The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patien
The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patients with non-severe COVID-19: A pilot, double-blind, placebo-controlled, randomized clinical trial
Background Ivermectin inhibits the replication of SARS-CoV-2 in vitro at concentrations not readily achievable with currently approved doses. There is limited evidence to support its clinical use in COVID-19 patients. We conducted a Pilot, randomized, double-blind, placebo-controlled trial to evaluate the efficacy of a single dose of ivermectin reduce the transmission of SARS-CoV-2 when administered early after disease onset. Methods Consecutive patients with non-severe COVID-19 and no risk factors for complicated disease attending the emergency room of the Clínica Universidad de Navarra between July 31, 2020 and September 11, 2020 were enrolled. All enrollments occurred within 72 h of onset of fever or cough. Patients were randomized 1:1 to receive ivermectin, 400 mcg/kg, single dose (n = 12) or placebo (n = 12). The primary outcome measure was the proportion of patients with detectable SARS-CoV-2 RNA by PCR from nasopharyngeal swab at day 7 post-treatment. The primary outcome was supported by determination of the viral load and infectivity of each sample. The differences between ivermectin and placebo were calculated using Fisher's exact test and presented as a relative risk ratio. This study is registered at ClinicalTrials.gov: NCT04390022. • View related content for this articleFindings All patients recruited completed the trial (median age, 26 [IQR 19–36 in the ivermectin and 21–44 in the controls] years; 12 [50%] women; 100% had symptoms at recruitment, 70% reported headache, 62% reported fever, 50% reported general malaise and 25% reported cough). At day 7, there was no difference in the proportion of PCR positive patients (RR 0·92, 95% CI: 0·77–1·09, p = 1·0). The ivermectin group had non-statistically significant lower viral loads at day 4 (p = 0·24 for gene E; p = 0·18 for gene N) and day 7 (p = 0·16 for gene E; p = 0·18 for gene N) post treatment as well as lower IgG titers at day 21 post treatment (p = 0·24). Patients in the ivermectin group recovered earlier from hyposmia/anosmia (76 vs 158 patient-days; p < 0.001). Interpretation Among patients with non-severe COVID-19 and no risk factors for severe disease receiving a single 400 mcg/kg dose of ivermectin within 72 h of fever or cough onset there was no difference in the proportion of PCR positives. There was however a marked reduction of self-reported anosmia/hyposmia, a reduction of cough and a tendency to lower viral loads and lower IgG titers which warrants assessment in larger trials.
This was a pilot, double-blind, placebo-controlled, single-center, parallel-arm, superiority, randomized clinical trial that compared a single dose of ivermectin with placebo in patients with non-severe COVID-19 and no risk factors. The trial protocol was published [ ], the last version of the protocol and statistical analysis plan are available as supplementary files. The protocol was approved by the Spanish national ethics committee for drug research (Hospital Puerta de Hierro Majadahonda) and by the Spanish Agency of Medicines and Medical Devices. All procedures were conducted in compliance with the latest revision of the Helsinki Declaration and Good Clinical Practice. All patients provided verbal informed consent at enrollment followed by written consent once their isolation was lifted in accordance to the EMA recommendations: “Guidance on the Management of Clinical Trials during the COVID-19 (Coronavirus) pandemic Version 2 (27/03/2020)” [ ]. This study is registered at ClinicalTrials.gov: NCT04390022. This study was funded by ISGlobal and the Clínica Universidad de Navarra. The funding sources had no role on the design, analysis or decision to publish the results of this study.
Consecutive outpatients attending the Emergency Room of the Clínica Universidad de Navarra (Pamplona, Spain) with symptoms compatible with COVID-19, no more than 72 h of fever or cough and a positive PCR for SARS-CoV-2 were enrolled. Patients with positive IgG against SARS-CoV-2, comorbidities considered risk factors for severe disease or COVID-19 pneumonia at baseline were excluded (detailed eligibility criteria are provided in the protocol -Supplementary file-).
2.2 Study design and oversight
The trial was conducted in the Pamplona metropolitan area (Navarra, Spain). Patients were enrolled between July 31, 2020 and September 11, 2020 and randomized in a 1:1 ratio to ivermectin (400 mcg/kg) single oral dose or placebo. The randomization sequence was computer-generated by the trial statistician using blocks of four to ensure balance. Allocation was made by the attending investigator using opaque envelopes. The placebo tablets did not match ivermectin in appearance, therefore, in order for the clinical trial team to remain blinded, treatment was administered under direct supervision by a non-participant nurse that picked up the opaque bottles directly from the pharmacy and administered the content behind closed doors. The clinical trial team had no contact with the investigational products. There was slow recruitment due to a sharp reduction in local transmission for 10 weeks after the lockdown of March-April 2020, the protocol was amended on September 2nd to extend the inclusion criteria from 48 to a maximum of 72 h of cough or fever.
The main objective was to determine the efficacy of a single dose of ivermectin, administered to low risk, non-severe COVID-19 patients in the first 72 h after fever or cough onset to reduce onward transmission.
2.3 Clinical, laboratory and virological monitoring
Assessments on enrollment and at days 4, 7, 14, 21 and 28 post treatment included: general symptoms report, physical examination (including respiratory rate, blood oxygen saturation and chest auscultation) and adverse events. All patients were asked to complete a daily online diary of symptoms from day 1 to 28 post treatment. On enrollment, as well as on days 7 and 14 blood samples were obtained to assess full blood count, C reactive protein, procalcitonin, ferritin, creatinine phosphokinase, lactic dehydrogenase, troponin T, D dimer, IL-6, and renal function.
