Impact of SARS-CoV-2 on the nervous system.

INTRODUCTION: The ongoing COVID-19 pandemic is the largest global public health struggle. The spread of the novel coronavirus had resulted in almost 7 million deaths worldwide by January 2023. STATE OF THE ART: The most common symptoms during the acute phase of COVID-19 are respiratory. However, many individuals present various neurological deficits at different stages of the infection. Furthermore, there are post-infectious complications that can be present within weeks after the initial symptoms. Both the central and peripheral nervous systems (CNS and PNS, respectively) can be affected. Many potential mechanisms and hypotheses regarding the neuropathology behind COVID-19 have been proposed. CLINICAL IMPLICATIONS: The distribution of neurological symptoms during COVID-19 infection among studies differs greatly, which is mostly due to differing inclusion criteria. One of the most significant is incidence involving CNS circulation. In this review, we present basic information regarding the novel coronavirus, the possible routes along which the pathogen can reach the nervous system, neuropathology mechanisms, and neurological symptoms following COVID-19. FUTURE DIRECTIONS: It seems that many factors, resulting both from the properties of the virus and from systemic responses to infection, play a role in developing neurological symptoms. The long-term effect of the virus on the nervous system is still unknown.

Case report: severe course of encephalitis of unknown origin.

The exact cause of encephalitis is still unclear in many cases, although the common etiological factors of this process are viruses such as herpes simplex virus and rabies virus, and also bacteria, fungi, parasites, several medicines and autoimmune diseases. Herein, we report a case of a 56-year-old man with a history of amnestic syndrome, impaired consciousness, somnolence throughout the day, headache, dizziness and hypertension, who was admitted to hospital with suspected neurological disease, and imaging features that were consistent with encephalitis of unknown etiology. Methods which were used to examine patient: cerebrospinal fluid testing, PCR examinations for viruses, testing of antibodies against surface antigens, magnetic resonance imaging of the head, psychiatric consultation, oncology consultation. The objective of this study is to demonstrate a case about an uncommon neurologic condition, which every clinician might meet in clinical practice. In this type of cases, the use of steroids such as dexamethasone and methylprednisolone might lead to a full recovery.

Evaluation of imaging methods in cerebral toxoplasmosis.

Cerebral toxoplasmosis is a parasitic disease resulting, in most cases, from a reactivation of a latent cyst with Toxoplasma gondii. The disease mainly affects immunosuppressed individuals, such as HIV (human immunodeficiency virus)-infected patients. Diagnosis is based on specialized antibody testing, clinical symptoms, neuroimaging methods, and histological examination. The gold standard for diagnosis is a brain biopsy, but more often the response to treatment seen in clinical symptoms and neuroimaging studies is sufficient. The imaging features support the diagnosis of cerebral toxoplasmosis and help assess the effectiveness of treatment.

COVID-19 pandemic in Podlaskie Voivodeship - analysis of infection and deaths in 2021.

INTRODUCTION: The course of COVID-19 pandemic in specific regions is affected by a variety of factors. Out of them, the one of the most significant is vaccination coverage among the population. This parameter in the first year of the vaccination program (2021), was particularly low in Podlaskie Voivodeship, compared to the whole country. AIM OF THE STUDY: The aim of this study is to trace the factors influencing the course of the COVID-19 pandemic in Podlaskie Voivodeship in 2021 in order to better prepare the region for possible future waves of COVID-19 infection. MATERIAL AND METHODS: The paper is based on a retrospective analysis of mortality and incidence of COVID-19 in the Podlaskie Voivodeship and Polish population in 2021. The data was obtained from the ministerial platform - BASiW, and Statistics Poland (GUS). A similar analysis was also performed for the University Clinical Hospital in Bialystok (later to be referred as USK), using its own patient data. RESULTS: COVID-19 mortality rate in 2021 was 10% higher in Podlaskie Voivodeship than in the general population of Poland. There is a slight positive correlation between this indicator and multigenerational family occurrence (i. e. proportion of households with inhabitants older than 65, which is significantly higher in Podlaskie). However, the main reason for the excess deaths was probably the lower immunization rate of Podlaskie Voivodeships inhabitants than of Poles in general - 41,6% at the beginning of autumn wave (37th week of the year) vs. 50,3%. It was also shown that the vaccine in the Podlaskie Voivodeship population is less effective in reducing the risk of infection and death from COVID-19 than in the nationwide population, but this is not due to the significant differences in Podlaskie Voivodeships health status or demographics. For unknown reasons, women of working age in Podlaskie Voivodeship turn out to be less likely to die from COVID-19 than a similar group in the entire Polish population, while men - more. In the autumn wave of cases in 2021 (the fourth wave of the pandemic in general) compared to the spring (III) wave, an increase in the mortality of patients hospitalized with COVID-19 in the USK in Bialystok was from 12% to 19%. CONCLUSIONS: Considering the weakened efficacy of the COVID-19 vaccine in Podlaskie Voivodeship, more emphasis should be placed on the prevention of civilisation diseases and the sanitary regime in the elderly population, so that the next waves of the pandemic do not bring excess deaths.

