This was a prospective cohort study.
The study was conducted in the outpatient department of the Aga Khan University Hospital, Karachi, Pakistan.
It is estimated that about 55% of patients with cirrhosis would have MHE based on a positive result for both Psychometric Hepatic Encephalopathy Scoring (PHES) and Critical Flicker Frequency (CFF) analysis16. Assuming the CFF to have a sensitivity of 87% and specificity of 82% for MHE, and a confidence interval of 5%, the estimated sample size for this study was calculated to be 16417. To achieve the study objectives, the sample size was inflated by 10% to 180.
Non-probability convenient sampling was used to identify the subjects.
Patients with liver cirrhosis (irrespective of cause), 18 years and above, and without prior history of overt hepatic encephalopathy were included in the study.
Patients currently receiving Hp eradication therapy, those on antibiotics for spontaneous bacterial peritonitis (SBP) prophylaxis or any other infection within last 4 weeks, or those who were on sedatives, rifaximin and/or lactulose (within 1 week) were excluded. Patients who had overt encephalopathy, severe cardiac, pulmonary, renal or cerebral disease, as well as those who had a history of recent upper gastrointestinal (GI) bleed (in last 6 weeks) were excluded from the study.
Ethical approval was obtained from the Ethical Review Committee (ERC) of the Aga Khan University, Karachi, Pakistan (ERC Reference #: 2873-Med-ERC-13). Written informed consent was obtained from all participants. Patients’ identification remained anonymous throughout the study. Patients were informed that the data will be used for research purpose and publication without revealing individual identification and information. Study was performed in accordance with the principles of good clinical practice from the Declaration of Helsinki.
Data collection procedure
All patients already diagnosed to have cirrhosis based on clinical signs and symptoms, laboratory parameters and imaging studies, meeting the inclusion criteria and agreeing to participate, were enrolled after written informed consent. The informed consent was translated in Urdu (native) language as well. Child Turcotte Pugh (CTP) and Model of End-stage Liver Disease (MELD) scores of individual patients were calculated in order to determine disease severity. These patients then underwent psychometric hepatic encephalopathy scoring (PHES) and critical flicker frequency (CFF) analysis to detect MHE. For the study purpose, a patient was diagnosed as having MHE if he tested positive for both CFF analysis and PHES testing. CFF analysis was incorporated as the examination is reliable, simple, easy to apply and can be performed without difficulty by patients with low educational background, such as patients from a country with relatively low levels of literacy.
Diagnosis of MHE
Psychometric hepatic encephalopathy scoring (PHES)
PHES is a battery of neuropsychological tests which has long been regarded as a ‘gold standard’ for the assessment of MHE18,19. Patients were classified as having MHE when the PHES score was less than −4 points. PHES comprises of five components:4
Number connection test A
The patient was instructed to join numbered circles in order on a piece of paper. The time required to complete the task was scored.
Number connection test B
The patient was instructed to join numbered circles and alphabets e.g. 1, A, 2, B and so on.
Line tracing (trail drawing test)
The patient was asked to trace a path, 5 mm wide, as fast as possible without touching the borders.
Serial dotting test
The patient was asked to dot the center of circles on a piece of paper.
Digit symbol test
The patient was asked to learn a code in which a digit is represented by a symbol. He/she then had to reproduce the symbol corresponding to the digit.
For calculation of PHES, individuals are supposed to complete all five components.
Critical flicker frequency (CFF) testing
The neurophysiologic CFF analysis is a tool that measures the ability of the central nervous system to detect flickering light, which is directly influenced by cortical activity5,20,21. In this method, an intermittent light stimulus appears as a flicker which is dependent on the frequency of light pulse presentation. The rate at which flicker just disappears is termed the critical flicker frequency (CFF). Patients were provided a CFF analyzer (a pair of spectacles shielding against outside light), and they were asked to concentrate on a red light, which was initially flickering. The frequency of the light was then gradually decreased by the operator until the patients perceived it as flickering, and they had to press a handgrip button when this happened. A lower level of 38 Hz was used as a cut-off for impaired CFF (and hence positive MHE).
Diagnosis of active H. pylori infection and SIBO
Diagnosis of active H. pylori infection was made by urea breath test (UBT) using Carbon 1322. Patients were asked to swallow 50 mg capsule of 13 C-urea. Breath samples were collected by exhaling into a 200 ml gas storage bag to be analyzed by an infrared spectrometer, and this was performed before and 15 minutes after the consumption of capsule. For the diagnosis of SIBO, hydrogen breath test (HBT) was utilized, in which 50 gram of lactulose was given to the subject, and an increase of ≥20 parts per million (p.p.m) in hydrogen production at 90 min from base-line was taken as a positive diagnosis for SIBO.
Treatment of active H. pylori infection and SIBO
In our study, all patients were subjected to UBT and HBT. Patients who were tested positive were given appropriate therapy. Triple medicine regimen which includes clarithromycin, amoxicillin (for 10 days) and a proton pump inhibitor (for another 4 weeks) was given for the eradication of H. pylori infection23. We chose the triple regime as it is easier to administer and because low recurrence rates of H. pylori infection (despite considerable resistance rates of approximately 36%) have been reported from our part of the world24.
With regards to treatment of patients with SIBO, rifaximin 1200 mg/day for 1 week, which has shown good efficacy for SIBO in various studies, was administered25,26. This dosage was selected as there had been substantial variation in the dose and duration of rifaximin at the time this study was conducted27. UBT and HBT were repeated after 6 weeks of completion of therapy28. CFF analysis and PHES were also repeated at the same time (i.e. after 6 weeks of completion of triple therapy and SIBO treatment) to detect an improvement in MHE. Rifaxamin was given to patients who came out as SIBO positive. Of note, none of the patients were given rifaximin as prophylaxis for hepatic encephalopathy during the follow-up period.
For comparison with healthy subjects, 90 age and gender-matched controls (e.g. patients’ relatives, healthy hospital employees, etc.) were also enrolled in the study after written informed consent and underwent UBT and HBT. Those who were found positive for Hp and SIBO were treated accordingly (Fig. 1).
Data analysis was done using IBM SPSS Statistics for Windows, version 19.0 (Armonk, NY: IBM Corp. IBM Corp. Released 2011). Initially, the frequency was generated for all the variables. Continuous variables were presented as mean ± SD and categorical variables were presented as a percentage. Continuous variables between groups were compared by using unpaired t test or Mann Whitney test where appropriate. Categorical variables were compared by using the chi-square test or Fisher exact test. A p-value <0.05 was considered significant.