A nasopharyngeal swab for SARS-CoV-2 PCR was taken at enrollment and on days 4, 7, 14 and 21 post treatment. For consistency, these samples were collected by three clinicians using the same technique. All samples were processed by PCR for genes N and E of SARS-CoV-2 (Real Time PCR SARS-CoV-2, Vircell SLU, Granada, Spain). For every sample, the viral load was calculated using standard reference curve (EDX Sars-Cov-2, Exact Diagnostics LLC, Fort Worth Texas). Additionally, all samples from day 4 post treatment were cultured in Vero cells for 7 days, after which the cytopathic effect was assessed and PCR conducted on the harvested cell-free supernatant. If the PCR from the supernatant was positive at day 4, the procedure was repeated on the samples of that patient for day 7. A semi-quantitative serology for IgG against SARS-CoV-2 (COVID-19 VIRCLIA IgG monotest, Vircell SLU, Granada, Spain) was done on samples from all patients on day 21 post-treatment.
2.4 Outcome measures
The primary outcome measure was the proportion of patients with detectable SARS-CoV-2 RNA by PCR from nasopharyngeal swab at day 7 post-treatment.
Relevant pre-specified secondary outcomes included viral load at days 4, 7, 14 and 21 post treatment; proportion of patients with symptoms (particularly fever and cough) at days 4, 7, 14 and 21 post-treatment as well as proportion of patients progressing to severe disease or death during the trial; proportion of patients with seroconversion at day 21 post-treatment and proportion of drug-related adverse events.
Of 94 patients assessed, 50 did not meet eligibility criteria, 20 declined to participate and 24 were randomized. All randomized patients received the corresponding study product and completed 28 days of follow-up (Fig. 1). The baseline characteristics of patients in both groups are presented in Table 1.
Genes E and N had comparable results at all time points. Patients in both study groups had similar viral load before treatment with median and interquartile range for genes E and N in the same orders of magnitude (Fig. 2 and Table S1). Although there was a consistent overlap in interquartile ranges and full ranges at all points, the median viral load for both genes was lower at days 4 and 7 post treatment in the ivermectin group with differences increasing from 3-fold lower at day 4 (p = 0·24 for gene E; p = 0·18 for gene N) to around 18-fold lower at day 7 (p = 0·16 for gene E; p = 0·18 for gene N) (Fig. 2 and Table S1). A similar tendency remained for the viral load at days 14 and 21, with values from patients in the ivermectin group consistently lower for at least one of the genes, the difference was not statistically significant at any single point (Fig. 2 and Table S1). The values of cycle thresholds had a very similar behavior (Figure S1). Summary statistics for viral kinetics are provided in Table S2.
3.4 Viral culture
At day 4 post-treatment, 7/12 samples in the ivermectin and 5/12 samples in the placebo group effectively replicated Vero cell culture; the median Ct values of replicating samples were 23·3 and 23·8 for genes N and E respectively, while the median Ct values of non-replicating samples were 27·6 and 27·9 for genes N and E respectively. By day 7 post treatment only 1/6 in the ivermectin (one previously positive sample was lost) and 1/5 in the placebo group replicated in the cell culture; the median Ct values of replicating samples were 25·1 and 26·0 for genes N and E respectively, while the median Ct values of non-replicating samples were 30·8 and 32·0 for genes N and E respectively.
There was good compliance with the daily online questionnaire with 282 patient-days reports (84%) and 295 patient-days reports (88%) in the ivermectin and placebo group respectively (Fig. S2).
Patients in the ivermectin group reported fewer patient-days of any symptoms than those in the placebo group (171 vs 255 patient-days). This difference is mostly driven by two symptoms, anosmia/hyposmia and cough. Patients in the ivermectin group reported 50% less anosmia/hyposmia than those in the placebo group (76 vs 158 patient-days of anosmia/hyposmia). The ivermectin group also reported 30% less cough (68 vs 97 patient-days of cough) (Fig. 3). Discussion
In spite of its partial antiviral properties, ivermectin received limited early attention in Europe or the US as a potential drug to be repurposed against COVID-19. This was largely based on one pharmacokinetic model stressing the inability of currently approved oral doses to reach lung tissue levels at the antiviral concentrations described by Caly et al., [ ] even if other, peer-reviewed models predict up to 10-fold accumulation of ivermectin in target tissue [ ]. There are additional reasons to avoid direct inferences from the results of in vitro experiments or pharmacokinetic models, these include the potential role of ivermectin metabolites, the potential immunomodulatory role of the drug, and questions about the virus/cell ratios and appropriateness of the Vero cellular lines used in the cultures [ ].
This pilot study was designed to assess the question of whether further investments in the potential repurposing of ivermectin were warranted. As such, we aimed at generating evidence on viral kinetics, antibody response and clinical efficacy in a cohort of patients at low risk of severe disease. Without a clearly defined mechanism of action, a sole signal in any of said parameters would not suffice to justify further efforts. This pilot shows a tendency to lower viral loads in the ivermectin group, a tendency to lower IgG titers that may reflect milder disease and clinical benefit in cardinal symptoms of COVID-19 associated with tissue damage: anosmia/hyposmia and cough. These results are in line with emerging evidence from trials in Bangladesh [ , ] and Argentina [ ] showing a faster viral clearance in treated participants, as well as with recent data from a SARS-CoV-2 hamster model from Institute Pasteur which also showed a marked sex dichotomy in the effect of ivermectin on anosmia/hyposmia [ ].
Pending confirmation of these results, this pilot sheds some light on the potential mechanism of action of ivermectin against COVID-19. Note the trial was not powered to detect modest differences in viral load, yet a small effect is suggested when viral load was ascertained directly by PCR and indirectly using IgG titers as markers of disease severity [ , ]. Also, in this pilot ivermectin has not shortened the duration of symptoms associated with systemic inflammation such as fever or malaise, nor has it had a measurable impact on systemic inflammatory markers.
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