Molnupiravir, Nirmatrelvir/Ritonavir, or Sotrovimab for High-Risk COVID-19 Patients Infected by the Omicron Variant: Hospitalization, Mortality, and Time until Negative Swab Test in Real Life

Background. Several drugs which are easy to administer in outpatient settings have been authorized and endorsed for high-risk COVID-19 patients with mild–moderate disease to prevent hospital admission and death, complementing COVID-19 vaccines. However, the evidence on the efficacy of COVID-19 antivirals during the Omicron wave is scanty or conflicting. Methods. This retrospective controlled study investigated the efficacy of Molnupiravir or Nirmatrelvir/Ritonavir (Paxlovid®) or Sotrovimab against standard of care (controls) on three different endpoints among 386 high-risk COVID-19 outpatients: hospital admission at 30 days; death at 30 days; and time between COVID-19 diagnosis and first negative swab test result. Multivariable logistic regression was employed to investigate the determinants of hospitalization due to COVID-19-associated pneumonia, whereas time to first negative swab test result was investigated by means of multinomial logistic analysis as well as Cox regression analysis. Results. Only 11 patients (overall rate of 2.8%) developed severe COVID-19-associated pneumonia requiring admission to hospital: 8 controls (7.2%); 2 patients on Nirmatrelvir/Ritonavir (2.0%); and 1 on Sotrovimab (1.8%). No patient on Molnupiravir was institutionalized. Compared to controls, hospitalization was less likely for patients on Nirmatrelvir/Ritonavir (aOR = 0.16; 95% CI: 0.03; 0.89) or Molnupiravir (omitted estimate); drug efficacy was 84% for Nirmatrelvir/Ritonavir against 100% for Molnupiravir. Only two patients died of COVID-19 (rate of 0.5%), both were controls, one (a woman aged 96 years) was unvaccinated and the other (a woman aged 72 years) had adequate vaccination status. At Cox regression analysis, the negativization rate was significantly higher in patients treated with both antivirals—Nirmatrelvir/Ritonavir (aHR = 1.68; 95% CI: 1.25; 2.26) or Molnupiravir (aHR = 1.45; 95% CI: 1.08; 1.94). However, COVID-19 vaccination with three (aHR = 2.03; 95% CI: 1.51; 2.73) or four (aHR = 2.48; 95% CI: 1.32; 4.68) doses had a slightly stronger effect size on viral clearance. In contrast, the negativization rate reduced significantly in patients who were immune-depressed (aHR = 0.70; 95% CI: 0.52; 0.93) or those with a Charlson index ≥5 (aHR = 0.63; 0.41; 0.95) or those who had started the respective treatment course 3+ days after COVID-19 diagnosis (aOR = 0.56; 95% CI: 0.38; 0.82). Likewise, at internal analysis (excluding patients on standard of care), patients on Molnupiravir (aHR = 1.74; 95% CI: 1.21; 2.50) or Nirmatrelvir/Ritonavir (aHR = 1.96; 95% CI: 1.32; 2.93) were more likely to turn negative earlier than those on Sotrovimab (reference category). Nonetheless, three (aHR = 1.91; 95% CI: 1.33; 2.74) or four (aHR = 2.20; 95% CI: 1.06; 4.59) doses of COVID-19 vaccine were again associated with a faster negativization rate. Again, the negativization rate was significantly lower if treatment started 3+ days after COVID-19 diagnosis (aHR = 0.54; 95% CI: 0.32; 0.92). Conclusions. Molnupiravir, Nirmatrelvir/Ritonavir, and Sotrovimab were all effective in preventing hospital admission and/or mortality attributable to COVID-19. However, hospitalizations also decreased with higher number of doses of COVID-19 vaccines. Although they are effective against severe disease and mortality, the prescription of COVID-19 antivirals should be carefully scrutinized by double opinion, not only to contain health care costs but also to reduce the risk of generating resistant SARS-CoV-2 strains. Only 64.7% of patients were in fact immunized with 3+ doses of COVID-19 vaccines in the present study. High-risk patients should prioritize COVID-19 vaccination, which is a more cost-effective approach than antivirals against severe SARS-CoV-2 pneumonia. Likewise, although both antivirals, especially Nirmatrelvir/Ritonavir, were more likely than standard of care and Sotrovimab to reduce viral shedding time (VST) in high-risk SARS-CoV-2 patients, vaccination had an independent and stronger effect on viral clearance. However, the effect of antivirals or COVID-19 vaccination on VST should be considered a secondary benefit. Indeed, recommending Nirmatrelvir/Ritonavir in order to control VST in high-risk COVID-19 patients is rather questionable since other cheap, large spectrum and harmless nasal disinfectants such as hypertonic saline solutions are available on the market with proven efficacy in containing VST.


