Hepatitis C virus (HCV) infection remains a major global health challenge, with approximately 1.5 million new cases per year and a worldwide prevalence of 58 million (WHO Global Hepatitis Report, 2022). In Egypt, a large-scale screening campaign led by the Ministry of Health identified 1.1 million individuals with HCV viremia; 92% of whom initiated treatment with DAAs (Waked et al., 2020), which has significantly improved cure rates up to 95%, even in complex cases (Elnadry et al., 2018; Martinello et al., 2017; Nawaz et al., 2023). DAAs have significantly improved cure rates to up to 95%, even in complex cases (Nawaz et al., 2023; Fathalla Khattab et al., 2021).

The gut-liver axis facilitates communication between the gut microbiota and the liver via the portal vein, systemic circulation, and biliary tract (Ladenheim et al., 1995). This connection allows the liver to handle potentially harmful substances from the gut while the liver secretes bile into the intestines, facilitating bidirectional communication (Guilliams et al., 2022). Most bile acids are reabsorbed in the terminal ileum and recycled to the liver, with some being converted into secondary bile acids by the colonic microbiota before being reabsorbed (Grüner and Mattner, 2021). This communication is crucial for gastrointestinal health and disease management. Studies indicate that liver diseases disrupt bile acid homeostasis, leading to proinflammatory bacterial overgrowth, alterations in the microbial community and disease progression (Mouzaki et al., 2016; Kakiyama et al., 2013; Qin et al., 2014).

HCV infection negatively affects gut health by reducing beneficial species that produce short-chain fatty acids essential for intestinal barrier integrity and immune regulation (Wong et al., 2006). This dysbiosis increases gut permeability, contributing to liver damage and potentially advancing HCV infection to cirrhosis and hepatocellular carcinoma (Aly et al., 2016; Frumento and Ţălu, 2024).

Research on the impact of HCV infection on the gut microbiota, particularly in developing countries, is still limited. The mechanisms through which microbial dysbiosis affects disease progression remain unclear (Aly et al., 2016; Bajaj et al., 2016; Zheng et al., 2020; Sultan et al., 2021). While achieving a SVR is linked to improved outcomes, the effect of HCV eradication on gut dysbiosis in chronic hepatitis C (CHC) patients is still debated because of challenges in controlling external factors affecting the microbiota in humans compared with those in animal models (Martinello et al., 2023). Furthermore, the role of DAAs in altering the microbiota, particularly among relapsed patients, has not been thoroughly investigated. Therefore, this study aimed to investigate the impact of CHC on the gut microbiota and compare microbiota changes in CHC patients who achieved SVR post-DAA treatment with those in patients who experienced relapse.

Changes in the gut microbiota play crucial roles in liver damage caused by various factors, including viruses, through the gut-liver axis (Cesaro et al., 2011). Although several studies have investigated the link between the gut microbiota composition and hepatitis C virus, the results have been inconsistent and influenced by treatment regimens (Sultan et al., 2021; Honda et al., 2021; Wellhöner et al., 2021). Research on the impact of DAAs on the gut microbiota, particularly among patients who relapse after treatment, is limited. This study aimed to evaluate the impact of CHC on the gut microbiota and to compare changes in the gut microbiota between those who achieved SVR after DAAs with those who experienced relapse.

Our findings revealed a significant increase in alpha diversity among Non-Treated CHC patients compared with other groups, which aligns with studies from Egypt and globally (Sultan et al., 2021; Hsu et al., 2022). However, several studies have shown decreased microbiota diversity in chronic HCV infections (Heidrich et al., 2018; Inoue et al., 2018). For example, Aly et al. (2016) reported lower alpha diversity in HCV patients with stage 4 disease than in eight healthy controls, whereas Ponziani et al. reported reduced alpha diversity in cirrhotic HCV patients than in healthy subjects before treatment (Ponziani et al., 2018).

In this work, the alpha diversity of patients treated with DAAs who achieved SVR significantly differed from that of patients in the nontreated CHC group and those who relapsed. The diversity of the gut microbiota was restored to a level comparable to that of the control group, with no significant difference, which is consistent with previous studies (Wellhöner et al., 2021; Hsu et al., 2022; Heidrich et al., 2018). On the other hand, DAAs did not significantly impact the gut microbiomes in other studies (Honda et al., 2021; Huang et al., 2023). The degree of underlying fibrosis plays a crucial role in gut microbiota restoration post-HCV eradication, with patients having lower fibrosis levels showing complete recovery, whereas those with higher fibrosis levels showed hindered recovery despite achieving SVR (Abd Alla et al., 2018; Pérez-Matute et al., 2019). Liver remodeling, which includes improved fibrosis and liver stiffness, occurs gradually in patients recovering from HCV, suggesting a link between liver changes and the gut microbiota (Wellhöner et al., 2021). Other studies have indicated no significant differences in gut dysbiosis between those who have cleared the virus and those who are still infected, emphasizing that HCV viremia alone does not dictate specific microbiota patterns when demographics and medical conditions, e.g., comorbidities, cirrhosis severity, and medication, are considered (Bajaj et al., 2016; Hsu et al., 2022).

