CYP2B6*6 genotype and high efavirenz plasma concentration but not nevirapine are associated with low lumefantrine plasma exposure and poor treatment response in HIV-malaria-coinfected patients.

We investigated the influence of efavirenz (EFV)- or nevirapine (NVP)-based antiretroviral therapy (ART) on lumefantrine plasma exposure in HIV-malaria-coinfected patients and implication of pharmacogenetic variations. A total of 269 HIV patients with uncomplicated falciparum malaria on NVP-based ART (NVP-arm), EFV-based ART (EFV-arm) or not receiving ART (control-arm) were enrolled and treated with artemether-lumefantrine. Day-7 lumefantrine, baseline EFV and NVP plasma concentrations, and CYP2B6*6,*18, CYP3A4*1B, CYP3A5*3,*6,*7, ABCB1 c.3435C>T and ABCB1 c.4036A>G genotypes were determined. The median day-7 lumefantrine plasma concentration was significantly lower in the EFV-arm compared with that in NVP- and control-arm. High EFV plasma concentrations and CYP2B6*6/*6 genotype significantly correlated with low lumefantrine plasma concentrations and high rate of recurrent parasitemia. No significant effect of NVP-based ART on lumefantrine exposure was observed. In conclusion, owing to long-term CYP3A induction, EFV-based ART cotreatment significantly reduces lumefantrine plasma exposure leading to poor malaria treatment response, which is more pronounced in CYP2B6 slow metabolizers.

Pharmacokinetic and pharmacogenomic modelling of the CYP3A activity marker 4ß-hydroxycholesterol during efavirenz treatment and efavirenz/rifampicin co-treatment.

OBJECTIVES: To assess the effect of the major efavirenz metabolizing enzyme (CYP2B6) genotype and the effects of rifampicin co-treatment on induction of CYP3A by efavirenz. PATIENTS AND METHODS: Two study arms (arm 1, n = 41 and arm 2, n = 21) were recruited into this study. In arm 1, cholesterol and 4ß-hydroxycholesterol were measured in HIV treatment-naive patients at baseline and then at 4 and 16 weeks after initiation of efavirenz-based antiretroviral therapy. In arm 2, cholesterol and 4ß-hydroxycholesterol were measured among patients taking efavirenz during rifampicin-based tuberculosis (TB) treatment (efavirenz/rifampicin) just before completion of TB treatment and then serially following completion of TB treatment (efavirenz alone). Non-linear mixed-effect modelling was performed. RESULTS: A one-compartment, enzyme turnover model described 4ß-hydroxycholesterol kinetics adequately. Efavirenz treatment in arm 1 resulted in 1.74 (relative standard error = 15%), 3.3 (relative standard error = 33.1%) and 4.0 (relative standard error = 37.1%) average fold induction of CYP3A for extensive (CYP2B6*1/*1), intermediate (CYP2B6*1/*6) and slow (CYP2B6*6/*6) efavirenz metabolizers, respectively. The rate constant of 4ß-hydroxycholesterol formation [mean (95% CI)] just before completion of TB treatment [efavirenz/rifampicin co-treatment, 7.40 × 10(-7) h(-1) (5.5 × 10(-7)-1.0 × 10(-6))] was significantly higher than that calculated 8 weeks after completion [efavirenz alone, 4.50 × 10(-7) h(-1) (4.40 × 10(-7)-4.52 × 10(-7))]. The CYP3A induction dropped to 62% of its maximum by week 8 of completion. CONCLUSIONS: Our results indicate that efavirenz induction of CYP3A is influenced by CYP2B6 genetic polymorphisms and that efavirenz/rifampicin co-treatment results in higher induction than efavirenz alone.

Simple and cost-effective liquid chromatographic method for determination of pyrimethamine in whole blood samples dried on filter paper.