Assessment of anti-HSV antibodies in patients with facial palsy in the course of neuroborreliosis.

AIM: There are many causes of facial nerve palsy. The most common causes are neuroborreliosis (NB), idiopathic paralysis or Herpes simplex virus (HSV) reactivation. The aim of this study was to characterize patients with facial palsy in the course of NB and to determine whether HSV-1 reactivation takes place during the acute phase of NB. METHODS: A retrospective analysis of 66 patients with facial nerve palsy was performed. In 38 patients, facial palsy was caused by Borrelia burgdorferi sl infection. Immunological tests for HSV-1, tick-borne encephalitis virus and B burgdorferi sl in serum and cerebrospinal fluid (CSF) were performed. RESULTS: In this analysis, 55.2% of NB patients had right nerve palsy and 21% bilateral palsy; 15.8% of patients had erythema migrans (EM). Lymphocytic meningitis was diagnosed in 92% of patients and Bannwarth's syndrome was diagnosed in 47% of patients. IgM anti-HSV-1 antibodies were detected in four patients with NB and two patients with facial nerve palsy of other origin. IgM anti-HSV-1 antibodies were detected in the CSF of three patients (7.9%) with NB, and one of them had bilateral VII paresis and EM simultaneously. Treatment with ceftriaxone or doxycycline led to complete recovery. CONCLUSIONS: Neuroborreliosis should always be considered as a cause of peripheral facial nerve palsy. Peripheral facial nerve palsy is a significant symptom in the course of NB, especially in patients accompanied by meningitis. Pathomechanism of facial nerve paresis has not been well explained so far and may depend on two independent mechanisms in NB, including HSV-1 reactivation.

Assessment of the potential effect of the implementation of serological testing tick-borne encephalitis on the detection of this disease on areas considered as non-endemic in Poland - preliminary report.

INTRODUCTION: In Poland, the number of reported cases of tick-borne encephalitis, and thus the designation of the regions of TBE occurrence, seems to be underestimated. AIM OF THE STUDY: The aim of the study was to evaluate the impact of the implementation of TBE virus infection tests in the routine diagnostics of patients with neuroinfections of undetermined viral etiology on the identification of TBE virus infections in areas considered non-endemic and finding new areas of TBE occurrence. MATERIAL AND METHODS: Twenty-nine departments in which patients with suspected neuroinfections are hospitalized participated in the study. The criterion for selecting the center was the location in an area considered non-endemic for TBE, where reporting is low or absent, and intermediate data indicate the possibility of undiagnosed disease (TBE). Diagnostics were performed in the Immunoserology Laboratory at the Department of Infectious Diseases and Neuroinfections of the Medical University of Bialystok using the ELISA method. The cooperation was undertaken with infectious wards or patients with suspected neuroinfection who are hospitalized and diagnosed (e.g., neurology ward). The supervising unit is the Department of Infectious Diseases and Neuroinfections of the Medical University of Bialystok, ul. Zurawia 14, 15-540 Bialystok. For testing the submitted serum and CSF samples by ELISA method were used commercial kits from Virotech (Germany). RESULTS: A total of 577 samples from 417 patients were tested, including 290 serum samples and 287 CSF samples. Serum antibodies against TBE were detected: IgM class in 27 samples, IgG class in 22 samples; in CSF: IgM class in 39 samples, IgG in 21 samples. The etiology of TBE was confirmed in 55 cases, i.e. in 13.19% of all tested people. CONCLUSIONS: 1. Detection of the presence of antibodies against TBE in samples of patients with meningitis reported as other neuroinfections indicates the etiology of TBE. 2. The number of TBE cases may be undiagnosed, and thus underestimated due to the failure to perform serological tests for TBE in areas considered non-endemic. 3. The diagnosis and reporting of neuroinfections caused by the TBE virus is essential for a proper risk assessment and in promoting prophylaxis in the form of vaccinations. 4. Preliminary results of the research indicate the need for their continuation in all voivodeships.

