Enlarged periventricular space and periventricular lesion extension on baseline brain MRI predicts poor neurological outcomes in cryptococcus meningoencephalitis

In Cryptococcus neoformans meningoencephalitis, brain MRI findings might reflect the phathomechanism of disease progression that is fungal accumulation in the peri-venular space and consequent invasion into the parenchyma. This study analyzed serial brain MRI findings of 76 patients with cryptococcus meningoencephalitis in association with the disease progression and outcomes. MRI parameters included the enlarged periventricular space (ePVS) score (range 0–8), periventricular lesion extension, cryptococcoma, and hydrocephalus. Clinical outcomes at 2-week, 10-week, and 6-month were evaluated using modified Rankin scale (mRS). At 6 months, 15 (19.7%) patients died and 34 (44.1%) had poor neurological outcomes (mRS scores > 2). At baseline, an ePVS score of ≥ 5 (Odds-ratio [OR]: 94.173, 95% confidence-interval [95%CI]: 7.507–1181.295, P < .001), periventricular lesion extension (OR: 51.965, 95%CI: 2.592–1041.673, P = .010), and presence of encephalitis feature (OR: 44.487, 95%CI: 1.689–1172.082, P = .023) were associated with 6-month poor outcomes. Presence of two or more risk factors among encephalitis feature, ePVS score ≥ 5, and periventricular lesion extension at baseline, was associated with 6-month poor outcomes (area under the curve [AUC]: 0.978, P < .001) and mortality (AUC: 0.836, P < .001). Disease progression was associated with interval development of cryptococcoma and hydrocephalus. Brain MRI findings might be useful in predicting outcomes and monitoring the progression of cryptococcus meningoencephalitis.


Materials and methods
Study subjects. This retrospective cohort study initially included all consecutive individuals admitted to the neurology department of the Seoul National University Hospital between January 2000 and December 2019 who were diagnosed with Cryptococcus meningoencephalitis. Among the initially included 117 individuals, the final study population was defined according to the following criteria: (1) underwent baseline brain MRI evaluation; (2) availability of clinical, treatment, laboratory, and long-term (> 6 months) neurological outcome data. According to the criteria, 33 patients without brain MRI evaluations and eight with inadequate data were sequentially excluded and the remaining 76 individuals were included in the study analysis. Diagnosis of Cryptococcus meningoencephalitis was based on the detection of the Cryptococcus antigen in CSF by latex agglutination or by lateral flow assay along with or without detecting Cryptococcus in CSF by culture or India ink assay 2,14,15 . The design of this study was approved by the institutional review board of the Seoul National University Hospital (SNUH) and the study was performed in compliance with the SNUH IRB regulations and the International Conference on Harmonisation guideline for Good Clinical Practice. Informed consent was waived by the IRB, as this study did not intervene with the evaluation or treatment process of the patients, and the patients were anonymized during the study process.
Clinical and laboratory evaluation. Along with the demographic information, patients' underlying immune status was reviewed and the causes of immunodeficiency were categorized as follows: Human Immunodeficiency Virus (HIV) infection, hematologic malignancy, solid organ cancer, post-transplant status, and longterm use of high-dose immune suppressants (for indications other than cancer treatment or post-transplantation immunosuppression) 4,16 . At baseline, encephalitis feature was defined according to the 2013 Consensus Statement of the International Encephalitis Consortium diagnostic criteria as: (1) altered mental status lasting more than 24 h without an alternative cause and (2) 3 or more of the followings: documented fever (> 38.0 °C); seizures not fully attributable to a preexisting seizure disorder; new onset of focal neurologic findings; CSF WBC count ≥ 5/mm 3 ; and abnormal brain MRI findings suggestive of encephalitis 17 . Baseline GCS score and modified Rankin Scale (mRS) score data were also obtained from the patients' medical records 1 . mRS is a widely used clinical scale for measuring the neurological function and the degree of disability in the daily activities after a stroke or other neurological diseases 18 . mRS ranges from 0 to 6, and each score represents neurological status as follows: 0, no symptoms or disabilities; 1, no significant disability despite some symptoms, which is able to perform all daily activities; 2, slight disability, which is able to carry out own daily activities without assistance, but not all previous activities; 3, moderate disability, which requires some help, but is able to walk unassisted; 4, moderately severe disability, which is unable to carry out own basic daily activities without assistance and unable to walk unassisted; 5, severe disability with bedridden status, which requires constant nursing care and attention; and 6, dead 18 . Nowadays, mRS is also widely used in measuring outcomes for encephalitis [19][20][21][22][23] .