A cost-effective HPLC method for determination of pyrimethamine (PYR) in human whole blood samples dried on filter paper (Whatman) is reported. Trimethoprim (TMP) was used as an internal standard. Whole blood spiked with PYR was transferred (100 microl) onto filter paper and dried at room temperature. Capillary blood samples (100 microl) after ingestion of three tablets of sulfadoxine-pyrimethamine (SP) by one subject were also tested. PYR and an internal standard (IS) TMP were extracted into di-isopropyl ether as bases and then re-extracted with 150 microl mobile phase. A C-18 column was used and the mobile phase consisted of phosphate buffer (0.05 M, pH 5):acetonitrile:concentrated perchloric acid (750:300:2.5, v/v/v). The absorbances of PYR and IS were monitored at 270 nm. The limit of quantification was 40 ng/ml. The within- and between-assay coefficient of variations were <10% at the limit of quantification.

High-performance liquid chromatographic method for determination of amodiaquine, chloroquine and their monodesethyl metabolites in biological samples.

A high-performance liquid chromatographic method for determination of amodiaquine (AQ), desethylamodiaquine (DAQ), chloroquine (CQ) and desethylchloroquine (DCQ) in human whole blood, plasma and urine is reported. 4-(4-Dimethylamino-1-methylbutylamino)-7-chloroquinoline was used as internal standard. The drugs and the internal standard were extracted into di-isopropyl ether as bases and then re-extracted into an acidic aqueous phase with 0.1 M phosphate buffer at pH 4.0 for AQ samples and at pH 2.5 for CQ filter paper samples. A C(18) column was used and the mobile phase consisted of methanol-phosphate buffer (0.1 M, pH 3)-perchloric acid (250: 747.5:2.5, v/v). The absorbance of the drugs was monitored at 333 nm and no endogenous compound interfered at this wavelength. The limit of quantification in whole blood, plasma and urine was 100 nM for AQ and DAQ (sample size 100 microliter) as well as for CQ and DCQ in blood samples dried on filter paper. For 1000 microliter AQ and DAQ samples, the limit of quantification was 10 nM in all three biological fluids. The within-assay and between-assay coefficients of variations were always <10% at the limits of quantification. Plasma should be preferred for the determination of AQ and DAQ since use of whole blood may be associated with stability problems.

Implementation of artemether-lumefantrine treatment policy for malaria at health facilities in Tanzania.

BACKGROUND: The purpose of this study was to compare knowledge on the part of health workers in public and private health facilities about prescribing and dispensing of an artemether-lumefantrine combination, 3 years after moving from sulfadoxine-pyrimethamine to artemether-lumefantrine as a first-line treatment for nonsevere malaria in Tanzania. METHODS: A cross-sectional survey of a convenience sample of 306 dispensaries and pharmacies was conducted in Dar Es Salaam and the Coast region of Tanzania. Of these, 122 were community pharmacies, 143 were private dispensaries, and 41 were public dispensaries. Specific outcome measures were health workers' knowledge of the new malaria treatment guidelines, recommended doses of artemether-lumefantrine, and food requirements. RESULTS: A total of 489 health workers were included in the study. The respondents were prescribers in private dispensaries, public dispensaries, and community pharmacies. Participants included medical officers (3.7%), clinical officers (38%), pharmacists (5.7%), and pharmaceutical technicians (3.9%). Nearly all workers in the public dispensaries and about 50% of workers in private dispensaries and community pharmacies were aware of recommended first-line malaria treatment. The difference in the proportion of health workers with adequate knowledge about the new recommended antimalarial medicine in public and private dispensaries was statistically significant (P < 0.0001). There was a higher proportion of workers in public dispensaries who had adequate knowledge about doses of artemether-lumefantrine for adults compared with workers in private dispensaries (P = 0.001). Only 58.0% of respondents were able to state correctly the recommended doses in private dispensaries as compared with 77.0% in public dispensaries. Knowledge about the requirement for a concomitant fatty meal was not significantly different between workers in public and private dispensaries (P = 0.280) or between those working in public dispensaries and pharmacies (P = 0.622). CONCLUSION: Knowledge about the use of artemether-lumefantrine was higher among health workers in public dispensaries than in their counterparts from private health care settings. The training organized by the Ministry of Health for workers in public health facilities in Tanzania contributed to such differences.

Effect of rifampicin and CYP2B6 genotype on long-term efavirenz autoinduction and plasma exposure in HIV patients with or without tuberculosis.