In contrast, Sultan et al. (2021) reported that healthy adults had lower microbial diversity than treated HCV patients did, suggesting that treatment affects the microbiome. Similarly, Ponziani et al. (2018) reported improved alpha diversity and gut microbial composition changes a year after HCV eradication in cirrhotic patients treated with DAA regimens, which were linked to pathophysiological improvements.

Dysbiosis severity correlates with disease stage and may influence progression, but it remains unclear whether HCV infection directly alters the gut microbiota, worsening liver inflammation and fibrosis through portal endotoxemia, or if gut dysbiosis results from chronic liver inflammation and dysfunction, creating a vicious cycle rather than stemming from the viral infection itself (Sultan et al., 2021; Heidrich et al., 2018; Ponziani et al., 2018; Pérez-Matute et al., 2019).

Owing to their specificity, high effectiveness, tolerability, and short duration, concerns about long-term effects of DAA regimens on microbial communities may be alleviated (Hsu et al., 2022; Götte and Feld, 2016).

In the present study, the relapsed group presented significantly lower gut microbiota diversity than the other groups did, likely because liver dysfunction affects bile acid synthesis and secretion, which inhibits beneficial bacteria. Persistent systemic inflammation can compromise gut barrier function, allowing lipopolysaccharides (LPSs) to enter the bloodstream and trigger inflammatory responses that disrupt the gut microbiota. This imbalance may exacerbate hepatitis viral infections (Xu et al., 2015). DAAs may alter the gut flora through liver metabolism changes or immune modulation (Trifan et al., 2023; Frumento, 2025). Relapsed HCV patients are more susceptible to secondary infections due to liver dysfunction and immune impairment, and frequent antibiotic use can further diminish beneficial bacteria, reducing microbial diversity. Additionally, dietary restrictions and nutrient malabsorption may impact gut health (Virseda-Berdices et al., 2025; Petersen and Round, 2014). Although DAAs can cure HCV, they may not fully reverse T-cell exhaustion, increasing the risk of reinfection (Thimme, 2021).

HCV RNA can reappear after a SVR is achieved because the virus resides in extrahepatic sites, such as gastrointestinal mucosa cells and peripheral blood mononuclear cells (PBMCs). Even after treatment with DAAs resulting in SVR, some patients have shown occult HCV infection with a negative-strand viral genome detected in PBMCs, indicating ongoing viral replication despite successful treatment (Frumento and Ţălu, 2024; Russelli et al., 2017; Elmasry et al., 2017).

We observed distinct clustering of the gut microbiome on the basis of disease status, which contrasts with the findings of Sultan et al. (2021), who reported no significant clustering. In this study, we identified 26 bacterial phyla, with Firmicutes and Bacteroidetes being the most dominant, followed by Proteobacteria, Spirochetes, and Cyanobacteria. Firmicutes were most abundant in Non-Treated CHC patients, whereas Bacteroidetes and Proteobacteria were enriched in the relapsed group. Notably, the Firmicutes/Bacteroidetes ratio significantly differed between HCV patients and controls, with a significant decrease in the relapsed group compared with untreated CHC patients and those who achieved SVR.

In Non-Treated CHC patients, a predominance of Firmicutes was noted, echoing findings by Pérez-Matute et al. (2019), who also reported higher Actinobacteria levels in HCV-infected subjects with less fibrosis. Our study revealed a significant decrease in Actinobacteria among those who achieved SVR, accompanied by an increase in Cyanobacteria. Firmicutes and Proteobacteria were previously linked to inflammatory effects in the gastrointestinal tract (Tecer et al., 2020).

Aly et al. (2016) reported increased levels of Bacteroidetes in HCV patients, whereas healthy individuals presented increased Firmicutes, Proteobacteria, and Actinobacteria. Inoue et al. (2018) reported similar findings, noting increased Bacteroidetes and decreased Firmicutes in CHC patients. An analysis of 14 HCV patients revealed fluctuations in the dominance of these phyla during treatment, with Bacteroidetes and Fusobacteria decreasing while Firmicutes and Verrucomicrobia increased (Honda et al., 2021). In the relapsed group in this study, Bacteroidetes and Proteobacteria were the most abundant phyla. Research on the microbiota patterns of HCV-relapsed patients remains limited.

In the present study, Bifidobacterium, Prevotella, Lactobacillus, Megasphaera, and Mitsuokella were significantly more prevalent in the relapsed group, whereas Faecalibacterium, Eubacterium coprostanoligenes, Asteroeplasma, Lachnospiraceae, Akkermansia, and Muribaculaceae were more prevalent in the Non-Treated CHC group. Similarly, Aly et al. (2016) reported that Faecalibacterium and Prevotella were more abundant in HCV patients than in healthy individuals, whereas Clostridium and Ruminococcus were more common in healthy controls. These findings suggest that the increased Bacteroidetes in HCV patients may stem from an overabundance of Prevotella, which is known for its proinflammatory properties (Larsen, 2017). The heightened levels of Prevotella in the relapsed group could be linked to a heightened inflammatory state, exacerbating intestinal inflammation and affecting liver and systemic inflammation through signaling metabolites (Midori et al., 2024; Iljazovic et al., 2020). Additionally, impaired digestion and absorption in HCV patients may lead to increased carbohydrate levels in the intestine, promoting Prevotella expansion (Aly et al., 2016). Notably, Aly et al. (2016) reported a greater abundance of Faecalibacterium prausnitzii, which is associated with anti-inflammatory effects whereas Akkermansia is crucial for maintaining the intestinal mucosal barrier (Ghotaslou et al., 2023). The predominance of many of these beneficial bacteria in the nontreated CHC and relapsed groups may act as a compensatory mechanism to combat chronic inflammation and support gut health. However, the impact of DAAs on microbiota patterns and their role in relapse remain unclear and require further investigation.

