Effectiveness of a sustained-release ammonium chloride formulation in reducing the viral load of patients with COVID-19 or influenza: A prospective, randomized, double-blind, placebo-controlled study

Effectiveness of a sustained-release ammonium chloride formulation in reducing the viral load of patients with COVID-19 or influenza: A prospective, randomized, double-blind, placebo-controlled study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effectiveness of a sustained-release ammonium chloride formulation in reducing the viral load of patients with COVID-19 or influenza: A prospective, randomized, double-blind, placebo-controlled study Helena C. Maltezou, Constantine Chalkias, Garyfalia Poulakou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9009421/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background We estimated the effectiveness of a novel sustained-release dietary supplement formulation containing 500 mg ammonium chloride and 2,000 IU vitamin D (ACF;) in reducing the viral load of patients with COVID-19 or influenza. Methods In this prospective, randomized, double-blind, placebo-controlled, study. Eligible patients with COVID-19 or influenza were randomized to receive ACF twice daily or placebo (2,000 IU vitamin D/twice daily; VDF) for 10 days. Nasopharyngeal swab samples were collected at Day 1, Day 3–5 and Day 10–11 and tested for SARS-CoV-2 and influenza via RT-PCR. Cycle threshold (Ct) values were measured. The study has been retrospectively registered in ClinicalTrials.gov (ClinicalTrials.gov identifier: NCT07254052) and the ISRCTN registry (ISRCTN study registration number: ISRCTN48259966). Results Thirty two patients were studied, 28 with COVID-19 and 4 with influenza. No patient developed severe disease, was hospitalized, or died. Sixteen patients received ACF and 16 VDF (mean age: 58.1 and 60.7 years, respectively; 68.8% and 25% with comorbidities, respectively). On Day 1, the mean Cts were 22.49 in ACF group and 21.01 in VDF group, on Day 3–5, the mean Cts were 33.20 and 30.82, respectively, and on Day 10–11, the mean Cts were 43.66 and 40.21, respectively. On Day 10–11 the adjusted mean difference was + 3.12 cycles (95% confidence interval: 0.22–6.02; p-value = 0.036). The Kaplan Meier analysis indicated faster clearance in the ACF group compared to the VDF group (p-value = 0.016). Conclusions Our data indicate that ACF-receiving patients had a statistically significant reduction in viral load compared to placebo-receiving patients. This is attributed to the pharmacodynamic action of ammonium chloride and the pharmacokinetic properties of ACF. Larger studies are needed to further investigate the role of ACF in various RNA-viral infections. Trial registration: This study was retrospectively registered in ClinicalTrials.gov (identifier: NCT07254052; registered on 22 October 2025) and in the ISRCTN registry (registration number: ISRCTN48259966; registered on 27 November 2025). Infectious Diseases ammonium chloride NH4Cl influenza SARS-CoV-2 RNA virus infections virus clearance Figures Figure 1 Figure 2 Introduction Respiratory viral infections are a leading cause of morbidity, mortality, and healthcare resources consumption globally. The World Health Organization estimates that seasonal influenza (hereafter referred to as influenza) affects up to 20% of the global population and causes up to 650,000 deaths due to respiratory diseases annually [ 1 ]. Almost six years after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the evolution of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2 remains a significant cause of morbidity and mortality globally, mainly among older adults and individuals with comorbidities, due to the emergence of new variants that escape vaccine-derived immunity or immunity due to past infections [2]. A meta-analysis of 21 studies conducted over two decades in seven high-income countries (France, Germany, Italy, Japan, Spain, the United Kingdom, and the United States) estimated that respiratory syncytial virus (RSV) will cause 5.7 million infections, 510,000 hospitalizations, and 37,000 deaths among adults ≥ 60 years in 2025 alone [3]. In addition to respiratory viruses, other RNA viruses are major causes of morbidity and mortality globally such as hepatitis A, C, D and E viruses [4]. Therefore, treatments to reduce the overall burden of RNA-viral infections are urgently needed. Ammonium chloride (NH 4 Cl), a lysosomotropic agent, demonstrates at specific concentrations a broad virostatic action through inhibition of virus uncoating and diminution or even inhibition of virus replication, and therefore delay of the progression of infection by various RNA viruses, including human and avian influenza A viruses, coronaviruses, and hepatitis A and C viruses [5–12]. This effect is achieved through a temporary and reversible increase of the pH of intracellular lysosomes which in turn prevents the fusion between viruses and the lysosomes’ membranes [5–12]. In vitro studies have demonstrated the importance of endosomal acidification for SARS-CoV-2 entry and infection [5]. Moreover, SARS-CoV-2-infected mice models have shown that ammonium chloride also possesses significant anti-SARS-CoV-2 activity, through inhibition of lysosomal acidification and therefore intracellular virus replication and alleviation of inflammation and infiltration in pulmonary tissues [13]. In addition, a model using data from 51 countries or regions across Europe collected as of 26 April 2020 confirmed the possible association between consumption of foods containing ammonium chloride and lower death rates from COVID-19 [14]. A randomized-controlled trial conducted in 2020 found that the time of recovery and SARS-CoV-2 loads were significantly reduced among COVID-19 patients who had received an ammonium chloride solution (125 mg/5 ml daily per os) compared with the placebo group (60 patients in each group), by odds ratios of 1.8 [95% confidence interval (CI), 1.15–2.83; p-value = 0.01] and 7.90 (95% CI, 1.62–14.17; p-value = 0.014), respectively [15]. The current study aimed to estimate the effectiveness of a sustained-release dietary supplement formulation containing ammonium chloride and vitamin D in reducing the viral loads of patients with COVID-19, influenza or RSV infection compared with the receipt of a vitamin D formulation only (placebo). Materials and Methods Setting This was a prospective, randomized, double-blind, placebo-controlled study. The study was conducted from September 1, 2024, to April 30, 2025, corresponding to the last follow-up visit of the final enrolled participant, at Sotiria Hospital for Respiratory and Thoracic Diseases and at En Ygeia Clinic in Athens, Greece. The study has been retrospectively registered in ClinicalTrials.gov ( identifier: NCT07254052, registered on 22-10-2025 https://clinicaltrials.gov/study/NCT07254052 ) and the ISRCTN registry (registration number: ISRCTN48259966, registered on 27-11-2025 https://www.isrctn.com/ISRCTN48259966 ). Study Population Patients with influenza-like illness (ILI) or acute respiratory infection (ARI) who attended the Infectious Diseases Unit of Sotiria Hospital for Respiratory and Thoracic Diseases or the Internal Medicine Outpatient Clinic of En Ygeia Clinic during the study period were eligible for the study. Eligible patients were tested with a combo rapid antigen test for SARS-CoV-2, influenza A and B, and RSV (CorDX, Inc., San Diego, CA, USA). Inclusion criteria were an age ≥ 18 years, laboratory-confirmed SARS-CoV-2, influenza virus or RSV infection, and written informed consent. Patients were excluded if they were < 18 years old, pregnant or lactating, had an organ transplantation, a frailty score ≥ 5, symptoms for ≥ 3–5 days, or a documented allergy to ammonium chloride, vitamin D or any excipient of the administered formulations. Participation in the study was discontinued when one of the following occurred: intolerance of the administered formulations, non-compliance with study requirements or withdrawal of informed consent. The authors did not participate in patients’ management decisions. Laboratory testing Nasopharyngeal swab samples were collected from each participant at three timepoints after diagnosis: Day 1 (baseline), Day 3–5 (intermediate), and Day 10–11 (final). All samples were transferred under ice-packed conditions to the Laboratory of the Research Group of Clinical Pharmacology and Pharmacogenomics at the Faculty of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens immediately after sampling, where they were kept at -20°C for a maximum of three days until processing. Total RNA was extracted using the ZYBIO Nucleic Acid Extraction Kit (B-200-20) on the Zybio EXM3000 system, according to the manufacturer’s protocol. Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using the Zybio SARS-CoV-2 & Influenza A/B Reagent Kit (catalogue number: SC2FA/B-96) on the Zybio ZIP 96V thermocycler. All primers required for the qPCR were included in this kit for clarity to the reader. This multiplex assay qualitatively detects SARS-CoV-2 RNA (N and S genes), influenza A (M gene), and influenza B (HA gene) through a one-step RT-PCR protocol. Cycle threshold (Ct) values were recorded for each sample. Each PCR run included both positive and negative controls, and at least one randomly selected sample from a prior run was included as a technical replicate for quality control purposes. According to the manufacturer’s instructions, samples that did not yield a valid Ct value within 45 amplification cycles were considered negative. Non-detects were therefore censored at the assay limit (Ct = 45), and samples without a valid Ct value at any time point were excluded from the analysis for the corresponding reagent. All PCR reactions were performed in a blinded manner with regard to group allocation, and blinding was maintained across laboratory technicians, case report form (CRF) handlers, and physicians until completion of the statistical analysis. Study procedures Participants were enrolled at Sotiria General Hospital outpatient clinic or the private outpatient clinic “En Ygeia”. Following provision of written informed consent, the treating physician dispensed the next available study medication card sequentially from the study supply box, without interfering with any concomitant treatment. The study tablets were manufactured by Help Pharmaceuticals, which also packaged the medication cards, prepared the dispensing order to ensure balanced allocation, and retained exclusive custody of the lot-to-treatment correspondence code until completion of the study. After baseline (Day 1) nasopharyngeal swab samples were collected, patients were randomly allocated in 1:1 ratio to receive either an enteric-coated sustained-release dietary supplement formulation containing 500 mg ammonium chloride, 2,000 IU vitamin D, and excipients (ACF group) or a physically identical sustained-release dietary supplement formulation containing the same amount of vitamin D (2,000 IU) and the same excipients only (VDF, placebo group). Randomization was implemented through the dispensing order of pre-packaged study medication cards prepared in advance by the manufacturer, ensuring an approximately balanced 1:1 allocation and preventing prediction of upcoming assignments by the enrolling clinician. ACF and VDF were administered twice daily (one tablet every 12 hours) for 10 consecutive days, alongside standard symptomatic supportive care. Both formulations were visually indistinguishable and with identical appearance, packaging, and labeling, each assigned a distinct LOT code corresponding to the respective formulation. The correspondence between LOT code and treatment assignment (ACF vs VDF) was maintained in a blinded treatment code held exclusively by the manufacturer and was not accessible to participants, enrolling clinicians, investigators, laboratory personnel, outcome assessors, or statisticians, until completion of the statistical analysis. The sustained-release ammonium chloride dietary supplement formulation (DIVIRNAM®, Metron Nutraceuticals, Cleveland, Ohio, USA) has been described in the United States Patent Application Publication No.: 2022/0160757 (Priority Date: November 20, 2020; Publication Date: May 26, 2022) and the International Patent Application Publication WO 2022/109393 (Priority Date: November 20, 2020; Publication Date: May 27, 2022). Ammonium chloride has been designated GRAS (Generally Recognized As Safe;) status by the Food and Drug Administration of the United States (21CFR 184.1138), and it is already under registration in the European Union. Both ACF and VDF dietary supplement formulations have been notified to the Hellenic National Organization for Medicines (EOF). Data collection Patients were prospectively followed for a period of up to 30 days following laboratory-confirmed diagnosis. Data collection was conducted in real time by trained healthcare professionals through structured patient interviews at the time of enrolment and comprehensive review of medical records