We performed a prospective comparative study to examine, from a pharmacogenetics perspective, the effect of rifampicin (RIF) on long-term efavirenz (EFV) autoinduction and kinetics. In a study population of patients with HIV receiving EFV with RIF (arm 2, n = 54) or without RIF (arm 1, n = 128 controls), intraindividual and interindividual plasma EFV and 8-hydroxyefavirenz levels were compared at weeks 4 and 16 of EFV therapy. In arm 2, RIF was initiated 4 weeks before starting EFV. In controls (arm 1), the plasma EFV was significantly lower whereas 8-hydroxyefavirenz was higher at week 16 as compared to week 4. By contrast, there were no significant differences in plasma EFV and 8-hydroxyefavirenz concentrations over time in arm 2. At week 4, the plasma EFV concentration was significantly lower in arm 2 as compared to arm 1, but no significant differences were observed by week 16. When stratified by CYP2B6 genotype, significant differences were observed only with respect to CYP2B6*1/*1 genotypes. Ours is the first report of the CYP2B6 genotype-dependent effect of RIF on long-term EFV autoinduction.

Long-term efavirenz autoinduction and its effect on plasma exposure in HIV patients.

We investigated the influence of gender and pharmacogenetic variations on long-term efavirenz autoinduction and disposition among patients with HIV in Tanzania (N = 129). Plasma concentrations (at 16 h) of efavirenz and 8-hydroxyefavirenz were quantified at weeks 4 and 16 of therapy. Genotyping was performed to identify cytochrome P450 (CYP) 2B6*6, CYP3A5*3, *6, and *7, and ABCB1-3435 C/T genotypes. There were reductions in the median efavirenz concentration (Wilcoxon matched-pair test P < 0.001) and efavirenz/8-hydroxyefavirenz ratio (P < 0.001) by 19 and 32%, respectively, at week 16 as compared with week 4. The proportion of patients with efavirenz concentration <1 µg/ml at week 16 was higher by 67, 25, and 5% in CYP2B6*1/*1, *1/*6, and *6/*6 genotypes, respectively. The defined therapeutic range based on observed plasma concentrations is affected by the time point of sampling and the CYP2B6 genotype. The effect of efavirenz autoinduction on reducing plasma exposure continues up to week 16 and predominantly affects CYP2B6 extensive metabolizers. Among CYP2B6 slow metabolizers, the presence of a CYP3A5 genotype allele is associated with greater effects of efavirenz autoinduction on plasma concentrations of the drug. The cumulative induction may influence the long-term antiretroviral therapy outcome, particularly in CYP2B6*1 carriers.

HIV patients presenting common adverse drug events caused by highly active antiretroviral therapy in Tanzania.

Antiretroviral (ARV) drug toxicities pose treatment challenges and contribute to poor adherence. This study was carried out to document the commonly reported adverse reactions caused by ARV drugs in HIV patients in Tanzania. Information on drug induced adverse reactions (ADRs) in patients using ARV drugs was collected from the databases maintained in HIV clinics of Dar es Salaam and Mbeya. A total of 7502 and 1234 records of patients under ARV therapy by December 2006 were analysed in Dar es Salaam and Mbeya, respectively. In May, 2008 a cross-sectional study was conducted in which, the association between nevirapine (NVP) plasma concentrations and skin rashes problems was determined in 50 patients put on NVP based HAART for less than 2 weeks. Determination of NVP plasma concentration was carried out using a validated HPLC method in which patients from Dar es Salaam were involved. The study revealed that, anaemia, liver toxicity, skin rash and peripheral neuropathy were the most reported ADRs. The NVP plasma level determination revealed that there was no difference between those who had experienced skin rashes and those who did not (mean of 6.05 and 5.5 microg/ml respectively). There was a slight increase in reported ADRs between 2005 and 2006. A total of 932 (12.4%) patients changed their regimen in Dar es Salaam between January 2005 and December, 2006. Similarly, a total of 542 (44%) patients in Mbeya changed their regimen during that period. It can be concluded that, in both Dar es Salaam and Mbeya patients developed ARV related ADRs which are similar to those reported elsewhere.

