key: cord-269838-1943g1ha
authors: Haffizulla, Jason; Hartman, Aaron; Hoppers, Melanie; Resnick, Harvey; Samudrala, Steve; Ginocchio, Christine; Bardin, Matthew; Rossignol, Jean-François
title: Effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial
date: 2014-05-19
journal: Lancet Infect Dis
DOI: 10.1016/s1473-3099(14)70717-0
sha: 
doc_id: 269838
cord_uid: 1943g1ha

BACKGROUND: Influenza is an important cause of morbidity and mortality worldwide. Treatment options are scarce, and new drugs with novel mechanisms of action are needed. We aimed to assess the efficacy and safety of nitazoxanide, a thiazolide anti-infective, for treatment of acute uncomplicated influenza. METHODS: We did a double-blind, randomised, placebo-controlled, phase 2b/3 trial in 74 primary care clinics in the USA between Dec 27, 2010, and April 30, 2011. We enrolled participants aged 12–65 years with fever, at least one respiratory symptom, and one constitutional symptom of influenza within 48 h of symptom onset. We randomly assigned participants to receive either nitazoxanide 600 mg, nitazoxanide 300 mg, or placebo twice daily for 5 days, (ratio 1:1:1) and followed them up for 28 days. Randomisation lists were computer generated and done in blocks of three. Sponsor, investigators, study monitors, patients, and laboratory personnel were all masked to treatment allocation in the study. The primary endpoint was the time from first dose to alleviation of symptoms. The primary analysis was by intention-to-treat for participants with influenza infection confirmed by RT-PCR or culture at baseline. This trial is registered with ClinicalTrials.gov, number NCT01227421. FINDINGS: Of 650 participants screened, 624 (96%) were enrolled. Of these, 212 were randomly assigned to receive placebo twice a day, 201 to receive nitazoxanide 300 mg twice a day, and 211 to receive nitazoxanide 600 mg a day. The median duration of symptoms for participants receiving placebo was 116·7 h (95% CI 108·1–122·1) compared with 95·5 h (84·0–108·0; p=0·0084) for those receiving 600 mg nitazoxanide and 109·1 h (96·1–129·5, p=0·52) for those receiving 300 mg nitazoxanide. Adverse events were similar between the three groups, the most common being headache reported by 24 (11%) of 212 patients enrolled in placebo group, 12 (6%) of 201 patients in the low-dose group, and 17 (8%) of 211 patients in the high-dose group, or diarrhoea, reported by seven (3%) patients in the placebo group, four (2%) patients enrolled in the low-dose group, and 17 (8%) patients in the high-dose group. INTERPRETATION: Treatment with nitazoxanide 600 mg twice daily for 5 days was associated with a reduction of the duration of symptoms in participants with acute uncomplicated influenza. Further studies are warranted to confirm these findings and to assess efficacy of the drug alone or in combination with existing drugs in seriously ill patients and those at risk of influenza complications. FUNDING: Romark Laboratories LC.

Infl uenza, a contagious respiratory illness caused by infl uenza A, B, or C viruses, is an infection of global concern resulting in about 3-5 million cases of severe disease and 250 000-500 000 deaths annually. 1 In the USA, seasonal infl uenza aff ects-on average-5-20% of the population per year leading to roughly 200 000 hospital admissions and 3000-49 000 deaths. 2, 3 The threat of pandemic infl uenza caused by emerging viruses such as the avian AH5N1 or more recently AH7N9 is a major concern because of potential eff ects on human life, economy, national security, and functioning of society. Because of widespread adamantine resistance, the treatment of infl uenza is at present limited to the neuraminidase inhibitors, oseltamivir and zanamivir. New drugs with novel mechanisms of action that can be used alone or in combination with neuraminidase inhibitors are urgently needed to improve treatment outcomes and mitigate risks of resistance.