Background
Several drugs which are easy to administer in outpatient settings-antivirals, human monoclonal antibodies, and immunomodulatory agents-have been authorized and recommended for high-risk COVID-19 patients with mild-moderate disease to prevent hospital admission and death, complementing COVID-19 vaccines [1][2][3][4].
In recent months, these drugs have been widely used worldwide for their supposed proven efficacy in real life [5], since they were endorsed by the respective randomized controlled trials (RCTs) to reduce the risk of hospitalization or death at 28-30 days attributable to COVID-19 in patients with risk factors such as age >50 years, immune depression, or other comorbidities [6][7][8][9][10][11][12][13]. However, most pharmaceutical COVID-19 treatments have been ruled out by the emergence of new variants or are way too expensive or impractical to treat large sectors of the general population [14].
The RCTs of these COVID-19 drugs were conducted on unvaccinated high-risk COVID-19 patients before the Omicron transmission period; hence when more virulent viral strains were circulating [6][7][8][9][10][11]. Omicron, classified as a variant of concern (VOC) on 26 November 2021, rapidly displaced previously circulating strains worldwide, dramatically changing the clinical and therapeutic evidence gathered over the previous two years. Omicron is associated with higher transmission rates, less severe disease, lower hospitalization rates, different clinical presentations and aberrant radiological features [15][16][17][18][19]. Whilst Delta VOC exhibited significant tropism for the ACE2-TMPRSS2 pathway associated with lung infection and increased disease severity, the Omicron BA.1 sub-lineage predominantly targets the upper respiratory tract [20].
Among novel human monoclonal antibodies, Sotrovimab gained attention for its neutralizing efficacy in reducing the risk of progression to severe disease in high-risk COVID-19 patients [21]. However, Sotrovimab needs intravenous (i.v.) administration and monitoring in a health care setting and its efficacy against emerging SARS-CoV-2 variants has been questioned [22][23][24][25].
Two oral antivirals-Molnupiravir and Nirmatrelvir/Ritonavir (Paxlovid ® )-reportedly more effective than human monoclonal antibodies and easier to administer than Remdesivir, which requires i.v. administration-were authorized between 2021 and 2022 for high-risk COVID-19 patients [9,26]. Molnupiravir is a ribonucleoside prodrug of Nhydroxycytidine (NHC), which forms after oral ingestion of Molnupiravir and circulates systemically to be phosphorylated intracellularly into NHC triphosphate. SARS-CoV-2 polymerase incorporates NHC triphosphate into the viral RNA, which then misguides the latter viral enzyme to incorporate either guanosine or adenosine during viral replication, heaping mutations into the viral genome eventually rendering SARS-CoV-2 unable to replicate [8]. By contrast, Nirmatrelvir/Ritonavir, a novel orally administered antiviral agent manufactured by Pfizer BionNTech, targets SARS-CoV-2 3-chymotrypsin-like cysteine protease enzyme (Mpro), which is essential for the viral replication cycle [7,14].
In addition to being effective and practical to administer, both Molnupiravir and Nirmatrelvir/Ritonavir significantly reduced the baseline viral load and the time between COVID-19 diagnosis and a negative swab test in the respective RCTs [7,8]. A recent observational multi-centric study conducted in France between 24 January 2022 and 5 May 2022 on 255 high-risk COVID-19 patients, all infected by Omicron BA.1 and BA.2, confirmed the effect of Nirmatrelvir/Ritonavir in reducing time until a negative test result [27]. The latter clearance effect against viral infection was reportedly lower for Molnupiravir in other observational studies [28,29].
Despite being effective against disease progression in the respective phase 3 RCTs and a number of observational studies [8,30], the efficacy of Molnupiravir was reportedly lower than that of Nirmatrelvir/Ritonavir [7,8]. In a prospective multi-centric open-label RCT conducted in the UK between December 2021 and April 2022, there was no evidence that Molnupiravir reduced the risk of hospitalization and death attributable to COVID-19 in high-risk patients [12]. The latter study was carried out on 26,411 community COVID-19 patients, largely vaccinated and aged > 50 years and/or with co-morbidities, who were randomly assigned to Molnupiravir plus standard of care (N = 12,821), standard of care alone (N = 12,962), or other treatments (N = 628). Vaccination uptake with the booster was 92% in the Molnupiravir group versus 93% in the control group. Hospitalizations or deaths at 28 days were observed in 1% (=105/12,529) of patients on Molnupiravir plus standard of care against 1% (=98/12,525) of those on standard of care, for an adjusted odds ratio of 1.06 (95% Bayesian credible interval 0.81; 1.41) [12].
Another retrospective observational study was conducted in Hong Kong between 26 February 2022 and 26 June 2022-during the Omicron sub-variant BA.2.2 wave-on 1,074,856 high-risk COVID-19 outpatients, less than half of whom fully vaccinated, who were treated with either Molnupiravir (N = 5383) or Nirmatrelvir/Ritonavir (N = 6464) and followed up for a median time of 103 and 99 days, respectively. Both antivirals were effective in reducing mortality and disease progression. However, while institutionalization was less likely in patients on Nirmatrelvir/Ritonavir (aHR = 0.76; 95% CI: 0.67; 0.86), there was no evidence of a difference between the patients on Molnupiravir and the controls (HR = 0.98; 95% CI: 0.89; 1.06) [31]. Nevertheless, patients on Molnupiravir were older and less vaccinated in the latter study [31].
Other observational studies did not find any efficacy difference between Molnupiravir or Nirmatrelvir/Ritonavir in terms of hospitalization or mortality rate [29,31]. A recent metanalysis, including 9 RCTs and 30,472 patients with mild-moderate COVID-19, reported mild evidence of lower rates of hospitalization, viral clearance, time until viral clearance, and time until symptoms resolution with Molnupiravir as compared to a placebo or standard of care [32].
However, the use of Molnupiravir in high-risk COVID-19 patients was downgraded with respect to Nirmatrelvir/Ritonavir, and following concerns about its clinical benefits expressed by the European Medicines Agency (EMA) on 24 February 2023, the Italian Medicines Agency (AIFA) eventually decided to suspend its use on 13 March 2023 [33].
The evidence on the efficacy of pharmaceutical interventions against hospitalization or mortality for COVID-19 in outpatients during the Omicron wave is scanty or conflicting; yet, it is difficult to disentangle the protective effect of early administration of these drugs from the effect of pre-existing humoral immunity or milder circulating viral strains. Population immunity can in fact be achieved through repeated natural infection and/or vaccination, which in high-income countries is mainly limited by vaccine hesitancy [34].
In view of the above, we conducted an observational controlled clinical study, investigating the efficacy of Molnupiravir or Nirmatrelvir/Ritonavir or Sotrovimab in reducing the risk of hospitalization and death and time until viral clearance when administered to high-risk COVID-19 outpatients, adjusting for potential confounders. Table 1 presents each treatment (Molnupiravir, Nirmatrelvir/Ritonavir, and Sotrovimab) by explanatory factors, contrasted with standard of care (controls). As can be seen, the distribution or treatments by sex was rather balanced and the mean age of COVID-19 patients was 68.1 ± 16.2 years (median: 72; IQR: 57; 80). Sixty-one percent of patients received a COVID-19 vaccine booster dose, 12.7% were immunized with two doses, and 18.1% were unvaccinated. Overall, 22.5% of patients were either unvaccinated or had received just one dose of vaccine. Patients affected by immune depression accounted for 23.6% (=91/386) of the total, and the percentage of those affected by severe co-morbidities (Charlson index ≥ 5) was 45.6% (=176/386), whereas 35.5% (=137/386) of patients had a moderate Charlson index (3)(4), and 18.9% (=73/386) a mild Charlson index (<3).

