Impact of direct-acting antiviral regimens on hepatic and extrahepatic manifestations of hepatitis C virus infection

Abstract

Hepatitis C virus (HCV) is a common cause of liver disease and is associated with various extrahepatic manifestations (EHMs). This mini-review outlines the currently available treatments for HCV infection and their prognostic effect on hepatic manifestations and EHMs. Direct-acting antiviral (DAA) regimens are considered pan-genotypic as they achieve a sustained virological response (SVR) > 85% after 12 wk through all the major HCV genotypes, with high percentages of SVR even in advanced fibrosis and cirrhosis. The risk factors for DAA failure include old males, cirrhosis, and the presence of resistance-associated substitutions (RAS) in the region targeted by the received DAAs. The effectiveness of DAA regimens is reduced in HCV genotype 3 with baseline RAS like A30K, Y93H, and P53del. Moreover, the European Association for the Study of the Liver recommended the identification of baseline RAS for HCV genotype 1a. The higher rate of hepatocellular carcinoma (HCC) after DAA therapy may be related to the fact that DAA regimens are offered to patients with advanced liver fibrosis and cirrhosis, where interferon was contraindicated to those patients. The change in the growth of pre-existing subclinical, undetectable HCC upon DAA treatment might be also a cause. Furthermore, after DAA therapy, the T cell-dependent immune response is much weaker upon HCV clearance, and the down-regulation of TNF-α or the elevated neutrophil to lymphocyte ratio might increase the risk of HCC. DAAs can result in reactivation of hepatitis B virus (HBV) in HCV co-infected patients. DAAs are effective in treating HCV-associated mixed cryoglobulinemia, with clinical and immunological responses, and have rapid and high effectiveness in thrombocytopenia. DAAs improve insulin resistance in 90% of patients, increase glomerular filtration rate, and decrease proteinuria, hematuria and articular manifestations. HCV clearance by DAAs allows a significant improvement in atherosclerosis and metabolic and immunological conditions, with a reduction of major cardiovascular events. They also improve physical function, fatigue, cognitive impairment, and quality of life. Early therapeutic approach with DAAs is recommended as it cure many of the EHMs that are still in a reversible stage and can prevent others that can develop due to delayed treatment.

Key Words: Hepatitis C virus; Hepatic; Extrahepatic; Direct-acting antivirals; Impact

Core Tip: Direct-acting antivirals (DAAs) are achieving an over 85% sustained virological response in treating hepatitis C virus (HCV) infection. The risk factors for DAAs failure include old males, cirrhosis, and the presence of resistance-associated substitutions mainly in genotypes 1a and 3. The higher rate of hepatocellular carcinoma after DAA therapy may be due to offering DAA regimens to patients with advanced liver fibrosis and cirrhosis, where using interferon was contraindicated. The change in the growth of pre-existing subclinical, undetectable hepatocellular carcinoma upon DAA treatment might be a cause. DAAs are effective in treating HCV-associated mixed cryoglobulinemia, thrombocytopenia, rheumatological, renal, and cardiovascular diseases.

INTRODUCTION

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be 58 million people, and 1.5 million individuals get new HCV infection annually. The World Health Organization stated that, about 290 thousand patients died from hepatitis C-related complications in 2019[1]. In 2016, the World Health Assembly adopted the Global Health Sector Strategy on viral hepatitis. This strategy is directed towards eliminating both viral hepatitis B and C infections. To achieve the target objective, this will require the diagnosis of 90% of the infected patients, followed by treatment of 80% of the diagnosed individuals[2]. HCV leads to acute and chronic hepatitis, progressing to lifelong liver cirrhosis and cancer, and is associated with several extrahepatic manifestations (EHMs)[1]. The aim of antiviral treatment is HCV eradication, thus preventing disease progression and reducing the EHMs. This mini-review outlines the currently available treatments for HCV infection and their prognostic effect on hepatic manifestations and EHMs.

HEPATITIS C VIRUS

HCV possesses a single-stranded RNA genome that encodes a polyprotein, which is processed into ten proteins: E1, E2, core, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B[3]. The structural proteins E1, E2, and core are components of the virion and the nonstructural proteins NS3/4A, NS5A, and NS5B are involved in viral genome replication[4,5]. To enter the host cell, HCV requires a cascade of synchronized and sequentially ordered events where the virus binds to many receptors. HCV particles circulate, as lipoviroparticles (LVPs), in association with low-density lipoprotein (LDL) and very-LDL (VLDL) components, including apolipoproteins (such as Apo-B, Apo-AI, Apo-CI, and Apo-E)[6]. HCV core, E1, E2, and P7 are essential for cell-free and cell-to-cell viral transmission[7]. HCV recognition is initiated by Toll-like receptor 3 and retinoic acid-inducible gene I[8].

HCV infects hepatocytes through cell-free and cell-to-cell viral transmission (Table 1). LVPs circulate in the sinusoidal blood, and through sinusoidal endothelial fenestration, they become in contact with receptors on the basolateral membrane of hepatocytes[9]. The virus envelope glycoproteins and virus-associated lipoprotein components (particularly apoE) of LVPs attach to hepatocyte basolateral membranes through interaction with highly sulfated proteoglycans, particularly syndecans, LDL receptor (LDLr), and scavenger receptor class B type I (SR-BI) on the cell surface[10]. SR-B1, as both an entry factor and an attachment factor, has been shown to bind viral envelope proteins[11]. Knockdown of individual gene of LDLr or SR-B1 had a moderate impact on HCV infection. While, knockdown of genes of both receptors resulted in a much more pronounced effect[12].

Table 1 Current diagnostic and tools to assess liver disease stages and severity in hepatitis C virus infected patients.