Nitazoxanide, a fi rst-in-class thiazolide anti-infective, inhibits replication of a broad range of infl uenza viruses, including neuraminidase inhibitor-resistant strains, blocking the maturation of viral haemagglutin at the posttranslational level. 4, 5 In cell culture studies, nitazoxanide acts synergistically with neuraminidase inhibitors. 5 Repeated passage of infl uenza viruses in subinhibitory concentrations of the drug have proven unsuccessful in selecting resistant strains suggesting a high barrier to resistance. 5 Nitazoxanide also inhibits replication of respiratory viruses including parainfl uenza virus, coronavirus, and respiratory syncytial virus in cell cultures. 5 Nitazoxanide (Alinia, Romark Laboratories LC) is licensed in the USA for treatment of diarrhoea caused by Cryptosporidium parvum and Giardia lamblia, 6 and has been widely used throughout Latin America for treatment of intestinal parasitic infections. A 300 mg controlled-release tablet was developed to deliver plasma con centrations suitable for treatment of viral respiratory infections. We aimed to assess safety and effi cacy of two diff erent doses of nitazoxanide in reducing the duration of symptoms of acute un complicated infl uenza.

We designed this phase 2b/3 randomised clinical trial in consultation with US Food and Drug Administration (FDA) Guidance for Industry 7 and published clinical trials of oseltamivir and zanamivir. [8] [9] [10] [11] [12] Effi cacy of nitazoxanide in treatment of acute uncomplicated infl uenza had to be established before doing studies in seriously ill patients or patients at high risk of complications of infl uenza. Because of the variability of disease and diffi culties defi ning margins of noninferiority to existing drugs, we used a placebo to clearly assess the activity of the drug. The 300 mg and 600 mg doses of nitazoxanide used in this study were selected on the basis of past experience with the pharmacokinetics and tolerability. 13 We recruited patients from 74 primary care clinics in the USA. The main eligibility criteria were participants aged 12-65 years, oral temperature greater than 38°C (>100·4°F), at least one respiratory symptom (cough, sore throat, nasal discharge, nasal congestion, sneezing) and one constitutional symptom (headache, myalgia, sweats or chills, fatigue), symptom duration of 48 h or less, and confi rmation of infl uenza by a laboratory in the local community (30 mile radius of the site). We excluded because of severity of illness requiring admission to hospital, high risk of infl uenza-related complications according to Infectious Diseases Society of America guidelines 14 or present US Centers for Disease Control and Prevention (CDC) criteria, 15 vaccination for seasonal infl uenza on or after Aug 1, 2010, treatment with any dose of oseltamivir, zanamivir, amantadine, rimantadine, nitazoxanide, or any investi gational drug therapy within 30 days before screening, and active respiratory allergies or pre-existing illnesses that could place the participant at an unreasonably increased risk.

The central institutional review board (IRB) or a local IRB at each centre approved the protocol. The trial was done under an investigational new drug application with the FDA and done in accordance with guidelines set by the World Medical Assembly (Declaration of Helsinki, last amendment in Seoul, 2008). Every participant or their guardian gave written informed consent. Participants younger than 18 years of age provided written assent.

Randomisation lists were computer generated. Every participant was randomly assigned (ratio 1:1:1) to receive either two bottles containing nitazoxanide 300 mg tablets (nitazoxanide 600 mg group), one bottle containing nitazoxanide 300 mg tablets plus one bottle containing placebo tablets (nitazoxanide 300 mg group), or two bottles of placebo (placebo group). Nitazoxanide tablets and placebo tablets were packaged in white high-density polyethylene bottles, each containing ten tablets. Participants were instructed to take two tablets, one from each bottle, twice daily with food for 5 days. The randomisation lists and medication packages were prepared by an independent third party who maintained the blinding of the trial until the database was locked. Randomisation was done in blocks of three. Masked study medication (three treatment kits per block) were assigned to every investigator who sequentially assigned treatment numbers to participants as they were enrolled. The randomisation list was masked to study participants such as sponsor, investigators, study monitors, patients, and laboratory personnel.

