Prediction of overt hepatic encephalopathy by the continuous reaction time method and the portosystemic encephalopathy syndrome test in clinically mentally unimpaired patients with cirrhosis.

BACKGROUND AND AIM: Predicting overt hepatic encephalopathy (OHE) is important because the condition is frequent, often requires hospitalization and is potentially preventable. The risk of OHE is related to pre-existing discrete cognitive defects, and for clinical practice it is recommended to apply two different psychometric tests to detect such deficits. We used the continuous reaction time test (CRT) and the portosystemic encephalopathy (PSE) syndrome test and examined their single and combined value for OHE prediction in cirrhosis patients. PATIENTS AND METHODS: We studied 130 clinically mentally unimpaired cirrhosis patients by the two tests and followed them for an average of 38.5 months. The CRT measures velocity and stability of motor reaction times to 150 repeated auditory signals. The PSE is a five sub-set paper-and-pencil test battery evaluating cognitive and psychomotor processing, speed and vision-motor coordination. We collected data on episodes of OHE during follow-up. The clinical course was analysed in patient groups according to the outcome of each test and of both tests together. No anti-HE treatment was initiated except for cases with OHE.
RESULTS: At baseline, the CRT test was abnormal in 74 patients and the PSE in 47. During follow-up 35 patients (27%) experienced 74 OHE events. 23 patients with abnormal CRT experienced OHE (prediction sensitivity 65%). The PSE predicted OHE in 14 patients (prediction sensitivity 40%). One or both tests were abnormal in 87/130 (67%) and this predicted OHE in 27 patients (21%) (prediction sensitivity 77%).
CONCLUSION: The CRT test was clinically useful in identifying two-thirds of clinically mentally unimpaired cirrhosis patients who later experienced OHE, and the use of both the CRT and PSE showed satisfactory prediction by identifying three-fourths of later OHE cases.

Introduction

Overt hepatic encephalopathy (OHE) requires treatment and is a frequent admission cause for patients with liver cirrhosis. Prognosticating OHE in order to prevent it is important because the condition impairs quality of life, often reoccurs, necessitates hospitalization, and is potentially preventable [1, 2].

It is known that the risk of developing OHE rises with decreasing liver function and portosystemic shunting [3]. Pre-existing discrete cognitive deficits caused by minimal hepatic encephalopathy (MHE), hyponatremia, and comorbidities are also associated with an increased risk of OHE [4-8]. The detection and quantification of such discrete cognitive deficits is done by psychometric testing [9]. International guidelines recommend patients with liver cirrhosis be cognitively evaluated by one or two validated psychometric tests meeting local preferences and resources [9]. A handful of tests are validated for the purpose. The continuous reaction time test (CRT) and the portosystemic encephalopathy (PSE) syndrome test are two such tests [9].

The CRT has been used for more than 3 decades, mostly in Scandinavia, for diagnosing and grading MHE and is validated regarding normal values, reproducibility, effects of age, gender, educational level, comorbid diseases, organic brain disease, sleep deprivation, and its ability to identify patients who will improve their CRT by anti-HE treatment [10-18]. In recent years the parallel use of CRT and the recommended surrogate gold standard test, the PSE, has gained momentum. The purpose of a two-test approach is to cover more cognitive domains and thus increase the collective test sensitivity [9, 19].

In spite of the availability of validated psychometric tests few centres routinely undertake psychometrics [20]. One important issue is that it is not clarified by the existing studies to what extent psychometric tests can predict OHE events. Such knowledge could motivate preventive anti-HE treatment and shed light on whether psychometric testing is worth the effort.

The aim of our study therefore is to examine the single and combined predictive value of the continuous reaction time test and the portosystemic encephalopathy syndrome test for OHE occurrence.

Our hypothesis is that each psychometric test has a good predictive value for future OHE and that their combined use offers the best predictive value. This assumption is based on the fact that a combined use detects a broader spectrum of cognitive deficits.

Patients and methods

Patients

The cohort consists of 130 patients with liver cirrhosis mainly of alcoholic aetiology (82%). The cohort was originally established to conduct a cross-sectional study, but with the approval of the local ethical committee we followed the cohort with the aim of registering later episodes of OHE, other admission causes, and death (10).

Inclusion criterions were: liver cirrhosis diagnosed by liver biopsy or by classic combinations of clinical, biochemical, image technique criteria; and the absence of OHE by the clinical assessment of the patient’s usual gastroenterologist.

Exclusion criteria were: HE West Haven Grade 1 or more, dementia (Mini Mental State Examination test score <24), prior cerebral insult, on-going alcohol use and acute intoxication at the day of inclusion, use of psychoactive medication (stable use of SSRI was allowed), severe hyponatraemia (p-Na < 125 mmol/L), renal failure (creatinine > 1.7 mg/dL), myxoedema, sepsis, or gastro-intestinal bleeding within a week before the tests. No one had diagnosed hepatocellular carcinoma at inclusion or at the time for psychometric testing. Inclusion and exclusion criteria and baseline data are previously published [12]. Regarding alcohol use we did not require a certain abstinence period but sobriety upon testing by simply asking patients if they had been drinking before commencing psychometric testing. Some patients had on/off alcohol use during follow up.