Diagnostic tool	Early fibrosis stages (METAVIR less than F2)	Fibrosis 2	Fibrosis 3	Compensated cirrhosis	Decompensated cirrhosis	Ref.
HCV antibody	Positive	Positive	Positive	Positive	Positive	AASLD and IDSA[30]
Quantitative HCV RNA (viral load)	Positive	Positive	Positive	Positive	Positive
Platelet count < 150000/mm3)	Normal	Normal	Normal	< 150000/mm3	< 150000/mm3
Total and direct bilirubin, ALT & AST	Normal/elevated	Normal/elevated	Normal/elevated	Elevated	Elevated
Child- Pugh	--------	--------	--------	Class A (scores 5-6)	Class B (scores 7-9); Class C (scores 10-15)
FIB-4 Score	< 1.45	≥ 1.45 but < 2.67	≥ 2.67 but < 3.25	≥ 3.25	> 3.25	Filozof et al[31]
Fibroscan by transient elastography	5.3 kPa	7.4 kPa	9.1 kPa	13.2 kPa	13.2 kPa	Platon et al[32]
Fibro test	< 0.48	0.48 - 0.58	> 0.58 but < 0.74	> 0.74	> 0.74	Laboratory Corporation of America[33]
Enhanced liver fibrosis test	< 7.7	7.7	9.8	11.3	11.3	Lichtinghagen et al[34]
Aspartate aminotransferase to platelet ratio index	< 0.77	0.77	0.77	≥ 0.83	≥ 0.83	Lin et al[35]
Liver nodularity and/or splenomegaly	Negative	Negative	Negative	Positive	Positive	AASLD and IDSA[30]
Prior liver biopsy	F0: No fibrosis; F1: Portal fibrosis without septa	F2: Portal Fibrosis with few septa	F3: Numerous septa without cirrhosis	F4: Cirrhosis	F4: Cirrhosis

HCV: Hepatitis C virus.

Attachment to SR-BI helps bind of LVPs to cluster of differentiation 81 (CD81), claudin-1 (CLDN1), and occludin (OCLN)[13]. Interaction of HCV with CD81 causes activation of epidermal growth factor receptor signaling and facilitates CD81 diffusion and formation of the HCV-CD81-CLDN1 complex[14]. This complex then interacts with OCLN, which mediates the clathrin-dependent internalization through interacting with GTPase dynamin[15]. Other entry factors have been demonstrated, such as CD36 which interacts directly with HCV E1 protein[16]. In addition, TIM-1/human hepatitis A virus cellular receptor 1/CD365 has been identified as a contributing factor to LVP attachment through interaction with phosphatidylserine exposed on the HCV envelope[17]. This interaction may enhance viral attachment and subsequent interaction with the main entry factors[18]. HCV uses cortactin (an actin-binding protein at the cell periphery) for its assembly to promote viral proliferation and controls cortactin phosphorylation to facilitate cell invasion. Cortactin may be involved in hepatic cell migration, so it may be a potential target to interfere with the HCV cellular pathogenesis[19].

SR-B1 has also a prominent role in cell-to-cell transmission. This type of transmission assists immune evasion and persistence. Cell-to-cell transmission may be the main route of HCV dissemination in chronically infected patients[20]. LIM and SH3 protein 1 (LASP-1) is a specific adhesion protein that plays an important role in the regulation of cell migration, proliferation, and protein-protein interactions. LASP-1 is an HCV NS5A-interacting partner. Both LASP-1 and NS5A are localized in the cytoplasm of HCV infected cells. RNA and protein levels of LASP-1 were increased in these cells, indicating that LASP-1 may be involved in HCV-induced liver pathogenesis[21].

HCV can also infect and replicate in other cell types, such as peripheral blood mononuclear cells (PBMCs) and bone marrow cells through cell-to-cell transmission[22]. HCV infects PBMCs and other cells through the interaction with CD81 molecules on the cell surface[23], allowing replication of HCV in the extrahepatic tissues, which is facilitated by the expression of miR-122[24]. B lymphocytes, particularly CD27+ memory B cells, can resist apoptosis and may serve as an HCV reservoir[25]. Infection of PBMCs with HCV leads to dysregulation of the signaling pathway mediators such as STAT-1 and IRF-1 and alterations in cytokine and chemokine production, including IL-1, IL-6, IL-8, and IL-10. Persistent HCV RNA and its antigens, combined with chronic immune activation, lead to exhaustion of PBMCs that become defective and more prone to programmed cell death[26]. Liu et al[27] prepared cell culture-derived infectious HCV particles (HCVcc) using Huh7 cells transfected with HCV RNA. They found that HCV entry into macrophages depends mainly on its phagocytic activity and does not depend on its cell receptors. Knockdown of CD81 had a minimal effect on the entry of HCVcc into macrophages. Exosomes have been demonstrated to contain HCV-RNA. However, the mechanism responsible for the transmission of HCV genomic RNA through exosomes is still not clarified[28].

EVALUATION OF SEVERITY OF LIVER DISORDERS BEFORE AND AFTER THERAPY

Before starting direct-acting antiviral (DAA) therapy, liver disease severity should be assessed to detect clinically unapparent advanced fibrosis (METAVIR score F3) or cirrhosis (METAVIR score F4). In patients with cirrhosis, portal hypertension and esophageal varices should also be assessed[29]. These are important steps, as the choice of DAA regimens, prognosis, and hepatocellular carcinoma (HCC) surveillance every 6 months depend on the stage of fibrosis. Table 1 summarizes some of the current available HCV diagnostic and staging tests according to AASLD and IDSA[30], Filozof et al[31], and other studies[32-35]. Liver stiffness measurement (LSM) using transient elastography can assess the degree of liver fibrosis and portal hypertension. Aspartate aminotransferase to platelet ratio index and fibrosis-4 (FIB-4) are simple, inexpensive, and reliable panels of fibrosis biomarkers that can be used. However, these panels may be less sensitive among African patients. Both LSM and biomarkers are expected to be efficient in distinguishing cirrhosis vs no fibrosis, with the lower ability for intermediate degrees of fibrosis. The combination of blood biomarkers or the combination of LSM and a blood test may improve accuracy[36].

LSM importance after sustained virological response (SVR) remains uncertain. Several studies have reported the significant regression of LSM after treatment of HCV infection with DAAs[37]. However, it is still debatable whether the decrease of LSM and post-DAA HCV eradication are due to the suppression of viral necro-inflammatory activity or regression of liver fibrosis[38]. It is recommended that assessing the fibrosis stage after therapy using non-invasive tools should not be endorsed as they are unreliable in this setting[29].

IMPACT OF DIRECT-ACTING ANTIVIRAL REGIMENS ON HCV INFECTION

Until 2011, pegylated interferon alpha (PEG-IFNα) with ribavirin (RBV) was the standard therapy for HCV infection, with an about 50% SVR[39]. The European Association for the Study of Liver Diseases (EASL)[29] recommended that the endpoint of therapy is undetectable HCV RNA either in serum or plasma by an assay with a lower limit of detection ≤ 15 IU/mL, 12 wk (SVR12) or 24 wk (SVR24) after the end of treatment[29]. In low-resource areas, as an alternative to HCV RNA, HCV antigen (HCV Ag) testing might be useful for diagnosis of active HCV infection and at the end of treatment[40].