CSF analysis included the evaluation of protein levels, WBC counts, and the elevation in the opening pressure (≥ 20 cmH2O) 8 . CSF Cryptococcus antigen titer was evaluated semi-quantitatively, and high antigen titer was defined as antigen detection in > 1:1 000 dilution 1 .
Treatment profile analysis. Intravenous amphotericin (0.7-1.0 mg/kg/day) with or without flucytosine (100 mg/kg/day) or fluconazole (400-800 mg/kg/day) was used for the induction treatment period (within 2 weeks from the treatment initiation). Oral fluconazole was used during the consolidation (8 weeks after the induction treatment) and long-term maintenance treatment period in most patients (74/76, 97.4%) [2][3][4][5] . Treatment profiles with the durations of each treatment regimen were reviewed.
Outcome analysis. The scores on mRS was obtained at the time of treatment initiation, at 2 weeks, Magnetic resonance imaging analysis. Baseline and follow-up MRI were performed using 1.5-T  Table 1 for the detailed MRI machines and parameters). All the image analysis was separately performed by a neurologist (W-J L., 9 years of experience) and a radiologist (Y R., 9 years of experience), blinded to all clinical data. Consensus was made by discussion for the discrepant cases. ePVS was defined as small, sharply delineated ovoid or linear lesions with T2-hyperintensity and T1-hypointensity. ePVS was semi-quantitatively rated in two brain regions-basal ganglia (BG) and centrum semiovale (CS)-using the scale introduced by Wardlaw et al., ranging 0-4 (0 for no, 1 for 1-10, 2 for 11-20, 3 for 21-40, and 4 for > 40 ePVS). BG-and the CS-ePVS scores were summed up to a total ePVS score ranging 0-8 24 .
Periventricular lesion extension was defined as T2/FLAIR hyperintensity along the lateral ventricular border extending to the periventricular or subcortical parenchyma. Cryptococcoma was defined as single or multiple discrete T2/FLAIR hyperintensity lesions with T1 hypointensity in brain parenchyma 24 . Hydrocephalus was defined as the Evans' index (the ratio of the maximal frontal horn width of lateral ventricle to the transverse inner skull diameter) of ≥ 0.3 (Fig. 1). For the 59 (77.6%) baseline MRIs with available T1 contrast enhancement images, the presence of an evident pathologic contrast enhancement in the brain parenchyma was also evaluated. To evaluate the serial changes in the MRI parameters, follow-up MRIs at 2 weeks (window time of ± 3 days), at 10 weeks (window time of ± 2 weeks), and at 6 months (window time of 5-10 months) were analyzed, if available. Statistical analysis. SPSS 25.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analyses. Data were reported as numbers (percentages), means ± standard deviations, or medians [interquartile ranges, IQR]. In univariate analyses, Pearson's chi-square test and Student's t-test were used. To evaluate factors associated with a poor neurological outcome, logistic regression analyses were performed including the parameters with a P < .10 in univariate analyses using a backward elimination method. Age was included in the final model of every regression analysis. Regression analyses were also performed separately for the subgroup without an encephalitis feature at baseline. Regression analysis for mortality was not performed due to its low frequency. Receiver operating characteristic (ROC) curve were drawn to evaluate the prognostic value of the factors derived from the regression analysis and to designate a cut-off value for predicting a poor outcome. For every analysis, a P value < .05 was considered statistically significant. Inter-reader reliability for the MRI parameters were evaluated using Cohen's κ values.   Table 1. Numbers of patients with magnetic resonance image (MRI) evaluations included at each time points were 76, 40, 49, and 59 at baseline, 2-week, 10-week, and 6-months, respectively.