Patients were recruited from the departments of hepatology out-patient clinics on the Hospital of Southwest Jutland and University Hospital of Odense (Denmark) between February 2013 and November 2014. All patients had a CRT and a PSE test on the day of inclusion. The study duration was from inclusion to study termination in May 2017. We collected data on all hospital contacts including hospital admissions for OHE, other admission causes, and death. Patients remained in the study after the first recorded OHE episode i.e. we collected data on repeated OHE events. At inclusion, only subjects that earlier had been diagnosed with OHE received anti-HE treatment according to standard protocol with lactulose. An exception was 6/130 patients (5%) who received preventive anti-HE treatment for investigative purpose, they received a combination of lactulose, branched-chain amino acids and rifaximin for 3 months[10].

All patients gave their written informed consent. The study was approved by the Danish Data Protection Agency and The Local Committee on Health Research Ethics, and the follow up analysis presented here was reported to the regional data protection office, all according to Danish law.

Methods

The CRT is a short, computerized test (equipment from EKHO, www.Bitmatic.com, Aarhus, Denmark). The test measures motor reaction speed, sustained attention, and inhibitory control, which are all key abilities of daily life functioning [14]. The test registers motor reaction time to 150 auditory stimuli (beeps at 500 Hz and 90 dB via headphones). The auditory stimuli are given at random intervals from 2 to 6 seconds and the patient is instructed to immediately press a handheld trigger button, in response to each beep. The software registers the response times and calculates the CRT index (the ratio: 50 percentile/ (90 minus 10 percentile)), which is the main test result and a measure of the patient’s reaction time stability. A CRT index value below 1.9 is abnormal and may indicate MHE. The test has been in use for 3 decades in an increasing number of hospitals and is validated in the target population. Reaction time stability (CRT index) is not influenced by gender, age or intelligence; and is able to identify patients who improve cognition by anti-HE treatment [10, 14, 15]. The CRT index may be adversely affected in chronic medical conditions i.e. severe obstructive lung disease and heart failure [11].

The PSE test is a widely used test battery that can be completed in 20 minutes and often serves as a comparator test between centres and have good external validity [21]. The PSE consists of five paper-pencil tests: Digit symbol test (DST), number connection test A and B (NCT A and B), serial dotting test (SDOT), and Line Tracing Test (LTT), which evaluate cognitive and psychomotor processing, speed and vision-motor coordination [19, 22]. The total score—portosystemic hepatic encephalopathy score (PHES)—ranges from -18 to 6 and summarizes the overall performance in the five tests relative to norm values. A result -5 or below is abnormal and may indicate MHE. Standard normative data of the PHES have been developed in German, Spanish and Italian [23]. We evaluated the test using the German normal values since Danish normal values are not yet available. There is no gender effect, and no significant effect of educational level. Four versions of the test battery are available to prevent the learning effect of repeated testing. The PSE test can be obtained from Hannover Medical School (Hannover, Germany), which holds the copyright (Weissenborn.karin@mh-hannover.de) [9].

Data collection and management

One observer reviewed the participants’ electronic medical files between March and May 2017 and collected data on OHE admissions and death, and other adverse outcomes i.e. orthopaedic injury, falls, infections, ascites, variceal bleeds and dehydration/signs of acute kidney injury. The available electronic medical file system is used at all hospitals in the Region of Southern Denmark. All admissions and out-patients visits in the region are therefore registered. Death outside of the hospitals is registered as well. Hospitalization outside of the region or in another country, without this being mentioned in the patient file, is unlikely in Denmark. If the patient’s admission was registered with more than one diagnosis, the main diagnosis was used. Whenever OHE was described in the discharge letters this was regarded as a study relevant diagnosis. OHE does not have a formal ICD-10 code, so the cases were sought out manually. The observer was blinded to the psychometric test results at inclusion. No subjects withdrew their consent, and none moved during study period. All were kept in the study after the first OHE event. Two were transplanted during follow up and 57 died and were withdrawn from our analysis from the time of transplantation/death.

Statistical analysis

Statistical analyses and graphics were performed with STATA (14.2 STATA Cooperation, College station, Texas, USA). Before analyses the patients were categorized according to their initial CRT and PSE test results into four test groups (“CRT and PSE normal”, “CRT abnormal and PSE normal”, “CRT normal and PSE abnormal”, “CRT and PSE abnormal”) to evaluate the predictive value for OHE of single and combined use of CRT and PSE tests.

Categorical variables were compared using the Fisher’s exact test (due to few observations in some cells when using X2); quantitative variables were compared using analysis of variance (ANOVA). Continuous variables that followed normal distribution were compared using student’s t-test. To compare admissions with OHE we performed robust regression analysis, which was tested appropriate due to some test groups showed variance, heterogeneity and couldn’t be considered normally distributed. All reported P-values are 2-tailed. P values ≤.05 were considered as statistically significant. Pairwise comparisons of significant p-values by the Tukey's HSD were made.

Results

Baseline characteristics of the whole cohort, and the sub-groups categorized by psychometric test results, are presented in Table 1. The total follow-up time was 5,000 months and the median follow-up 38.5 months (range 27–48 months). Out of 130, 29 (22%) had had their first OHE episode before entering the study, but none had OHE at the time of inclusion. Thirty-five patients (27%) experienced at least one admission with OHE during follow up, and the total number of observed episodes was 74–24/35 patients (69%) had one episode of OHE during follow-up while 11 patients (31%) had more than one OHE episode. OHE was together with infections the most frequent liver-related cause for hospital contact, followed by ascites (Table 2).

