Pooled Deep Sequencing of Drug Resistance Loci from Plasmodium falciparum Parasites across Ethiopia.

Although Ethiopia has an overall lower prevalence of Plasmodium falciparum among countries in Africa, the emergence of drug resistance could seriously hinder elimination efforts. Using samples collected from five therapeutic efficacy studies conducted in 2007-11, we evaluated the prevalence of putative drug resistance mutations in the pfcrt, pfmdr1, and kelch13 genes at the time of those studies, as well as the ama1 gene for genetic relatedness using a pooled amplicon deep sequencing approach. Among all sites, the kelch13 gene showed no mutations, whereas the pfcrt CVIET genotype was fixed in all populations. By contrast, the mdr1 gene demonstrated frequencies of resistant genotypes ranging from 10 to 100% at amino acid position 86 and from 0% to 57.8% at amino acid position 1246. Although we observed a low degree of haplotype sharing between sites, we did observe considerable haplotype sharing within sites over time. This suggests that P. falciparum populations in Ethiopia are isolated and able to persist through time.

Correction: Comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of Plasmodium vivax infection in Ethiopia: A randomized controlled trial.

[This corrects the article DOI: 10.1371/journal.pmed.1002299.].

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in Ethiopia: absence of common African and Mediterranean allelic variants in a nationwide study.

BACKGROUND: Building on the declining trend of malaria in Ethiopia, the Federal Ministry of Health aims to eliminate malaria by 2030. As Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia, the use of primaquine is indicated for both transmission interruption and radical cure, respectively. However, the limited knowledge of the local prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its associated variants has hindered the use of primaquine. METHODS: Some 11,138 dried blood spot (DBS) samples were collected in 2011 as part of a national, household Malaria Indicator Survey, a multi-stage nationally representative survey of all malaria-endemic areas of Ethiopia. A randomly selected sub-set of 1414 DBS samples was successfully genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Considering the geographical position and ethnic mix of the country, three common variants: G6PD*A (A376G), G6PD*A- (G202A) and Mediterranean (C563T) were investigated. RESULTS: Of the 1998 randomly selected individuals, 1429 (71.5%) DBS samples were genotyped and merged to the database, of which 53.5% were from females. G6PD*A (A376G) was the only genotype detected. No sample was positive for either G6PD*A- (G202A) or Mediterranean (C563T) variants. The prevalence of G6PD*A (A376G) was 8.9% [95% confidence interval (CI) 6.7-11.2] ranging from 12.2% in the Southern Nations, Nationalities and Peoples' (95% CI 5.7-18.7) to none in Dire Dawa/Harari Region. CONCLUSION: The common G6PD*A- (G202A) or Mediterranean (C563T) variants were not observed in this nationwide study. The observed G6PD*A (A376G) mutation has little or no clinical significance. These findings supported the adoption of primaquine for P. falciparum transmission interruption and radical cure of P. vivax in Ethiopia. As the presence of other clinically important, less common variants cannot be ruled out, the implementation of radical cure will be accompanied by active haematological and adverse events monitoring in Ethiopia.

Genetic diversity of the msp-1, msp-2, and glurp genes of Plasmodium falciparum isolates in Northwest Ethiopia.

