Serial Viral Load Analysis by Ddpcr to Evaluate Fnc Efficacy and Safety in the Treatment of Moderate Cases of Covid-19 (2024)

3.1 DEMOGRAPHIC ANALYSIS

A total of 476 participants were approached for this study between April 2021 and May 2022. Of these, 296 were excluded for not meeting the eligibility criteria, for worsening before being transferred to the ward of the research center or for withdrawal before participating in the clinical trial. A total of 180 participants were randomized, of whom 172 successfully completed treatment, 7 worsened due to natural disease progression and were referred to the ICU, 6 deaths and 1 hospital discharge in total. There was only 1 dropped out before completing treatment and 1 death (during follow-up) due to cardiac surgery refusal detected on admission (Figure 1).

Patient demographics and baseline characteristics were well matched between the FNC group and the control group at enrollment (Supplementary Table 1). The median age was 48 years (IQR 41-58), there was no significant difference between the age of participants who used the FNC and the placebo (p=0.135). The largest number of participants was male, totaling 104 individuals (58%), there were no significant differences in relation to gender (p=0.075), indicating that the results obtained were not influenced by the age of the individuals or by gender (Supplementary Table 1).

3.2 CLINICAL IMPROVIMENT

At the time of clinical discharge, except for one patient with withdrawal or seven patients who worsened, all ended up with a Clinical Score of 0 or 1, according to the WHO Ordinal Scale of Clinical¹⁴ Improvement. Participants who used the FNC had a final score of 0.02 ± 0.15, while those who participated in the control group had 0.11 ± 0.31, with a statistically significant difference between the groups (p=0.024) (Table 1; Supplementary Figure 1 and 2).

3.3 TIME OF THE NUCLEIC ACID NEGATIVE CONVERSION

The results indicate that participants treated with FNC had a significantly shorter time to first negative nucleic acid conversion (6.24 days; p=0.002) compared with participants treated with placebo (7.94 days) (Figure 2). The same was repeated in the results obtained for the second negative nucleic acid conversion, in which subjects treated with FNC had a shorter time to consecutive negative (7.73 days; p=0.028) compared to subjects treated with placebo (8.89 days) (Figure 2).

3.4 DETECTION OF SARS-COV-2 VIRAL LOAD BY RT-PCR AND DDPCR TECHNIQUE

Some reports regarding relationships between viral loads and disease severity have been reported^{16; 17; 18}. For example, Liu and colleagues¹⁰ showed that the viral load of severe cases was higher than in mild cases, and it’s also reported that the risk of incubation and death increased with higher viral loads¹¹. Moreover, Fajnzylber and colleagues¹² revealed that viral load was implicated in the severity and mortality of COVID-19. A univariate survival analysis revealed a significant difference in survival probability between those with high viral load and those with low viral load¹³.

Although it waspossible to observe a greater tendency of decrease in viral load quantified through the RT-PCR technique of the participants who used the FNC, it was not possible to identify a significant difference between them and the control group.Although this method is regarded as the gold standard for the etiological diagnosis of COVID-19 the sensitivity and reliability of RT-PCR were questioned due to the presence of negative results in some patients who were highly suspected of having the disease based on clinical presentation and exposure history, as well as positive results in some confirmed cases after recovery^{19; 2}. The results from RT‐PCR testing using primers in the ORF1ab gene and N genes can be affected by the variation of viral RNA sequences.

Although there was a significant reduction (p=0.028) in the viral load conversion time of the FNC group in relation to the PLACEBO group, it was expected to see the same in the quantification of viral load by RT-PCR in the standard curve calculation, however the logarithmic variability, despite demonstrating the course of the disease, is not reliable in quantification. Thus, after quantification of viral load by PCR, there was a significant difference between the two groups.(Table 2, Figure 3B). The high sensitivity of the DDPCR confronts the variability obtained by calculating the viral load by RT-PCR after treatment with FNC, showing a significant reduction in viral load in D3 (p<0.002), D5, D7 and D9 (p<0.001) and D11 (p<0.006). Several studies have shown that droplet digital PCR (ddPCR) has the advantages of absolute quantification and is more sensitive for virus detection than RT-PCR^20,21,22.

Notably, it was possible to observe significant differences in the time of improvement of fever in D1 (p<0,015), in D2 (p<0,040) and in D3 (p<0,026) and Chill (p=0.08) symptoms (Table 1). Other information is found in the supplementary material.

3.5 SEQUENCING OF SARS-COV 2 STRAINS

Here, genetic sequencing was performed to demonstrate the distribution of strains between the FNC and placebo groups.