throughout the follow-up period. For each participant, a standardized CRF was utilized to systematically document the following information: demographic data: age, sex medical history: presence of comorbid conditions, including chronic cardiovascular disease, chronic pulmonary disease, diabetes mellitus, chronic kidney disease, chronic neuromuscular disorders, obesity, malignancy, and immunosuppression infection and vaccination history: prior COVID-19 infections; COVID-19 vaccination status for the 2024–2025 season; influenza vaccination status for the 2024–2025 season laboratory-confirmed diagnosis: SARS-CoV-2, influenza or RSV clinical information: date of diagnosis, presenting symptoms, and subsequent clinical course, including hospitalization, need for supplemental oxygen, admission to an intensive care unit (ICU), and use of invasive mechanical ventilation clinical outcome: status at timepoint 2 (Day 3–5), timepoint 3 (Day 10–11), and 30 days after diagnosis. Outcomes The prespecified primary outcome was the reduction of the baseline viral load, assessed by cycle threshold (Ct) values measured by RT-PCR at Day 1, Day 3–5, and Day 10–11. Viral load was analysed using adjusted mean differences between groups and longitudinal comparisons across time points. Secondary outcomes included the severity of symptoms, documented prospectively over a 30-day follow-up period. Definitions ILI was defined as the sudden onset of at least one systemic symptom (fever, malaise, headache, myalgia) and at least one respiratory symptom (cough, sore throat, shortness of breath) [16]. ARI was defined as the sudden onset of at least one of the following symptoms: cough, sore throat, shortness of breath, and coryza [16]. COVID-19 was defined as a case with symptoms compatible with COVID-19 and laboratory-confirmed SARS-CoV-2 infection. Influenza was defined as a case with symptoms compatible with influenza and a laboratory-confirmed influenza infection. RSV infection was defined as a case with symptoms compatible with RSV respiratory illness and laboratory-confirmed RSV infection. Mild illness was defined as the presence of various signs and symptoms (e.g., fever, cough, sore throat, malaise, headache, myalgia, nausea, vomiting, diarrhoea) but no shortness of breath, dyspnoea or abnormal findings on chest imaging. Severe illness was defined as increasing needs for supplemental oxygen, admission to ICU, invasive mechanical ventilation and/or death. Death was defined as 30-day crude mortality. Virus clearance was defined as a Ct value of ≥ 40. Statistical analysis Baseline characteristics were statistically compared using independent-samples t-tests or Mann–Whitney U tests for continuous variables, and Fisher's exact test for categorical variables. Differences in Ct values between groups were assessed using parametric [Welch’s unequal-variance t-tests and analysis of covariance (ANCOVA)] and non-parametric (Mann–Whitney U tests, Hodges–Lehmann estimators and Cliff’s δ effect sizes) approaches [17]. The primary endpoint was analyzed using ANCOVA with baseline Ct as the covariate. Longitudinal trajectories were further evaluated using linear mixed-effects models for repeated measures (SPSS - MMRM), including fixed effects for group and visit, as well as their interaction. A random intercept was included for each subject, and the Kenward–Roger method was used to estimate the denominator degrees of freedom [18]. Time-to-viral clearance was examined using Kaplan–Meier survival curves and discrete-time complementary log–log models. Robustness was assessed using HC3 heteroskedasticity-consistent errors, robust regression, permutation testing with Freedman–Lane residualisation, bootstrap resampling and leave-one-out diagnostics [19–21]. All assumptions (normality, homoscedasticity and influence) were systematically checked. Analyses were conducted using IBM SPSS Statistics (version 26, IBM Corp., Armonk, NY, USA), Python (SciPy version 1.9) and GPower version 3.1. Statistical terms and abbreviations are presented in Appendix. Ethical issues The study protocol was approved by the Scientific Council of Sotiria Hospital for Respiratory and Thoracic Diseases (No. 14328/17-05-2024). All participants were informed about the study and were enrolled following written informed consent. The study was conducted in accordance with the Declaration of Helsinki. Data was managed in compliance with European Union General Data Protection Regulation standards. Results A total of 32 participants were analyzed, with 16 assigned to the ACF group and 16 assigned to the VDF group. Table 1 shows the characteristics of patients stratified per group. The mean age was 58.1 ± 17.6 years in the ACF group and 60.7 ± 17.2 years in the VDF group. ACF group patients more frequently had at least one comorbidity compared with VDF-treated patients (68.8% versus 25.0%; p-value=0.02). Overall, chronic cardiovascular disease, chronic neuromuscular disease, and diabetes mellitus were the prevalent comorbidities among studied patients (6, 5, and 4 patients each). Of the 32 patients studied, 28 had COVID-19 (14 in each group) and 4 had influenza A or B (2 in each group). No patient had RSV infection. All had a mild illness. No serious adverse events, treatment discontinuations, or safety concerns related to the study interventions were reported during the study period. No patient developed severe disease, was hospitalized, received oxygen, or died by 30 days after diagnosis. In general, the two groups did not differ regarding mean and median age, sex, past SARS-CoV-2 infection, and COVID-19 and influenza vaccination status for the 2024-2025 season. The only statistically significant difference was the prevalence of comorbidities, with the ACF group presenting a higher rate. Table 1. Baseline characteristics of participating patients by treatment group___ ACF: ammonium chloride formulation; VDF: vitamin D formulation; SD: standard deviation; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; COVID-19: coronavirus disease 2019 *11 ACF-treated patients had a total of 17 comorbidities: chronic cardiovascular disease: 5, chronic neuromuscular disease: 5, diabetes mellitus: 2, malignancy: 2, immunosuppression: 2, chronic renal disease: 1 **4 DVF-treated patients had 4 comorbidities: diabetes mellitus: 2; chronic cardiovascular disease: 1 ; obesity: 1 ***for the 2024-2025 season Figure 1. L ongitudinal Ct values trajectories per group Ct: cycle threshold; SD: standard deviation; ACF: ammonium chloride formulation; VDF: vitamin D formulation; RT-PCR: reverse-transcriptase polymerase chain reaction All 32 participants completed measurements on the three scheduled visits. Figure 1 summarizes longitudinal Ct trajectories per group. On Day 1, the mean Ct values were 22.49 ± 6.95 for the ACF group and 21.01 ± 8.29 for the VDF group. By Day 3–5, the mean Ct increased to 33.20 ± 8.10 in the ACF group and to 30.82 ± 5.68 in the VDF group. At Day 10–11, the difference widened, with the ACF group showing a mean Ct of 43.66 ± 2.57 compared to a mean of 40.21 ± 5.47 in the VDF group. The increasing Ct values over time in both groups is consistent with declining viral load, with the greatest separation observed at Day 10–11 in favor of ACF. These preliminary findings motivated baseline-adjusted modeling and censoring-aware analyses presented below. ANCOVA was conducted with treatment group as the main factor and baseline Ct as covariate. All 32 participants were included under the intention-to-treat principle. At Day 10–11, the adjusted mean difference (ACF – VDF) was +3.12 cycles (95% CI: 0.22–6.02), corresponding to a large, standardized effect size (Cohen’s d ≈ 0.81). The two-sided p-value was 0.036. In summary, the ACF formulation was associated with a statistically significant increase in Ct at Day 10–11 compared with VDF, indicating reduced viral load. Results from the MMRM analysis were consistent with the ANCOVA findings, confirming the longitudinal pattern of increasing Ct values and the treatment effect at Day 10–11 (Figure S1). Time-to-viral clearance was assessed by defining the first participant visit at which Ct ≥ 40 as the clearance event, with participants not cleared by Day 10–11 censored at that visit. The Kaplan Meier analysis indicated faster clearance in the ACF group compared to VDF (log-rank p-value = 0.016). By Day 10–11, clearance had been achieved in 14 of 16 ACF participants (87.5%) versus 8 of 16 VDF participants (50.0%). Effect measures derived at Day 10–11 showed a risk difference of +37.5 percentage points (95% CI: –8.0-68.5), a relative risk of 1.75 (95% CI: 1.04–2.95), and an odds ratio of 7.00 (95% CI: 1.18–41.36). These results are detailed in Table 2 and illustrated in Figure 2. Among the four influenza cases (n=2 per arm), Ct patterns were descriptively consistent with faster clearance in the ACF group; however, the influenza sample was too small for inference. Table 2. Viral clearance by Day 10–11 and effect measures (ITT*, Ct≥40) Measure Estimate 95% CI Cleared — ACF 14/16 = 87.5% [64.0%, 96.5%] (Wilson) Cleared — VDF 8/16 = 50.0% [28.0%, 72.0%] (Wilson) Risk difference (ACF−VDF) 0.375 (37.5 pp) [−0.080, 0.685] Relative risk (ACF vs VDF) 1.75 [1.04, 2.95] Odds ratio (ACF vs VDF) 7.00 [1.18, 41.36] NNT** 2.7 [1.5, ∞] ITT: Intention-to-Treat ; Ct: cycle threshold; ACF: ammonium chloride formulation; VDF: vitamin D formulation; CI: confidence interval; NNT: Number Needed to Treat *ITT: Intention-to-Treat, an analysis approach including all randomized participants in their originally assigned groups, regardless of adherence or protocol deviations. **NNT: Number Needed to Treat, the reciprocal of the absolute risk reduction, indicating how many patients must be treated with ACF instead of VDF for one additional patient to achieve viral clearance by Day 10–11. Figure 2. Clearance by Day 10–11 (Ct≥40): ACF vs VDF with 95% CI Ct: cycle threshold; ACF: ammonium chloride formulation; VDF: vitamin D formulation; vs: versus; 95% CI: confidence interval To evaluate the robustness of the Day 10–11 findings, we conducted a series of sensitivity analyses using alternative specifications and inference frameworks. These included robust standard errors, robust regression, permutation testing, bootstrap resampling, and leave-one-out diagnostics. The sensitivity analyses demonstrate that the observed effect of ACF on Day 10-11 is robust to heteroskedasticity, outliers, and small-sample distribution concerns. These results are summarized in Table S1 and Figure S2. Lastly, Table 3 compares the estimated differences between the ACF and VDF groups across multiple statistical approaches. Across all approaches, the Day 10–11 group effect consistently ranged from +2.6 to +3.1 cycles, with p-values around 0.03–0.04. The convergence of results across robust parametric and non-parametric frameworks increases confidence in the primary ANCOVA/MMRM signal. Table 3. Estimated differences between the ACF and VDF groups across multiple statistical approaches Analysis Effect size / Difference Significance ANCOVA (Day 10–11, adjusted) +3.12 Ct (Cohen’s d ≈ 0.81) p-value = 0.036 (nominal) q-value = 0.098 (FDR) MMRM (Day 10–11 LS-means) +3.1 Ct (consistent with ANCOVA) p-value ≈ 0.04 (nominal) q-value ≈ 0.10 (FDR) Unadjusted Welch t-test (Day 10–11) +3.45 Ct (Hedges’ g ≈ 0.81) p-value = 0.033 Non-parametric Hodges–Lehmann (Day 10–11) +3.45 Ct (median difference) 95% CI: 0.45 - 6.45 Time-to-clearance (Day 10–11, risk difference) +37.5 percentage points 95% CI: –8.0 - 68.5 Time-to-clearance (Day 10–11, OR) OR: 7.00 95% CI: 1.18–41.36 Significant ACF: ammonium chloride formulation; VDF: vitamin D formulation; ANCOVA: analysis of covariance; Ct: cycle threshold; FDR: False Discovery Rate; q: FDR adjusted p-value; MMRM: Mixed-Effects Model for Repeated Measures; LS: Least Squares means; CI: confidence interval; OR: odds ratio q-value: adjusted p-value after controlling for the false discovery rate (FDR) FDR: False Discovery Rate, a multiple-comparison correction method (e.g., Benjamini–Hochberg) controlling the expected proportion of false positives. MMRM: Mixed-Effects Model for Repeated Measures, a longitudinal model that accounts for within-subject correlations across timepoints. LS: Least Squares means (also called Estimated Marginal Means), model-adjusted group averages derived from MMRM rather than raw arithmetic means. Discussion The present prospective, randomized, double-blind, placebo-controlled study indicates that patients who received a sustained-release dietary supplement formulation containing 500 mg ammonium chloride and 2,000 IU vitamin D (ACF) twice daily had statistically significant lower viral loads compared to VDF, placebo-receiving patients. The largest consistent differences in Cts between the ACF and VDF groups were recorded on Day 10–11 and were confirmed by multiple statistical approaches. Moreover, the time-to-clearance analysis demonstrated that virus clearance occurred more rapidly and more frequently in the ACF group compared with the VDF