BACKGROUND: Determination of the genetic diversity of malaria parasites can inform the intensity of transmission and identify potential deficiencies in malaria control programmes. This study was conducted to characterize the genetic diversity and allele frequencies of Plasmodium falciparum in Northwest Ethiopia along the Eritrea and Sudan border. METHODS: A total of 90 isolates from patients presenting to the local health centre with uncomplicated P. falciparum were collected from October 2014 to January 2015. DNA was extracted and the polymorphic regions of the msp-1, msp-2 and glurp loci were genotyped by nested polymerase chain reactions followed by gel electrophoresis for fragment analysis. RESULTS: Allelic variation in msp-1, msp-2 and glurp were identified in 90 blood samples. A total of 34 msp alleles (12 for msp-1 and 22 for msp-2) were detected. For msp-1 97.8% (88/90), msp-2 82.2% (74/90) and glurp 46.7% (42/90) were detected. In msp-1, MAD20 was the predominant allelic family detected in 47.7% (42/88) of the isolates followed by RO33 and K1. For msp-2, the frequency of FC27 and IC/3D7 were 77% (57/74) and 76% (56/74), respectively. Nine glurp RII region genotypes were identified. Seventy percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 2.6 (95% CI 2.25-2.97). The heterozygosity index was 0.82, 0.62 and 0.20 for msp-1, msp-2 and glurp, respectively. There was no significant association between multiplicity of infection and age or parasite density. CONCLUSIONS: There was a high degree of genetic diversity with multiple clones in P. falciparum isolates from Northwest Ethiopia suggesting that there is a need for improved malaria control efforts in this region.

Comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of Plasmodium vivax infection in Ethiopia: A randomized controlled trial.

BACKGROUND: Recent efforts in malaria control have resulted in great gains in reducing the burden of Plasmodium falciparum, but P. vivax has been more refractory. Its ability to form dormant liver stages confounds control and elimination efforts. To compare the efficacy and safety of primaquine regimens for radical cure, we undertook a randomized controlled trial in Ethiopia. METHODS AND FINDINGS: Patients with normal glucose-6-phosphate dehydrogenase status with symptomatic P. vivax mono-infection were enrolled and randomly assigned to receive either chloroquine (CQ) or artemether-lumefantrine (AL), alone or in combination with 14 d of semi-supervised primaquine (PQ) (3.5 mg/kg total). A total of 398 patients (n = 104 in the CQ arm, n = 100 in the AL arm, n = 102 in the CQ+PQ arm, and n = 92 in the AL+PQ arm) were followed for 1 y, and recurrent episodes were treated with the same treatment allocated at enrolment. The primary endpoints were the risk of P. vivax recurrence at day 28 and at day 42. The risk of recurrent P. vivax infection at day 28 was 4.0% (95% CI 1.5%-10.4%) after CQ treatment and 0% (95% CI 0%-4.0%) after CQ+PQ. The corresponding risks were 12.0% (95% CI 6.8%-20.6%) following AL alone and 2.3% (95% CI 0.6%-9.0%) following AL+PQ. On day 42, the risk was 18.7% (95% CI 12.2%-28.0%) after CQ, 1.2% (95% CI 0.2%-8.0%) after CQ+PQ, 29.9% (95% CI 21.6%-40.5%) after AL, and 5.9% (95% CI 2.4%-13.5%) after AL+PQ (overall p < 0.001). In those not prescribed PQ, the risk of recurrence by day 42 appeared greater following AL treatment than CQ treatment (HR = 1.8 [95% CI 1.0-3.2]; p = 0.059). At the end of follow-up, the incidence rate of P. vivax was 2.2 episodes/person-year for patients treated with CQ compared to 0.4 for patients treated with CQ+PQ (rate ratio: 5.1 [95% CI 2.9-9.1]; p < 0.001) and 2.3 episodes/person-year for AL compared to 0.5 for AL+PQ (rate ratio: 6.4 [95% CI 3.6-11.3]; p < 0.001). There was no difference in the occurrence of adverse events between treatment arms. The main limitations of the study were the early termination of the trial and the omission of haemoglobin measurement after day 42, resulting in an inability to estimate the cumulative risk of anaemia. CONCLUSIONS: Despite evidence of CQ-resistant P. vivax, the risk of recurrence in this study was greater following treatment with AL unless it was combined with a supervised course of PQ. PQ combined with either CQ or AL was well tolerated and reduced recurrence of vivax malaria by 5-fold at 1 y. TRIAL REGISTRATION: ClinicalTrials.gov NCT01680406.

Genetic polymorphism of Merozoite Surface Protein-2 (MSP-2) in Plasmodium falciparum isolates from Pawe District, North West Ethiopia.