Table 1

Baseline characteristics.

	Overall cohort (n = 130)	CRT and PSE normal (n = 43)	CRT abnormal and PSE normal (n = 40)	CRT normal and PSE abnormal (n = 13)	CRT and PSE abnormal (n = 34)	P-value ANOVA
CRT index, mean (SE)	1.9 (0.06)	2.6 (0.11)	1.5 (0.05)	2.3 (0.08)	1.4 (0.05)	<0.001q
PHES, mean (SE)	-3.9 (0.40)	-0.9 (0.32)	-1.4 (0.37)	-8.2(0.85)	-9.1 (0.52)	<0.001x
Male gender % (n)	67.7 (88)	72 (31)	55 (22)	92 (12)	68 (23)	0.07
Age, median years (range)	59 (40–79)	57 (44–76)	59 (40–79)	65 (49–72)	62 (40–75)	0.25
MELD score, median (range)	10 (6–29)	10 (6–25)	12.5 (6–29)	10 (7–21)	13 (6–21)	0.24
Child-Pugh score, median (range)	6 (3–12)	6 (3–12)	6 (5–12)	6 (5–11)	7.5 (5–12)	<0.03y
Child-Pugh A % (n)	55.4 (72)	69.8 (30)	52.5 (21)	53.9 (7)	41.2 (14)	0.09
Child-Pugh B % (n)	33.1 (43)	20.9 (9)	40.0 (16)	38.5 (5)	38.2 (13)	0.23
Child-Pugh C % (n)	11.5 (15)	9.3 (4)	7.5 (3)	7.7 (1)	20.6 (7)	0.29
Previous HE % (n)	22.3 (29)	20.9 (9)	17.5 (7)	30.7 (4)	26.5 (9)	0.70
Ascites % (n)	48.5 (63)	34.9 (15)	55.0 (22)	46.2 (6)	58.8 (20)	0.15
TIPS % (n)	5.4 (7)	2.3(1)	0.0 (0)	23.1 (3)	8.8 (3)	<0.017z
Varices % (n)	59.2 (77)	65.1 (28)	60.0 (24)	53.9 (7)	53.0 (18)	0.73
Education, years (SD)	10.9 (2.6)	11.2 (2.6)	11.6 (2.8)	10.2 (2.7)	9.9 (1.8)	0.03 Δ
Charlson comorbidity index	3.5 (1.2)	3.3 (1.0)	3.3 (1.4)	3.5 (0.8)	3.8 (1.1)	0.30

130 patients with liver cirrhosis, tested for the presence of discrete cognitive deficits using the continuous reaction times test (CRT) and portosystemic encephalopathy (PSE) syndrome test. Data are expressed as median (range), unless specified otherwise. P-values in bold show significant outcome at a level below 0.05

Abbreviations: CRT: continuous reaction time test (CRT index abnormal if below 1.9), PSE: portosystemic encephalopathy syndrome test (PHES psychometric hepatic encephalopathy score, abnormal if below -4), MELD: model for end-stage liver disease, HE: hepatic encephalopathy, TIPS: trans jugular intrahepatic portosystemic shunt.

Statistical differences (P ≤ .05) between groups indicated as comparison between:

q: CRT and PSE normal vs. CRT abnormal and PSE normal; CRT abnormal and PSE normal vs. CRT normal and PSE abnormal; CRT and PSE normal vs. CRT and PSE abnormal; CRT normal and PSE abnormal vs. CRT and PSE abnormal

X: CRT abnormal and PSE normal vs. CRT normal and PSE abnormal; CRT and PSE normal vs. CRT normal and PSE abnormal; CRT and PSE normal vs. CRT and PSE abnormal; CRT abnormal and PSE normal vs. CRT and PSE abnormal

Y: CRT and PSE normal vs. CRT and PSE abnormal

Z: CRT and PSE normal vs. CRT normal and PSE abnormal; CRT abnormal and PSE normal vs. CRT normal and PSE abnormal

Δ: CRT abnormal and PSE normal vs. CRT and PSE abnormal

Table 2

Events requiring admissions during follow-up.

	Overall cohort (n = 130	CRT and PSE normal (n = 43)	CRT abnormal and PSE normal (n = 40)	CRT normal and PSE abnormal (n = 12)	CRT and PSE abnormal (n = 34)	P-value ANOVA
Cirrhosis related admissions
OHE % (n)	27 (35)	19 (8)	32 (13)	31 (4)	29 (10)	0.57
Number of events	74	14	29	6	25
Ascites % (n)	21 (27)	19 (8)	28 (11)	15 (2)	18 (6)	0.19
Number of events	70	15	31	15	9
Infection % (n)	28 (37)	33 (14)	25 (10)	0 (0)	35 (12)	0.15
Number of events	57	23	14	0	14
Variceal bleeding % (n)	12 (16)	21 (9)	10 (4)	15 (2)	3 (1)	0.10
Number of events	33	21	8	3	1
Alcohol related % (n)	8 (10)	5 (2)	10 (4)	8 (1)	9 (3)	0.45
Number of events	21	3	12	2	4
Other frequent admission causes
Orthopaedic* % (n)	18 (23)	14 (6)	18 (7)	15 (2)	24 (8)	0.51
Number of events	31	6	11	3	11
Dehydration % (n)	9 (12)	5 (2)	13 (5)	0 (0)	12 (4)	0.26
Number of events	22	2	9	0	5
Death	44 (57)	42 (18)	40 (16)	31 (4)	56 (19)	0.21

