A multicenter, randomized, open-label, controlled trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective study in adult patients with mild to moderate coronavirus disease 2019 (COVID-19).

OBJECTIVE: In this study, we evaluated the efficacy of hydroxychloroquine (HCQ) against coronavirus disease 2019 (COVID-19) via a randomized controlled trial (RCT) and a retrospective study.
METHODS: Subjects admitted to 11 designated public hospitals in Taiwan between April 1 and May 31, 2020, with COVID-19 diagnosis confirmed by pharyngeal real-time RT-PCR for SARS-CoV-2, were randomized at a 2:1 ratio and stratified by mild or moderate illness. HCQ (400 mg twice for 1 d or HCQ 200 mg twice daily for 6 days) was administered. Both the study and control group received standard of care (SOC). Pharyngeal swabs and sputum were collected every other day. The proportion and time to negative viral PCR were assessed on day 14. In the retrospective study, medical records were reviewed for patients admitted before March 31, 2020.
RESULTS: There were 33 and 37 cases in the RCT and retrospective study, respectively. In the RCT, the median times to negative rRT-PCR from randomization to hospital day 14 were 5 days (95% CI; 1, 9 days) and 10 days (95% CI; 2, 12 days) for the HCQ and SOC groups, respectively (p = 0.40). On day 14, 81.0% (17/21) and 75.0% (9/12) of the subjects in the HCQ and SOC groups, respectively, had undetected virus (p = 0.36). In the retrospective study, 12 (42.9%) in the HCQ group and 5 (55.6%) in the control group had negative rRT-PCR results on hospital day 14 (p = 0.70).
CONCLUSIONS: Neither study demonstrated that HCQ shortened viral shedding in mild to moderate COVID-19 subjects.

RCT Entities:   Population Interventions Outcomes

Introduction

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is an ongoing pandemic. The outbreak was first localized to Wuhan, Hubei Province, People’s Republic of China (PRC) on December 31, 2019 [1]. On January 30, 2020, the World Health Organization (WHO) declared that the outbreak was a public health emergency of international concern and thereafter recognized it as a pandemic [2, 3]. As of June 20, 2020, more than eight million cases of COVID-19 have been reported in 187 countries and territories. More than 450,000 deaths have been associated with this infection [4]. Taiwan is a close neighbor of PRC and reported its first COVID-19 case on January 21, 2020 [5]. As of June 20, 2020, there were 446 confirmed COVID-19 cases in Taiwan. As a result of the early implementation of social distancing, hand hygiene, and face masks, Taiwan has had a low incidence of domestic COVID-19 cases [6].

There is no known effective medical treatment against COVID-19. The mechanisms of potentially efficacious antiviral agents include the inhibition of RNA-dependent RNA polymerase (remdesivir [7-9] and favipiravir [10]), protease inhibition (lopinavir/ritonavir [9, 11, 12] and ivermectin [13]), the blockade of virus-cell membrane fusion (recombinant human angiotensin-converting enzyme 2 [14] and chloroquine and hydroxychloroquine (HCQ) [8, 15], and the modulation of the human immune system (interferon [9] and interleukin-6 blockers [16, 17]).

Chloroquine phosphate is a well-known antimalarial drug that has been on the market for several decades. An in vitro study showed that chloroquine is effective against SARS-CoV-2 at the entry and post-entry infection stages [8]. Chloroquine may either increase endosomal pH by blocking the fusion of the virus and the host cell membrane [18] or by interfering with cell receptor glycosylation [19]. Chloroquine may also repress proinflammatory signaling and cytokine (IL-1, IL-6, and TNF) production by inhibiting lysosome activity in antigen-presenting cells [20]. Compared to chloroquine, HCQ has an additional hydroxyl group, lower toxicity, and similar antiviral efficacy.

HCQ received emergency approval by the US Food and Drug Administration (FDA) for use in the treatment of COVID-19 [21]. However, the efficacy of HCQ against SARS-CoV-2 has been highly controversial. Certain elite journals have retracted influential papers published on the efficacy of HCQ against COVID-19 [22, 23]. HCQ is widely available in Taiwan and has become a potential candidate drug therapy against COVID-19 there. Therefore, an open-label randomized controlled trial (RCT) involving multiple centers was conducted to evaluate HCQ efficacy and tolerability in adult patients with mild to moderate COVID-19. These results would be compared with the standard of care treatment (SOC) in Taiwan. In addition, a retrospective observational study was also performed.

