Glucose-6-phosphate dehydrogenase activity measured by spectrophotometry and associated genetic variants from the Oromiya zone, Ethiopia.

BACKGROUND: The study aimed to gain first data on the prevalence of G6PD enzyme deficiency measured by spectrophotometry and associated genetic variants in Jimma and surroundings, Ethiopia. The area is a Plasmodium vivax endemic region, but 8-aminoquinolines such as primaquine are not recommended as G6PD testing is not available. METHODS: Healthy volunteers were recruited at Jimma University, Ethiopia. Enzyme activity was tested by spectrophotometry at the University of Ulm, Germany. A G6PD RDT (Binax NOW® G6PD, Alere, USA) was additionally performed. The G6PD gene was analysed for polymorphisms in a sub-population. Tests for haemoglobinopathies and the presence of malaria parasites were conducted. RESULTS: No severe or moderate (cut-off 60%) G6PD deficiency was found in 206 volunteers. Median male activity was 6.1 U/g Hb. Eleven participants (5.4%) showed activities between 70 and 80%. No haemoglobinopathy was detected. None of the subjects showed asymptomatic parasitaemia. One G6PD-A+ variant (A376G) and one new non-synonymous mutation (G445A) were found. CONCLUSIONS: As the prevalence of G6PD deficiency seems low in this area, the use of 8-aminoquinolines should be encouraged. However, reliable G6PD testing methods have to be implemented and safe cut-off levels need to be defined.

Similar trends of susceptibility in Anopheles arabiensis and Anopheles pharoensis to Plasmodium vivax infection in Ethiopia.

BACKGROUND: Around half of the global population is living in areas at risk of malaria infection. Plasmodium vivax malaria has become increasingly prevalent and responsible for a high health and socio-economic burden in Ethiopia. The availability of gametocyte carriers and mosquito species susceptible to P. vivax infection are vital for malaria transmission. Determining the susceptibility of vector species to parasite infection in space and time is important in vector control programs. This study assesses the susceptibility of Anopheles arabiensis, An. pharoensis and An. coustani group to Plasmodium vivax infection in Ethiopia. METHODS: Larvae of An. arabiensis, An. pharoensis and An. coustani group were collected from an array of breeding sites and reared to adult under controlled conditions. Batches of adult female mosquitoes of the three species were allowed to feed in parallel on the same infected blood with gametocytes drawn from Plasmodium vivax infected patients by Direct Membrane Feeding Assays (DMFA). Fed mosquitoes were kept in an incubator under controlled laboratory conditions. Seven days after each feeding assay, mosquitoes were dissected for midgut oocyst microscopy and enumeration. Data were analysed using R statistical software package version 3.1.0. RESULTS: Over all, 8,139 adult female mosquitoes were exposed to P. vivax infection. Of the exposed mosquitoes 16.64 % (95 % CI: 1,354-8,139) were properly fed and survived until dissection. The infection rate in An. arabiensis and An. pharoensis was 31.72 % (95 % CI: 28.35-35.08) and 28.80 % (95 % CI: 25.31-32.28), respectively. The intensity of infection for An. arabiensis and An. pharoensis was 2.5 (95 % CI: 1.9-3.2) and 1.4 (95 % CI: 1.1-1.8), respectively. Gametocyte density was positively correlated to infection rate and intensity of infection in An. arabiensis as well as An. pharoensis. No An. coustani group mosquitoes were found infected, though almost four hundred mosquitoes were successfully fed and dissected. All groups received blood from the same infected blood source containing gametocytes in parallel. There was no significant difference in susceptibility rates between An. arabiensis and An. pharoensis (P = 0.215). CONCLUSIONS: Anopheles arabiensis and An. pharoensis showed similar susceptibility to P. vivax infection. However, An. coustani group was not permissive for the development of P. vivax parasites.

Molecular markers of anti-malarial drug resistance in southwest Ethiopia over time: regional surveillance from 2006 to 2013.