The identification of HCV encoded proteins and their function allowed the development of highly effective DAA regimens against the NS3 protease, NS5A, and the NS5B polymerase[41]. The maximum effectiveness of therapy is obtained when the patients are treated at early stage before advanced liver fibrosis or cirrhosis[42,43]. According to the mechanism of action, DAAs can be classified into four different groups: NS3/4A protease inhibitors [Glecaprevir (GLE), Voxilaprevir (VOX), Grazoprevir, Paritaprevir (PTV), and Simeprevir (SIM)], NS5A protein inhibitors [Daclatasvir (DCV), Velpatasvir (VEL), Ledipasvir (LDV), Ombitasvir (OBV), Pibrentasvir (PIB), and Elbasvir], NS5B polymerase inhibitor-nucleoside analogue [Sofosbuvir (SOF)], and NS5B polymerase inhibitor-non-nucleoside analogue [Dasabuvir (DSV)]. These drugs are considered pan-genotypic as they achieve a SVR > 85% through all the major HCV genotypes[44]. All DAAs are effective for genotype 1 and 4 and SOF for genotype 2. While for genotype 3, SOF, DCV, and LDV are effective. For genotypes 5 and 6, a combination of two regimens (VEL/SOF and asunaprevir (ASV)/DCV/beclabuvir) is indicated. A review for 28 randomized clinical trials, enrolling more than 7000 HCV naïve patients, revealed that DAA regimens for 12 wk significantly increased SVR12 and SVR24 compared to placebo and HCV cure was achieved in about 90.5% of patients. DAAs were well tolerated with no increase in serious adverse effects[39].

DAAs are recommended for both naïve patients as well as those who failed to achieve SVR after prior treatment. In addition, treatment is recommended for patients with advanced fibrosis or cirrhosis, including decompensated cirrhosis. Guidelines for the global standard treatment established SOF + VEL or GLE/PIB as the first recommended drug regimen for naïve patients, irrespective to HCV genotype or the presence of compensated liver cirrhosis[29,45]. Moreover, lifelong monitoring for HCC is recommended for patients with advanced fibrosis and cirrhosis, even with SVR, as DAAs decrease, but does not eliminate the risk of HCC[29,46]. In a multicenter cohort study involving 868 HCV patients with liver cirrhosis treated with DAA regimens, SVR was attained at 90% in Child-Pugh A patients and 81% in Child-Pugh B/C patients. Within a median period of 28 months follow-up, 14% of patients with Child-Pugh A and 64% of those with Child-Pugh B/C developed disease progression[47]. The use of protease inhibitors is contraindicated in patients with decompensated cirrhosis or with prior episodes of decompensation. These inhibitors carry a substantially higher drug exposure and risk of toxicity due to their hepatic metabolization[48]. Thus, the fixed-dose combination of SOF and VEL is the treatment of choice for patients with decompensated (Child-Pugh B or C) cirrhosis or with compensated (Child-Pugh A) cirrhosis with prior episodes of decompensation[29]. Tables 2 summarizes the current recommended DAA regimens for treating HCV infection according to AASL/ADSA 2021[30].

Table 2 Recommended direct-acting antiviral regimens for treatment of hepatitis C virus infection according to AASL/ADSA 2021.

Treatment	No cirrhosis		Compensated cirrhosis		Decompensated cirrhosis
	Naïve HCV infected patient	Previously treated patients	Naïve HCV infected patients	Previously treated patients
Sofosbuvir (400 mg)/Velpatasvir (100 mg)	12 wk	Sofosbuvir (400 mg)/Velpatasvir (100 mg)/Voxilapevir (100 mg), 12 wk, for all genotypes. ALTERNATIVE: Glecaprevir (300 mg)/Pibrentasvir (120 mg), but not recommended for genotype 3 with Sofosbuvir/NS5A inhibitor	For genotypes 1, 2, 4, 5, and 6 & genotype 3 with NS5A-RAS Y93H negative, 12 wk, but not recommended for genotype 3 with NS5A-RAS Y93H positivity	Sofosbuvir (400 mg)/Velpatasvir (100 mg)/Voxilapevir (100 mg), 12 wk, for genotypes 1, 2, 4, 5, and 6; for genotype 3, 12 wk in addition to weight-based Ribavirin. ALTERNATIVE: Glecaprevir (300 mg)/Pibrentasvir (120 mg), but not recommended for genotype 3 with Sofosbuvir/NS5A inhibitor	Patients with HCV infection who have decompensated cirrhosis, i.e., Child-Pugh class B or class C, should be referred to a medical practitioner with expertise in that condition, ideally in a liver transplant center
Glecaprevir (300 mg)/Pibrentasvir (120 mg)	8 wk	16 wk in addition to Sofosbuvir (400 mg) + weight-based Ribavirin ALTERNATIVE: 12 wk of Sofosbuvir (400 mg)/Velpatasvir (100 mg)/Voxilapevir (100 mg)	8 wk	16 wk in addition to Sofosbuvir (400 mg) + weight-based Ribavirin. ALTERNATIVE: 12 wk of Sofosbuvir (400 mg)/Velpatasvir (100 mg)/Voxilapevir (100 mg) in addition to weight-based Ribavirin
Elbasvir (50 mg)/Grazoprevir (100 mg)	12 wk for genotype 1b	12 wk Sofosbuvir (400 mg)/Velpatasvir (100 mg)/Voxilapevir (100 mg). However, Glecaprevir/Pibrentasvir for 16 wk is not recommended as an alternative for this group of patients	12 wk for genotype 1B	NA

HCV: Hepatitis C virus; RAS: Resistance-associated substitutions; NA: Not applicable

