Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2023 Jan 23;18(1):e0280745. doi: 10.1371/journal.pone.0280745

Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: A randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU)

Raja Dhar 1,‡, John Kirkpatrick 2,‡, Laura Gilbert 3, Arjun Khanna 4, Mahavir Madhavdas Modi 5, Rakesh K Chawla 6, Sonia Dalal 7, Venkata Nagarjuna Maturu 8, Marcel Stern 9, Oliver T Keppler 9, Ratko Djukanovic 10,11,‡, Stephan D Gadola 10,11,12,‡,*
Editor: Davor Plavec13
PMCID: PMC9870104  PMID: 36689456

Abstract

Background

After admission to hospital, COVID-19 progresses in a substantial proportion of patients to critical disease that requires intensive care unit (ICU) admission.

Methods

In a pragmatic, non-blinded trial, 387 patients aged 40–90 years were randomised to receive treatment with SoC plus doxycycline (n = 192) or SoC only (n = 195). The primary outcome was the need for ICU admission as judged by the attending physicians. Three types of analyses were carried out for the primary outcome: “Intention to treat” (ITT) based on randomisation; “Per protocol” (PP), excluding patients not treated according to randomisation; and “As treated” (AT), based on actual treatment received. The trial was undertaken in six hospitals in India with high-quality ICU facilities. An online application serving as the electronic case report form was developed to enable screening, randomisation and collection of outcomes data.

Results

Adherence to treatment per protocol was 95.1%. Among all 387 participants, 77 (19.9%) developed critical disease needing ICU admission. In all three primary outcome analyses, doxycycline was associated with a relative risk reduction (RRR) and absolute risk reduction (ARR): ITT 31.6% RRR, 7.4% ARR (P = 0.063); PP 40.7% RRR, 9.6% ARR (P = 0.017); AT 43.2% RRR, 10.8% ARR (P = 0.007), with numbers needed to treat (NTT) of 13.4 (ITT), 10.4 (PP), and 9.3 (AT), respectively. Doxycycline was well tolerated with not a single patient stopping treatment due to adverse events.

Conclusions

In hospitalized COVID-19 patients, doxycycline, a safe, inexpensive, and widely available antibiotic with anti-inflammatory properties, reduces the need for ICU admission when added to SoC.

Introduction

The COVID-19 pandemic is an enormous burden on health care systems and economies around the globe. While effective vaccines directed against the SARS-CoV-2 spike protein are available, it will take much time and effort for worldwide immunization levels to be high enough to bring the COVID-19 pandemic to an end [1]. Variants of concern (VOC) of SARS-CoV-2 with increased transmissibility, such as the Delta (B.1.617.2) and, more recently, the Omicron (B.1.1.529) variants, have led to a substantial surge in the incidence rate of severe COVID-19 cases, particularly in the context of low vaccine coverage and especially in lower-middle and low-income countries. As a result, in many countries the saturation of intensive care units (ICU) has driven political decisions, including containment and segregation measures [2, 3].

A hyperinflammatory syndrome or cytokine storm contributes to severe disease [4], indicating a key role for dysregulated host innate immune mechanisms during advanced stages of COVID-19. Thus, major global efforts to develop effective treatments have targeted either the SARS-CoV-2 virus or the excessive inflammatory responses [5]. Conversely, the airway microbiome may also play a role in inflammatory responses, and bacterial superinfection of viral pneumonia is a well-known driver of severe disease and complications [6].

The development of new medicines and repurposing of existing drugs with potential value in COVID-19 has required large resources for the delivery of national and international trials. Complex networks have had to be set up, including the RECOVERY and PRINCIPLE platform trials in the UK and ACTIV in the USA, with additional international networks, such as the REMAP-CAP and the World Health Organisation (WHO) Solidarity Plus trial. This global effort has identified several effective treatments which feature in COVID-19 treatment guidelines [7]. Despite these advances, there remains a high unmet need for treatments that prevent COVID-19 progression in hospitalized patients to severe disease requiring transfer to an intensive care unit (ICU).

Doxycycline is an established, widely available and inexpensive oral drug with a good safety profile and pleiotropic therapeutic effects. It exerts broad-spectrum activity against both extra- and intracellular pathogens [8, 9], and is indicated for empiric treatment of community acquired pneumonia, including influenza-associated pneumonia [10, 11]. Doxycycline exhibits anti-inflammatory, anti-oxidative and tissue-protective effects, e.g. through potent inhibition of metalloproteinases, which are associated with the severity of COVID-19 [9, 12–14]. Therefore, doxycycline may be an effective treatment in hospitalized patients, who are at high risk for progressing to severe COVID-19 that requires treatment in ICU.

To test this hypothesis, we conducted a randomized, open-label, multi-centre trial based in 6 hospitals in India with high quality ICU facilities. The need for ICU admission was chosen as the primary outcome, and the study was powered based on the assumption that, with standard of care treatments (SoC) at the time of protocol development for this trial, up to 25% of hospitalized COVID-19 patients require ICU admission [15]. The trial followed all the principles of good clinical practice (GCP) but differed from standard clinical trials in respect of its low total cost (< USD 10,000), including free of charge participation of the study team and all study sites) and the use of an online application (developed by JK and LG) serving as the electronic case report form (eCRF) that enabled efficient screening, randomization and collection of outcomes data.

Methods

Study design

This was a parallel group, controlled (against standard of care, SoC), randomized trial, including a screening period (0–1 days), 14 days of treatment and 14 days of follow-up by telephone. The study was conducted at six sites across India. Using the screening data entered by the attending physician(s) into the online app designed specifically for this trial (see data collection below), patients were randomized by the online app within 24 hours of admission to hospital into one of two arms: SoC or SoC and doxycycline (SoC+Doxy). The study was approved by ethics committees from Fortis Hospital (EC Ref No:220/EC/PI/2020) on 30.09.2020, and from CMRI (Ref Nr: IEC/01/2021/ACD-CT/APRV/05). All patients gave their written informed consent. The trial was registered on the Clinical Trials Registry–India (final registration number CTRI202105033867). The full study protocol is available at: https://cmri.ckbirlahospitals.com/Study_Protocol_DOXPREVENT.pdf (S1 File).

Participants

We enrolled adult symptomatic patients with a proven diagnosis of COVID-19 who had been admitted to hospital within the last 24h. The following inclusion criteria had to be met: able to give informed consent, age ≥40 and <90 years, SARS-CoV-2 infection demonstrated by PCR, typical symptoms of COVID-19 (new onset or exacerbation of a pre-existing cough due to a chronic respiratory illness, dyspnea, increased body temperature (axillary T° > 37.6°C or oral T°>38°C), and admission to hospital within 10 days of onset of symptoms. Exclusion criteria were: hypersensitivity to doxycycline; myasthenia gravis, pregnancy, and hepatic failure (CHILD-Pugh score C).

Study endpoints

The question of scientific interest that the trial was designed to assess was whether treatment with doxycycline reduces the need (indication) for ICU care in newly hospitalized patients with symptomatic COVID-19. Therefore, the primary endpoint was the need for transfer to ICU within 14 days of admission, as judged by the attending physicians. Secondary endpoints included in the study protocol were: death, mechanical ventilation, time to discharge, recovery from symptoms, resolution of fever, prolonged hospital stay (>7days), and supplemental oxygen required.

Study treatment

All patients received SoC set for their hospital and in keeping with the guidelines in India. Patients randomized to the SoC+Doxy arm were given doxycycline 100 mg BID for 14 days. Patients discharged before the 14-day period of follow up were contacted by telephone two days after discharge to ensure they are well and convalescing; those assigned to the SoC+Doxy arm were asked to complete 14 days of treatment with doxycycline in addition to any other drugs they were discharged with.

Data collection and analysis

Data collection with an online app

An electronic case report form (eCRF) app was developed (by JK and LG) for online entry (by the attending physicians), randomization, stratification, patient screening, treatment and outcomes data. The eCRF app was hosted on a secure Amazon Web Services (AWS) server accessed by investigators via a user-specific username and password. The app consisted of 5 pages: screening, randomization, treatment, adverse events, and outcome. Instructions for its use (see supplemental appendix) were sent to all participants, and several online training sessions were held, including completion of mock patient data. All users had to be fully proficient before getting permission to enter real patient data. Specific instructions were issued for when the data were to be placed within each of the five pages.

