Abstract
Background. Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome (HFRS) also called nephropathia epidemica (NE). Recent case reports and retrospective studies suggest that NE may damage the pituitary gland. Based on these observations, our goal was to explore the nature of this complication prospectively.
Methods. A total of 58 hospitalized patients with acute NE volunteered to participate. Central nervous system (CNS) symptoms were recorded, cerebrospinal fluid (CSF) samples were collected, human leukocyte antigen (HLA) haplotype was analyzed, brain magnetic resonance imaging (MRI) was acquired, and electroencephalography (EEG) was recorded. Patients with abnormal pituitary MRI finding were examined by an endocrinologist.
Results. Most patients experienced CNS symptoms, and half of the CSF samples were positive for PUUV IgM, elevated protein level, or leukocyte count. CSF of patients negative for DR15(2)-DQ6 haplotype was less frequently affected. MRI revealed pituitary hemorrhage in two patients; these two patients suffered sudden loss of vision associated with headache, and they both developed hypopituitarism. Only one patient required long-term hormonal replacement therapy.
Conclusion. CNS-related symptoms and inflammation in the CSF are common in acute NE. Genetic properties of the host may predispose to CNS involvement. It does seem that pituitary injury and subsequent hormonal insufficiency may complicate the recovery.
Key words::
| Abbreviations | ||
| CNS | = | central nervous system |
| CSF | = | cerebrospinal fluid |
| EEG | = | electroencephalography |
| FSH | = | follicle-stimulating hormone |
| fT4v | = | free thyroid hormone |
| HFRS | = | hemorrhagic fever with renal syndrome |
| HLA | = | human leukocyte antigen |
| IGF-I | = | insulin-like growth factor |
| LH | = | luteinizing hormone |
| MRI | = | magnetic resonance imaging |
| NE | = | nephropathia epidemica |
| PCR | = | polymerase chain reaction |
| PUUV | = | Puumala virus |
| TSH | = | thyroid-stimulating hormone |
Key messages
Central nervous system-related findings are common in acute nephropathia epidemica.
Pituitary gland injury and hypopituitarism may complicate the recovery.
Genetic properties of the patient may predispose to the central nervous system involvement.
Introduction
Hantavirus infections causing hemorrhagic fever with renal syndrome (HFRS) are common in Europe and Asia (Citation1,Citation2). Puumala virus (PUUV) causes an HFRS also called nephropathia epidemica (NE) which is widely found in Europe (Citation1). NE is characterized by fever, headache, gastrointestinal symptoms, impaired renal function, blurred vision, and occasionally hemorrhagic complications. Genetic properties of the patient may influence the severity of the illness as demonstrated by HLA haplotype analysis (Citation3). Central nervous system (CNS) symptoms are common in acute NE (Citation4,Citation5), and direct PUUV infection of the CNS has been demonstrated (Citation6,Citation7). Case reports have claimed that pituitary gland injury followed by hypopituitarism is a potential complication of HFRS caused by PUUV or other hantaviruses (Citation6,Citation8–13). A recent retrospective study revealed that patients who had apparently recovered from HFRS may be at high risk of developing hypopituitarism (Citation14).
Nephropathia epidemica is endemic in northern Europe, for example about 5% of the population in Finland or northern Sweden are positive for PUUV antibodies (Citation15,Citation16). Importantly, over half of the elderly men living in rural parts of Finland are positive for PUUV antibodies, demonstrating that a PUUV infection in some cohorts within Europe may indeed be very common (Citation15). In addition, increasing numbers of NE patients are found in central and western Europe, possibly due to the climate change (Citation17,Citation18). This high frequency of the infection combined with the potential for causing serious complications, such as pituitary hemorrhage followed by hypopituitarism, is worrying; the burden of possible long-term sequelae or decreased quality of life may be significant.
The aims of our current study were to estimate the frequency and the nature of the CNS involvement in NE in a prospective manner. The major emphasis was to investigate the potential of hemorrhagic pituitary gland complications during the acute phase of the illness. In addition, we included HLA typing to the protocol for detection of possible associations with the CNS-related findings.