Immediately after obtaining informed consent and verifying eligibility, every patient had a baseline (day 1) physical examination and medical history recorded. We obtained two nasopharyngeal swabs (nylon fl ocked dry swabs, Copan Diagnostics, Murrieta, CA, USA), and blood and urine samples for laboratory safety testing and calculation of infl uenza antibody titres. Study drugs were dispensed, and participants were instructed to use a diary twice daily (roughly every 12 h) to record the time and amount of every dose of study drug, symptom severity, concomitant medications, and adverse events. At every diary entry, the participants graded all nine symptoms (runny nose, nasal congestion, sore throat, cough, headache, muscle aches, tiredness or fatigue, feverish, and sweats or chills) on a scale of 0 to 3 as absent (0), mild (1), moderate (2), or severe (3). Participants completed the diaries up to at least study day 7, or until all symptoms were either absent or mild and had remained so for at least 24 h.

A study nurse visited or telephoned every participant daily on study days 2-5 to review symptoms and check for infl uenza-related complications. Participants returned to the clinic for physical examination and diary review on day 7 and day 28. We obtained blood and urine samples for laboratory safety tests and nasopharyngeal swabs for virology testing on day 7. We obtained a blood sample for measurement of infl uenza antibody titres on day 28. At ten sites, a nurse visited participants on days 2, 3, 4, and 5 to collect two nasopharyngeal swabs. We selected 24 participants for 12 h assessment of pharmacokinetics immediately after their fi rst dose on the morning of day 2 (12 h after their last dose on day 1). Antiviral drugs for infl uenza, over-the-counter cough or cold medications, and anti-allergy drugs for respiratory allergies were prohibited during the study. Paracetamol was allowed as necessary for fever (oral temperature ≥38°C). To monitor compliance, participants returned the bottles in which the medication was dispensed along with any unused medication, and pill counts were recorded.

Laboratory safety tests were haematology tests (complete blood count with diff erential), blood chemistry tests (comprehensive metabolic panel with lipid profi le), and urinalysis at baseline and on day 7. North Shore-LIJ Health System Laboratories in collaboration with BARC USA (Lake Success, NY, USA) did virology tests on nasopharyngeal swab, which were placed into 3 mL of viral transport medium and transported refrigerated to a central laboratory within 24 h. The swab samples were eluted, divided into aliquots, and immediately frozen at -70°C. Naso pharyngeal swabs at baseline and on day 7 were cultured for infl uenza viruses and analysed by RT-PCR with the ProdesseProfl u+ (Gen-Probe Prodesse, Waukesha, WI, USA) for infl uenza A and B and the Luminex xTag RVP v1 assay (Luminex Corporation, Austin, TX, USA) to detect infl uenza A (non-specifi c for subtype), infl uenza A subtypes seasonal H1 and H3, infl uenza B, respiratory syncytial virus A, respiratory syncytial virus B, adenovirus, human meta-pneumovirus, enterovirus or rhinovirus, parainfl uenza viruses 1, 2, 3, and 4, and coronaviruses 229E, OC43, NL63, and HKU1. Samples positive for infl uenza A were also analysed by real-time RT-PCR for 2009 infl uenza A H1N1 on the basis of CDC protocol. 16 For participants positive for infl uenza A or B by culture or RT-PCR at baseline, viral titres were measured at baseline and all subsequent collection points. We calculated titres as log 10 tissue culture infective dose 50 (TCID 50 )/0·2 mL and as log 10 RNA copies with a quantitative RT-PCR method developed and validated by North Shore-LIJ Health System Laboratories. For participants diagnosed with infl uenza by culture or RT-PCR at baseline, we measured infl uenza antibody titres by haemagglutination inhibition assay from serum samples obtained at baseline and at day 28 to assess eff ect of treatment on humoral immune response. Reference antigens used for measurement of antibody titres were H1/A/ California/7/2009, H3/A/Perth/16/2009, and B/ Brisbane/60/2008. To assess potential for resistance, we tested the viruses cultured from nasopharyngeal swab samples of nitazoxanide-treated participants on day 5 or day 7 and the corresponding virus sample from baseline (day 1) for susceptibility to tizoxanide as previously described. 5

