This study was conducted in the Department of Medicine at Kasturba Hospital and Mahatma Gandhi Institute of Medical Sciences (MGIMS), Sevagram, India. Kasturba Hospital is a 920-bed rural teaching hospital of central India affiliated to MGIMS. Every year this hospital provides care to about 900,000 outpatients and 50,000 inpatients. We enrolled patients from inpatient settings in this study. Department of Medicine also has an outreach program where trained social workers screen individuals in the community for chronic diseases. Currently, this program runs in a total of 25 villages around our college. This case-control study was conducted from 1 November 2015 to 31 October 2017, after taking approval from the institutional ethics committee of MGIMS (IRB00003623). We obtained a written informed consent from all study participants before enrolling them in the study.
All patients aged 18 years and above admitted to the Medicine wards in Kasturba Hospital, were screened and with incident CLD were identified through a Hospital information system.
The diagnosis of CLD was based on the clinical profile, ultrasonography (USG) abdomen, and biochemical parameters. The cases were defined as those who have one or more of the following evidence suggestive of CLD irrespective of its etiology, i.e., hypoalbuminemia (serum albumin level < 3.5 mg/dl) along with any clinical evidence of portal hypertension (variceal bleed, ascites with or without spontaneous bacterial peritonitis, or recovered from hepatic encephalopathy) and/or a diagnostic imaging of abdomen showing raised, altered echo-texture suggestive of fatty liver, chronic hepatitis, or cirrhosis or biopsy result consistent with chronic hepatitis or cirrhosis [3].
Criteria for exclusion were non-cirrhotic portal fibrosis, obvious focal liver disease (hydatid cysts, liver abscess, cholangiocarcinoma, hepatocellular carcinoma, and multiple metastasis. Cases not consenting to the study and not a resident of Wardha district were also excluded. We enrolled consecutive inpatients with diagnosis of CLD according to our inclusion and exclusion criteria.
We recruited 200 cases of CLD as per case definition using consecutive sampling from our tertiary care hospital. Apparently, healthy controls with normal serum albumin levels, who are willing for participation in the study, were selected from the outreach service area and were identified only after the case-selection was completed. One healthy community-based control, frequency matched with the case and was within 5 years of age, and of same gender as the case selected. The exclusion criteria for controls were documented history of liver disease in the past (Fig. 1).
Data collection
Baseline characteristics and clinical data were recorded for all patients. All patients received the standard line of management for CLD. Patients with suspected CLD were undergone hemogram, liver function test (LFT) [alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, serum albumin, and globulin] in the central clinical laboratory. USG of the abdomen to observe liver architecture, liver size, and features of portal hypertension (splenomegaly, ascites, and portal vein diameter) were done by using a Logic GE machine ultrasound system, equipped with a 7.5 Megahertz (MHz) real-time, linear-array transducer B-mode scanner in patients of CLD by an experienced radiologist. Liver echo-texture was recorded as normal or coarse [4].
Measurement of risk factors
All cases and controls were enquired about the potential risk factors of CLD through a semi-structured pre-coded and pre-tested questionnaire by the research investigator. The interviews were conducted in their local language (Marathi) from all cases and controls in the same manner. The risk factors we enquired into were classified and presented into the following categories: demographic, socio-economic, anthropometric risk factors, occupational risk factors, past-morbidity related risk factors, and recreational substance use and laboratory measures.
BMI was calculated in all cases and control by the standard method. It was considered as normal from 18.5 to 24.49, underweight < 18.49, obese ≥ 25-29.99, and overweight ≥ 30 respectively. Blood pressure (BP) was measured in all cases and controls by Omron digital sphygmomanometer. Subjects were assigned as hypertensive if systolic BP was ≥ 140 mmHg or diastolic BP was ≥ 90 mmHg [5].
History of occupations like automobile or paint industry (lead, solvents, and engine fumes), mining (silica and sand blasting), construction (cement, asbestos), chemical industry (solvents, chlorine, nitrous gasses), and farming (insecticides, grain dust) were enquired. History of various known and documented diseases was also obtained from all cases and controls: diabetes mellitus, hypertension (HT), or on antihypertensive drugs, history suggestive of rapid weight loss (loss of > 10% of body weight within 6 months period), intestinal bypass surgery, biliary obstruction in past, polycystic liver disease, sickle cell disease and trait, known HIV infection.
History of chronic recreational substance use [alcohol, smoking tobacco (cigarettes or bidis), tobacco or beetle-nut chewing, intravenous or oral intake of a drug or abuse] with quantification was inquired among cases and controls [6, 7]. We also quantified alcohol intake by asking type of alcohol, frequency, and average amount of alcohol consumption and classified them as never, current, and past (those who had given up drinking for 6 months or more) users. Patients were defined as having non-alcoholic fatty liver disease (NAFLD) if they have a history of consumption of alcohol < 20 grams/day (g/day) [8,9,10,11,12,13]. Cases and controls were classified as never smoker (not smoked cigarette or bidi for at least three months in a lifetime), current smoker (smoked at least one cigarette or bidi in the last three months), and former smoker (one who does not meet both criteria).
Chronic consumption (˃ 3 months) of medications such as corticosteroids, methotrexate, amiodarone, methyldopa or herbal or folk remedies, or alternative medicine was also asked from all study participants.
In addition, venous blood samples after a 12-h overnight fast from all enrolled patients were collected for estimation of biochemical test. The collected sera were immediately refrigerated till they were tested by a serologist who was unaware of the clinical data of the patients. Hepatitis B surface antigen (HbsAg) and anti-hepatitis C antibodies qualitative detection (HCV) was done by rapid immuno-chromatographic assay. Venous blood samples transported within 6 h to central laboratory of hospital for lipid panel [total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG)] measurement by standard enzymatic tests. Levels were considered abnormal if total cholesterol > 200 mg/dl, LDL-C > 130 mg/dl, TG > 150 mg/dl, and HDL-C < 40 mg/dl respectively. Random glucose and serum creatinine were also obtained on day 1 in all enrolled participants.
Data management and analysis
We entered the data electronically by Microsoft Excel and analyzed by STATA software (Version 16, Stata Corporation, TX, USA). We analyzed normally distributed continuous variables by Student’s t test, proportions by a chi-square test and continuous variables with skewed distribution by a Mann-Whitney test. We considered a p value of less than 0.05 as significant. Exposure-related variables were included in the logistic regression analysis used in the multivariate model if they displayed a p value< 0.02 in the bivariate analysis. We calculated the odds ratios (ORs) as a measure of risk and 95% confidence interval (CI) associated with the independent variables.