The app was written in R Shiny and was hosted on shinyapps.io. Authentication based on user id and password was required to access the app. Data were stored in an AWS Aurora database instance, encrypted both at rest and in transit. It was only accessible via a secure, separately authenticated RestFUL API written in PHP and hosted on an AWS Elastic Compute server. All data entry and modification were audit trailed. The app downloaded and uploaded data to the database via the API securely via the SSL protocol. Data for analysis was also downloaded via the API interface. R version 4.0.3 was used to perform all analyses of data collected in the study. The quality of the data was controlled by JK and all queries were passed on to the individual sites, assisted by the study coordinator based in India.

Statistical analysis

The primary endpoint was the need for patient transfer to ICU within 14 days of hospital admission as judged by the attending physicians. Based on available data from the literature at the time of study planning in the first half of 2020, we assumed that up to 25% of patients admitted to hospital with COVID-19 would require ICU care within 14 days of admission [15]. The therapeutic effect size of doxycycline used for calculating the trial size was based on the pragmatic view that, in order to transform medical practice in hospitalized COVID-19 patients, addition of doxycycline to SoC should reduce the proportion of study participants meeting the primary outcome to 12.5%, a risk ratio of 0.500.

Initially, we planned a 2:1 (SoC+Doxy:SoC) randomization and interim analyses, allowing the possibility of stopping for both success and futility once the status of 50% and 75% of participants were known. In addition, we allowed for a futility-only interim once the status of 25% of participants was known. Loss to follow up was assumed to be ≤5%. The trial required 80% power to detect a reduction in the risk of needing admission to ICU from 25% to 12.5% using a one-sided significance level of 2.5%. These criteria gave a sample size of 347 in total, 231 in the doxycycline + SoC arm and 116 in the SoC arm. Due to an error during the implementation of the randomisation, the app used a 1:1 allocation ratio. It should be noted that, for any given sample size, the power of a study using a 1:1 allocation ratio is greater than that of a study that uses any other allocation ratio. For full details of the initial and revised sample size calculations, see the supplemental appendix (S2 File).

Stratification

As the benefits of COVID-19 treatments may be different depending on prior co-morbidities, study participants were stratified as follows: no relevant prior illness; pre-existing lung conditions (ILD, COPD, bronchiectasis, asthma); and other relevant non-respiratory comorbidities (e.g. diabetes, heart disease, uncontrolled hypertension, cancer). Participants with both pulmonary and non-pulmonary co-morbidities were included in the pulmonary stratum.

Generalised linear models (GLMs) with a binomial response and canonical link were used to analyse the primary endpoint. The predictor variables used were stratum, sex and treatment. As no interim analyses were conducted, p-values quoted are two-sided rather than one-sided. For more details of statistical analyses, please see the supplemental appendix. Other baseline and outcome variables were reported using summary statistics appropriate for either categorical or continuous variables, as needed.

In vitro studies

To test and compare the effects of doxycycline, tetracycline and remdesivir on SARS-CoV-2 infection of ACE-2-overexpressing lung-derived cell line A549-hACE2 and breast cancer cell line MDA-MB-231-hACE2, we carried out a standardized, luminometric viability assay following drug titration and exposure to six SARS-CoV-2 variants, in principle as reported [16]. For full details of the methods used see the supplemental appendix (S3 File).

Study sponsorship and coordination

This was an investigator-led study without formal sponsorship as no institution (national or international) was identified for this purpose. The pharmaceutical company, Cipla (Mumbai, India) generously provided Rs 500,000 (USD 6730) as an unrestricted grant to support a part-time salary of a study coordinator who assisted the Chief Investigator (RDh). Cipla had no role in the trial design, data collection, data analysis, data interpretation, or writing of the manuscript. The costs related to the secure AWS server hosting the eCRF app (USD 1000) were borne by JK, RD and SDG. JK, RD, RDh, and SDG had full access to all the data at the end of the study, and all authors had individual responsibility for the decision to submit for publication. The corresponding author (SDG) had the final responsibility to submit the paper for publication.

Results

Study conduct

Enrollment into the trial started on 1st November 2020 and continued for 27 weeks until 10 May 2021 at 6 individual study sites across India. In total, 387 patients participated, of whom 228 (58.9%) were enrolled from mid-March until early May 2021, during the second COVID-19 peak in India (Figs 1 and 2) [17]. During this period, extremely high hospital admission rates of COVID-19 resulted in rapid recruitment into the trial, but also placed additional pressure on clinical staff at trial sites. This created gaps in communication between the steering committee and the study sites, leading to the unintentional omission of the pre-specified interim analysis. On 14 May 2021, after 387 patients had been enrolled, the trial steering committee formally confirmed that enrollment should stop. As the number of patients enrolled exceed the number needed for a conventional (not group sequential) design, and since the randomization ratio actually used was 1:1 rather than 2:1, the study had clearly achieved its intended power. The trial was terminated, enrolment ceased, ongoing patients were followed to completion, all data queries were resolved, and statistical analyses completed.

Fig 1. Enrollment to analysis (CONSORT diagram).

Fig 1

Open in a new tab

Fig 2. Enrollment into the trial from 1st November 2020 until 10th May 2021.

Fig 2

Open in a new tab

(A) Enrollment over time. Cumulative number of enrolled patients shown on the y-axis. (B) Enrollment by week. Black columns represent one full week of enrollment.

Study participants

Of the 387 participants, 195 were randomized to the SoC arm and 192 to the SoC+Doxy arm. Baseline demographics were comparable between the two groups (Table 1). At study entry, all the participants had a positive PCR test for SARS-CoV-2 and were clinically diagnosed as having COVID-19. The median age (IQR, min, max) of participants was 58 (49–66, 40, 90) years, with a 2:1 ratio of males to females. The median axillary body temperature (IQR, min, max) at study entry was 38.2°C (38.0°C– 38.4°C, 37.8°C, 40.8°C) in the SoC+Doxy group and 38.3° C (37.6°C– 38.3°C, 37.6°C, 40.6°C) in the SoC group.

Table 1. Baseline characteristics and drug treatment during the study.


randomized (n)		 All Doxycyline group SoC group
387 192 195
Age, yrs (range) 58.6 (40–90) 58.6 (40–90) 58.6 (40–88)
n Female:Male (%) 140:247 (36.2:63.8) 69:123 (36:64) 71:124 (36.4:63.6)
n SARS-CoV-2 PCR+ (%) 387 (100) 192 (100) 195 (100)
Body temperature (°C), mean (range) 38.3 (38–41) 38.3 (38–41) 38.4 (38–41)
Comorbidities, n (%)
No comorbidity 97 (25.1) 48 (12.4) 49 (12.7)
Any comorbidity 290 (74.9)
Hypertension 206 (53.2) 101 (52.6) 105 (53.9)
Diabetes 138 (35.7) 65 (33.9) 73 (37.4)
Heart disease 52 (13.4) 28 (14.6) 24 (12.3)
Lung disease (all) 89 (23.0) 45 (23.4) 44 (22.6)
COPD 35 (9.0) 20 (10.4) 15 (7.7)
Asthma 29 (7.5) 13 (6.8) 16 (8.2)
ILD 5 (1.3) 3 (1.6) 2 (1.0)
Bronchiectasis 4 (1.0) 2 (1.0) 2 (1.0)
Other lung disease 16 (4.1) 7 (3.7) 9 (4.6)
Cancer 5 (1.3) 2 (1.0) 3 (1.5)
Drug treatment during study, n (%)
Doxycycline 183 (47.3) 178 (92.7) 5 (2.6)
Antibiotics other than doxycycline 314 (81.1) 158 (82.3) 156 (80.0)
Glucocorticoids 315 (81.4) 160 (83.3) 155 (79.5)
Antivirals 355 (91.7) 181 (94.3) 174 (89.2)
Anticoagulants 338 (87.3) 174 (90.6) 164 (84.1)
Antihypertensives 200 (51.7) 101 (52.6) 99 (50.8)
Antidiabetic drugs 144 (37.2) 70 (36.4) 74 (38.0)
Ivermectin 152 (39.3) 79 (41.2) 73 (37.4)
Tocilizumab 4 (1.3) 0 (0) 4 (2.1)
Open in a new tab

Among the 387 study participants, 290 (74.9%) had at least 2 pre-existing comorbidities associated with increased risk for severe COVID-19, including hypertension, diabetes, heart disease, lung diseases, and cancer, while 97 participants (25.1%) had no identifiable risk factors at study entry. A pre-existing lung disease was present in 84 of 387 patients (21.7%). The incidence of various risk factors (RF) was similar in the two treatment groups (Table 1), with total numbers of 579 RF in SoC and 570 in SoC+Doxy groups, and an average of 3 RF per study participant in both groups (S1 Table).

