1 BACKGROUND

Over a short time, the highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) viral infection went from being an outbreak in Wuhan, China to a worldwide pandemic with a mortality rate roughly 10 times greater than seasonal influenza.1, 2 Faced with this rapidly escalating crisis, clinicians sought therapies that could improve outcomes when combined with standard supportive treatments. Based on in vitro findings that aminoquinoline-4 agents, such as chloroquine (CQ) and hydroxychloroquine (HCQ), inhibited SARS-CoV2 at clinically achievable concentrations3-5 and on the widespread use of these agents as anti-malarial and anti-inflammatory agents, both were rapidly introduced into patient care.6, 7 Early small clinical trials suggested that these agents accelerated SARS-CoV2 clearance and time to clinical recovery.6, 7 However, observational and larger randomised studies have not consistently substantiated these earlier findings, and several indicate CQ and HCQ may have serious adverse effects in some SARS-CoV2-infected patients.8-11

The antiviral activity of aminoquinoline-4 agents is thought to be related to inhibition of virus entry and disruption of viral replication.12, 13 Based on in vitro and in vivo findings in virus infection models, CQ and HCQ were administered clinically in the past for up to nine different types of noncoronavirus viral infections.14-31

But at the outbreak of SARS-CoV2, neither agent was being recommended for any of these infections, and a recent narrative review, commentary and editorial suggested that prior clinical studies had not provided strong support for aminoquinoline-4 therapy for viral infection.32 To comprehensively examine the question of whether prior studies of HCQ and CQ had shown compelling evidence for their benefit in the treatment of other viral infections that would have supported their use for SARS-CoV-2, we performed a systematic review and planned a meta-analysis of clinical studies that were published before the SARS-CoV-2 outbreak. This analysis was designed to compare the effects of an aminoquinoline-4 agent to control for the treatment of any viral infection in earlier studies. Survival, a main therapeutic goal for these agents in COVID-19 patients, was to be the primary endpoint for the analysis, while viral clearance, biomarkers and organ injury were secondary endpoints.

2 METHODS

This systematic review was registered with PROSPERO on May 1 2020 (CRD42020183429) and prepared using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidance for literature review and data extraction (File S1).

2.1 Literature search and study selection

Using published guidelines33 and search strategies (File S2), two authors (P.T.P. and P.Q.E.) identified relevant studies in PubMed, EMBASE, Scopus and Web of Science from inception through 2 April 2020 without language restrictions. Published clinical studies in adults that compared the effects of CQ, HCQ or related compounds to a control group on survival (primary endpoint), viral clearance, organ injury or other biologic endpoints during viral infection other than coronavirus (secondary endpoint) were retrieved. Recovered reports were scanned for additional relevant ones.

2.2 Data extraction and quality assessment

Four investigators (P.T.P., C.X., Y.L. and P.Q.E.) extracted study data using a standardised extraction form (File S3). If primary and secondary endpoints were not clearly defined in the trial registration, we recorded measures that were most relevant for assessing the efficacy of tested treatments or those emphasised in figures or tables.

Risk of bias was assessed with the Cochrane Collaboration Risk of Bias Assessment Tool for randomised controlled trials and the Newcastle-Ottawa Tool for observational studies. Three investigators (P.T.P., J.S., P.Q.E.) assessed these risks independently and settled disagreements by consensus. Additional parameters of study weakness were also assessed and tabulated separately.

2.3 Data synthesis and analysis

Unless otherwise noted, levels of significance or non-significance (p = ns) reported in the text and/or figures and tables, are those provided by the investigators. In some cases, where analysis was not provided in a report but sufficient data available, we calculated the descriptive statistics and/or treatment differences. Median and interquartile ranges (IQRs) were converted to mean and SD using the method of Wan et al.34 For HIV, which included the greatest number of studies (n = 6), mean differences between treatment and control groups in changes in blood HIV RNA copy levels (log10-transformed) and CD4+ cell counts from pre- to posttreatment were calculated. Heterogeneity among studies was assessed using the Q statistic and I2 value.35 The plan was to analyse the mean differences using random-effect models,36 if appropriate. However, due to high heterogeneity among studies, we did not combine the studies. Analyses were performed using R37 (version 4.0.2) with packages meta38 (version 4.11-0). In studies also comparing nonaminoquinilone-4 agents to placebo, data are only presented for the comparison between the control and aminoquinilone-4 agent.

