A parallel randomized double blind placebo controlled trial with allocation ratio of 1:1 was planned at a single site.
The study was based in the pediatric and adult medicine wards of King George’s Medical University (KGMU) hospital in Lucknow - the capital city of Uttar Pradesh. This is a teaching hospital which caters mostly to the poor and seriously ill from the city and surrounding districts extending upto Nepal.
Prior approval for the trial was obtained from the Institutional Ethics Committee and Drug Controller General of India (DCGI). Consent and adverse event forms were filled up for each patient. A Data Safety Monitoring Board (DSMB) with nine members was formed.
Drug and placebo
Minocycline hydrochloride and placebo powder for suspension were obtained from Unimark Remedies, India in identical bottles. Placebo differed from drug in the active ingredient being absent. The bottles were supplied in lots – four bottles/lot for children upto 12 years old and three bottles/lot for older patients. All the bottle lots were labeled individually at INCLEN before sending to the study site for use. The bottles bearing same lot ID were used for only one patient.
Randomisation & masking
The bottles of minocycline drug and placebo powder were supplied to a 3rd party- an independent member from International Clinical Epidemiology Network (INCLEN), New Delhi, who generated the randomization list with blocks of four. Separate randomization was done for children aged < =12 years and older patients.
Contents of bottles (appearance, colour, smell) were similar. The randomization sequence and code of the bottle lots were kept in safe custody by INCLEN. The patient, personnel assessing outcomes and person entering data were blinded as to drug or placebo received.
From late August 2012 to May 2013, we actively screened patients hospitalized in pediatric (age < =12 years) and adult medicine wards with AES for enrollment in the study. Inclusion criteria for enrollment were i) age > three years but excluding women of child bearing age (between 16 and 44 years) ii) Presence of AES defined as fever with altered sensorium of < = seven days duration. Exclusion criteria were i) a firm alternative etiological diagnosis ii) consent for participation not obtained and iii) some contraindication to drug administration. Written informed consent for participation in the study was taken from the patient’s guardian. As patients were admitted, those meeting inclusion criteria without any exclusion criteria were enrolled and randomized to receive the next numbered bottle lot. No changes to eligibility criteria or outcome measures were made after commencement of the trial.
Enrollment, treatment and work up
At admission, a detailed history, physical and neurological examination was recorded on predesigned data collection forms. Intracranial tension could not be measured but presence of hypertension with bradycardia or decerebrate posturing or Cheyne Stokes type of breathing were taken as suggestive of raised intracranial tension. A standardized work up of patients of AES as used here was adopted - including blood counts, smear and rapid test for malaria, blood urea, blood culture, serum electrolytes, creatinine and liver function tests. Neuroimaging was done whenever possible. Cerebrospinal fluid (CSF) if obtained by lumbar puncture by the treating team was examined for cell count, sugar, protein, gram stain and bacterial culture. Supportive treatment with antipyretics, anticonvulsants, nursing care, intravenous fluids, cerebral dehydrants, oxygen , ventilation and vasopressors as needed was given to patients. Intravenous ceftriaxone was also usually administered for 7–10 days. Minocycline (or placebo) suspension was reconstituted and given through nasogastric tube for seven days at a loading dose of 5 mg/kg/day followed by 2 · 5 mg/kg 12 hourly in children upto 12 years old and 200 mg loading dose followed by 100 mg 12 hourly in older patients. The agent was started by the study personnel soon after the patient was enrolled and randomized. Code on the bottle was recorded. Every dose administered was entered on the case sheet and signed. If a dose was vomited out within half hour it was repeated. If an alternative diagnosis was made after starting the agent, it was continued but specific treatment was also given. Even if the agent was withdrawn due to some contraindication later on, the patient was followed up for outcome. Contraindications were abnormal liver enzymes greater than five times normal or abnormal renal function with blood urea exceeding 35 mmol/l, diarrhea, rash or severe gastric bleeding.
CSF and serum specimen were stored at 4 C and transported in ice to the Virology Laboratory, KGMU. IgM against JEV in CSF was tested by IgM Capture ELISA (MAC ELISA) using the National Institute of Virology (Pune) kit  and in serum, by the Panbio Combo kit (Australia). If a specimen collected within one week of illness onset was negative, the test was repeated in serum after an interval of 7–10 days.
Follow up and outcome
Patients were followed up daily till they left the hospital. Blood counts, liver and renal function tests were repeated after one week or earlier if indicated. Time taken to become convulsion free, afebrile, to start oral feeds and total hospital stay were recorded. Generally patients were discharged once they could be fed orally and were afebrile.
Outcome in hospital was classified as discharge or death. In many cases, the family of the patient preferred to take the patient home once the outcome appeared hopeless. This was called “leave against medical advice” or LAMA. Patients who just left the hospital without informing hospital staff were called ‘absconders’. Families of discharged patients were given appointments to revisit the hospital.
Three months after, the families were contacted on mobile phone and health of their patient was inquired into. The interviewer was unaware of the agent (drug/ placebo) received by the patient. Primary outcome in the study was cumulative mortality at 3 months from hospitalization. If the patient was alive, special questionnaires were administered telephonically. Questions included ability to walk, talk, epilepsy, mental functions, vision, hearing, focal deficits, rigidity, and abnormal movements. Overall outcome at 3 months was classified as normal/near normal, independent functioning, dependent, vegetative, or death as per the Glasgow Outcome Scale (GOS) . If the patient revisited the hospital, GOS was again assessed and full neurological examination done. In some patients who could not be contacted, home visits were made.
AES mortality in our hospital is 30 % (unpublished data). To detect lowering of mortality to 15 % with 95 % confidence, with sample of 135 patients per arm, power of the study came to roughly 80 % .
Standardized adverse event forms were filled up for each patient. Serious adverse events were reported within 24 hours and agent was discontinued.
Data management and analysis
Double data entry followed by data matching was done. Analysis was done by the 3rd party (INCLEN). Secondary outcome factors were days to become convulsion free, days to become afebrile, days to start feeding orally, duration of hospital stay in days and GOS at three months. Means were compared by ANOVA/student ‘t’ test and proportions by χ  test using Epi-info 3 · 5 · 4 software. Relative risks for death was calculated. Interim analysis was planned after each season and criteria to decode was observation of significant difference in primary outcome between the two groups. Kaplan Meier survival curves were generated. Analysis was by intention to treat.
Study personnel were given training in study procedures. Standard Operating Procedures were followed. A clinical research organization was hired.