Materials and methods
Study design and patients
The study patients were recruited among those hospitalized for acute NE at the Oulu University Hospital in northern Finland between September 2005 and February 2008, during which a total of 2851 patients were tested for PUUV antibodies (population 380,000). In all, 529 patients had an antibody finding consistent with acute NE, and 154 patients had been hospitalized according to the discharge register. The microbiology laboratory informed the clinician about hospitalized patients with an acute-phase PUUV antibody finding, and a clinician (TH or HK) contacted the newly diagnosed patients whenever this was possible to fit with their daily schedule. Some patients may not have been contacted because of the absence of the study doctors, a brief hospitalization period, or other technical reasons. A total of 58 patients with symptoms and serology consistent with acute NE volunteered to participate in the study.
The study protocol was explained to each patient, and they had the right to refuse or withdraw from the study according to the Helsinki Declaration. In addition, the patients had the possibility to take part in some components of the study; some patients, for example, chose not to undergo lumbar puncture or magnetic resonance imaging (MRI) of the brain. The study was approved by the Ethics Committee of the Oulu University Hospital, and all participants signed an informed consent form.
First, the patients were evaluated for their clinical history and symptoms. Symptoms potentially related with CNS involvement (headache, nausea/vomiting, dizziness, light sensitivity) were requested and recorded. MRI of the brain with a special emphasis on the pituitary gland was performed (45 patients). Cerebrospinal fluid (CSF) samples were obtained from 42 patients an average of 7 days (3–13 days) after the onset of fever. The lumbar puncture was delayed until the thrombocyte count had recovered to at least 50 × 109/L. Blood and urine samples (58 patients) were collected. Electroencephalography was recorded in 33 patients. The patients with an abnormal hypophyseal MRI finding were analyzed by an endocrinologist (6 patients).
Virological and immunological analysis
The laboratory diagnosis of NE was based on PUUV serology that was initially analyzed using a commercial enzyme-linked immunosorbent assay of IgM antibodies (Reagena Puumala IgM EIA Kit, Reagena, Toivala, Finland). In selected cases, the samples were also analyzed with an indirect immunofluorescence test for PUUV IgG which displayed a granular staining pattern in cases of typical acute infection (Citation19). In addition, the CSF and serum PUUV IgM and IgG antibody levels were later titrated as described below. The CSF samples were analyzed for cell count and also glucose and protein concentrations. The PUUV IgM levels were titrated starting with a dilution of 1:20 for CSF and 1:200 for serum, using the µ-capture IgM EIA test as previously described (Citation20), with the end-point titer calculated as the last dilution producing an absorbance value above the cut-off; and the IgG titer was determined with an immunofluorescence assay as previously described (Citation21,Citation22), with a starting dilution of 1:1 to 1:4 for CSF (depending on the amount of CSF available) and 1:20 for serum samples.
RNA was extracted from CSF and serum with QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The detection of PUUV S RNA was performed essentially as described earlier (Citation23) using reverse-transcriptase polymerase chain reaction (RT-PCR) and nested PCR with slightly modified primers (available upon request). The PCR amplicons were agarose-gel-purified with QIAquick Gel Extraction kit (Qiagen), and sequencing was performed automatically, using ABI PRISM™ Dye Terminator sequencing kit (Perkin Elmer, ABI, NJ).
Magnetic resonance imaging
Magnetic resonance imaging (MRI) of the brain and the pituitary gland was performed using a 1.5 T magnet. The T1-weighted sagittal and coronal images were obtained from the pituitary gland before and after intravenous administration of contrast agent. From the whole brain T2, T2-flair, and diffusion-weighted axial images were obtained together with post-contrast T1-weighted images. Two radiologists evaluated the images independently, after which a consensus reading was performed. Attention was paid to the pituitary gland, especially for signs of hemorrhage. Abnormal cerebral signal intensities and contrast enhancement were registered, as well as abnormal meningeal enhancement. The medical records of the patients were available for the radiologists.
Endocrinology
Patients with any abnormal pituitary imaging finding in the MRI were evaluated for the possibility of hormonal deficiency. The patients were evaluated for clinical history and signs of hormonal deficiencies, and they underwent laboratory investigation for thyroid, adrenal, and sex hormone concentrations. Serum free thyroid hormone (fT4v), serum thyroid-stimulating hormone (TSH), serum cortisol from an early morning sample, serum insulin-like growth factor (IGF-I), total serum testosterone, serum luteinizing hormone (LH), serum prolactin, and serum follicle-stimulating hormone (FSH) concentrations were analyzed.