Study treatment

Adherence to the protocol and study treatment per protocol was high, with 368 of 387 (95.1%) of participants receiving the allocated treatment based on randomisation. Of the 192 patients randomized to the SoC+Doxy arm, 178 (92.7%) received doxycycline, while 5 of 195 patients (2.6%) randomized to the SoC arm were prescribed doxycycline as an antibiotic following the site’s local guidelines for COVID-19 management (Table 1). Hence, 183 of the 387 (47.3%) patients in this trial were treated with SoC plus doxycycline, while 204 patients (52.7%) received treatment with SoC only.

Participants in the two treatment groups were similar with respect to concomitant medication (Table 1), with >80% of patients in both study arms receiving antibiotics other than doxycycline, glucocorticoids (dexamethasone or methylprednisolone), antivirals (remdesivir; favipiravir), and anticoagulants as SoC. Four patients in the SoC arm and none in the SoC+Doxy arm received tocilizumab. Other commonly used treatments included analgesics, antihypertensives, anti-diabetic drugs, and ivermectin.

Primary outcome

Of the 387 patients included in the trial, 77 (19.9%) reached the primary outcome, i.e. they developed COVID-19 symptoms deemed by the attending physicians severe enough to require critical care in the ICU (Fig 3). Among the 290 patients with pre-existing comorbidities, 65 (22.4%) needed ICU admission, compared to only 12 of 97 patients without risk factors (12.4%) (OR 2.0463, 95% CI 1.0529 to 3.9768, p = 0.0347). The proportions requiring ICU treatment were similar in patients with pre-existing lung disease (18 of 84; 21.4%) and those with other risk factors (47 of 206; 22.8%).

Fig 3. Outcomes (CONSORT diagram).

Fig 3

Open in a new tab

For comparison of the primary outcome between the two treatment arms in this trial we carried out three types of analysis: First, in the intention to treat analysis (ITT) of all 387 participants, 46 of 195 patients (23.6%) randomized to SoC reached the primary outcome compared to 31 of 192 patients (16.1%) randomized to SoC+Doxy (OR 0.617, 95% CI 0.369 to 1.027, p = 0.063) (Fig 4). As 19 of the 387 study participants (4.9%) had received a treatment that was contrary to the randomization, two additional analyses were carried out for the primary outcome. First, a “per protocol” (PP) analysis included only the 368 patients who had received study treatment in accordance with their randomization; among these, 45 of 190 patients (23.7%) in the SoC arm compared to 25 of 178 patients (14.0%) randomized to SoC+Doxy developed severe COVID-19 that met the criteria for ICU admission (OR 0.521, 95% CI 0.300 to 0.890, p = 0.017). Second, an “as treated” (AT) analysis of the primary outcome data was carried out after re-allocating the 19 patients to the two treatment arms by correcting for actual treatment. This showed that among the 183 participants who had actually received doxycycline in addition to SoC, a total of 26 of patients (14.2%) required ICU admission compared to 51 of the 204 (25.0%) participants treated with SoC only (OR 0.493, 95% CI 0.288 to 0.828, p = 0.007) (Figs 3 and 4). The observed effect of doxycycline on the requirement for ICU treatment was similar across different study sites, i.e., no batch effects were noted. Calculated relative risk reductions (RRR) were 31.6% (ITT), 40.7% (PP) and 43.2% (AT), with numbers needed to treat (NNT) ranging from 13.4 (ITT) to 9.3 (AT) (Table 2).

Fig 4. Summary of primary outcome data.

Fig 4

Open in a new tab

Analyses of primary outcome data (need for ICU admission). ITT, as randomized; PP, per protocol; AT, as treated. Red bars: SoC; green bars: SoC+Doxy.

Table 2. Relative Risk Reduction (RRR), Absolute Risk Reduction (ARR) and Number Needed to Treat (NNT) according to the three primary outcome analyses.

Analysis SoC SoC +Doxy All RRR (%) ARR (%) NNT (n)
Intention to treat (ITT) (n) 195 192 387 31.6 7.4 13.4
ICU required, n (%) 46 (23.6) 31 (16.1) 77 (19.9)
Per protocol (PP) (n) 190 178 368 40.7 9.6 10.4
ICU required, n (%) 45 (23.7) 25 (14.0) 70 (19.0)
As treated (AT) (n) 204 183 387 43.2 10.8 9.3
ICU required, n (%) 51 (25.0) 26 (14.2) 77 (19.9)
Open in a new tab

Key secondary outcomes

Of the 77 patients judged by their physicians to require admission to the local ICU during the study, 46 (59.7%) were actually admitted, the main reason being shortage of ICU beds, especially during the second wave of the pandemic which started in mid-March 2021. In some cases, patients were deemed unsuitable for ICU admission based on low likelihood of surviving subsequent weaning from ventilation. 30 of 195 patients randomized to SoC and 16 of 192 patients randomized to SoC+Doxy were actually admitted to ICU (OR 0.5000, 95% CI 0.2629 to 0.9510, p 0.0346). Among the 204 patients who were actually treated with SoC, 34 (16.7%) were admitted to ICU, compared to 12 of 183 (6.6%) patients treated with SoC plus doxycycline (OR 0.3509, 95% CI 0.1757 to 0.7006, p 0.003). Among patients requiring ICU treatment as judged by their treating physicians, 34 of 51 (66.7%) treated with SoC compared to 12 of 26 (46.2%) treated with SoC plus doxycycline were admitted to ICU (Fig 3). Thus, among patients requiring critical care, those treated with SoC plus doxycycline were less likely to be admitted to ICU than patients treated with SoC only, although the difference did not reach significance (OR for not being admitted to ICU 0.4286, 95% CI 0.1631 to 1.1262, p = 0.0856). We have no explanation for this observed difference in treatment.

Thirty-nine of 387 study participants (10.1%) died during the trial. All deaths occurred in hospital. No deaths were recorded after discharge during the 14 day telephone follow-up period. Thirty of the 31 patients (96.8%) who required intensive care but could not be admitted to an ICU died, compared to 9 of 46 patients (19.6%) who did receive critical care in ICU (Fig 3)(OR 0.008, 95% CI 0.001 to 0.068, p<0.0001). Death was significantly associated with pre-existing comorbidities with 35 of 290 (12.1%) patients with pre-existing comorbidities (known to be associated with severe COVID-19) died, as compared with only 4 of 97 (4.1%) without comorbidities (OR 0.3121, 95% CI 0.1080 to 0.9022, p 0.0316). Overall, no difference in death rate was observed between the two treatment arms in the ITT analysis of all 387 patients. Nineteen of 192 patients randomized to SoC+Doxy (9.9%) and 20 of 195 patients (10.3%) randomized to SoC died (p 0.90; OR 0.961, 95% CI 0.4348 to 1.6506; p = 0.91). In the AT analysis based on actual treatment, 22 of 204 patients who received SoC (10.8%) and 17 of 183 patients (9.3%) who received SoC+Doxy died.

