Azvudine therapy of common COVID‐19 in hemodialysis patients

There is no antiviral study on hemodialysis patients infected with coronavirus disease 2019 (COVID‐19), especially on the application of 2′‐deoxy‐2′‐β ‐fluoro‐4′‐azidocytidine (Azvudine, FNC) antiviral therapy. We conducted a multicenter observational study involving 1008 hemodialysis patients. After matching for age, sex, and other factors, 182 patients in the basic treatment group and 182 in the FNC group were included. The negative nucleic acid conversion rate of the FNC group was significantly higher than that of the basic treatment group, and viral loads, interleukin‐6, and C‐reactive protein were significantly lower than those of the basic treatment group (p < 0.05). There were no significant differences in liver function, renal function, or the number of adverse events between the two groups (p > 0.05). In conclusion, our study has provided novel evidence suggesting that the FNC scheme may be safe and effective compared to the basic treatment of hemodialysis patients with common COVID‐19.


| INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID- 19), has resulted in millions of fatalities and has had a catastrophic impact on society and the economy. 1 Research studies have demonstrated that hemodialysis patients who contract COVID-19 are at a higher risk of severe complications due to their compromised innate and adaptive immune systems, compared to individuals who are in good health. [2][3][4] Meanwhile, increasing evidence shows that the immune responses of hemohemodialysis patients after receiving the SARS-CoV-2 vaccine are delayed and sluggish. 5 In addition, compared with healthy individuals infected with COVID-19, hemodialysis patients have a higher mortality rate from COVID-19 infection. [5][6][7][8] remains a persistent and widespread epidemic across the globe, with the number of fatalities among hemodialysis patients due to the disease continuing to rise. Therefore, it is imperative to conduct research and develop effective antiviral treatments for hemodialysis patients infected with COVID- 19. 9 As an antiviral therapeutic drug, 2′-deoxy-2′-β-fluoro-4′azidocytidine (Azvudine, FNC) is a nucleoside analog that inhibits HIV-1 RNA-dependent RNA polymerase, 10,11 which can inhibit virus replication. In animal models of SARS-CoV-2 infection, azfudine significantly reduced the COVID-19 viral loads, improved the lymphocyte profile, reduced organ damage and inflammation, protected the immune function of the thymus, and quickly cured COVID- 19. 12 Antiviral research on hemodialysis patients infected with SARS-CoV-2 is rare, especially regarding the application of FNC antiviral therapy in such patients. The FNC user manual suggests that the drug should be used cautiously in patients with renal insufficiency.
Simultaneously, hemodialysis can partially replace the renal function of hemodialysis patients. Therefore, whether FNC can achieve safe and effective effects in anti-COVID-19 therapy for hemodialysis patients is controversial. Therefore, we evaluated the efficacy and safety of FNC in hemodialysis patients infected with SARS-CoV-2 for the first time, hoping to provide a reference for the antiviral treatment of these patients.

| Data collection
For each patient, we collected sociodemographic data from the patient's medical record. In addition, we recorded complications associated with high-risk and severe COVID-19, including diabetes, hypertension, and cardiovascular disease. 13 The patient's vital signs, respiratory symptoms, and signs were monitored and collected daily. Meanwhile, we collected the patient's liver function, kidney function, erythrocyte sedimentation, C-reactive protein, and other laboratory tests.
In addition, the lung CT image data carried out during the study were collated. According to the hospital's requirements, the hospital detects SARS-CoV-2 nucleic acid every 1-2 days after the patient starts treatment. The above nucleic acid detection continued until the patient had a negative PCR test.

| Grouping and analysis according to the treatment schemes
According to the different treatment schemes, the patients enrolled in this study were divided into the following two treatment groups for analysis, as follows.

| Basic treatment group
the patients received standard antiviral treatment (such as interferon), symptomatic treatment, nutritional support, and oxygen inhalation. The above treatment refers to the "Diagnosis and treatment program trial version 10 guidelines" issued by the National Health Commission of the People's Republic of China.