Methods

Clinical trial

Participants

The clinical trial was conducted at 11 public hospitals in northern, central, and southern Taiwan, including Keelung Hospital, Taipei Hospital, Taoyuan General Hospital, Miaoli General Hospital, Taichung Hospital, Feng Yuan Hospital, Nantou Hospital, Chang Hua Hospital, Chia Yi Hospital, Tainan Hospital, and Pintung Hospital, affiliated with the Ministry of Health and Welfare, Taiwan, between April 1 and May 31, 2020. Enrolled patients were aged 20–79 y and confirmed positive for SARS-CoV-2 infection by real-time reverse transcription polymerase chain reaction (rRT-PCR). They provided signed informed consent to participate in this study. Upon admission, the patients were stratified into three groups: (1) mild illness without evidence of infiltration according to chest X-ray; (2) moderate illness with evidence of infiltration according to chest X-ray but neither respiratory distress nor supplemental oxygen requirement; and (3) severe illness with respiratory distress, oxygen supplementation, and evidence of infiltration according to chest X-ray. Participants in group 3, presenting with severe illness, were excluded from this study. The following patients were excluded from the trial: (a) documented history of hypersensitivity to quinine derivatives; (b) retinal disease; (c) hearing loss; (d) severe neurological or mental illness; (e) pancreatitis; (f) lung disease; (g) liver disease (alanine aminotransferase/aspartate aminotransferase > 3× the normal upper limit); (h) kidney disease (estimated glomerular filtration rate < 30 mL/min/1.73 m2 according to MDRD or CKD-EPI); (i) hematological disease; (j) cardiac conduction abnormalities at electrocardiographic screening with long QT syndrome or QTcF interval > 450 msec for males and > 470 msec for females according to Fridericia’s correction at screening; (k) known HIV infection; (l) active hepatitis B or C without concurrent treatment (positive for hepatitis B [HBsAg and HBeAg] or hepatitis C ribonucleic acid [RNA] titer > 800,000 IU/mL); (m) G6PD; (n) psychiatric disorders and alcohol/substance dependence/abuse that may jeopardize patient safety; and (o) pregnant or breast-feeding women.

Clinical course

COVID-19 symptoms were recorded and followed up daily. Chest X-rays, electrocardiography, and the biomarkers complete blood count, white blood cell differential count, biochemistry, prothrombin time, activated partial thromboplastin time, ferritin, highly active troponin I, C-reactive protein, and erythrocyte sedimentation rate were tested upon admission and every 4 days after enrollment.

PCR assay

Nasopharyngeal swab and sputum were collected every other day until patient discharge following three consecutive negative results or day 14 of the study, depending upon which criterion was met first. SARS-CoV-2 RNA was assessed by rRT-PCR using a hydrolysis probe-based system targeting genes encoding envelope (E) protein and RNA-dependent RNA polymerase (R) as previously described [24]. Negative viral RNA detection was defined as cycle thresholds (Ct) values > 38 for the E gene and negative for the R gene. The PCR assay was conducted at the National Laboratory of the Taiwan Centers for Disease Control.

Study design

Eligible subjects were randomly assigned by an interactive web response system in a 2:1 ratio to receive either HCQ plus standard of care (SOC) or SOC alone. They were stratified by mild or moderate illnesses within 4 days of diagnosis. The incidence of domestic cases was low and the estimated case number was 45 (30:15). The HCQ administration plan was 400 mg b.i.d. on day 1 and 200 mg b.i.d. for 6 days on days 2–7. Both study group and comparison group received standard of care comprising supportive treatment without antibiotics for subjects with mild clinical COVID-19 symptoms and with antimicrobial therapy for subjects presenting with moderate clinical COVID-19 symptoms. The treatment consisted of: (1) ceftriaxone 2 g daily for 7 days ± azithromycin 500 mg on day 1 and 250 mg on days 2–5; or (2) levofloxacin 750 mg daily for 5 d; or (3) levofloxacin 500 mg daily; or (4) moxifloxacin 400 mg daily for 7–14 days for subjects allergic to ceftriaxone or azithromycin or according to physician discretion. Oseltamivir 75 mg b.i.d. was administered for 5 days to subjects presenting with concomitant influenza A or B infection. No HCQ dose reduction, modification, or change in administration frequency was recommended during the study period. This prospective randomized controlled clinical trial was registered at https://clinicaltrials.gov/ct2/show/NCT04384380?term=Hydroxychloroquine&cond=COVID-19&cntry=TW&draw=2&rank=1(NCT04384380) (Because the appendix of the protocol was in Mandarin, the text had to be translated to English to meet QC criteria. The registration of the clinical trial was accepted on May/12/2020, after the first enrollment on Apr/1/2020. However, the authors confirm that all ongoing and related trials for this drug/intervention were registered).

Outcome measurement

The primary endpoint was to evaluate the time to negative rRT-PCR assessments from randomization, up to 14 days, by arm. The secondary endpoints were to evaluate the proportion of negative viral rRT-PCR on hospital day 14, the resolution of clinical symptoms (time to clinical recovery), the proportion of discharges by day 14, and the mortality rate. HCQ safety and tolerability were also evaluated.