The primary endpoint for the clinical trial was time from fi rst dose to alleviation of symptoms based on patientreported symptom data. This method has been validated by clinical trials of oseltamivir and zanamivir. [8] [9] [10] [11] [12] We deemed the symptoms of the participants as alleviated at the beginning of the fi rst 24 h period throughout which each symptom was graded as either absent or mild (0 or 1). In addition to the seven symptoms (cough, nasal obstruction, sore throat, fatigue, headache, myalgia, and feverishness) monitored in the oseltamivir and zanamivir studies, participants enrolled in this study also monitored runny nose and sweats or chills. Secondary endpoints were change in infl uenza virus titre with time, time to cessation of viral shedding, time to alleviation of individual symptoms, symptom severity, complications of infl uenza, time to return to normal activity, time lost from work, and infl uenza antibody response.

We analysed data according to a statistical analysis plan developed before the start of the clinical trial. The plan called for two primary effi cacy analyses, each comparing the time from fi rst dose to alleviation of symptoms for nitazoxanide 300 mg versus placebo, and nitazoxanide 600 mg versus placebo using a Kaplan-Meier survival analysis, and a Cox proportional hazards test including geographic location as a factor, two-sided α=0·025. We checked the proportional hazards assumption by including interaction terms of all covariates with (log)time and by visual inspection of the graph of the log(-log(survival)) versus the log of time. If hazards were not proportional, we used a Prentice-Wilcoxon test. We did analyses for a population consisting of participants who received at least one dose of study drug and had laboratory-confi rmed infl uenza infection by RT-PCR or culture at baseline. We did sensitivity analyses to assess eff ects of paracetamol use (repeating the primary analysis including paracetamol use and its interaction with the treatment group) and censored data (assuming participants with censored data had 28 days to alleviation of symptoms). Sample size calculations assumed a median time to symptom alleviation for placebo group of 4·3 days, a diff erence in median time to symptom alleviation between placebo and nitazoxanide treatment groups of 1·5 days, and a dropout rate of 5%. With a log-rank test, with α=0·025 (due to two primary effi cacy analyses) and a power of 80%, a sample size of 146 randomly assigned and treated participants with laboratory-confi rmed infl uenza per group was calculated.

We also did secondary effi cacy analyses of time to alleviation of symptoms for all participants who received study drug irrespective of laboratory evidence of infection and for participants with or without confi rmed respiratory virus infections. We did statistical comparisons of changes in viral titre with a mixed model for repeated measures including baseline viral titre, treatment group, and geographic location. The safety population included all participants who received at least one dose of study medication. All analyses were done with SAS software (version 6.12; SAS Institute, Cary, NC, USA).

This trial is registered with ClinicalTrials.gov, number NCT01227421.

The sponsor designed the protocol and engaged a contract research organisation to select, initiate, monitor, and close out study sites and collect and analyse data. The sponsor wrote the study report. All authors participated in the interpretation of data, writing of the manuscript, and decision to submit the manuscript for publication. All authors had full access to the data.

Between Dec 27, 2010, and April 30, 2011, we enrolled 624 participants with infl uenza-like illness of 650 people screened at 74 centres in the USA (fi gure 1). The study was terminated with about 60% of the planned number of patients infected with infl uenza because of the end of the infl uenza season. After randomisation, patients' character istics were well balanced between groups (table 1). 386 (62%) of 624 participants enrolled were diagnosed with a respiratory virus infection at baseline including 257 (41%) with infl uenza A or B (table 2) . Demographic and disease-related characteristics of participants were similar for each of the three treatment groups (tables 1-3).