BACKGROUND: Drug resistance is one of the main reasons of anti-malarial treatment failures and impedes malaria containment strategies. As single nucleotide polymorphisms (SNPs) have been found to correlate with anti-malarial drug resistance, the surveillance strategy includes continuous monitoring of known molecular markers and detection of new mutation patterns. With the introduction of artemisinin-based combination therapy, selection of specific patterns has been observed worldwide. METHODS: From March to June 2013, whole blood was collected on filter paper from microscopically malaria positive patients in Jimma zone (District), southwestern Ethiopia. Plasmodium falciparum, Plasmodium vivax and mixed infections were included. SNPs were investigated by conventional or real-time PCR, restriction fragment length pattern analysis or sequencing. Results were compared to molecular patterns from Ethiopian isolates in 2004, 2006 and 2008/9. RESULTS: Plasmodium falciparum, P. vivax, and mixed infections were molecularly confirmed in 177, 80, and 14 samples, respectively. In P. falciparum, mutations in the pfcrt, pfmdr 1and pfATP 6 (SERCA) gene were investigated. Whereas the mutation in the pfcrt gene at codon 76 K was still found in 95.6% of all samples, the pfmdr 1 86 T mutation fell to 1.2% (2/163) in 2013 compared to 9% in 2008/9 and 86% in 2006 (P<0.001). The pfmdr 1 184 F mutation dominated with 100.0% (172/172) in 2013. Sequencing of the recently reported PF3D7_1343700 kelch propeller domain showed no mutation at codon 476. First sequencing data of the pvmdr 1 gene from Jimma region revealed a prevalence of the mutations 976 F and 1076 L in 72.7% (16/23) and 100.0% (19/19) of the isolates, respectively. CONCLUSION: Since the introduction of artemether-lumefantrine (AL) in Jimma, Ethiopia, in 2006, the prevalence of certain SNPs associated with AL use has increased. Markers for chloroquine resistance in P. vivax were highly frequent. Continuous molecular and clinical surveillance are of paramount importance.

Evaluation of Plasmodium falciparum gametocyte detection in different patient material.

BACKGROUND: For future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. Sensitive, non-invasive methods to detect gametocytes under field conditions can play a role in monitoring transmission potential. METHODS: Microscopically Plasmodium falciparum-positive patients from Jimma, Ethiopia donated finger-prick blood, venous blood, saliva, oral mucosa and urine samples that were spotted on filter paper or swabs. All samples were taken and stored under equal, standardized conditions. RNA was extracted from the filter paper and detected by real-time QT-NASBA. Pfs16-mRNA and Pfs25-mRNA were measured with a time to positivity to detect gametocyte specific mRNA in different gametocyte stages. They were compared to 18S-rRNA, which is expressed in all parasite stages. Results were quantified via a known dilution series of artificial RNA copies. RESULTS: Ninety-six samples of 16 uncomplicated malaria patients were investigated. 10 (66.7%) of the slides showed gametocyte densities between 0.3-2.9 gametocytes/µl. For all RNA-targets, molecular detection in blood samples was most sensitive; finger-prick sampling required significantly smaller amounts of blood than venous blood collection. Detection of asexual 18S-rRNA in saliva and urine showed sensitivities of 80 and 67%, respectively. Non-invasive methods to count gametocytes proved insensitive. Pfs16-mRNA was detectable in 20% of urine samples, sensitivities for other materials were lower. Pfs25-mRNA was not detectable in any sample. CONCLUSIONS: The sensitivity of non-invasively collected material such as urine, saliva or mucosa seems unsuitable for the detection of gametocyte-specific mRNA. Sensitivity in asymptomatic carriers might be generally even lower. Finger-prick testing revealed the highest absolute count of RNA copies per µL, especially for Pfs25-mRNA copies. The method proved to be the most effective and should preferably be applied in future transmission control and eradication plans. A rapid test for gametocyte targets would simplify efforts.