Hospitalization and Mortality Attributable to COVID-19
Overall, 3.1% (=12/384) of the patients were either admitted to hospital or died within the 30 days since COVID-19 diagnosis. Table 4 shows the distribution of the 11 hospital admissions attributable to severe COVID-19 pneumonia by explanatory factors. As can be seen, 7 patients were controls, two were treated with Nirmatrelvir/Ritonavir, and one with Sotrovimab. No patient treated with Molnupiravir was admitted to hospital. Compared to controls, hospitalization was less likely in patients on Nirmatrelvir/Ritonavir (aOR = 0.16; 95% CI: 0.03; 0.89) or Molnupiravir (omitted estimate, since zero patients of this group developed severe disease requiring institutionalization). This resulted in an efficacy against severe COVID-19 of 84% (1-aOR = 100%-16%) for Nirmatrelvir/Ritonavir versus 100% (1-aOR = 100%-0%) for Molnupiravir.  Furthermore, zero patients immunized with four doses of COVID-19 vaccine (N = 14) were admitted to hospital for COVID-19-associated pneumonia; yet, the risk of institutionalization also decreased for Sotrovimab treatment or 2-3 doses of COVID-19 vaccine, despite the fact that the numbers involved were too small to achieve enough statistical power. A progressively lower risk of hospital admission with increasing number of days before treatment start likely reflects a higher proportion of controls starting treatment (standard of care) on day 0 (Table 4). Figure 1 plots factors associated with hospitalization due to COVID-19 pneumonia according to results of the above multivariable logistic regression model (Table 4).

