Metabolic associated fatty liver disease (MAFLD) (formerly known as non-alcoholic fatty liver disease) is the most common liver disease that affects about 25-30% of the global population (1) (2) (3) . The MAFLD is a multisystem disease with wide consequences, as it not only increases the risk of liver cirrhosis and failure and hepatocellular carcinoma (HCC), but also increase the risk for various extra-hepatic complications such as cardiovascular disease, chronic kidney disease, type 2 diabetes, osteoporosis, and some types of cancers (4) .
The pathogenesis of the disease is complex and involves a dynamic interaction between environmental factors and genetic basis (5, 6) , with evidence of shared genetics between MAFLD and other metabolic diseases (7) . Although, classically, linked to obesity, there is an emerging evidence that a considerable proportion of patients are non-obese, as called lean MAFLD (8) . However, the characteristics of this group of patients are not well characterised, particularly in Caucasian white population.
Worryingly, the Middle East region has the highest prevalence of MAFLD globally, in parallel with dramatic nutrition transition and high rates of physical inactivity in this region (9, 10) .
However, the current "anti-definition" and linking to alcohol, the disease with labeling "NAFLD" represents a major lacuna that hinders the clinical and research progress(11).
Adding to the complexity, the Middle East region, and particularly Egypt had one of the highest prevalence of hepatitis C worldwide(12, 13) . The current definition of NAFLD that is based on exclusion of other diseases, was very problematic and limits the ability to assess the actual magnitude of both problems and proper management of liver diseases that patients have.
Recently, an international consensus proposed new diagnostic criteria for MAFLD that could potentially help to eliminate the current "anti-definition" (2) . The criteria are based on evidence of hepatic steatosis, in addition to one of the following three criteria, namely overweight/obesity, presence of type 2 diabetes mellitus, or evidence of metabolic dysregulation. However, the validity and robustness of these proposed criteria in a real-world cohorts are still unknown.
Thus, in this work, we examined the validity of the proposed criteria for MAFLD in a real-life cohort, characterize the characteristics of lean MAFLD and finally determine the characteristics of patients with MAFLD/HCV dual etiologies. Here, we report that the proposed criteria for diagnosis of MAFLD is well validated and easily applicable and capture the entire spectrum of disease including non-obese subjects.
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This is a prospective two centre cross-sectional study included 242 patients previously diagnosed with non-alcoholic fatty liver disease who met the inclusion criteria were consecutively included in the study. Patients were recruited from the outpatient clinics of Minia University Hospitals and Beni-Suef University Hospitals, Egypt. Individuals with alternative diagnoses were excluded including those with excess alcohol intake (>10 g per day for women; and >20 g per day for men), chronic viral hepatitis (hepatitis B and hepatitis C), decompensated liver disease, known active malignancy or an alternative cause for fatty liver. The validity of the novel criteria proposed by the international expert panel to diagnose MAFLD was investigated (2) . The novel criteria for diagnosis of MAFLD are shown in Figure 1 .
To investigate the impact of dual eitology on liver fibrosis, a second cohort of patients with dual pathology HCV and MAFLD criteria was included (n=78), based on the criteria mentioned above, apart from allowing for including HCV-positive patients.
The study was conducted in accordance to the ethics of good clinical practice and Helsinki Declaration after approval of the local ethical committee. A written informed consent was obtained from all patients.
All participants underwent a clinical history using a standardized questionnaire that collected information on age, gender, smoking, and alcohol intake. Weight (in kilograms) and height (in centimetres) were measured and the body mass index (BMI) was calculated and expressed as kg/m 2 .
Waist circumference was taken at the midpoint between the lowest rib and the top of the iliac crest and measured in centimetres. Hypertension was defined as a resting blood pressure of ≥ 130/80 mm Hg, or having any antihypertensive medication prescribed. Type 2 diabetes mellitus (T2DM) was defined as a fasting plasma glucose value ≥ 126 mg/dL or having any antidiabetic medication prescribed. The homeostasis model assessment (HOMA-IR) was calculated as (fasting serum insulin (μU ml −1 ) × fasting serum glucose (mmol l− ))/22.5. Fasting blood samples were taken in the morning for all participants and analyses for full blood count, liver biochemistry, lipid profile, fasting glucose and iron studies were performed.