BACKGROUND: In malaria endemic regions, Plasmodium falciparum infection is characterized by extensive genetic diversity. Describing this diversity provides important information about the local malaria situation. This study was conducted to evaluate the extent of genetic diversity of P. falciparum in Pawe district, North West Ethiopia, using the highly polymorphic merozoite surface protein 2 gene. METHODS: Atotal of 92 isolates from patients with uncomplicated P. falciparum attending Pawe Health Centre were collected from September to December 2013. Genomic DNA was extracted using the Chelex method and analyzed by length polymorphism following gel electrophoresis of DNA products from nested PCR of msp2 (block 3), targeting allelic families of FC27 and 3D7/IC. RESULTS: There were twenty-two different msp2 alleles, 11 corresponding to the 3D7/ IC and 11 to the FC27 allelic family. The frequency of isolates of the msp2 3D7/IC allelic familywas higher (51%) compared to FC27 (49%). The majority of the isolates (76%) contained multiple infections andthe overall mean multiplicity of infection was 2.8 (CI 95% 2.55-3.03). The heterozygosity index was 0.66 for msp2. There was no statically significant difference in the multiplicity of infection by age or parasite density. CONCLUSIONS: The results of this study show that P.falciparum polymorphismsare extensive in Northwest Ethiopia and most of the infections are composed of multiple clones.

Therapeutic efficacy of artemether-lumefantrine against uncomplicated Plasmodium falciparum malaria in a high-transmission area in northwest Ethiopia.

Malaria, particularly due to Plasmodium falciparum, remains a major public health threat in Ethiopia. Artemether-lumefantine (AL) has been the first-line antimalarial drug against uncomplicated P. falciparum malaria in the country since 2004. Regular monitoring of antimalarial drugs is recommended by the World Health Organization (WHO) to help early detection of drug resistant strains of the parasite and contain their rapid spread. The objective of this study was to assess the therapeutic efficacy of AL in a high-transmission setting in Ethiopia. The study site was Setit Humera, northwest Ethiopia. Single-arm prospective study of a 28-day follow-up was conducted from October 2014 to January 2015 according to the revised WHO 2009 drug efficacy study protocol. Study end-points were classified into primary end-point and secondary end-point. While the primary end-point was the day-28 adequate clinical and parasitological response the secondary end-points were clinical and parasitological evaluations (parasite, fever and gametocyte clearance rate, incidence of drug adverse events) and the relative increment in hemoglobin (Hb) level from baseline to day (D) 14 and D28. A total of 92 patients were enrolled and 79 had completed the 28-day follow-up period. The overall cure rate was 98.8% with 95% confidence interval of 0.915-0.998 without polymerase chain reaction correction. The parasite clearance rate was high with fast resolution of clinical symptoms; 100% of the study participants cleared parasitaemia and fever on D3. Gametocyte carriage was reduced from 7% on D0 to 1% on D3 and complete clearance was achieved on D14. Mean Hb concentration significantly increased on D28 compared to that on D14. There was no serious adverse event. AL was efficacious and safe in a high-transmission setting for treatment of uncomplicated falciparum malaria.

Open-label trial on efficacy of artemether/lumefantrine against the uncomplicated Plasmodium falciparum malaria in Metema district, Northwestern Ethiopia.