Other outcomes

No serious adverse events (SAE) related to doxycycline were observed, and there were no cases of discontinuations of doxycycline or SoC due to adverse events (AE). The mean duration of hospital stay in both groups was 9 days (range 5–40 days).

Discussion

The results of this randomized, controlled trial provide evidence which suggests that doxycycline reduces the need for ICU admission in hospitalized COVID-19 patients. The trial was carried out in challenging circumstances during the second wave of the COVID-19 pandemic in India defined by the rapid rise of the delta variant of SARS-CoV-2 [17]. All study participants had a positive SARS-CoV-2 PCR test, fever and additional clinical symptoms of COVID-19, and a high proportion of them had several risk factors for developing severe COVID-19, including diabetes in one third of patients. The consistency of the results in all three analytical approaches applied provides confidence that our conclusion is robust.

The main reasons for selecting doxycycline for this trial were its pleiotropic, anti-inflammatory and anti-microbial effects, robust safety profile and low cost. Approved in 1967, doxycycline shows minimal side effects [18], and is also safe in patients with severely impaired renal function [19]. The safety and tolerability in the current trial were excellent, with not a single patient stopping treatment because of an adverse event. Therefore, we conclude that the benefit:risk ratio of doxycycline in this population is highly positive.

No difference was seen in the secondary outcome measure of death between the two treatment arms. Possible reasons include the insufficient power of the trial to detect differences in mortality and the observed difference in ICU admission rates, which unintentionally disadvantaged patients treated with SoC plus doxycycline compared to those treated with SoC only.

This study suggests that the antimicrobial effects of doxycycline were not important in defining the need for ICU. Approximately 40% of patients were enrolled before and 60% after the start of the second wave in mid-March 2021 when the delta variant B.1.617.2 became dominant, suggesting that its therapeutic effect was independent of SARS-CoV-2 variants. Approximately 90% of patients in both study arms received proven antiviral drugs with activity against SARS-CoV-2. Our in vitro studies showed that neither doxycycline nor tetracycline had an inhibitory effects on different SARS-CoV-2 variants, including five variants of concern evaluated on two established ACE-2-overexpressing human cell lines (S1 Fig) [16]. As an antiviral positive control, the clinical RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir potently blocked SARS-CoV-2 replication and virus-induced cell death (S1 Fig). While studies by one laboratory in African green monkey-derived Vero-E6 cells suggested an antiviral effect of doxycycline in vitro [20, 21], another report has challenged this conclusion [22]. Potentially, the non-human origin of Vero-E6 cells may have been a confounder in the evaluation of anti-SARS-CoV-2-specific effects of doxycycline.

The UK platform trial, PRINCIPLE, to which some members of our study team contributed (SDG, JK, RD), found that doxycycline was not associated with reduced hospital admissions or death when used in the community in patients with suspected early COVID-19 [23]. This contrast in benefits of doxycycline with the current study of advanced hospital stages of COVID-19 further supports the notion that doxycycline has no direct antiviral activity against SARS-CoV-2. Of note, dependency on the stage of disease is also observed with monoclonal antibodies against the SARS-CoV-2 spike protein which act directly as anti-viral agents and are effective during early disease [24]. A high proportion of patients in both treatment groups received broad spectrum antibiotics, which argues against an important contribution of antibiotic properties of doxycycline to the observed benefit. Of note, current guidelines do not recommend antibiotics for COVID-19 unless there is a clinical suspicion of bacterial co-infection [17].

Severe COVID-19 that requires critical care is associated with a hyperinflammatory state [4], which provides the pathobiological substrate for glucocorticoids that have been shown to be effective in COVID-19 patients requiring oxygen treatment. The fact that approximately 80% of patients in both treatment groups of this trial received glucocorticoids suggests that doxycycline acts via different mechanisms. Doxycycline has well described anti-inflammatory effects in various human lung diseases, including cystic fibrosis [25], lung fibrosis [26], and sarcoidosis [27]. In patients with Dengue hemorrhagic fever and hyperinflammation, doxycycline substantially reduces mortality in association with a significant reduction in serum concentrations of interleukin-6, TNFα and interleukin-1 [28]. Various pathways promoting inflammation and oxidative cell stress are targeted by doxycycline, including mitogen-activated protein kinase (MAPK) and Smad pathways, matrix metalloproteinases (MMP) implicated in inflammatory lung injury, and malondialdehyde-acetaldehyde (MAA) activation of Nrf2 [9, 12, 13]. In addition, doxycycline has been shown to directly scavenge reactive oxygen species, such as superoxide [13], and to reduce the production of nitric oxide [29], both of which have been implicated in lung injury and endothelial dysfunction [30]. Hence, the beneficial effects of doxycycline observed in this study may have been related to its anti-inflammatory, anti-oxidative, and cell-protective properties.

In addition to its potential pharmacodynamic effects and safety profile, the pharmacokinetic (PK) properties of doxycycline made it a good choice for this trial, especially its high bioavailability after oral dosing, short time required to achieve effective blood levels, a half-life of 12-25h, and strong tissue penetration into respiratory tissues, with reach into the intracellular space [8]. Of note, an oral dose of 200mg doxycycline achieves sufficient concentrations in sputum to inhibit MMP-9 [31].

The selection of doxycycline for this trial was also strongly driven by our intent to identify cost-effective therapies that would benefit patients across the globe and not just those in affluent countries which can afford the effective, yet very expensive, drugs for which efficacy against COVID-19 has been reported so far. In the UK, a 14-day course of 200 mg doxycycline costs £ 6.50, while in India it is less than £5.00. The observed reduction in ICU admissions, when extrapolated to the large numbers of patients progressing to severe forms of COVID-19, also has the potential for major cost-savings. This is especially relevant in countries like India, where the cost of ICU–based care provided by private sector hospitals is estimated at $255 per day [32]. While this may not seem excessive and given that universal healthcare is guaranteed by the Indian constitution, the limited number of tertiary government-run teaching hospitals means that much of the ICU care is provided by the private sector for which costs are met by patients and their families. Inevitably, those patients and families without financial means to bear such costs may have no other choice but to decline ICU admission despite a strong clinical need. Doxycycline is produced by many companies and is included in the WHO 2019 core list of essential medicines [33], so there should be sufficient stocked drug available in case it becomes a standard treatment for COVID-19 in hospitalized patients.

This study has also shown that randomized controlled trials can be done at minimal cost during a pandemic, and it provides proof of concept for the use of an online app as a means of reliably collecting data in multi-centre trials. The only unavoidable, but ultimately trivial, cost was that of the server which enabled secure access to the application that served as an online eCRF. We sought help from a large Indian pharmaceutical company (Cipla) which generously provided a small unrestricted grant to support a research coordinator to help initiate the study by facilitating communication. None of the investigators or their institutions sought any compensation for the time and resources used. The study and the approach we took had its limitations, the main being the absence of the intensity of monitoring that drug trials usually entail. However, as a real-world study, this trial was just as determined and able as the major COVID-19 platform trials to obtain accurate data. We also recognize that we could not undertake the planned interim analyses because of strained communications during the second peak in India, something that may have been easier to cope with had more centres been recruited (meaning less burden per centre) and/or had trial monitors been assigned to each site, albeit raising the cost of the trial. Whilst we were confident about the main outcome (need for ICU admission) and mortality, we were less confident about collecting some of the secondary endpoints, so did not feel it justified to analyse these outputs. We conclude, therefore, that the approach taken, including the use of the eCRF, is less suited for collection of finer study details, although adjustments to improve the functionality of the app should make it possible to collect more complex datasets.

In summary, this study has shown that doxycycline prevents progression of COVID-19 to the point where ICU admission is needed. This comes with significant implications for both patients and health services, in particular in countries where, because of limited financial resources, ICU admission is dependent on accessibility and affordability rather than clinical indication. A similar study is needed to seek evidence of treatment on mortality, requiring large numbers of patients similar to those enrolled in well-funded, platform trials.

Supporting information

S1 Table. Number of Risk factors associated with severe COVID-19 in the study.

(DOCX)

Click here for additional data file. (13.9KB, docx)
S1 Fig. Antiviral effect of doxycycline, tetracycline and remdesivir.

