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Abstract
Background: In chronic hepatitis B virus (HBV) patients, fluctuations in HBV DNA serve as a “gray area” and impede the accurate identification of inactive carriers. We aimed to assess if such fluctuations impact the presence of significant hepatic fibrosis (Metavir F2-4) in chronic HBV patients.
Methods: Consecutive, untreated HBeAg-negative carriers (n = 234) with fluctuating HBV DNA (n = 73) above or below a level of 2000 IU/mL were included and compared to those without fluctuations (n = 161). Patients without fluctuating HBV DNA were further analyzed based on those with persistently low (<2,000 IU/mL, n = 137) and higher HBV DNA (2,000–20,000 IU/mL, n = 24). Hepatic fibrosis (assessed by transient elastography) was correlated with virologic and biochemical profiles.
Results: The mean age of the overall cohort was 47.8 ± 11.1 years, of whom 107 (45.7%) were male. During a median of 60 months (interquartile range [IQR] 34–82) of follow-up, 73 (31.2%) patients had a mean of 1.6 ± 0.9 fluctuations in HBV DNA. The median time to the first fluctuation was at 14.5 (IQR 5.0–33.7) months. Patients with fluctuating viremia had higher log10 qHBsAg (3.1 ± 0.8 vs. 2.7 ± 1.0, P = 0.022) and HBV DNA (3.4 ± 0.5 vs. 2.7 ± 0.8, P < 0.001) compared to those without fluctuations. Patients with fluctuant viremia were less likely to have F2-4 fibrosis (8.2%) compared to those without fluctuant viremia (18.2%, odds ratio [OR]: 0.407, 95% confidence interval [CI]: 0.161–1.030; P = 0.052). Males tended to have less fluctuation constituting 37.0% of patients with fluctuating HBV DNA (P = 0.071). Fluctuations occurred more frequently in those with predominantly higher HBV DNA levels (26.0%) compared to those without fluctuations (14.9%; P = 0.030).
Conclusions: Fluctuating HBV DNA levels occur frequently but are not associated with significant fibrosis. Minor fluctuations in HBV DNA levels are unlikely to be of clinical relevance.
Keywords: Fluctuation, gray zone, HBV DNA, hepatitis B, inactive carriers, significant fibrosis, viremia
How to cite this article:See accompanying Editorial
Introduction 
Hepatitis B virus (HBV) chronically infects approximately 292 million people globally and is a leading cause of cirrhosis, hepatocellular carcinoma (HCC), and liver-related death.[1] Assessment of hepatic fibrosis is of immense importance in managing chronic HBV patients. During the HBeAg-negative phase, treatment is recommended when alanine aminotransferase (ALT) is persistently or intermittently elevated above the upper limit of normal (ULN), the HBV DNA is over 2,000 IU/mL, and/or with significant fibrosis (for instance, METAVIR score F2-4).[2],[3] In contrast, given the good long-term prognosis, periodic monitoring is recommended in patients with chronic HBV infection (”inactive carriers”).[2],[3]
It is recognized that HBeAg-negative chronic hepatitis B (CHB) can run a fluctuating course[4],[5] with variability in HBV DNA and ALT levels, over time. Although there is a reasonable body of literature on liver histological changes in HBV cohorts with persistently normal ALT,[6],[7],[8] studies focusing on fluctuations in HBV DNA levels are scarce, with none assessing the development of liver fibrosis. Significant fibrosis in patients with low viremia may occur, with reports of progression to cirrhosis.[6],[7] Because fluctuations of ALT, serum HBV DNA or both may occur, the distinction of true inactive carriers from HBeAg-negative CHB may sometimes be difficult. These findings call into question the validity of conventional staging of HBV, in large part because of the substantial proportion of patients who do not fit readily into one of the usual stages or phases, and represent “gray zones” of disease classification.[2],[9],[10] We have previously reported fluctuations in HBV DNA levels above or below 2,000 IU/mL in 33.8% of HBeAg-negative patients[8] although the impact of this fluctuation was not studied, particularly in relation to the development of liver fibrosis.
