This was a prospective study conducted at the All India Institute of Medical Sciences (AIIMS), New Delhi between September, 2007 and March, 2011. Patients who tested positive for HIV by ELISA and were ART-naïve and presented with concomitant TB were enrolled as cases. Patients, who tested positive for HIV by ELISA, were ART-naïve and without TB were enrolled as controls. Only patients having CD4 count < 200 cells/mm3 and with normal renal and hepatic function (SGOT and SGPT ≤ 5 × upper normal limit, Serum Bilirubin ≤ 2.5 × upper normal limit and Creatinine ≤ 3 × upper normal limit) were included. The other inclusion criteria were age > 18 years, non-pregnant as confirmed by a negative urine pregnancy test, and absence of concomitant diabetes mellitus. Hepatitis B and C serologies were done and patients testing positive were excluded, as it could have a bearing on hepatotoxicity of study drugs which was one of the outcomes. Also, patients on anti-epileptic drugs, immunosuppressants and other drugs that induce liver microsomal enzyme systems were excluded. HIV infection was documented by licensed ELISA test kit (As per NACO guidelines). CD4/CD8 cell counts were determined by flow- cytometry (BD FACS CALIBUR). Viral load testing was done using AMPLICOR HIV-1 Monitor Test, version 1.5, manufactured by ROCHE Diagnostics. The protocol was approved by the institutional research Ethics Committee of the All India Institute of Medical Sciences, New Delhi. All participants gave signed informed consent to participate in this study.
All patients underwent a detailed physical examination. Their body weight and height were measured and their basal metabolic index (BMI) was calculated. Haemoglobin, complete blood counts, erythrocyte sedimentation rate, fasting blood glucose, renal function tests, liver function tests, serum albumin, serum uric acid and routine urinalysis were done for all patients. In addition, their CD4 counts and plasma HIV viral load were determined at baseline, six months and 12 months.
Cases were started first on anti-tuberculosis treatment (ATT) according to the Revised National Tuberculosis Control Programme (RNTCP) guidelines for directly observed therapy, short-course (DOTS) . After two to eight weeks of ATT, they were started on antiretroviral drug therapy, which consisted of zidovudine, lamivudine and nevirapine (fixed drug combination). The control group was started on ART when CD4 count < 200 cells/mm3. Those who had haemoglobin less than 8 g/dl were administered stavudine in place of zidovudine. The doses that were administered were in accordance with the NACO guidelines. Zidovudine was given in a dose of 300 mg twice a day, lamivudine 150 mg twice a day and stavudine 30 mg twice a day. Nevirapine was administered at a dose of 200 mg once a day for the first 14 days (called the lead-in dose) as per NACO guideline, and then the dose was escalated to 200 mg twice a day. The patients were advised to take the drug at 9 am for the first 14 days and at 9 am and 9 pm during the rest of the period of follow up.
Patients were assessed at day 14 after the start of ART, then at day 28, and every 4 weeks thereafter through 48 weeks. A complete haemogram and liver and kidney function tests were obtained at all these visits, and CD4 counts were measured at 8 weeks, 24 weeks and 48 weeks after the start of ART. HIV plasma viral load was measured at baseline, at 24 weeks and at the end of 48 weeks only in the cases. Trough nevirapine concentrations were assessed at day 14, day 28, day 42 and at day 180, 12 hours after the evening dose of nevirapine.
Vital status, clinical progression, Immunological and virological responses, mortality, and drug toxicity were assessed as outcome measures.
Immunological failure was defined as a fall in CD4 counts to baseline concentrations, a 50% fall from the peak CD4 count during treatment or persistent counts below 100 cells/mm3 at the end of 24 weeks. Disease progression was defined as a new or recurrent WHO stage 4 conditions, after at least 6 months of ART. Virologic response was defined as plasma viral load less than 400 copies/ml after 6 months of ART and drug toxicity was characterized as per the division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, December 2004.
Measurement of nevirapine concentrations
Blood samples for Nevirapine concentrations measurement were taken 12 hours after drug intake. Each patient was properly counselled about taking the drug in time so that the sample can be drawn exactly at 12 hours. Wherever feasible, the patient was asked to take the drug in front of the research staff and the sample was collected at 12 hours. In others, telephonic conversation was used to ensure that the patient has taken the drug on time. All samples were stored at -80°C until analyzed. At the time of processing of the sample, each plasma sample was allowed to reach room temperature. Nevirapine was procured from Indian Pharmacopoeia Commission IPC, Ghaziabad, India. Tablet Olanzapine (as internal standard) was procured commercially from Sun Pharmaceuticals Ltd., Mumbai, India. Thermo Finnigan High Performance Liquid Chromatographic system (Thermo Electron Corp, USA) with PDA detector controlled by ChromQuest (Ver.4.5) software was used to elude the analyte. Purospher Star RP-C18e, 55 × 4 mm, 3 μ particle (Merck, Germany) was used for analytical separation. Electron spray ionization technique in positive mode was applied using Tubo Ionspray source (ABS Biosystems, USA) in a 4000 Q trap MS/MS (MDS SCIEX, Applied Biosystems). Tandem Mass spectroscopy was controlled using Analyst (Ver.1.4.2) software. The same procedure for nevirapine measurement was used in controls as used in the cases.
Plasma spiking of nevirapine in the concentration of 0.07, 0.14, 0.28, 4.48 and 17.9 ng/ml was prepared by using blank plasma. For this, known amount of the nevirapine and olanzapine was added to blank human plasma (obtained from Blood Bank of AIIMS) and separate calibration curve was made. For the analysis of standard calibration curve, best fit was obtained with the inbuilt algorithm of Analysist. Ver. 1.4.2 was used. Best fit obtained for spiking was subjected to quantify unknown concentration. The best fit plotted the ratio of peak height between analyte and internal standard in "abscissa (Y)" axis and taking the ratio of their concentration in "ordinate (x)" axis.
Data were recorded on a pre-designed data sheet and managed on an 'Excel' spreadsheet. All entries were doubly checked for any possible recording error. Mean, frequency and medians were calculated for all quantitative variables along with the respective standard deviations and Interquartile ranges. Being a pilot study, initially sample size calculation was not planned and data was gathered as per the sample of convenience. On later calculation, sample size required was 127 patients per group for 80% power. All analysis was done by Intention to treat analysis principle. The Mann Whitney U-test was used to compare the mean nevirapine concentrations at day 28 in patients alive at 24 and 48 weeks, and those who died during the study period. The generalised estimation equations were used to find out the predictors of immunological response in terms of the increase in CD4. Statistical analysis was performed using statistical software package STATA version 11.0 [(intercooled version), Stata Corporation, Houston, Texas, USA].