| FNC group
Based on the above basic treatment, the patients in this group took FNC 5 mg orally according to the recommended dose. 10,12 FNC was administered daily until the patient was discharged from the hospital or transferred to the corresponding department to treat other diseases. 10 The criteria for hospital discharge were as follows: the patient's respiratory symptoms were improved significantly, pulmonary imaging showed obvious absorption of inflammation, and the patient had two consecutive negative respiratory pathogen nucleic acid tests (with a sampling interval of at least 1 day), and the patient's temperature had returned to normal for more than 3 days. 10  Safety evaluation: the statistical analysis of laboratory tests during treatments includes monitoring vital signs, changes in test sheets, and adverse events (including incidence rate, severity, time, and drug relevance). We collected adverse events in the digestive, urinary, nervous, circulation, blood, respiratory, and immune systems, focusing on carcinogenicity.

| RT-PCR for SARS-CoV-2 detection
We collected respiratory samples by taking throat swabs. RNA extraction was performed using a viral RNA extraction kit (Zybio Inc) according to the manufacturer's guidelines. The researchers then performed RT-PCR. RT-PCR was conducted with primers and probes targeting the ORF1ab and N genes and a positive reference gene.
The primer sequence information for the ORF and N genes is presented in Table S1. Following the manufacturer's specifications, the reaction system and amplification conditions were performed using a commercial detection kit from Biogerm Technology Co., Ltd.
The evaluation criteria of the test results were as follows: the result was considered positive when the Ct values of both target genes (ORF and N) were ≤ 35 and negative when they were both greater than 35. Samples were retested when the CT value was between 35 and 40. Repeated tests showed that a Ct value less than 40 was positive. 14

| Statistical analysis
Continuous variables with normal distributions were expressed as the mean ± SD. Comparisons between the two groups were conducted using a t-test: continuous variables with discrete distributions were expressed as the median (M), quartile spacing (Qu-Ql), and the mean ±SD and were tested using a nonparametric rank-sum test.
Categorical variables were expressed as absolute values (percentages). Comparisons between groups were conducted using the Pearson χ 2 test or Fisher's exact test. The propensity score matching (PSM) process was realized by using the PSM extension program of SPSS: taking whether to accept FNC treatment as the dependent variable and each covariate (such as age, systolic pressure, heart rate, white blood cell count, creatinine, and C-reactive protein) as the independent variables, the tendency score was estimated by logistic regression, and the 1:1 nearest neighbor matching method was used for matching. Each FNC group individual was matched with one of the basic treatment group individuals with the most similar tendency score. This process ensures the excellence of the matching results by defining the caliper value. Then, we compared the changes in the standard difference of the intergroup covariates before and after matching. The closer the standard difference after matching is to 0, the more satisfactory the matching result is. When the absolute value of the standard difference was less than 0.1, it was considered to be a better balance of the intergroup variables. The cumulative negative rate of nucleic acids and the pneumonia improvement rate were SHANG ET AL. | 3 of 11 analyzed by the log-rank test. All significance tests were two-tailed, and a p-value of less than 0.05 was considered statistically significant.
The vaccine's effect on these patients' prognoses was analyzed by Cox regression analysis. All data were analyzed using SPSS 26.0 software.

| RESULTS
There were 1008 hemodialysis patients infected with SARS-CoV-2. A total of 560 patients' information was collected and collated, including 260 patients in the basic treatment group and 300 patients in the FNC group. The details of the exclusion are shown in Figure 1.
Before using PSM, the FNC group was older and had higher systolic pressure, heart rate, white blood cell count, creatinine, and C-reactive protein than the baseline treatment group (specific information can be obtained from Supplementary Table 2). There were no significant differences in the other indicators. The 1:1 nearest neighbor matching method was adopted, and the caliper value was 0.15. The FNC group is the reference group for matching, and 182 pairs of the two groups were matched successfully. The unbalanced covariates between the two groups reached equilibrium after matching (p > 0.05, Table 1). Through PSM, the balance of each covariate was significantly improved.
Finally, a total of 364 patients completed the study. The screening flow chart is shown in Figure 1. 3.1 | The rate of nucleic acid negative conversion After 8 days of treatment, the nucleic acid negative conversion rate in the FNC group was 100%, while in the basic treatment group, it was only 44.5% (Figure 2A). The Kaplan-Meier curve significantly differed between the two groups (p < 0.001), indicating the tendency of earlier nucleic acid negativity in the FNC treatment patients. The FNC group received azvudine treatment for 7.97 ± 1.82 days.