3 RESULTS

We retrieved 18 studies (File S4) comparing the effects of CQ or HCQ to a control treatment in patients with either chronic HIV infection (n = 6 studies), mononucleosis ([EBV], n = 4), warts ([HPV], n = 2), acute dengue infection (n = 2), chronic HCV (n = 2), acute chikungunya infection (n = 1) and as prophylaxis for influenza A or B infection (n = 1). Tables 1–3 and Table S1 summarise the following: Table 1, patient populations targeted, study design, the 4-aminoquinilone and control agent studied, and number of control and treatment patients; Table 2, treatment regimens, length of observation and inclusion and exclusion criteria; Table 3, primary and secondary endpoints or measures investigated; Table S1, enrolment dates, comparisons between groups in baseline parameters and reported adverse events. Seventeen studies were prospective and one retrospective. No study examined the effect of CQ or HCQ on survival, our predefined primary outcome, and only four examined hospitalised patients. The variability of endpoints reported prevented us from proceeding with a meta-analysis of the studies retrieved. Additionally, because results reported varied dependent on the virus type studied, the studies are presented separately for each virus type.

TABLE 1. Summary of study characteristics Author (year) Patient population targeted Location Study design Treatment aPatients Referencea In- versus out-Pt Country CQ or HCQ Con Con Rx EBV (mononucleosis) Cowley (1962) Suspected infection In US C, DB CQ Plac 20 20 15 Schumacher (1963) Suspected infection In US C, DB CQ Plac 5 5 25 Talstad (1964) Suspected infection In Norway C CQ Plac (CaLac) 40 39 29 Updike (1967) Suspected infection Out US C, DB CQ Plac 19 21 31 Human papilloma virus (HPV, warts) Jacobs (1963) Warts Out US C, DB CQ Plac (Lac) 25 25 18 Murphy (1965) Warts Out US C, DB HCQ Plac 42 48 20 HIV Sperber (1995) Chronic HIV,CD4 200-500 cells/mm3 Out US R, C, DB HCQ Plac 19 19 28 Sperber (1997) Chronic HIV,CD4 200-500 cells/mm3 Out US R, C, DB HCQ ZDV 37 35 27 Semrau (2006) Chronic HIV, breast-feeding, treated for malaria Out Zambia Observational,CC CQ SP 12 18 26 Murray (2010) Chronic HIV,CD4>250 cells/mm3 Out US R, C, DB CQ Plac 3 8 21 Paton (2012) Chronic HIV,CD4>400 cells/mm3 Out US R, C, DB HCQ Plac (Lac) 41 42 22 Jacobson (2016) Chronic HIV, off or on-ART Out US R, C, DB, Cx CQ Plac 36b 34b 19 Chikungunya virus De Lamballerie (2008) Acute infection Out FRI R, C, DB CQ Plac 27 27 16 Dengue virus Tricou (2010) Acute infection In Vietnam R, C, DB CQ Plac 154 153 30 Borges (2013) Acute infection Out Brazil R, C, DB CQ Plac (Starch) 18 19 14 Hepatitis C virus Helal (2016) Chronic infection; failed IFN/RBV Out Egypt C, SB HCQ + standard Rx Standard Rx# 60 60 17 Peymani (2016) Chronic infection, failed standard Rx Out Iran R, C, TB CQ Plac 4 6 24 Influenza A and B virus Paton (2011) Subjects at risk of infection Out SG R, C, DB CQ Plac (Lac) 738 724 23 Abbreviations: ART, antiretroviral therapy; C, controlled; CaLac, calcium lactate; CC, cohort controlled; CD4, CD4+ T cells; Con, control; CQ, chloroquine; Cx, cross over; DB, double blind; FRI, French Reunion Island; HCQ, hydroxychloroquine; Lac, lactose; Plac, placebo; Pt, patient; R, randomised; Rx, treatment; SB, single blind; SG, Singapore; SP, sulfadoxine–pyrimethamine; TB, triple blinded. a Standard treatment = pegylated interferon and ribavirin, no placebo described. b In the initial 12 weeks of study, there were a combination of 36 patients in the off- and on-ART control groups and 34 in the CQ groups, see the text. TABLE 2. Summary of treatment regimens, observation, and inclusion and exclusion criteria in studies Author (year) Treatment regimen Observation time Inclusion criteria Exclusion criteria EBV (mononucleosis) Cowley (1962) CQ: 1g ×1; 500 mg at 8 h and daily ×9 days; 250 mg daily × 8 days LOS Fever; pharyngitis; ↑ WBC and atypical lymphocytes; lymphadenopathy; heterophile Ab ≥ 1:224 NR Schumacher (1963) CQ: 1 g ×1; 500 mg q6 h × 24 h; 250 mg every 6 h × 5 days NR >50% Lymphocytes; >20% atypical lymphocytes; heterophile Ab ≥ 1:56 NR Talstad (1964) CQ: 1 g ×1; 500 mg 6 h later; 500 mg daily × 2 days LOS NR NR Updike (1967) CQ: 250 mg twice daily × 7 days NR >20% Atypical lymphocytes;+heterophile Ab Illness besides mononucleosis Human papilloma virus (warts) Jacobs (1963) CQ: 250 mg daily for up to 60 days ≤60 days 2–50 verruca, plantaris, plana or accuminata lesions