Therapeutic efficacy of chloroquine for the treatment of Plasmodium vivax malaria among outpatients at Hossana Health Care Centre, southern Ethiopia.

BACKGROUND: Plasmodium vivax accounts for about 44 % of all malaria infection in Ethiopia. Chloroquine (CQ) is the first-line treatment for vivax malaria in Ethiopia. Chloroquine-resistant (CQR) P. vivax has been emerging in different parts of the world to compromise the efficacy of the drug and pose both health and economic impact in the developing world. The current study was aimed at assessing the therapeutic efficacy of CQ for the treatment of vivax malaria among outpatients at Hossana Health Care Centre, southern Ethiopia. METHODS: A one-arm, 28-day follow-up, in vivo therapeutic efficacy study was conducted from 5 April to 25 June, 2014. Sixty-three patients aged between four and 59 years were enrolled with microscopically confirmed P. vivax infection. All patients were treated with CQ 25 mg/kg for 3 days. Recurrence of parasitaemia and clinical conditions of patients were assessed on days 1, 2, 3, 7, 14, 21, and 28 during the 28-day follow-up period. Haemoglobin (Hb) level was determined on day 0, day 28 and on day of recurrence of parasitaemia by using portable spectrophotometer. RESULTS: Of the total 63 patients included in the study, 60 (95.2 %) completed their 28-day follow-up; three patients were excluded from the study: one patient due to vomiting of the second dose of drug, one patient due to Plasmodium falciparum infection and one patient lost to follow-up during the study. During enrolment, 35 (53.3 %) had a history of fever and 28 (46.7 %) had documented fever. The geometric mean of parasite density on day of enrolment was 3472 parasites/µl. Among these, two patients had recurrent parasitaemia within the 28-day follow-up. CQ was found to be efficacious in 96.7 % of the study participants except two treatment failures detected. The failure might be due to late parasitological failure among these two patients who had recurrent parasitaemia within the 28-day follow-up. CONCLUSION: The current study revealed that CQ showed a high rate of efficacy (96.7 %) among the study participants even though some reports from previous studies elsewhere in Ethiopia showed an increase in CQR P. vivax. Thus, CQR molecular markers and regular monitoring of the pattern of resistance to CQ is needed for rapid and effective control measures of possible spread of drug resistance in the study area.

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.

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 ≤ 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 ≤ 5 years was 9.4%. Therefore, further development should be monitored on a regular basis as recommended by WHO.

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°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°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 µl. 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.

Assessment of Intestinal Parasites, Enteric Bacterial Infections, and Antimicrobial Susceptibility among Street Food Handlers in Jimma Town, Southwest Ethiopia.

Background: Food-borne infections are common public health problems worldwide. A street food handler with poor personal hygiene contributes to the transmission of intestinal parasites and enteric bacteria to the public via contaminated foods. In Ethiopia, health risks associated with street food are common. Previous studies in this area are scanty. Hence, the aim of this study was to determine the prevalence of intestinal parasites, enteric bacterial infections, and antimicrobial susceptibility among street food handlers in Jimma town. Methods: A cross-sectional study was conducted from October to December 2020 among 260 street food handlers in Jimma town. A semi-structured questionnaire was used to collect data through face-to-face interviews. About 3 grams of the fecal specimen were collected from each food handler for bacterial culture and concentration techniques. The data were entered into Epi-Data 3.1 and analyzed by SPSS version 20. Associated factors were identified by using binary logistic regression analysis. A statistically significant association was determined at a p-value less than 0.05. Results: The overall prevalence of intestinal parasites and enteric bacterial pathogens was 39.2% (33.3%-45.2%) and 8.85% (5.4%-12.3%), respectively. Ascaris lumbricoides (18.5%) and Salmonella (8.1%) were the most predominant parasite and enteric bacterial isolates, respectively. Not trimming fingernails (AOR = 2.884; 95% CI: 1.682-4.945) and not washing hands with soap after toilet (AOR = 3.342; 95% CI: 1.939-5.761) were factors associated with increased risk of infection by intestinal parasites or enteric bacterial pathogens. All Salmonella and Shigella isolates were 100% resistant to ampicillin. Conclusion: The infection with intestinal parasites and enteric bacterial pathogens detected in this study indicated that street food handlers may serve as sources of pathogens/parasites for transmission and experience morbidities due to the infections. Therefore, periodic medical checkups and creating awareness of personal hygiene are mandatory to reduce the risk of infections.

