In a meta-analysis of 27 studies (5 RCTs) involving 44,684 COVID-19 patients, the use of HCQ was associated with a 22% lower risk of total mortality. The association was apparent by pooling 22 observational studies and was more evident in studies which used lower overall HCQ dosing. No association was found pooling 5 RCTs. Use of HCQ was not associated with severe adverse events.
The potential for selection bias in observational studies is an important issue. The decision from the clinician to utilize or not a drug may depend on comorbidities and baseline risk of the patient. However, in the pandemic, and in absence of guidelines and specific anti COVID-19 drugs, allocation of HCQ in observational studies was not associated systematically with a lower or higher baseline risk profile. For example, in the CORIST study  patients receiving HCQ were more likely younger and less likely had ischemic heart disease, cancer or chronic kidney disease, but, on the contrary, they were more likely men and had higher levels of C-reactive protein. As a consequence, it is not clear if in that particular study HCQ patients were potentially at higher or lower risk of a negative prognosis. In attempting to account for baseline differences between patients who received HCQ and those who did not, we used the results for adjusted measure of association for each study, and this was possible for 22 out of 27 studies included in the meta-analysis. After the exclusion of 3 unadjusted studies [21,24,35], the strength of the overall association of HCQ with mortality unchanged. Although we attempted to control for potential confounding factors inherent to patient and clinical characteristics, it is possible that unmeasurable confounding still remains, and this may explain the different finding between observational and RCT studies. However, it is hard to determine which are, if any, the unmeasured characteristics that have confused so strongly the association between HCQ and mortality in COVID-19 that was observed in observational studies. In fact, these features must be a) unmeasured in observational studies; b) associated with mortality in COVID-19 and c) associated with HCQ use, in a way that when the risky conditions are present the clinicians tend systematically to avoid using HCQ. For example, HCQ is contraindicated in patients with cardiomyopathy but this condition has been mostly measured in observational studies and was not recognised as a risk factor for mortality in COVID-19 patients.
The dissimilar findings between observational and RCTs we found might also be explained by differences in HCQ dosage . Interestingly, we observed that the reduced mortality associated with HCQ treatment is actually confined to studies that used a daily dose ≤400 mg, or a total dose ≤4,400 mg or which used HCQ for 5 or less days. Obviously these three conditions largely overlapped in studies, that we can now designate as “at low HCQ dosing”. Remarkably, 4 over 5 RCTs are in this category. In detail, the RECOVERY  and the SOLIDARITY study  used 800 mg/day for 9 or 10 days (after the first), respectively and a total dose of 9200 or 10000 mg of HCQ (including the dose at first day) respectively, a very high dose regimen as compared to the rest of studies, particularly of the observational ones.
The possibility that HCQ reduced the risk of negative prognosis in COVID-19 patients when only administered at “low dose” cannot be here undoubtedly proven starting from our findings, but it is a plausible hypothesis that may explain the different result between observational and RCT studies and, more importantly, might be useful in disentangling the debate on HCQ use in COVID-19. If our hypothesis of “low doses-short duration” is correct, it follows that immunomodulation and inflammation occurs quite early after infection with SARS-CoV-2, as also suggested by the benefit of HCQ treatment in patients with higher CRP . In this line, it is of interest that group of studies with low doses and long duration also provided an overall association against mortality. This finding would also support the immunomodulatory hypothesis as potential mechanism of HCQ action [37,39] as it implies a cytokine rebound when the treatment is stopped; if this happens at a critical moment, it could worsen the patient condition, thus vanishing the (potential) effect of HCQ .
High levels of HCQ administration were used in RCTs to maximise the antiviral activity of the drug that was considered to be the main mechanism of action of HCQ in this context. In some studies, the inverse association of HCQ with inpatient mortality was more evident in elderly, in patients who experienced a higher degree of COVID-19 severity or having elevated C-reactive protein levels , suggesting that the anti-inflammatory potential of HCQ may have had a more important role than its antiviral properties. HCQ, indeed, beside an antiviral activity, may have both anti-inflammatory and anti-thrombotic effects . This can justify its effect in reducing mortality risk, since Sars-Cov-2 can induce pulmonary microthrombi and coagulopathy, that are a possible cause of its severity [41, 42] and the lack in preventing SARS-CoV-2 infection after exposure . On the other end, National guidelines suggested to use HCQ 200 mg twice daily for 5-7 days probably to maintain a better risk benefit profile hypothesizing that low doses could be more effective and safer. Indeed, non-sigmoidal, bell-shaped dose-response curves are possible with drugs having complex biological effects, multiple-binding sites or cellular and organ targets. On the other hand, anti SARS-2-CoV-2 activity of HCQ has been confirmed in Vero cells . HCQ is also reported to reduce secretion of IFN-γ and IL-17 in activated Th1 and Th17 cells, respectively .
The concomitant use of azithromycin seems to not neither increase nor decrease the effect, if any, of the HCQ since the combination of the two drugs was associated with a lower mortality risk at very similar extent to that observed for HCQ alone, but the assumption is inconclusive because of the very large uncertainty in the findings.
A main concern with HCQ treatment have been its side effects, in particular a severe cardiovascular toxicity. Indeed, HCQ can cause prolongation of the QT interval on electrocardiogram , which could be exacerbated by coadministration with azithromycin, widely prescribed as co-treatment in Covid-19 treatment. Our meta-analysis of data from RCTs, that allowed a proper evaluation of side effects, shows that use of HCQ was associated with an increase in side effects of any type, but not of major type, including cardiovascular events. This despite the high prevalence of cardiovascular disease in patients with COVID-19 or the high dose used in RTCs.
This meta-analysis has the strength of including all available data recently published and that have not been included in previous meta-analyses [13,47], and of considering modification of effect by dosing of HCQ. As major limitations, we recognise that the majority of the primary studies were observational, the pooled findings suffer of a high degree of heterogeneity and that results in observational and RCT studies were different.