Statistical analysis

Data were entered into an electronic clinical trial information management system (CTIMeS; National Health Research Institutes, Taiwan) by study coordinators and summarized with SAS® v. 9.2 (SAS Institute Inc., Cary, NC, USA). All treatment data are summarized using descriptive statistics including continuous variables (number of non-missing observations, means, standard deviations (SD), medians, minima, and maxima), categorical variables (frequencies and percentages), and time to event variables (number of non-missing observations (N), medians, minima, and maxima). The negative rRT-PCR rates between treatment and control arms were compared using Fisher’s exact test and Cochran-Mantel-Haenszel (CMH) test, stratified by mild or moderate illness. The Kaplan-Meier method was used to estimate the distribution of time to a negative rRT-PCR test. The median time to a negative rRT-PCR and its 95% CI were calculated. The log-rank test with/without adjustment by disease severity was used to compare the distribution of time to negative rRT-PCR between arms. An analysis of covariance (ANCOVA), adjusted for mild/moderate diseases, and repeated measurements of analysis of variance (ANOVA) were performed to compare the area under the curve and Ct value across different time between arms. All tests were two-tailed. A p-value <0.05 was considered significant.

Ethical statement

The study protocol was approved by the Institutional Review Board of Taoyuan General Hospital (IRB No. TYGH109014). The study was performed according to Good Clinical Practices recommended by the Declaration of Helsinki and its amendments.

Retrospective observational study

The study was conducted at the same 11 hospitals. Cases were aged 20–79 y and confirmed positive for SARS-CoV-2 infection by rRT-PCR between January 25 and March 31, 2020. Medical registers were reviewed and clinical symptoms, laboratory data, and medications were recorded. Patients who had undetected virus within 2 days of hospitalization were excluded. The study protocol was approved by the Institutional Review Board of Taoyuan General Hospital (IRB No. TYGH109024).

Results

Thirty-three cases were enrolled in the RCT (Fig 1). The mean age (SD) of the subjects was 32.9 (10.7) y. Males comprised 57.6% of all subjects. A few individuals presented with underlying chronic illnesses. The initial presentation included anosmia (51.5%), cough (48.5%; 94% of these had mild cough and needed no symptomatic treatment), ageusia (30.0%), nasal obstruction (24.2%), and sore throat (21.2%). Of these, 12.1% of the cases had pneumonia according to the X-ray images (Table 1).

Fig 1

Patient disposition in the multicenter, open-label, randomized controlled trial (a) and the retrospective study (b) of hydroxychloroquine. Abbreviations: HCQ: hydroxychloroquine; SOC: standard of care.

Table 1

Baseline demographic and clinical characteristics of participants in the multi-center, open-label, randomized clinical trial and the retrospective observational study.

	Randomized controlled trial
	HCQa		SOCb		Overall
No. randomized patients	21		12		33
Mean age in years (sd)	33.0	(12.0)	32.8	(8.3)	32.9	(10.7)
Median (range)	30.0	(22–68)	33.5	(22–44)	31.0	(22–68)
Male (%)	11	(52.4%)	8	(66.7%)	19	(57.6%)
Stratification
Mild (%)	19	(90.5%)	10	(83.3%)	29	(87.9%)
Moderate (%)	2	(9.5%)	2	(16.7%)	4	(12.1%)
Symptoms
Median of QTc msec(range)	424	(356–453)	427.5	(392–458)	424	(356–458)
Anosmia (%)	11	(52.4%)	6	(50%)	17	(51.5%)
Cough (%)	9	(42.9%)	7	(63.6%)	16	(48.5%)
Ageusia (%)	4	(19.0%)	6	(50%)	10	(30.3%)
Nasal obstruction (%)	4	(19.0%)	4	(33.3%)	8	(24.2%)
Sore throat (%)	3	(14.3%)	4	(33.3%)	7	(21.2%)
Shortness of breath (%)	1	(4.8%)	1	(8.3%)	2	(6.1%)
Fever (%)c	1	(4.8%)	0	(0%)	1	(3.0%)
	Retrospective observational study
	HCQ		Control		Total
No. patients	28	9	37
Mean age (Std)	34.3	(14.5)	31.3	(18.0)	35.8	(14.5)
Median (range)	28	(20–66)	44	(21–56)	29	(20–66)
Male (%)	14	(50%)	3	(33.3%)	17	(45.9%)
Stratification
Mild (%)	23	(82.1%)	6	(66.7%)	29	(78.4%)
Moderate (%)	5	(17.9%)	3	(33.3%)	8	(21.6%)
Symptoms
Median of QTc msec(range)	NA		NA		NA
Anosmia (%)	8	(28.6%)	1	(11.1%)	9	(24.3%)
Cough (%)	18	(64.3%)	3	(33.3%)	21	(56.8%)
Ageusia (%)	5	(17.9%)	1	(11.1%)	6	(16.2%)
Sore throat (%)	8	(28.6%)	2	(22.2%)	10	(27.0%)
Shortness of breath (%)	0	(0%)	1	(11.1%)	1	(2.7%)
Fever (%)c	15	(53.6%)	3	(33.3%)	18	(48.6%)