Compared with healthy controls, Heidrich et al. (2018) reported that CHC patients had elevated levels of bacteria such as Escherichia Shigella, Akkermansia, Haemophilus, Bifidobacterium, Weissella, Micrococcus, Citrobacter, Pediococcus, and Clostridium sensu stricto. Similarly, Huang et al. (2023) noted relatively high levels of Eubacterium, Ruminococcaceae, Alistipes, Agathobacter, Klebsiella, and Bifidobacterium in the CHC group. In contrast, Ashour et al. (2022) reported that Bifidobacterium, Ruminococcus and some clostridia were more abundant in healthy controls than in HCV-infected patients. The variability among these studies may be attributed to the small sample sizes and factors influencing the gut microbiota, which are challenging to control in clinical studies, such as genetics, immune response, diet, and environmental microbial exposure (Lynch and Pedersen, 2016). In patients who achieved SVR, our findings revealed a significant increase in Clostridium sensu stricto, suggesting a restoration of gut homeostasis and microbial balance.

In the treatment-naive CHC group, many clusters presented significant positive correlations with gut microbiota members, including Escherichia Shigella, Veillonella, Streptococcus, Lactobacillus, and Bifidobacteria, which may contribute to disease pathogenesis. Yang et al. (2023) identified Lactobacillus, Butyricimonas, Veillonella, and Escherichia-Shigella as potential microbial markers for predicting the risk of developing viral hepatitis.

In our study, genera such as Lactobacillus, Bifidobacterium, Treponema, Parasutterella, Veillonella, and Streptococcus were positively correlated in the relapsed group, indicating disruption of intestinal integrity and increased liver inflammation. Conversely, the treated group that achieved SVR was positively correlated with beneficial bacteria responsible for intestinal integrity and health, such as Roseburia, Blautia, Catenibacteria, Prevotella, and Parabacteroides. Additionally, Bacteroides, Megasphaera, and Streptococci were negatively correlated with gut health-promoting bacteria such as Eubacterium eligens and Alloprevotella.

In the control group, several members, including the Eubacterium eligens group, Akkermansia, Christensenellaceae R-7, Butyrovibrio, and Asteroplasma, were positively correlated with maintaining intestinal integrity. A. muciniphila contributes to gut health by producing short-chain fatty acids (SCFAs), such as acetate and propionate (Lukovac et al., 2014), and enhances antimicrobial peptide synthesis and improves gut homeostasis (Ottman et al., 2017). Eubacterium genus members produce butyrate, which is essential for energy balance, immune regulation, colonic function, and inflammation control in the gut, whereas Butyrivibrio, another butyrate producer, also supports gut health (Mukherjee et al., 2020).

Correlation analyses revealed clinically relevant microbiota-host interactions. The positive association between Prevotella and relapse status aligns with its proinflammatory role, potentially exacerbating hepatic inflammation. Conversely, Faecalibacterium exhibited negative correlation with bilirubin and relapse which could be related to anti-inflammatory properties (Jiang et al., 2019; Lee et al., 2022). Intriguingly, Eubacterium coprostanoligenes showed dual associations, positively with AFP (a tumor marker) and negatively with PT/INR, which support its complex role in coagulation and oncogenesis. Similarly, Ruminococcaceae UCG-002 correlated negatively with PT/INR but positively with FIB-4, implying microbial involvement in fibrosis progression (Jinato et al., 2024).

Despite its strengths, this study has limitations. The single-center design and modest sample size may limit the generalizability of our findings, particularly across diverse geographic or dietary populations. While we controlled for antibiotic/probiotic use, unmeasured confounders such as dietary habits, regional microbiome variations, and lifestyle factors could influence microbial composition. Additionally, although we correlated microbial profiles with liver function tests (e.g., ALT, AST), future studies with longitudinal designs and expanded clinical metadata (e.g., detailed dietary records, inflammatory markers) could further elucidate causal relationships between microbiota shifts and host physiology. Larger, multi-center cohorts are needed to validate these preliminary observations and explore the long-term effects of DAAs on gut-liver axis dynamics.

The gut microbiota was grouped on the basis of disease status, with each group exhibiting distinct microbial patterns, biomarkers, and interactions. DAAs significantly influence the diversity of the gut microbiota, resulting in beneficial restoration and reconstitution of microbial communities in the SVR group, which resemble those observed in healthy individuals.