Asymptomatic Plasmodium infection and associated factors among pregnant women in the Merti district, Oromia, Ethiopia.

BACKGROUND: Asymptomatic Plasmodium infection (API) that occurs during pregnancy increases the risk of stillbirths, abortion, premature delivery, and low birth weight. API also hinders the control and prevention of malaria as infected hosts serve as silent reservoirs for transmission of Plasmodium species in the community. OBJECTIVE: The aim of this study was to determine the prevalence of API and associated factors among pregnant women. This community-based cross-sectional study was conducted at Merti district, Oromia, Ethiopia among 364 pregnant women from March to September 2018. METHODS: Sociodemographic and obstetrics features were collected using a structured questionnaire. About 2ml of blood was collected from participants to detect Plasmodium species, gametocyte carriage rate, parasite density, and anemia. RESULTS: The prevalence of API among pregnant women was 3.6%. The proportion of Plasmodium falciparum and Plasmodium vivax was 6(46.2%) and 7(53.8%) respectively. Out of 13 Plasmodium species identified, Gametocyte carriage rate was 4(30.7%). The geometric mean density of the asexual stage of the parasites was 994.7(interquartile [IQR], 320 to 2200) parasites/ul. The geometric mean gametocyte density was 303.3 (interquartile range [IQR], 160 to 600). The proportion of anemia among Plasmodium-infected participants was 12(92.3%). Previous infection by Plasmodium species (AOR = 5.42; 95% CI: 1.19-29.03, p = 0.047), lack of insecticide-treated bed net use (AOR = 6.52; 95% CI: 1.17-36.44, p = 0.032), and living close to stagnant water (AOR = 4.18; 95% CI (1.12-17.36, p = 0.049) were significantly associated with API. Anemia was significantly higher among Plasmodium-infected than non-infected pregnant women (x2 = 27.62, p <0.001). CONCLUSION: In the current study, a relatively high prevalence of API was detected among pregnant women. Identifying API in the community is important to prevent the unwanted outcomes of Plasmodium infection and its transmission.

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.

Anemia associated with asymptomatic malaria among pregnant women in the rural surroundings of Arba Minch Town, South Ethiopia.

BACKGROUND: Anemia during pregnancy is a well known medical condition most of the time under-recognized as it is overshadowed by the normal physiological condition during pregnancy. This study aimed at determining the prevalence and predictors of anemia among pregnant women residing in the rural surroundings of Arbaminch Town, south Ethiopia. METHODS: A cross-sectional community based study was conducted between April and June, 2013. A structured questionnaire was used to collect socio-economic and socio-demographic characteristics of the pregnant women. Hematocrit (HCT) level was determined to classify the pregnant women as anemic and non-anemic. Diagnosis of asymptomatic malaria parasitemia was done by Giemsa stained blood smear microscopy. HCT < 33%, (HCT ≥ 30% & < 33%), (HCT ≥ 21% & < 30%), and HCT < 21% was used to indicate anemia, mild anemia, moderate anemia, and severe anemia respectively. RESULTS: A total of 341 pregnant women participated in this study, out of which 118 (34.6%) were anemic. The median age of the pregnant women was 25 years (Inter-quartile range: 23-29). The mean HCT was 35.2% (95% CI: 34.6%-35.8%) with SD of ±5.5%. Of those 118 anemic women; 73(61.9%) were mildly anemic, 38(32.2%) were moderately anemic, and 7(5.9%) were found to be severely anemic. The prevalence of asymptomatic malaria parasitemia was 9.1% (31/341). The odds of being anemic were 15.72 times [AOR: 15.72, 95% CI (3.97, 62.22), P-value ≤ 0.001] more likely to occur in parasitemic individuals relative to the non parasitemic pregnant women. Not using insecticide treated bed net (ITN) was a significant predictor of anemia among the pregnant women [AOR: 3, 95% CI: (1.72, 5.22), P < 0.001]. CONCLUSION: This study highlighted the significant association between anemia and asymptomatic malaria among pregnant women in the study area. Therefore, the practice of routine screening for malaria and anemia followed by prompt management should be encouraged to curb the effect of malaria and anemia on the pregnant women as well as her fetus. Further studies should also be in place to test the effectiveness of routine screening for malaria and anemia followed by prompt management.

