Different mutation patterns of Plasmodium falciparum among patients in Jimma University Hospital, Ethiopia.

BACKGROUND: The emergence of drug resistance is a major problem in malaria control. Combination of molecular genotyping and characterization of mutations or single nucleotide polymorphisms (SNPs) correlated with drug resistance can provide information for subsequent surveillance of existing and developing drug resistance patterns. The introduction of artemether/lumefantrine (AL) as first-line treatment, never used before in Ethiopia, allowed the collection of baseline data of molecular polymorphisms before a selection due to AL could occur. METHOD: 97 patients with uncomplicated falciparum malaria were recruited from April to June 2006 and treated with either AL, quinine (Q) or atovaquone/proguanil (AP) in Jimma University Hospital, Ethiopia. Mutations or SNPs associated with resistance to these drugs were analysed by RFLP (pfdhfr, pfmdr1) and sequencing of the target genes (pfcytb, pfserca ). RESULTS: SNPs previously reported to be associated with resistance to the study drugs were identified in recrudescent and treatment sensitive isolates. A total of seven recrudescences were obtained. The pfmdr1 N86Y mutation was found in 84.5% of isolates. The triple mutation 51I,59R,108N of the pfdhfr gene occured in high frequency (83.3%) but no pfcytb mutation was detected. Sequencing showed a variety of previously described and new mutations in the pfserca gene. CONCLUSION: The prevalence of mutations was in accordance with the expected patterns considering recent drug regimens. The broad introduction of AL and the cessation of former drug regimens might probably change the current distribution of polymorphisms, possibly leading to decreased sensitivity to AL in future. Continuous surveillance of molecular patterns in this region is, therefore, recommended.

Ototoxicity of artemether/lumefantrine in the treatment of falciparum malaria: a randomized trial.

BACKGROUND: Due to increasing drug resistance, artemisinin-based combination chemotherapy (ACT) has become the first-line treatment of falciparum malaria in many endemic countries. However, irreversible ototoxicity associated with artemether/lumefantrine (AL) has been reported recently and suggested to be a serious limitation in the use of ACT. The aim of the study was to compare ototoxicity, tolerability, and efficacy of ACT with that of quinine and atovaquone/proguanil in the treatment of uncomplicated falciparum malaria. METHODS: Ninety-seven patients in south-west Ethiopia with slide-confirmed malaria were randomly assigned to receive either artemether/lumefantrine or quinine or atovaquone/proguanil and followed-up for 90 days. Comprehensive audiovestibular testing by pure tone audiometry (PTA), transitory evoked (TE) and distortion product (DP) otoacoustic emissions (OAE) and brain stem evoked response audiometry (BERA) was done before enrolment and after seven, 28 and 90 days. RESULTS: PTA and DP-OAE levels revealed transient significant cochlear hearing loss in patients treated with quinine but not in those treated with artemether/lumefantrine or atovaquone/proguanil. TE-OAE could be elicited in all examinations, except for three patients in the Q group on day 7, who suffered a transient hearing loss greater than 30 dB. There was no evidence of drug-induced brain stem lesions by BERA measurements. CONCLUSION: There was no detrimental effect of a standard oral regimen of artemether/lumefantrine on peripheral hearing or brainstem auditory pathways in patients with uncomplicated falciparum malaria. In contrast, transient hearing loss is common after quinine therapy and due to temporary outer hair cell dysfunction.

High prevalence of drug-resistance mutations in Plasmodium falciparum and Plasmodium vivax in southern Ethiopia.

BACKGROUND: In Ethiopia, malaria is caused by both Plasmodium falciparum and Plasmodium vivax. Drug resistance of P. falciparum to sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) is frequent and intense in some areas. METHODS: In 100 patients with uncomplicated malaria from Dilla, southern Ethiopia, P. falciparum dhfr and dhps mutations as well as P. vivax dhfr polymorphisms associated with resistance to SP and P. falciparum pfcrt and pfmdr1 mutations conferring CQ resistance were assessed. RESULTS: P. falciparum and P. vivax were observed in 69% and 31% of the patients, respectively. Pfdhfr triple mutations and pfdhfr/pfdhps quintuple mutations occurred in 87% and 86% of P. falciparum isolates, respectively. Pfcrt T76 was seen in all and pfmdr1 Y86 in 81% of P. falciparum. The P. vivax dhfr core mutations N117 and R58 were present in 94% and 74%, respectively. CONCLUSION: These data point to an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia, and strongly support that both SP and CQ are inadequate drugs for this region.

Validity of malaria diagnosis in nonimmune travelers in endemic areas.

BACKGROUND: Malaria has to be considered in all febrile travelers during or after a stay in endemic areas. However, malaria diagnosis in endemic countries may be inaccurate due to limited capacity and lack of resources of local health services. To assess the validity of malaria diagnosis in travelers in endemic areas, we investigated the retrospective confirmation of malaria by detection of specific antibodies. METHODS: Sera of 105 nonimmune travelers who presented between 2003 and 2005 with a history of diagnosis and treatment of malaria during a stay in malaria-endemic countries within the previous 6 months were analyzed for antibodies against Plasmodium falciparum and Plasmodium vivax blood forms by an indirect immunofluorescence test. About 241 follow-up sera from 176 nonimmune patients with microscopically confirmed malaria served as a control group. RESULTS: Antibodies against plasmodia were detectable within 180 days after reported date of diagnosis and treatment in 16 of 105 travelers (15.2%) only. In the control group, 71.6% of analyzed sera (151 of 211) showed positive results within this interval. Within 8 to 60 days after diagnosis of malaria, the seropositivity rates were 17.9% for travelers (n = 56) and 92.4% for controls (n = 92). CONCLUSIONS: Although the sensitivity of malaria serology for retrospective confirmation of malaria is limited, the results of this analysis strongly suggest that the majority of travelers with a recent history of malaria diagnosed and treated in endemic countries did not have malaria and that diagnosis of malaria during travel in endemic areas is frequently incorrect.

Viral NS3 helicase activity is inhibited by peptides reproducing the Arg-rich conserved motif of the enzyme (motif VI).

The NTPase/helicase of Flaviviridae viruses is one of the essential components of their replication complex. The enzyme is defined by the presence of seven highly conserved amino acid motifs. Random screening of numerous hepatitis C virus (HCV) derived peptides, revealed a basic amino acid stretch corresponding to motif VI of the HCV NTPase/helicase (amino acids 1487-1500 of the HCV polyprotein). This peptide inhibited the unwinding activity of the enzyme with an IC(50)=0.2 microM. Peptides corresponding to motif VI of HCV, West Nile virus (WNV) and Japanese encephalitis virus (JEV) were synthesized and tested as inhibitors of NTPase and unwinding reactions mediated by the viral enzymes. Peptides distinguished in regard to their length and structure. Between the peptides tested HCV(1487-1500) reproducing the sequence of motif VI was the most potent inhibitor of helicase activities of investigated enzymes. Other respective peptides were rather modest inhibitors. The examined peptides inhibited the Flaviviridae helicases in the following order of potency: HCV(1487-1500)>WNV(1959-1572)>JEV(1962-1975). Interestingly, the susceptibility of the helicase activity to the inhibition by the peptides was similar and in the row: HCV>WNV>JEV. The inhibition results from binding and blockade of the active site of the enzyme lyes beyond the NTP-binding and hydrolyzing site. The kinetic analyses indicated that the binding of the peptides do not interfere with the NTPase activity of the enzymes. The peptide may serve as effective and selective tool to reduce the virus propagation.