Little is known about the natural history of patients with fluctuant viremia because appropriate studies including large cohorts are lacking. Consequently, whether these patients simply deserve routine long-lasting monitoring or more intense monitoring with active intervention remains unclear. We aimed to assess the prevalence of HBV DNA fluctuation in an unselected cohort of HBeAg-negative patients with low viremia, and the validity of categorizing CHB into phases based upon measures of hepatic fibrosis and viral load, assuming that such determination will be useful for determining the need for antiviral therapy.
Patients and Methods Study patients
This study included all untreated, consecutive HBeAg-negative patients from the outpatient clinics of King Abdulaziz Medical City in Jeddah and Riyadh between May 2016 and January 2021. Patients were identified from a search of clinical records and the Saudi Observatory Liver Disease (SOLID) registry, which is a prospective, multicenter observational registry. The two recruiting centers provide healthcare to Saudi citizens exclusively, and to Saudi Arabian National Guard members and their families predominantly. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and the Principles of Good Clinical Practice and the Institutional Review Boards of both centers approved the study.
Eligible patients for the analysis had detectable HBsAg for at least 6 months before inclusion in the study. All patients were HBeAg-negative, had HBV DNA values predominantly <20,000 IU/mL (n = 234), and were between 16 and 80 years of age. The median duration of follow-up for the entire cohort was 60 months (interquartile range [IQR] 34–82). The exclusion criteria were (i) co-infection with hepatitis C, delta virus, or HIV; (ii) superimposed with other liver diseases; (iii) antiviral, immunosuppressive, or hepatotoxic medications; (iv) decompensated cirrhosis with a Child-Pugh score >6, or evidence of portal hypertension, variceal bleeding, laboratory findings of a platelet count <150 (109/L), an international normalized ratio ≥1.3; (v) creatinine >135 μmol/L; (vi) presence of hepato-biliary malignancy; (vii) alcohol consumption >20 g/day; and (viii) organ transplantation.
Study design and definitions
Patients were classified as either predominantly low HBV DNA where the majority of values were <2,000 IU/mL, or high when the majority of values were between 2,000 and 20,000 IU/mL. The fluctuation was defined as any ≥1 crossover of HBV DNA from a level above or below a threshold of 2,000 IU/mL. HBV DNA levels were defined as persistently low when all levels were <2,000 IU/mL and defined as persistently high when values were in the range of 2,000–20,000 IU/mL, over ≥12 months. A minimum of three HBV DNA measurements was required and the reference value utilized for analysis was the last level before the performance of transient elastography (TE) for liver stiffness measurement (LSM). A median number of 6 [IQR 4–9] measurements were made for 60 months.
A minimum number of three ALT measurements were required, and the last level before the performance of TE was used as the index value. A median number of 10 [IQR 7–15] ALT recordings were made for 60 months for the overall cohort. Routine liver biochemical tests were performed using commercially available autoanalyzers and hepatitis serological markers were assayed using commercially available enzyme-linked immunoassays. Quantitative HBV DNA levels were measured by Abbott Real-Time HBV assay (Abbott Molecular, Inc., Des Plaines, IL, USA), with a lower detection limit of 10 IU/mL. The serum quantitative HBsAg levels (qHBsAg) were measured by the Abbott ARCHITECT® Assay (Architect i2000SR, Abbott Diagnostics; Abbott Laboratories, Chicago, IL, USA). The detection value of qHBsAg ranged from 0.05 to 250 IU/mL and samples with HBsAg titers >250 IU/mL required a 1:500 dilution.
Liver fibrosis assessment
All patients underwent TE for LSM (FibroScan® 502 Touch, Echosens, Paris, France), and the procedure was performed by a single operator at each site with experience of over 500 procedures, according to instructions provided by the manufacturer. All TE assessments were made from January 2018 to December 2020. The median value of multiple successful measurements was recorded as the LSM value for each patient. Only LSM processes with at least 10 validated measurements, an IQR less than 30% of the median value, and a success rate of at least 60% were considered reliable.[11],[12] Stiffness is expressed in kilopascals (kPa). Any LSM value that did not satisfy the aforementioned conditions was considered unreliable and therefore excluded from further analysis. An XL probe was used as a second-line probe for patients for whom the regular M probe failed, such as some obese patients or patients with BMI ≥30 kg/m2.[13] The final controlled attenuation parameter (CAP) was taken as the median of individual CAP and is expressed in dB/m. Previously described cut-offs to discard moderate fibrosis (F ≥2) was TE ≤6 kPa. Steatosis scores of ≥2 were defined as moderate–severe corresponding to CAP values >260 dB/m.