PURPOSE: Following the increased Plasmodium falciparum resistance to chloroquine and sulfadoxine/pyrimethamine, Ethiopia adopted artemether/lumefantrine (AL) as the first-line treatment for uncomplicated P. falciparum in 2004. According to the recommendation of the World Health Organization, this study was carried out for regular monitoring of the efficacy of AL in treating the uncomplicated P. falciparum malaria in Metema district, Gondar Zone, Northwest Ethiopia. PATIENTS AND METHODS: This is a one-arm prospective 28-day in vivo therapeutic efficacy study among the uncomplicated P. falciparum malaria patients aged 6 months and older. The study was conducted from October 2014 to January 2015, based on the revised World Health Organization protocol of 2009 for surveillance of antimalarial drug therapeutic efficacy study. Standard six-dose regimen of AL was given twice daily for 3 days, and then the treatment outcomes were assessed on days 0, 1, 2, 3, 7, 14, 21, 28, and any other unscheduled day for emergency cases. RESULTS: There were 91 study subjects enrolled in this study, of whom 80 study subjects completed the full follow-up schedules and showed adequate clinical and parasitological responses on day 28, with no major adverse event. Per protocol analysis, the unadjusted cure rate of Coartem(®) was 98.8% (95% confidence interval: 93.3%-100%) in the study area. Recurrence of one P. falciparum case was detected on day 28, with a late parasitological failure rate of 1.2%. No early treatment failure occurred. Complete parasite and fever clearance was observed on day 3. Gametocyte carriage was 4.4% at enrollment that cleared on day 21. Although the difference is statistically not significant, a slight increase in the level of mean hemoglobin from baseline to day 28 was observed. CONCLUSION: The study showed high efficacy and tolerability of Coartem(®) against uncomplicated P. falciparum malaria, suggesting the continuation as a first-line drug in the study district. However, regular monitoring of the therapeutic efficacy of the drug, possibly with plasma drug-level measurement, is critical among the mobile border population.

Therapeutic Efficacy of Artemether-Lumefantrine (Coartem®) in Treating Uncomplicated P. falciparum Malaria in Metehara, Eastern Ethiopia: Regulatory Clinical Study.

BACKGROUND: As per the WHO recommendation, the development of resistance by P. falciparum to most artemisinin combination therapies (ACTs) triggered the need for routine monitoring of the efficacy of the drugs every two years in all malaria endemic countries. Hence, this study was carried out to assess the therapeutic efficacy of Artemether-Lumefantrine (Coartem®) in treating the uncomplicated falciparum malaria, after 9 years of its introduction in the Metehara, Eastern Ethiopia. METHOD: This is part of the therapeutic efficacy studies by the Federal Ministry of Health Ethiopia, which were conducted in regionally representative sentinel sites in the country from October 2014 to January 2015. Based on the study criteria set by WHO, febrile and malaria suspected outpatients in the health center were consecutively recruited to study. A standard six-dose regimen of AL was administered over three days and followed up for measuring therapeutic responses over 28 days. Data entry and analysis was done by using the WHO designed Excel spreadsheet and SPSS version 20 for Windows. Statistical significant was considered for P-value less than 0.05. RESULT: Of the 91 patients enrolled, the day-28 analysis showed 83 adequate clinical and parasitological responses (ACPRs). Per protocol analysis, PCR-uncorrected & corrected cure rates of Coartem® among the study participants were 97.6% (95%CI: 93.6-99.5) and 98.8% (CI: 93.5-100%), respectively. No parasite detected on day 3 and onwards. Fever clearance was above 91% on day-3. Mean hemoglobin was significantly increased (P<0.000) from 12.39 g/dl at day 0 to 13.45 g/dl on day 28. No serious adverse drug reactions were observed among the study participants. CONCLUSION: This study showed high efficacy of AL in the study area, which suggests the continuation of AL as first line drug for the treatment of uncomplicated P. falciparum malaria in the study area. This study recommends further studies on drug toxicity, particularly on repeated cough and oral ulceration.

Declining trend of Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles after the withdrawal of Sulfadoxine-Pyrimethamine in North Western Ethiopia.