In a cohort of 130 patients with liver cirrhosis, tested for the presence of discrete cognitive deficits using CRT and PSE test. Data are expressed as percentage of number of patients in each test group, Specific events or admission causes also has the total number of events specified under each group. P-values in bold show significant outcome at a level below 0.05. The reference value used in the ANOVA analysis were the group CRT and PSE normal.

*Acute orthopaedic injuries, fractures and falls

OHE: overt hepatic encephalopathy.

Predicting OHE using only the CRT test

Seventy-four out of the 130 patients (57%) exhibited an abnormal CRT and 23 of these (31%) experienced at least one episode of OHE—prediction sensitivity 65% (Table 2 and Fig 1). These 23 patients (31%) were responsible for 73% of all OHE admissions of the total cohort, reflecting that several of them experienced more than one OHE episode in spite of being discharged with preventive anti-HE treatment according to guideline.

Fig 1

Four scenarios for predicting OHE; with single or combined use of the two psychometric tests used.

All numbers inside the boxes (blue or white) represent the number of patients, having an abnormal (blue) or normal (white) test result. Panel a): A scenario with the single use of the continuous reaction time test (CRT), Panel b): Scenario with the single use of the PSE test. Panel c): Combined use where either abnormal test is diagnostic, Panel d): Combined use where two abnormal tests are diagnostic. PPV = positive predictive value; NPV = negative predictive value; Sens = sensitivity; Spec = specificity.

Predicting OHE using only the PSE test

Forty-seven of the 130 patients (36%) had an abnormal PHES and 14 of these (30%) developed at least one episode of OHE, yielding a prediction sensitivity of 40% (Table 2 and Fig 1). These 14 patients (30%) were responsible for 42% of all OHE admissions of the total cohort.

Predictive value of the combined use of CRT and PSE test

When combining the two tests, one or both tests were abnormal in 87/130 (67%) patients, and of these 27 patients (31%) developed at least one OHE episode, resulting in a prediction sensitivity of 77% (Table 2 and Fig 1). These 27 patients (31%) were responsible for 81% of OHE-admissions (Table 2).

Thirty-four out of 130 patients (26%) had abnormal results of both tests and of these 10 (29%) experienced at least on OHE episode. This gives a low prediction sensitivity of 29%

(Fig 1). These10 patients (29%) were responsible for only 34% of OHE admissions.

Eight of 43 patients (19%) with two normal tests experienced OHE and were responsible for 19% of OHE admissions (Table 2).

OHE admission patterns

Sixty-eight per cent (24/35) of patients experienced their first and only OHE admission during follow up. In the remaining 31% (11/35) OHE was relapsing. Most subjects relapsed 2–5 times, but two patients (2/35, 6%) experienced more relapses ascribed to uncontrolled recurring infections. There was a tendency that baseline CRT and PSE results were worse with increasing number of OHE events during follow up (S1 Table). Among the subjects with previous OHE at inclusion, 69% did not have another episode during follow up. For the OHE naïve subjects 74% still did not experience an episode during follow-up. For all test groups admission rates were higher among the groups with abnormal test results and highest in the group where CRT and PSE both were abnormal. (Fig 2). However, by multivariate analysis including potential known confounders (age, gender, MELD-score, Child-Pugh points), the higher admission rates in the groups with abnormal test results seen in the graphs (Fig 2) were not statistically significant (S2 Table).

Fig 2

The number of admissions with overt hepatic encephalopathy (OHE), per person year, in the four test groups.

In the group CRT and PSE normal (0.21), CRT abnormal and PSE normal (0.39), CRT normal and PSE abnormal (0.54) and CRT and PSE abnormal (0.81). Results are not statistically significantly different between groups after controlling for known confounders.

Discussion

The aim of this study was to examine the predictive value of the CRT and the PSE tests for OHE occurrence in a well-defined cohort of patients with liver cirrhosis. We assessed the predictive value of both single and combined use of the two tests for an average of 38.5 months follow up. Our hypothesis was that their combined use would offer the best predictive value since such an approach detects a broader spectrum of cognitive deficits i.e. the patients who are most cognitively impaired. Prediction was confirmed to be relevant, by 27% of the tested patients later developing OHE and thus being important to identify as prospective candidates for preventive anti-HE treatment.

We found that the CRT method alone predicted 65% of later OHE episodes, the PHES alone 40%, and both together 77%.