Factors
In Model 2, restricting the analysis to patients turning negative 5-9 days after COVID-19 diagnosis, the effect of the two antivirals vanished, whereas the vaccination's effect was reinforced for any dose number, particularly four (aHR = 5.55; 95% CI: 1.58; 19.45), although the estimates for the booster did not reach statistical significance (Table 7). Table 7. Multiple Cox regression model comparing the negativization rate of Nirmatrelvir/Ritonavir or Molnupiravir against Sotrovimab. Estimates expressed as adjusted hazard ratio (aHR) with 95% confidence interval (95% CI). Model 1 fitted to the entire cohort; Model 2 restricted to patients turning negative for SARS-CoV-2 in the first 5-9 days after COVID-19 diagnosis. Significantly lower negativization rates are highlighted in orange; significant higher negativization rates are marked in green.

Main Findings
In this retrospective real-world clinical study on 386 high-risk patients infected by the Omicron variant (mainly BA.1 and BA.2 like), only 11 cases (rate of 2.8%) of severe COVID-19-associated pneumonia requiring admission to hospital were observed. The mean age of the latter 11 institutionalized patients was 74.1 years. Eight (7.2%) of the latter 11 patients were controls, two (1.8%) were treated with Sotrovimab, and two (2.0%) were on Nirmatrelvir/Ritonavir. No patient on Molnupiravir was admitted to hospital. Therefore, patients receiving any of the above three treatment courses exhibited fewer hospitalizations for COVID-19-associated pneumonia with respect to controls; however, due to small numbers involved in the Sotrovimab group, the estimates were significant only for Molnupiravir (100% efficacy) or Nirmatrelvir/Ritonavir (84% efficacy). No adverse effects were reported in relation to any of the three pharmaceutical COVID-19 treatments under investigation.
Only five patients died (rate of 1.3%), but in only two cases death was attributable to COVID-19 (rate of 0.5%). The latter two patients were both controls, females, one 96 years old (unvaccinated for COVID-19) and the other was 72 years old (immunized with the booster dose 161 days before infection; hence, her vaccination status was considered adequate).
In the entire cohort, time between COVID-19 diagnosis and first negative test result ranged between 5 and 67 days (without the exclusion of any potential outlier), with a mean of 12.4 days and a median of 11 days. The median time until first negative swab test was 10 days for patients on Sotrovimab, 8 days for those on Molnupiravir, 7 days for those on Nirmatrelvir/Ritonavir, and 11 days for controls.
Patients on Nirmatrelvir/Ritonavir were more likely than patients on standard of care to turn negative during the first 5-9 days after COVID-19 diagnosis. In particular, according to NNT analysis, four patients had to be treated with Nirmatrelvir/Ritonavir to achieve one negative swab test result within the first 5-9 days after COVID-19 diagnosis. Multinomial logistic regression analysis as well as Cox regression analysis in the entire cohort confirmed higher negativization rates for patients on Nirmatrelvir/Ritonavir. However, COVID-19 vaccination status, particularly 3+ doses, had a more consistent and stronger effect size on negativization against SARS-CoV-2 infection.
At internal Cox regression analysis (excluding patients on standard of care), higher negativization was observed in patients on either antivirals compared to those on Sotrovimab. Nonetheless, 3+ doses of COVID-19 vaccines again exhibited a slightly stronger effect size than the two antivirals. Treatment starting at 3+ days after COVID-19 diagnosis, immune depression and 80+ years of age confirmed to be risk factors for longer viral shedding time (VST). Focusing on patients turning negative for SARS-CoV-2 during the first 5-9 days since COVID-19 diagnosis, the effect of both antivirals vanished, whereas COVID-19 vaccination's reinforced, particularly four vaccine doses.