Treatment with nitazoxanide 600 mg twice daily for 5 days signifi cantly decreased the time from fi rst dose to alleviation of symptoms compared with placebo in participants with confi rmed infl uenza (p=0·0084; fi gure 2, table 4). Time to symptom alleviation also decreased in patients given nitazoxanide 300 mg compared with those given placebo, but the diff erence was not signifi cant (p=0·52). We used Prentice-Wilcoxon test for these analyses because the proportional hazards assumption required for use of the Cox model was violated (interaction of the 600 mg nitazoxanide treatment group with log(time) was signifi cant [p=0·0294]). At the time of symptom alleviation, no participants were still receiving symptom relief medication (paracetamol). We did sensitivity analyses in the population with confi rmed infl uenza to assess the eff ects of censored data and use of paracetamol on the primary endpoint. For participants with censored data (dropouts or without symptom alleviation at last diary assessment, n=16), we assumed time to alleviation of symptoms of 28 days. In this analysis, results were similar to the primary effi cacy analysis favouring nitazoxanide 600 mg compared with placebo (p=0·0062 [data not shown).

212 (82%) of 257 participants enrolled in the population with confi rmed infl uenza reported taking paracetamol during the study as allowed by the study protocol. Proportions of participants taking paracetamol were similar in the three treatment groups: 73 (82%) of 89 participants in the placebo group, 74 (83%) of 89 in the nitazoxanide 300 mg group, and 65 (82%) of 79 in the nitazoxanide 600 mg group. When we repeated the primary analysis including paracetamol use and its interaction with the treatment group, participants receiving nitazoxanide 600 mg had a shorter time to symptom alleviation than did those receiving placebo (p=0·0085).

In this study, we enrolled participants within 48 h of symptom onset, whereas in studies of neuraminidase inhibitors participants were enrolled within 36 h. 8, 9, 11 To assess the eff ect of this variable, we analysed participants enrolled within 36 h of symptom onset. In these post-hoc analyses, diff erences (nitazoxanide 600 mg vs placebo) in Responses for subgroups infected with infl uenza A or B were similar (table 4). In analyses of 624 participants treated and 238 (38%) participants with no virus identifi ed at baseline, time from fi rst dose to alleviation of symptoms was signifi cantly lower in the 600 mg group than in the placebo group (table 4). We investigated too few participants infected with other individual viral infections to make meaningful analyses.

Another diff erence between the design of this study and earlier studies of the neuraminidase inhibitors was the scoring of nine symptoms instead of seven. 8, 9 Use of two additional symptoms, runny nose and sweats or chills, only aff ected the time to alleviation of symptoms for six participants (three given placebo, one given nitazoxanide 300 mg, and two given nitazoxanide 600 mg). Analysis based on the seven symptoms used in the oseltamivir studies showed median times to alleviation of symptoms of 115·9 h (95% CI 108-121) for the placebo group, 109·1 h (96-130, p=0·58) for the nitazoxanide 300 mg treatment group, and 95·5 h (84-108, p=0·0074) for the nitazoxanide 600 mg treatment group.

At baseline, cough and nasal congestion were the most persistent symptoms in participants infected with infl uenza. The median time to alleviation in the placebo group for cough was 104 h (95% CI 84-111) and for nasal congestion was 81 h (67-92). Treatment with nitazoxanide 600 mg was associated with a decrease in the median duration of these symptoms to 84 h (95% CI 60-104) for cough and to 60 h (48-74) for nasal congestion.

For nine symptoms, in participants infected with infl uenza, mean symptom score-hours (symptom severity score multiplied by hours) summed for every participant from baseline to alleviation of symptoms were 1221 (592 SD) for the placebo group, 1305 (765) for the nitazoxanide 300 mg treatment group, and 1125 (681) for the nitazoxanide 600 mg treatment group, whereas in all treated participants, the score-hours were 1232 (1028) for the placebo group, 1171 (972) for the 300 mg treatment group, and 1045 (661) for the 600 mg treatment group.