Direct-acting antiviral treatment failure and retreatment

Risk factors for DAA failure include males with advanced liver fibrosis/cirrhosis, the presence of resistance-associated substitutions (RAS) in the region targeted by the received DAAs, and inadequacy of treatment. RAS linked to the NS5A gene are present at higher levels and persist for longer duration than those linked to the NS3/4 gene[49]. The naturally occurring RAS do not affect treatment efficacy, as they are present in a minority of circulating HCV virions. RAS resulting from treatment are present in the majority of the circulating HCV quasispecies, which decrease the efficacy of re-treatment with the same DAA class[50]. For DAA regimens involving LDV/SOF and DCV/SOF, the identification of baseline RASs for HCV genotype, such as 1a, is recommended to decide the treatment duration or if RBV addition is needed[51]. Eventually, the adverse impact of baseline RAS could be decreased by increasing duration of treatment or optimizing DAA regimens. However, a considerable percent of treatment failures is triggered by RAS acquired during therapy[49]. This may be related to the relatively low barrier to resistance of the NS5A region and the high genetic barrier of SOF. Moreover, the third-generation NS3 inhibitors are expected to have intermediate genetic barrier in HCV genotypes 1a and 1b and very high in non-1 genotypes[52]. A meta-analysis on 6500 HCV infected patients, reported reduced effectiveness of GLE/PIB in HCV genotype 3 with baseline RAS like A30K, Y93H, and P53del. Testing RAS for genotype 3 HCV infection is mandatory to improve the prognosis of treatment outcome and selection of therapy[53].

Baseline RAS were only identified in the NS5A region in Iranian patients with HCV genotypes 1a and 3a with no RAS in the NS5B region[54]. Among 539 Italian HCV genotype 3 patients (417 DAA-naïve and 135 DAA-failed), Sanger sequencing of NS3/NS5A/NS5B at baseline samples showed a higher prevalence of NS5A RAS in DAA-failed (5/13, 38.5%) vs DAA-naïve (61/393, 15.5%, P = 0.04) patients. The presence of baseline Y93H and/or A30K was associated with SVR rate of 72.2% vs 95.7% among patients without NS5A RAS (P = 0.002). Chen et al[55] and Pisaturo et al[52] reported at least one RAS among over 85% out of the studied 220 HCV naïve patients with DAA-based treatment. However, according to the recommendation of international guidelines, massive testing for RAS detection before starting DAAs treatment is not needed, with some exceptions[29,45]. RAS testing before treatment is recommended for HCV genotype 3 infected patients with liver cirrhosis, as those without a baseline Y93H RAS in NS5A are eligible for SOF/VEL therapy. While, those with baseline Y93H RAS could be treated with SOF/VEL/VOX or SOF/VEL plus RBV[45]. However, according to EASL guidelines, the same therapeutic regimen should be used to all compensated cirrhotic patients regardless of viral genotype[29]. Currently, the US Food and Drug Administration (FDA) and European Medical Agency (EMA) approved two types of DAA regimens, SOF/VEL/VOX and GLE/PIB, to treat patients with previous experience of DAAs failure[36,46-48]. The effectiveness of the regimen SOF/VEL/VOX plus or minus RBV for 12 wk among patients with DAA failure revealed that SVR at 12 wk ranged from 91% to 100%. Most patients tolerated retreatment well[58].

Direct-acting antivirals and hepatocellular carcinoma

The impact of DAAs on the development of HCC is controversial. Meanwhile, it should be noted that in all studies, the risk of HCC remained even after successful HCV treatment. A meta-analysis study revealed that, the incidence rate for a new HCC was 3.3% (95% confidence interval: 1.2-9%) per year after DAA treatment[57]. Some studies reported that, the risk of de novo HCC after DAA therapy was reduced, while other studies noted a much higher HCC risk mainly within the first year after DAA therapy than later[58,59]. A retrospective cohort study was carried out on 243 consecutive HCV patients who received PEG-IFN/RBV and were followed for a median of 9.3 years, and 263 HCV patients who received DAA treatment and were followed for a median of 4.1 years. It revealed that a considerably increased hazard was associated with DAA treatment[60]. A French study conducted on 1270 HCV patients revealed that, the differences of the occurrence of HCC after IFN and DAA regimens could be explained by the higher prevalence of Child-Pugh class B, portal hypertension, and diabetes among DAA-treated patients vs IFN-induced SVR patients. A time-dependent Cox model weighted by inverse probability of treatment was used to overcome selection bias. This model shows that DAAs were not significantly associated with an increase in the risk of HCC occurrence (P = 0.73), nor with a more aggressive pattern of presentation[61].

The higher rate of HCC after DAA therapy may be because they are the drugs of choice for treating old patients and those with liver cirrhosis and end-stage liver disease as IFN was not indicated to treat such patients[62]. The possible clarification of the elevated incidence of HCC after the start of DAA therapy, might be the change in the growth of pre-existing subclinical and undetectable HCC upon DAA treatment[60]. A high HCC risk after DAA treatment was also reported, especially in individuals with uncharacterized liver nodules[63]. Owusu Sekyere et al[64] detected a reduced HCC specific tumor response upon DAA-induced HCV clearance. In HCV patients who subsequently developed HCC, the T cell-dependent immune response was much weaker, indicating their important role in inhibiting tumor growth. DAA therapy for HCV was associated with a weakening of the strength of HCC-specific CD8+ but not CD4+ T cell responses in cirrhotic patients in vitro. Moreover, a mechanism like cellular behavior after eradication of HCV by DAA therapy may increase the HCC growths as detected in early test models[59]. Recently, Lu et al[65] concluded that the down-regulation of tumor necrosis factor α (TNF-α) after successful DAA therapy increases the risk of HCC and the inhibition of TNF-α might attenuate the host immune surveillance against tumor cells. These findings might provide a clue for the pathogenesis of HCC and a strategy for HCC surveillance based on risk stratification. In Egypt, HCC was found to be significantly aggressive in HCV patients treated with DAAs, especially among those with an elevated neutrophil to lymphocyte ratio (P= 0.012)[66]. It is recommended to screen for HCV every 6 months for cases with cirrhosis and every 12 months for those without cirrhosis after DAA therapy[45].

DAAs appear safe for patients with a history of treated HCC and are not associated with an increased risk for cancer recurrence except for cases with vascular invasion, where aggressive HCC recurrence was reported[67]. For HCC patient candidates for a liver transplant, decisions regarding the timing of DAA treatment depend on organ availability and region wait times and should be individualized[68]. Moreover, Fouad et al[69] suggested that anti-HCV therapy in HCC patients should be postponed until further research for safety and effectiveness is carried out.