HCQ: hydroxychloroquine

bSOC: standard of care

central temperature

≥38°C; dNA: not available

Twenty-one cases were randomized to the HCQ group and 12 cases were randomized to the SOC group. However, two in the HCQ group and one in the SOC group had withdrawn consents before the first dose was administered. One (4.8%) in the HCQ group and two (16.7%) in the SOC group were concomitantly administered azithromycin. The median times to negative rRT-PCR assessment from randomization to hospital day 14 were 5 days (95% CI; 1, 9 days) for the HCQ group and 10 days (95% CI; 2, 12 days) for the SOC group (p = 0.40) (Fig 2; Table 2). By day 14, 81.0% (17/21) and 75.0% (9/12) of the subjects in the HCQ and SOC groups, respectively, had undetected virus (p = 0.36) (Table 2). The analysis of the area under the curve of the Ct value in the study interval showed that the least square mean (SD) was 501.7 (18.0) for the HCQ group and 496.6 (21.2) for the SOC group. The treatment difference was 5.1 (95% CI; -37.1, 47.2) (p = 0.81) (Table 3). The revolution of the mean Ct value in the study interval was also studied. The treatment effect on the Ct value was not significant (p = 0.4717) (Fig 3). For subjects presenting with mild illness, the median times to negative rRT-PCR assessment from randomization were 5 days (95% CI; 1, 11 days) for the HCQ group and 11 days (95% CI; 1, 12 days) for the SOC group (p = 0.31) (S1 Fig; S1 Table).

Fig 2

Probabilities of non-negative responses vs. time (days) for subjects in the HCQ and SOC groups in the multicenter, open-label, randomized controlled trial.

Abbreviations: HCQ: hydroxychloroquine; SOC: standard of care.

Table 2

Proportions of negative rRT-PCR assessments on day 14 and median times to negative rRT-PCR results after randomization in the multicenter, open-label, randomized controlled trial.

Group	N	Negativea	P-valueb	Median time to negativec (Days, 95% CId)	p-valuee
HCQf	21	17 (81.0%)	0.71	5 (1, 9)	0.40
SOCg	12	9 (75.0%)		10 (2, 12)

aNegative event: both pharyngeal swab and sputum showed negative results

bCMH test: stratified by clinical syndrome

cTime to negative = Event date or censored date–start day

dCI: confidence interval

eLog-rank test stratified by clinical syndromes

fHCQ: hydroxychloroquine

gSOC: standard of care.

Table 3

Comparison of area under curve of Ct value between subjects in the HCQ and SOC groups in the multicenter, open-label, randomized controlled trial.

	HCQa (N = 21)			SOCb (N = 12)			Treatment difference (95% CI)d	Treatment effect, p-valuee
	n	Mean (SD)c	Median (range)	n	Mean (SD)	Median (range)
Observed data	19	506.5 (43.7)	495.2 (424.0, 608.5)	11	501.6 (67.4)	495.1 (386.6, 594.5)	4.9 (-36.5, 46.3)
Least square mean (SE)f		501.7 (18.0)			496.6 (21.1)		5.1 (-37.1, 47.2)	0.81

aHCQ: hydroxychloroquine

bSOC: standard of care

cSD: standard deviation

dCI: confidence interval

ep-value: ANCOVA model

fLeast-square means based on ANCOVA model with treatment effect. SE is the standard error corresponding to the LSmean.

Fig 3

Mean Ct value of viral rRT-PCR vs. time (days) for subjects in the HCQ and SOC groups in the multicenter, open-label, randomized controlled trial.

Abbreviations: Ct: Cycle threshold; HCQ: hydroxychloroquine; rRT-PCR: real-time reverse transcription polymerase chain reaction; SOC: standard of care.

By day 14, 28.6% of the subjects in the HCQ group and 41.7% of the subjects in the SOC group presented with clinical recovery (p = 0.51) (S2 Fig; S2 Table). By day 14, 19.0% and 16.7% of the subjects in the HCQ and SOC groups, respectively, were no longer quarantined. There was no mortality in the present study.

No severe adverse events were reported in the clinical trial. Grades 1 and 2 HCQ-related adverse events included headache (21.1%), dizziness (5.3%), gastritis (5.3%), diarrhea (5.3%), nausea (5.3%), and photophobia (5.3%). The median QTc (ranges) were 429.5 msec (340–467) on day 4 and 421 msec (391–462) on day 8. No severe prolongation was noted.

Thirty-seven cases were enrolled in the observational study (Fig 1). The mean age (SD) of the subjects was 35.8 (14.5) years. There were 17 (45.9%) male subjects. Twenty-three (82.1%) in the HCQ group and zero (0%) in the control group were administered azithromycin concomitantly.