Poor knowledge on new malaria treatment guidelines among drug dispensers in private pharmacies in Tanzania: the need for involving the private sector in policy preparations and implementation.

OBJECTIVE: To assess the knowledge of dispensers in private pharmacies on new malaria treatment guidelines which involved switching from chloroquine (CQ) to sulfadoxine pyrimethamine (SP) and from SP to artemether-lumefantrine. METHODS: A structured questionnaire was used for data collection and the questions focused on whether the subjects were involved in the preparation or implementation of the guidelines or had undertaken any training on how to dispense new antimalarial medicines as recommended in the introduced new treatment guidelines. RESULTS: The study revealed that none of the participants had been involved in the preparation of the treatment guidelines, nor had they undertaken any training on their implementation. As many as 49% of the visited private pharmacies were found to continue stocking and selling CQ tablets and injections. Only 30% and 7% knew the correct dose regimen of SP and ALU respectively and none of them knew the condition of taking ALU with a fatty meal for improved absorption. CONCLUSION: Lack of involvement of the pharmaceutical personnel working in the private pharmacies, from the preparation of new malaria treatment guidelines to their implementation, contributed to their poor knowledge and skill on how to correctly dispense the medicines.

Validation of self-report and hospital pill count using unannounced home pill count as methods for determination of adherence to antiretroviral therapy.

Adherence to anti-retroviral therapy (ART) is very crucial for successful treatment outcomes. This study aimed to validate patient-self report (PSR) and hospital based pill count (HoPC) as adherence determination methods by using unannounced home visit pill count (HPC). The study was carried out at Muhimbili National Hospital in Dar es Salaam, Tanzania and 215 patients purposively selected were recruited. On refill day, the remaining pills were counted. They were also asked to report on the number of doses they missed during the past 28 days. They were later visited in their homes without appointment where the remaining pills were counted. Ninety-eight percent and 93% reported to adhere to ART by PSR method and HoPC, respectively. However, only 58% of the same study patients were found to be adherent by > or = 95% using HPC. In conclusion, PSR and HoPC do not always give reliable adherence data in patients undergoing ART. Therefore, we recommend application of combination methods for adherence measuring in patients starting to include patient self report and hospital based pill count in new patients and complementing them with unannounced home based pill count in experienced patients. Wherever possible, drug plasma concentration measurements should also be established.

Lack of impact of artesunate on the disposition kinetics of sulfadoxine/pyrimethamine when the two drugs are concomitantly administered.

OBJECTIVE: To determine the effect of artesunate (AT) on the disposition kinetics of sulfadoxine/pyrimethamine (SP) in humans. METHODS: In a randomized cross-over study, 16 healthy volunteers were given a dose of three SP tablets containing 500 mg of sulfadoxine (SDX) and 25 mg of pyrimethamine (PYR) (=SP group), while the second arm received three SP tablets + two AT tablets of 200 mg in total followed by 100 mg AT for the next 4 days (SP+AT group). Blood samples (100 microl) were collected by means of a finger prick and dried on filter paper. The blood spots were wrapped in polythene folders and stored at room temperature until analysis. The samples were assayed using high-performance liquid chromatographic methods. RESULTS: The peak concentration C(max)), time required to attain peak concentration (T(max)), half-life (t ((1/2))) and area under the plasma concentration-time curve (AUC) were determined. The C(max) of SDX were 92.9 and 98.9 microg/ml for the SP and SP+AT arms, respectively; for PYR, these were 0.86 and 0.79 microg/ml, respectively. The T(max) of SDX were 10 and 8 h for the SP and SP+AT arms, respectively; for PYR, these were 4.0 and 3.0 h, respectively. The AUC(0-288) of SDX were 15,840 and 18,876 microg/ml h for the SP and SP+AT arms, respectively; for PYR, they were 124 and 112 microg/ml h, respectively. The t ((1/2)) of values for SDX were 165 and 180 h for the SP and SP+AT arms, respectively; for PYR, these were 158 and 177 h, respectively. There was no statistically significant difference between the C(max), T(max), AUC(0-288) and t ((1/2)) between the two arms (p > 0.05). CONCLUSION: Taking AT concomitantly with SP does not have any impact in the disposition of SP.

