Abstract
Background
The human parechoviruses (HPeVs) were recently recognized as important viral pathogens involved in various illnesses in young children. However, routine detection is not performed in most clinical laboratories. Therefore, in this study, we aim to assess the relevance of HPeV detection in cerebrospinal fluid (CSF) of infants, according to clinical presentation.
Methods
A total of 120 CSF specimens collected during 2012 from infants aged less than 1 year and previously reported negative for Herpes simplex virus (HSV) and enterovirus were selected. HPeV detection was performed with a commercially available real‐time RT‐PCR and HPeV strains from positive samples were subsequently genotyped by sequencing.
Results
HPeV RNA was detected in nine (7.5%) CSF samples. The median age of infected children was 41 days (range: 19–122 days). HPeV genotyping could be performed on five samples and three HPeV‐3, one HPeV‐1, and one HPeV‐4 were identified. Hyperthermia associated with mottled skin was the predominant clinical presentation. Most clinical presentations of HPeV‐infected infants were mild with a final diagnosis of sepsis‐like illness. The median hospital stay was 3.5 days and five children received antibiotics.
Conclusion
Routine detection of HPeV in CSF may allow differential diagnosis of enterovirus infection and improve etiologic identification of sepsis‐like illness in children.
Keywords: human parechovirus, HPeV, cerebrospinal fluid, sepsis‐like illness
INTRODUCTION
Attention to the clinical significance of human parechoviruses (HPeVs) has increased over the last decade. HPeV are nonenveloped, single‐stranded, positive‐sense RNA viruses that were previously classified within the Enterovirus genus of the Picornaviridae family. However, regarding some distinct genetic and biological properties, they were re‐named and re‐classified in the specific distinct genus Parechovirus that clusters now up to 16 HPeV genotypes 1. The clinical importance of HPeV infection was extensively explored worldwide 2, 3, 4, 5. The majority of HPeV infections occur very early in life, mainly before the age of 1 year. HPeV infections are usually asymptomatic but may be associated to mild gastrointestinal and respiratory diseases or to encephalitis, meningitis, and sepsis‐like syndrome in neonates 5, 6, 7.
Developments of sensitive and specific molecular tests for HPeV RNA have recently improved HPeV detection and typing and have allowed to better understand HPeV epidemiology. Several recent studies have highlighted the great interest of a more systematic testing for HPeV infection in infants 2, 3, 4, 5, 6, 7. However, to date, few clinical laboratories have implemented HPeV molecular tests to their routine in addition to human enteroviruses 4, 8, 9. With the aim to set up the systematic detection of HPeV RNA in cerebrospinal fluid (CSF) collected from infants, we retrospectively investigated the presence of HPeV sequences in CSF specimens previously tested for routine diagnosis of viral infection.
MATERIAL AND METHODS
Patients and Samples
A total of 120 available CSF specimens collected during 2012 from infants aged less than 1 year were studied. These samples were previously tested and found negative for Herpes simplex virus (HSV) DNA and enterovirus RNA. Clinical and biological data were retrospectively recorded.
RNA was isolated from 200 μl of CSF spiked with the IC1 RNA internal control (bioMérieux—Argène, Verniolles, France) using the Nuclisens EasyMag extraction automated system (bioMérieux, Craponne, France). Extracts were stored at −80°C until use.
HPeV Detection by Real‐Time PCR
HPeV detection was performed on 10 μL of RNA extracts using a commercially available real‐time reverse‐transcription PCR assay targeting the 5′ nontranslated region (Parechovirus r‐gene, bioMérieux) on the LightCycler 480 system (Roche Diagnostics, Meylan, France) 4, 10.
HPeV Genotyping
HPeV genotyping was performed by VP1 sequencing 11. Sequences were blasted against GenBank for phylogenetic analysis.
RESULTS
HPeV RNA Detection and Genotypes
HPeV RNA was detected in nine (7.5%) CSF samples, with cycle threshold (C t) values ranging from 31.9 to 38.7 cycles. Median age of these infants was 41 days (range: 19–122 days), with a male/female ratio of 2. Six infants with HPeV infections were admitted during summer months (June–August) whereas the three other HPeV cases occurred later in November. Seven patients had specimens available for genotyping. In two patients, the virus could not be typed. Three patients were infected with HPeV‐3, one with HPeV‐4, and one with HPeV‐1.