Statistical analysis
The sampling technique was convenient sampling, and it was chosen to ensure the availability of required data. Patients with fluctuating HBV DNA above or below a level of 2,000 IU/mL were compared to those without fluctuations. Quantitative variables are expressed as the mean ± standard deviation or the median with IQR (25th–75th percentile), and categorical variables as frequencies and proportions. The unpaired Student's t-test was used to compare the means or log means of the variable, if the variable was normally or not normally distributed, respectively. Mann–Whitney U test was used to compare the medians of two groups when the variables were not normally distributed. The Chi-square or Fishers' exact test (α = 0.05) was used to compare frequencies and proportions in categorical variables, as appropriate. Odds ratios (OR) were calculated where appropriate, along with their 95% confidence intervals (CI). All analyses related to HBV DNA were conducted after logarithmically transforming the values to account for skewed distribution. Statistical Package for Social Sciences (SPSS, version 21.0; Chicago, IL, USA) was used for data analysis.
Results Baseline characteristics of patients
The mean age of the overall cohort was 47.8 ± 11.1 years, of whom 107 (45.7%) were males and 44 (18.8%) had elevated ALT. The mean HBV DNA log10 was 2.9 ± 0.8, and 137 (58.5%) had persistently low HBV DNA (<2,000 IU/mL) and 24 (10.3%) had persistently high HBV DNA (>2,000 IU/mL). During a median of 60 months (IQR 34–82) of follow-up, 73 (31.2%) patients had a mean of 1.6 ± 0.9 fluctuations in HBV DNA. Patients with fluctuations had more HBV DNA measurements (8 [IQR 5–9.5]) compared to those without fluctuations (6 [IQR 4–9]; P = 0.001). Throughout the follow-up, most patients exhibiting fluctuant viremia had a single fluctuation (n = 43, 18.8%), whereas two were seen in 16 (6.8%), three in 11 (4.7%), and four fluctuations in 3 patients (1.3%). Most fluctuations occurred early during the follow-up, with a median occurrence of 14.5 (IQR 5.0–33.7) months [Figure 1]. The mean magnitude of fluctuation was log10 3.5 ± 0.5 Fluctuations >20,000 IU/mL occurred in 11 (4.7%) patients, occurring usually in those with persistently higher (2,000–20,000 IU/mL) HBV DNA levels (9/11, 81.8%) and were mostly transient. Co-morbid conditions were observed in a majority of the patients, including diabetes in 19.7% and dyslipidemia in 24.9%. Moderate–severe steatosis (S2-3) was observed in 140 patients (61.1%) and 35 (15.0%) had significant fibrosis stage F2-4 (data not shown).
Figure 1: Kaplan–Meir curve displaying overall incidence of HBV DNA fluctuation. Median fluctuation occurred at 14.5 months (IQR: 5.0–33.7)Effect of fluctuating viremia
Overall, patients with fluctuation had a longer duration of follow-up (66.0 [IQR 43–84] months) compared to those without fluctuations (56.5 [IQR 28.3–80.0] months; P = 0.023). The presence of F2-4 fibrosis in those with fluctuation (8.2%) was lower but not significantly different (OR: 0.407, 95% CI: 0.161–1.030; P = 0.052) from those without fluctuation [18.0%, [Figure 2]]. Similarly, F2-4 fibrosis was not different (OR: 0.640, 95% CI: 0.275–1.487; P = 0.345) in those with predominantly lower (16.1%) or higher HBV DNA [9.5%, [Table 1]]. The presence of F2-4 fibrosis was not different in relation to the number of fluctuations [P = 0.346; [Table 2]]. Males tended to have less fluctuation constituting 37.0% of patients with fluctuating HBV DNA (P = 0.071). Patients with fluctuations tended to have higher qHBsAg log10 (3.1 ± 0.8 vs. 2.7 ± 1.0; P = 0.022) and were more likely to have predominantly higher HBV DNA levels (26.0% vs. 14.9%; P = 0.030) compared to those without fluctuations [Table 1]. These differences in qHBsAg log10 (P < 0.002) and HBV DNA log10 (P < 0.001) were retained when patients were categorized on the basis of fluctuating vs. persistently low or high HBV DNA [Supplementary Table 1[Additional file 1]].