The median times (ranges) to undetected virus were 15 (6–31) days for the HCQ group and 14 (7–22) days for the control group (p = 0.37) (S3 Table). On hospital day 14, the airway samples of 12 subjects (42.9%) in the HCQ group and 5 subjects (55.6%) in the control group turned negative in rRT-PCR (p = 0.70). On hospital day 14, the mean log change (SD) of the Ct value was 7.6 (4.8) in the HCQ group and 11.6 (5.6) in the control group, respectively (p = 0.0625).

Discussion

The present multicenter, randomized, open-label clinical trial showed that HCQ failed the primary endpoint of shortening the viral clearance interval. The retrospective study also demonstrated that HCQ conferred no therapeutic benefit to the COVID-19 cases investigated here.

Currently, there are > 1,000 ongoing COVID-19 clinical trials worldwide. In multicenter clinical trials conducted in China, chloroquine phosphate has demonstrated efficacy at preventing the progression of COVID-19-related pneumonia [25]. The Chinese guideline [26] recommendation for adults aged 18–65 y is 500 mg twice daily for 7 days in patients weighing > 50 kg and 500 mg twice daily for 2 days followed by 500 mg once daily for 5 days in patients weighing < 50 kg. A clinical trial in Italy planned to include 440 patients and test two different chloroquine doses but was suspended after 81 patients had been enrolled because of excessive QTc prolongation and high mortality rates in the high-dose (600 mg twice daily for 10 days) group [27]. Compared with the study of Borba et al. [27], the participants in our clinical trial were younger, did not receive high HCQ doses, and presented with a low incidence of QTc prolongation.

The first open-label, non-randomized study of HCQ treatment for COVID-19 was conducted in France [15]. Gautret et al. treated 20 patients with 200 mg HCQ thrice daily for 10 days. Six of these patients were administered concomitant azithromycin and 16 other patients received no HCQ therapy. The efficacy of HCQ at clearing the virus was remarkable: 70.0% by day 6 post-inclusion in treated patients vs. 12.5% at day 6 post-inclusion in untreated patients (p < 0.001). However, six of the patients being administered HCQ became clinically worse or were lost to follow-up. Consequently, they were excluded from the final analysis and interpretation of the data became very difficult. Hence, the same team performed an uncontrolled non-comparative observational study on a cohort comprising 80 patients presenting with mild COVID-19 symptoms who underwent HCQ and azithromycin treatment [28]. Subsequently, rapid declines in nasopharyngeal viral load were reported (83% and 93% of the treated patients at days 7 and 8, respectively). A large-scale observational study was conducted on 1,376 COVID-19 patients in New York [29] of whom 58.9% were administered HCQ 600 mg twice on day 1 and 400 mg daily thereafter for a median of 5 days. However, HCQ administration was not associated with the composite intubation or death endpoint (hazard ratio = 1.04; 95% CI = 0.82, 1.32). Another large-scale observational study, conducted on 2,541 COVID-19 patients in southeast Michigan [30], demonstrated that HCQ administration provides a 66% hazard ratio reduction in in-hospital mortality compared with no HCQ treatment (p < 0.001).

A pilot randomized controlled clinical trial (No. NCT04261517) on HCQ therapy for COVID-19 was performed at a single center in Shanghai. It enrolled 30 patients with 1:1 randomization [31]. The study did not reveal any significant difference between the two treatment groups. The viral clearance rates in the throat swab samples were relatively high by day 7 after enrolment in both groups (83.7% vs. 96.3%, respectively; p > 0.05). Moreover, the HCQ dose was comparatively low and the treatment interval was relatively short (400 mg daily for 5 days; no loading). All patients in this trial received aerosolized interferon alpha and most of them were also administered antiviral drugs that may have diminished or augmented the therapeutic efficacy of HCQ.

Another clinical trial (No. ChiCTR2000029559) enrolled 62 subjects of whom 31 received HCQ 400 mg/d for 5 days. The remaining 31 constituted the control group [32]. After 5 days, the clinical recovery time of the HCQ group was significantly shortened and fever and cough were alleviated relatively faster (p < 0.05). Pneumonia improved in 81% of the subjects in the HCQ group and 55% of the patients in the control group (p < 0.05). Although this study corroborated the therapeutic efficacy of HCQ, it did not measure or report viral clearance rates.

A recent multicenter, randomized, open-label clinical trial was conducted at 16 COVID-19 treatment centers in China and disclosed negative conversion rates of 85.4% and 81.3% for SARS-CoV-2 28 days after randomization into HCQ + SOC and SOC groups, respectively [33]. In Tang’s study, 98.6% (148/150) of the enrolled subjects were categorized as presenting with mild to moderate illness but 63% of the enrollees had also been treated with antiviral agents (arbidol, ribavirin, lopinavir/ritonavir, oseltamivir, or entecavir) which may have interfered with HCQ efficacy.