Biological and Clinical Data
Laboratory results are displayed in Table 1. Infants with HPeV infection had a median white blood cells' count of 6.6 × 103 cells/mm3 (range: 2.7–12.7 × 103 cells/mm3) with leukopenia (<5 × 103 cells/mm3) for only two patients. A mild inflammatory syndrome with a median C‐reactive protein (CRP) level at 21.5 mg/l (range: 2.7–121 mg/l) was observed, whereas the procalcitonine (PCT) ranged from 0.14 to 3.66 ng/ml with a median at 0.29 ng/ml. None but one CSF displayed a pleiocytosis (30 cells/mm3), the median CSF protein level was 0.53 g/l (range: 0.27–1.6 g/l, normal values: 0.15–0.45 g/l) and the median CSF glucose value was 2.9 mmol/l (range: 2.4–5.1 mmol/l, normal values: 2.5–4.5 mmol/l). Alpha‐interferon levels in the CSF were <2 UI/ml except a discrete detectable level of 4 UI/ml in one sample.
Table 1.
Laboratory Findings From HPeV‐Infected Children
| CSF | Blood | Viral data | |||||||
|---|---|---|---|---|---|---|---|---|---|
| ID | WBC (× 103 cells/μl) | Protein (g/l) | Glucose (mmol/l) | Interferon (UI/ml) | CRP (mg/l) | PCT (ng/ml) | WBC (× 103 cells/μl) | HPeV amplification threshold (C t) | HPeV genotype |
| 7 | nd | 1.6 | 5.1 | <2 | 2.7 | 0.17 | 2.9 | 32 | HPeV‐3 |
| 26 | 5 | 3.2 | 4 | <2 | 6.8 | 1.25 | 7.7 | 35 | HPeV‐1 |
| 28 | 5 | nd | nd | <2 | 21.5 | 0.23 | 6.6 | 37 | HPeV‐3 |
| 33 | 5 | nd | nd | <2 | 72.6 | 3.66 | 2.7 | 35 | HPeV‐4 |
| 34 | 5 | 0.27 | 2.6 | <2 | 101 | 0.29 | 12.7 | 38 | nd |
| 44 | nd | 0.57 | 2.9 | 4 | 14.3 | 0.14 | 6.4 | 37 | nd |
| 83 | 30 | 0.37 | 3 | <2 | 121 | 1.47 | 7.0 | 33 | Nontypable |
| 88 | nd | 1.02 | 4 | <2 | 2.9 | 0.15 | 6.6 | 36 | HPeV‐3 |
| 91 | 5 | 0.53 | 2.4 | <2 | 99 | 1.42 | nd | 36 | Nontypable |
nd, not determined; WBC, white blood cells; C t, cycle threshold.
Children were all hospitalized with a median hospital stay of 3.5 days (range: 2–8 days). At admission, all patients were febrile with a mean T max of 39.2 ± 0.8°C. The median duration of fever was 72 h (range: 48–96 h) and five infants received an antibiotics treatment. Hyperthermia was the predominant clinical presentation together with mottled skin. All children had neurological signs, seven of them had irritability, two of them had hypotonia, and one had tense fontanelle. Five patients presented a sepsis‐like syndrome, with tachycardia and mottled skin. Other observed presentations were mild gastrointestinal disorders including diarrhea, vomiting, or food refusal (n = 7), respiratory signs (n = 3), and rash (n = 1, Table 2).
Table 2.
Hospital Course and Clinical Characteristics of HPeV‐Infected Children
| ID | Age (days) | Gender | Fever (h) | Temp. max. (°C) | Hospital stay (days) | Sepsis signs | Neurologic signs | Respiratory signs | Gastrointestinal signs | Other signs | Atb (yes/no) | HPeV genotype | Diagnosis |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 7 | 20 | F | 48 | 38.2 | 3 | Irritability | Food refusal | No | HPeV‐3 | Sepsis‐like illness/meningitis | |||
| 26 | 98 | M | 72 | 39.2 | 3 | Tense fontanelle | Rhinopharyngitis | Yes | HPeV‐1 | Sepsis‐like illness/meningitis | |||
| 28 | 47 | F | 96 | 39.5 | 3 | Mottled skin | Irritability | Diarrhea | Yes | HPeV‐3 | Sepsis‐like illness nephritis | ||
| 33 | 84 | M | 96 | 40 | 4 | Mottled skin | Irritability | Food refusal | Yes | HPeV‐4 | Sepsis‐like illness | ||
| 34 | 122 | M | 72 | 39.5 | 2 | Irritability | Food refusal | No | nd | Febrile illness | |||
| 44 | 35 | M | 96 | 38.9 | 8 | Mottled skin | Irritability | Food refusal | Yes | nd | Sepsis‐like illness | ||
| 83 | 80 | M | 48 | 38.5 | 8 | Irritability | Bronchiolitis respiratory distress | Yes | Nontypable | Bronchiolitis with sepsis‐like illness | |||
| 88 | 19 | F | 72 | 38.6 | 5 | Mottled skin | Hypotonia | Emesis | No | HPeV‐3 | Sepsis‐like illness | ||
| 91 | 25 | M | 48 | 39.3 | 3 | Mottled skin | Irritability, hypotonia | Respiratory distress | Diarrhea | Rash | No | Nontypable | Enteroviral infection with sepsis‐like illness |
Atb, antibiotics.