Figure 2: Relationship of fluctuating HBV DNA levels with high quantitative HBsAg levels, moderate–severe steatosis, and significant fibrosis
Table 1: Baseline characteristics of chronic HBV patients in relation to fluctuating or non-fluctuating HBV DNA
Table 2: Distribution of significant fibrosis based on the presence and number of fluctuationsEffect and outcome of quantitative HBsAg levels
Higher qHBsAg levels were identified by younger age (P < 0.001), presence of diabetes (P = 0.002), and higher HBV DNA log10 (P < 0.001), >2,000 IU/mL (P = 0.001), and steatosis grade 2–3 (P = 0.042). Higher qHBsAg levels did not impact the presence of fluctuations in HBV DNA [P = 0.120, [Figure 3]]. Fibrosis 2–4 was not significantly different (OR: 0.807, 95% CI: 0.384–1.701; P = 0.574) in patients with high qHBsAg (14.1%) compared to lower levels [16.9%, [Table 3]].
Figure 3: Relationship of high quantitative HBsAg levels with serum ALT, fluctuating HBV DNA, HBV DNA levels, and significant fibrosisFactors identifying significant fibrosis
Patients with significant (F2-4) fibrosis were identified by lower serum albumin levels, higher BMI, and ALP, AST, and HBV DNA levels [P < 0.05 for all, [Supplementary Table 2[Additional file 2]]]. These patients had similar age, diabetes, dyslipidemia, hepatic steatosis, or HBV DNA values >2,000 IU/mL compared to those with non-significant fibrosis. The mean fluctuations were not different in those with significant fibrosis (1.83 ± 0.75) compared to those without (1.63 ± 0.90; P = 0.589).
Discussion In this real-life cohort study, we investigated for the first time the performance of repeated HBV DNA measurements for the identification of fluctuant viremia and its association with the presence of hepatic fibrosis. Although viral fluctuation at a threshold of 2,000 IU/mL was a frequent occurrence, occurring in one-third of the individuals, it was not associated with the presence of significant fibrosis. Despite fluctuations occurring frequently in those with low HBV DNA levels (<2,000 IU/mL), a substantially lesser number of such individuals harbored significant fibrosis (8.2%) compared to those with non-fluctuant, persistently low HBV DNA levels (18.1%).
Proper categorization of HBV-infected patients aids in the allocation of antiviral therapy and thus helps in reducing the associated long-term complications. Although the traditional classification of CHB is an important clinical distinction, some individuals fall into an indeterminate area, or “gray zone,” requiring treatment decisions to be individualized.[2] Patients with fluctuant viremia constitute one such category of “gray zone” HBV carriers where the clinical distinction between inactive carriers and HBeAg-negative CHB becomes blurred. In our study of this particular category of “gray zone” patients, we have shown that individuals with fluctuant viremia hold no special distinction and have clinical, biochemical, and histological characteristics similar to “inactive carriers.” Our findings are similar to other studies dealing with “gray zone” individuals. Bonacci et al.[9] described different categories of “gray zone” carriers including individuals falling between inactive carrier state and HBeAg-negative CHB, and reported excellent long-term outcomes in the absence of treatment, with a low rate of progression to HBeAg-negative CHB. Similarly, Oliveri et al.[14] also reported a benign outcome in a group of low viremic active carriers (HBV DNA ≤20,000 IU/mL), where only one patient (2.2%) progressed to the chronic hepatitis phase, and no major liver-related events were reported during the study.
The vast majority of our patients tended to exhibit early fluctuations, with roughly half occurring within the first 2 years of follow-up. Most (60%) demonstrated a single fluctuation over the median 5 years of follow-up, and with no disease impact demonstrated by multiple fluctuations.