NR Murphy (1965) HCQ: 200 mg twice daily maximum of 9 weeks ≤9 weeks NR NR HIV Sperber (1995) HCQ: 800 mg daily × 8 weeks 8 weeks Asymptomatic; no ART for 4 weeks; 200–500 CD4 cells/mm3; Hb, PMN, PLTS ≥ 8.5 g/dl, 1000 and 75,000 cells/mm3 respectively; ALT/AST and amylase <3X and 1.3X ULN respectively <18 years; pregnant; AIDS defining condition or malignancy, active ETOH/drug abuse, Stage 2 AIDS dementia, known G6PD deficiency Sperber (1997) HCQ: 800 mg daily × 16 weeks 16 weeks Asymptomatic; no ART for 4 weeks; 200–500 CD4 cells/mm3; Hb ≥ 8.5g/dl, PMN ≥ 1000 cells/mm3 respectively; ALT/AST <3X ULN <18 years; pregnant; AIDS defining condition or malignancy, Stage 2 AIDS dementia, known G6PD deficiency Semrau (2006) CQ: 600 mg Days 1, 2; 300 mg Day 3 ≤16 days NR NR Murray (2010) CQ: 250 mg daily in 6 pts, 500 mg daily in 3 pts, × 2 months 2 months Chronic HIV; >250 CD4 cells/mm3; off ART ≥ 16 months NR Paton (2012) HCQ: 400 mg daily × 48 weeks 48 weeks Chronic HIV; 18–65 years; no ART >12 months; >400 CD4 cells/mm3; HIV > 1000 copies/ml Psoriasis, epilepsy, arrhythmias, depression, diabetes, CA, chronic liver, retinal ds; pregnancy; primary HIV infection <12 months; infection/vaccination last 2 months; +HBV-Ag; +HCV PCR Jacobson (2016) CQ: 250 mg daily × 12 weeks 12 weeksa 18–55 years; off-ART – no ART 6 months, HIV ≥ 1000 copies, ≥400 CD4 cells/mm3; on-ART – on ART for 24 months, <350 CD4 cells/mm3, HIV copies undetectable NR Chikungunya virus De Lamballerie (2008) CQ: 600 mg Day 1; 300 mg twice daily Days 2, 3; 300 mg Days 4 and 5 25 days, phone follow-up at 200 days 18–65 years; >60 kg; <48 h acute febrile arthralgia; biologically confirmed diagnosis (viraemia quantified by real-time PCR and seroconversion between 1 and 16 days of study Pregnancy; renal, retinal, celiac ds; contraindications to CQ Dengue virus Tricou (2010) CQ: 600 mg Days 1, 2; 300 mg Day 3 ≥15 daysb >15 years; dengue symptoms <72 h Pregnant; therapy for chronic ds; CQ hypersensitivity; no consent Borges (2013) CQ: 500 mg twice daily × 3 days 7 daysc Enrolled if: fever and at least 2: headache, retro-orbital pain/muscle/joint pain, nausea/vomiting, rash for <72 h. Confirmed dengue by 2 of either PCR, ELISA or NS1 antigen detection assays <18 years; pregnant; CV or neurological ds Hepatitis C virus Helal (2016) HCQ: 200 mg twice daily × 12 weeks 12 weeks 18–60 years; HCV genotype-4; -HBV-Ag; +HCV-Ab; WBC, PMN, and PLTs > 3000, 1500, and 80,000 cells/mm3 respectively; Hb > 12 g/dl in males and 11 g/dl in females, Cr < 1.2; +HCV liver bx in <12 months. Pregnant; non-HCV liver ds; liver bx with F0 and F4 for necrosis, inflammation and fibrosis; BMI > 30; CV, thyroid, retinal ds; antiviral/immunosuppressive therapy past 6 months Peymani (2016) CQ: 150 mg daily × 8 weeks 8 weeks Males; 18–60 years; HCV genotype-1 unresponsive to pegIFN and RBV Decompensated cirrhosis; anti-neoplastic, anti-viral, immunomodulator therapy in prior 6 months; HAV, HBV, HDV, HIV; active ETOH use; mental impairment; LFTs > 5X ULN; adverse reaction to HCQ; HCC; already on therapy for HCV Influenza A and B virus Patton (2011) CQ: 500 mg daily × 1 week; once weekly × 11 weeks 12 weeks 18–65 years Pregnancy, breast feeding; psoriasis; porphyria; epilepsy; myopathy; depression; CV, retinal, hepatic, renal, ds; G6PD deficiency; hepatotoxic therapy; flu vaccination past 3 months; flu symptoms at screening Abbreviations: Ab, antibody; Ag, antigen; AIDS, acquired immunodeficiency syndrome; ALT, alanine amino-transferase; ART, antiretroviral therapy; AST, aspartate amino-transferase; BMI, body mass index; Cr, creatinine; CQ, chloroquine; CV, cardiovascular; ds, disease; ELISA, enzyme-linked immunosorbent assay; ETOH, alcohol; Flu, influenza; HAV, HBV, HDV, hepatitis A, B and D; Hb, haemoglobin; HCQ, hydroxychloroquine; HPV, human papilloma virus; LFT, liver function test; LOS, hospital length of stay; NR, not reported; pegIFN, pegylated interferon; PCR, polymerase chain reaction; PLT, platelet; PMN, polymorphonuclear cell; RBV, ribavirin; Rx, treatment; ULN, upper limit of normal. a Each of two cross-over periods was 12 weeks. b Blood drawn BID for 5 days in hospital and then at 10–14 days after discharge. c 7 days following confirmed infection. TABLE 3. Summary of endpoints or measures Author (year) Primary and secondary endpoints prospectively defined Endpoints or measures Prospective power analysis performed EBV (mononucleosis) Cowley (1962) UC