Electroencephalography
A 30-min period of electroencephalography (EEG) was recorded according to the international 10–20 system with additional T1 and T2 electrodes. The EEG system used was the NicoletOne (Nicolet Biomedical, Viasys Healthcare, San Diego, USA) with a sampling rate of 512 Hz. The band-pass filter was 0.5–70 Hz. Standard reference and bipolar montages were utilized during the EEG interpretation.
HLA typing
Class II HLA-DR-DQ haplotypes were defined using a low-resolution full-house genotyping method covering the common Caucasian class II haplotypes, including also DR4 subtypes. The method using lanthanide-labeled sequence-specific oligonucleotide-probe hybridization in the microtiter-plate format and time-resolved fluorometry-based reading has been previously described in detail (Citation24). The presence of the HLA-B27 allele was typed using an assay combining asymmetric PCR amplification and homogeneous hybridization (Citation25).
Ophthalmology
The two patients with pituitary hemorrhage were examined by an ophthalmologist since they experienced a sudden loss of vision. The ophthalmic examination consisted of evaluation of visual acuity and refraction, intraocular pressure measurement, slit-lamp examination, dilated funduscopy, and analysis of visual fields. The patients were re-examined after the clinical recovery.
Results
CNS symptoms and clinical character of the patients
In all, 51 of the 58 patients (87%) suffered symptoms suggestive of CNS involvement (headache, light sensitivity, nausea/vomiting, or dizziness), i.e. only 7 patients did not complain any of these symptoms. Severity of the CNS symptoms was not graded. Two patients experienced symptoms indicative of encephalitis (vomiting, headache, somnolence, confusion). Two patients experienced an abrupt and complete loss of vision from which they recovered spontaneously. Despite of the loss of vision, the clinical course of these two patients did not significantly differ from the rest of study population during the acute phase of the illness. Clinical parameters of the patients are summarized in .
Table I. Clinical characteristics of all 58 patients and those with normal (17 patients) or abnormal (25 patients) CSF findings or any MRI pituitary finding (MRI+, 6 patients). Hemoglobin concentration (Hb, g/L), thrombocyte count (×109/L), white blood cell (WBC) count (×109/L), and blood pressure (BP) at the time of hospital entry are shown. The highest values of plasma creatinine (mmol/L) and C-reactive protein (CRP, mg/L) during the hospitalization are given. None of the patients required dialysis.
MRI of the brain
MRI of the brain with a specific focus on the pituitary gland was used to screen the patients for a potential pituitary injury. The imaging was determined to be normal in 24 of the 45 patients that underwent the MRI. In 15 of the patients with an abnormal MRI finding, the imaging results were judged to be unspecific without an obvious connection to the acute NE, or the findings were attributed, for example, to a previous atherosclerotic event in two patients. None of the patients showed any signs of intracranial bleeding outside of the pituitary. In six patients, the MRI revealed an abnormality of the pituitary gland. In two patients, the MRI showed acute pituitary hemorrhage (). In four patients, the pituitary MRI findings were unspecific: uneven distribution of the contrast media (two patients), suspected pituitary edema (one patient), and a suspected pituitary cyst (one patient). None of the patients had imaging evidence of encephalitis. MRI was repeated in patients with any pituitary abnormality.
Figure 1. T1-weighted sagittal (A) and coronal (B) magnetic resonance images in a 34-year-old male patient displayed increased signal intensity in the pituitary gland consistent with hemorrhage. The patient developed hypopituitarism in the acute phase of the illness. Three months later (C and D) the pituitary gland had decreased in size, the signal intensity had normalized, as had his hormonal values.
CSF analysis and PUUV sequences
The CSF samples (n = 41) were analyzed for total cell count, protein concentration, PUUV IgM, PUUV IgG, and PUUV RNA. CSF pleocytosis was observed in 12 patients (mean 8.1/mm3, range 4–35/mm3), and the CSF protein concentration was elevated in 19 patients (mean 987 mg/L, range 519–3265 mg/L). In eight patients both pleocytosis and elevated protein concentration were seen. PUUV RNA was detected by RT-PCR in 1 CSF sample only, while the amplification of 12 serum samples was positive. White cell count and protein concentration were within normal limits in the CSF sample positive for PUUV RNA. The PUUV S segment sequences derived from these 12 patients were typical Finnish PUUV sequences, and no sequence fingerprints specific to these strains could be assigned.