group, which provides further evidence of antiviral effectiveness. Notably, despite a higher prevalence of baseline comorbidities, ACF-treated patients achieved faster and more frequent viral clearance, suggesting that the treatment effect was not simply due to baseline differences; however, this observation should be interpreted with caution due to the small sample size. To our knowledge, there is only one study published so far, which showed a significant reduction of the time of recovery as well as of SARS-CoV-2 loads among hospitalized COVID-19 patients who received an ammonium chloride solution, but not via a solid, sustained-release ammonium chloride formulation [15]. To our knowledge, there is no published data regarding the administration of ammonium chloride to patients with influenza or other respiratory viruses. Yet, recent studies have highlighted the potential of repurposed agents to exert antiviral activity against respiratory viruses, including influenza A, through inhibition of viral internalization and fusion, supporting the broader concept of non-virus-specific pharmacological interventions [22]. Monitoring of viral loads through PCR has become the standard of care for estimating the effectiveness of treatments for various viral infections [23–25]. In this context, recent experimental evidence has shown that pharmacological compounds with broad antiviral activity can significantly reduce viral loads and accelerate disease resolution in both SARS-CoV-2 and influenza models. Such findings support the biological plausibility that non-virus-specific agents may enhance viral clearance across multiple RNA viruses [26]. In our study, the gradual but consistent increase in Ct values observed among patients who received the ACF formulation is attributed to the combination of the pharmacodynamic action of ammonium chloride along with the pharmacokinetic properties of the ACF sustained-release formulation. The ACF dietary supplement formulation incorporates a 12-hour release mechanism to support prolonged immunomodulatory activity, therefore enabling the gradual and durable suppression of virus replication compared with the VDF formulation that lacks ammonium chloride. Studies are needed to investigate the effects of ammonium chloride on viral load of other RNA viruses that constitute public health threats, such as influenza A and H5N1. In our study, both patients with influenza who received the ammonium chloride formulation noted early viral clearance, as highlighted by Ct values ≥ 40 at Day 5. This pattern was not observed in the VDF-receiving influenza cases. While the number of patients with influenza was too small for statistical analysis, the trend could suggest a potentially enhanced antiviral effect of the ACF on influenza virus. Given the very small number of influenza patients, this observation is descriptive and hypothesis-generating rather than confirmatory. This finding may indicate a potentially enhanced antiviral effect of the ACF on influenza virus and can be elucidated through targeted studies in patients with influenza, particularly in light of shifting public health priorities, where COVID-19 concern is gradually subsiding and influenza remains a recurrent burden with significant clinical impact [27]. Prospective studies have demonstrated that vitamin D supplementation has a protective effect on ILI [28]. In addition, vitamin D supplementation, particularly when administered early in the course of COVID-19, ameliorated its clinical course and prognosis [29]. The positive effects of vitamin D are mediated by enhancing the innate antiviral immune response, facilitating the induction of antimicrobial peptides/autophagy, and modulating the host reactive hyperinflammatory phase during COVID-19 [29]. However, mixed results from other trials led to a lack of consensus on optimal dosing and timing of vitamin D supplementation in COVID-19. In our study, we assumed that vitamin D served as a booster for the intrinsic benefits conferred by ammonium chloride to the ACF-receiving patients. Both study formulations contained the same dose of vitamin D (2,000 IU twice daily); therefore, any between-group differences observed can be attributed to the presence or absence of ammonium chloride in the formulation. The main strength of the present study is its prospective, double-blind, placebo-controlled randomization design. This enabled the repeated nasopharyngeal swab sampling in predefined time points along with the study of the kinetics of Ct values. In addition, data about the clinical course and outcome were collected prospectively. The main limitation is the small number of study participants, which explains the wide CIs, and which did not allow the study of ACF on viral loads by virus, age groups, comorbidities, and settings (e.g. hospital versus ambulatory healthcare settings) [30]. Another limitation is that the precise time from the onset of symptoms to treatment was not available; nevertheless, all studied patients were enrolled 3–5 days after the onset of symptoms. The fact that Ct values concern viral nucleic acid and do not necessarily correspond to infectious virus should also be considered. Indeed, clinical studies evaluating established antivirals, such as favipiravir, have reported variable or inconclusive effects on viral clearance despite antiviral activity, underscoring the complexity of translating virological endpoints into consistent clinical outcomes [31]. Lastly, heterogeneity was present in our study but was adequately addressed through robust inference, and raised confidence regarding the primary ANCOVA results. Conclusions The findings of the present study indicate that a sustained-release ammonium chloride formulation (ACF - DIVIRNAM ® ) administered twice daily significantly reduced the viral load of patients with COVID-19 or influenza compared to placebo-receiving patients. This is attributed to the pharmacodynamic action of ammonium chloride and the pharmacokinetic properties of DIVIRNAM ® . Larger studies are needed to further investigate the effectiveness of DIVIRNAM ® in various RNA-viral infections, patient populations, and healthcare or non-healthcare settings. Abbreviations ACF (Ammonium Chloride Formulation): a novel sustained-release dietary supplement formulation containing 500 mg ammonium chloride and 2,000 IU vitamin D (DIVIRNAM®). ANCOVA (Analysis of Covariance): A general linear model that combines ANOVA and regression, used here to compare cycle threshold (Ct) values between groups while adjusting for baseline Ct. ARI (Acute Respiratory Infection): An acute infection of the upper and/or lower respiratory tract. ARR (Absolute Risk Reduction): The difference in event rates between two groups; used to calculate the NNT. Bootstrap (Percentile CI): A resampling method that estimates confidence intervals by repeatedly drawing samples with replacement from the data. CI (Confidence Interval): A statistical interval estimate that provides a range of values within which the true parameter is expected to lie with a specified probability (commonly 95%). Cliff’s δ (delta): A non-parametric effect size measure representing the probability that a randomly selected value from one group exceeds a randomly selected value from another group. Cohen’s d / Hedges’ g: Standardized effect size measures representing the difference between two means expressed in units of standard deviation. CRF (Case report form): A standardized document used to record study-related data for each participant in a clinical study. Ct (Cycle Threshold): The number of PCR cycles required for the fluorescent signal to cross the detection threshold; inversely proportional to the viral load. FDR (False Discovery Rate): A multiple-comparison correction method (e.g., Benjamini–Hochberg) that controls the expected proportion of false positives among results declared significant. GRAS (Generally Recognized As Safe): A regulatory designation indicating that a substance is considered safe for its intended use based on scientific evidence and/or a history of common use in food. HC3 robust SE (Heteroskedasticity-Consistent Standard Errors, type 3): A variance estimator that adjusts for heteroskedasticity and small-sample bias in OLS regression. Hodges–Lehmann Estimator: A non-parametric estimator of the median difference between two groups, robust to non-normal distributions. ICU (Intensive Care Unit): A specialized hospital unit providing intensive monitoring and treatment for critically ill patients. ILI (Influenza-Like Illness): An acute respiratory illness characterized by influenza-like symptoms. ITT (Intention-to-Treat): An analysis principle in which all randomized participants are analyzed in their originally assigned groups, regardless of adherence or protocol deviations. Kaplan–Meier Curve: A survival analysis method used to estimate the probability of an event (here, viral clearance) over time. LS (Least Squares Means or Estimated Marginal Means): Model-adjusted group averages derived from ANCOVA or MMRM, representing expected values after accounting for covariates, not simple arithmetic means. MMRM (Mixed-Effects Model for Repeated Measures): A longitudinal statistical model that accounts for correlations within subjects across repeated timepoints, including both fixed and random effects. NNT (Number Needed to Treat): The reciprocal of the absolute risk reduction; indicates how many patients must be treated with ACF instead of VDF for one additional patient to achieve viral clearance by Day 10–11. OLS (Ordinary Least Squares): The standard estimation method for linear regression and ANCOVA, minimizing the sum of squared residuals to obtain parameter estimates. OR (Odds Ratio): A measure of association representing the odds of an event occurring in one group compared to another. Permutation Test (Freedman–Lane): A non-parametric resampling method for testing significance by permuting residuals, robust to distributional assumptions. p-value: The probability of observing results as extreme as (or more extreme than) those obtained, under the null hypothesis of no difference. q-value: The false discovery rate (FDR)-adjusted p-value, representing the minimum FDR at which a particular test result would be considered significant. RR (Relative Risk): The ratio of the probability of an event in the treatment group to that in the control group. VDF (Vitamin D Formulation): sustained-release dietary supplement formulation containing 2,000 IU of vitamin D Declarations Ethics approval and consent to participate The study was approved by the Scientific Council of Sotiria Hospital for Respiratory and Thoracic Diseases (No. 14328/17-05-2024), which serves as the institution’s ethics and regulatory review body for clinical research in accordance with Greek national regulations. All participants were informed about the study and were enrolled following written informed consent. Patient consent for publication Participants provided written consent for publication. Availability of data The data that support the findings of this study are provided in the Supplementary Material (Tables S2–S3). The study protocol and additional information are available from the corresponding author upon reasonable request. Competening Interests Dr. Drakoulis has an interest in the intellectual property of DIVIRNAM ® . Dr. Tsirikos-Karapanos has an interest in the intellectual property of and direct financial interest in DIVIRNAM ® . Dr. Drakoulis’ and Dr. Tsirikos-Karapanos’ contribution in the study was only in the design of the study’s clinical research protocol. All other authors declare no conflict of interest. Funding This work was partially supported by Metron Nutraceuticals, LLC; Cleveland, OH, USA and HELP SA, Athens, Greece. Authors’ contributions HCΜ contributed to the conceptualization of the study, investigation, formal analysis, and drafted the original manuscript. CC and GP contributed to data curation and investigation, and participated in writing, review, and editing of the manuscript. AT and PX contributed to formal analysis and investigation, and participated in writing, review, and editing. AR performed the statistical analysis and contributed to writing, review, and editing. NS contributed to data curation and investigation, and participated in writing, review, and editing. NTK and ND contributed to the conceptualization of the study and participated in writing, review, and editing. All authors read and approved the final manuscript. Acknowledgements We are thankful to healthcare professionals in participating centers for their support. The opinions presented in this article are those of the authors and do not necessarily represent those of their institutions. References Burden of disease. https://www.who.int/teams/global-influenza-programme/surveillance-and-monitoring/burden-of-disease. Accessed 7 Jan 2026. Summary. datadot. https://data.who.int/dashboards/covid19/summary. Accessed 7 Jan 2026. Savic M, Penders Y, Shi T, Branche A, Pirçon J-Y. Respiratory syncytial virus disease burden in adults aged 60 years and older in high-income countries: A systematic literature review and meta-analysis. Influenza Other Respir Viruses. 