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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Method</p> <p>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 (<it>pfdhfr</it>, <it>pfmdr1</it>) and sequencing of the target genes (<it>pfcytb</it>, <it>pfserca </it>).</p> <p>Results</p> <p>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 <it>pfmdr1 </it>N86Y mutation was found in 84.5% of isolates. The triple mutation 51I,59R,108N of the <it>pfdhfr </it>gene occured in high frequency (83.3%) but no <it>pfcytb </it>mutation was detected. Sequencing showed a variety of previously described and new mutations in the <it>pfserca </it>gene.</p> <p>Conclusion</p> <p>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.</p

Open-label trial with artemether-lumefantrine against uncomplicated Plasmodium falciparum malaria three years after its broad introduction in Jimma Zone, Ethiopia

Background: In Jimma Zone, Ethiopia, the first-line treatment of uncomplicated falciparum malaria has been changed from sulphadoxine-pyrimethamine (SP) to artemether-lumefantrine (AL) in 2006. The objective of this study was to assess the effectiveness of AL in Jimma Zone two to three years after its broad introduction. Methods: An open-label, single-arm, 42-day study of AL against falciparum malaria was conducted in four areas with moderate transmission in Jimma Zone between November 2008 and January 2009 and between August and December 2009. Patients (one-81 years) with uncomplicated Plasmodium falciparum mono-infection were consecutively enrolled. Follow-up visits were at day 2, 3, 7, 28 and 42 or any other day if symptoms reoccurred. Primary and secondary endpoints were PCR-corrected and uncorrected cure rates (molecular differentiation between recrudescence and re-infection) on days 28 and 42. Other secondary endpoints were gametocytaemia at day 7 and day 28, parasitaemia at day 2 and 3, and re-infection rates at day 28 and day 42. Results: Of 348 enrolled patients, 313 and 301 completed follow-up at day 28 and at day 42, respectively. No early treatment failure occurred. For per protocol analysis, PCR-uncorrected cure rates at day 28 and 42 were 99.1% (95% CI 98.0-100.0) and 91.1% (95% CI 87.9-94.3), respectively. PCR-corrected cure rates at day 28 and 42 were 99.4% (95% CI 98.5-100.0) and 94.7% (95% CI 92.2-97.2), respectively. PCR-corrected cure rate at day 42 for children &lt;= 5 years was 90.6% (95% CI 82.4-98.7) only. Adverse events were in general mild to moderate. Incidence of new infections was 3.4% during 42 days, no new infections with Plasmodium vivax were observed. Microscopically detected gametocytaemia was reduced by 80% between day 0 and day 7. Conclusion: In general, AL was effective and well tolerated in Jimma Zone, Ethiopia. However, the PCR-corrected recrudescence rate per-protocol at day 42 for children &lt;= 5 years was 9.4%. Therefore, further development should be monitored on a regular basis as recommended by WHO

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/mul. 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 muL, 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

Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions

Background: Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. Methods: Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. Results: Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at -20 degrees C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37 degrees C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per mu l. Conclusions: The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations

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

Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 10 5 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases

Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 10 5 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases

Severe Plasmodium knowlesi infection with multi-organ failure imported to Germany from Thailand/Myanmar

During the last two decades human infections with Plasmodium knowlesi are increasingly diagnosed in South East Asia and have also been reported in travellers. A severe case of imported P. knowlesi infection in a 73-year old German is presented, who had been travelling through Myanmar and Thailand for three weeks. Microscopy showed a parasitaemia of 3% and different parasite stages including band-forms resembling Plasmodium malariae. Due to the clinical picture of severe malaria and the microscopical aspect (combination of parasites resembling P. malariae and Plasmodium falciparum), P. knowlesi was suspected. The patient was treated with intravenous quinine; he was put on mechanical ventilation and catecholamines due to cardiorespiratory failure. Parasitaemia was cleared rapidly but renal function deteriorated resulting in intermittent haemodialysis. The patient was hospitalized for six weeks but he recovered completely without any physical sequelae. Plasmodium knowlesi mono-infection was confirmed by molecular methods later on. Plasmodium knowlesi infection has to be taken into account in feverish travellers returning from Thailand/Myanmar. Moreover this species can cause life-threatening or even lethal complications. Accordingly severe P. knowlesi infection should be treated like severe P. falciparum infections