Effect of doxycycline and tetracycline compared to remdesivir on SARS-COV-2 infection of ACE-2-overexpressing lung-derived cell lines in vitro.

(TIF)

Click here for additional data file. (537.2KB, tif)
S1 File. Study protocol.

(PDF)

Click here for additional data file. (523.8KB, pdf)
S2 File. Sample size calculation.

(PDF)

Click here for additional data file. (252.5KB, pdf)
S3 File. Detailed material and methods of in vitro studies.

(PDF)

Click here for additional data file. (67.2KB, pdf)
S4 File. Reporting checklist for randomised trials (based on the CONSORT guidelines).

(PDF)

Click here for additional data file. (102.1KB, pdf)

Acknowledgments

We wish to acknowledge the following clinicians who, as members of the clinical teams attending to the patient participants on this trial, contributed to clinical management of the patients and data collection:

Dr Dipabali Acharjee, Research Associate, CMRI hospital, Kolkata, India.

Dr Virender Pratibh Prasad, Consultant Pulmonary Medicine, Yashoda Hospital, Somajiguda, India.

Dr Aditya K. Chawla, Junior Consultant, Saroj Superspeciality Hospital and Jaipur Golden Hospital, Delhi and ii. Dr Gaurav Chaudhary, DNB Resident (Final yr), Jaipur Golden Hospital, Delhi, India.

Dr Vishnu Gireesh, Resident, Department of Chest Medicine, Ruby Hall Clinic, Pune, India.

Dr Avhinav Bhosle, Consultant Pulmonologist, Kalyan Hospital, Vadodara, India.

Dr Viswesvaran Balasubramanian, Lead Consultant and Head, Department of Pulmonary and Sleep Medicine, Yashoda hospitals, Hyderabad

Data Availability

All data underlying the results presented in the study are available online on GitHub (https://github.com/PuzzledFace/DoxyICU_Results).

Funding Statement

RDh received an unrestricted grant (grant/study code: IIS/11/20) over Rs 500’000.00, corresponding to USD 6730.00) from Cipla (https://www.cipla.com) to finance a part-time study coordinator for this trial. Cipla had no role in the trial design, data collection, data analysis, data interpretation, or writing of the manuscript.

References

  • 1.Irwin A. What it will take to vaccinate the world against COVID-19. Nature 2021;592:176–178. doi: 10.1038/d41586-021-00727-3 [DOI] [PubMed] [Google Scholar]
  • 2.Truelove S, Smith CP, Qin M, et al. Projected resurgence of COVID-19 in the United States in July-December 2021 resulting from the increased transmissibility of the Delta variant and faltering vaccination. Preprint. medRxiv 2021;2021.08.28.21262748. doi: 10.1101/2021.08.28.21262748 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Tran Kiem C, Crépey P, Bosetti P, et al. Lockdown as a last resort option in case of COVID-19 epidemic rebound: a modelling study. Euro Surveill 2021;26:2001536. doi: 10.2807/1560-7917.ES.2021.26.22.2001536 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Gustine JN, Jones D. Immunopathology of Hyperinflammation in COVID-19. Am J Pathol 2021;191:4–17. doi: 10.1016/j.ajpath.2020.08.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Asselah T, Durantel D, Pasmant E, Lau G, Schinazi RF. COVID-19: Discovery, diagnostics and drug development. J Hepatol 2021;74:168–184. doi: 10.1016/j.jhep.2020.09.031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Morens DM, Taubenberger JK, Fauci AS. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: Implications for pandemic influenza preparedness. J Infect Dis 2008;198:962–70. doi: 10.1086/591708 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.COVID-19 rapid guideline: managing COVID-19. NICE guideline [NG191] Published: 23 March 2021 Last updated: 01 December 2021. https://www.nice.org.uk/guidance/ng191/chapter/Recommendations.
  • 8.Holmes NE, Charles PGP. Safety and Efficacy Review of Doxycycline. Clinical Medicine. Therapeutics 2009;1:471–482. doi: 10.4137/CMT.S2035 [DOI] [Google Scholar]
  • 9.Rothan HA, Mohamed Z, Paydar M, Abd Rahman N, Yusof R. Inhibitory effect of doxycycline against dengue virus replication in vitro. Arch Virol 2014;159:711–8. doi: 10.1007/s00705-013-1880-7 Epub 2013 Oct 19. [DOI] [PubMed] [Google Scholar]
  • 10.Grief SN, Loza JK. Guidelines for the Evaluation and Treatment of Pneumonia. Prim Care 2018;45:485–503. doi: 10.1016/j.pop.2018.04.001 ; PMCID: PMC7112285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.British Infection Society, British Thoracic Society, Health Protection Agency. Pandemic flu: clinical management of patients with an influenza-like illness during an influenza pandemic. Provisional guidelines from the British Infection Society, British Thoracic Society, and Health Protection Agency in collaboration with the Department of Health. Thorax 2007;62(Suppl 1):1e46. doi: 10.1136/thx.2006.073080 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kim HS, Luo L, Pflugfelder SC, Li D. Doxycycline inhibits TGF-β1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells. Invest Ophthalmol Vis Sci 2005;46:840–848. [DOI] [PubMed] [Google Scholar]
  • 13.Clemens DL, Duryee MJ, Sarmiento C, et al. Novel Antioxidant Properties of Doxycycline. Int J Mol Sci 2018;19:4078. doi: 10.3390/ijms19124078 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Syed F, Li W, Relich RF, Russell PM, Zhang S, Zimmerman MK, et al. Excessive matrix metalloproteinase-1 and hyperactivation of endothelial cells occurred in COVID-19 patients and were associated with the severity of COVID-19. J Infect Dis 2021;224:60–69. doi: 10.1093/infdis/jiab167 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wang D, Hu B, Hu C, et al. Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA 2020;323:1061–1069. doi: 10.1001/jama.2020.1585 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wratil PR, Stern M, Priller A, et al. Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern. Nat Med 2022. Mar; 28:496–503. doi: 10.1038/s41591-022-01715-4 [DOI] [PubMed] [Google Scholar]
  • 17.Choudhary OP, Priyanka, Singh I, Rodriguez-Morales AJ. Second wave of COVID-19 in India: Dissection of the causes and lessons learnt. Travel Med Infect Dis 2021;43:102126. doi: 10.1016/j.tmaid.2021.102126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Smith K, Leyden JJ. Safety of doxycycline and minocycline: a systematic review. Clin Ther 2005;27:1329–42. doi: 10.1016/j.clinthera.2005.09.005 [DOI] [PubMed] [Google Scholar]
  • 19.Ørnulf Stenbæk, Erik Myhre & Bjørn Peter Berdal. Doxycycline in renal failure. CMRO 1975;3(Suppl.2):24–30. [Google Scholar]
  • 20.Gendrot M, Andreani J, Jardot P, et al. In Vitro Antiviral Activity of Doxycycline against SARS-CoV-2. Molecules (Basel, Switzerland) 2020; 25:E5064. doi: 10.3390/molecules25215064 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Alexpandi R, Gendrot M, Abirami G, et al. Repurposing of Doxycycline to Hinder the Viral Replication of SARS-CoV-2: From in silico to in vitro Validation. Front Microbiol 2022; 13:757418. doi: 10.3389/fmicb.2022.757418 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Diomede L, Baroni S, De Luigi A, et al. Doxycycline Inhibition of a Pseudotyped Virus Transduction Does Not Translate to Inhibition of SARS-CoV-2 Infectivity. Viruses 2021; 13:1745. doi: 10.3390/v13091745 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Butler CC, Yu LM, Dorward J, et al. PRINCIPLE Trial Collaborative Group. Doxycycline for community treatment of suspected COVID-19 in people at high risk of adverse outcomes in the UK (PRINCIPLE): a randomised, controlled, open-label, adaptive platform trial. Lancet Respir Med 2021;9:1010–1020. doi: 10.1016/S2213-2600(21)00310-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Horby PW, Mafham M, Peto L, et al. Casirivimab and imdevimab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial. RECOVERY Collaborative Group. medRxiv 2021.06.15.21258542; doi: 10.1101/2021.06.15.21258542 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Xu X, Abdalla T, Bratcher PE, et al. Doxycycline improves clinical outcomes during cystic fibrosis exacerbations. Eur Respir J 2017;49:1601102. doi: 10.1183/13993003.01102-2016 [DOI] [PubMed] [Google Scholar]
  • 26.Bhattacharyya P, Nag S, Bardhan S, et al. The role of long-term doxycycline in patients of idiopathic pulmonary fibrosis: The results of an open prospective trial. Lung India 2009;26:81–85. doi: 10.4103/0970-2113.53231 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bachelez H, Senet P, Cadranel J, et al. The use of tetracyclines for the treatment of sarcoidosis. Arch Dermatol 2001;137:69–73. doi: 10.1001/archderm.137.1.69 [DOI] [PubMed] [Google Scholar]
  • 28.Fredeking TM, Zavala-Castro JE, González-Martínez P, et al. Dengue Patients Treated with Doxycycline Showed Lower Mortality Associated to a Reduction in IL-6 and TNF Levels. Recent Pat Antiinfect Drug Discov 2015;10:51–8. doi: 10.2174/1574891x10666150410153839 [DOI] [PubMed] [Google Scholar]
  • 29.Hoyt JC, Ballering J, Numanami H, Hayden JM, Robbins RA. Doxycycline modulates nitric oxide production in murine lung epithelial cells. J Immunol 2006;176:567–72. doi: 10.4049/jimmunol.176.1.567 [DOI] [PubMed] [Google Scholar]
  • 30.Mohiuddin M, Kasahara K. The emerging role of oxidative stress in complications of COVID-19 and potential therapeutic approach to diminish oxidative stress. Respir Med 2021;187:106605. doi: 10.1016/j.rmed.2021.106605 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Beringer PM, Owens H, Nguyen A, Benitez D, Rao A, D’Argenio DZ. Pharmacokinetics of doxycycline in adults with cystic fibrosis. Antimicrob Agents Chemother 2012;56:70–4. doi: 10.1128/AAC.05710-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Peter JV, Thomas K, Jeyaseelan L, et al. Cost of intensive care in India. Int J Technol Assess Health Care 2016;32:241–245. doi: 10.1017/S0266462316000398 [DOI] [PubMed] [Google Scholar]
  • 33.WHO model list of essential medicines - 22nd list, 2021. https://www.who.int/publications/i/item/WHO-MHP-HPS-EML-2021.02.