2023;17:e13031. https://doi.org/10.1111/irv.13031. Cooke GS, Andrieux-Meyer I, Applegate TL, Atun R, Burry JR, Cheinquer H, et al. Accelerating the elimination of viral hepatitis: a Lancet Gastroenterology & Hepatology Commission. Lancet Gastroenterol Hepatol. 2019;4:135–84. https://doi.org/10.1016/S2468-1253(18)30270-X. Prabhakara C, Godbole R, Sil P, Jahnavi S, Gulzar S-J, Zanten TS van, et al. Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors. PLOS Pathogens. 2021;17:e1009706. https://doi.org/10.1371/journal.ppat.1009706. Yoshimura A, Ohnishi S. Uncoating of influenza virus in endosomes. J Virol. 1984;51:497–504. https://doi.org/10.1128/JVI.51.2.497-504.1984. Di Trani L, Savarino A, Campitelli L, Norelli S, Puzelli S, D’Ostilio D, et al. Different pH requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza A viruses. Virol J. 2007;4:39. https://doi.org/10.1186/1743-422X-4-39. Superti F, Seganti L, Orsi N, Divizia M, Gabrieli R, Panà A. The effect of lipophilic amines on the growth of hepatitis A virus in Frp/3 cells. Arch Virol. 1987;96:289–96. https://doi.org/10.1007/BF01320970. Ashfaq UA, Javed T, Rehman S, Nawaz Z, Riazuddin S. Lysosomotropic agents as HCV entry inhibitors. Virol J. 2011;8:163. https://doi.org/10.1186/1743-422X-8-163. Helenius A, Marsh M, White J. Inhibition of Semliki forest virus penetration by lysosomotropic weak bases. J Gen Virol. 1982;58 Pt 1:47–61. https://doi.org/10.1099/0022-1317-58-1-47. Mizzen L, Hilton A, Cheley S, Anderson R. Attenuation of murine coronavirus infection by ammonium chloride. Virology. 1985;142:378–88. https://doi.org/10.1016/0042-6822(85)90345-9. Zeichhardt H, Wetz K, Willingmann P, Habermehl KO. Entry of poliovirus type 1 and Mouse Elberfeld (ME) virus into HEp-2 cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating. J Gen Virol. 1985;66 ( Pt 3):483–92. https://doi.org/10.1099/0022-1317-66-3-483. Shang C, Zhuang X, Zhang H, Li Y, Zhu Y, Lu J, et al. Inhibitors of endosomal acidification suppress SARS-CoV-2 replication and relieve viral pneumonia in hACE2 transgenic mice. Virol J. 2021;18:46. https://doi.org/10.1186/s12985-021-01515-1. Hidvégi M, Nichelatti M. Bacillus Calmette-Guerin vaccination Policy and Consumption of Ammonium Chloride-Enriched Confectioneries May Be Factors Reducing COVID-19 Death Rates in Europe. Isr Med Assoc J. 2020;22:501–4. Siami Z, Aghajanian S, Mansouri S, Mokhames Z, Pakzad R, Kabir K, et al. Effect of Ammonium Chloride in addition to standard of care in outpatients and hospitalized COVID-19 patients: A randomized clinical trial. Int J Infect Dis. 2021;108:306–8. https://doi.org/10.1016/j.ijid.2021.04.043. Operational considerations for respiratory virus surveillance in Europe. 2022. https://www.ecdc.europa.eu/en/publications-data/operational-considerations-respiratory-virus-surveillance-europe. Accessed 7 Jan 2026. Hess M, Kromrey J. Robust Confidence Intervals for Effect Sizes: A Comparative Study of Cohen’s d and Cliff’s Delta Under Non-normality and Heterogeneous Variances. Educ Psychol Meas. 2004;:699–719. Kenward MG, Roger JH. An improved approximation to the precision of fixed effects from restricted maximum likelihood. Computational Statistics & Data Analysis. 2009;53:2583–95. MacKinnon JG, White H. Some heteroskedasticity-consistent covariance matrix estimators with improved finite sample properties. Journal of Econometrics. 1985;29:305–25. https://doi.org/10.1016/0304-4076(85)90158-7. Hayes AF, Cai L. Using heteroskedasticity-consistent standard error estimators in OLS regression: an introduction and software implementation. Behav Res Methods. 2007;39:709–22. https://doi.org/10.3758/bf03192961. Anderson MJ, Robinson J. Permutation Tests for Linear Models. Aus NZ J of Statistics. 2001;43:75–88. https://doi.org/10.1111/1467-842X.00156. Li Y, Yang S, Jiang F, Luo S, Liang J, Jiang L, et al. Cilnidipine exerts antiviral effects in vitro and in vivo by inhibiting the internalization and fusion of influenza A virus. BMC Med. 2025;23:200. https://doi.org/10.1186/s12916-025-04022-0. Bakshi S, Chattopadhyay P, Ahammed M, Das R, Majumdar M, Dutta S, et al. Efficacy of Different Combinations of Direct-Acting Antivirals Against Different Hepatitis C Virus-Infected Population Groups: An Experience in Tertiary Care Hospitals in West Bengal, India. Viruses. 2025;17. https://doi.org/10.3390/v17020269. Shaikh SA, Kahn J, Aksentijevic A, Kawewat-Ho P, Bixby A, Rendulic T, et al. A multicenter evaluation of hepatitis B reactivation with and without antiviral prophylaxis after kidney transplantation. Transpl Infect Dis. 2022;24:e13751. https://doi.org/10.1111/tid.13751. Fougère Y, Brophy J, Hawkes MT, Lee T, Samson L, Gantt S, et al. Clinical and Immunologic Impact of CMV Coinfection Among Children Living With HIV in Canada. Pediatr Infect Dis J. 2025;44:764–71. https://doi.org/10.1097/INF.0000000000004811. Luo R, Shen B, Qian B, Fan L, Zhang J, Deng X, et al. Taurultam shows antiviral activity against SARS-CoV-2 and influenza virus. BMC Microbiol. 2025;25:292. https://doi.org/10.1186/s12866-025-03847-2. Christodoulou A, Katsarou M-S, Emmanouil C, Gavrielatos M, Georgiou D, Tsolakou A, et al. A Machine Learning-Based Web Tool for the Severity Prediction of COVID-19. BioTech. 2024;13. https://doi.org/10.3390/biotech13030022. Helmond N van, Brobyn TL, LaRiccia PJ, Cafaro T, Hunter K, Roy S, et al. Vitamin D3 Supplementation at 5000 IU Daily for the Prevention of Influenza-like Illness in Healthcare Workers: A Pragmatic Randomized Clinical Trial. Nutrients. 2022;15. https://doi.org/10.3390/nu15010180. Cicero AFG, Fogacci F, Borghi C, Cicero AFG, Fogacci F, Borghi C. Vitamin D Supplementation and COVID-19 Outcomes: Mounting Evidence and Fewer Doubts. Nutrients. 2022;14. https://doi.org/10.3390/nu14173584. Maltezou HC, Raftopoulos V, Vorou R, Papadima K, Mellou K, Spanakis N, et al. Association Between Upper Respiratory Tract Viral Load, Comorbidities, Disease Severity, and Outcome of Patients With SARS-CoV-2 Infection. J Infect Dis. 2021;223:1132–8. https://doi.org/10.1093/infdis/jiaa804. Luvira V, Schilling WHK, Jittamala P, Watson JA, Boyd S, Siripoon T, et al. Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial. BMC Infect Dis. 2024;24:89. https://doi.org/10.1186/s12879-023-08835-3. Additional Declarations The authors declare potential competing interests as follows: Competening Interests Dr. Drakoulis has an interest in the intellectual property of DIVIRNAM®. Dr. Tsirikos-Karapanos has an interest in the intellectual property of and direct financial interest in DIVIRNAM®. Dr. Drakoulis’ and Dr. Tsirikos-Karapanos’ contribution in the study was only in the design of the study’s clinical research protocol. All other authors declare no conflict of interest. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9009421","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599257836,"identity":"2284a6e2-5ed3-4855-a846-83f6f0a7f91a","order_by":0,"name":"Helena C. 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Longitudinal Ct values trajectories per group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCt: cycle threshold; SD: standard deviation; ACF: ammonium chloride formulation; VDF: vitamin D formulation; RT-PCR: reverse-transcriptase polymerase chain reaction\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9009421/v1/67b561bddccb0ce2440c5df7.png"},{"id":104178495,"identity":"a7c2b939-b80e-4319-aa6f-7919f63937ec","added_by":"auto","created_at":"2026-03-08 16:55:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2. Clearance by Day 10–11 (Ct≥40): ACF vs VDF with 95% CI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCt: cycle threshold; ACF: ammonium chloride formulation; VDF: vitamin D formulation; vs: versus; 95% CI: confidence interval\u003c/p\u003e","description":"","filename":"Figure2clean.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9009421/v1/3443bc564254a592257ec4cb.jpg"},{"id":104409004,"identity":"6f821552-fb40-4222-8f62-691d623bfa95","added_by":"auto","created_at":"2026-03-11 12:43:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1340259,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9009421/v1/c76406b5-7850-495d-b333-90fc6b20be46.pdf"},{"id":104178493,"identity":"cc7c9af5-a5d9-4fef-a218-0cfc531328c3","added_by":"auto","created_at":"2026-03-08 16:55:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":240745,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9009421/v1/e771a67e53cdadf5c645133c.docx"}],"financialInterests":"The authors declare potential competing interests as follows: Competening Interests\nDr. Drakoulis has an interest in the intellectual property of DIVIRNAM®. Dr. Tsirikos-Karapanos has an interest in the intellectual property of and direct financial interest in DIVIRNAM®. Dr. Drakoulis’ and Dr. Tsirikos-Karapanos’ contribution in the study was only in the design of the study’s clinical research protocol. All other authors declare no conflict of interest. \n","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffectiveness of a sustained-release ammonium chloride formulation in reducing the viral load of patients with COVID-19 or influenza: A prospective, randomized, double-blind, placebo-controlled study\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRespiratory viral infections are a leading cause of morbidity, mortality, and healthcare resources consumption globally. The World Health Organization estimates that seasonal influenza (hereafter referred to as influenza) affects up to 20% of the global population and causes up to 650,000 deaths due to respiratory diseases annually [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Almost six years after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the evolution of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2 remains a significant cause of morbidity and mortality globally, mainly among older adults and individuals with comorbidities, due to the emergence of new variants that escape vaccine-derived immunity or immunity due to past infections [2]. A meta-analysis of 21 studies conducted over two decades in seven high-income countries (France, Germany, Italy, Japan, Spain, the United Kingdom, and the United States) estimated that respiratory syncytial virus (RSV) will cause 5.7\u0026nbsp;million infections, 510,000 hospitalizations, and 37,000 deaths among adults\u0026thinsp;\u0026ge;\u0026thinsp;60 years in 2025 alone [3]. In addition to respiratory viruses, other RNA viruses are major causes of morbidity and mortality globally such as hepatitis A, C, D and E viruses [4]. Therefore, treatments to reduce the overall burden of RNA-viral infections are urgently needed.\u003c/p\u003e \u003cp\u003eAmmonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), a lysosomotropic agent, demonstrates at specific concentrations a broad virostatic action through inhibition of virus uncoating and diminution or even inhibition of virus replication, and therefore delay of the progression of infection by various RNA viruses, including human and avian influenza A viruses, coronaviruses, and hepatitis A and C viruses [5\u0026ndash;12]. This effect is achieved through a temporary and reversible increase of the pH of intracellular lysosomes which in turn prevents the fusion between viruses and the lysosomes\u0026rsquo; membranes [5\u0026ndash;12]. In vitro studies have demonstrated the importance of endosomal acidification for SARS-CoV-2 entry and infection [5]. Moreover, SARS-CoV-2-infected mice models have shown that ammonium chloride also possesses significant anti-SARS-CoV-2 activity, through inhibition of lysosomal acidification and therefore intracellular virus replication and alleviation of inflammation and infiltration in pulmonary tissues [13]. In addition, a model using data from 51 countries or regions across Europe collected as of 26 April 2020 confirmed the possible association between consumption of foods containing ammonium chloride and lower death rates from COVID-19 [14]. A randomized-controlled trial conducted in 2020 found that the time of recovery and SARS-CoV-2 loads were significantly reduced among COVID-19 patients who had received an ammonium chloride solution (125 mg/5 ml daily per os) compared with the placebo group (60 patients in each group), by odds ratios of 1.8 [95% confidence interval (CI), 1.15\u0026ndash;2.83; p-value\u0026thinsp;=\u0026thinsp;0.01] and 7.90 (95% CI, 1.62\u0026ndash;14.17; p-value\u0026thinsp;=\u0026thinsp;0.014), respectively [15].\u003c/p\u003e \u003cp\u003eThe current study aimed to estimate the effectiveness of a sustained-release dietary supplement formulation containing ammonium chloride and vitamin D in reducing the viral loads of patients with COVID-19, influenza or RSV infection compared with the receipt of a vitamin D formulation only (placebo).