Prevalence and predictors of asymptomatic malaria parasitemia among pregnant women in the rural surroundings of Arbaminch Town, South Ethiopia.

BACKGROUND: In Sub-Saharan African countries, including Ethiopia, malaria in pregnancy is a major public health threat which results in significant morbidities and mortalities among pregnant women and their fetuses. In malaria endemic areas, Plasmodium infections tend to remain asymptomatic yet causing significant problems like maternal anemia, low birth weight, premature births, and still birth. This study was conducted to determine the prevalence and predictors of asymptomatic Plasmodium infection among pregnant women in the rural surroundings of Arba Minch Town, Southern Ethiopia. METHODS: A community based cross-sectional study comprising multistage sampling was conducted between April and June, 2013. Socio-demographic data were collected by using a semi-structured questionnaire. Plasmodium infection was diagnosed by using Giemsa-stained blood smear microscopy and a rapid diagnostic test (SD BIOLINE Malaria Ag Pf/Pv POCT, standard diagnostics, inc., Korea). RESULTS: Of the total 341 pregnant women participated in this study, 9.1% (31/341) and 9.7% (33/341) were confirmed to be infected with Plasmodium species by microscopy and rapid diagnostic tests (RDTs), respectively. The geometric mean of parasite density was 2392 parasites per microliter (µl); 2275/ µl for P. falciparum and 2032/ µl for P. vivax. Parasitemia was more likely to occur in primigravidae (Adjusted odds ratio (AOR): 9.4, 95% confidence interval (CI): 4.3-60.5), secundigravidae (AOR: 6.3, 95% CI: 2.9-27.3), using insecticide treated bed net (ITN) sometimes (AOR: 3.2, 95% CI: 1.8- 57.9), not using ITN at all (AOR: 4.6, 95% CI: 1.4-14.4) compared to multigravidae and using ITN always, respectively. CONCLUSION: Asymptomatic malaria in this study is low compared to other studies' findings. Nevertheless, given the high risk of malaria during pregnancy, pregnant women essentially be screened for asymptomatic Plasmodium infection and be treated promptly via the antenatal care (ANC) services.

Parasitic Contamination of Fruits and Vegetables Collected from Selected Local Markets of Jimma Town, Southwest Ethiopia.

Background. A study aimed at determining the prevalence and predictors of parasitic contamination of fruits and vegetables collected from local markets in Jimma Town, Ethiopia, was conducted between April and May 2013. Methods. A total of 360 samples of fruits and vegetables were examined by sedimentation concentration after washing using normal saline. Results. The overall prevalence of parasitic contamination was 57.8%. Strongyloides like parasite (21.9%) was the most frequent parasitic contaminant followed by Toxocara Spp (14.7%), Cryptosporidium Spp (12.8%), H. nana (8.3%), G. lamblia (7.5%), A. lumbricoides (6.7%), E. histolytica/dispar (5.3%), Cyclospora spp (5.0%), and H. diminuta (1.4%). Washing of the fruits and vegetables before display for selling was significantly associated with decreased parasitic contamination (P < 0.001). Conclusion. Since fruits and vegetables are potential sources of transmission for intestinal parasites in the study area, consumers should always avoid acquiring parasitic infection from contaminated fruits and vegetables supplied in Jimma Town through proper cleaning and cooking.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.