The strength of our RCT lies in the fact that the enrollees were randomized within 4 days of diagnosis. Thus, the earliest possible intervention could be made. Clinical courses could be clearly and accurately monitored because of early diagnosis and treatment with SOC or HCQ. Furthermore, the HCQ treatment regimen used here consisted of loading twice with 400 mg followed by 200 mg twice daily for 7 days [34]. A physiologically based pharmacokinetic model indicated that the aforementioned dose used in the present study was ideal for HCQ therapy [35]. Third, no antiviral therapy was administered and only one case in the HCQ group and two cases in the SOC group had azithromycin treatment.

In contrast, there were limitations to the present study. Only patients presenting with mild to moderate disease symptoms were enrolled to determine the viral clearance efficacy of HCQ. Hence, there is relatively little data on the impact of severe disease in terms of intubation and mortality. At clinical trial launch, there were no indigenous and very few imported cases in Taiwan. Therefore, study enrollment was prematurely stopped. The low case numbers in the present study might account for the apparent lack of superior efficacy of HCQ, and only reflects the effects of HCQ in young COVID-19 patients with mild symptoms. However, 81% of the HCQ group and 75% of the SOC group had confirmed viral clearance on hospital day 14. Moreover, according to Taiwan CDC regulations, subjects could not leave quarantine until they presented with at least three consecutive negative rRT-PCR results. The outcome of this RCT may assist the Taiwan CDC in its decision to release quarantined patients when medical resources are in short supply. Another limitation of the study was that the mean patient age (SD) was 32.9 (10.7) y as opposed to 51.1 y (13.9) for Borba et al., 45.1 y for Gautret et al., 44.7 y (15.3) for Chen et al., 46.1 y (14.7) for Tang et al, and 63.7 (16.5) for Arshad et al. [15,27,30-32]. For this reason, the observed rates of cardiac and retinal toxicity were low in the present study. Electrocardiogram monitoring was performed frequently and close attention was paid to any changes in patient QTc interval, vision, and neurological symptoms. Lastly, readers might question the gap in median time to negative viral detection between the retrospective observational study and RCT. As the time from infection to admission, and the time from diagnosis to treatment were diverse, and the frequency of viral sampling was not the same in the retrospective observational cohort, the different outcome compared with the present RCT was not surprising.

Conclusions

Both the retrospective and randomized clinical studies performed here failed to demonstrate HCQ efficacy at shortening viral shedding in the majority of subjects presenting with mild COVID-19 symptoms. As the case number is small, the absence of evidence is not necessarily evidence of absence and, in future research, large-scale studies involving more patients should be conducted to investigate new agents or combinational therapy and explore viral dynamics.

CONSORT 2010 checklist of information to include when reporting a randomised trial*.

(DOC)

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Probabilities of non-negative responses with mild symptoms vs. time (days) for subjects in the HCQ and SOC groups in the multicenter, open-label, randomized controlled trial.

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Probabilities of non-clinical recovery vs. time (days) for subjects in the HCQ and SOC groups in the multicenter, open-label, randomized controlled trial.

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Comparison of median times and negative viral rRT-PCR results between subjects in the HCQ and SOC groups presenting with mild symptoms in the multicenter, open-label, randomized controlled trial.

(DOCX)

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Comparison of times to clinical recovery between subjects in the HCQ and SOC groups in the multicenter, open-label, randomized controlled trial.

(DOCX)

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Proportions of negative rRT-PCR assessments on day 14 and median times to negative rRT-PCR results in the multicenter, retrospective study.

(DOCX)

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(PDF)

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8 Sep 2020

PONE-D-20-20024

A Multicenter, randomized, open-label, controlled trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective study in adult patients with mild to moderate Coronavirus disease 2019 (COVID-19)

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study design or conduction, data collection, analysis or interpretation, writing of the

manuscript, or decision to submit it for publication. The authors also thank Taiwan Biotech

Co. Ltd. for their donation of investigational products..."

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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: Yes

**********

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: No

**********

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

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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: The authors present data from a Taiwan-based multi-center (11 publically funded hospitals) studies addressing the question of whether hydroxychloroquine has therapeutic efficacy for the treatment of COVID-19. Data is presented for an open label RCT (NCT04384380) and an observational retrospective study conducted for up to 14 days. There are limitations to the study that to a greater extent the authors have emphasized but a number of issues need to better emphasized and discussed.

1/ The authors have explained that recruitment was limited to few cases presenting in Taiwan. Only 33 were included for the RCT and 37 for the retrospective study. These numbers are very small. Furthermore, as can be seen from Table 1 there are very few who are classified as “moderate” – 2 for HCQ and 2 for SOC in the RCT and 5 and 3 respectively in the retrospective study. This needs to be better emphasized as the study data and conclusions really can only relate to mild cases AND comparatively also a young population ~30-35. Discussion could be added at lines 384/5 that due to few numbers the RCT data really only reflects that from young COVID-19 patients with mild symptoms.