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSetting\u003c/h2\u003e \u003cp\u003eThis was a prospective, randomized, double-blind, placebo-controlled study. The study was conducted from September 1, 2024, to April 30, 2025, corresponding to the last follow-up visit of the final enrolled participant, at Sotiria Hospital for Respiratory and Thoracic Diseases and at En Ygeia Clinic in Athens, Greece. The study has been retrospectively registered in ClinicalTrials.gov ( identifier: NCT07254052, registered on 22-10-2025 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/study/NCT07254052\u003c/span\u003e\u003cspan address=\"https://clinicaltrials.gov/study/NCT07254052\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) and the ISRCTN registry (registration number: ISRCTN48259966, registered on 27-11-2025 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.isrctn.com/ISRCTN48259966\u003c/span\u003e\u003cspan address=\"https://www.isrctn.com/ISRCTN48259966\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Population\u003c/h3\u003e\n\u003cp\u003ePatients with influenza-like illness (ILI) or acute respiratory infection (ARI) who attended the Infectious Diseases Unit of Sotiria Hospital for Respiratory and Thoracic Diseases or the Internal Medicine Outpatient Clinic of En Ygeia Clinic during the study period were eligible for the study. Eligible patients were tested with a combo rapid antigen test for SARS-CoV-2, influenza A and B, and RSV (CorDX, Inc., San Diego, CA, USA). Inclusion criteria were an age\u0026thinsp;\u0026ge;\u0026thinsp;18 years, laboratory-confirmed SARS-CoV-2, influenza virus or RSV infection, and written informed consent. Patients were excluded if they were \u0026lt;\u0026thinsp;18 years old, pregnant or lactating, had an organ transplantation, a frailty score\u0026thinsp;\u0026ge;\u0026thinsp;5, symptoms for \u0026ge;\u0026thinsp;3\u0026ndash;5 days, or a documented allergy to ammonium chloride, vitamin D or any excipient of the administered formulations. Participation in the study was discontinued when one of the following occurred: intolerance of the administered formulations, non-compliance with study requirements or withdrawal of informed consent. The authors did not participate in patients\u0026rsquo; management decisions.\u003c/p\u003e\n\u003ch3\u003eLaboratory testing\u003c/h3\u003e\n\u003cp\u003eNasopharyngeal swab samples were collected from each participant at three timepoints after diagnosis: Day 1 (baseline), Day 3\u0026ndash;5 (intermediate), and Day 10\u0026ndash;11 (final). All samples were transferred under ice-packed conditions to the Laboratory of the Research Group of Clinical Pharmacology and Pharmacogenomics at the Faculty of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens immediately after sampling, where they were kept at -20\u0026deg;C for a maximum of three days until processing.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using the ZYBIO Nucleic Acid Extraction Kit (B-200-20) on the Zybio EXM3000 system, according to the manufacturer\u0026rsquo;s protocol. Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using the Zybio SARS-CoV-2 \u0026amp; Influenza A/B Reagent Kit (catalogue number: SC2FA/B-96) on the Zybio ZIP 96V thermocycler. All primers required for the qPCR were included in this kit for clarity to the reader. This multiplex assay qualitatively detects SARS-CoV-2 RNA (N and S genes), influenza A (M gene), and influenza B (HA gene) through a one-step RT-PCR protocol. Cycle threshold (Ct) values were recorded for each sample. Each PCR run included both positive and negative controls, and at least one randomly selected sample from a prior run was included as a technical replicate for quality control purposes. According to the manufacturer\u0026rsquo;s instructions, samples that did not yield a valid Ct value within 45 amplification cycles were considered negative. Non-detects were therefore censored at the assay limit (Ct\u0026thinsp;=\u0026thinsp;45), and samples without a valid Ct value at any time point were excluded from the analysis for the corresponding reagent. All PCR reactions were performed in a blinded manner with regard to group allocation, and blinding was maintained across laboratory technicians, case report form (CRF) handlers, and physicians until completion of the statistical analysis.\u003c/p\u003e\n\u003ch3\u003eStudy procedures\u003c/h3\u003e\n\u003cp\u003eParticipants were enrolled at Sotiria General Hospital outpatient clinic or the private outpatient clinic \u0026ldquo;En Ygeia\u0026rdquo;. Following provision of written informed consent, the treating physician dispensed the next available study medication card sequentially from the study supply box, without interfering with any concomitant treatment. The study tablets were manufactured by Help Pharmaceuticals, which also packaged the medication cards, prepared the dispensing order to ensure balanced allocation, and retained exclusive custody of the lot-to-treatment correspondence code until completion of the study. After baseline (Day 1) nasopharyngeal swab samples were collected, patients were randomly allocated in 1:1 ratio to receive either an enteric-coated sustained-release dietary supplement formulation containing 500 mg ammonium chloride, 2,000 IU vitamin D, and excipients (ACF group) or a physically identical sustained-release dietary supplement formulation containing the same amount of vitamin D (2,000 IU) and the same excipients only (VDF, placebo group). Randomization was implemented through the dispensing order of pre-packaged study medication cards prepared in advance by the manufacturer, ensuring an approximately balanced 1:1 allocation and preventing prediction of upcoming assignments by the enrolling clinician. ACF and VDF were administered twice daily (one tablet every 12 hours) for 10 consecutive days, alongside standard symptomatic supportive care. Both formulations were visually indistinguishable and with identical appearance, packaging, and labeling, each assigned a distinct LOT code corresponding to the respective formulation. The correspondence between LOT code and treatment assignment (ACF vs VDF) was maintained in a blinded treatment code held exclusively by the manufacturer and was not accessible to participants, enrolling clinicians, investigators, laboratory personnel, outcome assessors, or statisticians, until completion of the statistical analysis. The sustained-release ammonium chloride dietary supplement formulation (DIVIRNAM\u0026reg;, Metron Nutraceuticals, Cleveland, Ohio, USA) has been described in the United States Patent Application Publication No.: 2022/0160757 (Priority Date: November 20, 2020; Publication Date: May 26, 2022) and the International Patent Application Publication WO 2022/109393 (Priority Date: November 20, 2020; Publication Date: May 27, 2022). Ammonium chloride has been designated GRAS (Generally Recognized As Safe;) status by the Food and Drug Administration of the United States (21CFR 184.1138), and it is already under registration in the European Union. Both ACF and VDF dietary supplement formulations have been notified to the Hellenic National Organization for Medicines (EOF).\u003c/p\u003e\n\u003ch3\u003eData collection\u003c/h3\u003e\n\u003cp\u003ePatients were prospectively followed for a period of up to 30 days following laboratory-confirmed diagnosis. Data collection was conducted in real time by trained healthcare professionals through structured patient interviews at the time of enrolment and comprehensive review of medical records throughout the follow-up period. For each participant, a standardized CRF was utilized to systematically document the following information:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003edemographic data: age, sex\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003emedical history: presence of comorbid conditions, including chronic cardiovascular disease, chronic pulmonary disease, diabetes mellitus, chronic kidney disease, chronic neuromuscular disorders, obesity, malignancy, and immunosuppression\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003einfection and vaccination history: prior COVID-19 infections; COVID-19 vaccination status for the 2024\u0026ndash;2025 season; influenza vaccination status for the 2024\u0026ndash;2025 season\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003elaboratory-confirmed diagnosis: SARS-CoV-2, influenza or RSV\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eclinical information: date of diagnosis, presenting symptoms, and subsequent clinical course, including hospitalization, need for supplemental oxygen, admission to an intensive care unit (ICU), and use of invasive mechanical ventilation\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eclinical outcome: status at timepoint 2 (Day 3\u0026ndash;5), timepoint 3 (Day 10\u0026ndash;11), and 30 days after diagnosis.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOutcomes\u003c/h2\u003e \u003cp\u003eThe prespecified primary outcome was the reduction of the baseline viral load, assessed by cycle threshold (Ct) values measured by RT-PCR at Day 1, Day 3\u0026ndash;5, and Day 10\u0026ndash;11. Viral load was analysed using adjusted mean differences between groups and longitudinal comparisons across time points. Secondary outcomes included the severity of symptoms, documented prospectively over a 30-day follow-up period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDefinitions\u003c/h3\u003e\n\u003cp\u003eILI was defined as the sudden onset of at least one systemic symptom (fever, malaise, headache, myalgia) and at least one respiratory symptom (cough, sore throat, shortness of breath) [16]. ARI was defined as the sudden onset of at least one of the following symptoms: cough, sore throat, shortness of breath, and coryza [16]. COVID-19 was defined as a case with symptoms compatible with COVID-19 and laboratory-confirmed SARS-CoV-2 infection. Influenza was defined as a case with symptoms compatible with influenza and a laboratory-confirmed influenza infection. RSV infection was defined as a case with symptoms compatible with RSV respiratory illness and laboratory-confirmed RSV infection. Mild illness was defined as the presence of various signs and symptoms (e.g., fever, cough, sore throat, malaise, headache, myalgia, nausea, vomiting, diarrhoea) but no shortness of breath, dyspnoea or abnormal findings on chest imaging. Severe illness was defined as increasing needs for supplemental oxygen, admission to ICU, invasive mechanical ventilation and/or death. Death was defined as 30-day crude mortality. Virus clearance was defined as a Ct value of \u0026ge;\u0026thinsp;40.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eBaseline characteristics were statistically compared using independent-samples t-tests or Mann\u0026ndash;Whitney U tests for continuous variables, and Fisher's exact test for categorical variables. Differences in Ct values between groups were assessed using parametric [Welch\u0026rsquo;s unequal-variance t-tests and analysis of covariance (ANCOVA)] and non-parametric (Mann\u0026ndash;Whitney U tests, Hodges\u0026ndash;Lehmann estimators and Cliff\u0026rsquo;s δ effect sizes) approaches [17]. The primary endpoint was analyzed using ANCOVA with baseline Ct as the covariate. Longitudinal trajectories were further evaluated using linear mixed-effects models for repeated measures (SPSS - MMRM), including fixed effects for group and visit, as well as their interaction. A random intercept was included for each subject, and the Kenward\u0026ndash;Roger method was used to estimate the denominator degrees of freedom [18]. Time-to-viral clearance was examined using Kaplan\u0026ndash;Meier survival curves and discrete-time complementary log\u0026ndash;log models. Robustness was assessed using HC3 heteroskedasticity-consistent errors, robust regression, permutation testing with Freedman\u0026ndash;Lane residualisation, bootstrap resampling and leave-one-out diagnostics [19\u0026ndash;21]. All assumptions (normality, homoscedasticity and influence) were systematically checked. Analyses were conducted using IBM SPSS Statistics (version 26, IBM Corp., Armonk, NY, USA), Python (SciPy version 1.9) and GPower version 3.1. Statistical terms and abbreviations are presented in Appendix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEthical issues\u003c/h2\u003e \u003cp\u003eThe study protocol was approved by the Scientific Council of Sotiria Hospital for Respiratory and Thoracic Diseases (No. 14328/17-05-2024). All participants were informed about the study and were enrolled following written informed consent. The study was conducted in accordance with the Declaration of Helsinki. Data was managed in compliance with European Union General Data Protection Regulation standards.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 32 participants were analyzed, with 16 assigned to the ACF group and 16 assigned to the VDF group. Table 1 shows the characteristics of patients stratified per group. The mean age was 58.1 \u0026plusmn; 17.6 years in the ACF group and 60.7 \u0026plusmn; 17.2 years in the VDF group. ACF group patients more frequently had at least one comorbidity compared with VDF-treated patients (68.8% versus 25.0%; p-value=0.02). Overall, chronic cardiovascular disease, chronic neuromuscular disease, and diabetes mellitus were the prevalent comorbidities among studied patients (6, 5, and 4 patients each).