Antimalarial drug resistance is one of the major challenges in global efforts of malaria control and elimination. In 1998, chloroquine was abandoned and replaced with sulfadoxine/pyrimethamine, which in turn was replaced with artemether/lumefantrine for the treatment of uncomplicated falciparum malaria in 2004. Sulfadoxine/pyrimethamine resistance is associated with mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes. The prevalence of mutation in Pfdhfr and Pfdhps genes were evaluated and compared for a total of 159 isolates collected in two different time points, 2005 and 2007/08, from Pawe hospital, in North Western Ethiopia. The frequency of triple Pfdhfr mutation decreased significantly from 50.8% (32/63) to 15.9% (10/63) (P<0.001), while Pfdhps double mutation remained high and changed only marginally from 69.2% (45/65) to 55.4% (40/65) (P = 0.08). The combined Pfdhfr/Pfdhps quintuple mutation, which is strongly associated with sulfadoxine/pyrimethamine resistance, was significantly decreased from 40.7% (24/59) to 13.6% (8/59) (P<0.0001). On the whole, significant decline in mutant alleles and re-emergence of wild type alleles were observed. The change in the frequency is explained by the reduction of residual drug-resistant parasites caused by the strong drug pressure imposed when sulfadoxine/pyrimethamine was the first-line drug, followed by lower fitness of these resistant parasites in the absence of drug pressure. Despite the decrease in the frequency of mutant alleles, higher percentages of mutation remain prevalent in the study area in 2007/08 in both Pfdhfr and Pfdhps genes. Therefore, further multi-centered studies in different parts of the country will be required to assess the re-emergence of sulfadoxine/pyrimethamine sensitive parasites and to monitor and prevent the establishment of multi drug resistant parasites in this region.

Genetic diversity of Plasmodium falciparum isolates based on MSP-1 and MSP-2 genes from Kolla-Shele area, Arbaminch Zuria District, southwest Ethiopia.

BACKGROUND: The genetic diversity of Plasmodium falciparum has been extensively studied in various countries. However, limited data are available from Ethiopia. This study was conducted to evaluate the extent of genetic diversity of P. falciparum in Kolla-Shele, in the southwest of Ethiopia. METHODS: A total of 88 isolates from patients with uncomplicated P. falciparum attending Kolla-Shele Health Centre was collected from September to December, 2008. After extraction of DNA by Chelex method, the samples were genotyped by using nested-PCR of msp1 (block 2) and msp2 (block 3) including their allelic families: K1, MAD20, RO33 and FC27, 3D7/IC1, respectively. RESULTS: Allelic variation in both msp1 and msp2 were identified in the 88 blood samples. For msp1 67% (59/88) and msp2 44% (39/88) were observed. K1 was the predominant msp1 allelic family observed in 33.9% (20/59) of the samples followed by RO33 and MAD20. Of the msp2 allelic family 3D7/IC1 showed higher frequency (21.5%) compared to FC27 (10.3%). A total of twenty-three alleles were detected; of which, eleven were from msp2 and twelve from msp2 genes. Fifty-nine percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 1.8 (95% CI: 1.48-2.04). The heterozygosity index was 0.79 and 0.54for msp1 and msp2, respectively. There was no statically significant difference in the multiplicity of infection by either age or parasite density (P > 0.05). CONCLUSION: This genetic diversity study showed the presence of five allelic types in the study area, with dominance K1 in the msp1 family and 3D7/IC1 in the msp2 family. Multiple infections were observed in nearly 60% of the samples.

Prevalence of sulfadoxine-pyrimethamine resistance-associated mutations in dhfr and dhps genes of Plasmodium falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia.