Hepatitis C virus and hepatitis B virus co-infection

Hepatitis B virus (HBV) and HCV are the major causes of liver disease worldwide. The administration of compulsory HBV vaccination is effective in providing long-term protection against infection, even with low seroprotection rate, proved by the presence of high anamnestic response rate after being given a HBV challenging dose[70,71]. However, poly-transfused vaccinated individuals, either with or without HCV infection, are at risk of HBV infection. In these patients, HBV-DNA was detected even among HBsAg negative patients (occult HBV infection)[72,73]. The co-infection with both viruses increases the rates of cirrhosis and HCC[74,75]. When both HBV and HCV are present in the same cell, reciprocal inhibition of one viral genome by the other virus takes place and leads to the dominance of one virus over the other. The dominant virus replicates more actively and inhibits the replication of the non-dominant virus. However, co-dominance may occur if there is nearly equal replication of both HBV and HCV[76]. In 2018, EASL recommended that hepatitis C patients should be tested for HB surface (HBs) antigen, HB core antibody (anti-HBc), and HBs antibody (anti-HBs) prior to starting DAA-based treatment. In HBs antigen positive patients, concurrent HBV nucleoside/nucleotide analogue therapy is indicated. For anti-HBc positive patients with negative HBsAg, serum alanine transaminase (ALT) levels should be monitored and both HBs antigen and HBV DNA should be tested, if ALT levels rise or do not return to normal during or after anti-HCV therapy. In anti-HBs and anti-HBc antibodies positive patients, monitoring of serum ALT levels is indicated[51].

Co-infected patients may experience HBV reactivation after the cure of their HCV by PEG-IFN or DAA-based therapy and anti-HBV therapy should be started if clinically indicated[77]. The US FDA warned of the higher risk and earlier onset of HBV reactivation with DAA treatment[78]. This can be expected since HCV DAAs have no direct or immunomodulatory effect on the replication of HBV. Close monitoring of HBV infection status is settled by all guidelines with the implementation of IFN-free regimens. A retrospective study revealed that only 9 out of 62290 patients treated with DAAs had HBV reactivation. Eight patients were known to be HBsAg positive, and one patient was known to be isolated anti-HBc-positive. Seventeen other patients had a small increase in HBV DNA levels that did not qualify as HBV reactivation[79].

EXTRAHEPATIC MANIFESTATIONS OF CHRONIC HEPATITIS C VIRUS INFECTION

HCV can cause extrahepatic diseases that lead to an increase in the overall mortality. Figure 1 shows the pathophysiology of HCV infection in hepatic and extrahepatic diseases. The following extrahepatic diseases are related to HCV infection:

Figure 1 Pathophysiology of hepatitis C virus infection in hepatic and extrahepatic diseases. IR: Insulin resistance; T2DM: Type 2 Diabetes Mellitus; IL-1β: Interleukin-1 beta; TNF-α: Tumor necrosis factor-alpha; RF: Rheumatoid factor; ASMA: Anti-smooth muscle antibody; B cell NHLs: B-cell non-Hodgkin lymphomas.

Mixed cryoglobulinemia and cryoglobulinemic vasculitis

Chronic HCV infection is a common cause of mixed cryoglobulinemic vasculitis (MCV). In about 40%-60% of patients with chronic HCV infection, circulating mixed cryoglobulins are detected. However, overt cryoglobulinemia vasculitis (CV) is observed in only 5%-10% of patients[80,81]. As shown in Figure 1, the pathogenesis of MCV involves viral-induced activation of B cell clones which generate pathogenic IgM with rheumatoid factor (RF) activity. Monoclonal IgM and polyclonal IgG bind together and recognize hepatitis C nucleocapsid and core antigens. The resulting circulating immune complexes deposit in vascular beds of small-to-medium vessels, enhancing complement activation, leukocyte recruitment, and vasculitis[82]. The clinical manifestations of the disease are variable, ranging from mild symptoms such as purpura, arthralgia, and fatigue to more serious life-threatening complications resulting from neurologic and renal involvement[83].

Treatment of HCV-MCV is challenging. The main goal is SVR in order to down-regulate the B-cell arm of autoimmunity that is triggered by the virus. DAA regimens are now the drug of choice for HCV-associated MCV. The combination of PEG-IFNα with RBV has been abandoned for their side-effects, including the immune-stimulatory effects[84]. With DAA therapy, the rate of SVR after 12 months of therapy was the same for HCV patients with and without mixed cryoglobulinemia (MC). However, MCV may persist or reappear in some patients after SVR[85]. Moreover, new onset cases of cryoglobulinemic glomerulonephritis were also reported[86]. After DAA therapy for HCV associated MC, 64% to 96% of the patients improved clinically; however, the immunological response (defined by marked reduction or disappearance of circulating cryoglobulins and normalization of the levels of RF and C4) was only from 48% to 89%[87].

Artemova et al[88] reported complete disappearance of cryoglobulinemia among 48% of HCV-CV patients and a decrease in cryoglobulins among 17% of them. Response rates of HCV-CV after DAA treatment vary according to the organ involvement. A higher response rate (75%-100%) was attained for cutaneous and musculoskeletal presentations, while lower response rates (30%-70%) were attained in peripheral nerve and renal involvement[89]. The lag in immunologic and/or clinical response behind the viral clearance may be due to delay in the clearance of cryoglobulins from the circulation after successful antiviral therapy or to the persistence of the RF-producing memory B-cell clones for at least 24 wk[90]. Occult HCV infection is another possible explanation especially in case of cryoglobulinemic glomerulonephritis[91]. Abdelhamid et al[92] suggested that some forms of alteration in the immune system can be responsible for the persistent HCV-related immune disease after viral clearance. The recommended drug for patients with persistent or recurrent MCV after SVR is Rituximab, which is a B-cell depleting monoclonal antibody. In rapidly progressing or fulminant cases or severe exacerbation of vasculitis causing life-threatening complications, plasmapheresis is added to remove the circulating cryoglobulins[93].

Thrombocytopenia

Thrombocytopenia is a common complication in chronic HCV infection, causing an increased risk of bleeding[94,95]. The prevalence and severity of thrombocytopenia increase with the progression of liver disease and the development of hepatocellular damage and hepatic fibrosis[96]. Its prevalence is 6% in chronic liver disease patients, while it is 24% in chronic HCV infected patients and increases to 78% in cirrhotic patients[94-97]. The pathophysiology of thrombocytopenia in chronic HCV is multifactorial and largely related to the severity of hepatic infection. It includes splenomegaly and the related hypersplenism causing platelet sequestration, auto-immunogenicity, impaired production of thrombopoietin due to advanced fibrosis, possible direct effect of HCV as direct bone marrow suppression, and therapeutic adverse effects[94-96].

