HPLC analysis of generic antiretroviral drugs purchased in Rwanda.

A reversed phase HPLC method using photo diode array detection for the simultaneous quantification of lamivudine, stavudine, nevirapine, zidovudine, methyl paraben and propyl paraben in solid and liquid drug formulations was developed and validated. The separation was achieved using a Waters Symmetry C8 column, using a mobile phase gradient comprising 50 mM NaH2PO4 (pH 3.8) and acetonitrile (95:5 to 45:55, v/v) and a flow gradient (0.5 to 1.0 ml/min). The limits of detection and quantification were below 19 ng/ml and 55 ng/ml respectively. The intra- and inter-day assay precisions were within 4.4% relative standard deviations. The developed method was applied to 12 different generic antiretroviral medications, consisting of tablets, capsules and solutions, produced by two Indian manufacturers and purchased by the Central Agency of Essential Drug Procurement of Rwanda for the ESTHER project in Rwanda. The average content of the antiretroviral agent(s) compared to the labeled amount(s) was 101.4%. Methyl paraben and propyl paraben, added to solutions as preservatives, were within the FDA recommended limits.

Evaluation of saliva as an alternative matrix for monitoring plasma Zidovudine, Lamivudine and nevirapine concentrations in Rwanda.

Saliva may provide interesting advantages as matrix for compliance measurements, pharmacokinetic studies and therapeutic drug monitoring in resource limited countries. We investigated the feasibility of using saliva for compliance monitoring of zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP) in 29 HIV-1 infected patients from Rwanda. ZDV, 3TC and NVP drug levels were quantified by an LC/MS-MS method in plasma and stimulated saliva samples and compared using Bland-Altman analysis. Seven patients demonstrated undetectable saliva ZDV levels while five out of these seven also showed no 3TC salivary concentrations. For the other samples, we observed a good agreement between salivary and plasma concentrations of each antiretroviral drug. A significant relation between the difference in saliva and plasma ZDV concentrations and the average ZDV concentration in the two matrices was deduced as follow: y = -380.15 + 1.79 x. The log saliva and plasma concentration difference of both 3TC and NVP was consistent across the range of average log concentration. Overall, we showed large agreement limits suggesting a wide inter patient variability that may result to non-reliable plasma level predictions from saliva drug measurements. Therefore, our results indicate that saliva may serve as a valuable tool only for NVP compliance testing because of its high salivary concentration.

Validation of 2006 WHO prediction scores for true HIV infection in children less than 18 months with a positive serological HIV test.

INTRODUCTION: All infants born to HIV-positive mothers have maternal HIV antibodies, sometimes persistent for 18 months. When Polymerase Chain Reaction (PCR) is not available, August 2006 World Health Organization (WHO) recommendations suggest that clinical criteria may be used for starting antiretroviral treatment (ART) in HIV seropositive children <18 months. Predictors are at least two out of sepsis, severe pneumonia and thrush, or any stage 4 defining clinical finding according to the WHO staging system. METHODS AND RESULTS: From January 2005 to October 2006, we conducted a prospective study on 236 hospitalized children <18 months old with a positive HIV serological test at the national reference hospital in Kigali. The following data were collected: PCR, clinical signs and CD4 cell count. Current proposed clinical criteria were present in 148 of 236 children (62.7%) and in 95 of 124 infected children, resulting in 76.6% sensitivity and 52.7% specificity. For 87 children (59.0%), clinical diagnosis was made based on severe unexplained malnutrition (stage 4 clinical WHO classification), of whom only 44 (50.5%) were PCR positive. Low CD4 count had a sensitivity of 55.6% and a specificity of 78.5%. CONCLUSION: As PCR is not yet widely available, clinical diagnosis is often necessary, but these criteria have poor specificity and therefore have limited use for HIV diagnosis. Unexplained malnutrition is not clearly enough defined in WHO recommendations. Extra pulmonary tuberculosis (TB), almost impossible to prove in young children, may often be the cause of malnutrition, especially in HIV-affected families more often exposed to TB. Food supplementation and TB treatment should be initiated before starting ART in children who are staged based only on severe malnutrition.

Efavirenz in human breast milk, mothers', and newborns' plasma.

BACKGROUND AND METHODS: Highly active antiretroviral therapy with efavirenz (EFV) has been prescribed to HIV-positive pregnant women in Rwanda (HIV status 1 and CD4 cell count > 350 cells/mm) during the last trimester of pregnancy and for 6 months after delivery. The EFV concentrations in maternal plasma, breast milk and in newborns' plasma of 13 women and their children between 6 weeks and 6 months post partum are reported. RESULTS: Results show a mean EFV plasma concentration of 6.55 mg/L in maternal plasma, 3.51 mg/L in skim milk, and 0.85 mg/L in infant plasma. Significant linear correlations between maternal plasma and skim milk (r = 0.8666, P < 0.0001) and between skim milk and infant plasma (r = 0.6646, P < 0.02) were found, but no significant correlation was observed between maternal and infant plasma concentrations (P > 0.05). CONCLUSIONS: After 6 months of breast-feeding, no child out of the 13 had been infected with HIV and all had good psychomotor and growth development. Our results suggest that EFV may be an alternative to nevirapine (NVP) during the third trimester of pregnancy and during the breast-feeding period. Further studies on larger groups of newborns will be necessary to get a better understanding of possible prophylactic protection of the newborns by highly active antiretroviral therapy with EFV given to the mothers.

Determination of nevirapine and efavirenz in plasma using GC/MS in selected ion monitoring mode.

An accurate, selective, and sensitive method for the determination of the nonnucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine (nvp) and efavirenz (efv) in human plasma using gas chromatography-mass spectroscopy in selected ion monitoring mode (GC/MS-SIM) was developed. Solid-phase extraction (SPE) gave extraction yields near 100% for both nvp and efv, and calibration curves were linear over the therapeutic concentration ranges. Variation of intraday and interday precision was below 5%. Intraday and interday inaccuracies varied between 0.6% and 10.4%. The lower limits of detection using a 200-microL plasma sample were 27 ng/mL for nvp and 26 ng/mL for efv, and the lower limits of quantification were 54 ng/mL and 72 ng/mL, respectively. The method was applied to the determination of nvp and efv in plasma specimens of 73 patients in HIV stage III or IV and on antiretroviral treatment in Kigali, Rwanda.