Analysis of CCL-4, CCL-17, CCL-20 and IL-8 concentrations in the serum of patients with tick-borne encephalitis and anaplasmosis.

PURPOSE: Tick-borne co-infections are a serious epidemiological and clinical problem. Only a few studies aimed to investigate the effect of tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) co-infection in the course of the inflammatory process and the participation of chemokines in the pathomechanism of these diseases. The aim of the study was to evaluate CCL-4, CCL-17, CCL-20, and IL-8 serum concentrations in patients with HGA, TBE and HGA + TBE co-infection. METHODS: Eighty-seven patients with HGA (n = 20), TBE (n = 49) and HGA + TBE (n = 18) were included to the study. The control group (CG) consisted of 20 healthy people. Concentrations of cytokines were measured in serum using commercial ELISA assays. In patients with TBE and HGA + TBE inflammatory markers were assessed during the acute and convalescent period. The results were analyzed using non-parametric tests with p < 0.05 considered as significant. RESULTS: Before treatment, significantly higher concentrations of IL-8, CCL-4 and CCL-20 were observed in HGA patients. CCL-4 and CCL-20 concentrations were significantly higher in TBE patients compared to CG. Concentrations of IL-8, CCL-4, and CCL-20 were significantly higher in HGA + TBE than in CG. After treatment, a significant reduction of IL-8, CCL-4, and CCL-20 concentrations in TBE patients and IL-8 in HGA + TBE co-infection was observed. CCL-4 concentration was higher in HGA + TBE co-infection in comparison to patients with TBE after treatment. CONCLUSIONS: Our study confirms that concentrations of IL-8, CCL-4, and CCL-20 are increased in the course of HGA and TBE. Their concentrations in serum may be used to monitor the course of TBE and HGA, as well as possibly detect co-infections with the diseases.

Assessment of the tau protein concentration in patients with tick-borne encephalitis.

There have been suggestions that tick-borne encephalitis (TBE) may cause neurodenenerative changes in the brain. The aim of this study was the assessment of the tau protein concentration in cerebrospinal fluid (CSF) of patients with different clinical forms of TBE. The concentration of tau protein in CSF was determined using Fujirebio tests (Ghent, Belgium) in 35 patients with TBE: group I-patients with meningitis (n = 16); group II-patients with meningoencephalitis (n = 19). None of the patients reported any neurodegenerative disorder that could affect the results of the study. The control group (CG) consisted of 10 patients in whom inflammatory process in central nervous system was excluded. Tau protein concentration in CSF before treatment did not differ significantly between the examined groups, while its concentration was significantly higher in encephalitis group than in CG after 14 days of treatment. Significant increase in tau protein concentration after treatment was observed in both examined groups. The comparison between the group of patients who fully recovered and patients who presented with persistent symptoms on discharge showed significant differences in tau protein concentration before and after treatment. ROC curve analysis indicates that CSF tau protein concentration before treatment may predict complicated course of the disease with 90.9% specificity and 80% sensitivity, while after treatment, specificity became 72.7% and 71.4% for sensitivity. Correlation analysis showed that in TBE patients (both meningoencephalitis and meningitis groups), CSF pleocytosis before treatment correlated negatively with tau protein concentration in CSF. (1) Neurodegeneration process is present in TBE encephalitis. (2) Tau protein concentration may be used as a predictor of complicated course of TBE.

Assessment of TLR-2 concentration in tick-borne encephalitis and neuroborreliosis.