In the IFN era, starting or maintaining IFN therapy was a great challenge in the treatment of chronic HCV patients with thrombocytopenia[96]. IFN causes a further decrease in the platelet count in up to 13% of patients[98]. On the other hand, DAA treatment achieve an over 95% SVR at 24 wk among HCV infected thrombocytopenic patients with advanced fibrosis and cirrhosis. Moreover, the platelet count showed statistically significant improvement[99,100]. Chen et al[97] found that 99.6% of chronic HCV infected patients with thrombocytopenia receiving DAA treatment achieved a SVR and thrombocytopenia improved significantly in 41.7% of them. Another study reported a highly effective and safe DAA regimen, with improvement of platelet count in 73% of thrombocytopenic patients, especially in mild to moderate stages of hepatic fibrosis[95].

Hepatitis C virus and glomerulopathies

HCV infection is associated with several glomerulopathies including membranoproliferative glomerulonephritis (MPGN). It is associated with MCV in 80%-95% of the cases. Other HCV glomerular diseases include membranous nephropathy, proliferative glomerulonephritis, focal segmental glomerulosclerosis, fibrillary glomerulonephritis, IgA nephropathy, immunotactoid glomerulopathy, and renal thrombotic microangiopathy. HCV infection also increases the risk of chronic kidney disease (CKD). The association between chronic HCV infection and CKD is more significant with high HCV viral load and HCV genotype 2[84,101,102].

In MPGN associated with MCV, the developed immune complex deposits in the mesangium, capillaries, and urinary space of glomeruli, which can be manifested as nephrotic and nephritic syndromes[91,103,104]. Furthermore, HCV can cause kidney damage through direct cytopathic effect by viral invasion of the renal parenchyma (mesangial, endothelial, and tubular cells of the kidney) and through nephrotoxicity of drugs used for its treatment. Additionally, non-immunological pathways as oxidative stress or pro-inflammatory cytokines help the development of renal disease by vascular injury as shown in Figure 1. In addition, HCV infected patients may have an increased risk of insulin resistance, which develops during the inflammation process and exacerbates renal damage[105-108]. In very few cases, viral NS3 was found in the glomerular deposits, capillary walls, and the mesangium[84].

Previously, HCV kidney manifestations were treated with PEG-IFN plus RBV; however, these drugs presented low efficacy, low SVR (< 50%), and severe side effects as acute renal failure, graft failure, and hemolytic anemia[109,110]. DAA therapy improves glomerular filtration rate, decreases proteinuria and hematuria, and shortens treatment duration to only 8-12 wk without significant side effects[111-113]. Delays in initiation of DAA therapy could have deleterious effects[114-116]. In patients with an estimated glomerular filtration rate (eGFR) > 30 ml/min/1.73 m², the use of SOF with SIM with or without ribavirin decreased proteinuria and improved eGFR[117,118]. There are different approved regimens for patients having an eGFR < 30 ml/min/1.73 m² or those on dialysis: (1) OBV + PTV + Ritonavir (RITV) + DSV; (2) RBV + Elbasvir + Grazoprevir; and (3) GLE + PIB. The side effects were only mild general symptoms like fatigue, insomnia, dizziness, and headache[119-121]. Furthermore, DCV and ASV are important options, especially for patients with renal impairment since both DCV and ASV have minimal renal excretion[122].

Hepatitis C virus and type 2 diabetes mellitus

HCV infected patients have impaired glucose metabolism with hyperinsulinemia due to decreased insulin catabolism or insulin resistance (IR). Up to 60%-80% of HCV cases have glucose intolerance, 20% of them develop type 2 diabetes (T2DM), and up to 41%-70% of them have IR. T2DM which develops as a complication of HCV infection is known as hepatogenous diabetes[123-125].

HCV can induce IR through direct and indirect ways as shown in Figure 1. The viral core protein can directly interfere with intracellular insulin signaling by inhibiting the expression of insulin receptor substrate (IRS)-1 and IRS-2. HCV replicates in the pancreatic β-cells, causing impairment of their function involved in glucose metabolism. In addition, HCV infection can indirectly induce IR due to oxidative stress, liver steatosis, release of inflammatory cytokines such as TNF-α, interleukin (IL)-1, IL-6, and leptin, phosphorylation of the insulin-1 receptor substrate and protein kinase B, and up-regulation of gluconeogenic genes such as glucose 6 phosphatase and phosphoenolpyruvate carboxy kinase[84,126,127].

SVR with PEG-IFN and RBV is associated with decreased IR after 24 wk of therapy[128,129]. DAAs improve the IR by 90%. Treatment with DAA regimens has ameliorated hyperglycemia, recovered pancreatic beta-cell function, and reduced cytokine production[130-132]. Furthermore, the eradication of HCV by DAAs such as SOF-based regimen led to the improvement in hemoglobin A1c percentage[133,134].

Hepatitis C virus and rheumatological manifestations

The rheumatologic and musculoskeletal manifestations are the most common EHMs, affecting 40-80% of HCV infected patients. MC is one of the causes of HCV associated rheumatologic manifestations (RM)[135]. Cryoglobulins were found to be deposited in small vessels of joints[136]. These manifestations are numerous and diverse, including fatigue, arthritis or arthralgia, myalgia, polyarthralgia, fibromyalgia, poly/dermato-myositis, sicca syndrome, and non-inflammatory musculoskeletal pain. The articular involvement is usually bilateral, symmetrical, and non-deforming, and it usually targets small joints such as the metacarpophalangeal joints, the proximal interphalangeal joints, wrists, and fingers. The knee, ankles, and back may be also affected[137,138]. RF is usually positive in these cases but anti-cyclic citrullinated peptide antibodies are negative and can be used to differentiate HCV arthropathy from early rheumatoid arthritis[139].