Hospitalization and Mortality Attributable to COVID-19
In the entire cohort, the rate of hospitalizations due to COVID-19 was 2.8% (1.8% in the Sotrovimab group; 2.0% in patients on Nirmatrelvir/Ritonavir; 0% with Molnupiravir; and 7.2 in patients on standard of care), and the death rate was 0.5% (two cases, both controls). These figures were similar to those of other real-world studies carried out during the Omicron transmission period [14,31,[35][36][37][38]. Moreover, only two deaths attributable to COVID-19 (rate of 0.5%) were observed; both were patients on standard of care (controls). Molnupiravir, Nirmatrelvir/Ritonavir, and Sotrovimab were therefore all effective in preventing hospital admission and/or mortality due to COVID-19.
Although the numbers involved were too small to draw significant estimates, COVID-19 vaccination (2+ doses) was also effective in preventing institutionalization for COVID-19associated pneumonia in the present study. Indeed, none of the 14 patients vaccinated with four doses were hospitalized. Moreover, the institutionalization rate was 2.1% in patients immunized with booster against 5.7% in those unvaccinated.
In an observational study conducted in Israel on 180,351 high-risk COVID-19 patients diagnosed between 1 January 2022 and 28 February 2022 (75.1% adequately vaccinated for COVID-19), though Nirmatrelvir/Ritonavir was associated with a lower rate of hospitalization or death at 28 days (HR = 0.54; 95% CI: 0.39; 0.75), adequate COVID-19 vaccination status had a stronger effect size than antiviral treatment on severe COVID-19 or associated mortality (HR = 0.20; 95% CI: 0.17; 0.22]) [14]. In line with other studies, the authors of the latter investigation concluded that COVID-19 vaccination had proven to be the most cost-effective approach to protect vulnerable patients from hospital admission and death due to COVID-19 [14].
In the present study, the efficacy estimates of the three pharmaceutical treatments under investigation were fairly in line with the respective phase 3 RCTs, although latter experimental studies were conducted on unvaccinated high-risk COVID-19 patients be-fore the Omicron transmission period, when more virulent SARS-CoV-2 variants were circulating [6][7][8].
The evidence on the comparative efficacy between the three treatments was rather conflicting in some subsequent observational studies, apart from the already-mentioned UK prospective open-label RCTs on 26,411 high-risk COVID-19 patients assessing the efficacy of Molnupiravir between December 2021 and April 2022 [12] and the large Hong Kong study on 1,074,856 COVID-19 outpatients receiving either Molnupiravir (N = 5383) or Nirmatrelvir/Ritonavir (N = 6464) between 26 February 2022 and 26 June 2022 [31].

