Amha Kebede

The use of real-time PCR to identify Entamoeba histolytica and E. dispar infections in prisoners and primary-school children in Ethiopia

Abstract In Ethiopia, it is generally unknown what proportion of the amoebic infections commonly found, by microscopy, in humans are caused by non-invasive Entamoeba dispar rather than the potentially invasive E. histolytica. Faecal samples were therefore collected from 363 primary-school students and 409 prisoners from various regions of Ethiopia. Each of these samples was checked for Entamoeba infection by the microscopical examination of formol-ether concentrates. DNA was then extracted from the 213 samples (27.6%) found Entamoeba-positive, and run in a real-time PCR with primers, based on the SSU-rRNA gene sequences of E. histolytica and E. dispar, that allow DNA from the two species to be distinguished. Although E. dispar DNA was identified in 195 (91.5%) of the 213 samples checked by PCR, no E. histolytica DNA was detected. This finding is consistent with the conclusion of a previous, smaller investigation: that many amoebic infections in Ethiopia are incorrectly attributed to E. histolytica and then treated, unnecessarily, with amoebicidal drugs.

Overdiagnosis of amoebiasis in the absence of Entamoeba histolytica among patients presenting with diarrhoea in Wonji and Akaki, Ethiopia

To confirm the high reported incidence of intestinal amoebiasis among study participants at 2 cohort sites in Ethiopia where an HIV/AIDS study is taking place, stool samples of 232 patients with complaints of diarrhoea were examined for the presence of Entamoeba histolytica and E. dispar DNA between April and December 2001. By microscopy, 91 (39%) of the study participants were reported to harbour Entamoeba trophozoites and/or four-nucleated cysts. Using specific E. histolytica and E. dispar DNA amplification and detection, none of the study participants were found to be infected with E. histolytica and only 21 (9%) with E. dispar. The consequences of the overdiagnosis of E. histolytica are briefly discussed.

Short communication: Misleading microscopy in amoebiasis

High prevalences of intestinal amoebiasis are commonly reported by microscopy in Ethiopia. In order to confirm the actual occurrence of Entamoeba histolytica we collected 108 stool specimens from different hospitals & health centers from patients in whom haematophagous trophozoites were believed to be found. We detected only a single E. histolytica case while 77 (71.3%) were E. dispar and the remaining 30 samples were negative for both species by real‐time PCR based on the small subunit ribosomal RNA gene sequence of E. histolytica and E. dispar. The tradition of microscopy in a routine diagnostic set‐up appears unsatisfactory to reliably differentiate rbc‐engulfing amoeba from non‐invasive amoeba in wet smears.

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

BackgroundThe 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.MethodsA 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.ResultsAllelic 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).ConclusionThis 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.

Comparison of Parascreen Pan/Pf, Paracheck Pf and light microscopy for detection of malaria among febrile patients, Northwest Ethiopia

Two malaria rapid diagnostic tests (RDT), Parascreen Pan/Pf and Paracheck Pf, were tested in rural health centres in Ethiopia against independent expert microscopy (the gold standard). Participants (n =1997) presented with presumptive malaria to ten health centers in Amhara Regional State during the 2007 peak malaria season (October to December). By microscopy, 475 (23.8%) suspected malaria cases were positive, of which 57.7% were P. falciparum; 24.6% P. vivax and 17.7% mixed infections. Parascreen and Paracheck were positive for 442 (22.1%) and 277 (13.9%) febrile patients, respectively. For Parascreen, P. falciparum sensitivity was 79.6%, specificity 97.4%, positive predictive value (PPV) 86.9%, and negative predictive value (NPV) 95.6%. For Parascreen, P. vivax sensitivity was 74.4%, specificity 98.6%, PPV 76.3% and NPV 98.4%. For Paracheck, P. falciparum sensitivity was 73.7%, specificity 99.2%, PPV 95.3%, NPV 94.5%. Sensitivity was significantly higher for both tests (P<0.05) when parasite density was >100/microl of blood; in these cases Parascreen was 90.7% and 91.5% sensitive for P. falciparum and P. vivax, respectively, while Paracheck was 87.9% sensitive for P. falciparum. Parascreen thus performed adequately for both P. falciparum and P. vivax compared to expert microscopy and is more useful than Paracheck where microscopy is unavailable.