2/ According to the study design lines 196-197 only those with moderate symptoms received antimicrobial treatment. Correct? If correct, emphasise that patients with mild symptoms only received HCQ or SOC.

2/ A clearer description of symptoms would be beneficial. For instance:

i Cough? How was this defined? The majority of the patients are listed as presenting with cough. Is this persistent cough, or --?

ii. Fever? How was this defined? Very few – one only had fever in the HCQ RCT.

3. PCR data for viral load was obtained every other day. Is it possible to compare the rates of decline of viral load for the RCT study HCQ vs SOC?

4. The authors have discussed their results in comparison with other published studies. I suggest adding comparison with the data from a large retrospective study from the Henry Ford Hospital in Detroit https://doi.org/10.1016/j.ijid.2020.06.099 published in the International J of Infectious Diseases (Dr Samia Arshad) et al that concludes that HCQ is effective and reduces mortality. However, note that the greater % of patients were also prescribed (cortico)steroids and median age was 64.

Reviewer #2: I would very much hope that the authors would consider making their data available in a de-identified manner in a simple spreadsheet readable format like a comma separated variable file that could be readily ingested into a stats package. I have made comments in a track changed format in the attachment. The only additional query I would raise is that the authors need to acknowledge the small sample sizes of the two study cohorts they have reported on and to maybe also acknowledge that absence of evidence is not necessarily evidence of absence and that this is especially the case in small studies.

**********

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Reviewer #1: Yes: Chris R Triggle

Reviewer #2: Yes: Greg Fegan

[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.]

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Submitted filename: Pone_hcq_multicentered_rct_retrospective_covid19_GFmods.docx

Click here for additional data file.

15 Oct 2020

To the editors and reviewers,

This rebuttal letter responds to each point raised by the academic editor and reviewers. The authors have prepared a marked-up copy that highlights the changes made to the original version, and also an unmarked version.

The authors have deposited the protocols in protocols.io. DOI: dx.doi.org/10.17504/protocol.io.bmfak3ie

The authors have ensured that the manuscript meets PLOS ONE's style requirements, including those for file naming. The item missed from the CONSORT flowchart was amended.

The authors understand the importance of trial registration before recruiting the first participant in a study. The reasons for the delay in registering this study (after enrolment of participants started) were that the appendix of the protocol was in Mandarin and had to be translated to English to meet QC criteria; the registration of the clinical trial was accepted on May/12/2020, after the first enrollment on Apr/1/2020. However, the authors confirm that all ongoing and related trials for this drug/intervention are registered. This statement has been added under the subheading “Study design.”

In the cover level, the corresponding author stated that she received research funding from the Hospital and Social Welfare Organizations Administration Commission, Ministry of Health and Welfare, Taiwan Biotech Co. Ltd. for their donation of investigational products. This funding source played no role in study design or conduction, data collection, analysis or interpretation, writing of the manuscript, or decision to submit it for publication. Furthermore, the commercial funding did not alter our adherence to PLOS ONE policies on the sharing of data and materials.

As there are no legal or ethical concerns, the authors have shared the data publicly on figshare (DOI: https://doi.org/10.6084/m9.figshare.12631403.v1).

Captions for Figure 3 and 4 have been added. Tables 3, 4, and 5 have also been added. Supporting information is provided at the end of the manuscript and the in-text citations have been updated accordingly.

Reviewer #1:

The authors present data from a Taiwan-based multi-center (11 publically funded hospitals) studies addressing the question of whether hydroxychloroquine has therapeutic efficacy for the treatment of COVID-19. Data is presented for an open label RCT (NCT04384380) and an observational retrospective study conducted for up to 14 days. There are limitations to the study that to a greater extent the authors have emphasized but a number of issues need to better emphasized and discussed.

1) The authors have explained that recruitment was limited to few cases presenting in Taiwan. Only 33 were included for the RCT and 37 for the retrospective study. These numbers are very small. Furthermore, as can be seen from Table 1 there are very few who are classified as “moderate” – 2 for HCQ and 2 for SOC in the RCT and 5 and 3 respectively in the retrospective study. This needs to be better emphasized as the study data and conclusions really can only relate to mild cases AND comparatively also a young population ~30-35. Discussion could be added at lines 384/5 that due to few numbers the RCT data really only reflects that from young COVID-19 patients with mild symptoms.

Response: The authors have emphasized the limitations of the young population and mild illness in the “Results (line 255-259 of Page 13), “Discussion” (lines 412-418 of page 23), and “Conclusion” (line 438-439 of page 25).