The management of HBeAg-negative CHB patients who have HBV DNA levels <20,000 IU/mL remains a matter of debate. A small proportion with HBV DNA <2,000 and those with HBV DNA between 2,000 and 20,000 IU/mL exhibit significant fibrosis and hence remain at a greater risk for disease progression.[6],[7],[8] In our study, the presence of hepatic fibrosis was similar in individuals with HBV DNA persistently <2,000 and 2,000–20,000 IU/mL, whereas it tended to be less frequent in those who demonstrated fluctuant levels. Despite a higher qHBsAg and HBV DNA log10 in patients with fluctuant viremia, significant fibrosis tended to be less frequent. Although this distinction remains a point of clinical interest, it is unclear what mechanisms are responsible for this observation. There were no obvious predictors of virologic fluctuation. Moreover, the only potential etiological basis for significant fibrosis was the presence of a greater BMI, hinting at the role of coexistent non-alcoholic fatty liver disease as a factor for the increased rates of hepatic fibrosis development. However, in patients with fluctuant and non-fluctuant viremia no differences in BMI, diabetes, or hepatic steatosis were seen. More relevantly, in multiple cohorts of HBV assessed histologically, the presence of hepatic steatosis revealed conflicting results on the occurrence of significant fibrosis.[8],[15],[16] Nevertheless, a greater prevalence of co-morbidities were observed in individuals with CHB correlated with liver disease-related sequelae in patients from Saudi Arabia.[17] As such, a longer duration of follow-up is warranted to further understand the role of underlying co-morbidities in hepatic disease progression.
Previous studies have evaluated the value of single-point or repeated qHBsAg measurements to determine the identification of the inactive carrier phase through long-term follow-up. However, reports from different regions have been conflicting in their outcomes. Previous analyses have suggested a qHBsAg of 1,000 IU/mL as an appropriate cutoff to distinguish active from inactive HBV.[18],[19] More recently, a multi-center analysis showed that the probability a patient would switch to active HBV at any following year was 24% for patients with initial qHBsAg levels >1,000 IU/mL.[20] However, in a cohort from Saudi Arabia, qHBsAg levels failed to accurately distinguish inactive carriers and were unable to identify significant histological disease.[21] Similarly, in this analysis, a threshold qHBsAg levels >1,000 IU/mL failed to distinguish individuals with significant fibrosis or identify those with HBV DNA fluctuations in a meaningful manner. Additionally, a substantial 73% of individuals with HBV DNA levels >2,000 IU/mL did not have qHBsAg levels >1,000 IU/mL, showing a poor correlation between these proposed thresholds. Nonetheless, it remains to be seen whether a long-term longitudinal follow-up at varying thresholds would be better for disease classification in Middle Eastern patients.
Our analysis must be viewed in the context of its limitations. First, this was a retrospective real-life cohort, limited by a lack of pre-defined serial performances of TE to assess the dynamic nature of liver fibrosis evolution. Second, it is our practice to monitor patients on a 6-month basis after an initial classification based on liver enzymes and HBV DNA levels.[3],[22] However, HBV DNA levels may fluctuate more frequently, especially those above or below a threshold of 2,000 IU/mL. As such, it is feasible that some HBV DNA fluctuations may have been missed, and some individuals potentially misclassified. Third, TE does not represent the gold standard for liver fibrosis assessment. However, we mitigated the role of inter-operator variability in LSM by restricting the performance of TE to single operators in either center throughout the study, each with vast experience in performing LSMs. In addition, it is noteworthy that in a recent prospective study, excellent diagnostic accuracy was reported using TE compared to biopsy with an area under the receiver operating characteristic curve of 0.81 for ≥F2, 0.93 for ≥F3, and 0.93 for F4.[23] Based on these findings, recent guidelines have suggested diagnostic algorithms that incorporate liver stiffness measurement as an important part rather than liver biopsy to facilitate the categorization of patients in a more rapid and less invasive way.[2],[3] This is especially relevant for patients who are thought to be “inactive carriers” in whom the risk of a liver biopsy may not be justified.
In conclusion, fluctuations in HBV DNA levels occur frequently but do not distinguish patients with a higher prevalence of significant fibrosis. Minor fluctuations are unlikely to be of clinical relevance, wherein such patients may be less likely to have significant fibrosis compared to those with non-fluctuant viremia. Such “gray zone” patients could potentially be classified as true inactive HBV carriers.
Financial support and sponsorship
Nil.
Conflicts of interest
Dr. Sanai is a consultant for, advises, and is on the speakers' bureau of, and received grants from Bristol-Myers Squibb and Gilead Sciences.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/sjg.sjg_48_22
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