and usually no clear epidemiologic clue is available at the moment of diagnosis. Thus, epidemiologic and virologic surveillance might ascertain that the true incidence of LCMV AME is more frequent than reported.
This work was fi nanced in part by Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (PI07/90154).