Time between COVID-19 Diagnosis and First Negative Swab Test
The present study found an earlier negativization of SARS-CoV-2 infection in patients treated with both antivirals, particularly Nirmatrelvir/Ritonavir. However, earlier negativization was also consistently observed for increasing number of doses of COVID-19 vaccine, particularly 3+ doses. The effect of both antivirals and vaccination on the negativization rate was observed during the first 5-9 days after COVID-19 diagnosis, waning afterwards. The latter interval fairly corresponds to the environmental temporal persistence of SARS-CoV-2 outside cells. Thereafter, the virus present in the nasal cavity and unable to replicate inside mucosal cells protected by antiviral treatment or pre-existing humoral immunity will progressively extinguish, also for the local response mounted by nasal mucosa of immunocompetent patients. However, patients affected by severe comorbidities or immune depression may struggle to clear SARS-CoV-2 even after 15+ days since infection, as found in the present study. A higher prevalence of immune depression and a Charlson score ≥5 for patients on Sotrovimab may at least partly explain a delayed negativization of this group compared to patients on standard of care, not to mention that the former group started the respective treatment course later, after a median time of 2 days after COVID-19 diagnosis.
Detection and quantification of SARS-CoV-2 load in nasopharyngeal swabs by RT-PCR was a secondary endpoint in the phase 3 RCT of Nirmatrelvir/Ritonavir [7]. Nasopharyngeal or nasal swabs were collected on day 1 (baseline) and days 3, 5, 10, and 14 after COVID-19 diagnosis in the latter experimental study [7]. Adjusting for baseline viral load, serology status and geographic region, the viral load at day 5 was significantly lower in patients on Nirmatrelvir/Ritonavir, by an adjusted mean of an additional 0.868 ± 0.105 log10 copies per milliliter (95% CI: -1.074; -0.6615; p < 0.001) if treatment started within 3 days after symptoms onset-a decrease in viral load by a factor of 10 compared to the placebo-and 0.695 ± 0.085 log10 copies per milliliter (95% CI: -0.861; -0.530; p < 0.001) if treatment was started within 5 days after symptoms onset. The latter results were also confirmed for patients receiving or assigned to receive treatment with human monoclonal antibodies [7].
Patients on Molnupiravir also exhibited a stronger reduction in viral load from the baseline levels at days 3, 5, and 10 compared to the controls in the respective phase 3 RCT [8]. Likewise, in the above systematic analysis on nine RCTs and 90,472 COVID-19 patients with mild-moderate disease, Molnupiravir was associated with shorter time through viral clearance (mean difference = −1.81 days, 95% CI: −3.31; −0.31) as well as increased rate of viral clearance at 7 days (RR = 3.47, 95% CI: 2.43; 4.96) [32].
The difference in the median time until a negative swab test between the above French multi-centric investigation (4 days) and the present study (7 days) may be explained by differences in the two populations, especially with regard to COVID-19 vaccination coverage. The proportion of patients immunized with 3+ doses of COVID-19 vaccines was 80% in the French study against 64.7% in the present investigation, where 18% of the patients were unvaccinated, 35.8% had an inadequate vaccination status, and 12.9% were immunized with just two doses. Considering only patients on Nirmatrelvir/Ritonavir, the latter percentage increased to 87% in the French study against 67.6% in the present study [27].
In a further observational study by the University of Naples (southern Italy) on 257 high-risk COVID-19 patients diagnosed between 18 February 2022 and 30 June 2022, the median time until a negative swab test was 8 days among 146 (56.8%) patients on Nirmatrelvir/Ritonavir against 10 days in 111 (43.2%) patients on Molnupiravir (p < 0.01); these estimates are in line with those of the present study [28]. The time until a negative swab test was 8 days among the 247 (96.1%) patients vaccinated with at least two doses versus 11 days in the 10 patients who were unvaccinated or immunized with only one dose (p = 0.306). The latter study reported only crude comparisons, though without controlling for potential confounders [28].
Virtually all authorized COVID-19 vaccines provide more protection against death and severe disease than asymptomatic infection or mild/moderate disease [31]. Nevertheless, the present study confirmed a consistent and stronger effect of COVID-19 vaccination in reducing VST compared to both antivirals under investigation. Recommending antivirals such as Nirmatrelvir/Ritonavir to stop VST in high-risk COVID-19 patients seems questionable, not only efficacy-wise but also from a cost-effectiveness perspective, considering the current cost of a Paxlovid ® treatment cycle (USD 530) compared to one dose of COVID-19 vaccine [39], albeit the current price of m-RNA COVID-19 vaccines is expected to be raised to USD 130 per single dose in the USA [40].
Furthermore, there is reasonable concern that antivirals may not remain effective with a rapidly evolving virus, thereby increasing the risk of generating an epidemic of drug resistance in the case of their indiscriminate use to control VST in high-risk COVID-19 patients. Nirmatrelvir/Ritonavir targets the SARS-CoV-2 3-chymotrypsin-like cysteine protease enzyme (Mpro), an essential protein conserved across various coronaviruses. The inhibitory effect of Nirmatrelvir/Ritonavir on the replication of various coronaviruses in vitro has been considered a barrier against antiviral resistance in newly emerging SARS-CoV-2 strains [7]. However, despite reassurances from the industry [7,36,41], in vitro experiments reported that SARS-CoV-2 resistance to Nirmatrelvir readily develops via multiple pathways [42], and several drug-resistant hot spots warranting close monitoring for possible clinical evidence of Paxlovid ® resistance have already been identified [43][44][45].
The issue of resistance also applies to human monoclonal antibodies such as Sotrovimab, which maintains neutralizing activity by targeting a conserved SARS-CoV-2 epitope outside the rapidly evolving domain interacting with the respective ACE-2 receptor [21]. As it is effective against all sarbecoviruses, it has been speculated that Sotrovimab, whose parental form S309 was isolated from a SARS-CoV-1 patient, "would target a highly conserved epitope that would be functionally retained as SARS-CoV-2 evolves" [21]. Nevertheless, the potential for a reduced efficacy of Sotrovimab against emerging SARS-CoV-2 variants has not been ruled out either [22][23][24][25][46][47][48]. Furthermore, there is theoretical concern about rapid SARS-CoV-2 sub-lineage evolution in the so-called "human culture medium" [49].
Other cheap, large spectrum, harmless agents including hypertonic saline solutions are available on the market as nasal disinfectants to effectively reduce VST in COVID-19 patients [50][51][52][53][54][55][56][57]. Hypertonic saline solutions inhibited SARS-CoV-2 replication in vitro yet stimulate the local nasal mucosa to produce hypochlorous acid (HOCl), the active principle of common bleach, a large spectrum agent recommended by the US Environmental Protection Agency for hand washing and fomites disinfection, irrespective of the emerging SARS-CoV-2 variants [58]. In a recent controlled clinical trial on low-risk COVID-19 patients with mild-moderate disease, daily treatment with hypertonic saline solutions sprayed inside the nasal cavity 3 times/day was the only factor independently associated with reduced VST during the first five days after COVID-19 diagnosis [50]

Strengths and Weaknesses
This observational, controlled real-world study assessed the efficacy of three different treatment courses on three separate endpoints among 386 high-risk COVID-19 patients, adjusting for a number of potential confounders. In contrast, some RCTs or observational studies estimated treatment efficacy of pharmacological COVID-19 treatments using composite outcomes combining hospital admission and mortality at 28-30 days.
A further important strength of this study is that all of the hospitalized patients were individually reviewed to exclude eventual institutionalizations and deaths not attributable to COVID-19.
Although randomization is impractical in real life, the baseline distribution of variables was fairly balanced, with the exception of age (relatively lower for the patients on both antivirals), immune depression (more prevalent in patients on Molnupiravir and Sotrovimab), time until treatment start (longer in Sotrovimab group, as expected considering the inclusion criteria), immune depression and Charlson index ≥ 5 (both more prevalent in patients on Sotrovimab).
One important limitation of this study is that since it was conducted in real life, swab tests could not be performed on a daily basis but according to variable time intervals after COVID-19 diagnosis; hence, patients may have turned negative earlier than observed. This means that our results on time until the negative swab test may have underestimated the true negativization rates.
It can be reasonably argued that a larger number of patients would have enabled some statistical risk estimates (such as for Sotrovimab or number doses of COVID-19 vaccines) to turn significant.
Finally, we did not have information on smoking, which is an additional risk factor for severe COVID-19 [7,59].