The aim of the study was to check whether measurement of TLR-2 in serum or cerebrospinal fluid (CSF) can help differentiate between neuroborreliosis (NB) and tick-borne encephalitis (TBE). Eighty patients with meningitis and meningoencephalitis were divided into two groups: Group I - patients with NB (n = 40) and Group II - patients with TBE (n = 40). Diagnosis was based on the clinical picture, CSF examination and presence of specific antibodies in serum and CSF. The control group (CG) consisted of healthy blood donors (n = 25) and patients in whom inflammatory process in central nervous system was excluded (n = 25). Concentration of TLR-2 was measured using a commercial kit [TLR-2 Elisa Kit (EIAab, China)]. The serum and CSF TLR-2 concentration of NB patients was significantly higher than in CG. The serum and CSF TLR-2 concentration in TBE patients was significantly higher than in the CG. Receiver operating characteristic analysis of the serum TLR-2 concentration showed significant differences between the group of patients with NB and a group of patients with TBE. TLR-2 is involved in the development of inflammatory process in the CNS caused by both tick-borne pathogens: viral and bacterial as TLR-2 concentration in both CSF and serum differentiates these groups from healthy patients. Although TLR-2 cannot be used as a sole and reliable biomarker differentiating NB from TBE, results of our study are a step forward toward discovering such biomarker in the future.

Sequelae of tick-borne encephalitis in retrospective analysis of 1072 patients.

Tick-borne encephalitis (TBE) is an emerging vector-borne disease in Europe. The aim of the study was to evaluate sequelae and to analyse the potential risk factors predisposing to sequelae development. We performed a retrospective analysis of medical records of 1072 patients who received a 1-month follow-up appointment after hospital discharge. Medical data, such as patients' age, gender, place of living, subjective complaints, neurological and psychiatric sequelae were evaluated twice: at the moment of discharge and at follow-up visits 1 month after discharge. We observed that sequelae may affect 20.6% of TBE patients. Subjective sequelae were more frequent than subjective complaints during the hospitalisation (P < 0.001), while objective neurological symptoms during the hospitalisation were more pronounced than neurological sequelae (P < 0.001). Patients with meningoencephalomyelitis were predisposed to neurological complications, while subjective symptoms were more common in meningoencephalitis. Independent risk factors for sequelae development were: age and cerebrospinal fluid (CSF) protein concentration. The risk of late neurological complications persisting was increased in patients with higher CSF protein concentration. Based on the results of our study we concluded that, there is a need for a better vaccination program, which would prevent the development of sequelae.

Herpesviridae Seropositivity in Patients with Multiple Sclerosis: First Polish Study.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system that leads to inflammation, demyelination and neurodegeneration. Viral aetiology has been suspected to be an MS trigger for a long time, and herpesviruses (HSs) are among the potential pathogens involved. OBJECTIVES: The present investigation aims to detect the presence of antibodies against the herpes simplex virus (HSV), varicella-zoster virus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV) and human herpesvirus 6 (HHV6) in the serum of MS patients and control individuals in north-eastern Poland. METHOD: Plasma was collected from 141 MS patients and 44 blood donors who served as the control group. These individuals were assessed for the presence of antibodies using an enzyme-linked immunosorbent assay. RESULTS: The statistical analysis showed a higher probability of EBV (p = 0.037, OR 4.359) and HHV6 (p = 0.020, OR 3.343) antibody presence in patients with MS compared to that in the control group. In the MS patient group, the prevalence of CMV IgG antibodies was significantly higher in females (p = 0.025). Patients who tested positive for anti-EBV IgG were diagnosed 7.9 years earlier than patients who tested negative for anti-EBV IgG (p = 0.048). CONCLUSIONS: The study showed that MS patients in north-eastern Poland were more likely to be seropositive for EBV and HHV6 than healthy individuals. Further work should be undertaken in other regions of Poland and other European countries with particular attention paid to testing seropositivity in all HSs, particularly in the MS patient population, to evaluate the impact of HSs on MS patients in different environments.

Tick paralysis

Tick paralysis is caused by neurotoxins secreted by adult female ticks, primarily in North America and on the east coast of Australia. Sporadic illness is also recorded in Europe and Africa. In the European countries, including Poland, there are 6 species of ticks capable of causing tick paralysis. The disease occurs in people of all ages, but is most commonly diagnosed in children under 8 years of age. Paralysis can take different forms - from rare isolated cranial nerve infections to quadriplegia and respiratory muscles paralysis. After the tick remove, the symptoms resolve spontaneously. In severe cases with paralysis of respiratory muscles, when there is no possibility of mechanical ventilation, the disease may lead to death.