For the use of any given predictive test the desirable level of predictive value is a decisive issue. Prediction of clinical events is never perfect. The desirable level varies with the specific clinical problem and is determined by a number of factors such as the frequency of the problem, if it is predicted by other factors, how serious it is, how complicated the test is, and the efficacy, cost and safety of the preventive measure. OHE, as noted above, is a frequent complication for mentally unimpaired cirrhosis patients; it was not satisfactory predicted by clinical data including prior HE episodes; it is a very serious complication; the psychometric tests are non-invasive, fast, and cheap [9]; OHE is preventable by lactulose treatment with a low number needed to be treated (NNT = 4), and lactulose is widely available, cheap, safe, and has negligible side effects [24]. These statements, although not giving a definite level, suggest that prediction of half of the later OHE cases by psychometry is meaningful, of two thirds useful, and of three fourths satisfactory.

According to such levels, the CRT method alone was useful (predicted 65%), the PHES alone not sufficient (predicted 40%), and their combined use gave satisfactory (predicted 77%).

A limitation of our study is that we primarily dealt with prediction of OHE occurrence because OHE is serious, not satisfactory predicted by clinical data and preventable. Prediction of non-occurrence might have been of interest if the preventive measure was expensive or had significant side effects. In that case, a high negative predictive value and a low number of false positives—a high specificity—would be of importance. The negative predictive value of the CRT alone was 78%, of the PHES 75%, and of the combined use 81%. These numbers suggest that the CRT and PSE, alone and combined, are fairly good at ruling out future OHE.

Further our study is limited by the fact that we did not systematically control for participants on/off alcohol consumption (we simply asked them if they had been drinking before commencing psychometric testing) or adherence/non-adherence to any anti-HE treatments. Also, the use of German PSE normal values may not me optimal although Germans and Danes hardly differ significantly.

Other recent studies [25-28] have dealt with prediction of OHE and one of them used a similar approach to ours [27] by evaluating single versus combined testing. A major difference from our approach is however that the study by Duarte-Rojo et. al [27] required both psychometric tests to be abnormal to give the diagnosis of MHE and accordingly found lower predictive values in the range 30–55%. Similarly, we found that in patients with abnormal CRT and PSE prediction sensitivity was only 29%. The reason for that is likely that by requiring two psychometric tests to be abnormal we overlook all the patients whose cognitive deficits are demonstrated in just one test but who still have an elevated risk for OHE as demonstrated by our findings. These results show very clearly that not all HE patients are affected by HE to the same degree and in the same cognitive functions and illustrate why sensitivity is increased by a combined use of tests requiring just one of them to be abnormal.

Duarte-Rojo et. al [27] found that the single use of the PSE test had a predictive value of 36% which is similar to our findings (40% PHES predictive value). Accordingly, both studies suggest that the predictive value of the PSE test is outperformed by that of the Stroop EncephalApp (55%) and the CRT test (65%).

Conclusion

The continuous reaction time test was clinically useful in identifying two-thirds of clinically mentally unimpaired cirrhosis patients who later experienced over hepatic encephalopathy, and the combined use of the continuous reaction time test and the portosystemic encephalopathy syndrome test showed satisfactory prediction by identifying three-fourths of later overt hepatic encephalopathy cases. We suggest our findings to imply that all patients with liver cirrhosis should be psychometrically tested and those with abnormal continuous reaction time test alone and abnormal continuous reaction time test and portosystemic encephalopathy syndrome test together; should be offered preventive lactulose treatment and close follow up regarding the effect on cognition.

Rawdata output on the cohort of 130 patients with cirrosis.

Data is anonymisized: gender, social security number, age and some other characteristics is deleted so that rare events will still not be traceable back to each subject.

(XLSX)

Click here for additional data file.

Number of admissions with overt hepatic encephalopathy (OHE), per person year, in subjects with normal or abnormal CRTindex.

Disregarding psychometric hepatic encephalopathy score.

(TIF)

Click here for additional data file.

Number of admissions with overt hepatic encephalopathy (OHE), per person year, in subjects with normal or abnormal PSE.

Disregarding CRT index.

(TIF)

Click here for additional data file.

Psychometric test results in patients with: No OHE event, a single OHE event and recurring OHE events.

(DOCX)

Click here for additional data file.

Analysis of association: Multivariable linear regression analysis on OHE admission per person year, coefficients and confidence intervals.

Supplementary statistical output—for linear logistic regression model with margins. Supplementary information to Fig 2.

(DOCX)

Click here for additional data file.

4 Oct 2019

PONE-D-19-25609

Prediction of overt hepatic encephalopathy by the continuous reaction time method and the portosystemic encephalopathy syndrome test in clinically mentally unimpaired patients with cirrhosis

PLOS ONE

Dear Ms Wernberg,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you can see, both reviewers evaluated your study as interesting and carefully performed, however, suggested additional information to be added/additional analyses to be performed.

We would appreciate receiving your revised manuscript by Nov 18 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Pavel Strnad

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In the Methods, please cite the original references describing the development of the PSE and CRT tests, or describe how others may gain access to them. If the PSE test was published under a copyright no more restrictive than CC-BY, please include a copy as Supporting Information.

3.  In the ethics statement in the Methods and online submission information, please ensure that you have specified whether consent was informed."

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Wernberg et al. investigated the predictive ability of CRT and PHES regarding the development of OHE in patients with liver cirrhosis. This study represents a lot of hard work that is carefully done. I have only a few comments:

1. Since medical files were reviewed retrospectively, how did you exclude that patients were hospitalized with OHE in different hospitals?