| Detection of viral loads
To explore the viral change, we measured the ORF and N gene values by RT-PCR of SARS-CoV-2. The CT values of the patients in the FNC and basic treatment groups were observed on the 0th, 7th, and 14th days after treatment ( Figure 2B,C). The results showed that the CT values of the ORF gene and N gene in the FNC group were higher than those in the control group on the 7th day (p < 0.05).

| Improvement rate of chest CT image
Before treatment, the lung CT of the two groups of patients showed abnormalities, mainly including ground-glass shadows, infiltrating shadows, patch shadows, and density shadows. After treatment, the lungs of the subjects were improved (the focus was reduced/ absorbed/without obvious abnormalities), as shown in Figure 3.
Compared with the basic treatment group, the improvement time of pneumonia in the FNC group was shorter (p < 0.001) ( Figure 2D). The improvement rate of pneumonia in the FNC group after 10 days of treatment was 100%, and that in the basic treatment group after 15 days of treatment was 100%.

| Time for the temperature and oxygen saturation to return to normal
During the study period, the daily temperature and oxygen saturation of the two groups of patients were recorded. As the patients recovered, the temperature and oxygen saturation tended to be normal. Among them, the temperature of the FNC group was significantly lower than that of the basic treatment group on the 4th day ( Figure 2E). Oxygen saturation increased in both groups compared to before treatment ( Figure 2F).

| Detection of relevant inflammatory indicators, immune cells, and D-dimer
The study found that compared with before treatment, the number of white blood cells and lymphocytes in the two groups increased continuously (p < 0.05), as shown in Figure 4A,B. The inflammatory factors (IL-6 and C-reactive protein) decreased continuously (p < 0.05), as shown in Figure 4C,D. The IL-6 level of the FNC group on the 8th day was significantly lower than that of the basic treatment group (p = 0.012). The C-reactive protein level of the FNC group on the 8th and 12th days was significantly lower than that of the basic treatment group (p < 0.05). In addition, D-dimer levels were continuously decreased in both groups compared with before treatment (p < 0.05), as shown in Figure 4E.

| Improvement in respiratory symptoms
Respiratory symptoms were fever, cough, phlegm, cold, or other

| Changes in liver function and renal function
The liver function (aspartate transaminase, alanine transaminase, and gamma-glutamyltransferase) of the two groups of patients was in the normal range, and there was no significant change during the treatment, as shown in Figure 5A-C. The serum albumin levels were not significantly different between the two groups before and after treatment (p > 0.05). The renal function (blood urea nitrogen and creatinine) of the two groups did not change significantly during the study and treatment, as shown in Figure 5E,F (p > 0.05).

| Safety assessment and adverse events
The vital signs of the FNC and basic treatment groups, including systolic and diastolic pressure, were within the normal range during treatment ( Figure 5G,H). During the study period, 10 adverse events occurred in the basic treatment group, with an incidence rate of 6.04%; four patients developed nausea and vomiting; two patients  Table 1.
Univariate Cox regression analysis was used to determine the influence of treatment schemes and vaccination status on the treatment effect of COVID-19. The results are shown in Table 2.
Single-factor Cox regression analysis was used to determine the effect of the treatment schemes (basic treatment or FNC) and vaccination status (0 doses of vaccine; 2 doses of vaccine; and 3 doses of vaccine) on the rate of nucleic acid negative conversion.
Compared with that without vaccination, the effect of two doses of vaccine and three doses of vaccine on the rate of nucleic acid negative conversion was not statistically significant (p > 0.05); F I G U R E 5 Liver function, serum albumin renal function, safety assessment, and adverse events. The two groups' AST, GGT, and ALT levels showed no significant change during the treatment, as shown in (A-C). The serum albumin of the two groups showed no significant change during the treatment, as shown in (D). The two groups' blood urea nitrogen and creatinine levels did not change significantly during the study and treatment, as shown in (E and F). (G) Indicated no significant difference in systolic pressure between the two groups during the treatment period (p > 0.05). (H) Showed no significant difference in diastolic pressure between the two groups. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase.  Table 2.
This study included treatment schemes and vaccination status for a multivariate Cox analysis. Through this analysis, we found that nucleic acid negative conversion rate in the two groups was not affected by vaccination or not, nor by vaccine dose (p > 0.05). See