In the dual eitology cohort, HCV was diagnosed using HCV RNA levels, carried out using the COBAS AmpliPrep /COBAS TaqMan HCV Quantitative Test, version 2.0, with a lower limit of quantification of 20 IU/m.
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The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20176214 doi: medRxiv preprint All patients received an abdominal ultrasonography using (Acuson X300, Siemens, USA) and CH5-2 probe. A single experienced examiner in each centre screened all patients in each centre and diagnosis of fatty liver was based upon ultrasound diagnostic criteria of fatty liver (14) (15) (16) .
For descriptive statistics, values were expressed as mean ± standard deviation, median and interquartile range as appropriate. The Mann-Whitney non-parametric test was used to obtain significance. For categorical variables, data were presented as frequency (percentage) and p-values.
Comparisons of distributions between groups was assessed using Fisher's exact test. Multivariable regression modelling with backward elimination was undertaken to test independent associations with significant fibrosis and FIB-4 score in the dual aetiologies (MAFLD and HCV). All statistical analyses were undertaken in Statistical analyses were performed using the statistical software package SPSS for Windows, version 21(SPSS, Chicago, IL).
We explored the validity of new "positive" criteria for diagnosis of MAFLD in a cohort of 242 patients with previously diagnosed NAFLD. Noticeably, in this cohort, the diagnostic criteria were captured in 240 patients (99.2%). The overweight criterion was met in 212 (87.6) patients, and either overweight or diabetes were met in 215 (88.8%) patients. The presence of 2 criterion of metabolic dysfunction were met in 25 (10.3%) patients. As, only two subjects (0.8%) who do not meet the MAFLD criteria, we were not able to investigate for their characteristics. In total, the newly proposed criteria are valid and capture virtually patients previously diagnosed to have NAFLD.
The prevalence and characteristics of subjects with lean MAFLD are still not completely known, particularly in other ethnicities apart from Caucasian. Thus, we next thought to investigate this in our cohort. Interestingly, 100 (41.3%) patients were non-obese (defined as BMI <30 kg/m 2 ), while 30 (12.3%) patients were lean (defined as BMI <25 kg/m 2 ),
The baseline characteristics of the nonobese MAFLD patients compared to their obese counterpart are depicted in Table 1 . Non-obese MAFLD patients had favourable metabolic profile, as by definition they had significantly lower BMI and waist circumference, as well as lower fasting blood sugar and HOMA-IR, while lipid profile and other characteristics were not significantly compared to their obese counterparts.
Non-obese MAFLD patients also have lower fibrosis, as measured by NFS (p=0.010), while FIB-4 was not significantly different ( Table 1) , while this association was less profound when we applied the low and high cut-off for each score. ( Table 2) All rights reserved. No reuse allowed without permission.
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Having confirmed the robustness of the MAFLD diagnostic criteria, we next explored the impact of HCV/MAFLD dual aetiologies compared to MAFLD alone. Baseline characteristics of the dual aetiologies are described in Table 3 . Interestingly the HOMA-IR, total cholesterol and LDLcholesterol were found to be are significantly higher in patients with dual aetiologies compared to MAFLD alone (p<0.001, for all comparisons). Notably, patients with the dual aetiologies have also significantly higher fibrosis compared to those with MAFLD alone, assessed by FIB-4 score (p=0.001) ( Table 4 ). This association remained significant after adjusting for gender, AST and ALT in multiple regression analysis (Odds ratio [95% confidence interval], 3.77 [1.49-9.48], P < 0.005) ( Table 5 ).
In total, patients with dual HCV/MAFLD aetiologies present with severe metabolic and fibrosis characteristics.