Sicca syndrome has been reported in 20 to 30% of patients with HCV infection. This may be due to the presence of the virus in the human salivary glands, where it can replicate. It is characterized by high RF titers, higher-frequency cryoglobulins, low antinuclear antibodies (ANA), hypocomplementemia, and a lower frequency of anti-Ro/SSA and anti-La/SSB autoantibodies[84]. Myalgia is a common finding in HCV infected patients, and it occurs in about 15% of cases. The mechanism of RM is possibly related to direct action of the virus as it was detected in muscle fibers. Figure 1 shows that these RM are mostly mediated by immunological mechanisms rather than being related to the infection of extra-hepatic tissues. HCV envelope E2 protein binds with CD81 expressed on the membrane of B-cells, forming a complex that decreases the threshold for activation of B-cells and also causes reduction of its apoptosis. These lead to aberrant activation of B-lymphocytes as well as their prolonged survival, therefore increasing the production of antibodies (including the auto-antibodies) and systemic inflammation[140]. Tissue damages, either directly by viruses or as a result of immune aggressions against infected cells, result in the release of a large number of tissue antigens. Additionally, it has been previously postulated that similarities between HCV antigens and host antigens are partly responsible for the development of ANA and anti-smooth muscle antibodies (ASMA)[141]. RF was detected in 70% of patients, followed by ANA (20 to 40%), anticardiolipin antibodies (15%), antithyroid antibodies (12%), and ASMA (7%)[140].

IFN-based regimens for HCV infection lead to exacerbation of rheumatic diseases and worsening of preexisting autoimmune disorders or even developing a new one[142]. DAAs reduce the viral load and therefore decrease the production of antibodies. The eradication of HCV with DAAs supports improving the articular manifestations[141]. SOF and DCV with or without ribavirin combination therapy are an effective and safe treatment with minimal side effects for eradication of HCV infection and amelioration of HCV related RM[143,144].

Hepatitis C virus and cardiovascular diseases

Chronic HCV infection has a significant, direct or indirect impact on the increased risk of cardiovascular diseases (CVD)[84]. HCV infection is associated with a 27% increase in risk of CVD and cerebrovascular atherosclerotic diseases including stroke events compared with uninfected controls[145,146]. The risk of death from cerebrovascular causes has been correlated with HCV RNA levels[147]. A meta-analysis of nine case-control studies showed a two-fold higher risk of carotid plaques in HCV infected individuals compared with uninfected controls[148]. Moreover, a higher prevalence of anti-HCV antibodies was detected in patients with cardiomyopathies and myocarditis than in the general population. The negative strand of HCV-RNA was detected in cardiac tissue, suggesting replication of the virus. These two findings indicate a direct association between HCV and cardiac injury, with CVD and heart failure seen in patients with HCV infection[145,149]. The effect of HCV infection on the risk of cardiovascular events was greater among older patients with hypertension or diabetes[146].

HCV infection leads to the development of atherosclerosis through different mechanisms as shown in Figure 1. A direct involvement of HCV in the induction of atherosclerosis, as the virus lives and replicates in thrombotic tissue, causes a chronic inflammatory reaction that participates in thrombus growth and instability[150]. Endothelial cells express HCV entry receptors which support viral replication. Moreover, HCV causes endothelial dysfunction through promoting migration and proliferation of smooth muscle cells from the tunica media to the intimal surface. HCV alters endothelial permeability, causes cell apoptosis and so, produces endothelial dysfunction[151]. Indirect mechanisms of atherosclerosis have been proposed, such as chronic low-grade systemic inflammation and activation of T helper cells with the release of pro-atherogenic cytokines and chemokines (e.g., IL-1, IL-6, and TNF)[152,153]. These in turn induce soluble vascular adhesion molecule 1 at the endothelial level, which has been found to be associated with endothelial dysfunction as well as the risk of CVD. HCV also interferes with glucose and lipid metabolism, leading to IR, diabetes, and liver steatosis, which are known factors that induce atherosclerosis[151]. A high TNF-α/ adiponectin ratio was found in HCV infected patients that is related to the development of IR and atherosclerosis[154].

Clearance of HCV by DAAs is associated with an improvement in atherosclerosis and metabolic and immunological conditions that promote the development of CVD[152]. Several studies reported the association between the achievement of SVR by DAAs and a significant reduction of the risk of acute coronary syndrome, CVD, and heart failure[84,155,156]. In pre-diabetic patients, Sasso et al[157] conducted a prospective multicenter study on prediabetic HCV positive cohort. They concluded that HCV eradication by DAAs allows a significant reduction of major CVD in the pre-diabetic population, regardless of the severity of liver disease and CV risk factors (age and hypercholesterolemia). This positive effect is mainly due to an improvement of serum markers of endothelial dysfunction and glucose metabolism[158,159].

Hepatitis C virus and non-Hodgkin lymphoma

A positive association was present between HCV and B-cell non-Hodgkin lymphoma (NHL) and it was detected among 5-15% of HCV patients. NHL includes marginal zone lymphoma, diffuse large B-cell lymphoma, lymphoplasmacytic lymphoma, follicular lymphoma, Burkitt’s lymphoma, non-Hodgkin T-cell lymphoma, and primary cutaneous T-cell lymphoma[160-162].

There are several HCV mechanisms determining neoplastic lymphoproliferative diseases. Bcell receptors are continuously stimulated by HCV viral antigens, leading to consecutive Bcell proliferation. HCV replication inside Bcells produces HCVderived viral proteins that induce genetic damage in the Bcells. The HCV envelope protein E3 binds to CD81 on the surface of Blymphocytes and forms a complex with CD19 and CD21, which in turn stimulates intracellular proliferative signals. When HCV enters B-cells, it causes oxidative stress that might result in mutations and defective DNA repair. Moreover, HCV infections are associated with increased frequencies of BCL6 and p53 gene mutations in Bcells. Accordingly, HCVrelated lymphomagenesis may be attributed to either chronic viral antigen stimulation or genetic mutations that lead to the clonal expansion and malignant transformation of Bcells[163-165]. In addition, MC is considered as a B-cell benign lymphoproliferative disorder frequently induced by HCV infection[166]. Figure 1 shows the pathophysiology of HCV infection in the occurrence of NHL.

DAA regimens in combination or after the completion of immunochemotherapy should be recommended. DAA treatment has been reported to reduce the frequency of the malignant B-cells in peripheral blood of patients affected by HCV-related lymphoproliferative disorders[167-169]. Moreover, DAAs might have a lower anti-lymphoma activity than IFN[84,170].

Hepatitis C virus and neuro-psychiatric manifestations

Up to 50% of patients with chronic HCV infection has neuropsychiatric symptoms. Among the reported psychiatric symptoms in chronic HCV patients are brain fog, depression, anxiety, weakness, and fatigue. These alterations lead to impaired quality of life[171]. The term HCV-associated neurocognitive disorder is used to refer to fundamental cognitive deficits unrelated to the severity of liver disease, viral load, and genotype and therefore distinct from the potentially reversible complications seen in patients with minimal hepatic encephalopathy (MHE)[172].