Evaluation of the quality of amodiaquine and sulphadoxine/pyrimethamine tablets sold by private wholesale pharmacies in Dar Es Salaam Tanzania.

OBJECTIVE: There are several independent reports in Tanzania of treatment failures with commercially available sulphadoxine/pyrimethamine (SP) and amodiaquine (AQ) brands. The aim of this work was to assess the quality of SP and AQ tablets marketed by wholesale pharmacies in Dar Es Salaam, Tanzania. METHODS: All eight wholesale pharmacies authorized to import medicines and located in Dar Es Salaam were included in the study. From each pharmacy, samples of all SP and AQ brands available at the time of sampling were bought, provided they had a shelf-life of at least 1 year. A sample was either an intact box of 100 tablets or a sealed tin of 100 tablets. To ensure blinding, 30 tablets of each sample were removed from their original containers, coded and sent to the quality control laboratory for analysis. The name, strength, batch number, manufacturer and the expiry dates of the tablets were recorded. In total 15 AQ and 18 SP samples were collected. Identity, assay for content of active ingredients and dissolution assay were performed as described in the United States Pharmacopoeia (USP). RESULTS: All samples passed the identity test. Among the AQ samples collected, two of 15 (13%) failed the dissolution test but all passed the assay for content, whereas two of 18 (11%) and eight of 18 (44%) SP samples failed the assay for content and dissolution tests, respectively. None of the pharmacies stocked all AQ and SP brands. CONCLUSION: This work reveals the availability of poor quality antimalarial drugs on the Tanzanian market. Use of substandard drugs could have serious clinical consequences to patients. The results support the need for continuous monitoring of the quality of marketed drugs to ensure safety and efficacy of these products in the treatment of malaria in endemic areas.

Poor Knowledge on New Malaria Treatment Guidelines among Drug Dispensers in Private Pharmacies in Tanzania: the Need for Involving the Private Sector in Policy Preparations and Implementation

Objective: To assess the knowledge of dispensers in private pharmacies on new malaria treatment guidelines which involved switching from chloroquine (CQ) to sulfadoxine pyrimethamine (SP) and from SP to artemether-lumefantrine. Methods: A structured questionnaire was used for data collection and the questions focused on whether the subjects were involved in the preparation or implementation of the guidelines or had undertaken any training on how to dispense new antimalarial medicines as recommended in the introduced new treatment guidelines. Results: The study revealed that none of the participants had been involved in the preparation of the treatment guidelines; nor had they undertaken any training on their implementation. As many as 49of the visited private pharmacies were found to continue stocking and selling CQ tablets and injections. Only 30 and 7knew the correct dose regimen of SP and ALU respectively and none of them knew the condition of taking ALU with a fatty meal for improved absorption. Conclusion: Lack of involvement of the pharmaceutical personnel working in the private pharmacies; from the preparation of new malaria treatment guidelines to their implementation; contributed to their poor knowledge and skill on how to correctly dispense the medicines

Validation of Self-Report and Hospital Pill Count using Unannounced Home Pill Count as Methods for Determination of Adherence to Antiretroviral Therapy

Adherence to anti-retroviral therapy (ART) is very crucial for successful treatment outcomes. This study aimed to validate patient-self report (PSR) and hospital based pill count (HoPC) as adherence determination methods by using unannounced home visit pill count (HPC). The study was carried out at Muhimbili National Hospital in Dar es Salaam; Tanzania and 215 patients purposively selected were recruited. On re?ll day; the remaining pills were counted. They were also asked to report on the number of doses they missed during the past 28 days. They were later visited in their homes without appointment where the remaining pills were counted. Ninety-eight percent and 93reported to adhere to ART by PSR method and HoPC; respectively. However; only 58of the same study patients were found to be adherent by = 95using HPC. In conclusion; PSR and HoPC do not always give reliable adherence data in patients undergoing ART. Therefore; we recommend application of combination methods for adherence measuring in patients starting to include patient self report and hospital based pill count in new patients and complementing them with unannounced home based pill count in experienced patients. Wherever possible; drug plasma concentration measurements should also be established