Acrodermatitis chronica atrophicans: various faces of the late form of Lyme borreliosis.

INTRODUCTION: Acrodermatitis chronica atrophicans (ACA) is probably the most common late and chronic manifestation of the Lyme borreliosis seen in European patients. AIM: To analyze epidemiological data, and to investigate the effects of treatment of patients with ACA. MATERIAL AND METHODS: Nine patients were included in the study. All patients had serological examinations (ELISA and Western blot) and histopathological examination of the skin lesions performed. Eight patients had PCR in the skin biopsy performed. RESULTS: The duration of symptoms ranged from 2 months to 2 years. In 7 patients, skin lesions were located on lower limbs, in 2 patients - in a non-typical body area - abdomen. In 1 patient, scleroderma and in 3 patients, diabetes mellitus was diagnosed. Borrelia burgdorferi DNA was detected in 25% of the skin biopsy specimens. IgG anti-B. burgdorferi specific antibodies were present in serum of all patients (confirmed by Western blot). In all cases, the diagnosis was confirmed by histopathological examination. The response to ceftriaxone therapy varied. In 5 cases, the lesions resolved completely, in others they faded. CONCLUSIONS: Despite raising awareness of Lyme borreliosis, late forms of the disease such as ACA are still observed. Acrodermatitis chronica atrophicans skin lesions may be located in non-characteristic areas, e.g. abdominal skin. Symptoms are not irritating or painful, therefore patients do not seek medical help. The effect of antibiotic treatment varies.

Evaluation of NF-κB concentration in patients with tick-borne encephalitis, neuroborreliosis, anaplasmosis and Anaplasma phagocythophilum with tick-borne encephalitis virus co-infection.

OBJECTIVES: The aim of the study was the evaluation of NF-κB concentration in serum and cerebrospinal fluid (CSF) of patients with diagnosis of tick-borne diseases: tick-borne encephalitis (TBE), neuroborreliosis (NB), anaplasmosis (ANA) and patients co-infected with tick-borne encephalitis virus and Anaplasma phagocythophilum (TBE+ANA). Additionally NF-κB concentration during acute and convalescent period was compared. METHODS: Sixty-seven patients with diagnosis of tick-borne diseases were included in the study. The control group (CG) consisted of 18 patients hospitalized because of headaches and had lumbar puncture performed. The NF-κB was measured by human inhibitory subunit of NF-κB ELISA Kit during acute and convalescent period. RESULTS: In serum the significant differences were observed only in patients with TBE+ANA co-infection. In CSF the concentration of NF-κB was significantly higher in patients with TBE, TBE+ANA co-infection, and patients with NB than in CG. Receiver operating characteristic (ROC) curves analysis showed that NF-κB concentration in CSF differentiated patients with NB with CG; patients co-infected with TBE and ANA with CG and patients with TBE with CG. NF-κB concentration in serum differentiated patients co-infected with TBE and ANA with NB and with ANA, with TBE and with CG. In TBE group the serum NF-κB concentration significantly decreased in convalescent period, while in NB and TBE groups significant CSF decrease of NF-κB concentration was observed.

Usefulness of copeptin as a potential biomarker in TBE.

OBJECTIVE: The aim of the study was to evaluate the usefulness of copeptin for differentiation of hyponatremia in the course of tick-borne encephalitis (TBE) and for being a prognostic marker of the severity of TBE. MATERIALS AND METHODS: One hundred and fourteen patients with TBE were included in the study. The control group consisted of 62 patients diagnosed with viral meningitis. RESULTS: Copeptin concentration did not differ in patients with hyponatremia and normonatremia. Urinary sodium excretion to plasma copeptin (copeptin/UNa Secretion) ratio was significantly lower in Syndrome of Inappropriate Antidiuretic Hormone (SIADH) Secretion patients than in patients with hyponatremia of other origin. Mean copeptin concentration in TBE patients was higher than in control group (VM) patients. There were no differences between patients with severe and mild course of TBE. CONCLUSIONS: Copeptin/UNa ratio may be used as a potential biomarker of SIADH in patients with TBE. Copeptin concentration is significantly higher in patients with TBE than in viral meningitis of other origin, especially in patients aged 18-34 and >49 years old. Copeptin does not differentiate TBE of mild and severe course.