Ethiopia changed the first-line anti-malarial drug for uncomplicated Plasmodium falciparum malaria from sulfadoxine-pyrimethamine (SP) to Coartem(®) in 2004 following nation-wide assessment of the efficacy of both drugs in 2003. This study was conducted to assess the prevalence of sulfadoxine-pyrimethamine resistance-associated mutations in dhfr and dhps genes of P. falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia. A total of 165 blood spot samples were collected from patients infected with P. falciparum in Bahir Dar Health Center in 2005 (n=78) and 2008 (n=87) using Whatman (3M) filter papers. The three dhfr codons (dhfr108, dhfr 51 and dhfr 59) and the two dhps codons (dhfr 437 and 540) which are believed to determine SP resistance were detected by using nested PCR-based dot blot-hybridization technique. In dhfr, only the dhfr59Arg mutant-type showed statistically significant reduction from 80.3% in 2005 to 56.4% in 2008 (p<0.01) with a significant increase of the wild type dhfr59Cys haplotypes from 4.9% in 2005 to 29.5% in 2008 (p<0.01). The double mutants dhfr108Asn/51Ile were detected at rate of 98.4% in 2005 and 98.7% in 2008. A significant decrease in the triple dhfr (108Asn/51Ile/59Arg) mutation was observed from 2005 (78.6%) to 2008(56.4%) (p<0.01). The quadruple mutations of dhfr (108Asn/51Ile/59Arg)/dhps437Gly were significantly declined from 78.6% in 2005 to 53.8% in 2008 (p<0.01) while quintuple mutations (dhfr (108Asn/51Ile/59Arg)/dhps437Gly/dhps540Glu) showed a reduction from 60.6% to 37.2% after three years (p<0.01). In conclusion, the decline in the prevalence of dhfr/dhps combination mutations might indicate the re-emergence of sensitive parasites in the population following SP withdrawal. Therefore, further monitoring and assessment is important to determine the feasibility of re-introduction of SP alone or in combination as a more affordable and safer drug in the future in Ethiopia.

In vivo efficacy of artemether-lumefantrine and chloroquine against Plasmodium vivax: a randomized open label trial in central Ethiopia.

BACKGROUND: In vivo efficacy assessments of antimalarials are essential for ensuring effective case management. In Ethiopia, chloroquine (CQ) without primaquine is the first-line treatment for Plasmodium vivax in malarious areas, but artemether-lumefantrine (AL) is also commonly used. METHODS AND FINDINGS: In 2009, we conducted a 42-day efficacy study of AL or CQ for P. vivax in Oromia Regional State, Ethiopia. Individuals with P. vivax monoinfection were enrolled. Primary endpoint was day 28 cure rate. In patients with recurrent parasitemia, drug level and genotyping using microsatellite markers were assessed. Using survival analysis, uncorrected patient cure rates at day 28 were 75.7% (95% confidence interval (CI) 66.8-82.5) for AL and 90.8% (95% CI 83.6-94.9) for CQ. During the 42 days of follow-up, 41.6% (47/113) of patients in the AL arm and 31.8% (34/107) in the CQ arm presented with recurrent P. vivax infection, with the median number of days to recurrence of 28 compared to 35 days in the AL and CQ arm, respectively. Using microsatellite markers to reclassify recurrent parasitemias with a different genotype as non-treatment failures, day 28 cure rates were genotype adjusted to 91.1% (95% CI 84.1-95.1) for AL and to 97.2% (91.6-99.1) for CQ. Three patients (2.8%) with recurrent parasitemia by day 28 in the CQ arm were noted to have drug levels above 100 ng/ml. CONCLUSIONS: In the short term, both AL and CQ were effective and well-tolerated for P. vivax malaria, but high rates of recurrent parasitemia were noted with both drugs. CQ provided longer post-treatment prophylaxis than AL, resulting in delayed recurrence of parasitemia. Although the current policy of species-specific treatment can be maintained for Ethiopia, the co-administration of primaquine for treatment of P. vivax malaria needs to be urgently considered to prevent relapse infections. TRIAL REGISTRATION: ClinicalTrials.gov NCT01052584.

Paracheck-pf® Test Versus Microscopy in the Diagnosis of Falciparum Malaria in Arbaminch Zuria Woreda of South Ethiopia.