Followed patients from hospital admission to discharge.

Factors assessed for degree of improvement—general condition, throat condition, lymphadenopathy, spleen and liver size, skin eruption, percent diet eaten and drug reaction.

Discharged 3–4 days after last temperature elevation and if no further symptoms occurred.

Lab data obtained but not presented—WBC and differential, haematocrit, ESR, serologic syphilis test, urinalysis, CXR, throat and blood cultures at admission and then later if needed; LFT twice weekly.

NR Schumacher (1963) UC

Followed patients from hospital admission to discharge.

Oral temperature > 99.6°F checked four times per day and signs or symptoms including sore throat, lymphadenopathy, hepatosplenomegaly, exanthem, enanthem reported over the 5 days after starting treatment.

Duration of illness prior to therapy, total hospitalisation time and time after therapy.

NR Talstad (1964) UC Mean temperature (°C) 1, 4 and 7 days; mean WBC and ESR 1–3 and 6–9 days; hospital LOS. NR Updike (1967) UC Disabling days—a combination of fully or partially disabling days students recorded in a log while on therapy based on whether they were too sick to attend class (1 point) or attended class but had symptoms (½ point) respectively, during the 7 days of treatment. NR Human papilloma virus (warts) Jacobs (1963) UC Wart clearance over 9 weeks; Warts mapped as to size and location at first visit;Baseline and monthly urinalysis, CBC and LFT. NR Murphy (1965) UC Wart clearance over 60 days. NR HIV Sperber (1995) UC

Compared changes within groups over 8 weeks of treatment.

Successful treatment defined as a reduction in levels of plasma HIV RNA, serum p24, or cultured virus from PBMC.

Other measures—CD4 count and %, CBC, β-microglobulin, IgG, IgM, IgA, IL-1α, IL-1β, IL-6 and TNF; antigen stimulation assays.

NR Sperber (1997) UC

Compared changes within and between groups over 16 weeks of treatment.

Successful treatment defined as a reduction in levels of plasma HIV RNA, serum p24, or cultured virus from PBMC.

Other measures—CD4 count and %, CBC, β-microglobulin, IgG, IL-6.