Complications of infl uenza (bronchitis, sinusitis, otitis, pneumonia, and pleurisy) were fairly uncommon in this population. The frequency was not signifi cantly diff erent between treatment groups: 15 events (7%) for placebo, compared with 20 (10%) for the nitazoxanide 300 mg treatment group (p=0·38), and 11 (5%) for the nitazoxanide 600 mg treatment group (p=0·54).

We obtained daily nasopharyngeal swabs at baseline and on days 2-5 for 113 participants (41 in the placebo group, 33 in the nitazoxanide 300 mg treatment group, and 39 in the nitazoxanide 600 mg treatment group). TCID 50 viral titres signifi cantly decreased during treatment in the nitazoxanide 600 mg group compared with placebo group (p=0·0006; fi gure 3 roughly 1 log 10 were apparent within 24 h after initiation of treatment for the nitazoxanide 600 mg group and continued until day 5 when viral shedding stopped for almost all participants in the placebo group. Reductions in TCID 50 viral titres were also recorded for the nitazoxanide 300 mg treatment group compared with the placebo group, although these diff erences were not as large as for the nitazoxanide 600 mg treatment group and were not signifi cant (fi gure 3). Reductions in viral titres detected by TCID 50 were similar to those dectected by RT-PCR except that the magnitude of reductions were somewhat smaller when measured by RT-PCR, which also detects non-infectious virus particles. Median times to cessation of viral shedding were 91·3 h (95% CI 67·5-96·0) for the placebo group, 77·0 h (48·0-95·5) for the nitazoxanide 300 mg treatment group, and 71·8 h (55·0-96·0) for the nitazoxanide 600 mg treatment group. At day 7, only one participant (placebo group) was positive for infl uenza infection by viral culture. Infl uenza virus was detected by RT-PCR in samples from day 7 for 23 (39%) of 59 patients in the placebo group, 17 (19%) of 73 patients in the nitazoxanide 300 mg treatment group, and 15 (24%) of 63 patients in the 600 mg treatment group. We cultured infl uenza viruses from nasopharyngeal swabs taken on day 5 from 13 participants treated with nitazoxanide. Viruses present in all 13 samples were inhibited by tizoxanide, with eff ective concentrations EC50 and EC90 similar to those inhibiting viruses present in the corresponding baseline samples (data not shown).

To assess potential eff ect of treatment on humoral immune response, we measured antibody titres at baseline and day 28 for participants with laboratory-confi rmed infl uenza by RT-PCR or viral culture at baseline. We noted no signifi cant diff erences in antibody titre change from baseline to day 28 or in the proportions of participants with laboratory-confi rmed infl uenza seroprotected (antibody titre ≥40) or seroconverted (four-fold increase in antibody titre) at day 28 between the treatment groups. Seroconversion rates were 34 (49%) of 70 participants for the placebo group, 36 (58%) of 62 for the nitazoxanide 300 mg treatment group, and 36 (55%) of 65 for the nitazoxanide 600 mg treatment groups (p=0·52).

We analysed plasma concentrations of tizoxanide during the fi rst 12 h after dosing on the morning of day 2 for six participants treated with nitazoxanide 300 mg and ten participants treated with nitazoxanide 600 mg. Mean maximum plasma concentrations of tizoxanide were 2·46 μg/mL (SD 1·36) for the nitazoxanide 300 mg group and 4·60 μg/mL (3·61) for the nitazoxanide 600 mg, and mean trough concentrations were 0·121 μg/mL (0·09) for the nitazoxanide 300 mg treatment group and 0·795 μg/mL (1·97) for the nitazoxanide 600 mg group. Mean area under the curve (AUC 0-12 h ) for tizoxanide plasma concentrations were 12·6 μg × h/mL (SD 9·2) for the nitazoxanide 300 mg group and 29·1 μg × h/mL (33·8) for the nitazoxanide 600 mg group. Analysis of change in TCID 50 viral titre for participants with confi rmed infl uenza that we took daily nasopharyngeal swabs from. Statistical comparison with mixed model for repeated measures including baseline viral titre, treatment group, and geographic location: p=0·0006 for the diff erence between nitazoxanide 600 mg and placebo, p=0·1553 for the diff erence between nitazoxanide 300 mg and placebo.