In half of the patients (n = 21), PUUV IgM antibodies were detectable in the CSF. The PUUV IgG titers in serum were very high (typically titers were thousands), and although IgG was also detected in the CSF, the serum/CSF ratio was not considered to be pathological. PUUV IgM was positive in 25 CSF samples. In 10 patients, in whom both serum and CSF PUUV IgM end-point titers were obtained, the serum/CSF PUUV IgM ratio was in the range of 80 to 1250. Normally, the ratio of serum/CSF IgM concentrations is about 7000 (Citation26). The CSF PUUV IgM positivity correlated with the presence of inflammatory cells (P = 0.008, chi-square test) and an elevated protein concentration (P < 0.0001, chi-square test) in the CSF. Any abnormality of the CSF (pleocytosis, elevated protein concentration, or presence of PUUV IgM) was associated with high plasma creatinine concentration ().
Endocrinology
The six patients with a pituitary abnormality in their brain MRI were evaluated for the possibility of hormonal dysfunction. Four of the six patients were determined to have completely normal hormonal values. Two patients had laboratory findings consistent with a pituitary injury and panhypopituitarism. These two male patients, in whom the MRI revealed hemorrhage of the pituitary gland, had suppressed TSH, fT4v, cortisol, and testosterone values. The hypopituitarism recovered spontaneously in one patient within 3 months (34-year-old male, ). The other patient (51-year-old male) has continued to require hormonal substitution for at least the following 2 years.
Ophthalmology
An ophthalmologist examined the two patients with the complete loss of vision. MRI examination revealed pituitary hemorrhage in both patients who also experienced a sudden, transient, and complete loss of vision for less than 5 minutes associated with severe headache, vomiting, and dizziness immediately before their hospitalization. At the time of the ophthalmic evaluation, their ocular symptoms had markedly improved. The slit-lamp examination, the dilated funduscopy, and the visual field test revealed normal findings in both patients. Acute phase examination revealed diminished visual acuity, myopization (−0.50/−0.50 and −1.0/−0.75 diopters), and a significant decrease in intraocular pressure (7/6 mmHg (acute phase), 14/16 mmHg (control); and 7/8 mmHg (acute phase), 12/12 mmHg (control)) compared to the control evaluation after clinical recovery. These kinds of ocular findings, however, are commonly observed in NE (Citation4,Citation27), and they did not explain the transient vision loss in the patients.
Electroencephalography
Electroencephalography was analyzed from 33 patients, and all of the traces were determined to be within normal limits. In three patients minor unspecific increases in delta and theta activity were recognized. These findings did not require control registration based on clinical judgment.
HLA genotypes
Analysis of class II HLA-DR-DQ haplotypes among patients revealed an increased frequency of HLA-(DR3)-DQA1*05-DQB1*02 or DR3-DQ2 haplotype (17/94, 0.181) compared to the frequency of 0.076 in 1244 control haplotypes from a diabetes family study in Finland (Citation24) (P = 0.001, chi-square test with Yates’ correction). A total of 16 of the 47 (34%) patients were positive for this haplotype compared to 91/622 (14.6%) among the affected family-based artificial controls (P = 0.001). The proportion of HLA-(DR15)-DQB1*0602 or DR15(2)-DQ6 was low among the patient haplotypes (0.085) compared to the control haplotypes (0.161), although this observation did not reach statistical significance (P = 0.071). The HLA-B27 allele was found in 5 of 44 successfully typed patients (11.4%). This figure is close to that reported in an extensive Finnish control series (14.4%) (Citation28).
No significant associations were found between positivity for DR3-DQ2 haplotype or B27 allele and gender, length of hospitalization, CNS involvement, abnormal CSF, presence of PUUV IgM in CSF, or CSF protein concentration. However, none of the 6 patients with DR15(2)-DQ6 haplotype had an abnormal CSF finding compared to 17 of 27 patients without this haplotype (P = 0.019); they had less often PUUV IgM in their CSF (P = 0.014) and had lower CSF protein concentrations (P = 0.003, Mann-Whitney U-test).