2. For how long did patients have to stop drinking before being included into the study?

3. Did you include patients with HCC?

4. Is it correct that no patient was lost to follow-up?

5. Is it correct that no patient required liver transplantation?

6. Currently, you are not dealing with competing events like death or liver transplantation in your analyses. I would recommend adding cumulative incidence plots for every testing strategy. Here death and liver transplantation can be considered as competing events for OHE. Additionally, please provide a table with the hazard ratios and confidence intervals of your multivariable analyses.

7. Some studies already investigated the predictive ability of PHES regarding development of OHE. Additionally, a recent study by Duarte-Rojo et al. tested the utility of single versus combined testing (Duarte-Rojo et al, Metab Brain Dis 2019). Here, the authors conclude that combined testing is not superior to single testing to predict OHE. This is somehow in contrast to your conclusion. Can the authors comment on this?

8. Line 25: “The negative predictive value of the CRT alone was 79%”. In figure 1 it is 78%. Please correct this.

9. Line 249:”of three fourths excellent”; Line 262 “showed excellent prediction”. A sensitivity of three fourths cannot be declared as excellent. Please tone these sentences down.

Reviewer #2: In this manuscript Wernberg et al. assessed the predictive value of the continuous reaction time method (CRT) and the portosystemic encephalopathy syndrome (PSE) test for episodes of overt hepatic encephalopathy in patients with liver cirrhosis. They assessed 130 patients with liver cirrhosis with both tests and followed them for an average of 38.5 months. Of the 74 patients with an abnormal CRT 23 patients developed an overt HE and of the 47 patients with an abnormal PSE test 14 patients developed overt HE. When one or both tests were abnormal the prediction sensitivity was 77%. The authors conclude that the CRT alone but especially the combination of CRT and PSE test are clinically useful to identify patients who are at risk to develop overt HE. The authors suggest that all patients with liver cirrhosis should be tested psychometrically and if pathological, preventive treatment should be initiated.

This manuscript addresses an important topic and I congratulate the authors for performing this follow-up study. Testing patients with liver cirrhosis psychometrically is important and has clinical impact.

Comments:

In general:

- The authors should provide references for statements where applicable.

- Please add “%” after all patient numbers throughout the manuscript where applicable.

Abstract:

- Please revise the first sentence. I suggest that the authors delete the word “unpleasant” and replace it with “impairment of health related quality of life” and “cognitive impairment”.

- Please correct “Porto Systemic Encephalopathy Test (PSE)” to “portosystemic encephalopathy (PSE) syndrome test” and use this spelling throughout the manuscript.

- Please provide a hypothesis.

- Lines 30/31: The description of the PSE is wrong, please correct it. (The PSE does not only measure time, e.g. digit symbol test, line tracing test)

Introduction:

- Line 47: Please revise as described above

- Lines 52-55: Please revise this sentence.

- Please provide a hypothesis at the end of the introduction

Patients and methods:

- Line 78: Please define “no clinically detectable cognitive deficit”. Did the authors use a test?

- The inclusion and exclusion criteria should be described

- Lines 89/90: This sentence is hard to understand, please revise it.

- Lines 92-97: move this paragraph further up. Delete “which none met at the time of inclusion” as it is self-explanatory.

- Line 101: spelling error

- Line 107: please revise this sentence

- Line 115: I am surprised that the CRT is not influenced by age as it is well known that age influences reaction time in general. Can the authors please specify their comment?

- A reference for the PSE test is missing

- Please correct the description of the PSE test

- Why did the authors use norm data of the German population and not those of the Danish population?

- Lines 146/147: Could you please make the names of the different groups easier to understand e.g. “CRT/PSE normal”, “CRT abnormal/PSE normal” etc.

Results:

- Table 1: Can you please provide the pairwise analysis for the significant p-values

- Line 178: The title of table 2 is misunderstanding. Had all patients 38 months follow up?

- Lines 186/187/201/205: Please choose a clearer title. For example “Prediction of OHE using only CRT test” etc.

- Please provide the measured mean values of the CRT and PHES.

Discussion:

- I recommend to provide a short introduction at the beginning of the discussion

- Lines 234-237: Please revise this paragraph as it is hard to understand

- Line 246: Reference is missing

- Line 250: Please discuss the results of the paper in more detail. Especially why the PHES alone was “not meaningful”. This result contradicts that of other papers and should be discussed.

- Line 253: please describe the problem

- Did patients with recurrent episodes of OHE have worse psychometric test results than those with only one episode?

- The authors should list limitations of the study.

- In general: Please critically discuss the results of this study in detail and place them in the context of previous literature.

Conclusions:

- Lines 262/263: Please revise this sentence.

- The last sentence (line 265) contradicts the results and should be revised: “in those with two abnormal tests…” In the results section these patients only have a predictive value of 29%.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Christian Labenz

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Nov 2019

Response to reviewers

Dear reviewers,

Thank you very much for your hard work; all comments and feedback! You have both made an effort and we have tried to address your comments to the best of our abilities and hope you feel the manuscript have improved.

Please see attached file: Response to reviewers.

The best of regards,

Charlotte Wernberg et al.

Text below is copy-pasted from the attached file: Response to reviewers. Format is therefore gone.

As to the journal comments:

1. In the Methods, please cite the original references describing the development of the PSE and CRT tests, or describe how others may gain access to them. If the PSE test was published under a copyright no more restrictive than CC-BY, please include a copy as Supporting Information.