Analysis of the relationship between single nucleotide polymorphism of the CD209, IL-10, IL-28 and CCR5 D32 genes with the human predisposition to developing tick-borne encephalitis.

<b>Introduction: </b>It is known that in the pathogenesis of tick-borne encephalitis (TBE) various molecules play a significant role. The most prominent factors include IL-10, IL-28B, CD-209 and CCR5. It is reasonable to search for genetic predispositions to the development of various clinical forms of TBE related to the genetic variation of IL-10, IL-28B, CD-209 and CCR5. In this study we aimed to search for the relationship between single nucleotide polymorphism in the promoter region of the CD209, IL-10, IL-28 and 32 base pair deletion in CCR5 coding region (Δ 32) with the human predisposition to development of various clinical presentations of TBE. We tried to assess the relation between the presence of particular alleles and genotypes with laboratory and clinical parameters. <b>Material/Methods </b>59 patients with TBE and 57 people, bitten by a tick who never developed TBE (Polish cohort), were included in the study. To assess the distribution of single nucleotide polymorphisms, TaqMan SNP genotyping assays were used for IL10: rs1800872 and rs1800896, for CD 209 rs4804803 and rs2287886, rs12979860 for IL 28B SNPs according to the manufacturer's protocol using real-time PCR technology on the StepOne thermal cycler. <b>Results </b>Comparison between TBE patients and CG showed that in SNP rs2287886 CD 209 AG heterozygotes were more frequent in the TBE group, while homozygotes GG were more frequent in the CG group. <b>Conclusions </b> SNP rs2287886 CD 209 AG heterozygotes predispose humans to develop TBE. Single nucleotide polymorphism in the promoter region of the CD209, IL-10, IL-28 and CCR5 D32 genes does not correlate with the severity of TBE.

Evaluation of Imaging Methods in Tick-Borne Encephalitis.

Tick-borne encephalitis (TBE) is caused by a virus that belongs to the Flaviviridae family and is transmitted by tick bites. The disease has a biphasic course. Diagnosis is based on laboratory examinations because of non-specific clinical features, which usually entails the detection of specific IgM antibodies in either blood or cerebrospinal fluid that appear in the second phase of the disease. Neurological symptoms, time course of the disease, and imaging findings are multifaceted. During the second phase of the disease, after the onset of neurological symptoms, magnetic resonance imaging (MRI) abnormalities are observed in a limited number of cases. However, imaging features may aid in predicting the prognosis of the disease.

Comparison of hyponatremia and SIADH frequency in patients with tick borne encephalitis and meningitis of other origin.

AIM: The aim of the study was the evaluation of frequency and origin of hyponatremia in tick borne encephalitis (TBE) in comparison to non-TBE viral meningitis and bacterial meningitis. METHODS: A total of 124 patients aged 18-80 years, with TBE were included to the study. The mild form of TBE was diagnosed in 59 patients, while the severe form was diagnosed in 65 patients. The first control group (VMG) consisted of 72 patients with viral meningitis, but excluded TBE. The second control group (BMG) consisted of 16 patients diagnosed with bacterial meningitis. RESULTS: Hyponatremia was diagnosed in 55 (44.4%) patients with TBE. In 12 (9.7%) patients (mean age 56.6 ± 19.9 years; 9 men, 3 women) syndrome of inappropriate secretion of antidiuretic hormone (SIADH) was diagnosed. In VMG hyponatremia was diagnosed in 7 (9.7%) patients. In the age group <35 years and in the age group of 50-64 years the frequency of hyponatremia and SIADH was higher in TBE than in VMG (p < 0.05). In BMG hyponatremia was diagnosed in 6 (37.5%) patients. No statistically significant differences in frequency of hyponatremia between BMG and TBE groups were observed. CONCLUSIONS: (1) Hyponatremia is a common disorder in TBE and is more frequent than in other viral types of meningitis, especially in young patients (< 35 years). (2) The most common cause of hyponatremia in TBE patients is dehydration and fluid supplementation should be a treatment of choice. (3) Overall, 16.9% of the patients with the severe form of TBE develop SIADH syndrome and they required treatment based on fluid restriction and hypertonic saline infusion.