group compared with none in the placebo group. This event was probably attributed to the colour of nitazoxanide metabolites. Because of the mild nature and relative infrequency of chromaturia (14 [2%] of 624 participants enrolled), the blinding of the study was not aff ected.

Treatment with nitazoxanide given orally 600 mg twice daily for 5 days beginning less than 48 h after symptom onset signifi cantly reduced the time from fi rst dose to alleviation of symptoms in participants aged 12-65 years with acute uncomplicated infl uenza. The 300 mg dose produced an intermediate response that did not signifi cantly diff er to that in the placebo group.

The trial was designed according to regulatory guidance and was consistent with previous trials of the neuraminidase inhibitors, oseltamivir and zanamivir (panel). [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] Reductions in time to alleviation of symptoms (median diff erence 21·2 h for the 600 mg group) were in the range of those recorded in trials of neuraminadase inhibitors. [8] [9] [10] [11] [12] [17] [18] [19] Diff erences in the size of treatment eff ect from one trial to another could arise as a result of diff erences in study design (eg, duration of symptoms at time of enrolment, populations selected for analysis, use of paracetamol) or characteristics of circulating infl uenza strains (eg, pathogenicity, drug susceptibility). In this study, we enrolled participants within 48 h of symptom onset, whereas most clinical trials of neuraminidase inhibitors enrolled participants within 36 h of symptom onset. Time to symptom alleviation was fastest in the subset enrolled within 36 h of symptom onset into the nitazoxanide 600 mg group.

By contrast with studies of neuraminidase inhibitors, the results from this study show a potential treatment benefi t in participants without infl uenza or other documented viral infection (17·3 h reduction in time to alleviation of symptoms for 600 mg vs placebo). Comparisons of treatment eff ect of nitazoxanide and neuraminidase inhibitors in participants with confi rmed infl uenza infection or with or without other documented viral infections will be an interesting goal for future trials.

The adverse event profi le of nitazoxanide is well known because it has been commercially available since 1996 in global markets for treatment of intestinal infections. This study used a new controlled-release formulation of nitazoxanide designed specifi cally to deliver drug into the blood and to the respiratory tract and to maintain blood concentrations for 12 h. In the 600 mg dose group, the drug was given at a dose that is 20% higher and for a duration of therapy that is 67% longer than the drug's approved dose for treatment of intestinal infections. 6 This increase in dose and duration did not result in any signifi cant change in the known side-eff ect profi le. Frequency or severity of adverse events did not signifi cantly diff er between participants in any group of the study.

The fi ndings of this clinical trial need to be confi rmed by other studies. Limitations of this study include the number of participants and the fact that they were all enrolled during a single infl uenza season. The absence of statistical signifi cance for the 300 mg treatment group might refl ect an absence of statistical power because only about 60% of the planned number of participants were recruited. Although this trial enrolled participants with infl uenza AH3N2, ApH1N1, and B, examination of the effi cacy and safety of the drug against infl uenza strains from another season would be benefi cial. The drug does not seem to be associated with resistance, as might be expected because of its mechanism of action, but further studies to assess the potential for resistance would be valuable. Furthermore, although this clinical trial has shown a treatment eff ect in otherwise healthy participants aged 12-65 years, the eff ect of the drug still needs to be studied in children and patients with serious illness or those at risk of infl uenza complications. 