We have added the appropriate references.

2. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether consent was informed."

We have specified in the manuscript that informed written consent was achieved from all participants and the collection of follow up data was approved by the regional data protection agency.

Reviewer #1:

1. Since medical files were reviewed retrospectively, how did you exclude that patients were hospitalized with OHE in different hospitals?

This is a very relevant point. The electronic medical file system was during the period used at all hospitals in the Region of Southern Denmark. All admissions and out-patients visits in the region are registered. Death outside of the hospitals is registered here as well. Hospitalization outside of the region, without this being mentioned in the patient file, is unlikely and a rare event. This has been clarified in the new manuscript page 7 lines 147-151.

2. For how long did patients have to stop drinking before being included into the study?

Ongoing alcohol abuse was an exclusion criterion, but we had no requirements as to the duration of abstinence. They were sober at the time of testing. Most patients had alcoholic cirrhosis and although they stated that they had stopped drinking at the time of inclusion into the cross-sectional cohort some of them had an on-off alcohol use during follow up. This has been mentioned in the manuscript page 5 line 90-93.

3. Did you include patients with HCC?

None had diagnosed hepatocellular carcinoma at inclusion or at the time of psychometric testing. This has been clarified in the new manuscript page 5 line 89-90.

4. Is it correct that no patient was lost to follow-up?

Thank you very much for bringing this up. We were in fact able to account for all patients in this cohort during follow up and only those who died or were transplanted (cf. below) were censured. We kept patients in the study after their first OHE episode.

5. Is it correct that no patient required liver transplantation?

Actually, two patients were transplanted. One of the tem died 4 months after due to complications to transplant. We had forgotten to exclude these persons in the admission causes analysis and admission causes statistics are now updated and new calculations are made.

6. Currently, you are not dealing with competing events like death or liver transplantation in your analyses. I would recommend adding cumulative incidence plots for every testing strategy. Here death and liver transplantation can be considered as competing events for OHE. Additionally, please provide a table with the hazard ratios and confidence intervals of your multivariable analyses.

Thank you for your comments and suggestions. We assume that you are referring to the analysis on admissions with OHE? We chose to split up the response here, as we read the comment as two separate issues. We hope we have addressed the issues accordingly and that we have interpreted the comments correctly.

With regards to “…Currently, you are not dealing with competing events like death or liver transplantation in your analyses. I would recommend adding cumulative incidence plots for every testing strategy. …”.

We have discussed this at an early phase with a statistician, before submitting the manuscript. We were advised against using classical survival analysis as seen in a cumulative incidence plot. This because some patients had several events with OHE and these needed to be treated as multiple failure-time data. We have therefore chosen not to use cumulative incidence plots for events with OHE in this manuscript.

With regards to “…Additionally, please provide a table with the hazard ratios and confidence intervals of your multivariable analyses…”

We have added a supplementary table with effect coefficients and 95%CI for the factors in the regression analysis and margins. There are however no hazard ratios when working with logistic regression. We have added a supplementary figure illustrating the admissions rates for a test strategy using only CRT and only PSE tests.

7. Some studies already investigated the predictive ability of PHES regarding development of OHE. Additionally, a recent study by Duarte-Rojo et al. tested the utility of single versus combined testing (Duarte-Rojo et al, Metab Brain Dis 2019). Here, the authors conclude that combined testing is not superior to single testing to predict OHE. This is somehow in contrast to your conclusion. Can the authors comment on this?

The Duarte-Rojo study is very interesting. We have focused on the comparison with the Duarte-Rojo study in our discussion lines 305-320.

8. Line 25: “The negative predictive value of the CRT alone was 79%”. In figure 1 it is 78%. Please correct this.

Thank you for noticing, typo corrected in text.

9. Line 249:”of three fourths excellent”; Line 262 “showed excellent prediction”. A sensitivity of three fourths cannot be declared as excellent. Please tone these sentences down.

We agree on this, and have toned it down.

Reviewer #2:

In general:

- The authors should provide references for statements where applicable.

Thank you, we agree that there were too few references. This has now been changed for the better, with relevant and accurate references. We hope you agree.

- Please add “%” after all patient numbers throughout the manuscript where applicable.

Thank you for noticing and make us aware of this, changes has been added all the way through.

Abstract:

- Please revise the first sentence. I suggest that the authors delete the word “unpleasant” and replace it with “impairment of health related quality of life” and “cognitive impairment”.

These suggestions improve the nuances to the text further, thank you.

- Please correct “Porto Systemic Encephalopathy Test (PSE)” to “portosystemic encephalopathy (PSE) syndrome test” and use this spelling throughout the manuscript.

Again, thank you very much. Spelling throughout the manuscript is corrected.

- Please provide a hypothesis.

Our hypothesis has been stated in the last section of the introduction.

- Lines 30/31: The description of the PSE is wrong, please correct it. (The PSE does not only measure time, e.g. digit symbol test, line tracing test).

We have changed the short description of PSE in the abstract:: The PSE is a five sub-set paper-and-pencil test battery evaluating cognitive and psychomotor processing, speed and vision-motor coordination.