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOf the 32 patients studied, 28 had COVID-19 (14 in each group) and 4 had influenza A or B (2 in each group). No patient had RSV infection. All had a mild illness. No serious adverse events, treatment discontinuations, or safety concerns related to the study interventions were reported during the study period. No patient developed severe disease, was hospitalized, received oxygen, or died by 30 days after diagnosis. In general, the two groups did not differ regarding mean and median age, sex, past SARS-CoV-2 infection, and COVID-19 and influenza vaccination status for the 2024-2025 season. The only statistically significant difference was the prevalence of comorbidities, with the ACF group presenting a higher rate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cu\u003eTable 1. Baseline characteristics of participating patients by treatment group___\u003c/u\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1772817092.png\" style=\"width: 582px;\"\u003e\u003c/p\u003e\n\u003cp\u003eACF: ammonium chloride formulation; VDF: vitamin D formulation; SD: standard deviation; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; COVID-19: coronavirus disease 2019\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*11 ACF-treated patients had a total of 17 comorbidities: chronic cardiovascular disease: 5, \u0026nbsp;chronic neuromuscular disease: 5, diabetes mellitus: 2, malignancy: 2, immunosuppression: 2, chronic renal disease: 1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e**4 DVF-treated patients had 4 comorbidities: diabetes mellitus: 2; chronic cardiovascular disease: 1 ; obesity: 1 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e***for the 2024-2025 season\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1. L\u003c/strong\u003e\u003cstrong\u003eongitudinal Ct values trajectories per group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCt: cycle threshold; SD: standard deviation; ACF: ammonium chloride formulation; VDF: vitamin D formulation; RT-PCR: reverse-transcriptase polymerase chain reaction \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll 32 participants completed measurements on the three scheduled visits.\u0026nbsp;Figure 1 summarizes longitudinal Ct trajectories per group. On Day 1, the mean Ct values were 22.49 \u0026plusmn; 6.95 for the ACF group and 21.01 \u0026plusmn; 8.29 for the VDF group. By Day 3\u0026ndash;5, the mean Ct increased to 33.20 \u0026plusmn; 8.10 in the ACF group and to 30.82 \u0026plusmn; 5.68 in the VDF group. At Day 10\u0026ndash;11, the difference widened, with the ACF group showing a mean Ct of 43.66 \u0026plusmn; 2.57 compared to a mean of 40.21 \u0026plusmn; 5.47 in the VDF group. The increasing Ct values over time in both groups is consistent with declining viral load, with the greatest separation observed at Day 10\u0026ndash;11 in favor of ACF. These preliminary findings motivated baseline-adjusted modeling and censoring-aware analyses presented below.\u003c/p\u003e\n\u003cp\u003eANCOVA was conducted with treatment group as the main factor and baseline Ct as covariate. All 32 participants were included under the intention-to-treat principle. At Day 10\u0026ndash;11, the adjusted mean difference (ACF \u0026ndash; VDF) was +3.12 cycles (95% CI: 0.22\u0026ndash;6.02), corresponding to a large, standardized effect size (Cohen\u0026rsquo;s d \u0026asymp; 0.81). The two-sided p-value was 0.036. In summary, the ACF formulation was associated with a statistically significant increase in Ct at Day 10\u0026ndash;11 compared with VDF, indicating reduced viral load.\u0026nbsp;Results from the MMRM analysis were consistent with the ANCOVA findings, confirming the longitudinal pattern of increasing Ct values and the treatment effect at Day 10\u0026ndash;11 (Figure S1).\u003c/p\u003e\n\u003cp\u003eTime-to-viral clearance was assessed by defining the first participant visit at which Ct \u0026ge; 40 as the clearance event, with participants not cleared by Day 10\u0026ndash;11 censored at that visit. The Kaplan Meier analysis indicated faster clearance in the ACF group compared to VDF (log-rank p-value = 0.016). By Day 10\u0026ndash;11, clearance had been achieved in 14 of 16 ACF participants (87.5%) versus 8 of 16 VDF participants (50.0%). Effect measures derived at Day 10\u0026ndash;11 showed a risk difference of +37.5 percentage points (95% CI: \u0026ndash;8.0-68.5), a relative risk of 1.75 (95% CI: 1.04\u0026ndash;2.95), and an odds ratio of 7.00 (95% CI: 1.18\u0026ndash;41.36). These results are detailed in Table 2 and illustrated in Figure 2. Among the four influenza cases (n=2 per arm), Ct patterns were descriptively consistent with faster clearance in the ACF group; however, the influenza sample was too small for inference.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eViral clearance by Day 10\u0026ndash;11 and effect measures (ITT*, Ct\u0026ge;40)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasure\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEstimate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eCleared \u0026mdash; ACF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e14/16 = 87.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e[64.0%, 96.5%] (Wilson)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eCleared \u0026mdash; VDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e8/16 = 50.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e[28.0%, 72.0%] (Wilson)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eRisk difference (ACF\u0026minus;VDF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e0.375 (37.5 pp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e[\u0026minus;0.080, 0.685]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eRelative risk (ACF vs VDF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e[1.04, 2.95]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eOdds ratio (ACF vs VDF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e7.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e[1.18, 41.36]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eNNT**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6495%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.1134%;\"\u003e\n \u003cp\u003e[1.5, \u0026infin;]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eITT: Intention-to-Treat ; Ct: cycle threshold; ACF: ammonium chloride formulation; VDF: vitamin D formulation; CI: confidence interval; NNT: Number Needed to Treat\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*ITT: Intention-to-Treat, an analysis approach including all randomized participants in their originally assigned groups, regardless of adherence or protocol deviations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e**NNT: Number Needed to Treat, the reciprocal of the absolute risk reduction, indicating how many patients must be treated with ACF instead of VDF for one additional patient to achieve viral clearance by Day 10\u0026ndash;11.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2. Clearance by Day 10\u0026ndash;11 (Ct\u0026ge;40): ACF vs VDF with 95% CI \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCt: cycle threshold; ACF: ammonium chloride formulation; VDF: vitamin D formulation; vs: versus; 95% CI: confidence interval \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the robustness of the Day 10\u0026ndash;11 findings, we conducted a series of sensitivity analyses using alternative specifications and inference frameworks. These included robust standard errors, robust regression, permutation testing, bootstrap resampling, and leave-one-out diagnostics. The sensitivity analyses demonstrate that the observed effect of ACF on Day 10-11 is robust to heteroskedasticity, outliers, and small-sample distribution concerns. These results are summarized in Table S1 and Figure S2. Lastly, Table 3 compares the estimated differences between the ACF and VDF groups across multiple statistical approaches. Across all approaches, the Day 10\u0026ndash;11 group effect consistently ranged from +2.6 to +3.1 cycles, with p-values around 0.03\u0026ndash;0.04. The convergence of results across robust parametric and non-parametric frameworks increases confidence in the primary ANCOVA/MMRM signal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Estimated differences between the ACF and VDF groups across multiple statistical approaches \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEffect size / Difference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignificance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eANCOVA (Day 10\u0026ndash;11, adjusted)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e+3.12 Ct (Cohen\u0026rsquo;s d \u0026asymp; 0.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003ep-value = 0.036 (nominal)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eq-value = 0.098 (FDR)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eMMRM (Day 10\u0026ndash;11 LS-means)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e+3.1 Ct (consistent with ANCOVA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003ep-value \u0026asymp; 0.04 (nominal) q-value \u0026asymp; 0.10 (FDR)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eUnadjusted Welch t-test (Day 10\u0026ndash;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e+3.45 Ct (Hedges\u0026rsquo; g \u0026asymp; 0.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003ep-value = 0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eNon-parametric Hodges\u0026ndash;Lehmann (Day 10\u0026ndash;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e+3.45 Ct (median difference)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e95% CI: 0.45 - 6.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eTime-to-clearance (Day 10\u0026ndash;11, risk difference)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e+37.5 percentage points\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e95% CI: \u0026ndash;8.0 - 68.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eTime-to-clearance (Day 10\u0026ndash;11, OR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eOR: 7.00\u003c/p\u003e\n \u003cp\u003e95% CI: 1.18\u0026ndash;41.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eACF: ammonium chloride formulation; VDF: vitamin D formulation; ANCOVA: analysis of covariance; Ct: cycle threshold; FDR: False Discovery Rate; q: FDR adjusted p-value; MMRM: Mixed-Effects Model for Repeated Measures; LS: Least Squares means; CI: confidence interval; OR: odds ratio \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eq-value: adjusted p-value after controlling for the false discovery rate (FDR)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFDR: False Discovery Rate, a multiple-comparison correction method (e.g., Benjamini\u0026ndash;Hochberg) controlling the expected proportion of false positives.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMMRM: Mixed-Effects Model for Repeated Measures, a longitudinal model that accounts for within-subject correlations across timepoints.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLS: Least Squares means (also called Estimated Marginal Means), model-adjusted group averages derived from MMRM rather than raw arithmetic means.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present prospective, randomized, double-blind, placebo-controlled study indicates that patients who received a sustained-release dietary supplement formulation containing 500 mg ammonium chloride and 2,000 IU vitamin D (ACF) twice daily had statistically significant lower viral loads compared to VDF, placebo-receiving patients. The largest consistent differences in Cts between the ACF and VDF groups were recorded on Day 10\u0026ndash;11 and were confirmed by multiple statistical approaches. Moreover, the time-to-clearance analysis demonstrated that virus clearance occurred more rapidly and more frequently in the ACF group compared with the VDF group, which provides further evidence of antiviral effectiveness. Notably, despite a higher prevalence of baseline comorbidities, ACF-treated patients achieved faster and more frequent viral clearance, suggesting that the treatment effect was not simply due to baseline differences; however, this observation should be interpreted with caution due to the small sample size. To our knowledge, there is only one study published so far, which showed a significant reduction of the time of recovery as well as of SARS-CoV-2 loads among hospitalized COVID-19 patients who received an ammonium chloride solution, but not via a solid, sustained-release ammonium chloride formulation [15]. To our knowledge, there is no published data regarding the administration of ammonium chloride to patients with influenza or other respiratory viruses. Yet, recent studies have highlighted the potential of repurposed agents to exert antiviral activity against respiratory viruses, including influenza A, through inhibition of viral internalization and fusion, supporting the broader concept of non-virus-specific pharmacological interventions [22].