BACKGROUND: Malaria is a major cause of morbidity and mortality in Ethiopia. Rapid diagnostic tests such as Paracheck Pf are the major tools for falciparum malaria diagnosis as an alternative to microscopy in peripheral health facilities. The objective of this study was to evaluate the sensitivity and specificity of Paracheck Pf against microscopy for diagnosis of P.falciparum infection and observe the persistence of the antigen for an elongated period. METHODS: Cross sectional study was undertaken in Arbaminch Zuria at Shele health center from October 2008 to January 2009. Paracheck-Pf versus microscopy comparison was done in conjunction with an artemisinin-based combination therapy efficacy monitoring for a period of 28 days. Standard microscopic procedures were done by experienced laboratory technicians and paracheck-Pf was performed in accordance with the manufacturer's instruction. RESULTS: out of 1293 examined blood films, 400(31%) were found to be malaria positive. Considering microscopy as the gold standard, paracheck-pf showed sensitivity of 94.1 %( 95%CI: 89.9-98.3%) and specificity of 80.0% (95%CI: 67.6-92.4%). The positive and negative predictive values were 93.3 %( 95%CI: 88.8-97.8%) and 82.1% (95%CI: 70-94.1%), respectively. Comparing microscopy results 98.7 % (79/80), 60% (48/80), 48.1% (37/77), and 44.6 %( 33/74) were also found to be positive by paracheck-pf at days7, 14, 21, and 28, respectively. CONCLUSION: Paracheck Pf® has a comparable diagnostic performance in detecting P. falciparum infections through the persistence of frequent false positivity is a limitation. Thus, this diagnostic test is not appropriate for monitoring of treatment effect.

In vivo efficacy of artemether-lumefantrine against uncomplicated Plasmodium falciparum malaria in Central Ethiopia.

BACKGROUND: In vivo efficacy assessments of the first-line treatments for Plasmodium falciparum malaria are essential for ensuring effective case management. In Ethiopia, artemether-lumefantrine (AL) has been the first-line treatment for uncomplicated P. falciparum malaria since 2004. METHODS: Between October and November 2009, we conducted a 42-day, single arm, open label study of AL for P. falciparum in individuals >6 months of age at two sites in Oromia State, Ethiopia. Eligible patients who had documented P. falciparum mono-infection were enrolled and followed according to the standard 2009 World Health Organization in vivo drug efficacy monitoring protocol. The primary and secondary endpoints were PCR uncorrected and corrected cure rates, as measured by adequate clinical and parasitological response on days 28 and 42, respectively. RESULTS: Of 4426 patients tested, 120 with confirmed falciparum malaria were enrolled and treated with AL. Follow-up was completed for 112 patients at day 28 and 104 patients at day 42. There was one late parasitological failure, which was classified as undetermined after genotyping. Uncorrected cure rates at both day 28 and 42 for the per protocol analysis were 99.1% (95% CI 95.1-100.0); corrected cure rates at both day 28 and 42 were 100.0%. Uncorrected cure rates at day 28 and 42 for the intention to treat analysis were 93.3% (95% CI 87.2-97.1) and 86.6% (95% CI 79.1-92.1), respectively, while the corrected cure rates at day 28 and 42 were 94.1% (95% CI 88.2-97.6) and 87.3% (95% CI 79.9-92.7), respectively. Using survival analysis, the unadjusted cure rate was 99.1% and 100.0% adjusted by genotyping for day 28 and 42, respectively. Eight P. falciparum patients (6.7%) presented with Plasmodium vivax infection during follow-up and were excluded from the per protocol analysis. Only one patient had persistent parasitaemia at day 3. No serious adverse events were reported, with cough and nausea/vomiting being the most common adverse events. CONCLUSIONS: AL remains a highly effective and well-tolerated treatment for uncomplicated falciparum malaria in the study setting after several years of universal access to AL. A high rate of parasitaemia with P. vivax possibly from relapse or new infection was observed. TRIAL REGISTRATION: NCT01052584.

Therapeutic efficacy of Artemether/Lumefantrine (Coartem(R)) against Plasmodium falciparum in Kersa, South West Ethiopia.