Decision Letter 0

Davor Plavec

8 Sep 2022

PONE-D-22-10587Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: a randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU)PLOS ONE

Dear Dr. Gadola,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR: Please make revisions as suggested by reviewers of write a detailed rebuttal on a point-by-point basis.

Please submit your revised manuscript by Oct 20 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Davor Plavec, MD, MSc, PhD, Prof.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments:

Dear Authors,

please make revisions as suggested by reviewers of write a detailed rebuttal on a point-by-point basis.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. PRINCIPLE, UK platform found that doxycycline was not associated with reduced hospital admission or death when used in the community in patients with suspected early COVID-19.

No difference was seen in the secondary outcome measure of death between the two treatment arms in this study. Also, results presented at the American College of Allergy, Asthma & Immunology (ACAAI) 2021 Annual Scientific Meeting are similar. Use of doxycycline is not associated with improved mortality rate in patients with COVID-19 pneumonia. How to explain these results?

2. Authors mentioned that doxycycline has no in vitro activity against SARS-CoV-2. This is in contrast with findings from other authors (e.g. Gendrot).

Reviewer #2: Mortality is not clearly defined. Is it measured at discharge or in follow-up? A survival analysis may be more suitable.

Most secondary outcomes were not analyzed or presented in the results section.

The results section was not clearly written. The primary analysis should focus on the comparison between two treatment groups. However, in “Primary Outcome”, primary findings were buried in subgroup analysis (Eg. Comorbidities, lung disease and etc). The subgroup analysis should be moved to another section. In “Key secondary Outcomes”, ICU admission were mentioned again. This is confusing as ICU is not secondary but primary.

Table 1 present either mean with SD or median with range. How are the two groups compared?

Add p values to Table 1 and 2.

Table 2 put n (%) in one cell.

Figure 2 a flow chart for outcome is not necessary and can be omitted.

“The two treatment groups were matched” on Page 7 study participants and Page 8 2nd paragraph. The authors may change the statement to “two groups are not significantly different”. Matching has different meaning in statistics.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Jan 23;18(1):e0280745. doi: 10.1371/journal.pone.0280745.r002

Author response to Decision Letter 0

29 Oct 2022

As requested by the academic editor, a rebuttal letter that responds to each point raised by the academic editor and the two reviewers of our manuscript has been uploaded as a separate file labeled 'Response to Reviewers'.

The responses contained in our letter are reproduced in the following:

II. Responses to reviewers’ comments on general aspects of the manuscript.

1) Is the manuscript technically sound, and do the data support the conclusions?

Response: We thank both reviewers for agreeing that our manuscript is technically sound and that the data support the conclusions.

2) Has the statistical analysis been performed appropriately and rigorously?

Response: Reviewer #2 raised a couple of points in relation to the statistical analysis. We will address these points below (in the section « 5. Response to reviewers’ comments on specific aspects of the manuscript »).

3) Have the authors made all data underlying the findings in their manuscript fully available?

Response: We thank both reviewers for appreciating our effort to make all data underlying our findings available to the public.

4) Is the manuscript presented in an intelligible fashion and written in standard English?

Response: Thank you for appreciating the clarity and writing style of our manuscript.

III. Point-by-point responses to reviewers’ comments on specific aspects of the manuscript.

Points raised by Reviewer #1

1) PRINCIPLE, UK platform found that doxycycline was not associated with reduced hospital admission or death when used in the community in patients with suspected early COVID-19.

Response: The main differences between the trial reported in the manuscript under consideration and that reported by the PRINCIPLE consortium, to which three of us contributed as co-authors, relate to the patient population enrolled in the respective trials. While PRINCIPLE was a community-based trial for all-comers from age 18 onwards, therefore including mostly people with mild COVID-19, our trial was designed to test the effect of doxycycline in patients who were severe enough to require hospital admission. Thus, the patients in the current trial had more advanced disease stage COVID-19 and were, therefore, at a much higher risk for severe outcome. Of note, during the 2nd wave of the pandemic in India, hospitals were at their capacity and stretched to their limit and only severely ill patients could be admitted. In fact, the situation was so dramatic that many COVID-19 patients died outside of these hospitals before they could be admitted). The differences in population between our trial and PRINCIPLE are evident from the very different (almost 20-fold different) death rate between the two trials, i.e. 0.6% death rate in PRINCIPLE versus 10.1% death rate in our trial. In conclusion, we argue that these two trials cannot be directly compared.

To emphasize this point more clearly in the manuscript, we have revised the text of the manuscript as follows:

Section: “Discussion”; 5th paragraph: «The UK platform trial, PRINCIPLE, to which some members of our study team contributed (SG, JK, RD), found that doxycycline was not associated with reduced hospital admissions or death when used in the community in patients with suspected early COVID-19.23 Of note, the population of this trial was much more severe than that studied in PRINCIPLE, a community-based platform trial. While most patients enrolled in PRINCIPLE had mild disease, all patients in this trial were severe enough to warrant hospital admission. This contrast in benefits of doxycycline between the PRINCIPLE and the current study of advanced hospital stages of COVID-19 further supports the notion that doxycycline has no direct antiviral activity against SARS-CoV-2. Of note, dependency on the stage of disease is also observed with monoclonal antibodies against the SARS-CoV-2 spike protein which act directly as anti-viral agents and are effective during early disease.20»

2) No difference was seen in the secondary outcome measure of death between the two treatment arms in this study.