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The clinical study was done in the USA under the US Food and Drug Administration IND #107 316. We thank Romark Laboratories LC, Tampa, FL, USA, for sponsoring the study; BARC USA, Lake Success, NY, USA, for serving as central laboratory for the study and doing laboratory safety and virology studies; SGS Life Science Services, Gaithersburg, MD, USA, for serving as contract research organisation for the study; and the laboratory of Gabriella Santoro, University of Rome Tor Vergata, Rome, Italy, for doing resistance testing. The international phase 3 clinical trial (NCT01610245) referred to in the Discussion section is funded in whole or in part with Federal funds from the US Department of Health and Human Services, Offi ce of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority under contract HHSO100201300004C.

An international phase 3 clinical trial in participants with acute uncomplicated infl uenza is ongoing (NCT01610245), which is designed to compare nitazoxanide given orally 600 mg twice daily for 5 days with placebo and to compare the combination of nitazoxanide and oseltamivir with oseltamivir or nitazoxanide given as monotherapy. Another trial in participants admitted to hospital with severe acute respiratory illness (NCT02057757) is in progress. These studies and others should provide additional information related to the value of the drug in the treatment of infl uenza and other viral respiratory infections.

J-FR designed the study. JH, AH, MH, HR, and SS recruited participants and collected data. CG supervised the clinical virology work. All authors reviewed and interpreted the data. MB and J-FR drafted the report. All authors approved the fi nal report. 

We searched PubMed for systematic reviews and meta-analyses published in English language between Jan 1, 2009, and Jan 29, 2014, with the search terms "infl uenza treatment", "infl uenza clinical trial", "oseltamivir", "zanamivir", and "neuraminidase inhibitor". With the same search terms, we also searched for articles published in English between Jan 1, 1990, and Jan 29, 2014, reporting placebo-controlled clinical trials of drugs for treatment of infl uenza. We identifi ed nine systematic reviews or meta-analyses within the past 5 years related to the use of the neuraminidase inhibitors, oseltamivir and zanamivir, and we identifi ed several articles reporting randomised placebo-controlled clinical trials reporting a treatment eff ect of oral oseltamivir, inhaled zanamivir, or intravenous peramivir 8-12,17-26 (another neuraminidase inhibitor) in reducing the time from fi rst dose to alleviation of symptoms in participants with acute uncomplicated infl uenza. Articles have also reported benefi cial treatment eff ect of oseltamivir and zanamivir in otherwise healthy children infected with infl uenza. Evidence to support treatment benefi ts of neuraminidase inhibitors in elderly and at-risk groups or their eff ects on rates of admission to hospital and mortality was insuffi cient.

In our trial, oral administration of nitazoxanide 600 mg twice daily for 5 days reduced the time to alleviation of symptoms of infl uenza in otherwise healthy adults and adolescents by roughly 1 day. Although many factors can aff ect the size of treatment eff ect observed in diff erent clinical trials, the eff ect recorded in this trial is similar to that previously reported for the neuraminidase inhibitors. [8] [9] [10] [11] [12] 17, 18, 27 Data from this trial suggest a treatment benefi t of nitazoxanide in participants without infl uenza or other documented viral infection-a fi nding that has not been reported for the neuraminidase inhibitors. Importantly, nitazoxanide possesses a novel mechanism of action against infl uenza viruses. A new drug with a diff erent mechanism of action could ultimately prove benefi cial in overcoming resistance to neuraminidase inhibitors or in providing more potent therapy when used in combination with existing drugs. As a fi rst step, it has been important to show eff ectiveness of nitazoxanide in a randomised placebo controlled trial by use of a method established for licensure of existing drugs. Further studies are warranted to confi rm our fi ndings and to assess effi cacy of nitazoxanide alone or in combination with existing drugs in seriously ill patients and those at risk of infl uenza complications.