\u003c/p\u003e \u003cp\u003eMonitoring of viral loads through PCR has become the standard of care for estimating the effectiveness of treatments for various viral infections [23\u0026ndash;25]. In this context, recent experimental evidence has shown that pharmacological compounds with broad antiviral activity can significantly reduce viral loads and accelerate disease resolution in both SARS-CoV-2 and influenza models. Such findings support the biological plausibility that non-virus-specific agents may enhance viral clearance across multiple RNA viruses [26]. In our study, the gradual but consistent increase in Ct values observed among patients who received the ACF formulation is attributed to the combination of the pharmacodynamic action of ammonium chloride along with the pharmacokinetic properties of the ACF sustained-release formulation. The ACF dietary supplement formulation incorporates a 12-hour release mechanism to support prolonged immunomodulatory activity, therefore enabling the gradual and durable suppression of virus replication compared with the VDF formulation that lacks ammonium chloride. Studies are needed to investigate the effects of ammonium chloride on viral load of other RNA viruses that constitute public health threats, such as influenza A and H5N1.\u003c/p\u003e \u003cp\u003eIn our study, both patients with influenza who received the ammonium chloride formulation noted early viral clearance, as highlighted by Ct values\u0026thinsp;\u0026ge;\u0026thinsp;40 at Day 5. This pattern was not observed in the VDF-receiving influenza cases. While the number of patients with influenza was too small for statistical analysis, the trend could suggest a potentially enhanced antiviral effect of the ACF on influenza virus. Given the very small number of influenza patients, this observation is descriptive and hypothesis-generating rather than confirmatory. This finding may indicate a potentially enhanced antiviral effect of the ACF on influenza virus and can be elucidated through targeted studies in patients with influenza, particularly in light of shifting public health priorities, where COVID-19 concern is gradually subsiding and influenza remains a recurrent burden with significant clinical impact [27].\u003c/p\u003e \u003cp\u003eProspective studies have demonstrated that vitamin D supplementation has a protective effect on ILI [28]. In addition, vitamin D supplementation, particularly when administered early in the course of COVID-19, ameliorated its clinical course and prognosis [29]. The positive effects of vitamin D are mediated by enhancing the innate antiviral immune response, facilitating the induction of antimicrobial peptides/autophagy, and modulating the host reactive hyperinflammatory phase during COVID-19 [29]. However, mixed results from other trials led to a lack of consensus on optimal dosing and timing of vitamin D supplementation in COVID-19. In our study, we assumed that vitamin D served as a booster for the intrinsic benefits conferred by ammonium chloride to the ACF-receiving patients. Both study formulations contained the same dose of vitamin D (2,000 IU twice daily); therefore, any between-group differences observed can be attributed to the presence or absence of ammonium chloride in the formulation.\u003c/p\u003e \u003cp\u003eThe main strength of the present study is its prospective, double-blind, placebo-controlled randomization design. This enabled the repeated nasopharyngeal swab sampling in predefined time points along with the study of the kinetics of Ct values. In addition, data about the clinical course and outcome were collected prospectively.\u003c/p\u003e \u003cp\u003eThe main limitation is the small number of study participants, which explains the wide CIs, and which did not allow the study of ACF on viral loads by virus, age groups, comorbidities, and settings (e.g. hospital versus ambulatory healthcare settings) [30]. Another limitation is that the precise time from the onset of symptoms to treatment was not available; nevertheless, all studied patients were enrolled 3\u0026ndash;5 days after the onset of symptoms. The fact that Ct values concern viral nucleic acid and do not necessarily correspond to infectious virus should also be considered. Indeed, clinical studies evaluating established antivirals, such as favipiravir, have reported variable or inconclusive effects on viral clearance despite antiviral activity, underscoring the complexity of translating virological endpoints into consistent clinical outcomes [31]. Lastly, heterogeneity was present in our study but was adequately addressed through robust inference, and raised confidence regarding the primary ANCOVA results.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings of the present study indicate that a sustained-release ammonium chloride formulation (ACF - DIVIRNAM\u003csup\u003e\u0026reg;\u003c/sup\u003e) administered twice daily significantly reduced the viral load of patients with COVID-19 or influenza compared to placebo-receiving patients. This is attributed to the pharmacodynamic action of ammonium chloride and the pharmacokinetic properties of DIVIRNAM\u003csup\u003e\u0026reg;\u003c/sup\u003e. Larger studies are needed to further investigate the effectiveness of DIVIRNAM\u003csup\u003e\u0026reg;\u003c/sup\u003e in various RNA-viral infections, patient populations, and healthcare or non-healthcare settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eACF (Ammonium Chloride Formulation):\u0026nbsp;\u003c/strong\u003ea novel sustained-release dietary supplement formulation containing 500 mg ammonium chloride and 2,000 IU vitamin D (DIVIRNAM\u0026reg;).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eANCOVA (Analysis of Covariance):\u003c/strong\u003e A general linear model that combines ANOVA and regression, used here to compare cycle threshold (Ct) values between groups while adjusting for baseline Ct.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eARI (Acute Respiratory Infection):\u003c/strong\u003e An acute infection of the upper and/or lower respiratory tract.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eARR (Absolute Risk Reduction):\u003c/strong\u003e The difference in event rates between two groups; used to calculate the NNT.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eBootstrap (Percentile CI):\u003c/strong\u003e A resampling method that estimates confidence intervals by repeatedly drawing samples with replacement from the data.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCI (Confidence Interval):\u003c/strong\u003e A statistical interval estimate that provides a range of values within which the true parameter is expected to lie with a specified probability (commonly 95%).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCliff\u0026rsquo;s \u0026delta;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(delta):\u003c/strong\u003e A non-parametric effect size measure representing the probability that a randomly selected value from one group exceeds a randomly selected value from another group.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCohen\u0026rsquo;s d / Hedges\u0026rsquo; g:\u003c/strong\u003e Standardized effect size measures representing the difference between two means expressed in units of standard deviation.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCRF\u003c/strong\u003e \u003cstrong\u003e(Case report form):\u003c/strong\u003e A standardized document used to record study-related data for each participant in a clinical study.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCt (Cycle Threshold):\u003c/strong\u003e The number of PCR cycles required for the fluorescent signal to cross the detection threshold; inversely proportional to the viral load.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFDR (False Discovery Rate):\u003c/strong\u003e A multiple-comparison correction method (e.g., Benjamini\u0026ndash;Hochberg) that controls the expected proportion of false positives among results declared significant.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eGRAS (Generally Recognized As Safe):\u003c/strong\u003e A regulatory designation indicating that a substance is considered safe for its intended use based on scientific evidence and/or a history of common use in food.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHC3 robust SE (Heteroskedasticity-Consistent Standard Errors, type 3):\u003c/strong\u003e A variance estimator that adjusts for heteroskedasticity and small-sample bias in OLS regression.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHodges\u0026ndash;Lehmann Estimator:\u003c/strong\u003e A non-parametric estimator of the median difference between two groups, robust to non-normal distributions.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eICU (Intensive Care Unit):\u003c/strong\u003e A specialized hospital unit providing intensive monitoring and treatment for critically ill patients.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eILI (Influenza-Like Illness):\u003c/strong\u003e An acute respiratory illness characterized by influenza-like symptoms.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eITT (Intention-to-Treat):\u003c/strong\u003e An analysis principle in which all randomized participants are analyzed in their originally assigned groups, regardless of adherence or protocol deviations.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eKaplan\u0026ndash;Meier Curve:\u003c/strong\u003e A survival analysis method used to estimate the probability of an event (here, viral clearance) over time.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLS (Least Squares Means or Estimated Marginal Means):\u003c/strong\u003e Model-adjusted group averages derived from ANCOVA or MMRM, representing expected values after accounting for covariates, not simple arithmetic means.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMMRM (Mixed-Effects Model for Repeated Measures):\u003c/strong\u003e A longitudinal statistical model that accounts for correlations within subjects across repeated timepoints, including both fixed and random effects.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eNNT (Number Needed to Treat):\u003c/strong\u003e The reciprocal of the absolute risk reduction; indicates how many patients must be treated with ACF instead of VDF for one additional patient to achieve viral clearance by Day 10\u0026ndash;11.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eOLS (Ordinary Least Squares):\u003c/strong\u003e The standard estimation method for linear regression and ANCOVA, minimizing the sum of squared residuals to obtain parameter estimates.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eOR (Odds Ratio):\u003c/strong\u003e A measure of association representing the odds of an event occurring in one group compared to another.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePermutation Test (Freedman\u0026ndash;Lane):\u003c/strong\u003e A non-parametric resampling method for testing significance by permuting residuals, robust to distributional assumptions.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ep-value:\u003c/strong\u003e The probability of observing results as extreme as (or more extreme than) those obtained, under the null hypothesis of no difference.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eq-value:\u003c/strong\u003e The false discovery rate (FDR)-adjusted p-value, representing the minimum FDR at which a particular test result would be considered significant.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eRR (Relative Risk):\u003c/strong\u003e The ratio of the probability of an event in the treatment group to that in the control group.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eVDF (Vitamin D Formulation):\u003c/strong\u003e sustained-release dietary supplement formulation containing 2,000 IU of vitamin D\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Scientific Council of Sotiria Hospital for Respiratory and Thoracic Diseases (No. 14328/17-05-2024), which serves as the institution’s ethics and regulatory review body for clinical research in accordance with Greek national regulations. All participants were informed about the study and were enrolled following written informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePatient consent for publication\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants provided written consent for publication. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are provided in the Supplementary Material (Tables S2–S3). The study protocol and additional information are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompetening Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Drakoulis has an interest in the intellectual property of DIVIRNAM\u003csup\u003e®\u003c/sup\u003e. Dr. Tsirikos-Karapanos has an interest in the intellectual property of and direct financial interest in DIVIRNAM\u003csup\u003e®\u003c/sup\u003e. Dr. Drakoulis’ and Dr. Tsirikos-Karapanos’ contribution in the study was only in the design of the study’s clinical research protocol. All other authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by Metron Nutraceuticals, LLC; Cleveland, OH, USA and HELP SA, Athens, Greece.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors’ contributions \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCΜ contributed to the conceptualization of the study, investigation, formal analysis, and drafted the original manuscript. CC and GP contributed to data curation and investigation, and participated in writing, review, and editing of the manuscript. AT and PX contributed to formal analysis and investigation, and participated in writing, review, and editing. AR performed the statistical analysis and contributed to writing, review, and editing. NS contributed to data curation and investigation, and participated in writing, review, and editing. NTK and ND contributed to the conceptualization of the study and participated in writing, review, and editing. All authors read and approved the final manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to healthcare professionals in participating centers for their support. The opinions presented in this article are those of the authors and do not necessarily represent those of their institutions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBurden of disease. https://www.who.int/teams/global-influenza-programme/surveillance-and-monitoring/burden-of-disease. Accessed 7 Jan 2026.\u003c/li\u003e\n\u003cli\u003eSummary. datadot. https://data.who.int/dashboards/covid19/summary. Accessed 7 Jan 2026.\u003c/li\u003e\n\u003cli\u003eSavic M, Penders Y, Shi T, Branche A, Pir\u0026ccedil;on J-Y. Respiratory syncytial virus disease burden in adults aged 60\u0026thinsp;years and older in high-income countries: A systematic literature review and meta-analysis. Influenza Other Respir Viruses. 2023;17:e13031. https://doi.org/10.1111/irv.13031.\u003c/li\u003e\n\u003cli\u003eCooke GS, Andrieux-Meyer I, Applegate TL, Atun R, Burry JR, Cheinquer H, et al. Accelerating the elimination of viral hepatitis: a Lancet Gastroenterology \u0026amp; Hepatology Commission. Lancet Gastroenterol Hepatol. 2019;4:135\u0026ndash;84. https://doi.org/10.1016/S2468-1253(18)30270-X.\u003c/li\u003e\n\u003cli\u003ePrabhakara C, Godbole R, Sil P, Jahnavi S, Gulzar S-J, Zanten TS van, et al. Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors. PLOS Pathogens. 2021;17:e1009706. https://doi.org/10.1371/journal.ppat.1009706.\u003c/li\u003e\n\u003cli\u003eYoshimura A, Ohnishi S. Uncoating of influenza virus in endosomes. J Virol. 1984;51:497\u0026ndash;504. https://doi.org/10.1128/JVI.51.2.497-504.1984.\u003c/li\u003e\n\u003cli\u003eDi Trani L, Savarino A, Campitelli L, Norelli S, Puzelli S, D\u0026rsquo;Ostilio D, et al. Different pH requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza A viruses. Virol J. 2007;4:39. https://doi.org/10.1186/1743-422X-4-39.\u003c/li\u003e\n\u003cli\u003eSuperti F, Seganti L, Orsi N, Divizia M, Gabrieli R, Pan\u0026agrave; A. The effect of lipophilic amines on the growth of hepatitis A virus in Frp/3 cells. Arch Virol. 1987;96:289\u0026ndash;96. https://doi.org/10.1007/BF01320970.\u003c/li\u003e\n\u003cli\u003eAshfaq UA, Javed T, Rehman S, Nawaz Z, Riazuddin S. Lysosomotropic agents as HCV entry inhibitors. Virol J. 2011;8:163. https://doi.org/10.1186/1743-422X-8-163.\u003c/li\u003e\n\u003cli\u003eHelenius A, Marsh M, White J. Inhibition of Semliki forest virus penetration by lysosomotropic weak bases. J Gen Virol. 1982;58 Pt 1:47\u0026ndash;61. https://doi.org/10.1099/0022-1317-58-1-47.\u003c/li\u003e\n\u003cli\u003eMizzen L, Hilton A, Cheley S, Anderson R. Attenuation of murine coronavirus infection by ammonium chloride. Virology. 1985;142:378\u0026ndash;88. https://doi.org/10.1016/0042-6822(85)90345-9.\u003c/li\u003e\n\u003cli\u003eZeichhardt H, Wetz K, Willingmann P, Habermehl KO. Entry of poliovirus type 1 and Mouse Elberfeld (ME) virus into HEp-2 cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating. J Gen Virol. 1985;66 ( Pt 3):483\u0026ndash;92. https://doi.org/10.1099/0022-1317-66-3-483.\u003c/li\u003e\n\u003cli\u003eShang C, Zhuang X, Zhang H, Li Y, Zhu Y, Lu J, et al. Inhibitors of endosomal acidification suppress SARS-CoV-2 replication and relieve viral pneumonia in hACE2 transgenic mice. Virol J. 2021;18:46. https://doi.org/10.1186/s12985-021-01515-1.\u003c/li\u003e\n\u003cli\u003eHidv\u0026eacute;gi M, Nichelatti M. Bacillus Calmette-Guerin vaccination Policy and Consumption of Ammonium Chloride-Enriched Confectioneries May Be Factors Reducing COVID-19 Death Rates in Europe. Isr Med Assoc J. 2020;22:501\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eSiami Z, Aghajanian S, Mansouri S, Mokhames Z, Pakzad R, Kabir K, et al. Effect of Ammonium Chloride in addition to standard of care in outpatients and hospitalized COVID-19 patients: A randomized clinical trial. Int J Infect Dis. 2021;108:306\u0026ndash;8. https://doi.org/10.1016/j.ijid.2021.04.043.\u003c/li\u003e\n\u003cli\u003eOperational considerations for respiratory virus surveillance in Europe. 2022. https://www.ecdc.europa.eu/en/publications-data/operational-considerations-respiratory-virus-surveillance-europe. Accessed 7 Jan 2026.\u003c/li\u003e\n\u003cli\u003eHess M, Kromrey J. Robust Confidence Intervals for Effect Sizes: A Comparative Study of Cohen\u0026rsquo;s d and Cliff\u0026rsquo;s Delta Under Non-normality and Heterogeneous Variances. Educ Psychol Meas. 2004;:699\u0026ndash;719.\u003c/li\u003e\n\u003cli\u003eKenward MG, Roger JH. An improved approximation to the precision of fixed effects from restricted maximum likelihood. Computational Statistics \u0026amp; Data Analysis. 2009;53:2583\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eMacKinnon JG, White H. Some heteroskedasticity-consistent covariance matrix estimators with improved finite sample properties. Journal of Econometrics. 1985;29:305\u0026ndash;25. https://doi.org/10.1016/0304-4076(85)90158-7.\u003c/li\u003e\n\u003cli\u003eHayes AF, Cai L. Using heteroskedasticity-consistent standard error estimators in OLS regression: an introduction and software implementation. Behav Res Methods. 2007;39:709\u0026ndash;22. https://doi.org/10.3758/bf03192961.\u003c/li\u003e\n\u003cli\u003eAnderson MJ, Robinson J. Permutation Tests for Linear Models. Aus NZ J of Statistics. 2001;43:75\u0026ndash;88. https://doi.org/10.1111/1467-842X.00156.\u003c/li\u003e\n\u003cli\u003eLi Y, Yang S, Jiang F, Luo S, Liang J, Jiang L, et al. Cilnidipine exerts antiviral effects in vitro and in vivo by inhibiting the internalization and fusion of influenza A virus. BMC Med. 2025;23:200. https://doi.org/10.1186/s12916-025-04022-0.\u003c/li\u003e\n\u003cli\u003eBakshi S, Chattopadhyay P, Ahammed M, Das R, Majumdar M, Dutta S, et al. Efficacy of Different Combinations of Direct-Acting Antivirals Against Different Hepatitis C Virus-Infected Population Groups: An Experience in Tertiary Care Hospitals in West Bengal, India. Viruses. 2025;17. https://doi.org/10.3390/v17020269.\u003c/li\u003e\n\u003cli\u003eShaikh SA, Kahn J, Aksentijevic A, Kawewat-Ho P, Bixby A, Rendulic T, et al. A multicenter evaluation of hepatitis B reactivation with and without antiviral prophylaxis after kidney transplantation. Transpl Infect Dis. 2022;24:e13751. https://doi.org/10.1111/tid.13751.\u003c/li\u003e\n\u003cli\u003eFoug\u0026egrave;re Y, Brophy J, Hawkes MT, Lee T, Samson L, Gantt S, et al. Clinical and Immunologic Impact of CMV Coinfection Among Children Living With HIV in Canada. Pediatr Infect Dis J. 2025;44:764\u0026ndash;71. https://doi.org/10.1097/INF.0000000000004811.\u003c/li\u003e\n\u003cli\u003eLuo R, Shen B, Qian B, Fan L, Zhang J, Deng X, et al. Taurultam shows antiviral activity against SARS-CoV-2 and influenza virus. BMC Microbiol. 2025;25:292. https://doi.org/10.1186/s12866-025-03847-2.\u003c/li\u003e\n\u003cli\u003eChristodoulou A, Katsarou M-S, Emmanouil C, Gavrielatos M, Georgiou D, Tsolakou A, et al. A Machine Learning-Based Web Tool for the Severity Prediction of COVID-19. BioTech. 2024;13. https://doi.org/10.3390/biotech13030022.\u003c/li\u003e\n\u003cli\u003eHelmond N van, Brobyn TL, LaRiccia PJ, Cafaro T, Hunter K, Roy S, et al. Vitamin D3 Supplementation at 5000 IU Daily for the Prevention of Influenza-like Illness in Healthcare Workers: A Pragmatic Randomized Clinical Trial. Nutrients. 2022;15. https://doi.org/10.3390/nu15010180.\u003c/li\u003e\n\u003cli\u003eCicero AFG, Fogacci F, Borghi C, Cicero AFG, Fogacci F, Borghi C. Vitamin D Supplementation and COVID-19 Outcomes: Mounting Evidence and Fewer Doubts. Nutrients. 2022;14. https://doi.org/10.3390/nu14173584.\u003c/li\u003e\n\u003cli\u003eMaltezou HC, Raftopoulos V, Vorou R, Papadima K, Mellou K, Spanakis N, et al. Association Between Upper Respiratory Tract Viral Load, Comorbidities, Disease Severity, and Outcome of Patients With SARS-CoV-2 Infection. J Infect Dis. 2021;223:1132\u0026ndash;8. https://doi.org/10.1093/infdis/jiaa804.\u003c/li\u003e\n\u003cli\u003eLuvira V, Schilling WHK, Jittamala P, Watson JA, Boyd S, Siripoon T, et al. Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial. BMC Infect Dis. 2024;24:89. https://doi.org/10.1186/s12879-023-08835-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ammonium chloride, NH4Cl, influenza, SARS-CoV-2, RNA virus infections, virus clearance","lastPublishedDoi":"10.21203/rs.3.rs-9009421/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9009421/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWe estimated the effectiveness of a novel sustained-release dietary supplement formulation containing 500 mg ammonium chloride and 2,000 IU vitamin D (ACF;) in reducing the viral load of patients with COVID-19 or influenza.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this prospective, randomized, double-blind, placebo-controlled, study. Eligible patients with COVID-19 or influenza were randomized to receive ACF twice daily or placebo (2,000 IU vitamin D/twice daily; VDF) for 10 days. Nasopharyngeal swab samples were collected at Day 1, Day 3\u0026ndash;5 and Day 10\u0026ndash;11 and tested for SARS-CoV-2 and influenza via RT-PCR. Cycle threshold (Ct) values were measured. The study has been retrospectively registered in ClinicalTrials.gov (ClinicalTrials.gov identifier: NCT07254052) and the ISRCTN registry (ISRCTN study registration number: ISRCTN48259966).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThirty two patients were studied, 28 with COVID-19 and 4 with influenza. No patient developed severe disease, was hospitalized, or died. Sixteen patients received ACF and 16 VDF (mean age: 58.1 and 60.7 years, respectively; 68.8% and 25% with comorbidities, respectively). On Day 1, the mean Cts were 22.49 in ACF group and 21.01 in VDF group, on Day 3\u0026ndash;5, the mean Cts were 33.20 and 30.82, respectively, and on Day 10\u0026ndash;11, the mean Cts were 43.66 and 40.21, respectively. On Day 10\u0026ndash;11 the adjusted mean difference was +\u0026thinsp;3.12 cycles (95% confidence interval: 0.22\u0026ndash;6.02; p-value\u0026thinsp;=\u0026thinsp;0.036). The Kaplan Meier analysis indicated faster clearance in the ACF group compared to the VDF group (p-value\u0026thinsp;=\u0026thinsp;0.016).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur data indicate that ACF-receiving patients had a statistically significant reduction in viral load compared to placebo-receiving patients. This is attributed to the pharmacodynamic action of ammonium chloride and the pharmacokinetic properties of ACF. Larger studies are needed to further investigate the role of ACF in various RNA-viral infections.\u003c/p\u003e\u003ch2\u003eTrial registration:\u003c/h2\u003e \u003cp\u003eThis study was retrospectively registered in ClinicalTrials.gov (identifier: NCT07254052; registered on 22 October 2025) and in the ISRCTN registry (registration number: ISRCTN48259966; registered on 27 November 2025).\u003c/p\u003e","manuscriptTitle":"Effectiveness of a sustained-release ammonium chloride formulation in reducing the viral load of patients with COVID-19 or influenza: A prospective, randomized, double-blind, placebo-controlled study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 16:55:42","doi":"10.21203/rs.3.rs-9009421/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90a8232c-1eb1-4106-a4a1-07410efe9a29","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63766120,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2026-03-08T16:55:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 16:55:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9009421","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9009421","identity":"rs-9009421","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}