DISCUSSION
The HPeV detection rate of 7.5% in CSF collected from young infants observed in this study is similar with previous observations 2, 4, 5. In agreement with these reports, most infections occurred during summer months, however a second cluster of cases was detected in November suggesting that the period of HPeV circulation might be more extended than previously thought and that viral detection should be driven yearlong. Infants with HPeV detection in CSF were predominantly very young boys (median age of 41 days, sex ratio of 2), in line with similar observations of HPeV‐3 infection affecting mainly male children under 3 months 4, 12, 13. This reinforces the interest for a focus on infants aged less than 3 months for HPeV testing.
In our retrospective study, a HPeV genotype could be identified in five of seven analyzed samples. As previously observed, HPeV‐3 was the most prevalent genotype in CSF. HPeV‐3 was recently shown to be a common cause of meningitis and neonatal sepsis in young infants and it is the most prevalent genotype detected in CSF samples 5, 12, 13, 14. Other genotypes in CSF appear to be more sporadic and are mainly described in children older (median age 6.6 months) than those infected by HPeV‐3 (median age <3 months) 1. The age of the two children infected with HPeV‐4 or HPeV‐1, although higher (84 and 98 days, respectively) than the median age was not significantly different from the other HPeV‐infected children. HPeV‐1, formerly echovirus 22, has been detected in CSF from infants with neurological diseases such as aseptic meningitis or encephalitis, or encephalomyelitis 15. Detection of HPeV‐4 genotype in CSF of children with febrile syndrome has been also previously reported 4, 16. HPeV4 was, furthermore, recently described in neonatal sepsis with similar clinical manifestations as those observed in this study 17. However, the detection of these HPeV genotypes is less frequently reported than that of HPeV‐3. Several studies have reported that HPeV‐3‐infected infants are younger and have more severe symptoms than those infected with other HPeV genotypes 7, 18. In this study, clinical presentations were mild for the three HPeV genotypes detected. Furthermore, the low HPeV viral loads in CSF observed in all patients are more likely to reflect a passive passage across the blood‐brain barrier than an active replication within the CSF. This could be as well corroborated by results from most CSF showing no detection of alpha‐interferon, absence of pleiocytosis, and a normal protein level. However, it must be specified that these markers are poor reliable indicators of CNS infection in children of less than 2 months 19.
Dominant clinical presentation of the nine children at admission was high body temperature frequently associated to mottled skin. Mild neurological signs like irritability were also frequently recorded. Few children exhibited additional signs of a more severe form such as occurrence of rash or symptoms compatible with a meningitis (hypotonia, tense fontanelle). A significant inflammatory syndrome with high CRP (70–121 mg/l) and high PCT (1.25–3.66 ng/ml) was observed in five infants, whereas only one had a documented associated bacterial infection (nephritis). Thus, for all these children, the final retained diagnosis was a sepsis‐like illness (not tolerated fever with peripheral vasoconstriction signs) and regarding current standard for care, hospitalization and administration of empiric antibiotics treatment were proposed. Furthermore, lumbar puncture was performed as part of workups to rule out sepsis and/or meningitis. Regarding the mild symptoms and presentations of HPeV‐infected infant, our study suggests that routine detection of HPeV in available CSF is worth considering as differential diagnosis of enterovirus infection, even in noncomplicated sepsis‐like illness of very young children. Furthermore, this diagnosis is now facilitated by the development of commercial solutions easy to set up in any clinical laboratory 4, 10. Length of hospital stay was not negligible for infants infected with HPeV and most of them received a likely useless antibiotics treatment. Therefore, detection of HPeV in such clinical situation of unknown etiology could reduce hospital stays, save antibiotics usage, and minimize overall care expenses.
ACKNOWLEDGMENT
We would like to thank BioMérieux/Argene for providing the Parechovirus r‐gene kits.
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