3. Materials and Methods

3.1. Search Strategy
According to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic literature search was performed in May 2020 and included all reports published to August 2020. The search was performed on specialized databases (PubMed and Scopus) using different combinations of HCQ and the following keywords: history, discovery, ethnomedical, synthesis, chemical structure, SAR, RAS, biological activity, approved biological activities, antiviral, antiviral mechanism, COVID-19, Q fever, Whipple’s disease, synergistic effects, synergic effects, toxicological effects, toxic effects, toxicity, animal model and antiviral activity, clinical study, preclinical study. We did not request full-text to investigators if not available and we did not try to find unpublished data.

3.2. Study Selection
The manuscript selection was based on the inclusion criteria: preclinical (in vivo) and clinical studies involving the use of HCQ and combinations, only articles published in English and containing keywords in the title or in the abstract were selected. Other review articles, meta-analysis, retrospective studies, abstracts, conferences, editorials, letters, conference proceedings, manuscripts without full text available or articles that did not meet the inclusion criteria were not included in this systematic review. For selecting the sources, three independent investigators (I.F., F.L., and M.P.) first selected the articles according to the title and abstract and then by analyzing the full-texts. In cases of non-consensus, authors tried to resolve any disagreements by discussion or, if necessary, two more independent reviewers were consulted (L.M. and N.D.T.). The selected articles were carefully reviewed with the aim of identify or exclude the manuscripts that did not fit the criteria described above. Additional papers were added to this review after the analysis of the bibliography from the included articles.

3.3. Data extraction
Data were collected and examined by the authors and information from the selected manuscripts on HCQ, as well as study design, experimental models, general mechanisms implicated in antiviral and biological activities, major outcomes doses or concentrations, and route of administration were extracted.

3.4. Methodological Quality Assessment
The risk of bias and the quality of the preclinical and clinical investigations were assessed independently by the authors, using a checklist adapted from Cochrane Handbook for Systematic Reviews of Interventions, specifically adjusted for animal intervention study (SYRCLE’s) [171,172] and clinical trials [97]. The evaluation of the selected studies’ methodological quality was based on the presence or absence of information regarding the main objectives and findings, randomization of the treatment allocation, blinded drug administration, blinded outcome assessment and outcome measurements, as reported in Table 3 and Table 4. Only studies that reported a positive judgment in all considered parameters were assessed to be of a higher methodological quality. In contrast, the studies that did not wholly fulfil the criteria were included in the medium risk of bias, while those that completely lacked this information were deemed to be at high risk of bias.