Discussion
Previous case reports and retrospective studies have revealed the possibility that pituitary hemorrhage followed by hormonal insufficiency may complicate acute HFRS (Citation6,Citation8–13). This complication may potentially be suffered by a substantial number of people living in or visiting endemic regions. Therefore, analysis of the frequency and nature of the complication is warranted. As far as we are aware, this report is the first to have explored the question in a prospective manner.
Our prospective analysis indicates that pituitary hemorrhage followed by panhypopituitarism is a possible complication of acute NE. Interestingly, one patient with pituitary hemorrhage made a full recovery (), and only one patient required long-term hormonal substitution. In addition, the patient with suspected pituitary edema in the acute-phase MRI did not develop any hormonal dysfunction. In contrast, Stojanovic et al. reported a high incidence of pituitary insufficiency in their previous retrospective analysis of HFRS survivors (Citation14). The type of hantavirus causing the HFRS was not specified in their study, and it seems possible that infection caused by Puumala and Dobrava viruses may have different outcomes (Citation1). For example, a rather high number of their patients required dialysis. Our patients, on the other hand, represented the severely ill NE patient population; they required treatment at a tertiary care hospital, and they had an increased frequency of the HLA-DR3 haplotype, which is associated with severe NE (Citation29).
We performed 45 brain MRI studies; a high number of them revealed abnormalities that were most likely not associated with any specific disease. Two hemorrhagic lesions were found, both in the pituitary gland. Both patients exhibiting the pituitary injury in their MRI suffered significant ocular problems: they had transient and complete loss of vision accompanied by severe headache. Our previously published fatal NE patient with pituitary hemorrhage, in whom we detected viral invasion of the gland, also suffered from transient vision loss in his left eye prior to death (Citation6). This kind of sudden and complete loss of vision with no light perception is completely different from the diminished visual acuity that is very typically seen during acute NE. This type of vision loss may be related to involvement of the chiasm itself, its blood supply, the adjacent optic nerve, or the optic tract. Importantly, a history of vision loss should alert the clinician to consider the possibility of pituitary injury in a NE patient.
We collected CSF samples from 42 patients, and 22 of them exhibited either pleocytosis or an elevated protein concentration. In addition, over half of the CSF samples were positive for PUUV IgM and the PUUV IgM serum/CSF ratio was high (Citation26), which may be a result of intrathecal antibody production although the possibility of leakage from the circulation cannot be ruled out. The elevated white cell count and the high protein level in the CSF had a correlation with the presence of CSF PUUV IgM, confirming that the CNS inflammation was definitely directly attributable to the PUUV infection. Furthermore, a significant negative correlation between the CSF inflammation and HLA-DR15(2)-DQ6 haplotype was found, indicating that the genetic properties of the host may have an important role in the CNS involvement. However, this finding will need to be confirmed in a larger patient population. PUUV RNA was found in only one CSF sample which was obtained from a mildly symptomatic patient 5 days after the onset of fever (Citation7). Lumbar puncture was delayed in many patients with severe symptoms and significant thrombocytopenia. For example, the CSF sample was obtained 10 days after the onset of fever from the patient that developed lasting hormonal deficiency. It is possible that the presence of the virus in the CSF should be sought during the very early phases of the illness.
Our current study suggests that the number of patients with acute pituitary injury leading to long-term hormonal substitution may not be very high after HFRS caused by PUUV. Nonetheless, it is possible that direct PUUV invasion of the pituitary gland seen in our previous study may play a role in the hormonal dysfunction (Citation6). It can only be speculated that the PUUV invasion without hemorrhage during the acute phase of the illness could injure the neuroendocrine cells, e.g. through inflammatory mechanisms and consequently lead to a slowly developing hormonal deficiency. There is some evidence to suggest that viral or bacterial meningitis, for example, may evoke this kind of phenomenon (Citation30). Slowly developing pituitary damage would also explain the high number of patients requiring hormonal substitution in the previous retrospective study (Citation14). A longer-term follow-up and investigation of patients recovering from HFRS caused by PUUV will need to be undertaken to answer this question.
Acknowledgements
The study was supported by the Finnish Cultural Foundation, Paulo Foundation, Sigrid Jusélius Foundation, EVO grant from Oulu University Hospital, and a grant from the Hospital District of Helsinki and Uusimaa (EVO/TYH6215 and TYH 2008309).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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