In the univariate analysis for the factors associated with 6-month mortality, the mortality of 6-month was associated with baseline encephalitis feature (P = .013), elevated CSF opening pressure (P = .032), periventricular lesion extension (P < .001), cryptococcoma (P = .013), and higher scores of GB-, CS-, and total ePVS (all P < .001) in the baseline MRI (Table 3). Due to low frequency (n = 15), multivariate analysis for mortality was not performed.
We compared the clinical profiles and the outcomes between the groups with or without baseline encephalitis feature. The group with baseline encephalitis feature was associated with a higher frequency of HIV infection (P = .015), GCS score < 15 (P < .001), elevated CSF opening pressure (P < .001), the presence of periventricular extension (P < .001), cryptococcoma (P = .015), and hydrocephalus (P = .021) in the baseline MRI, and lower baseline mRS scores and higher scores of GB-, CS-, and total ePVS, compared to the group without encephalitis feature (all P < .001). The 6-month mRS score was also lower in the subgroup with baseline encephalitis feature (P < .001, Supplemental Table 2).
Risk score for a poor 6-month outcome was calculated by summing up the number of the factors associated with poor outcomes (encephalitis feature, ePVS score ≥ 5, and periventricular lesion extension), with a score range of 0-3. In ROC curve analysis for the total study population, the risk score predicted a poor 6-month outcome with area under the curve (AUC) of 0.978 (95% CI: 0.950-1.000, P < .001) and 6-month mortality with AUC of 0.836 (95% CI: 0.745-0.927, P < .001, Fig. 2A and B). The risk score of 2 predicted a poor 6-month outcome with a sensitivity of 94.1% and a specificity of 95.2%, and 6-month mortality with a sensitivity of 93.3% and a specificity of 67.2%. For the subgroup without baseline encephalitis feature, the risk score predicted a poor 6-month outcome with AUC of 0.952 (95% CI: 0.896-1.000, P < .001) and 6-month mortality with AUC of 0.870 (95% CI: 0.764-0.978, P = .003, Fig. 2C and D). In this subgroup, the risk score of 2 predicted a poor 6-month outcome with a sensitivity of 85.7% and a specificity of 95.0%, and 6-month mortality with a sensitivity of 83.3% and a specificity of 81.2%.
Two-week follow-up MRI evaluation data were available for 40 (59.7%), 10-week follow-up MRI for 49 (64.5%), and 6-month follow-up MRI for 59 (77.6%) patients. The median number of MRI evaluations analyzed per patient was 3 . When the serial changes in the MRI parameters were analyzed in association with the changes in mRS scores, the subgroup with baseline ePVS score ≥ 5 showed gradual deterioration in the mRS score along with progressive increment of the frequency of cryptococcoma and hydrocephalus (Fig. 3A), whereas the subgroup with baseline ePVS score < 5 showed gradual improvement in the mRS score and maintained a low frequency of periventricular lesion extension, cryptococcoma, and hydrocephalus in the follow-up MRIs (Fig. 3B). A similar trend was observed in the subgroup without baseline encephalitis feature ( Fig. 3C and D www.nature.com/scientificreports/ Fig. 4 for representative cases). The profiles of the MRI parameters between the groups evaluated using 1.5-T or 3.0-T MRI machines were comparable (Supplemental Table 4).

Discussion
This study demonstrated clinical and MRI parameters associated with the progression and the poor outcomes of Cryptococcus meningoencephalitis. Along with the baseline encephalitis feature, a high ePVS score and periventricular lesion extension were independently associated with poor outcomes. Especially, presence of two or more risk factors at baseline showed high association with poor neurological outcomes and mortality, indicating that these might serve as prognostic markers. Given that the association was still valid for the subgroups without baseline encephalitis feature, these prognostic factors not only reflect the disease severity but also predict the www.nature.com/scientificreports/ www.nature.com/scientificreports/ risk of progression. Additionally, neurological deterioration manifested in brain MRI data as development of cryptococcoma and progression of hydrocephalus, which suggests that these MRI markers can also be used for monitoring the progression of disease. According to a large-sized prospective study of 501 patients with HIV infection, high fungal burden in the CSF, altered mental status, old age, high peripheral WBC count, low body weight, anemia, and low CSF opening pressure were associated with 10-week mortality 1 . Further, combination treatment of amphotericin and flucytosine at induction period reduced 10-week mortality while fluconazole-based induction treatment was associated with higher mortality 3 . However, these studies mainly focused on mortality or fungal clearance in the CSF, while the neurological outcome of Cryptococcus meningoencephalitis has not been investigated in depth. In this regard, this is the first study to describe the dynamic neurologic course of the disease, and demonstrate the early accessible MRI factors that are useful to predict or monitor the neurological outcomes.
