- Cytotoxicity was not associated with drugs in the control wells (without viruses)
- Cell lyses at 60 hours was associated with viral production 
- AZM + HCQ led to significant inhibition of viral replication in wells with 5 μM HCQ and either 10 or 5 μM AZM
- Cytopathic effect was observed in 5/31 wells at 60 hours after infection as compared to the 13/13 positive control wells 


Smith College

In a French study, researchers isolated SARS-CoV-2-IHUMI-3, a strain of the coronavirus, and tested various combinations of HCQ and AZM with Vero E6 cells. 

- Isolation of the viral strain and viral stock
- In vitro testing procedure 
- In vitro testing results of combined HCQ and AZM on viral strain  
- Discussion of in vitro testing results of combined HCQ and AZM on viral strain  

In vitro testing revealed synergistic effect of combined hydroxychloroquine (HCQ) and azithromycin (AZM) on  SARS-CoV-2

- Full procedure of isolation of SARS-CoV-2 strain IHUMU-3 can be found at  https://doi.org/10.1016/j.ijantimicag.2020.105949
- Cultures of Vero E6 cells in Minimum Essential Media (MEM) with 4% of fetal bovine serum and 1% glutamine were used
- An inverted microscope was used daily to monitor cytopathic effect
- After total cell lysis, which took about 96 hours, viral supernatant was used for inoculation on a 96 well plate
- The TCID50 of the strain was found at 5.105 infections particles per mL


Isolation of the viral strain and viral stock

- 96 well plates with 5.105 cells/mL of Vero E6 were prepared and incubated overnight at 37 degrees Celsius in a carbon dioxide atmosphere
- 1, 2, and 5 μM of HCQ were tested with 5 and 10μM of AZM 
- Each test was completed at least three times and was repeated twice (5 μM of HCQ with 5 and 10 μM of AZM was repeated three times)  
- Cell culture supernatant was extracted and replaced by drugs diluted in the culture medium four hours prior to infection
- At time zero virus suspension in culture medium was added to all wells except in controls where medium was added instead
- Multiplicity of infection was 0.25
- RT-PCR was performed 30 min after infection in one plate and again at 60 hours after infection on a second plate 


In vitro testing procedure 

In vitro testing results of combined HCQ and AZM on viral strain 

- Results support the clinical use of AZM + HCQ, especially during the earlier stages of infection before patients develop respiratory distress syndrome 
- AZM + HCQ has a synergistic effect on SARS-CoV-2 at concentrations compatible with the human lung 


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