After the genetic sequencing, it was possible to observe that the volunteers who participated in the study were infected by three strains of SARS-COV-2, namely: ALPHA, DELTA and GAMMA (Table 3 and Figure 4). The strain with the lowest prevalence was ALPHA, which affected 7.8% and 18.8% of the volunteers who used FNC and placebo, respectively (Figure 4). The DELTA strain affected 37.7% of both volunteers who used FNC and placebo (Figure 4). The strain with the highest incidence during the research was GAMMA, which affected 54.5% and 43.8% of the volunteers who used FNC and placebo respectively (Figure 4). The data suggest that there is no difference in the distribution of strains between both groups, FNC and placebo.

This study had only 06 vaccinated participants, 03 from placebo group and 03 from FNC group. This study was carried out in a period when vaccines were not widely available for the population, and therefore vaccine interference may exist in only 03 vaccinated participants, infected by the DELTA strain variants AY.99.1, AY .99.1, AY.99.2, respectively.

3.6 CHANGES IN KIDNEY AND LIVER FUNCTIONS BASELINES

In this study, the treatment with FNC was well tolerated by patients. Vital signs, liver function and kidney function in both groups were normal. The results of the exams referring to the renal function of the individuals distributed in FNC and placebo groups, including creatinine and blood urea nitrogen, showed profiles of similar values, within the normal parameters throughout the treatment and without significant differences between the groups during the days of treatment (Figure 5A and B). These data reinforce what was observed in thepilotclinical trial previously performed with FNC, in which hepatic and renal functions do not change between the FNC and control group, indicating non-toxicity of the drug. This is not the case for many antivirals, in studies with remdesivir, for example, nephrotoxicity and hepatoxicity were reported as adverse drug events in patients with Covid-19^{5; 6}. It was reported that similar type of antiviral drugs may cause mitochondrial injury in renal tubular epithelial cells²³^;⁶. Therefore, our results highlight the safety of FNC since no changes were observed in markers of kidney and liver damage when the two groups are compared.

Regarding the results of the tests referring to the liver function of the individuals distributed in FNC and placebo groups, including aspartate aminotransferase, alanine aminotransferase, glutamyl transpeptidase and total bilirubin, they presented values within the normal range, with the groups presenting similar results profiles and without statistically significant changes during the days of treatment, as well as the results observed in exams referring to renal function (Figure 5C, D, E, and F).

3.7 ADVERSE EVENTS AND CLINICAL SAFETY OF FNC

A total of 112 cases of adverse events were observed in this study, of which 105 were considered non-serious adverse events and only 7 were considered serious adverse events. These 7 SAEs were not related to the use of FNC, being caused as a consequence of the rapid progress of the disease (Table 5).

The adverse events observed in this study were mainly related to the increase in ALT (45 cases), GGT (13 cases), AST (10 cases) and GRADE 1 HEADACHE (8 cases), with normalization of these events until the end of treatment (Table 4). Under these conditions, it was possible to observe an increase as well as a reduction in GGT, however the values tended to reduce even under normal conditions. The adverse reactions observed in this study were the same as those related to antiviral drugs, with no unexpected adverse reactions occurring (Table 4).

The phlebitis that occurred during the study is due to the administration of intravenous antibiotic, which was later changed to oral. There was also no significant change in urinary phosphorus. In preliminary studies, vertigo (incidence ≥ 5%) is attributed to FNC, however in this study there were only 2 reported cases of dizziness related to Labyrinthitis (Past history) and hypoglycemia (due to loss of taste). It should also be considered that the participants were bedridden, which could potentiate these events.

There were 7 exclusions due to disease worsening, progressing to ICU. There were 6 deaths and 1 recovery where participants received adequate care and support during hospitalization. In the case of deaths, 3 participants arrived at the hospital with a worsening condition, since after admission they were transferred to the ICU within 1 to 3 days (Table 5).

When comparing the adverse events of the FNC and placebo groups, there is an equivalence without predominance in either group, as would be expected, showing that adverse events are also due to the disease.

The analysis of the viral load, every 48 hours, served as a safety examination that could identify how much the individual is infected, being a marker in the prevention of worsening (a condition that, when it occurs, excludes the participant from the study). The management avoided aggravation and allowing the safety parameters to be better evaluated, keeping more participants in the study.

Other data from the study are described in detail in the supplementary material, such as the presence of comorbidities among the participants (Supplementary Table 5), Demonstration of inflammatory marker (Supplementary Table 9, 10 and 11), Demonstration of lung imaging values (Supplementary Table 12), Demonstration of respiratory symptoms and O2 saturation (Supplementary Table 13, 14 and 15), Demonstration of the distribution of participants in the type of ventilatory support and O2 consumption during ventilatory support (Supplementary Table 16 and 17), Demonstration of the use of mechanical ventilation (Supplementary Table 18) and other variables during the days of study (Supplementary Table 6, 7 and 8).