And in the “methods” section: The total score — portosystemic hepatic encephalopathy score (PHES)— ranges from -18 to 6 and summarizes the overall performance in the five tests relative to norm values.

Introduction:

- Line 47: Please revise as described above.

This has been revised; please see manuscript, lines 47-49.

- Lines 52-55: Please revise this sentence.

The passage has been revised to “It is known that the risk of developing OHE rises with decreasing liver function and portosystemic shunting. Pre-existing discrete cognitive deficit caused by minimal hepatic encephalopathy (MHE), hyponatremia, and comorbidities are also associated with an increased risk of OHE. The detection and quantification of such discrete cognitive deficits is done by psychometric testing.”

- Please provide a hypothesis at the end of the introduction

Our hypothesis has been stated in the last section of the introduction.

Patients and methods:

- Line 78: Please define “no clinically detectable cognitive deficit”. Did the authors use a test?

“no clinically detectable cognitive deficit” simply means no overt HE i.e. HE that can be detected clinically without the use of psychometric tests. This has been clarified in the exclusion criterions cf. below.

- The inclusion and exclusion criteria should be described.

We agree. These have been added.

- Lines 89/90: This sentence is hard to understand, please revise it.

Thank you, this has been revised.

- Lines 92-97: move this paragraph further up. Delete “which none met at the time of inclusion” as it is self-explanatory.

Paragraph is moved to a position further up in the paragraph.

- Line 101: spelling error.

This has been corrected.

- Line 107: please revise this sentence.

This has been done.

- Line 115: I am surprised that the CRT is not influenced by age as it is well known that age influences reaction time in general. Can the authors please specify their comment?

The crude reaction times are very influenced by age and gender but he CRT measures reactions time stability as given by the CRT index and the reaction time stability is not influenced by age. It is however influenced by other comorbid diseases such as heart failure, diabetes and COPD.

- A reference for the PSE test is missing.

Reference is added.

- Please correct the description of the PSE test.

This has been done.

- Why did the authors use norm data of the German population and not those of the Danish population?

There is no norm data in Danish yet. These are under development.

- Lines 146/147: Could you please make the names of the different groups easier to understand e.g. “CRT/PSE normal”, “CRT abnormal/PSE normal” etc.

We welcome the purposed names and will change the groups for these more logical and easier names, thank you!

Results:

- Table 1: Can you please provide the pairwise analysis for the significant p-values

We have now added pairwise analysis for the significant p-values to Table 1and hope that the way we have presented it is logical and satisfying.

- Line 178: The title of table 2 is misunderstanding. Had all patients 38 months follow up?

“38 months” has been deleted since not all participants have 38 months of follow up time. Thank you for that comment.

- Lines 186/187/201/205: Please choose a clearer title. For example “Prediction of OHE using only CRT test” etc.

Good idea. This has been corrected.

- Please provide the measured mean values of the CRT and PHES.

This has been added in Table 1.

Discussion:

- I recommend to provide a short introduction at the beginning of the discussion

This has been added.

- Lines 234-237: Please revise this paragraph as it is hard to understand

The paragraph has been divided and altered

- Line 246: Reference is missing

Reference is added.

- Line 250: Please discuss the results of the paper in more detail. Especially why the PHES alone was “not meaningful”. This result contradicts that of other papers and should be discussed.

We have changed the phrasing of this section and discussed results from other papers.

- Line 253: please describe the problem

This has been done: This work primarily dealt with prediction of OHE occurrence because OHE is serious, not satisfactory predicted by clinical data including prior HE episodes and preventable.

- Did patients with recurrent episodes of OHE have worse psychometric test results than those with only one episode?

Thank you for raising this question. We have chosen to add this data in the text in the results section, paragraph OHE admission patterns. There does not seem to be significant differences in the values of CRT index and PHES when we look at no/ one/ or >1 event with OHE.

- The authors should list limitations of the study.

These have been added.

- In general: Please critically discuss the results of this study in detail and place them in the context of previous literature.

We have chosen to discuss our findings in relation to the recent and very similar study by Duarte-Rojo as suggested by reviewer #2 and rewritten the discussion to a large extent.

Conclusions:

- Lines 262/263: Please revise this sentence.

This has been done regarding the term “excellent” which has been changed to “satisfactory”.

- The last sentence (line 265) contradicts the results and should be revised: “in those with two abnormal tests…” In the results section these patients only have a predictive value of 29%.

Thanks for pointing this out. The sentence has been altered.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Nov 2019

Prediction of overt hepatic encephalopathy by the continuous reaction time method and the portosystemic encephalopathy syndrome test in clinically mentally unimpaired patients with cirrhosis

PONE-D-19-25609R1

Dear Dr. Wernberg,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Pavel Strnad

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper has been further improved and my points were addressed accordingly. Congratulations on this fine piece of work!

Reviewer #2: Thank you very much for addressing all comments! The authors have done an excellent job responding to the questions.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Christian Labenz, MD

Reviewer #2: Yes: Henning Pflugrad, MD

5 Dec 2019

PONE-D-19-25609R1

Prediction of overt hepatic encephalopathy by the continuous reaction time method and the portosystemic encephalopathy syndrome test in clinically mentally unimpaired patients with cirrhosis

Dear Dr. Wernberg:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Pavel Strnad

Academic Editor

PLOS ONE