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

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.

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.

Human resource capacity to effectively implement malaria elimination: A policy brief for Ethiopia

OBJECTIVE The aim of this study was to investigate malaria elimination in Ethiopia. Ethiopia has planned to eliminate malaria by 2015 in areas of unstable malaria transmission and in the entire country by 2020. However, there is a shortage and maldistribution of the health workforce in general and malaria experts in particular. Training, motivating, and retaining the health workforce involved in malaria control is one strategy to address the shortage and maldistribution of the health workforce to achieve the goal of elimination. METHODS Policy options include the following: (i) in-service training (educational outreach visits, continuing education meetings and workshops, audit and feedback, tailored interventions, and guideline dissemination) may improve professional practice; (ii) recruiting and training malaria specialists together with academic support, career guidance, and social support may increase the number of malaria experts; and (iii) motivation and retention packages (such as financial, educational, personal, and professional support incentives) may help motivate and retain malaria professionals. RESULTS Implementation strategies include the following: (i) massive training of health personnel involved in malaria elimination and malaria experts (requiring special training) at different levels (national, sub-national, District & community levels), and (ii) recruiting highly qualified health personnel and retention and motivation mechanisms are needed. CONCLUSIONS The lack of adequately trained human resources and personnel attrition are major challenges to effectively implement the planned multi-faceted malaria elimination by 2020 strategy in Ethiopia. Although a reduction in malaria incidence has been observed in the last 3-4 years, maintaining this success and achieving the malaria elimination goal with the present human resource profile will be impossible. A clear strategy for developing the capacity of the health workers in general, and malaria experts in particular, and retaining and motivating staff are crucial for malaria control and elimination.

In Vivo and In Vitro Cross Neutralization Studies of Local Rabies Virus Isolates with ERA Based Cell Culture Anti-Rabies Vaccine Produced In Ethiopia

Rabies is a worldwide problem, and the case is most severe in developing countries where cell culture anti-rabies vaccines are unaffordable or the available nervous tissue-derived vaccines are of questionable immunogenicity and may cause neurological complications. The aim of this research was to study cross protection of local rabies virus isolates with Evenyl Roktincki Abelseth (ERA) based cell culture anti-rabies vaccine produced in Ethiopia and to develop challenge virus from local isolates. The viruses were isolated from rabid dogs’ brains and human saliva, and adapted on Swiss albino mice and cell lines. Cross protection with ERA based vaccine was studied by in vivo and in vitro methods. For in vivo method, a group of mice were immunized on day zero and seven with 0.5 ml (1:5 dilutions) of ERA based cell culture anti-rabies vaccine produced locally. On day fourteenth, mice were challenged with working dilution of each local isolates and one group with challenge virus standard (CVS-11), and observed for further 14 days. High protection was recorded in CVS-11 challenged mice and low protection in all local isolates (p=0.045); specifically protection to HOS challenged mice was very low. In vitro test was done by fluorescent antibody virus neutralization (FAVN) test on BHK-21 cell lines. Sera from dog immunized with locally produced vaccine and OIE serum were incubated with local virus isolates and CVS-11 for 48 hours in the presence of cell lines. Maximum antibody titer (2.74 IU/ml) was obtained with CVS-11 challenge virus and minimum antibody titer (1.55 IU/ml) was obtained with cow origin (CO) virus isolate. All locally isolated rabies virus show low antibody titer when compared to CVS-11 and PV-12 (p=0.000). From the results, it can be concluded that local isolates have some genetic variation from fixed virus strain which can affect efficacy of the candidate vaccine and potency value should be set in-terms of local isolate using as challenge virus. Generally, the exact genetic relationship should be studied by molecular techniques and locally isolated virus should be used as challenge virus for vaccine quality control.