Notably, the outcome predictors in the current study reflect the distinct pathomechanism underlying the progression of Cryptococcus meningoencephalitis and can therefore be related with the previously reported prognostic factors for mortality. High fungal antigen titer and larger fungus capsule size might also contribute to a mechanical stagnation of CSF flow, especially in the peri-venular space which has small diameter 1,9,10 . Therefore, these factors can be correlated with the enlarged PVS in the baseline MRI which reflects the degree of CSF stasis. www.nature.com/scientificreports/ Altered mental status is a factor constituting a baseline encephalitis feature and is also related to MRI indicators of the parenchymal invasion of the Cryptococcus, such as periventricular lesion extension and cryptococcoma 1 .
Increased ICP might also be the consequence of CSF recirculation failure resulting from the wide-spread cryptococcus accumulation over the perivascular space and manifest as the progression of hydrocephalus in MRI 8,9 .
In addition to outcome prediction and disease monitoring, the findings of the current study can also be useful for risk estimation and deciding the treatment strategy. For the patients with ≥ 2 baseline risk factors, higher combination or higher dose of anti-fungal treatment could be used to prevent the patient deterioration [25][26][27] . Additionally, frequent follow-up brain imaging to monitor the progression might be beneficial for the timely detection and early intervention to lower the ICP or other neurological complications. www.nature.com/scientificreports/ ). The patient was treated with intravenous amphotericin combined with fluconazole for two weeks. However, follow-up MRI at 2 week showed interval development of parenchymal T2 high-signal intensity (HSI) at the right frontal cortex on T2 fluid attenuated inversion recovery (FLAIR) images (Panels (B)). Despite the amphotericin-based combination treatment for 40 days and oral fluconazole for 176 days, the mRS score at 6 month was 4. A 72-year-old immunocompetent woman presented with fever and headache (initial mRS score = 1). At baseline MRI, CS-EPVS score was 3 and BG-EPVS score was 2 (Panels (C)). The patient was treated with intravenous amphotericin combined with flucytosine and maintained with oral fluconazole for 214 days. However, gait disturbance and cognitive impairment progressed (6-month mRS score = 4). Follow-up MRI at 6 month showed interval development of periventricular extension of the T2 HSI and slight hydrocephalus on T2 FLAIR images (Evans ratio = 0.35, Panels (D)). www.nature.com/scientificreports/ The current study has several limitations. First, as a retrospective study, the number and the interval of CSF and MRI evaluations, and treatment regimen were heterogeneous and not standardized. Second, the study population with baseline MRI evaluation might bear a potential source of selection bias, as this criteria might exclude patients with severe or unstable baseline clinical status. Different subpopulations included in each time point of the serial MRI data analysis also warrant a careful interpretation of the result. Third, this study included both 1.5-T and 3.0-T machines, although baseline and follow-up images were obtained from the same scanner and the MRI parameter profiles between the groups with different MRI powers were comparable. Fourth, the rate of fungal clearance in CSF, one of the major parameters for determining treatment outcome, was not analyzed in this study 28 . Fifth, although the presence of pathological parenchyma contrast enhancement might be a potential outcome predictor as it represents a significant breakdown of blood-brain barrier, it was excluded from the outcome analysis due to the low frequency and the significant portion of patients without baseline T1 contrast enhancement images. Additionally, careful discrimination ePVS and periventricular lesion extension from aging-related cerebral small-vessel disease is warranted, although their associations with outcomes were significant after adjusting for age.