NR Semrau (2006) UC Compared breast milk and plasma HIV RNA, and CD4 counts in blood obtained 3–16 days after patients received treatment. NR Murray (2010) UC

Blood sampled at baseline, 1 and 2 months of treatment.

Measured CD8CD38+HLA+ cell frequency and Ki-67 expression, CD4 counts, plasma HIV RNA, preservation of CD4 and CD8 cell producing TNF, IFN-γ, and IL-2 in response to Gag, Pol, Enc or NEF HIV antigens, LPS levels

NR Paton (2012) Yes

Primary endpoint—Change in proportion of activated T-cells measured by expression of CD38 and HLA-DR surface markers from baseline to 48 weeks.

Secondary endpoint—Change in CD4 cell activation and counts, plasma HIV RNA and IL-6 levels from baseline to 48 weeks.

Changes calculated based on samples obtained at baseline, 4, 12, 24, 26 and 48 weeks.

Yes:80 patients Jacobson (2016) Yes for 1° endpoint Primary endpoint—changes in %CD8CD38+HLA-DR+ T-cells from baseline to the first 12 weeks of study.Other measures—%CD8CD38+HLA-DR+ T-cells, over the second 12 weeks of study and CD4 counts, HIV RNA, IL-6 and LPS over the first and second 12 weeks of study and over the two 12 weeks periods combined. NR Chikungunya virus De Lamballerie (2008) UC

Primary (‘main criterion’) duration of febrile arthralgia.

Other measures—change in viral genome level from 1 to 3 days and presence of viraemia.

Clinical exam was performed by a general practitioner at Days 1, 7 and 25 and patients monitored their symptoms twice daily on Days 1–5 and qd on Days 6–14.

Biologic investigations were performed at Days 1, 3, 6 and 16.

Yes:250 patients required. Only 54 enrolled due to decrease in epidemic Dengue virus Tricou (2010) Yes

Primary endpoint—time to resolution of viraemia or antigenemia defined as the time from the initiation of treatment until the first two consecutive plasma samples were RT-PCR negative or NS1 ELISA negative, respectively.

Blood samples obtained at hospital admission before drug administration and then twice daily for a minimum of 5 days and again 10–14 days after hospital discharge.

Secondary endpoints—fever clearance time defined as the time from the initiation of treatment to the beginning of the first 48-h period the temperature remained <37.5°C, platelet count nadir, mean maximum % haemoconcentration based on haematocrit values, proportion of patients in either arm requiring intravenous fluids or developing dengue hemorrhagic fever (DHF).

Yes:213 patients Borges (2013) UC

Duration of dengue disease and duration and intensity of fever up to 7 days after treatment.

Possible dengue symptoms including fever (axillary temperature ≥ 37.8°C), headache, and retro-orbital, muscle, bone or joint pain were determined at baseline and after 1 week of therapy.

NR Hepatitis C virus Helal (2016) Yes

Primary—early virological response (EVR) defined as either undetectable HCV RNA tested at 12 weeks (complete EVR) or ≥2 log drop in HCV RNA from baseline to 12 weeks (partial EVR).

Other measure—early biochemical response (EBR) defined as an aspartate aminotransferase (ALT) < 40 IU after 12 weeks of therapy.

NR Peymani (2016) UC Plasma HCV RNA and ALT were measured at baseline, 4, 8 and 12 weeks. NR Influenza virus A and B Paton (2011) Yes

Primary endpoint—combination of laboratory-confirmed and clinical influenza.Laboratory-confirmed influenza infection based on one of the following test results—PCR confirmation on a nasal swab taken by the participant, PCR confirmation or positive influenza culture on a nasal swab taken by a health-care worker, or serological confirmation by at least a fourfold increase in antibody titre on a haemoagglutinin-inhibition or microneutralisation assay for H1N1, H3N2 or influenza B infection from baseline to 12 weeks.

Clinical influenza based on the development of influenza-like symptoms including reported temperature of at least 37.2°C with at least one respiratory symptom (sneezing, runny nose, blocked nose, sore throat, dry cough, coughing up phlegm, wheezing, shortness of breath) and at least one constitutional symptom (feverish feeling, muscle aches, fatigue, headache, diarrhoea) occurring on the same day.

Main secondary endpoint—laboratory confirmed influenza alone.

Patients completed weekly diaries if asymptomatic or daily ones if symptomatic via a password-protected trial website that presented symptom checklists.

Blood was drawn at baseline and at 12 weeks; patients were followed by their primary care givers.

Yes:1500 patients