Response: We have discussed this in the manuscript (Discussion, 3rd paragraph), based on the results described in the section “Key secondary outcomes”. Due to space constraints and the fact that it is a secondary outcome measure, we kept this part of the discussion brief. However, if the reviewer insists and the editor agrees, we would be happy to expand in a further revision. In our view, two main aspects of this trial may explain why no statistically significant difference in death rate was observed between the two treatment arms:

1. Our prospective randomised clinical trial was not powered to see differences in death rate between the two study arms: A retrospective power calculation, based on the observed death rate of 10% in the SoC group, shows that a sample size of 868 patients (i.e. more than 2x the number of patients included in our trial) would have been required to observe (with alpha 0.05 and power 80%) a (quite dramatic) 50% reduction of the death rate. In order to observe a lesser, albeit still highly relevant 25% reduction of the death rate, a sample size of 4008 patients (i.e. >10x the number of patients in our trial) would have been required. A trial of that size was simply beyond our means.

2. Differences in ICU admission rates for patients of the two study arms who reached the primary outcome of “need for ICU admission”:

� A notable result of our study was that all but 1 of the 31 patients, judged by their doctors to require ICU treatment but could not be admitted to ICU, died (96.8%), while only 1 of 5 patients (9 of 46, 19.6%) admitted to ICU died. Thus, those patients in our trial whose COVID-19 deteriorated to the extent that they reached the primary outcome “need for ICU treatment” and could be admitted to ICU stood a much better chance of survival than those who reached the primary outcome but were not admitted to ICU for reasons that we explain in the paper.

� By chance (for unknown reasons), only 46% (12 of 26) of patients in the Doxycyline arm who reached the primary outcome were actually admitted to ICU, compared to 67% (34 of 51) of patients in the SoC arm. This bias in ICU admission rates, for which we have no explanation, may have confounded the secondary outcome of death in our study.

3) Results presented at the American College of Allergy, Asthma & Immunology (ACAAI) 2021 Annual Scientific Meeting are similar (to results of the PRINCIPLE trial). Use of doxycycline is not associated with improved mortality rate in patients with COVID-19 pneumonia. How to explain these results?

Response: The study presented at the American College of Allergy, Asthma & Immunology (ACAAI) 2021 Annual Scientific Meeting was a retrospective chart study in 110 patients with COVID-19 pneumonia. The limited information provided in the brief study abstract does not allow us to assess the patient population or other relevant aspects of the study. It is, therefore, not possible to conduct a fair comparison between this the ACAAI study and our prospective randomised controlled trial.

4) Authors mentioned that doxycycline has no in vitro activity against SARS-CoV-2. This is in contrast with findings from other authors (e.g. Gendrot).

Response: Thank you for raising this difference between our own in vitro study and that of Gendrot et al. For additional clarity, we have added text in the methods section of the main manuscript, adding an appropriate reference, and have expanded the discussion on previous in vitro studies, also adding three references.

(Both the numbering of references and the list of references were updated accordingly).

Please note the following edits in the revised manuscript:

Section: “Methods”; subsection “In vitro studies”, 1st sentence: «To test and compare the effects of doxycycline, tetracycline and remdesivir on SARS-CoV-2 infection of ACE-2-overexpressing lung-derived cell line A549-hACE2 and breast cancer cell line MDA-MB-231-hACE2, we carried out a standardized, luminometric viability assay following drug titration and exposure to six SARS-CoV-2 variants, in principle as reported.16»

Section: “Discussion”, 4th paragraph, edited sentences 4 and new sentences 5-7: «Our in vitro studies showed that neither doxycycline nor tetracycline had an inhibitory effect on different SARS-CoV-2 variants, including five variants of concern evaluated on two established ACE-2-overexpressing human cell lines (PMID: 35090165) (Suppl. Figure 1). As an antiviral positive control, the clinical RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir potently blocked SARS-CoV-2 replication and virus-induced cell death (Suppl. Figure 1).16 While studies by one laboratory in African green monkey-derived Vero-E6 cells suggested an antiviral effect of doxycycline in vitro,20, 21, a report by another group has challenged this conclusion.22 Potentially, the non-human origin of Vero-E6 cells may have been a confounder in the evaluation of anti-SARS-CoV-2-specific effects of doxycycline.»

Points raised by Reviewer #2:

1) Mortality is not clearly defined. Is it measured at discharge or in follow-up? A survival analysis may be more suitable.

Response: Thank you for seeking more clarification. Survival analysis is normally conducted on data for which the period of observation is long. Given the relatively short duration over which the primary outcome was assessed (14 days) we did not, and still do not, think that a time-to-event analysis would add any useful insight.

We are grateful for the question about the time-point at which mortality was measured and have, therefore, edited the text as follows:

Section: “Discussion”; subsection “Key secondary Outcomes”, New 2nd sentence in 2nd paragraph: «All deaths occurred in hospital. No deaths were recorded after discharge during the 14-day telephone follow-up period.»

2) Most secondary outcomes were not analyzed or presented in the results section.

Response: In this paper we have focused on the primary outcome “need for ICU admission”. The secondary outcome data are available for independent analysis via the supplementary information. However, any analysts seeking to undertake this should be aware that data collection for some secondary endpoints was not as complete as we had hoped due to pressures caused by the pandemic wave that India was going through at the time.

3) The results section was not clearly written. The primary analysis should focus on the comparison between two treatment groups. However, in “Primary Outcome”, primary findings were buried in subgroup analysis (Eg. Comorbidities, lung disease and etc). The subgroup analysis should be moved to another section.

Response:

We agree with the reviewer that the clarity of this section can be improved. We have, therefore, edited the text in the “Primary Outcome” section as shown below.

We also agree with the reviewer that the focus of the primary analysis must be on the comparison between treatment groups, although we argue that we have actually built the section in this way. The first, introductory paragraph of the “Primary Outcome” section provides the relevant primary outcome data of the whole trial population and of the clinically important strata. As these are actual primary outcome data, we argue they should stay in this (“Primary Outcome”) section of the manuscript. On the other hand, to put them at the end of this section, which (in the actual version) ends with the most exciting result of the trial, seems inappropriate.

Edited text as follows:

Section: Results; subsection “Primary Outcome”, 1st paragraph, first sentence:

«Of the 387 patients included in the trial, 77 (19.9%) reached the primary outcome, i.e., they developed COVID-19 symptoms deemed by the attending physicians severe enough to require critical care in the ICU (Figure 3).»

Section: Results; subsection “Primary Outcome”, 2nd paragraph, a new, first sentence and a slightly modified, second sentence as follows:

«For comparison of the primary outcome between the two treatment arms in this trial we carried out three types of analysis: First, in the intention to treat analysis (ITT) of all 387 participants, 46 of 195 patients (23.6%) randomized to SoC reached the primary outcome compared to 31 of 192 patients (16.1%) randomized to SoC+Doxy (OR 0.617, 95% CI 0.369 to 1.027, p=0.063) (Figure 4).»

4) In “Key secondary Outcomes”, ICU admission were mentioned again. This is confusing as ICU is not secondary but primary.

Response: Thank you for giving us the opportunity to clarify. The primary endpoint of our trial is need for ICU admission. However, as explained in the manuscript, not all patients reaching this (primary) endpoint could actually be admitted to ICU, for reasons explained in the manuscript. Thus, actual ICU admission was included as a secondary endpoint and is discussed under key secondary outcomes in the manuscript. The reference of the primary outcome of need for ICU admission is to contextualise the secondary (but closely related) endpoint of actual admission.

5) Table 1 present either mean with SD or median with range. How are the two groups compared?

Response: We present means and ranges for continuous variables and n (%) for categorical ones. We can amend to mean (SD) if the editor/reviewers consider this essential for acceptance, but we feel the current presentations provide an equivalent level of detail. Interested readers have access to the raw data via the paper’s supplements if they are sufficiently motivated to derive alternative summaries of their own.

6) Add p values to Table 1 and 2.