Ethical Considerations
Ethical approval to conduct this observational clinical study was received by the Regional Ethic Committee of Friuli Venezia Giulia Region (CEUR, No. CEUR 2020-OS-072).
Patient consent was waived because according to Italian privacy law (Legislative Decree 101/2018, D. Lgs. 101/2018) patients' data routinely collected by the Italian National Health Service (Italian NHS) can be used for scientific purposes within the frame of approved studies/protocols, provided that sensitive information is anonymized.

Study Design
A retrospective clinical study was conducted on 386 high-risk COVID-19 patients diagnosed between 1 February 2022 and 31 May 2022 (Omicron circulation > 90%). Patients were infected mainly by BA.1 and BA.2-like variants and were referred by general practitioners or other specialist consultants to the outpatient infectious disease service of Trieste (North-eastern Italy) due to high risk of developing severe COVID-19. High-risk patients were considered those with comorbidities and/or immune depression.
The above high-risk COVID-19 patients were offered treatment with Molnupiravir, Sotrovimab, or Nirmatrelvir/Ritonavir, according to the following criteria:

Confounding Factors
In addition to age and sex of patients, information was available on number of doses of COVID-19 vaccine, number of positive swab tests undertaken before the first negative swab result, number of days between COVID-19 diagnosis and treatment start, immune depression and Charlson index.
Immune depression was defined as an immune condition sustained by either cancer, leukemia, lymphoma, AIDS, or immunosuppressive medications.
A binary term (yes vs. no) combining number of doses of COVID-19 vaccines with number of days since last dose received was created to account for adequate COVID-19 vaccination status, as follows:

•
Patients were considered inadequately vaccinated if they were fully unvaccinated (0 doses); OR immunized with just with one dose of COVID-19 vaccine; OR immunized with 2+ vaccine doses, but last vaccine dose was received >180 days before COVID-19 diagnosis.
• Patients were considered adequately vaccinated if they were immunized with 2+ vaccine doses; AND last vaccine dose was received <181 days before SARS-CoV-2 infection.

Charlson Index
The Charlson co-morbidity index (CCI) was developed by Dr. Mary Charlson in 1987 to classify co-morbidities increasing mortality risk; it was estimated from a cohort study on 604 subjects in a New York hospital over the course of one month back in 1984. CCI was subsequently used to estimate the risk of death associated with breast cancer in 685 patients treated at Yale New Haven hospital between 1962 and 1969 [63]. In its classical form, CCI considers 16 medical conditions stratified by an indicator ranging from 1 to 6, for a final score ranging from 0 to 33. Each score is assigned according to the severity of the condition, particularly the risk of death in one year. CCI was subsequently updated to also include the patient's age, thus expanding the final score from 0 to 37 [64].

Statistical Analysis
Three separate endpoints were investigated: The rate of hospital admissions at 30 days due to COVID-19 was reviewed in depth to exclude any institutionalization not attributable to COVID-19. Hospitalizations for COVID-19 were then investigated by multiple logistic regression analysis, reporting odds ratio (aOR) with a 95% confidence interval (95% CI), adjusting for age, sex, number of doses of COVID-19 vaccine, immune depression, Charlson index, and time between COVID-19 diagnosis and treatment start.
As there were only five deaths in total-and only two being attributable to COVID-19only descriptive analysis was used, reporting frequencies and percentages.