BACKGROUND: Artemether/Lumefantrine (Coartem(R)) has been used as a first-line treatment for uncomplicated Plasmodium falciparum infection since 2004 in Ethiopia. In the present study the therapeutic efficacy of artemether/lumefantrine for the treatment of uncomplicated P. falciparum infection in Kersa, Jima zone, South-west Ethiopia, has been assessed. METHODS: A 28 day therapeutic efficacy study was conducted between November 2007 and January 2008, in accordance with the 2003 WHO guidelines. Outcomes were classified as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) and adequate clinical and parasitological response (ACPR). RESULTS: 90 patients were enrolled and completed the 28 day follow-up period after treatment with artemether/lumefantrine. Cure rate was very high, 96.3%, with 95% CI of 0.897-0.992 (PCR uncorrected). Age-stratified data showed adequate clinical and parasitological response (ACPR) to be 100% for children under 5 and 97.4% and 87.3% for children aged 5-14, and adults, respectively. There was no early treatment failure (ETF) in all age groups. Fever was significantly cleared on day 3 (P<0.05) and 98% of parasites where cleared on day 1 and almost all parasites were cleared on day 3. 72.5% of gametocytes were cleared on day 1, the remaining 27.5% of gametocytes were maintained up to day 3 and total clearance was observed on day 7. Hemoglobin concentration showed a slight increase with parasitic clearance (P>0.05). No major side effect was observed in the study except the occurrence of mouth ulcers in 7% of the patients. CONCLUSIONS: The current study proved the excellent therapeutic efficacy of artemether/lumefantrine in the study area and the value of using it. However, the proper dispensing and absorption of the drug need to be emphasized in order to utilize the drug for a longer period of time. This study recommends further study on the toxicity of the drug with particular emphasis on the development of oral ulcers in children.

Anti-malarial activity of withania somnifera L. Dunal extracts in mice.

OBJECTIVE: This study aims at investigating the in vivo antiplasmodial activity of a traditionally used medicinal plant, Withania somnifera, L. Dunal, (Solanaceae). METHODS: Rodent malaria parasite, Plasmodium berghei, 0.2 ml of x l0(7) parasites, was inoculated into Swiss albino mice intraperitoneally. Extracts were administered by intra gastric tube daily for four days starting from the day of parasite inoculation. Negative controls received the same amount of solvent used to suspend the exracts and the positive controls were given chloroquine by the same route. Parasite suppressive effect and effects on body weight, packed cell volume (PCV) and body temperature were determined. RESULTS: Parasitemia percent inhibition of W. somnifera roots and root barks were 50.43% and 29.13% respectively, with 600 mg/kg dose. Inhibition was statistically significant at all dose levels, compared to the negative controls (p < 0.05), and maximum inhibition was seen at 600 mg/kg. CONCLUSION: Extracts of the leaves and root barks of W. somnifera showed parasite suppressive effect and a protective effect on PCV drop (at higher doses), both in dose-related fashions. However, the effects on body weight and body temperature falls are inconclusive.

Development of resistance by Plasmodium falciparum to sulfadoxine/pyrimethamine in Amhara Region, Northwestern Ethiopia.

Following the high rate of chloroquine-resistant Plasmodium falciparum, Ethiopia changed the national drug policy in 1999 from chloroquine to sulfadoxine/pyrimethamine (SP) as first line. However, the useful therapeutic lifespan of SP may be limited by the rapid emergence of resistance. We conducted a study between October and November 2001 to examine the current extent of SP resistance to P. falciparum in Amhara Region, Northern Ethiopia. A total of 93 patients with uncomplicated P. faliciparum malaria were studied from Habru (n=50) and Weizazirt (n=43) localities. Drug resistance was evaluated using the 14-day WHO in vivo test protocol. Parasitological resistance to SP was found to be 32.0% (16/50) and 4.7% (2/43) in Harbu and Weizazirt localities, respectively. The corresponding clinical failure rates were 20.0% (10/50) and 4.7% (2/43). Of the parasitological failure at Harbu, 9 patients were classified as RI, 6 as RII and one as RIII type response. Among the clinical failures, 7 were LTF while the remaining 3 were ETF. Nevertheless, there was hardly any patient with RII/RIII or ETF response, and only two patients each with RI and LTF response were seen from Weizazirt locality. Therefore, the study underscores the presence of varying level of SP resistance to P. falicparum in the areas studied. Whereas SP remains quite effective in the treatment of uncomplicated P. falciparum in Weizazirt locality, the observed high rate of resistance from Harbu is alarming and an area of concern.