This is the first evaluation of the diagnostic accuracy of the novel criteria proposed by the international expert panel to diagnose MAFLD (2) in a real-world cohort of patients previously diagnosed with NAFLD. We demonstrate that the criteria were easy to apply and fit to the diagnosis in nearly all patients (99%), have an excellent diagnostic property with virtually all patients (99%) were identified, with sensitivity (98.4%) and negative predictive value (96.1%). The fact that there is no need to depend on exclusion of other causes of chronic liver disease enhances the feasibility of these criteria.
The new name, MAFLD, was distinguished by several benefits, including accurate and easy diagnosis of the disease in addition to providing appropriate health care and avoidance of stigmatization, trivialization and proper catalyst to increase funding and heath plan action (11).
It was not long before the new nomenclature, MAFLD, was published until the researchers grabbed it.
Applying the MAFLD criteria in studying a cohort of patients with COVID-19 infection in China was published recently (17) . The investigators found that the presence of MAFLD increased the risk obesity on severity of illness with COVID-19infection. In the same context, other researcher, in a multicenter study, found a severe form of Covid-19 in younger patients with MAFLD in the preliminary analysis (18) . This finding was attributed to the systemic and hepatic immune response caused by MAFLD contributing to cytokine storm in patients with COVID-19.
Another intriguing finding in our study, non-obese MAFLD patients represent more than 40% of all patients with a higher ratio than other areas in the world and 12.3% were lean MAFLD. Patients with lean MAFLD have favourable metabolic characteristics and fibrosis scores compared to their All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 22, 2020. Though the pathogenesis of lean MAFLD is not completely understood, a recent study that is shaped by differential metabolic adaptation, that is mediated by increased bile acids and Farnesoid X receptor (FXR) activity compared to the obese counterparts (20) . Lean MAFLD patients also tends to have distinct microbiota profile and frequency of genetic variants, such as Transmembrane 6 superfamily 2 and (TM6SF2) interferon lambda 3 (IFNL3) compared to obese subjects (20) (21) (22) .
Noticeably, the new MAFLD criteria allowed us to better define patients with dual aetiologies. Herein, we characterised the nature of (MAFLD-HCV) instead of exclusion of HCV in previous NAFLD name leading to a drop of large number of patients with actual metabolic dysfunction especially in countries with high prevalence of HCV like Egypt. When comparing MAFLD patients with patients with dual pathology (MAFLD-HCV) we found not only a significant increase in the metabolic parameters but also a severer fibrosis in patients with dual pathology. This association remained significant after adjusting gender, ALT and AST in multiple regression analysis.
Identification of MAFLD and dual pathologies may lead to a notable change in the liver disease map globally. In a recent report revising data of 2.14 million liver related death, NAFLD was diagnosed in 9% of patients while HCV was diagnosed in 26% and alcoholic liver disease was diagnosed in 25% who died of chronic liver diseases (23). We think that health care and research could get benefit from distinguishing metabolic associated fatty liver diseases and dual pathologies for better management and decreasing the mortality.
The study could have some limitation, the cohort was recruited in a tertiary centre, so it might be subject of selection bias and lack of liver biopsy hinders the details assessment of histological features. Further studies would be required to ascertain that these proposed criteria would achieve the claimed high utility in other communities with different genetic backgrounds, environmental factors, and health care systems than those in the Egypt.
In conclusion, the diagnostic criteria proposed by the international consensus panel for the diagnosis of MAFLD EDCT are easy to apply, and have excellent diagnostic properties, which make them useful for routine clinical practice, large population-based epidemiological studies or for, clinical trials. In addition, application of these criteria allowed to capture the entire spectrum of All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20176214 doi: medRxiv preprint disease including non-obese subjects and to identify a previously largely overlooked group who are having dual etiologies of MAFLD with other liver diseases. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 22, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 22, 2020 All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. T  a  b  l  e  3  :  a  s  s  o  c  i  a  t  i  o  n  b  e  t  w  e  e  n  t  h  e  d  i  f  f  e  r  e  n  t  f  i  b  r  o  s  i  s  s  c  o  r  e  s  a  n  d  t  h  e  M  A  F  L All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20176214 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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