Time between COVID-19 Diagnosis and First Negative Swab Test
The effectiveness of Nirmatrelvir/Ritonavir, which exhibited the shortest time through first negative swab test result was investigated by calculating the following proportions: • Rate of events in the experimental arm (EER) = number of events/number of patients in the experimental arm (Nirmatrelvir/Ritonavir course); • Rate of events in the control arm (CER) = number of events/number of patients in the control arm (standard of care).
Using the above data and given that patients receiving Nirmatrelvir/Ritonavir benefited from an early negativization of SARS-CoV-2 infection, the following measurements of clinical significance were estimated for each consecutive day as well as by time intervals ( The time between COVID-19 diagnosis and first negative swab test result was further investigated by multinomial logistic regression analysis, contrasting the risk of turning negative at 10-14 or 15+ days after COVID-19 diagnosis compared to 5-9 days (base), adjusting for potential confounders displayed in Table 1. The latter analysis yielded an adjusted relative risk ratio (aRRR) with a 95% CI.
The negativization rate was then investigated by multivariable Cox proportional regression analysis, controlling for the explanatory factors displayed in Table 1 and reporting adjusted hazard ratios (aHR) with a 95% CI. The latter approach was applied to the entire cohort of patients and, again, was also broken down by time interval between COVID-19 diagnosis and first negative swab test (5-9 vs. 10-14 vs. 15+ days).
All of the above multinomial and Cox regression models were adjusted for age, sex, number of doses of COVID-19 vaccine, immune depression, Charlson index, time between COVID-19 diagnosis and treatment start, and number of positive swab tests undertaken until negativization. Due to collinearity issue, adequate COVID-19 vaccination status was dropped in order to retain number of doses of COVID-19 vaccines (a more informative term).
Finally, in order to compare the viral clearance efficacy of both antivirals against Sotrovimab, a multivariable Cox proportional regression model was fitted excluding patients on standard of care, investigating the negativization rate of Molnupiravir and Nirmatrelvir/Ritonavir against Sotrovimab (reference category) and adjusting for the same explanatory factors of previous multivariable regression models. The latter Cox model was fitted on the entire cohort (MODEL 1) and restricted to patients testing negative against SARS-CoV-2 during the first 5-9 days since diagnosis (MODEL 2). The results were expressed as aHR (95% CI).
Missing values were excluded and a complete case analysis was performed. The analysis was conducted by Stata 16 (Stata Corp, College Station, TX, USA).

Conclusions
In this real-world clinical study on 386 high-risk COVID-19 patients, only 2.8% developed severe COVID-19-associated pneumonia requiring admission to hospital: 1.8% patients on Sotrovimab, 2.0% of those on Nirmatrelvir/Ritonavir, 0% of those on Molnupiravir and 72.7% of patients without treatment. Therefore, patients receiving any of the three latter pharmaceutical interventions had fewer COVID-19 hospitalizations than patients on standard of care, although the risk estimates were significant only for Molnupiravir (100% efficacy) or Nirmatrelvir/Ritonavir (84% efficacy). No adverse effects were reported in relation to any of the three COVID-19 pharmaceutical treatments under investigation.
Only five patients died (rate of 1.3%), and in only two cases death was attributable to COVID-19 (rate of 0.5%). The latter two patients were both controls, one unvaccinated and one adequately immunized against COVID-19.
Although numbers were small, Molnupiravir, Nirmatrelvir/Ritonavir and Sotrovimab were therefore all more effective than standard of care in preventing hospital admission and/or mortality due to COVID-19. However, hospitalizations also decreased also with number of doses of COVID-19 vaccine received.
The median time between COVID-19 diagnosis and first negative swab test was 10 days for Sotrovimab, 8 days for Molnupiravir, 7 days for Nirmatrelvir/Ritonavir and 11 days for standard of care. Patients on both antivirals, particularly Nirmatrelvir/Ritonavir, were more likely to turn negative earlier than patients on standard of care or those on Sotrovimab.
Although they are effective against severe disease and mortality, prescription of antivirals should be carefully scrutinized by double opinion, not only to contain health care costs but also to reduce the risk of generating resistant SARS-CoV-2 strains. Only 64.7% patients were immunized with 3+ doses of COVID-19 vaccine in the present study. High-risk patients should prioritize COVID-19 vaccination, a more cost-effective approach than antivirals against severe COVID-19 pneumonia. Moreover, recommending Nirmatrelvir/Ritonavir in order to control VST in high-risk COVID-19 patients is rather questionable, since vaccination exhibited a consistent and stronger effect size in reducing the time until negative swab test; furthermore, a reduction in VST should in any case be considered a secondary benefit of either COVID-19 vaccines or antivirals. Other cheap, large spectrum, harmless agents such as hypertonic saline solutions are available on the market as powerful nasal disinfectants to effectively reduce VST.
Funding: This research received no external funding.
Institutional Review Board Statement: Ethical approval to conduct this observational clinical study was received by the Regional Ethic Committee of Friuli Venezia (CEUR, (No. CEUR 2020-OS-072)).
Informed Consent Statement: Patient consent was waived because according to Italian privacy law (Legislative Decree 101/2018, D. Lgs. 101/2018) patients' data routinely collected by the Italian National Health Service (NHS) can be used for scientific purposes within the frame of approved studies/protocols, provided that the sensitive information is anonymized.

Data Availability Statement:
The datasets generated and analyzed during the current study are not publicly available since they were purposively collected by the authors for the present study, but they may be available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.