Chronic HCV patients exhibit prevalent involvement of the frontal lobe, which is responsible for alterations of executive functions[173] and the posterior regions of the cerebral cortex, particularly the occipital and parietal lobes[174]. Therefore, they exhibit difficulties in problem solving, monitoring one’s own behavior, self-control, cognitive flexibility, working memory, volition, sustained attention, and logical reasoning, in addition to verbal learning and verbal recall[175,176]. On the other hand, cognitive domains related to posterior brain regions, primarily involved in visuospatial, visual perceptual abilities, and constructive practice regions are mainly altered in patients with MHE[172]. Moreover, T2DM as an EHM in HCV infected patients may be associated with cognitive impairments[177]. Conversely, some studies did not confirm the association between chronic HCV infection and neurologic disorders[178]. Direct neuroinvasion changes in metabolic pathways and cerebral and systemic inflammation have been proposed as pathogenetic mechanisms[179]. Central fatigue and depression may share the same neurobiological causal pathways triggered by HCV infection[180]. Lower levels of dopamine were found in the ascending reticular activating and limbic systems among fatigued patients with HCV infection possibly from cytokine-induced reduction in tetrahydrobiopterin, which is an enzyme involved in the dopamine synthesis[181,182]. Impaired serotonin transmission implicated in depression has also been correlated with increased fatigue[183].

The prevalence of depression in HCV patients ranges from 20 to 50%, compared to a 10% prevalence in the general population[184]. In the US National Health and Nutrition Examination Survey, a cross-sectional study involving 10231 patients suffering from various liver diseases, only HCV was found to be independently associated with depression. The presence and severity of depression were independent of cirrhotic status, viral load, degree of hepatic inflammation, and use of IFN^[185]. They suggested the presence of another exclusive HCV-mediated mechanism contributing to depression. Depression among people with HCV infection may be partially attributed to social and occupational limitations that may precede the infection, often causing viral acquisition, e.g., through intravenous drug use. Awareness of infection with subsequent poor acceptance and social stigma contributes to depressive symptoms independent of socioeconomic status or educational level[186]. Associated HCV complications such as cirrhosis, ascites, and encephalopathy, and comorbidities such as IR, RM, and CVD lead to limitation in physical function, and thus create higher physical load leading to increased depression[187].

The use of IFN was poorly tolerated as it was associated with neuropsychiatric disorders and impaired health related quality of life (HRQOL) in up to 70% of patients[172]. These disorders induced depression during and at the end of IFN treatment[188]. On the other hand, DAA regimens showed no significant psychiatric side effects and patients experience an improved HRQOL while on treatment, regardless of the stage of liver disease[189-192]. Viral clearance attained by DAAs improves fatigue, physical function, mental health, cognitive functions, and quality of life[193-196]. Several studies reported a significant reduction in the choline/creatine and myo-inositol/creatine ratios as well as an increase in cognitive functions in HCV infected patients who reached SVR compared to untreated patients or patients who did not achieve SVR^[173,195]. It is suggested that the persistence of some cognitive symptoms at the end of therapy with DAAs can be attributed to compartmentalization of virus in the central nervous system that may represent a potential source of its reactivation[182,197].

Mazzaro et al[84] recommended starting DAAs as early as possible in the natural history of HCV infection. Early therapeutic approach not only will cure many of the EHMs that are still in a reversible stage, but it also can prevent those that develop due to delayed treatment.

CONCLUSION

HCV is a common cause of liver disease and is associated with a variety of EHMs. Among these manifestations are the rheumatologic diseases, T2DM, IR, several glomerulopathies, cardiovascular diseases, neuropsychiatric, and cognitive disorders.

DAA regimens are considered pan-genotypic as they achieve a SVR of > 85% at 12 wk through all the major HCV genotypes as well as a very high percentage of SVR even in advanced fibrosis and cirrhosis. DAAs improved the symptoms of EHMs and reduced the risk of complications. The risk factors for DAA failure include advanced liver fibrosis/cirrhosis and the presence of RAS in the region targeted by the received DAAs. The effectiveness of GLE/PIB is reduced in HCV genotype 3 with baseline RAS like A30K, Y93H, and P53del. Baseline RAS testing is recommended for HCV genotype 3 infected patients with liver cirrhosis, as those without a baseline Y93H RAS in NS5A are eligible for SOF/VEL therapy. While, those with baseline Y93H RAS could be treated with SOF/VEL/VOX or SOF/VEL plus RBV. Moreover, EASL recommended the identification of baseline RAS for HCV genotype 1a. No RAS was reported in the NS5B region. The higher rate of HCC after DAA therapy may be explained by the fact that DAA regimens are offered to patients with advanced liver fibrosis and compensated or decompensated cirrhosis, where IFN was contraindicated. In addition, the change in the growth of pre-existing subclinical undetectable HCC upon DAA treatment might be a cause. Furthermore, after DAA therapy, the T cell-dependent immune response is much weaker upon HCV clearance, and the down-regulation of TNF-α or the elevated neutrophil to lymphocyte ratio might increase the risk of HCC. DAAs appear to be safe for patients with a history of treated HCC except for cases with vascular invasion. DAAs can result in reactivation of HBV in HCV co-infected patients.

Concerning EHMs, DAAs are now the drug of choice for HCV-associated MCV, and they can achieve clinical and immunological responses for cutaneous and musculoskeletal manifestations, and peripheral nerve and renal involvement. DAAs have rapid and high effectiveness in thrombocytopenia. They also improve IR by 90%, increased glomerular filtration rate, and decrease proteinuria, hematuria, articular manifestations, and lymphoproliferative disorders. Moreover, HCV clearance by DAAs allows a significant improvement in atherosclerosis and metabolic and immunological conditions with a reduction of major cardiovascular events, regardless of the severity of liver disease. DAA treatment also improves physical function, fatigue, and HRQOL greatly during and at the end of treatment as well as at SVR. Viral clearance attained by DAAs improves also cognitive functions in patients with subtle cognitive defects independent of their liver condition. Early therapeutic approach with DAAs not only will cure many of the EHMs that are still in a reversible stage, but it can also prevent those develop due to delayed treatment.

ACKNOWLEDGMENT

The authors would like to acknowledge El-Atroush ES and Kamal OA for designing and drawing the figure presented in the minireview.