2) According to the study design lines 196-197 only those with moderate symptoms received antimicrobial treatment. Correct? If correct, emphasise that patients with mild symptoms only received HCQ or SOC.

Response: The subjects with mild symptoms did not receive antimicrobial treatment. The authors have clarified this in “Study design” (lines 196-198 of page 10).

3) A clearer description of symptoms would be beneficial. For instance:

i Cough? How was this defined? The majority of the patients are listed as presenting with cough. Is this persistent cough, or --?

ii. Fever? How was this defined? Very few – one only had fever in the HCQ RCT.

Response: 1. The severity of cough was defined as mild (no symptomatic treatment), moderate (responsive to symptomatic treatment), and severe (symptomatic treatment given but without response). Most subjects had mild cough and the authors have added the data in “Results” (line 257 of page 13). 2. A central temperature of more than 38 °C is defined as fever (Table 1).

4) PCR data for viral load was obtained every other day. Is it possible to compare the rates of decline of viral load for the RCT study HCQ vs SOC?

Response: The rates of decline are presented in Fig. 3 and the difference was not significant between the groups.

5) The authors have discussed their results in comparison with other published studies. I suggest adding comparison with the data from a large retrospective study from the Henry Ford Hospital in Detroit https://doi.org/10.1016/j.ijid.2020.06.099 published in the International J of Infectious Diseases (Dr Samia Arshad) et al that concludes that HCQ is effective and reduces mortality. However, note that the greater % of patients were also prescribed (cortico)steroids and median age was 64.

Response: The retrospective study of Arshad et al. from Henry Ford Hospital was cited and discussed (lines 372-375 of page 20 and line 425-426 of page 22).

Reviewer #2: I would very much hope that the authors would consider making their data available in a de-identified manner in a simple spreadsheet readable format like a comma separated variable file that could be readily ingested into a stats package. I have made comments in a track changed format in the attachment. The only additional query I would raise is that the authors need to acknowledge the small sample sizes of the two study cohorts they have reported on and to maybe also acknowledge that absence of evidence is not necessarily evidence of absence and that this is especially the case in small studies.

Response:

1) In figshare (DOI: https://doi.org/10.6084/m9.figshare.12631403.v1), the authors have provided a simple spreadsheet readable format that can be readily ingested into a stats package.

2) The authors acknowledge that “Both the retrospective and randomized clinical studies performed here failed to demonstrate HCQ efficacy at shortening viral shedding in the majority of subjects presenting with mild COVID-19 symptoms. As the case number is small, absence of evidence is not necessarily evidence of absence…” and have added this statement in the “Conclusions” (lines 439-430 of pages 23 ).

3) The comments in the “track changed format” were replied as follows:

i. What’s wrong with X-ray?

Response: The authors would like to change them.

ii. These adjacent statements seem contradictory.

Response: The authors have clarified that cases of severe illness were not enrolled (lines 159-160 of page 8).

iii. Is this simply related to study size given the allocation ratio?

Response: The estimated sample size was 45 (Lane 194 of page 10).

iv. Tense needs changing

Response: Thank you. We have changed a number of phrases in the Methods from the future to the past and present tense, as appropriate.

v. Suggest you rephrase to the trial was prospectively/retrospectively (delete accordingly) registered at (give URL)

Response: The authors have stated that “this prospective randomized controlled clinical trial was registered at https://clinicaltrials.gov/ct2/show/NCT04384380?term=Hydroxychloroquine&cond=COVID-19&cntry=TW&draw=2&rank=1(NCT04384380). (lines 205-207 of page 10).

vi. bit picky - but no hospitals are mentioned just geographic locales across Taiwan.

Response: We have clarified that the study was conducted at the same 11 hospitals (line 148-151 of page 8).

vii. Suggest you combine this into 3 columns eg mean and (sd) in one cell

Response: Table 1 has been adjusted to use only three columns.

viii. I take it that the 95% CI is (-37.1, 47.2) This needs clarifying. Would suggest you report all CIs as LB,UB not LB-UB) as you do in Table below

Response: Your interpretation was correct. The data have been adjusted.

Submitted filename: Pone_D_20_20024_Reply_reviewerscomments_nontrack.docx

Click here for additional data file.

10 Nov 2020

A multicenter, randomized, open-label, controlled trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective study in adult patients with mild to moderate Coronavirus disease 2019 (COVID-19)

PONE-D-20-20024R1

Dear Dr. Cheng,

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.

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Kind regards,

Stephen L Atkin, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

**********

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 #2: (No Response)

**********

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

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 #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 #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 #2: (No Response)

**********

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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 #2: Yes: Greg Fegan

18 Nov 2020

PONE-D-20-20024R1

A multicenter, randomized, open-label, controlled trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective study in adult patients with mild to moderate coronavirus disease 2019 (COVID-19)

Dear Dr. Cheng:

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.

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on behalf of

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Academic Editor

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