Response: We respectfully disagree with the suggestion from reviewer 2 that p-values should be added to this table (and to table 2). Such an action is contrary to guidance from the CONSORT Consortium which states that "such significance tests assess the probability that observed baseline differences could have occurred by chance; however, we already know that any differences are caused by chance. Tests of baseline differences are not necessarily wrong, just illogical"

(for full guidance please see https://www.consort-statement.org/checklists/view/32--consort-2010/510-baseline-data, second para) See also deBoer et al (2015) Int J Behav Nutr Phys Act https://doi.org/10.1186%2Fs12966-015-0162-z

7) Table 2 put n (%) in one cell.

Response: Table 2 has been edited according to the reviewer’s suggestion (please see revised manuscript).

8) Figure 2 a flow chart for outcome is not necessary and can be omitted.

Response: We believe Reviewer #2 is actually referring to Figure 3, as Figure 1 is the CONSORT diagram which needs to be included in the manuscript as per guidance of the journal, and Figure 2 is showing enrollment into the trial. The flow chart in Figure 3 provides the reader with an overview of the main results. We agree that it could be omitted from the main manuscript text and propose (to the academic editor) that, if he so chooses, the current Figure 3 can be moved to the supplementary section.

9) “The two treatment groups were matched” on Page 7 study participants and Page 8 2nd paragraph. The authors may change the statement to “two groups are not significantly different”. Matching has different meaning in statistics.

Response: We thank the editor for this comment and have edited, accordingly, the text in the manuscript.

Please see the following edited text in the manuscript:

Section: “Results”, subsection “Study participants”, 2nd paragraph, third sentence, as follows:

«The incidence of various risk factors (RF) was similar in the two treatment groups (Table 1).»

Section: “Results”; subsection “Study treatment”, 2nd paragraph, first sentence, as follows:

«Participants in the two treatment groups were similar with respect to concomitant medication (Table 1), …»

Final note to academic editor

On review of our manuscript, we have deleted the following sentence from the manuscript as it is redundant:

Section: “Methods”, subsection “Data collection and analysis”, 2nd paragraph, deleted the following (second) sentence «Data could not be persisted on the app and, therefore, needed to be downloaded on every load, and new data needed to be uploaded before exit; thus, the app synchronized all changes in real time with a remote database in the cloud.»

Thank You for considering our revised manuscript for publication in PLoS One.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (32.5KB, docx)

Decision Letter 1

Davor Plavec

15 Nov 2022

PONE-D-22-10587R1Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: a randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU)PLOS ONE

Dear Dr. Gadola,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: Please revise as suggested by the reviewers.==============================

Please submit your revised manuscript by Dec 30 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Davor Plavec, MD, MSc, PhD, Prof.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Please revise as suggested by the reviewer.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for your response. In my opinion, all major and minor comments have been adequately addressed.

Reviewer #2: For table 1, please follow the PLOS ONE guidelines

https://www.equator-network.org/wp-content/uploads/2013/03/SAMPL-Guidelines-3-13-13.pdf

Summarize data that are approximately normally

distributed with means and standard deviations

(SD). Use the form: mean (SD), not mean ± SD.

Summarize data that are not normally distributed

with medians and interpercentile ranges, ranges, or

both. Report the upper and lower boundaries of

interpercentile ranges and the minimum and

maximum values of ranges, not just the size of the

range

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Jan 23;18(1):e0280745. doi: 10.1371/journal.pone.0280745.r004

Author response to Decision Letter 1

18 Dec 2022

Response to Reviewer 2

PONE-D-22-10587

Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: a randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU)

Dear Editor and reviewers,

We thank Reviewer #1 for his conclusion that all major and minor comments have been adequately addressed in our previously submitted revised manuscript.

Reviewer #2 raised the following point to which we will respond in the following.

Points raised by Reviewer #2 (in response to submitted 1st version of our revised manuscript):

1) For table 1, please follow the PLOS ONE guidelines

(https://www.equator-network.org/wp-content/uploads/2013/03/SAMPL-Guidelines-3-13-13.pdf):

Summarize data that are approximately normall distributed with means and standard deviations (SD). Use the form: mean (SD), not mean ± SD.

Summarize data that are not normally distributed with medians and interpercentile ranges, ranges, or both. Report the upper and lower boundaries of interpercentile ranges and the minimum and maximum values of ranges, not just the size of the range

Response: As it is unusual, in our experience, to distinguish between normal and non-Normal data rather than continuous and discrete data, it would be helpful if Reviewer 2 could indicate which continuous variables they regard as “approximately normally distributed” and those which they do not.

We are also unsure about the comment of Reviewer 2 in relation to “interpercentile range” without specification of the percentiles that define the lower and upper boundaries of the range. Reporting interquartile ranges as well as minima, maxima and medians makes no sense for dichotomous and other categorical variables (which are by definition not Normal), such as gender, baseline PCR test results, the presence or absence of comorbidities and the use of concomitant medications.

In an effort to oblige Reviewer 2’s comment, we have made the following changes to the text of the manuscript on page 7, “Results”, first paragraph of “Study participants”, 3rd and 4th sentence, to include median, IQR, min and max for age and temperature:

“The median age (IQR, min, max) of participants was 58 (49 – 66, 40, 90) years, with a 2:1 ratio of males to females. The median axillary body temperature (IQR, min, max) at study entry was 38.2°C (38.0°C – 38.4°C, 37.8°C, 40.8°C) in the SoC+Doxy group and 38.3° C (37.6°C – 38.3°C, 37.6°C, 40.6°C) in the SoC group.”

In the revised Table 1, we summarise the following variables:

Variable Comment

Age Continuous, but not Normal. We currently report mean and range. I’ve added the requested summaries in reviewer2-requests.pdf

Sex Dichotomous. We currently report n and percentage. I suggest no change

Baseline PCR status Dichotomous. We currently report n and percentage. I suggest no change

Body temp Continuous. Is it Normal? Probably not. I’ve added the requested summaries in reviewer2-requests.pdf

Comorbidities Dichotomous. We currently report n and percentage. I suggest no change

Con meds Dichotomous. We currently report n and percentage. I suggest no change

Table 2: Suggest no changes: all variables are dichotomous.

Final note to academic editor

Thank You for all your efforts with our manuscript and for considering this revised version for publication in PLoS One.

Attachment

Submitted filename: 2nd Response to Reviewers_Dec 2022.docx

Click here for additional data file. (20KB, docx)

Decision Letter 2

Davor Plavec

8 Jan 2023

Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: a randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU)

PONE-D-22-10587R2

Dear Dr. Gadola,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Davor Plavec, MD, MSc, PhD, Prof.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript is acceptable in its current form.

Reviewers' comments:

Acceptance letter

Davor Plavec

13 Jan 2023

PONE-D-22-10587R2

Doxycycline for the prevention of progression of COVID-19 to severe disease requiring intensive care unit (ICU) admission: a randomized, controlled, open-label, parallel group trial (DOXPREVENT.ICU)

Dear Dr. Gadola:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Davor Plavec

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Number of Risk factors associated with severe COVID-19 in the study.

    (DOCX)

    Click here for additional data file. (13.9KB, docx)
    S1 Fig. Antiviral effect of doxycycline, tetracycline and remdesivir.

    Effect of doxycycline and tetracycline compared to remdesivir on SARS-COV-2 infection of ACE-2-overexpressing lung-derived cell lines in vitro.

    (TIF)

    Click here for additional data file. (537.2KB, tif)
    S1 File. Study protocol.

    (PDF)

    Click here for additional data file. (523.8KB, pdf)
    S2 File. Sample size calculation.

    (PDF)

    Click here for additional data file. (252.5KB, pdf)
    S3 File. Detailed material and methods of in vitro studies.

    (PDF)

    Click here for additional data file. (67.2KB, pdf)
    S4 File. Reporting checklist for randomised trials (based on the CONSORT guidelines).

    (PDF)

    Click here for additional data file. (102.1KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (32.5KB, docx)
    Attachment

    Submitted filename: 2nd Response to Reviewers_Dec 2022.docx

    Click here for additional data file. (20KB, docx)

    Data Availability Statement

    All data underlying the results presented in the study are available online on GitHub (https://github.com/PuzzledFace/DoxyICU_Results).

    Articles from PLOS ONE are provided here courtesy of PLOS

    RESOURCES