In most cases, the aetiological background of the acute inflammation of the central nervous system (CNS) remains unknown [1]. The lymphocytic choriomeningitis virus (LCMV) (genus Mammarenavirus, family Arenaviridae) is one of the causative agents of acute (meningo)encephalitis in humans worldwide and the only known arenavirus in Europe [2;3]. LCMV is an enveloped virus with a segmented, single-stranded ambisense RNA genome. LCMVs are classified into four different genetic lineages [4].
The lymphocytic choriomeningitis (LCM) caused by LCMV is a rodent-borne viral zoonotic infection, which is “neglected” in the mirror of epidemiological, clinical and laboratory diagnostic aspects. The main reservoir of LCMV is the house mouse (Mus musculus), but other rodents (e.g. hamsters) can also be hosts [4]. Human-to-human transmission has not yet been confirmed, but transplacental transmission and transmission with infected organs have been registered [5;6]. Acquired LCMV infection in immunocompetent persons may be asymptomatic or present as a non-specific [5], self-limited febrile disease [7]. However, the illness can progress to meningitis or meningoencephalitis. No specific treatment or antiviral drug is available [8]. The LCMV seroprevalence is estimated between 1.7 and 13% [4]. The LCMV can be investigated with serological (ELISA), immunofluorescence assay (IFA) and PCR-based methods [5].
LCM is underdiagnosed in both clinical and laboratory practice. The aim of this retrospective clinical and laboratory study was to detect LCMV viral RNA using the RT-PCR method from cerebrospinal fluid samples collected from CNS infections of unknown aetiology over the past 12 years, between 2009 and 2020, in Hungary.
A total of 74 blood and cerebrospinal fluid samples were collected from hospitalized patients (in Baranya, Tolna and Pest counties) in Hungary with clinical diagnoses of encephalitis and tested simultaneously for routine serological and molecular diagnostic methods for human herpesvirus types 1 and 2 (HHV1/2), between 2009 and 2020. Blood samples were screened for HHV1/2 IgM/IgG ELISA kits (HSV-IgM/IgG ELISA, Dia.Pro, Italy), then total nucleic acids were extracted from blood, cerebrospinal fluid samples with High Pure Viral Nucleic Acid kit (Roche, Switzerland). Samples were tested prospectively by PCR for HHV1/2 [9], afterward retrospectively by “in-house” RT-semi-nested-PCR for LCMV using LCMV-screen-3670-F (5'-ACNTGGCAYATGCAYAA-3'), LCMV-screen-3840-F (5'-AGYCTHATTGAYATGGG-3') and LCMV-screen-4140-R (5'-ACYTCYTCNCCCCANACATA-3') primers designed in this study for the LCMV L segment (RdRp). PCR products were run on agarose gel and directly sequenced and run on an automated sequencer (3500 Genetic Analyzer, Japan).
Out of the 74 sample pairs, 61 came from the University of Pécs mostly from the Department of Paediatrics (N=38) and the Department of Neurology (N=16). The average age of the patients was 24 years [min. 5 – max. 74] with the predominance of men [44 (59.5%); women: 30 (40.5%)]. Two-two (2.7%-2.7%) cerebrospinal fluid samples were revealed to be positive for HHV1/2 DNA and LCMV RNA by (RT)-PCR and sequencing (Figure 1.).
Case 1. A 5-year-old preschool boy had a hamster bite on his left-hand finger 2 months before the symptoms appeared on May 12, 2020 (Figure 2.). The accident happened in a nursery, where the pet was brought in for "petting". The child presented to the Emergency Department with fever (>38.5°C) lasting for 5 days, loss of appetite, lethargy, weakness, and repeated vomiting. On the day after hospital admission, his vomiting complaints worsened, and neck stiffness also appeared and was transferred to the Intensive Care Unit (ICU). The detected LCMV (HUN7862/2020/HUN, MW558451) belongs to the genetic lineage I and showed 89/95% nucleotide/amino acid identities to the corresponding region of the LCMV strain JX31/China Jilin (MG554172) detected in a tick in China [10] (Figure 1.).
Case 2. A 74-year-old man who was living in a village had incipient dementia and a previous permanent functional CNS impairment (vascular encephalopathy, ischemia) because of a stroke. He was transported to the Emergency Department with confusion, weakness, fatigue, fever, aphasia and difficulty in moving his left arm on Oct 8, 2020 (Figure 3.). He had no fever, but psychomotorically was restless and convulsions appeared. The detected LCMV (HUN11808/2020/HUN, OM648933) belongs to the genetic lineage II and showed 82/90% nucleotide/amino acid identities to the corresponding region of LCMV strain SK1194 (MZ558313) detected in mice in the Czech Republic [11] (Figure 1.).
The nucleotide/amino-acid identities between the two Hungarian LCMV strains in partial LCMV L segment (RdRp) were 85% and 91%, respectively.
The LCMV is a rodent-borne zoonotic pathogen [4], but the infection is “neglected” both internationally and nationally. In Hungary, a total of 1,424 cases of acute encephalitis were reported between 2009 and 2020, of which 622 cases (43.7%) were of unknown aetiology by laboratory methods. During this period, a total of two LCMV cases were confirmed using RT-PCR method among 74 patients with suspected CNS infection, uncovering the aetiology of the disease. Interestingly, HHV1/2 and LCMV viral nucleic acids were obtained from equal frequency, which can certainly draw attention to the important, but the hidden prevalence of LCMV.
In our study, a young child, and an elderly man, were affected by LCMV which may indicate a wide age susceptibility to the disease. The possible source of infections is always an important question. In Case 1, there was a contact with hamster and even an animal bite in the patient’s medical history at the onset before the disease began. In Case 2, repeated (hetero)anamnesis was no longer possible due to the patient's condition; however, he lived in a rural environment and his family also raised animals. It can be assumed that in a rural environment where feed is also stored, rodents are also more common. The man's underlying medical condition presumably also made it more difficult to maintain good hygiene. The clinical suspicion of LCM may be facilitated by an adequate anamnesis (e.g., animal contact), clinical symptoms of CNS inflammation and integrated interpretation of the results of laboratory chemistry tests. In the presented cases, the clinical diagnosis of "viral encephalitis" was already made during patients care based on the clinical picture and partly the results of instrumental investigations. In Case 2, the history of stroke made it difficult to interpret clinical symptoms. The results of laboratory chemistry tests of cerebrospinal fluid and sera were characterised by markedly increased biomarkers including total protein, which reached extreme values in Case 2.
The two LCMVs cases from Baranya County in the year 2020 belonged to two different genetic lineages (I and II), unrelated to each other, therefore a common source of infection can be excluded. The habitat zones of one of the main host species Mus musculus musculus and M. domesticus run through Central and Eastern Europe including Hungary [11]. If the LCMV genetic lineages have host-specific associations [11], LCMV variants of lineages I and II specific to the two mice or other species may be mixed and thus present in Hungary. The fact that both human cases occurred in 2020 is an interesting observation, raising the question of whether it is a coincidence or there is a significant but unknown environmental factor in the background.
In our study, a “neglected” LCM was reported from two cases with a CNS inflammation of unknown origin representing the first human LCMV infections confirmed by molecular methods in Hungary. Our results confirm that the pathogenic role of LCMV in encephalitis/meningitis should be considered in clinical practice and microbiological laboratories must have appropriate diagnostic tools to confirm the infection. Although LCMV is presently the only known endemic arenavirus in Europe, its true human pathogenic role, significance, and genetic diversity are still far from known.